Predictive value of KRAS/NRAS, IL-4 VNTR and HPV alterations in metastases of colorectal cancer.

PURPOSE: The main aim of the study was to evaluate the potential roles of KRAS/NRAS proto-oncogenes, IL-4 VNTR variants and HPV prevalence in colorectal cancer metastasis. As the second aim, the interactions of the analyzed genes and viral sequences with both clinicopathological variables and each other were targeted. METHODS: DNA was extracted using AmoyDx FFPE DNA Extraction kit from paraffin-embedded colorectal tumor tissue samples (n = 60). NRAS/KRAS mutational profiles were determined with real-time polymerase chain reaction using AmoyDx KRAS/NRAS Mutation Detection Kit. Genotyping of IL-4 VNTR was made with PCR. HPV detection was analyzed by PCR with both GP5+/GP6+ consensus primers and type-specific primers for HPV-16 and HPV-18. SPSS v22 (IBM) statistics software was used for all statistical analyses. RESULTS: From the demographical/clinicopathological parameters, age and biopsy specimens revealed an association with metastasis. KRAS mutation rate was as high as 65 % in the patients and the most prevalent mutation type was G12D. Metastasis risk was 3.19-fold increased in KRAS-mutated patients compared to KRAS-negative ones. IL-4 VNTR genotypes/alleles were not associated with metastasis in our analysis.  The frequency of HPVs in our colorectal cancer cohort was 36.7 %, but HPV positivity was not found to be associated with metastasis.  A significant association was found between HPV and NRAS mutations; NRAS wild-type status acted as a protective factor by 7.5-fold for HPV negativity. CONCLUSION: Our study comprehensively and concomitantly evaluated several potential molecular risk factors. Future studies designed in such combined approaches will substantially contribute to better manage colorectal cancer tumorigenesis from molecular biological perspective (Tab. 6, Fig. 2, Ref. 40).

Detection of multidrug-resistant Pseudomonas isolates and distribution of denitrifying functional genes.

The aim of this study was to characterize 59 Pseudomonas strains isolated from soil samples in terms of Inducible Beta-Lactamase (IBL), fluorescence production, antibiotic resistance, presence of plasmids and genetic diversity, as well as denitrification functional genes. Fourteen out of fifty-nine (23.7%) Pseudomonas isolates were identified as multidrug-resistant. To evaluate the contribution of denitrification functional genes to genetic diversity, PCR products were screened by RFLP. It was determined that the 18, 6 and 22 out of 59 isolate harbored nirS, nirK and nosZ genes, respectively. It was found that the 37 isolates were nosZ-negative. Thus, these results suggest that nosZ gene-missing pseudomonad denitrifiers are partly contribute to N2O emission. Moreover, nirS, nirK and nosZ genes were found to be positive with IBL and negatively correlated with fluorescence production. These results suggest that Pseudomonas species have important roles in soil and even in biosphere due to their diversity and genetic factors.

Frequency of retrotransposon human endogenous retrovirus-K113 and a preliminary analysis of some microbial clues in bipolar disorder.

Aim: The aim of our study was to investigate whether the retrotransposon human endogenous retrovirus (HERV)-K113 could be related with bipolar disorder or not. As a second and a preliminary aim, we also conducted bacterial screening in whole blood in a limited number of samples. Patients & methods: Three separate PCR reactions including the preintegration sites and sites within the viral sequences were performed for HERV-K113 detection. Bacterial screening was performed with SSCP/sequencing analysis. Results & conclusion: No difference was observed in terms of the frequency of retrotransposon HERV-K113 in Turkish bipolar disorder patients and healthy controls. SSCP/sequencing and alignment analysis for bacterial screening reflected the possible presence of different bacteria. We strongly recommend the broadened retrotransposon and microbial diversity analyses in bipolar disorder for future studies.

Quantification of denitrifier genes population size and its relationship with environmental factors.

The objectives of this study were to use real-time PCR for culture-independent quantification of the copy numbers of 16S rRNA and denitrification functional genes, and also the relationships between gene copy numbers and soil physicochemical properties. In this study, qPCR analysis of the soil samples showed 16S rRNA, nirS, nirK, nosZI and nosZII average densities of 3.0 × 108, 2.25 × 107, 2.9 × 105, 4.0 × 106 and 1.75 × 107 copies per gram of dry soil, respectively. In addition, the abundances of (nirS + nirK), nosZI and nosZII relative to 16S rRNA genes were 1.4-34.1%, 0.06-3.95% and 1.3-39%, respectively, confirming the low proportion of denitrifiers to total bacteria in soil. This showed that the non-denitrifying nosZII-type bacteria may contribute significantly to N2O consumption in the soil. Furthermore, the shifts in abundance and diversity of the total bacteria and denitrification functional gene copy numbers correlated significantly with the various soil factors. It is the first study in Turkey about the population size of denitrification functional genes in different soil samples. It also aims to draw attention to nitrous oxide-associated global warming.

Community structures and comparison of nosZ and 16S rRNA genes from culturable denitrifying bacteria.

The objectives of this study were (i) to isolate and characterize of cultivable denitrifying bacteria using classic microbiological and molecular methods, (ii) to compare of 16S rRNA and nosZ genes as molecular markers, (iii) to determine bacterial community structure and diversity in soil samples using single-strand conformation polymorphism (SSCP) analysis. In this study, 49 bacterial isolates were cultivated and phylogenetic analyses grouped them into two phyla: Proteobacteria (37 species) and Firmicutes (12 species). Our study showed that the nosZ functional gen could be used to identify denitrifying bacteria abundance in environment but could not be used to identify pure bacterial cultures. In addition, the bacterial community structure showed significant differences among the various soil types. Phylogenetic analysis of community structure indicated that 51 clones could be divided into 2 phylotypes. Uncultured bacteria (80.4%) and Gammaproteobacteria (19.6%) were the dominant components of the soil bacterial community. For 16S rRNA, PCR products of 49 bacteria were obtained with 27F-1492R primer pairs. For nosZ, PCR products were obtained with primers 1F-1R (259 bp), 2F-2R (267 bp), and F-1622R (453 bp) of 39 bacteria that the single nosZ band provided on the agarose gel. The bacterial 16S rRNA gene clone library was dominated by Gammaproteobacteria and Bacilli. The nosZ clone sequences did not represent the bacteria from which they were obtained but were found to be closer to the environmental clones. Our study showed that the nosZ functional gene could be used to identify denitrification abundance in environment but could not be used to identify pure bacterial cultures. It was also found that the nosZ sequences showed uncultured denitrifier species.

Detection of Cryptosporidium species in the sea and tap water samples of Black Sea, Turkey.

The aim of this study was to evaluate Cryptosporidium spp. contamination of sea and tap water samples from Sinop and Ordu Provinces, Black Sea, Turkey. The samples (10 L) were collected in spring, summer, autumn, and winter in 2011. A total of 128 water samples was analyzed using an immunofluorescence test (IFT), as well as loop-mediated isothermal amplification (LAMP) and nested polymerase chain reaction (PCR). Cryptosporidium spp. oocysts were detected by IFT in 43 of the 70 samples (61.4%; 1-40 oocysts per 0.5 L) and 35 of the 58 samples (60.3%; 1-23 oocysts per 0.5 L) in the sea water samples from Ordu and Sinop, respectively. The highest number of oocysts by IFT were detected in spring and winter in Ordu and Sinop, respectively. The results of the S-adenosylmethionine synthetase (SAM) gene LAMP assays were 65.5% positive for Cryptosporidium parvum , Cryptosporidium hominis , and Cryptosporidium meleagridis in all examined samples, while the SSUrRNA gene nested PCR assay was 31.0% positive. Six C. parvum nested PCR products from all positive samples were successfully sequenced.

[Diagnosis of protozoa by loop-mediated isothermal amplification: (LAMP)]. / Protozoonlarin tanisinda ilmige Dayali Izotermal Çogaltma Yöntemi (LAMP).

Plasmodium, Isospora, Toxoplasma, Babesia and Cryptosporidium are parasites which have a significant role in human health. The location in which the protozoon settles in the body, the way that it leaves the host, and the sample which is send from the clinic are the elements of the general methods used in the diagnosis of the ailments caused from protozoan. Classical methods are not adequate for the identification of some protozoon; also, molecular methods have to be used in designating the distinctions between the species. In recent years, one of the molecular methods which has been used frequently in the identification of protozoon is the technique of LAMP. With the aid of the LAMP technique, from which it is possible to obtain reliable outcomes without the contribution of technical skills and professional equipment, in constant temperature it is possible to have a great number of copies from the targeted DNA in a short period. The aim of this collation is to give information about the usage of the technique of LAMP in the identification of the protozoa which are important for human health and the comparison between the technique of LAMP and other molecular methods.