आण्विक जीवविज्ञान: खुली पहुंच

A cornerstone of autologous cell therapy for Duchenne muscular dystrophy is the engineering of suitable cells to express dystrophin in a stable fashion upon differentiation to muscle fibers. Most viral transduction methods are typically restricted to the expression of truncated recombinant dystrophin derivatives and by the risk of insertional mutagenesis, while non-viral vectors often suffer from inefficient transfer, expression and/or silencing. Here we addressed such limitations by using plasmid vectors containing nuclear matrix attachment regions (MAR). Using in vitro transfection and intra muscular transplantation in nude and immunosuppressed mdx mice, we show that clones of mesoangioblast skeletal muscle progenitors can be generated to mediate stable expression from MAR-containing vectors, while maintaining their ability to differentiate in vitro and in vivo and to express dystrophin after transplantation in dystrophic mouse muscles. We conclude that the incorporation of MARs into plasmid vectors may improve non-viral plasmid-based cell therapy feasibility.

Background: Molecular genetic techniques have greatly improved the understanding of pediatric illnesses. Based on our own experience, the aim of this review is to illustrate the importance of such molecular studies for the development of the pediatric surgery.

Methods: Various molecular techniques for the investigation of DNA, RNA, Chromosomes, gene sequences, genomic rearrangements and gene amplifications have been employed to answer the clinical and therapeutic questions. Suitable statistic methods allowed the comparison of the results between patients and controls whenever possible.

Results: We demonstrated that essential information for diagnostics and therapy of pediatric illnesses can be obtained through molecular genetic testing, including (but not limited to) translocations and consequent protein chimera expression in infantile lung tumors, the occurrence of genomic variants associated to neuroblastomas, the occurrence of microdeletions and insertions in target genes for autonomic diseases, the significantly higher occurrence of target gene mutations and polymorphisms in hypertrophic pyloric stenosis, the identification of a somatic gonosomal mosaicism and uniparental disomy in a complex disorder of sex differentiation and finally the simultaneous occurrence of embryonal tumors as early consequences of genomic instability.

Conclusions: The importance of molecular genetic research for the development of pediatric surgery is evident from the multiple findings hereby described, demonstrating that the application of molecular genetic techniques and the development of a “genetically oriented” thinking for diagnostic and therapy strategies may indeed broaden the expertise and knowledge of pediatric surgeons, ultimately resulting in a better quality of care and higher rate of success for the pediatric patients.

About 95% of multi-exonic genes express more than one mRNA and downstream proteins by alternative splicing (AS) through the inclusion or exclusion of specific exons. Although AS provides a significant advantage in human evolution by increasing proteomic diversity, deregulation of AS can result in various pathologic conditions. The androgen receptor (AR), encoded by AR gene, is a steroid receptor transcription factor which mediates the cellular functions of androgen. The AR-mediated androgen actions play important and dual roles in the human reproduction development and function. Dysregulation of AR will result in human infertility. Multiple AR alternative splicing variants have been identified in different pathologies conditions, including androgen insensitivity syndrome, which will cause male infertility. More recently, our group has identified two AR splice variants expressed in granulosa cells from patients with polycystic ovary syndrome, which is one of the most common causes of female infertility. All of the aforementioned indicate that androgen receptor alternative splicing may be an important pathogenic mechanism in human infertility. The purpose of this review is to summarize the various alternatively spliced AR variants that have been discovered, with a focus on their role and origin in the pathologies of the human infertility diseases, including polycystic ovary syndrome and androgen insensitivity syndrome.

Background: Thyroid transcription factor gene (FOXE1) and Foxp3 play important roles in autoimmune and inflammatory disease as well as human malignancies. We aimed to investigate the association of FOXE1 and Foxp3 polymorphism with the susceptibility to differentiated thyroid cancers (DTC).

Methods: Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 350 DTC patients and 306 healthy controls.

Results: AA/AG genotype of FOXE1-rs1867277 and AA/AC genotype of Foxp3-rs3761548 were associated with a higher risk of DTC. The frequency of Foxp3-rs2280883 CC/CT genotype was lower in DTC patients. Besides, the AA/AC genotype of rs3761548 was more frequent in female DTC than the male. We further analyzed the association between 3 single nucleotide polymorphisms (SNPs) and DTC. We found that rs3761548 AA/AC genotype was more frequent in severe DTC patients (tumor diameter >1 cm) compared with the relative tender DTC patients (tumor diameter <1 cm). On the contrast, the frequency of rs2280883 CC/CT genotype was lower in severe DTC patients.

Conclusion: Our findings suggested that FOXE1 and Foxp3 polymorphism were associated with the risk of DTC in Chinese Han population. Besides, rs3761548 AA/CC genotype was a potential risk factor for the susceptibility to DTC and rs2280883 CC/CT was a protective factor.

Providencia rettgeri (P. rettgeri) is a clinically significant Gram-negative bacterium of genus Providencia, and commonly associated with hospital-acquired infection like urinary tract infection (UTI), gastroenteritis, and ocular infections. Present study was designed to evaluate the effect of biofield treatment on P. rettgeri against antimicrobial susceptibility, biochemical reaction pattern, biotype number, and 16S rDNA sequence. The samples of P. rettgeri (ATCC 9250) were divided into three groups: Gr.I (control), Gr.II (treatment, revived), and Gr.III (treatment, lyophilized). The Gr.II and III were treated with Mr. Trivedi’s biofield, and then subsequently characterized for antimicrobial susceptibility, minimum inhibitory concentration (MIC), biochemical reactions, and biotype numbering. The 16S rDNA sequencing was carried out to correlate the phylogenetic relationship of P. rettgeri with other bacterial species. The treated cells of P. rettgeri showed an alteration in susceptibility of about 50% and 53.3% tested antimicrobials of Gr.II on day 5 and 10, respectively; and 53.3% of tested antimicrobials of Gr.III on day 10. MIC results showed a significant decrease in MIC values of 53.1, 56.3, and 56.3% antimicrobials in Gr.II on day 5, Gr.II on day 10, and Gr.III on day 10, respectively, as compared to control. The significant changes in biochemical reactions and biotype numbers were also observed in all the treated groups of P. rettgeri. Based on nucleotides homology and phylogenetic analysis the P. rettgeri was found to be Proteus mirabilis (GenBank Accession Number: AY820623) and nearest homolog species was found to be Proteus vulgaris (Accession No. DQ499636). These findings suggest that biofield treatment can prevent the emergence of absolute resistance of existing antimicrobials to P. rettgeri.

Aberrant regulation of rRNA genes has been reported in human cancers. The aim of this study was to analyze the epigenetic regulation of rRNA genes in lung cancer and to correlate methylation levels of the promoter, 18S and 28S regions with different chromatin states. Here we report, that methylation of rDNA ranged from 10% to 30% at individual CpG sites in the promoter region. In primary lung cancers a 1.2-fold increased rDNA methylation was observed at 19 analyzed CpGs (p < 0.001). Moreover, we report an increased methylation level of rDNA towards the 28S region of rRNA genes. This hypermethylation was more pronounced in cancer cell lines compared to primary tissue. To analyze the methylation status of protein-enriched rDNA, we utilized a technique that combines ChIP and bisulfite sequencing. Pol I- and CTCF-associated rDNA exhibited reduced methylation levels compared to global rDNA. Histone H1- associated promoter regions showed a 1.5-fold increase in methylation levels compared to H3-associated rDNA. In DNMT1 knock out cells a strong reduction of methylation of 18S and 28S regions was found in comparison to wild type HCT116 cells (2.7- and 1.4-fold reduction, respectively). In double DNMT1 and DNMT3B knock out cells no substantial rDNA methylation was detected. Independent of this hypomethylation, 28S level and number of rDNA repeats were constant in wild type, single knock out and double knockout HCT116 cells. Our data suggest that aberrant methylation of rDNA occurs in human cancer and that rRNA gene activity can be modulated in a constant manner independent of the level of methylated rDNA.

Adiponectin (ADIPOQ) is an abundant protein hormone which belongs to a family of adipokines. It is expressed mostly by adipocytes and is an important regulator of lipid and glucose metabolism. It was shown that decreased serum adiponectin concentration indicated insulin resistance in type 2 diabetes mellitus (T2DM) with the risk of cardiovascular complications. The fact that adiponectin is an insulin-sensitizing hormone with anti-diabetic, anti-inflammatory and anti-atherogenic properties. In this short commentary we are trying to confirm the association of ADIPOQ gene polymorphisms in subjects with T2DM. Furthermore, genetic study in a larger population will address the answer.

The Affymetrix DMETT^MM plus platform (Affymetrix, Santa Clara, CA, USA) is a GeneChip where 1936 SNPs can be genotyped in any given sample at once. The 1936 SNPs were distributed cross 225 genes in the genome. Thirtynine genes on the chip belong to phase I–II drug metabolism, disposition and drug transport gene family. These genes are functional in metabolizing the most widely prescribed anticancer drugs in the world including aromatase inhibitors, tamoxifen and thiopurines groups. In a high-throughput GeneChip array which is based on hybridization with allelespecific probes; genotyping errors are very common which limits the technologies applications; in addition missing calls for many SNPs on the chip immerge as a bigger and more serious problem in high- throughput genotyping methods. This study focuses on re-genotyping these No-call genotypes to maintain sample sizes that are already genotyped by Affymetrix DMETT^MM plus platform. Sixty six different variations were identified, 39 of them had No-call genotypes; our re-genotyping resulted in increasing calling rate from 89.08% to 95.56%. Furthermore, Among the variations that were identified, 8 were non-reported: C.-209T > G in UGT2B7 and -698°C > A in CYP1A2 as promoter variants, c.252G > T in SLC22A6 and c.1356T > C in SLC15A1 as silent mutation, c.1277 + 69°C > T and c.1277 + 82°C > T in SLC22A1 as intronic variants associated with each other and the most important variations are two missense (non- synonymous) in NAT2 gene: D20N c.58G > A and G11S c.31G > A. Affymetrix DMETTMM plus platform.