bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of female sexual development‐1 (fsd‐1) Transcript Structure, Expression, and Localization in the Fungus Neurospora crassa
Authors: Hurysz Thomas , Gebhardt Bryce , Pyatt Mary , Emmens Kathryn , Toufexis Christine , Hutchison Elizabeth
Published: June 2021

Neurospora crassa is a filamentous ascomycete fungus that can reproduce sexually and asexually. The signaling pathways that control sexual development in N. crassa are not well characterized. FSD‐1 is an N. crassa transcription factor that controls both early and late stages of sexual development. Although we know that fsd‐1 regulates female sexual development (including fertility and spore maturation), little is known about (1) its transcriptional regulation, (2) RNA isoforms, (3) protein expression, and (4) localization. Intron and exon boundaries of fsd‐1 were mapped using reverse transcription (RT)‐PCR and sequencing and we determined that fsd‐1 can be expressed as three different transcripts, which differ in length and intron/exon structure. Quantitative RT‐PCR during a time course of sexual development showed that expression of fsd‐1 transcripts is not detectable until N. crassa initiates sexual development. In addition, one transcript variant is predominantly expressed during sexual development, and expression of this transcript peaks approximately five days after crossing (mating). It is unknown whether all three fsd‐1 transcripts are translated into protein, and thus we will monitor FSD‐1 protein expression using immunoprecipitation and western blotting. The three fsd‐1 transcript variants encode for predicted proteins of slightly different sizes, which can be visualized via western blot. To investigate FSD‐1 localization, we constructed a GFP‐tagged version of FSD‐1 using overlap PCR and targeted this construct to the native locus. FSD‐1‐GFP localization was monitored via fluorescence microscopy throughout a time course of development. We found that FSD‐1‐GFP localized to the nuclei of female reproductive tissues, such as paraphyses, but not to tissues where meiosis occurs. In addition, we confirmed that the FSD‐1‐GFP strain complements the sexual development‐related phenotypes that occur in an fsd‐1 knockout strain, such as sterility and lack of ascospore melanization. Finally, we constructed strains where fsd‐1 has an overexpression promoter, and we will determine whether overexpression of fsd‐1 has a dominant negative effect on wild‐type strains during mating. Little is known about the regulatory pathways that function in non‐meiotic filamentous fungal female sexual tissues, and our work on fsd‐1 provides an important first step in characterizing this aspect of fungal development.

Support or Funding Information

Geneseo Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Systematic Screening for Transcriptional Regulators of Adult Myogenesis in Drosophila by RNAi
Authors: Soudachanh Tommy , Oas Sandy , Mendes Tyler , Byrantsev Anton , Cripps Richard
Published: June 2021

The muscles of Drosophila show similar structure and pathway of development to the muscles of humans, and allows Drosophila to model human muscle disorders. However, little is known about many of the genes and their potential role in muscle development. Thus, the focus of this project is to efficiently screen for potential transcriptional regulators of muscle development and identify associated disorder phenotypes. Genes were selected based on having a potential role in transcription, which were silenced using RNA interference (RNAi). The 1151‐gal4 driver was utilized to induce RNAi in developing adult muscles. Combined with the driver, the flies harbored enhancers of either Flightin‐LacZ or TpnC41C‐LacZ. The resulting transgenic flies were collected and assayed to measure the effects of this genetic loss. All 101 genes in the latest set have been assayed for a change of enhancer activity of less than 70% or greater than 130%, in Flightin and TpnC41C; the genes selected for immunostaining exhibited semi‐lethality or lethality, and indicated a loss or gain of LacZ. The immunostaining would reveal several factors: muscle morphology, and expansion or loss of enhancer activity associated with specific flight and jump muscle genes. The resulting data was classified into three categories, severe loss, moderate loss, or no change in muscle development. Genes with a severe loss indicates a potential issue with myoblasts fusion, and genes with a moderate loss indicates issues with adult muscle formation and maturation. Therefore, the results demonstrate that the screen is efficient in identifying potential transcriptional regulators and muscle disorder phenotypes.

Support or Funding Information

National Institute of Health ROI GM061738

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of Positive Coactivator 4 with Histone 3.3 Protein is Essential for Transcriptional Activation of the Luteinizing Hormone Receptor Gene
Authors: Kavarthapu Raghuveer , Zhao Peng , Liao Mingjuan , Dufau Maria
Published: June 2021

The luteinizing hormone receptor (LHR) is a member of the G protein‐coupled receptor family which is essential for sexual development and reproduction in mammals. Previous studies from our laboratory established that Sp1 is important for basal LHR promoter activity and has central role in derepression of LHR gene transcription induced by the histone deacetylase inhibitor Trichostatin A (TSA) in MCF‐7 cancer cells. Moreover, our studies revealed that the co‐activator PC4 which associates directly with Sp1 at the LHR promoter is essential for TSA‐mediated LHR transcription. The present study explores interactions of PC4 with histone proteins which presumably triggers chromatin modifications during LHR transcriptional activation induced by TSA. TSA treatment of MCF‐7 cells transiently expressing PC4‐Flag protein induces acetylation of H3 and immunoprecipitation (IP) studies revealed its interaction with PC4‐Flag protein. Mass spectrometry analysis from the protein complex obtained after IP with PC4 flag in TSA treated MCF‐7 samples detected H3.3 acetylated at lysine residues (K9, K14, K18, K23, K27 and K36) as a PC4 interacting protein. This association of PC4 with H3.3 was corroborated by re‐ChIP. In subsequent studies by IP and reChip analysis using specific antibodies against acetylated H3 protein for the lysine residues, also confirmed PC4 interaction with acetylated H3 protein. Knockdown of PC4 in MCF‐7 cells significantly reduced H3 protein acetylation at these sites and LHR promoter activity in TSA treated cells despite an increase in H3 and H3.3, linking PC4 expression to H3.3 recruitment and its acetylation. Depletion of H3.3 A/B in MCF7 cells significantly impair recruitment of Pol II and TFIIB to the LHR promoter and its activation induced by TSA. In this study, we have shown interaction between PC4 and acetylated H3.3 in LHR induced derepression by TSA. Together these findings point to the critical role of PC4 and it is associated acetylated H3.3 protein in TSA‐induced LHR gene transcription.

Support or Funding Information

Intramural Research Program, National Institute of Child Health and Human Development, NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Regulatory Mechanism of the Transcription Factor Krüppel homolog 1 in Mosquito Reproduction
Authors: Ahmed Tahmina Hossain , Zhu Jinsong
Published: June 2021

Insect juvenile hormone (JH) plays a crucial role during reproduction in adult mosquitoes. Krüppel homolog 1 (Kr‐h1) is a transcription factor that is upregulated by JH. However, the molecular action of AaKr‐h1 in the JH‐regulated gene expression has not yet been well studied in adult mosquitoes. Our recent ChIP‐seq study has indicated that Kr‐h1 in Aedes aegypti binds to the promoter regions of genes that are under the control of JH or another crucial insect hormone, 20‐hydroxyecdysone (20E). Therefore, we hypothesize that AaKr‐h1 modulates the JH and 20E responses in adult mosquitoes. We have performed RNAi mediated gene silencing to examine the regulatory function of AaKr‐h1 on the selected JH (Met, Early Trypsin) and 20E (EcRA, HR3, E75A, βFTZ‐F1, and E93) responsive genes. Successful knockdown at the mRNA and protein levels was confirmed by Quantitative PCR (qPCR) and Western blot analyses respectively. In the AaKr‐h1 depleted mosquitoes, most of the selected JH and 20E responsive genes showed considerable alteration in their expression. Knockdown of AaKr‐h1 led to an increase in the expression of Early Trypsin and a significant reduction in the expression of Met, EcRA, HR3, and E93. In conclusion, our study showed that AaKr‐h1 regulates the expression of diverse genes that are crucial for egg maturation in adult female mosquitoes. Ae. aegypti is a vector of several tropical diseases including yellow fever, dengue, chikungunya and zika fever. The growing resistance to many of the currently used insecticides in mosquitoes creates an urgent need to develop novel insecticides with unique modes of action. This study will advance our understanding of the regulatory mechanism of AaKr‐h1 during reproduction and might provide a new target for novel insecticides to suppress mosquito population.

Support or Funding Information

NIH

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 Rapid, Simple, High‐throughput Compatible Approach to Generating CRISPR/Cas9 Knock‐out Cell Lines
Authors: Wu Meiye , Okino Steven , Uy Gerald , Woo Deanna , Shulewitz Mark , Wang Yan
Published: June 2021

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated endonuclease Cas9 have emerged as a revolutionary genome engineering tool. Using CRISPR/Cas9 to induce mutations in protein encoding genomic DNA regions is a powerful method for studying protein function in the native cellular context. However, the existing workflows for generating CRISPR/Cas9 knock‐out cell lines are cumbersome and labor intensive, comprising of arduous rounds of cloning, limiting dilutions, and surveyor assays performed on dozens if not hundreds of clones. In order for large scale CRISPR/Cas9 studies to become practical and accurate for the genomics community, a streamlined process must be developed and validated at both genomic and protein levels. Here, we report a novel workflow that leverages the power of droplet digital PCR (ddPCR) and high‐resolution melt analysis (HRM) to produce DNA sequence and western blot validated CRISPR/Cas9 knock‐out cell lines in under 5 weeks.

Support or Funding Information

Bio‐Rad, Inc Internal R&D support.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fluorescence Kinetic Studies of DNA Unwrapping in Transcription Initiation with NTP addition and in Open Complex Dissociation by high salt
Authors: Callies Katelyn , McNerney Christina , Cimperman Clare Kai , Xue Andrew , Kan Hertina , Sreenivasan Raashi , Chhabra Munish , Record Tom
Published: June 2021

λPR promoter DNA is bent and wrapped around E. coli RNA polymerase (RNAP) in the stable open complex (RPo), as demonstrated by Cy3‐Cy5 fluorescent energy transfer (FRET) between dyes attached 115 bp apart on the far‐upstream (−100) and downstream (+14) position of DNA. Here we report the use of FRET and PIFE (single‐dye fluorescence enhancements that indicate close contacts between RNAP and the far‐upstream or downstream DNA) to investigate unwrapping of upstream DNA after NTP addition to initiate transcription, form a short RNA‐DNA hybrid and escape from the promoter. We observe two kinetic phases of roughly equal amplitude in the decrease in FRET in transcription initiation. The first kinetic phase (0.1 s to 10 s) of DNA unbending/unwrapping appears slower than the rate of NTP binding and dinucleotide synthesis, indicating that this first phase of unbending/unwrapping occurs in the processes of translocation and hybrid extension. The second kinetic phase (at times > 10 s) corresponds to that of production of full‐length (14 bp) RNA transcripts, as determined in parallel single‐round transcription assays. These results indicate that the second phase of DNA unbending and unwrapping occurs in promoter escape. In addition, we report the kinetics of unwrapping in dissociation by salt‐upshifts. These experiments reveal that salt‐induced unwrapping occurs during the conversion of the stable open complex to the unstable open intermediate I2, prior to DNA closing.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
USP24 Induces IL‐6 in Tumor‐associated Microenvironment through stabilizing p300 and β‐TrCP to Promote the Malignancy of Lung Cancer
Authors: HUNG Jan‐Jong
Published: June 2021

Our previous study indicated that increase in USP24 level in lung cancer facilitates lung cancer metastasis. Our recent study found that higher USP24 level was found in tumor‐associated microenvironment (TAM) than surrounding lung cancer cells in lung cancer patient specimens. USP24 level was significantly increased in M2‐macrophages. Knockdown of USP24 in M2‐macrophages decreased the migratory and chemotactic activity of lung cancer cells and angiogenetic ability of human umbilical vein endothelial cells (HUVEC) induced by M2‐derived conditioned medium. In vivo experiments also showed USP24 knockdown decreased M2‐macrophages induced lung cancer cells metastasis and angiogenesis. IL‐6 mRNA and protein levels were significantly decreased in USP24 knockdown M2‐macrophages and lung cancer cells. IL‐6 replenishment can rescue migratory and chemotactic ability of conditioned medium derived from USP24 knockdown M2‐macrophages, suggesting that increase in USP24 in M2‐macrophages induce the metastasis activity of lung through promoting IL‐6 expression. To address the molecular mechanism of how USP24 regulated IL‐6 in M2‐macrophage and lung cancer cells, we found that USP24 stabilized p300, thereby increased the transcriptional activity of IL‐6. USP24 could also decreased DNM1 and IkB level through stabilizing β‐TrCP and resulted in upregulated IL‐6. Metastatic cytokines in tumor‐associated microenvironment are important for cancer progression, in this study, we provide the direct evidence to support that USP24 increased in M2‐macrophages and lung cancer cells positive regulates the metastasis of lung cancer through enhancing the IL‐6 expression, which might be beneficial for the drug development for cancer therapy in the future.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing the Spatial Variance of Autophagic Cargo Receptor, OPTN
Authors: Najera Susana Sarai , Heo Jin‐Mi , Harper Wade
Published: June 2021

The mitochondria are required for various cellular processes spanning from cellular energy generation to signal transduction, growth and survival. Not surprisingly, cumulative mitochondrial damage is tightly linked to the development of multiple human diseases including various neurodegenerative diseases such as Parkinson's and Huntington's disease. In order to maintain functional mitochondria, cells have coped with multiple mitochondrial quality control pathways such as mitochondrial Unfolded Protein Response (mt‐UPR), the ubiquitin proteasome system, and mitochondrial autophagy (mitophagy). The most well‐characterized mitophagy is the PINK1‐PARKIN pathway, where damaged mitochondria are selectively marked with ubiquitin chains by the PINK1 kinase and PARKIN ubiquitin E3 ligase, engulfed by autophagosome, and cleared by the lysosome. While we now have a better understanding on this pathway, it is still unclear how autophagic cargo adaptor proteins, such as optineurin (OPTN), get recruited to damaged mitochondria, synthesized or recycled throughout the process. We hypothesized that the availability of OPTN would be the rate‐limiting factor to achieve complete removal of damaged mitochondria and this would require synthesis of new OPTN molecule. To test this hypothesis, we generated a cell line containing a genomic Halo tag at OPTN N‐terminus by utilizing CRISPR/cas9, where differentially fluorescent labeled Halo tag can distinguish older versus newer OPTN populations over time. After successfully generating a CRISPR/Cas9 knock‐in cell line for OPTN, single cell imaging was performed to determine the spatial variance and emergence of OPTN recruited to mitochondria. We were able to conclude that OPTN localizes to a fraction of damaged mitochondria despite the fact that the entire mitochondria are coated with ubiquitin chains, suggesting specificity of this recruitment. Additionally, our preliminary results indicated that newly synthesized OPTN is recruited to mitochondria for the continuation of mitophagy. Together, these observations suggest that OPTN is the rate‐limiting factor and that in order to completely remove all dysfunctional mitochondria, cells need to synthesize new OPTN. In the future, we would like to further explore the underlying mechanism of OPTN's binding specificity and determine what drives this selective recruitment of OPTN to mitochondria.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating putative key catalytic residues and uncoupled hydroperoxyflavin formation in the mechanism of 6‐hydroxynicotinate‐3‐monooxygenase, a decarboxylative‐hydroxylase in bacterial nicotinate catabolism
Authors: Perkins Scott W. , Snider Mark J.
Published: June 2021

Agrochemicals, pharmaceuticals and personal care products (APPCPs) are contaminants of emerging concern that are ineffectively remediated by current methodologies. Many APPCPs contain N‐heterocyclic aromatic constituents (NHACs) that are persistent contaminants in aquatic environments and cause adverse effects in non‐target organisms. Understanding bacterial degradation of NHACs may assist future bioremediation techniques. Aerobic bacterial catabolism of nicotinic acid (NA) serves as a model for understanding the biodegradation of NHACs, and a key enzyme of this pathway is 6‐hydroxynicotinate 3‐monooxygenase (NicC). We recently published the first crystal structure and two postulated mechanisms for NicC. Both mechanisms involve acid‐base catalysis, in which active site residue His211 may serve as a key H‐bond donor. Additionally, the Cys202 active site residue may donate a thiol group, playing a role in one postulated mechanism. Kinetic characterization of the H211A and C202A variants shows that the apparent substrate affinity and catalytic turnover are largely unaffected by mutagenesis of these bases (H211A: KM of 6HNA increases 2.1–fold, KM of NADH increases 2.3–fold, kcat increases 30% compared to WT; C202A: KM of 6HNA increases 2.4–fold, kcat drops 15‐fold). These data suggest that the His211 and Cys202 residues are not critical for catalysis. 6‐Hydroxynicotinaldehyde (6‐HNAH), a competitive inhibitor of NicC with respect to 6‐HNA, exhibits tight binding (KD of 6HNAH = 36 ± 2 μM) compared to 6HNA (KD of 6HNA = 190 ± 25 μM), but shows weak inhibition (Ki of 6HNAH = 8.5 ± 2.4 mM). These results are consistent with the possibility that the formation of hydroperoxyflavin (FADHOOH) is uncoupled from the oxidative decarboxylation reaction, and subsequently produces hydrogen peroxide. Hydrogen peroxide production was detected for reactions of WT+6HNAH and H211A+6HNAH, but not detected for WT+6HNA and H211A+6HNA, suggesting that 6HNAH causes uncoupling and His211 is not a key catalytic residue in the mechanism of NicC.

Support or Funding Information

College of Wooster Sophomore Research Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In vitro Biochemical Studies of the Decarboxylase Domain of ArnA from Pseudomonas aeruginosa
Authors: de Miranda Rodger , Conrad Laura Miller
Published: June 2021

Pseudomonas aeruginosa is an opportunistic, Gram‐negative pathogen that is resistant to many classes of antibiotics. One pathway leading to resistance to cationic antimicrobial peptides like colistin is upregulation of the arnBCADTEF operon. With the biosynthetic enzymes encoded in this pathway, P. aeruginosa modifies its outer membrane to decrease its net negative charge. This neutralization of charge lessens the driving force for electrostatic interactions with positively charged antibiotics such as colistin, hindering their entry into the cell. The first committed step of this pathway is catalyzed by the decarboxylase domain of ArnA in which UDP‐glucuronic acid undergoes a redox reaction with NAD+. Due to its role in the arnB pathway of antibiotic resistance, we seek to characterize the kinetics of ArnA using in vitro biochemical studies. To do this we measured the change in absorbance at 340 nm allowing us to calculate the reduction of NAD+ to NADH. These data were analyzed to produce the Michaelis‐Menten kinetics of ArnA from P. aeruginosa. Elucidating the kinetics of ArnA will enable analysis of small molecule inhibitors for changes in the activity of this enzyme. This knowledge will enable future work to design strategies to inhibit ArnA and thus block the resistance pathway.

Support or Funding Information

National Institutes of Health (NIH) Grant # 5SC3GM118199

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pomegranate phenolics inhibit type I collagen cross‐linking induced by glycative stress
Authors: Cai Ang , Liu Weixi , Ma Hang , Dombi George W. , Dain Joel A. , Seeram Navindra P.
Published: June 2021

Collagen is a class of long‐lived structural proteins in the extracellular space in various fibrous tissues including skin, tendons, and ligaments. Collagen fibrils are subjected to metabolic stresses including oxidation and glycation, which lead to its structural modification including non‐enzymatic glycation and excess cross‐linking. These modifications are critical factors in microstructural cross‐link associated disorders including skin aging, Alzheimer's disease and cardiovascular disease. Many natural products including phenolics are regarded as promising inhibitors that prevent the formation of advanced glycation endrpoducts (AGE). Previously, we reported that hydrolyzable tannins including punicalagin (PA) and ellagic acid (EA) from pomegranate extract, along with their gut microbial metabolites, namely, urolithins, can inhibit the formation of AGE in a bovine serum albumin model. The beneficial inhibitory effects of these phenolics on the glycative stress‐induced collagen cross‐linking remain unknown. Therefore, in this current study, we examined the protective effects of pomegranate phenolics on collagen glycation. Pomegranate phenolics (at a concentration of 200 μM) including PA, EA, and urolithin A (UA) inhibited the fructose‐induced collagen glycation by 64.4, 15.0, and 17.3%, respectively, and the methylglyoxal (MGO)‐induced collagen glycation by 70.6, 17.6, and 14.4%, respectively. Furthermore, PA, EA, and UA (each at a concentration of 200 μM) inhibited the formation of Amadori products by 45.7, 31.5, and 40.0%, respectively. In addition, circular dichroism (CD) analysis showed that PA, EA, and UA exhibited protective effects on the secondary structure of collagen on the denaturing effect of glycation. The inhibitory effects of hydrolyzable tannins on collagen glycation warrants further investigation as they may provide a novel therapy to complications related to collagen glycation and crosslinking as in diabetes and hypertrophic scarring.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Expanding the Toolbox of Chemoenzymatically Synthesized Peptidoglycan Probes and Investigating their Interaction with Nucleotide Sugar Transporters”
Authors: Jones Zachary Scott , DeMeester Kristen Elizabeth , Liang Hai , Jensen Matthew , D'Ambrosio Elizabeth , Grimes Catherine Leimkuher
Published: June 2021

Introduction

The microbiome has gained increasing attention due to its relevance to human health, particularly in the intestinal tract. Bacteria possess a unique structure, peptidoglycan (PG), which is known to illicit an innate immune response by inflammation. Within the peptidoglycan, uridine diphosphate N‐acetyl muramic acid (UDP‐NAM) is the first committed step to PG biosynthesis. Previously, NAM probes with functional moieties on the 2‐position of the sugar have been used to label bacteria on the PG via copper catalyzed click chemistry. Using a chemoenzymatic synthesis method, a UDP moiety can be attached to achieve this first committed PG substrate.

Glycan chemical synthesis of UDP sugar probes, has proven difficult to achieve synthetically. Currently, the goal is to optimize large‐scale quantities of UDP‐NAM and derivatives and to synthesize chemical probes with expanding functionalities in addition to fluorescent labelling for incorporation into the PG of bacteria. By tracking the dynamics of PG, specifically the breakdown of these chemical probes, innate immune reactions and responses may be better understood.

Methods

The method developed utilizes the recycling enzymes anomeric NAM/NAG kinase (AmgK) and uridylyltransferase (MurU) in order to chemoenzymatically synthesize UDP‐NAM derivatives. Due to the relaxed substrate specificity of these enzymes, the NAM derivatives are able to be accepted into the active site so that the muramyl lactol will be converted to the charged UDP sugar donor. AmgK and MurU were expressed and purified from Escherichia coli.

Purification was carried out by high‐pressure liquid chromatography (HPLC) coupled to liquid chromatography mass spectrometry (LC/MS) on a hydrophilic liquid chromatography column. Fractions were collected and the products were characterized by LC/MS and nuclear magnetic resonance (NMR) imaging.

Results and Discussion

Currently, large scale synthesis, purification, and characterization has been completed of the natural UDP‐NAM substrate and approximately 8 derivatives on a high mg scale. Also, probes with a CF2H and a diazo moiety have been synthesized to be tested with the recycling enzymes. 2‐azido‐UDP‐NAM has successfully been incorporated into the PG of Lactobacillus acidophilus, but was unsuccessful in labelling other strains of gram positive bacteria.

It is proposed that a nucleotide sugar transporter exists in L. acidophilus, and there have been several candidates found by BLAST sequencing. The presence of nucleotide sugar transporter proteins will be explored, and their homology and binding specificity to the UDP‐NAM derivatives will be examined.

From these results, the library and functionality of UDP‐NAM probes has been expanded, and future work involves elucidating the mechanism by which the probes pass through the cell membrane.
Interleukin‐like EMT Inducer ILEI mediates breast cancer stem cell formation and tumorigenesis through LIF receptor signaling
Authors: Woosley Alec , Dalton Annamarie , Howe Philip
Published: June 2021

During the epithelial‐mesenchymal transition (EMT), cells undergo a switch from a polarized, epithelial state to a more fibroblastic and mesenchymal phenotype. This process is associated with the formation of highly tumorigenic cancer stem cells (CSC). Our lab has identified a regulatory mechanism by which Transforming Growth Factor‐β (TGFβ) induces EMT at the translational step of gene expression. Among gene products controlled by this post‐translational mechanism is the cytokine Interleukin‐like EMT Inducer (ILEI), a secreted factor belonging to the FAM3 family that is involved in EMT induction and tumorigenesis. The biochemical mechanism of the ILEI signaling and identification of an ILEI receptor has remained elusive. Preliminary studies from our lab indicate that modulation of ILEI in several cell lines results in altered stemness potential, giving insight into the possible role of ILEI in the formation of CSCs. The basis of this study aims to investigate the mechanism of the ILEI signaling pathway and its contribution to CSC formation. In order to identify potential ILEI binding partners, we utilized yeast two‐hybrid screening and observed that ILEI interacts with the 190kD leukemia inhibitory factor receptor (LIFR). LIFR serves as a signaling platform for several cytokine ligands and forms intricate complexes with an array of other ligand‐specific co‐receptors during its activation. Our data indicates that ILEI functions through LIFR, inducing downstream CSC phenotypes by activating PI3k/Akt and Jak/STAT signaling pathways. Insight into the TGFβ/ILEI/LIFR pathway may serve as a platform for development of a novel targeted therapy against metastatic breast cancer.

Support or Funding Information

NIH F31CA213627NIH R01CA555536

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Leptin alleviates the saturated fatty acid‐induced increase in BACE1 expression and Amyloid‐β production ‐ Relevance to Alzheimer's disease pathogenesis
Authors: Marwarha Gurdeep , Ghribi Othman
Published: June 2021

Epidemiological studies implicate diets rich in saturated free fatty acids (sFFA) as a potential risk factor for developing Alzheimer's disease (AD). In particular, high plasma levels of the sFFA palmitic acid (palmitate) have been shown to inversely correlate with cognitive function. AD is histo‐pathologically characterized by the deposition of aggregated Amyloid‐β (Aβ) peptide as extracellular amyloid plaques and the intracellular accumulation of aggregated hyper‐phosphorylated tau protein as neurofibrillary tangles. Our recent studies have demonstrated that palmitate‐enriched diet increases Amyloid‐β (Aβ) genesis in the hippocampus by increasing the expression and activity of the aspartyl protease BACE1 which catalyzes the rate limiting step in Aβ (Marwarha et al. 2017, Ghribi & Marwarha 2017). We have also shown that palmitate‐enriched diet decreases the expression of leptin, an adipokine that functions as a key neurotrophic cytokine in the brain (Marwarha et al. 2016). However, the role of palmitate‐induced mitigation in leptin expression and dysregulation of leptin signaling in the palmitate‐induced increase in BACE1 expression and the ensuing Aβ genesis has not been examined. Herein, we determined the role of leptin in modulating the palmitate‐induced increase in BACE1 expression and the ensuing Aβ genesis in human neuroblastoma SH‐SY5Y cells. Concomitant treatment of the cells with exogenous recombinant leptin significantly decreased the exogenous palmitate‐induced increase in BACE1 expression and enzymatic activity that culminated into a reduction in intracellular and secreted Aβ levels. Additionally, human SH‐SY5Y neuroblastoma cells stably overexpressing endogenous leptin, exhibited a lower increase in BACE1 expression and enzymatic activity as well as the ensuing Aβ accumulation in response to exogenous palmitate. Our unprecedented study illuminates a novel role of leptin in the brain and provides a unique insight into the mechanisms that underlie palmitate induced effects on molecular players involved in the etio‐pathogenesis of Alzheimer's disease.

Support or Funding Information

This work was supported by a Grant from the NIH (R01AG045264) to Dr. Othman Ghribi.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detection of Novel Rho GAP/GTPase Selectivities Using In Vitro Split‐luciferase Assays
Authors: Wilander Benjamin A. , Hamann Michael J.
Published: June 2021

Rho GTPases regulate the assembly of cellular actin structures for a wide range of dynamic activities, including actin‐dependent migration, constriction/scission and transport‐related processes. These events are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase‐activating proteins (GAPs). Although most research has tended to focus on the role of GEF‐catalyzed GTPase signal activation, GAP inactivation of GTPase signaling pathways needs to be better characterized since inactivation is required to avoid prolonged and possibly detrimental cellular responses. To this end, the GTPase selectivity of several novel hematopoietically‐expressed RhoGAPS, specifically ARHGAP1, 9, 12, 17, 25, and 26, were tested by generating MBP fusion proteins of the GAP domains of these proteins and testing their activities with Cdc42, Rac1 and RhoA. Activities were assayed using a previously described in vitro split‐luciferase assay with bacterially‐expressed purified proteins, and GAPs with known selectivities were used as positive controls. These results will provide an important first step in linking these RhoGAPs to particular GTPase pathways and eventually linking them to cellular functions.

Support or Funding Information

Support was provided by the Nielson Foundation, Bemidji, MN and a grant from Regenerative Medicine Minnesota (RMM‐2017‐EP‐04)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
: Atypical G protein β5 promotes cardiac oxidative stress and fibrotic remodeling in response to multiple cancer chemotherapeutics
Authors: Chakraborti Sreemoyee , Pramanick Arnab , Maity Biswanath
Published: June 2021

The clinical use of multiple classes of cancer chemotherapeutics is limited by irreversible, dose‐dependent, and sometimes life‐threatening cardiotoxicity. Though distinct in their mechanisms of action, doxorubicin, paclitaxel, and 5‐FU all induce rapid and robust upregulation of atypical G protein Gβ5 in the myocardium correlating with oxidative stress, myocyte apoptosis, and the accumulation of pro‐inflammatory and pro‐fibrotic cytokines. In ventricular cardiac myocytes (VCM) Gβ5 deficiency provided substantial protection against the cytotoxic actions of chemotherapeutics including reductions in oxidative stress and simultaneous attenuation of ROS‐dependent activation of the ATM and CaMKII pro‐apoptotic signaling cascades. In addition, Gβ5 loss allowed for maintenance of Δψm, basal MCU and mitochondrial Ca2+ levels, effects likely to preserve functional myocyte excitation‐contraction coupling. The deleterious effects of Gβ5 are not restricted to VCM, however, as Gβ5 knockdown also reduces chemotherapy‐induced release of pro‐inflammatory cytokines (e.g. TNFα), hypertrophic factors (e.g. ANP) and pro‐fibrotic factors (e.g. TGFβ1) from both VCM and VCF with the most dramatic reduction occurring in co‐cultured cells. Our experiments suggest that Gβ5 facilitates the myofibroblast transition, the persistence of which contributes to pathological remodeling and heart failure. The convergence of Gβ5‐mediated, ROS‐dependent signaling pathways in both cell types represents a critical etiological factor in the pathogenesis of chemotherapy‐induced cardiotoxicity. Indeed, intracardiac injection of Gβ5‐targeted shRNA allowed for heart specific protection against the damaging impact of chronic chemotherapy. Together, our results suggest that inhibition of Gβ5 might represent a novel means to circumvent cardiotoxicity in cancer patients whose treatment regimens include anthracyclines, taxanes or fluoropyrimidines.

Support or Funding Information

Ramalingaswami Fellowship (BT/RLF/Re‐entry/15/2013), Department of Biotechnology, and Department of Science and Technology, SERB (EMR/2016/006873), Government of India to Dr. B Maity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Blended Instruction in Introduction to Biochemistry Class
Authors: Nicholas Timothy , Reddy Neha , Srinivasan Rekha
Published: June 2021

A challenge for biochemistry instructors is dealing with the volume and difficulty of concepts typically taught during semester‐long introductory biochemistry courses. Students struggle to grasp topics because they may not have enough in‐class instruction and practice time to process the new information. Blended learning strategies, such as the flipped classroom approach along with traditional lectures, may offer a solution to this concern. Herein we present the creation of a blended learning introductory biochemistry course. The course used online modules consisting of concept videos tied to active learning problem sets, followed by in‐class sessions of small groups to discuss the application of these concepts. Each topic was covered with a video presentation suited for display on mobile devices that focused on problem solving and an associated active learning project that encouraged practice and application of skills developed in that topic. Students were encouraged to take an online quiz after each set of core topics to assess their readiness in applying the concept. The initial implementation of this module was tested in the Introduction to Biochemistry class and the preliminary data is presented herein

Support or Funding Information

Nord Grant, UCITE, Case Western Reserve University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Triclosan Exposures on Interleukin 6 and Interleukin 1 Beta Secretion from Human Immune Cells
Authors: Wilburn Wendy JoAnne , Brooks Dylan , Whalen Margaret
Published: June 2021

Triclosan (TCS) is an antimicrobial compound that is widely used in personal hygiene products such as mouthwash and toothpaste. TCS can be ingested or absorbed through the skin and has been found in human blood, breast milk, and urine. Interleukin (IL)‐6 and IL‐1 beta (IL‐1β) are important pro‐inflammatory cytokines produced by lymphocytes, monocytes, and other cells. Both regulate cell growth, tissue repair, and immune function, increased levels of each have been associated with a number of diseases including rheumatoid arthritis and certain cancers. TCS has been shown to inhibit the lytic function of human natural killer (NK) lymphocytes and to decrease expression of key cell surface proteins on NK cells. Here we examine whether TCS alters the secretion of IL‐1β and IL‐6 from human immune cell preparations. Human peripheral blood mononuclear cells (PBMCs) and monocyte‐depleted (MD)‐PBMCs were exposed to TCS at concentrations of 0–5 μM. Cytokine secretion was measured at 24 h, 48 h, and 6 days using enzyme‐linked immunosorbent assay (ELISA). Both IL‐1β and IL‐6 secretion were increased at one or more concentration of TCS at one or more length of exposure. These results indicate that TCS has the capacity to disrupt secretion of these two important pro‐inflammatory cytokines.

Support or Funding Information

Supported by NIH grant U54CA163066

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 Basis of Heme Acquisition from Human Hemoglobin by the Gram‐Positive Pathogen Streptococcus pyogenes
Authors: Macdonald Ramsay , Phillips Martin , Cascio Duilio , Collazo Michael , Clubb Robert T.
Published: June 2021

Our research seeks to learn how Gram‐positive pathogens acquire iron from the human host, a key process required for bacterial pathogenesis and a potential drug target. In particular, the focus of our work is the mechanism of heme extraction from hemoglobin (Hb), the main source of iron in the human body. In an effort to decipher this process, we are using a combination of biochemical and biophysical methods including X‐ray crystallography, isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), analytical ultra‐centrifugation (AUC), and heme transfer assays. Current efforts are devoted to studying this process in Streptococcus pyogenes (S. pyogenes) given its relevance as a human pathogen. We have recently characterized a novel Hb receptor from S. pyogenes ‐ the Streptococcal hemoprotein receptor (Shr). Shr appears to contain a similar domain architecture to the well‐characterized Hb receptors IsdH and IsdB found in Staphylococcus aureus. Unlike IsdH and IsdB, Shr mediates Hb binding through a domain of unknown function (DUF). Here we present the structure of this DUF domain in isolation and progress toward determining its structure in complex with Hb. The crystal structure of the DUF domain in isolation reveals a novel Hb‐binding domain structurally unrelated to Hb‐binding domains from other Gram‐positive pathogens. In addition, we have collected biochemical data on this domain suggesting that it slows the rate of heme release from Hb. We hypothesize that the DUF domain may slow the spontaneous process of heme release by Hb after erythrocyte lysis, allowing heme to be subsequently utilized downstream in the S. pyogenes heme acquisition pathway. This work is significant as it describes a novel mechanism of heme acquisition by a Gram‐positive pathogen. Future studies will be devoted to understanding the mechanism of heme extraction by the full‐length Shr protein.

Support or Funding Information

This work was supported by National Institutes of Health Grant AI52217 (to R.T.C.). R.M. was supported by a Cellular and Molecular Biology Training Grant (Ruth L. Kirschstein National Research Service Award GM007185). NMR equipment used in this research was purchased using funds from shared equipment grant NIH S10OD016336.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ni(II) Uptake by Yersiniabactin, a Metallophore Produced by Uropathogenic E. coli
Authors: Robinson Anne E. , Lowe Jessica E. , Koh Eun‐Ik , Henderson Jeffrey P.
Published: June 2021

Metal ions can play dual roles as nutrients and toxins in the interaction between bacteria and their environments. E. coli isolates associated with urinary tract infections (UTIs) deploy siderophores, small molecule chelators defined by their ability to competitively bind iron(III) and deliver it to the cell. Yersiniabactin (Ybt) is a virulence‐associated siderophore of uropathogenic E. coli (UPEC) and was recently appreciated to also bind copper during UTIs in humans, forming Cu(II)‐Ybt complexes. Copper binding by Ybt was found to protect E. coli from copper toxicity and also to mediate controlled copper import for nutritional purposes. We have termed this process nutritional passivation. Ni(II) is generally less abundant than copper but is also a nutrient for E. coli with the potential for toxicity. We show here that the Ybt system of UPEC engages in an analogous nutritional passivation activity with Ni(II). Ybt passivates Ni(II) ions, protecting against Ni(II) toxicity in vitro. We also observe that UPEC can use Ni(II)‐Ybt as a nutrient source for nickel. Using quantitative LC‐MS/MS and isotope labeling, we detect import of Ni(II)‐Ybt and dissociation of the nickel, providing a nickel source for cellular enzymes. The metal‐free Ybt is secreted back outside of the cell to bind other metal ions. These findings broaden the spectrum of Ybt activity, reinforcing its value to UPEC as a true metallophore, capable of binding and importing not only iron, but also copper and nickel.

Support or Funding Information

J.P.H. acknowledges National Institute of Diabetes and Digestive and Kidney Diseases grant R01DK099534. A.E.R. was supported by the Mr. and Mrs. Spencer T. Olin Fellowship for Women in Graduate Study.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Janus‐Faced Role of ATP1B4 Gene Co‐option in Mammalian Evolution
Authors: Modyanov Nikolai
Published: June 2021

ATP1B4 genes represent a rare instance of orthologous vertebrate gene co‐option (evolutionary changes of functions of the genes, which are present in a single unambiguous copy in all known vertebrate genomes) that radically changed functions of the encoded BetaM proteins, which are the Na, K‐ATPase subunits in lower vertebrates. In placental mammals, BetaM proteins gained entirely different properties through changes in the structure of N‐terminal domains. As a result of these evolutionary alterations, eutherian BetaM completely lost its ancestral role and became the only currently known skeletal and atrial cardiac muscle‐specific protein of the inner nuclear membrane, where it functions as a regulator of gene expression. Expression of BetaM is the highest in myocytes during late fetal and early postnatal development and is also robustly induced in regenerating adult muscle.

Evolutionarily acquired new functions and a unique pattern of expression of BetaM proteins suggest that those new physiological functions are essential, might be necessary for survival of placental mammals in natural conditions, and provide an evolutionary advantage.

To characterize BetaM function directly in vivo, the Atp1b4 knockout mouse model was generated. We have shown that loss of BetaM results in significantly lower body size and weight, growth retardation and high mortality of Atp1b4 knockout neonates. Transcriptome analysis by mRNA sequencing of skeletal muscle from neonatal wild type and knockout littermates revealed strong down‐regulation of fast‐twitch and up‐regulation of slow‐twitch muscle genes, and broad changes in expression of genes regulating lipid metabolism. These results imply that BetaM plays an important role during a critical period of perinatal and neonatal development of placental mammals and its damage or malfunction can cause birth and growth defects in humans.

Most importantly, our experiments revealed a somewhat unexpected and profound beneficial effect of Atp1b4 disruption on metabolic parameters in adult male mice. We have shown that Atp1b4 knockout males, which survived to adulthood, have a significantly lower percentage of body fat, exhibit enhanced metabolic rate and insulin sensitivity, and are resistant to high‐fat diet‐induced obesity. These robust changes in mouse metabolic parameters induced by Atp1b4 disruption clearly demonstrate that eutherian BetaM plays the important role in development of pathways regulating metabolism of adult mice.

Finally, ablation of the evolutionarily acquired functions of Atp1b4 gene in mice is in fact a simulation of the alternative pathway of mammalian evolution.

Support or Funding Information

This work was supported by Department of Physiology & Pharmacology and Center for Diabetes and Endocrine Research, University of Toledo College of Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.