bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Analysis of the Role of Phosphorylated PCNA in Cancer Development and its Potential in Cancer Therapy
Authors: Peng Bo , Jiang Qingwen , Ortega Janice , Gu Liya , Dai Junbiao , Li Guo‐Min 
Published: November 2023

Proliferating cell nuclear antigen (PCNA), a ring‐shaped homotrimer, is an essential factor in DNA metabolism including DNA replication and repair. PCNA functions in these reactions appear to be regulated by posttranslational modifications, such as ubiquitylation, sumoylation and phosphorylation. Recently, we have shown that PCNA phosphorylation by EGFR at tyrosine 211 (Y211) disrupts PCNA interactions with mismatch recognition proteins, thereby inhibiting DNA mismatch repair (MMR). In addition, phosphorylated PCNA promotes misincorporation during DNA replication. However, it is unknown whether one, two or all three phosphorylated subunits are responsible for MMR inhibition and error‐prone DNA synthesis. To answer this question, we created a series of phosphorylation‐mimicking and non‐phosphorylation‐mimicking PCNA molecules with variously phosphorylated subunits, and examined their roles in MMR and DNA synthesis in vitro and in vivo. Our biochemical experiments reveal that all forms of phosphorylated PCNA inhibit MMR, with the inhibition activity proportional to the increased levels of phosphorylation; all forms of phosphorylated PCNA slow down the speed of DNA synthesis while conducting error‐prone DNA synthesis. Our in vivo data show that high‐levels of PCNA phosphorylation induce cell death. This study therefore provides novel mechanisms for phosphorylated PCNA in cancer development and its potential role in cancer therapy.Support or Funding InformationThis work is supported in part by grants from National Natural Science Foundation of China (31370766), by Tsinghua‐Peking Joint Center for Life Sciences, and by National Institutes of Health of the United States (GM089684).
Using Transcriptome Analysis to Solve the Puzzle of DNA Repair in Bdelloid Rotifers
Authors: Huber Christa Caye , Glazko Galina , Rahmatallah Yasir , MacLeod Stewart , Boerma Marjan , Schurko Andrew 
Published: November 2023

Bdelloid rotifers are microscopic aquatic animals that have apparently survived for more than 40 million years without sex, males, or meiosis. Bdelloids have an efficient DNA repair system that gives them the ability to recover from levels of ionizing radiation that are lethal to other eukaryotes. In bdelloids, DNA double strand breaks induced by high doses (>1000 Gray) of ionizing radiation are repaired without significant loss of viability. The objective of this project is to gain insight into the mechanisms of DNA repair in bdelloids by identifying genes involved in this process. This was done using transcriptome sequencing (RNA‐seq) to characterize genes that are differentially expressed during DNA repair in the bdelloid Adineta vaga. To induce DNA double‐strand breaks, bdelloid cultures were irradiated with a total dose of 280 Gray of ionizing radiation using a cesium‐137 source. RNA was then isolated from these cultures at three time points (0, 30 and 60 minutes) post‐irradiation and also from non‐irradiated controls. RNA‐seq was done using the TruSeq RNA Library Preparation Kit and the Illumina HiSeq 2500 platform. From this data, over 500 genes were differentially expressed during DNA repair. Real‐time PCR was done to validate some differentially expressed genes from RNA‐seq. Gene ontology enrichment analysis was carried out to identify biological processes and pathways that were differentially expressed in irradiated bdelloids. Many upregulated genes were related to DNA repair functions (e.g. ligation, non‐homologous end‐joining, DNA polymerases) while the majority of downregulated genes were associated with transcription, translation and cell cycle regulation. Interestingly, over 20% of differentially expressed genes are apparent “bdelloid‐specific genes,” meaning that they lack homologs in other eukaryotes. However, in some cases these bdelloid‐specific genes represent inadequately annotated gene models; for example, the Random Amplification of cDNA Ends (RACE) method was used to identify a previously unannotated map kinase‐activated protein kinase 2 homolog among these genes. Conversely, other differentially expressed genes appear to be genuine bdelloid‐specific genes since further investigation using computational and molecular methods failed to identify their homolog. Overall, this ongoing gene expression analysis has identified several candidate genes for having functions in DNA repair, which will provide further insight into the mechanism of DNA repair in bdelloid rotifers.Support or Funding InformationThis work was supported by grants from the National Center for Research Resources (5P20RR016460) & National Institute of General Medical Sciences (8P20GM103429) from the NIH.
An Investigation of Protein Function During DNA Repair in Bdelloid Rotifers
Authors: McReynolds Jeanita Cherise , Boerma Marjan , Byrum Stephanie D , Orr Lisa M , Tackett Alan J , Schurko Andrew 
Published: November 2023

Bdelloid rotifers are a class of aquatic microinvertebrates that have seemingly flourished for over 40 million years without sex, meiosis, or males. The long‐term success of this ancient ameiotic lineage is attributed, in part, to the extraordinary ability of bdelloids to repair DNA double strand breaks induced by desiccation. Our objective is to characterize proteins and their interactions during DNA repair in bdelloid rotifers. We first targeted meiotic proteins as a group of candidate DNA repair proteins. Despite the longstanding absence of meiosis in bdelloids, their genomes contain four genes that code for proteins (SPO11, HOP1, MSH4, MSH5) specific to meiosis in other eukaryotes. Western blot assays using custom antibodies for SPO11, HOP1, MSH4 and MSH5 were done to confirm that their corresponding genes were expressing proteins in the bdelloid Adineta vaga. To characterize binding partners of these meiotic proteins, protein pull‐down assays were then done. We generated 6×histidine‐tagged copies of each meiotic protein (and also of DNA repair proteins RAD51 and MRE11) and these were used in pull‐down assays with Dynabeads and protein lysates from A. vaga. SDS‐PAGE and mass spectrometry were used to identify potential binding partners. Next, to further our investigation into DNA repair proteins, we also identified other candidate DNA repair proteins using a whole proteome analysis of irradiated and non‐irradiated cultures of A. vaga with high‐resolution mass spectrometry. To induce DNA double‐strand breaks, bdelloid cultures were irradiated with a total dose of 280 Gray of ionizing radiation using a cesium‐137 source. Proteins were then isolated from these bdelloids and identified using tandem mass tag labeling and high‐resolution mass spectrometric analysis with a Thermo Velos Orbitrap mass spectrometer. These proteins will the targeted for future analyses using pull‐downs, yeast two‐hybrid and other techniques to further examine their role in DNA repair. Overall, this study aims to identify candidate proteins and characterize their roles in DNA repair in bdelloids, which will further our understanding of this process in rotifers and other eukaryotes.Support or Funding InformationThis work was supported by grants from the National Center for Research Resources (5P20RR016460) & National Institute of General Medical Sciences (8P20GM103429) from the NIH.
Sensitive and Specific Detection of Ligands Using Engineered Riboswitches
Authors: Laney Jessilyn P , Morse Daniel P 
Published: November 2023

Riboswitches are RNA elements found in non‐coding regions of messenger RNAs that regulate gene expression through a ligand‐triggered conformational change (for review, see ref. 1). In, B. subtilis and other gram positive bacteria, purine metabolism is regulated by riboswitches that bind to guanine (2). The best studied of the guanine riboswitches regulates the transcription of the B. subtilis xpt‐pbuX operon. Guanine binding to this riboswitch inhibits transcription by stabilizing an alternative conformation that allows formation of a premature transcriptional terminator. In order to study guanine‐triggered structure switching, we have developed a fluorescence quenching assay for the function of the guanine riboswitch. Our system consists of the guanine‐binding domain (aptamer domain) of the riboswitch (xpt RNA), and two DNA oligonucleotides that are related to the 5′ and 3′ halves of the terminator stem (5′T and 3′T). xpt RNA is labeled at its 3′ end with a fluorophore and 5′T is labeled at its 5′ end with a fluorescence quencher. When 5′T is annealed to a complementary sequence at the 3′ end of xpt RNA, fluorescence is quenched. Guanine binding causes 5′T to dissociate from xpt RNA and anneal to 3′T. This strand‐exchange reaction causes an increase in fluorescence intensity as the quencher moves away from the fluorophore. Using this assay, we reproducibly detected as little as 5 nM guanine. We are currently using the same strategy to adapt a 2′‐deoxyguanosine riboswitch (3) for use as a probe that can detect its ligand in vitro. In addition, we have used our strand‐exchange reaction to develop a method for in vitro selection of variant riboswitches with altered ligand specificity. The results of our latest experiments will be discussed.Support or Funding InformationDefense Threat Reduction Agency (DTRA), Office of Naval Research (ONR), Chemistry Department, U.S, Naval Academy
SOX18 transcription factor interactome: Protein‐Protein interaction a new road for anti‐cancer drug discovery
Authors: Moustaqil Mehdi Denis , Fontaine Frank , Overman Jeroen , Giles Nichole Denis , Bhumkar Akshay Denis , O'Caroll Ailis Denis , Francois Mathias , Gambin Yann Denis , Sierecki Emma Denis 
Published: November 2023

Deciphering how transcription factors (TFs) coordinate genes expression is fundamental to understand biological processes and further develop new therapeutic approaches. A single transcription factor has a multiple transcriptional effects that are context‐dependent. This versatility of activity is thought to be mediated by different protein‐protein interactions (PPIs). These PPIs offer a new avenue for the selective pharmacological modulation of transcription factor activity, which has proven to be a challenging endeavour using standard drug discovery approaches centred on blocking protein/DNA binding or interfering with post‐translational modifications.In this study we show that SOX18, a transcription factor a molecular switch of embryonic lymphatic vascular development and neo‐lymphangiogenesis during cancer metastasis, interacts with multiple protein partners specific to endothelial cells. Using a systematic truncation analysis of SOX18 domains, we propose a comprehensive map of the protein binding domains, revealing the existence of different interacting regions specific to subsets of SOX18 interactors. Using mutant forms of SOX18 protein associated with human rare disease Hypotrichosis‐Lymphedema‐Telangiectasia (HLT) we validate domains essential to drive specific SOX18‐dependent PPIs and correlate these variations with phenotypic variations observed in HLT.Using a focused library of small molecules, we are able to show that distinct SOX18 protein partner recruitment can be disrupted. On this basis we propose an initial Structure‐Activity Relationship study, paving the way to the development of specific inhibitors to SOX18‐dependent protein‐protein interactions. This approach has enabled us to better understand the mode of action of a compound currently used in clinic to manage HLT condition. Finally, we show that one of our newly identified SOX18 small molecule inhibitor is able to prevent metastasis in a pre‐clinical model of breast cancer, further validating the value of targeting SOX18‐dependent transcription factor complexes.This work opens up a new molecular strategy to target transcription factor activity and reposition this class of protein as viable drug target.
Selective Regulation of Hypoxia Inducible Factors by Endogenous Ligands
Authors: Romero Claudia Moreno , Wang Hanzhi , Bruick Richard 
Published: November 2023

Hypoxia inducible factors (HIFs) are transcription factors that regulate hundreds of genes that are important in a multitude of pathways under low oxygen conditions. HIFs are heterodimers composed of an alpha subunit (HIF‐1a, HIF‐2a, or HIF‐3a) and a beta subunit, also known as the aryl hydrocarbon nuclear translocator (ARNT). The alpha subunit is stabilized under hypoxic conditions and regulates a number of genes involved in oxygen adaption and recovery. Once oxygen levels are normalized, the protein is ubiquitinated and degraded. However, in many cancers there is a defect in HIF regulation such as the inability to be inactivated, therefore HIF activity levels are always high which promotes tumor progression. Two members of the HIF family, HIF‐1 and HIF‐2, have been the focus of our research. Though these two factors are very similar, they activate certain unique genes, such as PGK1 and EPO, respectively. It is now evident that there are key structural differences among these two HIF isomers, most notably the presence of a significantly larger cavity within the Per‐ARNT‐Sim (PAS) protein‐protein interaction domain of HIF‐2a. It is hypothesized that this cavity could confer the ability of an endogenous ligand(s) to bind and regulate HIFs differentially, though the nature of those ligands remains unknown. In order to test the hypothesis, we first need to develop a model system in cultured cells which will allow us to explore the different regulation of the HIF isomers. In doing so, we will identify the HIF‐1 and HIF‐2 expression profile of different cell lines. We are specifically looking for cell lines that express both HIFs, with each having unique target genes that can serve as diagnostic reporters. Currently, we are looking at Hep3B cells, SW156, and SV582 to see if they meet these criteria. Once we identify a suitable cell line, we will attempt to introduce mutations via state of the art manipulation techniques such as the CRISPR/Cas‐9 system, in order to interfere with the ligand binding activity of these factors.Support or Funding InformationSupported by the Cancer Prevention and Research Institute of Texas (RP130513)
Probing the Role of Dynamics in Allosteric Signal Transduction in the CO‐Sensing Transcription Factor CooA Using Site‐Directed Spin Label EPR Spectroscopy
Authors: Dent Matthew R. , Hines Judy P , Stevens Daniel J. , Burstyn Judith N 
Published: November 2023

CooA (CO oxidation activator) is a CO‐sensing transcription factor that is a member of the cyclic AMP receptor protein/fumarate and nitrate reductase regulatory protein (CRP/FNR) superfamily. The CO‐sensing function of CooA is carried out by a heme cofactor: the heme can be reduced from Fe(III) to Fe(II) under anaerobic conditions, and binding of CO to Fe(II) heme results in allosteric activation of DNA binding activity. While crystal structures of CooA in both active and inactive states exist, there is no clear consensus on how the signal of CO binding propagates from the regulatory domain to the DNA binding domain. Growing evidence supports the notion that dynamics, in addition to structure, plays a crucial role in allosteric regulation. In CRP, NMR experiments highlighted the importance of entropy‐driven dynamics in effector‐mediated allostery. To understand the role of dynamics in propagating the effector‐binding signal in CooA, we employed site‐directed spin label electron paramagnetic resonance (SDSL‐EPR) spectroscopy. Through site‐directed mutagenesis of a fully active protein missing four native Cys residues, we reintroduced Cys residues in key locations for attachment of spin labels: the surface of the heme domain (K26C), the top of the 4/5 loop (E60C), the domain‐linking hinge (F132C, D134C) and the end of the DNA‐binding helix (S175C). All variants exhibited heme characteristics, and most exhibited CO‐dependent DNA binding affinities, nearly identical to those of wild‐type CooA. The variant F132C, missing a phenyl ring believed to make a key allosteric contact with the DNA‐binding helix, exhibits weak, constitutive DNA binding. Multicomponent signals were observed in EPR spectra of all Fe(III) variants labelled with the nitroxide probes MTSL and MAL‐6. EPR spectra of these probes are sensitive to variations on the nanosecond timescale, consistent with large‐scale motions of entire protein domains, and report the same multiplicity of dynamic populations. The presence of multicomponent signals that are responsive to osmolytes implicate multiple dynamic populations that arise from different local conformational states. The relative intensities of the component signals varied with the site of label attachment, demonstrating that local dynamics vary as a function of position in the CooA protein. These observations suggest that interconversion among different dynamic populations may play a role in the allosteric mechanism of signal transduction in CooA.Support or Funding InformationNational Science Foundation supported these studies under CHE‐1213739 (to J.N.B.)
Characterization of Transcriptional Changes in a Vimentin Knock‐out Mouse Model Following Influenza A Infection Using RNAseq
Authors: Koch Clarissa M , Davis Jennifer M , Abdala‐Valencia Hiam , Misharin Alexander V , Ridge Karen M 
Published: November 2023

Introduction/RationaleOur group has previously demonstrated that vimentin, an intermediate filament, is required for the assembly and activation of the NLRP3 inflammasome in an infection model of Influenza A virus (IAV). To further investigate the role of vimentin in the response to IAV, our aim was to compare wild‐type and vimentin‐null mice and study changes in specific cell populations of the lung following IAV infection using RNAseq as an unbiased approach to study transcriptional changes.MethodsWild‐type and vimentin‐null 129S mice were infected with 200pfu of Influenza A/WSN/33 virus. Lungs were collected on days 0, 2, 3, 4, and 6 post‐infection (p.i.) and tissue was processed into single‐cell suspension. AM, MoDC, and ATII cells were flow‐sorted and total RNA was subsequently isolated using an RNeasy Plus Mini extraction kit (Qiagen). Following mRNA‐enrichment, libraries were prepared and sequenced using Illumina NextSeq 500.ResultsRNA sequencing data demonstrated significant differences in gene expression between the wild‐type and vimentin‐null ATII, AM, and MoDC cells post‐IAV infection in three cell sub‐populations of the lung. Differential gene expression analysis and pairwise comparison between wild‐type and vimentin‐null cells identified a significant number of differentially expressed genes between the two groups. The newly identified gene clusters were grouped into multiple GO processes involved in the innate immune response and cellular structure.ConclusionsOur RNAseq data demonstrated that following IAV infection, vimentin‐null mice have a strikingly different response compared to the wild‐type animals, which was evident in the alveolar macrophages (AM), monocyte‐derived cells (MoDC), and to a lesser extent in the alveolar type II (ATII) cell subpopulations and their respective differential gene analyses.Support or Funding InformationFunding: HL128194 and HL071643
Cas9: The Gateway to Genome Engineering
Authors: Schroeder Alexandra , Mendoza Naomi , Mahesh Rhea , Lim Shulammite , Howard Ashley 
Published: November 2023

Bacterial defense mechanisms employ an adaptive immune system encoded by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated (cas) genes to acquire immunity against phage infection and plasmid transfer. A CRISPR array is a series of repeated segments separated by unique spacer sequences. Immunity is gained after a short sequence of foreign DNA is inserted in a CRISPR locus as a new spacer. The entire CRISPR locus is transcribed into a long precursor‐CRISPR RNA (pre‐crRNA), which is processed into a mature form containing a spacer region and portions of the CRISPR repeat sequence. The crRNA serves as a template with which complementary sequences in foreign DNA are recognized and cut by Cas proteins, allowing bacteria to fend off invaders. In nature, the CRISPR‐Cas system makes site‐specific double‐strand breaks in target DNA to prevent viral infection, a function that is being repurposed as a powerful means of genome editing among researchers. There are six types of CRISPR‐Cas systems, each employing a unique set of Cas proteins. Type II‐A CRISPR systems use a single DNA endonuclease, Cas9, for the targeting and degradation of target DNA. Streptococcus pyogenes Cas9 (SpyCas9) is a 1368‐amino acid DNA endonuclease which has two distinct lobes: the alpha‐helical recognition lobe (REC), which is responsible for holding the DNA in place, and the nuclease lobe (NUC) containing the HNH and RuvC nuclease domains that snip the target double‐stranded DNA (dsDNA). When Cas9 is assembled with guide RNA, either a native CRISPR RNA (crRNA) with trans‐activating crRNA (tracrRNA) or a synthetic single guide RNA (sgRNA) in which the 3′ end of the crRNA is linked to the 5′ end of the tracrRNA, it begins DNA interrogation. Upon finding a proper protospacer adjacent motif (PAM), a three‐nucleotide sequence present only in targets and not in the host CRISPR array, Cas9 checks the flanking DNA for possible guide RNA complementarity. The 20‐nucleotide spacer sequence of the sgRNA can be changed easily, allowing scientists to make double‐stranded breaks to sequences of interest for editing. Although the CRISPR‐Cas9 system remains susceptible to error and the reasons behind undesired off‐target cleavage are not yet fully understood, the high specificity of Cas9 makes it a powerful genome editing tool. Our literature review and structural analysis using PyMol software and 3D printing of a physical model reveal how the structure of the Cas9 protein allows it to function as a programmable, RNA‐guided DNA endonuclease.Support or Funding InformationThis is a SMART Team project supported through the contributions of Dr. Megan Hochstrasser of The University of California, Berkeley, the Milwaukee School of Engineering, and the Olathe Medical Professions 21st Century Academy
Postmortem study on psychiatric disorders
Authors: Bao Ai‐Min 
Published: November 2023

With the postmortem brain material obtained from the Netherlands Brain Bank, we have carried on a series of studies with the aim to elucidate the specific changes in the stress‐related brain systems in relation to special subtypes of mood disorders. Our final destination is to set up tailor‐made treatment for psychiatric disorder patients on the basis of his/her developmental history, genetic and epigenetic background, the vulnerability in particular neurobiological systems.Support or Funding InformationAAA Symposium: Progress of Human Brain Banking in China: Construction and Research. Supported by AAA (American Association of Anatomists) and CSAS (Chinese Society for Anatomical Sciences), and by the National Natural Science Foundation of China (91332102 and 31271130).