CRISPR/dCas9-mediated transposition with specificity and efficiency of site-directed genomic insertions.

The ability and efficiency of targeted nucleases to perform sequence replacements or insertions into the genome are limited. This limited efficiency for sequence replacements or insertions can be explained by the dependency on DNA repair pathways, the possibility of cellular toxicity, and unwanted activation of proto-oncogenes. The piggyBac (PB) transposase uses a very efficient enzymatic mechanism to integrate DNA fragments into the genome in a random manner. In this study, we fused an RNA-guided catalytically inactive Cas9 (dCas9) to the PB transposase and used dual sgRNAs to localize this molecule to specific genomic targets. We designed and used a promoter/reporter complementation assay to register and recover cells harboring-specific integrations, where only by complementation upon correct genomic integration, the reporter can be activated. Using an RNA-guided piggyBac transposase and dual sgRNAs, we were able to achieve site-directed integrations in the human ROSA26 safe harbor region in 0.32% of cells. These findings show that the methodology used in this study can be used for targeting genomic regions. An application for this finding could be in cancer cells to insert sequences into specific target regions that are intended to be destroyed, or to place promoter cargos behind the tumor suppressor genes to activate them.

Architectural Control of Isosorbide-Based Polyethers via Ring-Opening Polymerization.

Isosorbide is a rigid, sugar-derived building block that has shown promise in high-performance materials, albeit with a lack of available controlled polymerization methods. To this end, we provide mechanistic insights into the cationic and quasi-zwitterionic ring-opening polymerization (ROP) of an annulated isosorbide derivative (1,4:2,5:3,6-trianhydro-d-mannitol, 5). Ring-opening selectivity of this tricyclic ether was achieved, and the polymerization is selectively directed toward different macromolecular architectures, allowing for formation of either linear or cyclic polymers. Notably, straightforward recycling of unreacted monomer can be accomplished via sublimation. This work provides the first platform for tailored polymer architectures from isosorbide via ROP.

Targeting the death receptor signaling pathway as a potential therapeutic target in the treatment of colorectal cancer.

Despite advances in the diagnosis and treatment of colorectal cancer (CRC), it remains a major cause of cancer related death globally. There are currently no chemotherapeutic agents that have been found to eradicate the disease without adverse effects. A defect in the death receptor signaling pathway is a feature of CRC. The ligand of these receptors belongs to the tumor necrosis factor family, and that are particularly expressed by cells of the immune system, and that induce apoptosis in a caspase dependent manner. The fact that malignant cells are particularly sensitive to these ligands, compared to normal cells, has led to work on the assessment of compounds that activate this pathway in the treatment of CRC. Phase I trials have shown that these death receptor agonists are safe. Phase II and III trials are currently investigating the efficacy of these therapeutic agents in the treatment of CRC. In this review, we describe the biochemical death receptor signaling pathway and its relationship to CRC. We also summarize the current clinical studies that are targeting this signaling pathway in CRC treatment.

Bioinformatics analysis and machine learning approach applied to the identification of novel key genes involved in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) comprises a range of chronic liver diseases that result from the accumulation of excess triglycerides in the liver, and which, in its early phases, is categorized NAFLD, or hepato-steatosis with pure fatty liver. The mortality rate of non-alcoholic steatohepatitis (NASH) is more than NAFLD; therefore, diagnosing the disease in its early stages may decrease liver damage and increase the survival rate. In the current study, we screened the gene expression data of NAFLD patients and control samples from the public dataset GEO to detect DEGs. Then, the correlation betweenbetween the top selected DEGs and clinical data was evaluated. In the present study, two GEO datasets (GSE48452, GSE126848) were downloaded. The dysregulated expressed genes (DEGs) were identified by machine learning methods (Penalize regression models). Then, the shared DEGs between the two training datasets were validated using validation datasets. ROC-curve analysis was used to identify diagnostic markers. R software analyzed the interactions between DEGs, clinical data, and fatty liver. Ten novel genes, including ABCF1, SART3, APC5, NONO, KAT7, ZPR1, RABGAP1, SLC7A8, SPAG9, and KAT6A were found to have a differential expression between NAFLD and healthy individuals. Based on validation results and ROC analysis, NR4A2 and IGFBP1b were identified as diagnostic markers. These key genes may be predictive markers for the development of fatty liver. It is recommended that these key genes are assessed further as possible predictive markers during the development of fatty liver.

A genetic polymorphism in the CYP1B1 gene in patients with squamous cell carcinoma of the esophagus: an Iranian Mashhad cohort study recruited over 10 years.

AIM: Esophageal cancer is the eighth most common cancer globally and the seventh most common cause of cancer-related deaths in men. Recent studies have shown that CYP450, family 1, subfamily B, polypeptide 1, which plays a role in the metabolism of xenobiotics, is associated with several cancers. Therefore, in the present study we investigated the association between a genetic variant, CYP1B1-rs1056836 gene, with the clinical characteristics of patients with squamous cell carcinoma of the esophagus (ESCC). METHOD: In this study, 117 patients with ESCC and 208 healthy controls were recruited. DNA was extracted and genotyped using real-time PCR-based TaqMan. Kaplan-Meier curves were utilized to assess overall and progression-free survival. To evaluate the relationship between clinicopathological data, genotypic frequencies, disease prognosis and survival, Pearson's χ2 and t-test were used. Logistic regression was utilized to assess the association between the risk of ESCC and genotypes. RESULTS: The genotypic frequency for GG, GC and CC were 58.6, 29.8 and 11.5%, respectively, in the healthy subjects and 51.8, 36.14 and 12% in the ESCC group. An association between the GG genotype and stage of ESCC was found. Also, statistically significant results were not found for this variation and risk of ESCC. CONCLUSION: Our findings suggest a relationship between the CYP1B1-rs1056836 genetic polymorphism and clinical features of ESCC, supporting further studies in larger populations in different ethnic groups, taking into account potentially important environmental factors such as diet.

Evaluating the expression of cyclooxygenase-2 enzyme by immunohistochemistry in normal and tumoral tissue before and after neoadjuvant chemoradiotherapy in patients with esophageal cancer in Khorasan Province.

BACKGROUND: Esophageal cancer is the third most common cancer in Iran. Neoadjuvant chemoradiotherapy (NCRT) is the appropriate treatment for esophageal cancer. AIM: This study investigated the expression of cyclooxygenase (COX)-2 enzyme in normal and tumoral tissues before any treatment in patients with esophageal cancer, this study also assessed the effect of NCRT on the expression of COX-2 enzyme in normal and tumoral tissue in samples derived by surgery furthermore, and this study investigated the relationship between expression of COX-2 enzyme and the pathologic tumor regression grade (PTRG) patients. MATERIALS AND METHODS: In this study, a total of 120 patients admitted to Omid Hospital, Imam Reza Hospitals, and Reza-Mashhad Medical Center, who were treated with NCRT, were recruited and the expression of the COX-2 enzyme in normal and tumoral tissues was assessed by immunohistochemistry before and after treatment by an expert pathologist between zero and 300. PTRG was determined by a pathologist after treatment. RESULTS: The mean levels of COX-2 expression, obtained from tumoral and normal tissue baseline biopsy in patients, were 177.69 and 64.29, respectively, while in surgical specimen were 177.25 and 49.84, respectively. A significant association was found between PTRG of surgical specimen and COX-2 expression in normal tissue (baseline biopsy) at diagnosis (P = 0.034). CONCLUSIONS: The results indicated that expression of COX-2 in tumoral tissues exceeds the expression of COX-2 in normal tissue of the baseline biopsy. Patients with a high expression of COX-2 in baseline tumor biopsies had less response to treatment of pathology compared to patients with lower expression of COX-2 in baseline tumor biopsies.

Expression of p53 in colorectal carcinoma: correlation with clinicopathologic features.

BACKGROUND: The p53 gene mutation is closely related to carcinogenesis in most malignant diseases. The main function of wild p53 protein is to maintain the integrity of genes by detecting mutations and preventing the division of cells with damaged DNA. The mutated form of p53 protein is overexpressed due to an extended half-life and can be easily detected by immunohistochemistry. OBJECTIVE: To estimate the frequency of p53 protein overexpression in colorectal carcinoma and its correlation with some clinicopathologic variables. METHODS: One hundred paraffin-preserved colorectal carcinoma samples were collected randomly from patients undergoing tumor resection from April 1995 through April 2001 in Omid Hospital, affiliated to Mashhad University of Medical Sciences, Mashhad, Iran. The overexpression of p53 protein was studied using a monoclonal antibody (clone DO-7; Dako). The number of cells stained were classified semiquantitatively as (-): <5% positive cells, (+): 5 - 25% positive cells, (++): 25 - 75% positive cells, and (+++): >75% positive cells. Clinicopathologic data including gender, age, tumor location, histologic type, and stage (Astler-Coller) were collected from the files maintained at the Department of Pathology. The correlation between p53 protein overexpression and each variable was evaluated using Chi-square analysis. RESULTS: p53 staining was positive in 59 of 100 specimens. Out of these 100 specimens, 16 were weekly (+), 16 moderately (++), and 27 intensely (+++) positive for p53 protein over-expression. There was no significant correlation between p53 staining and gender (P = 0.34), age (< 40 vs. > or = 40 yr; P = 0.74), site of tumor (right vs. left colon and rectum; P = 0.26), pathologic type (mucinous vs. nonmucinous; P = 0.63), and stage of the disease (P = 0.12). CONCLUSION: Considering the p53 protein overexpression in a relatively high percentage of patients, it seems that p53 mutation plays an important role in development of colorectal carcinoma. There was no significant association between p53 protein expression and some common clinicopathologic variables such as age, gender, site of tumor, pathologic type, and stage of the disease.

Comparative In silico Study of Sex-Determining Region Y (SRY) Protein Sequences Involved in Sex-Determining.

BACKGROUND: The SRY gene (SRY) provides instructions for making a transcription factor called the sex-determining region Y protein. The sex-determining region Y protein causes a fetus to develop as a male. In this study, SRY of 15 spices included of human, chimpanzee, dog, pig, rat, cattle, buffalo, goat, sheep, horse, zebra, frog, urial, dolphin and killer whale were used for determine of bioinformatic differences. METHODS: Nucleotide sequences of SRY were retrieved from the NCBI databank. Bioinformatic analysis of SRY is done by CLC Main Workbench version 5.5 and ClustalW (http:/www.ebi.ac.uk/clustalw/) and MEGA6 softwares. RESULTS: The multiple sequence alignment results indicated that SRY protein sequences from Orcinus orca (killer whale) and Tursiopsaduncus (dolphin) have least genetic distance of 0.33 in these 15 species and are 99.67% identical at the amino acid level. Homosapiens and Pantroglodytes (chimpanzee) have the next lowest genetic distance of 1.35 and are 98.65% identical at the amino acid level. CONCLUSION: These findings indicate that the SRY proteins are conserved in the 15 species, and their evolutionary relationships are similar.

Polymeric nanocylinders by combining block copolymer self-assembly and nanoskiving.

A new facile fabrication approach to generate polymeric nanostructures is described. Block copolymers containing immiscible segments can self-assemble to generate ordered nanostructures, such as cylinders of one block in a matrix of the other in the bulk, which can then be sectioned on the nanoscale using a microtome (nanoskiving). Dispersing these sections in a selective solvent for the matrix block results in nanocylinders. In one example, we utilized a poly(N,N-dimethylacrylamide)-block-poly(styrene) (PDMA-PS) copolymer containing 36% by volume of PS. This composition was selected as it self-assembles into cylinders of PS in a matrix of PDMA. Following a previously described procedure, the cylinders were aligned using a channel die. The aligned samples were subsequently sectioned using a microtome containing a diamond knife and dispersed in water, a selective solvent for the PDMA matrix, affording PS nanocylinders with a PDMA corona. This technique allows tuning of nanocylinders without the requirement of specialty fabrication equipment.

Comparison of hormone receptor status in primary and recurrent breast cancer.

BACKGROUND: Systematic treatments such as hormone and chemotherapy are selected according to tumor characteristic after major therapeutic approaches such as surgery. This study attempted to analyze and compare the status of Estrogen Receptor (ER) and Progesterone Receptor (PR) in primary and recurrent sites of breast cancer in patients. METHODS: We reviewed all medical records of breast cancer women who were treated between January 1995 and December 2008. One hundred eighty two out of 2241 patients (8.12%) had a metastatic breast cancer. Amongst them 48 patients had tumor and biopsy-driven samples, however 13 samples were destroyed and only 35 samples were investigated in this study, therefore 35 malignant biopsy specimens of breast cancer patients were examined by immunohistochemistry essay for ER and PR. Binominal proportional test and Chi square test were conducted to determine the significant correlation between positive cases of hormone receptors among primary and metastases sites. RESULTS: Hormone Receptor in the primary tumor (HR1) of 9 patients (25.7%) was positive (ER1 and/or PR1) and in the recurrent areas (HR2) of 8 patients (22.9%) was positive (either ER2 or PR2 positive). Kappa coefficients of diagnostic agreement in primary and recurrent cases were 0.077 and 0.125 for estrogen and progesterone, respectively which indicated that the amount of coefficient of agreement is not considerable between primary and recurrent sites. CONCLUSION: The current study indicated that receptor status in recurrent tumors did not pose predictable value based on the analysis of hormone receptors in primary stage, so it is not an appropriate basis to set up therapeutic protocol in the metastatic patients. Therefore, tissue sampling and hormone receptor re-analyzing of metastatic sites should be considered in these cases.