Cellular and Molecular Science

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21731


Recent Submissions

Now showing 1 - 6 of 6
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    Omega-3 Content of Fish Oil Supplements as Monitored by Benchtop Time-domain NMR
    (2015-03) Robinson, Michelle D.; Atkinson, Stephen; Cistola, David P.
    Benchtop time-domain nuclear magnetic resonance (TD-NMR) is well suited for probing the visco-elastic and phase properties of samples of biological and industrial interest. Unlike conventional NMR spectroscopy, TD-NMR relaxometry does not require homogeneous magnetic fields and can be performed using relatively simple low-field instruments. Using TD-NMR, we observed that the T2 values of hydrocarbon chains in non-esterified fatty acids were linearly correlated with fluidity and dependent on cis-double bond content (Robinson, M.D, & Cistola, D.P., 2014, Biochemistry 53, 7515-7522. DOI:10.1021/bi5011859). Here we apply this method to analyze the omega-3 fatty acid content of triglyceride-based fish-oil supplements. The standard chemical methods for assessing the purity and potency of commercial fish oil products are expensive and tedious. Therefore, there is a need for simpler, non-invasive methods for quality control during manufacturing and for consumer safety monitoring. Using TD-NMR, the T2 profiles for pure triglycerides and mixtures reveal four resolved domains, corresponding to different segments of the hydrocarbon chain as well as the glycerol backbone. The T2 value for each resolved domain increases with the number of cis-double bonds. Across a wide series of pure triglycerides, mixtures and commercial fish-oil supplements, the T2 values are linearly correlated with percent ω-3 content, as quantified by gas-liquid chromatography. These results provide a framework for developing a quick, non-invastive benchtop TD-NMR method for analyzing the quality and potency of fish oil products.
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    Inhibition of the Glutaredoxin System Increases Doxorubicin Sensitivity in Hepatocellular Carcinoma by Impairing the Nrf2-dependent Antioxidant Response
    (2015-03) Xavier, Christy; Wu, Hongli; Liu, Xiaobin; Nguyen, Benjamin
    Purpose: Hepatocellular carcinoma (HepG2) is the most common type of liver cancer, causing approximately 1.25 million deaths annually. Even with premier anti-cancer drugs like doxorubicin, the lethality of hepatocellular carcinoma has increased and is mainly attributed to growing drug resistance. Specifically, overexpression of key antioxidant enzymes such as the glutaredoxin system (Grxs) may enable drug resistance. Glutaredoxin is a powerful protective thiol repair enzyme that increases cancer cell survival. In this study, we explored a new anti-cancer strategy, the inhibition of Grxs, as a way to both increase doxorubicin sensitivity and reverse resistance in HepG2 by impairing the Nrf2-dependent antioxidant response. Methods: HepG2 cells (Sigma) were transfected with Grxs or scramble shRNA vector. HepG2 was treated with doxorubicin in a dose and time-dependent manner. Cell viability was measured by the WST-8 colorimetric assay. Western blot was performed to test expression levels of pro- and anti-apoptotic proteins like Bax, Bcl2, and cleaved caspase-3. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Western blot was used to also examine the expression levels of Nrf2 and its downstream genes in Grxs-inhibited cells before and after doxorubicin treatment. Nrf2 translocation assay and co-immunoprecipitation with Grxs and PSSG was also performed. Antioxidant gene screening for 91 Nrf2-pathway related genes for scramble and Grxs shRNA after doxorubicin treatment was analyzed. Results: shRNA transfection gave a 50-70% Grxs knockdown. Grxs inhibition caused increased doxorubicin sensitivity with lower cell viabilities, higher pro-apoptotic protein expression levels, and increased glutathionylation than control. Grxs inhibition also decreased the expression of antioxidant enzyme transcription factor regulator, Nrf2, and its downstream antioxidant genes like HO-1, catalase, thioredoxin, and NQO1 especially after doxorubicin treatment. Nrf2’s presence in the nucleus and cytoplasm decreased with glutaredoxin knockdown. Glutaredoxin inhibition also significantly increased Nrf2 glutathionylation. Gene screening also showed significant decrease in mRNA levels of Nrf2-pathway related genes with Grxs inhibition after doxorubicin treatment. Conclusions: Grxs inhibition causes increased doxorubicin sensitivity and apoptosis of hepatocellular carcinoma by attenuating Nrf2 and its downstream antioxidant genes activation.
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    METH-induced, TAAR1-associated CREB signaling serves as a master regulator for astrocyte EAAT-2
    (2015-03) Cisneros, Irma E.; Ghorpade, Anuja
    Methamphetamine (METH) abuse accelerates the onset and severity of HIV-associated neurocognitive disorders (HAND) and astrocyte-mediated excitotoxicity. METH targets several receptors, particularly astrocyte trace amine associated receptor 1 (TAAR1), as we have previously reported. Molecular alterations of astrocyte TAAR1 correspond to changes in astrocyte excitatory amino acid transporter-2 (EAAT-2) levels and function; however, the signaling pathways downstream of METH-induced TAAR1 activation remain unclear. Astrocyte EAAT-2 is tightly regulated at the transcriptional and translational levels by cAMP and calcium, yet METH-mediated increases in these second messengers have not been shown to directly modulate astrocyte EAAT-2. Furthermore, HIV-1 relevant stimuli and IL-1b, increase TAAR1 and may exacerbate METH-mediated excitotoxicity via MAPK/ERK and NF-kB. We propose CREB activation serves as a master regulator of astrocyte EAAT-2. To investigate the temporal order of CREB activation we utilized genetically encoded calcium indicators, or GCaMPs, to visualize and quantify METH-induced calcium signaling. RNA interference targeting PKA and NF-kB subunit p65, in addition to PKA and MAPK/ERK specific inhibitors support their involvement in astrocyte EAAT-2 regulation. Furthermore, we investigated CREB phosphorylation at serine 133/142, the co-activator and co-repressor forms, respectively, following METH-induced activation. Overall, this work identifies critical signaling pathways and therapeutic targets for astrocyte EAAT-2 recovery.
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    Improvement of thailanstatins production through metabolic engineering
    (2015-03) Zhu, Hui; Liu, Xiangyang; Patel, Rinkal; Cheng, Yiqiang E.
    Thailanstatin A and thailanstatin D are potent antiproliferative natural products discovered by our group from the fermentation broth of Burkholderia thailandensis MSMB43 through a genome-guided approach. Large-scale production of thailanstatin A and thailanstatin D for animal studies, however, has not been possible due to their low titer in fermentation broth and extremely low yield after multiple steps of purification. To address this technical obstacle, we metabolically engineered the thailanstatin biosynthetic pathway through targeted-gene deletion. Deletion of tstP, which encodes a dioxygenase involved in converting thailanstatin A to another compound, resulted in 58% increase of thailanstatin A and 132% increase of thailanstatin D. Deletion of tstR, which encodes a cytochrome P450 involved in converting thailanstatin D to thailanstatin A, resulted in more than 7 fold increase of thailanstatin D. Further metabolic engineering and fermentation optimization to drastically increase the production of those promising experimental therapeutic compounds are in progress.
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    A comparison of photophysical characteristics of rHDL encapsulated anti-cancer drug valrubicin and free valrubicin.
    (2015-03) Shah, Sunil Ajit; Chib, Rahul; Raut, Sangram; Bermudez, Jaclyn Y.; Sabnis, Nirupama; Duggal, Divya; Kimball, Joseph; Lacko, Andras G.; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy
    Nanotechnology as a channel for drug delivery is one of the rapidly developing fields in cancer therapeutics. Targeted drug delivery has the advantage of having minimal interaction with healthy tissue, thereby reducing the toxicity of the drug to the rest of the body. rHDL nanoparticles have been found to be an efficient delivery system for highly lipophilic anti-cancer drugs. This is achieved through the interaction of scavenger receptors class B type I (SR-BI), which are highly expressed on cancer cells interact with rHDL nanoparticles for effective drug delivery to the cancer cell and tumor. The drug under investigation is Valrubicin, which, apart from being an effective anti-cancer drug, also has intrinsic fluorescence. This allowed for the comparison of photophysical properties of free Valrubicin and rHDL Valrubicin via steady state and time resolved fluorescence measurements. The steady-state anisotropy of rHDL Valrubicin is higher as compared to free Valrubicin, suggesting its encapsulation in rHDL nanoparticles. A longer rotational correlation time was observed for rHDL Valrubicin in time resolved anisotropy measurements compared to free Valrubicin, further supporting steady state anisotropy data.. We also studied the cellular internalization of free Valrubicin and rHDL Valrubicin using confocal microscopy. This could help track the movement of rHDL nanoparticles within the cancer cells.
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    Post-Transcriptional Regulation of Astrocyte-Tissue Inhibitor Metalloproteinase-1 (TIMP-1) in HAND
    (2015-03) Thete, Mayuri V.; Ghorpade, Anuja
    Purpose (a): HIV-1 can lead to several central nervous system impairments together termed HIV-1-associated neurocognitive disorders (HAND). In acute versus chronic neuroinflammation, differential regulation of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) is relevant to HAND neuropathogenesis. However, the underlying mechanisms are still being uncovered. In our study, we investigated the post-transcriptional regulation of TIMP-1 3’UTR via miRNAs. Method (b): Microarray analysis was used to analyze miRNA changes in IL-1β-activated astrocytes. To investigate miRNA-mediated TIMP-1 3’UTR post-transcriptional regulation, TIMP-1 3’UTR and specific miRNA overexpression constructs were used. Primary human astrocytes were nucleofected with TIMP-1 3’UTR and Pmir 146b/Pmir 155, and treated with IL-1b 24 h post-transfection. Firefly luciferase activity and astrocyte TIMP-1 levels were analyzed in parallel experiments 24 and 72 hours after IL-1β treatment. Result (c): Microarray analysis showed an increase in 12 miRNAs and decrease in 4 miRNAs. Seven of those were further confirmed by RT-PCR. The most consistent increase was observed in miRNA 155 and miRNA 146b. Overexpression of miRNA 155 and miRNA 146b in IL-1β-activated astrocytes decreased both luciferase activity and endogenous TIMP-1 levels. Conclusion (d): In summary, our preliminary studies suggest that astrocyte-TIMP-1 may be regulated post-transcriptionally by miRNAs (146b and 155) during HAND.