bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Calcitriol in the DNA Damage by Molecular Subtypes of Breast Cancer Cell Lines
Authors: Padilla Luis , Vergne Ralphdy , Ortiz Carmen , Encarnación Jarline , Matta Jaime
Published: June 2021

Calcitriol is the physiologically‐active form of vitamin D which can regulate different cellular mechanisms, such as cell cycle arrest, differentiation, migration, invasion, and proliferation of some tumor cell types. Fluorouracil (5‐FU) is one of the most commonly used drugs to treat several types of cancer, including breast cancer. Its main mechanism of action is based on interfering with DNA or RNA synthesis. Breast cancer is a heterogenous disease in which the behavior and biological characteristic of tumors can vary widely, affecting the prognosis and the treatment of the patient. Breast tumors are classified into four principal molecular subtypes: Luminal A, Luminal B, HER2+ and triple‐negative. Our main objective is to assess the DNA damaging effects of calcitriol pre‐ and post‐treatment in cells treated with 5‐FU in breast cancer cells with different molecular subtypes. We hypothesize that calcitriol will induce DNA damage alone and in combination with 5‐FU in Luminal A cells when compared with Luminal B cells. Breast cancer cell lines resembling Luminal A (MCF‐7) and Luminal B (BT474) were grown with their respective media. To select the appropriate doses of 5‐FU and calcitriol, the MTT assay was used to calculate the inhibitory concentration 50 (IC50). Protein was extracted to assess changes in expression of key proteins involved in DNA damage checkpoint and repair. DNA damage for each treatment was assessed using the comet assay. For 5‐FU, the IC50 was calculated to be 40 μM while for calcitriol it was 1 μM in cells treated for 24 hours. Our preliminary results show that when MCF‐7 cells were treated with calcitriol, a higher DNA damage index was observed when compared to untreated cells (p=0.0351). Moreover, when MCF‐7 were treated with calcitriol before 5‐FU treatment a higher DNA damage index was observed when compared with calcitriol only (p=0.0172). However, when comparing the extent of DNA damage in cells treated with calcitriol after 5‐FU treatment no significant differences were found (p=0.999) when compared calcitriol only. In BT474 cells, no statistically significant differences were observed when comparing calcitriol treated with untreated cells. Currently, we are performing additional experiments to confirm these preliminary results. Also, we are assessing expression changes in protein related to DNA damage and cell cycle checkpoints.

Support or Funding Information

This study was supported by UPR‐Ponce RISE NIH‐NIGMS R25GM096955. This study was supported by NIH‐NIGMS #2R25GM096955, #S06GM008239‐20, 9SC1CA182846‐04, #U54CA163068, and G12‐MD007579. We appreciate the technical support of the Molecular and Genomics Core Laboratory at Ponce Health Sciences University‐Ponce Research Institute (Grant R003050/MD007579).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cancer‐associated variants of POLQ induce genomic instability
Authors: Headrick Ashley , Wanchu Sahaj , Avalos Lisbeth , Victorino Jorge , Towle‐Weicksel Jamie , Ray Sreerupa
Published: June 2021

Melanoma is the most severe form of skin cancer and it develops when unrepaired DNA damage in skin cells causes mutations which further leads to genomic instability. The widely accepted environmental cause of melanoma is ultra‐violet (UV) radiation, which leads to DNA damage in skin cells. Failure to repair DNA, or its error‐prone repair, can lead to mutagenesis which can propagate further through rounds of replication, eventually resulting in uncontrolled cell growth leading to cancer. However, the cell has a mechanism for repair using a variety of highly specialized enzymes known as DNA polymerases. DNA Polymerase theta (Pol θ or POLQ) is hypothesized to be an important DNA polymerase involved in double‐strand break (DSB) repair through microhomology‐mediated end‐joining (MMEJ), an alternative non‐homologous end‐joining (alt‐NHEJ) pathway, however it is a low fidelity enzyme that often leads to numerous errors during repair. There is evidence that breast cancer patients that overexpress POLQ tend to have poorer survival rate suggesting a potential role for POLQ in cancer. In collaboration with the Yale SPORE in Skin Cancer and Rhode Island College, we have identified several variants of POLQ in melanoma patients. Here we demonstrate that the cancer‐associated variants of POLQ induce genomic instability and have accumulation of DNA DSBs. In addition, biochemical studies indicate that these variants display an inherently slower polymerization rate compared to wild‐type (WT) suggesting a different mechanism for nucleotide incorporation. Together, this suggests that the cancer‐associated variants lead to genomic instability and may play a role in cancer. Our studies suggest that variants of POLQ alter the DNA repair and has potential to impact the treatment of cancer.

Support or Funding Information

This research was supported by Linfield College Start‐Up Funds and Linfield College Faculty Student Research Collaborative Grant (FSRCG) to SR.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Post‐Translational Modifications of Mitochondrial Proteins MRPL12 and POLRMT as a Potential Regulatory Mechanism for Mitochondrial DNA Transcription
Authors: Bostwick Alicia M , Senti Mackenna L , Dittenhafer‐Reed Kristin E.
Published: June 2021

Mitochondrial DNA (mtDNA) encodes 13 of the protein subunits of the oxidative phosphorylation pathway, the pathway that produces the majority of ATP in eukaryotic cells. Mitochondrial dysfunction is associated with neurodegenerative disorders, cancer, inherited mitochondrial diseases, and other pathologies, making mitochondrial biochemistry an important area of investigation. However, there lacks an understanding of key elements of this biochemistry, including the mechanisms that regulate mtDNA transcription. mtDNA is known to be complexed to various proteins in structures known as nucleoids. Some nucleoid proteins are key members of the mtDNA transcription machinery; these include the mitochondrial RNA polymerase (POLRMT) and mitochondrial ribosomal protein L12 (MRPL12), which maintains POLRMT stability and promotes mtDNA transcription. We hypothesize that reversible post‐translational modifications (PTMs), such as phosphorylation of threonine, serine, or tyrosine residues or acetylation of lysine residues within these and other nucleoid proteins, may play a role in regulating mtDNA transcription. The objective of our study is to determine the effects of MRPL12 and POLRMT post‐translational modifications on the activities of these proteins in the context of mtDNA transcription in order to assess the roles of such modifications in regulating this process. To test this, we performed mutagenesis PCR on MRPL12 and POLRMT genes within bacterial expression vectors to replace the sequence encoding an amino acid at a PTM site with a sequence encoding a modified or unmodified amino acid mimic (T, S, Y → E/A, phosphorylated/dephosphorylated mimic; K → Q/R, acetylated/deacetylated mimic). We optimized bacterial expression and protein purification conditions and purified wild‐type and mutant MRPL12 proteins, as well as wild‐type POLRMT. We performed preliminary in vitro transcription assays to assess POLRMT functionality and found that our protein successfully transcribed short mtDNA templates. Going forward, we will perform protein‐protein binding assays to determine the effects of MRPL12 modifications on MRPL12's ability to bind POLRMT, as well as POLRMT mutagenesis and more in vitro transcription assays to measure the effects of MRPL12 and POLRMT modifications on mtDNA transcription.

Support or Funding Information

This research is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845, with additional funding from the Arnold and Mabel Beckman Foundation Beckman Scholars Award and the ASBMB Undergraduate Research award.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the Effects of Androgen and Glucocorticoid Receptor Sequences on DNA Binding Preferences
Authors: Boulanger Kyle R. , Miller Austin M , Pufall Miles A , Hall Bonnie L.
Published: June 2021

Prostate cancer is the second leading cause of cancer among men and although survival rates are high for regional tumors, it is estimated that almost 30,000 men will succumb to their diagnosis in 2018. Androgen hormones fuel prostate tumor growth by signaling through the androgen receptor (AR), a member of the nuclear receptor superfamily of transcription factors. Blocking androgen signaling through administration of antagonists (e.g bicalutamide, enzalutamide) or halting production of androgens through chemical or surgical castration arrests progression of the disease. Patients often relapse with castration resistant prostate cancer (CRPC) that continues to grow despite the lack of androgens. One mechanism of resistance is due to upregulation of a related nuclear receptor, the glucocorticoid receptor (GR). Because AR and GR have overlapping DNA binding specificities, GR is able to functionally substitute for AR at key disease genes. We have shown previously, using SELEX (Selective Evolution of Ligands by Exponential Enrichment) coupled with a novel computational algorithm (SelexGLM), that although AR and GR bind similar sequences, they recognize DNA differently. GR is less specific than AR, allowing it to accommodate predominantly AR sequences. To better understand the selectivity of AR and GR, and to identify the determinants of their specificity, we are measuring the sequence preference of AR and GR mutants using the No Read Left Behind (NRLB) method. As opposed to traditional SELEX, NRLB yields high resolution models of specificity with a single round of selection, which allows rapid screening of our mutants. We have over‐expressed and purified both the AR and GR DNA binding domains, and have designed hybrid mutants. Comparison of the DNA binding preferences of AR, GR and the hybrid molecules by NRLB should help uncover how GR might be playing a role in CRPC.

Support or Funding Information

This work was supported in part by the FUTURE program of the University of Iowa Carver College of Medicine.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the Role of Piccolo Splice Variants in Regulated Secretion of Insulin from Rat Pancreatic Cells
Authors: Quintana Victoria G , Fenster Steven D.
Published: June 2021

Secretion of insulin from pancreatic beta‐cells is required for proper glucose homeostasis; however, the molecular mechanism of insulin granule exocytosis has not been fully elucidated. In pancreatic beta‐cells, glucose‐induced insulin secretion is synergistically potentiated through an Epac2‐Rim2‐Piccolo signaling response mechanism. Both Piccolo and Rim2 were originally identified as active zone proteins in neurons and have been shown to regulate synaptic vesicle cycling and neurotransmitter signaling. A recent study showed that a number of active zone proteins, including Rim2 and Piccolo, are downregulated in human pancreatic islet cells isolated from patients with type 2 diabetes (T2D). Understanding the role of Piccolo in regulated secretion of insulin may provide valuable insights into the causes of T2D. The C‐terminal region of Piccolo contains a Ca2+‐binding C2A domain. It is likely that Piccolo may regulate secretion from insulin granule vesicles by serving as a Ca2+‐sensor. Alternative splicing of exons within the coding region of the C2A domain results in an exon‐skipping event generating either a splice variant that lacks the 27‐nucleotide exon 16 (short C2A) or a variant that retains this exon (long C2A). Exclusion of the 9 amino acid residue sequence in the short C2A domain results in enhanced Ca2+‐binding affinity. In this study, we sought to examine the differential roles of the short C2A and long C2A variants in Ca2+‐mediated insulin secretion from rat INS‐832/13 pancreatic beta‐cells. We demonstrate that INS‐832/13 cells express both long and short variants of the C2A domain and real‐time qPCR demonstrated differential expression of the variants. In addition, we examined the effects of overexpression of the short and long C2A domains on glucose‐stimulated insulin secretion from INS‐832/13 cells.

Support or Funding Information

Maximizing Access to Research Careers Program at Fort Lewis College (NIH‐T34GM092711)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Role of Histone H4 Acetylation in RNA Splicing in Saccharomyces cerevisiae
Authors: Kopew Jessica S , Mendizabal Adriana G , Lin Mengjia M , Kress Tracy L
Published: June 2021

The ability of RNA splicing and transcription to take place simultaneously allows for efficient coordination and regulation within cells. Few known connections exist between chromatin modifications, which regulate transcription, and RNA splicing in the yeast Sacchraomyces cerevisiae, as well as in metazoa1,2, leaving much to explore. Our previous research has shown that genetic interactions exist between splicing protein genes and the NuA4 complex genes in S. cerevisiae. In addition, we found that deletion or mutation of these NuA4 complex genes impact RNA splicing. Nua4 plays a key role in regulating transcription by acetylating histone H4; following this, the Swr1 chromatin remodeling complex is recruited to the acetylated histone and ultimately deposits H2A.Z in place of H2A, allowing for transcriptional activity3–5. Given these findings, our current research is focused on elucidating whether specific acetylation of histone H4 is required for coordinating splicing with transcription. We constructed an array of double mutant yeast strains comprising a deletion of an essential splicing factor gene and a point mutation in either H4K5, H4K8, H4K12, or H4K16 thus preventing a chemical modification. We conducted genetic interaction studies to identify synthetic sick/lethal, or suppressive genetic interactions between the two genes. The majority of the double mutant growth phenotypes reveal synthetic sick/lethal interactions when compared to single mutant strains, suggesting a functional link between H4 acetylation and RNA splicing. Using RT‐qPCR we determined that mutation of H4 resulted in a reduction in pre‐mRNA splicing. In addition, mutation of H4 exacerbates the splicing defects observed in splicing factor deletion strains, which is consistent with the genetic interactions. Our future research will focus on identifying the specific mechanism by which the histone H4 acetylation is functioning to regulate RNA splicing. To this end we are currently using chromatin immunoprecipitation to examine the impact of these H4 mutations on the recruitment of splicing factors.

Support or Funding Information

Research Corporation for the Advancement of Science (Cottrell College Science award no. 20186) National Institutes of Health (R15GM122026).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The RS/RGG Domain of the SR‐like Protein Npl3 is Required for Efficient RNA Splicing in Yeast
Authors: Lin Mengjia M , Gala Kinisha , Lobby Jenna , Moazamian Aron , Kress Tracy L
Published: June 2021

Npl3 is an SR‐like protein found in Saccharomyces cerevisiae that functions in co‐transcriptional pre‐mRNA splicing by promoting the recruitment of splicing factors to the pre‐mRNA during transcription1. It contains two RNA recognition motifs (RRMs), an RS/RGG domain rich in arginine and serine repeats, and an uncharacterized N‐terminus. While Npl3 binds RNA2–4 via the RRM domains5, the role of each of the functional domains in splicing is currently unknown. Here we have generated a panel of flag‐tagged Npl3 proteins lacking one or more of the protein domains. We identified genetic interactions between Npl3 mutations lacking the RS/RGG domain and mutations in genes that encode RNA splicing factors, suggesting that the RS/RGG domain is important for RNA splicing. Indeed, we show that deletion of the RS/RGG domain of Npl3 causes a significant decrease in RNA splicing efficiency of Npl3‐dependent pre‐mRNAs. In addition, deletion of the RS/RGG domain abolishes the ability of Npl3 to interact with splicing factors, which strongly suggest that the RS/RGG domain is required to recruit the splicing machinery during transcription. Interestingly, we found that phosphorylation of the RS/RGG domain is not important for RNA splicing. Replacing the Npl3 RS/RGG domain with the RS/RGG domain from the Candida albicans Npl3 ortholog6 restores growth, interaction with splicing proteins, and RNA splicing. Together this work demonstrates that the RS/RGG domain is required for the activity of Npl3 in RNA 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.
Investigation of the ERK/MAP kinase long first intron and its possible role in gene regulation and germ cell development
Authors: McCloskey John , Robinson‐Thiewes Sarah , Kimble Judith
Published: June 2021

One distinguishing feature of eukaryotic genomes is the presence of introns within nascent transcripts. Genomic analyses of distantly related species, spanning yeast to humans, reveal conservation of intron size and position, suggesting an intron can be advantageous. Many studies have examined the relationship between short introns and gene expression; however, the effect of long introns and gene expression is not well understood. Critical developmental regulators often have an unusually long intron that is conserved in size and position and typically one of the first introns. We hypothesize that long first introns of developmental regulators affect gene expression. To investigate the relationship between a long first intron and gene expression, we have focused on the Caenorhabditis elegans gene encoding ERK/MAP kinase (mpk‐1). Like other metazoan ERK/MAP kinases, mpk‐1 harbors a long first intron in one of its isoforms. The mpk‐1b nascent transcripts include a small first exon and an 8.2 kb first intron, while mpk‐1a lacks that first exon and intron but shares other exons and introns with mpk‐1b. To visualize RNAs, we designed single molecule in situ hybridization (smFISH) probes targeting the 8.2 kb intron to visualize nascent transcripts as well as other probes targeting the shared exons to detect the mRNAs. We use confocal microscopy and MATLAB image processing to assess mpk‐1 transcription from germline stem cell self‐renewal to meiotic pachytene of differentiating cells. In parallel, we are using CRISPR to delete regions of the long mpk‐1b intron to assess its functional importance. Our plan is to compare mpk‐1 nascent and mature transcripts during germline development, both in wild type and in in mutants that lack part of most of that large intron. Our analysis will tell us if the mpk‐1b 8.2 intron is critical for the mpk‐1 regulatory program during germ cell development.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the molecular mechanism of ProQ‐RNA interactions using a bacterial three‐hybrid assay
Authors: Pandey Smriti , Gravel Chandra , Berry Katherine
Published: June 2021

In bacteria, small RNAs (sRNAs) play important roles in gene regulation; sRNAs can regulate the translation and stability of target mRNAs via imperfect base pairing. These sRNA‐mRNA interactions are often facilitated by RNA chaperone proteins. ProQ has recently been identified as a global RNA‐binding protein that binds to dozens of sRNAs and hundreds of mRNAs in multiple proteobacteria; this observation has led to the proposal that ProQ may act as a widespread regulator of bacterial gene expression.

Our goal is to understand the molecular mechanisms of ProQ's interaction with regulatory RNAs, mapping the amino acids on ProQ's surface and nucleotides of RNAs that contribute to binding and regulation by using a bacterial three‐hybrid (B3H) assay to genetically detect ProQ‐RNA interactions. In the B3H assay, ProQ is fused to RNA polymerase (RNAP) and a hybrid RNA containing either an sRNA or 3′UTR of interest is tethered to a DNA sequence upstream of a test promoter. Interaction of ProQ with the RNA stabilizes the binding of RNAP to the test promoter and activates transcription of a reporter gene.

We have detected preliminary B3H interactions of ProQ with several of its RNA partners. Our data supports a model where the conserved N‐terminal‐domain (NTD) is the primary region for RNA binding. Further, we have identified point mutations in ProQ that affect its RNA interactions and have used immunodetection to determine ProQ expression levels to ensure that these mutations are not destabilizing. Current efforts are focused on screening for additional ProQ point mutations to locate the binding interface(s) for the sRNAs and mRNAs with which it interacts as well as exploring the structure and sequences of RNA that are required for ProQ interaction.

Support or Funding Information

Research supported by Mount Holyoke College and the Luce Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNA binding properties of the Lsm1–7 ring from Schizosaccharomyces pombe
Authors: Virta Johanna M , Hayes Samuel M , Montemayor Eric J , Butcher Samuel E
Published: June 2021

Lsm proteins form heptameric rings that bind RNA. Eukaryotes contain Lsm1–7 and Lsm2–8 ring structures, which are comprised of Lsm2, Lsm3, Lsm4, Lsm5, Lsm6, Lsm7 and either Lsm1 or Lsm8. Thus, the two rings differ by only one component. This small change in composition has a profound effect on the biological function of the two rings. Lsm1–7 is located in the cytosol and plays an essential role in the decay of messenger RNA, whereas Lsm2–8 is located in the nucleus and specifically binds to the 3′ end of U6 RNA. It is important to understand the RNA binding properties of Lsm rings. For example, the Lsm1–7 mRNA decay pathway regulates a large proportion of gene expression (Garneau et al., 2007).

It is known that Lsm2–8 binds to U6 snRNA via a 3′ polyuridine stretch and a terminal phosphate group (Licht et al., 2008; Didychuk et al., 2017). In contrast, the RNA binding properties of the Lsm1–7 ring are not well understood. Free Lsm1–7 rings in S. cerevisiae lack tight binding affinity for polyuridine tracts (Zhou and Zhou, et al., 2014). The cognate RNA sequence for the free Lsm1–7 ring is unknown, although association of the ring with the Pat1 protein can stimulate binding of the ring to polyuridine tracts (Wu et al., 2014). Crystal structures of the free Lsm1–7 complex from S. cerevisiae show that the C‐terminal region of Lsm1 impinges on the uridine‐phosphate binding pocket observed in the S. cerevisiae Lsm2–8 ring with U6 snRNA (Zhou and Zhou, et al., 2014; Sharif and Conti, 2013; Montemayor et al., 2018). We hypothesized that Lsm1 is a central regulator of RNA binding activity in the Lsm1–7 complex and that Pat1 is an allosteric regulator of Lsm1.

We have purified Lsm rings from the fission yeast S. pombe, which closely resemble humans with respect to Lsm ring sequence and known binding properties. We have made several novel observations. First, free Lsm1–7 is capable of binding polyuridine tracts in the absence of the Pat1 cofactor if the polyuridine tract is followed by an adenosine residue. This is the first observation of tight RNA binding affinity for Lsm1–7. Second, we find that deletion of the C‐terminal region of Lsm1 allows Lsm1–7 to bind polyuridine tracts 10‐fold more tightly in the absence of Pat1 or the single adenosine residue. These data support our hypothesis that the C‐terminal region of Lsm1 inhibits association of the ring with RNA, and as such is a negative regulator of RNA binding activity. Finally, we were able to determine a 1.9 Å resolution crystal structure of the S. pombe Lsm1–7 ring (lacking the C‐terminal region of Lsm1) in complex with a polyuridine RNA. This structure shows that the uridine nucleotides bind into the same binding pockets as in Lsm2–8, as hypothesized previously (Montemayor et al., 2018). This work expands our understanding of how the Lsm1–7 complex binds RNA.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RaPID Analysis of Cytomegalovirus of Long Noncoding RNA Binding Proteins
Authors: Lee Jessica E. , Kulesza Caroline
Published: June 2021

Human Cytomegalovirus (HCMV) is a ubiquitous herpes virus that is widespread and usually asymptomatic among healthy individuals. Congenital HCMV infections are the leading cause of virally induced birth defects. HCMV also causes severe disease in immunocompromised patients. Previous work in our laboratory has shown that CMV encodes a large noncoding RNA called RNA5.0. Using an animal model, the homologous RNA in the murine model called RNA7.2 has been shown to help the virus persist in host salivary glands, but it is unclear how this works at the molecular level. To identify the proteins attaching to RNA5.0, the RaPID system will be used. The RaPID system uses a highly active biotin ligase that tags proteins which bind to specific RNA sequences in cells. The RaPID system was pioneered in Paul Khavari's lab to demonstrate a small cellular RNA (EDEN15) binds to the CELF1 protein. We have reproduced their data in our laboratory and will use this system to identify RNA5.0 binding proteins. We will be looking for proteins that bind to the 3′ end of RNA5.0 because there is evidence this sequence is related to processing and stability of the RNA. Moving forward different length fragments spanning the 3′ end of RNA5.0 will be used in the RaPID system to identify which proteins interact with the sequence. The identified proteins might point out specific pathways to target for a potential treatment for CMV.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Uncovering Caprin1's biological role to understand it's function in autism
Authors: Martinez Criseyda , Pani Bibhusita , Nudler Evgeny
Published: June 2021

Deregulation of various cellular pathways such as the immune, mRNA translation and cell cycle pathways have been linked to autism disorders, yet the cause of deregulation remains unclear. Here we study the Cytoplasmic Activation/Proliferation‐Associated Protein‐1 (Caprin1), an RNA binding protein implicated in autism. In neurons, Caprin1 binds and represses the translation of selective mRNAs at RNA granules. Upon various stimulations such as with brain derived neurotropic factor (BDNF), mRNAs dissociate from Caprin1 and shift into actively translating ribosomes. In addition, studies identified Caprin1 as a positive cell cycle regulator, allowing cells to transition between G1 and S phase by binding to upregulated cell cycle mRNAs. Yet, how Caprin1 mediates repression or promotion of different mRNAs is not well understood. To identify and understand the global mechanism of Caprin1 we conducted a series of high throughput assays including mass‐spectrometry‐based quantitative proteomics and RNAseq. We show that in the absence of Caprin1, cell cycle and cell metabolic genes were significantly decreased. On the other hand, absence of Caprin1 significantly increased genes involved in immune responses, including antigen processing. To further identify a mechanism we conducted a crosslinking‐immunoprecipitation (CLIP) assay to identify mRNAs bound directly to Caprin1. We find that Caprin1 mainly binds to mRNAs upstream of cytokine signaling and cell cycle pathways at the 3′UTR. We further show that Caprin1 degrades or stabilizes different bound mRNAs. Thus, we conclude that Caprin1 has a role in RNA stability, particularly in genes involved in cell cycle and immune response pathways, which are deregulated and implicated in autism.

Support or Funding Information

Robertson Foundation, NIH R01AI090110 Supplement, Howard Hughes Medical Institute (HHMI)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human Tat‐specific Factor 1 Binds and Exports HIV‐1 RNA to the Cytoplasm
Authors: Miller Heather
Published: June 2021

Human immunodeficiency virus type 1 (HIV‐1) requires many human cofactors for propagation. Multiple groups have shown that human Tat‐specific factor 1 (Tat‐SF1) is an HIV‐1 dependency factor. This protein's knockdown does not affect the overall levels of viral RNA, however it does regulate the relative size classes (unspliced, singly spliced, and fully spliced). The molecular mechanism behind this is unknown, but data is consistent with roles in enhancing splicing, export, and/or stability of the viral RNA. We hypothesized that if Tat‐SF1 plays any one of these roles, then we should be able to demonstrate binding of the protein to the RNA genome. Furthermore, if Tat‐SF1 is critical for nuclear export of the viral RNAs, then knocking down Tat‐SF1 should result in more nuclear retention of the viral genome. Fragments of the HIV‐1 genome were synthesized by in vitro transcription and end‐labelled with fluorescein. These RNA probes were used in both electrophoretic mobility shift assays (EMSA) and florescence polarization (FP) assays. To analyze RNA export, HeLa cells with an shRNA‐expressing plasmid targeting Tat‐SF1 were also transfected with pSG3Δenv, a non‐infectious HIV‐1 plasmid. RNA from nuclear and cytoplasmic fractions was purified, followed by RT‐qPCR analysis. Our results suggest that Tat‐SF1 plays a role in binding the HIV‐1 genome and exporting the unspliced transcripts to the cytoplasm. This work provides additional molecular details on how Tat‐SF1 acts as an HIV‐1 dependency factor.

Support or Funding Information

High Point University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Stress Modulates Elongator Protein 3 (ELP3) Function to Enhance Dopaminergic Neurotoxicity in Cell Culture and Animal Models of Parkinson's Disease
Authors: Abdalla Ahmed , Charli Adhithiya , Ghaisas Shivani , Kondru Naveen , Malovic Emir , Jin Huajun , Anantharam Vellareddy , Kanthasamy Arthi , Kanthasamy Anumantha
Published: June 2021

Mitochondrial dysfunction is well established in Parkinson's disease (PD). Exposure to mitochondria‐targeting neurotoxicants, e.g., those neurotoxic pesticides that act as mitochondrial complex I inhibitors, has been linked to the pathogenesis of environmentally linked PD, but the molecular mechanisms underlying the enhanced vulnerability of dopaminergic neurons to mitochondrial toxicants are not fully understood. The histone acetyltransferase ELP3 (Elongator Protein 3) is the catalytic subunit of the RNA polymerase II elongator complex and plays an essential role in transcript elongation and maturation of projection neurons. Dysregulation of ELP3 has been implicated in a number of human disorders, but its potential role in the pathogenesis of PD remains elusive. In a serendipitous finding, we observed that mitochondria‐targeting pesticides can disrupt ELP3's function in dopaminergic neurons in cell culture and rodent models of PD. In this study, we further characterized ELP3 expression in the mouse brain and found high expression of full‐length ELP3 (62 kDa) in cytosolic fractions from different brain regions, while high levels of short‐form ELP3 (~40 kDa) were detected in the olfactory bulb (OB). Interestingly, immunohistochemical analyses revealed that ELP3 is highly expressed in TH‐positive neurons of the OB. Furthermore, OB slice cultures treated with the mitochondrial complex‐I inhibiting pesticide tebufenpyrad (Tebu) showed a reduced level of short‐form ELP3 in mitochondrial fractions. Consistently, we also found that ELP3 levels were significantly downregulated in the OB of transgenic MitoPark mice, a model which recapitulates mitochondrial dysfunction in PD. Collectively, our results indicate that ELP3 is a novel cellular target that can modulate the susceptibility of dopaminergic neurons to neurotoxic mitochondria‐targeting stressors

Support or Funding Information

NIH grants ES026892 and ES027245 and Eugene and Linda Lloyd Endowed Chair

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Acetylation on Beta‐2‐Microglobulin on Amyloid Formation
Authors: Ballard Abigail Rene , Richardson John
Published: June 2021

Beta‐2‐Microglobulin (B2m) is a small 99 residue globular protein that is a part of the type I major histocompatibility complex found on all nucleated cells. It is shed from the surface of cells into the bloodstream as cells undergo apoptosis then is degraded and excreted from the body in people with normal functioning kidneys. However, B2m is not effectively removed from the blood by hemodialysis causing systemic problems for patients in end‐stage kidney disease (ESKD). B2m serum levels in patients with ESKD are up to 60 times greater than healthy individuals. Dialysis related amyloidosis (DRA), is a disease that is often experienced by those with ESKD. DRA results from the accumulation of misfolded B2m and is characterized by carpal tunnel like symptoms coupled with chronic joint inflammation. DRA is a result of the deposition of misfolded B2m in the musculoskeletal system due to B2m amyloid plaque formation. However, the increased concentration of B2m alone is not sufficient to trigger the pathogenic amyloidosis at physiological pH. Thus, a secondary factor must be acting as the trigger of misfolding. This study is investigating the effect of acetylation as a non‐natural post translational modification on B2m. Acetylation is a practicable suspect as aspirin consumption is known to acetylate a variety of proteins non‐specifically. The acetylation of lysines would alter the overall charge and possibly disrupt ability of B2m to maintain its native state conformation thus increasing the aggregation propensity of B2m. We are primarily testing the susceptibility of B2m to acetylation via protocol established for acetylation in bovine carboxy anhydrase. Additionally, growth of the amyloid fibrils of the acetylated B2m will be monitored with thioflavin‐T fluorescence to assess the effects of lysine acetylation on B2m amyloid formation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism of gasdermin D recognition by inflammatory caspases and their inhibition by a gasdermin D‐derived peptide inhibitor
Authors: YANG JIE , Liu Zhonghua , Wang Chuanping , Yang Rui , Rathkey Joseph K. , Shi Wuxian , Chen Yinghua , Dubyak George R. , Abbott Derek W. , Xiao Tsan Sam
Published: June 2021

The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases‐1/4/5/11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N‐terminal domain to associate with membrane lipids and form pores, which induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full‐length GSDMD and its cleavage site peptide FLTD. A GSDMD‐derived inhibitor N‐Acetyl‐Phe‐Leu‐Thr‐Asp‐Chloromethylketone (Ac‐FLTD‐CMK) inhibits GSDMD cleavage by caspases‐1/4/5/11 in vitro, suppresses pyroptosis downstream of both canonical and non‐canonical inflammasomes, as well as reduces IL‐1β release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase‐3 or apoptotic cell death, suggesting that Ac‐FLTD‐CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase‐1 in complex with Ac‐FLTD‐CMK reveals extensive enzyme‐inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase‐1 structures demonstrates drastic conformational changes at the four active‐site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases, which is a novel tool for investigating inflammasome signaling.

Support or Funding Information

T.S.X. is supported by NIH grant AR069908 and the Case Research Institute. Z.L. is supported by a postdoctoral fellowship from the American Association of Immunologists. J.K.R. is supported by NIH grant T32 GM007250. D.W.A. is funded by NIH grants DK091222 and GM086550.

This 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 Photoswitchable c‐Raf Inhibitor
Authors: Weiblen Emilee Ann , Ranger Marah Noel , Streu Craig Nolan
Published: June 2021

Cancer is one of the leading causes of death in the United States. As a result of its staggering impact, it is unsurprising that scientists have been working diligently to find treatments. Chemotherapeutics and other anticancer drugs have come a long way since their discovery, however the vast majority of them still cause unintended side effects. The main issue with many cancer medications at the moment is the high rate at which healthy, noncancerous cells are damaged along alongside the cancer cells. Highly desirable new treatments for cancer are ones that can selectively target cancerous cells while excluding healthy cells. One way that we can address the problems with the current therapies is by using photoisomerizable compounds, more specifically azo‐stilbenes.

Photoisomerizable compounds are molecules that are isomerized by light. In other words, their structure changes when exposed to specific light wavelengths. The azo‐stilbenes, in particular, undergo a trans to cis isomerization when hit with blue or UV light. This isomerization is easily reversible and can be accomplished quickly by irradiating the molecule with a longer wavelength of light (typically >500 nm) or more slowly at room temperature in the absence of light.

Photoisomerizable compounds like the azo‐stilbene group have become widely used in many fields, such as functional materials, yet these azo‐compounds have more recently found utility in cancer research due to their ability to be isomerized. These azo‐compounds take one conformation in their trans form and when hit with light isomerize into the cis conformation, thus taking a very different shape. Given that shape complementarity is critical for many drug‐target interactions, this isomerization results in a change in the bioactivity of these molecules. When in their trans form, the azo‐compounds cannot bind to the same target that they can in their cis form. Judicious application of the appropriate wavelengths of light can be used as a switch that will allow for selectivity in cytotoxic treatments. With the use of azo‐stilbenes, off‐target side effects could be greatly reduced and potentially eradicated.

One example of a potential target for photo‐switchable therapeutics is c‐Raf. This kinase plays a key role in a pathway that regulates the cellular cycle including the production of new cells and apoptosis, which is dysregulated in some cancers. This work will describe the design, completed synthesis, photokinetic analysis, and preliminary bioactivity measurements for two lead molecules with potential as photoswitchable drugs based upon a previously reported c‐Raf inhibitor.

Support or Funding Information

Albion College Foundation for Undergraduate Research Scholarship and Creativity (FURSCA)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological activities of Nepenthes miranda extracts
Authors: Lien Yi
Published: June 2021

Carnivorous Nepenthes plants exhibit many ethnobotanical uses and their extracts possess many pharmacological activities. In this study, we prepared different extracts from leaves, stems, pitchers, and flowers of N. miranda (Nm) and investigated the antioxidant, antibacterial, anticancer, and anti‐inflammatory properties. To evaluate extraction efficiency, different solvents such as methanol, ethanol, acetone, and distilled water were used individually for Nm extract preparations. Through DPPH radical scavenging assay, Folin‐Ciocalteu method, and aluminium chloride calorimetric analysis, the stems extract obtained by 100% acetone (Nm‐s‐a) showed the highest total antioxidant activity, total phenolic content, and total flavonoid content, respectively. In addition, we found that the same extract also exhibited the highest antibacterial activity against three pathogens, namely, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The inhibition zones of Nm‐s‐a determined by agar‐well diffusion assay ranged from 9–29 mm and were in the following order: E. coli > S. aureus > P. aeruginosa. To demonstrate the cytotoxicity effect of Nm‐s‐a, human embryonic kidney 293 cells (HEK 293) and melanoma cells were cultured with various concentrations of Nm‐s‐a. Furthermore, inhibition of human neutrophil elastase by Nm‐s‐a was also found, suggesting the anti‐inflammatory activity. These results indicated that N. miranda may have some medicinal potentials. To further elucidate the structure‐activity relationship, our laboratory is currently using LC‐MS to isolate and identify the active compounds from Nm‐s‐a.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a reference panel as a way to harmonize data of genotyped Sclerotinia sclerotiorum
Authors: Persson Rachel
Published: June 2021

Several population studies of S. sclerotiorum have been conducted and resulted in important publications furthering our knowledge of this pathogen. Genotype data from those studies has been published with the notion that this data could be re‐used by other scientists. However, amplicon sizes were known to vary across labs due to primer modifications for fluorophore labeling and the instrument used to perform fragment analysis. Thus, it is necessary to create a reference panel of DNA that represents allele sizes known to be present within the population, which is a technique used in human DNA forensics for the same type of genetic marker. Given recent completion of genotyping of more than 366 S. sclerotiorum isolates from across the U.S., development of a DNA Reference Panel that represents most alleles within S. sclerotiorum loci isis now possible. Here, we identified 22 S. sclerotiorum isolates that were collected and genotyped previously and represented all known possible alleles among the 366 S. sclerotiorum. Then, DNA was isolated from those 22 S. sclerotiorum strains and genotyping was performed using 11 previously developed SSR markers. PCR used a 3‐primer indirect fluorescent labeling system that was slightly different from the system used to genotype the 366 isolates, which used direct primer labeling. Amplicons were sized using capillary electrophoresis and raw chromatograms imported into R for sizing and analysis. This data was used to determine differences in allele sizes and create an R function to automatically re‐classify observed data so that it could be seamlessly integrated into our 366 genotype database. Using results of 22 isolates, harmonization of data from two previous studies that genotyped 95 isolates mainly from Brazil and 366 isolates from U.S. took place. From this data, estimation of population genetic statistics, such as genetic/genotypic diversity, evenness, and richness, and test for evidence of sexual recombination was done. Taken together, the DNA reference panel represents most alleles within S. sclerotiorum and allows for integration of previously published population genetic data of this plant pathogen.

Support or Funding Information

Supported by Margarita Marroquin‐Guzman and Sydney E. Everhart. Funded by Undergraduate Creative Activities and Research Experience at UNL

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The landscape of aging and maturation signatures of mouse oocyte development
Authors: Lee Tin Lap , Qian Yan , Cao Qin , Liao Jingyue , Lee Annie Wing Tung , Yip Yuk Lap , Leung Tak Yeung , Zhao Ming Peng , Chan David YL , Lee Tin Chiu , Chan Wai Yee
Published: June 2021

Female fertility declines dramatically with age, and childbearing at advanced maternal age is a big health concern due to greater risk of early pregnancy‐related complications such as miscarriage, genetic disorders, and ectopic pregnancy. The negative impact on fertility caused by advanced maternal aging is also implicated in assisted reproduction technologies. A major factor that accounts for fertility deterioration in older women is poor oocyte quality. By applying RNA‐seq, we compared transcriptome profiles of GV and MII oocytes from the young and old group. We identified 4613 maturation‐related DEGs at the young age and 4223 maturation‐related DEGs at old age. Then, we compared the maturation‐related dynamics at the young age against the maturation‐related dynamics at old age. These DEGs showed high Pearson's correlation (r = 0.8181), suggesting that most of them shared the same maturational expression pattern. To characterize DEGs whose maturational expression changes were affected by age, we established a linear regression model and used a statistical cutoff (95% prediction interval) to define 260 DMRs. The signatures will contribute to novel knowledge essential to the development of assays and treatments for female reproductive aging.

Support or Funding Information

Hong Kong Collaborative Research Fund C4054‐16G

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.