Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “PCR”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 847 records · Page 47
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Selective incorporation of vRNP into influenza A virions determined by its specific interaction with M1 protein

Influenza A viruses contain eight single-stranded, negative-sense RNA segments as viral genomes in the form of viral ribonucleoproteins (vRNPs). During genome replication in the nucleus, positive-sense complementary RNPs (cRNPs) are produced as replicative intermediates, which are not incorporated into progeny virions. To analyze the mechanism of selective vRNP incorporation into progeny virions, we quantified vRNPs and cRNPs in the nuclear and cytosolic fractions of infected cells, using a strand-specific qRT-PCR. Unexpectedly, we found that cRNPs were also exported to the cytoplasm. This export was chromosome region maintenance 1 (CRM1)-independent unlike that of vRNPs. Although both vRNPs and cRNPs were present in the cytosol, viral matrix (M1) protein, a key regulator for viral assembly, preferentially bound vRNPs over cRNPs. These results indicate that influenza A viruses selectively uptake cytosolic vRNPs through a specific interaction with M1 during viral assembly. - Highlights: •Influenza cRNPs are exported from the nucleus of an infected cell via a CRM1-independent pathway. •Influenza A viruses selectively incorporate cytosolic vRNPs through a specific interaction with M1 during viral assembly. •M1 dissociates from vRNP export complex after nuclear export, and is re-associated with vRNPs at the plasma membrane.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Dengue virus replicates and accumulates in Aedes aegypti salivary glands

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidence that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. - Highlights: •Strand-specific RT-qPCR allows accurate quantification of DENV (-) RNA in mosquito tissues. •Detection of DENV (-) RNA in salivary glands provides evidence of viral replication in this tissue. •Viral replication in salivary glands likely replenishes DENV genetic diversity prior to transmission.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Production of high-quality polyethylene (PE) films from post-consumer shrink wrap with solvent targeted recovery and precipitation (STRAP)

The solvent-targeted recovery and precipitation (STRAP™) process separates polymers from contaminants present in the feedstock. In this work, we demonstrate the recyclability of a post-consumer waste (PCW) shrink wrap that contains inks, adhesives, and paper using STRAP. We produced a clear rPE cast film from the PCR shrink wrap with no visual contamination from paper or inks. STRAP decreased the ash content from 0.96 wt% to 0.24 wt%. A techno-economic analysis (TEA) shows that a STRAP plant processing 50,000 metric tons annually of shrink wrap waste (purchased at $\$0.25$/kg) containing 85 wt% PE can achieve an IRR of 16.2% when selling rPE at $\$1.30$/kg, an IRR of 10.7% with an rPE price of $\$1.00$/kg, or an IRR of 3.79% with an rPE price of $\$0.70$/kg. Furthermore, the life cycle assessment (LCA) shows a 64% reduction in global warming potential (kg CO 2 equivalent) when compared to the production of virgin LDPE. As a result, this work shows how feedstocks with insoluble impurities can be recycled using STRAP on a large scale.

Radkevich, Elizaveta [University of Wisconsin-Madi↗
DOE OSTI2013

The role of 3D microenvironmental organization in MCF-7 epithelial–mesenchymal transition after 7 culture days

We present a multi-technique study on in vitro epithelial–mesenchymal transition (EMT) in human MCF-7 cells cultured on electrospun scaffolds of poly(L-lactic acid) (PLA), with random and aligned fiber orientations. Our aim is to investigate the morphological and genetic characteristics induced by extracellular matrix in tumor cells cultured in different 3D environments, and at different time points. Cell vitality was assessed with AlamarBlue at days 1, 3, 5 and 7. Scanning electron microscopy was performed at culture days 3 and 7. Immunohistochemistry (for E-cadherin, β-catenin, cytokeratins, nucleophosmin, tubulin, Ki-67 and vimentin), immunofluorescence (for F-actin) western blot (for E-cadherin, β-catenin and vimentin) and transmission electron microscopy were carried out at day 7. An EMT gene array followed by PCR analysis confirmed the regulation of selected genes. At day 7, scanning electron microscopy on aligned-PLA revealed spindle-shaped cells gathered in buds and ribbon-like structures, with a higher nucleolar/nuclear ratio and a loss in E-cadherin and β-catenin at immunohistochemistry and western blot. An up-regulation of SMAD2, TGF-β2, TFPI2 and SOX10 was found in aligned-PLA compared to random-PLA cultured cells. The topography of the extracellular matrix has a role in tumor EMT, and a more aggressive phenotype characterizes MCF-7 cells cultured on aligned-PLA scaffold. -- Highlights: • After 7 culture days an aligned-PLA scaffold induces a spindle shape to MCF-7 cells. • Despite these changes, the aligned MCF-7 cells keep an epithelial phenotype. • The extracellular environment alone influences the E-cadherin/β-catenin axis. • The extracellular environment can promote the epithelial–mesenchymal transition.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Migration-stimulating factor (MSF) is over-expressed in non-small cell lung cancer and promotes cell migration and invasion in A549 cells over-expressing MSF

Migration-stimulating factor (MSF), an oncofetal truncated isoform of fibronectin, is a potent stimulator of cell invasion. However, its distribution and motogenic role in non-small cell lung cancer (NSCLC) have never been identified. In this study, real-time PCR and immunohistochemical staining (IHC) were performed to detect MSF mRNA and protein levels in tumor tissues and matched adjacent tumor-free tissues. Furthermore, to examine the effect of MSF on invasiveness, MSF was upregulated in A549 cells. The invasiveness and viability of A549 cells were then determined using a transwell migration assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays, respectively. The expression level of MSF in NSCLC tissue was markedly higher than in matched adjacent tumor-free tissue. Additionally, the level of MSF protein expression in stage III and IV NSCLC samples was higher than in stage I and II NSCLC samples. More importantly, we also demonstrated that migration and invasion of A549 cells increased substantially after upregulating MSF, although proliferation remained unchanged. Meanwhile, we found no correlation between increasing motility and invasiveness of MSF-overexpressing cells and expression levels and activities of matrix metalloprotease MMP-2 and MMP-9. Our current study shows that MSF plays a role in migration and invasion of A549 cells and suggests that MSF may be a potential biomarker of NSCLC progression. - Highlights: • MSF expression was upregulated in NSCLC and correlated with TNM stages. • MSF may be a new biomarker for NSCLC progression. • MSF promoted migration and invasion in A549 cells, independent of MMP-2/MMP-9 expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Tetramethylpyrazine attenuates TNF-α-induced iNOS expression in human endothelial cells: Involvement of Syk-mediated activation of PI3K-IKK-IκB signaling pathways

Endothelial cells produce nitric oxide (NO) by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NO synthase (iNOS). We explored the effect of tetramethylpyrazine (TMP), a compound derived from chuanxiong, on tumor necrosis factor (TNF)-α-induced iNOS in human umbilical vein endothelial cells (HUVECs) and explored the signal pathways involved by using RT-PCR and Western blot. TMP suppressed TNF-α-induced expression of iNOS by inhibiting IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor κB (NF-κB) nuclear translocation, which were required for NO gene transcription. Exposure to wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression, suggesting activation of such a signal pathway might be phosphoinositide-3-kinase (PI3K) dependent. Spleen tyrosine kinase (Syk) inhibitor piceatannol significantly inhibited NO production. Furthermore, piceatannol obviously suppressed TNF-α-induced IκB phosphorylation and the downstream NF-κB activation, suggesting that Syk is an upstream key regulator in the activation of PI3K/IKK/IκB-mediated signaling. TMP significantly inhibited TNF-α-induced phosphorylation of Syk and PI3K. Our data indicate that TMP might repress iNOS expression, at least in part, through its inhibitory effect of Syk-mediated PI3K phosphorylation in TNF-α-stimulated HUVECs. -- Highlights: •TMP suppressed TNF-α-induced expression of iNOS by inhibiting IKK/IκB/NF-κB pathway. •PI3K inhibitor wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression. •Syk inhibitor piceatannol repressed PI3K/IKK/IκB mediated NO production. •Syk is an upstream regulator in the activation of PI3K/IKK/IκB-mediated signaling. •TMP might repress iNOS expression through Syk-mediated PI3K pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Mitochondrial-related gene expression profiles suggest an important role of PGC-1alpha in the compensatory mechanism of endemic dilated cardiomyopathy

Keshan disease (KD) is an endemic dilated cardiomyopathy with unclear etiology. In this study, we compared mitochondrial-related gene expression profiles of peripheral blood mononuclear cells (PBMCs) derived from 16 KD patients and 16 normal controls in KD areas. Total RNA was isolated, amplified, labeled and hybridized to Agilent human 4×44k whole genome microarrays. Mitochondrial-related genes were screened out by the Third-Generation Human Mitochondria-Focused cDNA Microarray (hMitChip3). Quantitative real-time PCR, immunohistochemical and biochemical parameters related mitochondrial metabolism were conducted to validate our microarray results. In KD samples, 34 up-regulated genes (ratios≥2.0) were detected by significance analysis of microarrays and ingenuity systems pathway analysis (IPA). The highest ranked molecular and cellular functions of the differentially regulated genes were closely related to amino acid metabolism, free radical scavenging, carbohydrate metabolism, and energy production. Using IPA, 40 significant pathways and four significant networks, involved mainly in apoptosis, mitochondrion dysfunction, and nuclear receptor signaling were identified. Based on our results, we suggest that PGC-1alpha regulated energy metabolism and anti-apoptosis might play an important role in the compensatory mechanism of KD. Our results may lead to the identification of potential diagnostic biomarkers for KD in PBMCs, and may help to understand the pathogenesis of KD. Highlights: • Thirty-four up-regulated genes were detected in KD versus health controls. • Forty pathways and four networks were detected in KD. • PGC-1alpha regulated energy metabolism and anti-apoptosis in KD.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

CHIR99021 promotes self-renewal of mouse embryonic stem cells by modulation of protein-encoding gene and long intergenic non-coding RNA expression

Embryonic stem cells (ESCs) can proliferate indefinitely in vitro and differentiate into cells of all three germ layers. These unique properties make them exceptionally valuable for drug discovery and regenerative medicine. However, the practical application of ESCs is limited because it is difficult to derive and culture ESCs. It has been demonstrated that CHIR99021 (CHIR) promotes self-renewal and enhances the derivation efficiency of mouse (m)ESCs. However, the downstream targets of CHIR are not fully understood. In this study, we identified CHIR-regulated genes in mESCs using microarray analysis. Our microarray data demonstrated that CHIR not only influenced the Wnt/β-catenin pathway by stabilizing β-catenin, but also modulated several other pluripotency-related signaling pathways such as TGF-β, Notch and MAPK signaling pathways. More detailed analysis demonstrated that CHIR inhibited Nodal signaling, while activating bone morphogenetic protein signaling in mESCs. In addition, we found that pluripotency-maintaining transcription factors were up-regulated by CHIR, while several developmental-related genes were down-regulated. Furthermore, we found that CHIR altered the expression of epigenetic regulatory genes and long intergenic non-coding RNAs. Quantitative real-time PCR results were consistent with microarray data, suggesting that CHIR alters the expression pattern of protein-encoding genes (especially transcription factors), epigenetic regulatory genes and non-coding RNAs to establish a relatively stable pluripotency-maintaining network. - Highlights: • Combined use of CHIR with LIF promotes self-renewal of J1 mESCs. • CHIR-regulated genes are involved in multiple pathways. • CHIR inhibits Nodal signaling and promotes Bmp4 expression to activate BMP signaling. • Expression of epigenetic regulatory genes and lincRNAs is altered by CHIR.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Overexpression of FABP3 inhibits human bone marrow derived mesenchymal stem cell proliferation but enhances their survival in hypoxia

Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, and the differential expression of FABP3 was tested by quantitative {sup RT}PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions. - Highlights: • FABP3 expression pattern was studied in 12 human hypoxic-MSCs. • FABP3 mRNA and proteins are upregulated in the MSCs under hypoxic conditions. • Overexpression of FABP3 inhibits cell growth but advanced the MSC survival under hypoxia. • Overexpression of FABP3 down-regulate the cell cycle and stem cell signaling pathways.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion

We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer. - Highlights: • BAD and p-BAD expressions are decreased in breast cancer compared with normal breast tissue. • BAD impedes breast cancer invasion and migration. • BAD inhibits the EMT and transcription factors that promote cancer cell migration. • Invasion and migration functions of BAD are distinct from the BAD's role in apoptosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Different origin of adipogenic stem cells influences the response to antiretroviral drugs

Lipodystrophy (LD) is a main side effect of antiretroviral therapy for HIV infection, and can be provoked by nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). LD exists in different forms, characterized by fat loss, accumulation, or both, but its pathogenesis is still unclear. In particular, few data exist concerning the effects of antiretroviral drugs on adipocyte differentiation. Adipose tissue can arise either from mesenchymal stem cells (MSCs), that include bone marrow-derived MSCs (hBM-MSCs), or from ectodermal stem cells, that include dental pulp stem cells (hDPSCs). To analyze whether the embryonal origin of adipocytes might impact the occurrence of different phenotypes in LD, we quantified the effects of several antiretroviral drugs on the adipogenic differentiation of hBM-MSCs and hDPSCs. hBM-MSCs and hDPSCs were isolated from healthy donors. Cells were treated with 10 and 50 μM stavudine (d4T), efavirenz (EFV), atazanavir (ATV), ritonavir (RTV), and ATV-boosted RTV. Viability and adipogenesis were evaluated by staining with propidium iodide, oil red, and adipoRed; mRNA levels of genes involved in adipocyte differentiation, i.e. CCAAT/enhancer-binding protein alpha (CEBPα) and peroxisome proliferator-activated receptor gamma (PPARγ), and in adipocyte functions, i.e. fatty acid synthase (FASN), fatty acid binding protein-4 (FABP4), perilipin-1 (PLIN1) and 1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2), were quantified by real time PCR. We found that ATV, RTV, EFV, and ATV-boosted RTV, but not d4T, caused massive cell death in both cell types. EFV and d4T affected the accumulation of lipid droplets and induced changes in mRNA levels of genes involved in adipocyte functions in hBM-MSCs, while RTV and ATV had little effects. All drugs stimulated the accumulation of lipid droplets in hDPSCs. Thus, the adipogenic differentiation of human stem cells can be influenced by antiretroviral drugs, and depends, at least in part, on their embryonal origin. - Highlights: • ATV, RTV, EFV and ATV-boosted RTV induce massive cell death in hBM-MSCs and hDPSCs. • EFV and d4T strongly affect the accumulation of lipid droplets in hBM-MSCs. • All drugs stimulate the accumulation of lipid droplets in hDPSCs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

MiR-145, a microRNA targeting ADAM17, inhibits the invasion and migration of nasopharyngeal carcinoma cells

MiR-145 is downregulated and functions as a tumor suppressor in many malignancies. In this study, the biological function, molecular mechanism, and direct target genes of miR-145 in nasopharyngeal carcinoma (NPC) cells were investigated. Cell survival was detected by cell viability assay, and cell cycle was determined through flow cytometry. Invasion and migration of NPC cells were examined using cell invasion and wound healing assays, respectively. A disintegrin and metalloproteinase 17 (ADAM17) was verified as the target of miR-145 through luciferase reporter assay, qRT-PCR, and Western blot analysis. In NPC cell lines, miR-145 expression was significantly downregulated and ADAM17 protein expression was upregulated. ADAM17 was downregulated at the post-transcriptional level by miR-145 via the binding site of ADAM17-3′UTR. Transfection with miR-145 mimic suppressed cell growth and induced cell cycle arrest in the G0/G1 phase by upregulating key G0/G1 phase regulators, namely, p53 and p21. MiR-145 also inhibited cellular migration and invasion through targeting ADAM17 involving the regulation of EGFR and E-cadherin. Knockdown of ADAM17 elicited similar effects to that of miR-145 on NPC cells. This study reveals that miR-145 suppressed the invasion and migration of NPC cells by targeting ADAM17. Thus, miR-145 could be a therapeutic target for NPC. - Highlights: • MiR-145 inhibits proliferation, invasion, and migration in NPC cells. • Knockdown of ADAM17 inhibits invasion and migration in NPC cells. • MiR-145 inhibits invasion and migration in NPC cells by targeting ADAM17.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

MicroRNA-126 suppresses proliferation of undifferentiated (BRAF{sup V600E} and BRAF{sup WT}) thyroid carcinoma through targeting PIK3R2 gene and repressing PI3K-AKT proliferation-survival signalling pathway

Background: The objectives of this study are to investigate the expression of miR-126 and evaluate its effect on proliferation in undifferentiated thyroid carcinoma. Methods: miR-126 expression of undifferentiated thyroid carcinoma cell lines 8505C (BRAF{sup V600E/V600E}), BHT-101 (BRAF{sup V600E/WT}) and MB-1 (BRAF{sup WT/WT}) were quantified with q-PCR. These cell lines were transiently transfected with exogenous miR-126 (mimic). Following transfection, proliferation effects were observed through MTS proliferation assay and colony formation abilities. Immunofluorescence imaging and Western blot assay were also done to check target proteins expression. Results: Under-expression (p<0.05) of miR-126 was noted in BRAF{sup V600E} mutated undifferentiated thyroid carcinoma cells (8505C and BHT-101), but no change in expression was noted in non BRAF{sup V600E} mutated undifferentiated thyroid carcinoma cells (MB-1). In addition, a 30–50% drop in proliferation ability and a 35–45% reduction in colony formation capability were noticed in miR-126 mimic transfected group when compared to control group. Furthermore, immunofluorescence images showed reduced expression of p85β and p-AKT protein in miR-126 mimic transfected cells when compared to un-transfected cells. Also, Western blot analysis revealed a 34–40% suppression of p85β protein and a 21–53% drop in active AKT kinase (p-AKT) protein in miR-126 mimic transfected group when compared to control group. Conclusions: Expression of miR-126 was down-regulated in BRAF{sup V600E} mutated undifferentiated thyroid carcinoma. In addition, miR-126 was found to act as proliferation suppressor targeting PIK3R2 gene and reducing p85β (a regulatory subunit of PI3K kinase) protein translation and lower AKT kinase activity. Therefore, miR-126 could be a potential therapeutic tool in the treatment of undifferentiated thyroid carcinoma. - Highlights: • miR-126 expression was low in BRAF mutated undifferentiated thyroid carcinoma. • miR-126 suppresses p85β expression in undifferentiated thyroid carcinoma. • miR-126 suppresses AKT kinase activity in undifferentiated thyroid carcinoma. • miR-126 suppresses proliferation of undifferentiated thyroid carcinoma.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS. • Different serum conditions influence epigentic patterns of genes. • Osteogenic genes specifically up-regulate in AS in response to differentiation signals.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

MicroRNA library screening identifies growth-suppressive microRNAs that regulate genes involved in cell cycle progression and apoptosis

Micro(mi)RNAs play important and varied roles in tumorigenesis; however, the full repertoire of miRNAs that affect cancer cell growth is not known. In this study, an miRNA library was screened to identify those that affect the growth of A549 tumor cells. Among 300 miRNAs, miR-28-5p, -323-5p, -510-5p, -552-3p, and -608 were the most effective in inhibiting cell growth. More specifically, overexpressing miR-28-5p, -323-5p, and -510-5p induced G1 arrest, as determined by flow cytometry, whereas that of miR-608 induced cell death in a caspase-dependent manner. Moreover, several genes involved in apoptosis and cell cycle progression were downregulated upon overexpression of each of the five miRNAs, with the functional targets of miR-552-3p and miR-608 confirmed by microarray, quantitative real-time PCR, and luciferase reporter assay. In miR-608-transfected cells, B cell lymphoma 2-like 1 (BCL2L1), D-type cyclin 1 (CCND1), CCND3, cytochrome b5 reductase 3 (CYB5R3), phosphoinositide 3-kinase regulatory subunit 2 (PIK3R2), specificity protein 1 (SP1), and phosphorylated Akt were all downregulated, while Bcl-2-interacting killer (BIK) was upregulated. Moreover, miR-608 was determined to have a suppressive function on tumor growth in an NCI-H460 xenograft model. These findings provide insights into the roles of five miRNAs in growth inhibition and their potential function as cancer therapeutics. - Highlights: • MicroRNAs (miRNAs) affecting cancer cell growth were identified through a screen. • Five miRNAs that inhibit cell proliferation and survival were identified. • MiRNAs modulate various target genes to inhibit cancer cell growth. • These five miRNAs can potentially serve as new agents for cancer treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

TIGIT overexpression diminishes the function of CD4 T cells and ameliorates the severity of rheumatoid arthritis in mouse models

Rheumatoid arthritis (RA) is an immune-mediated disease with a pathogenesis that involves CD4 T cell activation. Multiple immune regulatory molecules expressed on CD4{sup +} T cells were involved in RA pathogenesis. In this study, we investigated the role of T cell immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibition motif) domain (TIGIT) in RA. The frequency of TIGIT-positive CD4{sup +} T cells in the synovial fluid (SF) of active RA patients was lower than that of inactive RA patients. And a negative correlation between RA disease activity and TIGIT expression was found. In CD4{sup +} T cells isolated from SF of active RA patients, TIGIT upregulation significantly decreased cell proliferation, as shown by MTT assay. TIGIT overexpression also significantly decreased the production of IFN-γ and IL-17, and increased that of IL-10, as determined by ELISA and qRT-PCR. In CD4{sup +} T cells isolated from SF of inactive RA patients, TIGIT was silenced by siRNA transfection. As expected, TIGIT knockdown resulted in an opposite effect on cell proliferation and the production of cytokines, including IFN-γ, IL-17 and IL-10. A RA mouse model was established using type II collagen induction. TIGIT was upregulated in RA mouse by lentivector infection. As expected, TIGIT overexpression in vivo significantly alleviated the disease severity and deceased the levels of anti-collagen II antibodies. TIGIT upregulation in the early stage was more effective to alleviate disease severity. Our data suggested the potential therapeutic role of TIGIT in RA patients. - Highlights: • TIGIT expression on SF CD4{sup +} T cells was decreased in active RA patients. • TIGIT overexpression diminished the function of CD4{sup +} T cells. • TIGIT-siRNA transfection promotes the function of CD4{sup +} T cells. • Mice with TIGIT overexpression were resistant to collagen induced RA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Apoptosis in human myelodysplastic syndrome CD34+ cells is modulated by the upregulation of TLRs and histone H4 acetylation via a β-arrestin 1 dependent mechanism

Excessive apoptosis of hematopoietic precursors in the bone marrow underlies the ineffective hematopoiesis characteristic of myelodysplastic syndrome (MDS). Toll-like receptor (TLR) signaling is abnormally activated in MDS and may be involved in excessive programmed cell death in the pathogenesis of MDS. TLRs expression and global histone H3/H4 acetylation were analyzed in bone marrow (BM) CD34+ cells from 20 lower-risk and 20 higher-risk MDS patients and 10 healthy controls. Apoptosis of BM CD34+ cells was examined by flow cytometry, and its correlation to histone acetylation and the expression of TLR2 and β-arrestin1 (β-arr1), measured by enzyme-linkedimmunosorbent assay and qRT-PCR, was assessed. TLR1, TLR2 and TLR6 expression and H4 acetylation levels were higher in lower-risk MDS patients than in higher-risk MDS patients or controls, and TLR2 expression and H4 acetylation levels were positively correlated with an increased rate of apoptosis. Lower-risk MDS was associated with increased β-arr1 expression and histone acetyltransferase p300 activity. In in vitro-cultured primary normal and lower-risk MDS CD34+ cells, TLR2 activation-induced apoptosis was mediated by the upregulation of β-arr1 leading to the recruitment of p300 and increased histone H4 acetylation. The nuclear accumulation of βarr1 following TLR2 activation promote H4 acetylation at specific target gene promoters and may thus affect transcription of target genes in BM CD34+ cells. The mechanisms underlying the deregulation of TLR2 and increased apoptosis in MDS may involve the β-arr1 mediated recruitment of p300 leading to increased levels of histone H4 acetylation. - Highlights: • TLR2 is upregulated in lower-risk MDS patients. • TLR2 expression and H4 acetylation correlated with increased CD34+ cell apoptosis. • TLR2-induced apoptosis was mediated by β-arr1 upregulation in normal CD34+ cells. • β-arr1 mediated recruitment of p300 lead to increased histone H4 acetylation levels.

60 APPLIED LIFE SCIENCES↗