bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of an Evolutionarily Conserved RNA Polymerase II‐Associated Factor 1 (Paf1) Involved in Pancreatic Oncogenesis
Authors: Barman Priyanka , Ferdoush Jannatul , Kaja Amala , Karmakar Saswati , Uprety Bhawana , Batra Surinder K. , Bhaumik Sukesh R. 
Published: June 2021

The multifunctional RNA polymerase II‐associated factor (Paf1) is highly conserved from yeast to humans and plays an important role in regulation of transcription and its associated processes. Hence, malfunctions and/or misregulations of Paf1 are associated with cellular pathologies. Recent studies found that Paf1 (also known as PD2 or pancreatic differentiation 2) is upregulated in poorly differentiated cancer cells, and such upregulation is involved in cellular transformation or oncogenesis. However, the basis for Paf1 upregulation in these cells remains largely unknown. In light of this, we have tested here the idea that the ubiquitin‐proteasome system (UPS) regulates the cellular abundance of Paf1. In this direction, we analyzed the role of UPS in regulation of Paf1's abundance in yeast. We find that Paf1 undergoes ubiquitylation and is degraded by the 26S proteasome in yeast, thus deciphering UPS regulation of an evolutionarily conserved factor, Paf1, involved in various cellular processes at the crossroads of the cancer networks. Likewise, Paf1 undergoes proteasomal degradation in well‐differentiated, but not poorly differentiated, pancreatic cancer cells, hence pointing to the UPS in upregulation of Paf1 in poorly differentiated cancers. Thus, our results reveal UPS regulation of Paf1 and suggest downregulation of UPS in elevating Paf1's abundance in poorly differentiated cancers. Further, we find that misregulation of Paf1's abundance alters transcription. Collectively, our results demonstrate that Paf1 is regulated by UPS to control transcription, thus providing a new regulatory mechanism of gene expression.Support or Funding InformationNIH‐2R15GM088798‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of genome‐engineered mesenchymal stem cells secreting angiogenic or anti‐inflammatory factors for the treatment of acute kidney injury
Authors: Park Hye‐Jeong , Cho Jeong‐In , Jang Hyo‐Ju , Park Eui‐Jung , Choi Hyo‐Jung , Kwon Tae‐Hwan 
Published: June 2021

Stem cell therapy has been proposed as a potential therapeutic strategy for acute kidney injury (AKI). By exploiting genome editing and cell sheet technology, we aimed to generate mesenchymal stem cells (MSCs) secreting angiogenic factors [vascular endothelial growth factor (VEGF) and angiopoietin‐1 (ANG1)] or anti‐inflammatory factors [erythropoietin (EPO) and a‐melanocyte stimulating hormone (a‐MSH)] for therapeutic application in AKI. To integrate each gene expression cassette into the safe harbor locus, AAVS1, of the human umbilical cord‐derived MSCs (hUC‐MSCs) chromosome, AAVS1‐targeting Zinc Finger Nuclease (ZFN) or AAVS1‐targeting CRISPR/Cas9 system was exploited. Junction PCR analysis demonstrated the ZFN‐ or CRISPR/Cas9‐aided gene integration in hUC‐MSCs. Flow cytometry and osteogenic and adipogenic differentiation assay revealed that stemness was maintained despite genome engineering. Protein measurement in conditioned media by ELISA and immunoblotting confirmed the production and secretion of each integrated gene product. In vitro assay demonstrated the angiogenic functions (increased HUVEC migration and increased mRNA expression of MMP‐9 and Tie‐2 in co‐cultured HUVEC) of genome‐engineered hUC‐MSCs secreting VEGF or ANG1. For the stem cell therapy in AKI, a scaffold‐free cell sheet system was established using a temperature‐responsive polymer (poly(N‐isopropylacrylamide). Taken together, cell sheet system of hUC‐MSCs secreting angiogenic or anti‐inflammatory factors is successfully established. This is to be examined in animal models of AKI to demonstrate the therapeutic effects of stem cell‐based regenerative strategy against AKI.Support or Funding InformationThis study was supported by the National Research Foundation of Korea Grants 2014R1A5A2009242 and 2016R1A2B4009365 funded by the Ministry of Science, ICT and Future Planning, KoreaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An mRNA‐rRNA Base Pairing Model for Efficient Protein Translation
Authors: Barr Will , Sheth Ruchi , Chatterji Om , Moon Hye Kyung , Weir Michael 
Published: June 2021

The idea that base pairing between mRNAs and structural rRNAs of ribosomes might contribute to protein translation has long been an intriguing possibility. The 530 loop of 16S rRNA has been implicated in translation initiation, elongation and termination. This loop and the corresponding highly‐conserved loop in 18S rRNA are located in the mRNA entrance tunnel of ribosomes. As noted before high‐resolution ribosome structures were described, the 530 loop contains a 3‐nucleotide‐repeating pattern complementary to the 3‐nucleotide periodicity of protein open reading frames characterized by overrepresentation of (GCN)n. We find that the 3‐nucleotide periodicity is significantly enhanced downstream of translation start codons of highly expressed Saccharomyces cerevisiae genes with significant depression of G at nucleotides 2 and 3 of these codons. We propose that during translocation steps, exposed rRNA nucleotides of the 530 loop can transiently base pair with the second and third mRNA nucleotides of the incoming A‐site codon before engagement of the A‐site tRNA and that this promotes efficient launching of the ribosome through the start region codons permitting high protein expression. This cooperation between the A‐site and 530 loop requires a precise positioning of the mRNA reading frame, which if compromised in several adjacent codons leads to low translation levels.Support or Funding InformationNational Institutes of Health 1R15GM096228Beckman Scholars Program, Arnold & Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Atypical Binding Behavior of Epsin‐like Clathrin Adaptor 1 to Diacylglycerol Pyrophosphate
Authors: Putta Priya , Kooijman Edgar E. 
Published: June 2021

Epsin N‐terminal homology (ENTH) and AP‐180 N‐terminal homology (ANTH) domain proteins are membrane binding proteins known to interact with polyphosphoinositides (PIPs). They share function, structure and sequence similarity, and are grouped together as A/ENTH domain proteins. The ENTH domain contains a distinctive N‐terminal helix (Ho) region that plays a crucial role in membrane bending. This Ho helix is not usually found in ANTH domains. Epsin‐like Clathrin Adaptor 1 (ECA1) is one such A/ENTH protein that acts as an adaptor protein in clathrin mediated endocytosis. It interacts with clathrin via its C‐terminus, and PIPs via its N‐terminus. In addition to having ANTH domain sequence similarity, ECA1 also contains the distinctive Ho helix region that is generally found in the ENTH domain. ECA1 is therefore best characterized as an N‐ANTH domain protein with a unique combination of both ANTH and ENTH regions. ECA1 is known to play a role in stress signaling in plants and also binds to phosphatidic acid (PA) upon salt stress. Like proteins, membrane lipids undergo drastic changes during stress stimuli. PA is a lipid second messenger that rapidly and transiently increases under stress stimuli. Upon rise of PA levels another lipid, diacylglycerol pyrophosphate (DGPP) starts to accumulate. DGPP is phosphorylated PA. It is suggested to be synthesized at higher levels as a signal for PA attenuation during stress. We recently showed in vitro that ECA1‐PA ‐affinity is modulated as a function of membrane curvature stress. In this research, we investigate ECA1 affinity to DGPP. We hypothesize that the PA target ECA1 has affinity for its metabolite DGPP which is highly negatively charged and this affinity is variable at varying pH. We tested ECA1 affinity to DGPP using liposome binding assay along with other anionic lipids; i.e., phosphatidylserine, phosphatidylinositol (4)‐phosphate and phosphatidylinositol (4,5)‐bisphosphate in the presence and absence of phosphatidylethanolamine. Our results show striking evidence that protein‐lipid interactions are not simply based on electrostatics. We also show that higher charge density on membranes does not always correspond to higher protein affinity. In fact, DGPP showed lesser protein binding to ECA1 compared to PA although DGPP has a higher negative charge. This study brings us a step closer towards understanding the complexity of protein interactions with anionic lipids and opens the discussion to lipid homeostasis upon salt stress.Support or Funding InformationDepartment of Biological Science, Kent State University and NSF CHE 1412920This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interactome mapping of the pathogen Helicobacter pylori using All‐vs‐All sequencing (AVA‐seq)
Authors: Ramadan Stephanie , Andrews Simeon , Al‐Thani Nayra , Ahmed Ikhlak , Malek Joel 
Published: June 2021

A first step in discovering new drug targets is to identify protein‐protein interactions specific to pathogenic bacteria that could be disrupted by small molecules (Valkov et al., 2011; Kuzmanov and Emili 2013). We recently developed a novel method for studying protein‐protein interactions, termed All‐vs‐All sequencing (AVA‐seq), which is rapid and sensitive. This new technique dramatically reduces the effort needed to map all protein interactions in an organism and, for the first time in protein interaction mapping, utilizes the full power of next‐generation sequencing.While this method is new, the bacterial two‐hybrid system it is based on is well validated (Dove et al., 2004). Our innovations on this original system are multiple. They include the fusion of two vectors to allow the genes encoding interacting proteins to remain fused to each other. This critical change is what allows the system to be compatible with next‐generation sequencing. Further changes include a liquid based selection rather than colony based. This allows us to detect protein interactions by changes in a population of potential interactors much the same way that RNA‐seq detect differentially expressed genes. The method returns very rich interaction data including the ability to localize the interacting domains between two proteins, quantitate their interaction strength and observe the interaction in multiple fusion forms for validation. AVA‐seq will revolutionize the way genome‐wide protein interaction maps (Interactomes) are produced.Here we show method validation using Helicobacter pylori, both for its clinical importance and, more importantly, because its interactome has been mapped with the old, labor‐intensive yeast two‐hybrid approach so it offers an excellent validation set. This older method took 14 years to complete for H. pylori and we are close to completing a similar map in just a matter of months. Additionally, our preliminary results with known interactors in H. pylori attest to the sensitivity of the method.Support or Funding InformationFunding for this research was provided by the Biomedical Research Program Grant from Qatar Foundation Research to Weill Cornell 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.
The Related Roles of a Ubiquitin Processing Protease, Nutrient Sensor, and Cytidine Deaminase in the Growth‐to‐Development Transition of Dictyostelium Development
Authors: Nelson Brianna , Pandoy Ronald , Cook Kelia , Lindsey David 
Published: June 2021

The ubiquitin processing protease, UbpA (a homolog of yeast Ubp14 and human IsoT/USP5), is required for the growth‐to‐development transition of Dictyostelium. To understand the role of UbpA in regulating this transition, ubpA− cells were used to elucidate UbpA‐dependent pathways. ubpA− cells were hypersensitive to oxidative and nitrosative stresses, and miss‐regulated genes at the growth‐to‐development transition, including lmcB. ubpA is required for growth‐stage expression of lmcB which is involved in sensing nutrient levels and signaling cells to develop upon starvation. Exogenous expression of ubpA in ubpA− cells restored lmcB transcript levels, while exogenous expression of lmcB partially restored the developmental defect in ubpA− cells. Interestingly, lmcB is downregulated in wild‐type cells once the growth‐to‐development transition has occurred. Pull down experiments utilizing His‐tagged LmcB and TAP‐tagged LmcB expressed in Dictyostelium identified putative interacting proteins that included LmcA, PsmC5, CdaA, and a cAMP‐inducible protein. Direct binding experiments confirmed the interaction between LmcB and LmcA, and between LmcB and CdaA, a cytidine deaminase. To investigate cytidine deaminase (CDA) activity levels at the growth‐to‐development transition, a CDA assay was adapted for Dictyostelium. Wild‐type cells showed relatively low levels of CDA activity when growing and substantially increased levels upon starvation. Both growing and starving ubpA− cells have high CDA levels similar to the starving wild type cells. It is possible that LmcB binds CdaA to repress CDA activity during growth; then, upon starvation, LmcB is downregulated releasing CdaA. These results will lead to a better understanding of the mechanisms cells use to sense starvation stress and respond by making the transition from growth to development.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical Characterization of DszD, the Flavin Reductase Involved in Bacterial Biodesulfurization
Authors: Mendez Gilberto 
Published: June 2021

Dibenzothiophene (DBT) is a compound found in crude oil that upon combustion releases sulfur dioxide. The current chemical process used to remove sulfur from crude oil, hydrodesulfurization (HDS), is inefficient because it fails to work effectively on heterocyclic organosulfur compounds, such as DBT. Biodesulfurization (BDS) is a promising alternative to HDS for industrial sulfur removal. Microorganisms, like the bacterium Rhodococcus erythropolis, utilize BDS enzymes to remove sulfur from these organosulfur compounds while retaining the hydrocarbon structure of the fossil fuel components. In our work, we focus on the flavin reductase from the 4S BDS pathway, dibenzothiophene flavin reductase (DszD) from R. erythropolis, which catalyzes the reduction of flavin for subsequent use of dibenzothiophene monooxygenases DszC and DszA. This project entails the use of biochemical and structural experimentation to fully comprehend catalysis of DszD. DszD has been successfully overexpressed and purified to homogeneity from E. coli. Kinetic parameters have been determined using steady‐state and transient‐state kinetics. Weare currently looking at binding interactions between DszD and its substrate, DBT. Our preliminary experiments suggest that DBT may be acting as an effector; which means that DBT is selectively binding to DszD and regulating its biological activity. We plan to follow similar experiments with other sulfur‐containing compounds found in crude oil to see there is a direct interaction between substrate and DszD. Our current work will ultimately propel our research towards the long‐term goal of improving the BDS pathway for widespread industrial use.Support or Funding InformationThis work was funded by the Research Corporation through CCSA 22672 and the NIH through Grant 5SC2AI109500This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Mutant Sunflower Acetoacetyl CoA Thiolase
Authors: Dyer James 
Published: June 2021

Sunflower (Helianthus annus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. Crystal structures of thiolase from other organisms have identified conserved cysteines and a histidine present in the active site of the enzyme indicating that these residues probably play a key role in the reaction mechanism and other residues have been implicated in the stabilization of the enolate intermediate in the synthetic reaction. Site‐directed mutagenesis of the various amino acid residues was done and the effect of these mutations on the catalytic activity was measured.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinetic characterization of Staphylococcus aureus SpeG polyamine N‐acetyltransferase
Authors: Boeck Paloma , Renolo Rossellini , Forwood Jade , Kuhn Misty L. 
Published: June 2021

SpeG is a bacterial polyamine N‐acetyltransferase that belongs to the Gcn5‐related N‐acetyltransferase (GNAT) superfamily. Members of this family have a diverse set of functions and are present in all domains of life. Polyamines are cationic molecules present in all organisms, with roles in nucleic acid and protein synthesis, the cell cycle, stress responses, and biofilm regulation. While SpeGs from Gram‐negative bacteria have been studied previously, SpeGs from Gram‐positive bacteria have not been kinetically characterized. Here, we present the kinetic characterization of SpeG from Staphylococcus aureus and compare our results with those of other bacterial polyamine acetyltransferases. Additionally, we present a structural comparison of all SpeG proteins that have been deposited into the Protein Data Bank from both Gram‐positive and Gram‐negative bacteria. An understanding of the structure/function relationships of these enzymes is critical to advancing our understanding of how polyamine concentrations are regulated across both Gram‐positive and Gram‐negative bacteria. This may lead to a deeper understanding of the evolution of polyamine acetyltransferases across domains of life and offer opportunities to apply this knowledge toward production of effective pharmaceuticals for infectious bacteria 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.
Resolving the mechanism of glycine oxidation by GoxA using X‐ray crystallography
Authors: Avalos Dante Merced , Mamounis Kyle , Davidson Victor L. , Yukl Erik T. 
Published: June 2021

GoxA, responsible for the oxidation of glycine, is the second oxidase observed to utilize a cysteine tryptophylquinone (CTQ) cofactor after the lysine oxidase LodA. All other known enzymes containing a CTQ cofactor are involved in dehydrogenation reactions. The CTQ cofactor is produced in a post‐translation modification on Trp697 of Pseudoalteromonas luteoviolacea GoxA by the flavoenzyme GoxB. GoxA catalyzes an oxidative reaction, resulting in a deamination of glycine, producing glyoxalate, H2O2, and ammonia. The mechanism of action is unknown for this process, and revealing it could provide insight for amino acids involved in an oxidative active site containing a CTQ cofactor. A proposed mechanism is supported by using high resolution X‐ray crystal structures of GoxA in its native and substrate bound forms. Overall, the crystal structure of GoxA is observed to be a dimer of dimers, with two chains working cooperatively in the active site of each other. Native GoxA crystals are yellow in color due to the CTQ cofactor. Upon anaerobic soaking in glycine, these crystals turned a blue color, suggesting a change in the electronic structure of CTQ. Indeed, the crystal structure of this intermediate reveals the substrate glycine covalently bound to the CTQ cofactor. The bond angles of the covalently bound intermediate as well as the color change upon its formation allow us to suggest the nature of this species along the GoxA catalytic pathway. The active site environments of these two crystal structures reveals residues that may provide key interactions with the glycine for the completion of the reaction. The importance of each residue will be confirmed in future work using site‐directed mutagenesis. Other intermediates in the reaction will be investigated using methods such as resonance Raman spectroscopy. These results will shed light on the vital amino acids necessary for oxidation by a CTQ cofactor. Comparing these results with those of LodA could determine how specificity arises in these active sites.Support or Funding InformationThis work was supported by National Institutes of Health Grant 1SC2GM111170‐01 from NIGMS. Dante Avalos Supported by New Mexico State University's Howard Hughes Medical Institute Research Scholars 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.
Safety and Efficacy of a Novel Curculigo orchioides Extract in Boosting Testosterone Levels in Male Rats
Authors: Swaroop Anand , Preuss Harry G. , Bagchi Manashi , Bagchi Debasis 
Published: June 2021

Curculigo orchioides Gaertn (family Hypoxidaceae, also known as black or kali musli), is an endangered medicinal plant used for diverse medicinal purposes including impotency, aphrodisiac, diuretic, tonic, jaundice, and skin ailments. Phytochemical investigations of rhizomes revealed the presence of a novel phenolic glycoside, curculigoside, triterpenoid, saponins, flavones, cellulose, hemicellulose, and calcium oxalate. We developed a novel extract of Curculigo orchioides (Blamus™, standardized to 30% curculigosides) and assessed its dose‐and time‐dependent efficacy (0, 10, 25 and 50 mg/kg body weight p.o.) on body weight, serum free and total testosterone levels in male rats (200–230 grams; n = 6) over a period of 28 days. Blamus™ didn't cause any marked elevation in serum free testosterone levels at either10 or 25 mg/kg body weight doses, however, a 50 mg/kg body weight dose of Blamus™ induced a significant increase in serum free testosterone level (*p < 0.0001). However, no significant increases were observed in serum total testosterone levels at 0, 10, 25 or 50 mg/kg body weight doses of Blamus™. Extensive testicular histopathological analyses including investigations on the seminiferous tubules, spermatogenesis, sperm cell morphology, Leydig cells and Sertoli cells following treatment with either 0, 10, 25 or 50 mg/kg body weight doses of Blamus™ demonstrated dose‐dependent improvement in structural integrity. Furthermore, no significant changes were observed in serum SGOT, SGPT, BUN and creatinine levels following treatment with any of the given doses, which demonstrated its safety profiles. These data demonstrated that Blamus™ may serve as a safe and novel, natural testosterone booster and provide broad spectrum application is sports nutrition, muscle building and exercise pathophysiology.Support or Funding InformationThis Project was Funded by Cepham Inc., Piscataway, NJThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.