Co-expression of CXCR4 and CD133 in gastric neoplastic tissue and their correlation with clinicopathological factors and prognosis in gastric cancer

Purpose: Worldwide Gastric carcinoma considered as the second most common cause of cancer related death. Cancer stem cell plays significant role in prognosis and invasion of gastric cancer. CXCR4 is a chemokine receptor and plays an important role in self renewal, differentiation potential, and cell adhesion of cancer stem cell (CSC). On the other hand CD133 is a cell surface glycoprotein and could serve as a prognostic indicator for tumor re-growth, malignant progression, and patient survival. The aim of this study was to establish the expression pattern of CXCR4 and CD133 in gastric cancer tissues; and their correlation with clinicopathological factors like age and gender of patients, position, size and depth of tumor, lymphatic invasion and node metastasis.Methods: Expression of CXCR4 and CD133 proteins were assessed by immunohistochemical and immunofluorescence staining of paraffin–embedded tissues, and followed by RT-PCR in 90 tumors (observed group) and 30 normal gastric samples. The clinical pathological data was statistically analyzed by chi-square methods. Results: The positive rate of CXCR4 and CD133 expression in the observed group was 94.44 (85/90) and 95.55 (86/90) respectively. The expression of CXCR4 and CD133 were correlated with age and gender of patients, and position, size, & depth of the tumor, lymphatic invasion and node metastasis (p < 0.05). While CXCR4 was positive, CD133 had a positive rate of 92.22% but the positive rate was 2.22% when CXCR4 expression was negative (χ2 = 58.657; p < 0.001). Conclusion: Overall this data suggests that increased expression of CXCR4 and CD133 might be attributed with disease progression and malignant transformation of gastric epithelium cells. A significant correlation was found in between CXCR4 and CD133 expression and their co-expression may play significant role in invasiveness of gastric cancer

Selection of chromosomal DNA libraries using a multiplex CRISPR system.

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function

Co-expression of CXCR4 and CD133 in gastric neoplastic tissue and their correlation with clinicopathological factors and prognosis in gastric cancer

Purpose: Worldwide Gastric carcinoma considered as the second most common cause of cancer related death. Cancer stem cell plays significant role in prognosis and invasion of gastric cancer. CXCR4 is a chemokine receptor and plays an important role in self renewal, differentiation potential, and cell adhesion of cancer stem cell (CSC). On the other hand CD133 is a cell surface glycoprotein and could serve as a prognostic indicator for tumor re-growth, malignant progression, and patient survival. The aim of this study was to establish the expression pattern of CXCR4 and CD133 in gastric cancer tissues; and their correlation with clinicopathological factors like age and gender of patients, position, size and depth of tumor, lymphatic invasion and node metastasis.Methods: Expression of CXCR4 and CD133 proteins were assessed by immunohistochemical and immunofluorescence staining of paraffin–embedded tissues, and followed by RT-PCR in 90 tumors (observed group) and 30 normal gastric samples. The clinical pathological data was statistically analyzed by chi-square methods. Results: The positive rate of CXCR4 and CD133 expression in the observed group was 94.44 (85/90) and 95.55 (86/90) respectively. The expression of CXCR4 and CD133 were correlated with age and gender of patients, and position, size, &amp; depth of the tumor, lymphatic invasion and node metastasis (p &lt; 0.05). While CXCR4 was positive, CD133 had a positive rate of 92.22% but the positive rate was 2.22% when CXCR4 expression was negative (χ2 = 58.657; p &lt; 0.001). Conclusion: Overall this data suggests that increased expression of CXCR4 and CD133 might be attributed with disease progression and malignant transformation of gastric epithelium cells. A significant correlation was found in between CXCR4 and CD133 expression and their co-expression may play significant role in invasiveness of gastric cancer.</p

Improvement of Gene Delivery and Mutation Efficiency in the CRISPR-Cas9 Wheat (Triticum aestivum L.) Genomics System via Biolistics

Discovery of the CRISPR-Cas9 gene editing system revolutionized the field of plant genomics. Despite advantages in the ease of designing gRNA and the low cost of the CRISPR-Cas9 system, there are still hurdles to overcome in low mutation efficiencies, specifically in hexaploid wheat. In conjunction with gene delivery and transformation frequency, the mutation efficiency bottleneck has the potential to slow down advancements in genomic editing of wheat. In this study, nine bombardment parameter combinations using three gold particle sizes and three rupture disk pressures were tested to establish optimal stable transformation frequencies in wheat. Utilizing the best transformation protocol and a knockout cassette of the phytoene desaturase gene, we subjected transformed embryos to four temperature treatments and compared mutation efficiencies. The use of 0.6 &mu;m gold particles for bombardment increased transformation frequencies across all delivery pressures. A heat treatment of 34 &deg;C for 24 h resulted in the highest mutation efficiency with no or minimal reduction in transformation frequency. The 34 &deg;C treatment produced two M0 mutant events with albino phenotypes, requiring biallelic mutations in all three genomes of hexaploid wheat. Utilizing optimal transformation and heat treatment parameters greatly increases mutation efficiency and can help advance research efforts in wheat genomics

Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

BackgroundAn efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day.ResultsHere we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1&nbsp;day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0&nbsp;days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo.ConclusionThe current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice

Pressurized intraperitoneal aerosol chemotherapy, a new surgical technique for the treatment of unresectable peritoneal carcinomatosis

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications