Experiment study on the transfection of exogenous genes promoted by ultrasound-targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal / 中华超声影像学杂志

Objective To increase the transfection of EGFP-N3 plasmids into 293T cells using ultrasound-targeted microbubbles delivery(UTMD) mediated a peptide nucleic acid (PNA) binding nuclear localization signal (NLS).Methods Antibody-targeted microbubbles were used in the experiments which can specifically recognize the SV40T antigen receptor.The SV40T antigen receptors were expressed on the membranes of 293T cells.The PNA containing the NLS were inserted in the EGFP-N3 plasmid DNA,which increased nuclear localization.Ultrasound-targeted microbubble delivery (UTMD) and the PNA binding NLS were utilized to improve the cytoplasmic import of plasmids and the nuclear intake of the plasmid from the cytoplasm,respectively.The study was divided into five groups:Contrast (group A),Common microbubble + DNA (group B),Antibody-targeted microbubbles + DNA (group C),Common microbubbles + NLS-PNA-DNA (group D),Antibody-targeted microbubbles + NLS-PNA-DNA (group E).Fluorescence microscope was used to observe the fluorescent light in each group;flow cytometry to test the transfection;RT-PCR and Western blot to detect genes' mRNA and protein expression level.Results Ultrasound and antibody-targeted microbubble delivery (UTMD) significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability(＞80％).Fluorescent microscope showed that the quantities of green fluorescence in cells were increased successfully.The transfection results of flow cytometry were 0,(9.30 ± 0.46)％,(26.46 ± 2.01)％,(29.54 ± 0.62)％,(45.72 ± 1.86)％,respectively,and the differences were statistically significant(P ＜0.05).The relative mRNA and protein expression in group E were greater than those in group C and D respectively (P ＜0.05).Conclusions UTMD combined with antibody-targeted microbubbles and a PNA binding NLS plasmid can significantly improve transfection efficiency of exogenous genes by enhancing both cytoplasmic and nuclear DNA import.

Progress of synthesis of modified peptide nucleic acid / 中国药学杂志

Peptide nucleic acid (PNA) is DNA mimic, in which the sugar-phosphate backbone is replaced by polyamide structure. Its basic framework is N-(2-aminoethyl) glycine. PNA has good chemical, physical, and biological properties, such as high stability, strong specificity, not hybridization by nuclease and protease. But it has poor water solubility, low membrane permeability. In this paper, the synthesis of modified peptide nucleic acid is reviewed from the backbone, bases and the linker of backbone and base, which makes PNA have better application prospect.

Epidermal growth factor receptor mutation in non-small-cell lung carcinomas: A retrospective analysis of 1036 lung cancer specimens from a network of tertiary cancer care centers in India.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutation plays a vital role in the prognosis of patients with lung cancer. However, there is a dearth of studies on EGFR mutation in Indian population. In this retrospective study conducted at a network of tertiary cancer care centers across India, we evaluated the proportion of EGFR mutation in patients with non‑small‑cell lung carcinomas (NSCLC). MATERIALS AND METHODS: A total of 1036 cases of non‑small lung cancer were assessed for EGFR mutation status using Scorpion amplified refractory mutation system real time polymerase chain reaction method from fine needle aspiration cytology core biopsy, pleural fluid and cell blocks. For a few cases, macro dissection of tumor from H and E slides was also performed for EGFR analysis. EGFR Status was assessed for the most commonly known driver mutations in Exons 18, 19, 20 and 21, which contributes to a total of 29 somatic mutations including the resistance mutation T790M. RESULTS: Around 39% of the cohort was female and 61% were male. Mutation was positive in 40.3% and negative (wild type) in 59.7%. There was 1.8% mutation in exon 18, 24.6% in exon 19, 1.6% in exon 20 and 12.8% in exon 21. 38.2% had a mutation in a single site and 1.1% had a mutation in two sites. Overall mutation was significant in females (50.5% vs. 33.9%) compared with males (c2 = 28.3, P < 0.001). Mutation was significant in exon 21 (16.8% vs. 10.3%, c2 = 9.44, P = 0.002) and exon 19 (30.7% vs. 20.7%, c2 = 13.2, P < 0.001) in females compared with males. CONCLUSION: EGFR is expressed differentially/ mutated in patients with NSCLC. Further studies to unravel the predictors for acquired genetic alterations of EGFR are needed.

Applications of antisense technology in discovery and development of novel antibacterial drugs / 国际药学研究杂志

Antibiotic-resistance has become a serious problem to human health. Innovative approaches are urgently required for both antimicrobial drug discovery and reversal of resistance. Great success has been achieved since the introduction of antisense technology. This review focuses on the applications of antisense technology in the aspects including identification and validation of novel antibacterial drug targets, screening of antibacterial drugs from natural products using hypersensitized strains engineered by inducible antisense RNA, and inhibition of antibiotic-resistance genes to reverse susceptibility of antibiotic-resistant strains to currently used antibiotics. In addition, the antibacterial activities, the shortcomings and prospects as antibacterial drugs of synthetic antisense oligodeoxynucleotides and their analogs are also assessed in this review.

Peptide nucleic acid-mediated one-step PCR assay in detection of K-ras mutation / 第二军医大学学报

Objective: To establish a peptide nucleic acid-mediated one-step PCR assay for detecting K-ras mutation, and to evaluate its diagnostic value. Methods: We developed a one-step polymerase chain reaction (PCR) approach with melting curve analysis using wild-type specific peptide nucleic acid (PNA) and fluorescent dye SYBR Green I to determine the genotypes in codon 12 and 13 of K-ras oncogene. The result of our method was compared with that of restriction fragment length polymorphism (RFLP) analysis. Results: Our method simultaneously examined condon 12 and 13 of K-ras oncogene, and was easy to perform. The sensitivity of our method was 0.001% when in a 105-fold excess of wild-type K-ras DNA. The detection limit was 102 copies. Our method could be used for large sample test, with the time of a single test being 1 hour. The limit of traditional method was 103 copies and the sensitivity was 0.01%. The testing time was about 2 days for samples which needed only 1 hour using our method. Conclusion: Our method has obvious advantage over RFLP analysis; it is suitable for detecting K-ras mutation in large samples and can be used as an effective method for early diagnosis of pancreatic cancer.

Effects of Peptide Nucleic Acids against Ki-67 Gene on the Proliferation and Apoptosis of Human Renal Carcinoma Cell Line / 华中科技大学学报（医学）（英德文版）

To investigate the effects of anti-sense peptide nucleic acids (PNAs) targeting Ki-67gene on modulation of the proliferation and apoptosis of human renal carcinoma cell lines, human renal carcinoma cell line 786-0 cells were treated with anti-sense PNAs at different concentrations (1.0 μmol/L, 2.0 μmol/L, 10.0 μmol/L). The Ki-67 expression of 786-0 cells was detected by immunohistochemical technique and Western blot method respectively. The proliferation of 786-0 cells was studied by cell growth curves and 3H-thymidine incorporation. The apoptosis of 786-0 cells was detected by TUNEL assay. The control groups were treated with anti-sense oligonucleotide (ASODNs)targeting Ki-67 gene. Our results showed that the Ki-67 expression of 786-0 cells treated with anti-sense PNAs (16.9±0.7) was significantly inhibited as compared with that of the control groups (28.6±0.4) (P＜0.01). The Ki-67 protein rate of 786-0 cells treated with anti-sense PNAs (42.1±2.2)was significantly reduced when compared with that of the control groups (83.6±1.4) (P＜0.01). Proliferation of 786-0 cells treated with anti-sense PNAs (20.7±1.5) was significantly inhibited as compared with that of the control groups (58.6±1.4) (P＜0.01). The apoptosis rate of 786-0 cells treated with anti-sense PNAs (28.7±2.3) was significantly increased higher compared with that of the control groups (13.8±1.0) (P＜0.01). From these finds we are led to conclude that anti-sense PNAs targeting Ki-67 gene have stronger effects on the inhibition of the proliferation and induction of apoptosis of human renal carcinoma cells than ASODNs targeting Ki-67 gene. The strategies using anti-sense PNAs targeting Ki-67 gene may be a promising approach for the treatment of renal cell carcinoma.

Blocking the CC chemokine receptor 5 pathway by antisense peptide nucleic acid prolongs islet allograft survival / 中国普通外科杂志

Objective To identify the effect of peptide nucleic acid of CC chemokine receptor 5 on acute rejection of islet allograft.Methods Mice islet transplant models were used to test the effect of PNA CCR5 by targeting CCR5 in acute allograft rejection.In vitro T cell proliferative responses were assessed by mixed lymphocyte response(MLR).RT-PCR and Western blot were used to detect the expression of mRNA and protein.Results PNA CCR5-treated recipients demonstrated statistically significant prolongation(12.00?1.75)d in functional allograft survival when compared with saline(6.50?0.58)d or PNA mismatch-treated recipients(6.50?0.50)d.The CCR5 mRNA expression level of PNA CCR5,control,and PNA mismatch treatment recipients at day 7 posttransplant was 0.56?0.05,1.68?0.07 and 1.80?0.14,respectively.The data showed that CCR5 protein was significantly down-regulated in PNA CCR5 treatment allografts compared with saline and PNA mismatch treatment allografts(P