SIMULTANEOUS OVER-EXPRESSION OF INSULIN-LIKE GROWTH FACTOR- Ⅱ (IGF- Ⅱ ) AND IGF- Ⅱ RECEPTOR(IGF- Ⅱ R) GENES IN HUMAN PRIMARY CANCER-IMPLICATION OF AUTOCRINE AND PARACRINE MECHANISM IN AUTONOMOUS GROWTH OF HEPATIC CANCER

This is first report about the simultaneous over-expression of both Insulin-like growth factor (IGF- I ) and its receptor (IGF- I R) at mRNA level in human primary hepatic Cancer (PHC). In 10 PHC samples from China, IGF-I and IGF- I R were both over-expressed, whereas only a background signal was detected in normal liver. In 5 pairs of PHC and its non- tumorous adjacent liver tissues from South Africa, IGF- I and IGF- I R were also over-expressed in PHC. mRNA expression of IGF- I in all 5 cases and IGF- I R in 4 of 5 cases were higher in cancer than non- tumorous adjacent liver tissues. These results strongly implicate that an autocrine and/ or paracrine mechanism might be Involved in formation and progression of PHC.

Protocol for Artificial MicroRNA Mediated Over-Expression of miR820 in Indica Rice

In the present study, we illustrate the strategy and protocol required to generate rice transgenics over-expressing the 21-nt form of Osa-miR820. The miR exists in two size variants of 21-nt and 24-nt so the natural precursor cannot be employed for the purpose of miR over-expression as the cellular machinery can process both size variants thereby masking the role of PTGS regulation. Hence, we adopted the artificial miR technology to specifically over-express the 21-nt species in the transgenics. During the course of experiments it was observed that the amiR constructs probably interfered with the regeneration of the transformed callus, necessitating protocol modifications. The results indicate the successful over-expression of the 21-nt miR species. These plants can serve as a useful source for the functional dissection of the role played by the 21-nt Osa-miR820 species. They will also be valuable in highlighting the importance for the existence of a dual mode of miR mediated target regulation.

Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector

Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to construct a lentiviral VEGF165 expression vector and then infect the ADSCs to produce therapeutic seed cells.Methods EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF165 transcript （NM_001025368）. Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T cells.And then the ADSCs （multiplicity of infection=20） were transfected with the vectors after titer determination. Stable expression of VEGF165 in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis.Results DNA sequencing and 293T transfection verified VEGF165 was linked to the GFP fused vector. The virus titer is up to 2x10a determined by quantitative PCR. VEGF165 transduced cells could show green fluorescence confirmed by immunofluorescence staining （almost 95%）. ELISA analyses could detect out the density of VEGF was 850.86-1202.13pg/ml （mean （923.00±31.22） pg/ml） in the supernatant of VEGF16s-transduced cells but not detected in the GFP-transduced cells （P 〈0.001） and the Western blotting analyses also confirmed VEGF165 expression in VEGF165-transduced cells.Conclusions The VEGF165 over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.

Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis

The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway,the HMGR gene from Glycyrrhiza uralensis Fisch.(GuHMGR)was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC.The results showed that all the recombinant P.pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains.However,as the copy number increased,the content of ergosterol showed an increasing–decreasing–increasing pattern.This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G.uralensis.

Over-expression of CD163, CD169, and CD151 is not sufficient to improve the susceptibility to porcine reproductive and respiratory syndrome virus infection in transgenic mice

Porcine reproductive and respiratory syndrome virus(PRRSV)is a major pathogen that causes reproductive failure and respiratory disease in pigs,resulting in devastating economic losses worldwide[1].Porcine alveolar macrophages(PAMs)are the primary target cells of PRRSV[2],and the putative receptors,including CD163,CD169,and CD151,play key roles during infection[3–6].However,the understanding of PRRSV infection and pathogenesis is

Improving the accumulation of 18α-and 18β-glycyrrhizins by over-expressing GuHMGR, GuSQS1, and GuBAS genes in Glycyrrhiza uralensis

Objective:To study the influence of over-expression of three functional genes involved in GC biosynthetic pathway,GuHMGR,GuSQS1,and GuBAS on GC production.Methods:Three plant expression vectors were constructed and transformed into Agrobacterium tumefaciens EHA105,which were used to infect Glycyrrhiza uralensis hypocotyls explants.After induction,selection,differentiation,culture,and transplantation,12,15,and 5 regenerated plants over-expressing GuHMGR,GuSQS1,and GuBAS,were obtained,respectively.Results:RT-PCR analysis showed these transgenic regenerated G.uralensis plants had 2-6 copies of GuHMGR,GuSQS1,or GuBAS.HPLC analysis showed the contents of 18α-and 18β-GC in all transgenic regenerated samples were both higher than that in the blank control.With the increase of copy numbers of GuHMGR,GuSQS1,and GuBAS,the contents of 18α-and 18β-GC were both increased in most samples.The highest 18α-and 18β-GC contents in transgenic regenerated plants were about 3.05 times and 2.80 times higher than that in the blank control,respectively.Conclusion:Over-expression of the GuHMGR,GuSQS1,and GuBAS genes enhance the accumulation of 18α-and 18β-GC in the roots and rhizomes of G.uralensis.We hope this work can lay a foundation for the molecular breeding research of G.uralensis and improving the quality of the roots and rhizomes of G.uralensis cultivars.

Characterization of NPR1 Genes from Norton and Cabernet Sauvignon Grapevine

Non-expressor of pathogenesis-related genes 1 （NPR1） plays a significant role in the defense responses of plants to pathogens by regulating the expression of defense-related genes. In the present study, we isolated two NPR1 genes from Vitis aestivalis cv. Norton and Vitis vinifera cv. Cabernet Sauvignon, which were referred to as VaNPR1.1 and VvNPR1. 1-CS, respectively. They encode a protein of 584 amino acids with a predicted molecular weight of 64.8 kDa and a theoretical isoelectric point （pI） of 5.74. The predicted amino acid sequences of VaNPR1.1 and VvNPR1.1-CS differ by only one amino acid. Over-expression of VaNPR1.1 gene in Arabidopsis npr1-1 mutant plants restores the transcriptional expression of AtPR-1 gene, though not to the full scale. This result demonstrated that a grapevine VaNPR1.1 possesses a similar function to the Arabidopsis NPR1 in the regulation of defense-related genes. Over-expression of VaNPR1.1 in transgenic Arabidopsis plant increased tolerance to salinity, but had no effect on the drought tolerance. We conclude that VaNPR1.1 is a functional ortholog of AtNPR1 and also involved in grapevine＇s response to the salt stress.

Lentivirus-mediated Persephin overexpression in Parkinson's disease rats

Persephin, together with glial cell line-derived neurotrophic factor and neurturin, has a neurotrophic effect and promotes the survival of motor neurons cultured in vitro. In this study, dopaminergic neurons in the substantia nigra of rats were transfected with the Persephin gene. One week later 6-hydroxydopamine was injected into the anterior medial bundle to establish a Parkinson＇s disease model in the rats. Results found that the number of dopaminergic neurons in the substantia nigra increased, tyrosine hydroxylase expression was upregulated and concentrations of dopamine and its metabolites in corpus striatum were increased after pretreatment with Persephin gene. In addition, the rotating effect of the induced Parkinson＇s disease rats was much less in the group pretreated with the Persephin gene. Persephin has a neuroprotective effect on the 6-hydroxydopamine-induced Parkinson＇s disease through protecting dopaminergic neurons.

Bzw2 Promotes Proliferation and Lactation of Mammary Epithelial Cell in Dairy Goat

Mitosis of mammary epithelial cell is foundation of mammal lactation. We developed a strategy of combined application of generation of longer cDNA fragments from the serial analysis of gene expression （SAGE） tags for gene identification （GLGI） to screen and identify genes influencing lactating ability of mammary epithelial cell in dairy goat. GLGI as a new tag identification technique was brought about with SAGE. Bzw2 was found as a candidate gene related to lactation by screening Long-SAGE library of mammary gland in dairy goat. Bzw2 cDNA was synthesized by switching mechanism at 5＂-end of RNA transcript （SMART） technology. The mRNA level of Bzw2 was relatively higher in early lactation than in other development stages of mammary gland. The proliferation of mammary epithelial cell was inhibited by transfecting specific shRNA of Bzw2. The mRNA levels of Stat5, Csn2 and Prlr were also down-regulated, suggesting the lactating ability of mammary epithelial cell was attenuated after Bzw2 RNAi. The reduction of mammary epithelial cell growth and lactation by Bzw2 RNAi was rescued through over-expression of Bzw2. These results revealed that Bzw2 might play an important role in lactation though the molecular mechanism was still unclear.

An engineered <i>Phlebia radiata</i>manganese peroxidase: expression, refolding, purification and preliminary characterization

Manganese peroxidases (MnPs) are interesting enzymes in protein engineering, aimed at maximizing industrial bioprocesses such as lignin degradation and biofuel production. cDNA of the secreted short-type of MnP from Phlebia radiata (Pr-MnP3) has been successfully engineered and amplified by polymerase chain reaction (PCR). Five mutant genes (E40H, E44H, E40H/E44H, D186H and D186N) of recombinant Phlebia radiata MnP3 (rPr-MnP3) were generated. The wild-type and the mutant genes were expressed in Escherichia coli (W3110 strain) and the resultant body proteins were lysed, purified and refolded into active enzymes. 6% - 7% recovery of pure and fully active rPr-MnP3 for wild-type and mutants were obtained and the availability of rPr-MnP3 enzymes will greatly facilitate its structure-function relationships studies. rPr-MnP3 mass was characterised using SDS-PAGE and MALDI-TOF mass spectrometry. Molecular weight of both the wild-type and mutant rPr-MnP3 enzymes was approximately 36 kDa. This describes the spectral characterization of the wild-type and mutant rPr-MnP3 enzymes with are very close similarities;substantially high spin haem enzymes. Therefore we report the engineering, cloning, expression, refolding/activation of MnP3 genes and preliminary characterization of the wild-type and mutant Phlebia radiata MnP3 enzymes.

Overexpression of aspen sucrose synthase gene promotes growth and development of transgenic Arabidopsis plants

In plants, sucrose synthase (SUS) enzymes catalyze conversion of sucrose into fructose and UDP-glucose in the presence of UDP. To investigate the impact of overexpression of heterologous SUS on the growth and development of Arabidopsis, we transformed Arabidopsis plants with an overexpression vector containing an aspen SUS gene (PtrSUS1). The genomic PCR confirmed the successful integration of PtrSUS1 transgene in the Arabidopsis genome. PtrSUS1 expression in transgenic Arabidopsis plants was confirmed by RT-PCR. The SUS activity was dramatically increased in all transgenic lines examined. The three selected transgenic PtrSUS1 lines exhibited faster growth and flowered about 10 days earlier compared to untransformed controls, and also possessed 133%, 139%, and 143% SUS activity compared to controls. Both fresh weights and dry biomass yields of the whole plants from these three selected transgenic lines were significantly increased to 125% of the controls. Transgenic PtrSUS1 lines also had a higher tolerance to higher concentration of sucrose which was reflective of the increased SUS activity in transgenic versus wild-type plants. The growth differences between wild-type and transgenic plants, either in root and hypocotyl length or in fresh and dry weight of whole plant, became more pronounced on the media containing higher sucrose concentrations. Taken together, these results showed that the early flowering, faster growth and increased tolerance to higher sucrose in transgenic lines were caused by the genome integration and constitutive expression of the aspen PtrSUS1 gene in transgenic Arabidopsis.

Overexpression of the PdpapERF109 gene enhances resistance of Populus davidiana×P.alba var.pyramidalis to Fusarium oxysporum infection

The key transcription factor gene PdP apE RF109 was cloned from Populus davidiana×P.alba var.pyramidalis(Pdpap),and after overexpression of P dP ap ERF109 in transformants,the gene functions in the resistance response to Fusarium oxysporum infection.Compared with the wild Pdpap,after inoculation with F.oxysporum,the physiological and biochemical characteristics,including relative fresh weight,peroxidase activity,and the percentage of electrolyte leakage showed that,after overexpression of the PdPapERF109 gene,the transformants grew well and displayed significant resistance to F.oxysporum infection.By comparing the reactive oxygen species scavenging capacity of Pdpap plants after pathogen infection,the P dPapERF109-overexpressing plants had significantly better reactive oxygen species scavenging ability than the wild plants.Comprehensive analysis of plant morphology and various physiological and biochemical parameters showed that the overexpression of the P dpapERF109 gene significantly improved the resistance of Pdpap plants to F.oxysporum root rot.Therefore,increasing the expression of the homologous ERF109 gene can be an effective strategy to increase disease resistance in hybrid poplars.

Over-expressing root-specific β-amyrin synthase gene increases glycyrrhizic acid content in hairy roots of glycyrrhiza uralensis

Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene(GuBAS).Methods: Tobacco root-specific promoter TobRB7 and Gu BAS c DNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G.uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC.Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of Gu BAS in these transgenic hairy roots was intended by q RT-PCR to be 3, 7,and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots.Conclusion: Over-expressing Gu BAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.

Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii

Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.

Enhancing ergosterol production in Pichia pastoris GS115 by overexpressing squalene synthase gene from Glycyrrhiza uralensis

The present study was designed to determine the effects of copy number variations(CNVs) of squalene synthase 1(SQS1) gene on the mevalonate(MVA) pathway.SQS1 gene from G.uralensis(Gu SQS1) was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy number of Gu SQS1 were constructed.HPLC was used to assay the level of ergosterol in all transgenic P.pastoris strains containing Gu SQS1.HPLC analysis showed that the contents of ergosterol in all of the transgenic P.pastoris containing Gu SQS1 were higher than that in the negative control.And with the increase of copy number of Gu SQS1,the content of ergosterol showed an increasing-decreasing-increasing pattern.The contents of ergosterol in 10-copy-Gu SQS1 P.pastoris and 47-copy-Gu SQS1 P.pastoris were significantly higher than that in the rest recombinant P.pastoris strains.In conclusion,the CNVs of Gu SQS1 influence the content of secondary metabolites in the MVA pathway.The present study provides a basis for over-expressing Gu SQS1 and increasing the content of glycyrrhizin in G.uralensis cultivars.

Phage display screening of therapeutic peptide for cancer targeting and therapy

Recently,phage display technology has been announced as the recipient of Nobel Prize in Chemistry 2018.Phage display technique allows high affinity target-binding peptides to be selected from a complex mixture pool of billions of displayed peptides on phage in a combinatorial library and could be further enriched through the biopanning process;proving to be a powerful technique in the screening of peptide with high affinity and selectivity.In this review,we will first discuss the modifications in phage display techniques used to isolate various cancer-specific ligands by in situ,in vitro,in vivo,and ex vivo screening methods.We will then discuss prominent examples of solid tumor targeting-peptides;namely peptide targeting tumor vasculature,tumor microenvironment(TME)and overexpressed receptors on cancer cells identified through phage display screening.We will also discuss the current challenges and future outlook for targeting peptidebased therapeutics in the clinics.