bluuebunny/crossref_metadata_2025_split huggingface dataset:
New strategies for the expression of human P‐glycoprotein (MDR1) in the yeast Pichia pastoris
Authors: de Oliveira Maisa C. , Lavigne Collette A. , Thornton Henry A. , Tran Brandon M. , Wise John G. , Vogel Pia D. 
Published: November 2023

P‐glycoprotein (P‐gp) overexpression is often correlated with multidrug‐resistance (MDR) of cancer. The search for P‐gp inhibitors as co‐therapeutics to combat MDR has had little success after decades of studies. We previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. Effects on P‐gp ATPase hydrolysis activity was used as an initial biochemical screen for inhibitor effectiveness followed by assays of MDR‐reversal of a prostate cancer cell line ((Brewer et al. in Mol Pharmacol. 2014; Follit et al in Pharmacol Res Perspect. 2015). More recently, computationally identified compounds were shown to reverse MDR in cancer cells, but did not appear to inhibit ATP hydrolysis by isolated P‐gp. One reason may be that mouse P‐gp rather than human P‐gp was previously used in our biochemical tests. Expression of human P‐gp in Pichia pastoris in a biologically active form was previously reported, but unfortunately, we experienced very modest expression levels of the human P‐gp expression construct for P. pastoris available in our lab. Codon optimization of mouse P‐gp was previously shown by others to result in increased protein yield, therefore a gene for human P‐gp was designed with optimized codon usage for translation in P. pastoris. A strong Kozak sequence was incorporated at the 5′ end of the coding frame. Unique internal restriction sites were incorporated that allow subcloning of individual sections of the gene, if necessary for troubleshooting and future mutagenesis. The DNA was synthesized using standard Gibson assembly methods and cloned into a pUC57 cloning vector. When transformed into E. coli, DNA rearrangement was observed. To identify whether a specific section of the insert was the cause of counter‐selection in E. coli, the insert DNA was digested into 5 component pieces and each one was subcloned. One of the sections was found to be more susceptible to rearrangements. We report here our attempts at constructing a stable, codon‐optimized, human P‐gp gene for high yield protein production in Pichia pastoris.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to JGW/PDV, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.
Optimization of a Medium‐Throughput Cathepsin K Inhibition Assay for NAAA Inhibitors
Authors: Golden Emily Rose , Miyabe Christina , Wood JodiAnne , Malamas Michael , Alapafuja Shakiru , Makriyannis Alexandros 
Published: November 2023

Cathepsin K and N‐acylethanolamine‐hydrolyzing acid amidase (NAAA) are members of the cysteine protease family. While NAAA is a metabolic enzyme in the endocannabinoid system, Cathepsin K is involved in bone reabsorption and the release of calcium into the bloodstream. Our interest in the endocannabinoid system has resulted in significant NAAA inhibitor development for the treatment of inflammation and the resulting chronic pain and has prompted our interest in Cathepsin K for off‐target selectivity screening. A medium‐throughput fluorescent assay to determine Cathepsin K inhibition was optimized to screen NAAA‐lead compounds using Omnicathepsin (aka Z‐Phe‐Arg‐AMC) as the fluorogenic substrate. The concentrations of enzyme (2 nM Cathepsin K) and substrate (25 μM Omnicathepsin) were optimized and the Km of Cathepsin K and Omnicathepsin was found to be 9.8 μM. Two published compounds, Odanacatib and BML‐244 with IC50 values of 0.54 ± 0.4 nM and 317 ± 41 nM respectively, were developed to be reference compounds in the inhibition 3‐point screen. The optimized assay was then used to screen NAAA‐inhibitors for competitive inhibition of Cathepsin K in order to create more potent, specific drugs for NAAA.Support or Funding InformationThis project was funded by NIH: 2‐R44‐DK104636, NAAA Inhibitors as Anti‐Inflammatory Agents, Shakiru Alapafuja, PI; Northeastern PI, JodiAnne T. Wood.
Viable Strategy for Inhibition of Death Receptor 5 Signaling by Disrupting Receptor‐Receptor Interactions
Authors: Vunnam Nagamani , Lo Chih Hung , Grant Benjamin D. , Thomas David D. , Sachs Jonathan N. 
Published: November 2023

Therapeutic targeting of TNFR signaling in inflammatory diseases is a billion‐dollar industry. Current treatments for TNF‐related diseases function either by sequestering ligand or blocking ligand‐receptor interactions, which can cause adverse effects by inhibiting the receptors that are not involved in the disease condition. In the current study, we prove the viability of an alternative approach, namely the targeting of receptor‐receptor interactions. We use the soluble extracellular domain of Death Receptor 5 to block endogenous receptor assembly, mimicking the biological effect of decoy receptors that lack the intracellular signaling machinery to trigger apoptosis. Such a strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.Support or Funding InformationThis work is supported by National Institutes of Health Grant R01 GM107175
Improving the Sensitivity of P‐Glycoprotein to Drug‐Like Inhibitors in ATPase Assays and ESR Studies
Authors: Chen Gang , Ballou‐Crawford Jett , Wise John G. , Vogel Pia D. 
Published: November 2023

Multidrug resistance (MDR) in cancers is frequently associated with transmembrane efflux proteins including P‐glycoprotein (P‐gp), multidrug resistance associated protein (MRP1), and breast cancer resistance protein (BCRP). Over‐expression of these transporters in cancer cells increases efflux of therapeutic drugs rendering them ineffective. In order to re‐sensitize multidrug resistant cancers to chemotherapy, we have found inhibitors of P‐gp by in silico screening methods (Brewer et al, Mol. Pharmacol. 2014). To investigate the mechanism underlying the inhibition, we have employed biochemical assays and electron spin resonance spectroscopy (ESR), and have established nanodisc technologies that incorporate and stabilize P‐gp in native‐like phospholipid bilayer environments for our drug finding and mechanistic studies. In ATP hydrolysis assays using P‐gp in micelles, we found that the concentration of detergents used to solubilize P‐gp during purification had a significant impact on the sensitivity of P‐gp to the putative inhibitors. We hypothesized that partitioning of the compounds into empty micelles may decrease the effective concentration of the potential inhibitors. Carefully decreasing the amount of detergents used during purification resulted in significantly increased sensitivity of P‐gp to the inhibitors, while overall protein activity and stability were not affected. Similarly, excess lipids used during reconstitution of P‐gp into nanodisc may cause partitioning of inhibitors into empty nanodiscs or empty lipid vesicles, resulting in lowered sensitivity of P‐gp to the inhibitors. By optimizing the amount of lipids used during nanodisc assembly, we restored the sensitivity of P‐gp to the inhibitory compounds. Biophysical studies using ESR and a spin‐labeled analog of ATP showed that the computationally identified compounds inhibited P‐gp by binding to the ATP binding domain of P‐gp, which was consistent with our predictions by in silico screening method.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM09477102] to JGW/PDV, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.
Analysis of the Stability of Natural and Unnatural Amino Acids in Extraterrestrial Conditions
Authors: Mammoser Claire , Brown Bayland , Dhar Shilpa , Rowe Laura 
Published: November 2023

In addition to the 20 natural amino acids used in the genetic codes of most biological organisms, there are hundreds more non‐proteinogenic, or “unnatural” amino acids which could be used in the genetic codes of organisms evolving under conditions different than those on Earth. However, in order for a protein based extraterrestrial life‐form to have evolved in the first place, the amino acids within the organism must be relatively stable in the extraterrestrial environmental conditions In this work, the stability of ten unnatural amino acids and five representative natural amino acids has been tested under a variety of extraterrestrial conditions, such as heat, cold, pH variation, and UV radiation. The environmental conditions selected were intended to potentially mimic the conditions on the three most likely locations of life development within our Solar System: the ice caps of Mars, and the subsurface oceans of the moons Europa and Enceladus. After the amino acids were subjected to these conditions while in solution, their stability was quantified using Ultra‐Performance Liquid Chromatography with UV‐Vis and MS detectors. The area of the parent amino acid peak (determined through MS) on the UV‐Vis chromatogram could then be described as a percentage of the area of all non‐solvent peaks, with any non‐solvent, nonparent peaks representing degradation of the original amino acid. Ultimately, trends in stability could indicate favorable traits for amino acids to possess in order to survive on other planets and moons.Support or Funding InformationThis work has been supported by an Indiana Space Grant consortium grant.
An interdisciplinary investigation of antimicrobial peptides
Authors: Wadhwa Raoul Rajesh , Stevens‐Truss Regina 
Published: November 2023

Antimicrobial peptides (AMPs) have become a subject of research interest as a novel class of antimicrobial agents to complement conventional antibiotics. Because of their versatility and the low rate at which resistance against them develops, new research interest in them has been spurred. This study used an interdisciplinary approach to investigate the effect that the primary sequence of a short, cationic, alpha‐helical peptide has on antimicrobial activity. Computational and statistical methods were used to design peptide sequences that were complemented by experimental methods designed to assess antimicrobial activity. An analysis of data contained in two AMP databases (Antimicrobial Peptide Database (APD) and Collection of Anti‐Microbial Peptides (CAMP)) revealed that AMPs possess a number of factors in common that can explain their actions. Based on these factors, two test peptides were designed based on hydrophobic moment optimization and overall hydrophobicity, and tested for their ability to prevent bacterial cell growth, lyse red blood cells (RBCs), and liposomes. Software created for peptide structural analysis allowed for prediction of short AMP secondary structure through periodicity in the hydrophobicity as a function of the angle subtended. The experimentally tested peptides were both found to possess antimicrobial properties, but were also found to lyse RBCs and liposomes making them unfit for clinical use in their current state. However, the results of this study demonstrate that one can design short peptide sequences that mimic the actions of natural AMPs. With additional manipulation of specific properties, the primary sequence of the AMPs could be potentially tweaked to affect only microbes. This could open up a world of potential therapeutic agents useful in combating a host of microbial organisms, in particular multidrug‐resistant bacterial strains.Support or Funding InformationF.W. & Elsie Heyl Foundation Summer Grant.
Lasertherapy after induction of myocardial infarction by coronary artery occlusion in rats: a genetic signature
Authors: Feliciano Regiane Santos , Manchini Martha T , Santana Eduardo T , Maretti Joao V , Caproni Larissa I , Antonio Ednei , Serra Andrey J , Tucci Paulo J , Silva Jose A 
Published: November 2023

Myocardial infarction (MI) promoted by occlusion of the anterior descending coronary artery is the most widely used model in experimental research that seeks to elucidate the functional, structural and molecular changes associated with ischemic heart disease. Previously, we observed that low‐level laser therapy (LLLT) was able to decrease MI area and ameliorate cardiac function pot‐MI. The aim of this study was to assess a molecular signature regarding to mRNA expression of several signal transduction pathways after lasertherapy. Thus, we analyzed 48 rats that underwent to left ventricle coronary artery occlusion followed by laser irradiation in the occlusion site. Three days after surgery, we measured the infarct area and function by echocardiography followed by euthanasia. Three groups were studied as followed: Sham (n=5), MI (n=23) and MI+Laser (n=20). Remote area from MI site (septum) was removed from each rat and changes in mRNA expression were analyzed by microarray TaqMan Real‐time PCR. In‐silico analyses were used to identify genes involved in metabolic pathways related to MI in rats resulting in 47 genes of 9 metabolic pathways (angiogenesis and cell survival, hypertrophy, oxidative stress, apoptosis, extracellular matrix, Ca2+ kinetics, cell metabolism and inflammation). MI and the MI+Laser groups presented 22 (44.60%) and 12 (25.53%) differentially expressed genes, respectively, compared to Sham group. 15 genes (71.42%) of the IM group were upregulated and 6 (28.57%) were downregulated. In the IM+Laser group, 6 genes (50%) were upregulated and 6 (50%) were downregulated. IM + Laser group, when compared to the IM Group, presented 14 differentially expressed genes, all of them downregulated. Our results suggest that lasertherapy was able to modulate several post‐MI processes, including apoptosis, inflammation, cardiac hypertrophy, calcium kinetics and especially those related to cardiac remodeling. To the best of our knowledge, this was the first study that comprised a global analysis of several mRNA expression post‐MI and lasertherapy. Thus, the screening of initial mRNA expression profiles after laser irradiation in acute MI contributed to better understanding the beneficial effect of lasertherapy in the myocardium.Support or Funding InformationThis work was supported by grants number 2009/54225‐8, 2012/15808‐0, and 2015/11028‐9 from São Paulo Research Foundation (FAPESP) and National Council for Scientific and Technological (CNPq grant numbers 477458/2009‐2 and 479395/2012‐8). The authors would like FAPESP, CNPq, UNINOVE and UNIFESP for all support.
Using Transcriptomics for Biomarker Discovery Enabling High‐Throughput Drug Screening of iPSC‐Cardiomyocytes Modeling a Cardiomyopathy
Authors: Rowland Teisha J , Hashem Sherin I. , Jones Kenneth , Bonham Andrew J. , Jett Susan , Adler Eric , LaBarbera Daniel , Mestroni Luisa , Taylor Matthew 
Published: November 2023

Danon disease is an untreatable and severe X‐linked form of cardiomyopathy that leads to profound cardiac hypertrophy, causing death or a need for cardiac transplantation in males by the third decade. Danon disease is known to be caused by loss of the lysosome‐associated membrane protein‐2 (LAMP‐2) protein, which is a membrane glycoprotein likely involved in lysosome‐autophagosome fusion and vesicle trafficking during autophagy. However, it is unknown how LAMP‐2 protein deficiency leads to the development of Danon disease. To better characterize the molecular and cellular pathogenic mechanisms involved and enable biomarker discovery for high‐throughput screening (HTS) of compound libraries, human induced pluripotent stem cells (iPSCs) were generated from patients with Danon disease and differentiated into cardiomyocytes. RNA transcription levels from the Danon iPSC‐derived cardiomyocytes (iPSC‐CMs) were analyzed using RNA‐Sequencing (RNA‐Seq) and expression of key genes confirmed via quantitative real‐time PCR (qRT‐PCR). The data show significant upregulation of genes involved in cellular respiration and sarcomere expression in Danon iPSC‐CMs compared to unaffected control iPSC‐CMs. The upregulation of cellular respiration transcripts in our data align with published data suggesting mitochondrial function is affected in these cells, including increased levels of mitochondrial oxidative stress. While biomarker discovery has previously been a hurdle for successfully undertaking HTS drug discovery using these cells, our RNA‐Seq findings of potentially impacted cellular functions in the Danon iPSC‐CM model can be used to enable HTS drug discovery by identifying novel biomarkers related to mitochondrial function and mitophagy. Following development and validation of a primary HTS assay, a pilot HTS using a library of 355 kinase inhibitors and then a HTS drug discovery using a custom designed diversity library of 20,000 compounds will be performed and hits confirmed using secondary and tertiary assays. After additional in vivo validation, these confirmed hits may offer novel potential therapeutic treatments for individuals with this currently devastating and untreatable cardiomyopathy.Support or Funding InformationThis work was supported by European Union grant FP7‐PEOPLE‐2011‐IRSES 291834 SarcoSI and U.S. National Institutes of Health grants UL1 RR025780, UL1 TR001082, R01 HL69071, and R01 116906 (to Dr. Mestroni); and grants CCTSI K23 JL067915 and R01HL109209 (to Dr. Taylor). This work was supported in part by a Trans‐Atlantic Network of Excellence grant from the Leducq Foundation (14‐CVD 03).
Assessing Social Phenotypes of M. xanthus Following Homologous Recombination of Arg‐Kinase Genes
Authors: Grady Mark , Fraga Dean , Kratt Shelby 
Published: November 2023

In many eukaryotic cell systems, phosphagen kinases help modulate ATP levels during rapid energy consumption. In the past decade, more than two dozen bacterial species have been identified which express Arginine Kinases (AKs) that are homologous to those of invertebrate eukaryotes. Myxococcus xanthus is a model single‐celled myxosporulating organism which demonstrates social cooperation to acquire and conserve resources; this is exemplified by fruiting bodies which sustain spores, and rippling bodies which cover new territory and swarm around prey. The gene MXAN2252 has been identified in M. xanthus and Bragg et al (2012) determined that it expresses a protein “Ark” that can act to buffer arginine and ATP levels. While the deletion of MXAN2252 was nonfatal and did not impair vegetative growth in strain MS2252‐ΔAK, colonies are noticeably impaired at fruiting body formation when rapidly starved.The main hypothesis of this work is that closely homologous AK genes will facilitate the social competence that is characteristic among Myxococcus strains. Homologous recombination has been used to insert the wildtype Ark gene (MXAN2252) and a homologous AK gene from invertebrate L. polyphemus. Strains with these recombined genes will then be tested to determine if and to what extent, relative to the wild type and the ΔAK strain, they are restored in developmental phenotypes of fruiting body formation and recovery from environmental stressors like acidity and osmolarity changes.Support or Funding InformationThis research was conducted with frequent collaboration with Dr Mitch Singer and expertise from his laboratory at UC Davis. Summer research in this subject was funded by the Copeland Fund for from the College of Wooster.
MiRNA‐21 mediates the antiangiogenic activity of metformin through targeting PTEN and SMAD7 expression and PI3K/AKT pathway
Authors: Luo Mao , Tan Xiao Yong , Wan Qin , Wu Jianbo 
Published: November 2023

BackgroundMetformin, an anti‐diabetic drug commonly used for type 2 diabetes therapy, is associated with anti‐angiogenic effects in conditions beyond diabetes. miR‐21 has been reported to be involved in the process of angiogenesis. However, the precise regulatory mechanisms by which the metformin‐induced endothelial suppression and its effects on miR‐21‐dependent pathways are still unclear.MethodsBioinformatic analysis and identification of miR‐21 and its targets and their effects on metformin‐induced antiangiogenic activity were assessed using luciferase assays, quantitative real‐time PCR, western blots, scratch assays and CCK‐8 assays.ResultsIn this study, miR‐21 was strikingly downregulated by metformin in a time‐ and dose‐dependent manner. miR‐21 directly targeted the 3′‐UTR of PTEN and SMAD7, and negatively regulated their expression. Overexpression of miR‐21 abrogated the metformin‐mediated inhibition of endothelial cells proliferation, migration, and the TGF‐β‐induced AKT/GSK‐3β, SMAD‐ and ERK‐dependent phosphorylations, and conversely, down‐regulation of miR‐21 aggravated metformin's action and revealed significant promotion effects.ConclusionsOur study broadens our understanding of the regulatory mechanism of miR‐21 mediating metformin‐induced anti‐angiogenic effects, providing important implications regarding the design of novel miRNA‐based therapeutic strategies against angiogenesis.Support or Funding InformationThis work was supported by the American Heart Association Scientist Development Grant (10SDG2570037), National Natural Science Foundation of China Grant (81172050, 81570263)
Encoding the Human Phosphoproteome in an Engineered Bacterial System
Authors: Barber Karl , Rinehart Jesse 
Published: November 2023

Phosphorylation can potently alter protein folding and function, such as by modulating enzymatic activity, exposing scaffolding domains, or directly recruiting interaction partners. Although serine phosphorylation is the most common post‐translational modification in eukaryotes, the significance of the vast majority of phosphorylation sites across the human proteome remains unknown. Using an orthogonal translation system for phosphoserine (SepOTS), we are able to co‐translationally incorporate phosphoserine as a non‐standard amino acid in E. coli. We demonstrate the ability to synthesize and observe phosphoproteome‐scale peptide libraries representing tens of thousands of human phosphoproteins in a single experiment. We then deploy this massive‐scale peptide library to identify physiologically‐relevant phosphobinding protein interaction partners. Our synthetic human phosphoproteome is uniquely poised to answer lingering questions regarding the functional role of phosphorylation in signal transduction pathways by simultaneously monitoring thousands of human phosphosites.Support or Funding InformationK.W.B. is supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1122492. J.R. is supported by NIH R01 GM117230.