Browsing by Author "Wang, Degeng"
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Item Characterization of serotonin and gut microbiota following exposure to antibiotics in white-tailed deer (Odocoileus virginianus)(2019-05) Martinez, Audrey N.; Smith, Ernest E.; Cañas-Carrell, Jaclyn E.; Wang, DegengIn this thesis we will be discussing the importance of developing baseline serotonin concentrations in white-tailed deer serum and urine samples, both are minimally invasive tissues. An analytical method for the detection and confirmation of serotonin, 5-hydroxytryptamine (5-HT), in white-tailed deer tissues was developed and validated. Serum and urine samples were extracted with acetonitrile. Liquid chromatography separation was attained on a Phenomenex C18 column with a Security Guard ULTRA guard column with gradient elution using a mobile phase of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. This methodology was applied to baseline (control), chlortetracycline (CTC) treated, florfenicol treated and tulathromycin treated white-tailed deer serum and urine samples. We will also be discussing the impact chlortetracycline, an antibiotic growth promoter (AGP), has on serotonin concentrations as well as on the white-tailed deer gut microbiota. Florfenicol and tulathromycin impacts on serotonin concentration changes were also investigated. Next-generation sequencing of 16S rRNA was used to characterize the bacterial communities in the rumen and fecal samples of white-tailed deer treated with chlortetracycline. This research will aid the white-tailed deer breeders and other researchers toward the characterization of “normal” concentrations of serotonin in healthy animals. This characterization will allow for the development of a biomarker using non-invasive sample tissues in sick animals, for example, non-clinical cases of chronic wasting disease. It will also allow some further insight into whether the use of antibiotics as growth promoters (AGP), such as chlortetracycline, is affecting serotonin concentrations in white-tailed deer. If serotonin concentrations are being affected by the use of AGPs, it could indirectly affect behaviors that are associated with serotonin and overall herd health. Antibiotics inhibit the growth of microorganisms so characterizing the gut microbiota in white-tailed deer in control and chlortetracycline treated samples will also allow insight into whether the use of AGPs impact the abundance and/or diversity of bacterial communities in the gut of white-tailed deer.Item Effect of select perfluorinated compounds on hatching success of, and biomass accumulation in, the house cricket (Acheta domesticus).(2017-11-27) Mauricio, Jessica; Anderson, Todd A.; Wang, Degeng; Crago, JordanPerfluoroalkyls (PFAS) have been widely used since their discovery in the 1940s. PFAS can be separated into two main divisions, polyfluorinated and perfluorinated compounds (PFCs). PFASs are known to be persistent organic pollutants. PFASs are used in a variety of industries including aerospace, automotive, manufacturing, electronics, and textiles. PFASs are also used in a variety of household products. Multiple research studies have provided information regarding concentrations of PFASs in human blood, various mammals, and aquatic life. There is a noted lack of studies regarding the potential terrestrial impacts of PFAS contamination. In order to fill this gap, the effect of perfluoro-1-butanesulfonate (PFBS), perfluoro-1-octanesulfonate (PFOS), perfluoroheptanoic acid (PFHpA), perfluorohexanesulfonic acid (PFHxS), perfluorononanoic acid (PFNA) and perfluro-n-octanoic acid (PFOA) on biomass accumulation and hatching success in the house cricket (Acheta domesticus) was studied. Cricket eggs (n=20/replicate) were exposed to select PFASs in lab-grade sand to evaluate a scenario in which PFASs were readily bioavailable. Liquid chromatography coupled with an electrospray ionization-triple stage quadrupole mass spectrometer was used for confirmatory analysis of the 6 different PFASs. Treatment concentrations (n = 5) were from 0 to 42 µg/g. Previous unpublished results suggest that these selected PFASs are toxic to cricket eggs at concentrations ≥ 42 µg/g; these toxic effects are manifested as a decrease in hatching success. Cohorts that successfully hatched were observed to determine gender ratio, functional development, and PFAS body burdens in adults. Understanding PFAS body burdens in adult crickets could aid toxicologists and ecologists in understanding the dispersion of PFASs through terrestrial food webs via insect predation. However, further work is necessary to understand the potential long-term impacts of PFAS on terrestrial systems.Item Enabling in vivo Signaling Analyses via Chemical Genetics and Nanoparticle-mediated Probe Delivery(2020-03-17) Chen, Fengqian; Wang, Degeng; Crago, Jordan; Liang, Hongjun; Gao, Weimin; Mayer, GregCell signaling transduction is a critical cellular process for various diseases. Mechanistic understanding of functional proteins will improve our fundamental understanding of the versatile signaling modules and is also vital for further therapeutic development targeting cell signaling pathways. This study is to apply nanotechnology in developing a critical but never-been-accomplished approach: for the in vivo study of cellular signaling pathways. Protein kinases mediate cell signaling by pinching a γ-phosphate off ATP and sticking the phosphate onto serine, threonine, or tyrosine substrate proteins. This process is called the phosphorylation, an approach by which proteins transmit chemical signals from the external environment into the cell, ultimately down-regulating or up-regulating the activities of their substrates, thus to regulate many essential cellular processes such as cell cycles, division, migration, and apoptosis. The kinase-substrate interaction is a fundamental regulatory mechanism in cellular signaling transduction. Many diseases are related to over-activating or over-repressing kinases and substrates, thus offering great therapeutic opportunities. For instance, type II diabetic treatment include the regulation of overreacting AMPK protein kinases’ activities. Various cancer treatments include the control of oncogenic and tumor-suppressive kinases’ activities. The analog chemical-genetic strategy offers a powerful tool to study protein kinase activity under in vitro test tube conditions. However, significant limitations have persisted in conducting studies based on this approach in intact living cells, such as xxx and xxx. Herein, we have established a method for study under in vivo conditions, which is using the analog chemical-genetic strategy in intact living cells for studying AKT1 and AKT2 kinases. We intracellularly delivered bulk A*TP with a tagged γ-phosphate group using a nanoparticle delivery system, and uniquely transfer the γ-phosphate tag into the substrates of the AKT1 or AKT2 kinases using the analog chemical-genetic strategy. With this methodology, kinase-substrate interactions are entirely intracellular, and kinase substrates can be exclusively labeled in intact living cells with the thiophosphorylation of substrates, which differ from other normal substrates. It allows for the following purification and identification of those substrates from the kinase of interest, by immunoprecipitation method using thiophosphopeptides. Our study of RAS and AKTs is essential for cell signaling, and it presents as a “prove of concept” method for the study of various kinases targeting various disease treatments. With the help of our research, it opens up tremendous a novel investigative opportunity in cell signaling studies.Item In vitro toxicology and hormone assessment in cetaceans(2020-12) Wittmaack, Christiana; Godard-Codding, Céline; Goa, Weimin; Anderson, Todd A.; Wang, DegengEndocrinological and toxicological data is imperative to the conservation of marine mammals. Because of their protected status, it is necessary to conduct both these analyses using minimally invasive biopsies and in vitro techniques. Therefore, it is critical that tissue be used to gather the largest amount of data possible. Steroid hormone analysis has been used to assess reproduction and stress in multiple species. Minimally invasive blubber biopsies can be obtained from free ranging animals and split for numerous investigations. We developed methodology and analyzed several sex and stress steroid hormones via liquid chromatography mass spectrometry (LC-MS). The hormone panel consisted of, aldosterone, androstenedione, cortisol, cortisone, corticosterone, 17ß-estradiol, estrone, 17α-hydroxyprogesterone, progesterone, and testosterone. We verified the optimal, yet minimal tissue mass required, by comparing results between paired 50 mg and 150 mg samples and then paired 150 mg and 400 mg samples. In addition, we identified the optimal method of homogenization by comparing lysing (via Fast Prep) and shredding (via polytron) techniques. Our results indicated that 50 mg of tissue was enough for hormone analysis. We also identified significant differences in the blubber matrices between species. These differences have possible implications for not only homogenization technique but also matrix match calibrations. Although hormone data is imperative to conservation, identifying the toxicants of concern is just as vital. Currently there is very little toxicological data in gray whales. Therefore, we tested several toxicants that animals are potentially exposed to. Those included polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), per/poly-fluorinated compounds (PFASs), crude oil, and crude oil dispersant (COREXIT™). Despite the presence of these xenobiotics in the marine environment very little is understood about their effects in the species. Therefore, we tested these xenobiotics on gray whale skin cell fibroblasts. Our aim was to determine whether these xenobiotics were of concern. Contaminants selected for analysis included benzo[a]pyrene (B[a]P), PCB 126, perfluorooctanoic acid (PFOA), or media accommodated fractions of crude oil and/or Corexit™ 9500A.We measured cytotoxicity via both MTT (methylthiazolyldiphenyl-tetrazolium bromide) and LDH (lactate dehydrogenase) assays. We found that all toxicants significantly reduced cellular viability. In addition, we pinpointed the toxicants that may pose the most risk. In this dissertation, we present and describe a methodology for the simultaneous analysis of multiple sex and stress steroid hormones from small blubber masses using liquid chromatography mass spectrometry. We also describe the establishment of skin fibroblasts, propagated from skin biopsies, and the results from subsequent toxicity testing. Our results will help aid future science-based conservation efforts as well as expand our knowledge regarding gray whale endocrinology and toxicology.Item Overcoming the drug resistance of epidermal growth factor receptor-tyrosine kinase inhibitors in lung cancer cell lines(2018-08) Liu, Zhongwei; Gao, Weimin; Singh, Kamaleshwar P.; Mayer, Gregory D.; Wang, DegengEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib has demonstrated dramatic clinical efficacy in non-small cell lung cancer (NSCLC) patients. However, its therapeutic efficacy is ultimately limited by the development of acquired drug resistance. The aim of this study was to explore the potential utility of chromosome region maintenance 1 (CRM1) inhibitor leptomycin B (LMB) in combination with gefitinib to overcome primary and acquired gefitinib resistance in NSCLC cells. The combinative effects of gefitinib and LMB were evaluated by MTT and its underlining mechanism was assessed by flow cytometry and Western blot. LMB displayed a synergistic effect on gefitinib-induced cytotoxicity in A549 (IC50: 25.0 ± 2.1 μM of gefitinib + LMB vs. 32.0 ± 2.5 μM of gefitinib alone, p < 0.05). Gefitinib+ LMB caused a significantly different cell cycle distribution and signaling pathways involved in EGFR/survivin/p21 compared with gefitinib. A549 cells then were treated with progressively increased concentrations of gefitinib (A549GR) or in combination with LMB (A549GLR) over 10 months to generate gefitinib resistance. IC50 of gefitinib in A549GLR (37.0 ± 2.8 μM) was significantly lower than that in A549GR (53.0 ± 3.0 μM, p < 0.05), which indicates that LMB could reverse gefitinib-induced resistance in A549. Further mechanism investigation revealed that the expression patterns of EGFR pathway and epithelial-mesenchymal transition (EMT) markers in A549, A549GR, and A549GLR were significantly different. It is noticeable that the p-Akt level of A549GLR is significantly lower than that of A549GR, which may explain why the A549GLR is more sensitive to gefitinib or afatinib compared with A549GR. In conclusion, LMB at a very low concentration (0.5 nM) combined with gefitinib showed synergistic therapeutic effects and ameliorated the development of gefitinib-induced resistance in lung cancer cells. In addition, the knockdown of twist-1 expression in A549GR by shRNA plasmid was found to significantly increase the sensitivity of A549GR to gefitinib or AZD9291. In our next study, gefitinib-resistant H1650 (H1650GR) or AZD9291-resistant H1975 (H1975AR) was generated by exposing H1650 or H1975 to progressively increased concentrations of gefitinib or AZD9291 over 11 months. IC50 of gefitinib in H1650GR (50±3.0 µM) significantly increased compared with that of H1650 (31±1.0 µM) (p<0.05). Similarly, the IC50 of AZD9291 in H1975AR (10.3± 0.9 µM) significantly increased compared with H1975 (5.5±0.6 µM) (p<0.05). However, it was found that IC50 (8.5 ± 0.5µM) of H1650GR to AZD9291, a third generation EGFR-TKI, did not increase compared with that of H1650 (9.7 ± 0.7 µM). On the other hand, IC50 of A549GR to AZD9291 (12.7 ± 0.8 µM) was significantly increased compared with that of A549 (7.00 ± 1.00 µM) (p<0.05). Western blot analyses revealed that p-Akt may play a key role in determine the sensitivities of A549, A549GR, H1650, and H1650GR to gefitinib or AZD9291 treatment. Further luminescent caspase-Glo 3/7 assay showed that 10 µM AZD9291 treatment induced the increased caspase 3/7 activities in A549GR but not A549, H1650, and H1650GR. Since genetic analyses demonstrated that there were delE746-A750 in EGFR exon 19 but no T790M detected in EGFR exon20 of H1650GR, the results of our study may explain the observations in the clinical trial showing that AZD9291 was effective in overcoming the acquired resistance of the first generation of EGFR-TKI in the treatment of T790-negative NSCLC patients with EGFR mutations. In addition, it was found that knockdown of Twist1 by shRNA could significantly enhance the sensitivities of A549GR to gefitinib and AZD9291 via reversing EMT and downregulating p-Akt. However, knockdown of Twist1 in H1975AR without apparent EMT change compared to H1975 did not sensitize H1975AR to AZD9291, suggesting that different therapeutic strategies should be adopted to overcome the acquired resistance of EGFR-TKIs based on the different resistant mechanisms. Finally, we attempted to elucidate the mode of action of a traditional Chinese medicine prescription with a total of 14 components, named Lian-Jia-San-Jie-Fang (LJSJF, in Chinese), where SB works as the "principle" against non-small cell lung cancer (NSCLC) cells. Four different NSCLC cell lines (A549, H460, H1650, and H1975) were used. Cytotoxicity, in vitro tumorigenicity, gene expression, and protein expression were analyzed by MTT assay, real-time PCR, and Western blots, respectively. Among the 14 components in LJSJF, SB was the only one to possess cytotoxic effects at its pharmacologically relevant doses. Additionally, we observed synergistically dose-dependent cytotoxic effects of SB in combination with other LJSJF components. After SB or LJSJF treatment, a notable dose-dependent decrease in EGFR was observed in A549, H460, and H1650; significant downregulation in EGFR and its downstream signaling targets mTOR and p38MAPK were also observed in A549 and H460; and p53 and p21 were significantly increased while survivin, cyclin D1, and MDM2 were significantly decreased in A549. Additionally, p53, p21, and Mettl7b were decreased, but p73 was increased in H460. Neither EGFR nor p53 was changed in H1975. Therefore, SB or LJSJF may induce cytotoxic effects by regulating multiple and/or distinct apoptotic pathways in different NSCLC cells. LJSJF exerts more pronounced cytotoxic effects against NSCLC cells than SB does by synergistically regulating the underlining molecular mechanisms including EGFR and/or p53 signaling pathways.Item The Pattern of microRNA Binding Site Distribution(MDPI, 2017-10-27) Zhang, Fangyuan; Wang, DegengMicro-RNA (miRNA or miR) regulates at least 60% of the genes in the human genome through their target sites at mRNA 3’-untranslated regions (UTR), and defects in miRNA expression regulation and target sites are frequently observed in cancers. We report here a systematic analysis of the distribution of miRNA target sites. Using the evolutionarily conserved miRNA binding sites in the TargetScan database (release 7.1), we constructed a miRNA co-regulation network by connecting genes sharing common miRNA target sites. The network possesses characteristics of the ubiquitous small-world network. Non-hub genes in the network—those sharing miRNA target sites with small numbers of genes—tend to form small cliques with their neighboring genes, while hub genes exhibit high levels of promiscuousness in their neighboring genes. Additionally, miRNA target site distribution is extremely uneven. Among the miRNAs, the distribution concentrates on a small number of miRNAs, in that their target sites occur in an extraordinarily large number of genes, that is, they have large numbers of target genes. The distribution across the genes follows a similar pattern; the mRNAs of a small proportion of the genes contain extraordinarily large numbers of miRNA binding sites. Quantitatively, the patterns fit into the P(K) ∝ K−α relationship (P(K): the number of miRNAs with K target genes or genes with K miRNA sites; α: a positive constant), the mathematical description of connection distribution among the nodes and a defining characteristic of the so-called scale-free networks—a subset of small-world networks. Notably, well-known tumor-suppressive miRNAs (Let-7, miR-15/16, 26, 29, 31, 34, 145, 200, 203–205, 223, and 375) collectively have more than expected target genes, and well-known cancer genes contain more than expected miRNA binding sites. In summary, miRNA target site distribution exhibits characteristics of the small-world network. The potential to use this pattern to better understand miRNA function and their oncological roles is discussed.Item Protein Kinase Assay Using AKT1 And AKT2 In Colorectal Cancer Cells(2023-08) HIlliard, Terrell A.; Wang, Degeng; Singh, Kameleshwar; Smith, Ernest; Liang, HongjunProtein kinases form the backbone of the cellular signaling networks, and their aberrancy is a major underpinning of many diseases. The human kinome contains >500 protein kinases and they act by transferring the γ-phosphate group of the ATP molecules covalently onto their substrates to activate or repress their activities. The issue is that kinase studies are hindered by a lack of in vivo analysis approaches due to two factors: the inability to distinguish the kinase reaction of interest from other kinases and the cell impermeability of the ATP analogs. This project focuses on AKT1 and AKT2, two prominent protein kinases and therapeutic targets for multiple diseases as our initial model to develop a system for kinase-substrate relationship analysis and kinase activity measurement, in cells. AKT1 and AKT2 are a part of the PI3K-AKT/PKB-PTEN signaling module that integrates signals for cell growth, cell cycle, metabolism and many other cellular processes. In summary, we addressed these issues by combining chemical genetics with nanoparticle mediated intracellular delivery of the ATP analog. Enlargement of the ATP binding pocket, by mutating the gate-keeper Methionine residue to a Glycine, prompted the mutant AKT1 and AKT2 to preferentially use the bulky ATP analog Trinitrophenol-N6-Benzyl-ATP (TNP-A*TP) and, thus, differentiating AKT1 and AKT2 phosphorylation events. The lipid/calcium/phosphate (LCP) nanoparticle was used for efficient intracellular delivery of TNP-A*TP, overcoming the cell impermeability issue. Only mutant, not wildtype, kinases are able to use the TNP-A*TP analog. This approach will help to improve the understanding of the PI3K-AKT/PKB-PTEN signaling pathway.Item Uncovering the cellular capacity for intensive and specific feedback self-control of the argonautes and MicroRNA targeting activity(2020) Wang, Degeng; Wang, Tingzeng; Gill, Audrey (TTU); Hilliard, Terrell; Chen, Fengqian; Karamyshev, Andrey L. (TTUHSC); Zhang, Fangyuan (TTU)The miRNA pathway has three segments - biogenesis, targeting and downstream regulatory effectors. We aimed to better understand their cellular control by exploring the miRNA-mRNA-targeting relationships. We first used human evolutionarily conserved sites. Strikingly, AGOs 1-3 are all among the top 14 mRNAs with the highest miRNA site counts, along with ANKRD52, the phosphatase regulatory subunit of the recently identified AGO phosphorylation cycle; and the AGO phosphorylation cycle mRNAs share much more than expected miRNA sites. The mRNAs for TNRC6, which acts with AGOs to channel miRNA-mediated regulatory actions onto specific mRNAs, are also heavily miRNA-targeted. In contrast, upstream miRNA biogenesis mRNAs are not, and neither are downstream regulatory effectors. In short, binding site enrichment in miRNA targeting machinery mRNAs, but neither upstream biogenesis nor downstream effector mRNAs, was observed, endowing a cellular capacity for intensive and specific feedback control of the targeting activity. The pattern was confirmed with experimentally determined miRNA-mRNA target relationships. Moreover, genetic experiments demonstrated cellular utilization of this capacity. Thus, we uncovered a capacity for intensive, and specific, feedback-regulation of miRNA targeting activity directly by miRNAs themselves, i.e. segment-specific feedback auto-regulation of miRNA pathway, complementing miRNAs pairing with transcription factors to form hybrid feedback-loop.