Restriction Enzyme Digestion Analysis Research Articles

Prokaryotic expression, purification and characterization of tissue inhibitor of metalloproteinase-2

Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.

CLIA Laboratory Testing for Facioscapulohumeral Dystrophy: A Retrospective Analysis.

ObjectiveTo summarize facioscapulohumeral muscular dystrophy (FSHD) diagnostic testing results from the University of Iowa Molecular Pathology Laboratory.MethodsAll FSHD tests performed in the diagnostic laboratory from January 2015 to July 2019 were retrospectively reviewed. Testing was by restriction enzyme digestion and Southern blot analysis with sequencing of SMCHD1, if indicated. Cases were classified as FSHD1 (4q35 EcoRI size ≤40 kb; 1–10 D4Z4 repeats), FSHD2 (permissive 4q35A allele, D4Z4 hypomethylation, and pathogenic SMCHD1 variant), or non-FSHD1,2. We also noted cases with borderline EcoRI fragment size (41–43 kb; 11 D4Z4 repeats), cases that meet criteria for both FSHD1 and FSHD2, somatic mosaicism, and cases with hybrid alleles that add complexity to test interpretation.ResultsOf the 1,594 patients with FSHD tests included in the analysis, 703 (44.1%) were diagnosed with FSHD. Among these positive tests, 664 (94.5%) met criteria for FSHD1 and 39 (5.5%) met criteria for FSHD2. Of all 1,594 cases, 20 (1.3%) had a 4q35 allele of borderline size, 23 (1.5%) were somatic mosaics, and 328 (20.9%) had undergone translocation events. Considering only cases with at least 1 4q35A allele, D4Z4 repeat number differed significantly among groups: FSHD1 cases median 6.0 (interquartile range [IQR] 4–7) repeats, FSHD2 cases 15.0 (IQR 12–22) repeats, and non-FSHD1,2 cases 28.0 (IQR 19–40) repeats.ConclusionFSHD1 accounts for 94.5% of genetically confirmed cases of FSHD. The data show a continuum of D4Z4 repeat numbers with FSHD1 samples having the fewest, FSHD2 an intermediate number, and non-FSHD1,2 the most.

A Closed-Tube Loop-Mediated Isothermal Amplification Assay for the Visual Detection of Staphylococcus aureus.

Food-borne diseases induced by Staphylococcus aureus contamination seriously affect human health and food safety. Therefore, a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus was developed in this study. Firstly, two pairs of outer and inner primers were designed targeting on a conserved fragment of gyrB gene in different S. aureus strains. Secondly, the weakly buffered gyrB-LAMP assays were optimized under various pH values and other conditions, followed by the visual evaluation of five pH-sensitive indicators, and the cresol-red was chosen as the best dye for the best visual performance. Thirdly, the cresol-red-based LAMP assay showed good sensitivity with the detection limit of 5.4copies/μL for purified DNAs, and good specificity with no cross-reaction with other related species. The specificity of the amplified products was further confirmed by XbaI restriction enzyme digestion analysis. Finally, the cresol-red-based LAMP assay was validated by the clinical-dried fish samples inoculated with known numbers of S. aureus and further validated by 20 blind samples. To our knowledge, this is the first report of a closed-tube LAMP assay based on pH-sensitive indicators for the visual detection of the food-borne S. aureus by the gyrB gene.

Construction of nitrogen-fixing Klebsiella variicola GN02 expression vector pET28a-Lac-EGFP and its colonization of Pennisetum giganteum z.x.lin roots

To explore the colonization of endophytic nitrogen-fixing bacterium Klebsiella variicola in Penniseum sinense, pET28a and the enhanced green fluorescent protein (EGFP) were used as basic elements to construct a recombinant expression vector pET28a-Lac-EGFP with the addition of starter Lac in pUC18 vector using enzymatic digestion and splicing methods. The constructed recombinant vector was transformed into K. variicola GN02 cells with electroporation method and the colonization of EGFP-tagged K. variicola GN02 in the roots of Pennisetum sp. was observed using laser confocal scanning microscopy. The results of restriction enzyme digestion and sequencing analysis indicated that pET28a-Lac-EGFP was successfully constructed and the target gene was successfully transferred into K. variicola GN02 cells. Positive colonies with obvious green fluorescence were observed under ultraviolet (UV) light. Sodium dodecyl sulphate-polyacrylamide gel-electrophoresis (SDS-PAGE) showed that expression of the EGFP-labelled target protein was also successfully induced in K. variicola GN02 cells. Root scanner and confocal microscopy showed that inoculation of K. variicola GN02 strain was more conducive to the root growth of P. sinense and K. variicola GN02 mainly colonized the endothelial layer of Pennisetum sp. roots. However, colonization with a small amount of K. variicola GN02 led to its concentration in the epidermis, middle column and ducts. Our findings suggest that the endophytic nitrogen-fixing bacterium K. variicola accumulates in plant roots and is subsequently dispersed, aggregated or even colonized in susceptible plants. Furthermore, we provided a theoretical basis for the practical used of K. variicola GN02-based microbial fertilizers in P. sinense and gramineous plants.

Generation of a Novel Gene Therapy Vector Encoding Beta-Cell–Specific Insulin for Diabetes Treatment

Insulin injection is the main treatment modality especially for patients with type 1 diabetes. Despite the development of several insulin analogs, recent meta-analysis indicated that rapid and long acting insulin analogs provide little benefit compared to conventional insulins regarding to glycemic control. Therefore, gene therapy methods are investigated to develop a novel treatment approach to reconstitute a natural endogenous insulin expression profile in diabetic patients. For this purpose, Multisite Gateway Technology was employed to generate a transfer vector carrying lentiviral backbone with minimal insulin promotor hooked up to proinsulin gene sequence. Following confirmation of the transfer plasmid by restriction enzyme digestion and DNA sequence analysis, high-titer LentiINS vectors at a concentration of 10e9 TU/ml were successfully produced by CaPO4 cotransfection of 293T cells with packaging and transfer plasmids. While transduction of NIT1 mouse pancreatic beta cell lines produced 3 fold increase in insulin gene expression compared to controls, no insulin expression was detected in 293T kidney cell line. These results indicate that we successfully constructed an HIV-based lentiviral gene therapy vector being capable of beta cell-specific insulin gene expression and secretion (TUBITAK 215S820). Disclosure E.O. Sahin: None. F. Erendor: None. M. Balci: None. S. Sanlioglu: None.

Immunization with r-Lactococcus lactis expressing outer membrane protein A of Shigella dysenteriae type-1: evaluation of oral and intranasal route of administration.

To evaluate the comparative immunogenic potential of food grade Lactococcus lactis expressing outer membrane protein A (OmpA) of Shigella dysenteriae type-1 (SD-1) when administered either orally or intranasally. OmpA of SD-1 was cloned and expressed first in Escherichia coli and then in L. lactis. Presence of recombinant gene was confirmed by restriction enzyme digestion and immunoblot analysis. Using immobilized metal affinity chromatography, OmpA was purified from recombinant E.coliBL21 (DE3) and subcutaneously administered in BALB/c mice. Detection of OmpA-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA) confirmed the immunogenicity of OmpA. In order to establish r-L. lactis as a mucosal delivery vehicle, it was administered orally and nasally in BALB/c mice. Serum IgG and faecal IgA were assessed through ELISA to compare the relative potential of immunization routes and immunogenic potential of r-L. lactis. Immunization via the oral route proved superior to intranasal exposure. Recombinant L. lactis expressing OmpA of SD-1 was found to be immunogenic. Oral administration of r-L. lactis elicited higher systemic and mucosal immune response when compared with the nasal route. Using food grade recombinant L. lactis has implications in the development of a prophylactic against multidrug-resistant Shigella, which can be used as a prospective vaccine candidate. Evaluating mucosal routes of immunization demonstrated that the oral route of administration elicited better immune response against OmpA of Shigella.

Prokaryotic expression of a multi-epitope gene of Xinjiang hemorrhagic fever virus and development of an indirect ELISA for virus detection

Objective To express and purify the multi-epitope peptide of Xinjiang hemorrhagic fever virus (XHFV) in a prokaryotic expression system and to further use it as diagnostic antigen to establish an indirect ELISA for the detection of XHFV in animal sera. Methods A double copy multi-epitope gene (MEPX2) segment with repeated tandem including six highly conservative and dominant B cell epitopes was designed based on the analysis of hydrophilicity and antigenic determinant sites in amino acid sequences of nucleoprotein and glycoprotein from XHFV strain YL04057. The multi-epitope gene MEPX2 was synthesized by chemical method and then inserted into the prokaryotic expression vector pET-32a (+ ) to construct the prokaryotic expression plasmid pET-32a (+ )-MEPX2. The recombinant expression plasmid was transformed into E.coli BL21 (DE3) to induce the expression of rMEPX2 by IPTG. The expressed products were purified by Ni-NTA purification system. The antigenicity of rMEPX2 was identified by Western blot assay. The purified rMEPX2 was used as coating antigen to establish an indirect ELISA by checkerboard for the detection of XHFV in animal serum samples. The results of virus detection were compared with those by using commercial ELISA kit. Results The recombinant expression plasmid pET-32a (+ )-MEPX2 was constructed successfully as confirmed by restriction enzyme digestion and DNA sequencing analysis. Results of the SDS-PAGE and Western blot assay confirmed that the rMEPX2 was expressed correctly with a relative molecular mass (Mr) of about 49×103. The rMEPX2 was recognized specifically by His-tag mouse monoclonal antibody and by positive serum samples of sheep naturally infected with XHFV. The indirect ELISA for the detection of XHFV IgG antibody was established by using the purified rMEPX2 as antigen. Compared with the commercial ELISA kit, the established indirect ELISA showed good specificity for the detection of XHFV in 108 sheep serum samples. Conclusion The recombinant multi-epitope protein rMEPX2 with good antigenicity was obtained successfully. The established indirect ELISA based on the rMEPX2 could be used for the detection of specific antibodies against XHFV. Key words: Xinjiang hemorrhagic fever virus; Multi-epitope peptide; Prokaryotic expression; Indirect ELISA

167 Long-term in vivo correction of a recessive dystrophic epidermolysis bullosa phenotype using RNA trans-splicing repair

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene

The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homology-directed repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa.

165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

169 Regeneration of a functional epidermis at a large, long-standing wound by gene-corrected autologous epidermal stem cells

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

FABP4-mediated homocysteine-induced cholesterol accumulation in THP-1 monocyte-derived macrophages and the potential epigenetic mechanism

Hyperhomocysteinemia (HHcy) is an independent risk factor for the development of atherosclerosis (AS), according to overwhelming number of clinical and epidemiological studies. However, the underlying pathogenic molecular mechanisms by which HHcy promotes AS remain to be fully elucidated. Fatty acid binding protein 4 (FABP4) has been shown to be important in macrophage cholesterol trafficking. The objective of the present study was to determine whether homocysteine (Hcy) accelerates AS through regulating FABP4, and then mediates cholesterol accumulation in macrophages. Hcy concentrations of 0, 50, 100, 200 and 500µM, and 100µM Hcy+30µMvitamin B12 (VB12)+30µM folic acid (FA) were respectively added to cultured THP‑1 monocyte‑derived macrophages for 24h. The levels of FABP4, which acts as a key factor connecting cellular lipid accumulation to inflammation, were determined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses in the macrophages. The present study used a nested touchdown methylation‑specific PCR assay to detect the DNA methylation status of the FABP4 promoter region. In addition, the FABP4 gene fragment was inserted into the cloning vector, pcDNA3.1‑EGFP, to construct the recombinant plasmid, pcDNA3.1‑EGFP/FABP4, which was identified using restriction endonuclease digestion analysis and DNA sequencing. The pcDNA3.1‑EGFP/FABP4 expression plasmid was transfected into THP‑1 monocyte‑derived macrophages, mediated by liposome reagent, following which the expression levels of FABP4 were detected using RT‑qPCR and western blot analyses. The present study also determined the intracellular accumulation of total cholesterol in the macrophages. The results indicated that Hcy decreased the levels of FABP4 promoter methylation, but increased the mRNA and protein expression levels of FABP4 in the macrophages, compared with the control group (0µMHcy). However, no dose‑dependent changes were observed with increasing concentrations of Hcy. The recombinant fluorescent eukaryotic expression vector, pcDNA3.1‑EGFP/FABP4, was successfully constructed and effectively expressed in the THP‑1 macrophages. The results also showed that FABP4 accelerated the accumulation of cholesterol in the macrophages. Taken together, the results of the present study suggested that FABP4 DNA hypomethylation induced by Hcy may be involved in the overexpression of FABP4, thereby inducing cholesterol accumulation in macrophages.

CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes

AbstractHuman platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the PlA1/PlA2 polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin β3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33+ megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41+ megakaryocyte progenitors derived from these cells expressed the HPA-1b (PlA2) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b–specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use.

Isolation and characterization of T7-like lytic bacteriophages infecting multidrug resistant Pseudomonas aeruginosa isolated from Egypt.

In this study, two lytic phages designated as ϕPSZ1 and ϕPSZ2 infecting multidrug resistant Pseudomonas aeruginosa were isolated from sewage samples collected in Zagazig, Egypt. Morphological analysis by transmission electron microscopy revealed that both phages belong to the podoviridae family and resembles typical T7-like phages. ϕPSZ1 has a head of about 60 ± 5 nm in diameter with a short tail of 19 ± 2 nm in length, while ϕPSZ2 has a head of about 57 ± 5 nm in diameter with a short tail of 14 ± 2 nm in length. Both phages were shown to be able to infect 13 different P. aeruginosa strains and has no effect on other tested bacteria. In spite of morphological similarity, these phages showed diverged genomic sequences revealed by restriction enzyme digestion analysis. One-step growth curves of bacteriophages revealed eclipse and latent periods of 12 min for ϕPSZ1 and 15 min for ϕPSZ2, respectively, with burst sizes of about 100 per infected cell. Phage treatment prevented the growth of P. aeruginosa for up to 18 h with multiplicity of infection ratios of 1. These results suggest that both phages have a high potential for phage application to control P. aeruginosa.

DKK1 eukaryotic expression plasmid and expression product identification.

We constructed the human dickkopf 1 (DKK1) eukaryotic expression plasmid and expressed, purified, and identified its expression product. We extracted cancer cells from cervical cancer tissue, followed by extraction of mRNA. Reverse transcription-polymerase chain reaction was conducted to obtain DKK1 gene fragments. Using these fragments, we prepared the recombinant plasmid pCMV-HA2/DKK1. The recombinant plasmid was restriction enzyme-digested and sequenced, and using liposome vectors, was transiently transfected into Free-Style 293-F cells (serum-free medium). DKK1 protein was detected by western blotting. The amplification product showed the expected size. Restriction enzyme digestion and sequence analysis showed that the recombinant plasmid was PCMV-HA2/DKK1. The expression product was verified properly by western blotting using an anti-DKKI antibody. The successful cloning of the DKKI gene and expression of DKKI protein will be useful for studying the biological activity of tumorigenesis.

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

A Loop-Mediated Isothermal Amplification (LAMP) Assay for Strongyloides stercoralis in Stool That Uses a Visual Detection Method with SYTO-82 Fluorescent Dye

An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to < 10 copies of target sequence in a plasmid and up to a 10(-2) dilution of DNA extracted from a Strongyloides ratti larva spiked into stool. Sensitivity was increased when further dilutions were made in water, indicative of reduced reaction inhibition. Twenty-seven of 28 stool samples microscopy and polymerase chain reaction positive for S. stercoralis were positive with the LAMP method. On the basis of these findings, the assay warrants further clinical validation.

16S rRNA PCR followed by restriction endonuclease digestion: A rapid approach for genus level identification of important enteric bacterial pathogens

The study describes a rapid approach for detection of common enteric bacterial pathogens, which involves partial amplification of the 16S rRNA gene by PCR using a colony from selective medium followed by restriction enzyme (RE) digestion using the EcoRI, HindIII and SalI enzymes. On the basis of RE digestion analysis different genera namely, Escherichia, Salmonella, Shigella, Vibrio, Campylobacter, Arcobacter, Yesinia and Listeria were differentiated.

High prevalence of azithromycin resistance to Treponema pallidum in geographically different areas in China

AbstractTreatment with effective antibiotics is one important strategy for syphilis control in China. This study aimed to evaluate the prevalence of azithromycin resistance to T. pallidum in China. A cross-sectional study was conducted among 391 patients with early syphilis recruited from STD clinics in eight cities during October 2008 and October 2011. The swabs were obtained from the moist lesions of the participating patients. A touchdown/nested PCR of the 23S ribosomal RNA (rRNA) gene was performed on DNA samples extracted from these specimens. The presence or absence of the A2058G point mutation, conferring resistance to azithromycin, was determined by restriction enzyme digestion analysis of the PCR amplicon by MboII. Two hundred and eleven patients with primary or secondary syphilis were found to have T. pallidum DNA in their moist lesions by PCR assays. The A2058G mutation was present in 91.9% (194/211, 95% CI, 87.2–95.1%) of these patients, with no significant differences noted between patients from the eastern part (93.8%), southern part (88.6%) and northern part (95.2%) of China (v2 = 2.303, p 0.316). Compared with patients who had not taken macrolides in previous years before study entry, the patients who had taken the antibiotics had a significantly higher prevalence of azithromycin resistance (97.0% vs. 62.5%), with an odds ratio of 19.65 (95% CI, 5.77–66.93). It can be concluded that prevalence of azithromycin resistance is substantial in China and consequently that the macrolides should not be used as a treatment option for early or incubating syphilis in China.

English

An effective method for plant rhizosphere microbial genomic DNA extraction was established. High-purity nucleic acids extracted from soil samples, with considerable yield, could be used for study of soil microorganism molecular ecology. Genomic DNAs were extracted from 12 soil samples of different crop roots. Above 10 &mu;g of genomic DNA with approximately 20-kb fragment length was isolated from 1 g of soil sample. The average value of A260/A230 of the genomic DNA was 1.505, while that of A260/A280 was 1.780. The absorption curves of the full wavelengths of the DNA extracted were consistent with that of pure nucleic acid. DNAs from different dilutions of pollutants were applicable for restriction enzyme digestion analysis, and the lambda DNA in 100-fold diluted soil samples had the same restriction enzyme digestion results as done in ddH2O. Amplicons was produced with the expected molecular size by polymerase chain reaction (PCR).Therefore, the DNA extracted by this method was suitable to be used in analysis of bacterial ecology composition by denaturing gradient gel electrophoresis. &nbsp; Key words:&nbsp;DNA extraction, DNA quality, PCR amplification, soil microbial community diversity, DGGE.