bluuebunny/crossref_metadata_2025_split huggingface dataset:
Whole transcriptomic evaluation of SK‐MEL‐3 melanoma cells in response to histone deacetylase inhibitor trichostatin A.
Authors: Mazzio Elizabeth A. , Soliman Karam F. 
Published: June 2021

Malignant melanoma has a proclivity for resistance to radiation and chemotherapy drugs such as dacarbazine or kinase inhibitors that target RAS‐independent auto‐activated serine/threonine kinase, a consequence of somatic missense mutant BRAF v 599E. An underlying controlling component of aggressive melanoma is a change to the epigenetic histone landscape to which overexpression of histone deacetylases (HDACs) has been attributed. The full effects of direct inhibition of HDAC on the entire transcriptome are not well understood, yet there is growing number of synthetic drugs classified as HDAC inhibitors (HDACis). In this work, we evaluated the whole transcriptome response in SK‐MEL‐3 human melanoma to trichostatin A using both Affymetrix 2.1 Human Arrays and 4.1 miRNA chips. TSA was confirmed as a potent HDAC inhibitor in nuclear lysates, and in cells trichostatin, A (TSA) [10nM] at sublethal concentrations were used for to evaluate transcriptomic shifts at 24 hours exposure. Of interest, the data show no changes to the expression of 12 HDAC transcripts (HDACs subtypes 1–12) or B‐raf protooncogene V599E by TSA. In contrast, TSA evoked differential expressed changes with 833 up‐regulated and 810 down‐regulated genes. The top 10 up‐regulated genes were TSPAN13, SERPINI1, ATP1B2, NMNAT2, PDGFRL, CYP1A1, PARM1, SCG2, SYT11, ROPN1L and top 10 down‐regulated genes GALNT3, CA14, BCL2A1, TRPM1, PYCARD, UBAP1L, GPX8, IL‐6, TP53, SERPINH1. Functional pathway analysis shows TSA effects to negatively impact cell survival systems, cell cycle progression, migration and biological pH buffering specifically that of carbonic anhydrase 14. These changes occurred concomitant to an elevation of miRs 185‐3p, 4783‐3p, 6716‐3p and reduction of miR‐6795‐5P. In conclusion, data from this work provide an overall effect of TSA on the whole transcriptome of coding and non‐coding RNA in malignant melanomaSupport or Funding InformationThis study was supported by the National Institute on Minority Health and Health Disparities (NIMHD) awards G12MD007582 and P20 MD 00738.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rapid flow‐induced Gαq/11 activation occurs upstream of Piezo1 activation
Authors: dela Paz Nathaniel G. , Frangos John A. 
Published: June 2021

Endothelial mechanotransduction is the process by which mechanical stimuli are sensed by endothelial cells (ECs) and transduced into biochemical signals and ultimately into physiological responses. Identifying the primary mechanosensor(s) and describing the mechanism(s) by which they receive and transmit the signals has remained a central focus within the field. The heterotrimeric G protein, Gαq/11, is proposed to be part of a macromolecular complex together with PECAM‐1 at the cell‐cell junction of ECs and may serve as a primary mechanosensor as it is rapidly activated within seconds of flow onset. The mechanically‐activated cation channel, Piezo1, has recently been implicated due in part to its involvement in mediating early responses, such as calcium influx and ATP release. In this study, we used in situ proximity ligation assay (PLA) to investigate the role of Piezo1 in the rapid shear stress‐induced activation of Gαq/11. We show that the flow‐induced dissociation of Gαq/11 from PECAM‐1 in ECs at 15 sec is abrogated by BIM‐46187, a specific inhibitor of Gαq/11 activation, suggesting that Gαq/11 activation is a prerequisite for PECAM‐1/Gαq/11 dissociation. Although siRNA knockdown of Piezo1 caused a dramatic decrease in PECAM‐1/Gαq/11 association in the basal condition, it had no effect on their flow‐induced dissociation. Interestingly, siRNA knockdown of Piezo1 caused a marked decrease in PECAM‐1 expression. Selective blockade of Piezo1 with ion channel inhibitors also had no effect on flow‐induced PECAM‐1/Gαq/11 dissociations despite having an inhibitory effect on Akt phosphorylation. Rapid flow (15 sec) also led to increased association of Gβ1 with Piezo1 as well as with the p101 subunit of phosphoinositide 3‐kinase (PI3K), which were both blocked in the presence of the Gβγ inhibitor, gallein. Taken together, our results indicate that flow‐induced activation of Piezo1 is downstream of G protein activation.Support or Funding InformationThis work was supported by NIH MERIT Award R37 HL040696 to J.A.F.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insulin and ketones elicit disparate effects on mitochondrial uncoupling in adipose tissue
Authors: Bikman Benjamin T. 
Published: June 2021

ObjectiveInsulin is a powerful regulator of metabolic function, promoting a general storage of nutrients. In contrast, ketones, inversely regulated by insulin, lead to enhanced energy use and wasting. The purpose of this study was to explore the effects of insulin and ketones on mitochondrial coupling status in adipose tissue.MethodsWe used cell, rodent, and human models, to varying degrees. 3T3‐L1 adipocytes were treated at various times with insulin or ketones. Similarly, rodents were chronically injected with insulin or placed on ketogenic diets. Finally, human fat biopsies were obtained from in states of ketosis. In all models, mitochondrial respiration was determined and, where available, uncoupling protein (UCP) 1 was measured.ResultsIn cells and rodents, insulin reduced mitochondrial respiration and, in rodents, reduced indirect calorimetry. In all models, including human adipose, ketones increased respiration rates and UCP1 levels.ConclusionsInsulin and ketones elicit disparate effects on mitochondrial uncoupling in adipose tissue. Whereas insulin reduces uncoupling, resulting in more tightly coupled electron transport, ketones enhanced uncoupling, resulting in energy wasting.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Misregulation of Polycomb Repressive Complexes by Oncohistones
Authors: Lewis Peter W. 
Published: June 2021

Covalent modifications to DNA and the histone packaging proteins allow chromatin to act as a dynamic signaling platform to regulate genomic DNA access and ultimately establish and maintain cellular phenotypes. Moreover, there is increasing appreciation that chromatin alterations per se, including DNA and histone modifications, are involved in the pathogenesis of cancer. Nowhere is this better supported than with the groundbreaking discoveries of high‐frequency, somatic mutations in histones that are drivers of oncogenesis. These mutations (collectively called ‘oncohistones') cause amino acid substitutions that localize to conserved residues in the N‐terminal tail of histone H3 and all seem to be linked, either directly or indirectly, to disruption of normal levels and distribution of histone H3 lysine 27 methylation and thus transcription regulation. We are currently using a combination of biochemical, chromatin and transcriptomic approaches, along with defined cell lines to understand how histone H3 lead to altered chromatin states that profoundly influence gene expression patterns. I will discuss some of our recent work on how histone mutations perturb cellular differentiation through inhibition of specific chromatin‐modifying enzymes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glutamine Synthetase Serves as an Accurate Immunohistological Marker for Perivenular Hepatocytes in the Liver
Authors: Mak Ki M. 
Published: June 2021

In the mouse liver, Axin2 (axis inhibition protein 2) is selectively expressed in hepatocytes that form a single layer surrounding the central vein, defined as perivenular hepatocytes. Axin2 hepatocytes display hepatocyte stem cell‐like properties. These cells self‐renew and their progenies migrate from the centrilobular zone towards the periportal area, replacing lobular uninjured or senescent hepatoytes and contributing to physiological turnover of parenchymal cells. In one year, Axin2 cells replenish on average 40% of hepatocytes in the liver under normal conditions. Axin2 cells also express glutamine synthetase (GS), which detoxifies the ammonia entering the liver circulation. However, both Axin2 and GS expression is maintained by the Wnt/β‐catenin pathway derived from the central vein endothelium. Because no suitable antibody that detects perivenular Axin2 expression by immunohistochemistry is available to date, GS has been used as a surrogate marker for Axin2 to stain perivenular hepatocytes. The present study tested whether GS serves as a marker for perivenular hepatocytes and evaluated the effects of fibrogenesis on the lobular distribution of GS‐positive cells in the human liver using cadaveric liver samples. Immunoperoxidase staining with a polyclonal GS antibody revealed that in the liver with minimal fibrosis (N = 8), GS selectively stained perivenular hepatocytes generally arranged in 1–3 layers surrounding the central veins. GS‐positive cells were also seen as a single layer along the larger collecting veins. There was a variable presence of small aggregates of GS‐positive non‐perivenular hepatocytes in the centrilobular zone. The GS immunoreactivity in hepatocytes was cytoplasmic. No GS staining of periportal hepatocytes was seen. A similar distribution of GS‐positive hepatocytes was observed in the liver showing advanced stages of septal fibrosis (N = 8), bridging fibrosis (N = 8) and incomplete cirrhosis (N = 6). In end‐stage cirrhosis (N = 4), GS expression could not be observed around the central veins in the nodular parenchyma.ConclusionsGS serves as an accurate immunohistological marker for perivenular hepatocytes that are considered as hepatocyte stem cells in the mouse. GS expression in non‐perivenular hepatocytes in the centrilobular zone suggests an expansion of perivenular hepatocytes in response to hepatic fibrogenesis. Loss of perivenular GS expression in cirrhosis may reflect a disruption of contact between the central vein endothelium and perivenular hepatocytes. Understanding the histogenesis of GS expressing perivenular hepatocytes may help in therapeutic planning for enhancing hepatic regeneration in liver fibrosis disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective Effect of Beta vulgaris on Carbon tetrachloride induced Hepatotoxicity in adult Wistar Rat
Authors: EGEONU SOLOMON , IHENTUGE CHURCHILL , OKECHUKWU HOPE , ANIBEZE CHIKE , AKPUAKA FRANK 
Published: June 2021

Beta vulgaris also known as beet root has been used to treat a wide range of symptoms and diseases, such as fever, constipation, cancer, diabetes, liver and heart diseases. The aim of this study was to investigate the protective ability of beta vulgaris on the liver following Carbon tetrachloride (CCl4) intoxication. Twenty five adult albino wistar weighing between 150g and 200g were used in this study. The animals were divided into five groups A, B, C, D, and E. Groups A and B served as control were injected with paraffin oil for two consecutive days and group B fed with Beta vulgaris extract alone. Groups C and D were injected with CCl4 (1.0ml/kg b.w i.p) for two consecutive days and group D fed with BE throughout the experimental days. Group E was fed with Beta vulgaris extract throughout the experiment and injected CCl4 on the last two days of the experimental period (28 days).At the end of the experimental period animals were reweighed and sacrificed using chloroform vapour. Blood was collected for serum enzyme analysis and liver was harvested and trimmed for histological studies. The result showed that, there was a decrease in mean body weight of 5% in group C animals while there was an increase in other groups. The liver weight were significantly higher in group C (p<0.05) when compared with control groups and other experimental groups. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in group C were significantly higher (p<0.05) when compared with the control group while groups B, D and E were normal.Histological examination of liver (H&E) showed numerous fatty changes, distortion of hepatocytes, focal necrosis, loss of nuclei and cytoplasmic shrinkage in group C animals, groups A, B and E appear normal, while group D showed mild focal inflammation and areas of regeneration of tissuesThese results shows that Beta vulgaris extract has the ability to prevent and also restore histological and biochemical changes in the liver following CCl4 injection.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immunolocalization of Hsc70 at Bacterially‐Generated Actin‐Rich Structures
Authors: Walker Brittany Dominique , Chua Michael Dominic , Guttman Julian Andrew 
Published: June 2021

The actin cytoskeleton is commonly hijacked by bacterial pathogens. Listeria commandeers this filament system during their internalization, and for actin‐rich comet tail formation that enable the microbes to move within the infected cells as well as transfer from cell to cell. Enteropathogenic Escherichia coli (EPEC), an extracellular microbe, controls this cytoskeletal system when they generate E. coli pedestals as part of their firm docking to the plasma membrane of their host's cells and as they “surf” atop the infected cells. Finally, Salmonella exploit the host actin cytoskeleton to enter eukaryotic cells by forcing the generation of actin‐based membrane ruffling. We recently identified Hsc70 in a proteomics screen of EPEC pedestal components. This protein is normally involved with chaperoning functions within the cell. Despite there being a lack of evidence for an association of Hsc70 and actin, due to our proteomics identification of Hsc70 at EPEC pedestals we hypothesized that Hsc70 could be a common target of bacterial pathogens that control the actin cytoskeleton during their disease processes. To test this hypothesis we immunolocalized Hsc70 during Listeria, EPEC and Salmonella infections and found it concentrated at all actin structures generated by the microbes, demonstrating that this protein is a universal component for bacterial actin‐based structures.Support or Funding InformationFunding provided through NSERC. (Grant No. 355316 to J. A. G.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Plant PCR inhibitor release as a function of sample dissociation method
Authors: Easparro Brandon , Morehouse Zachary , Proctor Caleb , Atwood James 
Published: June 2021

The polymerase chain reaction (PCR) is an invaluable technique used in a wide range of molecular biology applications across multiple disciplines ranging from medical diagnosis and forensic analysis to botanical research. Since PCR is an enzymatic reaction, it is sensitive to inhibitors. PCR inhibitors may interfere with multiple steps in the PCR process leading to reduced sensitivity or even false negatives through inhibition of the amplification of nucleic acids by enzyme binding, nucleic acid cross‐linking/interaction, or through mimicking the nucleic acid structure. Inhibitors result from the original sample or during the extraction process. For plant studies, PCR inhibition may result from contamination by acidic polysaccharides and phenols including xylan, dextran sulfate, and flavonol; among others. While PCR inhibition has been demonstrated for multiple plant‐based compounds and procedural approaches have been recommended for removal of these inhibitors, to our knowledge, there has not been a study to compare the release of these compounds and the subsequent inhibitory effect based on different methods of sample dissociation.Herein, we evaluate three different dissociation techniques, including bead milling, cryo‐grinding and rotor‐stator dissociation for the extraction of genomic DNA and total RNA from sweet basil (Ocimum basilicum) and blueberries (Vaccinium corymbosum) and the release of plant PCR inhibitors. Polysaccharide, phenol, nucleic acid, and protein content was quantified and compared between each approach. qPCR was then employed to monitor the impact of PCR inhibitor content for both classic PCR and RT‐PCRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diet Restriction Induces Heat Shock Gene Expression While Tempering Heat Stress Response in Drosophila melanogaster
Authors: Tresser Jason W. , Bliss Megan , Mayweather Hailey , Rustand Emerson , Sears Alex , Varamini Behzad 
Published: June 2021

Diet restriction (DR) in fruit flies mimics the longevity effects that calorie reduction has in other organisms. Long‐lived flies are smaller, have global down regulation of protein levels, and lower fecundity. We predicted that flies on DR food would have a limited ability to respond to heat stress. We used qPCR to measure expression levels of heat shock proteins in Drosophila melanogaster fed control food or diet restricted food at standard or high temperatures. HSP22, HSP70 and HSP83 are known to be upregulated in flies in response to various stressors. We show here that flies under diet restriction have elevated HSP22 and HSP70 expression at standard temperatures and upregulate HSP 83 in response to heat stress.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The subcellular distribution of the human Fic protein, HYPE/FicD, using a high resolution, tomography compatible electron microscopic method
Authors: Mattoo Seema , Sengupta Ranjan 
Published: June 2021

Localization of membrane proteins via electron microscopy (EM) at high resolution is dependent on a robust detection technology and a sample preparation method that confers superior ultrastructural preservation of membranes. Current methods for precise localization rely on chemical fixation of the sample followed by alcohol dehydration at room temperature. The combination of these traditional methods exhibit poor preservation of membrane, induces membrane artifacts and affects resolution. High pressure freezing (HPF) of cells followed by heavy metal staining via freeze substitution (FS) in acetone at sub‐zero temperatures are techniques that exhibit the best possible membrane preservation. Therefore, we have developed a hybrid method that combines high pressure freezing with an existing peroxide based detection technique typically used with chemical fixation methods. As proof of concept, we assessed the localization of a fusion construct, consisting of the N‐terminus of HMG CO‐A protein fused with a peroxidase tag (that faces the cytosol). We exploited the induction of OSERs (organized smooth ER) in cells by the over‐expression of this N‐terminal fragment, as a metric for specific staining and membrane preservation. We next extended our studies to assess the localization of the sole human Fic (filament induced by cyclic AMP) protein, HYPE. We previously showed that HYPE resides in the ER lumen, but various global transcriptomic and proteomic studies suggest the existence of cytosolic and extracellular substrates for HYPE. Using our hybrid methodology, here we provide unequivocal evidence that the HYPE is compartmentalized within the various subdomains of the ER. We further demonstrate via serial sectioning, that our technique enables the localization of HYPE through a large volume of the cell. Our work thus aligns well with the three‐dimensional mapping of membrane proteins in large organelles using EM‐tomography in a near‐native cellular context.Support or Funding InformationThis work was supported by funding from 1) an Indiana CTSI (Clinical and Translational Sciences Institute) Project Development Team Award and 2) the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R01GM100092.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The polypeptide exit tunnel of the ribosomal large subunit requires assembly factors for proper construction and function
Authors: Wilson Daniel , LaPeruta Amber , Woolford John 
Published: June 2021

The final product of ribosome assembly consists of two ribosomal subunits capable of functioning with high fidelity. In eukaryotes, this process is facilitated by protein assembly factors that can promote proper formation of functional centers through structural probing, functional test‐driving, acting as a scaffold, or protecting the site from premature activity. The polypeptide exit tunnel (PET) of the ribosomal large subunit (LSU) is the conduit through which nascent polypeptide chains are passed as they are synthesized. Protein and RNA components of the PET lumen actively participate in translation by interacting with the nascent chain in order to regulate ribosome stalling events, thus regulating translation speed and gene expression. Recent cryo‐EM structures of immature LSU particles from different stages of assembly spanning the nucleoplasm to the cytoplasm have shown that the PET is sequentially probed by the C‐termini of three assembly factors: first, Nog1, then Rei1, and finally Reh1. The C‐termini of these three assembly factors mimic nascent chains, plugging the PET for almost the entirety of LSU assembly. No biological function, however, has been assigned to this redundant occupation of the PET. Here, we show that truncating PET‐inclusive residues from the C‐terminus of Nog1 causes LSU assembly defects that are exacerbated by analogous truncations to one or both of the Rei1 and Reh1 C‐termini. These defects manifest in processing of late immature ribosomal RNA intermediates. Interestingly, these assembly defects are further exacerbated at lower temperatures. Despite these mutants displaying slow growth at permissive temperatures, however, mature ribosomes are produced that may be defective in translation. Our data supports the model that Nog1 is necessary for efficient construction of the PET during early stages of assembly while Rei1 and Reh1 carry out checkpoints during later stages.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.