Detection of mosaicism for the polymorphic variants in the 5′‐UTR of hOGG1 by cloning and sequence analysis and pyrosequencing

Mosaicism refers to the presence of genetically distinct cell lines within an organism or a tissue. Somatic mosaicism exists in distinct populations of somatic cells and commonly arises as a result of somatic mutations, mainly in early embryonic development. SNPs are important markers that distinguish between different individuals in heterogeneous biological samples and contribute greatly to disease risk association studies. In this work, we investigated the relationship between the functional variants in the 5′‐UTR of the hOGG1 gene and the risk of type 2 diabetes. Upon detection of the polymorphisms c.‐53G>C, c.‐23A>G, and c.‐18G>T in the hOGG1 gene, we found that mosaicism was present in 3/28 (10.71%), 7/51 (13.73%), and 1/44 (2.27%) patients respectively, who were carriers of these single nucleotide variations, by cloning and sequence analysis and pyrosequencing. Statistical analysis showed that the frequency of the variation c.‐23A>G in the hOGG1 5′‐UTR in type 2 diabetic patients was significantly higher than that in healthy controls. However, sequencing of the mutant alleles in mosaic individuals showed weak peaks that may affect detection of the SNPs and impair association‐based investigations.     

Metabolic heat and CO2 evolution rates measured by calorimetry during different in vitro induction processes of cineraria

Explanted cotyledons from cineraria ‘Jester Pink’ seeds were cultured in vitro on a modified Murashige-Skoog medium containing (in μM): 13.5 NAA, 13.5 2,4-D, 4.4 BAP, 13.5 NAA + 4.4 BAP, 13.5 2,4-D + 4.4 BAP or no plant growth regulators (control). Photographs were taken at 7-day intervals for 35 days to follow the embryogenic/organogenic development of the explanted cotyledons. Calorespirometric (metabolic heat rate, R_q and respiration rate, RCO₂) measurements were made in triplicate on the cultured cotyledons at the same interval as the photographs. Results showed a close correlation between the embryogenic/organogenic response of the tissues and R_q values. Cotyledons on a medium containing 2,4-D + BAP had the highest R_q values while those cotyledons that failed to develop or those of control, that suffered deterioration, had the lowest. This work showed that calorespirometric data may be useful in estimating early responses of tissues/organs in vitro embryogenic/organogenic culture systems.     

Imaging the stomatal physiology of somatic embryo-derived peanut leaves by scanning electrochemical microscopy

The stomatal physiology, chlorophyll distribution and photosynthetic activity of somatic embryo (SE)- and seedling-derived peanut plants grown in vitro (test tube-grown) and extra vitrum (soil-grown) are investigated using scanning electrochemical microscopy (SECM). This SECM imaging is performed in two different feedback modes, corresponding to oxygen evolution and chlorophyll distribution. More specifically, the oxygen evolution profiles of the in vitro leaves indicate important differences in leaf anatomy between the SE- and seedling-derived leaves. On the other hand, the chlorophyll distribution images show individual stomata of size ca. 27 ± 5μm. Further studies on senescing (aged) leaves reveal interesting voltammograms that vary widely over the stomatal complexes and the surrounding tissues, probably due to the release of electroactive metabolites during chlorophyll breakdown when the leaves turn yellow. Thus, the present investigation could open up new opportunities for characterizing botanical systems using electroanalytical techniques. In addition, it could provide further insights into various areas of current relevance, including signal transduction, cell fate/differentiation and developmental biology. Schematic representation of SECM imaging used in this investigation. The SECM probe is a Pt UME disk (25 μm diameter) embedded in an insulating glass sheath so that the ratio of the diameter of the death to that of the electrode surface (RG) is 7. RE denotes the reference electrode Ag/AgCl, sat. KCl and CE refers to the counter electrode, a Pt wire. Oxygen evolving from the leaf surface during photosynthesis diffuses into the electrolyte (0.1 M KCl) and gets reduced at the Pt UME, biased to a potential of −0.5 V, at a diffusion-limited rate to produce a change in the tip-current     

CDH1/E-cadherin and solid tumors. An updated gene-disease association analysis using bioinformatics tools

Cancer is a group of diseases that causes millions of deaths worldwide. Among cancers, Solid Tumors (ST) stand-out due to their high incidence and mortality rates. Disruption of cell–cell adhesion is highly relevant during tumor progression. Epithelial-cadherin (protein: E-cadherin, gene: CDH1) is a key molecule in cell–cell adhesion and an abnormal expression or/and function(s) contributes to tumor progression and is altered in ST. A systematic study was carried out to gather and summarize current knowledge on CDH1/E-cadherin and ST using bioinformatics resources. The DisGeNET database was exploited to survey CDH1-associated diseases. Reported mutations in specific ST were obtained by interrogating COSMIC and IntOGen tools. CDH1 Single Nucleotide Polymorphisms (SNP) were retrieved from the dbSNP database.DisGeNET analysis identified 609 genes annotated to ST, among which CDH1 was listed. Using CDH1 as query term, 26 disease concepts were found, 21 of which were neoplasms-related terms. Using DisGeNET ALL Databases, 172 disease concepts were identified. Of those, 80 ST disease-related terms were subjected to manual curation and 75/80 (93.75%) associations were validated. On selected ST, 489 CDH1 somatic mutations were listed in COSMIC and IntOGen databases. Breast neoplasms had the highest CDH1-mutation rate. CDH1 was positioned among the 20 genes with highest mutation frequency and was confirmed as driver gene in breast cancer. Over 14,000 SNP for CDH1 were found in the dbSNP database.This report used DisGeNET to gather/compile current knowledge on gene-disease association for CDH1/E-cadherin and ST; data curation expanded the number of terms that relate them. An updated list of CDH1 somatic mutations was obtained with COSMIC and IntOGen databases and of SNP from dbSNP. This information can be used to further understand the role of CDH1/E-cadherin in health and disease.     

Structural prediction,whole exome sequencing and molecular dynamics simulation confirms p.G118D somatic mutation of PIK3CA as functionally important in breast cancer patients

To understand the structural and functional importance of PIK3CA somatic mutations, whole exome sequencing, molecular dynamics simulation techniques in combination with in silico prediction algorithms such as SIFT, PolyPhen, Provean and CADD were employed. Twenty out of eighty missense somatic mutations in PIK3CA gene were found to be pathogenic by all the four algorithms. Most recurrent mutations found were known hotspot PIK3CA mutations with known clinical significance like p.E545 K, p.E545A, p.E545 G and p.C420R. A missense mutation p.G118D was found to be recurrently mutated in 5 cases. Interestingly, this mutation was observed in one of the patients who underwent whole exome sequencing and was completely absent from the controls. To see the effect of this mutation on the structure of PIK3CA protein, molecular dynamics simulation was performed. By molecular dynamics approach, we have shown that p.G118D mutation deviated from the native structure which was supported by the decrease in the number of hydrogen bonds, difference in hydrogen bond distance and angle, difference in root mean square deviation between the native and the mutant structures.