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Browsing Journal Articles, BioMed Central and SpringerOpen by Department "Chemistry"
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- Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutantTemeyer, Kevin B.; Tong, Fan; Totrov, Maxim M.; Tuckow, Alexander P.; Chen, Qiao-Hong; Carlier, Paul R.; Pérez de León, Adalberto A.; Bloomquist, Jeffrey R. (2014-12-10)Background Phlebotomus papatasi vectors zoonotic cutaneous leishmaniasis. Previous expression of recombinant P. papatasi acetylcholinesterase (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that effectively inhibited PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation causing high level resistance to organophosphate insecticides in mosquitoes may occur in PpAChE1 and may reduce sensitivity to inhibition. We report construction, expression, and biochemical properties of rPpAChE1 containing the G119S orthologous mutation. Methods Targeted mutagenesis introduced the G119S orthologous substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S mutation were expressed in the baculoviral system. Biochemical assays were conducted to determine altered catalytic properties and inhibitor sensitivity resulting from the G119S substitution. A molecular homology model was constructed to examine the modeled structural interference with docking of inhibitors of different classes. Genetic tests were conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi. Results Recombinant PpAChE1 containing the G119S substitution exhibited altered biochemical properties, and reduced inhibition by compounds that bind to the acylation site on the enzyme (with the exception of eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good agreement with modeled inhibitor docking. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect the G119S mutation in a laboratory colony of P. papatasi but did reveal that the G119S codon existed in polymorphic form (GGA + GGC). Conclusions The finding of G119S codon polymorphism in a laboratory colony of P. papatasi suggests that a single nucleotide transversion (GGC → AGC) may readily occur, causing rapid development of resistance to organophosphate and phenyl-substituted carbamate insecticides under strong selection. Careful management of pesticide use in IPM programs is important to prevent or mitigate development and fixation of the G119S mutation in susceptible pest populations. Availability of recombinant AChEs enables identification of novel inhibitory ligands with improved efficacy and specificity for AChEs of arthropod pests.
- Antiproliferative triterpenoid saponins from Leptaulus citroides Baill. from the Madagascar rain forestSu, Qingxi; Brodie, Peggy J.; Liu, Yixi; Miller, James S.; Andrianjafy, Naina M.; Antsiferana, Rabodo; Rasamison, Vincent E.; Kingston, David G. I. (Springer, 2016)Bioassay-guided fractionation of EtOH extracts obtained from the roots and wood of the Madagascan plant Leptaulus citroides Baill. (Cardiopteridaceae) led to the isolation of ethyl esters of three new triterpenoid saponins (1–3) and the known sesquiterpenoid cinnamosmolide (4). The structures of 1–3 were elucidated by extensive 1D and 2D NMR experiments and mass spectrometry. Compounds 1, 2, and 4 showed moderate cytotoxicity against the A2780 human ovarian cancer cell line with IC50 values of 2.8, 10.2 and 2.0 lM, respectively.
- Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR–Cas9 plasmidJo, Ami; Ringel-Scaia, Veronica M.; McDaniel, Dylan K.; Thomas, Cassidy A.; Zhang, Rui; Riffle, Judy S.; Allen, Irving C.; Davis, Richey M. (2020-01-20)Background The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy. Despite promising reports of the utility of CRISPR–Cas9 for in vivo gene editing, a principal problem in implementing this new process is delivery of high molecular weight DNA into cells. Results Using poly(lactic-co-glycolic acid) (PLGA), a nanoparticle carrier was designed to deliver a model CRISPR–Cas9 plasmid into primary bone marrow derived macrophages. The engineered PLGA-based carriers were approximately 160 nm and fluorescently labeled by encapsulation of the fluorophore 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene). An amine-end capped PLGA encapsulated 1.6 wt% DNA, with an encapsulation efficiency of 80%. Release studies revealed that most of the DNA was released within the first 24 h and corresponded to ~ 2–3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24 h of treatment with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells. Conclusions In this work, plasmids for the CRISPR–Cas9 system were encapsulated in nanoparticles comprised of PLGA and were shown to induce expression of bacterial Cas9 in murine bone marrow derived macrophages in vitro. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR–Cas9 system.
- Peripheral loss of EphA4 ameliorates TBI-induced neuroinflammation and tissue damageKowalski, Elizabeth A.; Chen, Jiang; Hazy, Amanda; Fritsch, Lauren E.; Gudenschwager-Basso, Erwin K.; Chen, Michael; Wang, Xia; Qian, Yun; Zhou, Mingjun; Byerly, Matthew; Pickrell, Alicia M.; Matson, John B.; Allen, Irving C.; Theus, Michelle H. (2019-11-11)Background The continuum of pro- and anti-inflammatory response elicited by traumatic brain injury (TBI) is suggested to play a key role in the outcome of TBI; however, the underlying mechanisms remain ill -defined. Methods Here, we demonstrate that using bone marrow chimeric mice and systemic inhibition of EphA4 receptor shifts the pro-inflammatory milieu to pro-resolving following acute TBI. Results EphA4 expression is increased in the injured cortex as early as 2 h post-TBI and on CX3CR1gfp-positive cells in the peri-lesion. Systemic inhibition or genetic deletion of EphA4 significantly reduced cortical lesion volume and shifted the inflammatory profile of peripheral-derived immune cells to pro-resolving in the damaged cortex. These findings were consistent with in vitro studies showing EphA4 inhibition or deletion altered the inflammatory state of LPS-stimulated monocyte/macrophages towards anti-inflammatory. Phosphoarray analysis revealed that EphA4 may regulate pro-inflammatory gene expression by suppressing the mTOR, Akt, and NF-κB pathways. Our human metadata analysis further demonstrates increased EPHA4 and pro-inflammatory gene expression, which correlates with reduced AKT concurrent with increased brain injury severity in patients. Conclusions Overall, these findings implicate EphA4 as a novel mediator of cortical tissue damage and neuroinflammation following TBI.
- Proteomic study reveals a functional network of cancer markers in the G1-Stage of the breast cancer cell cycleTenga, Milagros J.; Lazar, Iuliana M. (BioMed Central, 2014)Background: Cancer cells are characterized by a deregulated cell cycle that facilitates abnormal proliferation by allowing cells to by-pass tightly regulated molecular checkpoints such as the G1/S restriction point. To facilitate early diagnosis and the identification of new drug targets, current research efforts focus on studies that could lead to the development of protein panels that collectively can improve the effectiveness of our response to the detection of a life-threatening disease. Methods: Estrogen-responsive MCF-7 cells were cultured and arrested by serum deprivation in the G1-stage of the cell cycle, and fractionated into nuclear and cytoplasmic fractions. The protein extracts were trypsinized and analyzed by liquid chromatography - mass spectrometry (MS), and the data were interpreted with the Thermo Electron Bioworks software. Biological characterization of the data, selection of cancer markers, and identification of protein interaction networks was accomplished with a combination of bioinformatics tools provided by GoMiner, DAVID and STRING. Results: The objective of this work was to explore via MS proteomic profiling technologies and bioinformatics data mining whether randomly identified cancer markers can be associated with the G1-stage of the cell cycle, i.e., the stage in which cancer cells differ most from normal cells, and whether any functional networks can be identified between these markers and placed in the broader context of cell regulatory pathways. The study enabled the identification of over 2000 proteins and 153 cancer markers, and revealed for the first time that the G1-stage of the cell cycle is not only a rich source of cancer markers, but also a host to an intricate network of functional relationships within the majority of these markers. Three major clusters of interacting proteins emerged: (a) signaling, (b) DNA repair, and (c) oxidative phosphorylation. Conclusions: The identification of cancer marker regulatory components that act not alone, but within networks, represents an invaluable resource for elucidating the moxlecular mechanisms that govern the uncontrolled proliferation of cancer cells, as well as for catalyzing the development of protein panels with biomarker and drug target potential, screening tests with improved sensitivity and specificity, and novel cancer therapies aimed at pursuing multiple drug targets.
- A Review on Nano-/Microstructured Materials Constructed by Electrochemical Technologies for SupercapacitorsLv, Huizhen; Pan, Qing; Song, Yu; Liu, Xiao-Xia; Liu, Tianyu (2020-05-30)The article reviews the recent progress of electrochemical techniques on synthesizing nano-/microstructures as supercapacitor electrodes. With a history of more than a century, electrochemical techniques have evolved from metal plating since their inception to versatile synthesis tools for electrochemically active materials of diverse morphologies, compositions, and functions. The review begins with tutorials on the operating mechanisms of five commonly used electrochemical techniques, including cyclic voltammetry, potentiostatic deposition, galvanostatic deposition, pulse deposition, and electrophoretic deposition, followed by thorough surveys of the nano-/microstructured materials synthesized electrochemically. Specifically, representative synthesis mechanisms and the state-of-the-art electrochemical performances of exfoliated graphene, conducting polymers, metal oxides, metal sulfides, and their composites are surveyed. The article concludes with summaries of the unique merits, potential challenges, and associated opportunities of electrochemical synthesis techniques for electrode materials in supercapacitors.