An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves.

PubMed

Rosenthal, Sun Hee; Diamos, Andrew G; Mason, Hugh S

2018-03-01

We have found interesting features of a plant gene (extensin) 3' flanking region, including extremely efficient polyadenylation which greatly improves transient expression of transgenes when an intron is removed. Its use will greatly benefit studies of gene expression in plants, research in molecular biology, and applications for recombinant proteins. Plants are a promising platform for the production of recombinant proteins. To express high-value proteins in plants efficiently, the optimization of expression cassettes using appropriate regulatory sequences is critical. Here, we characterize the activity of the tobacco extensin (Ext) gene terminator by transient expression in Nicotiana benthamiana, tobacco, and lettuce. Ext is a member of the hydroxyproline-rich glycoprotein (HRGP) superfamily and constitutes the major protein component of cell walls. The present study demonstrates that the Ext terminator with its native intron removed increased transient gene expression up to 13.5-fold compared to previously established terminators. The enhanced transgene expression was correlated with increased mRNA accumulation and reduced levels of read-through transcripts, which could impair gene expression. Analysis of transcript 3'-ends found that the majority of polyadenylated transcripts were cleaved at a YA dinucleotide downstream from a canonical AAUAAA motif and a UG-rich region, both of which were found to be highly conserved among related extensin terminators. Deletion of either of these regions eliminated most of the activity of the terminator. Additionally, a 45 nt polypurine sequence ~ 175 nt upstream from the polyadenylation sites was found to also be necessary for the enhanced expression. We conclude that the use of Ext terminator has great potential to benefit the production of recombinant proteins in plants.

Polyethyleneimine Coating Enhances the Cellular Uptake of Mesoporous Silica Nanoparticles and Allows Safe Delivery of siRNA and DNA Constructs

PubMed Central

Xia, Tian; Kovochich, Michael; Liong, Monty; Meng, Huan; Kabehie, Sanaz; Zink, Jeffrey I.; Nel, Andre E.

2014-01-01

Surface-functionalized mesoporous silica nanoparticles (MSNP) can be used as an efficient and safe carrier for bioactive molecules. In order to make the MSNP a more efficient delivery system, we modified the surface of the particles by a functional group that enhances cellular uptake and allows nucleic acid delivery in addition to traditional drug delivery. Non-covalent attachment of polyethyleneimine (PEI) polymers to the surface not only increases MSNP cellular uptake, but also generates a cationic surface to which DNA and siRNA constructs could be attached. While efficient for intracellular delivery of these nucleic acids, the 25 KD PEI polymer unfortunately changes the safety profile of the MSNP that is otherwise very safe. By experimenting with several different polymer molecular weights, it was possible to retain high cellular uptake and transfection efficiency while reducing or even eliminating cationic MSNP cytotoxicity. The particles coated with the 10 KD PEI polymer was particularly efficient for transducing HEPA-1 cells with a siRNA construct that was capable of knocking down GFP expression. Similarly, transfection of a GFP plasmid induced effective expression of the fluorescent protein in > 70% cells in the population. These outcomes were quantitatively assessed by confocal microscopy and flow cytometry. We also demonstrated that the enhanced cellular uptake of the non-toxic cationic MSNP enhance the delivery of the hydrophobic anticancer drug, paclitaxel, to pancreatic cancer cells. In summary, we demonstrate that by a careful selection of PEI size, it is possible to construct cationic MSNP that are capable of nucleotide and enhanced drug delivery with minimal or no cytotoxicity. This novel use of a cationic MSNP extends its therapeutic use potential. PMID:19739605

Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system.

PubMed

Peng, Feng; Wang, Xinyue; Sun, Yang; Dong, Guibin; Yang, Yankun; Liu, Xiuxia; Bai, Zhonghu

2017-11-14

Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.

The automaticity of emotion recognition.

PubMed

Tracy, Jessica L; Robins, Richard W

2008-02-01

Evolutionary accounts of emotion typically assume that humans evolved to quickly and efficiently recognize emotion expressions because these expressions convey fitness-enhancing messages. The present research tested this assumption in 2 studies. Specifically, the authors examined (a) how quickly perceivers could recognize expressions of anger, contempt, disgust, embarrassment, fear, happiness, pride, sadness, shame, and surprise; (b) whether accuracy is improved when perceivers deliberate about each expression's meaning (vs. respond as quickly as possible); and (c) whether accurate recognition can occur under cognitive load. Across both studies, perceivers quickly and efficiently (i.e., under cognitive load) recognized most emotion expressions, including the self-conscious emotions of pride, embarrassment, and shame. Deliberation improved accuracy in some cases, but these improvements were relatively small. Discussion focuses on the implications of these findings for the cognitive processes underlying emotion recognition.

Activation tagging in indica rice identifies ribosomal proteins as potential targets for manipulation of water-use efficiency and abiotic stress tolerance in plants.

PubMed

Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Udaya Kumar, M; Reddy, Attipalli R; Rao, K V; Siddiq, E A; Kirti, P B

2016-11-01

We have generated 3900 enhancer-based activation-tagged plants, in addition to 1030 stable Dissociator-enhancer plants in a widely cultivated indica rice variety, BPT-5204. Of them, 3000 were screened for water-use efficiency (WUE) by analysing photosynthetic quantum efficiency and yield-related attributes under water-limiting conditions that identified 200 activation-tagged mutants, which were analysed for flanking sequences at the site of enhancer integration in the genome. We have further selected five plants with low Δ 13 C, high quantum efficiency and increased plant yield compared with wild type for a detailed investigation. Expression studies of 18 genes in these mutants revealed that in four plants one of the three to four tagged genes became activated, while two genes were concurrently up-regulated in the fifth plant. Two genes coding for proteins involved in 60S ribosomal assembly, RPL6 and RPL23A, were among those that became activated by enhancers. Quantitative expression analysis of these two genes also corroborated the results on activating-tagging. The high up-regulation of RPL6 and RPL23A in various stress treatments and the presence of significant cis-regulatory elements in their promoter regions along with the high up-regulation of several of RPL genes in various stress treatments indicate that they are potential targets for manipulating WUE/abiotic stress tolerance. © 2016 John Wiley & Sons Ltd.

Performance-enhanced mesenchymal stem cells via intracellular delivery of steroids

NASA Astrophysics Data System (ADS)

Ankrum, James A.; Dastidar, Riddhi G.; Ong, Joon Faii; Levy, Oren; Karp, Jeffrey M.

2014-04-01

Inadequate immunomodulatory potency of mesenchymal stem cells (MSC) may limit their therapeutic efficacy. We report glucocorticoid steroids augment MSC expression and activity of indoleamine-2,3-dioxygenase (IDO), a primary mediator of MSC immunomodulatory function. This effect depends on signaling through the glucocorticoid receptor and is mediated through up-regulation of FOXO3. Treatment of MSCs with glucocorticoids, budesonide or dexamethasone, enhanced IDO expression following IFN-γ stimulation in multiple donors and was able to restore IDO expression in over-passaged MSCs. As IDO enhancement was most notable when cells were continuously exposed to budesonide, we engineered MSC with budesonide loaded PLGA microparticles. MSC efficiently internalized budesonide microparticles and exhibited 4-fold enhanced IDO activity compared to budesonide preconditioned and naïve MSC, resulting in a 2-fold improvement in suppression of stimulated peripheral blood mononuclear cells in an IDO-dependent manner. Thus, the augmentation of MSC immune modulation may abrogate challenges associated with inadequate potency and enhance their therapeutic efficacy.

Performance-enhanced mesenchymal stem cells via intracellular delivery of steroids

PubMed Central

Ankrum, James A.; Dastidar, Riddhi G.; Ong, Joon Faii; Levy, Oren; Karp, Jeffrey M.

2014-01-01

Inadequate immunomodulatory potency of mesenchymal stem cells (MSC) may limit their therapeutic efficacy. We report glucocorticoid steroids augment MSC expression and activity of indoleamine-2,3-dioxygenase (IDO), a primary mediator of MSC immunomodulatory function. This effect depends on signaling through the glucocorticoid receptor and is mediated through up-regulation of FOXO3. Treatment of MSCs with glucocorticoids, budesonide or dexamethasone, enhanced IDO expression following IFN-γ stimulation in multiple donors and was able to restore IDO expression in over-passaged MSCs. As IDO enhancement was most notable when cells were continuously exposed to budesonide, we engineered MSC with budesonide loaded PLGA microparticles. MSC efficiently internalized budesonide microparticles and exhibited 4-fold enhanced IDO activity compared to budesonide preconditioned and naïve MSC, resulting in a 2-fold improvement in suppression of stimulated peripheral blood mononuclear cells in an IDO-dependent manner. Thus, the augmentation of MSC immune modulation may abrogate challenges associated with inadequate potency and enhance their therapeutic efficacy. PMID:24717973

Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes.

PubMed

Haga, K; Lemp, N A; Logg, C R; Nagashima, J; Faure-Kumar, E; Gomez, G G; Kruse, C A; Mendez, R; Stripecke, R; Kasahara, N; Kasahara, N A; Cicciarelli, J C

2006-12-01

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

Structure-function correlation of chloroquine and analogues as transgene expression enhancers in nonviral gene delivery.

PubMed

Cheng, Jianjun; Zeidan, Ryan; Mishra, Swaroop; Liu, Aijie; Pun, Suzie H; Kulkarni, Rajan P; Jensen, Gregory S; Bellocq, Nathalie C; Davis, Mark E

2006-11-02

To understand how chloroquine (CQ) enhances transgene expression in polycation-based, nonviral gene delivery systems, a number of CQ analogues with variations in the aliphatic amino side chain or in the aromatic ring are synthesized and investigated. Our studies indicate that the aliphatic amino moiety of CQ is essential to provide increased gene expression. Further, the enhancements are more dramatically affected by changes to the aromatic ring and are positively correlated to the strength of intercalation between DNA and the CQ analogues. Quinacrine (QC), a CQ analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to CQ at a concentration 10 times lower, while N(4)-(4-pyridinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CP), a CQ analogue that has a weakly intercalating pyridinyl ring, shows no effect on gene expression. Subtle change on the 7-substituent of the chloroquine aromatic structure can also greatly affect the ability of the CQ analogues to enhance transgene expression. Transfection in the presence of N(4)-(7-trifluoromethyl-4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at same concentration, while transfection in the presence of N(4)-(4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CQ7b) does not reveal any enhancing effects on expression. Through a number of comparative studies with CQ and its analogues, we conclude that there are at least three mechanistic features of CQ that lead to the enhancement in gene expression: (i) pH buffering in endocytic vesicles, (ii) displacement of polycations from the nucleic acids in polyplexes, and (iii) alteration of the biophysical properties of the released nucleic acid.

The Absence of NOD1 Enhances Killing of Aspergillus fumigatus Through Modulation of Dectin-1 Expression.

PubMed

Gresnigt, Mark S; Jaeger, Martin; Subbarao Malireddi, R K; Rasid, Orhan; Jouvion, Grégory; Fitting, Catherine; Melchers, Willem J G; Kanneganti, Thirumala-Devi; Carvalho, Agostinho; Ibrahim-Granet, Oumaima; van de Veerdonk, Frank L

2017-01-01

One of the major life-threatening infections for which severely immunocompromised patients are at risk is invasive aspergillosis (IA). Despite the current treatment options, the increasing antifungal resistance and poor outcome highlight the need for novel therapeutic strategies to improve outcome of patients with IA. In the current study, we investigated whether and how the intracellular pattern recognition receptor NOD1 is involved in host defense against Aspergillus fumigatus . When exploring the role of NOD1 in an experimental mouse model, we found that Nod1 -/- mice were protected against IA and demonstrated reduced fungal outgrowth in the lungs. We found that macrophages derived from bone marrow of Nod1 -/- mice were more efficiently inducing reactive oxygen species and cytokines in response to Aspergillus . Most strikingly, these cells were highly potent in killing A. fumigatus compared with wild-type cells. In line, human macrophages in which NOD1 was silenced demonstrated augmented Aspergillus killing and NOD1 stimulation decreased fungal killing. The differentially altered killing capacity of NOD1 silencing versus NOD1 activation was associated with alterations in dectin-1 expression, with activation of NOD1 reducing dectin-1 expression. Furthermore, we were able to demonstrate that Nod1 -/- mice have elevated dectin-1 expression in the lung and bone marrow, and silencing of NOD1 gene expression in human macrophages increases dectin-1 expression. The enhanced dectin-1 expression may be the mechanism of enhanced fungal killing of Nod1 -/- cells and human cells in which NOD1 was silenced, since blockade of dectin-1 reversed the augmented killing in these cells. Collectively, our data demonstrate that NOD1 receptor plays an inhibitory role in the host defense against Aspergillus . This provides a rationale to develop novel immunotherapeutic strategies for treatment of aspergillosis that target the NOD1 receptor, to enhance the efficiency of host immune cells to clear the infection by increasing fungal killing and cytokine responses.

Sox2 regulatory region 2 sequence works as a DNA nuclear targeting sequence enhancing the efficiency of an exogenous gene expression in ES cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Funabashi, Hisakage; Takatsu, Makoto; Saito, Mikako

2010-10-01

Research highlights: {yields} SV40-DTS worked as a DTS in ES cells as well as other types of cells. {yields} Sox2 regulatory region 2 worked as a DTS in ES cells and thus was termed as SRR2-DTS. {yields} SRR2-DTS was suggested as an ES cell-specific DTS. -- Abstract: In this report, the effects of two DNA nuclear targeting sequence (DTS) candidates on the gene expression efficiency in ES cells were investigated. Reporter plasmids containing the simian virus 40 (SV40) promoter/enhancer sequence (SV40-DTS), a DTS for various types of cells but not being reported yet for ES cells, and the 81 basemore » pairs of Sox2 regulatory region 2 (SRR2) where two transcriptional factors in ES cells, Oct3/4 and Sox2, are bound (SRR2-DTS), were introduced into cytoplasm in living cells by femtoinjection. The gene expression efficiencies of each plasmid in mouse insulinoma cell line MIN6 cells and mouse ES cells were then evaluated. Plasmids including SV40-DTS and SRR2-DTS exhibited higher gene expression efficiency comparing to plasmids without these DTSs, and thus it was concluded that both sequences work as a DTS in ES cells. In addition, it was suggested that SRR2-DTS works as an ES cell-specific DTS. To the best of our knowledge, this is the first report to confirm the function of DTSs in ES cells.« less

E-cadherin expression increases cell proliferation by regulating energy metabolism through nuclear factor-κB in AGS cells.

PubMed

Park, Song Yi; Shin, Jee-Hye; Kee, Sun-Ho

2017-09-01

β-Catenin is a central player in Wnt signaling, and activation of Wnt signaling is associated with cancer development. E-cadherin in complex with β-catenin mediates cell-cell adhesion, which suppresses β-catenin-dependent Wnt signaling. Recently, a tumor-suppressive role for E-cadherin has been reconsidered, as re-expression of E-cadherin was reported to enhance the metastatic potential of malignant tumors. To explore the role of E-cadherin, we established an E-cadherin-expressing cell line, EC96, from AGS cells that featured undetectable E-cadherin expression and a high level of Wnt signaling. In EC96 cells, E-cadherin re-expression enhanced cell proliferation, although Wnt signaling activity was reduced. Subsequent analysis revealed that nuclear factor-κB (NF-κB) activation and consequent c-myc expression might be involved in E-cadherin expression-mediated cell proliferation. To facilitate rapid proliferation, EC96 cells enhance glucose uptake and produce ATP using both mitochondria oxidative phosphorylation and glycolysis, whereas AGS cells use these mechanisms less efficiently. These events appeared to be mediated by NF-κB activation. Therefore, E-cadherin re-expression and subsequent induction of NF-κB signaling likely enhance energy production and cell proliferation. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Elongin B-mediated epigenetic alteration of viral chromatin correlates with efficient human cytomegalovirus gene expression and replication.

PubMed

Hwang, Jiwon; Saffert, Ryan T; Kalejta, Robert F

2011-01-01

Elongins B and C are members of complexes that increase the efficiency of transcriptional elongation by RNA polymerase II (RNAPII) and enhance the monoubiquitination of histone H2B, an epigenetic mark of actively transcribed genes. Here we show that, in addition to its role in facilitating transcription of the cellular genome, elongin B also enhances gene expression from the double-stranded DNA genome of human cytomegalovirus (HCMV), a pathogenic herpesvirus. Reducing the level of elongin B by small interfering RNA- or short hairpin RNA-mediated knockdown decreased viral mRNA expression, viral protein accumulation, viral DNA replication, and infectious virion production. Chromatin immunoprecipitation analysis indicated viral genome occupancy of the elongating form of RNAPII, and monoubiquitinated histone H2B was reduced in elongin B-deficient cells. These data suggest that, in addition to the previously documented epigenetic regulation of transcriptional initiation, HCMV also subverts cellular elongin B-mediated epigenetic mechanisms for enhancing transcriptional elongation to enhance viral gene expression and virus replication. The genetic and epigenetic control of transcription initiation at both cellular and viral promoters is well documented. Recently, the epigenetic modification of histone H2B monoubiquitination throughout the bodies of cellular genes has been shown to enhance the elongation of RNA polymerase II-initiated transcripts. Mechanisms that might control the elongation of viral transcripts are less well studied. Here we show that, as with cellular genes, elongin B-mediated monoubiquitination of histone H2B also facilitates the transcriptional elongation of human cytomegalovirus genes. This and perhaps other epigenetic markings of actively transcribed regions may help in identifying viral genes expressed during in vitro latency or during natural infections of humans. Furthermore, this work identifies a novel, tractable model system to further study the regulation of transcriptional elongation in living cells.

Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex

PubMed Central

Gerits, Annelies; Vancraeyenest, Pascaline; Vreysen, Samme; Laramée, Marie-Eve; Michiels, Annelies; Gijsbers, Rik; Van den Haute, Chris; Moons, Lieve; Debyser, Zeger; Baekelandt, Veerle; Arckens, Lutgarde; Vanduffel, Wim

2015-01-01

Abstract. Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area. PMID:26839901

Unprecedented enhancement of transient gene expression from minimal cassettes using a double terminator.

PubMed

Beyene, Getu; Buenrostro-Nava, Marco T; Damaj, Mona B; Gao, San-Ji; Molina, Joe; Mirkov, T Erik

2011-01-01

The potential of using vector-free minimal gene cassettes (MGCs) with a double terminator for the enhancement and stabilization of transgene expression was tested in sugarcane biolistic transformation. The MGC system used consisted of the enhanced yellow fluorescent protein (EYFP) reporter gene driven by the maize ubiquitin-1 (Ubi) promoter and a single or double terminator from nopaline synthase (Tnos) or/and Cauliflower mosaic virus 35S (35ST). Transient EYFP expression from Tnos or 35ST single terminator MGC was very low and unstable, typically peaking early (8-16 h) and diminishing rapidly (48-72 h) after bombardment. Addition of a ~260 bp vector sequence (VS) to the single MGC downstream of Tnos (Tnos + VS) or 35ST (35ST + VS) enhanced EYFP expression by 1.25- to 25-fold. However, a much more significant increase in EYFP expression was achieved when the VS in 35ST + VS was replaced by Tnos to generate a 35ST-Tnos double terminator MGC, reaching its maximum at 24 h post-bombardment. The enhanced EYFP expression from the double terminator MGC was maintained for a long period of time (168 h), resulting in an overall increase of 5- to 65-fold and 10- to 160-fold as compared to the 35ST and Tnos single terminator MGCs, respectively. The efficiency of the double terminator MGC in enhancing EYFP expression was also demonstrated in sorghum and tobacco, suggesting that the underlying mechanism is highly conserved among monocots and dicots. Our results also suggest the involvement of posttranscriptional gene silencing in the reduced and unstable transgene expression from single terminator MGCs in plants.

Radiofrequency-enhanced vascular gene transduction and expression for intravascular MR imaging-guided therapy: feasibility study in pigs.

PubMed

Du, Xiangying; Qiu, Bensheng; Zhan, Xiangcan; Kolmakova, Antonina; Gao, Fabao; Hofmann, Lawrence V; Cheng, Linzhao; Chatterjee, Subroto; Yang, Xiaoming

2005-09-01

To evaluate the feasibility of radiofrequency (RF)-enhanced vascular gene transduction and expression by using a magnetic resonance (MR) imaging-heating guidewire as an intravascular heating vehicle during MR imaging-guided therapy. The institutional committee for animal care and use approved the experimental protocol. The study included in vitro evaluation of the use of RF energy to enhance gene transduction and expression in vascular cells, as well as in vivo validation of the feasibility of intravascular MR imaging-guided RF-enhanced vascular gene transduction and expression in pig arteries. For in vitro experiments, approximately 10(4) vascular smooth muscle cells were seeded in each of four chambers of a cell culture plate. Next, 1 mL of a green fluorescent protein gene (gfp)-bearing lentivirus was added to each chamber. Chamber 4 was heated at approximately 41 degrees C for 15 minutes by using an MR imaging-heating guidewire connected to a custom RF generator. At day 6 after transduction, the four chambers were examined and compared at confocal microscopy to determine the efficiency of gfp transduction and expression. For the in vivo experiments, a lentivirus vector bearing a therapeutic gene, vascular endothelial growth factor 165 (VEGF-165), was transferred by using a gene delivery balloon catheter in 18 femoral-iliac arteries (nine artery pairs) in domestic pigs and Yucatan pigs with atherosclerosis. During gene infusion, one femoral-iliac artery in each pig was heated to approximately 41 degrees C with RF energy transferred via the intravascular MR imaging-heating guidewire, while the contralateral artery was not heated (control condition). At day 6, the 18 arteries were harvested for quantitative Western blot analysis to compare VEGF-165 transduction and expression efficiency between RF-heated and nonheated arterial groups. Confocal microscopy showed gfp expression in chamber 4 that was 293% the level of expression in chamber 1 (49.6% +/- 25.8 vs 16.8% +/- 8.0). Results of Western blot analysis showed VEGF-165 expression for normal arteries in the RF-heated group that was 300% the level of expression in the nonheated group (70.4 arbitrary units [au] +/- 107.1 vs 23.5 au +/- 29.8), and, for atherosclerotic arteries in the RF-heated group, 986% the level in the nonheated group (129.2 au +/- 100.3 vs 13.1 au +/- 4.9). Simultaneous monitoring and enhancement of vascular gene delivery and expression is feasible with the MR imaging-heating guidewire.

A high-level prokaryotic expression system: synthesis of human interleukin 1 alpha and its receptor antagonist.

PubMed

Birikh, K R; Lebedenko, E N; Boni, I V; Berlin, Y A

1995-10-27

Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene. Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression.

A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5’UTRs of Selected Tumor Suppressors

PubMed Central

Master, Adam; Wójcicka, Anna; Giżewska, Kamilla; Popławski, Piotr; Williams, Graham R.; Nauman, Alicja

2016-01-01

Background Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. Findings We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5’-untranslated region (5’UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5’UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5’UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5’UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5’UTRs of complex structure. Significance This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5’UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene and may have future application in gene therapy strategies to enhance expression of proteins including tumor suppressors. PMID:27171412

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Yarimizu, Tohru; Iwakiri, Ryo; Hoshida, Hisashi; Akada, Rinji

2015-08-01

Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.

Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection.

PubMed

Dietrich, Isabelle; McMonagle, Elizabeth L; Petit, Sarah J; Vijayakrishnan, Swetha; Logan, Nicola; Chan, Chi N; Towers, Greg J; Hosie, Margaret J; Willett, Brian J

2011-06-01

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and "OrfA" proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.

Ultrasonic destruction of albumin microbubbles enhances gene transfection and expression in cardiac myocytes.

PubMed

Wang, Guo-zhong; Liu, Jing-hua; Lü, Shu-zheng; Lü, Yun; Guo, Cheng-jun; Zhao, Dong-hui; Fang, Dong-ping; He, Dong-fang; Zhou, Yuan; Ge, Chang-jiang

2011-05-01

It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes. The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed. The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P < 0.01), and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced β-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05). Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.

Localized epigenetic silencing of a damage-activated WNT enhancer limits regeneration in mature Drosophila imaginal discs

PubMed Central

Harris, Robin E; Setiawan, Linda; Saul, Josh; Hariharan, Iswar K

2016-01-01

Many organisms lose the capacity to regenerate damaged tissues as they mature. Damaged Drosophila imaginal discs regenerate efficiently early in the third larval instar (L3) but progressively lose this ability. This correlates with reduced damage-responsive expression of multiple genes, including the WNT genes wingless (wg) and Wnt6. We demonstrate that damage-responsive expression of both genes requires a bipartite enhancer whose activity declines during L3. Within this enhancer, a damage-responsive module stays active throughout L3, while an adjacent silencing element nucleates increasing levels of epigenetic silencing restricted to this enhancer. Cas9-mediated deletion of the silencing element alleviates WNT repression, but is, in itself, insufficient to promote regeneration. However, directing Myc expression to the blastema overcomes repression of multiple genes, including wg, and restores cellular responses necessary for regeneration. Localized epigenetic silencing of damage-responsive enhancers can therefore restrict regenerative capacity in maturing organisms without compromising gene functions regulated by developmental signals. DOI: http://dx.doi.org/10.7554/eLife.11588.001 PMID:26840050

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

1998-10-13

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

1998-01-01

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous gene

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

2007-03-20

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

2000-08-22

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Laboratory formulated magnetic nanoparticles for enhancement of viral gene expression in suspension cell line.

PubMed

Bhattarai, Shanta Raj; Kim, Sun Young; Jang, Kyu Yun; Lee, Ki Chang; Yi, Ho Keun; Lee, Dae Yeol; Kim, Hak Yong; Hwang, Pyoung Han

2008-02-01

One factor critical to successful gene therapy is the development of efficient delivery systems. Although advances in gene transfer technology including viral and non-viral vectors have been made, an ideal vector system has not yet been constructed. Due to the growing concerns over the toxicity and immunogenicity of viral DNA delivery systems, DNA delivery via improve viral routes has become more desirable and advantageous. The ideal improve viral DNA delivery system should be a synthetic materials plus viral vectors. The materials should also be biocompatible, efficient, and modular so that it is tunable to various applications in both research and clinical settings. The successful steps towards this improve viral DNA delivery system is demonstrated: a magnetofection system mediated by modified cationic chitosan-coated iron oxide nanoparticles. Dense colloidal cationic iron oxide nanoparticles serve as an uptake-enhancing component by physical concentration at the cell surface in presence of external magnetic fields; enhanced viral gene expression (3-100-fold) due to the particles is seen as compared to virus vector alone with little virus dose.

Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli.

PubMed

Link, A James; Skretas, Georgios; Strauch, Eva-Maria; Chari, Nandini S; Georgiou, George

2008-10-01

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.

An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20.

PubMed

Zhao, Huimin; Tan, Zilong; Wen, Xuejing; Wang, Yucheng

2017-02-14

Syringe infiltration is an important transient transformation method that is widely used in many molecular studies. Owing to the wide use of syringe agroinfiltration, it is important and necessary to improve its transformation efficiency. Here, we studied the factors influencing the transformation efficiency of syringe agroinfiltration. The pCAMBIA1301 was transformed into Nicotiana benthamiana leaves for investigation. The effects of 5-azacytidine (AzaC), Ascorbate acid (ASC) and Tween-20 on transformation were studied. The β-glucuronidase ( GUS ) expression and GUS activity were respectively measured to determine the transformation efficiency. AzaC, ASC and Tween-20 all significantly affected the transformation efficiency of agroinfiltration, and the optimal concentrations of AzaC, ASC and Tween-20 for the transgene expression were identified. Our results showed that 20 μM AzaC, 0.56 mM ASC and 0.03% ( v / v ) Tween-20 is the optimal concentration that could significantly improve the transformation efficiency of agroinfiltration. Furthermore, a combined supplement of 20 μM AzaC, 0.56 mM ASC and 0.03% Tween-20 improves the expression of transgene better than any one factor alone, increasing the transgene expression by more than 6-fold. Thus, an optimized syringe agroinfiltration was developed here, which might be a powerful method in transient transformation analysis.

Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

PubMed

White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

2008-12-01

Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

The Absence of NOD1 Enhances Killing of Aspergillus fumigatus Through Modulation of Dectin-1 Expression

PubMed Central

Gresnigt, Mark S.; Jaeger, Martin; Subbarao Malireddi, R. K.; Rasid, Orhan; Jouvion, Grégory; Fitting, Catherine; Melchers, Willem J. G.; Kanneganti, Thirumala-Devi; Carvalho, Agostinho; Ibrahim-Granet, Oumaima; van de Veerdonk, Frank L.

2017-01-01

One of the major life-threatening infections for which severely immunocompromised patients are at risk is invasive aspergillosis (IA). Despite the current treatment options, the increasing antifungal resistance and poor outcome highlight the need for novel therapeutic strategies to improve outcome of patients with IA. In the current study, we investigated whether and how the intracellular pattern recognition receptor NOD1 is involved in host defense against Aspergillus fumigatus. When exploring the role of NOD1 in an experimental mouse model, we found that Nod1−/− mice were protected against IA and demonstrated reduced fungal outgrowth in the lungs. We found that macrophages derived from bone marrow of Nod1−/− mice were more efficiently inducing reactive oxygen species and cytokines in response to Aspergillus. Most strikingly, these cells were highly potent in killing A. fumigatus compared with wild-type cells. In line, human macrophages in which NOD1 was silenced demonstrated augmented Aspergillus killing and NOD1 stimulation decreased fungal killing. The differentially altered killing capacity of NOD1 silencing versus NOD1 activation was associated with alterations in dectin-1 expression, with activation of NOD1 reducing dectin-1 expression. Furthermore, we were able to demonstrate that Nod1−/− mice have elevated dectin-1 expression in the lung and bone marrow, and silencing of NOD1 gene expression in human macrophages increases dectin-1 expression. The enhanced dectin-1 expression may be the mechanism of enhanced fungal killing of Nod1−/− cells and human cells in which NOD1 was silenced, since blockade of dectin-1 reversed the augmented killing in these cells. Collectively, our data demonstrate that NOD1 receptor plays an inhibitory role in the host defense against Aspergillus. This provides a rationale to develop novel immunotherapeutic strategies for treatment of aspergillosis that target the NOD1 receptor, to enhance the efficiency of host immune cells to clear the infection by increasing fungal killing and cytokine responses. PMID:29326692

Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression

PubMed Central

Moreno, J.C.; Cerda, A.; Simpson, K.; Lopez-Diaz, I.; Carrera, E; Handford, M.; Stange, C.

2016-01-01

Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene β-cyclase (LCYB) that catalyzes the conversion of lycopene into β-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, β-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition. PMID:26893492

Unraveling molecular mechanistic differences in liver metabolism between lean and fat lines of Pekin duck (Anas platyrhynchos domestica): a proteomic study.

PubMed

Zheng, Aijuan; Chang, Wenhuan; Hou, Shuisheng; Zhang, Shu; Cai, Huiyi; Chen, Guilan; Lou, Ruiying; Liu, Guohua

2014-02-26

Duck is one of the major poultry meat sources for human consumption. To satisfy different eating habits, lean and fat strains of Pekin ducks have been developed. The objective of this study was to determine the molecular mechanistic differences in liver metabolism between two duck strains. The liver proteome of the Pekin duck lines was compared on days 1, 14, 28, and 42 posthatching using 2-DE based proteomics. There was a different abundance of 76 proteins in the livers of the two duck lines. Fat ducks strongly expressed proteins related to pathways of glycolysis, ATP synthesis, and protein catabolism, suggesting enhanced fat deposition rather than protein retention. In contrast, highly expressed proteins in lean ducks improved protein anabolism and reduced protein catabolism, resulting in an enhancement of lean meat deposition. Along with the decrease in fat deposition, the immune system of the lean duck strain may be enhanced by enhanced expression of proteins involved in stress response, immune defense, and antioxidant functions. These results indicate that selection pressure has shaped the two duck lines differently resulting in different liver metabolic capacities. These observed variations between the two strains at the molecular level are matched with physiological changes in growth performance and meat production. This information may have beneficial impacts in areas such as genetic modification through the manipulation of target proteins or genes in specific pathways to improve the efficiency of duck meat production. The objective of this study was to unravel molecular mechanistic differences in liver metabolism between lean and fat Pekin duck (Anas platyrhynchos domestica) strains. There was a different abundance of 76 proteins in the livers of the two duck lines. Enhanced protein expression in the fat ducks related to pathways of glycolysis, ATP synthesis and protein catabolism suggesting increased fat deposition rather than protein retention. In contrast, highly expressed proteins in the lean ducks facilitated protein deposition by increasing protein anabolism and reducing protein catabolism to enhance the lean meat percentage. Along with the decrease of fat deposition, the immunity of lean duck appeared to be enhanced by increased expression of proteins involved in stress response, defense and antioxidant function. This study provides potential target proteins or genes for further functional analysis and genetic manipulation to increase the efficiency of duck meat production and help satisfy the global demand for poultry meat. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents

PubMed Central

Hunt, Michelle A.; Currie, Margaret J.; Robinson, Bridget A.; Dachs, Gabi U.

2010-01-01

Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for transfection, followed by flow cytometry of transfected HUVEC to determine the proportion of EGFP-expressing cells as a measure of transfection efficiency. Lipofectamine 2000 and Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) gave the highest transfection efficiencies of the reagents tested. Lipofectamine LTX was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited, allowing sufficient yield of transfected HUVEC for use in subsequent assays. PMID:20592869

Feline Tetherin Efficiently Restricts Release of Feline Immunodeficiency Virus but Not Spreading of Infection▿

PubMed Central

Dietrich, Isabelle; McMonagle, Elizabeth L.; Petit, Sarah J.; Vijayakrishnan, Swetha; Logan, Nicola; Chan, Chi N.; Towers, Greg J.; Hosie, Margaret J.; Willett, Brian J.

2011-01-01

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and “OrfA” proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread. PMID:21490095

Antisense Treatments for Biothreat Agents

DTIC Science & Technology

2006-08-01

2001) 19(4):360-364. 82. Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L: Inhibition of Staphylococcus aureus gene expression and growth using...to PNA enhanced the entry of the antisense molecules and reduced expression of the bacterial target genes both in E coli [81] and Staphylococcus ... aureus [82]. Peptide-tagged PMOs can also efficiently inhibit bacterial growth in pure and infected cultures [75]. In a recent study, we observed that

Genetic engineering of stem cells for enhanced therapy.

PubMed

Nowakowski, Adam; Andrzejewska, Anna; Janowski, Miroslaw; Walczak, Piotr; Lukomska, Barbara

2013-01-01

Stem cell therapy is a promising strategy for overcoming the limitations of current treatment methods. The modification of stem cell properties may be necessary to fully exploit their potential. Genetic engineering, with an abundance of methodology to induce gene expression in a precise and well-controllable manner, is particularly attractive for this purpose. There are virus-based and non-viral methods of genetic manipulation. Genome-integrating viral vectors are usually characterized by highly efficient and long-term transgene expression, at a cost of safety. Non-integrating viruses are also highly efficient in transduction, and, while safer, offer only a limited duration of transgene expression. There is a great diversity of transfectable forms of nucleic acids; however, for efficient shuttling across cell membranes, additional manipulation is required. Both physical and chemical methods have been employed for this purpose. Stem cell engineering for clinical applications is still in its infancy and requires further research. There are two main strategies for inducing transgene expression in therapeutic cells: transient and permanent expression. In many cases, including stem cell trafficking and using cell therapy for the treatment of rapid-onset disease with a short healing process, transient transgene expression may be a sufficient and optimal approach. For that purpose, mRNA-based methods seem ideally suited, as they are characterized by a rapid, highly efficient transfection, with outstanding safety. Permanent transgene expression is primarily based on the application of viral vectors, and, due to safety concerns, these methods are more challenging. There is active, ongoing research toward the development of non-viral methods that would induce permanent expression, such as transposons and mammalian artificial chromosomes.

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

PubMed

Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

2017-08-01

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Liposome-chaperoned cell-free synthesis for the design of proteoliposomes: Implications for therapeutic delivery.

PubMed

Lu, Mei; Zhao, Xiaoyun; Xing, Haonan; Xun, Zhe; Yang, Tianzhi; Cai, Cuifang; Wang, Dongkai; Ding, Pingtian

2018-04-03

Cell-free (CF) protein synthesis has emerged as a powerful technique platform for efficient protein production in vitro. Liposomes have been widely studied as therapeutic carriers due to their biocompatibility, biodegradability, low toxicity, flexible surface manipulation, easy preparation, and higher cargo encapsulation capability. However, rapid immune clearance, insufficient targeting capacity, and poor cytoplasmic delivery efficiency substantially restrict their clinical application. The incorporation of functional membrane proteins (MPs) or peptides allows the transfer of biological properties to liposomes and imparts them with improved circulation, increased targeting, and efficient intracellular delivery. Liposome-chaperoned CF synthesis enables production of proteoliposomes in one-step reaction, which not only substantially simplifies the production procedure but also keeps protein functionality intact. Building off these observations, proteoliposomes with integrated MPs represent an excellent candidate for therapeutic delivery. In this review, we describe recent advances in CF synthesis with emphasis on detailing key factors for improving CF expression efficiency. Furthermore, we provide insights into strategies for rational design of proteoliposomal nanodelivery systems via CF synthesis. Liposome-chaperoned CF synthesis has emerged as a powerful approach for the design of recombinant proteoliposomes in one-step reaction. The incorporation of bioactive MPs or peptides into liposomes via CF synthesis can facilitate the development of proteoliposomal nanodelivery systems with improved circulation, increased targeting, and enhanced cellular delivery capacity. Moreover, by adapting lessons learned from natural delivery vehicles, novel bio-inspired proteoliposomes with enhanced delivery properties could be produced in CF systems. In this review, we first give an overview of CF synthesis with focus on enhancing protein expression in liposome-chaperoned CF systems. Furthermore, we intend to provide insight into harnessing CF-synthesized proteoliposomes for efficient therapeutic delivery. Copyright © 2018. Published by Elsevier Ltd.

The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

PubMed

de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

2015-11-01

In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects.

Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fink, J.K.; Correll, P.H.; Perry, L.K.

1990-03-01

Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, the authors developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py{sup +}/Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hemaptopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeatmore » (Mo-MLV LTR) and levels of Py{sup +}/Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py{sup +}/Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity has been normalized.« less

Optimization of the transductional efficiency of lentiviral vectors: effect of sera and polycations

PubMed Central

Denning, Warren; Das, Suvendu; Guo, Siqi; Xu, Jun; Kappes, John C.; Hel, Zdenek

2012-01-01

Lentiviral vectors are widely used as effective gene-delivery vehicles. Optimization of the conditions for efficient lentiviral transduction is of a high importance for a variety of research applications. Presence of positively-charged polycations reduces the electrostatic repulsion forces between a negatively-charged cell and an approaching enveloped lentiviral particle resulting in an increase in the transduction efficiency. Although a variety of polycations are commonly used to enhance the transduction with retroviruses, the relative effect of various types of polycations on the efficiency of transduction and on the potential bias in the determination of titer of lentiviral vectors is not fully understood. Here we present data suggesting that DEAE-dextran provides superior results in enhancing lentiviral transduction of most tested cell lines and primary cell cultures. Specific type and source of serum affects the efficiency of transduction of target cell populations. Non-specific binding of enhanced green fluorescent protein (EGFP)-containing membrane aggregates in the presence of DEAE-dextran does not significantly affect the determination of the titer of EGFP-expressing lentiviral vectors. In conclusion, various polycations and types of sera should be tested when optimizing lentiviral transduction of target cell populations. PMID:22407723

Optimization of the transductional efficiency of lentiviral vectors: effect of sera and polycations.

PubMed

Denning, Warren; Das, Suvendu; Guo, Siqi; Xu, Jun; Kappes, John C; Hel, Zdenek

2013-03-01

Lentiviral vectors are widely used as effective gene-delivery vehicles. Optimization of the conditions for efficient lentiviral transduction is of a high importance for a variety of research applications. Presence of positively charged polycations reduces the electrostatic repulsion forces between a negatively charged cell and an approaching enveloped lentiviral particle resulting in an increase in the transduction efficiency. Although a variety of polycations are commonly used to enhance the transduction with retroviruses, the relative effect of various types of polycations on the efficiency of transduction and on the potential bias in the determination of titer of lentiviral vectors is not fully understood. Here, we present data suggesting that DEAE-dextran provides superior results in enhancing lentiviral transduction of most tested cell lines and primary cell cultures. Specific type and source of serum affects the efficiency of transduction of target cell populations. Non-specific binding of enhanced green fluorescent protein (EGFP)-containing membrane aggregates in the presence of DEAE-dextran does not significantly affect the determination of the titer of EGFP-expressing lentiviral vectors. In conclusion, various polycations and types of sera should be tested when optimizing lentiviral transduction of target cell populations.

Enhancement and optimization of plasmid expression in femtosecond optical transfection.

PubMed

Praveen, Bavishna B; Stevenson, David J; Antkowiak, Maciej; Dholakia, Kishan; Gunn-Moore, Frank J

2011-04-01

Cell transfection using femtosecond lasers is gaining importance for its proven ability to achieve selective transfection in a sterile and relatively non-invasive manner. However, the net efficiency of this technique is limited due to a number of factors that ultimately makes it difficult to be used as a viable and widely used technique. We report here a method to achieve significant enhancement in the efficiency of femtosecond optical transfection. The transfection procedure is modified by incorporating a suitable synthetic peptide containing nuclear localization and DNA binding sequences, assisting DNA import into the nucleus. We achieved a 3-fold enhancement in the transfection efficiency for adherent Chinese Hamster Ovary (CHO-K1) cells with this modified protocol. Further, in the presence of this biochemical reagent, we were able to reduce the required plasmid concentration by ~70% without compromising the transfection efficiency. Also, we report for the first time the successful photo-transfection of recently trypsinised cells with significantly high transfection efficiency when transfected with modified plasmid. This paves the way for the development of high throughput microfluidic optical transfection devices. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cytomegalovirus vector expressing RAE-1γ induces enhanced anti-tumor capacity of murine CD8+ T cells.

PubMed

Tršan, Tihana; Vuković, Kristina; Filipović, Petra; Brizić, Ana Lesac; Lemmermann, Niels A W; Schober, Kilian; Busch, Dirk H; Britt, William J; Messerle, Martin; Krmpotić, Astrid; Jonjić, Stipan

2017-08-01

Designing CD8 + T-cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE-1γ as CD8 + T cell-based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE-1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD-1. CMV vector expressing RAE-1γ potentiated expansion of KLRG1 + CD8 + T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long-term maintenance of epitope-specific CD8 + T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8 + T-cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8 + T-cell sensitive tumors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins

NASA Astrophysics Data System (ADS)

Kamau Chapman, Sarah W.; Hassa, Paul O.; Koch-Schneidemann, Sabine; von Rechenberg, Brigitte; Hofmann-Amtenbrink, Margarethe; Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich; Hottiger, Michael O.

Primary cell lines are more difficult to transfect when compared to immortalized/transformed cell lines, and hence new techniques are required to enhance the transfection efficiency in these cells. We isolated and established primary cultures of synoviocytes, chondrocytes, osteoblasts, melanocytes, macrophages, lung fibroblasts, and embryonic fibroblasts. These cells differed in several properties, and hence were a good representative sample of cells that would be targeted for expression and delivery of therapeutic genes in vivo. The efficiency of gene delivery in all these cells was enhanced using polyethylenimine-coated polyMAG magnetic nanoparticles, and the rates (17-84.2%) surpassed those previously achieved using other methods, especially in cells that are difficult to transfect. The application of permanent and pulsating magnetic fields significantly enhanced the transfection efficiencies in synoviocytes, chondrocytes, osteoblasts, melanocytes and lung fibroblasts, within 5 min of exposure to these magnetic fields. This is an added advantage for future in vivo applications, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

PubMed Central

Gupta, Manoj K.; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F.; Windmueller, Rebecca; Wagers, Amy J.

2015-01-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. PMID:26253715

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

PubMed

Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo

2006-04-01

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

Region of Herpes Simplex Virus Type 1 Latency-Associated Transcript Sufficient for Wild-Type Spontaneous Reactivation Promotes Cell Survival in Tissue Culture

PubMed Central

Inman, Melissa; Perng, Guey-Chuen; Henderson, Gail; Ghiasi, Homayon; Nesburn, Anthony B.; Wechsler, Steven L.; Jones, Clinton

2001-01-01

The latency-associated transcript (LAT) is the only abundant herpes simplex virus type 1 (HSV-1) transcript expressed during latency. In the rabbit eye model, LAT null mutants do not reactivate efficiently from latency. We recently demonstrated that the LAT null mutant dLAT2903 induces increased levels of apoptosis in trigeminal ganglia of infected rabbits compared to LAT+ strains (G.-C. Perng, C. Jones, J. Ciacci-Zarella, M. Stone, G. Henderson, A. Yokht, S. M. Slanina, F. M. Hoffman, H. Ghiasi, A. B. Nesburn, and C. S. Wechsler, Science 287:1500–1503, 2000).The same study also demonstrated that a plasmid expressing LAT nucleotides 301 to 2659 enhanced cell survival of transfected cells after induction of apoptosis. Consequently, we hypothesized that LAT enhances spontaneous reactivation in part, because it promotes survival of infected neurons. Here we report on the ability of plasmids expressing different portions of the 5′ end of LAT to promote cell survival after induction of apoptosis. A plasmid expressing the first 1.5 kb of LAT (LAT nucleotides 1 to 1499) promoted cell survival in neuro-2A (mouse neuronal) and CV-1 (monkey fibroblast) cells. A plasmid expressing just the first 811 nucleotides of LAT promoted cell survival less efficiently. Plasmids expressing the first 661 nucleotides or less of LAT did not promote cell survival. We previously showed that a mutant expressing just the first 1.5 kb of LAT has wild-type spontaneous reactivation in rabbits, and a mutant expressing just the first 811 nucleotides of LAT has a reactivation frequency higher than that of dLAT2903 but lower than that of wild-type virus. In addition, mutants reported here for the first time, expressing just the first 661 or 76 nucleotides of LAT, had spontaneous reactivation indistinguishable from that of the LAT null mutant dLAT2903. In summary, these studies provide evidence that there is a functional relationship between the ability of LAT to promote cell survival and its ability to enhance spontaneous reactivation. PMID:11264353

Genome and epigenome engineering CRISPR toolkit for in vivo modulation of cis-regulatory interactions and gene expression in the chicken embryo.

PubMed

Williams, Ruth M; Senanayake, Upeka; Artibani, Mara; Taylor, Gunes; Wells, Daniel; Ahmed, Ahmed Ashour; Sauka-Spengler, Tatjana

2018-02-23

CRISPR/Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present an optimised, adaptable toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise novel efficient guide-RNA mini expression vectors utilising chick U6 promoters, provide a strategy for rapid somatic gene knockout and establish a protocol for evaluation of mutational penetrance by targeted next-generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo . © 2018. Published by The Company of Biologists Ltd.

Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent.

PubMed

Chernousova, S; Epple, M

2017-05-01

The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2-3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application.

Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent

PubMed Central

Chernousova, S; Epple, M

2017-01-01

The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2–3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application. PMID:28218744

High-Efficiency Promoter-Dependent Transduction by Adeno-Associated Virus Type 6 Vectors in Mouse Lung

PubMed Central

HALBERT, CHRISTINE L.; LAM, SIU-LING; MILLER, A. DUSTY

2014-01-01

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues has been shown to depend heavily on the AAV type from which the vector capsid proteins are derived. Among the AAV types studied, AAV6 efficiently transduces cells of the airway epithelium, making it a good candidate for the treatment of lung diseases such as cystic fibrosis. Here we have evaluated the effects of various promoter sequences on transduction rates and gene expression levels in the lung. Of the strong viral promoters examined, the Rous sarcoma virus (RSV) promoter performed significantly better than a human cytomegalovirus (CMV) promoter in the airway epithelium. However, a hybrid promoter consisting of a CMV enhancer, β-actin promoter and splice donor, and a β-globin splice acceptor (CAG promoter) exhibited even higher expression than either of the strong viral promoters alone, showing a 38-fold increase in protein expression over the RSV promoter. In addition, we show that vectors containing either the RSV or CAG promoter expressed well in the nasal and tracheal epithelium. Transduction rates in the 90% range were achieved in many airways with the CAG promoter, showing that with the proper AAV capsid proteins and promoter sequences, highly efficient transduction can be achieved. PMID:17430088

Temperature regulates methane production through the function centralization of microbial community in anaerobic digestion.

PubMed

Lin, Qiang; De Vrieze, Jo; He, Guihua; Li, Xiangzhen; Li, Jiabao

2016-09-01

Temperature is crucial for the performance of anaerobic digestion process. In this study of anaerobic digestion of swine manure, the relationship between the microbial gene expression and methane production at different temperatures (25-55°C) was revealed through metatranscriptomic analysis. Daily methane production and total biogas production increased with temperature up to 50°C, but decreased at 55°C. The functional gene expression showed great variation at different temperatures. The function centralization (opposite to alpha-diversity), assessed by the least proportions of functional pathways contributing for at least 50% of total reads positively correlated to methane production. Temperature regulated methane production probably through reducing the diversity of functional pathways, but enhancing central functional pathways, so that most of cellular activities and resource were invested in methanogenesis and related pathways, enhancing the efficiency of conversion of substrates to methane. This research demonstrated the importance of function centralization for efficient system functioning. Copyright © 2016 Elsevier Ltd. All rights reserved.

Overexpression of Tet3 in donor cells enhances goat somatic cell nuclear transfer efficiency.

PubMed

Han, Chengquan; Deng, Ruizhi; Mao, Tingchao; Luo, Yan; Wei, Biao; Meng, Peng; Zhao, Lu; Zhang, Qing; Quan, Fusheng; Liu, Jun; Zhang, Yong

2018-05-23

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency. © 2018 Federation of European Biochemical Societies.

Survival of the Fittest: How Bacterial Pathogens Utilize Bile To Enhance Infection

PubMed Central

Sistrunk, Jeticia R.; Nickerson, Kourtney P.; Chanin, Rachael B.; Rasko, David A.

2016-01-01

SUMMARY Bacterial pathogens have coevolved with humans in order to efficiently infect, replicate within, and be transmitted to new hosts to ensure survival and a continual infection cycle. For enteric pathogens, the ability to adapt to numerous host factors under the harsh conditions of the gastrointestinal tract is critical for establishing infection. One such host factor readily encountered by enteric bacteria is bile, an innately antimicrobial detergent-like compound essential for digestion and nutrient absorption. Not only have enteric pathogens evolved to resist the bactericidal conditions of bile, but these bacteria also utilize bile as a signal to enhance virulence regulation for efficient infection. This review provides a comprehensive and up-to-date analysis of bile-related research with enteric pathogens. From common responses to the unique expression of specific virulence factors, each pathogen has overcome significant challenges to establish infection in the gastrointestinal tract. Utilization of bile as a signal to modulate virulence factor expression has led to important insights for our understanding of virulence mechanisms for many pathogens. Further research on enteric pathogens exposed to this in vivo signal will benefit therapeutic and vaccine development and ultimately enhance our success at combating such elite pathogens. PMID:27464994

Vaccinia Virus-mediated Expression of Human Erythropoietin in Tumors Enhances Virotherapy and Alleviates Cancer-related Anemia in Mice

PubMed Central

Nguyen, Duong H; Chen, Nanhai G; Zhang, Qian; Le, Ha T; Aguilar, Richard J; Yu, Yong A; Cappello, Joseph; Szalay, Aladar A

2013-01-01

Recombinant human erythropoietin (rhEPO), a glycoprotein hormone regulating red blood cell (RBC) formation, is used for the treatment of cancer-related anemia. The effect of rhEPO on tumor growth, however, remains controversial. Here, we report the construction and characterization of the recombinant vaccinia virus (VACV) GLV-1h210, expressing hEPO. GLV-1h210 was shown to replicate in and kill A549 lung cancer cells in culture efficiently. In mice bearing A549 lung cancer xenografts, treatment with a single intravenous dose of GLV-1h210 resulted in tumor-specific production and secretion of functional hEPO, which exerted an effect on RBC progenitors and precursors in the mouse bone marrow, leading to a significant increase in the number of RBCs and in the level of hemoglobin. Furthermore, virally expressed hEPO, but not exogenously added rhEPO, enhanced virus-mediated green fluorescent protein (GFP) expression in tumors and subsequently accelerated tumor regression when compared with the treatment with the parental virus GLV-1h68 or GLV-1h209 that expressed a nonfunctional hEPO protein. Moreover, intratumorally expressed hEPO caused enlarged tumoral microvessels, likely facilitating virus spreading. Taken together, VACV-mediated intratumorally expressed hEPO not only enhanced oncolytic virotherapy but also simultaneously alleviated cancer-related anemia. PMID:23765443

Encouraging expressions affect the brain and alter visual attention.

PubMed

Martín-Loeches, Manuel; Sel, Alejandra; Casado, Pilar; Jiménez, Laura; Castellanos, Luis

2009-06-17

Very often, encouraging or discouraging expressions are used in competitive contexts, such as sports practice, aiming at provoking an emotional reaction on the listener and, consequently, an effect on subsequent cognition and/or performance. However, the actual efficiency of these expressions has not been tested scientifically. To fill this gap, we studied the effects of encouraging, discouraging, and neutral expressions on event-related brain electrical activity during a visual selective attention task in which targets were determined by location, shape, and color. Although the expressions preceded the attentional task, both encouraging and discouraging messages elicited a similar long-lasting brain emotional response present during the visuospatial task. In addition, encouraging expressions were able to alter the customary working pattern of the visual attention system for shape selection in the attended location, increasing the P1 and the SP modulations while simultaneously fading away the SN. This was interpreted as an enhancement of the attentional processes for shape in the attended location after an encouraging expression. It can be stated, therefore, that encouraging expressions, as those used in sport practice, as well as in many other contexts and situations, do seem to be efficient in exerting emotional reactions and measurable effects on cognition.

Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers.

PubMed Central

Kukowska-Latallo, J F; Bielinska, A U; Johnson, J; Spindler, R; Tomalia, D A; Baker, J R

1996-01-01

Starburst polyamidoamine dendrimers are a new class of synthetic polymers with unique structural and physical characteristics. These polymers were investigated for the ability to bind DNA and enhance DNA transfer and expression in a variety of mammalian cell lines. Twenty different types of polyamidoamine dendrimers were synthesized, and the polymer structure was confirmed using well-defined analytical techniques. The efficiency of plasmid DNA transfection using dendrimers was examined using two reporter gene systems: firefly luciferase and bacterial beta-galactosidase. The transfections were performed using various dendrimers, and levels of expression of the reporter protein were determined. Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using the DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers and in some situations required the presence of additional compounds, such as DEAE-dextran, that appeared to alter the nature of the complex. A few cell lines demonstrated enhanced transfection with the addition of chloroquine, indicating endosomal localization of the complexes. The capability of a dendrimer to transfect cells appeared to depend on the size, shape, and number of primary amino groups on the surface of the polymer. However, the specific dendrimer most efficient in achieving transfection varied between different types of cells. These studies demonstrate that Starburst dendrimers can transfect a wide variety of cell types in vitro and offer an efficient method for producing permanently transfected cell lines. Images Fig. 1 Fig. 2 Fig. 4 PMID:8643500

Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression.

PubMed

Moreno, J C; Cerda, A; Simpson, K; Lopez-Diaz, I; Carrera, E; Handford, M; Stange, C

2016-04-01

Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene β-cyclase (LCYB) that catalyzes the conversion of lycopene into β-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, β-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity.

PubMed

Dent, Matthew; Hurtado, Jonathan; Paul, Amber M; Sun, Haiyan; Lai, Huafang; Yang, Ming; Esqueda, Adrian; Bai, Fengwei; Steinkellner, Herta; Chen, Qiang

2016-12-01

The mAb E60 has the potential to be a desirable therapeutic molecule since it efficiently neutralizes all four serotypes of dengue virus (DENV). However, mammalian-cell-produced E60 exhibits antibody-dependent enhancement of infection (ADE) activity, rendering it inefficacious in vivo, and treated animals more susceptible to developing more severe diseases during secondary infection. In this study, we evaluated a plant-based expression system for the production of therapeutically suitable E60. The mAb was transiently expressed in Nicotiana benthamianaWT and a ∆XFT line, a glycosylation mutant lacking plant-specific N-glycan residues. The mAb was efficiently expressed and assembled in leaves and exhibited highly homogenous N-glycosylation profiles, i.e. GnGnXF3 or GnGn structures, depending on the expression host. Both E60 glycovariants demonstrated equivalent antigen-binding specificity and in vitro neutralization potency against DENV serotypes 2 and 4 compared with their mammalian-cell-produced counterpart. By contrast, plant-produced E60 exhibited reduced ADE activity in Fc gamma receptor expressing human cells. Our results suggest the ability of plant-produced antibodies to minimize ADE, which may lead to the development of safe and highly efficacious antibody-based therapeutics against DENV and other ADE-prone viral diseases. Our study provides so far unknown insight into the relationship between mAb N-glycosylation and ADE, which contributes to our understanding of how sugar moieties of antibodies modulate Fc-mediated functions and viral pathogenesis.

A recombinant adenovirus bicistronically expressing porcine interferon-α and interferon-γ enhances antiviral effects against foot-and-mouth disease virus.

PubMed

Kim, Su-Mi; Kim, Se-Kyung; Park, Jong-Hyeon; Lee, Kwang-Nyeong; Ko, Young-Joon; Lee, Hyang-Sim; Seo, Min-Goo; Shin, Yeun-Kyung; Kim, Byounghan

2014-04-01

Foot-and-mouth disease (FMD) is a virulent and economically costly disease in domestic livestock. Since the current vaccine available against FMD provides no protection until 7days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is by the application of anti-viral agents. The combination of recombinant adenovirus expressing type I interferon (IFN-α) and adenovirus expressing type II IFN (IFN-γ) has been reported to be an effective anti-viral treatment strategy against FMDV. Nevertheless, the recombinant adenovirus mixture may be inefficient because of the low anti-viral efficiency of IFN-γ compared to that of IFN-α. In this study, we generated a recombinant adenovirus co-expressing porcine IFN-α and IFN-γ in tandem using an FMDV 2A sequence to mediate effective cleavage of the two proteins (referred to as Ad-porcine IFN-αγ). We demonstrated that both recombinant porcine IFN-α and IFN-γ were expressed and interferon stimulated gene (ISG)s related with IFN-α and IFN-γ were induced in porcine kidney (IBRS-2) cells infected with Ad-porcine IFN-αγ. Additionally, the anti-viral effects of Ad-porcine IFN-αγ against FMDV were enhanced both in IBRS-2 cells and in CD-1 (ICR) suckling mice compared to that of adenovirus expressing only a single protein. We propose that Ad-porcine IFN-αγ could be a rapid, highly efficient, convenient anti-viral agent against FMDV. Copyright © 2014 Elsevier B.V. All rights reserved.

Heterologous expression of Talaromyces emersonii cellobiohydrolase Cel7A in Trichoderma reesei increases the efficiency of corncob residues saccharification.

PubMed

Sun, Ningning; Qian, Yuanchao; Wang, Weiwei; Zhong, Yaohua; Dai, Meixue

2018-07-01

Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii. Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65 °C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction. The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.

Fungal Gene Mutation Analysis Elucidating Photoselective Enhancement of UV-C Disinfection Efficiency Toward Spoilage Agents on Fruit Surface.

PubMed

Zhu, Pinkuan; Li, Qianwen; Azad, Sepideh M; Qi, Yu; Wang, Yiwen; Jiang, Yina; Xu, Ling

2018-01-01

Short-wave ultraviolet (UV-C) treatment represents a potent, clean and safe substitute to chemical sanitizers for fresh fruit preservation. However, the dosage requirement for microbial disinfection may have negative effects on fruit quality. In this study, UV-C was found to be more efficient in killing spores of Botrytis cinerea in dark and red light conditions when compared to white and blue light. Loss of the blue light receptor gene Bcwcl1 , a homolog of wc-1 in Neurospora crassa , led to hypersensitivity to UV-C in all light conditions tested. The expression of Bcuve1 and Bcphr1 , which encode UV-damage endonuclease and photolyase, respectively, were strongly induced by white and blue light in a Bcwcl1 -dependent manner. Gene mutation analyses of Bcuve1 and Bcphr1 indicated that they synergistically contribute to survival after UV-C treatment. In vivo assays showed that UV-C (1.0 kJ/m 2 ) abolished decay in drop-inoculated fruit only if the UV-C treatment was followed by a dark period or red light, while in contrast, typical decay appeared on UV-C irradiated fruits exposed to white or blue light. In summary, blue light enhances UV-C resistance in B. cinerea by inducing expression of the UV damage repair-related enzymes, while the efficiency of UV-C application for fruit surface disinfection can be enhanced in dark or red light conditions; these principles seem to be well conserved among postharvest fungal pathogens.

Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

PubMed Central

Gao, Xuefei; Tsang, Jason C.H.; Gaba, Fortis; Wu, Donghai; Lu, Liming; Liu, Pentao

2014-01-01

The transcription activator–like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas9) utlilize distinct molecular mechanisms in targeting site recognition. The two proteins can be modified to carry additional functional domains to regulate expression of genomic loci in mammalian cells. In this study, we have compared the two systems in activation and suppression of the Oct4 and Nanog loci by targeting their enhancers. Although both are able to efficiently activate the luciferase reporters, the CRISPR/dCas9 system is much less potent in activating the endogenous loci and in the application of reprogramming somatic cells to iPS cells. Nevertheless, repression by CRISPR/dCas9 is comparable to or even better than TALE repressors. We demonstrated that dCas9 protein binding results in significant physical interference to binding of native transcription factors at enhancer, less efficient active histone markers induction or recruitment of activating complexes in gene activation. This study thus highlighted the merits and drawbacks of transcription regulation by each system. A combined approach of TALEs and CRISPR/dCas9 should provide an optimized solution to regulate genomic loci and to study genetic elements such as enhancers in biological processes including somatic cell reprogramming and guided differentiation. PMID:25223790

Enhancing soybean photosynthetic CO2 assimilation using a cyanobacterial membrane protein, ictB

USDA-ARS?s Scientific Manuscript database

Soybean C3 photosynthesis can suffer a severe loss in efficiency due to photorespiration and the lack of a carbon concentrating mechanism (CCM) such as those present in other plant species or cyanobacteria. Transgenic soybean (Glycine max cv. Thorne) plants constitutively expressing cyanobacterial i...

Conducting Research on the International Space Station Using the EXPRESS Rack Facilities

NASA Technical Reports Server (NTRS)

Thompson, Sean W.; Lake, Robert E.

2014-01-01

EXPRESS Racks provide capability for payload access to ISS resources. The successful on-orbit operations and versatility of the EXPRESS Rack has facilitated the operations of many scientific areas, with the promise of continued payload support for years to come. EXPRESS Racks are currently deployed in the US Lab, Columbus and JEM. Process improvements and enhancements continue to improve the accommodations and make the integration and operations process more efficient. Payload Integration Managers serve as the primary interface between the ISS Program and EXPRESS Payload Developers. EXPRESS Project coordinates across multiple functional areas and organizations to ensure integrated EXPRESS Rack and subrack products and hardware are complete, accurate, on time, safe, and certified for flight. NASA is planning to expand the EXPRESS payload capacity by developing new Basic Express Racks expected to be on ISS in 2018.

Functional FRIGIDA allele enhances drought tolerance by regulating the P5CS1 pathway in Arabidopsis thaliana.

PubMed

Chen, Qian; Zheng, Yan; Luo, Landi; Yang, Yongping; Hu, Xiangyang; Kong, Xiangxiang

2018-01-01

Flowering at the right time is important for the reproductive success of plants and their response to environmental stress. In Arabidopsis, a major determinant of natural variation in flowering time is FRIGIDA (FRI). In the present study, we show that overexpression of the functional FRIGIDA gene in wild-type Col background (ColFRI) positively enhances the drought tolerance by activating P5CS1 expression and promoting proline accumulation during water stress. Furthermore, no significant changes in FRI gene and protein expression levels were observed with drought treatment, whereas P5CS1 protein expression significantly increased. In contrast, vernalization treatment efficiently reduced P5CS1 expression levels and resulted in a decrease in drought tolerance in the ColFRI plants. The flc mutants with a functional FRI background also relieved FRI-mediated activation of P5CS1 during drought tolerance. Taken together, our findings reveal the novel function of FRI in enhancing drought resistance through its downstream P5CS1 pathway during water-deficit stress, which is dependent on its target, the FLC gene. Copyright © 2017 Elsevier Inc. All rights reserved.

Chemotherapy Enhances Cross-Presentation of Nuclear Tumor Antigens

PubMed Central

Anyaegbu, Chidozie C.; Lake, Richard A.; Heel, Kathy; Robinson, Bruce W.; Fisher, Scott A.

2014-01-01

Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8+ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens. PMID:25243472

Strain Breeding Enhanced Heterologous Cellobiohydrolase Secretion by Saccharomyces cerevisiae in a Protein Specific Manner.

PubMed

Kroukamp, Heinrich; den Haan, Riaan; la Grange, Daniël C; Sibanda, Ntsako; Foulquié-Moreno, Maria R; Thevelein, Johan M; van Zyl, Willem H

2017-10-01

The yeast Saccharomyces cerevisiae has a long association with alcoholic fermentation industries and has received renewed interest as a biocatalyst for second-generation bioethanol production. Rational engineering strategies are used to create yeast strains for consolidated bioprocessing of lignocellulosic biomass. Although significant progress is made in this regard with the expression of different cellulolytic activities in yeast, cellobiohydrolase (CBH) titers remain well below ideal levels. Through classical breeding, S. cerevisiae strains with up to twofold increased CBH secretion titers is obtained in strains expressing a single gene copy. An increase of up to 3.5-fold in secreted cellobiohydrolase activity is subsequently shown for strains expressing the heterologous gene on a high copy episomal vector. To our knowledge, this is the first report of classical breeding being used to enhance heterologous protein secretion and also the most significant enhancement of CBH secretion in yeast yet reported. This enhanced secretion phenotype is specific for cellobiohydrolase I secretion, indicating that reporter protein properties might be a major determining factor for efficient protein secretion in yeast. By exploring the latent potential of different S. cerevisiae strains, the authors show that the allele pool of various strains is a valuable engineering resource to enhance secretion in yeast. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular evolution of Theta-class glutathione transferase for enhanced activity with the anticancer drug 1,3-bis-(2-chloroethyl)-1-nitrosourea and other alkylating agents.

PubMed

Larsson, Anna-Karin; Shokeer, Abeer; Mannervik, Bengt

2010-05-01

Glutathione transferase (GST) displaying enhanced activity with the cytostatic drug 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and structurally related alkylating agents was obtained by molecular evolution. Mutant libraries created by recursive recombination of cDNA coding for human and rodent Theta-class GSTs were heterologously expressed in Escherichia coli and screened with the surrogate substrate 4-nitrophenethyl bromide (NPB) for enhanced alkyltransferase activity. A mutant with a 70-fold increased catalytic efficiency with NPB, compared to human GST T1-1, was isolated. The efficiency in degrading BCNU had improved 170-fold, significantly more than with the model substrate NPB. The enhanced catalytic activity of the mutant GST was also 2-fold higher with BCNU than wild-type mouse GST T1-1, which is 80-fold more efficient than wild-type human GST T1-1. We propose that GSTs catalyzing inactivation of anticancer drugs may find clinical use in protecting sensitive normal tissues to toxic side-effects in treated patients, and as selectable markers in gene therapy. Copyright 2010 Elsevier Inc. All rights reserved.

Enhanced expression of the urokinase-type plasminogen activator gene and reduced colony formation in soft agar by ectopic expression of PU.1 in HT1080 human fibrosarcoma cells.

PubMed Central

Kondoh, N.; Yamada, T.; Kihara-Negishi, F.; Yamamoto, M.; Oikawa, T.

1998-01-01

To investigate the cell biological function of PU.1, a member of the Ets family of transcription factors, a vector capable of expressing the protein was transfected into HT1080 human fibrosarcoma cells. Exogenous expression of PU.1 in HT1080 cells reduced colony-forming efficiency but stimulated cell migration in soft agar, although it did not affect cell growth in adherent culture. Expression of the urokinase-type plasminogen activator (uPA) mRNA, which is known to be correlated with cell migration and invasion, was enhanced in PU.1 transfectants compared with mock transfectants. Run-on analysis demonstrated that uPA transcription was unaffected by PU.1, suggesting that this enhancement mainly occurs at a post-transcriptional level. On the other hand, treatment of HT1080 cells with the synthetic glucocorticoid dexamethasone (DEX; 10(-7) M) significantly reduced uPA gene expression at a transcriptional level. Furthermore, DEX inhibited cell migration in soft agar without affecting cell growth. These negative effects of DEX on uPA expression and cell migration were alleviated by the expression of PU.1 in HT1080 cells, whereas expression of the N-ras oncogene, which is responsible for maintenance of the transformed phenotypes in HT1080 cells, was unaffected by PU.1 expression or DEX treatment in the cells. Our results suggest that expression of PU.1 can stimulate uPA gene expression at the post-transcriptional level, which may subsequently lead to activation of cell motility and/or reduced cell-cell adhesion, but reduces anchorage-independent growth of HT1080 cells. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9743289

[Construction of transgenic tobacco expressing popW and analysis of its biological phenotype].

PubMed

Wang, Cui; Liu, Hongxia; Cao, Jing; Wang, Chao; Guo, Jianhua

2014-04-01

In a previous study, we cloned popW from Ralstonia solanacearum strain ZJ3721, coding PopW, a new harpin protein. The procaryotically expressed PopW can induce resistance to Tobacco mosaic virus (TMV), enhance growth and improve quality of tobacco, when sprayed onto tobacco leaves. Here, we constructed an expression vector pB- popW by cloning popW into the bionary vector pBI121 and transformed it into Agrobacterium tumefaciens strain EHA105 via freeze-thaw method. Tobacco (Nicotiana tobacum cv. Xanthi nc.) transformation was conducted by infection of tobacco leaf discs with recombinant A. tumefaciens. After screening on MS medium containing kanamycin, PCR and RT-PCR analysis, 21 T3 lines were identified as positive transgenic. Genomic intergration and expression of the transferred gene were determined by PCR and RT-PCR. And GUS staining analysis indicated that the protein expressed in transgenic tobacco was bioactive and exhibited different expression levels among lines. Disease bioassays showed that the transgenic tobacco had enhanced resistance to TMV with biocontrol efficiency up to 54.25%. Transgenic tobacco also exhibited enhanced plant growth, the root length of 15 d old seedlings was 1.7 times longer than that of wild type tobacco. 60 d after transplanting to pots, the height, fresh weight and dry weight of transgenic tobacco were 1.4, 1.7, 1.8 times larger than that of wild type tobacco, respectively.

Effects of nanoscale vacuum gap on photon-enhanced thermionic emission devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yuan; Liao, Tianjun; Zhang, Yanchao

2016-01-28

A new model of the photon-enhanced thermionic emission (PETE) device with a nanoscale vacuum gap is established by introducing the quantum tunneling effect and the image force correction. Analytic expressions for both the thermionic emission and tunneling currents are derived. The electron concentration and the temperature of the cathode are determined by the particle conservation and energy balance equations. The effects of the operating voltage on the maximum potential barrier, cathode temperature, electron concentration and equilibrium electron concentration of the conduction band, and efficiency of the PETE device are discussed in detail for different given values of the vacuum gapmore » length. The influence of the band gap of the cathode and flux concentration on the efficiency is further analyzed. The maximum efficiency of the PETE and the corresponding optimum values of the band gap and the operating voltage are determined. The results obtained here show that the efficiency of the PETE device can be significantly improved by employing a nanoscale vacuum gap.« less

Separation of natural product using columns packed with Fused-Core particles.

PubMed

Yang, Peilin; Litwinski, George R; Pursch, Matthias; McCabe, Terry; Kuppannan, Krishna

2009-06-01

Three HPLC columns packed with 3 microm, sub-2 microm, and 2.7 microm Fused-Core (superficially porous) particles were compared in separation performance using two natural product mixtures containing 15 structurally related components. The Ascentis Express C18 column packed with Fused-Core particles showed an 18% increase in column efficiency (theoretical plates), a 76% increase in plate number per meter, a 65% enhancement in separation speed and a 19% increase in back pressure compared to the Atlantis T3 C18 column packed with 3 microm particles. Column lot-to-lot variability for critical pairs in the natural product mixture was observed with both columns, with the Atlantis T3 column exhibiting a higher degree of variability. The Ascentis Express column was also compared with the Acquity BEH column packed with sub-2 microm particles. Although the peak efficiencies obtained by the Ascentis Express column were only about 74% of those obtained by the Acquity BEH column, the 50% lower back pressure and comparable separation speed allowed high-efficiency and high-speed separation to be performed using conventional HPLC instrumentation.

Use of lipolanthionine peptide, a toll-like receptor 2 inhibitor, enhances transdermal delivery efficiency.

PubMed

Chen, Bin; Liu, Da-Lie; Pan, Wen-Yan; Yang, Xiao-Hui; Shou, Jia-Bao; Wu, Ju-Hua; Mao, Qing-Long; Wang, Jia

2014-08-01

The transdermal delivery system (TDS) is able to obtain a systemic therapeutic effect by administration through the skin, which has low side effects and is able to maintain a sustained blood concentration. However, due to the barrier presented by the stratum corneum, numerous drugs have poor percutaneous permeability. Therefore, the improvement of skin permeability is key to TDS. The main method of promoting transdermal absorption is through the usage of penetration enhancers. Dimethyl sulfoxide (DMSO) is a commonly used penetration enhancer, which has anti‑inflammatory analgesic effects and is able to penetrate the skin. Retinoic acid (RA) and lipolanthionine peptide (LP) may also benefit the permeation efficiency of TDS. Therefore, the present study examined the function of DMSO, RA and LP as penetration enhancers in TDS. Firstly, the optimum concentration of DMSO was confirmed by detecting the expression of the LacZ gene in vitro. Secondly, different combinations of LP, RA and DMSO were applied to mouse skin to analyze the penetration enhancer combination with the greatest efficacy. All the animals were divided into five groups: The RA + LP + DMSO + pORF‑LacZ group, the RA + DMSO + pORF‑LacZ group, the LP + DMSO + pORF‑LacZ group, the DMSO + pORF-LacZ group and the control group. Skin was soaked in combinations of LP, RA and DMSO for seven days and then the pORF‑LacZ plasmids were daubed onto the skin once daily three days. On the 11th day, all the animals were sacrificed by cervical dislocation and the skin and blood samples were collected. The blood samples were used to detect the expression of the LacZ gene by quantitative polymerase chain reaction and the skin samples were used to detect the expression of claudin‑4 and zonula occluden‑1 (ZO‑1) proteins by immunohistochemistry and western blot analysis. The results demonstrated that the combination of LP, RA and DMSO exhibited the greatest transdermal delivery efficiency, which verified that RA and LP were able to increase the penetration effects. Following treatment with LP, the symptoms of dermal edema were relieved and the capillaries contracted, which suggested that LP was a safe and effective penetration enhancer able to reduce the side‑effects caused by DMSO. The present study provides a guideline for the synthesis of novel penetration enhancers.

Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

PubMed

Maggi, Maristella; Scotti, Claudia

2017-08-01

Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

Overexpression of SOS genes in ciprofloxacin resistant Escherichia coli mutants.

PubMed

Pourahmad Jaktaji, Razieh; Pasand, Shirin

2016-01-15

Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation. The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin. Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing. Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated. It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

Design of Fusion Proteins for Efficient and Soluble Production of Immunogenic Ebola Virus Glycoprotein in Escherichia coli.

PubMed

Ji, Yang; Lu, Yuan; Yan, Yishu; Liu, Xinxin; Su, Nan; Zhang, Chong; Bi, Shengli; Xing, Xin-Hui

2018-03-03

The Ebola hemorrhagic fever caused by Ebola virus is an extremely dangerous disease, and effective therapeutic agents are still lacking. Platforms for the efficient production of vaccines are crucial to ensure quick response against an Ebola virus outbreak. Ebola virus glycoprotein (EbolaGP) on the virion surface is responsible for membrane binding and virus entry, thus becoming the key target for vaccine development. However, heterologous expression of this protein still faces engineering challenges such as low production levels and insoluble aggregation. Here, the authors design and compare various fusion strategies, attaching great importance to the solubility-enhancing effect, and tag removal process. It is found that a C-terminal intein-based tag greatly enhances the solubility of EbolaGP and allows one-step chromatographic purification of the untagged EbolaGP through thiol-catalyzed self-cleavage. The purified untagged EbolaGP alone or with Freund's adjuvant are highly immunogenic, as confirmed in a mouse model. Consequently, the present study puts forward a new strategy for the efficient and soluble expression of untagged immunogenic EbolaGP. The intein-based protein fusion approach may be of importance for the large-scale production of Ebola virus subunit vaccine. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparation of a Nanoscaled Poly(vinyl alcohol)/Hydroxyapatite/DNA Complex Using High Hydrostatic Pressure Technology for In Vitro and In Vivo Gene Delivery.

PubMed

Kimura, Tsuyoshi; Nibe, Yoichi; Funamoto, Seiichi; Okada, Masahiro; Furuzono, Tsutomu; Ono, Tsutomu; Yoshizawa, Hidekazu; Fujisato, Toshiya; Nam, Kwangwoo; Kishida, Akio

2011-01-01

Our previous research showed that poly(vinyl alcohol) (PVA) nanoparticles incorporating DNA with hydrogen bonds obtained by high hydrostatic pressurization are able to deliver DNA without any significant cytotoxicity. To enhance transfection efficiency of PVA/DNA nanoparticles, we describe a novel method to prepare PVA/DNA nanoparticles encapsulating nanoscaled hydroxyapatites (HAps) prepared by high hydrostatic pressurization (980 MPa), which is designed to facilitate endosomal escape induced by dissolving HAps in an endosome. Scanning electron microscopic observation and dynamic light scattering measurement revealed that HAps were significantly encapsulated in PVA/HAp/DNA nanoparticles. The cytotoxicity, cellular uptake, and transgene expression of PVA/HAp/DNA nanoparticles were investigated using COS-7 cells. It was found that, in contrast to PVA/DNA nanoparticles, their internalization and transgene expression increased without cytotoxicity occurring. Furthermore, a similar level of transgene expression between plasmid DNA and PVA/HAp/DNA nanoparticles was achieved using in vivo hydrodynamic injection. Our results show a novel method of preparing PVA/DNA nanoparticles encapsulating HAp nano-crystals by using high hydrostatic pressure technology and the potential use of HAps as an enhancer of the transfection efficiency of PVA/DNA nanoparticles without significant cytotoxicity.

Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus.

PubMed

Kurena, Baiba; Vežāne, Aleksandra; Skrastiņa, Dace; Trofimova, Olga; Zajakina, Anna

2017-07-01

Semliki Forest virus (SFV) is a potential cancer gene therapy vector capable of providing high and transient expression of heterologous proteins in mammalian cells. However, SFV has shown suboptimal transduction levels in several cancer cell types as well as wide biodistribution of SFV has been observed after in vivo applications. Magnetic nanoparticles (MNPs) have been shown to increase cell transduction with several viral vectors in vitro under an external magnetic field and enhance magnetically guided viral vector delivery. Here, we examined a panel of MNPs for enhanced cancer cell transduction with SFV vector. Magneto-transduction using positively charged MNPs increased Semliki Forest virus transduction in TS/A mouse mammary carcinoma cells in vitro in the presence of fetal bovine serum. Positively charged MNPs efficiently captured SFV particles independently of capturing medium, and MNPs-SFV complexes were successfully separated from suspension by magnetic precipitation. These results reveal the potential application of MNPs for enhanced gene delivery by SFV vector as well as proposes magnetic precipitation for efficient concentration of SFV particles from different media. Copyright © 2017 Elsevier B.V. All rights reserved.

DNase I hypersensitivity and epsilon-globin transcriptional enhancement are separable in locus control region (LCR) HS1 mutant human beta-globin YAC transgenic mice.

PubMed

Shimotsuma, Motoshi; Okamura, Eiichi; Matsuzaki, Hitomi; Fukamizu, Akiyoshi; Tanimoto, Keiji

2010-05-07

Expression of the five beta-like globin genes (epsilon, Ggamma, Agamma, delta, beta) in the human beta-globin locus depends on enhancement by the locus control region, which consists of five DNase I hypersensitive sites (5'HS1 through 5'HS5). We report here a novel enhancer activity in 5'HS1 that appears to be potent in transfected K562 cells. Deletion analyses identified a core activating element that bound to GATA-1, and a two-nucleotide mutation that disrupted GATA-1 binding in vitro abrogated 5'HS1 enhancer activity in transfection experiments. To determine the in vivo role of this GATA site, we generated multiple lines of human beta-globin YAC transgenic mice bearing the same two-nucleotide mutation. In the mutant mice, epsilon-, but not gamma-globin, gene expression in primitive erythroid cells was severely attenuated, while adult beta-globin gene expression in definitive erythroid cells was unaffected. Interestingly, DNaseI hypersensitivity near the 5'HS1 mutant sequence was eliminated in definitive erythroid cells, whereas it was only mildly affected in primitive erythroid cells. We therefore conclude that, although the GATA site in 5'HS1 is critical for efficient epsilon-globin gene expression, hypersensitive site formation per se is independent of 5'HS1 function, if any, in definitive erythroid cells.

Alteration in cardiac uncoupling proteins and eNOS gene expression following high-intensity interval training in favor of increasing mechanical efficiency.

PubMed

Fallahi, Ali Asghar; Shekarfroush, Shahnaz; Rahimi, Mostafa; Jalali, Amirhossain; Khoshbaten, Ali

2016-03-01

High-intensity interval training (HIIT) increases energy expenditure and mechanical energy efficiency. Although both uncoupling proteins (UCPs) and endothelial nitric oxide synthase (eNOS) affect the mechanical efficiency and antioxidant capacity, their effects are inverse. The aim of this study was to determine whether the alterations of cardiac UCP2, UCP3, and eNOS mRNA expression following HIIT are in favor of increased mechanical efficiency or decreased oxidative stress. Wistar rats were divided into five groups: control group (n=12), HIIT for an acute bout (AT1), short term HIIT for 3 and 5 sessions (ST3 and ST5), long-term training for 8 weeks (LT) (6 in each group). The rats of the training groups were made to run on a treadmill for 60 min in three stages: 6 min running for warm-up, 7 intervals of 7 min running on treadmill with a slope of 5° to 20° (4 min with an intensity of 80-110% VO2max and 3 min at 50-60% VO2max), and 5-min running for cool-down. The control group did not participate in any exercise program. Rats were sacrificed and the hearts were extracted to analyze the levels of UCP2, UCP3 and eNOS mRNA by RT-PCR. UCP3 expression was increased significantly following an acute training bout. Repeated HIIT for 8 weeks resulted in a significant decrease in UCPs mRNA and a significant increase in eNOS expression in cardiac muscle. This study indicates that Long term HIIT through decreasing UCPs mRNA and increasing eNOS mRNA expression may enhance energy efficiency and physical performance.

Alteration in cardiac uncoupling proteins and eNOS gene expression following high-intensity interval training in favor of increasing mechanical efficiency

PubMed Central

Fallahi, Ali Asghar; Shekarfroush, Shahnaz; Rahimi, Mostafa; Jalali, Amirhossain; Khoshbaten, Ali

2016-01-01

Objective(s): High-intensity interval training (HIIT) increases energy expenditure and mechanical energy efficiency. Although both uncoupling proteins (UCPs) and endothelial nitric oxide synthase (eNOS) affect the mechanical efficiency and antioxidant capacity, their effects are inverse. The aim of this study was to determine whether the alterations of cardiac UCP2, UCP3, and eNOS mRNA expression following HIIT are in favor of increased mechanical efficiency or decreased oxidative stress. Materials and Methods: Wistar rats were divided into five groups: control group (n=12), HIIT for an acute bout (AT1), short term HIIT for 3 and 5 sessions (ST3 and ST5), long-term training for 8 weeks (LT) (6 in each group). The rats of the training groups were made to run on a treadmill for 60 min in three stages: 6 min running for warm-up, 7 intervals of 7 min running on treadmill with a slope of 5° to 20° (4 min with an intensity of 80-110% VO2max and 3 min at 50-60% VO2max), and 5-min running for cool-down. The control group did not participate in any exercise program. Rats were sacrificed and the hearts were extracted to analyze the levels of UCP2, UCP3 and eNOS mRNA by RT-PCR. Results: UCP3 expression was increased significantly following an acute training bout. Repeated HIIT for 8 weeks resulted in a significant decrease in UCPs mRNA and a significant increase in eNOS expression in cardiac muscle. Conclusion: This study indicates that Long term HIIT through decreasing UCPs mRNA and increasing eNOS mRNA expression may enhance energy efficiency and physical performance. PMID:27114795

OsACA6, a P-type IIB Ca²⁺ ATPase promotes salinity and drought stress tolerance in tobacco by ROS scavenging and enhancing the expression of stress-responsive genes.

PubMed

Huda, Kazi M K; Banu, M Sufara Akhter; Garg, Bharti; Tula, Suresh; Tuteja, Renu; Tuteja, Narendra

2013-12-01

Calcium (Ca²⁺) regulates several signalling pathways involved in growth, development and stress tolerance. Cellular Ca²⁺ homeostasis is achieved by the combined action of channels, pumps and antiporters, but direct evidence for a role of Ca²⁺ATPase pumps in stress tolerance is lacking. Here we report the characterization of a Ca²⁺ ATPase gene (OsACA6) from Oryza sativa, and elucidate its functions in stress tolerance. OsACA6 transcript levels are enhanced in response to salt, drought, abscisic acid and heat. In vivo localization identified plasma membranes as an integration site for the OsACA6-GFP fusion protein. Using transgenic tobacco lines, we demonstrate that over-expression of OsACA6 is triggered during salinity and drought stresses. The enhanced tolerance to these stresses was confirmed by changes in several physiological indices, including water loss rate, photosynthetic efficiency, cell membrane stability, germination, survival rate, malondialdehyde content, electrolyte leakage and increased proline accumulation. Furthermore, over-expressing lines also showed higher leaf chlorophyll and reduced accumulation of H₂O₂ and Na⁺ ions compared to the wild-type. Reduced accumulation of reactive oxygen species (ROS) was observed in transgenic lines. The increased proline accumulation and ROS scavenging enzyme activities in transgenic plants over-expressing OsACA6 efficiently modulate the ROS machinery and proline biosynthesis through an integrative mechanism. Transcriptional profiling of these plants revealed altered expression of genes encoding many transcription factors, stress- and disease-related proteins, as well as signalling components. These results suggest that Ca²⁺ ATPases have diverse roles as regulators of many stress signalling pathways, leading to plant growth, development and stress tolerance. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Enhancing the performance of Escherichia coli-inoculated microbial fuel cells by introduction of the phenazine-1-carboxylic acid pathway.

PubMed

Feng, Jiao; Qian, Ying; Wang, Zhen; Wang, Xin; Xu, Sheng; Chen, Kequan; Ouyang, Pingkai

2018-06-10

Microbial fuel cells (MFCs) are a renewable green energy source that uses microorganisms to catalytically convert chemical energy into electrical energy. The efficiency of extracellular electron transfer (EET) from the microbe cell to the anode electrode plays a key role in the MFC. However, the insulating properties of the cell membrane limit the efficiency of EET. Herein, EET efficiency was improved by introducing a phenazine synthesis pathway into Escherichia coli. Through the heterologous expression of phzA1B1C1D1E1F1G1, phenazine-1-carboxylic acid production increased, and the maximum power density increased from 16.7 mW/m 2 to 181.1 mW/m 2 . Furthermore, the charge transfer resistance of 6.7 Ω decreased to 4.2 Ω, which reflected the enhancement of the EET efficiency and the electricity power output. Our results imply that introducing a heterologous electron shuttle into E. coli could be an efficient approach to improving the EET efficiency and performance of an MFC. Copyright © 2018 Elsevier B.V. All rights reserved.

N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.

PubMed

Jornot, L; Morris, M A; Petersen, H; Moix, I; Rochat, T

2002-01-01

It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N-acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of beta-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. Pretreatment of cells with NAC prior to Ad infection enhanced beta-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in beta-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, beta-Gal activity was further enhanced, by 15-fold. Augmentation of beta-Gal activity was paralleled by an increase in beta-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases. Copyright 2001 John Wiley & Sons, Ltd.

Oral Delivery of Probiotics Expressing Dendritic Cell-Targeting Peptide Fused with Porcine Epidemic Diarrhea Virus COE Antigen: A Promising Vaccine Strategy against PEDV.

PubMed

Wang, Xiaona; Wang, Li; Huang, Xuewei; Ma, Sunting; Yu, Meiling; Shi, Wen; Qiao, Xinyuan; Tang, Lijie; Xu, Yigang; Li, Yijing

2017-10-25

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, is the causative agent of porcine epidemic diarrhea (PED) that damages intestinal epithelial cells and results in severe diarrhea and dehydration in neonatal suckling pigs with up to 100% mortality. The oral vaccine route is reported as a promising approach for inducing protective immunity against PEDV invasion. Furthermore, dendritic cells (DCs), professional antigen-presenting cells, link humoral and cellular immune responses for homeostasis of the intestinal immune environment. In this study, in order to explore an efficient oral vaccine against PEDV infection, a mucosal DC-targeting oral vaccine was developed using Lactobacillus casei to deliver the DC-targeting peptide (DCpep) fused with the PEDV core neutralizing epitope (COE) antigen. This probiotic vaccine could efficiently elicit secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in vivo. Significant differences ( p < 0.05) in the immune response levels were observed between probiotics expressing the COE-DCpep fusion protein and COE antigen alone, suggesting better immune efficiency of the probiotics vaccine expressing the DC-targeting peptide fused with PEDV COE antigen. This mucosal DC-targeting oral vaccine delivery effectively enhances vaccine antigen delivery efficiency, providing a useful strategy to induce efficient immune responses against PEDV infection.

Efficient gusA transient expression in Porphyra yezoensis protoplasts mediated by endogenous beta-tubulin flanking sequences

NASA Astrophysics Data System (ADS)

Gong, Qianhong; Yu, Wengong; Dai, Jixun; Liu, Hongquan; Xu, Rifu; Guan, Huashi; Pan, Kehou

2007-01-01

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'-and 3'-flanking regions ( Tub5' and Tub3') up-and down-stream of β-glucuronidase (GUS) gene ( gusA), respectively, into pA, a derivative of pCAT®3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3'. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

Process optimization for enhancing production of cis-4-hydroxy-L-proline by engineered Escherichia coli.

PubMed

Chen, Kequan; Pang, Yang; Zhang, Bowen; Feng, Jiao; Xu, Sheng; Wang, Xin; Ouyang, Pingkai

2017-11-22

Understanding the bioprocess limitations is critical for the efficient design of biocatalysts to facilitate process feasibility and improve process economics. In this study, a proline hydroxylation process with recombinant Escherichia coli expressing L-proline cis-4-hydroxylase (SmP4H) was investigated. The factors that influencing the metabolism of microbial hosts and process economics were focused on for the optimization of cis-4-hydroxy-L-proline (CHOP) production. In recombinant E. coli, SmP4H synthesis limitation was observed. After the optimization of expression system, CHOP production was improved in accordance with the enhanced SmP4H synthesis. Furthermore, the effects of the regulation of proline uptake and metabolism on whole-cell catalytic activity were investigated. The improved CHOP production by repressing putA gene responsible for L-proline degradation or overexpressing L-proline transporter putP on CHOP production suggested the important role of substrate uptake and metabolism on the whole-cell biocatalyst efficiency. Through genetically modifying these factors, the biocatalyst activity was significantly improved, and CHOP production was increased by twofold. Meanwhile, to further improve process economics, a two-strain coupling whole-cell system was established to supply co-substrate (α-ketoglutarate, α-KG) with a cheaper chemical L-glutamate as a starting material, and 13.5 g/L of CHOP was successfully produced. In this study, SmP4H expression, and L-proline uptake and degradation, were uncovered as the hurdles for microbial production of CHOP. Accordingly, the whole-cell biocatalysts were metabolically engineered for enhancing CHOP production. Meanwhile, a two-strain biotransformation system for CHOP biosynthesis was developed aiming at supplying α-KG more economically. Our work provided valuable insights into the design of recombinant microorganism to improve the biotransformation efficiency that catalyzed by Fe(II)/α-KG-dependent dioxygenase.

Generation of enhanced definitive endoderm from human embryonic stem cells under an albumin/insulin-free and chemically defined condition.

PubMed

Qu, Su; Yan, Liang; Fang, Bo; Ye, Shoudong; Li, Ping; Ge, Shengyang; Wu, Jian; Qu, Di; Song, Houyan

2017-04-15

To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system. Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test. Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition. This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation. Copyright © 2017 Elsevier Inc. All rights reserved.

Intracellular spectral recompositioning of light enhances algal photosynthetic efficiency

PubMed Central

Fu, Weiqi; Chaiboonchoe, Amphun; Khraiwesh, Basel; Sultana, Mehar; Jaiswal, Ashish; Jijakli, Kenan; Nelson, David R.; Al-Hrout, Ala’a; Baig, Badriya; Amin, Amr; Salehi-Ashtiani, Kourosh

2017-01-01

Diatoms, considered as one of the most diverse and largest groups of algae, can provide the means to reach a sustainable production of petrochemical substitutes and bioactive compounds. However, a prerequisite to achieving this goal is to increase the solar-to-biomass conversion efficiency of photosynthesis, which generally remains less than 5% for most photosynthetic organisms. We have developed and implemented a rapid and effective approach, herein referred to as intracellular spectral recompositioning (ISR) of light, which, through absorption of excess blue light and its intracellular emission in the green spectral band, can improve light utilization. We demonstrate that ISR can be used chemogenically, by using lipophilic fluorophores, or biogenically, through the expression of an enhanced green fluorescent protein (eGFP) in the model diatom Phaeodactylum tricornutum. Engineered P. tricornutum cells expressing eGFP achieved 28% higher efficiency in photosynthesis than the parental strain, along with an increased effective quantum yield and reduced nonphotochemical quenching (NPQ) induction levels under high-light conditions. Further, pond simulator experiments demonstrated that eGFP transformants could outperform their wild-type parental strain by 50% in biomass production rate under simulated outdoor sunlight conditions. Transcriptome analysis identified up-regulation of major photosynthesis genes in the engineered strain in comparison with the wild type, along with down-regulation of NPQ genes involved in light stress response. Our findings provide a proof of concept for a strategy of developing more efficient photosynthetic cell factories to produce algae-based biofuels and bioactive products. PMID:28879232

Enhanced pathogenicity of diabetogenic T cells escaping a non-MHC gene-controlled near death experience.

PubMed

Choisy-Rossi, Caroline-Morgane; Holl, Thomas M; Pierce, Melissa A; Chapman, Harold D; Serreze, David V

2004-09-15

For unknown reasons, the common MHC class I variants encoded by the H2g7 haplotype (Kd, Db) aberrantly elicit autoreactive CD8 T cell responses essential to type 1 diabetes development when expressed in NOD mice, but not other strains. In this study, we show that interactive non-MHC genes allow a NOD-derived diabetogenic CD8 T cell clonotype (AI4) to be negatively selected at far greater efficiency in C57BL/6 mice congenically expressing H2g7 (B6.H2g7). However, the few AI4 T cells escaping negative selection in B6.H2g7 mice are exported from the thymus more efficiently, and are more functionally aggressive than those of NOD origin. This provides mechanistic insight to previous findings that resistant mouse strains carry some genes conferring greater diabetes susceptibility than the corresponding NOD allele. In the B6.H2g7 stock, non-MHC gene-controlled elevations in TCR expression are associated with both enhanced negative selection of diabetogenic CD8 T cells and increased aggressiveness of those escaping this process. An implication of this finding is that the same phenotype, in this case relatively high TCR expression levels, could have double-edged sword effects, contributing to type 1 diabetes resistance at one level of T cell development, but at another actually promoting pathogenesis. Copyright 2004 The American Association of Immunologists, Inc.

Selection for Improved Energy Use Efficiency and Drought Tolerance in Canola Results in Distinct Transcriptome and Epigenome Changes.

PubMed

Verkest, Aurine; Byzova, Marina; Martens, Cindy; Willems, Patrick; Verwulgen, Tom; Slabbinck, Bram; Rombaut, Debbie; Van de Velde, Jan; Vandepoele, Klaas; Standaert, Evi; Peeters, Marrit; Van Lijsebettens, Mieke; Van Breusegem, Frank; De Block, Marc

2015-08-01

To increase both the yield potential and stability of crops, integrated breeding strategies are used that have mostly a direct genetic basis, but the utility of epigenetics to improve complex traits is unclear. A better understanding of the status of the epigenome and its contribution to agronomic performance would help in developing approaches to incorporate the epigenetic component of complex traits into breeding programs. Starting from isogenic canola (Brassica napus) lines, epilines were generated by selecting, repeatedly for three generations, for increased energy use efficiency and drought tolerance. These epilines had an enhanced energy use efficiency, drought tolerance, and nitrogen use efficiency. Transcriptome analysis of the epilines and a line selected for its energy use efficiency solely revealed common differentially expressed genes related to the onset of stress tolerance-regulating signaling events. Genes related to responses to salt, osmotic, abscisic acid, and drought treatments were specifically differentially expressed in the drought-tolerant epilines. The status of the epigenome, scored as differential trimethylation of lysine-4 of histone 3, further supported the phenotype by targeting drought-responsive genes and facilitating the transcription of the differentially expressed genes. From these results, we conclude that the canola epigenome can be shaped by selection to increase energy use efficiency and stress tolerance. Hence, these findings warrant the further development of strategies to incorporate epigenetics into breeding. © 2015 American Society of Plant Biologists. All Rights Reserved.

Direct comparison of administration routes for AAV8-mediated ocular gene therapy.

PubMed

Igarashi, Tsutomu; Miyake, Koichi; Asakawa, Nagisa; Miyake, Noriko; Shimada, Takashi; Takahashi, Hiroshi

2013-05-01

We recently demonstrated that direct subretinal (SR) injection of adeno-associated virus (AAV) type 8 (AAV8) into photoreceptor cells and retinal pigment epithelium (RPE) is a highly efficient model of gene delivery. The current study compared transduction efficiency and expression patterns associated with various routes of vector administration. The efficacy of intravitreal (VT), SR and subconjunctival (SC) injections for delivery of AAV8-derived vectors, i.e. those expressing luciferase (Luc) and enhanced green fluorescent protein (GFP) - AAV8/Luc and AAV8/GFP, respectively - were compared in an animal (mouse) model (n = 8 mice/group). Transduction efficiency and expression patterns were examined at post-injection weeks 1 and 2, and months 1, 3, 6 and 12 via in vivo imaging. One year after AAV injection, AAV8/Luc-treated mice exhibited stable and sustained high expression of vector in the VT and SR groups, but not in the SC group (VT:SR:SC = 3,218:2,923:115; 1 × 10(5 )photons/s). Histological analysis showed that GFP expression was observed in the inner retina of VT group mice, and in photoreceptor cells and RPE of SR group mice, whereas no GFP expression was noted in the SC group. Electroretinography (ERG) revealed adverse effects following SR delivery. Results suggest that both SR and VT injections of AAV8 vectors are useful routes for administering ocular gene therapy, and stress the importance of selecting an appropriate administration route, i.e. one that targets specific cells, for treating ocular disorders.

Plasmid DNA-encapsulating liposomes: effect of a spacer between the cationic head group and hydrophobic moieties of the lipids on gene expression efficiency.

PubMed

Obata, Yosuke; Saito, Shunsuke; Takeda, Naoya; Takeoka, Shinji

2009-05-01

We have synthesized a series of cationic amino acid-based lipids having a spacer between the cationic head group and hydrophobic moieties and examined the influence of the spacer on a liposome gene delivery system. As a comparable spacer, a hydrophobic spacer with a hydrocarbon chain composed of 0, 3, 5, 7, or 11 carbons, and a hydrophilic spacer with an oxyethylene chain (10 carbon and 3 oxygen molecules) were investigated. Plasmid DNA (pDNA)-encapsulating liposomes were prepared by mixing an ethanol solution of the lipids with an aqueous solution of pDNA. The zeta potentials and cellular uptake efficiency of the cationic liposomes containing each synthetic lipid were almost equivalent. However, the cationic lipids with the hydrophobic spacer were subject to fuse with biomembrane-mimicking liposomes. 1,5-Dihexadecyl-N-lysyl-N-heptyl-l-glutamate, having a seven carbon atom spacer, exhibited the highest fusogenic potential among the synthetic lipids. Increased fusion potential correlated with enhanced gene expression efficiency. By contrast, an oxyethylene chain spacer showed low gene expression efficiency. We conclude that a hydrophobic spacer between the cationic head group and hydrophobic moieties is a key component for improving pDNA delivery.

Progress and challenges in bioinformatics approaches for enhancer identification

PubMed Central

Kleftogiannis, Dimitrios; Kalnis, Panos

2016-01-01

Enhancers are cis-acting DNA elements that play critical roles in distal regulation of gene expression. Identifying enhancers is an important step for understanding distinct gene expression programs that may reflect normal and pathogenic cellular conditions. Experimental identification of enhancers is constrained by the set of conditions used in the experiment. This requires multiple experiments to identify enhancers, as they can be active under specific cellular conditions but not in different cell types/tissues or cellular states. This has opened prospects for computational prediction methods that can be used for high-throughput identification of putative enhancers to complement experimental approaches. Potential functions and properties of predicted enhancers have been catalogued and summarized in several enhancer-oriented databases. Because the current methods for the computational prediction of enhancers produce significantly different enhancer predictions, it will be beneficial for the research community to have an overview of the strategies and solutions developed in this field. In this review, we focus on the identification and analysis of enhancers by bioinformatics approaches. First, we describe a general framework for computational identification of enhancers, present relevant data types and discuss possible computational solutions. Next, we cover over 30 existing computational enhancer identification methods that were developed since 2000. Our review highlights advantages, limitations and potentials, while suggesting pragmatic guidelines for development of more efficient computational enhancer prediction methods. Finally, we discuss challenges and open problems of this topic, which require further consideration. PMID:26634919

A Chimeric HS4-SAR Insulator (IS2) That Prevents Silencing and Enhances Expression of Lentiviral Vectors in Pluripotent Stem Cells

PubMed Central

Gutierrez-Guerrero, Alejandra; Cobo, Marién; Muñoz, Pilar

2014-01-01

Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3′ LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells. PMID:24400083

Development of a CRISPR-Cas9 System for Efficient Genome Editing of Candida lusitaniae.

PubMed

Norton, Emily L; Sherwood, Racquel K; Bennett, Richard J

2017-01-01

Candida lusitaniae is a member of the Candida clade that includes a diverse group of fungal species relevant to both human health and biotechnology. This species exhibits a full sexual cycle to undergo interconversion between haploid and diploid forms. C. lusitaniae is also an emerging opportunistic pathogen that can cause serious bloodstream infections in the clinic and yet has often proven to be refractory to facile genetic manipulations. In this work, we develop a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (Cas9) system to enable genome editing of C. lusitaniae . We demonstrate that expression of CRISPR-Cas9 components under species-specific promoters is necessary for efficient gene targeting and can be successfully applied to multiple genes in both haploid and diploid isolates. Gene deletion efficiencies with CRISPR-Cas9 were further enhanced in C. lusitaniae strains lacking the established nonhomologous end joining (NHEJ) factors Ku70 and DNA ligase 4. These results indicate that NHEJ plays an important role in directing the repair of DNA double-strand breaks (DSBs) in C. lusitaniae and that removal of this pathway increases integration of gene deletion templates by homologous recombination. The described approaches significantly enhance the ability to perform genetic studies in, and promote understanding of, this emerging human pathogen and model sexual species. IMPORTANCE The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. Candida lusitaniae is an important member of the Candida clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in C. lusitaniae and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of C. lusitaniae biology.

Enhanced retroviral gene delivery in ultrasonic standing wave fields.

PubMed

Lee, Y-H; Peng, C-A

2005-04-01

Enhancement of retroviral transduction efficiency has been achieved by several physical and chemical approaches. However, the application of those methods is hampered by not easily scalable configurations. In this study, instead of looking into the effect of sonoporation, the potential of ultrasonic standing wave fields (USWF) to facilitate retroviral transduction rate was explored. We reasoned that, driven by the primary acoustic radiation force, suspended cells moved to the pressure nodal planes first and formed cell bands. Nanometer-sized retroviruses, circulated between nodal planes by acoustic microstreaming, then used the preformed cell bands as the nucleating sites to attach on. As a result, the encounter opportunity between retroviruses and cells was increased and further facilitated the gene delivery efficiency. Our results showed that mega-Hertz USWF brought K562 erythroleukemia cells (10(6) cells/ml) and vesicular stomatitis virus G-protein (VSV-G) pseudotyped retroviruses (titer of 5 x 10(6) CFU/ml) into close contact at the pressure nodal planes, yielding a four-fold increment of enhanced green fluorescent protein transgene expression after 5-min USWF exposure in the presence of Polybrene. Furthermore, with a fixed titer of retrovirus, the transduction rate was augmented with the increase of cell concentration. In summary, USWF offer a feasible means to enhance retroviral transduction efficiency in large-scale settings.

Enhancement of bioelectricity generation via heterologous expression of IrrE in Pseudomonas aeruginosa-inoculated MFCs.

PubMed

Luo, Jianmei; Wang, Tingting; Li, Xiao; Yang, Yanan; Zhou, Minghua; Li, Ming; Yan, Zhongli

2018-05-30

Low electricity power output (EPT) is still the main bottleneck limited the industrial application of microbial fuel cells (MFCs). Herein, EPT enhancement by introducing an exogenous global regulator IrrE derived from Deinococcus radiodurans into electrochemically active bacteria (EAB) was explored using Pseudomonas aeruginosa PAO1 as a model strain, achieving a power density 71% higher than that of the control strain. Moreover, IrrE-expressing strain exhibited a remarkable increase in the total amount of electron shuttles (majorly phenazines compounds) and a little decrease in internal resistance, which should underlie the enhancement in extracellular electron transfer (EET) efficiency and EPT. Strikingly, IrrE significantly affected substrate utilization profiling, improved cell growth characterization and cell tolerance to various stresses. Further quantitative RT-PCR analysis revealed that IrrE led to many differentially expressed genes, which were responsible for phenazines core biosynthesis, biofilm formation, QS systems, transcriptional regulation, glucose metabolism and general stress response. The results substantiated that targeting cellular regulatory network by the introduction of exogenous global regulators could be a facile and promising approach for the enhancement of bioelectricity generation and cell multiple phenotypes, and thus would be of great potential application in the practical MFCs. Copyright © 2018 Elsevier B.V. All rights reserved.

Eight Nucleotide Substitutions Inhibit Splicing to HPV-16 3′-Splice Site SA3358 and Reduce the Efficiency by which HPV-16 Increases the Life Span of Primary Human Keratinocytes

PubMed Central

Li, Xiaoze; Johansson, Cecilia; Cardoso Palacios, Carlos; Mossberg, Anki; Dhanjal, Soniya; Bergvall, Monika; Schwartz, Stefan

2013-01-01

The most commonly used 3′-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16. PMID:24039800

Death receptor 3 signaling enhances proliferation of human regulatory T cells.

PubMed

Bittner, Sebastian; Knoll, Gertrud; Ehrenschwender, Martin

2017-04-01

Exploiting regulatory T cells (Tregs) to control aberrant immune reactions is a promising therapeutic approach, but is hampered by their relative paucity. In mice, activation of death receptor 3 (DR3), a member of the TNF-receptor superfamily (TNFRSF), increases Treg frequency and efficiently controls exuberant immune activation. For human Tregs, neither DR3 expression nor potential functions have been described. Here, we show that human Tregs express DR3 and demonstrate DR3-mediated activation of p38, ERK, and NFκB. DR3 stimulation enhances Treg expansion ex vivo while retaining their suppressive capacity. In summary, our results establish a functional role for DR3 signaling in human Tregs and could potentially help to tailor Treg-based therapies. © 2017 Federation of European Biochemical Societies.

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

PubMed

Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State.

PubMed

Gao, Wen; Yin, Jun; Bao, Lichen; Wang, Qun; Hou, Shan; Yue, Yali; Yao, Wenbing; Gao, Xiangdong

2018-05-18

Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.

A new maltose-inducible high-performance heterologous expression system in Bacillus subtilis.

PubMed

Yue, Jie; Fu, Gang; Zhang, Dawei; Wen, Jianping

2017-08-01

To improve heterologous proteins production, we constructed a maltose-inducible expression system in Bacillus subtilis. An expression system based on the promoter for maltose utilization constructed in B. subtilis. Successively, to improve the performance of the P malA -derived system, mutagenesis was employed by gradually shortening the length of P malA promoter and altering the spacing between the predicted MalR binding site and the -35 region. Furthermore, deletion of the maltose utilization genes (malL and yvdK) improved the P malA promoter activity. Finally, using this efficient maltose-inducible expression system, we enhanced the production of luciferase and D-aminoacylase, compared with the P hpaII system. A maltose-inducible expression system was constructed and evaluated. It could be used for high level expression of heterologous proteins production.

75 FR 13416 - Federal Acquisition Regulation; FAR Case 2008-006, Enhanced Competition for Task- and Delivery...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-19

.... ``Unusual and Compelling Urgency.'' In the event of a FAR subpart 6.3 exception for a single contract award over $100 million including an unusual and compelling urgency exception, the commenter expresses... work. In those situations, it does not make sense in terms of costs and efficiency to require the...

A review of therapeutic prospects of non-viral gene therapy in the retinal pigment epithelium

PubMed Central

Koirala, Adarsha; Conley, Shannon M.; Naash, Muna I.

2013-01-01

Ocular gene therapy has been extensively explored in recent years as a therapeutic avenue to target diseases of the cornea, retina and retinal pigment epithelium (RPE). Adeno-associated virus (AAV)-mediated gene therapy has shown promise in several RPE clinical trials but AAVs have limited payload capacity and potential immunogenicity. Traditionally however, non-viral alternatives have been plagued by low transfection efficiency, short-term expression and low expression levels. Recently, these drawbacks have begun to be overcome by the use of specialty carriers such as polylysine, liposomes, or polyethyleneimines, and by inclusion of suitable DNA elements to enhance gene expression and longevity. Recent advancements in the field have yielded non-viral vectors that have favorable safety profiles, lack immunogenicity, exhibit long-term elevated gene expression, and show efficient transfection in the retina and RPE, making them poised to transition to clinical applications. Here we discuss the advancements in nanotechnology and vector engineering that have improved the prospects for clinical application of non-viral gene therapy in the RPE. PMID:23796578

Engineering crop nutrient efficiency for sustainable agriculture.

PubMed

Chen, Liyu; Liao, Hong

2017-10-01

Increasing crop yields can provide food, animal feed, bioenergy feedstocks and biomaterials to meet increasing global demand; however, the methods used to increase yield can negatively affect sustainability. For example, application of excess fertilizer can generate and maintain high yields but also increases input costs and contributes to environmental damage through eutrophication, soil acidification and air pollution. Improving crop nutrient efficiency can improve agricultural sustainability by increasing yield while decreasing input costs and harmful environmental effects. Here, we review the mechanisms of nutrient efficiency (primarily for nitrogen, phosphorus, potassium and iron) and breeding strategies for improving this trait, along with the role of regulation of gene expression in enhancing crop nutrient efficiency to increase yields. We focus on the importance of root system architecture to improve nutrient acquisition efficiency, as well as the contributions of mineral translocation, remobilization and metabolic efficiency to nutrient utilization efficiency. © 2017 Institute of Botany, Chinese Academy of Sciences.

In Vitro Comparison of Adipokine Export Signals.

PubMed

Sharafi, Parisa; Kocaefe, Y Çetin

2016-01-01

Mammalian cells are widely used for recombinant protein production in research and biotechnology. Utilization of export signals significantly facilitates production and purification processes. 35 years after the discovery of the mammalian export machinery, there still are obscurities regarding the efficiency of the export signals. The aim of this study was the comparative evaluation of the efficiency of selected export signals using adipocytes as a cell model. Adipocytes have a large capacity for protein secretion including several enzymes, adipokines, and other signaling molecules, providing a valid system for a quantitative evaluation. Constructs that expressed N-terminal fusion export signals were generated to express Enhanced Green Fluorescence Protein (EGFP) as a reporter for quantitative and qualitative evaluation. Furthermore, fluorescent microscopy was used to trace the intracellular traffic of the reporter. The export efficiency of six selected proteins secreted from adipocytes was evaluated. Quantitative comparison of intracellular and exported fractions of the recombinant constructs demonstrated a similar efficiency among the studied sequences with minor variations. The export signal of Retinol Binding Protein (RBP4) exhibited the highest efficiency. This study presents the first quantitative data showing variations among export signals, in adipocytes which will help optimization of recombinant protein distribution.

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators.

PubMed

Cui, Yulin; Zhao, Jialin; Hou, Shichang; Qin, Song

2016-05-01

On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5'RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.

Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules

PubMed Central

Tajima, Yosuke; Yoshida, Koki; Oikawa, Mami; Azuma, Rika; Allen, George E.; Tsujikawa, Tomomi; Tsukaguchi, Tomomasa; Bradshaw, Charles R.; Jullien, Jerome; Yamagata, Kazuo; Matsumoto, Kazuya; Anzai, Masayuki; Imai, Hiroshi; Gurdon, John B.; Yamada, Masayasu

2017-01-01

ABSTRACT Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency. PMID:28412714

LY294002 enhances expression of proteins encoded by recombinant replication-defective adenoviruses via mTOR- and non-mTOR-dependent mechanisms.

PubMed

Shepelev, Mikhail V; Korobko, Elena V; Vinogradova, Tatiana V; Kopantsev, Eugene P; Korobko, Igor V

2013-03-04

Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replication-defective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirus-delivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery.

Autologous implantation of BMP2-expressing dermal fibroblasts to improve bone mineral density and architecture in rabbit long bones.

PubMed

Ishihara, Akikazu; Weisbrode, Steve E; Bertone, Alicia L

2015-10-01

Cell-mediated gene therapy may treat bone fragility disorders. Dermal fibroblasts (DFb) may be an alternative cell source to stem cells for orthopedic gene therapy because of their rapid cell yield and excellent plasticity with bone morphogenetic protein-2 (BMP2) gene transduction. Autologous DFb or BMP2-expressing autologous DFb were administered in twelve rabbits by two delivery routes; a transcortical intra-medullar infusion into tibiae and delayed intra-osseous injection into femoral drill defects. Both delivery methods of DFb-BMP2 resulted in a successful cell engraftment, increased bone volume, bone mineral density, improved trabecular bone microarchitecture, greater bone defect filling, external callus formation, and trabecular surface area, compared to non-transduced DFb or no cells. Cell engraftment within trabecular bone and bone marrow tissue was most efficiently achieved by intra-osseous injection of DFb-BMP2. Our results suggested that BMP2-expressing autologous DFb have enhanced efficiency of engraftment in target bones resulting in a measurable biologic response by the bone of improved bone mineral density and bone microarchitecture. These results support that autologous implantation of DFb-BMP2 warrants further study on animal models of bone fragility disorders, such as osteogenesis imperfecta and osteoporosis to potentially enhance bone quality, particularly along with other gene modification of these diseases. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos

PubMed Central

Ding, Biao; Zuo, Xiaoyuan; Li, Hui; Ding, Jianping; Li, Yunsheng; Huang, Weiping; Zhang, Yunhai

2017-01-01

The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells’ chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, co-treatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression. PMID:28114389

TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos.

PubMed

Cao, Zubing; Hong, Renyun; Ding, Biao; Zuo, Xiaoyuan; Li, Hui; Ding, Jianping; Li, Yunsheng; Huang, Weiping; Zhang, Yunhai

2017-01-01

The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells' chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, co-treatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression.

Introducing an algal carbon-concentrating mechanism into higher plants: location and incorporation of key components.

PubMed

Atkinson, Nicky; Feike, Doreen; Mackinder, Luke C M; Meyer, Moritz T; Griffiths, Howard; Jonikas, Martin C; Smith, Alison M; McCormick, Alistair J

2016-05-01

Many eukaryotic green algae possess biophysical carbon-concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO2 concentrations. They are thus an attractive option for improving productivity in higher plants. In this study, the intracellular locations of ten CCM components in the unicellular green alga Chlamydomonas reinhardtii were confirmed. When expressed in tobacco, all of these components except chloroplastic carbonic anhydrases CAH3 and CAH6 had the same intracellular locations as in Chlamydomonas. CAH6 could be directed to the chloroplast by fusion to an Arabidopsis chloroplast transit peptide. Similarly, the putative inorganic carbon (Ci) transporter LCI1 was directed to the chloroplast from its native location on the plasma membrane. CCP1 and CCP2 proteins, putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas and tobacco, suggesting that the algal CCM model requires expansion to include a role for mitochondria. For the Ci transporters LCIA and HLA3, membrane location and Ci transport capacity were confirmed by heterologous expression and H(14) CO3 (-) uptake assays in Xenopus oocytes. Both were expressed in Arabidopsis resulting in growth comparable with that of wild-type plants. We conclude that CCM components from Chlamydomonas can be expressed both transiently (in tobacco) and stably (in Arabidopsis) and retargeted to appropriate locations in higher plant cells. As expression of individual Ci transporters did not enhance Arabidopsis growth, stacking of further CCM components will probably be required to achieve a significant increase in photosynthetic efficiency in this species. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium.

PubMed

Peng, Lin; Yu, Xiao; Li, Chengyuan; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

2016-04-01

Signal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k "Helper" Proteins.

PubMed

Gwiazda, Kamila S; Grier, Alexandra E; Sahni, Jaya; Burleigh, Stephen M; Martin, Unja; Yang, Julia G; Popp, Nicholas A; Krutein, Michelle C; Khan, Iram F; Jacoby, Kyle; Jensen, Michael C; Rawlings, David J; Scharenberg, Andrew M

2016-09-29

Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.

Efficient transduction of equine adipose-derived mesenchymal stem cells by VSV-G pseudotyped lentiviral vectors.

PubMed

Petersen, Gayle F; Hilbert, Bryan; Trope, Gareth; Kalle, Wouter; Strappe, Padraig

2014-12-01

Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomatitis virus (V-GFP) or, for the first time, the baculovirus gp64 envelope protein (G-GFP). In this study, we produced similarly high titre V-GFP and G-GFP lentiviral vectors. Flow cytometric analysis showed efficient transduction using V-GFP; however G-GFP exhibited a poor ability to transduce EADMSC. Transduction resulted in sustained GFP expression over four passages, with minimal effects on cell viability and doubling time, and an unaltered chondrogenic differentiation potential. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enhanced cellulase producing mutants developed from heterokaryotic Aspergillus strain.

PubMed

Kaur, Baljit; Oberoi, H S; Chadha, B S

2014-03-01

A heterokaryon 28, derived through protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis (Dal8), was subjected cyclic mutagenesis followed by selection on increasing levels of 2-deoxy glucose (2-DG) as selection marker. The derived deregulated cellulase hyper producing mutant '64', when compared to fusant 28, produced 9.83, 7.8, 3.2, 4.2 and 19.74 folds higher endoglucanase, β-glucosidase, cellobiohydrolase, FPase and xylanase, respectively, under shake cultures. The sequence analysis of PCR amplified β-glucosidase gene from wild and mutant showed nucleotide deletion/substitution. The mutants showed highly catalytic efficient β-glucosidase as evident from low Km and high Vmax values. The expression profiling through zymogram analysis also indicated towards over-expression of cellulases. The up/down regulated expressed proteins observed through SDS-PAGE were identified by Peptide mass fingerprinting The cellulase produced by mutants in conjunction with cellulase free xylanase derived from Thermomyces lanuginosus was used for efficient utilization of alkali treated rice straw for obtaining xylo-oligosaccharides and ethanol. Copyright © 2014 Elsevier Ltd. All rights reserved.

eMBI: Boosting Gene Expression-based Clustering for Cancer Subtypes.

PubMed

Chang, Zheng; Wang, Zhenjia; Ashby, Cody; Zhou, Chuan; Li, Guojun; Zhang, Shuzhong; Huang, Xiuzhen

2014-01-01

Identifying clinically relevant subtypes of a cancer using gene expression data is a challenging and important problem in medicine, and is a necessary premise to provide specific and efficient treatments for patients of different subtypes. Matrix factorization provides a solution by finding checker-board patterns in the matrices of gene expression data. In the context of gene expression profiles of cancer patients, these checkerboard patterns correspond to genes that are up- or down-regulated in patients with particular cancer subtypes. Recently, a new matrix factorization framework for biclustering called Maximum Block Improvement (MBI) is proposed; however, it still suffers several problems when applied to cancer gene expression data analysis. In this study, we developed many effective strategies to improve MBI and designed a new program called enhanced MBI (eMBI), which is more effective and efficient to identify cancer subtypes. Our tests on several gene expression profiling datasets of cancer patients consistently indicate that eMBI achieves significant improvements in comparison with MBI, in terms of cancer subtype prediction accuracy, robustness, and running time. In addition, the performance of eMBI is much better than another widely used matrix factorization method called nonnegative matrix factorization (NMF) and the method of hierarchical clustering, which is often the first choice of clinical analysts in practice.

eMBI: Boosting Gene Expression-based Clustering for Cancer Subtypes

PubMed Central

Chang, Zheng; Wang, Zhenjia; Ashby, Cody; Zhou, Chuan; Li, Guojun; Zhang, Shuzhong; Huang, Xiuzhen

2014-01-01

Identifying clinically relevant subtypes of a cancer using gene expression data is a challenging and important problem in medicine, and is a necessary premise to provide specific and efficient treatments for patients of different subtypes. Matrix factorization provides a solution by finding checker-board patterns in the matrices of gene expression data. In the context of gene expression profiles of cancer patients, these checkerboard patterns correspond to genes that are up- or down-regulated in patients with particular cancer subtypes. Recently, a new matrix factorization framework for biclustering called Maximum Block Improvement (MBI) is proposed; however, it still suffers several problems when applied to cancer gene expression data analysis. In this study, we developed many effective strategies to improve MBI and designed a new program called enhanced MBI (eMBI), which is more effective and efficient to identify cancer subtypes. Our tests on several gene expression profiling datasets of cancer patients consistently indicate that eMBI achieves significant improvements in comparison with MBI, in terms of cancer subtype prediction accuracy, robustness, and running time. In addition, the performance of eMBI is much better than another widely used matrix factorization method called nonnegative matrix factorization (NMF) and the method of hierarchical clustering, which is often the first choice of clinical analysts in practice. PMID:25374455

A multifunctional lipid nanoparticle for co-delivery of paclitaxel and curcumin for targeted delivery and enhanced cytotoxicity in multidrug resistant breast cancer cells

PubMed Central

Baek, Jong-Suep; Cho, Cheong-Weon

2017-01-01

The objective of the work was to develop a multifunctional nanomedicine based on a folate-conjugated lipid nanoparticles loaded with paclitaxel and curcumin. The novel system combines therapeutic advantageous of efficient targeted delivery via folate and timed-release of curcumin and paclitaxel via 2-hydroxypropyl-ß-cyclodextrin, thereby overcoming multidrug resistance in breast cancer cells (MCF-7/ADR). The faster release of curcumin from the folate-conjugated curcumin and paclitaxel-loaded lipid nanoparticles enables sufficient p-glycoprotein inhibition, which allows increased cellular uptake and cytotoxicity of paclitaxel. In western blot assay, curcumin can efficiently inhibit the expression of p-glycoprotein, conformed the enhancement of cytotoxicity by paclitaxel. Furthermore, folate-conjugated curcumin and paclitaxel-loaded lipid nanoparticles exhibited increased uptake of paclitaxel and curcumin into MCF-7/ADR cells through the folate receptor-mediated internalization. Taken together, these results indicate that folate-conjugated curcumin and paclitaxel-loaded lipid nanoparticles enables the enhanced, folate-targeted delivery of multiple anticancer drugs by inhibiting the multi-drug resistance efficiently, which may also serve as a useful nano-system for co-delivery of other anticancer drugs. PMID:28423731

The use of biodegradable PLGA nanoparticles to mediate SOX9 gene delivery in human mesenchymal stem cells (hMSCs) and induce chondrogenesis.

PubMed

Kim, Jae-Hwan; Park, Ji Sun; Yang, Han Na; Woo, Dae Gyun; Jeon, Su Yeon; Do, Hyun-Jin; Lim, Hye-Young; Kim, Jung Mo; Park, Keun-Hong

2011-01-01

In stem cell therapy, transfection of specific genes into stem cells is an important technique to induce cell differentiation. To perform gene transfection in human mesenchymal stem cells (hMSCs), we designed and fabricated a non-viral vector system for specific stem cell differentiation. Several kinds of gene carriers were evaluated with regard to their transfection efficiency and their ability to enhance hMSCs differentiation. Of these delivery vehicles, biodegradable poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles yielded the best results, as they complexed with high levels of plasmid DNA (pDNA), allowed robust gene expression in hMSCs, and induced chondrogenesis. Polyplexing with polyethylenimine (PEI) enhanced the cellular uptake of SOX9 DNA complexed with PLGA nanoparticles both in vitro and in vivo. The expression of enhanced green fluorescent protein (EGFP) and SOX9 increased up to 75% in hMSCs transfected with PEI/SOX9 complexed PLGA nanoparticles 2 days after transfection. SOX9 gene expression was evaluated by RT-PCR, real time-qPCR, glycosaminoglycan (GAG)/DNA levels, immunoblotting, histology, and immunofluorescence. Copyright © 2010 Elsevier Ltd. All rights reserved.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

PubMed Central

Migita, M; Medin, J A; Pawliuk, R; Jacobson, S; Nagle, J W; Anderson, S; Amiri, M; Humphries, R K; Karlsson, S

1995-01-01

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected. Images Fig. 2 Fig. 3 PMID:8618847

Orlistat Reduces Proliferation and Enhances Apoptosis in Human Pancreatic Cancer Cells (PANC-1).

PubMed

Sokolowska, Ewa; Presler, Malgorzata; Goyke, Elzbieta; Milczarek, Ryszard; Swierczynski, Julian; Sledzinski, Tomasz

2017-11-01

Pancreatic cancer is a disease with very poor prognosis, and none of currently available pharmacotherapies have proven to be efficient in this indication. The aim of this study was to analyze the expression of fatty acid synthase (FASN) gene as a potential therapeutic target in proliferating human pancreatic cancer cells (PANC-1), and verify if orlistat, originally developed as an anti-obesity drug, inhibits PANC-1 proliferation. The effects of orlistat on gene expression, lipogenesis, proliferation and apoptosis was studied in PANC-1 cell culture. Expression of FASN increased during proliferation of PANC-1. Inhibition of FASN by orlistat resulted in a significant reduction of PANC-1 proliferation and enhanced apoptosis of these cells. This study showed, to our knowledge for the first time, that orlistat exhibits significant antitumor activity against PANC-1 cells. This implies that orlistat analogs with good oral bioavailability may find application in pharmacotherapy of pancreatic cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

ABCG2 Is a Selectable Marker for Enhanced Multilineage Differentiation Potential in Periodontal Ligament Stem Cells

PubMed Central

Szepesi, Áron; Matula, Zsolt; Szigeti, Anna; Várady, György; Szabó, Gyula; Uher, Ferenc; Sarkadi, Balázs

2015-01-01

Periodontal ligament stem cells (PDLSCs) provide an important source for tissue regeneration and may become especially useful in the formation of osteogenic seeds. PDLSCs can be cultured, expanded, and differentiated in vitro; thus, they may be applied in the long-term treatment of the defects in the dental regions. Here we studied numerous potential markers allowing the selection of human PDLSCs with a maximum differentiation potential. We followed the expression of the ATP-binding cassette subfamily G member 2 (ABCG2) membrane transporter protein and isolated ABCG2-expressing cells by using a monoclonal antibody, recognizing the transporter at the cell surface in intact cells. The expression of the ABCG2 protein, corresponding to the so-called side-population phenotype in various tissue-derived stem cells, was found to be a useful marker for the selection of PDLSCs with enhanced osteogenic, chondrogenic, and adipogenic differentiation. These findings may have important applications in achieving efficient dental tissue regeneration by using stem cells from extracted teeth. PMID:25101689

Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

PubMed

Gupta, Sanjeev K; Shukla, Pratyoosh

2016-12-01

Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.

Inhibition of master transcription factors in pluripotent cells induces early stage differentiation

PubMed Central

De, Debojyoti; Jeong, Myong-Ho; Leem, Young-Eun; Svergun, Dmitri I.; Wemmer, David E.; Kang, Jong-Sun; Kim, Kyeong Kyu; Kim, Sung-Hou

2014-01-01

The potential for pluripotent cells to differentiate into diverse specialized cell types has given much hope to the field of regenerative medicine. Nevertheless, the low efficiency of cell commitment has been a major bottleneck in this field. Here we provide a strategy to enhance the efficiency of early differentiation of pluripotent cells. We hypothesized that the initial phase of differentiation can be enhanced if the transcriptional activity of master regulators of stemness is suppressed, blocking the formation of functional transcriptomes. However, an obstacle is the lack of an efficient strategy to block protein–protein interactions. In this work, we take advantage of the biochemical property of seventeen kilodalton protein (Skp), a bacterial molecular chaperone that binds directly to sex determining region Y-box 2 (Sox2). The small angle X-ray scattering analyses provided a low resolution model of the complex and suggested that the transactivation domain of Sox2 is probably wrapped in a cleft on Skp trimer. Upon the transduction of Skp into pluripotent cells, the transcriptional activity of Sox2 was inhibited and the expression of Sox2 and octamer-binding transcription factor 4 was reduced, which resulted in the expression of early differentiation markers and appearance of early neuronal and cardiac progenitors. These results suggest that the initial stage of differentiation can be accelerated by inhibiting master transcription factors of stemness. This strategy can possibly be applied to increase the efficiency of stem cell differentiation into various cell types and also provides a clue to understanding the mechanism of early differentiation. PMID:24434556

SS-mPEG chemical modification of recombinant phospholipase C for enhanced thermal stability and catalytic efficiency.

PubMed

Fang, Xian; Wang, Xueting; Li, Guiling; Zeng, Jun; Li, Jian; Liu, Jingwen

2018-05-01

PEGylation is one of the most promising and extensively studied strategies for improving the properties of proteins as well as enzymic physical and thermal stability. Phospholipase C, hydrolyzing the phospholipids offers tremendous applications in diverse fields. However, the poor thermal stability and higher cost of production have restricted its industrial application. This study focused on improving the stabilization of recombinant PLC by chemical modification with methoxypolyethylene glycol-Succinimidyl Succinate (SS-mPEG, MW 5000). PLC gene from isolate Bacillus cereus HSL3 was fused with SUMO, a novel small ubiquitin-related modifier expression vector and over expressed in Escherichia coli. The soluble fraction of SUMO-PLC reached 80% of the total recombinant protein. The enzyme exhibited maximum catalytic activity at 80 °C and was relatively thermostable at 40-70 °C. It showed extensive substrate specificity pattern and marked activity toward phosphatidylcholine, which made it a typical non-specific PLC for industrial purpose. SS-mPEG-PLC complex exhibited an enhanced thermal stability at 70-80 °C and the catalytic efficiency (K cat /K m ) had increased by 3.03 folds compared with free PLC. CD spectrum of SS-mPEG-PLC indicated a possible enzyme aggregation after chemical modification, which contributed to the higher thermostability of SS-mPEG-PLC. The increase of antiparallel β sheets in secondary structure also made it more stable than parallel β sheets. The presence of SS-mPEG chains on the enzyme molecule surface somewhat changed the binding rate of the substrates, leading to a significant improvement in catalytic efficiency. This study provided an insight into the addition of SS-mPEG for enhancing the industrial applications of phospholipase C at higher temperature. Copyright © 2018 Elsevier B.V. All rights reserved.

Regulation of Plant Growth, Photosynthesis, Antioxidation and Osmosis by an Arbuscular Mycorrhizal Fungus in Watermelon Seedlings under Well-Watered and Drought Conditions.

PubMed

Mo, Yanling; Wang, Yongqi; Yang, Ruiping; Zheng, Junxian; Liu, Changming; Li, Hao; Ma, Jianxiang; Zhang, Yong; Wei, Chunhua; Zhang, Xian

2016-01-01

Drought stress has become an increasingly serious environmental issue that influences the growth and production of watermelon. Previous studies found that arbuscular mycorrhizal (AM) colonization improved the fruit yield and water use efficiency (WUE) of watermelon grown under water stress; however, the exact mechanisms remain unknown. In this study, the effects of Glomus versiforme symbiosis on the growth, physio-biochemical attributes, and stress-responsive gene expressions of watermelon seedlings grown under well-watered and drought conditions were investigated. The results showed that AM colonization did not significantly influence the shoot growth of watermelon seedlings under well-watered conditions but did promote root development irrespective of water treatment. Drought stress decreased the leaf relative water content and chlorophyll concentration, but to a lesser extent in the AM plants. Compared with the non-mycorrhizal seedlings, mycorrhizal plants had higher non-photochemical quenching values, which reduced the chloroplast ultrastructural damage in the mesophyll cells and thus maintained higher photosynthetic efficiency. Moreover, AM inoculation led to significant enhancements in the enzyme activities and gene expressions of superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase in watermelon leaves upon drought imposition. Consequently, AM plants exhibited lower accumulation of MDA, H2O2 and [Formula: see text] compared with non-mycorrhizal plants. Under drought stress, the soluble sugar and proline contents were significantly increased, and further enhancements were observed by pre-treating the drought-stressed plants with AM. Taken together, our findings indicate that mycorrhizal colonization enhances watermelon drought tolerance through a stronger root system, greater protection of photosynthetic apparatus, a more efficient antioxidant system and improved osmoregulation. This study contributes to advances in the knowledge of AM-induced drought tolerance.

Regulation of Plant Growth, Photosynthesis, Antioxidation and Osmosis by an Arbuscular Mycorrhizal Fungus in Watermelon Seedlings under Well-Watered and Drought Conditions

PubMed Central

Mo, Yanling; Wang, Yongqi; Yang, Ruiping; Zheng, Junxian; Liu, Changming; Li, Hao; Ma, Jianxiang; Zhang, Yong; Wei, Chunhua; Zhang, Xian

2016-01-01

Drought stress has become an increasingly serious environmental issue that influences the growth and production of watermelon. Previous studies found that arbuscular mycorrhizal (AM) colonization improved the fruit yield and water use efficiency (WUE) of watermelon grown under water stress; however, the exact mechanisms remain unknown. In this study, the effects of Glomus versiforme symbiosis on the growth, physio-biochemical attributes, and stress-responsive gene expressions of watermelon seedlings grown under well-watered and drought conditions were investigated. The results showed that AM colonization did not significantly influence the shoot growth of watermelon seedlings under well-watered conditions but did promote root development irrespective of water treatment. Drought stress decreased the leaf relative water content and chlorophyll concentration, but to a lesser extent in the AM plants. Compared with the non-mycorrhizal seedlings, mycorrhizal plants had higher non-photochemical quenching values, which reduced the chloroplast ultrastructural damage in the mesophyll cells and thus maintained higher photosynthetic efficiency. Moreover, AM inoculation led to significant enhancements in the enzyme activities and gene expressions of superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase in watermelon leaves upon drought imposition. Consequently, AM plants exhibited lower accumulation of MDA, H2O2 and O2− compared with non-mycorrhizal plants. Under drought stress, the soluble sugar and proline contents were significantly increased, and further enhancements were observed by pre-treating the drought-stressed plants with AM. Taken together, our findings indicate that mycorrhizal colonization enhances watermelon drought tolerance through a stronger root system, greater protection of photosynthetic apparatus, a more efficient antioxidant system and improved osmoregulation. This study contributes to advances in the knowledge of AM-induced drought tolerance. PMID:27242845

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM--role of satellite cells in anabolic steroid-induced muscle growth in feedlot steers.

PubMed

Dayton, W R; White, M E

2014-01-01

Both androgenic and estrogenic steroids are widely used as growth promoters in feedlot steers because they significantly enhance feed efficiency, rate of gain, and muscle growth. However, despite their widespread use relatively little is known about the biological mechanism by which androgenic and estrogenic steroids enhance rate and efficiency of muscle growth in cattle. Treatment of feedlot steers with a combined estradiol (E2) and trenbolone acetate (TBA) implant results in an increased number of muscle satellite cells, increased expression of IGF-1 mRNA in muscle tissue, and increased levels of circulating IGF-1. Similarly, treatment of bovine satellite cell (BSC) cultures with either TBA or E2 results in increased expression of IGF-1 mRNA, increased rates of proliferation and protein synthesis, and decreased rates of protein degradation. Effects of E2 on BSC are mediated at least in part through the classical E2 receptor, estrogen receptor-α (ESR1), the IGF-1 receptor (IGFR1), and the G protein-coupled estrogen receptor-1 (GPER-1), formerly known as G protein-coupled receptor-30 (GPR30). The effects of TBA appear to be primarily mediated through the androgen receptor. Based on current research results, it is becoming clear that anabolic steroid-enhanced bovine muscle growth involves a complex interaction of numerous pathways and receptors. Consequently, additional in vivo and in vitro studies are necessary to understand the mechanisms involved in this complex process. The fundamental information generated by this research will help in developing future, safe, and effective strategies to increase rate and efficiency of muscle growth in beef cattle.

Foxtail Millet [Setaria italica (L.) Beauv.] Grown under Low Nitrogen Shows a Smaller Root System, Enhanced Biomass Accumulation, and Nitrate Transporter Expression.

PubMed

Nadeem, Faisal; Ahmad, Zeeshan; Wang, Ruifeng; Han, Jienan; Shen, Qi; Chang, Feiran; Diao, Xianmin; Zhang, Fusuo; Li, Xuexian

2018-01-01

Foxtail millet (FM) [ Setaria italica (L.) Beauv.] is a grain and forage crop well adapted to nutrient-poor soils. To date little is known how FM adapts to low nitrogen (LN) at the morphological, physiological, and molecular levels. Using the FM variety Yugu1, we found that LN led to lower chlorophyll contents and N concentrations, and higher root/shoot and C/N ratios and N utilization efficiencies under hydroponic culture. Importantly, enhanced biomass accumulation in the root under LN was in contrast to a smaller root system, as indicated by significant decreases in total root length; crown root number and length; and lateral root number, length, and density. Enhanced carbon allocation toward the root was rather for significant increases in average diameter of the LN root, potentially favorable for wider xylem vessels or other anatomical alterations facilitating nutrient transport. Lower levels of IAA and CKs were consistent with a smaller root system and higher levels of GA may promote root thickening under LN. Further, up-regulation of SiNRT1.1, SiNRT2.1, and SiNAR2.1 expression and nitrate influx in the root and that of SiNRT1.11 and SiNRT1.12 expression in the shoot probably favored nitrate uptake and remobilization as a whole. Lastly, more soluble proteins accumulated in the N-deficient root likely as a result of increases of N utilization efficiencies. Such "excessive" protein-N was possibly available for shoot delivery. Thus, FM may preferentially transport carbon toward the root facilitating root thickening/nutrient transport and allocate N toward the shoot maximizing photosynthesis/carbon fixation as a primary adaptive strategy to N limitation.

Design and testing of regulatory cassettes for optimal activity in skeletal and cardiac muscles.

PubMed

Himeda, Charis L; Chen, Xiaolan; Hauschka, Stephen D

2011-01-01

Gene therapy for muscular dystrophies requires efficient gene delivery to the striated musculature and specific, high-level expression of the therapeutic gene in a physiologically diverse array of muscles. This can be achieved by the use of recombinant adeno-associated virus vectors in conjunction with muscle-specific regulatory cassettes. We have constructed several generations of regulatory cassettes based on the enhancer and promoter of the muscle creatine kinase gene, some of which include heterologous enhancers and individual elements from other muscle genes. Since the relative importance of many control elements varies among different anatomical muscles, we are aiming to tailor these cassettes for high-level expression in cardiac muscle, and in fast and slow skeletal muscles. With the achievement of efficient intravascular gene delivery to isolated limbs, selected muscle groups, and heart in large animal models, the design of cassettes optimized for activity in different muscle types is now a practical goal. In this protocol, we outline the key steps involved in the design of regulatory cassettes for optimal activity in skeletal and cardiac muscle, and testing in mature muscle fiber cultures. The basic principles described here can also be applied to engineering tissue-specific regulatory cassettes for other cell types.

Heterologous co-expression of a yeast diacylglycerol acyltransferase (ScDGA1) and a plant oleosin (AtOLEO3) as an efficient tool for enhancing triacylglycerol accumulation in the marine diatom Phaeodactylum tricornutum.

PubMed

Zulu, Nodumo Nokulunga; Popko, Jennifer; Zienkiewicz, Krzysztof; Tarazona, Pablo; Herrfurth, Cornelia; Feussner, Ivo

2017-01-01

Microalgae are promising alternate and renewable sources for producing valuable products such as biofuel and essential fatty acids. Although this is the case, there are still challenges impeding on the effective commercial production of microalgal products. For instance, their product yield is still too low. Therefore, this study was oriented towards enhancing triacylglycerol (TAG) accumulation in the diatom Phaeodactylum tricornutum (strain Pt4). To achieve this, a type 2 acyl-CoA:diacylglycerol acyltransferase from yeast ( ScDGA1 ) and the lipid droplet (LD) stabilizing oleosin protein 3 from Arabidopsis thaliana ( AtOLEO3 ) were expressed in Pt4. The individual expression of ScDGA1 and AtOLEO3 in Pt4 resulted in a 2.3- and 1.4-fold increase in TAG levels, respectively, in comparison to the wild type. The co-expression of both, ScDGA1 and AtOLEO3 , was accompanied by a 3.6-fold increase in TAG content. On the cellular level, the lines co-expressing ScDGA1 and AtOLEO3 showed the presence of the larger and increased numbers of lipid droplets when compared to transformants expressing single genes and an empty vector. Under nitrogen stress, TAG productivity was further increased twofold in comparison to nitrogen-replete conditions. While TAG accumulation was enhanced in the analyzed transformants, the fatty acid composition remained unchanged neither in the total lipid nor in the TAG profile. The co-expression of two genes was shown to be a more effective strategy for enhancing TAG accumulation in P. tricornutum strain Pt4 than a single gene strategy. For the first time in a diatom, a LD protein from a vascular plant, oleosin, was shown to have an impact on TAG accumulation and on LD organization.

Creation of High Efficient Firefly Luciferase

NASA Astrophysics Data System (ADS)

Nakatsu, Toru

Firefly emits visible yellow-green light. The bioluminescence reaction is carried out by the enzyme luciferase. The bioluminescence of luciferase is widely used as an excellent tool for monitoring gene expression, the measurement of the amount of ATP and in vivo imaging. Recently a study of the cancer metastasis is carried out by in vivo luminescence imaging system, because luminescence imaging is less toxic and more useful for long-term assay than fluorescence imaging by GFP. However the luminescence is much dimmer than fluorescence. Then bioluminescence imaging in living organisms demands the high efficient luciferase which emits near infrared lights or enhances the emission intensity. Here I introduce an idea for creating the high efficient luciferase based on the crystal structure.

Translational initiation in Leishmania tarentolae and Phytomonas serpens (Kinetoplastida) is strongly influenced by pre-ATG triplet and its 5' sequence context.

PubMed

Lukes, Julius; Paris, Zdenek; Regmi, Sandesh; Breitling, Reinhard; Mureev, Sergey; Kushnir, Susanna; Pyatkov, Konstantin; Jirků, Milan; Alexandrov, Kirill A

2006-08-01

To investigate the influence of sequence context of translation initiation codon on translation efficiency in Kinetoplastida, we constructed a library of expression plasmids randomized in the three nucleotides prefacing ATG of a reporter gene encoding enhanced green fluorescent protein (EGFP). All 64 possible combinations of pre-ATG triplets were individually stably integrated into the rDNA locus of Leishmania tarentolae and the resulting cell lines were assessed for EGFP expression. The expression levels were quantified directly by measuring the fluorescence of EGFP protein in living cells and confirmed by Western blotting. We observed a strong influence of the pre-ATG triplet on the level of protein expression over a 20-fold range. To understand the degree of evolutionary conservation of the observed effect, we transformed Phytomonas serpens, a trypanosomatid parasite of plants, with a subset of the constructs. The pattern of translational efficiency mediated by individual pre-ATG triplets in this species was similar to that observed in L. tarentolae. However, the pattern of translational efficiency of two other proteins (red fluorescent protein and tetracycline repressor) containing selected pre-ATG triplets did not correlate with either EGFP or each other. Thus, we conclude that a conserved mechanism of translation initiation site selection exists in kinetoplastids that is strongly influenced not only by the pre-ATG sequences but also by the coding region of the gene.

Drosophila social clustering is disrupted by anesthetics and in narrow abdomen ion channel mutants.

PubMed

Burg, E D; Langan, S T; Nash, H A

2013-04-01

Members of many species tend to congregate, a behavioral strategy known as local enhancement. Selective advantages of local enhancement range from efficient use of resources to defense from predators. While previous studies have examined many types of social behavior in fruit flies, few have specifically investigated local enhancement. Resource-independent local enhancement (RILE) has recently been described in the fruit fly using a measure called social space index (SSI), although the neural mechanisms remain unknown. Here, we analyze RILE of Drosophila under conditions that allow us to elucidate its neural mechanisms. We have investigated the effects of general volatile anesthetics, compounds that compromise higher order functioning of the type typically required for responding to social cues. We exposed Canton-S flies to non-immobilizing concentrations of halothane and found that flies had a significantly decreased SSI compared with flies tested in air. Narrow abdomen (na) mutants, which display altered responses to anesthetics in numerous behavioral assays, also have a significantly reduced SSI, an effect that was fully reversed by restoring expression of na by driving a UAS-NA rescue construct with NA-GAL4. We found that na expression in cholinergic neurons fully rescued the behavioral defect, whereas expression of na in glutamatergic neurons did so only partially. Our results also suggest a role for na expression in the mushroom bodies (MBs), as suppressing na expression in the MBs of NA-GAL4 rescue flies diminishes SSI. Our data indicate that RILE, a simple behavioral strategy, requires complex neural processing. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Transgenic plants with enhanced growth characteristics

DOEpatents

Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

2016-09-06

The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.

Dipolar induced para-hydrogen-induced polarization.

PubMed

Buntkowsky, Gerd; Gutmann, Torsten; Petrova, Marina V; Ivanov, Konstantin L; Bommerich, Ute; Plaumann, Markus; Bernarding, Johannes

2014-01-01

Analytical expressions for the signal enhancement in solid-state PHIP NMR spectroscopy mediated by homonuclear dipolar interactions and single pulse or spin-echo excitation are developed and simulated numerically. It is shown that an efficient enhancement of the proton NMR signal in solid-state NMR studies of chemisorbed hydrogen on surfaces is possible. Employing typical reaction efficacy, enhancement-factors of ca. 30-40 can be expected both under ALTADENA and under PASADENA conditions. This result has important consequences for the practical application of the method, since it potentially allows the design of an in-situ flow setup, where the para-hydrogen is adsorbed and desorbed from catalyst surfaces inside the NMR magnet. Copyright © 2014 Elsevier Inc. All rights reserved.

Expression systems for therapeutic glycoprotein production.

PubMed

Durocher, Yves; Butler, Michael

2009-12-01

There are slightly over 165 recombinant pharmaceuticals currently approved for human use. Another 500 protein candidates are in preclinical and clinical development, about 70% of these being glycosylated proteins. The need for expression systems allowing the efficient manufacturing of high quality glycoproteins is thus becoming imperative. Recent developments with CHO cells, the predominant mammalian expression system, have focused on either increasing cell specific productivity or prolonging the life span of cells in culture that translates to high integrated viable cell densities. These two factors have allowed volumetric productivities in excess of 5 g/L under conditions of controlled nutrient feeding. In addition to glycoengineering strategies, which are offering considerable advantage in producing proteins with enhanced therapeutic properties, several alternative expression systems are being developed for their manufacture, each with their advantages and limitations.

Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

PubMed

Nair, Nisha R; Chidambareswaren, M; Manjula, S

2014-09-01

Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

Differential Effects of Methyl Jasmonate on the Expression of the Early Light-Inducible Proteins and Other Light-Regulated Genes in Barley1

PubMed Central

Wierstra, Inken; Kloppstech, Klaus

2000-01-01

The effects of methyl jasmonate (JA-Me) on early light-inducible protein (ELIP) expression in barley (Hordeum vulgare L. cv Apex) have been studied. Treatment of leaf segments with JA-Me induces the same symptoms as those exhibited by norflurazon bleaching, including a loss of pigments and enhanced light stress that results in increased ELIP expression under both high- and low-light conditions. The expression of both low- and high-molecular-mass ELIP families is considerably down-regulated by JA-Me at the transcript and protein levels. This repression occurs despite increased photoinhibition measurable as a massive degradation of D1 protein and a delayed recovery of photosystem II activity. In JA-Me-treated leaf segments, the decrease of the photochemical efficiency of photosystem II under high light is substantially more pronounced as compared to controls in water. The repression of ELIP expression by JA-Me is superimposed on the effect of the increased light stress that leads to enhanced ELIP expression. The fact that the reduction of ELIP transcript levels is less pronounced than those of light-harvesting complex II and small subunit of Rubisco transcripts indicates that light stress is still affecting gene expression in the presence of JA-Me. The jasmonate-induced protein transcript levels that are induced by JA-Me decline under light stress conditions. PMID:11027731

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair

PubMed Central

Nowarski, Roni; Wilner, Ofer I.; Cheshin, Ori; Shahar, Or D.; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S.; Goldberg, Michal; Willner, Itamar

2012-01-01

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy. PMID:22645179

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

PubMed

Nowarski, Roni; Wilner, Ofer I; Cheshin, Ori; Shahar, Or D; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S; Goldberg, Michal; Willner, Itamar; Kotler, Moshe

2012-07-12

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis

PubMed Central

Pandey, Shiv S.; Singh, Sucheta; Babu, C. S. Vivek; Shanker, Karuna; Srivastava, N. K.; Shukla, Ashutosh K.; Kalra, Alok

2016-01-01

Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229–403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant. PMID:27220774

Prostaglandin E2 modulates dendritic cell function via EP2 and EP4 receptor subtypes.

PubMed

Harizi, Hedi; Grosset, Christophe; Gualde, Norbert

2003-06-01

We have reported previously that PGE(2) inhibits dendritic cells (DC) functions. Because E prostanoid receptor (EPR) subtypes involved in this action are unknown, expression and functions of these receptors were examined in DC. Western blot and flow cytometry analyses showed that all EPRs were coexpressed in DC. In a dose-dependent manner, lipopolysaccharide (LPS) enhanced EP(2)R/EP(4)R but not EP(1)R/EP(3)R expressions. NS-398, a cyclooxygenase (COX)-2-selective inhibitor, suppressed LPS-enhanced EP(2)R/EP(4)R expression, suggesting that COX-2-issued prostaglandin E(2) (PGE(2)) modulates DC function through stimulation of specific EPR subtypes. Using selective agonists, we found that butaprost, an EP(2)R agonist, and PGE(1) alcohol, an EP(2)R and EP(2)R/EP(4)R agonist, inhibited major histocompatibility complex class II expression and enhanced interleukin-10 production from DC. However, no effect was observed with sulprostone and 17-phenyl-omega-trinor-PGE(2), selective agonists for EP(1)R and EP(1)R/EP(3)R, respectively. Treatment of DC with dibutyryl cyclic adenosine monophosphate (cAMP), an analog of cAMP, mimics PGE(2)-induced, inhibitory effects. Taken together, our data demonstrate that EP(2)R/EP(4)R are efficient for mediating PGE(2)-induced modulation of DC functions.

Potent CD4+ T-cell epitope P30 enhances HER2/neu-engineered dendritic cell-induced immunity against Tg1-1 breast cancer in transgenic FVBneuN mice by enhanced CD4+ T-cell-stimulated CTL responses.

PubMed

Xie, Y; Chen, Y; Ahmed, K A; Li, W; Ahmed, S; Sami, A; Chibbar, R; Tang, X; Tao, M; Xu, J; Xiang, J

2013-10-01

One of the major obstacles in human epidermal growth factor receptor (HER)-2/neu-specific trastuzumab immunotherapy of HER2/neu-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Although dendritic cell (DC) vaccines have been extensively applied in clinical trials for cancer treatment, the vaccination efficacy is still limited, mostly because DC vaccines are not sufficient to break tumor-associated antigen-specific self-immune tolerance in cancer patients. P30 (FNNFTVSFWLRVPKVSASHLE) derived from tetanus toxin is a universally potent CD4(+) T helper epitope capable of enhancing CD8(+) cytotoxic T-lymphocyte (CTL) responses. In this study, we constructed two recombinant adenoviral vectors (AdVs), AdVOVA-P30 and AdVHER2/neu-P30, expressing ovalbumin (OVA)-P30 and HER2/neu-P30. In order to enhance DC vaccine efficacy, we transfected mouse bone marrow (BM)-derived DCs with AdVOVA-P30 and AdVHER2/neu-P30 to generate engineered DCOVA-P30 and DCHER2/neu-P30 vaccines, respectively. We, then, compared CD4(+) and CD8(+) T-cell responses and antitumor immunity derived from DCOVA-P30 and DCHER2/neu-P30 vaccination in wild-type C57BL/6 and transgenic FVBneuN mice, respectively. We demonstrate that engineered DCOVA-P30 vaccine stimulates more efficient CD4(+) and CD8(+) T-cell responses than DCOVA in C57BL/6 mice. Interestingly, the increased DCOVA-P30-induced CTL responses are mainly contributed by enhanced CD4(+) T-cell-stimulated CTL proliferation. We show that DCOVA-P30 vaccine also stimulates more efficient therapeutic immunity against OVA-expressing BL6-10OVA melanoma than DCOVA in C57BL/6 mice. In addition, we demonstrate that DCHER2/neu-P30 vaccine stimulates more efficient CD4(+) and CD8(+) T-cell responses and protective immunity against HER2/neu-expressing Tg1-1 breast cancer than DCHER2/neu in transgenic FVBneuN mice with HER2/neu-specific self-immune tolerance. Therefore, the engineered DCHER2/neu-P30 vaccine may provide a new immunotherapy alternative for women with HER2/neu(+) breast cancer, especially for trastuzumab-resistant HER2/neu(+) breast cancer patients.

Differential allelic expression of IL13 and CSF2 genes associated with asthma.

PubMed

Burkhardt, Jana; Kirsten, Holger; Wolfram, Grit; Quente, Elfi; Ahnert, Peter

2012-07-01

An important area of genetic research is the identification of functional mechanisms in polymorphisms associated with diseases. A highly relevant functional mechanism is the influence of polymorphisms on gene expression levels (differential allelic expression, DAE). The coding single nucleotide polymorphisms (SNPs) CSF2(rs25882) and IL13(rs20541) have been associated with asthma. In this work, we investigated whether the mRNA expression levels of CSF2 or IL13 were correlated with these SNPs. Samples were analyzed by mass spectrometry-based quantification of gene expression. Both SNPs influenced gene expression levels (CSF2(rs25882): p(overall) = 0.008 and p(DAE samples) = 0.00006; IL13(rs20541): p(overall) = 0.059 and p(DAE samples) = 0.036). For CSF2, the expression level was increased by 27.4% (95% CI: 18.5%-35.4%) in samples with significant DAE in the presence of one copy of risk variant CSF2(rs25882-T). The average expression level of IL13 was increased by 29.8% (95% CI: 3.1%-63.4%) in samples with significant DAE in the presence of one copy of risk variant IL13(rs20541-A). Enhanced expression of CSF2 could stimulate macrophages and neutrophils during inflammation and may be related to the etiology of asthma. For IL-13, higher expression could enhance the functional activity of the asthma-associated isoform. Overall, the analysis of DAE provides an efficient approach for identifying possible functional mechanisms that link disease-associated variants with altered gene expression levels.

Cationic star-shaped polymer as an siRNA carrier for reducing MMP-9 expression in skin fibroblast cells and promoting wound healing in diabetic rats.

PubMed

Li, Na; Luo, Heng-Cong; Yang, Chuan; Deng, Jun-Jie; Ren, Meng; Xie, Xiao-Ying; Lin, Diao-Zhu; Yan, Li; Zhang, Li-Ming

2014-01-01

Excessive expression of matrix metalloproteinase-9 (MMP-9) is deleterious to the cutaneous wound-healing process in the context of diabetes. The aim of the present study was to explore whether a cationic star-shaped polymer consisting of β-cyclodextrin (β-CD) core and poly(amidoamine) dendron arms (β-CD-[D₃]₇) could be used as the gene carrier of small interfering RNA (siRNA) to reduce MMP-9 expression for enhanced diabetic wound healing. The cytotoxicity of β-CD-(D₃)₇ was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MMT) method in the rat CRL1213 skin fibroblast cell line. The transfection efficiency of β-CD-(D₃)₇/MMP-9-small interfering RNA (siRNA) complexes was determined by confocal microscopy and flow cytometry. Quantitative real time (RT) polymerase chain reaction was performed to measure the gene expression of MMP-9 after the transfection by β-CD-(D₃)₇/MMP-9-siRNA complexes. The β-CD-(D₃)₇/MMP-9-siRNA complexes were injected on the wounds of streptozocin-induced diabetic rats. Wound closure was measured on days 4 and 7 post-wounding. β-CD-(D₃)₇ exhibited low cytotoxicity in fibroblast cells, and easily formed the complexes with MMP-9-siRNA. The β-CD-(D₃)₇/MMP-9-siRNA complexes were readily taken up by fibroblast cells, resulting in the downregulation of MMP-9 gene expression (P<0.01). Animal experiments revealed that the treatment by β-CD-(D₃)₇/MMP-9-siRNA complexes enhanced wound closure in diabetic rats on day 7 post-wounding (P<0.05). β-CD-(D₃)₇ may be used as an efficient carrier for the delivery of MMP-9-siRNA to reduce MMP-9 expression in skin fibroblast cells and promote wound healing in diabetic rats.

Efficient Translation of Epstein-Barr Virus (EBV) DNA Polymerase Contributes to the Enhanced Lytic Replication Phenotype of M81 EBV.

PubMed

Church, Trenton Mel; Verma, Dinesh; Thompson, Jacob; Swaminathan, Sankar

2018-03-15

Epstein-Barr virus (EBV) is linked to the development of both lymphoid and epithelial malignancies worldwide. The M81 strain of EBV, isolated from a Chinese patient with nasopharyngeal carcinoma (NPC), demonstrates spontaneous lytic replication and high-titer virus production in comparison to the prototype B95-8 EBV strain. Genetic comparisons of M81 and B95-8 EBVs were previously been performed in order to determine if the hyperlytic property of M81 is associated with sequence differences in essential lytic genes. EBV SM is an RNA-binding protein expressed during early lytic replication that is essential for virus production. We compared the functions of M81 SM and B95-8 SM and demonstrate that polymorphisms in SM do not contribute to the lytic phenotype of M81 EBV. However, the expression level of the EBV DNA polymerase protein was much higher in M81- than in B95-8-infected cells. The relative deficiency in the expression of B95-8 DNA polymerase was related to the B95-8 genome deletion, which truncates the BALF5 3' untranslated region (UTR). Similarly, the insertion of bacmid DNA into the widely used recombinant B95-8 bacmid creates an inefficient BALF5 3' UTR. We further showed that the while SM is required for and facilitates the efficient expression of both M81 and B95-8 mRNAs regardless of the 3' UTR, the BALF5 3' UTR sequence is important for BALF5 protein translation. These data indicate that the enhanced lytic replication and virus production of M81 compared to those of B95-8 are partly due to the robust translation of EBV DNA polymerase required for viral DNA replication due to a more efficient BALF5 3' UTR in M81. IMPORTANCE Epstein-Barr virus (EBV) infects more than 90% of the human population, but the incidence of EBV-associated tumors varies greatly in different parts of the world. Thus, understanding the connection between genetic polymorphisms from patient isolates of EBV, gene expression phenotypes, and disease is important and may help in developing antiviral therapy. This study examines potential causes of the enhanced lytic replicative properties of M81 EBV isolated from a nasopharyngeal carcinoma (NPC) patient and provides new evidence for the role of the BALF5 gene 3' UTR sequence in DNA polymerase protein expression during lytic replication. Variation in the gene structure of the DNA polymerase gene may therefore contribute to lytic virus reactivation and pathogenesis. Copyright © 2018 American Society for Microbiology.

Nutrients Can Enhance the Abundance and Expression of Alkane Hydroxylase CYP153 Gene in the Rhizosphere of Ryegrass Planted in Hydrocarbon-Polluted Soil

PubMed Central

Arslan, Muhammad; Afzal, Muhammad; Amin, Imran; Iqbal, Samina; Khan, Qaiser M.

2014-01-01

Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K) in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum) was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination. PMID:25360680

pEGFP transfection into murine skeletal muscle by electrosonoporation

NASA Astrophysics Data System (ADS)

Tamošiūnas, Mindaugas; Jakovels, Dainis; Rubins, Uldis; Kadikis, Roberts; Petrovska, Ramona; Šatkauskas, Saulius

2017-12-01

In this study, we aimed to determine whether the combination of electroporation (EP) and ultrasound (US) waves (sonoporation) can affect the plasmid DNA transfection to mice tibialis cranialis muscle. Multispectral imaging technique combined with fluorescence spectroscopy point measurements has been used for the transcutaneous detection of enhanced green fluorescent protein (EGFP) fluorescence, providing information on location and duration of EGFP expression. We found that electrosonoporation, commonly enhancing pDNA transfection in vitro, had no positive effect on EGFP transfection efficiency increase in vivo with respect to electroporation alone. We presume that this may be associated with decreased viability of transfected fibers.

Systemic delivery of shRNA by AAV9 provides highly efficient knockdown of ubiquitously expressed GFP in mouse heart, but not liver.

PubMed

Piras, Bryan A; O'Connor, Daniel M; French, Brent A

2013-01-01

AAV9 is a powerful gene delivery vehicle capable of providing long-term gene expression in a variety of cell types, particularly cardiomyocytes. The use of AAV-delivery for RNA interference is an intense area of research, but a comprehensive analysis of knockdown in cardiac and liver tissues after systemic delivery of AAV9 has yet to be reported. We sought to address this question by using AAV9 to deliver a short-hairpin RNA targeting the enhanced green fluorescent protein (GFP) in transgenic mice that constitutively overexpress GFP in all tissues. The expression cassette was initially tested in vitro and we demonstrated a 61% reduction in mRNA and a 90% reduction in GFP protein in dual-transfected 293 cells. Next, the expression cassette was packaged as single-stranded genomes in AAV9 capsids to test cardiac GFP knockdown with several doses ranging from 1.8×10(10) to 1.8×10(11) viral genomes per mouse and a dose-dependent response was obtained. We then analyzed GFP expression in both heart and liver after delivery of 4.4×10(11) viral genomes per mouse. We found that while cardiac knockdown was highly efficient, with a 77% reduction in GFP mRNA and a 71% reduction in protein versus control-treated mice, there was no change in liver expression. This was despite a 4.5-fold greater number of viral genomes in the liver than in the heart. This study demonstrates that single-stranded AAV9 vectors expressing shRNA can be used to achieve highly efficient cardiac-selective knockdown of GFP expression that is sustained for at least 7 weeks after the systemic injection of 8 day old mice, with no change in liver expression and no evidence of liver damage despite high viral genome presence in the liver.

[RS-1 enhanced the efficiency of CRISPR-Cas9 mediated knock-in of human lactoferrin].

PubMed

Zhou, Wenjun; Guo, Rihong; Deng, Mingtian; Wang, Feng; Zhang, Yanli

2017-08-25

This study aims to knock out the goat β-lactoglobulin (BLG) gene using CRISPR-Cas9 system and knock in human lactoferrin (hLF) at the BLG locus, and further study the effect of RAD51 stimulatory compound (RS-1) on homologous recombination efficiency. First, we designed an sgRNA targeting the first exon of goat BLG gene and constructed a co-expression vector pCas9-sgBLG. This sgRNA vector was then transfected into goat ear fibroblasts (GEFs), and the target region was examined by T7EN1 assay and sequencing. Second, we constructed a targeting vector pBHA-hLF-NIE including NEO and EGFP genes based on BLG gene locus. This targeting vector together with pCas9-sgBLG expression vector was co-transfected into GEFs. Transfected cells were then treated with 0, 5, 10 and 20 μmol/L RS-1 for 72 h to analyse the EGFP expression efficiency. Next, we used 800 μg/mL G418 to screen G418-resistent cell clones, and studied hLF site-specific knock-in cell clones by PCR and sequencing. The editing efficiency of sgBLG was between 25% and 31%. The EGFP expression efficiency indicated that the gene knock-in efficiency was improved by RS-1 in a dose-dependent manner, which could reach 3.5-fold compared to the control group. The percentage of positive cells with hLF knock-in was increased to 32.61% when 10 μmol/L RS-1 was used. However, when the concentration of RS-1 increased to 20 μmol/L, the percentage of positive cells decreased to 22.22% and resulted in an increase of senescent cell clone number. These results suggested that hLF knock-in and BLG knock-out in GEFs were achieved by using CRISPR/Cas9 system, and optimum concentration of RS-1 could improve knock-in efficiency, which provides a reference for efficiently obtaining gene knock-in cells using CRISPR/Cas9 in the future.

Targeted gene delivery to the synovial pannus in antigen-induced arthritis by ultrasound-targeted microbubble destruction in vivo.

PubMed

Xiang, Xi; Tang, Yuanjiao; Leng, Qianying; Zhang, Lingyan; Qiu, Li

2016-02-01

The purpose of this study was to optimize an ultrasound-targeted microbubble destruction (UTMD) technique to improve the in vivo transfection efficiency of the gene encoding enhanced green fluorescent protein (EGFP) in the synovial pannus in an antigen-induced arthritis rabbit model. A mixture of microbubbles and plasmids was locally injected into the knee joints of an antigen-induced arthritis (AIA) rabbits. The plasmid concentrations and ultrasound conditions were varied in the experiments. We also tested local articular and intravenous injections. The rabbits were divided into five groups: (1) ultrasound+microbubbles+plasmid; (2) ultrasound+plasmid; (3) microbubble+plasmid; (4) plasmid only; (5) untreated controls. EGFP expression was observed by fluorescent microscope and immunohistochemical staining in the synovial pannus of each group. The optimal plasmid dosage and ultrasound parameter were determined based on the results of EGFP expression and the present and absent of tissue damage under light microscopy. The irradiation procedure was performed to observe the duration of the EGFP expression in the synovial pannus and other tissues and organs, as well as the damage to the normal cells. The optimal condition was determined to be a 1-MHz ultrasound pulse applied for 5 min with a power output of 2 W/cm(2) and a 20% duty cycle along with 300 μg of plasmid. Under these conditions, the synovial pannus showed significant EGFP expression without significant damage to the surrounding normal tissue. The EGFP expression induced by the local intra-articular injection was significantly more increased than that induced by the intravenous injection. The EGFP expression in the synovial pannus of the ultrasound+microbubbles+plasmid group was significantly higher than that of the other four groups (P<0.05). The expression peaked on day 5, remained detectable on day 40 and disappeared on day 60. No EGFP expression was detected in the other tissues and organs. The UTMD technique can significantly enhance the in vivo gene transfection efficiency without significant tissue damage in the synovial pannus of an AIA model. Thus, this could become a safe and effective non-viral gene transfection procedure for arthritis therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

The secreted protein ANGPTL2 promotes metastasis of osteosarcoma cells through integrin α5β1, p38 MAPK, and matrix metalloproteinases.

PubMed

Odagiri, Haruki; Kadomatsu, Tsuyoshi; Endo, Motoyoshi; Masuda, Tetsuro; Morioka, Masaki Suimye; Fukuhara, Shigetomo; Miyamoto, Takeshi; Kobayashi, Eisuke; Miyata, Keishi; Aoi, Jun; Horiguchi, Haruki; Nishimura, Naotaka; Terada, Kazutoyo; Yakushiji, Toshitake; Manabe, Ichiro; Mochizuki, Naoki; Mizuta, Hiroshi; Oike, Yuichi

2014-01-21

The tumor microenvironment can enhance the invasive capacity of tumor cells. We showed that expression of angiopoietin-like protein 2 (ANGPTL2) in osteosarcoma (OS) cell lines increased and the methylation of its promoter decreased with time when grown as xenografts in mice compared with culture. Compared with cells grown in normal culture conditions, the expression of genes encoding DNA demethylation-related enzymes increased in tumor cells implanted into mice or grown in hypoxic, serum-starved culture conditions. ANGPTL2 expression in OS cell lines correlated with increased tumor metastasis and decreased animal survival by promoting tumor cell intravasation mediated by the integrin α5β1, p38 mitogen-activated protein kinase, and matrix metalloproteinases. The tolloid-like 1 (TLL1) protease cleaved ANGPTL2 into fragments in vitro that did not enhance tumor progression when overexpressed in xenografts. Expression of TLL1 was weak in OS patient tumors, suggesting that ANGPTL2 may not be efficiently cleaved upon secretion from OS cells. These findings demonstrate that preventing ANGPTL2 signaling stimulated by the tumor microenvironment could inhibit tumor cell migration and metastasis.

Formation of friable embryogenic callus in cassava is enhanced under conditions of reduced nitrate, potassium and phosphate

PubMed Central

Utsumi, Yoshinori; Utsumi, Chikako; Tanaka, Maho; Ha, Vu The; Matsui, Akihiro; Takahashi, Satoshi; Seki, Motoaki

2017-01-01

Agrobacterium-mediated transformation is an important research tool for the genetic improvement of cassava. The induction of friable embryogenic callus (FEC) is considered as a key step in cassava transformation. In the present study, the media composition was optimized for enhancing the FEC induction, and the effect of the optimized medium on gene expression was evaluated. In relative comparison to MS medium, results demonstrated that using a medium with reducing nutrition (a 10-fold less concentration of nitrogen, potassium, and phosphate), the increased amount of vitamin B1 (10 mg/L) and the use of picrolam led to reprogram non-FEC to FEC. Gene expression analyses revealed that FEC on modified media increased the expression of genes related to the regulation of polysaccharide biosynthesis and breakdown of cell wall components in comparison to FEC on normal CIM media, whereas the gene expression associated with energy flux was not dramatically altered. It is hypothesized that we reprogram non-FEC to FEC under low nitrogen, potassium and phosphate and high vitamin B1. These findings were more effective in inducing FEC formation than the previous protocol. It might contribute to development of an efficient transformation strategy in cassava. PMID:28806727

Pulsed electromagnetic fields promote osteogenesis and osseointegration of porous titanium implants in bone defect repair through a Wnt/β-catenin signaling-associated mechanism

PubMed Central

Jing, Da; Zhai, Mingming; Tong, Shichao; Xu, Fei; Cai, Jing; Shen, Guanghao; Wu, Yan; Li, Xiaokang; Xie, Kangning; Liu, Juan; Xu, Qiaoling; Luo, Erping

2016-01-01

Treatment of osseous defects remains a formidable clinical challenge. Porous titanium alloys (pTi) have been emerging as ideal endosseous implants due to the excellent biocompatibility and structural properties, whereas inadequate osseointegration poses risks for unreliable long-term implant stability. Substantial evidence indicates that pulsed electromagnetic fields (PEMF), as a safe noninvasive method, inhibit osteopenia/osteoporosis experimentally and clinically. We herein investigated the efficiency and potential mechanisms of PEMF on osteogenesis and osseointegration of pTi in vitro and in vivo. We demonstrate that PEMF enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton for in vitro osteoblasts seeded in pTi. PEMF promoted gene expressions in Runx2, OSX, COL-1 and Wnt/β-catenin signaling. PEMF-stimulated group exhibited higher Runx2, Wnt1, Lrp6 and β-catenin protein expressions. In vivo results via μCT and histomorphometry show that 6-week and 12-week PEMF promoted osteogenesis, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defect. PEMF promoted femoral gene expressions of Runx2, BMP2, OCN and Wnt/β-catenin signaling. Together, we demonstrate that PEMF improve osteogenesis and osseointegration of pTi by promoting skeletal anabolic activities through a Wnt/β-catenin signaling-associated mechanism. PEMF might become a promising biophysical modality for enhancing the repair efficiency and quality of pTi in bone defect. PMID:27555216

The drought response of Theobroma cacao (cacao) and the regulation of genes involved in polyamine biosynthesis by drought and other stresses.

PubMed

Bae, Hanhong; Kim, Soo-Hyung; Kim, Moon S; Sicher, Richard C; Lary, David; Strem, Mary D; Natarajan, Savithiry; Bailey, Bryan A

2008-02-01

Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined: an ornithine decarboxylase (TcODC), an arginine decarboxylase (TcADC), an S-adenosylmethionine decarboxylase (TcSAMDC), a spermidine synthase (TcSPDS), and a spermine synthase (TcSPMS). Expression analysis using quantitative real-time reverse transcription-PCR (QPCR) results showed that the PA biosynthesis genes were expressed in all plant tissues examined. Constitutive expression of PA biosynthesis genes was generally highest in mature leaves and open flowers. Expression of TcODC, TcADC, and TcSAMDC was induced with the onset of drought and correlated with changes in stomatal conductance, photosynthesis, photosystem II efficiency, leaf water potential and altered emission of blue-green fluorescence from cacao leaves. Induction of TcSAMDC in leaves was most closely correlated with changes in water potential. The earliest measured responses to drought were enhanced expression of TcADC and TcSAMDC in roots along with decreases in stomatal conductance, photosynthesis, and photosystem II efficiency. Elevated levels of putrescine, spermidine, and spermine were detected in cacao leaves 13days after the onset of drought. Expression of all five PA associated transcripts was enhanced (1.5-3-fold) in response to treatment with abscisic acid. TcODC and TcADC, were also responsive to mechanical wounding, infection by Phytophthora megakarya (a causal agent of black pod disease in cacao), the necrosis- and ethylene-inducing protein (Nep1) of Fusarium oxysporum, and flower abscission. TcSAMDC expression was responsive to all stresses except flower abscission. TcODC, although constitutively expressed at much lower levels than TcADC, TcSAMDC, TcSPDS, and TcSPMS, was highly inducible by the fungal protein Nep1 (135-fold) and the cacao pathogen Phytophthora megakarya (671-fold). The full length cDNA for ODC was cloned and characterized. Among the genes studied, TcODC, TcADC, and TcSAMDC were most sensitive to induction by drought in addition to other abiotic and biotic stresses. TcODC, TcADC, and TcSAMDC may share signal transduction pathways and/or the stress induced signal induction pathways may converge at these three genes leading to similar although not identical patterns of expression. It is possible altering PA levels in cacao will result in enhanced tolerance to multiple stresses including drought and disease as has been demonstrated in other crops.

Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology.

PubMed

Tanaka, Tsutomu; Kondo, Akihiko

2015-02-01

In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often utilized as a cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Highly efficient local delivery of endothelial progenitor cells significantly potentiates angiogenesis and full-thickness wound healing.

PubMed

Wang, Chenggui; Wang, Qingqing; Gao, Wendong; Zhang, Zengjie; Lou, Yiting; Jin, Haiming; Chen, Xiaofeng; Lei, Bo; Xu, Huazi; Mao, Cong

2018-03-15

Wound therapy with a rapid healing performance remains a critical clinical challenge. Cellular delivery is considered to be a promising approach to improve the efficiency of healing, yet problems such as compromised cell viability and functionality arise due to the inefficient delivery. Here, we report the efficient delivery of endothelial progenitor cells (EPCs) with a bioactive nanofibrous scaffold (composed of collagen and polycaprolactone and bioactive glass nanoparticles, CPB) for enhancing wound healing. Under the stimulation of CPB nanofibrous system, the viability and angiogenic ability of EPCs were significantly enhanced through the activation of Hif-1α/VEGF/SDF-1α signaling. In vivo, CPB/EPC constructs significantly enhanced the formation of high-density blood vessels by greatly upregulating the expressions of Hif-1α, VEGF, and SDF-1α. Moreover, owing to the increased local delivery of cells and fast neovascularization within the wound site, cell proliferative activity, granulation tissue formation, and collagen synthesis and deposition were greatly promoted by CPB/EPC constructs resulting in rapid re-epithelialization and regeneration of skin appendages. As a result, the synergistic enhancement of wound healing was observed from CPB/EPC constructs, which suggests the highly efficient delivery of EPCs. CPB/EPC constructs may become highly competitive cell-based therapeutic products for efficient impaired wound healing application. This study may also provide a novel strategy to develop bioactive cell therapy constructs for angiogenesis-related regenerative medicine. This paper reported a highly efficient local delivery of EPCs using bioactive glass-based CPB nanofibrous scaffold for enhancing angiogenesis and wound regeneration. In vitro study showed that CPB can promote the proliferation, migration, and tube formation of EPCs through upregulation of the Hif-1α/VEGF/SDF-1α signaling pathway, indicating that the bioactivity and angiogenic ability of EPCs can be highly maintained and promoted by the CPB scaffold. Moreover, CPB/EPC constructs effectively stimulated the regeneration of diabetic wounds with satisfactory vascularization and better healing outcomes in a full-thickness wound model, suggesting that the highly efficient delivery of EPCs to wound site facilitates angiogenesis and further leads to wound healing. The high angiogenic capacity and excellent healing ability make CPB/EPC constructs highly competitive in cell-based therapeutic products for efficient wound repair application. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Layer-by-layer assembly of small interfering RNA and poly(ethyleneimine) for substrate-mediated electroporation with high efficiency.

PubMed

Fujimoto, Hiroyuki; Kato, Koichi; Iwata, Hiroo

2010-05-01

Electroporation microarrays have been developed for the high-throughput transfection of expression constructs and small interfering RNAs (siRNAs) into living mammalian cells. These techniques have potential to provide a platform for the cell-based analysis of gene functions. One of the key issues associated with microarray technology is the efficiency of transfection. The capability of attaining reasonably high transfection efficiency is the basis for obtaining functional data without false negatives. In this study, we aimed at improving the transfection efficiency in the system that siRNA loaded on an electrode is electroporated into cells cultured directly on the electrode. The strategy we adopted here is to increase the surface density of siRNA loaded onto electrodes. For this purpose, the layer-by-layer assembly of siRNA and cationic polymers, branched or linear form of poly(ethyleneimine), was performed. The multilayer thus obtained was characterized by infrared reflection-adsorption spectroscopy and surface plasmon resonance analysis. Transfection efficiency was evaluated in a system that siRNA specific for enhanced green fluorescent protein (EGFP) was electroporated on the electrode into human embryonic kidney cells stably transformed with the EGFP gene. The suppression of EGFP expression was assessed by fluorescence microscopy and flow cytometry. Our data showed that the layer-by-layer assembly of siRNA with branched poly(ethyleneimine) facilitated to increase the surface density of loaded siRNA. As a result, the expression of EGFP gene in the electroporated cells was suppressed much more on the electrodes with the multilayer of siRNA than that with the monolayer.

Construction and evaluation of pH-sensitive immunoliposomes for enhanced delivery of anticancer drug to ErbB2 over-expressing breast cancer cells.

PubMed

Li, Tianshu; Amari, Takuya; Semba, Kentaro; Yamamoto, Tadashi; Takeoka, Shinji

2017-04-01

1,5-Dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG) liposomes were previously developed to enhance drug delivery efficiency in tumor cells owing to its pH-responsive properties. Herein, we report the modification of GGLG liposomes by conjugating a Fab' fragment of an ErbB2 antibody to the terminus of PEG (polyethylene glycol)-lipid (Fab'-GGLG liposomes). The conjugation of Fab' fragments did not affect the antibody activity, drug (doxorubicin, DOX) encapsulation efficiency, stability during storage or pH-sensitivity. However, the binding affinity of Fab'-GGLG liposomes was enhanced to ErbB2-overexpressing HCC1954 cells specifically, and the cell association increased 10-fold in comparison to GGLG liposomes. Consequently, intracellular DOX delivery was enhanced, with an increased cytotoxicity in HCC1954 cells (i.e., IC 50 of 1.17 and 3.08 μg/mL for Fab'-GGLG-DOX and GGLG-DOX liposomes, respectively). Further, a significantly enhanced tumor growth inhibition was obtained in an ErbB2-overexpressing breast cancer-bearing mouse model. Therefore, a potent anticancer drug delivery system was constructed by the immunological modification of pH-sensitive liposomes. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhanced transfection by antioxidative polymeric gene carrier that reduces polyplex-mediated cellular oxidative stress.

PubMed

Lee, Min Sang; Kim, Nak Won; Lee, Kyuri; Kim, Hongtae; Jeong, Ji Hoon

2013-06-01

To test the hypothesis in which polyplex-induced oxidative stress may affect overall transfection efficiency, an antioxidative transfection system minimizing cellular oxidative stress was designed for enhanced transfection. An amphiphilic copolymer (PEI-PLGA) was synthesized and used as a micelle-type gene carrier containing hydrophobic antioxidant, α-tocopherol. Cellular oxidative stress and the change of mitochondrial membrane potential after transfection was measured by using a fluorescent probe (H₂DCFDA) and lipophilic cationic probe (JC-1), respectively. Transfection efficiency was determined by measuring a reporter gene (luciferase) expression level. The initial transfection study with conventional PEI/plasmid DNA polyplex showed significant generation of reactive oxygen species (ROS). The PEI-PLGA copolymer successfully carried out the simultaneous delivery of α-tocopherol and plasmid DNA (PEI-PLGA/Toco/pDNA polyplex) into cells, resulting in a significant reduction in cellular ROS generation after transfection and helped to maintain the mitochondrial membrane potential (ΔΨ). In addition, the transfection efficiency was dramatically increased using the antioxidative transfection system. This work showed that oxidative stress would be one of the important factors that should be considered in designing non-viral gene carriers and suggested a possible way to reduce the carrier-mediated oxidative stress, which consequently leads to enhanced transfection.

Bolstering cholesteryl ester hydrolysis in liver: A hepatocyte-targeting gene delivery strategy for potential alleviation of atherosclerosis

PubMed Central

He, Hongliang; Lancina, Michael G.; Wang, Jing; Korzun, William J.; Yang, Hu; Ghosh, Shobha

2017-01-01

Current atherosclerosis treatment strategies primarily focus on limiting further cholesteryl esters (CE) accumulation by reducing endogenous synthesis of cholesterol in the liver. No therapy is currently available to enhance the removal of CE, a crucial step to reduce the burden of the existing disease. Given a central role of hepatic cholesteryl ester hydrolase (CEH) in intrahepatic hydrolysis of CE and subsequent removal of the resulting free cholesterol, in this work, we applied galactose-functionalized polyamidoamine (PAMAM) dendrimer G5 (Gal-G5) for hepatocyte-specific delivery of CEH expression vector. The data presented herein show increased specific uptake of Gal-G5 by the hepatocytes in vitro and in vivo. Furthermore, the upregulated CEH expression in the hepatocytes significantly enhanced intracellular hydrolysis of HDL-CE and subsequent conversion/secretion of hydrolyzed free cholesterol (FC) as bile acids. Increased CEH expression in the liver significantly increased the flux of HDL-CE to biliary as well as fecal FC and bile acids. Meanwhile, Gal-G5 did not induce hepatic or renal toxicity. It was not immunotoxic. Because of these encouraging pre-clinical testing results, using this safe and highly efficient hepatocyte-specific gene delivery platform to enhance the hepatic processes involved in cholesterol elimination is a promising strategy for alleviation of atherosclerosis. PMID:28349866

Arabidopsis and tobacco plants ectopically expressing the soybean antiquitin-like ALDH7 gene display enhanced tolerance to drought, salinity, and oxidative stress.

PubMed

Rodrigues, Simone M; Andrade, Maxuel O; Gomes, Ana Paula Soares; Damatta, Fabio M; Baracat-Pereira, Maria C; Fontes, Elizabeth P B

2006-01-01

Despite extensive studies in eukaryotic aldehyde dehydrogenases, functional information about the ALDH7 antiquitin-like proteins is lacking. A soybean antiquitin homologue gene, designated GmTP55, has been isolated which encodes a dehydrogenase motif-containing 55 kDa protein induced by dehydration and salt stress. GmTP55 is closely related to the stress-induced plant antiquitin-like proteins that belong to the ALDH7 family. Transgenic tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) plants constitutively expressing GmTP55 have been obtained in order to examine the physiological role of this enzyme under a variety of stress conditions. Ectopic expression of GmTP55 in both Arabidopsis and tobacco conferred tolerance to salinity during germination and to water deficit during plant growth. Under salt stress, the germination efficiency of both transgenic tobacco and Arabidopsis seeds was significantly higher than that of their control counterparts. Likewise, under progressive drought, the transgenic tobacco lines apparently kept the shoot turgidity to a normal level, which contrasted with the leaf wilt phenotype of control plants. The transgenic plants also exhibited an enhanced tolerance to H(2)O(2)- and paraquat-induced oxidative stress. Both GmTP55-expressing Arabidopsis and tobacco seeds germinated efficiently in medium supplemented with H(2)O(2), whereas the germination of control seeds was drastically impaired. Similarly, transgenic tobacco leaf discs treated with paraquat displayed a significant reduction in the necrotic lesions as compared with control leaves. These transgenic lines also exhibited a lower concentration of lipid peroxidation-derived reactive aldehydes under oxidative stress. These results suggest that antiquitin may be involved in adaptive responses mediated by a physiologically relevant detoxification pathway in plants.

Analysis of whitefly transcriptional responses to Beauveria bassiana infection reveals new insights into insect-fungus interactions.

PubMed

Xia, Jun; Zhang, Chang-Rong; Zhang, Shan; Li, Fang-Fang; Feng, Ming-Guang; Wang, Xiao-Wei; Liu, Shu-Sheng

2013-01-01

The fungal pathogen, Beauveria bassiana, is an efficient biocontrol agent against a variety of agricultural pests. A thorough understanding of the basic principles of insect-fungus interactions may enable the genetic modification of Beauveria bassiana to enhance its virulence. However, the molecular mechanism of insect response to Beauveria bassiana infection is poorly understood, let alone the identification of fungal virulent factors involved in pathogenesis. Here, next generation sequencing technology was applied to examine the expression of whitefly (Bemisia tabaci) genes in response to the infection of Beauveria bassiana. Results showed that, compared to control, 654 and 1,681genes were differentially expressed at 48 hours and 72 hours post-infected whiteflies, respectively. Functional and enrichment analyses indicated that the DNA damage stimulus response and drug metabolism were important anti-fungi strategies of the whitefly. Mitogen-activated protein kinase (MAPK) pathway was also likely involved in the whitefly defense responses. Furthermore, the notable suppression of general metabolism and ion transport genes observed in 72 hours post-infected B. tabaci might be manipulated by fungal secreted effectors. By mapping the sequencing tags to B. bassiana genome, we also identified a number of differentially expressed fungal genes between the early and late infection stages. These genes are generally associated with fungal cell wall synthesis and energy metabolism. The expression of fungal cell wall protein genes might play an important role in fungal pathogenesis and the dramatically up-regulated enzymes of carbon metabolism indicate the increasing usage of energy during the fungal infection. To our knowledge, this is the first report on the molecular mechanism of fungus-whitefly interactions. Our results provide a road map for future investigations on insect-pathogen interactions and genetically modifying the fungus to enhance its efficiency in whitefly control.

Optimisation of intradermal DNA electrotransfer for immunisation.

PubMed

Vandermeulen, Gaëlle; Staes, Edith; Vanderhaeghen, Marie Lise; Bureau, Michel Francis; Scherman, Daniel; Préat, Véronique

2007-12-04

The development of DNA vaccines requires appropriate delivery technologies. Electrotransfer is one of the most efficient methods of non-viral gene transfer. In the present study, intradermal DNA electrotransfer was first optimised. Strong effects of the injection method and the dose of DNA on luciferase expression were demonstrated. Pre-treatments were evaluated to enhance DNA diffusion in the skin but neither hyaluronidase injection nor iontophoresis improved efficiency of intradermal DNA electrotransfer. Then, DNA immunisation with a weakly immunogenic model antigen, luciferase, was investigated. After intradermal injection of the plasmid encoding luciferase, electrotransfer (HV 700 V/cm 100 micros, LV 200 V/cm 400 ms) was required to induce immune response. The response was Th1-shifted compared to immunisation with the luciferase recombinant protein. Finally, DNA electrotransfer in the skin, the muscle or the ear pinna was compared. Muscle DNA electrotransfer resulted in the highest luciferase expression and the best IgG response. Nevertheless electrotransfer into the skin, the muscle and the ear pinna all resulted in IFN-gamma secretion by luciferase-stimulated splenocytes suggesting that an efficient Th1 response was induced in all case.

Next gen perception and cognition: augmenting perception and enhancing cognition through mobile technologies

NASA Astrophysics Data System (ADS)

Goma, Sergio R.

2015-03-01

In current times, mobile technologies are ubiquitous and the complexity of problems is continuously increasing. In the context of advancement of engineering, we explore in this paper possible reasons that could cause a saturation in technology evolution - namely the ability of problem solving based on previous results and the ability of expressing solutions in a more efficient way, concluding that `thinking outside of brain' - as in solving engineering problems that are expressed in a virtual media due to their complexity - would benefit from mobile technology augmentation. This could be the necessary evolutionary step that would provide the efficiency required to solve new complex problems (addressing the `running out of time' issue) and remove the communication of results barrier (addressing the human `perception/expression imbalance' issue). Some consequences are discussed, as in this context the artificial intelligence becomes an automation tool aid instead of a necessary next evolutionary step. The paper concludes that research in modeling as problem solving aid and data visualization as perception aid augmented with mobile technologies could be the path to an evolutionary step in advancing engineering.

Using rabies virus vaccine strain SRV9 as viral vector to express exogenous gene.

PubMed

Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Li, Ling; Qi, Yinglin; Liang, Meng; Zhao, Yongkun; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Jin, Ningyi; Yang, Songtao; Xia, Xianzhu

2015-04-01

Rabies virus (RABV) can cause a fatal neurological disease in human and animals, and vaccines were generally applied for the immunoprophylaxis of rabies. Here, a recombinant viral vector carrying the exogenous gene expression component between phosphoprotein (P) and matrix protein (M) genes of RABV was constructed based on the vaccine strain SRV9 used in China. To develop a reverse genetic system, the full-length cDNA plasmids of SRV9 were constructed using the eukaryotic expression vector pCI or pcDNA3.1(+). However, recovery efficiency based on the pcDNA3.1 vector was significantly higher than that of the pCI vector. The exogenous gene expression component PE-PS-BsiWI-PmeI or PS-BsiWI-PmeI-PE was introduced in different locations between the P and M genes of SRV9. When the enhanced green fluorescent protein (eGFP) was used as a reporter gene, both locations could rescue recombinant RABV (rRABV) expressing eGFP with high efficiency. Characterization of rRABV expressing eGFP in vitro revealed that its growth was similar to that of the parental virus. Animal experiments showed that rRABV expressing eGFP could replicate and express eGFP in the brains of suckling mice. Furthermore, rRABV of SRV9 was nonpathogenic for 3-week-old mice and could be cleared from the central nervous system at 5 days post-inoculation. Our results showed that the recombinant SRV9 virus could be used as a useful viral vector for exogenous gene expression.

Pantropic retroviruses as a transduction tool for sea urchin embryos

PubMed Central

Core, Amanda B.; Reyna, Arlene E.; Conaway, Evan A.; Bradham, Cynthia A.

2012-01-01

Sea urchins are an important model for experiments at the intersection of development and systems biology, and technical innovations that enhance the utility of this model are of great value. This study explores pantropic retroviruses as a transduction tool for sea urchin embryos, and demonstrates that pantropic retroviruses infect sea urchin embryos with high efficiency and genomically integrate at a copy number of one per cell. We successfully used a self-inactivation strategy to both insert a sea urchin-specific enhancer and disrupt the endogenous viral enhancer. The resulting self-inactivating viruses drive global and persistent gene expression, consistent with genomic integration during the first cell cycle. Together, these data provide substantial proof of principle for transduction technology in sea urchin embryos. PMID:22431628

Transgenic plants with enhanced growth characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of themore » double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.« less

The competitor-introduced Ggamma recruitment system, a new approach for screening affinity-enhanced proteins.

PubMed

Fukuda, Nobuo; Ishii, Jun; Tanaka, Tsutomu; Kondo, Akihiko

2010-04-01

We have developed a new approach based on the Ggamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Ggamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein gamma subunit (Ggamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered Ggamma that lacks membrane localization upon deletion of the lipid modification site (Ggamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with Ggamma(cyto) are expressed. As protein-protein interactions bring Ggamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-Ggamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced Ggamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.

Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme.

PubMed

Jalomo-Khayrova, Ekaterina; Mares, Rosa E; Muñoz, Patricia L A; Meléndez-López, Samuel G; Rivero, Ignacio A; Ramos, Marco A

2018-04-03

Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases. Using E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile. We report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding.

Efficient osmolyte-based procedure to increase expression level and solubility of infectious hematopoietic necrosis virus (IHNV) nucleoprotein in E. coli.

PubMed

Mohammadinezhad, Rezvan; Farahmand, Hamid; Jalali, Seyed Amir Hossein; Mirvaghefi, Alireza

2018-05-01

The nucleoprotein of infectious hematopoietic necrosis virus (IHNV) is considered as the main target antigen for detection of IHNV infection in salmonid fish. This study aimed at improving the expression and solubility of IHNV nucleoprotein (IHNV-NP) in E. coli expression system. The effects of several expression strategies including host strain type, protein expression temperature, heat-shock treatment prior to protein induction, and additives in the growth medium and in the cell lysis buffer were examined. Results showed that bacterial strain type had a great impact on protein expression level, whereas it was not effective in preventing protein aggregation. Production of soluble IHNV-NP was proportionally increased with decreased incubation temperature. Heat-shock treatment prior to protein induction did not change the percent of solubility. For cells grown at low temperature, the presence of additives in the lysis buffer enhanced the solubility of IHNV-NP up to 24%. The highest yield of soluble protein was obtained via incorporation of osmolytes in the growth medium of cells exposed to a mild salt stress, in the following order: sucrose > sorbitol > glycerol > glycine. Soluble protein obtained by the optimized condition was efficiently purified in high yield and successfully detected by two monoclonal antibodies in a sandwich ELISA. Taken together, a combination of proper host strain, low-temperature expression, and timely application of osmolytes in the growth medium provided sufficient quantities of soluble recombinant IHNV-NP that has the potential to be used for diagnostic purposes.

A caspase 8-based suicide switch induces apoptosis in nanobody-directed chimeric receptor expressing T cells.

PubMed

Khaleghi, Sepideh; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J; Pognonec, Philippe

2012-04-01

In accordance with the two-step hypothesis of T cell activation and the observation that stimulation through the T cell receptor (TCR) alone may lead to anergy, we focused on the introduction of co-stimulatory signaling to this type of receptors to achieve optimal activation. Enhanced mRNA and cell surface receptor expression via the co-stimulatory gene fragment (OX40) was confirmed by RT-PCR and flow cytometry. Inclusion of the OX40 co-stimulatory signaling region in series with the TCR led to enhanced antigen-induced IL-2 production after stimulation by MUC1-expressing cancer cell lines as compared to the chimeric receptor without OX40. Moreover, with the aim of maintaining high efficiency, while providing a means of controlling any possible unwanted proliferation in vivo, a regulation system was used. This controls the dimerization of a membrane-bound caspase 8 protein. Toward that goal, pFKC8 and CAR constructs were co-transfected into Jurkat cells, and the level of apoptosis was measured. 24 h after addition of the dimerizer, a 91% decrease in transfected cells was observed.

Prostaglandin E2 induces expression of P-selectin (CD62P) on cultured human umbilical vein endothelial cells and enhances endothelial binding of CD4-T-cells.

PubMed

Hailer, N P; Oppermann, E; Leckel, K; Cinatl, J; Markus, B H; Blaheta, R A

2000-07-15

Interaction of endothelial P-selectin with sialyl Lewis(x)-glycoprotein or P-selectin glycoprotein ligand (PSGL)-1 on leukocytes represents an early step in leukocyte recruitment. Redistribution of P-selectin to the endothelial cell surface occurs rapidly after challenge with several proinflammatory agents, for example, histamine, leucopterins, or lipopolysaccharide. We present evidence that prostaglandin E2 (PGE2) is an efficient inductor of surface P-selectin on cultured human umbilical vein endothelial cells (HUVEC). The increase in P-selectin-immunoreactivity coincided with redistribution of cytoplasmic P-selectin-reactive granulae to the endothelial cell surface, as visualized by confocal laser microscopic examination. CD4-T-cell adhesion to PGE2-stimulated HUVEC was also enhanced by a factor of 4, and blocking mAb directed against the binding site of P-selectin almost completely abrogated this increase in CD4-T-cell adhesion. In summary, our findings show that liberation of PGE2 is an important inductor of P-selectin surface expression on endothelial cells, resulting in enhanced recruitment of inflammatory cells.

Increasing plant growth by modulating omega-amidase expression in plants

DOEpatents

Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

2015-06-30

The present disclosure relates to compositions and methods for increasing the leaf-to-root ratio of the signal metabolite 2-oxoglutaramate and related proline molecules in plants by modulating levels of .omega.-amidase to increase nitrogen use efficiency, resulting in enhanced growth, faster growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, increased tolerance to high salt conditions, and increased biomass yields.

Enhancing soybean photosynthetic CO2 assimilation using a cyanobacterial membrane protein, ictB.

PubMed

Hay, William T; Bihmidine, Saadia; Mutlu, Nedim; Hoang, Khang Le; Awada, Tala; Weeks, Donald P; Clemente, Tom E; Long, Stephen P

2017-05-01

Soybean C 3 photosynthesis can suffer a severe loss in efficiency due to photorespiration and the lack of a carbon concentrating mechanism (CCM) such as those present in other plant species or cyanobacteria. Transgenic soybean (Glycine max cv. Thorne) plants constitutively expressing cyanobacterial ictB (inorganic carbon transporter B) gene were generated using Agrobacterium-mediated transformation. Although more recent data suggest that ictB does not actively transport HCO3-/CO 2 , there is nevertheless mounting evidence that transformation with this gene can increase higher plant photosynthesis. The hypothesis that expression of the ictB gene would improve photosynthesis, biomass production and seed yield in soybean was tested, in two independent replicated greenhouse and field trials. Results showed significant increases in photosynthetic CO 2 uptake (A net ) and dry mass in transgenic relative to wild type (WT) control plants in both the greenhouse and field trials. Transgenic plants also showed increased photosynthetic rates and biomass production during a drought mimic study. The findings presented herein demonstrate that ictB, as a single-gene, contributes to enhancement in various yield parameters in a major commodity crop and point to the significant role that biotechnological approaches to increasing photosynthetic efficiency can play in helping to meet increased global demands for food. Copyright © 2017 Elsevier GmbH. All rights reserved.

Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.

PubMed

Kim, Young Mi; Kang, Yun Gyeong; Park, So Hee; Han, Myung-Kwan; Kim, Jae Ho; Shin, Ji Won; Shin, Jung-Woog

2017-06-08

Mechanical stimuli play important roles in the proliferation and differentiation of adult stem cells. However, few studies on their effects on induced pluripotent stem cells (iPSCs) have been published. Human dermal fibroblasts were seeded onto flexible membrane-bottom plates, and infected with retrovirus expressing the four reprogramming factors OCT4, SOX2, KLF, and c-MYC (OSKM). The cells were subjected to equiaxial stretching (3% or 8% for 2, 4, or 7 days) and seeded on feeder cells (STO). The reprogramming into iPSCs was evaluated by the expression of pluripotent markers, in vitro differentiation into three germ layers, and teratoma formation. Equiaxial stretching enhanced reprogramming efficiency without affecting the viral transduction rate. iPSCs induced by transduction of four reprogramming factors and application of equiaxial stretching had characteristics typical of iPSCs in terms of pluripotency and differentiation potentials. This is the first study to show that mechanical stimuli can increase reprogramming efficiency. However, it did not enhance the infection rate, indicating that mechanical stimuli, defined as stretching in this study, have positive effects on reprogramming rather than on infection. Additional studies should evaluate the mechanism underlying the modulation of reprogramming of somatic cells into iPSCs.

The B7-1 Cytoplasmic Tail Enhances Intracellular Transport and Mammalian Cell Surface Display of Chimeric Proteins in the Absence of a Linear ER Export Motif

PubMed Central

Lin, Yi-Chieh; Chen, Bing-Mae; Lu, Wei-Cheng; Su, Chien-I; Prijovich, Zeljko M.; Chung, Wen-Chuan; Wu, Pei-Yu; Chen, Kai-Chuan; Lee, I-Chiao; Juan, Ting-Yi; Roffler, Steve R.

2013-01-01

Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells. PMID:24073236

Correction of Murine Sickle Cell Disease Using γ-Globin Lentiviral Vectors to Mediate High-level Expression of Fetal Hemoglobin

PubMed Central

Pestina, Tamara I; Hargrove, Phillip W; Jay, Dennis; Gray, John T; Boyd, Kelli M; Persons, Derek A

2008-01-01

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, γ-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different γ-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the γ-globin gene driven by 3.1 kb of β-globin regulatory sequences and a 130-bp β-globin promoter. The second vector, V5m3, was identical except that the γ-globin 3′-untranslated region (3′-UTR) was replaced with the β-globin 3′-UTR. Adult erythroid cells have β-globin mRNA 3′-UTR-binding proteins that enhance β-globin mRNA stability and we postulated this design might enhance γ-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human γ-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of γ-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a γ-globin lentiviral vector. PMID:19050697

Bolstering cholesteryl ester hydrolysis in liver: A hepatocyte-targeting gene delivery strategy for potential alleviation of atherosclerosis.

PubMed

He, Hongliang; Lancina, Michael G; Wang, Jing; Korzun, William J; Yang, Hu; Ghosh, Shobha

2017-06-01

Current atherosclerosis treatment strategies primarily focus on limiting further cholesteryl esters (CE) accumulation by reducing endogenous synthesis of cholesterol in the liver. No therapy is currently available to enhance the removal of CE, a crucial step to reduce the burden of the existing disease. Given the central role of hepatic cholesteryl ester hydrolase (CEH) in the intrahepatic hydrolysis of CE and subsequent removal of the resulting free cholesterol (FC), in this work, we applied galactose-functionalized polyamidoamine (PAMAM) dendrimer generation 5 (Gal-G5) for hepatocyte-specific delivery of CEH expression vector. The data presented herein show the increased specific uptake of Gal-G5/CEH expression vector complexes (simply Gal-G5/CEH) by hepatocytes in vitro and in vivo. Furthermore, the upregulated CEH expression in the hepatocytes significantly enhanced the intracellular hydrolysis of high density lipoprotein-associated CE (HDL-CE) and subsequent conversion/secretion of hydrolyzed FC as bile acids (BA). The increased CEH expression in the liver significantly increased the flux of HDL-CE to biliary as well as fecal FC and BA. Meanwhile, Gal-G5 did not induce hepatic or renal toxicity. It was also not immunotoxic. Because of these encouraging pre-clinical testing results, using this safe and highly efficient hepatocyte-specific gene delivery platform to enhance the hepatic processes involved in cholesterol elimination is a promising strategy for the alleviation of atherosclerosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of the soybean GmALMT family genes and the function of GmALMT5 in response to phosphate starvation.

PubMed

Peng, Wenting; Wu, Weiwei; Peng, Junchu; Li, Jiaojiao; Lin, Yan; Wang, Yanan; Tian, Jiang; Sun, Lili; Liang, Cuiyue; Liao, Hong

2018-03-01

A potential mechanism to enhance utilization of sparingly soluble forms of phosphorus (P) is the root secretion of malate, which is mainly mediated by the ALMT gene family in plants. In this study, a total of 34 GmALMT genes were identified in the soybean genome. Expression patterns diverged considerably among GmALMTs in response to phosphate (Pi) starvation in leaves, roots and flowers, with expression altered by P availability in 26 of the 34 GmALMTs. One root-specific GmALMT whose expression was significantly enhanced by Pi-starvation, GmALMT5, was studied in more detail to determine its possible role in soybean P nutrition. Analysis of GmALMT5 tissue expression patterns, subcellular localization, and malate exudation from transgenic soybean hairy roots overexpressing GmALMT5, demonstrated that GmALMT5 is a plasma membrane protein that mediates malate efflux from roots. Furthermore, both growth and P content of transgenic Arabidopsis overexpressing GmALMT5 were significantly increased when sparingly soluble Ca-P was used as the external P source. Taken together, these results indicate that members of the soybean GmALMT gene family exhibit diverse responses to Pi starvation. One member of this family, GmALMT5, might contribute to soybean P efficiency by enhancing utilization of sparingly soluble P sources under P limited conditions. © 2017 Institute of Botany, Chinese Academy of Sciences.

Fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C.

PubMed

Lin, Zhaoyu; Liu, Fei; Shi, Peiliang; Song, Anying; Huang, Zan; Zou, Dayuan; Chen, Qin; Li, Jianxin; Gao, Xiang

2018-02-26

Changes in metabolic pathway preferences are key events in the reprogramming process of somatic cells to induced pluripotent stem cells (iPSCs). The optimization of metabolic conditions can enhance reprogramming; however, the detailed underlying mechanisms are largely unclear. By comparing the gene expression profiles of somatic cells, intermediate-phase cells, and iPSCs, we found that carnitine palmitoyltransferase (Cpt)1b, a rate-limiting enzyme in fatty acid oxidation, was significantly upregulated in the early stage of the reprogramming process. Mouse embryonic fibroblasts isolated from transgenic mice carrying doxycycline (Dox)-inducible Yamanaka factor constructs were used for reprogramming. Various fatty acid oxidation-related metabolites were added during the reprogramming process. Colony counting and fluorescence-activated cell sorting (FACS) were used to calculate reprogramming efficiency. Fatty acid oxidation-related metabolites were measured by liquid chromatography-mass spectrometry. Seahorse was used to measure the level of oxidative phosphorylation. We found that overexpression of cpt1b enhanced reprogramming efficiency. Furthermore, palmitoylcarnitine or acetyl-CoA, the primary and final products of Cpt1-mediated fatty acid oxidation, also promoted reprogramming. In the early reprogramming process, fatty acid oxidation upregulated oxidative phosphorylation and downregulated protein kinase C activity. Inhibition of protein kinase C also promoted reprogramming. We demonstrated that fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C activity in the early stage of the reprogramming process. This study reveals that fatty acid oxidation is crucial for the reprogramming efficiency.

Heavy metal accumulation and signal transduction in herbaceous and woody plants: Paving the way for enhancing phytoremediation efficiency.

PubMed

Luo, Zhi-Bin; He, Jiali; Polle, Andrea; Rennenberg, Heinz

2016-11-01

Heavy metal (HM)-accumulating herbaceous and woody plants are employed for phytoremediation. To develop improved strategies for enhancing phytoremediation efficiency, knowledge of the microstructural, physiological and molecular responses underlying HM-accumulation is required. Here we review the progress in understanding the structural, physiological and molecular mechanisms underlying HM uptake, transport, sequestration and detoxification, as well as the regulation of these processes by signal transduction in response to HM exposure. The significance of genetic engineering for enhancing phytoremediation efficiency is also discussed. In herbaceous plants, HMs are taken up by roots and transported into the root cells via transmembrane carriers for nutritional ions. The HMs absorbed by root cells can be further translocated to the xylem vessels and unloaded into the xylem sap, thereby reaching the aerial parts of plants. HMs can be sequestered in the cell walls, vacuoles and the Golgi apparatuses. Plant roots initially perceive HM stress and trigger the signal transduction, thereby mediating changes at the molecular, physiological, and microstructural level. Signaling molecules such as phytohormones, reactive oxygen species (ROS) and nitric oxide (NO), modulate plant responses to HMs via differentially expressed genes, activation of the antioxidative system and coordinated cross talk among different signaling molecules. A number of genes participated in HM uptake, transport, sequestration and detoxification have been functionally characterized and transformed to target plants for enhancing phytoremediation efficiency. Fast growing woody plants hold an advantage over herbaceous plants for phytoremediation in terms of accumulation of high HM-amounts in their large biomass. Presumably, woody plants accumulate HMs using similar mechanisms as herbaceous counterparts, but the processes of HM accumulation and signal transduction can be more complex in woody plants. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhanced thermoelectric response in the fractional quantum Hall effect

NASA Astrophysics Data System (ADS)

Roura-Bas, Pablo; Arrachea, Liliana; Fradkin, Eduardo

2018-02-01

We study the linear thermoelectric response of a quantum dot embedded in a constriction of a quantum Hall bar with fractional filling factors ν =1 /m within Laughlin series. We calculate the figure of merit Z T for the maximum efficiency at a fixed temperature difference. We find a significant enhancement of this quantity in the fractional filling in relation to the integer-filling case, which is a direct consequence of the fractionalization of the electron in the fractional quantum Hall state. We present simple theoretical expressions for the Onsager coefficients at low temperatures, which explicitly show that Z T and the Seebeck coefficient increase with m .

Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

PubMed

Zhang, Xiaoyue; Xu, Keyan; Ou, Yanmei; Xu, Xiaodong; Chen, Hongying

2018-05-02

The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.

Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surface.

PubMed

Balyasnikova, Irina V; Franco-Gou, Rosa; Mathis, J Michael; Lesniak, Maciej S

2010-06-01

Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright 2009 John Wiley & Sons, Ltd.

Histone-Targeted Nucleic Acid Delivery for Tissue Regenerative Applications

NASA Astrophysics Data System (ADS)

Munsell, Erik V.

Nucleic acid delivery has garnered significant attention as an innovative therapeutic approach for treating a wide variety of diseases. However, the design of non-viral delivery systems that negotiate efficient intracellular trafficking and nuclear entry represents a significant challenge. Overcoming these hurdles requires a combination of well-controlled materials approaches with techniques to understand and direct cellular delivery. Recent investigations have highlighted the roles histone tail sequences play in directing nuclear delivery and retention, as well as activating DNA transcription. We established the ability to recapitulate these natural histone tail activities within non-viral gene nanocarriers, driving gene transfer/expression by enabling effective navigation to the nucleus via retrograde vesicular trafficking. A unique finding of this histone-targeted approach was that nanocarriers gained enhanced access to the nucleus during mitosis. The work described in this dissertation builds off of these fundamental insights to facilitate the translation of this histone-targeted delivery approach toward regenerative medicine applications. During native tissue repair, actively proliferating mesenchymal stem cells (MSCs) respond to a complex series of growth factor signals that direct their differentiation. Accordingly, the investigations in this work focused on utilizing the histone-targeted nanocarriers to enhance osteogenic growth factor gene transfer in dividing MSCs leading to augmented MSC chondrogenic differentiation, an essential first step in skeletal tissue repair. Concurrently, additional studies focused on optimizing the histone-targeted nanocarrier design strategy to enable improved plasmid DNA (pDNA) binding stability and tunable harnessing of native cellular processing pathways for enhanced gene transfer. Overall, the work presented herein demonstrated substantial increases in growth factor expression following histone-targeted gene transfer. This enhanced expression enabled more robust levels of chondrogenesis in MSCs than treatments with equivalent amounts of recombinant growth factor protein. Additionally, nanocarrier design optimization provided effective pDNA condensation and controllable interactions with native histone effectors. Importantly, these optimized nanocarriers conferred stable nanoplex formation and maintained transfection efficiency under physiologically relevant conditions. Taken together, these advances may help drive the clinical translation of histone-targeted nucleic acid delivery strategies for the regeneration of damaged tissue following traumatic injury.

Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes

NASA Astrophysics Data System (ADS)

Xu, Shujuan; Li, Qian; Xiang, Junfeng; Yang, Qianfan; Sun, Hongxia; Guan, Aijiao; Wang, Lixia; Liu, Yan; Yu, Lijia; Shi, Yunhua; Chen, Hongbo; Tang, Yalin

2016-04-01

RNA G-quadruplexes (G4s) play important roles in translational regulation, mRNA processing events and gene expression. Therefore, a fluorescent probe that is capable of efficiently recognizing RNA G-quadruplex structures among other RNA forms is highly desirable. In this study, a water-soluble fluorogenic dye (i.e., Thioflavin T (ThT)) was employed to recognize RNA G-quadruplex structures using UV-Vis absorption spectra, fluorescence spectra and emission lifetime experiments. By stacking on the G-tetrad, the ThT probe exhibited highly specific recognition of RNA G-quadruplex structures with striking fluorescence enhancement compared with other RNA forms. The specific binding demonstrates that ThT is an efficient fluorescence sensor that can distinguish G4 and non-G4 RNA structures.

Escherichia coli as a production host for novel enzymes from basidiomycota.

PubMed

Zelena, Katerina; Eisele, Nadine; Berger, Ralf G

2014-12-01

Many enzymes from basidiomycota have been identified and more recently characterized on the molecular level. This report summarizes the potential biotechnological applications of these enzymes and evaluates recent advances in their heterologous expression in Escherichia coli. Being one of the most widely used hosts for the production of recombinant proteins, there are, however, recurrent problems of recovering substantial yields of correctly folded and active enzymes. Various strategies for the efficient production of recombinant proteins from basidiomycetous fungi are reviewed including the current knowledge on vectors and expression strains, as well as methods for enhancing the solubility of target expression products and their purification. Research efforts towards the refolding of recombinant oxidoreductases and hydrolases are presented to illustrate successful production strategies. Copyright © 2014 Elsevier Inc. All rights reserved.

Tree genetic engineering and applications to sustainable forestry and biomass production.

PubMed

Harfouche, Antoine; Meilan, Richard; Altman, Arie

2011-01-01

Forest trees provide raw materials, help to maintain biodiversity and mitigate the effects of climate change. Certain tree species can also be used as feedstocks for bioenergy production. Achieving these goals may require the introduction or modified expression of genes to enhance biomass production in a sustainable and environmentally responsible manner. Tree genetic engineering has advanced to the point at which genes for desirable traits can now be introduced and expressed efficiently; examples include biotic and abiotic stress tolerance, improved wood properties, root formation and phytoremediation. Transgene confinement, including flowering control, may be needed to avoid ecological risks and satisfy regulatory requirements. This and stable expression are key issues that need to be resolved before transgenic trees can be used commercially. Copyright © 2010 Elsevier Ltd. All rights reserved.

Ganoderic acid Me induces the apoptosis of competent T cells and increases the proportion of Treg cells through enhancing the expression and activation of indoleamine 2,3-dioxygenase in mouse lewis lung cancer cells.

PubMed

Que, Zujun; Zou, Fangyuan; Zhang, Anle; Zheng, Yuanhong; Bi, Ling; Zhong, Jianjiang; Tian, Jianhui; Liu, Jianwen

2014-11-01

The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. It is known that ganoderic acid Me can enhance IFN-γ expression and IDO is preferentially induced by IFN-γ. However, whether GA-Me can induce IDO expression has not been clarified yet. We established stable clones of IDO-overexpressing 2 LL cells (2LL-EGFP-IDO). After co-culturing with IDO expressing or control vector-transfected 2LL-EGFP cells, T cell apoptosis was determined and the proportion of the regulatory T cells (Tregs) and CD8+ T cell subset was measured. The total cellular protein samples of 2 LL-EGFP-IDO cells were isolated for detecting JAK-STAT1 signalling pathway. Co-culture supernatants were used to detect amino acids and cytokines. IDO transfected 2 LL cells yielded high level of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in T cells after cocultured with IDO+2LL cells and the proportion of CD4+CD25+ cells and FoxP3+ cells increased while CD8+ cells decreased. The specific inhibitor of IDO, 1-D-MT and GA-Me efficiently enhanced T cell apoptosis, increased Tregs, and reduced CD8+ T cells in vitro. Increased expression of IDO, p-JAK1 and p-STAT1 were confirmed by Western blot analysis. The levels of IFN-γ, IL-10, LDH and kynurenine in co-culture supernatant correspondingly increased, while tryptophan reduced. These results suggest that GA-Me contributing to IDO helps to create a tolerogenic milieu in lung tumors by directly inducing T cell apoptosis, restraining CD8+ T cell activation, and enhancing Treg-mediated immunosuppression. Copyright © 2014 Elsevier B.V. All rights reserved.

Responsive Proteins in Wheat Cultivars with Contrasting Nitrogen Efficiencies under the Combined Stress of High Temperature and Low Nitrogen

PubMed Central

Abd_Allah, Elsayed Fathi; Nauman, Mohd; Asif, Ambreen; Hashem, Abeer; Alqarawi, Abdulaziz A.

2017-01-01

Productivity of wheat (Triticum aestivum) is markedly affected by high temperature and nitrogen deficiency. Identifying the functional proteins produced in response to these multiple stresses acting in a coordinated manner can help in developing tolerance in the crop. In this study, two wheat cultivars with contrasting nitrogen efficiencies (N-efficient VL616 and N-inefficient UP2382) were grown in control conditions, and under a combined stress of high temperature (32 °C) and low nitrogen (4 mM), and their leaf proteins were analysed in order to identify the responsive proteins. Two-dimensional electrophoresis unravelled sixty-one proteins, which varied in their expression in wheat, and were homologous to known functional proteins involved in biosynthesis, carbohydrate metabolism, energy metabolism, photosynthesis, protein folding, transcription, signalling, oxidative stress, water stress, lipid metabolism, heat stress tolerance, nitrogen metabolism, and protein synthesis. When exposed to high temperature in combination with low nitrogen, wheat plants altered their protein expression as an adaptive means to maintain growth. This response varied with cultivars. Nitrogen-efficient cultivars showed a higher potential of redox homeostasis, protein stability, osmoprotection, and regulation of nitrogen levels. The identified stress-responsive proteins can pave the way for enhancing the multiple-stress tolerance in wheat and developing a better understanding of its mechanism. PMID:29186028

Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

PubMed Central

Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

2015-01-01

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications. PMID:26670247

In Vivo Bio-distribution and Efficient Tumor Targeting of Gelatin/Silica Nanoparticles for Gene Delivery

NASA Astrophysics Data System (ADS)

Zhao, Xueqin; Wang, Jun; Tao, SiJie; Ye, Ting; Kong, Xiangdong; Ren, Lei

2016-04-01

The non-viral gene delivery system is an attractive alternative to cancer therapy. The clinical success of non-viral gene delivery is hampered by transfection efficiency and tumor targeting, which can be individually overcome by addition of functional modules such as cell penetration or targeting. Here, we first engineered the multifunctional gelatin/silica (GS) nanovectors with separately controllable modules, including tumor-targeting aptamer AGRO100, membrane-destabilizing peptide HA2, and polyethylene glycol (PEG), and then studied their bio-distribution and in vivo transfection efficiencies by contrast resonance imaging (CRI). The results suggest that the sizes and zeta potentials of multifunctional gelatin/silica nanovectors were 203-217 nm and 2-8 mV, respectively. Functional GS-PEG nanoparticles mainly accumulated in the liver and tumor, with the lowest uptake by the heart and brain. Moreover, the synergistic effects of tumor-targeting aptamer AGRO100 and fusogenic peptide HA2 promoted the efficient cellular internalization in the tumor site. More importantly, the combined use of AGRO100 and PEG enhanced tumor gene expression specificity and effectively reduced toxicity in reticuloendothelial system (RES) organs after intravenous injection. Additionally, low accumulation of GS-PEG was observed in the heart tissues with high gene expression levels, which could provide opportunities for non-invasive gene therapy.

Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents.

PubMed

Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

2015-12-08

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications.

Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons

NASA Astrophysics Data System (ADS)

Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Zhao, Ping; Xia, Qingyou

2015-11-01

With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a “safe harbor” locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-Invasive Multielectrode Array

PubMed Central

Guo, Siqi; Israel, Annelise L.; Basu, Gaurav; Donate, Amy; Heller, Richard

2013-01-01

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after gene electrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest gene electrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient. PMID:24015305

Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

PubMed

Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

2008-12-01

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

Sensitivity of GBM cells to cAMP agonist-mediated apoptosis correlates with CD44 expression and agonist resistance with MAPK signaling.

PubMed

Daniel, Paul M; Filiz, Gulay; Mantamadiotis, Theo

2016-12-01

In some cell types, activation of the second messenger cAMP leads to increased expression of proapoptotic Bim and subsequent cell death. We demonstrate that suppression of the cAMP pathway is a common event across many cancers and that pharmacological activation of cAMP in glioblastoma (GBM) cells leads to enhanced BIM expression and apoptosis in specific GBM cell types. We identified the MAPK signaling axis as the determinant of cAMP agonist sensitivity in GBM cells, with high MAPK activity corresponding to cAMP resistance and low activity corresponding to sensitization to cAMP-induced apoptosis. Sensitive cells were efficiently killed by cAMP agonists alone, while targeting both the cAMP and MAPK pathways in resistant GBM cells resulted in efficient apoptosis. We also show that CD44 is differentially expressed in cAMP agonist-sensitive and -resistant cells. We thus propose that CD44 may be a useful biomarker for distinguishing tumors that may be sensitive to cAMP agonists alone or cAMP agonists in combination with other pathway inhibitors. This suggests that using existing chemotherapeutic compounds in combination with existing FDA-approved cAMP agonists may fast track trials toward improved therapies for difficult-to-treat cancers, such as GBM.

Increased tumor localization and reduced immune response to adenoviral vector formulated with the liposome DDAB/DOPE.

PubMed

Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Abu-Asab, Mones S; Tsokos, Maria; Morris, John C; Kalle, Wouter H J

2007-04-01

We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB-DOPE liposomes to form adenovirus-liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of beta-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) (P<0.05). The tumor to non-tumor ratio of beta-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes (P<0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more beta-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus. Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

Boronic acid-tethered amphiphilic hyaluronic acid derivative-based nanoassemblies for tumor targeting and penetration.

PubMed

Jeong, Jae Young; Hong, Eun-Hye; Lee, Song Yi; Lee, Jae-Young; Song, Jae-Hyoung; Ko, Seung-Hak; Shim, Jae-Seong; Choe, Sunghwa; Kim, Dae-Duk; Ko, Hyun-Jeong; Cho, Hyun-Jong

2017-04-15

(3-Aminomethylphenyl)boronic acid (AMPB)-installed hyaluronic acid-ceramide (HACE)-based nanoparticles (NPs), including manassantin B (MB), were fabricated for tumor-targeted delivery. The amine group of AMPB was conjugated to the carboxylic acid group of hyaluronic acid (HA) via amide bond formation, and synthesis was confirmed by spectroscopic methods. HACE-AMPB/MB NPs with a 239-nm mean diameter, narrow size distribution, negative zeta potential, and >90% drug encapsulation efficiency were fabricated. Exposed AMPB in the outer surface of HACE-AMPB NPs (in the aqueous environment) may react with sialic acid of cancer cells. The improved cellular accumulation efficiency, in vitro antitumor efficacy, and tumor penetration efficiency of HACE-AMPB/MB NPs, compared with HACE/MB NPs, in MDA-MB-231 cells (CD44 receptor-positive human breast adenocarcinoma cells) may be based on the CD44 receptor-mediated endocytosis and phenylboronic acid-sialic acid interaction. Enhanced in vivo tumor targetability, infiltration efficiency, and antitumor efficacies of HACE-AMPB NPs, compared with HACE NPs, were observed in a MDA-MB-231 tumor-xenografted mouse model. In addition to passive tumor targeting (based on an enhanced permeability and retention effect) and active tumor targeting (interaction between HA and CD44 receptor), the phenylboronic acid-sialic acid interaction can play important roles in augmented tumor targeting and penetration of HACE-AMPB NPs. STATEMENT OF SIGNIFICANCE: (3-Aminomethylphenyl)boronic acid (AMPB)-tethered hyaluronic acid-ceramide (HACE)-based nanoparticles (NPs), including manassantin B (MB), were fabricated and their tumor targeting and penetration efficiencies were assessed in MDA-MB-231 (CD44 receptor-positive human adenocarcinoma) tumor models. MB, which exhibited antitumor efficacies via the inhibition of angiogenesis and hypoxia inducible factor (HIF)-1, was entrapped in HACE-AMPB NPs in this study. Phenylboronic acid located in the outer surface of HACE-AMPB/MB NPs (in the aqueous milieu) may react with the sialic acid over-expressed in cancer cells and intramolecular B‒O bond can be formed. This phenylboronic acid-sialic acid interaction may provide additional tumor targeting and penetration potentials together with an enhanced permeability and retention (EPR) effect (passive tumor targeting) and HA-CD44 receptor interaction (active tumor targeting). Developed HACE-AMPB NP may be one of promising nanocarriers for the imaging and therapy of CD44 receptor-expressed cancers. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Inhibition of alpha interferon (IFN-α)-induced microRNA-122 negatively affects the anti-hepatitis B virus efficiency of IFN-α.

PubMed

Hao, Junli; Jin, Wensong; Li, Xinghui; Wang, Saifeng; Zhang, Xiaojun; Fan, Hongxia; Li, Changfei; Chen, Lizhao; Gao, Bin; Liu, Guangze; Meng, Songdong

2013-01-01

Alpha interferon (IFN-α)-based therapy can effectively treat chronic hepatitis B virus (HBV) infection, which causes life-threatening complications. Responses to IFN-α therapy vary greatly in chronic hepatitis B (CHB) patients, but underlying mechanisms are almost unknown. In this study, we found that IFN-α treatment induced a marked decrease of microRNA-122 (miR-122) expression in hepatocytes. We next showed that IFN-α-induced miR-122 downregulation was only partly due to transcriptional suppression. One IFN-stimulated gene (ISG), NT5C3, which was identified as a miR-122 target, efficiently inhibited miR-122 by binding and sequestering miR-122 with its mRNA 3'-untranslated region (3'-UTR), indicating that this ISG is involved in IFN-α-mediated miR-122 suppression. Notably, the inhibitory effect of IFN-α on miR-122 was completely abolished by blocking IFN-α-induced upregulation of NT5C3 mRNA expression by RNA interference (RNAi). Meanwhile, we observed that miR-122 dramatically inhibited HBV expression and replication. Finally, we showed that IFN-α-mediated HBV-inhibitory effects could be enhanced significantly by blocking IFN-α-induced downregulation of miR-122. We therefore concluded that IFN-α-induced inhibition of miR-122 may negatively affect the anti-HBV function of IFN-α. These data provide valuable insights for a better understanding of the antiviral mechanism of IFN-α and raise further potential interest in enhancing its anti-HBV efficacy.

Inhibition of Alpha Interferon (IFN-α)-Induced MicroRNA-122 Negatively Affects the Anti-Hepatitis B Virus Efficiency of IFN-α

PubMed Central

Hao, Junli; Jin, Wensong; Li, Xinghui; Wang, Saifeng; Zhang, Xiaojun; Fan, Hongxia; Li, Changfei; Chen, Lizhao; Gao, Bin

2013-01-01

Alpha interferon (IFN-α)-based therapy can effectively treat chronic hepatitis B virus (HBV) infection, which causes life-threatening complications. Responses to IFN-α therapy vary greatly in chronic hepatitis B (CHB) patients, but underlying mechanisms are almost unknown. In this study, we found that IFN-α treatment induced a marked decrease of microRNA-122 (miR-122) expression in hepatocytes. We next showed that IFN-α-induced miR-122 downregulation was only partly due to transcriptional suppression. One IFN-stimulated gene (ISG), NT5C3, which was identified as a miR-122 target, efficiently inhibited miR-122 by binding and sequestering miR-122 with its mRNA 3′-untranslated region (3′-UTR), indicating that this ISG is involved in IFN-α-mediated miR-122 suppression. Notably, the inhibitory effect of IFN-α on miR-122 was completely abolished by blocking IFN-α-induced upregulation of NT5C3 mRNA expression by RNA interference (RNAi). Meanwhile, we observed that miR-122 dramatically inhibited HBV expression and replication. Finally, we showed that IFN-α-mediated HBV-inhibitory effects could be enhanced significantly by blocking IFN-α-induced downregulation of miR-122. We therefore concluded that IFN-α-induced inhibition of miR-122 may negatively affect the anti-HBV function of IFN-α. These data provide valuable insights for a better understanding of the antiviral mechanism of IFN-α and raise further potential interest in enhancing its anti-HBV efficacy. PMID:23055569

Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Ke, E-mail: dingke@med.uestc.edu.cn; Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu 610072; Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038

Multipotent muscle satellite cells (MuSCs) have been identified as potential seed cells for bone tissue engineering. However, MuSCs exhibit a rapid loss of stemness after in vitro culturing, thereby compromising their therapeutic efficiency. Muscle segment homeobox gene 1 (msx1) has been found to induce the dedifferentiation of committed progenitor cells, as well as terminally differentiated myotubes. In this study, a Tet-off retroviral gene delivery system was used to modulate msx1 expression. After ten passages, MuSCs that did not express msx-1 (e.g., the non-msx1 group) were compared with MuSCs with induced msx-1 expression (e.g., the msx1 group). The latter group exhibitedmore » a more juvenile morphology, it contained a significantly lower percentage of senescent cells characterized by positive β-galactosidase staining, and it exhibited increased proliferation and a higher proliferation index. Immunocytochemical stainings further detected a more primitive gene expression profile for the msx1 group, while osteogenic differentiation assays and ectopic bone formation assays demonstrated an improved capacity for the msx1 group to undergo osteogenic differentiation. These results suggest that transient expression of msx1 in MuSCs can retain a primitive state, thereby enhancing their capacity for osteogenic differentiation and restoring the potential for MuSCs to serve as seed cells for bone tissue engineering.« less

MACF1 Overexpression by Transfecting the 21 kbp Large Plasmid PEGFP-C1A-ACF7 Promotes Osteoblast Differentiation and Bone Formation.

PubMed

Zhang, Yan; Yin, Chong; Hu, Lifang; Chen, Zhihao; Zhao, Fan; Li, Dijie; Ma, Jianhua; Ma, Xiaoli; Su, Peihong; Qiu, Wuxia; Yang, Chaofei; Wang, Pai; Li, Siyu; Zhang, Ge; Wang, Liping; Qian, Airong; Xian, Cory J

2018-02-01

Microtubule actin crosslinking factor 1 (MACF1) is a large spectraplakin protein known to have crucial roles in regulating cytoskeletal dynamics, cell migration, growth, and differentiation. However, its role and action mechanism in bone remain unclear. The present study investigated optimal conditions for effective transfection of the large plasmid PEGFP-C1A-ACF7 (∼21 kbp) containing full-length human MACF1 cDNA, as well as the potential role of MACF1 in bone formation. To enhance MACF1 expression, the plasmid was transfected into osteogenic cells by electroporation in vitro and into mouse calvaria with nanoparticles. Then, transfection efficiency, osteogenic marker expression, calvarial thickness, and bone formation were analyzed. Notably, MACF1 overexpression triggered a drastic increase in osteogenic gene expression, alkaline phosphatase activity, and matrix mineralization in vitro. Mouse calvarial thickness, mineral apposition rate, and osteogenic marker protein expression were significantly enhanced by local transfection. In addition, MACF1 overexpression promoted β-catenin expression and signaling. In conclusion, MACF1 overexpression by transfecting the large plasmid containing full-length MACF1 cDNA promotes osteoblast differentiation and bone formation via β-catenin signaling. Current data will provide useful experimental parameters for the transfection of large plasmids and a novel strategy based on promoting bone formation for prevention and therapy of bone disorders.

Maternal N-Carbamylglutamate Supply during Early Pregnancy Enhanced Pregnancy Outcomes in Sows through Modulations of Targeted Genes and Metabolism Pathways.

PubMed

Cai, Shuang; Zhu, Jinlong; Zeng, Xiangzhou; Ye, Qianhong; Ye, Changchuan; Mao, Xiangbing; Zhang, Shihai; Qiao, Shiyan; Zeng, Xiangfang

2018-06-13

Reducing pregnancy loss is important for improving reproductive efficiency for both human and mammalian animals. Our previous study demonstrates that maternal N-carbamylglutamate (NCG) supply during early pregnancy enhances embryonic survival in gilts. However, whether maternal NCG supply improves the pregnancy outcomes is still not known. Here we found maternal NCG supply during early pregnancy in sows significantly increased the numbers of total piglets born alive per litter ( P < 0.05) and significantly changed the levels of metabolites in amniotic fluid and serum involved in metabolism of energy, lipid, and glutathione and immunological regulation. The expression of endometrial progesterone receptor membrane component 1 (PGRMC1) was significantly increased by NCG supplementation ( P < 0.05) as well as the expression of PGRMC1, endothelial nitric oxide synthesases (eNOS), and lamin A/C in fetuses and placentae ( P < 0.05). Among the NCG-associated amino acids, arginine and glutamine, markedly increased PGRMC1 and eNOS expression in porcine trophectoderm cells ( P < 0.05), whereas glutamate could stimulate the expression of vimentin and lamin A/C in porcine trophectoderm (pTr) cells ( P < 0.05) and proline stimulated lamin A/C expression ( P < 0.05). Collectively, these data reveal the mechanisms of NCG in reducing early embryo loss. These findings have important implications that NCG has great potential to improve pregnancy outcomes in human and mammalian animals.

Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation.

PubMed

Ding, Ke; Liu, Wen-Ying; Zeng, Qiang; Hou, Fang; Xu, Jian-Zhong; Yang, Zhong

2017-03-01

Multipotent muscle satellite cells (MuSCs) have been identified as potential seed cells for bone tissue engineering. However, MuSCs exhibit a rapid loss of stemness after in vitro culturing, thereby compromising their therapeutic efficiency. Muscle segment homeobox gene 1 (msx1) has been found to induce the dedifferentiation of committed progenitor cells, as well as terminally differentiated myotubes. In this study, a Tet-off retroviral gene delivery system was used to modulate msx1 expression. After ten passages, MuSCs that did not express msx-1 (e.g., the non-msx1 group) were compared with MuSCs with induced msx-1 expression (e.g., the msx1 group). The latter group exhibited a more juvenile morphology, it contained a significantly lower percentage of senescent cells characterized by positive β-galactosidase staining, and it exhibited increased proliferation and a higher proliferation index. Immunocytochemical stainings further detected a more primitive gene expression profile for the msx1 group, while osteogenic differentiation assays and ectopic bone formation assays demonstrated an improved capacity for the msx1 group to undergo osteogenic differentiation. These results suggest that transient expression of msx1 in MuSCs can retain a primitive state, thereby enhancing their capacity for osteogenic differentiation and restoring the potential for MuSCs to serve as seed cells for bone tissue engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Silencing Hsp25/Hsp27 gene expression augments proteasome activity and increases CD8+ T-cell-mediated tumor killing and memory responses.

PubMed

Nagaraja, Ganachari M; Kaur, Punit; Neumann, William; Asea, Edwina E; Bausero, María A; Multhoff, Gabriele; Asea, Alexzander

2012-01-01

Relatively high expression of Hsp27 in breast and prostate cancer is a predictor of poor clinical outcome. This study elucidates a hitherto unknown mechanism by which Hsp27 regulates proteasome function and modulates tumor-specific T-cell responses. Here, we showed that short-term silencing of Hsp25 or Hsp27 using siRNA or permanent silencing of Hsp25 using lentivirus RNA interference technology enhanced PA28α mRNA expression, PA28α protein expression, and proteasome activity; abrogated metastatic potential; induced the regression of established breast tumors by tumor-specific CD8(+) T cells; and stimulated long-lasting memory responses. The adoptive transfer of reactive CD8(+) T cells from mice bearing Hsp25-silenced tumors efficiently induced the regression of established tumors in nontreated mice which normally succumb to tumor burden. The overexpression of Hsp25 and Hsp27 resulted in the repression of normal proteasome function, induced poor antigen presentation, and resulted in increased tumor burden. Taken together, this study establishes a paradigm shift in our understanding of the role of Hsp27 in the regulation of proteasome function and tumor-specific T-cell responses and paves the way for the development of molecular targets to enhance proteasome function and concomitantly inhibit Hsp27 expression in tumors for therapeutic gain. ©2011 AACR.

Silencing hsp25/hsp27 gene expression augments proteasome activity and increases CD8+ T cell-mediated tumor killing and memory responses

PubMed Central

Nagaraja, Ganachari M.; Kaur, Punit; Neumann, William; Asea, Edwina E.; Bausero, María A.; Multhoff, Gabriele; Asea, Alexzander

2011-01-01

Relatively high expression of Hsp27 in breast and prostate cancer is a predictor of poor clinical outcome. This study elucidates a hitherto unknown mechanism by which Hsp27 regulates proteasome function and modulates tumor-specific T cell responses. Here we demonstrated that short term silencing of Hsp25 or Hsp27 using siRNA or permanent silencing of Hsp25 using lentivirus-RNAi technology enhanced PA28α mRNA expression, PA28α protein expression, proteasome activity, abrogated metastatic potential, induced the regression of established breast tumors by tumor-specific CD8+ T cells and stimulated long-lasting memory responses. The adoptive transfer of reactive CD8+ T cells from mice bearing Hsp25-silenced tumors efficiently induced the regression of established tumors in non-treated mice which normally succumb to tumor burden. The overexpression of Hsp25 and Hsp27 resulted in the repression of normal proteasome function, induced poor antigen presentation and resulted in increased tumor burden. Taken together, this study establishes a paradigm shift in our understanding of the role of Hsp27 in the regulation of proteasome function and tumor-specific T cell responses and paves the way for the development of molecular targets to enhance proteasome function and concomitantly inhibit Hsp27 expression in tumors for therapeutic gain. PMID:22185976

Defective CXCR4 expression in aged bone marrow cells impairs vascular regeneration

PubMed Central

Shao, Hongwei; Xu, Qiyuan; Wu, Qiuling; Ma, Qi; Salgueiro, Luis; Wang, Jian’An; Eton, Darwin; Webster, Keith A; Yu, Hong

2011-01-01

The chemokine stromal cell-derived factor-1 (SDF-1) plays a critical role in mobilizing precursor cells in the bone marrow and is essential for efficient vascular regeneration and repair. We recently reported that calcium augments the expression of chemokine receptor CXCR4 and enhances the angiogenic potential of bone marrow derived cells (BMCs). Neovascularization is impaired by aging therefore we suggested that aging may cause defects of CXCR4 expression and cellular responses to calcium. Indeed we found that both the basal and calcium-induced surface expression of CXCR4 on BMCs was significantly reduced in 25-month-old mice compared with 2-month-old mice. Reduced Ca-induced CXCR4 expression in BMC from aged mice was associated with defective calcium influx. Diminished CXCR4 surface expression in BMC from aged mice correlated with diminished neovascularization in an ischemic hindlimb model with less accumulation of CD34+ progenitor cells in the ischemic muscle with or without local overexpression of SDF-1. Intravenous injection of BMCs from old mice homed less efficiently to ischemic muscle and stimulated significantly less neovascularization compared with the BMCs from young mice. Transplantation of old BMCs into young mice did not reconstitute CXCR4 functions suggesting that the defects were not reversible by changing the environment. We conclude that defects of basal and calcium-regulated functions of the CXCR4/SDF-1 axis in BMCs contribute significantly to the age-related loss of vasculogenic responses. PMID:21143386

Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins.

PubMed

Kessels, Jana Elena; Wessels, Inga; Haase, Hajo; Rink, Lothar; Uciechowski, Peter

2016-09-01

The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2'-deoxycytidine (AZA) increased intracellular (after 24 and 48h) and total cellular zinc levels (after 48h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48h. MT mRNA was significantly enhanced after 24h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells. Copyright © 2016 Elsevier GmbH. All rights reserved.

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis

PubMed Central

Alton, Eric W F W; Beekman, Jeffery M; Boyd, A Christopher; Brand, June; Carlon, Marianne S; Connolly, Mary M; Chan, Mario; Conlon, Sinead; Davidson, Heather E; Davies, Jane C; Davies, Lee A; Dekkers, Johanna F; Doherty, Ann; Gea-Sorli, Sabrina; Gill, Deborah R; Griesenbach, Uta; Hasegawa, Mamoru; Higgins, Tracy E; Hironaka, Takashi; Hyndman, Laura; McLachlan, Gerry; Inoue, Makoto; Hyde, Stephen C; Innes, J Alastair; Maher, Toby M; Moran, Caroline; Meng, Cuixiang; Paul-Smith, Michael C; Pringle, Ian A; Pytel, Kamila M; Rodriguez-Martinez, Andrea; Schmidt, Alexander C; Stevenson, Barbara J; Sumner-Jones, Stephanie G; Toshner, Richard; Tsugumine, Shu; Wasowicz, Marguerite W; Zhu, Jie

2017-01-01

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air–liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and ‘benchmarked’ against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90–100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017. PMID:27852956

The effect of environmental pH on polymeric transfection efficiency.

PubMed

Kang, Han Chang; Samsonova, Olga; Kang, Sun-Woong; Bae, You Han

2012-02-01

Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6-7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1-2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers. Copyright © 2011 Elsevier Ltd. All rights reserved.

Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

PubMed

Gan, Rui; Perez, Jessica G; Carlson, Erik D; Ntai, Ioanna; Isaacs, Farren J; Kelleher, Neil L; Jewett, Michael C

2017-05-01

The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Controlled expression of pectic enzymes in Arabidopsis thaliana enhances biomass conversion without adverse effects on growth.

PubMed

Tomassetti, Susanna; Pontiggia, Daniela; Verrascina, Ilaria; Reca, Ida Barbara; Francocci, Fedra; Salvi, Gianni; Cervone, Felice; Ferrari, Simone

2015-04-01

Lignocellulosic biomass from agriculture wastes is a potential source of biofuel, but its use is currently limited by the recalcitrance of the plant cell wall to enzymatic digestion. Modification of the wall structural components can be a viable strategy to overcome this bottleneck. We have previously shown that the expression of a fungal polygalacturonase (pga2 from Aspergillus niger) in Arabidopsis and tobacco plants reduces the levels of de-esterified homogalacturonan in the cell wall and significantly increases saccharification efficiency. However, plants expressing pga2 show stunted growth and reduced biomass production, likely as a consequence of an extensive loss of pectin integrity during the whole plant life cycle. We report here that the expression in Arabidopsis of another pectic enzyme, the pectate lyase 1 (PL1) of Pectobacterium carotovorum, under the control of a chemically inducible promoter, results, after induction of the transgene, in a saccharification efficiency similar to that of plants expressing pga2. However, lines with high levels of transgene induction show reduced growth even in the absence of the inducer. To overcome the problem of plant fitness, we have generated Arabidopsis plants that express pga2 under the control of the promoter of SAG12, a gene expressed only during senescence. These plants expressed pga2 only at late stages of development, and their growth was comparable to that of WT plants. Notably, leaves and stems of transgenic plants were more easily digested by cellulase, compared to WT plants, only during senescence. Expression of cell wall-degrading enzymes at the end of the plant life cycle may be therefore a useful strategy to engineer crops unimpaired in biomass yield but improved for bioconversion. Copyright © 2014 Elsevier Ltd. All rights reserved.

One Size Does Not Fit All: The Effect of Chain Length and Charge Density of Poly(ethylene imine) Based Copolymers on Delivery of pDNA, mRNA, and RepRNA Polyplexes.

PubMed

Blakney, Anna K; Yilmaz, Gokhan; McKay, Paul F; Becer, C Remzi; Shattock, Robin J

2018-05-03

Nucleic acid delivery systems are commonly translated between different modalities, such as DNA and RNA of varying length and structure, despite physical differences in these molecules that yield disparate delivery efficiency with the same system. Here, we synthesized a library of poly(2-ethyl-2-oxazoline)/poly(ethylene imine) copolymers with varying molar mass and charge densities in order to probe how pDNA, mRNA, and RepRNA polyplex characteristics affect transfection efficiency. The library was utilized in a full factorial design of experiment (DoE) screening, with outputs of luciferase expression, particle size, surface charge, and particle concentration. The optimal copolymer molar mass and charge density was found as 83 kDa/100%, 72 kDa/100%, and 45 kDa/80% for pDNA, RepRNA, and mRNA, respectively. While 10 of the synthesized copolymers enhanced the transfection efficiency of pDNA and mRNA, only 2 copolymers enhanced RepRNA transfection efficiency, indicating a narrow and more stringent design space for RepRNA. These findings suggest that there is not a "one size fits all" polymer for different nucleic acid species.

RAD6 promotes DNA repair and stem cell signaling in ovarian cancer and is a promising therapeutic target to prevent and treat acquired chemoresistance.

PubMed

Somasagara, R R; Spencer, S M; Tripathi, K; Clark, D W; Mani, C; Madeira da Silva, L; Scalici, J; Kothayer, H; Westwell, A D; Rocconi, R P; Palle, K

2017-11-30

Ovarian cancer (OC) is the most deadly gynecological cancer and unlike most other neoplasms, survival rates for OC have not significantly improved in recent decades. We show that RAD6, an ubiquitin-conjugating enzyme, is significantly overexpressed in ovarian tumors and its expression increases in response to carboplatin chemotherapy. RAD6 expression correlated strongly with acquired chemoresistance and malignant behavior of OC cells, expression of stem cell genes and poor prognosis of OC patients, suggesting an important role for RAD6 in ovarian tumor progression. Upregulated RAD6 enhances DNA damage tolerance and repair efficiency of OC cells and promotes their survival. Increased RAD6 levels cause histone 2B ubiquitination-mediated epigenetic changes that stimulate transcription of stem cell genes, including ALDH1A1 and SOX2, leading to a cancer stem cell phenotype, which is implicated in disease recurrence and metastasis. Downregulation of RAD6 or its inhibition using a small molecule inhibitor attenuated DNA repair signaling and expression of cancer stem cells markers and sensitized chemoresistant OC cells to carboplatin. Together, these results suggest that RAD6 could be a therapeutic target to prevent and treat acquired chemoresistance and disease recurrence in OC and enhance the efficacy of standard chemotherapy.

Generation of an Adenovirus-Parvovirus Chimera with Enhanced Oncolytic Potential

PubMed Central

El-Andaloussi, Nazim; Bonifati, Serena; Kaufmann, Johanna K.; Mailly, Laurent; Daeffler, Laurent; Deryckère, François; Nettelbeck, Dirk M.; Rommelaere, Jean

2012-01-01

In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells. PMID:22787235

An interferon regulatory factor binding site in the U5 region of the bovine leukemia virus long terminal repeat stimulates Tax-independent gene expression.

PubMed

Kiermer, V; Van Lint, C; Briclet, D; Vanhulle, C; Kettmann, R; Verdin, E; Burny, A; Droogmans, L

1998-07-01

Bovine leukemia virus (BLV) replication is controlled by both cis- and trans-acting elements. The virus-encoded transactivator, Tax, is necessary for efficient transcription from the BLV promoter, although it is not present during the early stages of infection. Therefore, sequences that control Tax-independent transcription must play an important role in the initiation of viral gene expression. This study demonstrates that the R-U5 sequence of BLV stimulates Tax-independent reporter gene expression directed by the BLV promoter. R-U5 was also stimulatory when inserted immediately downstream from the transcription initiation site of a heterologous promoter. Progressive deletion analysis of this region revealed that a 46-bp element corresponding to the 5' half of U5 is principally responsible for the stimulation. This element exhibited enhancer activity when inserted upstream or downstream from the herpes simplex virus thymidine kinase promoter. This enhancer contains a binding site for the interferon regulatory factors IRF-1 and IRF-2. A 3-bp mutation that destroys the IRF recognition site caused a twofold decrease in Tax-independent BLV long terminal repeat-driven gene expression. These observations suggest that the IRF binding site in the U5 region of BLV plays a role in the initiation of virus replication.

Generation of an adenovirus-parvovirus chimera with enhanced oncolytic potential.

PubMed

El-Andaloussi, Nazim; Bonifati, Serena; Kaufmann, Johanna K; Mailly, Laurent; Daeffler, Laurent; Deryckère, François; Nettelbeck, Dirk M; Rommelaere, Jean; Marchini, Antonio

2012-10-01

In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.

Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating gene dosage, co-expressing with chaperones and fermenting in fed-batch mode.

PubMed

Wang, Qing-Hua; Liang, Lan; Liu, Wan-Cang; Gong, Ting; Chen, Jing-Jing; Hou, Qi; Yang, Jin-Ling; Zhu, Ping

2017-06-01

The scorpion peptide BmK AngM1 was reported to exhibit evident analgesic effect, but its yield by extraction from scorpion venom limits the research and application. The heterologous expression of BmK AngM1 was achieved in Pichia pastoris in our previous study. In order to realize high-level expression of recombinant BmK AngM1 (rBmK AngM1), the gene dosage of BmK AngM1 was optimized in engineered strains. The yield of rBmK AngM1 in the four-copy strain reached up to 100 mg/L, which was further enhanced to 190 mg/L by co-expressing with chaperones of PDI, BiP, and HAC1. Moreover, the yield of rBmK AngM1 was up to 1200 mg/L by high-density fermentation in 10 L fermenter. Finally, 360 mg rBmK AngM1 was purified from 1 L cultures by a two-step purification method. The efficient and convenient techniques presented in this study could facilitate further scale-up for industrial production of rBmK AngM1.

Semianalytical model for the electromagnetic enhancement by a rectangular nanowire optical antenna on metallic substrate.

PubMed

Wan, Jianing; Zhu, Junda; Zhong, Ying; Liu, Haitao

2018-06-01

The electromagnetic enhancement by a metallic nanowire optical antenna on metallic substrate is investigated theoretically. By considering the excitation and multiple scattering of surface plasmon polaritons in the nanogap between the antenna and the substrate, we build up an intuitive and comprehensive model that provides semianalytical expressions for the electromagnetic field in the nanogap to achieve an understanding of the mechanism of electromagnetic enhancement. Our results show that antennas with short lengths that support the lowest order of resonance can achieve a high electric-field enhancement factor over a large range of incidence angles. Two phase-matching conditions are derived from the model for predicting the antenna lengths at resonance. Excitation of symmetric or antisymmetric localized surface plasmon resonance is further explained with the model. The model also shows superior computational efficiency compared to the full-wave numerical method when scanning the antenna length, the incidence angle, or the wavelength.

Expression of β-glucosidase increases trichome density and artemisinin content in transgenic Artemisia annua plants.

PubMed

Singh, Nameirakpam Dolendro; Kumar, Shashi; Daniell, Henry

2016-03-01

Artemisinin is highly effective against multidrug-resistant strains of Plasmodium falciparum, the aetiological agent of the most severe form of malaria. However, a low level of accumulation of artemisinin in Artemisia annua is a major limitation for its production and delivery to malaria endemic areas of the world. While several strategies to enhance artemisinin have been extensively explored, enhancing storage capacity in trichome has not yet been considered. Therefore, trichome density was increased with the expression of β-glucosidase (bgl1) gene in A. annua through Agrobacterium-mediated transformation. Transgene (bgl1) integration and transcript were confirmed by molecular analysis. Trichome density increased up to 20% in leaves and 66% in flowers of BGL1 transgenic plants than Artemisia control plants. High-performance liquid chromatography, time of flight mass spectrometer data showed that artemisinin content increased up to 1.4% in leaf and 2.56% in flowers (per g DW), similar to the highest yields achieved so far through metabolic engineering. Artemisinin was enhanced up to five-fold in BGL1 transgenic flowers. This study opens the possibility of increasing artemisinin content by manipulating trichomes' density, which is a major reservoir of artemisinin. Combining biosynthetic pathway engineering with enhancing trichome density may further increase artemisinin yield in A. annua. Because oral feeding of Artemisia plant cells reduced parasitemia more efficiently than the purified drug, reduced drug resistance and cost of prohibitively expensive purification process, enhanced expression should play a key role in making this valuable drug affordable to treat malaria in a large global population that disproportionally impacts low-socioeconomic areas and underprivileged children. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Expression of Beta-glucosidase increases trichome density and artemisinin content in transgenic Artemisia annua plants

PubMed Central

Singh, Nameirakpam Dolendro; Kumar, Shashi; Daniell, Henry

2015-01-01

Artemisinin is highly effective against multidrug-resistant strains of Plasmodium falciparum, the etiological agent of the most severe form of malaria. However, a low level of accumulation of artemisinin in Artemisia annua is a major limitation for its production and delivery to malaria endemic areas of the world. While several strategies to enhance artemisinin have been extensively explored, enhancing storage capacity in trichome has not yet been considered. Therefore, trichome density was increased with the expression of β glucosidase (bgl1) gene in A. annua through Agrobacterium-mediated transformation. Transgene (bgl1) integration and transcript was confirmed by molecular analysis. Trichome density increased up to 20% in leaves and 66% in flowers of BGL1 transgenic plants than Artemisia control plants. High-performance liquid chromatography (HPLC, MS-TOF) data showed that artemisinin content increased up to 1.4% in leaf and 2.56% in flowers (g-1DW), similar to the highest yields achieved so far through metabolic engineering. Artemisinin was enhanced up to 5-fold in BGL1 transgenic flowers. The present study opens the possibility of increasing artemisinin content by manipulating trichomes density, which is a major reservoir of artemisinin. Combining biosynthetic pathway engineering with enhancing trichome density may further increase artemisinin yield in A. annua. Because oral feeding of Artemisia plant cells reduced parasitemia more efficiently than the purified drug, reduced drug resistance and cost of prohibitively expensive purification process, enhanced expression should play a key role in making this valuable drug affordable to treat malaria in a large global population that disproportionally impacts low-socioeconomic areas and underprivileged children. PMID:26360801

Arctigenin Efficiently Enhanced Sedentary Mice Treadmill Endurance

PubMed Central

Chen, Jing; Yu, Liang; Hu, Lihong; Jiang, Hualiang; Shen, Xu

2011-01-01

Physical inactivity is considered as one of the potential risk factors for the development of type 2 diabetes and other metabolic diseases, while endurance exercise training could enhance fat oxidation that is associated with insulin sensitivity improvement in obesity. AMP-activated protein kinase (AMPK) as an energy sensor plays pivotal roles in the regulation of energy homeostasis, and its activation could improve glucose uptake, promote mitochondrial biogenesis and increase glycolysis. Recent research has even suggested that AMPK activation contributed to endurance enhancement without exercise. Here we report that the natural product arctigenin from the traditional herb Arctium lappa L. (Compositae) strongly increased AMPK phosphorylation and subsequently up-regulated its downstream pathway in both H9C2 and C2C12 cells. It was discovered that arctigenin phosphorylated AMPK via calmodulin-dependent protein kinase kinase (CaMKK) and serine/threonine kinase 11(LKB1)-dependent pathways. Mice treadmill based in vivo assay further indicated that administration of arctigenin improved efficiently mice endurance as reflected by the increased fatigue time and distance, and potently enhanced mitochondrial biogenesis and fatty acid oxidation (FAO) related genes expression in muscle tissues. Our results thus suggested that arctigenin might be used as a potential lead compound for the discovery of the agents with mimic exercise training effects to treat metabolic diseases. PMID:21887385

Enhanced pyruvate production in Candida glabrata by carrier engineering.

PubMed

Luo, Zhengshan; Liu, Song; Du, Guocheng; Xu, Sha; Zhou, Jingwen; Chen, Jian

2018-02-01

Pyruvate is an important organic acid that plays a key role in the central metabolic pathway. Manipulating transporters is an efficient strategy to enhance production of target organic acids and a means to understand the effects of altered intracellular pyruvate content on global metabolic networks. Efforts have been made to manipulate mitochondrial pyruvate carrier (MPC) to transport pyruvate into different subcellular compartments in Candida glabrata to demonstrate the effects of the subcellular distribution of pyruvate on central carbon metabolism. By increasing the mitochondrial pyruvate content through enhancing the rate of pyruvate transport into mitochondria, a high central carbon metabolism rate, specific growth rate and specific pyruvate production rate were obtained. Comparing the intracellular pyruvate content of engineered and control strains showed that higher intracellular pyruvate levels were not conducive to improving pyruvate productivity or central carbon metabolism. Plasma membrane expression of MPCs significantly increased the expression levels of key rate-limiting glycolytic enzymes. Moreover, pyruvate production of CGΔura3-Sp-MPC1, CGΔura3-Sp-MPC2, and CGΔura3-Sp-MPC1-Sp-MPC2 increased 134.4%, 120.3%, and 30.0%, respectively. In conclusion, lower intracellular pyruvate content enhanced central carbon metabolism and provided useful clues for improving the production of other organic acids in microorganisms. © 2017 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacob, Reed B.; Ding, Feng; Ye, Dongmei

Organophosphates are widely used for peaceful (agriculture) and military purposes (chemical warfare agents). The extraordinary toxicity of organophosphates and the risk of deployment, make it critical to develop means for their rapid and efficient deactivation. Organophosphate hydrolase (OPH) already plays an important role in organophosphate remediation, but is insufficient for therapeutic or prophylactic purposes primarily due to low substrate affinity. Current efforts focus on directly modifying the active site to differentiate substrate specificity and increase catalytic activity. Here, we present a novel strategy for enhancing the general catalytic efficiency of OPH through computational redesign of the residues that are allostericallymore » coupled to the active site and validated our design by mutagenesis. Specifically, we identify five such hot-spot residues for allosteric regulation and assay these mutants for hydrolysis activity against paraoxon, a chemical-weapons simulant. A high percentage of the predicted mutants exhibit enhanced activity over wild-type (k cat =16.63 s -1), such as T199I/T54I (899.5 s -1) and C227V/T199I/T54I (848 s -1), while the Km remains relatively unchanged in our high-throughput cell-free expression system. Further computational studies of protein dynamics reveal four distinct distal regions coupled to the active site that display significant changes in conformation dynamics upon these identified mutations. These results validate a computational design method that is both efficient and easily adapted as a general procedure for enzymatic enhancement.« less

Targeting the Intratumoral Dendritic Cells by the Oncolytic Adenoviral Vaccine Expressing RANTES Elicits Potent Antitumor Immunity

PubMed Central

Lapteva, Natalia; Aldrich, Melissa; Weksberg, David; Rollins, Lisa; Goltsova, Tatiana; Chen, Si-Yi; Huang, Xue F.

2014-01-01

Summary Dendritic cells (DCs) are professional antigen (Ag)-presenting cells capable of inducing immune responses to tumor Ags and, therefore, play a central role in the induction of antitumor immunity. There is a large amount of evidence, however, about paucity of tumor-associated DCs and that DCs’ immunogenic functions are suppressed in a tumor environment. Here we describe a potent in situ vaccine targeting tumoral DCs in vivo. This vaccine comprised of an oncolytic adenovirus expressing RANTES (regulated upon activation, normally T expressed, and presumably secreted) (Ad-RANTES-E1A), enhanced tumor infiltration, and maturation of Ag-presenting cells in vivo. In this study, we show that intratumoral vaccinations with Ad-RANTES-E1A induced significant primary tumor growth regression and blocked metastasis formation in JC and E.G-7 murine tumor models. This vaccine recruited DCs, macrophages, natural killer cells, and CD8+ T cells to the tumor site, and thus enhanced Ag-specific cytotoxic T lymphocyte responses and natural killer cell responses. DCs purified from the Ad-RANTES-E1A–treated E.G-7 tumors secreted significantly higher levels of interferon-γ and interleukin-12, as compared with control groups and more efficiently enhanced CD8+ T-cell response. This in situ immunization strategy could be a potent antitumor immunotherapy approach for aggressive established tumors. PMID:19238013

Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies Black-footed cat cloned embryos

USGS Publications Warehouse

Gómez, M. C.; Biancardi, M.N.; Jenkins, J.A.; Dumas, C.; Galiguis, J.; Wang, G.; Earle Pope, C.

2012-01-01

Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

Nanochelating based nanocomplex, GFc7, improves quality and quantity of human mesenchymal stem cells during in vitro expansion.

PubMed

Hafizi, Maryam; Hajarizadeh, Atena; Atashi, Amir; Kalanaky, Somayeh; Fakharzadeh, Saideh; Masoumi, Zahra; Nazaran, Mohammad Hassan; Soleimani, Masoud

2015-11-23

Human mesenchymal stem cells (hMSCs) have been approved for therapeutic applications. Despite the advances in this field, in vitro approaches are still required to improve the essential indices that would pave the way to a bright horizon for an efficient transplantation in the future. Nanotechnology could help to improve these approaches. Studies signified the important role of iron in stem cell metabolism and efficiency of copper chelation application for stem cell expansion For the first time, based on novel Nanochelating technology, we design an iron containing copper chelator nano complex, GFc7 and examined on hMSCs during in vitro expansion. In this study, the hMSCs were isolated, characterized and expanded in vitro in two media (with or without GFc7). Then proliferation, cell viability, cell cycle analysis, surface markers, HLADR, pluripotency genes expression, homing and antioxidative defense at genes and protein expression were investigated. Also we analyzed the spontaneous differentiation and examined osteogenic and lipogenic differentiation. GFc7 affected the expression of key genes, improving both the stemness and fitness of the cells in a precise and balanced manner. We observed significant increases in cell proliferation, enhanced expression of pluripotency genes and homing markers, improved antioxidative defense, repression of genes involved in spontaneous differentiation and exposing the hMSCs to differentiation medium indicated that pretreatment with GFc7 increased the quality and rate of differentiation. Thus, GFc7 appears to be a potential new supplement for cell culture medium for increasing the efficiency of transplantation.

Protein Engineering and Homologous Expression of Serratia marcescens Lipase for Efficient Synthesis of a Pharmaceutically Relevant Chiral Epoxyester.

PubMed

Chen, Ke-Cai; Zheng, Ming-Min; Pan, Jiang; Li, Chun-Xiu; Xu, Jian-He

2017-10-01

The lipase isolated from Serratia marcescens (LipA) is a useful biocatalyst for kinetic resolution of a pharmaceutically relevant epoxyester, (±)-3-(4'-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM], to afford optically pure (-)-MPGM, a key intermediate for the synthesis of diltiazem hydrochloride. Two mutants, LipA L315S and LipA S271F , were identified from the combinatorial saturation mutation library of 14 amino acid residues lining the substrate-binding pocket. LipA L315S , LipA S271F , and their combination LipA L315S/S271F showed 2.6-, 2.2-, and 4.6-fold improvements in their specific activities towards para-nitrophenyl butyrate (pNPB), respectively. Among these positive mutants, LipA S271F displayed a 3.5-fold higher specific activity towards the pharmaco substrate (±)-MPGM. Kinetic study showed that the improvement in catalytic efficiency of LipA S271F against (±)-MPGM was mainly resulted from the enhanced affinity between substrate and enzyme, as indicated by the decrease of K m . Furthermore, to address the insoluble expression issue in Escherichia coli, the homologous expression of LipA gene in S. marcescens was achieved by introducing it into an expression vector pUC18, resulting in ca. 20-fold higher lipase production. The significantly improved volumeric production and specific activity of S. marcescens lipase make it very attractive as a new-generation biocatalyst for more efficient and economical manufacturing of (-)-MPGM.

The Expression of TaRca2-α Gene Associated with Net Photosynthesis Rate, Biomass and Grain Yield in Bread Wheat (Triticum aestivum L.) under Field Conditions

PubMed Central

Saeed, Iqbal; Bachir, Daoura Goudia; Chen, Liang; Hu, Yin-Gang

2016-01-01

Improvement in activation of Rubisco by Rubisco activase can potentially enhance CO2 assimilation and photosynthetic efficiency in plants. The three homoeologous copies of TaRca2-α were identified on chromosomes 4AL, 4BS and 4DS (TaRca2-α-4AL, TaRca2-α-4BS, and TaRca2-α-4DS) in bread wheat. Expression patterns of the three copies at heading (Z55), anthesis (Z67) and grain-filling (Z73) stages were investigated through qRT-PCR analyses in a panel of 59 bread wheat genotypes and their effects on net photosynthesis rate (Pn), biomass plant-1 (BMPP) and grain yield plant-1 (GYPP) were further explored. Different but similar expression patterns were observed for the three copies of TaRca2-α at the three growth stages with highest expression at grain-filling stage. TaRca2-α-4BS expressed higher at the three stages than TaRca2-α-4AL and TaRca2-α-4DS. The 59 genotypes could be clustered into three groups as high (7 genotypes), intermediate (41 genotypes) and low (11 genotypes) expression based on the expression of the three copies of TaRca2-α at three growth stages. Significant variations (P<0.01) were observed among the three groups of bread wheat genotypes for Pn, BMPP and GYPP. Generally, the genotypes with higher TaRca2-α expression also showed higher values for Pn, BMPP and GYPP. The expressions of the three copies of TaRca2-α at heading, anthesis and grain-filling stages were positively correlated with Pn, BMPP and GYPP (P<0.01) with stronger association for TaRca2-α-4BS at grain-filling stage. These results revealed that the expression of TaRca2-α contribute substantially to Pn, BMPP and GYPP, and suggested that manipulating TaRca-α expression may efficiently improve Pn, BMPP and GYPP in bread wheat and detecting TaRca-α expression levels with emphasis on TaRca2-α-4BS may be a positive strategy for selection in improving photosynthetic efficiency and grain yield of bread wheat. PMID:27548477

Utilization of recombinant Trichoderma reesei expressing Aspergillus aculeatus β-glucosidase I (JN11) for a more economical production of ethanol from lignocellulosic biomass.

PubMed

Treebupachatsakul, Treesukon; Shioya, Koki; Nakazawa, Hikaru; Kawaguchi, Takashi; Morikawa, Yasushi; Shida, Yosuke; Ogasawara, Wataru; Okada, Hirofumi

2015-12-01

The capacity of Trichoderma reesei cellulase to degrade lignocellulosic biomass has been enhanced by the construction of a recombinant T. reesei strain expressing Aspergillus aculeatus β-glucosidase I. We have confirmed highly efficient ethanol production from converge-milled Japanese cedar by recombinant T. reesei expressing A. aculeatus β-glucosidase I (JN11). We investigated the ethanol productivity of JN11 and compared it with the cocktail enzyme T. reesei PC-3-7 with reinforced cellobiase activity by the commercial Novozyme 188. Results showed that the ethanol production efficiency under enzymatic hydrolysis of JN11 was comparable to the cocktail enzyme both on simultaneous saccharification and fermentation (SSF) or separate hydrolysis and fermentation (SHF) processes. Moreover, the cocktail enzyme required more protein loading for attaining similar levels of ethanol conversion as JN11. We propose that JN11 is an intrinsically economical enzyme that can eliminate the supplementation of BGL for PC-3-7, thereby reducing the cost of industrial ethanol production from lignocellulosic biomass. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Concomitant Intravenous Nitroglycerin With Intracoronary Delivery of AAV1.SERCA2a Enhances Gene Transfer in Porcine Hearts

PubMed Central

Karakikes, Ioannis; Hadri, Lahouaria; Rapti, Kleopatra; Ladage, Dennis; Ishikawa, Kiyotake; Tilemann, Lisa; Yi, Geng-Hua; Morel, Charlotte; Gwathmey, Judith K; Zsebo, Krisztina; Weber, Thomas; Kawase, Yoshiaki; Hajjar, Roger J

2012-01-01

SERCA2a gene therapy improves contractile and energetic function of failing hearts and has been shown to be associated with benefits in clinical outcomes, symptoms, functional status, biomarkers, and cardiac structure in a phase 2 clinical trial. In an effort to enhance the efficiency and homogeneity of gene uptake in cardiac tissue, we examined the effects of nitroglycerin (NTG) in a porcine model following AAV1.SERCA2a gene delivery. Three groups of Göttingen minipigs were assessed: (i) group A: control intracoronary (IC) AAV1.SERCA2a (n = 6); (ii) group B: a single bolus IC injection of NTG (50 µg) immediately before administration of intravenous (IV) AAV1.SERCA2a (n = 6); and (iii) group C: continuous IV NTG (1 µg/kg/minute) during the 10 minutes of AAV1.SERCA2a infusion (n = 6). We found that simultaneous IV infusion of NTG and AAV1.SERCA2a resulted in increased viral transduction efficiency, both in terms of messenger RNA (mRNA) as well as SERCA2a protein levels in the whole left ventricle (LV) compared to control animals. On the other hand, IC NTG pretreatment did not result in enhanced gene transfer efficiency, mRNA or protein levels when compared to control animals. Importantly, the transgene expression was restricted to the heart tissue. In conclusion, we have demonstrated that IV infusion of NTG significantly improves cardiac gene transfer efficiency in porcine hearts. PMID:22215018

Enhanced visible light generation in an active microcavity via third-harmonic conversion beyond the non-depletion approximation

NASA Astrophysics Data System (ADS)

Yu, Rong; Ding, Chunling; Wang, Jiangpeng; Zhang, Duo

2017-12-01

We explore the possibility of using an active doubly resonant microtoroid resonator to produce high-efficiency third-harmonic generation (THG) by exploiting optical third-order nonlinearity. In a microresonator, the active fundamental mode is coherently driven with a continuous-wave input laser at the telecommunication wavelength (1550 nm), and then, the visible THG signal (517 nm) is monitored via an individual bus waveguide. We thoroughly compare our results with those obtained from the conventional passive (i.e., loss) microtoroid resonator by a systematic analysis and detailed numerical simulations based on the Heisenberg-Langevin equations of motion. It is shown that the achievable THG spectrum features an ultralow critical input power. The THG power transmission can be significantly enhanced by about three orders of magnitude at a low input power of 0.1 μ W as compared with the obtained results in the passive microtoroid resonator THG system. Moreover, the THG efficiency can reach up to 100% with optical critical input power as low as a few microwatts. In turn, the analytical expressions of the critical intracavity intensity of the light in the microcavity, the critical input pump power, and the maximum THG efficiency are obtained. The enhanced THG power transmission and high conversion efficiency are attributed to a gain-induced loss compensation in the microtoroid resonator, reducing the effective loss felt by the resonator photons. With state-of-the art technologies in the field of solid-state resonators, including but not limited to microtoroids, the proposed THG scheme is experimentally realizable.

Enhanced Osteogenic and Vasculogenic Differentiation Potential of Human Adipose Stem Cells on Biphasic Calcium Phosphate Scaffolds in Fibrin Gels

PubMed Central

2016-01-01

For bone tissue engineering synthetic biphasic calcium phosphate (BCP) with a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ratio of 60/40 (BCP60/40) is successfully clinically applied, but the high percentage of HA may hamper efficient scaffold remodelling. Whether BCP with a lower HA/β-TCP ratio (BCP20/80) is more desirable is still unclear. Vascular development is needed before osteogenesis can occur. We aimed to test the osteogenic and/or vasculogenic differentiation potential as well as degradation of composites consisting of human adipose stem cells (ASCs) seeded on BCP60/40 or BCP20/80 incorporated in fibrin gels that trigger neovascularization for bone regeneration. ASC attachment to BCP60/40 and BCP20/80 within 30 min was similar (>93%). After 11 days of culture BCP20/80-based composites showed increased alkaline phosphatase activity and DMP1 gene expression, but not RUNX2 and osteonectin expression, compared to BCP60/40-based composites. BCP20/80-based composites also showed enhanced expression of the vasculogenic markers CD31 and VEGF189, but not VEGF165 and endothelin-1. Collagen-1 and collagen-3 expression was similar in both composites. Fibrin degradation was increased in BCP20/80-based composites at day 7. In conclusion, BCP20/80-based composites showed enhanced osteogenic and vasculogenic differentiation potential compared to BCP60/40-based composites in vitro, suggesting that BCP20/80-based composites might be more promising for in vivo bone augmentation than BCP60/40-based composites. PMID:27547223

Strategies for microbial synthesis of high-value phytochemicals

NASA Astrophysics Data System (ADS)

Li, Sijin; Li, Yanran; Smolke, Christina D.

2018-03-01

Phytochemicals are of great pharmaceutical and agricultural importance, but often exhibit low abundance in nature. Recent demonstrations of industrial-scale production of phytochemicals in yeast have shown that microbial production of these high-value chemicals is a promising alternative to sourcing these molecules from native plant hosts. However, a number of challenges remain in the broader application of this approach, including the limited knowledge of plant secondary metabolism and the inefficient reconstitution of plant metabolic pathways in microbial hosts. In this Review, we discuss recent strategies to achieve microbial biosynthesis of complex phytochemicals, including strategies to: (1) reconstruct plant biosynthetic pathways that have not been fully elucidated by mining enzymes from native and non-native hosts or by enzyme engineering; (2) enhance plant enzyme activity, specifically cytochrome P450 activity, by improving efficiency, selectivity, expression or electron transfer; and (3) enhance overall reaction efficiency of multi-enzyme pathways by dynamic control, compartmentalization or optimization with the host's metabolism. We also highlight remaining challenges to — and future opportunities of — this approach.

Systems analysis of the CO2 concentrating mechanism in cyanobacteria

PubMed Central

Mangan, Niall M; Brenner, Michael P

2014-01-01

Cyanobacteria are photosynthetic bacteria with a unique CO2 concentrating mechanism (CCM), enhancing carbon fixation. Understanding the CCM requires a systems level perspective of how molecular components work together to enhance CO2 fixation. We present a mathematical model of the cyanobacterial CCM, giving the parameter regime (expression levels, catalytic rates, permeability of carboxysome shell) for efficient carbon fixation. Efficiency requires saturating the RuBisCO reaction, staying below saturation for carbonic anhydrase, and avoiding wasteful oxygenation reactions. We find selectivity at the carboxysome shell is not necessary; there is an optimal non-specific carboxysome shell permeability. We compare the efficacy of facilitated CO2 uptake, CO2 scavenging, and HCO3− transport with varying external pH. At the optimal carboxysome permeability, contributions from CO2 scavenging at the cell membrane are small. We examine the cumulative benefits of CCM spatial organization strategies: enzyme co-localization and compartmentalization. DOI: http://dx.doi.org/10.7554/eLife.02043.001 PMID:24842993

Tyro3 Family-Mediated Cell Entry of Ebola and Marburg Viruses

PubMed Central

Shimojima, Masayuki; Takada, Ayato; Ebihara, Hideki; Neumann, Gabriele; Fujioka, Kouki; Irimura, Tatsuro; Jones, Steven; Feldmann, Heinz; Kawaoka, Yoshihiro

2006-01-01

Filoviruses, represented by the genera Ebolavirus and Marburgvirus, cause a lethal hemorrhagic fever in humans and in nonhuman primates. Although filovirus can replicate in various tissues or cell types in these animals, the molecular mechanisms of its broad tropism remain poorly understood. Here we show the involvement of members of the Tyro3 receptor tyrosine kinase family—Axl, Dtk, and Mer—in cell entry of filoviruses. Ectopic expression of these family members in lymphoid cells, which otherwise are highly resistant to filovirus infection, enhanced infection by pseudotype viruses carrying filovirus glycoproteins on their envelopes. This enhancement was reduced by antibodies to Tyro3 family members, Gas6 ligand, or soluble ectodomains of the members. Live Ebola viruses infected both Axl- and Dtk-expressing cells more efficiently than control cells. Antibody to Axl inhibited infection of pseudotype viruses in a number of Axl-positive cell lines. These results implicate each Tyro3 family member as a cell entry factor in filovirus infection. PMID:17005688

In Vivo Assessment of Cold Tolerance through Chlorophyll-a Fluorescence in Transgenic Zoysiagrass Expressing Mutant Phytochrome A

PubMed Central

Gururani, Mayank Anand; Venkatesh, Jelli; Ganesan, Markkandan; Strasser, Reto Jörg; Han, Yunjeong; Kim, Jeong-Il; Lee, Hyo-Yeon; Song, Pill-Soon

2015-01-01

Chlorophyll-a fluorescence analysis provides relevant information about the physiology of plants growing under abiotic stress. In this study, we evaluated the influence of cold stress on the photosynthetic machinery of transgenic turfgrass, Zoysia japonica, expressing oat phytochrome A (PhyA) or a hyperactive mutant phytochrome A (S599A) with post-translational phosphorylation blocked. Biochemical analysis of zoysiagrass subjected to cold stress revealed reduced levels of hydrogen peroxide, increased proline accumulation, and enhanced specific activities of antioxidant enzymes compared to those of control plants. Detailed analyses of the chlorophyll-a fluorescence data through the so-called OJIP test exhibited a marked difference in the physiological status among transgenic and control plants. Overall, these findings suggest an enhanced level of cold tolerance in S599A zoysiagrass cultivars as reflected in the biochemical and physiological analyses. Further, we propose that chlorophyll-a fluorescence analysis using OJIP test is an efficient tool in determining the physiological status of plants under cold stress conditions. PMID:26010864

Novel transgenic pigs with enhanced growth and reduced environmental impact

PubMed Central

Yang, Huaqiang; Liu, Dewu; Cai, Gengyuan; Li, Guoling; Mo, Jianxin; Wang, Dehua; Zhong, Cuili; Wang, Haoqiang; Sun, Yue; Shi, Junsong; Zheng, Enqin; Meng, Fanming; Zhang, Mao; He, Xiaoyan; Zhou, Rong; Zhang, Jian; Huang, Miaorong; Zhang, Ran; Li, Ning; Fan, Mingzhe; Yang, Jinzeng

2018-01-01

In pig production, inefficient feed digestion causes excessive nutrients such as phosphorus and nitrogen to be released to the environment. To address the issue of environmental emissions, we established transgenic pigs harboring a single-copy quad-cistronic transgene and simultaneously expressing three microbial enzymes, β-glucanase, xylanase, and phytase in the salivary glands. All the transgenic enzymes were successfully expressed, and the digestion of non-starch polysaccharides (NSPs) and phytate in the feedstuff was enhanced. Fecal nitrogen and phosphorus outputs in the transgenic pigs were reduced by 23.2–45.8%, and growth rate improved by 23.0% (gilts) and 24.4% (boars) compared with that of age-matched wild-type littermates under the same dietary treatment. The transgenic pigs showed an 11.5–14.5% improvement in feed conversion rate compared with the wild-type pigs. These findings indicate that the transgenic pigs are promising resources for improving feed efficiency and reducing environmental impact. PMID:29784082

Magnetic nanoparticles enhance adenovirus transduction in vitro and in vivo.

PubMed

Sapet, Cédric; Pellegrino, Christophe; Laurent, Nicolas; Sicard, Flavie; Zelphati, Olivier

2012-05-01

Adenoviruses are among the most powerful gene delivery systems. Even if they present low potential for oncogenesis, there is still a need for minimizing widespread delivery to avoid deleterious reactions. In this study, we investigated Magnetofection efficiency to concentrate and guide vectors for an improved targeted delivery. Magnetic nanoparticles formulations were complexed to a replication defective Adenovirus and were used to transduce cells both in vitro and in vivo. A new integrated magnetic procedure for cell sorting and genetic modification (i-MICST) was also investigated. Magnetic nanoparticles enhanced viral transduction efficiency and protein expression in a dose-dependent manner. They accelerated the transduction kinetics and allowed non-permissive cells infection. Magnetofection greatly improved adenovirus-mediated DNA delivery in vivo and provided a magnetic targeting. The i-MICST results established the efficiency of magnetic nanoparticles assisted viral transduction within cell sorting columns. The results showed that the combination of Magnetofection and Adenoviruses represents a promising strategy for gene therapy. Recently, a new integrated method to combine clinically approved magnetic cell isolation devices and genetic modification was developed. In this study, we validated that magnetic cell separation and adenoviral transduction can be accomplished in one reliable integrated and safe system.

Enhancement of efficiency of chitosan-based complexes for gene transfection with poly(γ-glutamic acid) by augmenting their cellular uptake and intracellular unpackage.

PubMed

Liao, Zi-Xian; Peng, Shu-Fen; Chiu, Ya-Ling; Hsiao, Chun-Wen; Liu, Hung-Yi; Lim, Woon-Hui; Lu, Hsiang-Ming; Sung, Hsing-Wen

2014-11-10

As a cationic polysaccharide, chitosan (CS) has been identified for its potential use as a non-viral vector for exogenous gene transfection. However, owing to their electrostatic interactions, CS complexes may cause difficulties in gene release upon their arrival at the site of action, thus limiting their transfection efficiency. In this work, an attempt is made to facilitate the release of a gene by incorporating a negatively-charged poly(γ-glutamic acid) (γPGA) into CS complexes in order to diminish their attractive interactions. The mechanisms of exploiting γPGA to enhance the transfection efficiency of CS complexes are elucidated. The feasibility of using this CS/γPGA-based system for DNA or siRNA transfer is explored as well. Additionally, potential of the CS/γPGA formulation to deliver disulfide bond-conjugated dual PEGylated siRNAs for multiple gene silencing is also examined. Moreover, the genetic use of pKillerRed-mem, delivered using complexes of CS and γPGA, to express a membrane-targeted KillerRed as an intrinsically generated photosensitizer for photodynamic therapy is described. Copyright © 2014 Elsevier B.V. All rights reserved.

A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong

2012-11-15

Highlights: ► An easy and direct way to prepare QDs–DNA complexes was developed. ► Surface charge of QDs was tuned with different ratio of amino and glycolate. ► Transfection efficiency was dependent on the surface zeta potentials of QDs. ► Cellular toxicity of this gene vectors is much lower than commercial liposome. ► Whole intracellular behavior of QDs–DNA complexes can be monitored in real time. -- Abstract: Nanoparticle carrier has been developed by combining water-soluble quantum dots and plasmid DNA expressed enhanced green fluorescent protein (EGFP) in a convenient and direct way. First the QDs with different surface charges weremore » obtained by coating with amino and carboxyl terminals at different ratios. Then plasmid DNA was conjugated to QDs via electrostatic interaction. The resultant QDs–DNA complexes showed enhanced resistance to DNase I digestion. The following transfection experiments demonstrated that the transfection efficiency was dependent on the surface charges on QDs. The real time imaging of the transfection process showed that the nanoparticles experienced binding, penetrating the cell membrane and entering cytoplasm in the first 6 h of transfection. The green fluorescence of EGFP began to appear after 18 h transfection and plasmid DNA was fully expressed in the following 6 h. This new QDs–DNA platform showed great potential as new gene delivery carrier.« less

A regulatory sequence from the retinoid X receptor γ gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina.

PubMed

Blixt, Maria K E; Hallböök, Finn

2016-01-01

Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors. A 208 bp gene regulatory sequence from the chicken retinoid X receptor γ gene (RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures. Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRγ gene expression in the early retina. In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRγ-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma

PubMed Central

2013-01-01

Background Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells’ ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. Methods A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Results Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells. Conclusions Our study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment. PMID:23656794

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma.

PubMed

Shen, Chan-Juan; Yang, Yu-Xiu; Han, Ethan Q; Cao, Na; Wang, Yun-Fei; Wang, Yi; Zhao, Ying-Ying; Zhao, Li-Ming; Cui, Jian; Gupta, Puja; Wong, Albert J; Han, Shuang-Yin

2013-05-09

Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells' ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells. Our study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment.

Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells

PubMed Central

Tielen, Frans; Elstak, Edo; Benschop, Julian; Grimbergen, Max; Stallen, Jan; Janssen, Richard; van Marle, Andre; Essrich, Christian

2017-01-01

Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single “all-in-one” Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities. PMID:28800587

Arabidopsis Enhanced Drought Tolerance1/HOMEODOMAIN GLABROUS11 Confers Drought Tolerance in Transgenic Rice without Yield Penalty1[W][OA

PubMed Central

Yu, Linhui; Chen, Xi; Wang, Zhen; Wang, Shimei; Wang, Yuping; Zhu, Qisheng; Li, Shigui; Xiang, Chengbin

2013-01-01

Enhancing drought tolerance without yield decrease has been a great challenge in crop improvement. Here, we report the Arabidopsis (Arabidopsis thaliana) homodomain-leucine zipper transcription factor Enhanced Drought Tolerance/HOMEODOMAIN GLABROUS11 (EDT1/HDG11) was able to confer drought tolerance and increase grain yield in transgenic rice (Oryza sativa) plants. The improved drought tolerance was associated with a more extensive root system, reduced stomatal density, and higher water use efficiency. The transgenic rice plants also had higher levels of abscisic acid, proline, soluble sugar, and reactive oxygen species-scavenging enzyme activities during stress treatments. The increased grain yield of the transgenic rice was contributed by improved seed setting, larger panicle, and more tillers as well as increased photosynthetic capacity. Digital gene expression analysis indicated that AtEDT1/HDG11 had a significant influence on gene expression profile in rice, which was consistent with the observed phenotypes of transgenic rice plants. Our study shows that AtEDT1/HDG11 can improve both stress tolerance and grain yield in rice, demonstrating the efficacy of AtEDT1/HDG11 in crop improvement. PMID:23735506

[Knockdown of ATG5 enhances the sensitivity of human renal carcinoma cells to sunitinib].

PubMed

Li, Peng; Han, Qi; Tang, Ming; Zhang, Keqin

2017-03-01

Objective To investigate the expression levels of autophagy-related gene 5 (ATG5) and microtubule-associated protein 1 light chain 3 (LC3) and their effects on sunitinib resistance in human renal carcinoma cells. Methods After clinic-pathologic feature and survival analysis, 99 renal clear cell carcinoma tissues with different histological grades were used to detect the expression of ATG5 and LC3 by immunohistochemistry. Renal carcinoma cell line A-498 was infected with lentivirus-mediated ATG5 shRNA. Western blot analysis was performed to confirm the efficiency of ATG5 knockdown. Proliferation rate of A-498 cells in control group and ATG5 low expression group was determined by flow cytometry. Finally, the survival rate was detected by MTT assay after A-498 cells were treated with different concentrations of sunitinib. Results The expression levels of ATG5 and LC3 in renal clear cell carcinoma tissues were significantly higher than those in para-tumor tissues. The expression levels of ATG5 and LC3 were associated with classification, histological grade, TNM stage and survival rate, rather than gender, age, location, tumor size. Compared with the control group, the protein expressions of ATG5 and LC3 significantly decreased in A-498 cells with ATG5 low expression. The cell proliferation rate in ATG5 downregulation group was lower than that in the control group. Compared with control group, the survival rate in ATG5 low expression group were significantly reduced in a dose-dependent manner after sunitinib treatment. Conclusion Autophagy is active in renal clear cell carcinoma, and the drug sensitivity to sunitinib in renal cancer cells can be enhanced by the downregulation of ATG5.

Folic acid-decorated polyamidoamine dendrimer mediates selective uptake and high expression of genes in head and neck cancer cells.

PubMed

Xu, Leyuan; Kittrell, Shannon; Yeudall, W Andrew; Yang, Hu

2016-11-01

Folic acid (FA)-decorated polyamidoamine dendrimer G4 (G4-FA) was synthesized and studied for targeted delivery of genes to head and neck cancer cells expressing high levels of folate receptors (FRs). Cellular uptake, targeting specificity, cytocompatibility and transfection efficiency were evaluated. G4-FA competes with free FA for the same binding site. G4-FA facilitates the cellular uptake of DNA plasmids in a FR-dependent manner and selectively delivers plasmids to FR-high cells, leading to enhanced gene expression. G4-FA is a suitable vector to deliver genes selectively to head and neck cancer cells. The fundamental understandings of G4-FA as a vector and its encouraging transfection results for head and neck cancer cells provided support for its further testing in vivo.

Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

NASA Astrophysics Data System (ADS)

Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

2013-12-01

First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

PubMed Central

Yin, Linlin; Maddison, Lisette A.; Li, Mingyu; Kara, Nergis; LaFave, Matthew C.; Varshney, Gaurav K.; Burgess, Shawn M.; Patton, James G.; Chen, Wenbiao

2015-01-01

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. PMID:25855067

Au@Pt nanoparticles as catalase mimics to attenuate tumor hypoxia and enhance immune cell-mediated cytotoxicity

NASA Astrophysics Data System (ADS)

Liang, Hong; Wu, Ying; Ou, Xiang-Yu; Li, Jing-Ying; Li, Juan

2017-11-01

Hypoxic tumor microenvironment (TME) is closely linked to tumor progression, heterogeneity and immune suppression. Therefore, the development of effective methods to overcome hypoxia and substantially enhance the immunotherapy efficacy remains a desirable goal. Herein, we engineered a biocompatible Au core/Pt shell nanoparticles (Au@Pt NPs) to reoxygenate the TME by reacting with endogenous H2O2. Treatment with Au@Pt NPs appeared to improve oxygen in intracellular environments and decrease hypoxia-inducible factor-1α expression. Furthermore, the integration of high catalytic efficiency of Au@Pt NPs with cytokine-induced killer (CIK) cell immunotherapy, could lead to significantly improve the effect of CIK cell-mediated cytotoxicity. These results suggest great potential of Au@Pt NPs for regulation of the hypoxic TME and enhance immune cell mediated anti-tumor immunity.

Human Embryonic and Induced Pluripotent Stem Cells Express TRAIL Receptors and Can Be Sensitized to TRAIL-Induced Apoptosis

PubMed Central

Vinarsky, Vladimir; Krivanek, Jan; Rankel, Liina; Nahacka, Zuzana; Barta, Tomas; Jaros, Josef; Andera, Ladislav

2013-01-01

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis. PMID:23806100

Transgenic mice that accept Luciferase- or GFP-expressing syngeneic tumor cells at high efficiencies.

PubMed

Aoyama, Naoki; Miyoshi, Hiroyuki; Miyachi, Hitoshi; Sonoshita, Masahiro; Okabe, Masaru; Taketo, Makoto Mark

2018-05-11

Jellyfish green fluorescent protein (GFP) and firefly luciferase can serve as versatile tracking markers for identification and quantification of transplanted cancer cells in vivo. However, immune reactions against these markers can hamper the formation of syngraft tumors and metastasis that follows. Here, we report two transgenic (Tg) mouse lines that express nonfunctional mutant marker proteins, namely modified firefly luciferase (Luc2) or enhanced GFP (EGFP). These mice, named as Tg-mLuc2 and Tg-mEGFP, turned out to be immunologically tolerant to the respective tracking markers and thus efficiently accepted syngeneic cancer cells expressing the active forms of the markers. We then injected intrarectally the F 1 hybrid Tg mice (BALB/c × C57BL/6J) with Colon-26 (C26) colon cancer cells that originated from a BALB/c mouse. Even when C26 cells expressed active Luc2 or EGFP, they formed primary tumors in the Tg mice with only 10 4 cells per mouse compared with more than 10 6 cells required in the nontransgenic BALB/c hosts. Furthermore, we detected metastatic foci of C26 cells in the liver and lungs of the Tg mice by tracking the specific reporter activities. These results show the usefulness of the Tg mouse lines as recipients for transplantation experiments with the non-self tracking marker-expressing cells. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

NASA Astrophysics Data System (ADS)

Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

2008-08-01

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

CADM1/TSLC1 Identifies HTLV-1-Infected Cells and Determines Their Susceptibility to CTL-Mediated Lysis

PubMed Central

Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.

2016-01-01

Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax–at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax–HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax−CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+CADM1– cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax–CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus. PMID:27105228

Analysis of Whitefly Transcriptional Responses to Beauveria bassiana Infection Reveals New Insights into Insect-Fungus Interactions

PubMed Central

Zhang, Shan; Li, Fang-Fang; Feng, Ming-Guang; Wang, Xiao-Wei; Liu, Shu-Sheng

2013-01-01

Background The fungal pathogen, Beauveria bassiana, is an efficient biocontrol agent against a variety of agricultural pests. A thorough understanding of the basic principles of insect-fungus interactions may enable the genetic modification of Beauveria bassiana to enhance its virulence. However, the molecular mechanism of insect response to Beauveria bassiana infection is poorly understood, let alone the identification of fungal virulent factors involved in pathogenesis. Methodology/Principal Findings Here, next generation sequencing technology was applied to examine the expression of whitefly (Bemisia tabaci) genes in response to the infection of Beauveria bassiana. Results showed that, compared to control, 654 and 1,681genes were differentially expressed at 48 hours and 72 hours post-infected whiteflies, respectively. Functional and enrichment analyses indicated that the DNA damage stimulus response and drug metabolism were important anti-fungi strategies of the whitefly. Mitogen-activated protein kinase (MAPK) pathway was also likely involved in the whitefly defense responses. Furthermore, the notable suppression of general metabolism and ion transport genes observed in 72 hours post-infected B. tabaci might be manipulated by fungal secreted effectors. By mapping the sequencing tags to B. bassiana genome, we also identified a number of differentially expressed fungal genes between the early and late infection stages. These genes are generally associated with fungal cell wall synthesis and energy metabolism. The expression of fungal cell wall protein genes might play an important role in fungal pathogenesis and the dramatically up-regulated enzymes of carbon metabolism indicate the increasing usage of energy during the fungal infection. Conclusions/Significance To our knowledge, this is the first report on the molecular mechanism of fungus-whitefly interactions. Our results provide a road map for future investigations on insect-pathogen interactions and genetically modifying the fungus to enhance its efficiency in whitefly control. PMID:23861870

Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

PubMed

Liu, Xin; Li, Yun-Pan; Zhong, Zhen-Min; Tan, Hui-Qi; Lin, Hao-Peng; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

2017-02-01

The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

Novel Nano-Therapeutic Approach Actively Targets Human Ovarian Cancer Stem Cells after Xenograft into Nude Mice.

PubMed

Abou-ElNaga, Amoura; Mutawa, Ghada; El-Sherbiny, Ibrahim M; Abd-ElGhaffar, Hassan; Allam, Ahmed A; Ajarem, Jamaan; Mousa, Shaker A

2017-04-12

The power of tumorigenesis, chemo-resistance and metastasis in malignant ovarian tumors resides in a tiny population of cancer cells known as ovarian cancer stem cells (OCSCs). Developing nano-therapeutic targeting of OCSCs is considered a great challenge. The potential use of poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) was investigated as a drug delivery system for paclitaxel (PTX) against OCSCs in vitro and in vivo. PTX-loaded PLGA NPs were prepared by an emulsion solvent evaporation method, supported by incorporation of folic acid (FA) as the ligand. NPs were characterized for size, surface morphology, drug loading, and encapsulation efficiency. In vitro cytotoxicity of PTX-loaded FA/PLGA NPs was tested against OCSCs with MTT assay. In vivo anti-tumoral efficiency and active targeting potential of prepared NPs against tumors in nude mice were investigated. In vitro results revealed that IC 50 of PTX was significantly reduced after loading on PLGA NPs. On the other hand, in vivo results showed that PLGA NPs enhanced the tumor suppression efficiency of PTX. Investigation with real time quantitative PCR analysis revealed the limiting expression of chemo-resistant genes ( ABCG2 and MDR1 ) after applying PLGA NPs as a drug delivery system for PTX. Histopathological examination of tumors showed the effective biological influence of PTX-loaded FA/PLGA NPs through the appearance of reactive lymphoid follicles. Targeting potential of PTX was activated by FA/PLGA NPs through significant preservation of body weight ( p < 0.0001) and minimizing the systemic toxicity in healthy tissues. Immunohistochemical investigation revealed a high expression of apoptotic markers in tumor tissue, supporting the targeting effect of FA/PLGA NPs. A drug delivery system based on FA/PLGA NPs can enhance PTX's in vitro cytotoxicity and in vivo targeting potential against OCSCs.

Gene therapy for ocular diseases meditated by ultrasound and microbubbles (Review)

PubMed Central

WAN, CAIFENG; LI, FENGHUA; LI, HONGLI

2015-01-01

The eye is an ideal target organ for gene therapy as it is easily accessible and immune-privileged. With the increasing insight into the underlying molecular mechanisms of ocular diseases, gene therapy has been proposed as an effective approach. Successful gene therapy depends on efficient gene transfer to targeted cells to prove stable and prolonged gene expression with minimal toxicity. At present, the main hindrance regarding the clinical application of gene therapy is not the lack of an ideal gene, but rather the lack of a safe and efficient method to selectively deliver genes to target cells and tissues. Ultrasound-targeted microbubble destruction (UTMD), with the advantages of high safety, repetitive applicability and tissue targeting, has become a potential strategy for gene- and drug delivery. When gene-loaded microbubbles are injected, UTMD is able to enhance the transport of the gene to the targeted cells. High-amplitude oscillations of microbubbles act as cavitation nuclei which can effectively focus ultrasound energy, produce oscillations and disruptions that increase the permeability of the cell membrane and create transient pores in the cell membrane. Thereby, the efficiency of gene therapy can be significantly improved. The UTMD-mediated gene delivery system has been widely used in pre-clinical studies to enhance gene expression in a site-specific manner in a variety of organs. With reasonable application, the effects of sonoporation can be spatially and temporally controlled to improve localized tissue deposition of gene complexes for ocular gene therapy applications. In addition, appropriately powered, focused ultrasound combined with microbubbles can induce a reversible disruption of the blood-retinal barrier with no significant side effects. The present review discusses the current status of gene therapy of ocular diseases as well as studies on gene therapy of ocular diseases meditated by UTMD. PMID:26151686

Dietary silymarin supplementation promotes growth performance and improves lipid metabolism and health status in grass carp (Ctenopharyngodon idellus) fed diets with elevated lipid levels.

PubMed

Xiao, Peizhen; Ji, Hong; Ye, Yuantu; Zhang, Baotong; Chen, Yongsheng; Tian, Jingjing; Liu, Pin; Chen, Liqiao; Du, Zhenyu

2017-02-01

This study was carried out to evaluate whether silymarin supplementation influences growth, lipid metabolism, and health status in grass carp fed elevated dietary lipid levels. The juvenile fish (27.43 ± 0.17 g/tail) were fed six isonitrogenous and isocaloric diets in a factorial design containing 0, 100, or 200 mg kg -1 silymarin (SM0, SM100, SM200) associated with either 4 or 8 % lipid level (low lipid, LL, and high lipid, HL, respectively) for 82 days. The results showed that both dietary silymarin supplementation and high lipid level significantly enhanced growth performance (WG, SGR), protein efficiency ratio, and feed utilization. Silymarin supplementation significantly reduced the VSI, hepatic lipid content, and the total bilirubin concentration in the serum. The gallbladdersomatic index displayed higher in the SM100 groups than SM200 groups. Serum total cholesterol content exhibited lower in the SM100 groups than SM0 groups. Meanwhile, significant interactions were shown for hepatic gene expression of HSL and CPT1 by two factors, and SM100 group had higher hepatic gene expression of HSL and CPT1 in fish fed with the HL diets. The SM100 groups up-regulated hepatic gene expressions of HMGCR and CYP7A1 compared with the SM0 groups. Silymarin supplementation notably reduced the elevated serum MDA content induced by HL treatments. Thus, silymarin supplementation markedly promoted growth and protein efficiency, suppressed lipid accumulation, and improved health status in grass carp fed with high-lipid diets, which might be associated with its enhancement of lipolysis and β-oxidation, antioxidant capacity.

A lentiviral vector with a short troponin-I promoter for tracking cardiomyocyte differentiation of human embryonic stem cells.

PubMed

Gallo, P; Grimaldi, S; Latronico, M V G; Bonci, D; Pagliuca, A; Gallo, P; Ausoni, S; Peschle, C; Condorelli, G

2008-02-01

Human embryonic stem cells (hESCs) may become important for cardiac repair due to their potentially unlimited ability to generate cardiomyocytes (CMCs). Moreover, genetic manipulation of hESC-derived CMCs would be a very promising technique for curing myocardial disorders. At the present time, however, inducing the differentiation of hESCs into CMCs is extremely difficult and, therefore, an easy and standardizable technique is needed to evaluate differentiation strategies. Vectors driving cardiac-specific expression may represent an important tool not only for monitoring new cardiac-differentiation strategies, but also for the manipulation of cardiac differentiation of ESCs. To this aim, we generated cardiac-specific lentiviral vectors (LVVs) in which expression is driven by a short fragment of the cardiac troponin-I proximal promoter (TNNI3) with a human cardiac alpha-actin enhancer, and tested its suitability in inducing tissue-specific gene expression and ability to track the CMC lineage during differentiation of ESCs. We determined that (1) TNNI3-LVVs efficiently drive cardiac-specific gene expression and mark the cardiomyogenic lineage in human and mouse ESC differentiation systems (2) the cardiac alpha-actin enhancer confers a further increase in gene-expression specificity of TNNI3-LVVs in hESCs. Although this technique may not be useful in tracking small numbers of cells, data suggested that TNNI3-based LVVs are a powerful tool for manipulating human ESCs and modifying hESC-derived CMCs.

Heterologous expression of Homo sapiens alpha-folate receptors in E. coli by fusion with a trigger factor for enhanced solubilization.

PubMed

Miranda, Beatriz Nogueira Messias; Fotoran, Wesley Luzetti; Canduri, Fernanda; Souza, Dulce Helena Ferreira; Wunderlich, Gerhard; Carrilho, Emanuel

2018-02-01

The role of Alpha folate receptors (FRα) in folate metabolism and cancer development has been extensively studied. The reason for this is not only associated to its direct relation to disease development but also to its potential use as a highly sensitive and specific biomarker for cancers therapies. Over the recent years, the crystal structures of human FRα complexed with different ligands were described relying on an expensive and time-consuming production process. Here, we constructed an efficient system for the expression and purification of a human FRα in E. coli. Unlike a conventional expression method we used a specific protein fusion expressing the target protein together with a trigger factor (TF). This factor is a chaperone from E. coli that assists the correct folding of newly synthesized polypeptide chains. The activity of rTFFRα was comparable to glycosylphosphatidylinositol (GPI) anchored proteins extracted from HeLa tumor cells. Our work demonstrates a straightforward and versatile approach for the production of active human FRα by heterologous expression; this approach further enhances the development of inhibition studies and biotechnological applications. The purified product was then conjugated to liposomes, obtaining a 35% higher signal from densitometry measurement on the immunoblotting assay in the contruct containing the Ni-NTA tag, as a mimesis of an exosome, which is of vital importance to nanotherapeutic techniques associated to treatment and diagnosis of tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

[Spectral scatter correction of coal samples based on quasi-linear local weighted method].

PubMed

Lei, Meng; Li, Ming; Ma, Xiao-Ping; Miao, Yan-Zi; Wang, Jian-Sheng

2014-07-01

The present paper puts forth a new spectral correction method based on quasi-linear expression and local weighted function. The first stage of the method is to search 3 quasi-linear expressions to replace the original linear expression in MSC method, such as quadratic, cubic and growth curve expression. Then the local weighted function is constructed by introducing 4 kernel functions, such as Gaussian, Epanechnikov, Biweight and Triweight kernel function. After adding the function in the basic estimation equation, the dependency between the original and ideal spectra is described more accurately and meticulously at each wavelength point. Furthermore, two analytical models were established respectively based on PLS and PCA-BP neural network method, which can be used for estimating the accuracy of corrected spectra. At last, the optimal correction mode was determined by the analytical results with different combination of quasi-linear expression and local weighted function. The spectra of the same coal sample have different noise ratios while the coal sample was prepared under different particle sizes. To validate the effectiveness of this method, the experiment analyzed the correction results of 3 spectral data sets with the particle sizes of 0.2, 1 and 3 mm. The results show that the proposed method can eliminate the scattering influence, and also can enhance the information of spectral peaks. This paper proves a more efficient way to enhance the correlation between corrected spectra and coal qualities significantly, and improve the accuracy and stability of the analytical model substantially.

The Y-located gonadoblastoma gene TSPY amplifies its own expression through a positive feedback loop in prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kido, Tatsuo; Lau, Yun-Fai Chris, E-mail: Chris.Lau@UCSF.edu

2014-03-28

Highlights: • Y-encoded proto-oncoprotein TSPY amplifies its expression level via a positive feedback loop. • TSPY binds to the chromatin/DNA at exon 1 of TSPY gene. • TSPY enhances the gene expression in a TSPY exon 1 sequence dependent manner. • The conserved SET/NAP-domain is essential for TSPY transactivation. • Insights on probable mechanisms on TSPY exacerbation on cancer development in men. - Abstract: The testis-specific protein Y-encoded (TSPY) is a repetitive gene located on the gonadoblastoma region of the Y chromosome, and has been considered to be the putative gene for this oncogenic locus on the male-only chromosome. Itmore » is expressed in spermatogonial cells and spermatocytes in normal human testis, but abundantly in gonadoblastoma, testicular germ cell tumors and a variety of somatic cancers, including melanoma, hepatocellular carcinoma and prostate cancer. Various studies suggest that TSPY accelerates cell proliferation and growth, and promotes tumorigenesis. In this report, we show that TSPY could bind directly to the chromatin/DNA at exon 1 of its own gene, and greatly enhance the transcriptional activities of the endogenous gene in the LNCaP prostate cancer cells. Domain mapping analyses of TSPY have localized the critical and sufficient domain to the SET/NAP-domain. These results suggest that TSPY could efficiently amplify its expression and oncogenic functions through a positive feedback loop, and contribute to the overall tumorigenic processes when it is expressed in various human cancers.« less

Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche

PubMed Central

Han, Xue; Ma, Shurong; Kong, Xianghui; Takano, Tetsuo; Liu, Shenkui

2013-01-01

Poplar is a model organism for high in vitro regeneration in woody plants. We have chosen a hybrid poplar Populus davidiana Dode × Populus bollena Lauche. By optimizing the Murashige and Skoog medium with (0.3 mg/L) 6-benzylaminopurine and (0.08 mg/L) naphthaleneacetic acid, we have achieved the highest frequency (90%) for shoot regeneration from poplar leaves. It was also important to improve the transformation efficiency of poplar for genetic breeding and other applications. In this study, we found a significant improvement of the transformation frequency by controlling the leaf age. Transformation efficiency was enhanced by optimizing the Agrobacterium concentration (OD600 = 0.8–1.0) and an infection time (20–30 min). According to transmission electron microscopy observations, there were more Agrobacterium invasions in the 30-day-old leaf explants than in 60-day-old and 90-day-old explants. Using the green fluorescent protein (GFP) marker, the expression of MD–GFP fusion proteins in the leaf, shoot, and root of hybrid poplar P. davidiana Dode × P. bollena Lauche was visualized for confirmation of transgene integration. Southern and Northern blot analysis also showed the integration of T-DNA into the genome and gene expression of transgenic plants. Our results suggest that younger leaves had higher transformation efficiency (~30%) than older leaves (10%). PMID:23354481

Insights into Resistance to Fe Deficiency Stress from a Comparative Study of In Vitro-Selected Novel Fe-Efficient and Fe-Inefficient Potato Plants

PubMed Central

Boamponsem, Georgina A.; Leung, David W. M.; Lister, Carolyn

2017-01-01

Iron (Fe) deficiency induces chlorosis (IDC) in plants and can result in reduced plant productivity. Therefore, development of Fe-efficient plants is of great interest. To gain a better understanding of the physiology of Fe-efficient plants, putative novel plant variants were regenerated from potato (Solanum tubersosum L. var. ‘Iwa’) callus cultures selected under Fe deficient or low Fe supply (0–5 μM Fe). Based on visual chlorosis rating (VCR), 23% of callus-derived regenerants were classified as Fe-efficient (EF) and 77% as Fe-inefficient (IFN) plant lines when they were grown under Fe deficiency conditions. Stem height was found to be highly correlated with internodal distance, leaf and root lengths in the EF plant lines grown under Fe deficiency conditions. In addition, compared to the IFN plant lines and control parental biotype, the EF plants including the lines named A1, B2, and B9, exhibited enhanced formation of lateral roots and root hairs as well as increased expression of ferritin (fer3) in the leaf and iron-regulated transporter (irt1) in the root. These morphological adaptations and changes in expression the fer3 and irt1 genes of the selected EF potato lines suggest that they are associated with resistance to low Fe supply stress. PMID:28955367

Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System.

PubMed

Stachler, Aris-Edda; Marchfelder, Anita

2016-07-15

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System*

PubMed Central

Stachler, Aris-Edda; Marchfelder, Anita

2016-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. PMID:27226589

A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen

PubMed Central

Chan, Kok-Fei; Oveissi, Sara; Jackson, Heather M.; Dimopoulos, Nektaria; Guillaume, Philippe; Knights, Ashley J.; Lowen, Tamara; Robson, Neil C.; Russell, Sarah E.; Scotet, Emmanuel; Davis, Ian D.; Maraskovsky, Eugene; Cebon, Jonathan; Luescher, Immanuel F.; Chen, Weisan

2012-01-01

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8+ T cell epitope, NY-ESO-188–96 (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1157–165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-188–96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1157–165. On the other hand, NY-ESO-1157–165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A26–35; whereas NY-ESO-188–96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-188–96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-188–96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8+ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed. PMID:22970293

Development of "Purple Endosperm Rice" by Engineering Anthocyanin Biosynthesis in the Endosperm with a High-Efficiency Transgene Stacking System.

PubMed

Zhu, Qinlong; Yu, Suize; Zeng, Dongchang; Liu, Hongmei; Wang, Huicong; Yang, Zhongfang; Xie, Xianrong; Shen, Rongxin; Tan, Jiantao; Li, Heying; Zhao, Xiucai; Zhang, Qunyu; Chen, Yuanling; Guo, Jingxing; Chen, Letian; Liu, Yao-Guang

2017-07-05

Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice (Oryza sativa L.), could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes (two regulatory genes from maize and six structural genes from Coleus) driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm "Purple Endosperm Rice" (called "Zijingmi" in Chinese), which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation (or enhancement) of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Modification of the hTERT promoter by heat shock elements enhances the efficiency and specificity of cancer targeted gene therapy.

PubMed

Wang, Xiaolong; Zhou, PeiHua; Sun, XueJun; Wei, GuangBing; Zhang, Li; Wang, Hui; Yao, JianFeng; Jia, PengBo; Zheng, JianBao

2016-05-01

One of the current challenges facing cancer gene therapy is the tumour-specific targeting of therapeutic genes. Effective targeting in gene therapy requires accurate spatial and temporal control of gene expression. To develop a sufficient and accurate tumour-targeting method for cancer gene therapy, we have investigated the use of hyperthermia to control the expression of a transgene under the control of the human telomerase reverse transcriptase (hTERT) promoter and eight heat shock elements (8HSEs). Luciferase reporters were constructed by inserting eight HSEs and the hTERT promoter (8HSEs-hTERTp) upstream of the pGL4.20 vector luciferase gene. The luciferase activity of the hTERT promoter and 8HSEs-hTERT promoter were then compared in the presence and absence of heat. The differences in luciferase activity were analysed using dual luciferase assays in SW480 (high hTERT expression), MKN28 and MRC-5 cells (low hTERT expression). The luciferase activity of the Hsp70B promoter was also compared to the 8HSEs-hTERT promoter in the above listed cell lines. Lentiviral vector and heat-induced expression of EGFP expression under the control of the 8HSEs-hTERT promoter in cultured cells and mouse tumour xenografts was measured by reverse transcription polymerase (RT-PCR), Western blot and immunofluorescence assays. hTERT promoter activity was higher in SW480 cells than in MKN28 or MRC-5 cells. At 43 °C, the luciferase activity of the 8HSEs-hTERT promoter was significantly increased in SW480 cells, but not in MKN28 or MRC-5 cells. Importantly, the differences in luciferase activity were much more obvious in both high (SW480) and low (MKN28 and MRC-5) hTERT expressing cells when the activity of the 8HSEs-hTERT promoter was compared to the Hsp70B promoter. Moreover, under the control of 8HSEs-hTERT promoter in vitro and in vivo, EGFP expression was obviously increased by heat treatment in SW480 cells but not in MKN28 or MRC-5 cells, nor was expression increased under normal temperature conditions. The hTERT promoter is a potentially powerful tumour-specific promoter and gene therapy tool for cancer treatment. Incorporating heat-inducible therapeutic elements (8HSEs) into the hTERT promoter may enhance the efficiency and specificity of cancer targeting gene therapy under hyperthermic clinical conditions.

A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, Trichoderma reesei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subramanian, Venkataramanan; Schuster, Logan A.; Moore, Kyle T.

Here, the industrial workhorse fungus, Trichoderma reesei, is typically exploited for its ability to produce cellulase enzymes, whereas use of this fungus for over-expression of other proteins (homologous and heterologous) is still very limited. Identifying transformants expressing target protein is a tedious task due to low transformation efficiency, combined with highly variable expression levels between transformants. Routine methods for identification include PCR-based analysis, western blotting, or crude activity screening, all of which are time-consuming techniques. To simplify this screening, we have adapted the 2A peptide system from the foot-and-mouth disease virus (FMDV) to T. reesei to express a readily screenablemore » marker protein that is co-translated with a target protein. The 2A peptide sequence allows multiple independent genes to be transcribed as a single mRNA. Upon translation, the 2A peptide sequence causes a 'ribosomal skip' generating two (or more) independent gene products. When the 2A peptide is translated, the 'skip' occurs between its two C-terminal amino acids (glycine and proline), resulting in the addition of extra amino acids on the C terminus of the upstream protein and a single proline addition to the N terminus of the downstream protein. To test this approach, we have cloned two heterologous proteins on either side of a modified 2A peptide, a secreted cellobiohydrolase enzyme (Cel7A from Penicillium funiculosum) as our target protein, and an intracellular enhanced green fluorescent protein (eGFP) as our marker protein. Using straightforward monitoring of eGFP expression, we have shown that we can efficiently monitor the expression of the target Cel7A protein.« less

A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, Trichoderma reesei

DOE PAGES

Subramanian, Venkataramanan; Schuster, Logan A.; Moore, Kyle T.; ...

2017-02-06

Here, the industrial workhorse fungus, Trichoderma reesei, is typically exploited for its ability to produce cellulase enzymes, whereas use of this fungus for over-expression of other proteins (homologous and heterologous) is still very limited. Identifying transformants expressing target protein is a tedious task due to low transformation efficiency, combined with highly variable expression levels between transformants. Routine methods for identification include PCR-based analysis, western blotting, or crude activity screening, all of which are time-consuming techniques. To simplify this screening, we have adapted the 2A peptide system from the foot-and-mouth disease virus (FMDV) to T. reesei to express a readily screenablemore » marker protein that is co-translated with a target protein. The 2A peptide sequence allows multiple independent genes to be transcribed as a single mRNA. Upon translation, the 2A peptide sequence causes a 'ribosomal skip' generating two (or more) independent gene products. When the 2A peptide is translated, the 'skip' occurs between its two C-terminal amino acids (glycine and proline), resulting in the addition of extra amino acids on the C terminus of the upstream protein and a single proline addition to the N terminus of the downstream protein. To test this approach, we have cloned two heterologous proteins on either side of a modified 2A peptide, a secreted cellobiohydrolase enzyme (Cel7A from Penicillium funiculosum) as our target protein, and an intracellular enhanced green fluorescent protein (eGFP) as our marker protein. Using straightforward monitoring of eGFP expression, we have shown that we can efficiently monitor the expression of the target Cel7A protein.« less

An estrogen receptor targeted ruthenium complex as a two-photon photodynamic therapy agent for breast cancer cells.

PubMed

Zhao, Xueze; Li, Mingle; Sun, Wen; Fan, Jiangli; Du, Jianjun; Peng, Xiaojun

2018-06-21

In this study, we reported a tamoxifen modified Ru(ii) polypyridyl complex (Ru-tmxf) as an estrogen receptor (ER) targeted photosensitizer. Ru-tmxf displays enhanced cellular uptake and PDT efficiency toward breast cancer cells with high ER expression due to the specific targeting of tamoxifen to ER and finally localizes in lysosomes. Moreover, Ru-tmxf can be activated by two-photon excitation, generating 1O2 to damage lysosomes and result in cell death.

Glycoprotein-Based Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Infectious Laryngotracheitis

PubMed Central

Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta

2015-01-01

For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. PMID:25694519

High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize.

PubMed

Qi, Weiwei; Zhu, Tong; Tian, Zhongrui; Li, Chaobin; Zhang, Wei; Song, Rentao

2016-08-11

CRISPR/Cas9 genome editing strategy has been applied to a variety of species and the tRNA-processing system has been used to compact multiple gRNAs into one synthetic gene for manipulating multiple genes in rice. We optimized and introduced the multiplex gene editing strategy based on the tRNA-processing system into maize. Maize glycine-tRNA was selected to design multiple tRNA-gRNA units for the simultaneous production of numerous gRNAs under the control of one maize U6 promoter. We designed three gRNAs for simplex editing and three multiple tRNA-gRNA units for multiplex editing. The results indicate that this system not only increased the number of targeted sites but also enhanced mutagenesis efficiency in maize. Additionally, we propose an advanced sequence selection of gRNA spacers for relatively more efficient and accurate chromosomal fragment deletion, which is important for complete abolishment of gene function especially long non-coding RNAs (lncRNAs). Our results also indicated that up to four tRNA-gRNA units in one expression cassette design can still work in maize. The examples reported here demonstrate the utility of the tRNA-processing system-based strategy as an efficient multiplex genome editing tool to enhance maize genetic research and breeding.

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells.

PubMed

Müller, Tina; Uherek, Christoph; Maki, Guitta; Chow, Kai Uwe; Schimpf, Annemarie; Klingemann, Hans-Georg; Tonn, Torsten; Wels, Winfried S

2008-03-01

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcgammaRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3zeta chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.

Drosophila mini-white model system: new insights into positive position effects and the role of transcriptional terminators and gypsy insulator in transgene shielding

PubMed Central

Silicheva, Margarita; Golovnin, Anton; Pomerantseva, Ekaterina; Parshikov, Aleksander; Georgiev, Pavel; Maksimenko, Oksana

2010-01-01

The white gene, which is responsible for eye pigmentation, is widely used to study position effects in Drosophila. As a result of insertion of P-element vectors containing mini-white without enhancers into random chromosomal sites, flies with different eye color phenotypes appear, which is usually explained by the influence of positive/negative regulatory elements located around the insertion site. We found that, in more than 70% of cases when mini-white expression was subject to positive position effects, deletion of the white promoter had no effect on eye pigmentation; in these cases, the transposon was inserted into the transcribed regions of genes. Therefore, transcription through the mini-white gene could be responsible for high levels of its expression in most of chromosomal sites. Consistently with this conclusion, transcriptional terminators proved to be efficient in protecting mini-white expression from positive position effects. On the other hand, the best characterized Drosophila gypsy insulator was poorly effective in terminating transcription and, as a consequence, only partially protected mini-white expression from these effects. Thus, to ensure maximum protection of a transgene from position effects, a perfect boundary/insulator element should combine three activities: to block enhancers, to provide a barrier between active and repressed chromatin, and to terminate transcription. PMID:19854952

Selective Detoxification of Phenols by Pichia pastoris and Arabidopsis thaliana Heterologously Expressing the PtUGT72B1 from Populus trichocarpa

PubMed Central

Xu, Zhi-Sheng; Lin, Ya-Qiu; Xu, Jing; Zhu, Bo; Zhao, Wei; Peng, Ri-He; Yao, Quan-Hong

2013-01-01

Phenols are present in the environment and commonly in contact with humans and animals because of their wide applications in many industries. In a previous study, we reported that uridine diphosphate-glucose-dependent glucosyltransferase PtUGT72B1 from Populus trichocarpa has high activity in detoxifying trichlorophenol by conjugating glucose. In this study, more experiments were performed to determine the substrate specificity of PtUGT72B1 towards phenolic compounds. Among seven phenols tested, three were glucosylated by PtUGT72B1 including phenol, hydroquinone, and catechol. Transgenic Arabidopsis plants expressing the enzyme PtUGT72B1 showed higher resistance to hydroquinone and catechol but more sensitivity to phenol than wild type plants. Transgenic Pichia pastoris expressing PtUGT72B1 showed enhanced resistance to all three phenols. Compared with wild type Arabidopsis plants, transgenic Arabidopsis plants showed higher removal efficiencies and exported more glucosides of phenol, phenyl β-D-glucopyranoside, to the medium after cultured with the three phenols. Protein extracts from transgenic Arabidopsis plants showed enhanced conjugating activity towards phenol, hydroquinone and catechol. PtUGT72B1 showed much higher expression level in Pichia pastoris than in Arabidopsis plants. Kinetic analysis of the PtUGT72B1 was also performed. PMID:23840543

Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity.

PubMed

Wagner, Waldemar; Kania, Katarzyna D; Ciszewski, Wojciech M

2017-04-01

Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

Transgenic barley (Hordeum vulgare L.) expressing the wheat aluminium resistance gene (TaALMT1) shows enhanced phosphorus nutrition and grain production when grown on an acid soil.

PubMed

Delhaize, Emmanuel; Taylor, Phillip; Hocking, Peter J; Simpson, Richard J; Ryan, Peter R; Richardson, Alan E

2009-06-01

Barley (Hordeum vulgare L.), genetically modified with the Al(3+) resistance gene of wheat (TaALMT1), was compared with a non-transformed sibling line when grown on an acidic and highly phosphate-fixing ferrosol supplied with a range of phosphorus concentrations. In short-term pot trials (26 days), transgenic barley expressing TaALMT1 (GP-ALMT1) was more efficient than a non-transformed sibling line (GP) at taking up phosphorus on acid soil, but the genotypes did not differ when the soil was limed. Differences in phosphorus uptake efficiency on acid soil could be attributed not only to the differential effects of aluminium toxicity on root growth between the genotypes, but also to differences in phosphorus uptake per unit root length. Although GP-ALMT1 out-performed GP on acid soil, it was still not as efficient at taking up phosphorus as plants grown on limed soil. GP-ALMT1 plants grown in acid soil possessed substantially smaller rhizosheaths than those grown in limed soil, suggesting that root hairs were shorter. This is a probable reason for the lower phosphorus uptake efficiency. When grown to maturity in large pots, GP-ALMT1 plants produced more than twice the grain as GP plants grown on acid soil and 80% of the grain produced by limed controls. Expression of TaALMT1 in barley was not associated with a penalty in either total shoot or grain production in the absence of Al(3+), with both genotypes showing equivalent yields in limed soil. These findings demonstrate that an important crop species can be genetically engineered to successfully increase grain production on an acid soil.

Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates.

PubMed

Reimer, D L; Kong, S; Monck, M; Wyles, J; Tam, P; Wasan, E K; Bally, M B

1999-05-01

The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.

One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

PubMed Central

Nakano, Masashi; Kino, Kuniki

2015-01-01

We developed a novel process for efficient synthesis of l-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of l-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize l-THA. By using these recombinant enzymes, l-THA was obtained by l-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. l-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased l-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the l-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, l-THA was successfully obtained from l-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of l-THA. PMID:25795668

GARP-TGF-β complexes negatively regulate regulatory T cell development and maintenance of peripheral CD4+ T cells in vivo.

PubMed

Zhou, Angela X; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2013-05-15

The role of surface-bound TGF-β on regulatory T cells (Tregs) and the mechanisms that mediate its functions are not well defined. We recently identified a cell-surface molecule called Glycoprotein A Repetitions Predominant (GARP), which is expressed specifically on activated Tregs and was found to bind latent TGF-β and mediate a portion of Treg suppressive activity in vitro. In this article, we address the role of GARP in regulating Treg and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. We found that, despite forced expression of GARP on all T cells, stimulation through the TCR was required for efficient localization of GARP to the cell surface. In addition, IL-2 signals enhanced GARP cell surface expression specifically on Tregs. GARP-transgenic CD4(+) T cells and Tregs, especially those expressing higher levels of GARP, were significantly reduced in the periphery. Mature Tregs, but not conventional CD4(+) T cells, were also reduced in the thymus. CD4(+) T cell reduction was more pronounced within the effector/memory subset, especially as the mouse aged. In addition, GARP-overexpressing CD4(+) T cells stimulated through the TCR displayed reduced proliferative capacity, which was restored by inhibiting TGF-β signaling. Furthermore, inhibiting TGF-β signals greatly enhanced surface expression of GARP on Tregs and blocked the induction of Foxp3 in activated CD4(+) T cells overexpressing GARP. These findings suggest a role for GARP in natural and induced Treg development through activation of bound latent TGF-β and signaling, which negatively regulates GARP expression on Tregs.

Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producing γ-aminobutyrate in Corynebacterium glutamicum.

PubMed

Shi, Feng; Luan, Mingyue; Li, Yongfu

2018-04-18

Glutamate decarboxylase (GAD) converts L-glutamate (Glu) into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene, gadB2 or gadB1, can synthesize GABA from its own produced Glu. To enhance GABA production in C. glutamicum, ribosomal binding site (RBS) sequence and promoter were searched and optimized for increasing the expression efficiency of gadB2. R4 exhibited the highest strength among RBS sequences tested, with 6 nt the optimal aligned spacing (AS) between RBS and start codon. This combination of RBS sequence and AS contributed to gadB2 expression, increased GAD activity by 156% and GABA production by 82% compared to normal strong RBS and AS combination. Then, a series of native promoters were selected for transcribing gadB2 under optimal RBS and AS combination. P dnaK , P dtsR , P odhI and P clgR expressed gadB2 and produced GABA as effectively as widely applied P tuf and P cspB promoters and more effectively than P sod promoter. However, each native promoter did not work as well as the synthetic strong promoter P tacM , which produced 20.2 ± 0.3 g/L GABA. Even with prolonged length and bicistronic architecture, the strength of P dnaK did not enhance. Finally, gadB2 and mutant gadB1 were co-expressed under the optimal promoter and RBS combination, thus converted Glu into GABA completely and improved GABA production to more than 25 g/L. This study provides useful promoters and RBS sequences for gene expression in C. glutamicum.

A real-time control system of gene expression using ligand-bound nucleic acid aptamer for metabolic engineering.

PubMed

Wang, Jing; Cui, Xun; Yang, Le; Zhang, Zhe; Lv, Liping; Wang, Haoyuan; Zhao, Zhenmin; Guan, Ningzi; Dong, Lichun; Chen, Rachel

2017-07-01

Artificial control of bio-functions through regulating gene expression is one of the most important and attractive technologies to build novel living systems that are useful in the areas of chemical synthesis, nanotechnology, pharmacology, cell biology. Here, we present a novel real-time control system of gene regulation that includes an enhancement element by introducing duplex DNA aptamers upstream promoter and a repression element by introducing a RNA aptamer upstream ribosome binding site. With the presence of ligands corresponding to the DNA aptamers, the expression of the target gene can be potentially enhanced at the transcriptional level by strengthening the recognition capability of RNAP to the recognition region and speeding up the separation efficiency of the unwinding region due to the induced DNA bubble around the thrombin-bound aptamers; while with the presence of RNA aptamer ligand, the gene expression can be repressed at the translational level by weakening the recognition capability of ribosome to RBS due to the shielding of RBS by the formed aptamer-ligand complex upstream RBS. The effectiveness and potential utility of the developed gene regulation system were demonstrated by regulating the expression of ecaA gene in the cell-free systems. The realistic metabolic engineering application of the system has also tested by regulating the expression of mgtC gene and thrombin cDNA in Escherichia coli JD1021 for controlling metabolic flux and improving thrombin production, verifying that the real-time control system of gene regulation is able to realize the dynamic regulation of gene expression with potential applications in bacterial physiology studies and metabolic engineering. Copyright © 2017. Published by Elsevier Inc.

Protection of pigs against pandemic swine origin H1N1 influenza A virus infection by hemagglutinin- or neuraminidase-expressing attenuated pseudorabies virus recombinants.

PubMed

Klingbeil, Katharina; Lange, Elke; Blohm, Ulrike; Teifke, Jens P; Mettenleiter, Thomas C; Fuchs, Walter

2015-03-02

Influenza is an important respiratory disease of pigs, and may lead to novel human pathogens like the 2009 pandemic H1N1 swine-origin influenza virus (SoIV). Therefore, improved influenza vaccines for pigs are required. Recently, we demonstrated that single intranasal immunization with a hemagglutinin (HA)-expressing pseudorabies virus recombinant of vaccine strain Bartha (PrV-Ba) protected pigs from H1N1 SoIV challenge (Klingbeil et al., 2014). Now we investigated enhancement of efficacy by prime-boost vaccination and/or intramuscular administration. Furthermore, a novel PrV-Ba recombinant expressing codon-optimized N1 neuraminidase (NA) was included. In vitro replication of this virus was only slightly affected compared to parental virus. Unlike HA, the abundantly expressed NA was efficiently incorporated into PrV particles. Immunization of pigs with the two PrV recombinants, either singly or in combination, induced B cell proliferation and the expected SoIV-specific antibodies, whose titers increased substantially after boost vaccination. After immunization of animals with either PrV recombinant H1N1 SoIV challenge virus replication was significantly reduced compared to PrV-Ba vaccinated or naïve controls. Protective efficacy of HA-expressing PrV was higher than of NA-expressing PrV, and not significantly enhanced by combination. Despite higher serum antibody titers obtained after intramuscular immunization, transmission of challenge virus to naïve contact animals was only prevented after intranasal prime-boost vaccination with HA-expressing PrV-Ba. Copyright © 2015 Elsevier B.V. All rights reserved.

Cyclodextrin based ternary system of modafinil: Effect of trimethyl chitosan and polyvinylpyrrolidone as complexing agents.

PubMed

Patel, Parth; Agrawal, Y K; Sarvaiya, Jayrajsinh

2016-03-01

Modafinil is an approved drug for the treatment of narcolepsy and have a strong market presence in many countries. The drug is widely consumed for off-label uses and currently listed as a restricted drug. Modafinil has very low water solubility. To enhance the aqueous solubility of modafinil by the formation of a ternary complex with Hydroxypropyl-β-cyclodextrin and two hydrophilic polymers was the main objective of the present study. Pyrrolidone (PVP K30) and a water soluble chitosan derivative, trimethyl chitosan (TMC) were studied by solution state and solid state characterization methods for their discriminatory efficiency in solubility enhancement of modafinil. Phase solubility study depicted the highest complexation efficiency (2.22) of cyclodextrin derivative in the presence of TMC compared to the same in the presence of PVP K30 (0.08) and in the absence of any polymer (0.92). FT-IR analysis of binary and ternary complex expressed comparable contribution of both polymers in formation of inclusion complex. The thermal behaviour of binary and ternary complex, involving individual polymers disclosed the influence of TMC on polymorphism of the drug. DSC study revealed efficiency of TMC to prevent conversion of metastable polymorphic form to stable polymorphic form. Ternary complex, involving TMC enhanced water solubility of the drug 1.5 times more compared to the binary complex of the drug whereas PVP K30 reduced the Solubility. Copyright © 2015 Elsevier B.V. All rights reserved.

The upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in murine glycogen storage disease type Ia.

PubMed

Lee, Young Mok; Pan, Chi-Jiunn; Koeberl, Dwight D; Mansfield, Brian C; Chou, Janice Y

2013-11-01

Glycogen storage disease type-Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α (G6Pase-α or G6PC) manifest impaired glucose homeostasis characterized by fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. Two efficacious recombinant adeno-associated virus pseudotype 2/8 (rAAV8) vectors expressing human G6Pase-α have been independently developed. One is a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer (rAAV8-GPE) and the other is a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer (rAAV8-miGPE). To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-α expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia. © 2013.

Elevated CO2 increases R gene-dependent resistance of Medicago truncatula against the pea aphid by up-regulating a heat shock gene.

PubMed

Sun, Yucheng; Guo, Huijuan; Yuan, Erliang; Ge, Feng

2018-03-01

Resistance against pathogens and herbivorous insects in many plant results from the expression of resistance (R) genes. Few reports, however, have considered the effects of elevated CO 2 on R gene-based resistance in plants. The current study determined the responses of two near isogenic Medicago truncatula genotypes (Jester has an R gene and A17 does not) to the pea aphid and elevated CO 2 in open-top chambers in the field. Aphid abundance, mean relative growth rate and feeding efficiency were increased by elevated CO 2 on A17 plants but were reduced on Jester plants. According to proteomic and gene expression data, elevated CO 2 enhanced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) but decreased the effector-triggered immunity (ETI) in aphid-infested A17 plants. For aphid-infested Jester plants, by contrast, elevated CO 2 enhanced the ETI-related heat shock protein (HSP) 90 and its co-chaperones, the jasmonic acid (JA) signaling pathway, and ubiquitin-mediated proteolysis. In a loss-of-function experiment, silencing of the HSP90 gene in Jester plants impaired the JA signaling pathway and ubiquitin-mediated proteolysis against the aphid under ambient CO 2 , and negated the increased resistance against the aphid under elevated CO 2 . Our results suggest that increases in expression of HSP90 are responsible for the enhanced resistance against the aphid under elevated CO 2 . © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Retinaldehyde dehydrogenase 2 as a molecular adjuvant for enhancement of mucosal immunity during DNA vaccination.

PubMed

Holechek, Susan A; McAfee, Megan S; Nieves, Lizbeth M; Guzman, Vanessa P; Manhas, Kavita; Fouts, Timothy; Bagley, Kenneth; Blattman, Joseph N

2016-11-04

In order for vaccines to induce efficacious immune responses against mucosally transmitted pathogens, such as HIV-1, activated lymphocytes must efficiently migrate to and enter targeted mucosal sites. We have previously shown that all-trans retinoic acid (ATRA) can be used as a vaccine adjuvant to enhance mucosal CD8 + T cell responses during vaccination and improve protection against mucosal viral challenge. However, the ATRA formulation is incompatible with most recombinant vaccines, and the teratogenic potential of ATRA at high doses limits its usage in many clinical settings. We hypothesized that increasing in vivo production of retinoic acid (RA) during vaccination with a DNA vector expressing retinaldehyde dehydrogenase 2 (RALDH2), the rate-limiting enzyme in RA biosynthesis, could similarly provide enhanced programming of mucosal homing to T cell responses while avoiding teratogenic effects. Administration of a RALDH2- expressing plasmid during immunization with a HIVgag DNA vaccine resulted in increased systemic and mucosal CD8 + T cell numbers with an increase in both effector and central memory T cells. Moreover, mice that received RALDH2 plasmid during DNA vaccination were more resistant to intravaginal challenge with a recombinant vaccinia virus expressing the same HIVgag antigen (VACVgag). Thus, RALDH2 can be used as an alternative adjuvant to ATRA during DNA vaccination leading to an increase in both systemic and mucosal T cell immunity and better protection from viral infection at mucosal sites. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

NASA Astrophysics Data System (ADS)

Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad

2016-05-01

Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and 1H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol40 %) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor.

PubMed

Florez, Sergio L; Erwin, Rachel L; Maximova, Siela N; Guiltinan, Mark J; Curtis, Wayne R

2015-05-16

Theobroma cacao, the chocolate tree, is an important economic crop in East Africa, South East Asia, and South and Central America. Propagation of elite varieties has been achieved through somatic embryogenesis (SE) but low efficiencies and genotype dependence still presents a significant limitation for its propagation at commercial scales. Manipulation of transcription factors has been used to enhance the formation of SEs in several other plant species. This work describes the use of the transcription factor Baby Boom (BBM) to promote the transition of somatic cacao cells from the vegetative to embryonic state. An ortholog of the Arabidopsis thaliana BBM gene (AtBBM) was characterized in T. cacao (TcBBM). TcBBM expression was observed throughout embryo development and was expressed at higher levels during SE as compared to zygotic embryogenesis (ZE). TcBBM overexpression in A. thaliana and T. cacao led to phenotypes associated with SE that did not require exogenous hormones. While transient ectopic expression of TcBBM provided only moderate enhancements in embryogenic potential, constitutive overexpression dramatically increased SE proliferation but also appeared to inhibit subsequent development. Our work provides validation that TcBBM is an ortholog to AtBBM and has a specific role in both somatic and zygotic embryogenesis. Furthermore, our studies revealed that TcBBM transcript levels could serve as a biomarker for embryogenesis in cacao tissue. Results from transient expression of TcBBM provide confirmation that transcription factors can be used to enhance SE without compromising plant development and avoiding GMO plant production. This strategy could compliment a hormone-based method of reprogramming somatic cells and lead to more precise manipulation of SE at the regulatory level of transcription factors. The technology would benefit the propagation of elite varieties with low regeneration potential as well as the production of transgenic plants, which similarly requires somatic cell reprogramming.

Ternary particles for effective vaccine delivery to the pulmonary system

NASA Astrophysics Data System (ADS)

Terry, Treniece La'shay

Progress in the fields of molecular biology and genomics has provided great insight into the pathogenesis of disease and the defense mechanisms of the immune system. This knowledge has lead to the classification of an array of abnormal genes, for which, treatment relies on cellular expression of proteins. The utility of DNA-based vaccines hold great promise for the treatment of genetically based and infectious diseases, which ranges from hemophilia, cystic fibrosis, and HIV. Synthetic delivery systems consisting of cationic polymers, such as polyethylenimine (PEI), are capable of condensing DNA into compact structures, maximizing cellular uptake of DNA and yielding high levels of protein expression. To date, short term expression is a major obstacle in the development of gene therapies and has halted their expansion in clinical applications. This study intends to develop a sustained release vaccine delivery system using PLA-PEG block copolymers encapsulating PEI:DNA polyplexes. To enhance the effectiveness of such DNA-based vaccines, resident antigen presenting cells, macrophages and dendritic cells, will be targeted within the alveoli regions of the lungs. Porous microspheres will be engineered with aerodynamic properties capable of achieving deep lung deposition. A fabrication technique using concentric nozzles will be developed to produce porous microspheres. It was observed that modifications in the dispersed to continuous phase ratios have the largest influence on particle size distributions, release rates and encapsulation efficiency which ranged form 80--95% with fourteen days of release. Amphiphilic block copolymers were also used to fabricate porous microspheres. The confirmation of PEG within the biodegradable polymer backbone was found to have a tremendous impact on the microsphere morphology and encapsulation efficiency which varied from 50--90%. Porous microspheres were capable of providing sustained gene expression when tested in vitro using the luciferase reporter gene plasmid DNA. Prolonged expression was obtained for 9 days. PLGA and PLA-PEG microspheres were administered in vivo by intra-tracheal instillation and produced an acute inflammatory response, as observed from the large presence of neutrophils. The response using PLA-PEG microspheres yielded a lower total cell count signifying the incorporation of PEG into the copolymer backbone enhances the biocompatibility of the delivery system.

Matrix Metallopeptidase 14 Plays an Important Role in Regulating Tumorigenic Gene Expression and Invasion Ability of HeLa Cells.

PubMed

Zhang, Ying-Hui; Wang, Juan-Juan; Li, Min; Zheng, Han-Xi; Xu, Lan; Chen, You-Guo

2016-03-01

The objectives of this study were to investigate the functional effect of matrix metallopeptidase 14 (MMP14) on cell invasion in cervical cancer cells (HeLa line) and to study the underlying molecular mechanisms. Expression vector of short hairpin RNA targeting MMP14 was treated in HeLa cells, and then, transfection efficiency was verified by a florescence microscope. Transwell assay was used to investigate cell invasion ability in HeLa cells. Quantitative polymerase chain reaction and Western blotting analysis were used to detect the expression of MMP14 and relative factors in messenger RNA and protein levels, respectively. Matrix metallopeptidase 14 short hairpin RNA expression vector transfection obviously decreased MMP14 expression in messenger RNA and protein levels. Down-regulation of MMP14 suppressed invasion ability of HeLa cells and reduced transforming growth factor β1 and vascular endothelial growth factor B expressions. Furthermore, MMP14 knockdown decreased bone sialoprotein and enhanced forkhead box protein L2 expression in both RNA and protein levels. Matrix metallopeptidase 14 plays an important role in regulating invasion of HeLa cells. Matrix metallopeptidase 14 knockdown contributes to attenuating the malignant phenotype of cervical cancer cell.

Over-Expression of a Tobacco Nitrate Reductase Gene in Wheat (Triticum aestivum L.) Increases Seed Protein Content and Weight without Augmenting Nitrogen Supplying

PubMed Central

Zhao, Xiao-Qiang; Nie, Xuan-Li; Xiao, Xing-Guo

2013-01-01

Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, “Nongda146” and “Jimai6358”, by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying. PMID:24040315

Over-expression of a tobacco nitrate reductase gene in wheat (Triticum aestivum L.) increases seed protein content and weight without augmenting nitrogen supplying.

PubMed

Zhao, Xiao-Qiang; Nie, Xuan-Li; Xiao, Xing-Guo

2013-01-01

Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, "Nongda146" and "Jimai6358", by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying.

New and improved tools and methods for enhanced biosynthesis of natural products in microorganisms.

PubMed

Wang, Zhiqing; Cirino, Patrick C

2016-12-01

Engineering efficient biosynthesis of natural products in microorganisms requires optimizing gene expression levels to balance metabolite flux distributions and to minimize accumulation of toxic intermediates. Such metabolic optimization is challenged with identifying the right gene targets, and then determining and achieving appropriate gene expression levels. After decades of having a relatively limited set of gene regulation tools available, metabolic engineers are recently enjoying an ever-growing repertoire of more precise and tunable gene expression platforms. Here we review recent applications of natural and designed transcriptional and translational regulatory machinery for engineering biosynthesis of natural products in microorganisms. Customized trans-acting RNAs (sgRNA, asRNA and sRNA), along with appropriate accessory proteins, are allowing for unparalleled tuning of gene expression. Meanwhile metabolite-responsive transcription factors and riboswitches have been implemented in strain screening and evolution, and in dynamic gene regulation. Further refinements and expansions on these platform technologies will circumvent many long-term obstacles in natural products biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency.

PubMed

Pang, Yun-Wei; An, Lei; Wang, Peng; Yu, Yong; Yin, Qiu-Dan; Wang, Xiao-Hong; Xin-Zhang; Qian-Zhang; Yang, Mei-Ling; Min-Guo; Wu, Zhong-Hong; Tian, Jian-Hui

2013-05-01

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Hemagglutinating virus of Japan-artificial viral envelope liposome-mediated cotransfer of bag-1 and bcl-2 genes protects hepatic cells against ischemic injury through BAG-1-assisted preferential enhancement of bcl-2 protein expression.

PubMed

Yanada, Shinobu; Sasaki, Masao; Takayama, Shinichi; Kaneda, Yasufumi; Miwa, Nobuhiko

2005-05-01

Hepatic injury subsequent to ischemia-reperfusion (I/R) was demonstrated in our previous study to be prevented by hemagglutinating virus of Japan (HVJ)-artificial viral envelope (AVE) liposome-mediated gene transfer of the antiapoptotic gene, human bcl-2 (h-bcl-2). In the present study, we introduced simultaneously both mouse Bcl-2-associated athanogene 1 (m-bag-1) and the h-bcl-2 gene by the same HVJ-AVE liposome transfection method, and found that I/R-induced hepatic injuries such as release of hepatic marker enzymes into blood, cell morphological degeneration, and cellular DNA strand cleavage were suppressed more effectively than by transfection with either gene singly. In addition, the h-Bcl-2 expression level in the ischemic state, but not in the nonischemic state, was markedly higher in h-bcl-2/m-bag-1-cotransfected liver than in h-bcl-2-transfected liver. In contrast, the m-BAG-1 expression level in the ischemic state, but not in the nonischemic state, was only slightly higher in h-bcl-2/m-bag-1-cotransfected liver than in m-bag-1-transfected liver. Thus, with dual gene cotransfer, coexistent Bcl-2 protein exerts no activity to assist a marked enhancement of BAG-1 protein, whereas the function of overexpressed BAG-1 as a Bcl-2-binding protein may lead to the enhancement of efficient expression of h-Bcl-2 in I/R-treated liver as compared with nonischemic liver, which results in repression of diverse I/R-induced cell death symptoms, presumably through the formation of functional complexes of BAG-1 and Bcl-2.

Production of caffeoylmalic acid from glucose in engineered Escherichia coli.

PubMed

Li, Tianzhen; Zhou, Wei; Bi, Huiping; Zhuang, Yibin; Zhang, Tongcun; Liu, Tao

2018-07-01

To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli. We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli-E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L. Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

PubMed

Srivastava, Preeti; Deb, J K

2002-07-02

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

Simultaneous and Sequential Integration by Cre/loxP Site-Specific Recombination in Saccharomyces cerevisiae.

PubMed

Choi, Ho-Jung; Kim, Yeon-Hee

2018-05-28

A Cre/ loxP -δ-integration system was developed to allow sequential and simultaneous integration of a multiple gene expression cassette in Saccharomyces cerevisiae . To allow repeated integrations, the reusable Candida glabrata MARKER ( CgMARKER ) carrying loxP sequences was used, and the integrated CgMARKER was efficiently removed by inducing Cre recombinase. The XYLP and XYLB genes encoding endoxylanase and β-xylosidase, respectively, were used as model genes for xylan metabolism in this system, and the copy number of these genes was increased to 15.8 and 16.9 copies/cell, respectively, by repeated integration. This integration system is a promising approach for the easy construction of yeast strains with enhanced metabolic pathways through multicopy gene expression.

Exendin-4 enhances the migration of adipose-derived stem cells to neonatal rat ventricular cardiomyocyte-derived conditioned medium via the phosphoinositide 3-kinase/Akt-stromal cell-derived factor-1α/CXC chemokine receptor 4 pathway

PubMed Central

ZHOU, HAO; YANG, JUNJIE; XIN, TING; ZHANG, TAO; HU, SHUNYIN; ZHOU, SHANSHAN; CHEN, GUANGHUI; CHEN, YUNDAI

2015-01-01

Adipose-derived stem cells (ADSCs) are considered a suitable source of cells for the repair of tissue following acute myocardial infarction (AMI); however, the transplantation efficiency of ADSCs remains low. Therefore, identification of an efficient method to enhance the migration of engrafted cells to the target site is required. The present study used exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, to optimize the migratory capacity of ADSCs. The aim was to determine the effect and mechanisms of Ex-4 on the migration of ADSCs to neonatal rat ventricular cardiomyocyte-derived conditioned medium (NRVC-CM). The ADSCs and cardiomyocytes were cultured in vitro. Following incubation of the ADSCs with Ex-4, cell proliferation was measured using an MTT assay and the expression levels of CXC chemokine receptor 4 (CXCR4) were investigated by reverse transctiption quantitative polymerase chain reaction (RT-qPCR), western blot analysis and flow cytometry. In addition, the expression levels of stromal cell-derived factor-1α (SDF-1α) were evaluated in the NRVC-CM treated with Ex-4 by ELISA, RT-qPCR and western blot analysis. The migration of the ADSCs to the NRVC-CM was examined using a Transwell assay. Changes in the protein expression levels of phosphorylated (p−)Akt were examined in the two types of cell by western blot analysis. The results suggested that Ex-4 promoted the proliferation and expression of CXCR4 in the ADSCs, increased the secretion of SDF-1α in the cardiomyocytes and increased the expression levels of p-Akt in both cells. However, the alterations to the SDF-1α/C XC R4 cascade in the cells were abrogated following pretreatment with LY-294002, a phosphoinositide 3-kinase(PI3K) inhibitor. Furthermore, a Transwell migration assay revealed marked translocation of the ADSCs through the membranes, towards the NRVC-CM, following treatment with Ex-4. However, these effects were reduced significantly by pretreatment of the cells with the SDF-1α/CXCR4 cascade antagonist, AMD3100, and the PI3K inhibitor, LY-294002. These results indicated that Ex-4 augmented the SDF-1α/CXCR4 cascade by activating the PI3K/Akt pathways in the ADSCs and NRVCs. Furthermore, enhancement of the PI3K/Akt-SDF-1α/CXCR4 pathway may be important in the migratory response of ADSCs to NRVC-CM in vitro. PMID:25625935

A Simple Method to Increase the Transduction Efficiency of Single-Stranded Adeno-Associated Virus Vectors In Vitro and In Vivo

PubMed Central

Ma, Wenqin; Li, Baozheng; Ling, Chen; Jayandharan, Giridhara R.; Byrne, Barry J.

2011-01-01

Abstract We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors. PMID:21219084

Photosynthetic behavior of woody species under high ozone exposure probed with the JIP-test: a review.

PubMed

Bussotti, Filippo; Strasser, Reto J; Schaub, Marcus

2007-06-01

Visible ozone symptoms on leaves are expressions of physiological mechanisms to cope with oxidative stresses. Often, the symptoms consist of stippling, which corresponds to localized cell death (hypersensitive response, HR), separated from healthy cells by a layer of callose. The HR strategy tends to protect the healthy cells and in most cases the efficiency of chlorophyll to trap energy is not affected. In other cases, the efficiency of leaves to produce biomass declines and the plant loses its photosynthetic apparatus replacing it with a new, more efficient one. Another strategy consists of the production of pigments (anthocyanins), and leaves become reddish. In these cases, the most significant physiological manifestation consists of the enhanced dissipation of energy. These different behavior patterns are reflected in the initial events of photosynthetic activity, and can be monitored with techniques based on the direct fluorescence of chlorophyll a in photosystem II, applying the JIP-test.

Cre-mediated recombination in pituitary somatotropes

PubMed Central

Nasonkin, Igor O.; Potok, Mary Anne; Camper, Sally A.

2009-01-01

We report a transgenic line with highly penetrant cre recombinase activity in the somatotrope cells of the anterior pituitary gland. Expression of the cre transgene is under the control of the locus control region of the human growth hormone gene cluster and the rat growth hormone promoter. Cre recombinase activity was assessed with two different lacZ reporter genes that require excision of a floxed stop sequence for expression: a chick β-actin promoter with the CMV enhancer transgene and a ROSA26 knock-in. Cre activity is detectable in the developing pituitary after initiation of Gh transcription and persists through adulthood with high penetrance in Gh expressing cells and lower penetrance in lactotropes, a cell type that shares a common origin with somatotropes. This Gh-cre transgenic line is suitable for efficient, cell-specific deletion of floxed regions of genomic DNA in differentiated somatotropes and a subset of lactotrope cells of the anterior pituitary gland. PMID:19039787

Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Dell, William B.; Swartz, Paul D.; Weiss, Kevin L.

Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and themore » production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.« less

The Effect of α-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris

PubMed Central

Lin-Cereghino, Geoff P.; Stark, Carolyn M.; Kim, Daniel; Chang, Jennifer; Shaheen, Nadia; Poerwanto, Hansel; Agari, Kimiko; Moua, Pachai; Low, Lauren K.; Tran, Namphuong; Huang, Amy D.; Nattestad, Maria; Oshiro, Kristin T.; Chang, John William; Chavan, Archana; Tsai, Jerry W.; Lin-Cereghino, Joan

2013-01-01

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. PMID:23454485

A Protein Chimera Strategy Supports Production of a Model "Difficult-to-Express" Recombinant Target.

PubMed

Hussain, Hirra; Fisher, David I; Roth, Robert G; Abbott, W Mark; Carballo-Amador, Manuel Alejandro; Warwicker, Jim; Dickson, Alan J

2018-06-22

Due in part to the needs of the biopharmaceutical industry, there has been an increased drive to generate high quality recombinant proteins in large amounts. However, achieving high yields can be a challenge as the novelty and increased complexity of new targets often makes them 'difficult-to-express'. This study aimed to define the molecular features that restrict the production of a model 'difficult-to-express' recombinant protein, Tissue Inhibitor Metalloproteinase-3 (TIMP-3). Building from experimental data, computational approaches were used to rationalise the re-design of this recombinant target to generate a chimera with enhanced secretion. The results highlight the importance of early identification of unfavourable sequence attributes, enabling the generation of engineered protein forms that bypass 'secretory' bottlenecks and result in efficient recombinant protein production. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Combination of Garcinia cambogia Extract and Pear Pomace Extract Additively Suppresses Adipogenesis and Enhances Lipolysis in 3T3-L1 Cells.

PubMed

Sharma, Kushal; Kang, Siwon; Gong, Dalseong; Oh, Sung-Hwa; Park, Eun-Young; Oak, Min-Ho; Yi, Eunyoung

2018-01-01

Inhibition of adipogenesis has been a therapeutic target for reducing obesity and obesity-related disorders such as diabetes, hypertension, atherosclerosis, and cancer. For decades, anti-adipogenic potential of many herbal extracts has been investigated. One example is Garcinia cambogia extract (GE) containing (-)-hydroxycitric acid as an active ingredient. GE is currently marketed as a weight loss supplement, used alone or with other ingredients. Pear pomace extract (PE), another natural product, has been also shown to have anti-adipogenic activity in a recent report. It was tested if the mixture of PE and GE (MIX) would produce more effective anti-adipogenic activity than PE or GE alone. Differentiation of 3T3-L1 preadipocyte was induced by adding insulin, dexamethasone, and isobutylmethylxanthine and lipid accumulation was measured by Oil Red O staining. Cellular markers for adipogenesis and lipolysis such as CCAAT/enhancer binding protein (C/EBP-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), fatty acid synthase (FAS), and hormone-sensitive lipase (HSL) was measured using immunocytochemistry. MIX, compared to PE or GE alone, showed greater inhibition of lipid accumulation. Furthermore, MIX reduced the expression of adipogenesis-related factors C/EBP-α, PPAR-γ, and FAS more than PE or GE alone did. In contrast, the expression of HSL the enzyme required for lipolysis was further enhanced in MIX-treated adipocytes compared to the PE or GE alone treated groups. Anti-adipogenic effect of PE and GE appears synergistic, and the MIX may be a useful therapeutic combination for the treatment of obesity and obesity-related diseases. PE and GE efficiently inhibited adipocyte differentiation by suppressing the expression of adipogenic transcription factor CEBP-α and PPAR-γ.PE and GE significantly decreased the expression of adipogenic enzyme FAS.PE and GE increased the expression of lipid degrading enzyme HSL.Mixture of PE and GE exhibited additive or moderately synergistic effect on adipocyte differentiation and lipid accumulation. Abbreviations used: CEBP-a: CCAT/enhancer binding protein alpha, CI: Combination Index, FAS: Fatty acid synthase, GE: Garcinia cambogia extract, HSL: Hormone sensitive lipase, PE: Pear pomace extract, PPAR-γ: Peroxisome proliferator-activated receptor gamma.

Enhancing Bidirectional Electron Transfer of Shewanella oneidensis by a Synthetic Flavin Pathway.

PubMed

Yang, Yun; Ding, Yuanzhao; Hu, Yidan; Cao, Bin; Rice, Scott A; Kjelleberg, Staffan; Song, Hao

2015-07-17

Flavins regulate the rate and direction of extracellular electron transfer (EET) in Shewanella oneidensis. However, low concentration of endogenously secreted flavins by the wild-type S. oneidensis MR-1 limits its EET efficiency in bioelectrochemical systems (BES). Herein, a synthetic flavin biosynthesis pathway from Bacillus subtilis was heterologously expressed in S. oneidensis MR-1, resulting in ∼25.7 times' increase in secreted flavin concentration. This synthetic flavin module enabled enhanced bidirectional EET rate of MR-1, in which its maximum power output in microbial fuel cells increased ∼13.2 times (from 16.4 to 233.0 mW/m(2)), and the inward current increased ∼15.5 times (from 15.5 to 255.3 μA/cm(2)).

Morinda citrifolia leaf enhanced performance by improving angiogenesis, mitochondrial biogenesis, antioxidant, anti-inflammatory & stress responses.

PubMed

Mohamad Shalan, Nor Aijratul Asikin; Mustapha, Noordin M; Mohamed, Suhaila

2016-12-01

Morinda citrifolia fruit, (noni), enhanced performances in athletes and post-menopausal women in clinical studies. This report shows the edible noni leaves water extract enhances performance in a weight-loaded swimming animal model better than the fruit or standardized green tea extract. The 4weeks study showed the extract (containing scopoletin and epicatechin) progressively prolonged the time to exhaustion by threefold longer than the control, fruit or tea extract. The extract improved (i) the mammalian antioxidant responses (MDA, GSH and SOD2 levels), (ii) tissue nutrient (glucose) and metabolite (lactate) management, (iii) stress hormone (cortisol) regulation; (iv) neurotransmitter (dopamine, noradrenaline, serotonin) expressions, transporter or receptor levels, (v) anti-inflammatory (IL4 & IL10) responses; (v) skeletal muscle angiogenesis (VEGFA) and (v) energy and mitochondrial biogenesis (via PGC, UCP3, NRF2, AMPK, MAPK1, and CAMK4). The ergogenic extract helped delay fatigue by enhancing energy production, regulation and efficiency, which suggests benefits for physical activities and disease recovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Next generation bone tissue engineering: non-viral miR-133a inhibition using collagen-nanohydroxyapatite scaffolds rapidly enhances osteogenesis

NASA Astrophysics Data System (ADS)

Mencía Castaño, Irene; Curtin, Caroline M.; Duffy, Garry P.; O'Brien, Fergal J.

2016-06-01

Bone grafts are the second most transplanted materials worldwide at a global cost to healthcare systems valued over $30 billion every year. The influence of microRNAs in the regenerative capacity of stem cells offers vast therapeutic potential towards bone grafting; however their efficient delivery to the target site remains a major challenge. This study describes how the functionalisation of porous collagen-nanohydroxyapatite (nHA) scaffolds with miR-133a inhibiting complexes, delivered using non-viral nHA particles, enhanced human mesenchymal stem cell-mediated osteogenesis through the novel focus on a key activator of osteogenesis, Runx2. This study showed enhanced Runx2 and osteocalcin expression, as well as increased alkaline phosphatase activity and calcium deposition, thus demonstrating a further enhanced therapeutic potential of a biomaterial previously optimised for bone repair applications. The promising features of this platform offer potential for a myriad of applications beyond bone repair and tissue engineering, thus presenting a new paradigm for microRNA-based therapeutics.

Intermittent fasting induces hypothalamic modifications resulting in low feeding efficiency, low body mass and overeating.

PubMed

Chausse, Bruno; Solon, Carina; Caldeira da Silva, Camille C; Masselli Dos Reis, Ivan G; Manchado-Gobatto, Fúlvia B; Gobatto, Claudio A; Velloso, Licio A; Kowaltowski, Alicia J

2014-07-01

Intermittent fasting (IF) is an often-used intervention to decrease body mass. In male Sprague-Dawley rats, 24 hour cycles of IF result in light caloric restriction, reduced body mass gain, and significant decreases in the efficiency of energy conversion. Here, we study the metabolic effects of IF in order to uncover mechanisms involved in this lower energy conversion efficiency. After 3 weeks, IF animals displayed overeating during fed periods and lower body mass, accompanied by alterations in energy-related tissue mass. The lower efficiency of energy use was not due to uncoupling of muscle mitochondria. Enhanced lipid oxidation was observed during fasting days, whereas fed days were accompanied by higher metabolic rates. Furthermore, an increased expression of orexigenic neurotransmitters AGRP and NPY in the hypothalamus of IF animals was found, even on feeding days, which could explain the overeating pattern. Together, these effects provide a mechanistic explanation for the lower efficiency of energy conversion observed. Overall, we find that IF promotes changes in hypothalamic function that explain differences in body mass and caloric intake.

Transgenic Cotton Plants Expressing the HaHR3 Gene Conferred Enhanced Resistance to Helicoverpa armigera and Improved Cotton Yield

PubMed Central

Han, Qiang; Wang, Zhenzhen; He, Yunxin; Xiong, Yehui; Lv, Shun; Li, Shupeng; Zhang, Zhigang; Qiu, Dewen; Zeng, Hongmei

2017-01-01

RNA interference (RNAi) has been developed as an efficient technology. RNAi insect-resistant transgenic plants expressing double-stranded RNA (dsRNA) that is ingested into insects to silence target genes can affect the viability of these pests or even lead to their death. HaHR3, a molt-regulating transcription factor gene, was previously selected as a target expressed in bacteria and tobacco plants to control Helicoverpa armigera by RNAi technology. In this work, we selected the dsRNA-HaHR3 fragment to silence HaHR3 in cotton bollworm for plant mediated-RNAi research. A total of 19 transgenic cotton lines expressing HaHR3 were successfully cultivated, and seven generated lines were used to perform feeding bioassays. Transgenic cotton plants expressing dsHaHR3 were shown to induce high larval mortality and deformities of pupation and adult eclosion when used to feed the newly hatched larvae, and 3rd and 5th instar larvae of H. armigera. Moreover, HaHR3 transgenic cotton also demonstrated an improved cotton yield when compared with controls. PMID:28867769

PML IV/ARF interaction enhances p53 SUMO-1 conjugation, activation, and senescence

PubMed Central

Ivanschitz, Lisa; Takahashi, Yuki; Jollivet, Florence; Ayrault, Olivier; Le Bras, Morgane; de Thé, Hugues

2015-01-01

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) recruit multiple partners, including p53 and many of its regulators. NBs are believed to facilitate several posttranslational modifications and are key regulators of senescence. PML, the organizer of NBs, is expressed as a number of splice variants that all efficiently recruit p53 partners. However, overexpression of only one of them, PML IV, triggers p53-driven senescence. Here, we show that PML IV specifically binds ARF, a key p53 regulator. Similar to ARF, PML IV enhances global SUMO-1 conjugation, particularly that of p53, resulting in p53 stabilization and activation. ARF interacts with and stabilizes the NB-associated UBC9 SUMO-conjugating enzyme, possibly explaining PML IV-enhanced SUMOylation. These results unexpectedly link two key tumor suppressors, highlighting their convergence for global control of SUMO conjugation, p53 activation, and senescence induction. PMID:26578773

PML IV/ARF interaction enhances p53 SUMO-1 conjugation, activation, and senescence.

PubMed

Ivanschitz, Lisa; Takahashi, Yuki; Jollivet, Florence; Ayrault, Olivier; Le Bras, Morgane; de Thé, Hugues

2015-11-17

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) recruit multiple partners, including p53 and many of its regulators. NBs are believed to facilitate several posttranslational modifications and are key regulators of senescence. PML, the organizer of NBs, is expressed as a number of splice variants that all efficiently recruit p53 partners. However, overexpression of only one of them, PML IV, triggers p53-driven senescence. Here, we show that PML IV specifically binds ARF, a key p53 regulator. Similar to ARF, PML IV enhances global SUMO-1 conjugation, particularly that of p53, resulting in p53 stabilization and activation. ARF interacts with and stabilizes the NB-associated UBC9 SUMO-conjugating enzyme, possibly explaining PML IV-enhanced SUMOylation. These results unexpectedly link two key tumor suppressors, highlighting their convergence for global control of SUMO conjugation, p53 activation, and senescence induction.

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH.

PubMed

Kumar, V V; Pichon, C; Refregiers, M; Guerin, B; Midoux, P; Chaudhuri, A

2003-08-01

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

Expression of human factor IX gene in murine plasma through lentiviral vector-infected haematopoietic stem cells.

PubMed

Chen, Haoming; Yao, Hengmei; Huang, Lu; Shen, Qi; Jia, William; Xue, Jinglun

2006-12-01

1. Haematopoietic stem cells (HSC) are an attractive target for gene therapy. Gene transfer to HSC can provide a potential cure for many inherited diseases. Moreover, recombinant lentiviral vectors can transfer genes efficiently to HSC. In the present study, we used the recombinant lentiviruses FUGW (Flip, ubiquitin promoter, GFP and WRE vector) and FUXW (Flip, ubiquitin promoter, F IX and WRE vector), which carry the enhanced green fluorescent protein (EGFP) and human factor IX (hFIX) gene, respectively, to infect HSC. 2. High titres of recombinant lentivirus were prepared from 293T cells by calcium phosphate-mediated transient cotransfection. Murine mononuclear cells (MNC) separated from murine bone marrow and HSC separated by magnetic cell sorting were cultured in vitro. Cells they were infected by the recombinant lentiviruses FUGW and FUXW. The expression of EGFP was observed under a fluorescent microscope and was analysed by fluorescence-activated cell sorting, whereas the expression of hFIX was detected by ELISA. 3. The results show that the lentiviral vectors can efficiently infect murine HSC in vitro and that transduction was more efficient following cytokine treatment with interleukin (IL)-3, IL-6 and stem cell factor. 4. Haematopoietic stem cells infected with lentivirus FUXW were transplanted into [(60)Co]-irradiated non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice. The expression of hFIX in the blood plasma of the transplanted mice reached a peak of 44.9 +/- 7.6 ng/mL on Day 7. An assay of transaminase levels and a histological study of the liver showed that there was no significant damage following HSC transplantation to mice. 5. The results of the present study suggest that transplantation of HSC results in the persistant expression of hFIX in mice, which may be useful in haemophilia B gene therapy.

Evolving phage vectors for cell targeted gene delivery.

PubMed

Larocca, David; Burg, Michael A; Jensen-Pergakes, Kristen; Ravey, Edward Prenn; Gonzalez, Ana Maria; Baird, Andrew

2002-03-01

We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

PubMed

Raisin, Sophie; Morille, Marie; Bony, Claire; Noël, Danièle; Devoisselle, Jean-Marie; Belamie, Emmanuel

2017-08-22

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

Improving cluster-based missing value estimation of DNA microarray data.

PubMed

Brás, Lígia P; Menezes, José C

2007-06-01

We present a modification of the weighted K-nearest neighbours imputation method (KNNimpute) for missing values (MVs) estimation in microarray data based on the reuse of estimated data. The method was called iterative KNN imputation (IKNNimpute) as the estimation is performed iteratively using the recently estimated values. The estimation efficiency of IKNNimpute was assessed under different conditions (data type, fraction and structure of missing data) by the normalized root mean squared error (NRMSE) and the correlation coefficients between estimated and true values, and compared with that of other cluster-based estimation methods (KNNimpute and sequential KNN). We further investigated the influence of imputation on the detection of differentially expressed genes using SAM by examining the differentially expressed genes that are lost after MV estimation. The performance measures give consistent results, indicating that the iterative procedure of IKNNimpute can enhance the prediction ability of cluster-based methods in the presence of high missing rates, in non-time series experiments and in data sets comprising both time series and non-time series data, because the information of the genes having MVs is used more efficiently and the iterative procedure allows refining the MV estimates. More importantly, IKNN has a smaller detrimental effect on the detection of differentially expressed genes.

PAMAM dendrimer-baculovirus nanocomplex for microencapsulated adipose stem cell-gene therapy: in vitro and in vivo functional assessment.

PubMed

Paul, Arghya; Shao, Wei; Abbasi, Sana; Shum-Tim, Dominique; Prakash, Satya

2012-09-04

The present study aims to develop a new stem cell based gene delivery system consisting of human adipose tissue derived stem cells (hASCs) genetically modified with self-assembled nanocomplex of recombinant baculovirus and PAMAM dendrimer (Bac-PAMAM) to overexpress the vascular endothelial growth factor (VEGF). Cells were enveloped into branched PEG surface functionalized polymeric microcapsules for efficient transplantation. In vitro analysis confirmed efficient transduction of hASCs expressing 7.65 ± 0.86 ng functionally active VEGF per 10(6) microencapsulated hASCs (ASC-VEGF). To determine the potential of the developed system, chronically infarcted rat hearts were treated with either empty microcapsules (MC), microencapsulated hASCs expressing MGFP reporter protein (MC+ASC-MGFP), or MC+ASC-VEGF, and analyzed for 10 weeks. Post-transplantation data confirmed higher myocardial VEGF expressions with significantly enhanced neovasculature in the MC+ASC-VEGF group. In addition, the cardiac performance, as measured by percentage ejection fraction, also improved significantly in the MC+ASC-VEGF group (48.6 ± 6.1%) compared to that in MC+ASC-MGFP (38.8 ± 5.3%) and MC groups (31.5 ± 3.3%). Collectively, these data demonstrate the feasibility of this system for improved stem cell therapy applications.

Plant growth responses to elevated atmospheric CO2 are increased by phosphorus sufficiency but not by arbuscular mycorrhizas.

PubMed

Jakobsen, Iver; Smith, Sally E; Smith, F Andrew; Watts-Williams, Stephanie J; Clausen, Signe S; Grønlund, Mette

2016-11-01

Capturing the full growth potential in crops under future elevated CO 2 (eCO 2 ) concentrations would be facilitated by improved understanding of eCO 2 effects on uptake and use of mineral nutrients. This study investigates interactions of eCO 2 , soil phosphorus (P), and arbuscular mycorrhizal (AM) symbiosis in Medicago truncatula and Brachypodium distachyon grown under the same conditions. The focus was on eCO 2 effects on vegetative growth, efficiency in acquisition and use of P, and expression of phosphate transporter (PT) genes. Growth responses to eCO 2 were positive at P sufficiency, but under low-P conditions they ranged from non-significant in M. truncatula to highly significant in B. distachyon Growth of M. truncatula was increased by AM at low P conditions at both CO 2 levels and eCO 2 ×AM interactions were sparse. Elevated CO 2 had small effects on P acquisition, but enhanced conversion of tissue P into biomass. Expression of PT genes was influenced by eCO 2 , but effects were inconsistent across genes and species. The ability of eCO 2 to partly mitigate P limitation-induced growth reductions in B. distachyon was associated with enhanced P use efficiency, and requirements for P fertilizers may not increase in such species in future CO 2 -rich climates. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Interleukin-6 and vascular endothelial growth factor release by renal cell carcinoma cells impedes lymphocyte-dendritic cell cross-talk.

PubMed

Cabillic, F; Bouet-Toussaint, F; Toutirais, O; Rioux-Leclercq, N; Fergelot, P; de la Pintière, C Thomas; Genetet, N; Patard, J-J; Catros-Quemener, V

2006-12-01

Anti-tumour T cell response requires antigen presentation via efficient immunological synapse between antigen presenting cells, e.g. dendritic cells (DC), and specific T cells in an adapted Th1 cytokine context. Nine renal cell carcinoma (RCC) primary culture cells were used as sources of tumour antigens which were loaded on DC (DC-Tu) for autologous T cell activation assays. Cytotoxic activity of lymphocytes stimulated with DC-Tu was evaluated against autologous tumour cells. Assays were performed with 75 grays irradiated tumour cells (Tu irr) and with hydrogen peroxide +/- heat shock (Tu H(2)O(2) +/- HS) treated cells. DC-Tu irr failed to enhance cytotoxic activity of autologous lymphocytes in seven of 13 assays. In all these defective assays, irradiated tumour cells displayed high interleukin (IL)-6 and vascular endothelial growth factor (VEGF) release. Conversely, when tumour cells released low IL-6 levels (n = 4), DC-Tu irr efficiently enhanced CTL activity. When assays were performed with the same RCC cells treated with H(2)O(2) + HS, DC-Tu stimulation resulted in improved CTL activity. H(2)O(2) + HS treatment induced post-apoptotic cell necrosis of tumour cells, totally abrogated their cytokine release [IL-6, VEGF, transforming growth factor (TGF)-beta1] and induced HSP70 expression. Taken together, data show that reduction in IL-6 and VEGF release in the environment of the tumour concomitantly to tumour cell HSP expression favours induction of a stronger anti-tumour CTL response.

Oil Body-Bound Oleosin-rhFGF-10: A Novel Drug Delivery System that Improves Skin Penetration to Accelerate Wound Healing and Hair Growth in Mice

PubMed Central

Yang, Jing; Cai, Jingbo; Wang, Hongyu; Tian, Haishan; Huang, Jian; Qiang, Weidong; Zhang, Linbo; Li, Haiyan; Li, Xiaokun; Jiang, Chao

2017-01-01

Recombinant human fibroblast growth factor 10 (rhFGF-10) is frequently used to treat patients with skin injuries. It can also promote hair growth. However, the effective application of rhFGF-10 is limited because of its poor stability and transdermal absorption. In this study, polymerase chain reaction (PCR) and Southern blotting were used to identify transgenic safflowers carrying a gene encoding an oleosin-rhFGF-10 fusion protein. The size and structural integrity of oleosin-rhFGF-10 in oil bodies extracted from transgenic safflower seeds was characterized by polyacrylamide gel electrophoresis and western blotting. Oil body extracts containing oleosin-rhFGF-10 were topically applied to mouse skin. The absorption of oleosin-rhFGF-10 was studied by immunohistochemistry. Its efficiency in promoting wound healing and hair regeneration were evaluated in full thickness wounds and hair growth assays. We identified a safflower line that carried the transgene and expressed a 45 kDa oleosin-rhFGF-10 protein. Oil body-bound oleosin-rhFGF-10 was absorbed by the skin with higher efficiency and speed compared with prokaryotically-expressed rhFGF-10. Oleosin-rhFGF-10 also enhanced wound closure and promoted hair growth better than rhFGF-10. The application of oleosin-rhFGF-10 in oil bodies promoted its delivery through the skin, providing a basis for improved therapeutic effects in enhancing wound healing and hair growth. PMID:29057820

Effect of low-level mechanical vibration on osteogenesis and osseointegration of porous titanium implants in the repair of long bone defects

NASA Astrophysics Data System (ADS)

Jing, Da; Tong, Shichao; Zhai, Mingming; Li, Xiaokang; Cai, Jing; Wu, Yan; Shen, Guanghao; Zhang, Xuhui; Xu, Qiaoling; Guo, Zheng; Luo, Erping

2015-11-01

Emerging evidence substantiates the potential of porous titanium alloy (pTi) as an ideal bone-graft substitute because of its excellent biocompatibility and structural properties. However, it remains a major clinical concern for promoting high-efficiency and high-quality osseointegration of pTi, which is beneficial for securing long-term implant stability. Accumulating evidence demonstrates the capacity of low-amplitude whole-body vibration (WBV) in preventing osteopenia, whereas the effects and mechanisms of WBV on osteogenesis and osseointegration of pTi remain unclear. Our present study shows that WBV enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton of primary osteoblasts in pTi. WBV upregulated osteogenesis-associated gene and protein expression in primary osteoblasts, including OCN, Runx2, Wnt3a, Lrp6 and β-catenin. In vivo findings demonstrate that 6-week and 12-week WBV stimulated osseointegration, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defects via μCT, histological and histomorphometric analyses. WBV induced higher ALP, OCN, Runx2, BMP2, Wnt3a, Lrp6 and β-catenin, and lower Sost and RANKL/OPG gene expression in rabbit femora. Our findings demonstrate that WBV promotes osteogenesis and osseointegration of pTi via its anabolic effect and potential anti-catabolic activity, and imply the promising potential of WBV for enhancing the repair efficiency and quality of pTi in osseous defects.

Oil Body-Bound Oleosin-rhFGF-10: A Novel Drug Delivery System that Improves Skin Penetration to Accelerate Wound Healing and Hair Growth in Mice.

PubMed

Li, Wenqing; Yang, Jing; Cai, Jingbo; Wang, Hongyu; Tian, Haishan; Huang, Jian; Qiang, Weidong; Zhang, Linbo; Li, Haiyan; Li, Xiaokun; Jiang, Chao

2017-10-18

Recombinant human fibroblast growth factor 10 (rhFGF-10) is frequently used to treat patients with skin injuries. It can also promote hair growth. However, the effective application of rhFGF-10 is limited because of its poor stability and transdermal absorption. In this study, polymerase chain reaction (PCR) and Southern blotting were used to identify transgenic safflowers carrying a gene encoding an oleosin-rhFGF-10 fusion protein. The size and structural integrity of oleosin-rhFGF-10 in oil bodies extracted from transgenic safflower seeds was characterized by polyacrylamide gel electrophoresis and western blotting. Oil body extracts containing oleosin-rhFGF-10 were topically applied to mouse skin. The absorption of oleosin-rhFGF-10 was studied by immunohistochemistry. Its efficiency in promoting wound healing and hair regeneration were evaluated in full thickness wounds and hair growth assays. We identified a safflower line that carried the transgene and expressed a 45 kDa oleosin-rhFGF-10 protein. Oil body-bound oleosin-rhFGF-10 was absorbed by the skin with higher efficiency and speed compared with prokaryotically-expressed rhFGF-10. Oleosin-rhFGF-10 also enhanced wound closure and promoted hair growth better than rhFGF-10. The application of oleosin-rhFGF-10 in oil bodies promoted its delivery through the skin, providing a basis for improved therapeutic effects in enhancing wound healing and hair growth.

Expression and characterization of an enhanced recombinant heparinase I with chitin binding domain.

PubMed

Xu, Shuqin; Qiu, Meiling; Zhang, Xuanyue; Chen, Jinghua

2017-12-01

Heparinase I (Hep I) can efficiently depolymerize heparin and heparin sulfate to oligosaccharides or unsaturated disaccharides, which resulted in loss of physiological function such as blood coagulation. In order to realize the immobilization of Hep I on chitin carriers, we cloned Hep I with the chitin binding domain (ChBD) as a chitin-affinity tag, and the Small Ubiquitin-like MOdifier (SUMO) linker as a solvation enhancer in different fusion sequence. DNA and protein gels suggested that 4 kinds of recombinants were successfully constructed and expressed in Escherichia coli (E. coli). And the triple functional heparinases isolated from cell lysate could be efficiently purified by chitin beads. After optimizing fermentation conditions, it gave the specific enzyme activities of 1.88±0.11, 3.69±0.45, 3.44±0.38, and 2.73±0.29IU/mg total proteins for ChBD-Hep I, ChBD-SUMO-Hep I, SUMO-ChBD-Hep I, and ChBD-Hep I-SUMO, respectively, with unfractionated heparin as substrate. The optimal reaction temperature and pH were determined to be 30°C and 7.0 for all the fusion enzymes. ChBD-SUMO-Hep I exhibited the maximum half-life (48min) at 30°C and best thermo-stability under 15-50°C. All the fusion enzymes showed broad pH-stability in the range of 5.4-9.0. Copyright © 2017 Elsevier B.V. All rights reserved.

Treatment of Parkinson's disease in rats by Nrf2 transfection using MRI-guided focused ultrasound delivery of nanomicrobubbles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Long, Ling; Cai, Xiaodong; Guo, Ruomi

Parkinson's disease (PD) is a very common neurological disorder. However, effective therapy is lacking. Although the blood-brain-barrier (BBB) protects the brain, it prevents the delivery of about 90% of drugs and nucleotides into the brain, thereby hindering the development of gene therapy for PD. Magnetic resonance imaging (MRI)-guided focused ultrasound delivery of microbubbles enhances the delivery of gene therapy vectors across the BBB and improves transfection efficiency. In the present study, we delivered nuclear factor E2-related factor 2 (Nrf2, NFE2L2) contained in nanomicrobubbles into the substantia nigra of PD rats by MRI-guided focused ultrasound, and we examined the effect ofmore » Nrf2 over-expression in this animal model of PD. The rat model of PD was established by injecting 6-OHDA in the right substantia nigra stereotactically. Plasmids (pDC315 or pDC315/Nrf2) were loaded onto nanomicrobubbles, and then injected through the tail vein with the assistance of MRI-guided focused ultrasound. MRI-guided focused ultrasound delivery of nanomicrobubbles increased gene transfection efficiency. Furthermore, Nrf2 gene transfection reduced reactive oxygen species levels, thereby protecting neurons in the target region. - Highlights: • MRI-guided focused ultrasound enhances gene transfection into the brain of rats. • Increased Nrf2 expression protects neurons in the rat model of PD. • Nrf2 protects neurons in PD by inhibiting ROS production.« less

Effect of low-level mechanical vibration on osteogenesis and osseointegration of porous titanium implants in the repair of long bone defects

PubMed Central

Jing, Da; Tong, Shichao; Zhai, Mingming; Li, Xiaokang; Cai, Jing; Wu, Yan; Shen, Guanghao; Zhang, Xuhui; Xu, Qiaoling; Guo, Zheng; Luo, Erping

2015-01-01

Emerging evidence substantiates the potential of porous titanium alloy (pTi) as an ideal bone-graft substitute because of its excellent biocompatibility and structural properties. However, it remains a major clinical concern for promoting high-efficiency and high-quality osseointegration of pTi, which is beneficial for securing long-term implant stability. Accumulating evidence demonstrates the capacity of low-amplitude whole-body vibration (WBV) in preventing osteopenia, whereas the effects and mechanisms of WBV on osteogenesis and osseointegration of pTi remain unclear. Our present study shows that WBV enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton of primary osteoblasts in pTi. WBV upregulated osteogenesis-associated gene and protein expression in primary osteoblasts, including OCN, Runx2, Wnt3a, Lrp6 and β-catenin. In vivo findings demonstrate that 6-week and 12-week WBV stimulated osseointegration, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defects via μCT, histological and histomorphometric analyses. WBV induced higher ALP, OCN, Runx2, BMP2, Wnt3a, Lrp6 and β-catenin, and lower Sost and RANKL/OPG gene expression in rabbit femora. Our findings demonstrate that WBV promotes osteogenesis and osseointegration of pTi via its anabolic effect and potential anti-catabolic activity, and imply the promising potential of WBV for enhancing the repair efficiency and quality of pTi in osseous defects. PMID:26601709

The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

PubMed Central

Papanikolaou, Eleni; Georgomanoli, Maria; Stamateris, Evangelos; Panetsos, Fottes; Karagiorga, Markisia; Tsaftaridis, Panagiotis; Graphakos, Stelios

2012-01-01

Abstract To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated self-inactivating (SIN) lentiviral vector, termed GGHI, carrying the Aγ-globin gene with the −117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of Aγ-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34+ hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with β-thalassemia. Our results show that GGHI increased the production of γ-globin by 32.9% as measured by high-performance liquid chromatography (p=0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of γ-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro. PMID:21875313

Internal quantum efficiency enhancement of GaInN/GaN quantum-well structures using Ag nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iida, Daisuke; Department of Photonics Engineering, Technical University of Denmark, 2800 Lyngby; Faculty of Science and Technology, Meijo University, 1-501 Shiogamaguchi Tempaku, 468-8502 Nagoya

2015-09-15

We report internal quantum efficiency enhancement of thin p-GaN green quantum-well structure using self-assembled Ag nanoparticles. Temperature dependent photoluminescence measurements are conducted to determine the internal quantum efficiency. The impact of excitation power density on the enhancement factor is investigated. We obtain an internal quantum efficiency enhancement by a factor of 2.3 at 756 W/cm{sup 2}, and a factor of 8.1 at 1 W/cm{sup 2}. A Purcell enhancement up to a factor of 26 is estimated by fitting the experimental results to a theoretical model for the efficiency enhancement factor.

Variability of interferon-λ induction and antiviral activity in Nipah virus infected differentiated human bronchial epithelial cells of two human donors.

PubMed

Sauerhering, Lucie; Müller, Helena; Behner, Laura; Elvert, Mareike; Fehling, Sarah Katharina; Strecker, Thomas; Maisner, Andrea

2017-10-01

Highly pathogenic Nipah virus (NiV) generally causes severe encephalitis in humans. Respiratory symptoms are infrequently observed, likely reflecting variations in infection kinetics in human airways. Supporting this idea, we recently identified individual differences in NiV replication kinetics in cultured airway epithelia from different human donors. As type III interferons (IFN-λ) represent major players in the defence mechanism against viral infection of the respiratory mucosa, we studied IFN-λ induction and antiviral activity in NiV-infected primary differentiated human bronchial epithelial cells (HBEpCs) cultured under air-liquid interface conditions. Our studies revealed that IFN-λ was upregulated in airway epithelia upon NiV infection. We also show that IFN-λ pretreatment efficiently inhibited NiV replication. Interestingly, the antiviral activity of IFN-λ varied in HBEpCs from two different donors. Increased sensitivity to IFN-λ was associated with higher expression levels of IFN-λ receptors, enhanced phosphorylation of STAT1, as well as enhanced induction of interferon-stimulated gene expression. These findings suggest that individual variations in IFN-λ receptor expression affecting IFN responsiveness can play a functional role for NiV replication kinetics in human respiratory epithelial cells of different donors.

Disruption of pknG enhances production of gamma-aminobutyric acid by Corynebacterium glutamicum expressing glutamate decarboxylase.

PubMed

Okai, Naoko; Takahashi, Chihiro; Hatada, Kazuki; Ogino, Chiaki; Kondo, Akihiko

2014-01-01

Gamma-aminobutyric acid (GABA), a building block of the biodegradable plastic polyamide 4, is synthesized from glucose by Corynebacterium glutamicum that expresses Escherichia coli glutamate decarboxylase (GAD) B encoded by gadB. This strain was engineered to produce GABA more efficiently from biomass-derived sugars. To enhance GABA production further by increasing the intracellular concentration of its precursor glutamate, we focused on engineering pknG (encoding serine/threonine protein kinase G), which controls the activity of 2-oxoglutarate dehydrogenase (Odh) in the tricarboxylic acid cycle branch point leading to glutamate synthesis. We succeeded in expressing GadB in a C. glutamicum strain harboring a deletion of pknG. C. glutamicum strains GAD and GAD ∆pknG were cultured in GP2 medium containing 100 g L(-1) glucose and 0.1 mM pyridoxal 5'-phosphate. Strain GAD∆pknG produced 31.1 ± 0.41 g L(-1) (0.259 g L(-1) h(-1)) of GABA in 120 hours, representing a 2.29-fold higher level compared with GAD. The production yield of GABA from glucose by GAD∆pknG reached 0.893 mol mol(-1).

Exosomes derived from human mesenchymal stem cells promote gastric cancer cell growth and migration via the activation of the Akt pathway.

PubMed

Gu, Hongbing; Ji, Runbi; Zhang, Xu; Wang, Mei; Zhu, Wei; Qian, Hui; Chen, Yongchang; Jiang, Pengcheng; Xu, Wenrong

2016-10-01

Mesenchymal stem cells (MSCs) are a component of the tumor microenvironment and can promote the development of gastric cancer through paracrine mechanism. However, the effects of MSC‑exosomes (MSC‑ex) on gastric cancer are less clear. The present study reported that MSC‑ex promoted the proliferative and metastatic potential of gastric cancer cells ex vivo. It was found that MSC‑ex enhanced the migration and invasion of HGC‑27 cells via the induction of the epithelial‑mesenchymal transition. MSC‑ex increased the expression of mesenchymal markers and reduced the expression of epithelial markers in gastric cancer cells. MSC‑ex also enhanced the tumorigenicity of gastric cancer cells ex vivo. MSC‑ex induced the stemness of gastric cancer cells. The expression of octamer‑binding transcription factor 4, ex determining region Y‑box 2 and Lin28B significantly increased in gastric cancer cells treated with MSC‑ex. The present study further demonstrated that MSC‑ex elicited these biological effects predominantly via the activation of the protein kinase B signaling pathway. Taken together, the present findings provided novel evidence for the role of MSC‑ex in gastric cancer and a new opportunity for improving the efficiency of gastric cancer treatment by targeting MSC‑ex.

Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts.

PubMed

Nagaoka, Aya; Yoshida, Hiroyuki; Nakamura, Sachiko; Morikawa, Tomohiko; Kawabata, Keigo; Kobayashi, Masaki; Sakai, Shingo; Takahashi, Yoshito; Okada, Yasunori; Inoue, Shintaro

2015-12-25

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Nonorthogonal orbital based N-body reduced density matrices and their applications to valence bond theory. I. Hamiltonian matrix elements between internally contracted excited valence bond wave functions

NASA Astrophysics Data System (ADS)

Chen, Zhenhua; Chen, Xun; Wu, Wei

2013-04-01

In this series, the n-body reduced density matrix (n-RDM) approach for nonorthogonal orbitals and their applications to ab initio valence bond (VB) methods are presented. As the first paper of this series, Hamiltonian matrix elements between internally contracted VB wave functions are explicitly provided by means of nonorthogonal orbital based RDM approach. To this end, a more generalized Wick's theorem, called enhanced Wick's theorem, is presented both in arithmetical and in graphical forms, by which the deduction of expressions for the matrix elements between internally contracted VB wave functions is dramatically simplified, and the matrix elements are finally expressed in terms of tensor contractions of electronic integrals and n-RDMs of the reference VB self-consistent field wave function. A string-based algorithm is developed for the purpose of evaluating n-RDMs in an efficient way. Using the techniques presented in this paper, one is able to develop new methods and efficient algorithms for nonorthogonal orbital based many-electron theory much easier than by use of the first quantized formulism.

Enhanced Development of Skeletal Myotubes from Porcine Induced Pluripotent Stem Cells

PubMed Central

Genovese, Nicholas J.; Domeier, Timothy L.; Telugu, Bhanu Prakash V. L.; Roberts, R. Michael

2017-01-01

The pig is recognized as a valuable model in biomedical research in addition to its agricultural importance. Here we describe a means for generating skeletal muscle efficiently from porcine induced pluripotent stem cells (piPSC) in vitro thereby providing a versatile platform for applications ranging from regenerative biology to the ex vivo cultivation of meat. The GSK3B inhibitor, CHIR99021 was employed to suppress apoptosis, elicit WNT signaling events and drive naïve-type piPSC along the mesoderm lineage, and, in combination with the DNA methylation inhibitor 5-aza-cytidine, to activate an early skeletal muscle transcription program. Terminal differentiation was then induced by activation of an ectopically expressed MYOD1. Myotubes, characterized by myofibril development and both spontaneous and stimuli-elicited excitation-contraction coupling cycles appeared within 11 days. Efficient lineage-specific differentiation was confirmed by uniform NCAM1 and myosin heavy chain expression. These results provide an approach for generating skeletal muscle that is potentially applicable to other pluripotent cell lines and to generating other forms of muscle. PMID:28165492

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

NASA Astrophysics Data System (ADS)

Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

2017-01-01

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode.

PubMed

Rezaie, F; Davami, F; Mansouri, K; Agha Amiri, S; Fazel, R; Mahdian, R; Davoudi, N; Enayati, S; Azizi, M; Khalaj, V

2017-05-08

The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase ® cultivation mode and a redox mutant strain in the E. coli expression system. A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle ® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase ® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis ® . This study demonstrated that the combination of SUMO fusion technology, EnBase ® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments. © 2017 The Society for Applied Microbiology.

Poly (dopamine) coated superparamagnetic iron oxide nanocluster for noninvasive labeling, tracking, and targeted delivery of adipose tissue-derived stem cells.

PubMed

Liao, Naishun; Wu, Ming; Pan, Fan; Lin, Jiumao; Li, Zuanfang; Zhang, Da; Wang, Yingchao; Zheng, Youshi; Peng, Jun; Liu, Xiaolong; Liu, Jingfeng

2016-01-05

Tracking and monitoring of cells in vivo after transplantation can provide crucial information for stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be an effective and non-invasive technique for cell tracking in living bodies. However, commercial superparamagnetic iron oxide nanoparticles (SPIONs) applied to label cells suffer from shortages such as potential toxicity, low labeling efficiency, and low contrast enhancing. Herein, the adipose tissue-derived stem cells (ADSCs) were efficiently labeled with SPIONs coated with poly (dopamine) (SPIONs cluster@PDA), without affecting their viability, proliferation, apoptosis, surface marker expression, as well as their self-renew ability and multi-differentiation potential. The labeled cells transplanted into the mice through tail intravenous injection exhibited a negative enhancement of the MRI signal in the damaged liver-induced by carbon tetrachloride, and subsequently these homed ADSCs with SPIONs cluster@PDA labeling exhibited excellent repair effects to the damaged liver. Moreover, the enhanced target-homing to tissue of interest and repair effects of SPIONs cluster@PDA-labeled ADSCs could be achieved by use of external magnetic field in the excisional skin wound mice model. Therefore, we provide a facile, safe, noninvasive and sensitive method for external magnetic field targeted delivery and MRI based tracking of transplanted cells in vivo.

Defined Host–Guest Chemistry on Nanocarbon for Sustained Inhibition of Cancer

PubMed Central

Ostadhossein, Fatemeh; Misra, Santosh K.; Mukherjee, Prabuddha; Ostadhossein, Alireza; Daza, Enrique; Tiwari, Saumya; Mittal, Shachi; Gryka, Mark C.; Bhargava, Rohit

2017-01-01

Signal transducer and activator of transcription factor 3 (STAT-3) is known to be overexpressed in cancer stem cells. Poor solubility and variable drug absorption are linked to low bioavailability and decreased efficacy. Many of the drugs regulating STAT-3 expression lack aqueous solubility; hence hindering efficient bioavailability. A theranostics nanoplatform based on luminescent carbon particles decorated with cucurbit[6]uril is introduced for enhancing the solubility of niclosamide, a STAT-3 inhibitor. The host–guest chemistry between cucurbit[6]uril and niclosamide makes the delivery of the hydrophobic drug feasible while carbon nanoparticles enhance cellular internalization. Extensive physicochemical characterizations confirm successful synthesis. Subsequently, the host–guest chemistry of niclosamide and cucurbit[6]uril is studied experimentally and computationally. In vitro assessments in human breast cancer cells indicate approximately twofold enhancement in IC50 of drug. Fourier transform infrared and fluorescence imaging demonstrate efficient cellular internalization. Furthermore, the catalytic biodegradation of the nanoplatforms occur upon exposure to human myeloperoxidase in short time. In vivo studies on athymic mice with MCF-7 xenograft indicate the size of tumor in the treatment group is half of the controls after 40 d. Immunohistochemistry corroborates the downregulation of STAT-3 phosphorylation. Overall, the host–guest chemistry on nanocarbon acts as a novel arsenal for STAT-3 inhibition. PMID:27545321

Poly (dopamine) coated superparamagnetic iron oxide nanocluster for noninvasive labeling, tracking, and targeted delivery of adipose tissue-derived stem cells

NASA Astrophysics Data System (ADS)

Liao, Naishun; Wu, Ming; Pan, Fan; Lin, Jiumao; Li, Zuanfang; Zhang, Da; Wang, Yingchao; Zheng, Youshi; Peng, Jun; Liu, Xiaolong; Liu, Jingfeng

2016-01-01

Tracking and monitoring of cells in vivo after transplantation can provide crucial information for stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be an effective and non-invasive technique for cell tracking in living bodies. However, commercial superparamagnetic iron oxide nanoparticles (SPIONs) applied to label cells suffer from shortages such as potential toxicity, low labeling efficiency, and low contrast enhancing. Herein, the adipose tissue-derived stem cells (ADSCs) were efficiently labeled with SPIONs coated with poly (dopamine) (SPIONs cluster@PDA), without affecting their viability, proliferation, apoptosis, surface marker expression, as well as their self-renew ability and multi-differentiation potential. The labeled cells transplanted into the mice through tail intravenous injection exhibited a negative enhancement of the MRI signal in the damaged liver-induced by carbon tetrachloride, and subsequently these homed ADSCs with SPIONs cluster@PDA labeling exhibited excellent repair effects to the damaged liver. Moreover, the enhanced target-homing to tissue of interest and repair effects of SPIONs cluster@PDA-labeled ADSCs could be achieved by use of external magnetic field in the excisional skin wound mice model. Therefore, we provide a facile, safe, noninvasive and sensitive method for external magnetic field targeted delivery and MRI based tracking of transplanted cells in vivo.

Enhanced disease resistance and drought tolerance in transgenic rice plants overexpressing protein elicitors from Magnaporthe oryzae.

PubMed

Wang, Zhenzhen; Han, Qiang; Zi, Qian; Lv, Shun; Qiu, Dewen; Zeng, Hongmei

2017-01-01

Exogenous application of the protein elicitors MoHrip1 and MoHrip2, which were isolated from the pathogenic fungus Magnaporthe oryzae (M. oryzae), was previously shown to induce a hypersensitive response in tobacco and to enhance resistance to rice blast. In this work, we successfully transformed rice with the mohrip1 and mohrip2 genes separately. The MoHrip1 and MoHrip2 transgenic rice plants displayed higher resistance to rice blast and stronger tolerance to drought stress than wild-type (WT) rice and the vector-control pCXUN rice. The expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes was also increased, suggesting that these two elicitors may trigger SA signaling to protect the rice from damage during pathogen infection and regulate the ABA content to increase drought tolerance in transgenic rice. Trypan blue staining indicated that expressing MoHrip1 and MoHrip2 in rice plants inhibited hyphal growth of the rice blast fungus. Relative water content (RWC), water usage efficiency (WUE) and water loss rate (WLR) were measured to confirm the high capacity for water retention in transgenic rice. The MoHrip1 and MoHrip2 transgenic rice also exhibited enhanced agronomic traits such as increased plant height and tiller number.

Prolonging the expression duration of ultrasound-mediated gene transfection using PEI nanoparticles.

PubMed

Lee, Jyun-Lin; Lo, Chia-Wen; Ka, Shuk-Man; Chen, Ann; Chen, Wen-Shiang

2012-05-30

Ultrasound (US) irradiation has been found to facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, its transfection efficiency is generally low, and the expression duration of transfected gene is short. Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. The purpose of this study is to determine whether PEI can also prolong the expression duration after US-mediated transfection. A mixture of pCMViLUC and 22-kDa linear PEI was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulsed US. The duration of expression was assessed periodically following US treatment. As expected, strong expression of luciferase could be found 30days after the treatment of DNA-PEI complex with US exposure, both in vitro and in vivo. However, without US, only very low transfection level could be obtained in vivo. The DNA/PEI complex showed protective effect against digestion of DNase I enzymes as compared with groups without PEI or to which PEI was added following the mixing of plasmid DNA with DNase I. PEI enhanced the US transfection efficiency by increasing both the intracellular uptake of plasmid DNA and the percentage of transfected cells. Most of the DNA uptake occurred at 3h after US exposure, suggesting that endocytosis took place. Moreover, the PEI-facilitated US gene transfection depended on the ratio of PEI and DNA (N/P ratio), which was different for in-vitro and in-vivo conditions. This system could be applied in future human gene therapies. Copyright © 2012 Elsevier B.V. All rights reserved.

Partially Redundant Enhancers Cooperatively Maintain Mammalian Pomc Expression Above a Critical Functional Threshold

PubMed Central

Lam, Daniel D.; de Souza, Flavio S. J.; Nasif, Sofia; Yamashita, Miho; López-Leal, Rodrigo; Meece, Kana; Sampath, Harini; Mercer, Aaron J.; Wardlaw, Sharon L.

2015-01-01

Cell-specific expression of many genes is conveyed by multiple enhancers, with each individual enhancer controlling a particular expression domain. In contrast, multiple enhancers drive similar expression patterns of some genes involved in embryonic development, suggesting regulatory redundancy. Work in Drosophila has indicated that functionally overlapping enhancers canalize development by buffering gene expression against environmental and genetic disturbances. However, little is known about regulatory redundancy in vertebrates and in genes mainly expressed during adulthood. Here we study nPE1 and nPE2, two phylogenetically conserved mammalian enhancers that drive expression of the proopiomelanocortin gene (Pomc) to the same set of hypothalamic neurons. The simultaneous deletion of both enhancers abolished Pomc expression at all ages and induced a profound metabolic dysfunction including early-onset extreme obesity. Targeted inactivation of either nPE1 or nPE2 led to very low levels of Pomc expression during early embryonic development indicating that both enhancers function synergistically. In adult mice, however, Pomc expression is controlled additively by both enhancers, with nPE1 being responsible for ∼80% and nPE2 for ∼20% of Pomc transcription. Consequently, nPE1 knockout mice exhibit mild obesity whereas nPE2-deficient mice maintain a normal body weight. These results suggest that nPE2-driven Pomc expression is compensated by nPE1 at later stages of development, essentially rescuing the earlier phenotype of nPE2 deficiency. Together, these results reveal that cooperative interactions between the enhancers confer robustness of Pomc expression against gene regulatory disturbances and preclude deleterious metabolic phenotypes caused by Pomc deficiency in adulthood. Thus, our study demonstrates that enhancer redundancy can be used by genes that control adult physiology in mammals and underlines the potential significance of regulatory sequence mutations in common diseases. PMID:25671638

Advantages and applications of CAR-expressing natural killer cells

PubMed Central

Glienke, Wolfgang; Esser, Ruth; Priesner, Christoph; Suerth, Julia D.; Schambach, Axel; Wels, Winfried S.; Grez, Manuel; Kloess, Stephan; Arseniev, Lubomir; Koehl, Ulrike

2015-01-01

In contrast to donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR) expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy. PMID:25729364

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.

PubMed

Yin, Linlin; Maddison, Lisette A; Li, Mingyu; Kara, Nergis; LaFave, Matthew C; Varshney, Gaurav K; Burgess, Shawn M; Patton, James G; Chen, Wenbiao

2015-06-01

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. Copyright © 2015 by the Genetics Society of America.

Therapy of pancreatic cancer via an EphA2 receptor-targeted delivery of gemcitabine

PubMed Central

Barile, Elisa; Das, Swadesh K.; Emdad, Luni; Sarkar, Devanand; De, Surya K.; Kharagh, Susan Morvaridi; Stebbins, John L.; Pandol, Stephen J.; Fisher, Paul B.; Pellecchia, Maurizio

2016-01-01

First line treatment for pancreatic cancer consists of surgical resection, if possible, and a subsequent course of chemotherapy using the nucleoside analogue gemcitabine. In some patients, an active transport mechanism allows gemcitabine to enter efficiently into the tumor cells, resulting in a significant clinical benefit. However, in most patients, low expression of gemcitabine transporters limits the efficacy of the drug to marginal levels, and patients need frequent administration of the drug at high doses, significantly increasing systemic drug toxicity. In this article we focus on a novel targeted delivery approach for gemcitabine consisting of conjugating the drug with an EphA2 targeting agent. We show that the EphA2 receptor is highly expressed in pancreatic cancers, and accordingly, the drug-conjugate is more effective than gemcitabine alone in targeting pancreatic tumors. Our preliminary observations suggest that this approach may provide a general benefit to pancreatic cancer patients and offers a comprehensive strategy for enhancing delivery of diverse therapeutic agents to a wide range of cancers overexpressing EphA2, thereby potentially reducing toxicity while enhancing therapeutic efficacy. PMID:26959746

CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity.

PubMed

Xu, Xiang-Ru Shannon; Gantz, Valentino Matteo; Siomava, Natalia; Bier, Ethan

2017-12-23

The knirps ( kni ) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila . Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these ' in locus ' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis. © 2017, Xu et al.

CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity

PubMed Central

Siomava, Natalia

2017-01-01

The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these ‘in locus’ mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis. PMID:29274230

Supplementation of chitosan alleviates high-fat diet-enhanced lipogenesis in rats via adenosine monophosphate (AMP)-activated protein kinase activation and inhibition of lipogenesis-associated genes.

PubMed

Chiu, Chen-Yuan; Chan, Im-Lam; Yang, Tsung-Han; Liu, Shing-Hwa; Chiang, Meng-Tsan

2015-03-25

This study investigated the role of chitosan in lipogenesis in high-fat diet-induced obese rats. The lipogenesis-associated genes and their upstream regulatory proteins were explored. Diet supplementation of chitosan efficiently decreased the increased weights in body, livers, and adipose tissues in high-fat diet-fed rats. Chitosan supplementation significantly raised the lipolysis rate; attenuated the adipocyte hypertrophy, triglyceride accumulation, and lipoprotein lipase activity in epididymal adipose tissues; and decreased hepatic enzyme activities of lipid biosynthesis. Chitosan supplementation significantly activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and attenuated high-fat diet-induced protein expressions of lipogenic transcription factors (PPAR-γ and SREBP1c) in livers and adipose tissues. Moreover, chitosan supplementation significantly inhibited the expressions of downstream lipogenic genes (FAS, HMGCR, FATP1, and FABP4) in livers and adipose tissues of high-fat diet-fed rats. These results demonstrate for the first time that chitosan supplementation alleviates high-fat diet-enhanced lipogenesis in rats via AMPK activation and lipogenesis-associated gene inhibition.

Rerouting Carbon Flux To Enhance Photosynthetic Productivity

PubMed Central

Ducat, Daniel C.; Avelar-Rivas, J. Abraham; Way, Jeffrey C.

2012-01-01

The bioindustrial production of fuels, chemicals, and therapeutics typically relies upon carbohydrate inputs derived from agricultural plants, resulting in the entanglement of food and chemical commodity markets. We demonstrate the efficient production of sucrose from a cyanobacterial species, Synechococcus elongatus, heterologously expressing a symporter of protons and sucrose (cscB). cscB-expressing cyanobacteria export sucrose irreversibly to concentrations of >10 mM without culture toxicity. Moreover, sucrose-exporting cyanobacteria exhibit increased biomass production rates relative to wild-type strains, accompanied by enhanced photosystem II activity, carbon fixation, and chlorophyll content. The genetic modification of sucrose biosynthesis pathways to minimize competing glucose- or sucrose-consuming reactions can further improve sucrose production, allowing the export of sucrose at rates of up to 36.1 mg liter−1 h illumination−1. This rate of production exceeds that of previous reports of targeted, photobiological production from microbes. Engineered S. elongatus produces sucrose in sufficient quantities (up to ∼80% of total biomass) such that it may be a viable alternative to sugar synthesis from terrestrial plants, including sugarcane. PMID:22307292

Cloning, Expression, and Biochemical Characterization of an Enantioselective Lipase, YLIP9, from Yarrowia lipolytica MSR80.

PubMed

Syal, Poonam; Gupta, Rani

2015-05-01

A novel lipase gene, ylip9, of Yarrowia lipolytica MSR80 was cloned and expressed in pEZZ18-HB101 system and was 99% identical to YLIP9 of Y. lipolytica CLIB122. It was purified using IgG-Sepharose as ZZ fused YLIP9 and had specific activity of 0.8 U/mg. ZZ-YLIP9 was most active at pH 8.0 and 70 °C. It was stable over a wide pH range of 3.0-11.0 and 100 % active at 70 °C up to 2 h and had t1/2 of 286.42 min at 80 °C. It showed high specificity toward p-nitrophenyldecanoate with kcat and catalytic efficiency of 30.17 s(-1) and 16.67 mM(-1) s(-1), respectively. It was non-regioselective, but an S-enantioselective lipase and the percentage conversion were enhanced in presence of hexane. ZZ-YLIP9 was stable in all of the organic solvents used, and its activity was enhanced by solvents having logP value less than 2.

Immunoglobulin-like transcript receptors on human dermal CD14+ dendritic cells act as a CD8-antagonist to control cytotoxic T cell priming

PubMed Central

Banchereau, Jacques; Zurawski, Sandra; Thompson-Snipes, LuAnn; Blanck, Jean-Philippe; Clayton, Sandra; Munk, Adiel; Cao, Yanying; Wang, Zhiqing; Khandelwal, Sunaina; Hu, Jiancheng; McCoy, William H.; Palucka, Karolina A.; Reiter, Yoram; Fremont, Daved H.; Zurawski, Gerard; Colonna, Marco; Shaw, Andrey S.; Klechevsky, Eynav

2012-01-01

Human Langerhans cells (LCs) are highly efficient at priming cytolytic CD8+ T cells compared with dermal CD14+ dendritic cells (DCs). Here we show that dermal CD14+ DCs instead prime a fraction of naïve CD8+ T cells into cells sharing the properties of type 2 cytokine-secreting CD8+ T cells (TC2). Differential expression of the CD8-antagonist receptors on dermal CD14+ DCs, the Ig-like transcript (ILT) inhibitory receptors, explains the difference between the two types of DCs. Inhibition of CD8 function on LCs inhibited cytotoxic T lymphocytes (CTLs) and enhanced TC2 generation. In addition, blocking ILT2 or ILT4 on dermal CD14+ DCs enhanced the generation of CTLs and inhibited TC2 cytokine production. Lastly, addition of soluble ILT2 and ILT4 receptors inhibited CTL priming by LCs. Thus, ILT receptor expression explains the polarization of CD8+ T-cell responses by LCs vs. dermal CD14+ DCs. PMID:23112154

Rerouting Carbon Flux To Enhance Photosynthetic Productivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ducat, DC; Avelar-Rivas, JA; Way, JC

2012-03-23

The bioindustrial production of fuels, chemicals, and therapeutics typically relies upon carbohydrate inputs derived from agricultural plants, resulting in the entanglement of food and chemical commodity markets. We demonstrate the efficient production of sucrose from a cyanobacterial species, Synechococcus elongatus, heterologously expressing a symporter of protons and sucrose (cscB). cscB-expressing cyanobacteria export sucrose irreversibly to concentrations of >10 mM without culture toxicity. Moreover, sucrose-exporting cyanobacteria exhibit increased biomass production rates relative to wild-type strains, accompanied by enhanced photosystem II activity, carbon fixation, and chlorophyll content. The genetic modification of sucrose biosynthesis pathways to minimize competing glucose-or sucrose-consuming reactions can furthermore » improve sucrose production, allowing the export of sucrose at rates of up to 36.1 mg liter(-1) h illumination(-1). This rate of production exceeds that of previous reports of targeted, photobiological production from microbes. Engineered S. elongatus produces sucrose in sufficient quantities (up to similar to 80% of total biomass) such that it may be a viable alternative to sugar synthesis from terrestrial plants, including sugarcane.« less

Dual RNA-seq transcriptional analysis of wheat roots colonized by Azospirillum brasilense reveals up-regulation of nutrient acquisition and cell cycle genes.

PubMed

Camilios-Neto, Doumit; Bonato, Paloma; Wassem, Roseli; Tadra-Sfeir, Michelle Z; Brusamarello-Santos, Liziane C C; Valdameri, Glaucio; Donatti, Lucélia; Faoro, Helisson; Weiss, Vinicius A; Chubatsu, Leda S; Pedrosa, Fábio O; Souza, Emanuel M

2014-05-16

The rapid growth of the world's population demands an increase in food production that no longer can be reached by increasing amounts of nitrogenous fertilizers. Plant growth promoting bacteria (PGPB) might be an alternative to increase nitrogenous use efficiency (NUE) in important crops such wheat. Azospirillum brasilense is one of the most promising PGPB and wheat roots colonized by A. brasilense is a good model to investigate the molecular basis of plant-PGPB interaction including improvement in plant-NUE promoted by PGPB. We performed a dual RNA-Seq transcriptional profiling of wheat roots colonized by A. brasilense strain FP2. cDNA libraries from biological replicates of colonized and non-inoculated wheat roots were sequenced and mapped to wheat and A. brasilense reference sequences. The unmapped reads were assembled de novo. Overall, we identified 23,215 wheat expressed ESTs and 702 A. brasilense expressed transcripts. Bacterial colonization caused changes in the expression of 776 wheat ESTs belonging to various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones and phytochemicals. In addition, genes encoding proteins related to bacterial chemotaxi, biofilm formation and nitrogen fixation were highly expressed in the sub-set of A. brasilense expressed genes. PGPB colonization enhanced the expression of plant genes related to nutrient up-take, nitrogen assimilation, DNA replication and regulation of cell division, which is consistent with a higher proportion of colonized root cells in the S-phase. Our data support the use of PGPB as an alternative to improve nutrient acquisition in important crops such as wheat, enhancing plant productivity and sustainability.

N-methylpurine DNA glycosylase and DNA polymerase β modulate BER inhibitor potentiation of glioma cells to temozolomide

PubMed Central

Tang, Jiang-bo; Svilar, David; Trivedi, Ram N.; Wang, Xiao-hong; Goellner, Eva M.; Moore, Briana; Hamilton, Ronald L.; Banze, Lauren A.; Brown, Ashley R.; Sobol, Robert W.

2011-01-01

Temozolomide (TMZ) is the preferred chemotherapeutic agent in the treatment of glioma following surgical resection and/or radiation. Resistance to TMZ is attributed to efficient repair and/or tolerance of TMZ-induced DNA lesions. The majority of the TMZ-induced DNA base adducts are repaired by the base excision repair (BER) pathway and therefore modulation of this pathway can enhance drug sensitivity. N-methylpurine DNA glycosylase (MPG) initiates BER by removing TMZ-induced N3-methyladenine and N7-methylguanine base lesions, leaving abasic sites (AP sites) in DNA for further processing by BER. Using the human glioma cell lines LN428 and T98G, we report here that potentiation of TMZ via BER inhibition [methoxyamine (MX), the PARP inhibitors PJ34 and ABT-888 or depletion (knockdown) of PARG] is greatly enhanced by over-expression of the BER initiating enzyme MPG. We also show that methoxyamine-induced potentiation of TMZ in MPG expressing glioma cells is abrogated by elevated-expression of the rate-limiting BER enzyme DNA polymerase β (Polβ), suggesting that cells proficient for BER readily repair AP sites in the presence of MX. Further, depletion of Polβ increases PARP inhibitor-induced potentiation in the MPG over-expressing glioma cells, suggesting that expression of Polβ modulates the cytotoxic effect of combining increased repair initiation and BER inhibition. This study demonstrates that MPG overexpression, together with inhibition of BER, sensitizes glioma cells to the alkylating agent TMZ in a Polβ-dependent manner, suggesting that the expression level of both MPG and Polβ might be used to predict the effectiveness of MX and PARP-mediated potentiation of TMZ in cancer treatment. PMID:21377995

A silencing-mediated enhancement of osteogenic differentiation by supramolecular ternary siRNA polyplexes comprising biocleavable cationic polyrotaxanes and anionic fusogenic peptides.

PubMed

Inada, Takasuke; Tamura, Atsushi; Terauchi, Masahiko; Yamaguchi, Satoshi; Yui, Nobuhiko

2018-01-30

Gene silencing of noggin by small interfering RNA (siRNA) is a promising approach for the treatment of bone defects, because noggin deactivates bone morphogenetic protein-2 (BMP-2) and suppresses osteogenic differentiation. Here, we demonstrated the silencing of the noggin gene by siRNA polyplexes composed of noggin-targeted siRNA and biocleavable cationic polyrotaxanes (DMAE-SS-PRX). To improve the endosomal escape efficiencies of the DMAE-SS-PRX/siRNA polyplexes, anionic and fusogenic GALA peptides were integrated onto the DMAE-SS-PRX/siRNA polyplexes via simple electrostatic interactions. The formation of ternary complexes was confirmed by gel electrophoresis, dynamic light scattering, and zeta-potential measurements. Although the association of GALA peptides with the DMAE-SS-PRX/siRNA polyplexes did not remarkably affect the cellular uptake efficiency of siRNA, the endosomal escape efficiency was remarkably increased for GALA/DMAE-SS-PRX/siRNA ternary polyplexes because of the endosomal and lysosomal membrane destabilization by GALA peptides. Consequently, GALA/DMAE-SS-PRX/siRNA ternary polyplexes showed significantly higher gene silencing efficiency against noggin and enhanced the BMP-2-mediated osteogenic differentiation efficiency. Therefore, we concluded that GALA/DMAE-SS-PRX/siRNA ternary polyplexes can be effective siRNA carriers for suppressing the expression of specific endogenous genes. Consequently, we believe that a more practical approach in vivo will be the combined use of BMP-2 and GALA/DMAE-SS-PRX/siRNA ternary polyplexes, because it will improve the efficacy of bone regeneration therapy.

Thiolated chitosan/DNA nanocomplexes exhibit enhanced and sustained gene delivery.

PubMed

Lee, Dongwon; Zhang, Weidong; Shirley, Shawna A; Kong, Xiaoyuan; Hellermann, Gary R; Lockey, Richard F; Mohapatra, Shyam S

2007-01-01

Thiolated chitosan appears to possess enhanced mucoadhesiveness and cell penetration properties, however, its potential in gene-drug delivery remains unknown. Herein, we report on a highly effective gene delivery system utilizing a 33-kDa thiol-modified chitosan derivative. Thiolated chitosan was prepared by the reaction with thioglycolic acid. Nanocomplexes of unmodified chitosan or thiolated chitosan with plasmid DNA encoding green fluorescenct protein (GFP) were characterized for their size, zeta potential, their ability to bind and protect plasmid DNA from degradation. The transfection efficiency of thiolated chitosan and sustained gene expression were evaluated in various cell lines in vitro and in Balb/c mice in vivo. Thiolated chitosan-DNA nanocomplexes ranged in size from 75 to 120 nm in diameter and from +2.3 to 19.7 mV in zeta potential, depending on the weight ratio of chitosan to DNA. Thiolated chitosan, CSH360, exhibited effective physical stability and protection against DNase I digestion at a weight ratio>or=2.5:1. CSH360/DNA nanocomplexes induced significantly (P<0.01) higher GFP expression in HEK293, MDCK and Hep-2 cell lines than unmodified chitosan. Nanocomplexes of disulphide-crosslinked CSH360/DNA showed a sustained DNA release and continuous expression in cultured cells lasting up to 60 h post transfection. Also, intranasal administration of crosslinked CSH360/DNA nanocomplexes to mice yielded gene expression that lasted for at least 14 days. Thiolated chitosans condense pDNA to form nanocomplexes, which exhibit a significantly higher gene transfer potential and sustained gene expression upon crosslinking, indicating their great potential for gene therapy and tissue engineering.

Activated STAT5 promotes long-lived cytotoxic CD8+ T cells that induce regression of autochthonous melanoma.

PubMed

Grange, Magali; Buferne, Michel; Verdeil, Grégory; Leserman, Lee; Schmitt-Verhulst, Anne-Marie; Auphan-Anezin, Nathalie

2012-01-01

Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs. ©2011 AACR.

Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

PubMed Central

Li, Dong; Secher, Jan O.; Mashayekhi, Kaveh; Nielsen, Troels T.; Hyttel, Poul; Freude, Kristine K.

2017-01-01

ABSTRACT Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore, reprogramming of SSEA-1+ sorted pEFs led to higher reprogramming efficiency. Subsequent transcriptome profiling of the SSEA-1+ vs. the SSEA-1neg cell fraction revealed highly comparable gene signatures. However several genes that were found to be upregulated in the SSEA-1+ cells were similarly expressed in mesenchymal stem cells (MSCs). We therefore termed these cells SSEA-1 Expressing Enhanced Reprogramming (SEER) cells. Interestingly, SEER cells were more effective at differentiating into osteocytes and chondrocytes in vitro. We conclude that SEER cells are more amenable for reprogramming and that the expression of mesenchymal stem cell genes is advantageous in the reprogramming process. This data provides evidence supporting the elite theory and helps to delineate which cell types and specific genes are important for reprogramming in the pig. PMID:28426281

The same enhancer regulates the earliest Emx2 expression in caudal forebrain primordium, subsequent expression in dorsal telencephalon and later expression in the cortical ventricular zone.

PubMed

Suda, Yoko; Kokura, Kenji; Kimura, Jun; Kajikawa, Eriko; Inoue, Fumitaka; Aizawa, Shinichi

2010-09-01

We have analyzed Emx2 enhancers to determine how Emx2 functions during forebrain development are regulated. The FB (forebrain) enhancer we identified immediately 3' downstream of the last coding exon is well conserved among tetrapods and unexpectedly directed all the Emx2 expression in forebrain: caudal forebrain primordium at E8.5, dorsal telencephalon at E9.5-E10.5 and the cortical ventricular zone after E12.5. Otx, Tcf, Smad and two unknown transcription factor binding sites were essential to all these activities. The mutant that lacked this enhancer demonstrated that Emx2 expression under the enhancer is solely responsible for diencephalon development. However, in telencephalon, the FB enhancer did not have activities in cortical hem or Cajal-Retzius cells, nor was its activity in the cortex graded. Emx2 expression was greatly reduced, but persisted in the telencephalon of the enhancer mutant, indicating that there exists another enhancer for Emx2 expression unique to mammalian telencephalon.

Methamphetamine-induced stereotypy correlates negatively with patch-enhanced prodynorphin and arc mRNA expression in the rat caudate putamen: the role of mu opioid receptor activation.

PubMed

Horner, Kristen A; Noble, Erika S; Gilbert, Yamiece E

2010-06-01

Amphetamines induce stereotypy, which correlates with patch-enhanced c-Fos expression the patch compartment of caudate putamen (CPu). Methamphetamine (METH) treatment also induces patch-enhanced expression of prodynorphin (PD), arc and zif/268 in the CPu. Whether patch-enhanced activation of any of these genes correlates with METH-induced stereotypy is unknown, and the factors that contribute to this pattern of expression are poorly understood. Activation of mu opioid receptors, which are expressed by the neurons of the patch compartment, may underlie METH-induced patch-enhanced gene expression and stereotypy. The current study examined whether striatal mu opioid receptor blockade altered METH-induced stereotypy and patch-enhanced gene expression, and if there was a correlation between the two responses. Animals were intrastriatally infused with the mu antagonist CTAP (10 microg/microl), treated with METH (7.5 mg/kg, s.c.), placed in activity chambers for 3h, and then sacrificed. CTAP pretreatment attenuated METH-induced increases in PD, arc and zif/268 mRNA expression and significantly reduced METH-induced stereotypy. Patch-enhanced PD and arc mRNA expression in the dorsolateral CPu correlated negatively with METH-induced stereotypy. These data indicate that mu opioid receptor activation contributes to METH-induced gene expression in the CPu and stereotypy, and that patch-enhanced PD and arc expression may be a homeostatic response to METH treatment. Copyright 2010 Elsevier Inc. All rights reserved.

Methamphetamine-Induced Stereotypy Correlates Negatively with Patch-Enhanced Prodynorphin and ARC mRNA Expression in the Rat Caudate Putamen: The Role of Mu Opioid Receptor Activation

PubMed Central

Horner, Kristen A.; Noble, Erika S.; Gilbert, Yamiece E.

2010-01-01

Amphetamines induce stereotypy, which correlates with patch-enhanced c-Fos expression the patch compartment of caudate putamen (CPu). Methamphetamine (METH) treatment also induces patch-enhanced expression of prodynorphin (PD), arc and zif/268 in the CPu. Whether patch-enhanced activation of any of these genes correlates with METH-induced stereotypy is unknown, and the factors that contribute to this pattern of expression are poorly understood. Activation of mu opioid receptors, which are expressed by the neurons of the patch compartment, may underlie METH-induced patch-enhanced gene expression and stereotypy. The current study examined whether striatal mu opioid receptor blockade altered METH-induced stereotypy and patch-enhanced gene expression, and if there was a correlation between the two responses. Animals were intrastriatally infused with the mu antagonist CTAP (10 μg/μl), treated with METH (7.5 mg/kg, s.c.), placed in activity chambers for 3h, and then sacrificed. CTAP pretreatment attenuated METH-induced increases in PD, arc and zif/268 mRNA expression and significantly reduced METH-induced stereotypy. Patch-enhanced PD and arc mRNA expression in the dorsolateral CPu correlated negatively with METH-induced stereotypy. These data indicate that mu opioid receptor activation contributes to METH-induced gene expression in the CPu and stereotypy, and that patch-enhanced PD and arc expression may be a homeostatic response to METH treatment. PMID:20298714

Potato Annexin STANN1 Promotes Drought Tolerance and Mitigates Light Stress in Transgenic Solanum tuberosum L. Plants.

PubMed

Szalonek, Michal; Sierpien, Barbara; Rymaszewski, Wojciech; Gieczewska, Katarzyna; Garstka, Maciej; Lichocka, Malgorzata; Sass, Laszlo; Paul, Kenny; Vass, Imre; Vankova, Radomira; Dobrev, Peter; Szczesny, Pawel; Marczewski, Waldemar; Krusiewicz, Dominika; Strzelczyk-Zyta, Danuta; Hennig, Jacek; Konopka-Postupolska, Dorota

2015-01-01

Annexins are a family of calcium- and membrane-binding proteins that are important for plant tolerance to adverse environmental conditions. Annexins function to counteract oxidative stress, maintain cell redox homeostasis, and enhance drought tolerance. In the present study, an endogenous annexin, STANN1, was overexpressed to determine whether crop yields could be improved in potato (Solanum tuberosum L.) during drought. Nine potential potato annexins were identified and their expression characterized in response to drought treatment. STANN1 mRNA was constitutively expressed at a high level and drought treatment strongly increased transcription levels. Therefore, STANN1 was selected for overexpression analysis. Under drought conditions, transgenic potato plants ectopically expressing STANN1 were more tolerant to water deficit in the root zone, preserved more water in green tissues, maintained chloroplast functions, and had higher accumulation of chlorophyll b and xanthophylls (especially zeaxanthin) than wild type (WT). Drought-induced reductions in the maximum efficiency and the electron transport rate of photosystem II (PSII), as well as the quantum yield of photosynthesis, were less pronounced in transgenic plants overexpressing STANN1 than in the WT. This conferred more efficient non-photochemical energy dissipation in the outer antennae of PSII and probably more efficient protection of reaction centers against photooxidative damage in transgenic plants under drought conditions. Consequently, these plants were able to maintain effective photosynthesis during drought, which resulted in greater productivity than WT plants despite water scarcity. Although the mechanisms underlying this stress protection are not yet clear, annexin-mediated photoprotection is probably linked to protection against light-induced oxidative stress.

Potato Annexin STANN1 Promotes Drought Tolerance and Mitigates Light Stress in Transgenic Solanum tuberosum L. Plants

PubMed Central

Szalonek, Michal; Sierpien, Barbara; Rymaszewski, Wojciech; Gieczewska, Katarzyna; Garstka, Maciej; Lichocka, Malgorzata; Sass, Laszlo; Paul, Kenny; Vass, Imre; Vankova, Radomira; Dobrev, Peter; Szczesny, Pawel; Marczewski, Waldemar; Krusiewicz, Dominika; Strzelczyk-Zyta, Danuta; Hennig, Jacek; Konopka-Postupolska, Dorota

2015-01-01

Annexins are a family of calcium- and membrane-binding proteins that are important for plant tolerance to adverse environmental conditions. Annexins function to counteract oxidative stress, maintain cell redox homeostasis, and enhance drought tolerance. In the present study, an endogenous annexin, STANN1, was overexpressed to determine whether crop yields could be improved in potato (Solanum tuberosum L.) during drought. Nine potential potato annexins were identified and their expression characterized in response to drought treatment. STANN1 mRNA was constitutively expressed at a high level and drought treatment strongly increased transcription levels. Therefore, STANN1 was selected for overexpression analysis. Under drought conditions, transgenic potato plants ectopically expressing STANN1 were more tolerant to water deficit in the root zone, preserved more water in green tissues, maintained chloroplast functions, and had higher accumulation of chlorophyll b and xanthophylls (especially zeaxanthin) than wild type (WT). Drought-induced reductions in the maximum efficiency and the electron transport rate of photosystem II (PSII), as well as the quantum yield of photosynthesis, were less pronounced in transgenic plants overexpressing STANN1 than in the WT. This conferred more efficient non-photochemical energy dissipation in the outer antennae of PSII and probably more efficient protection of reaction centers against photooxidative damage in transgenic plants under drought conditions. Consequently, these plants were able to maintain effective photosynthesis during drought, which resulted in greater productivity than WT plants despite water scarcity. Although the mechanisms underlying this stress protection are not yet clear, annexin-mediated photoprotection is probably linked to protection against light-induced oxidative stress. PMID:26172952

Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri

PubMed Central

van Pijkeren, Jan-Peter; Neoh, Kar Mun; Sirias, Denise; Findley, Anthony S.; Britton, Robert A.

2012-01-01

Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution. PMID:22750793

Effective Targeted Gene Delivery to Dendritic Cells via Synergetic Interaction of Mannosylated Lipid with DOPE and BCAT

PubMed Central

Kim, Hee-Kwon; Wei, Huiling; Kulkarni, Aditya; Pogranichniy, Roman M.; Thompson, David H.

2012-01-01

The efficient delivery of plasmids encoding antigenic determinants into dendritic cells (DCs) that control immune response is a promising strategy for rapid development of new vaccines. In this study, we prepared a series of targeted cationic lipoplex based on two synthetic lipid components, mannose-poly(ethylene glycol, MW3000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (Mannose-PEG3000-DSPE) and O-(2R-1,2-di-O-(1'Z,9'Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), that were formulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for evaluation as non-viral vectors for transgene expression in DCs. First, we optimized the N:P ratio for maximum transfection and then screened the effects of mannose targeting for further enhancement of transfection levels. Our results indicate that efficient delivery of gWIZ GFP plasmid into DCs was observed for mannose compositions of ~10%, whereas low transfection efficiencies were observed with non-targeted formulations. Mannose-targeted lipofectamine complexes also showed high GFP expression levels in DCs relative to non-targeted lipofectamine controls. The best transfection performance was observed using 10 mol % Mannose-PEG3000-DSPE, 60 mol% BCAT, and 30 mol % DOPE, indicating that the most efficient delivery into DCs occurs via synergistic interaction between mannose targeting and acid-labile, fusogenic BCAT:DOPE formulations. Our data suggest that mannose-PEG3000-DSPE:BCAT:DOPE formulations may be effective gene delivery vehicles for the development of DC-based vaccines. PMID:22229467

An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.

PubMed

Zhang, Jin jing; Shi, Liang; Chen, Hui; Sun, Yun qi; Zhao, Ming wen; Ren, Ang; Chen, Ming jie; Wang, Hong; Feng, Zhi yong

2014-01-01

Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species. Copyright © 2014 Elsevier GmbH. All rights reserved.

A DNA replicon system for rapid high-level production of virus-like particles in plants.

PubMed

Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles; Mason, Hugh

2009-07-01

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this article, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without P19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. (c) 2009 Wiley Periodicals, Inc.

A DNA replicon system for rapid high-level production of virus-like particles in plants

PubMed Central

Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles

2009-01-01

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low level antigen accumulation and long time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this paper, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within five days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without p19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. PMID:19309755

Electroluminescence Efficiency Enhancement using Metal Nanoparticles

DTIC Science & Technology

2008-06-22

ABSTRACT We apply the “effective mode volume” theory to evaluate enhancement of the electroluminescence efficiency of semiconductor emitters placed in... Electroluminescence efficiency enhancement using metal nanoparticles J. B. Khurgin,1 G. Sun,2,a and R. A. Soref3 1Department of Electrical and Computer...published online 17 July 2008 We apply the “effective mode volume” theory to evaluate enhancement of the electroluminescence efficiency of semiconductor

Enhanced Cell Capture on Functionalized Graphene Oxide Nanosheets through Oxygen Clustering.

PubMed

Bardhan, Neelkanth M; Kumar, Priyank V; Li, Zeyang; Ploegh, Hidde L; Grossman, Jeffrey C; Belcher, Angela M; Chen, Guan-Yu

2017-02-28

With the global rise in incidence of cancer and infectious diseases, there is a need for the development of techniques to diagnose, treat, and monitor these conditions. The ability to efficiently capture and isolate cells and other biomolecules from peripheral whole blood for downstream analyses is a necessary requirement. Graphene oxide (GO) is an attractive template nanomaterial for such biosensing applications. Favorable properties include its two-dimensional architecture and wide range of functionalization chemistries, offering significant potential to tailor affinity toward aromatic functional groups expressed in biomolecules of interest. However, a limitation of current techniques is that as-synthesized GO nanosheets are used directly in sensing applications, and the benefits of their structural modification on the device performance have remained unexplored. Here, we report a microfluidic-free, sensitive, planar device on treated GO substrates to enable quick and efficient capture of Class-II MHC-positive cells from murine whole blood. We achieve this by using a mild thermal annealing treatment on the GO substrates, which drives a phase transformation through oxygen clustering. Using a combination of experimental observations and MD simulations, we demonstrate that this process leads to improved reactivity and density of functionalization of cell capture agents, resulting in an enhanced cell capture efficiency of 92 ± 7% at room temperature, almost double the efficiency afforded by devices made using as-synthesized GO (54 ± 3%). Our work highlights a scalable, cost-effective, general approach to improve the functionalization of GO, which creates diverse opportunities for various next-generation device applications.

Notch Signaling Augments BMP9-Induced Bone Formation by Promoting the Osteogenesis-Angiogenesis Coupling Process in Mesenchymal Stem Cells (MSCs).

PubMed

Liao, Junyi; Wei, Qiang; Zou, Yulong; Fan, Jiaming; Song, Dongzhe; Cui, Jing; Zhang, Wenwen; Zhu, Yunxiao; Ma, Chao; Hu, Xue; Qu, Xiangyang; Chen, Liqun; Yu, Xinyi; Zhang, Zhicai; Wang, Claire; Zhao, Chen; Zeng, Zongyue; Zhang, Ruyi; Yan, Shujuan; Wu, Tingting; Wu, Xingye; Shu, Yi; Lei, Jiayan; Li, Yasha; Luu, Hue H; Lee, Michael J; Reid, Russell R; Ameer, Guillermo A; Wolf, Jennifer Moriatis; He, Tong-Chuan; Huang, Wei

2017-01-01

Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into several lineages including bone. Successful bone formation requires osteogenesis and angiogenesis coupling of MSCs. Here, we investigate if simultaneous activation of BMP9 and Notch signaling yields effective osteogenesis-angiogenesis coupling in MSCs. Recently-characterized immortalized mouse adipose-derived progenitors (iMADs) were used as MSC source. Transgenes BMP9, NICD and dnNotch1 were expressed by adenoviral vectors. Gene expression was determined by qPCR and immunohistochem¡stry. Osteogenic activity was assessed by in vitro assays and in vivo ectopic bone formation model. BMP9 upregulated expression of Notch receptors and ligands in iMADs. Constitutively-active form of Notch1 NICD1 enhanced BMP9-induced osteogenic differentiation both in vitro and in vivo, which was effectively inhibited by dominant-negative form of Notch1 dnNotch1. BMP9- and NICD1-transduced MSCs implanted with a biocompatible scaffold yielded highly mature bone with extensive vascularization. NICD1 enhanced BMP9-induced expression of key angiogenic regulators in iMADs and Vegfa in ectopic bone, which was blunted by dnNotch1. Notch signaling may play an important role in BMP9-induced osteogenesis and angiogenesis. It's conceivable that simultaneous activation of the BMP9 and Notch pathways should efficiently couple osteogenesis and angiogenesis of MSCs for successful bone tissue engineering. © 2017 The Author(s)Published by S. Karger AG, Basel.

The GARP/Latent TGF-β1 complex on Treg cells modulates the induction of peripherally derived Treg cells during oral tolerance.

PubMed

Edwards, Justin P; Hand, Timothy W; Morais da Fonseca, Denise; Glass, Deborah D; Belkaid, Yasmine; Shevach, Ethan M

2016-06-01

Treg cells can secrete latent TGF-β1 (LTGF-β1), but can also utilize an alternative pathway for transport and expression of LTGF-β1 on the cell surface in which LTGF-β1 is coupled to a distinct LTGF-β binding protein termed glycoprotein A repetitions predominant (GARP)/LRRC32. The function of the GARP/LTGF-β1 complex has remained elusive. Here, we examine in vivo the roles of GARP and TGF-β1 in the induction of oral tolerance. When Foxp3(-) OT-II T cells were transferred to wild-type recipient mice followed by OVA feeding, the conversion of Foxp3(-) to Foxp3(+) OT-II cells was dependent on recipient Treg cells. Neutralization of IL-2 in the recipient mice also abrogated this conversion. The GARP/LTGF-β1 complex on recipient Treg cells, but not dendritic cell-derived TGF-β1, was required for efficient induction of Foxp3(+) T cells and for the suppression of delayed hypersensitivity. Expression of the integrin αvβ8 by Treg cells (or T cells) in the recipients was dispensable for induction of Foxp3 expression. Transient depletion of the bacterial flora enhanced the development of oral tolerance by expanding Treg cells with enhanced expression of the GARP/LTGF-β1 complex. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Quantifying translational coupling in E. coli synthetic operons using RBS modulation and fluorescent reporters.

PubMed

Levin-Karp, Ayelet; Barenholz, Uri; Bareia, Tasneem; Dayagi, Michal; Zelcbuch, Lior; Antonovsky, Niv; Noor, Elad; Milo, Ron

2013-06-21

Translational coupling is the interdependence of translation efficiency of neighboring genes encoded within an operon. The degree of coupling may be quantified by measuring how the translation rate of a gene is modulated by the translation rate of its upstream gene. Translational coupling was observed in prokaryotic operons several decades ago, but the quantitative range of modulation translational coupling leads to and the factors governing this modulation were only partially characterized. In this study, we systematically quantify and characterize translational coupling in E. coli synthetic operons using a library of plasmids carrying fluorescent reporter genes that are controlled by a set of different ribosome binding site (RBS) sequences. The downstream gene expression level is found to be enhanced by the upstream gene expression via translational coupling with the enhancement level varying from almost no coupling to over 10-fold depending on the upstream gene's sequence. Additionally, we find that the level of translational coupling in our system is similar between the second and third locations in the operon. The coupling depends on the distance between the stop codon of the upstream gene and the start codon of the downstream gene. This study is the first to systematically and quantitatively characterize translational coupling in a synthetic E. coli operon. Our analysis will be useful in accurate manipulation of gene expression in synthetic biology and serves as a step toward understanding the mechanisms involved in translational expression modulation.

Enhanced expression of EGFP gene in CHSE-214 cells by an ARS element from mud loach (Misgurnus mizolepis).

PubMed

Kim, Moo-Sang; Lim, Hak-Seob; Ahn, Sang Jung; Jeong, Yong-Kee; Kim, Chul Geun; Lee, Hyung Ho

2007-11-01

The origins of replication are associated with nuclear matrices or are found in close proximity to matrix attachment regions (MARs). In this report, fish MARs were cloned into an autonomously replicating sequence (ARS) cloning vector and were screened for ARS elements in Saccharomyces cerevisiae. Sixteen clones were isolated that were able to grow on the selective plates. In particular, an ARS905 that shows high efficiency among them was selected for this study. Southern hybridization indicated the autonomous replication of the transformation vector containing the ARS905 element. DNA sequences analysis showed that the ARS905 contained two ARS consensus sequences as well as MAR motifs, such as AT tracts, ORI patterns, and ATC tracts. In vitro matrix binding analysis, major matrix binding activity and ARS function coincided in a subfragment of the ARS905. To analyze the effects of ARS905 on expression of a reporter gene, an ARS905(E1158) with ARS activity was inserted into pBaEGFP(+) containing mud loach beta-actin promoter, EGFP as a reporter gene, and SV40 poly(A) signal. The pBaEGFP(+)-ARS905(E1158) was transfected into a fish cell line, CHSE-214. The intensity of EGFP transfected cells was a 7-fold of the control at 11days post-transfection. These results indicate that ARS905 enhances the expression of the EGFP gene and that it should be as a component of expression vectors in further fish biotechnological studies.

Expression of β-glucuronidase on the surface of bacteria enhances activation of glucuronide prodrugs.

PubMed

Cheng, C-M; Chen, F M; Lu, Y-L; Tzou, S-C; Wang, J-Y; Kao, C-H; Liao, K-W; Cheng, T-C; Chuang, C-H; Chen, B-M; Roffler, S; Cheng, T-L

2013-05-01

Extracellular activation of hydrophilic glucuronide prodrugs by β-glucuronidase (βG) was examined to increase the therapeutic efficacy of bacteria-directed enzyme prodrug therapy (BDEPT). βG was expressed on the surface of Escherichia coli by fusion to either the bacterial autotransporter protein Adhesin (membrane βG (mβG)/AIDA) or the lipoprotein (lpp) outermembrane protein A (mβG/lpp). Both mβG/AIDA and mβG/lpp were expressed on the bacterial surface, but only mβG/AIDA displayed enzymatic activity. The rate of substrate hydrolysis by mβG/AIDA-BL21cells was 2.6-fold greater than by pβG-BL21 cells, which express periplasmic βG. Human colon cancer HCT116 cells that were incubated with mβG/AIDA-BL21 bacteria were sensitive to a glucuronide prodrug (p-hydroxy aniline mustard β-D-glucuronide, HAMG) with an half maximal inhibitory concentration (IC50) value of 226.53±45.4 μM, similar to the IC50 value of the active drug (p-hydroxy aniline mustard, pHAM; 70.6±6.75 μM), indicating that mβG/AIDA on BL21 bacteria could rapidly and efficiently convert HAMG to an active anticancer agent. These results suggest that surface display of functional βG on bacteria can enhance the hydrolysis of glucuronide prodrugs and may increase the effectiveness of BDEPT.

Glycoprotein-based enzyme-linked immunosorbent assays for serodiagnosis of infectious laryngotracheitis.

PubMed

Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta; Samal, Siba K

2015-05-01

For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer

NASA Astrophysics Data System (ADS)

Kos, Petra; Lächelt, Ulrich; Herrmann, Annika; Mickler, Frauke Martina; Döblinger, Markus; He, Dongsheng; Krhač Levačić, Ana; Morys, Stephan; Bräuchle, Christoph; Wagner, Ernst

2015-03-01

Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06556e

Hypoxia enhances periodontal ligament stem cell proliferation via the MAPK signaling pathway.

PubMed

He, Y; Jian, C X; Zhang, H Y; Zhou, Y; Wu, X; Zhang, G; Tan, Y H

2016-11-21

There is high incidence of periodontal disease in high-altitude environments; hypoxia may influence the proliferation and clone-forming ability of periodontal ligament stem cells (PDLSCs). The MAPK signaling pathway is closely correlated with cell proliferation, differentiation, and apoptosis. Thus, we isolated and cultured PDLSCs under hypoxic conditions to clarify the impact of hypoxia on PDLSC proliferation and the underlying mechanism. PDLSCs were separated and purified by the limiting dilution method and identified by flow cytometry. PDLSCs were cultured under hypoxic or normoxic conditions to observe their cloning efficiency. PDLSC proliferation at different oxygen concentrations was evaluated by MTT assay. Expression of p38/MAPK and MAPK/ERK signaling pathway members was detected by western blotting. Inhibitors for p38/MAPK or ERK were applied to PDLSCs to observe their impacts on clone formation and proliferation. Isolated PDLSCs exhibited typical stem cell morphological characteristics, strong abilities of globular clone formation and proliferation, and upregulated expression of mesenchymal stem cell markers. Stem cell marker expression was not statistically different between PDLSCs cultured under hypoxia and normoxia (P > 0.05). The clone number in the hypoxia group was significantly higher than that in the control (P < 0.05). PDLSC proliferation under hypoxia was higher than that of the control (P < 0.001). p38 and ERK1/2 phosphorylation in hypoxic PDLSCs was markedly enhanced compared to that in the control (P < 0.05). Either P38/MAPK inhibitor or ERK inhibitor treatment reduced clone formation and proliferation. Therefore, hypoxia enhanced PDLSC clone formation and proliferation by activating the p38/MAPK and ERK/MAPK signaling pathways.

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors

PubMed Central

Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

2014-01-01

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around −120 to −80 bp, while highly effective sgRNAs targeted from −147 to −89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells. PMID:24500196

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

PubMed

Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

2014-04-01

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

RNA-Seq Analysis of Abdominal Fat in Genetically Fat and Lean Chickens Highlights a Divergence in Expression of Genes Controlling Adiposity, Hemostasis, and Lipid Metabolism

PubMed Central

Resnyk, Christopher W.; Chen, Chuming; Huang, Hongzhan; Wu, Cathy H.; Simon, Jean; Le Bihan-Duval, Elisabeth; Duclos, Michel J.; Cogburn, Larry A.

2015-01-01

Genetic selection for enhanced growth rate in meat-type chickens (Gallus domesticus) is usually accompanied by excessive adiposity, which has negative impacts on both feed efficiency and carcass quality. Enhanced visceral fatness and several unique features of avian metabolism (i.e., fasting hyperglycemia and insulin insensitivity) mimic overt symptoms of obesity and related metabolic disorders in humans. Elucidation of the genetic and endocrine factors that contribute to excessive visceral fatness in chickens could also advance our understanding of human metabolic diseases. Here, RNA sequencing was used to examine differential gene expression in abdominal fat of genetically fat and lean chickens, which exhibit a 2.8-fold divergence in visceral fatness at 7 wk. Ingenuity Pathway Analysis revealed that many of 1687 differentially expressed genes are associated with hemostasis, endocrine function and metabolic syndrome in mammals. Among the highest expressed genes in abdominal fat, across both genotypes, were 25 differentially expressed genes associated with de novo synthesis and metabolism of lipids. Over-expression of numerous adipogenic and lipogenic genes in the FL chickens suggests that in situ lipogenesis in chickens could make a more substantial contribution to expansion of visceral fat mass than previously recognized. Distinguishing features of the abdominal fat transcriptome in lean chickens were high abundance of multiple hemostatic and vasoactive factors, transporters, and ectopic expression of several hormones/receptors, which could control local vasomotor tone and proteolytic processing of adipokines, hemostatic factors and novel endocrine factors. Over-expression of several thrombogenic genes in abdominal fat of lean chickens is quite opposite to the pro-thrombotic state found in obese humans. Clearly, divergent genetic selection for an extreme (2.5–2.8-fold) difference in visceral fatness provokes a number of novel regulatory responses that govern growth and metabolism of visceral fat in this unique avian model of juvenile-onset obesity and glucose-insulin imbalance. PMID:26445145

Enhanced microglial pro-inflammatory response to lipopolysaccharide correlates with brain infiltration and blood-brain barrier dysregulation in a mouse model of telomere shortening.

PubMed

Raj, Divya D A; Moser, Jill; van der Pol, Susanne M A; van Os, Ronald P; Holtman, Inge R; Brouwer, Nieske; Oeseburg, Hisko; Schaafsma, Wandert; Wesseling, Evelyn M; den Dunnen, Wilfred; Biber, Knut P H; de Vries, Helga E; Eggen, Bart J L; Boddeke, Hendrikus W G M

2015-12-01

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self-renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as 'priming'. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first-generation G1 mTerc(-/-) )- and late-generation (third-generation G3 and G4 mTerc(-/-) ) telomerase-deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late-generation mTerc(-/-) microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc(-/-) microglia are comparable with microglia derived from G1 mTerc(-/-) mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc(-/-) microglia mice show an enhanced pro-inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age-associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood-brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

PubMed Central

2011-01-01

Background Huntington's disease is caused by aggregation of mutant huntingtin (mHtt) protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease activities may contribute to toxicity whereas others are consistent with protection. These discrepancies may be due to a number of mechanisms including distinct effects of the specific intermediate digestion products of mutant huntingtin generated by different proteases. These observations suggested a critical need to investigate the consequence of upregulation of individual lysosomal enzyme in mutant huntingtin accumulation and toxicity. Results In this study, we used molecular approaches to enhance lysosomal protease activities and examined their effects on mutant huntingtin level and toxicity. We found that enhanced expression of lysosomal cathepsins D and B resulted in their increased enzymatic activities and reduced both full-length and fragmented huntingtin in transfected HEK cells. Furthermore, enhanced expression of cathepsin D or B protected against mutant huntingtin toxicity in primary neurons, and their neuroprotection is dependent on macroautophagy. Conclusions These observations demonstrate a neuroprotective effect of enhancing lysosomal cathepsins in reducing mutant huntingtin level and toxicity in transfected cells. They highlight the potential importance of neuroprotection mediated by cathepsin D or B through macroautophagy. PMID:21631942

Mouse alpha1(I)-collagen promoter is the best known promoter to drive efficient Cre recombinase expression in osteoblast.

PubMed

Dacquin, Romain; Starbuck, Michael; Schinke, Thorsten; Karsenty, Gérard

2002-06-01

Cell- and time-specific gene inactivation should enhance our knowledge of bone biology. Implementation of this technique requires construction of transgenic mouse lines expressing Cre recombinase in osteoblasts, the bone forming cell. We tested several promoter fragments for their ability to drive efficient Cre expression in osteoblasts. In the first mouse transgenic line, the Cre gene was placed under the control of the 2.3-kb proximal fragment of the alpha1(I)-collagen promoter, which is expressed at high levels in osteoblasts throughout their differentiation. Transgenic mice expressing this transgene in bone were bred with the ROSA26 reporter (R26R) strain in which the ROSA26 locus is targeted with a conditional LacZ reporter cassette. In R26R mice, Cre expression and subsequent Cre-mediated recombination lead to expression of the LacZ reporter gene, an event that can be monitored by LacZ staining. LacZ staining was detected in virtually all osteoblasts of alpha1(I)-Cre;R26R mice indicating that homologous recombination occurred in these cells. No other cell type stained blue. In the second line studied, the 1.3-kb fragment of osteocalcin gene 2 (OG2) promoter, which is active in differentiated osteoblasts, was used to drive Cre expression. OG2-Cre mice expressed Cre specifically in bone. However, cross of OG2-Cre mice with R26R mice did not lead to any detectable LacZ staining in osteoblasts. Lastly, we tested a more active artificial promoter derived from the OG2 promoter. The artificial OG2-Cre transgene was expressed by reverse transcriptase-polymerase chain reaction in cartilage and bone samples. After cross of the artificial OG2-Cre mice with R26R mice, we detected a LacZ staining in articular chondrocytes but not in osteoblasts. Our data suggest that the only promoter able to drive Cre expression at a level sufficient to induce recombination in osteoblasts is the alpha1(I)-collagen promoter. Copyright 2002 Wiley-Liss, Inc.

Subtilisin QK-2: secretory expression in Lactococcus lactis and surface display onto gram-positive enhancer matrix (GEM) particles.

PubMed

Mao, Ruifeng; Zhou, Kangping; Han, Zhenwei; Wang, Yefu

2016-05-12

Purified from the supernatant of Bacillus subtilis QK02 culture broth, Subtilisin QK-2 is a type of effective thrombolytic reagent that has great exploitable potential. However, the unbearable flavor that occurs with fermentation and the complicated methods that are required to obtain pure products limit the application of this enzyme. Lactic acid bacteria (LAB)-based delivery vehicles are promising as cheap and safe options for medicinal compounds. The secretory expression and surface display using LAB may popularize Subtilisin QK-2 more easily and conveniently with minimal adverse effects. Subtilisin QK-2 was expressed successfully in two forms using lactic acid bacteria. For the secretory expression in Lactococcus lactis, Subtilisin QK-2 was efficiently secreted into the culture using the promoter P nisA and signal peptide SPUsp. The expression levels were not different in L. lactis NZ9000 and NZ3900 without the effect of different selection markers. However, leaky expression was only detected in L. lactis NZ3900. The biological activity of this secreted Subtilisin QK-2 was enhanced by modulating the pH of medium to slightly alkaline during induction and by codon optimization of either the entire gene sequence (qk') or only the propeptide gene sequence (qkpro'). For surface display onto gram-positive enhancer matrix (GEM) particles, n LysM repeats from the C-terminal region of the major autolysin AcmA of L. lactis were fused to either the C-terminus (n = 1, 3, 5) or the N-terminus (n = 1) of the Subtilisin QK-2. These fusion proteins were secreted into the culture medium, and the QK-3LysM was able to bind to the surface of various LAB GEM particles without a loss of fibrinolytic activity. Furthermore, the binding capacity significantly increased with a higher concentration of QK-3LysM. Compared to the free-form Subtilisin QK-2, the QK-3LysM displayed on the surface of GEM particles was more stable in the simulated gastric juice. Combined with the safety and popularity of LAB, Subtilisin QK-2 may be easily applied worldwide to prevent and control thrombosis diseases.

Improving membrane protein expression by optimizing integration efficiency

PubMed Central

2017-01-01

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

SUMOylation of TARBP2 regulates miRNA/siRNA efficiency

PubMed Central

Chen, Cheng; Zhu, Changhong; Huang, Jian; Zhao, Xian; Deng, Rong; Zhang, Hailong; Dou, Jinzhuo; Chen, Qin; Xu, Ming; Yuan, Haihua; Wang, Yanli; Yu, Jianxiu

2015-01-01

Small RNA-induced gene silencing is essential for post-transcriptional regulation of gene expression; however, it remains unclear how miRNA/siRNA efficiency is regulated. Here we show that TARBP2 is SUMOylated at K52, which can be enhanced by its phosphorylation. This modification can stabilize TARBP2 via repressing its K48-linked ubiquitination. We find that TARBP2 SUMOylation does not influence the overall production of mature miRNAs, but it regulates miRNA/siRNA efficiency. SUMOylated TARBP2 recruits Ago2 to constitute the RNA-induced silencing complex (RISC)-loading complex (RLC), and simultaneously promotes more pre-miRNAs to load into the RLC. Consequently, Ago2 is stabilized and miRNAs/siRNAs bound by TARBP2/Dicer is effectively transferred to Ago2. Thus, these processes lead to the formation of the effective RISC for RNA interference (RNAi). Collectively, our data suggest that SUMOylation of TARBP2 is required for regulating miRNA/siRNA efficiency, which is a general mechanism of miRNA/siRNA regulation. PMID:26582366

Comparative Analysis of Two Flavonol Synthases from Different-Colored Onions Provides Insight into Flavonoid Biosynthesis.

PubMed

Park, Sangkyu; Kim, Da-Hye; Lee, Jong-Yeol; Ha, Sun-Hwa; Lim, Sun-Hyung

2017-07-05

We isolated cDNAs encoding flavonol synthase (FLS) from the red onion "H6" (AcFLS-H6) and the yellow onion "Hwangryongball" (AcFLS-HRB). We found three amino acid variations between the two sequences. Kinetic analysis with recombinant proteins revealed that AcFLS-HRB exhibited approximately 2-fold higher catalytic efficiencies than AcFLS-H6 for dihydroflavonol substrates and that both proteins preferred dihydroquercetin to dihydrokaempferol. The expression patterns of flavonoid biosynthesis genes corresponded to the accumulation patterns of flavonoid aglycones in both onions. Whereas the other flavonoid biosynthesis genes were weakly expressed in the HRB sheath compared to that of H6, the expression of FLS was similar in both onions. This relatively enhanced FLS expression, along with the higher activity of AcFLS-HRB, could increase the quercetin production in the HRB sheath. The quercetin content was approximately 12-fold higher than the cyanidin content in the H6 sheath, suggesting that FLS has priority in the competition between FLS and dihydroflavonol 4-reductase (DFR) for their substrate dihydroquercetin.

An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

PubMed

Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

2015-09-01

A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhanced phytoremediation of mixed heavy metal (mercury)-organic pollutants (trichloroethylene) with transgenic alfalfa co-expressing glutathione S-transferase and human P450 2E1.

PubMed

Zhang, Yuanyuan; Liu, Junhong; Zhou, Yuanming; Gong, Tingyun; Wang, Jing; Ge, Yinlin

2013-09-15

Soil contamination is a global environmental problem and many efforts have been made to find efficient remediation methods over the last decade. Moreover, remediation of mixed contaminated soils are more difficult. In the present study, transgenic alfalfa plants pKHCG co-expressing glutathione S-transferase (GST) and human P450 2E1 (CYP2E1) genes were used for phytoremediation of mixed mercury (Hg)-trichloroethylene (TCE) contaminants. Simultaneous expression of GST and CYP2E1 may produce a significant synergistic effect, and leads to improved resistance and accumulation to heavy metal-organic complex contaminants. Based on the tolerance and accumulation assays, pKHCG transgenic plants were more resistant to Hg/TCE complex pollutants and many folds higher in Hg/TCE-accumulation than the non-transgenic control plants in mixed contaminated soil. It is confirmed that GST and CYP2E1 co-expression may be a useful strategy to help achieve mixed heavy metal-organic pollutants phytoremediation. Copyright © 2013 Elsevier B.V. All rights reserved.

Targeting ferritin receptors for the selective delivery of imaging and therapeutic agents to breast cancer cells

NASA Astrophysics Data System (ADS)

Geninatti Crich, S.; Cadenazzi, M.; Lanzardo, S.; Conti, L.; Ruiu, R.; Alberti, D.; Cavallo, F.; Cutrin, J. C.; Aime, S.

2015-04-01

In this work the selective uptake of native horse spleen ferritin and apoferritin loaded with MRI contrast agents has been assessed in human breast cancer cells (MCF-7 and MDA-MB-231). The higher expression of L-ferritin receptors (SCARA5) led to an enhanced uptake in MCF-7 as shown in T2 and T1 weighted MR images, respectively. The high efficiency of ferritin internalization in MCF-7 has been exploited for the simultaneous delivery of curcumin, a natural therapeutic molecule endowed with antineoplastic and anti-inflammatory action, and the MRI contrast agent Gd-HPDO3A. This theranostic system is able to treat selectively breast cancer cells over-expressing ferritin receptors. By entrapping in apoferritin both Gd-HPDO3A and curcumin, it was possible to deliver a therapeutic dose of 167 μg ml-1 (as calculated by MRI) of this natural drug to MCF-7 cells, thus obtaining a significant reduction of cell proliferation.In this work the selective uptake of native horse spleen ferritin and apoferritin loaded with MRI contrast agents has been assessed in human breast cancer cells (MCF-7 and MDA-MB-231). The higher expression of L-ferritin receptors (SCARA5) led to an enhanced uptake in MCF-7 as shown in T2 and T1 weighted MR images, respectively. The high efficiency of ferritin internalization in MCF-7 has been exploited for the simultaneous delivery of curcumin, a natural therapeutic molecule endowed with antineoplastic and anti-inflammatory action, and the MRI contrast agent Gd-HPDO3A. This theranostic system is able to treat selectively breast cancer cells over-expressing ferritin receptors. By entrapping in apoferritin both Gd-HPDO3A and curcumin, it was possible to deliver a therapeutic dose of 167 μg ml-1 (as calculated by MRI) of this natural drug to MCF-7 cells, thus obtaining a significant reduction of cell proliferation. Electronic supplementary information (ESI) available: Competition studies with free apoferritin, Fig. S1; APO-FITC intracellular distribution by immunofluorescence, Fig. S2. See DOI: 10.1039/c5nr00352k

pOsNAR2.1:OsNAR2.1 expression enhances nitrogen uptake efficiency and grain yield in transgenic rice plants.

PubMed

Chen, Jingguang; Fan, Xiaoru; Qian, Kaiyun; Zhang, Yong; Song, Miaoquan; Liu, Yu; Xu, Guohua; Fan, Xiaorong

2017-10-01

The nitrate (NO3-) transporter has been selected as an important gene maker in the process of environmental adoption in rice cultivars. In this work, we transferred another native OsNAR2.1 promoter with driving OsNAR2.1 gene into rice plants. The transgenic lines with exogenous pOsNAR2.1:OsNAR2.1 constructs showed enhanced OsNAR2.1 expression level, compared with wild type (WT), and 15 N influx in roots increased 21%-32% in response to 0.2 mm and 2.5 mm 15NO3- and 1.25 mm 15 NH 4 15 NO 3 . Under these three N conditions, the biomass of the pOsNAR2.1:OsNAR2.1 transgenic lines increased 143%, 129% and 51%, and total N content increased 161%, 242% and 69%, respectively, compared to WT. Furthermore in field experiments we found the grain yield, agricultural nitrogen use efficiency (ANUE), and dry matter transfer of pOsNAR2.1:OsNAR2.1 plants increased by about 21%, 22% and 21%, compared to WT. We also compared the phenotypes of pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines in the field, found that postanthesis N uptake differed significantly between them, and in comparison with the WT. Postanthesis N uptake (PANU) increased approximately 39% and 85%, in the pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines, respectively, possibly because OsNRT2.1 expression was less in the pOsNAR2.1:OsNAR2.1 lines than in the pOsNAR2.1:OsNRT2.1 lines during the late growth stage. These results show that rice NO 3 - uptake, yield and NUE were improved by increased OsNAR2.1 expression via its native promoter. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

The feasibility of using magnetic nanoparticles modified as gene vector.

PubMed

Chen, D; Tang, Q; Xue, W; Wang, X

2010-06-01

To evaluate the feasibility of using magnetic nanoparticles (MNPs) as gene vector and the effect of magnetic field on efficiency of transfection. Magnetic nanoparticles were prepared by controlling some chemical reaction parameters through a partially reduction precipitation method with ferric chloride aqueous solution as precursor material. The surface of particles was modified by polyethyleneimine (PEI) agents. The appearance, the size distribution, structure and phase constitute of MNPs were characterized by Transmission electron microscope (TEM), X-ray diffraction (XRD); the potential of absorbing DNA of MNPs was analysed by electrophoresis. Transfection was determined by delivering reporter gene, PGL2-control encoding luciferase, to different cell lines using MNPs-PLL as vector. The effect of magnetic field on the efficiency of transfection was determined using Nd-Fe-B permanent magnet. Foreign gene could be delivered to various cell lines by MNPs-PLL and expressed with high efficiency but the transfection efficiency and time course varied in the different cell lines studied. Magnetic field could enhance the efficiency of transfection by 5-10 fold. MNPs- PLL can be used as a novel non-viral gene vector in vitro, which offers a basis for gene delivery in vivo.

Optimization of Light-Harvesting Pigment Improves Photosynthetic Efficiency.

PubMed

Jin, Honglei; Li, Mengshu; Duan, Sujuan; Fu, Mei; Dong, Xiaoxiao; Liu, Bing; Feng, Dongru; Wang, Jinfa; Wang, Hong-Bin

2016-11-01

Maximizing light capture by light-harvesting pigment optimization represents an attractive but challenging strategy to improve photosynthetic efficiency. Here, we report that loss of a previously uncharacterized gene, HIGH PHOTOSYNTHETIC EFFICIENCY1 (HPE1), optimizes light-harvesting pigments, leading to improved photosynthetic efficiency and biomass production. Arabidopsis (Arabidopsis thaliana) hpe1 mutants show faster electron transport and increased contents of carbohydrates. HPE1 encodes a chloroplast protein containing an RNA recognition motif that directly associates with and regulates the splicing of target RNAs of plastid genes. HPE1 also interacts with other plastid RNA-splicing factors, including CAF1 and OTP51, which share common targets with HPE1. Deficiency of HPE1 alters the expression of nucleus-encoded chlorophyll-related genes, probably through plastid-to-nucleus signaling, causing decreased total content of chlorophyll (a+b) in a limited range but increased chlorophyll a/b ratio. Interestingly, this adjustment of light-harvesting pigment reduces antenna size, improves light capture, decreases energy loss, mitigates photodamage, and enhances photosynthetic quantum yield during photosynthesis. Our findings suggest a novel strategy to optimize light-harvesting pigments that improves photosynthetic efficiency and biomass production in higher plants. © 2016 American Society of Plant Biologists. All Rights Reserved.

Optimization of Light-Harvesting Pigment Improves Photosynthetic Efficiency1[OPEN

PubMed Central

Jin, Honglei; Li, Mengshu; Duan, Sujuan; Fu, Mei; Dong, Xiaoxiao; Feng, Dongru; Wang, Jinfa

2016-01-01

Maximizing light capture by light-harvesting pigment optimization represents an attractive but challenging strategy to improve photosynthetic efficiency. Here, we report that loss of a previously uncharacterized gene, HIGH PHOTOSYNTHETIC EFFICIENCY1 (HPE1), optimizes light-harvesting pigments, leading to improved photosynthetic efficiency and biomass production. Arabidopsis (Arabidopsis thaliana) hpe1 mutants show faster electron transport and increased contents of carbohydrates. HPE1 encodes a chloroplast protein containing an RNA recognition motif that directly associates with and regulates the splicing of target RNAs of plastid genes. HPE1 also interacts with other plastid RNA-splicing factors, including CAF1 and OTP51, which share common targets with HPE1. Deficiency of HPE1 alters the expression of nucleus-encoded chlorophyll-related genes, probably through plastid-to-nucleus signaling, causing decreased total content of chlorophyll (a+b) in a limited range but increased chlorophyll a/b ratio. Interestingly, this adjustment of light-harvesting pigment reduces antenna size, improves light capture, decreases energy loss, mitigates photodamage, and enhances photosynthetic quantum yield during photosynthesis. Our findings suggest a novel strategy to optimize light-harvesting pigments that improves photosynthetic efficiency and biomass production in higher plants. PMID:27609860

LincRNA-p21 activates p21 in cis to promote Polycomb target gene expression and to enforce the G1/S checkpoint

PubMed Central

Dimitrova, Nadya; Zamudio, Jesse R.; Jong, Robyn M.; Soukup, Dylan; Resnick, Rebecca; Sarma, Kavitha; Ward, Amanda J.; Raj, Arjun; Lee, Jeannie; Sharp, Phillip A.; Jacks, Tyler

2014-01-01

SUMMARY The p53-regulated long non-coding RNA lincRNA-p21 has been proposed to act in trans via several mechanisms ranging from repressing genes in the p53 transcriptional network to regulating mRNA translation and protein stability. To further examine lincRNA-p21 function we generated a conditional knockout mouse model. We find that lincRNA-p21 predominantly functions in cis to activate expression of its neighboring gene, p21. Mechanistically, we show that lincRNA-p21 acts in concert with hnRNP-K as a co-activator for p53-dependent p21 transcription. Additional phenotypes of lincRNA-p21 deficiency could be attributed to diminished p21 levels, including deregulated expression and altered chromatin state of some Polycomb target genes, defective G1/S checkpoint, increased proliferation rates, and enhanced reprogramming efficiency. These findings indicate that lincRNA-p21 affects global gene expression and influences the p53 tumor suppressor pathway by acting in cis as a locus-restricted co-activator for p53-mediated p21 expression. PMID:24857549

Expression of Arabidopsis Hexokinase in Citrus Guard Cells Controls Stomatal Aperture and Reduces Transpiration.

PubMed

Lugassi, Nitsan; Kelly, Gilor; Fidel, Lena; Yaniv, Yossi; Attia, Ziv; Levi, Asher; Alchanatis, Victor; Moshelion, Menachem; Raveh, Eran; Carmi, Nir; Granot, David

2015-01-01

Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1) under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species.

Expression of Arabidopsis Hexokinase in Citrus Guard Cells Controls Stomatal Aperture and Reduces Transpiration

PubMed Central

Lugassi, Nitsan; Kelly, Gilor; Fidel, Lena; Yaniv, Yossi; Attia, Ziv; Levi, Asher; Alchanatis, Victor; Moshelion, Menachem; Raveh, Eran; Carmi, Nir; Granot, David

2015-01-01

Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1) under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species. PMID:26734024

Primate Lentiviruses Modulate NF-κB Activity by Multiple Mechanisms to Fine-Tune Viral and Cellular Gene Expression

PubMed Central

Heusinger, Elena; Kirchhoff, Frank

2017-01-01

The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays a complex role during the replication of primate lentiviruses. On the one hand, NF-κB is essential for induction of efficient proviral gene expression. On the other hand, this transcription factor contributes to the innate immune response and induces expression of numerous cellular antiviral genes. Recent data suggest that primate lentiviruses cope with this challenge by boosting NF-κB activity early during the replication cycle to initiate Tat-driven viral transcription and suppressing it at later stages to minimize antiviral gene expression. Human and simian immunodeficiency viruses (HIV and SIV, respectively) initially exploit their accessory Nef protein to increase the responsiveness of infected CD4+ T cells to stimulation. Increased NF-κB activity initiates Tat expression and productive replication. These events happen quickly after infection since Nef is rapidly expressed at high levels. Later during infection, Nef proteins of HIV-2 and most SIVs exert a very different effect: by down-modulating the CD3 receptor, an essential factor for T cell receptor (TCR) signaling, they prevent stimulation of CD4+ T cells via antigen-presenting cells and hence suppress further induction of NF-κB and an effective antiviral immune response. Efficient LTR-driven viral transcription is maintained because it is largely independent of NF-κB in the presence of Tat. In contrast, human immunodeficiency virus type 1 (HIV-1) and its simian precursors have lost the CD3 down-modulation function of Nef and use the late viral protein U (Vpu) to inhibit NF-κB activity by suppressing its nuclear translocation. In this review, we discuss how HIV-1 and other primate lentiviruses might balance viral and antiviral gene expression through a tight temporal regulation of NF-κB activity throughout their replication cycle. PMID:28261165

Expression profiling of Ribosomal Protein gene family in dehydration stress responses and characterization of transgenic rice plants overexpressing RPL23A for water-use efficiency and tolerance to drought and salt stresses

NASA Astrophysics Data System (ADS)

Moin, Mazahar; Bakshi, Achala; Madhav, M. S.; Kirti, P. B.

2017-11-01

Our previous findings on the screening of a large-pool of activation tagged rice plants grown under limited water conditions revealed the activation of Ribosomal Protein Large (RPL) subunit genes, RPL6 and RPL23A in two mutants that exhibited high water-use efficiency (WUE) with the genes getting activated by the integrated 4x enhancers (Moin et al., 2016a). In continuation of these findings, we have comprehensively characterized the Ribosomal Protein (RP) gene family including both small (RPS) and large (RPL) subunits, which have been identified to be encoded by at least 70 representative genes; RP-genes exist as multiple expressed copies with high nucleotide and amino acid sequence similarity. The differential expression of all the representative genes in rice was performed under limited water and drought conditions at progressive time intervals in the present study. More than 50% of the RP genes were upregulated in both shoot and root tissues. Some of them exhibited an overlap in the upregulation under both the treatments indicating that they might have a common role in inducing tolerance under limited water and drought conditions. Among the genes that became significantly upregulated in both the tissues and under both the treatments are RPL6, 7, 23A, 24 and 31 and RPS4, 10 and 18a. To further validate the role of RP genes in WUE and inducing tolerance to other stresses, we have raised transgenic plants overexpressing RPL23A in rice. The high expression lines of RPL23A exhibited low Δ13C, increased quantum efficiency along with suitable growth and yield parameters with respect to negative control under the conditions of limited water availability. The constitutive expression of RPL23A was also associated with transcriptional upregulation of many other RPL and RPS genes. The seedlings of RPL23A high expression lines also showed a significant increase in fresh weight, root length, proline and chlorophyll contents under simulated drought and salt stresses. Taken together, our findings provide a secure basis for the RPL gene family expression as a potential resource for exploring abiotic stress tolerant properties in rice.

Suppression of HLA Expression by Lentivirus-mediated Gene Transfer of siRNA Cassettes and In Vivo Chemoselection to Enhance Hematopoietic Stem Cell Transplantation

PubMed Central

Hacke, Katrin; Falahati, Rustom; Flebbe-Rehwaldt, Linda; Kasahara, Noriyuki; Gaensler, Karin M. L.

2010-01-01

Current approaches for hematopoietic stem cell (HSC) and organ transplantation are limited by donor and host-mediated immune responses to allo-antigens. Application of these therapies is limited by the toxicity of preparative and post-transplant immunosuppressive regimens and a shortage of appropriate HLA-matched donors. We have been exploring two complementary approaches for genetically modifying donor cells that achieve long-term suppression of cellular proteins that elicit host immune responses to mismatched donor antigens, and provide a selective advantage to genetically engineered donor cells after transplantation. The first approach is based on recent advances that make feasible targeted down-regulation of HLA expression. Suppression of HLA expression could help to overcome limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors. Accordingly, we have recently investigated whether knockdown of HLA by RNA interference (RNAi) enables allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors that integrate into genomic DNA, thereby permanently modifying transduced donor cells. Lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA achieved efficient and dose-dependent reduction in surface expression of HLA in human cells, and enhanced resistance to allo-reactive T lymphocyte-mediated cytotoxicity, while avoiding non-MHC restricted killing. Complementary strategies for genetic engineering of HSC that would provide a selective advantage for transplanted donor cells and enable successful engraftment with less toxic preparative and immunosuppressive regimens would increase the numbers of individuals to whom HLA suppression therapy could be offered. Our second strategy is to provide a mechanism for in vivo selection of genetically modified HSC and other donor cells. We have uniquely combined transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of mechanisms of tolerance induction to neo-antigens, and allogeneic stem cells during immune ontogeny. HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O6-methylguanine-methyltransferase (MGMTP140K). The MGMTP140K DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. In vivo chemoselection enables enrichment of donor cells at the stem cell level. Using complementary approaches of in vivo chemoselection and RNAi-induced silencing of HLA expression may enable the generation of histocompatibility-enhanced, and eventually, perhaps “universally” compatible cellular grafts. PMID:19048410

Fiber-optic multiphoton flow cytometry in whole blood and in vivo

NASA Astrophysics Data System (ADS)

Chang, Yu-Chung; Ye, Jing Yong; Thomas, Thommey P.; Cao, Zhengyi; Kotlyar, Alina; Tkaczyk, Eric R.; Baker, James R.; Norris, Theodore B.

2010-07-01

Circulating tumor cells in the bloodstream are sensitive indicators for metastasis and disease prognosis. Circulating cells have usually been monitored via extraction from blood, and more recently in vivo using free-space optics; however, long-term intravital monitoring of rare circulating cells remains a major challenge. We demonstrate the application of a two-photon-fluorescence optical fiber probe for the detection of cells in whole blood and in vivo. A double-clad fiber was used to enhance the detection sensitivity. Two-channel detection was employed to enable simultaneous measurement of multiple fluorescent markers. Because the fiber probe circumvents scattering and absorption from whole blood, the detected signal strength from fluorescent cells was found to be similar in phosphate-buffered saline (PBS) and in whole blood. The detection efficiency of cells labeled with the membrane-binding dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindoldicarbocyanine, 4-chlorobenzenesulfonate (DiD) was demonstrated to be the same in PBS and in whole blood. A high detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was also demonstrated. To characterize in vivo detection, DiD-labeled untransfected and GFP-transfected cells were injected into live mice, and the cell circulation dynamics was monitored in real time. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed ex vivo in whole blood.

Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production.

PubMed

Jo, Suah; Yoon, Jinkyung; Lee, Sun-Mi; Um, Youngsoon; Han, Sung Ok; Woo, Han Min

2017-09-20

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization. Copyright © 2017 Elsevier B.V. All rights reserved.

High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2.

PubMed

Abécassis, V; Pompon, D; Truan, G

2000-10-15

The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.

An electrospun scaffold integrating nucleic acid delivery for treatment of full thickness wounds

PubMed Central

Kobsa, Serge; Kristofik, Nina J.; Sawyer, Andrew J.; Bothwell, Alfred L.M.; Kyriakides, Themis R.; Saltzman, W. Mark

2013-01-01

We developed a multi-functional construct capable of controlled delivery of bioactive substances that can improve wound repair by supporting the intrinsic ability of the skin to heal. We synthesized electrospun scaffolds—composed of a blend of the degradable polymers poly(L-lactide) (PLA) or polycaprolactone (PCL)—that produce highly efficient non-viral in vivo gene delivery to cells in the wound bed, provide a protective barrier during early wound healing, and support cell migration and growth. This multi-functional material was tested for its influence on wound healing: scaffolds were loaded with plasmids encoding keratinocyte growth factor (KGF) and applied to full thickness wounds in mice. Compared to scaffolds with control plasmids, animals receiving the KGF plasmid-loaded scaffold produced significant enhancements in wound healing, which was quantified by improvements in the rate of wound re-epithelialization, keratinocyte proliferation, and granulation response. Further, we quantified the expression level of endogenous and plasmid-derived KGF in wound samples: qRT-PCR on wound sections revealed a correlation between the levels of plasmid-derived protein expression and histological analysis of wound healing, revealing an inverse relationship between the expression level of exogenous KGF and the size of the unhealed epithelial layer in wounds. Our findings suggest that engineered nanofiber PLA/PCL scaffolds are capable of highly efficient controlled DNA delivery and are promising materials for treatment of cutaneous wounds. PMID:23453058

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation.

PubMed

Lee, Mi-Sook; Kim, Sudong; Kim, Baek Gil; Won, Cheolhee; Nam, Seo Hee; Kang, Suki; Kim, Hye-Jin; Kang, Minkyung; Ryu, Jihye; Song, Haeng Eun; Lee, Doohyung; Ye, Sang-Kyu; Jeon, Noo Li; Kim, Tai Young; Cho, Nam Hoon; Lee, Jung Weon

2014-09-01

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents. Copyright © 2014 Elsevier B.V. All rights reserved.

Shine-Dalgarno sequence enhances the efficiency of lacZ repression by artificial anti-lac antisense RNAs in Escherichia coli.

PubMed

Stefan, Alessandra; Schwarz, Flavio; Bressanin, Daniela; Hochkoeppler, Alejandro

2010-11-01

Silencing of the lacZ gene in Escherichia coli was attempted by means of the expression of antisense RNAs (asRNAs) in vivo. A short fragment of lacZ was cloned into the pBAD expression vector, in reverse orientation, using the EcoRI and PstI restriction sites. This construct (pBAD-Zcal1) was used to transform E. coli cells, and the antisense transcription was induced simply by adding arabinose to the culture medium. We demonstrated that the Zcal1 asRNA effectively silenced lacZ using β-galactosidase activity determinations, SDS-PAGE, and Western blotting. Because the concentration of the lac mRNA was always high in cells that expressed Zcal1, we hypothesize that this antisense acts by inhibiting messenger translation. Similar analyses, performed with a series of site-specific Zcal1 mutants, showed that the Shine-Dalgarno sequence, which is conferred by the pBAD vector, is an essential requisite for silencing competence. Indeed, the presence of the intact Shine-Dalgarno sequence positively affects asRNA stability and, hence, silencing effectiveness. Our observations will contribute to the understanding of the main determinants of silencing as exerted by asRNAs as well as provide useful support for the design of robust and efficient prokaryotic gene silencers. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Modulation of extracellular matrix in cancer is associated with enhanced tumor cell targeting by bacteriophage vectors.

PubMed

Yata, Teerapong; Lee, Eugene L Q; Suwan, Keittisak; Syed, Nelofer; Asavarut, Paladd; Hajitou, Amin

2015-06-03

Gene therapy has been an attractive paradigm for cancer treatment. However, cancer gene therapy has been challenged by the inherent limitation of vectors that are able to deliver therapeutic genes to tumors specifically and efficiently following systemic administration. Bacteriophage (phage) are viruses that have shown promise for targeted systemic gene delivery. Yet, they are considered poor vectors for gene transfer. Recently, we generated a tumor-targeted phage named adeno-associated virus/phage (AAVP), which is a filamentous phage particle whose genome contains the adeno-associated virus genome. Its effectiveness in delivering therapeutic genes to tumors specifically both in vitro and in vivo has been shown in numerous studies. Despite being a clinically useful vector, a multitude of barriers impede gene transduction to tumor cells. We hypothesized that one such factor is the tumor extracellular matrix (ECM). We used a number of tumor cell lines from different species and histological types in 2D monolayers or 3D multicellular tumor spheroid (MCTS) models. To assess whether the ECM is a barrier to tumor cell targeting by AAVP, we depleted the ECM using collagenase, hyaluronidase, or combination of both. We employed multiple techniques to investigate and quantify the effect of ECM depletion on ECM composition (including collagen type I, hyaluronic acid, fibronectin and laminin), and how AAVP adsorption, internalisation, gene expression and therapeutic efficacy are subsequently affected. Data were analyzed using a student's t test when comparing two groups or one-way ANOVA and post hoc Tukey tests when using more than two groups. We demonstrate that collagenase and hyaluronidase-mediated degradation of tumor ECM affects the composition of collagen, hyaluronic acid and fibronectin. Consequently, AAVP diffusion, internalisation, gene expression and tumor cell killing were enhanced after enzymatic treatment. Our data suggest that enhancement of gene transfer by the AAVP is solely attributed to ECM depletion. We provide substantial evidence that ECM modulation is relevant in clinically applicable settings by using 3D MCTS, which simulates in vivo environments more accurately. Our findings suggest that ECM depletion is an effective strategy to enhance the efficiency of viral vector-guided gene therapy.

Featured Article: Downregulation of transgelin blocks interleukin-8 utilization and suppresses vasculogenic mimicry in breast cancer cells

PubMed Central

Aikins, Anastasia R; Kim, MiJung; Raymundo, Bernardo

2017-01-01

Vasculogenic mimicry (VM) is a non-classical mechanism recently described in many tumors, whereby cancer cells, rather than endothelial cells, form blood vessels. Transgelin is an actin-binding protein that has been implicated in multiple stages of cancer development. In this study, we investigated the role of transgelin in VM and assessed its effect on the expression of endothelial and angiogenesis-related genes during VM in MDA-MB-231 breast cancer cells. We confirmed the ability of MDA-MB-231 cells to undergo VM through a tube formation assay. Flow cytometry analysis revealed an increase in the expression of the endothelial-related markers VE-cadherin and CD34 in cells that underwent VM, compared with those growing in a monolayer, which was confirmed by immunocytochemistry. We employed siRNA to silence transgelin, and knockdown efficiency was determined by western blot analyses. Downregulation of transgelin suppressed cell proliferation and tube formation, but increased IL-8 levels in Matrigel cultures. RT-PCR analyses revealed that the expression of IL-8, VE-cadherin, and CD34 was unaffected by transgelin knockdown, indicating that increased IL-8 expression was not due to enhanced transcriptional activity. More importantly, the inhibition of IL-8/CXCR2 signaling also resulted in suppression of VM with increased IL-8 levels, confirming that increased IL-8 levels after transgelin knockdown was due to inhibition of IL-8 uptake. Our findings indicate that transgelin regulates VM by enhancing IL uptake. These observations are relevant to the future development of efficient antivascular agents. Impact statement Vasculogenic mimicry (VM) is an angiogenic-independent mechanism of blood vessel formation whereby aggressive tumor cells undergo formation of capillary-like structures. Thus, interventions aimed at angiogenesis might not target the entire tumor vasculature. A more holistic approach is therefore needed in the development of improved antivascular agents. Transgelin, an actin-binding protein, has been associated with multiple stages of cancer development such as proliferation, migration and invasion, but little is known about its role in vasculogenic mimicry. We present here, an additional mechanism by which transgelin promotes malignancy by way of its association with the occurrence of VM. Although transgelin knockdown did not affect the transcript levels of most of the angiogenesis-related genes in this study, it was associated with the inhibition of the uptake of IL-8, accompanied by suppressed VM, indicating that transgelin is required for VM. These observations are relevant to the future development of efficient antivascular agents. PMID:28058861

Enhanced in Vitro Anti-Tumor Activity of 5-Azacytidine by Entrapment into Solid Lipid Nanoparticles

PubMed Central

Jahanfar, Farhad; Hasani, Akbar; Shanebandi, Dariush; Rahmati, Mohammad; Hamishehkar, Hamed

2016-01-01

Purpose: In this study the effectiveness of encapsulating of 5-azacytidine into the lipid nanoparticles was investigated and in vitro effect of encapsulated 5-azacytidine studied on MCF-7 cell lines Methods: 5-azacytidine -loaded solid lipid nanoparticles were produced by double emulsification (w/o/w) method by using stearic acid as lipid matrix, soy lecithin and poloxamer 407 as surfactant and co-surfactant respectively. Particle size, zeta potential, surface morphology, entrapment efficiency and kinetic of drug release were studied. In vitro effect of 5-azacytidine on MCF-7 cell line studied by MTT assay, DAPI staining, Rhodamine B relative uptake, and also Real time RT-PCR was performed for studying difference effect of free and encapsulated drug on expression of RARß2 gene. Results: The formulation F5 with 55.84±0.46 % of entrapment efficiency shows zero order kinetic of drug release and selected for in vitro studies; the cytotoxicity of free drug and encapsulated drug in 48 h of incubation have significant difference. DAPI staining shows morphology of apoptotic nucleus in both free and encapsulated drug, Rhodamine B labeled SLNs show time dependency and accumulation of SLNs in cytoplasm. Real time qRT-PCR doesn’t show any significant difference (p>0.05) in expression of RARß2 gene in both cells treated with free or encapsulated drug. Conclusion: The results of the present study indicated that the entrapment of 5-azacytidine into SLNs enhanced its cytotoxicity performance and may pave a way for the future design of a desired dosage form for 5-azacytidine. PMID:27766220

Enhanced crude oil biodegradation in soil via biostimulation.

PubMed

Al-Saleh, Esmaeil; Hassan, Ali

2016-08-02

Research on feasible methods for the enhancement of bioremediation in soil contaminated by crude oil is vital in oil-exporting countries such as Kuwait, where crude oil is a major pollutant and the environment is hostile to biodegradation. This study investigated the possibility of enhancing crude oil bioremediation by supplementing soil with cost-effective organic materials derived from two widespread locally grown trees, Conocarpus and Tamarix. Amendments in soils increased the counts of soil microbiota by up to 98% and enhanced their activity by up to 95.5%. The increase in the biodegradation of crude oil (75%) and high levels of alkB expression substantiated the efficiency of the proposed amendment technology for the bioremediation of hydrocarbon-contaminated sites. The identification of crude-oil-degrading bacteria revealed the dominance of the genus Microbacterium (39.6%), Sphingopyxis soli (19.3%), and Bordetella petrii (19.6%) in unamended, Conocarpus-amended, and Tamarix-amended contaminated soils, respectively. Although soil amendments favored the growth of Gram-negative bacteria and reduced bacterial diversity, the structures of bacterial communities were not significantly altered.

Combined Approach for Government E-Tendering Using GA and TOPSIS with Intuitionistic Fuzzy Information.

PubMed

Wang, Yan; Xi, Chengyu; Zhang, Shuai; Zhang, Wenyu; Yu, Dejian

2015-01-01

As E-government continues to develop with ever-increasing speed, the requirement to enhance traditional government systems and affairs with electronic methods that are more effective and efficient is becoming critical. As a new product of information technology, E-tendering is becoming an inevitable reality owing to its efficiency, fairness, transparency, and accountability. Thus, developing and promoting government E-tendering (GeT) is imperative. This paper presents a hybrid approach combining genetic algorithm (GA) and Technique for Order Preference by Similarity to an Ideal Solution (TOPSIS) to enable GeT to search for the optimal tenderer efficiently and fairly under circumstances where the attributes of the tenderers are expressed as fuzzy number intuitionistic fuzzy sets (FNIFSs). GA is applied to obtain the optimal weights of evaluation criteria of tenderers automatically. TOPSIS is employed to search for the optimal tenderer. A prototype system is built and validated with an illustrative example from GeT to verify the feasibility and availability of the proposed approach.

Combined Approach for Government E-Tendering Using GA and TOPSIS with Intuitionistic Fuzzy Information

PubMed Central

Zhang, Wenyu; Yu, Dejian

2015-01-01

As E-government continues to develop with ever-increasing speed, the requirement to enhance traditional government systems and affairs with electronic methods that are more effective and efficient is becoming critical. As a new product of information technology, E-tendering is becoming an inevitable reality owing to its efficiency, fairness, transparency, and accountability. Thus, developing and promoting government E-tendering (GeT) is imperative. This paper presents a hybrid approach combining genetic algorithm (GA) and Technique for Order Preference by Similarity to an Ideal Solution (TOPSIS) to enable GeT to search for the optimal tenderer efficiently and fairly under circumstances where the attributes of the tenderers are expressed as fuzzy number intuitionistic fuzzy sets (FNIFSs). GA is applied to obtain the optimal weights of evaluation criteria of tenderers automatically. TOPSIS is employed to search for the optimal tenderer. A prototype system is built and validated with an illustrative example from GeT to verify the feasibility and availability of the proposed approach. PMID:26147468

Expansion of the gateway multisite recombination cloning toolkit.

PubMed

Shearin, Harold K; Dvarishkis, Alisa R; Kozeluh, Craig D; Stowers, R Steven

2013-01-01

Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters.

Expansion of the Gateway MultiSite Recombination Cloning Toolkit

PubMed Central

Shearin, Harold K.; Dvarishkis, Alisa R.; Kozeluh, Craig D.; Stowers, R. Steven

2013-01-01

Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters. PMID:24204935

Forecasting Nonlinear Chaotic Time Series with Function Expression Method Based on an Improved Genetic-Simulated Annealing Algorithm

PubMed Central

Wang, Jun; Zhou, Bi-hua; Zhou, Shu-dao; Sheng, Zheng

2015-01-01

The paper proposes a novel function expression method to forecast chaotic time series, using an improved genetic-simulated annealing (IGSA) algorithm to establish the optimum function expression that describes the behavior of time series. In order to deal with the weakness associated with the genetic algorithm, the proposed algorithm incorporates the simulated annealing operation which has the strong local search ability into the genetic algorithm to enhance the performance of optimization; besides, the fitness function and genetic operators are also improved. Finally, the method is applied to the chaotic time series of Quadratic and Rossler maps for validation. The effect of noise in the chaotic time series is also studied numerically. The numerical results verify that the method can forecast chaotic time series with high precision and effectiveness, and the forecasting precision with certain noise is also satisfactory. It can be concluded that the IGSA algorithm is energy-efficient and superior. PMID:26000011

Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils.

PubMed

Zimmermann, Maili; Aguilera, Francisco Bianchetto; Castellucci, Monica; Rossato, Marzia; Costa, Sara; Lunardi, Claudio; Ostuni, Renato; Girolomoni, Giampiero; Natoli, Gioacchino; Bazzoni, Flavia; Tamassia, Nicola; Cassatella, Marco A

2015-01-23

Controversy currently exists about the ability of human neutrophils to produce IL-6. Here, we show that the chromatin organization of the IL-6 genomic locus in human neutrophils is constitutively kept in an inactive configuration. However, we also show that upon exposure to stimuli that trigger chromatin remodelling at the IL-6 locus, such as ligands for TLR8 or, less efficiently, TLR4, highly purified neutrophils express and secrete IL-6. In TLR8-activated neutrophils, but not monocytes, IL-6 expression is preceded by the induction of a latent enhancer located 14 kb upstream of the IL-6 transcriptional start site. In addition, IL-6 induction is potentiated by endogenous TNFα, which prolongs the synthesis of the IκBζ co-activator and sustains C/EBPβ recruitment and histone acetylation at IL-6 regulatory regions. Altogether, these data clarify controversial literature on the ability of human neutrophils to generate IL-6 and uncover chromatin-dependent layers of regulation of IL-6 in these cells.

A transposable element in a NAC gene is associated with drought tolerance in maize seedlings

PubMed Central

Mao, Hude; Wang, Hongwei; Liu, Shengxue; Li, Zhigang; Yang, Xiaohong; Yan, Jianbing; Li, Jiansheng; Tran, Lam-Son Phan; Qin, Feng

2015-01-01

Drought represents a major constraint on maize production worldwide. Understanding the genetic basis for natural variation in drought tolerance of maize may facilitate efforts to improve this trait in cultivated germplasm. Here, using a genome-wide association study, we show that a miniature inverted-repeat transposable element (MITE) inserted in the promoter of a NAC gene (ZmNAC111) is significantly associated with natural variation in maize drought tolerance. The 82-bp MITE represses ZmNAC111 expression via RNA-directed DNA methylation and H3K9 dimethylation when heterologously expressed in Arabidopsis. Increasing ZmNAC111 expression in transgenic maize enhances drought tolerance at the seedling stage, improves water-use efficiency and induces upregulation of drought-responsive genes under water stress. The MITE insertion in the ZmNAC111 promoter appears to have occurred after maize domestication and spread among temperate germplasm. The identification of this MITE insertion provides insight into the genetic basis for natural variation in maize drought tolerance. PMID:26387805

Tandem SUMO fusion vectors for improving soluble protein expression and purification.

PubMed

Guerrero, Fernando; Ciragan, Annika; Iwaï, Hideo

2015-12-01

Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin. Copyright © 2015 Elsevier Inc. All rights reserved.

Intracellular regulation of the insect chemoreceptor complex impacts odour localization in flying insects.

PubMed

Getahun, Merid N; Thoma, Michael; Lavista-Llanos, Sofia; Keesey, Ian; Fandino, Richard A; Knaden, Markus; Wicher, Dieter; Olsson, Shannon B; Hansson, Bill S

2016-11-01

Flying insects are well known for airborne odour tracking and have evolved diverse chemoreceptors. While ionotropic receptors (IRs) are found across protostomes, insect odorant receptors (ORs) have only been identified in winged insects. We therefore hypothesized that the unique signal transduction of ORs offers an advantage for odour localization in flight. Using Drosophila, we found expression and increased activity of the intracellular signalling protein PKC in antennal sensilla following odour stimulation. Odour stimulation also enhanced phosphorylation of the OR co-receptor Orco in vitro, while site-directed mutation of Orco or mutations in PKC subtypes reduced the sensitivity and dynamic range of OR-expressing neurons in vivo, but not IR-expressing neurons. We ultimately show that these mutations reduce competence for odour localization of flies in flight. We conclude that intracellular regulation of OR sensitivity is necessary for efficient odour localization, which suggests a mechanistic advantage for the evolution of the OR complex in flying insects. © 2016. Published by The Company of Biologists Ltd.

The Ars insulator facilitates I-SceI meganuclease-mediated transgenesis in the sea urchin embryo.

PubMed

Ochiai, Hiroshi; Sakamoto, Naoaki; Suzuki, Kenichi; Akasaka, Koji; Yamamoto, Takashi

2008-09-01

For the efficient generation of transgenic sea urchins, we have adopted an I-SceI meganuclease-mediated transgenesis method. Several types of promoter-GFP gene constructs flanked by two I-SceI recognition sequences were co-injected with I-SceI into sea urchin fertilized eggs. Using cell-lineage-specific promoter constructs, the frequency of transgene expression was elevated, and their level of mozaicism was reduced. The addition of the Ars insulator sequence, which is known to block the enhancer activity and protect transgenes from position effects, led to a reduction in ectopic transgene expression and an elevation of transgene expression frequency in this I-SceI-mediated system. However, the magnitude of the effects of the Ars insulator was dependent upon the promoter constructs. QPCR analysis also showed that the Ars insulator increases the transgene copy number. These results suggest that the I-SceI-mediated method using the Ars insulator is advantageous for transgenesis in the sea urchin embryo.

Differentially expressed genes in healthy and plum pox virus-infected Nicotiana benthamiana plants.

PubMed

Vozárová, Z; Žilová, M; Šubr, Z

2015-12-01

Viruses use both material and energy sources of their hosts and redirect the production of disposable compounds in order to make viral replication more efficient. Metabolism of infected organisms is modified by these enhanced requirements as well by their own defense response. Resulting complex story consists of many regulation events on various gene expression levels. Elucidating these processes may contribute to the knowledge on virus-host interactions and to evolving new antiviral strategies. In our work we applied a subtractive cloning technique to compare the transcriptomes of healthy and plum pox virus (PPV)-infected Nicotiana benthamiana plants. Several genes were found to be induced or repressed by the PPV infection. The induced genes were mainly related to general stress response or photosynthesis, several repressed genes could be connected with growth defects evoked by the infection. Interestingly, some genes usually up-regulated by fungal or bacterial infection were found repressed in PPV-infected plants. Potential involvement of particular differently expressed genes in the process of PPV infection is discussed.

Forecasting nonlinear chaotic time series with function expression method based on an improved genetic-simulated annealing algorithm.

PubMed

Wang, Jun; Zhou, Bi-hua; Zhou, Shu-dao; Sheng, Zheng

2015-01-01

The paper proposes a novel function expression method to forecast chaotic time series, using an improved genetic-simulated annealing (IGSA) algorithm to establish the optimum function expression that describes the behavior of time series. In order to deal with the weakness associated with the genetic algorithm, the proposed algorithm incorporates the simulated annealing operation which has the strong local search ability into the genetic algorithm to enhance the performance of optimization; besides, the fitness function and genetic operators are also improved. Finally, the method is applied to the chaotic time series of Quadratic and Rossler maps for validation. The effect of noise in the chaotic time series is also studied numerically. The numerical results verify that the method can forecast chaotic time series with high precision and effectiveness, and the forecasting precision with certain noise is also satisfactory. It can be concluded that the IGSA algorithm is energy-efficient and superior.

Establishment of an efficient fermentation system of gamma-aminobutyric acid by a lactic acid bacterium, Enterococcus avium G-15, isolated from carrot leaves.

PubMed

Tamura, Takayoshi; Noda, Masafumi; Ozaki, Moeko; Maruyama, Masafumi; Matoba, Yasuyuki; Kumagai, Takanori; Sugiyama, Masanori

2010-01-01

In the present study, we successfully isolated a carrot leaf-derived lactic acid bacterium that produces gamma-aminobutyric acid (GABA) from monosodium L-glutamate (L-MSG) at a hyper conversion rate. The GABA-producing bacterium, identified as Enterococcus (E.) avium G-15, produced 115.7±6.4 g/l GABA at a conversion rate of 86.0±5.0% from the added L-MSG under the optimum culture condition by a continuous L-MSG feeding method using a jar-fermentor, suggesting that the bacterium displays a great potential ability for the commercial-level fermentation production of GABA. Using the reverse transcription polymerase chain reaction (RT-PCR) method, we analyzed the expression of genes for the GABA transporter and glutamate decarboxylase, designated gadT and gadG, respectively, which were cloned from the E. avium G-15 chromosome. Both genes were expressed even without the added L-MSG, but their expression was enhanced by the addition of L-MSG.

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.

PubMed

Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea; Burdick, Ryan C; Levine, Louis; Li, Kelvin; Rein, Alan; Pathak, Vinay K; Hu, Wei-Shau

2017-08-15

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication. Copyright © 2017 American Society for Microbiology.

TRAIL overexpression co-regulated by Egr1 and HRE enhances radiosensitivity of hypoxic A549 cells depending on its apoptosis inducing role.

PubMed

Yang, Yan-Ming; Fang, Fang; Li, Xin; Yu, Lei; Wang, Zhi-Cheng

2017-01-01

Ionizing radiation can upregulate the expression levels of TRAIL and enhance tumor cell apoptosis. While Early growth response 1 (Egr1) gene promoter has radiation inducible characteristics, the expression for exogenous gene controlled by Egr1 promoter could be enhanced by ionizing radiation, but its efficiency is limited by tissue hypoxia. Hypoxia response elements (HREs) are important hypoxic response regulatory sequences and sensitivity enhancers. Therefore, we chose TRAIL as the gene radiotherapy to observe whether it is regulated by Egr1 and HER and its effects on A549 cells and its mechanism. The pcDNA3.1-Egr1-TRAIL (pc-E-hsT) and pcDNA3.1-HRE/Egr1-TRAIL (pc-H/E-hsT) plasmids containing Egr1-hsTRAIL and HRE/Egr1-hsTRAIL were transfected into A549 cells, the cells were treated by hypoxia and radiation. The TRAIL mRNA in the cells and protein concentration in the culture supernatants were measured by RT-PCR and ELISA, respectively. Mean lethal dose D0 value was evaluated with colony forming assay. The cell apoptotic rates were analyzed by FCM and TUNEL assay. Expression of DR4, DR5 and cleaved caspase-3 proteins were analyzed by western blotting. It showed that TRAIL mRNA expression and TRAIL concentration all significantly increased under hypoxia and/or radiation. D0 value of pc-H/E‑hsT transfected cells under hypoxia was lowest, indicating more high radiosensitivity. Hypoxia could not cause the pc-E-hsT transfected cell apoptotic rate increase, but there were promoting effects in pc-H/E-hsT transfected cells. DR4 had not obvious change in pc-E-hsT and pc-H/E-hsT transfected cells under normoxic and hypoxic condition, otherwise, DR5 and cleaved caspase-3 increased mostly in pc-H/E-hsT transfected cells under hypoxic condition. TRAIL overexpression was co-regulated by Egr1 and HRE. TRAIL might promote hypoxic A549 cell radiosensitivity and induce apoptosis depending on DR5 to caspase-3 pathways.

Convection-enhanced delivery of an anti-miR is well-tolerated, preserves anti-miR stability and causes efficient target de-repression: a proof of concept.

PubMed

Halle, Bo; Marcusson, Eric G; Aaberg-Jessen, Charlotte; Jensen, Stine S; Meyer, Morten; Schulz, Mette K; Andersen, Claus; Kristensen, Bjarne W

2016-01-01

Over-expressed microRNAs (miRs) are promising new targets in glioblastoma (GBM) therapy. Inhibition of over-expressed miRs has been shown to diminish GBM proliferation, invasion and angiogenesis, indicating a significant therapeutic potential. However, the methods utilized for miR inhibition have had low translational potential. In clinical trials convection-enhanced delivery (CED) has been applied for local delivery of compounds in the brain. The aim of this study was to determine if safe and efficient miR inhibition was possible by CED of an anti-miR. We used a highly invasive GBM orthotopic xenograft model and targeted a well-validated miR, let-7a, with a 2'-O-methoxyethyl anti-miR with a combined phosphodiester/phosphorothioate backbone to establish an initial proof of concept. In vitro, anti-let-7a was delivered unassisted to the patient-derived T87 glioblastoma spheroid culture. In vivo, anti-let-7a or saline were administered by CED into orthotopic T87-derived tumors. After 1 month of infusion, tumors were removed and tumor mRNA levels of the target-gene High-mobility group AT-hook 2 (HMGA2) were determined. In vitro, 5 days inhibition was superior to 1 day at de-repressing the let-7a target HMGA2 and the inhibition was stable for 24 h. In vivo, anti-miR integrity was preserved in the pumps and no animals showed signs of severe adverse effects attributable to the anti-miR treatment. HMGA2 tumor level was significantly de-repressed in the anti-miR treated animals. The results showed-as an initial proof of concept-that miRs can be efficiently inhibited using CED delivery of anti-miR. The next step is to apply CED for anti-miR delivery focusing on key oncogenic miRs.

A novel CXCL10-based GPI-anchored fusion protein as adjuvant in NK-based tumor therapy.

PubMed

Muenchmeier, Niklas; Boecker, Sophia; Bankel, Lorenz; Hinz, Laura; Rieth, Nicole; Lapa, Constantin; Mendler, Anna N; Noessner, Elfriede; Mocikat, Ralph; Nelson, Peter J

2013-01-01

Cellular therapy is a promising therapeutic strategy for malignant diseases. The efficacy of this therapy can be limited by poor infiltration of the tumor by immune effector cells. In particular, NK cell infiltration is often reduced relative to T cells. A novel class of fusion proteins was designed to enhance the recruitment of specific leukocyte subsets based on their expression of a given chemokine receptor. The proteins are composed of an N-terminal chemokine head, the mucin domain taken from the membrane-anchored chemokine CX3CL1, and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor replacing the normal transmembrane domain allowing integration of the proteins into cell membranes when injected into a solid tumor. The mucin domain in conjunction with the chemokine head acts to specifically recruit leukocytes expressing the corresponding chemokine receptor. A fusion protein comprising a CXCL10 chemokine head (CXCL10-mucin-GPI) was used for proof of concept for this approach and expressed constitutively in Chinese Hamster Ovary cells. FPLC was used to purify proteins. The recombinant proteins efficiently integrated into cell membranes in a process dependent upon the GPI anchor and were able to activate the CXCR3 receptor on lymphocytes. Endothelial cells incubated with CXCL10-mucin-GPI efficiently recruited NK cells in vitro under conditions of physiologic flow, which was shown to be dependent on the presence of the mucin domain. Experiments conducted in vivo using established tumors in mice suggested a positive effect of CXCL10-mucin-GPI on the recruitment of NK cells. The results suggest enhanced recruitment of NK cells by CXCL10-mucin-GPI. This class of fusion proteins represents a novel adjuvant in cellular immunotherapy. The underlying concept of a chemokine head fused to the mucin domain and a GPI anchor signal sequence may be expanded into a broader family of reagents that will allow targeted recruitment of cells in various settings.

Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

PubMed

Sharma, Manoj Kumar; Jani, Dewal; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

2008-05-20

In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.

Biochemical mechanisms of imidacloprid resistance in Nilaparvata lugens: over-expression of cytochrome P450 CYP6AY1.

PubMed

Ding, Zhiping; Wen, Yucong; Yang, Baojun; Zhang, Yixi; Liu, Shuhua; Liu, Zewen; Han, Zhaojun

2013-11-01

Imidacloprid is a key insecticide extensively used for control of Nilaparvata lugens, and its resistance had been reported both in the laboratory selected strains and field populations. A target site mutation Y151S in two nicotinic acetylcholine receptor subunits and enhanced oxidative detoxification have been identified in the laboratory resistant strain, contributing importantly to imidacloprid resistance in N. lugens. To date, however, imidacloprid resistance in field population is primarily attributable to enhanced oxidative detoxification by over-expressed P450 monooxygenases. A resistant strain (Res), originally collected from a field population and continuously selected in laboratory with imidacloprid for more than 40 generations, had 180.8-fold resistance to imidacloprid, compared to a susceptible strain (Sus). Expression of different putative P450 genes at mRNA levels was detected and compared between Res and Sus strains, and six genes were found expressed significantly higher in Res strain than in Sus strain. CYP6AY1 was found to be the most different expressed P450 gene and its mRNA level in Res strain was 17.9 times of that in Sus strain. By expressing in E. coli cells, CYP6AY1 was found to metabolize imidacloprid efficiently with initial velocity calculated of 0.851 ± 0.073 pmol/min/pmol P450. When CYP6AY1 mRNA levels in Res strain was reduced by RNA interference, imidacloprid susceptibility was recovered. In four field populations with different resistance levels, high levels of CYP6AY1 transcript were also found. In vitro and in vivo studies provided evidences that the over-expression of CYP6AY1 was one of the key factors contributing to imidacloprid resistance in the laboratory selected strain Res, which might also be the important mechanism for imidacloprid resistance in field populations, when the target site mutation was not prevalent at present. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and polyphosphate kinase.

PubMed

Ruiz, Oscar N; Alvarez, Derry; Gonzalez-Ruiz, Gloriene; Torres, Cesar

2011-08-12

The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1) and polyphosphate kinase (ppk) genes in bacteria in order to provide high mercury resistance and accumulation. In this study, bacterial transformation with transcriptional and translational enhanced vectors designed for the expression of metallothionein and polyphosphate kinase provided high transgene transcript levels independent of the gene being expressed. Expression of polyphosphate kinase and metallothionein in transgenic bacteria provided high resistance to mercury, up to 80 μM and 120 μM, respectively. Here we show for the first time that metallothionein can be efficiently expressed in bacteria without being fused to a carrier protein to enhance mercury bioremediation. Cold vapor atomic absorption spectrometry analyzes revealed that the mt-1 transgenic bacteria accumulated up to 100.2 ± 17.6 μM of mercury from media containing 120 μM Hg. The extent of mercury remediation was such that the contaminated media remediated by the mt-1 transgenic bacteria supported the growth of untransformed bacteria. Cell aggregation, precipitation and color changes were visually observed in mt-1 and ppk transgenic bacteria when these cells were grown in high mercury concentrations. The transgenic bacterial system described in this study presents a viable technology for mercury bioremediation from liquid matrices because it provides high mercury resistance and accumulation while inhibiting elemental mercury volatilization. This is the first report that shows that metallothionein expression provides mercury resistance and accumulation in recombinant bacteria. The high accumulation of mercury in the transgenic cells could present the possibility of retrieving the accumulated mercury for further industrial applications.

Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and polyphosphate kinase

PubMed Central

2011-01-01

Background The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1) and polyphosphate kinase (ppk) genes in bacteria in order to provide high mercury resistance and accumulation. Results In this study, bacterial transformation with transcriptional and translational enhanced vectors designed for the expression of metallothionein and polyphosphate kinase provided high transgene transcript levels independent of the gene being expressed. Expression of polyphosphate kinase and metallothionein in transgenic bacteria provided high resistance to mercury, up to 80 μM and 120 μM, respectively. Here we show for the first time that metallothionein can be efficiently expressed in bacteria without being fused to a carrier protein to enhance mercury bioremediation. Cold vapor atomic absorption spectrometry analyzes revealed that the mt-1 transgenic bacteria accumulated up to 100.2 ± 17.6 μM of mercury from media containing 120 μM Hg. The extent of mercury remediation was such that the contaminated media remediated by the mt-1 transgenic bacteria supported the growth of untransformed bacteria. Cell aggregation, precipitation and color changes were visually observed in mt-1 and ppk transgenic bacteria when these cells were grown in high mercury concentrations. Conclusion The transgenic bacterial system described in this study presents a viable technology for mercury bioremediation from liquid matrices because it provides high mercury resistance and accumulation while inhibiting elemental mercury volatilization. This is the first report that shows that metallothionein expression provides mercury resistance and accumulation in recombinant bacteria. The high accumulation of mercury in the transgenic cells could present the possibility of retrieving the accumulated mercury for further industrial applications. PMID:21838857

Sequential anaerobic/aerobic digestion for enhanced sludge stabilization: comparison of the process performance for mixed and waste sludge [corrected].

PubMed

Tomei, M Concetta; Carozza, Nicola Antonello

2015-05-01

Sequential anaerobic-aerobic digestion has been demonstrated as a promising alternative for enhanced sludge stabilization. In this paper, a feasibility study of the sequential digestion applied to real waste activated sludge (WAS) and mixed sludge is presented. Process performance is evaluated in terms of total solid (TS) and volatile solid (VS) removal, biogas production, and dewaterability trend in the anaerobic and double-stage digested sludge. In the proposed digestion lay out, the aerobic stage was operated with intermittent aeration to reduce the nitrogen load recycled to the wastewater treatment plant (WWTP). Experimental results showed a very good performance of the sequential digestion process for both waste and mixed sludge, even if, given its better digestibility, higher efficiencies are observed for mixed sludge. VS removal efficiencies in the anaerobic stage were 48 and 50% for waste and mixed sludge, respectively, while a significant additional improvement of the VS removal of 25% for WAS and 45% for mixed sludge has been obtained in the aerobic stage. The post-aerobic stage, operated with intermittent aeration, was also efficient in nitrogen removal, providing a significant decrease of the nitrogen content in the supernatant: nitrification efficiencies of 90 and 97% and denitrification efficiencies of 62 and 70% have been obtained for secondary and mixed sludges, respectively. A positive effect due to the aerobic stage was also observed on the sludge dewaterability in both cases. Biogas production, expressed as Nm(3)/(kgVSdestroyed), was 0.54 for waste and 0.82 for mixed sludge and is in the range of values reported in the literature in spite of the low anaerobic sludge retention time of 15 days.

Gene expression analysis of growth factor receptors in human chondrocytes in monolayer and 3D pellet cultures

PubMed Central

Witt, Anika; Salamon, Achim; Boy, Diana; Hansmann, Doris; Büttner, Andreas; Wree, Andreas; Bader, Rainer; Jonitz-Heincke, Anika

2017-01-01

The main goal of cartilage repair is to create functional tissue by enhancing the in vitro conditions to more physiological in vivo conditions. Chondrogenic growth factors play an important role in influencing cartilage homeostasis. Insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β1 affect the expression of collagen type II (Col2) and glycosaminoglycans (GAGs) and, therefore, the targeted use of growth factors could make chondrogenic redifferentiation more efficient. In the present study, human chondrocytes were postmortally isolated from healthy articular cartilage and cultivated as monolayer or 3D pellet cultures either under normoxia or hypoxia and stimulated with IGF-1 and/or TGF-β1 to compare the impact of the different growth factors. The mRNA levels of the specific receptors (IGF1R, TGFBR1, TGFBR2) were analyzed at different time points. Moreover, gene expression rates of collagen type 1 and 2 in pellet cultures were observed over a period of 5 weeks. Additionally, hyaline-like Col2 protein and sulphated GAG (sGAG) levels were quantified. Stimulation with IGF-1 resulted in an enhanced expression of IGF1R and TGFBR2 whereas TGF-β1 stimulated TGFBR1 in the monolayer and pellet cultures. In monolayer, the differences reached levels of significance. This effect was more pronounced under hypoxic culture conditions. In pellet cultures, increased amounts of Col2 protein and sGAGs after incubation with TGF-β1 and/or IGF-1 were validated. In summary, constructing a gene expression profile regarding mRNA levels of specific growth factor receptors in monolayer cultures could be helpful for a targeted application of growth factors in cartilage tissue engineering. PMID:28534942

Establishment of a Novel Permissive Cell Line for the Propagation of Hepatitis C Virus by Expression of MicroRNA miR122

PubMed Central

Kambara, Hiroto; Fukuhara, Takasuke; Shiokawa, Mai; Ono, Chikako; Ohara, Yuri; Kamitani, Wataru

2012-01-01

The robust cell culture systems for hepatitis C virus (HCV) are limited to those using cell culture-adapted clones (HCV in cell culture [HCVcc]) and cells derived from the human hepatoma cell line Huh7. However, accumulating data suggest that host factors, including innate immunity and gene polymorphisms, contribute to the variation in host response to HCV infection. Therefore, the existing in vitro systems for HCV propagation are not sufficient to elucidate the life cycle of HCV. A liver-specific microRNA, miR122, has been shown to participate in the efficient replication of HCV. In this study, we examined the possibility of establishing a new permissive cell line for HCV propagation by the expression of miR122. A high level of miR122 was expressed by a lentiviral vector placed into human liver cell lines at a level comparable to the endogenous level in Huh7 cells. Among the cell lines that we examined, Hep3B cells stably expressing miR122 (Hep3B/miR122) exhibited a significant enhancement of HCVcc propagation. Surprisingly, the levels of production of infectious particles in Hep3B/miR122 cells upon infection with HCVcc were comparable to those in Huh7 cells. Furthermore, a line of “cured” cells, established by elimination of HCV RNA from the Hep3B/miR122 replicon cells, exhibited an enhanced expression of miR122 and a continuous increase of infectious titers of HCVcc in every passage. The establishment of the new permissive cell line for HCVcc will have significant implications not only for basic HCV research but also for the development of new therapeutics. PMID:22114337

Amplification of ABA biosynthesis and signaling through a positive feedback mechanism in seeds.

PubMed

Nonogaki, Mariko; Sall, Khadidiatou; Nambara, Eiji; Nonogaki, Hiroyuki

2014-05-01

Abscisic acid is an essential hormone for seed dormancy. Our previous study using the plant gene switch system, a chemically induced gene expression system, demonstrated that induction of 9-cis-epoxycarotenoid dioxygenase (NCED), a rate-limiting ABA biosynthesis gene, was sufficient to suppress germination in imbibed Arabidopsis seeds. Here, we report development of an efficient experimental system that causes amplification of NCED expression during seed maturation. The system was created with a Triticum aestivum promoter containing ABA responsive elements (ABREs) and a Sorghum bicolor NCED to cause ABA-stimulated ABA biosynthesis and signaling, through a positive feedback mechanism. The chimeric gene pABRE:NCED enhanced NCED and ABF (ABRE-binding factor) expression in Arabidopsis Columbia-0 seeds, which caused 9- to 73-fold increases in ABA levels. The pABRE:NCED seeds exhibited unusually deep dormancy which lasted for more than 3 months. Interestingly, the amplified ABA pathways also caused enhanced expression of Arabidopsis NCED5, revealing the presence of positive feedback in the native system. These results demonstrated the robustness of positive feedback mechanisms and the significance of NCED expression, or single metabolic change, during seed maturation. The pABRE:NCED system provides an excellent experimental system producing dormant and non-dormant seeds of the same maternal origin, which differ only in zygotic ABA. The pABRE:NCED seeds contain a GFP marker which enables seed sorting between transgenic and null segregants and are ideal for comparative analysis. In addition to its utility in basic research, the system can also be applied to prevention of pre-harvest sprouting during crop production, and therefore contributes to translational biology. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

AMT1;1 transgenic rice plants with enhanced NH4(+) permeability show superior growth and higher yield under optimal and suboptimal NH4(+) conditions.

PubMed

Ranathunge, Kosala; El-Kereamy, Ashraf; Gidda, Satinder; Bi, Yong-Mei; Rothstein, Steven J

2014-03-01

The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4(+)). The NH4(+) uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4(+) transport in rice plants. However, little is known about its involvement in NH4(+) uptake in rice roots and subsequent effects on NH4(+) assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4(+) permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4(+) content in the shoots and roots than the WT. Direct NH4(+) fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4(+) contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4(+) levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions.

PtrA/NINV, an alkaline/neutral invertase gene of Poncirus trifoliata, confers enhanced tolerance to multiple abiotic stresses by modulating ROS levels and maintaining photosynthetic efficiency.

PubMed

Dahro, Bachar; Wang, Fei; Peng, Ting; Liu, Ji-Hong

2016-03-29

Alkaline/neutral invertase (A/N-INV), an enzyme that hydrolyzes sucrose irreversibly into glucose and fructose, is essential for normal plant growth,development, and stress tolerance. However, the physiological and/or molecular mechanism underpinning the role of A/N-INV in abiotic stress tolerance is poorly understood. In this report, an A/N-INV gene (PtrA/NINV) was isolated from Poncirus trifoliata, a cold-hardy relative of citrus, and functionally characterized. PtrA/NINV expression levels were induced by cold, salt, dehydration, sucrose, and ABA, but decreased by glucose. PtrA/NINV was found to localize in both chloroplasts and mitochondria. Overexpression of PtrA/NINV conferred enhanced tolerance to multiple stresses, including cold, high salinity, and drought, as supported by lower levels of reactive oxygen species (ROS), reduced oxidative damages, decreased water loss rate, and increased photosynthesis efficiency, relative to wild-type (WT). The transgenic plants exhibited higher A/N-INV activity and greater reducing sugar content under normal and stress conditions. PtrA/NINV is an important gene implicated in sucrose decomposition, and plays a positive role in abiotic stress tolerance by promoting osmotic adjustment, ROS detoxification and photosynthesis efficiency. Thus, PtrA/NINV has great potential to be used in transgenic breeding for improvement of stress tolerance.

Efficient modulation of γ-aminobutyric acid type A receptors by piperine derivatives.

PubMed

Schöffmann, Angela; Wimmer, Laurin; Goldmann, Daria; Khom, Sophia; Hintersteiner, Juliane; Baburin, Igor; Schwarz, Thomas; Hintersteininger, Michael; Pakfeifer, Peter; Oufir, Mouhssin; Hamburger, Matthias; Erker, Thomas; Ecker, Gerhard F; Mihovilovic, Marko D; Hering, Steffen

2014-07-10

Piperine activates TRPV1 (transient receptor potential vanilloid type 1 receptor) receptors and modulates γ-aminobutyric acid type A receptors (GABAAR). We have synthesized a library of 76 piperine analogues and analyzed their effects on GABAAR by means of a two-microelectrode voltage-clamp technique. GABAAR were expressed in Xenopus laevis oocytes. Structure-activity relationships (SARs) were established to identify structural elements essential for efficiency and potency. Efficiency of piperine derivatives was significantly increased by exchanging the piperidine moiety with either N,N-dipropyl, N,N-diisopropyl, N,N-dibutyl, p-methylpiperidine, or N,N-bis(trifluoroethyl) groups. Potency was enhanced by replacing the piperidine moiety by N,N-dibutyl, N,N-diisobutyl, or N,N-bistrifluoroethyl groups. Linker modifications did not substantially enhance the effect on GABAAR. Compound 23 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dipropyl-2,4-pentadienamide] induced the strongest modulation of GABAA (maximal GABA-induced chloride current modulation (IGABA-max = 1673% ± 146%, EC50 = 51.7 ± 9.5 μM), while 25 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dibutyl-2,4-pentadienamide] displayed the highest potency (EC50 = 13.8 ± 1.8 μM, IGABA-max = 760% ± 47%). Compound 23 induced significantly stronger anxiolysis in mice than piperine and thus may serve as a starting point for developing novel GABAAR modulators.

Efficient Modulation of γ-Aminobutyric Acid Type A Receptors by Piperine Derivatives

PubMed Central

2014-01-01

Piperine activates TRPV1 (transient receptor potential vanilloid type 1 receptor) receptors and modulates γ-aminobutyric acid type A receptors (GABAAR). We have synthesized a library of 76 piperine analogues and analyzed their effects on GABAAR by means of a two-microelectrode voltage-clamp technique. GABAAR were expressed in Xenopus laevis oocytes. Structure–activity relationships (SARs) were established to identify structural elements essential for efficiency and potency. Efficiency of piperine derivatives was significantly increased by exchanging the piperidine moiety with either N,N-dipropyl, N,N-diisopropyl, N,N-dibutyl, p-methylpiperidine, or N,N-bis(trifluoroethyl) groups. Potency was enhanced by replacing the piperidine moiety by N,N-dibutyl, N,N-diisobutyl, or N,N-bistrifluoroethyl groups. Linker modifications did not substantially enhance the effect on GABAAR. Compound 23 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dipropyl-2,4-pentadienamide] induced the strongest modulation of GABAA (maximal GABA-induced chloride current modulation (IGABA-max = 1673% ± 146%, EC50 = 51.7 ± 9.5 μM), while 25 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dibutyl-2,4-pentadienamide] displayed the highest potency (EC50 = 13.8 ± 1.8 μM, IGABA-max = 760% ± 47%). Compound 23 induced significantly stronger anxiolysis in mice than piperine and thus may serve as a starting point for developing novel GABAAR modulators. PMID:24905252

Astragaloside IV improves lipid metabolism in obese mice by alleviation of leptin resistance and regulation of thermogenic network

PubMed Central

Wu, Hui; Gao, Yan; Shi, Hai-Lian; Qin, Li-Yue; Huang, Fei; Lan, Yun-Yi; Zhang, Bei-Bei; Hu, Zhi-Bi; Wu, Xiao-Jun

2016-01-01

Obesity is a worldwide threat to public health in modern society, which may result from leptin resistance and disorder of thermogenesis. The present study investigated whether astragaloside IV (ASI) could prevent obesity in high-fat diet (HFD)-fed and db/db mice. In HFD-fed mice, ASI prevented body weight gain, lowered serum triglyceride and total cholesterol levels, mitigated liver lipid accumulation, reduced fat tissues and decreased the enlargement of adipose cells. In metabolic chambers, ASI lessened appetite of the mice, decreased their respiratory exchange ratio and elevated VCO2 and VO2 without altering circadian motor activity. Moreover, ASI modulated thermogenesis associated gene expressions in liver and brawn fat tissues, as well as leptin resistance evidenced by altered expressions of leptin, leptin receptor (ObR) or appetite associated genes. In SH-SY5Y cells, ASI enhanced leptin signaling transduction. However, in db/db mice, ASI did not change body weight gain and appetite associated genes. But it decreased serum triglyceride and total cholesterol levels as well as liver triglyceride. Meanwhile, it significantly modulated gene expressions of PPARα, PGC1-α, UCP2, ACC, SCD1, LPL, AP2, CD36 and SREBP-1c. Collectively, our study suggested that ASI could efficiently improve lipid metabolism in obese mice probably through enhancing leptin sensitivity and modulating thermogenic network. PMID:27444146

Astragaloside IV improves lipid metabolism in obese mice by alleviation of leptin resistance and regulation of thermogenic network.

PubMed

Wu, Hui; Gao, Yan; Shi, Hai-Lian; Qin, Li-Yue; Huang, Fei; Lan, Yun-Yi; Zhang, Bei-Bei; Hu, Zhi-Bi; Wu, Xiao-Jun

2016-07-22

Obesity is a worldwide threat to public health in modern society, which may result from leptin resistance and disorder of thermogenesis. The present study investigated whether astragaloside IV (ASI) could prevent obesity in high-fat diet (HFD)-fed and db/db mice. In HFD-fed mice, ASI prevented body weight gain, lowered serum triglyceride and total cholesterol levels, mitigated liver lipid accumulation, reduced fat tissues and decreased the enlargement of adipose cells. In metabolic chambers, ASI lessened appetite of the mice, decreased their respiratory exchange ratio and elevated VCO2 and VO2 without altering circadian motor activity. Moreover, ASI modulated thermogenesis associated gene expressions in liver and brawn fat tissues, as well as leptin resistance evidenced by altered expressions of leptin, leptin receptor (ObR) or appetite associated genes. In SH-SY5Y cells, ASI enhanced leptin signaling transduction. However, in db/db mice, ASI did not change body weight gain and appetite associated genes. But it decreased serum triglyceride and total cholesterol levels as well as liver triglyceride. Meanwhile, it significantly modulated gene expressions of PPARα, PGC1-α, UCP2, ACC, SCD1, LPL, AP2, CD36 and SREBP-1c. Collectively, our study suggested that ASI could efficiently improve lipid metabolism in obese mice probably through enhancing leptin sensitivity and modulating thermogenic network.

Efficient Generation of Human Embryonic Stem Cell-Derived Cardiac Progenitors Based on Tissue-Specific Enhanced Green Fluorescence Protein Expression

PubMed Central

Szebényi, Kornélia; Péntek, Adrienn; Erdei, Zsuzsa; Várady, György; Orbán, Tamás I.; Sarkadi, Balázs

2015-01-01

Cardiac progenitor cells (CPCs) are committed to the cardiac lineage but retain their proliferative capacity before becoming quiescent mature cardiomyocytes (CMs). In medical therapy and research, the use of human pluripotent stem cell-derived CPCs would have several advantages compared with mature CMs, as the progenitors show better engraftment into existing heart tissues, and provide unique potential for cardiovascular developmental as well as for pharmacological studies. Here, we demonstrate that the CAG promoter-driven enhanced green fluorescence protein (EGFP) reporter system enables the identification and isolation of embryonic stem cell-derived CPCs. Tracing of CPCs during differentiation confirmed up-regulation of surface markers, previously described to identify cardiac precursors and early CMs. Isolated CPCs express cardiac lineage-specific transcripts, still have proliferating capacity, and can be re-aggregated into embryoid body-like structures (CAG-EGFPhigh rEBs). Expression of troponin T and NKX2.5 mRNA is up-regulated in long-term cultured CAG-EGFPhigh rEBs, in which more than 90% of the cells become Troponin I positive mature CMs. Moreover, about one third of the CAG-EGFPhigh rEBs show spontaneous contractions. The method described here provides a powerful tool to generate expandable cultures of pure human CPCs that can be used for exploring early markers of the cardiac lineage, as well as for drug screening or tissue engineering applications. PMID:24734786

Gene therapy for prostate cancer: where are we now?

PubMed

Steiner, M S; Gingrich, J R

2000-10-01

The ability to recombine specifically and alter DNA sequences followed by techniques to transfer these sequences or even whole genes into normal and diseased cells has revolutionized medical research and ushered the clinicians of today into the age of gene therapy. We provide urologists a review of relevant background information, outline current treatment strategies and clinical trials, and delineate current challenges facing the field of gene therapy for advanced prostate cancer. We comprehensively reviewed the literature, including PubMed and recent abstract proceedings from national meetings, relevant to gene therapy and advanced prostate cancer. We selected for review literature representative of the principal scientific background for current gene therapy strategies and National Institutes of Health Recombinant DNA Advisory Committee approved clinical trials. Current prostate cancer gene therapy strategies include correcting aberrant gene expression, exploiting programmed cell death pathways, targeting critical cell biological functions, introducing toxic or cell lytic suicide genes, enhancing the immune system antitumor response and combining treatment with conventional cytotoxic chemotherapy or radiation therapy. Many challenges lie ahead for gene therapy, including improving DNA transfer efficiency to cells locally and at distant sites, enhancing levels of gene expression and overcoming immune responses that limit the time that genes are expressed. Nevertheless, despite these current challenges it is almost certain that gene therapy will be part of the urological armamentarium against prostate cancer in this century.

Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-x(L)

NASA Technical Reports Server (NTRS)

Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy; Chatterjee, A. (Principal Investigator)

2002-01-01

Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in DSB repair in human cells. However, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We demonstrated previously that overexpression of BCL-2 or BCL-x(L) enhanced the frequency of X-ray-induced TK1 mutations, including loss of heterozygosity events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells and to determine whether ectopic expression of BCL-x(L) affects HDR. Using TK6-neo cells, we find that a single DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold, demonstrating efficient DSB repair by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3-4-fold more frequent in TK6 cells that stably overexpress the antiapoptotic protein BCL-X(L). Thus, HDR plays an important role in maintaining genomic integrity in human cells, and ectopic expression of BCL-x(L) enhances HDR of DSBs. This is the first study to highlight a function for BCL-x(L) in modulating DSB repair in human cells.

Enhanced tolerance and remediation to mixed contaminates of PCBs and 2,4-DCP by transgenic alfalfa plants expressing the 2,3-dihydroxybiphenyl-1,2-dioxygenase.

PubMed

Wang, Yan; Ren, Hejun; Pan, Hongyu; Liu, Jinliang; Zhang, Lanying

2015-04-09

Polychlorinated biphenyls (PCBs) and 2,4-dichlorophenol (2,4-DCP) generally led to mixed contamination of soils as a result of commercial and agricultural activities. Their accumulation in the environment poses great risks to human and animal health. Therefore, the effective strategies for disposal of these pollutants are urgently needed. In this study, genetic engineering to enhance PCBs/2,4-DCP phytoremediation is a focus. We cloned the 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC.B) from a soil metagenomic library, which is the key enzyme of aerobic catabolism of a variety of aromatic compounds, and then it was expressed in alfalfa driven by CaMV 35S promoter using Agrobacterium-mediated transformation. Transgenic line BB11 was selected out through PCR, Western blot analysis and enzyme activity assays. Its disposal and tolerance to both PCBs and 2,4-DCP were examined. The tolerance capability of transgenic line BB11 towards complex contaminants of PCBs/2,4-DCP significantly increased compared with non-transgenic plants. Strong dissipation of PCBs and high removal efficiency of 2,4-DCP were exhibited in a short time. It was confirmed expressing BphC.B would be a feasible strategy to help achieving phytoremediation in mixed contaminated soils with PCBs and 2,4-DCP. Copyright © 2014 Elsevier B.V. All rights reserved.

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

PubMed Central

WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

2013-01-01

Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

Physiological and molecular alterations in plants exposed to high [CO2] under phosphorus stress.

PubMed

Pandey, Renu; Zinta, Gaurav; AbdElgawad, Hamada; Ahmad, Altaf; Jain, Vanita; Janssens, Ivan A

2015-01-01

Atmospheric [CO2] has increased substantially in recent decades and will continue to do so, whereas the availability of phosphorus (P) is limited and unlikely to increase in the future. P is a non-renewable resource, and it is essential to every form of life. P is a key plant nutrient controlling the responsiveness of photosynthesis to [CO2]. Increases in [CO2] typically results in increased biomass through stimulation of net photosynthesis, and hence enhance the demand for P uptake. However, most soils contain low concentrations of available P. Therefore, low P is one of the major growth-limiting factors for plants in many agricultural and natural ecosystems. The adaptive responses of plants to [CO2] and P availability encompass alterations at morphological, physiological, biochemical and molecular levels. In general low P reduces growth, whereas high [CO2] enhances it particularly in C3 plants. Photosynthetic capacity is often enhanced under high [CO2] with sufficient P supply through modulation of enzyme activities involved in carbon fixation such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). However, high [CO2] with low P availability results in enhanced dry matter partitioning towards roots. Alterations in below-ground processes including root morphology, exudation and mycorrhizal association are influenced by [CO2] and P availability. Under high P availability, elevated [CO2] improves the uptake of P from soil. In contrast, under low P availability, high [CO2] mainly improves the efficiency with which plants produce biomass per unit P. At molecular level, the spatio-temporal regulation of genes involved in plant adaptation to low P and high [CO2] has been studied individually in various plant species. Genome-wide expression profiling of high [CO2] grown plants revealed hormonal regulation of biomass accumulation through complex transcriptional networks. Similarly, differential transcriptional regulatory networks are involved in P-limitation responses in plants. Analysis of expression patterns of some typical P-limitation induced genes under high [CO2] suggests that long-term exposure of plants to high [CO2] would have a tendency to stimulate similar transcriptional responses as observed under P-limitation. However, studies on the combined effect of high [CO2] and low P on gene expression are scarce. Such studies would provide insights into the development of P efficient crops in the context of anticipated increases in atmospheric [CO2]. Copyright © 2015 Elsevier Inc. All rights reserved.

Large-scale expansion of Wharton's jelly-derived mesenchymal stem cells on gelatin microbeads, with retention of self-renewal and multipotency characteristics and the capacity for enhancing skin wound healing.

PubMed

Zhao, Guifang; Liu, Feilin; Lan, Shaowei; Li, Pengdong; Wang, Li; Kou, Junna; Qi, Xiaojuan; Fan, Ruirui; Hao, Deshun; Wu, Chunling; Bai, Tingting; Li, Yulin; Liu, Jin Yu

2015-03-19

Successful stem cell therapy relies on large-scale generation of stem cells and their maintenance in a proliferative multipotent state. This study aimed to establish a three-dimension culture system for large-scale generation of hWJ-MSC and investigated the self-renewal activity, genomic stability and multi-lineage differentiation potential of such hWJ-MSC in enhancing skin wound healing. hWJ-MSC were seeded on gelatin microbeads and cultured in spinning bottles (3D). Cell proliferation, karyotype analysis, surface marker expression, multipotent differentiation (adipogenic, chondrogenic, and osteogenic potentials), and expression of core transcription factors (OCT4, SOX2, NANOG, and C-MYC), as well as their efficacy in accelerating skin wound healing, were investigated and compared with those of hWJ-MSC derived from plate cultres (2D), using in vivo and in vitro experiments. hWJ-MSC attached to and proliferated on gelatin microbeads in 3D cultures reaching a maximum of 1.1-1.30×10(7) cells on 0.5 g of microbeads by days 8-14; in contrast, hWJ-MSC derived from 2D cultures reached a maximum of 6.5 -11.5×10(5) cells per well in a 24-well plate by days 6-10. hWJ-MSC derived by 3D culture incorporated significantly more EdU (P<0.05) and had a significantly higher proliferation index (P<0.05) than those derived from 2D culture. Immunofluorescence staining, real-time PCR, flow cytometry analysis, and multipotency assays showed that hWJ-MSC derived from 3D culture retained MSC surface markers and multipotency potential similar to 2D culture-derived cells. 3D culture-derived hWJ-MSC also retained the expression of core transcription factors at levels comparable to their 2D culture counterparts. Direct injection of hWJ-MSC derived from 3D or 2D cultures into animals exhibited similar efficacy in enhancing skin wound healing. Thus, hWJ-MSC can be expanded markedly in gelatin microbeads, while retaining MSC surface marker expression, multipotent differential potential, and expression of core transcription factors. These cells also efficiently enhanced skin wound healing in vivo, in a manner comparable to that of hWJ-MSC obtained from 2D culture.

Constitutive expression of a plant ferredoxin-like protein (pflp) enhances capacity of photosynthetic carbon assimilation in rice (Oryza sativa).

PubMed

Chang, Hsiang; Huang, Hsiang-En; Cheng, Chin-Fu; Ho, Mei-Hsuan; Ger, Mang-Jye

2017-04-01

The plant ferredoxin-like protein (PFLP) gene, cloned from sweet peppers predicted as an electron carrier in photosynthesis, shows high homology to the Fd-I sequence of Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Spinacia oleracea. Most of pflp related studies focused on anti-pathogenic effects, while less understanding for the effects in photosynthesis with physiological aspects, such as photosynthesis rate, and levels of carbohydrate metabolites. This project focuses on the effects of pflp overexpression on photosynthesis by physiological evaluations of carbon assimilation with significant higher levels of carbohydrates with higher photosynthesis efficiency. In this report, two independent transgenic lines of rice plants (designated as pflp-1 and pflp-2) were generated from non-transgenic TNG67 rice plant (WT). Both transgenic pflp rice plants exhibited enhanced photosynthesis efficiency, and gas exchange rates of photosynthesis were 1.3- and 1.2-fold higher for pflp-1 and pflp-2 than WT respectively. Significantly higher electron transport rates of pflp rice plants were observed. Moreover, photosynthetic products, such as fructose, glucose, sucrose and starch contents of pflp transgenic lines were increased accordingly. Molecular evidences of carbohydrate metabolism related genes activities (osHXK5, osHXK6, osAGPL3, osAGPS2α, osSPS, ospFBPase, oscFBPase, and osSBPase) in transgenic lines were higher than those of WT. For performance of crop production, 1000-grain weight for pflp-1 and pflp-2 rice plants were 52.9 and 41.1 g that were both significantly higher than 31.6 g for WT, and panicles weights were 1.4- and 1.2-fold higher than WT. Panicle number, tiller number per plants for pflp rice plants were all significantly higher compared with those of WT where there was no significant difference observed between two pflp rice plants. Taken altogether; this study demonstrated that constitutive pflp expression can improve rice production by enhancing the capacity of photosynthetic carbon assimilation.

Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts

PubMed Central

Weiskirchen, Ralf; Kneifel, Jens; Weiskirchen, Sabine; van de Leur, Eddy; Kunz, Dagmar; Gressner, Axel M

2000-01-01

Background The hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer. Results With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE™6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively. Conclusions Our results indicate that FuGENE™6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells. PMID:11178102

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

PubMed

Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

2017-01-01

There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide

Highlights: Black-Right-Pointing-Pointer We use MEL-A-containing cationic liposomes for siRNA delivery. Black-Right-Pointing-Pointer MEL-A-containing cationic liposomes can efficiently and rapidly deliver siRNA into the cytoplasm. Black-Right-Pointing-Pointer Rapid delivery of siRNA is due to the membrane fusion between liposomes and plasma membrane. -- Abstract: The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easiermore » to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine Trade-Mark-Sign RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.« less

Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha

PubMed Central

2012-01-01

Background Currently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA) and streptokinase (SK). Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK), or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study’s goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system. Results The development of an efficient large scale (80 L) Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.). Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth) of biologically active rSAK-2 of expected molecular weight was achieved. Conclusion The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent. PMID:23253823

Poly (dopamine) coated superparamagnetic iron oxide nanocluster for noninvasive labeling, tracking, and targeted delivery of adipose tissue-derived stem cells

PubMed Central

Liao, Naishun; Wu, Ming; Pan, Fan; Lin, Jiumao; Li, Zuanfang; Zhang, Da; Wang, Yingchao; Zheng, Youshi; Peng, Jun; Liu, Xiaolong; Liu, Jingfeng

2016-01-01

Tracking and monitoring of cells in vivo after transplantation can provide crucial information for stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be an effective and non-invasive technique for cell tracking in living bodies. However, commercial superparamagnetic iron oxide nanoparticles (SPIONs) applied to label cells suffer from shortages such as potential toxicity, low labeling efficiency, and low contrast enhancing. Herein, the adipose tissue-derived stem cells (ADSCs) were efficiently labeled with SPIONs coated with poly (dopamine) (SPIONs cluster@PDA), without affecting their viability, proliferation, apoptosis, surface marker expression, as well as their self-renew ability and multi-differentiation potential. The labeled cells transplanted into the mice through tail intravenous injection exhibited a negative enhancement of the MRI signal in the damaged liver-induced by carbon tetrachloride, and subsequently these homed ADSCs with SPIONs cluster@PDA labeling exhibited excellent repair effects to the damaged liver. Moreover, the enhanced target-homing to tissue of interest and repair effects of SPIONs cluster@PDA-labeled ADSCs could be achieved by use of external magnetic field in the excisional skin wound mice model. Therefore, we provide a facile, safe, noninvasive and sensitive method for external magnetic field targeted delivery and MRI based tracking of transplanted cells in vivo. PMID:26728448

Defined Host-Guest Chemistry on Nanocarbon for Sustained Inhibition of Cancer.

PubMed

Ostadhossein, Fatemeh; Misra, Santosh K; Mukherjee, Prabuddha; Ostadhossein, Alireza; Daza, Enrique; Tiwari, Saumya; Mittal, Shachi; Gryka, Mark C; Bhargava, Rohit; Pan, Dipanjan

2016-08-22

Signal transducer and activator of transcription factor 3 (STAT-3) is known to be overexpressed in cancer stem cells. Poor solubility and variable drug absorption are linked to low bioavailability and decreased efficacy. Many of the drugs regulating STAT-3 expression lack aqueous solubility; hence hindering efficient bioavailability. A theranostics nanoplatform based on luminescent carbon particles decorated with cucurbit[6]uril is introduced for enhancing the solubility of niclosamide, a STAT-3 inhibitor. The host-guest chemistry between cucurbit[6]uril and niclosamide makes the delivery of the hydrophobic drug feasible while carbon nanoparticles enhance cellular internalization. Extensive physicochemical characterizations confirm successful synthesis. Subsequently, the host-guest chemistry of niclosamide and cucurbit[6]uril is studied experimentally and computationally. In vitro assessments in human breast cancer cells indicate approximately twofold enhancement in IC 50 of drug. Fourier transform infrared and fluorescence imaging demonstrate efficient cellular internalization. Furthermore, the catalytic biodegradation of the nanoplatforms occur upon exposure to human myeloperoxidase in short time. In vivo studies on athymic mice with MCF-7 xenograft indicate the size of tumor in the treatment group is half of the controls after 40 d. Immunohistochemistry corroborates the downregulation of STAT-3 phosphorylation. Overall, the host-guest chemistry on nanocarbon acts as a novel arsenal for STAT-3 inhibition. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Osteopontin-deficient progenitor cells display enhanced differentiation to adipocytes.

PubMed

Moreno-Viedma, Veronica; Tardelli, Matteo; Zeyda, Maximilian; Sibilia, Maria; Burks, J Deborah; Stulnig, Thomas M

2018-03-06

Osteopontin (OPN, Spp1) is a protein upregulated in white adipose tissue (WAT) of obese subjects. Deletion of OPN protects mice from high-fat diet-induced WAT inflammation and insulin resistance. However, the alterations mediated by loss of OPN in WAT before the obesogenic challenge have not yet been investigated. Therefore, we hypothesised that the lack of OPN might enhance the pro-adipogenic micro environment before obesity driven inflammation. OPN deficiency was tested in visceral (V) and subcutaneous (SC) WAT from WT and Spp1 -/- female mice. Gene expression for hypoxia, inflammation and adipogenesis was checked in WT vs. Spp1 -/- mice (n=15). Adipocytes progenitor cells (APC) were isolated by fluorescence cell sorting and role of OPN deficiency in adipogenesis was investigated by cell images and RT-PCR. We show that Spp1 -/- maintained normal body and fat-pad weights, although hypoxia and inflammation markers were significantly reduced. In contrast, expression of genes involved in adipogenesis was increased in WAT from Spp1 -/- mice. Strikingly, APC from Spp1 -/- were diminished but differentiated more efficiently to adipocytes than those from control mice. APC from SC-WAT of lean OPN-deficient mice display an enhanced capacity for differentiating to adipocytes. These alterations may explain the healthy expansion of WAT in the OPN-deficient model which is associated with reduced inflammation and insulin resistance. Copyright © 2018. Published by Elsevier Ltd.

Enhanced generation of perfluoroalkyl carboxylic acids (PFCAs) from fluorotelomer alcohols (FTOHs) via ammonia-oxidation process.

PubMed

Yu, Xiaolong; Nishimura, Fumitake; Hidaka, Taira

2018-05-01

With the phase-out of persistent, bioaccumalative, and toxic perfluoroalkyl carboxylic acids (PFCAs), it is needed to explore the potential release of PFCAs from precursors being emitted into the environment. Biotransformation of fluorotelomer alcohols (FTOHs) via biological processes in wastewater treatment plants (WWTPs) leads to discharge of PFCAs into receiving waters. However, the commonly existed microbial activity that can impact on FTOHs biodegradation in WWTPs remains unclear. The objective of present research was to explore the relationship between ammonia-oxidation process and the enhanced PFCAs generation from FTOHs biodegradation under aerobic activated sludge. The obtained results indicate that the cometabolism process performed by nitrifying microorganisms (NMs) was responsible for enhanced PFCAs generation. Among NMs, the ammonia-oxidation bacteria that can express non-specific enzyme of ammonia monooxygenases resulted in the enhanced PFCAs generation from FTOHs. Meanwhile, the different addition amount of ammonia contributed to different defluorination efficiency of FTOHs. The present study further correlated the enhanced PFCAs generation from FTOHs biodegradation with ammonia-oxidation process, which can provide practical information on effective management of PFCAs generation in WWTPs. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enhancement of caffeic acid phenethyl ester on all-trans retinoic acid-induced differentiation in human leukemia HL-60 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuo, H.-C.; Kuo, W.-H.; Lee, Y.-J.

2006-10-01

All-trans retinoic acid (ATRA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL); however, the response is sometimes very slow. Furthermore, relapse and resistance to treatment often occur despite continued treatment with ATRA. Thereafter, combination treatment strategies have been suggested to circumvent these problems. The present study demonstrates that caffeic acid phenethyl ester (CAPE), a major component of honeybee propolis, enhanced ATRA-induced granulocytic differentiation in HL-60, a human promyelocytic cell line. The differentiation was assessed by Wright-Giemsa stain, nitroblue tetrazolium reduction, and membrane differentiation marker CD11b. In addition, CAPE enhanced ATRA-induced cell cycle arrest atmore » the G1 phase by decreasing the association of cdk2-cyclin E complex. Finally, it was demonstrated that CAPE promoted the ATRA-mediated nuclear transcription activation of RAR{alpha} assessed by EMSA assay and enhanced the expression of target genes including RAR{alpha}, C/EBP{epsilon}, and p21 protein resulting in the differentiation development of leukemia. It is suggested that CAPE possesses the potential to enhance the efficiency of ATRA in the differentiation therapy of APL.« less

Exploring advantages/disadvantages and improvements in overcoming gene delivery barriers of amino acid modified trimethylated chitosan.

PubMed

Zheng, Hao; Tang, Cui; Yin, Chunhua

2015-06-01

Present study aimed at exploring advantages/disadvantages of amino acid modified trimethylated chitosan in conquering multiple gene delivery obstacles and thus providing comprehensive understandings for improved transfection efficiency. Arginine, cysteine, and histidine modified trimethyl chitosan were synthesized and employed to self-assemble with plasmid DNA (pDNA) to form nanocomplexes, namely TRNC, TCNC, and THNC, respectively. They were assessed by structural stability, cellular uptake, endosomal escape, release behavior, nuclear localization, and in vitro and in vivo transfection efficiencies. Besides, sodium tripolyphosphate (TPP) was added into TRNC to compromise certain disadvantageous attributes for pDNA delivery. Optimal endosomal escape ability failed to bring in satisfactory transfection efficiency of THNC due to drawbacks in structural stability, cellular uptake, pDNA liberation, and nuclear distribution. TCNC evoked the most potent gene expression owing to multiple advantages including sufficient stability, preferable uptake, efficient pDNA release, and high nucleic accumulation. Undesirable stability and insufficient pDNA release adversely affected TRNC-mediated gene transfer. However, incorporation of TPP could improve such disadvantages and consequently resulted in enhanced transfection efficiencies. Coordination of multiple contributing effects to conquer all delivery obstacles was necessitated for improved transfection efficiency, which would provide insights into rational design of gene delivery vehicles.

HCV T Cell Receptor Chain Modifications to Enhance Expression, Pairing, and Antigen Recognition in T Cells for Adoptive Transfer.

PubMed

Foley, Kendra C; Spear, Timothy T; Murray, David C; Nagato, Kaoru; Garrett-Mayer, Elizabeth; Nishimura, Michael I

2017-06-16

T cell receptor (TCR)-gene-modified T cells for adoptive cell transfer can mediate objective clinical responses in melanoma and other malignancies. When introducing a second TCR, mispairing between the endogenous and introduced α and β TCR chains limits expression of the introduced TCR, which can result in impaired efficacy or off-target reactivity and autoimmunity. One approach to promote proper TCR chain pairing involves modifications of the introduced TCR genes: introducing a disulfide bridge, substituting murine for human constant regions, codon optimization, TCR chain leucine zipper fusions, and a single-chain TCR. We have introduced these modifications into our hepatitis C virus (HCV) reactive TCR and utilize a marker gene, CD34t, which allows us to directly compare transduction efficiency with TCR expression and T cell function. Our results reveal that of the TCRs tested, T cells expressing the murine Cβ2 TCR or leucine zipper TCR have the highest levels of expression and the highest percentage of lytic and interferon-γ (IFN-γ)-producing T cells. Our studies give us a better understanding of how TCR modifications impact TCR expression and T cell function that may allow for optimization of TCR-modified T cells for adoptive cell transfer to treat patients with malignancies.

Differentially Expressed Genes Associated with Low-Dose Gamma Radiation

NASA Astrophysics Data System (ADS)

Hegyesi, Hargita; Sándor, Nikolett; Schilling, Boglárka; Kis, Enikő; Lumniczky, Katalin; Sáfrány, Géza

We have studied low dose radiation induced gene expression alterations in a primary human fibroblast cell line using Agilent's whole human genome microarray. Cells were irradiated with 60Co γ-rays (0; 0.1; 0.5 Gy) and 2 hours later total cellular RNA was isolated. We observed differential regulation of approximately 300-500 genes represented on the microarray. Of these, 126 were differentially expressed at both doses, among them significant elevation of GDF-15 and KITLG was confirmed by qRT-PCR. Based on the transcriptional studies we selected GDF-15 to assess its role in radiation response, since GDF-15 is one of the p53 gene targets and is believed to participate in mediating p53 activities. First we confirmed gamma-radiation induced dose-dependent changes in GDF-15 expression by qRT-PCR. Next we determined the effect of GDF-15 silencing on radiosensitivity. Four GDF-15 targeting shRNA expressing lentiviral vectors were transfected into immortalized human fibroblast cells. We obtained efficient GDF-15 silencing in one of the four constructs. RNA interference inhibited GDF-15 gene expression and enhanced the radiosensitivity of the cells. Our studies proved that GDF-15 plays an essential role in radiation response and may serve as a promising target in radiation therapy.

[Knockdown of NEDD9 inhibits the proliferation, invasion and migration of esophageal carcinoma EC109 cells].

PubMed

Zhang, Wen; Li, Shaojun; Zhao, Yunlong; Guo, Nannan; Li, Yingjie

2016-12-01

Objective To observe the expression of the neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) in esophageal cancer, to investigate the impact of decreased expression of NEDD9 on invasion and migration, and to explicit the function of NEDD9 in EC109 human esophageal cancer cell line. Methods Immunohistochemical staining was used to detect the expression of NEDD9 in human esophageal cancer tissues and paracancerous normal tissues. RNA interfering (RNAi) was used to knockdown NEDD9 in EC109 cells. The interference efficiency was detected by reverse transcription PCR (RT-PCR) and Western blot analysis. Cell proliferation was determined by MTT assay and the invasion and migration abilities of EC109 cells were monitored by Transwell TM assay. The protein levels of proliferating cell nuclear antigen (PCNA), Bax and Bcl-2 were tested by Western blotting. Results The positive expression rate of NEDD9 in esophageal carcinoma tissues was significantly higher compared with that in the paracancerous tissues. After NEDD9 expression was successfully downregulated in EC109 cells by siRNA, the proliferation, invasion and migration rates in transfection group were significantly lower than those in control group; meanwhile, the expression of Bcl-2 was reduced and Bax expression was enhanced. Conclusion The protein expression level of NEDD9 is higher in esophageal carcinoma tissues than that in adjacent normal tissues. Knockdown of NEDD9 expression can restrain the proliferation, invasion and migration of EC109 cells.

Cytotoxic T lymphocyte antigen 4 decreases humoral and cellular immunity by adenovirus to enhance target GFP gene transfer in C57BL/6 mice.

PubMed

Bai, Dou; Zhu, Wei; Zhang, Yu; Long, Ling; Zhu, Naishuo

2015-01-01

Adenoviruses (Ad) are once potential and promising vectors for gene delivery, but the immunogenicity attenuates its transfer efficiency. Cytotoxic T lymphocyte antigen 4 (CTLA-4) can inhibit T cell immunity. Thus, we aimed to study the effect of CTLA-4 in the process of Ad-mediated gene transfer. The C57BL/6 mice were injected by Ad vectors at twice, and CTLA-4 was administrated after the first Ad injection. Then, the CD3(+)CD4(+) T cells and circulating levels of IL-2, IL-4, and anti-Ad IgG were decreased by CTLA-4, while Ad generated immune responses. The green fluorescence protein (GFP) expressions of tissues were enhanced by CTLA-4 till injection of Ad at twice. Our results indicate that CTLA-4 can inhibit humoral and cellular immunity by adenovirus generation to enhance GFP delivery, and provide a potential way to assist in Ad-mediated gene transfer.

Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity

PubMed Central

Kanayama, Masashi; Inoue, Makoto; Danzaki, Keiko; Hammer, Gianna; He, You-Wen; Shinohara, Mari L.

2014-01-01

Immune responses must be well restrained in a steady state to avoid excessive inflammation. However, such restraints are quickly removed to exert anti-microbial responses. Here, we report a role of autophagy in an early host anti-fungal response by enhancing NFκB activity through A20 sequestration. Enhancement of NFκB activation is achieved by autophagic depletion of A20, an NFκB inhibitor, in F4/80hi macrophages in the spleen, peritoneum, and kidney. We show that p62, an autophagic adaptor protein, captures A20 to sequester it in the autophagosome. This allows the macrophages to release chemokines to recruit neutrophils. Indeed, mice lacking autophagy in myeloid cells show higher susceptibility to Candida albicans infection due to impairment in neutrophil recruitment. Thus, at least in the specific aforementioned tissues, autophagy appears to break A20-dependent suppression in F4/80hi macrophages, which express abundant A20 and contribute to the initiation of efficient innate immune responses. PMID:25609235