Understanding the role of transmembrane 9 superfamily member 1 in bladder cancer pathogenesis

In this editorial we comment on the article by Wei et al,published in the recent issue of the World Journal of Clinical Oncology.The authors investigated the role of Transmembrane 9 superfamily member 1(TM9SF1)protein in bladder cancer(BC)carcinogenesis.Lentiviral vectors were used to achieve silencing or overexpression of TM9SF1 gene in three BC cell lines.These cell lines were then subject to cell counting kit 8,wound-healing assay,transwell assay,and flow cytometry.Proliferation,migration,and invasion of BC cells were increased in cell lines subjected to TM9SF1 overexpression.TM9SF1 silencing inhibited proliferation,migration and invasion of BC cells.The authors conclude that TM9SF1 may be an oncogene in bladder cancer pathogenesis.

Biotin-modified Galactosylated Chitosan-gene Carrier in Hepatoma Cells Targeting Delivery

Our previous studies have successfully grafted biotin and galactose onto chitosan(CS)and synthesized biotin modified galactosylated chitosan(Bio-GC).The optimum N/P ratio of Bio-GC and plasmid DNA was 3:1.At this N/P ratio,the transfection efficiency in the hepatoma cells was the highest with a slow release effect.Bio-GC nanomaterials exhibit the protective effect of preventing the gene from nuclease degradation,and can target the transfection into hepatoma cells by combination with galactose and biotin receptors.The transfection rate was inhibited by the competition of galactose and biotin.Bio-GC nanomaterials were imported into cells’cytoplasm by their receptors,followed by the imported exogenous gene transfected into the cells.Bio-GC nanomaterials can also cause inhibitory activity in the hepatoma cells in the model of orthotopic liver transplantation in mice,by carrying the gene through the blood to the hepatoma tissue.Taken together,bio-GC nanomaterials act as gene vectors with the activity of protecting the gene from DNase degradation,improving the rate of transfection in hepatoma cells,and transporting the gene into the cytoplasm in vitro and in vivo.Therefore,they are efficient hepatoma-targeting gene carriers.

Increasingβ-hexosaminidase A activity using genetically modified mesenchymal stem cells

GM2 gangliosidoses are a group of autosomal-recessive lysosomal storage disorde rs.These diseases result from a deficiency of lysosomal enzymeβ-hexosaminidase A(HexA),which is responsible for GM2 ganglioside degradation.HexA deficiency causes the accumulation of GM2-gangliosides mainly in the nervous system cells,leading to severe progressive neurodegeneration and neuroinflammation.To date,there is no treatment for these diseases.Cell-mediated gene therapy is considered a promising treatment for GM2 gangliosidoses.This study aimed to evaluate the ability of genetically modified mesenchymal stem cells(MSCs-HEXA-HEXB)to restore HexA deficiency in Tay-Sachs disease patient cells,as well as to analyze the functionality and biodistribution of MSCs in vivo.The effectiveness of HexA deficiency cross-correction was shown in mutant MSCs upon intera ction with MSCs-HEXA-HEXB.The results also showed that the MSCs-HEXA-HEXB express the functionally active HexA enzyme,detectable in vivo,and intravenous injection of the cells does not cause an immune response in animals.These data suggest that genetically modified mesenchymal stem cells have the potentials to treat GM2 gangliosidoses.

Enhancing Cancer Classification through a Hybrid Bio-Inspired Evolutionary Algorithm for Biomarker Gene Selection

In this study,our aim is to address the problem of gene selection by proposing a hybrid bio-inspired evolutionary algorithm that combines Grey Wolf Optimization(GWO)with Harris Hawks Optimization(HHO)for feature selection.Themotivation for utilizingGWOandHHOstems fromtheir bio-inspired nature and their demonstrated success in optimization problems.We aimto leverage the strengths of these algorithms to enhance the effectiveness of feature selection in microarray-based cancer classification.We selected leave-one-out cross-validation(LOOCV)to evaluate the performance of both two widely used classifiers,k-nearest neighbors(KNN)and support vector machine(SVM),on high-dimensional cancer microarray data.The proposed method is extensively tested on six publicly available cancer microarray datasets,and a comprehensive comparison with recently published methods is conducted.Our hybrid algorithm demonstrates its effectiveness in improving classification performance,Surpassing alternative approaches in terms of precision.The outcomes confirm the capability of our method to substantially improve both the precision and efficiency of cancer classification,thereby advancing the development ofmore efficient treatment strategies.The proposed hybridmethod offers a promising solution to the gene selection problem in microarray-based cancer classification.It improves the accuracy and efficiency of cancer diagnosis and treatment,and its superior performance compared to other methods highlights its potential applicability in realworld cancer classification tasks.By harnessing the complementary search mechanisms of GWO and HHO,we leverage their bio-inspired behavior to identify informative genes relevant to cancer diagnosis and treatment.

大鼠Neurogenesin-1基因真核表达载体的构建及在cos-7细胞中的表达

目的克隆大鼠海马中Neurogenesin-1(Ng1)基因片段,构建pSecTag2/HygroB-Ng1真核表达载体,并检测其在cos-7细胞中的表达,为进一步研究该基因对脊髓神经干细胞分化的影响提供实验依据。方法在无RNA酶污染的条件下提取出大鼠海马总RNA。利用逆转录聚合酶链反应扩增出Ng1基因片段。将该基因片段连接到真核表达载体pSecTag2/HygroB,聚合酶链反应初步筛选,双酶切鉴定后送测序。将构建成功的重组真核表达载体转染入cos-7细胞,Westernblot鉴定重组Ng1蛋白的表达。结果逆转录聚合酶链反应成功获得大鼠Ng1cDNA。随机挑选10个重组真核表达载体的克隆,聚合酶链反应筛选出阳性克隆2个,经双酶切鉴定、测序及Blast分析鉴定重组质粒构建成功。脂质体介导转染cos-7细胞48h后,Westernblot鉴定重组Ng1蛋白在cos-7细胞中的表达,在46ku处出现阳性条带。结论大鼠海马Ng1基因的真核表达载体pSecTag2/HygroB-Ng1构建成功,转染cos-7细胞后能够表达重组Ng1蛋白。

NS特异性干扰载体pGenesil-1-NS的构建及鉴定

目的核干细胞因子(nucleostemin,NS)是维持干细胞和癌细胞增殖所必需的蛋白质,可能成为肿瘤基因治疗的潜在靶点.本文旨在构建靶向NS干扰载体p Genesil-1-NS,为后续实验奠定基础.方法基于已发布的NS mRNA序列(NM_206825),选取5'-AAGCCTA GGAAAGACCCAGG-3'(397-416)作为候选靶序列,按shRNA载体设计原则,设计合成两条互补DNA链,退火后插入载体,并以酶切和测序鉴定重组转化子.结果经酶切及测序鉴定证实合成序列正确插入载体.结论成功构建NS特异性干扰载体p Genesil-1-NS,可用于NS在肿瘤中的功能研究.

Construction of eukaryotic expression vector of HBV x gene

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Parameters selection in gene selection using Gaussian kernel support vector machines by genetic algorithm

In microarray-based cancer classification, gene selection is an important issue owing to the large number of variables and small number of samples as well as its non-linearity. It is difficult to get satisfying results by using conventional linear sta- tistical methods. Recursive feature elimination based on support vector machine (SVM RFE) is an effective algorithm for gene selection and cancer classification, which are integrated into a consistent framework. In this paper, we propose a new method to select parameters of the aforementioned algorithm implemented with Gaussian kernel SVMs as better alternatives to the common practice of selecting the apparently best parameters by using a genetic algorithm to search for a couple of optimal parameter. Fast implementation issues for this method are also discussed for pragmatic reasons. The proposed method was tested on two repre- sentative hereditary breast cancer and acute leukaemia datasets. The experimental results indicate that the proposed method per- forms well in selecting genes and achieves high classification accuracies with these genes.

A Non-Marker Mutagenesis Strategy to Generate Poly-hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.

Recent advances in liver-directed gene transfer vectors

BACKGROUND: Gene therapy as part of modern molecu-lar medicine holds great promise for the treatment of hepa-tocellular carcinoma (HCC) and has the potential to bringa revolutionary era to cancer treatment. For the past de-cade various viral and non-viral vectors have been engi-neered for improved liver gene therapy.DATA RESOURCES: An English-language literature searchusing MEDLINE (2004), Index Medicus (2004) and biblio-graphic reviews of books and review articles. Liver-directedgene transfer vectors and their history and recent clinicalapplications.RESULTS: The ultimate goal of liver-directed gene therapyfor HCC is the stable expression of a therapeutic transgenein a significant proportion of hepatocytes. The design of avector system providing efficient and stable gene engraft-ment and expression in human hepatocytes is still a chal-lenging issue. The advantages and disadvantages of the ge-netically engineered vector of viral or non-viral origin arediscussed with respect to their essential relevance.CONCLUSION: Liver gene therapy has a long way to goand efficient and innocuous liver-directed gene transfer vec-tors are therefore urgently required.

Alterations of tumor-related genes do not exactly match the histopathological grade in gastric adenocarcinomas

AIM:To investigate the diverse characteristics of different pathological gradings of gastric adenocarcinoma (GA) using tumor-related genes.METHODS:GA tissues in different pathological gradings and normal tissues were subjected to tissue arrays.Expressions of 15 major tumor-related genes were detected by RNA in situ hybridization along with 3' terminal digoxin-labeled anti-sense single strandedoligonucleotide and locked nucleic acid modifying probe within the tissue array.The data obtained were processed by support vector machines by four different feature selection methods to discover the respective critical gene/gene subsets contributing to the GA activities of different pathological gradings.RESULTS:In comparison of poorly differentiated GA with normal tissues,tumor-related gene TP53 plays a key role,although other six tumor-related genes could also achieve the Area Under Curve (AUC) of the receiver operating characteristic independently by more than 80%.Comparing the well differentiated GA with normal tissues,we found that 11 tumor-related genes could independently obtain the AUC by more than 80%,but only the gene subsets,TP53,RB and PTEN,play a key role.Only the gene subsets,Bcl10,UVRAG,APC,Beclin1,NM23,PTEN and RB could distinguish between the poorly differentiated and well differentiated GA.None of a single gene could obtain a valid distinction.CONCLUSION:Different from the traditional point of view,the well differentiated cancer tissues have more alterations of important tumor-related genes than the poorly differentiated cancer tissues.

HBeAg gene expression with baculovirus vector in silk worm cells

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An improved protein expression system for T3SS genes regulation analysis in Xanthomonas oryzae pv. oryzae

Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo.Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium.The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo.Thus,an efficient and reliable genetic tool system is required for detection of the T3SS proteins.In this study,we constructed a protein expression vector pH3-flag based on the backbone of pHM1,a most widely used vector in Xoo strains,especially a model strain PXO99A.This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site(MCS),containing a modified pUC18 polylinker,and Flag as a C-terminal tag.The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression.We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo.This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.

Surface Modification of Biomimetic PLGA-(ASP-PEG) Matrix with RGD-Containing Peptide:a New Non-Viral Vector for Gene Transfer and Tissue Engineering

RGD-containing peptide （ K16-GRGDSPC） , characterized as non-viral gene vectors, was fabricated to modify the surface of PLGA-[ASP- PEG] matrix, which offered the foundation for gene transfer with porous matrix of gene activated later. Peptide was synthesized and matrix was executed into chips A, B and chip C. Chip C was regarded as control. Chips A and B were reacted with cross-linker. Then chip A was reacted with peptide. MS and HPLC were ased to detect the .14W and purity of peptide. Sulphur, existing on the surface of biomaterials, was detected by XPS. The purity of un-reacted peptide in residual solution was detected by a spectrophotometer. HPLC shows that the peptide purity was 94%- 95% , and MS shows that the MW was 2 741. 3307. XPS reveals that the binding energy of sulphur was 164 eV and the ratio of carbon to sulphur （C/S） was 99. 746 ：0. 1014 in reacted chip A. The binding energy of sulphur in reacted chip B was 164 eV and 162 eV, C/ S was 99.574：0.4255, aM there was no sulphur in chip C. Peptide was manufactured and linked to the surface of biomimetic and 3-D matrix, which offered the possibilities for gene transfer and tissue engineering with this new kind of non-viral gene vector.

Characteristics and advantages of adeno-associated virus vector-mediated gene therapy for neurodegenerative diseases

Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to delay or possibly stop further progression of the neurodegenerative disease in affected patients. Adeno-associated virus has been the vector of choice in recent clinical trials of therapies for neurodegenerative diseases due to its safety and efficiency in mediating gene transfer to the central nervous system. This review aims to discuss and summarize the progress and clinical applications of adeno-associated virus in neurodegenerative disease in central nervous system. Results from some clinical trials and successful cases of central neurodegenerative diseases deserve further study and exploration.

Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.

Internal ribosome entry site-based vectors for combined gene therapy

Gene therapy appears as a promising strategy to treatincurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites(IRESs), RNA elements naturally present in the 5' untranslated regions of a few m RNAs, constitute a powerful tool to co-express several genes of interest. IRESs are translational enhancers allowing the translational machinery to start protein synthesis by internal initiation. This feature allowed the design of multi-cistronic vectors expressing several genes from a single m RNA. IRESs exhibit tissue specificity, and drive translation in stress conditions when the global cell translation is blocked, which renders them useful for gene transfer in hypoxic conditions occurring in ischemic diseases and cancer. IRES-based viral and non viral vectors have been used successfully in preclinical and clinical assays of combined gene therapy and resulted in therapeutic benefits for various pathologies including cancers, cardiovascular diseases and degenerative diseases.

Gene Therapy of HSV-TK Transferred by the EBV based Expression Vector on Experimental Hepatocellular Carcinoma

To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatocellular carcinoma cell line SMMC 7721 by using liposome mediated transfection technique,and then gene expression was detected by RT PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model,the antitumor effects of pDR2 TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2 TK /GCV had cytotoxic effect and about 70 % SMMC 7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2 TK gene was significantly smaller than that in control group . On the 10th day the tumor in 3 mice (60 %) disappeared completely after GCV treatment. It is concluded that the pDR2 TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.

Functionality of a bicistronic construction containing HEXA and HEXB genes encoding β-hexosaminidase A for cell-mediated therapy of GM2 gangliosidoses

Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency ofβ-hexosaminidase A(HexA)enzyme,which results in the accumulation of GM2 gangliosides in the nervous system cells.In this work,we analyzed the efficacy and safety of cell-mediated gene therapy for Sandhoff disease and Sandhoff disease using a bicistronic lentiviral vector encoding cDNA of HexAα-andβ-subunit genes separated by the nucleotide sequence of a P2A peptide(HEXA-HEXB).The functionality of the bicistronic construct containing the HEXA-HEXB genetic cassette was analyzed in a culture of HEK293T cells and human umbilical cord blood mononuclear cells(hUCBMCs).Our results showed that the enzymatic activity of HexA in the conditioned medium harvested from genetically modified HEK293T-HEXA-HEXB and hUCBMCs-HEXA-HEXB was increased by 23 and 8 times,respectively,compared with the conditioned medium of native cells.Western blot analysis showed that hUCBMCs-HEXA-HEXB secreted both completely separated HEXA and HEXB proteins,and an uncleaved protein containing HEXA+HEXB linked by the P2A peptide.Intravenous injection of genetically modified hUCBMCs-HEXA-HEXB to laboratory Wistar rats was carried out,and the HexA enzymatic activity in the blood plasma of experimental animals,as well as the number of live cells of immune system organs(spleen,thymus,bone marrow,lymph nodes)were determined.A significant increase in the enzymatic activity of HexA in the blood plasma of laboratory rats on days 6 and 9(by 2.5 and 3 times,respectively)after the administration of hUCBMCsHEXA-HEXB was shown.At the same time,the number of live cells in the studied organs remained unchanged.Thus,the functionality of the bicistronic genetic construct encoding cDNA of the HEXA and HEXB genes separated by the nucleotide sequence of the P2A peptide was shown in vitro and in vivo.We hypothesize that due to the natural ability of hUCBMCs to overcome biological barriers,such a strategy can restore the activity of the missing enzyme in the central nervous system of patients with GM2 gangliosidoses.Based on the obtained data,it can be concluded that intravenous administration of hUCBMCs with HexA overexpression is a promising method of the therapy for GM2 gangliosidoses.The animal protocol was approved by the Animal Ethics Committee of the Kazan Federal University(No.23)on June 30,2020.