bluuebunny/crossref_metadata_2025_split huggingface dataset:
Histone deacetylases in axonal growth and regeneration and their relevance to Parkinson's disease.
Authors: O'Keeffe Gerard 
Published: June 2021

Functional decline in Parkinson's disease (PD) is the cumulative result of regulatory alterations affecting large numbers of genes involved in various aspects of neuronal maintenance and function, and it has recently emerged that axonal degeneration plays a key role. Central to the alterations in gene expression that underlie this failure of axon maintenance, is the regulation of gene transcription by enzymes known as histone deacetylases (HDAC) which promote the de‐acetylation of histone proteins to inhibit transcription. alpha‐synuclein, a key protein that accumulates in the brains of patients with the disease, binds histone proteins in vitro & in vivo, and nuclear a‐synuclein reduces acetylated histone 3 (AcH3) levels. However while these molecular insights are important, how they regulate the neuronal structure and axonal growth in neurons affected by Parkinson's, and the relevance of this cell biology approach to the human brain is unknown.Here we used a network approach to identify all genes that had a significant positive (r+) or negative (r−) correlation with the expression of all HDACs using microarray data from the human substantia nigra (SN; GSE 60863). We then conducted a detailed enrichment analysis to identify biological pathways that were over‐represented and may be linked to Parkinson's Disease. These analyses showed that Class‐II but not Class‐I HDACs were significantly correlated with functional pathways linked to Parkinson's Disease (p<0.0001). These included genes linked to familial Parkinson's Disease and oxidative phosphorylation, including those associated with mitochondrial complex I.To link these data with changes in neuronal structure, we next examined the effects of class specific HDAC inhibitors on neuronal structure by examining a range of HDAC inhibitors for their ability to promote axonal growth and branching in dopaminergic and sympathetic neurons which are the two main cell types that undergo progressive axonal degeneration in Parkinson's Disease. We found that a MC1568 (a class II inhibitor) but not MS275 (a class I inhibitor) promoted significant increases in axon growth and branching (p<0.01). Therefore we further examined the therapeutic potential of MC1568, and found it protected against the detrimental effects of MPP+‐ and alpha‐synuclein induced axonal degeneration in a models of Parkinson's disease.This study therefore provides new insights into the regulator mechanisms that control axonal growth in neurons affects by Parkinson's, and highlights the potential of using this approach to identify therapeutic targets for neuroprotection and axonal regeneration.Support or Funding InformationThis work was funded by a Career Development Award from Science Foundation Ireland under the grant number 15/CDA/3498.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epigenetic Regulation of Oncogenes in Leukemia by the Ikaros Tumor Suppressor
Authors: Payne Jonathon Lee , Dovat Elanora , Song Chunhua , Dovat Sinisa 
Published: June 2021

The IKZF1 gene controls expression of the Ikaros protein which functions as a tumor suppressor in pediatric acute lymphoblastic leukemia (ALL). Dysfunction of Ikaros due to deletion or an inactivating mutation of a single allele is associated with high‐risk ALL, increased risk for relapse, and poor prognosis. Ikaros is a transcriptional regulator of a large set of target genes; however, the mechanism by which Ikaros regulates gene expression in leukemia and mediates its tumor‐suppressor effect has yet to be fully elucidated. In this study, chromatin immunoprecipitation with next‐generation sequencing (ChIP‐Seq) was used to identify Ikaros' target genes and their epigenetic signature. ChIP‐Seq results were confirmed using quantitative chromatin immunoprecipitation (qChIP). Ikaros gain‐ and loss‐of‐function assays were used to determine the role of Ikaros in the epigenetic regulation of gene expression which was assessed by quantitative, real‐time PCR (qRT‐PCR) and western blot. ChIP‐Seq and qChIP assays identified a large number of target genes that are regulated by Ikaros. Ikaros gain‐ and loss‐of‐function experiments in combination with qChIP “scanning” at Ikaros target gene promoters revealed that Ikaros can repress transcription by inducing the formation of heterochromatin at gene promoters. Ikaros overexpression in ALL results in an increase in H3K27me3 and H3K9me3 repressive chromatin markers at the promoters of genes that support cell cycle progression and the phosphoinositide 3‐kinase (PI3K) pathway—both pathways are frequently activated in cancer. We also demonstrate that Ikaros overexpression results in the transcriptional repression of the JARID1B/KDM5B histone demethylase. Decreased KDM5B expression is accompanied by a genome‐wide increase in H3K4me3 as evidenced by western blot. In conclusion, our results show that Ikaros' tumor suppressor activity in high‐risk ALL involves epigenetic regulation at the promoters of its target genes and regulation of H3K4me3 levels via transcriptional repression of the JARID1B histone demethylase. These data give further insight into the mechanisms underlying Ikaros' tumor suppressor function in acute lymphoblastic leukemia.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) Grant R01 HL095120, a St. Baldrick's Foundation Career Development Award, a Hyundai Hope on Wheels Scholar Grant Award, the Four Diamonds Fund of the Pennsylvania State University College of Medicine, and the John Wawrynovic Leukemia Research Scholar Endowment.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of HDAC inhibitor Vorinostat and radiation on HTB4 and MDA‐MB‐435 cancer cells
Authors: Wing Joseph Francis , Maini Trisha , O'Donnell Robert Watts 
Published: June 2021

HDAC inhibitors have shown promise in the treatment of various types of cancer. The HDAC inhibitor vorinostat was first introduced during treatments for cutaneous T‐cell lymphoma and has been shown to cause an increase in the effectiveness of radiation on multiple cancer cell lines. For the purpose of these experiments, the effects of vorinostat and radiation on HTB4 cells (a human bladder cancer cell line) and MDA‐MB‐435 cells (a human melanoma cell line) are being investigated in order to determine the efficacy of this combined treatment. Using cell cycle analysis and MTT assays, we were able to analyze the effects of radiation and vorinostat alone compared to the combination of both. For cell cycle analysis, we plated the cells in four flasks and treated three of them with either vorinostat (2.5 μM) alone, radiation (~20Gy) alone or the combination of the two. The cells were harvested, stained and analyzed five hours after radiation and 24 hours after vorinostat treatment by flow cytometry. Preliminary results for the HTB4 cells treated with both radiation and vorinostat showed an increase in the percent of cells in G2 and a reduction in the percent of those in both G1 and S phase compared to either treatment alone or the control population. Results for the 435's did not show a similar trend with distribution of cells between the control and three treatments. The MTT assay was performed using two 96‐well microtiter plates with 16μM vorinostat serially diluted 1:2 across 10 columns, and only cells in the 11th column and medium alone in the 12th column. One plate was irradiated using a lower dose of 5 Gy based on a literature survey. MTT was added to both plates directly after the one plate was irradiated. Results of the MTT assay for HTB4's and 435's showed that increasing concentrations of vorinostat alone and when coupled with radiation resulted in less MTT product produced compared to the radiation treatment alone. Based on the MTT data, the HTB4 cell line appears to be more sensitive than the 435's to the effects of vorinostat. Future experiments will focus on the effects of radiation on multiple tumor cell lines and investigate the molecular mechanisms by which HDAC inhibition influences radiation‐based cancer treatment.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Bacterial Transcriptomics to Investigate Targets of Host‐Bacterial Interactions in C. elegans
Authors: Wong Hoi Tong , Chan Jason P. , Brumbaugh Jamey T. , Ardasheva Anastasia , McLimans Christopher J. , Wright Justin , Lamendella Regina 
Published: June 2021

The interactions between a host and its resident microbes form complicated networks and disentangling these interactions can help us better understand mechanisms of pathogenesis while also defining the integral commensal protection that host‐associated microbiota offer to promote health. Here we utilize a tractable model organism, C. elegans, to study the mechanisms involved in host‐microbe interactions and to identify different expression profiles under different host genotypes to increase our knowledge of host‐bacteria interspecies interactions. We developed an RNAseq protocol and bioinformatics pipeline to elucidate 1) bacterial gene expression differences in host associated versus in vitro E. coli OP50 and 2) differential bacterial gene expression in known models of aging within C. elegans (long‐lived daf‐2/InsR mutant versus short‐lived daf‐16/FOXO transcription factor mutant). Briefly, RNA extracts (n=18) were subject to ribosomal RNA subtraction, cDNA synthesis, Nextera library preparation and sequencing using the Illumina Hiseq platform. Our bioinformatics pipeline utilizes the most robust programs for quality filtering, assembly, annotation, and differential gene analysis. Our analysis revealed a variety of differentially expressed genes in plated E. coli OP50 versus E. coli OP50 fed to C. elegans. Differential gene expression analysis revealed that expression of biosynthetic genes was enriched in host‐associated E. coli, and several of these expressed genes code for the precursors and products needed for the synthesis of Lipopolysaccharides (LPS), which are important in innate immune and stress responses, as well as pathogenicity. Preliminary bacterial transcriptome data also indicate differences in bacterial expression of several biosynthetic pathways and virulence mechanisms, which may contribute to the aging phenotypes of the long‐lived daf‐2/InsR mutant and the short‐lived daf‐16/FOXO transcription factor mutant. Our research has employed a robust RNA sequencing and bioinformatics approach to study differential gene expression of bacteria in C. elegans, and will enable us to better test how gut bacteria and their gene regulatory networks regulate host physiology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Caloric restriction impacts plasma microRNAs in rhesus monkeys
Authors: Clark Josef , Schneider Augusto , Dhahbi Joseph , Atamna Hani , Colman Ricki , Anderson Rozalyn 
Published: June 2021

Caloric restriction (CR) is one of the most robust interventions shown to delay aging in diverse species, including rhesus monkeys (Macaca mulatta). Although the mechanisms have yet to be elucidated, identification of factors involved in delayed aging by CR brings a promise of translatability to human health and human aging. Here we show that CR in rhesus monkeys induced a profound change in abundance of circulating microRNAs (miRNAs) linked to growth and insulin signaling pathway, suggesting that miRNAs are involved in CR's mechanisms of action in primates. Deep sequencing of plasma RNA extracts enriched for short species revealed a total of 243 unique species of miRNAs including 47 novel species. Approximately 70% of the plasma miRNAs detected were conserved between rhesus monkeys and humans. CR induced or repressed 24 known and 10 novel miRNA species. Regression analysis revealed correlations between bodyweight, adiposity, and insulin sensitivity for 10 of the CR‐regulated known miRNAs. Sequence alignment and target identification for these 10 miRNAs identifies a role in signaling downstream of the insulin receptor. The highly abundant miR‐125a‐5p correlated positively with adiposity and negatively with insulin sensitivity, and was negatively regulated by CR. Putative target pathways of CR‐associated miRNAs were highly enriched for growth and insulin signaling that have been previously been implicated in delayed aging. Clustering analysis further pointed to CR‐induced miRNA regulation of ribosomal, mitochondrial, and spliceosomal pathways. These data are consistent with a model where CR recruits miRNA‐based homeostatic mechanisms to coordinate a program of delayed aging.Support or Funding InformationNIH/NIA AG040178, AG047358, and the Glenn Foundation for Medical Research. JPC is a T32 Fellow NIH/NIA AG000213This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interactions between Divalent Cations and a Periplasmic Lipoprotein involved in Salmonella Magnesium Homeostasis
Authors: Soens Ross , May John 
Published: June 2021

All living cells require magnesium to generate biochemical energy during metabolism and for maintenance of essential structures. A recently discovered protein of unknown function, DcrB, plays a role in magnesium homeostasis within Salmonella enterica, which must adapt to low external levels of Mg2+ to cause food‐borne illness. DcrB is a small protein attached to the periplasmic face of the cytoplasmic membrane. Given this location, one possible model for the function of DcrB is that DcrB brings Mg2+ to membrane Mg2+ transporters in the cytoplasmic membrane. Currently, Salmonella phenotypic assays have shown DcrB to promote growth in low Mg2+. The hypothesis of this project is that DcrB binds magnesium and possibly other divalent cations. To test this hypothesis, we examined whether Mg2+ and other divalent cations affected the thermal stability of purified DcrB protein. We examined the effect of divalent cations using a fluorescence‐based thermal shift assay and circular dichroism techniques. Furthermore, we are using our recent crystallographic structure of DcrB to identify and test specific metal binding sites. Our results have established the specificity of DcrB for binding divalent cations, which we anticipate will shed light on the role of DcrB in metal homeostasis in pathogenic bacteria.Support or Funding InformationThis research is supported by a University of Wisconsin‐‐La Crosse Undergraduate Research and Creativity Grant (RS) and a University of Wisconsin‐‐La Crosse Faculty Research Grant (JM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Analysis of Cortactin After Anion Exchange Chromatography
Authors: Graves Gabriel , Kruchten Anne 
Published: June 2021

Cortactin is a cytoskeletal actin‐binding protein that is upregulated in several types of metastatic cancers. Further research on its three‐dimensional structure may allow for greater understanding of its role in cancers. Purification of our in vitro expressed His‐tagged cortactin from E. coli was done through affinity purification with Ni2+ resin followed by anion exchange chromatography. Elution fractions of the protein were analyzed via polyacrylamide gels both in native and SDS environments to analyze both length and hydrodynamic size of the protein. Silver staining of subsequent gels allowed for precise analysis of banding pattern and relative strength. Our results demonstrate variations in banding patterns with increasing concentration of KCl elution on native‐PAGE, suggesting cortactin has a variable three dimensional structure in vitro. Future work will determine if these structural differences are maintained in vivo.Support or Funding InformationThis work is supported by the College of Saint Scholastica SSC Summer Research Fellowships.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying protein‐protein interactions in fatty acid and polyketide biosynthetic pathways via site‐specific vibrational spectroscopy
Authors: Chioti Vasiliki T. , Londergan Casey H. , Charkoudian Louise K. 
Published: June 2021

By virtue of their structural diversity and striking potency, natural products have long been used as therapeutic agents. Natural products are biosynthesized by assemblies of enzymes encoded in gene clusters. Repurposing the natural product biosynthetic repertoire to assemble “unnatural” natural products would have profound implications for human health. Our current lack of understanding of how these biosynthetic enzymes operate at a molecular level, which is partially attributed to the lack of tools that can capture the weak and transient interactions of the enzyme assemblies, stymies such engineering endeavors. Our lab previously showed that labeling acyl carrier proteins, which are central hubs of natural product assemblies, with a thiocyanate (SCN) vibrational probe provides an innovative method to study the proteins' conformational dynamics and interactions with ketosynthases. In this work, we evaluate the general utility of the probe by assessing ACP‐SCN interactions with other partner enzymes. The SCN probe was loaded onto the 4′‐phosphopantetheine (Ppant) arm of ACPs involved in fatty acid and actinorhodin type II polyketide biosynthetic pathways, AcpP and actACP, respectively. Changes in the CN stretching frequency were monitored as the ACPs were titrated with partner enzymes, 3‐hydroxyacyl‐ACP dehydrase (FabA) and actinorhodin polyketide ketoreductase (actKR). Upon a productive interaction of the ACP with its respective partner enzyme, the CN absorption band shifts to lower wavenumbers, which could correlate to the probe accessing the partner enzyme's hydrophobic catalytic active site. In light of these findings, the SCN probe appears to report on a wide range of ACP‐partner protein interactions. As a result, the probe could be a useful tool for future engineering studies.Support or Funding InformationWe would like to acknowledge an NIH Award #CHE‐1652424 to Louise K. Charkoudian as a source of funding for this project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A role for the N‐terminal domain in modulating the activities of the nucleotide exchange factor Sil1
Authors: Pareja Kristeen Alcaide , Sevier Carolyn 
Published: June 2021

The Hsp70 BiP is an essential molecular chaperone in the endoplasmic reticulum (ER) and is highly involved in maintaining the quality of proteins in the secretory pathway. Using yeast as our model system, we have previously shown that BiP is also involved in ER oxidative stress signaling. BiP contains a conserved cysteine located in its nucleotide binding domain, and this cysteine can be modified by peroxide (sulfenylated) and/or glutathione (glutathionylated) when the ER experiences an overly‐oxidizing environment. Normally, BiP undergoes ATP‐dependent conformational changes that drive the association and dissociation of BiP with polypeptides. We have shown that the cysteine modification limits BiP's ATP hydrolysis yet BiP maintains its ability to bind substrates. We have demonstrated that this ATP‐independent holdase activity of modified BiP is beneficial to cells under conditions of ER oxidative stress, likely a consequence of limiting polypeptide aggregation under suboptimal folding conditions. When cells go back to non‐stress conditions, reversibility of BiP's cysteine modification is also important for normal cellular function and survival. The protein Sil1, a known nucleotide exchange factor (NEF) of BiP, can remove the modification on BiP's cysteine. Sil1's unexpected activity as a reductant depends on two cysteines within its N‐terminal domain: Cys‐52 and Cys‐57. Although the N‐terminal domain containing the cysteines is required for Sil1 activity as a reductant, Sil1 is competent as a NEF in the absence of the N‐terminal domain. Mutations in the Sil1 gene are major cause of Marinesco‐Sjögren Syndrome (MSS), a rare autosomal disease characterized by early onset cataracts, cerebellar ataxia, and delayed mental development. Interestingly, we found a patient mutation that maps within the N‐terminal region which not only influences Sil1's reductant activity but also dramatically alters Sil1's activity as a NEF. We are in the process of further elucidating the molecular details relating to how the N‐terminal region of Sil1 impacts both its NEF and reductant activity by utilizing a variety of biochemical and biophysical approaches. Our current data suggest that the N‐terminal domain adopts multiple conformations, allowing for regulation of Sil1's NEF activity and potentially its activity as a reductant.Support or Funding InformationNSF GRFP, SUNY Diversity Fellowship, NIGMSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Complex Effects of J‐protein Alterations on Hsp104‐mediated Curing of Prion [PSI+]
Authors: Berger Scott E. , Kamiya Erina , Astor Michael T. , Hines Justin K. 
Published: June 2021

The amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded protein, passed to daughter cells following fragmentation by a set of molecular chaperones which includes the J‐protein Sis1, Hsp70, and Hsp104. Overexpression of Hsp104 efficiently cures the prion [PSI+], a phenomenon which has promoted the exploration of Hsp104 as a potential therapeutic agent for neurodegenerative diseases. However, the mechanism of [PSI+] elimination by Hsp104 overexpression has been the subject of significant debate for the past two decades and has garnered significant interest in the recent literature as multiple conflicting models have been proposed. Yeast prion propagation is inexorably reliant on the function of molecular chaperones of the Hsp100, Hsp70, and Hsp40 classes. Specifically, four Hsp40s (also called J‐proteins) have been implicated in various aspects of yeast prion biology: Sis1, Ydj1, Apj1, and Swa2. We found that overexpression of Sis1 or Apj1 accelerates strong [PSI+] elimination by Hsp104 overexpression, yet Ydj1 overexpression has a profound and opposing effect, completely blocking Hsp104‐mediated curing, indicating that Apj1 and Sis1 likely have similar and partially overlapping roles in this process. Interestingly results for weak variants of [PSI+] indicated potentially no role for J‐proteins in curing as no J‐protein alteration whatsoever affected the ability of Hsp104 to cure these variants. Additional experiments to determine the specific J‐protein domains responsible for various effects, as well as J‐protein requirements in cell backgrounds harboring both [PSI+] and [RNQ+] are underway. Overall our data support the hypothesis that Hsp104‐mediated curing may occur by biochemically distinct, variant‐specific mechanisms, only some of which involve J‐proteins.Support or Funding InformationThis work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.