Oxidative Stress and Disease

Typically in aerobic metabolism, organic compounds such as nucleic acids, proteins and lipids can undergo structural damage by oxidative reactions. This damage caused by reactive oxygen/nitrogen species has been recognized as “oxidative stress”. Despite the biological systems present efficient enzymatic and nonenzymatic antioxidant systems, oxidative stress indicates a pro-oxidant/antioxidant imbalance in favor of excessive generation of free radicals or decrease in the removal rate. Various diseases such as cancer, diabetes, cardiovascular diseases and neurodegenerative clearly exemplify the chronic oxidative stress. Therefore, it is important to consider that at low and moderate ROS levels, it can, for example, act as signaling molecules that support cell proliferation and differentiation and activate survival pathways in response to stress. Correlations between oxidative stress and disease should be carefully investigated in order to understand whether oxidative stress actually increases susceptibility to a particular disease or opposite

Putative biomarkers for cervical cancer: SNVs, methylation and expression profiles

Cervical cancer is primarily caused by Human papillomavirus (HPV) infection, but other factors such as smoking habits, co-infections and genetic background, can also contribute to its development. Although this cancer is avoidable, it is the fourth most frequent type of cancer in females worldwide and can only be treated with chemotherapy and radical surgery. There is a need for biomarkers that will enable early diagnosis and targeted therapy for this type of cancer. Therefore, a systems biology pipeline was applied in order to identify potential biomarkers for cervical cancer, which show significant reports in three molecular aspects: DNA sequence variants, DNA methylation pattern and alterations in mRNA/protein expression levels. CDH1, CDKN2A, RB1 and TP53 genes were selected as putative biomarkers, being involved in metastasis, cell cycle regulation and tumour suppression. The other ten genes (CDH13, FHIT, PTEN, MLH1, TP73, CDKN1A, CACNA2D2, TERT, WIF1, APC) seemed to play a role in cervical cancer, but the lack of studies prevented their inclusion as possible biomarkers. Our results highlight the importance of these genes. However, further studies should be performed to elucidate the impact of DNA sequence variants and/or epigenetic deregulation and altered expression of these genes in cervical carcinogenesis and their potential as biomarkers for cervical cancer diagnosis and prognosis.This work was funded by the Foundation for Science and Technology of the state of Pernambuco (FACEPE). The authors are grateful to Vinicius Albertin Tigre da Costa for his contributions to the construction of the figures.info:eu-repo/semantics/publishedVersio

Influence of pH on cellular growth of Pichia pastoris KM71H by fed-batch process

Pichia pastoris is a methylotrophic yeast that can be genetically engineered to express proteins for industrial use. One of the most important advantages of protein expression in P. pastoris is its capability of growing on minimal medium and efficiently secreting heterologous proteins with low secretion levels of endogenous proteins. Operational variables such as pH, temperature, stirring rate, among others, usually affect the microorganism’s growth during the fermentation processes. Therefore, the present work aimed to evaluate the influence of pH on cellular growth of P. pastoris KM71H by fed‐batch process. The fermentation run was carried out in a 1.6 L (total volume) bioreactor, being performed in two phases: In the first stage (24 h), the yeast was batchcultured in BMGH medium; while in the second stage (72 h), it was cultivated by feed‐batch operation with a feeding medium containing 50% glycerol and 12ml/l of trace metal solution. During the overall process, which lasted after 96 h, the aeration and temperature conditions were fixed at 10 ml\L.h, 1.5 vvm and 30°C, respectively. Different pH values were evaluated: 5.0, 5.5 and 6.0. Cellular growth was determined by measuring the fermentation broth UVspectrophotometric absorbance at 600 nm, which was correlated to a calibration curve (dry weight ´ optical density). Glycerol consumption was detected by HPLC analysis. P. pastoris KM71H successfully grew in all the evaluated pH values; but the highest biomass production was observed at pH 5.0 (98.79 g/L). Although P. pastoris is reported as being a microorganism able to grow over a wide pH range (from 3 to 7); it was not observed high cell density of P. pastoris KM71H strain when cultivated at pHs 5.5 and 6.0. High cellular growth is especially important for proteins secretion, as the concentration of product in the medium is roughly proportional to the concentration of cells in culture. Finally, these results reveal the possibility of obtaining high cell density of P. pastoris KM71H by fed‐bach cultivation at pH 5.0, which can be a suitable condition for the yeast application in heterologous proteins production.Conselho Nacional de Desenvolvimento Científico e Tecnológico Brazil (CNPq)Improving Skills Across Continents (ISAC ) - Erasmus Mundus External Cooperation Window (ERASMUS

In silico study for cervical cancer diagnosis: a novel gene panel

Cervical cancer (CC) is the second cause of death cancer related among women worldwide. Nowadays,it is also done visual inspection with acetic acid to aid in detection of cervical lesions.Pap smear exam is insensitive, and false negatives occur. So, more accurate methods are needed to avoid problems with late diagnosis, once that it makes more likely the tumour spread, making the treatment more challenging. Genetic aspects like SNPs (single nucleotide polymorphisms) presence and methylation events can be related to CC development. SNP presence also can affect gene expression by altering promoter activity or by changing a coding codon. Methylation is related to gene expression, once the hypermethylation of the promoter region prevents gene transcription, while hypomethylation increases gene expression. This information can be used to have a more reliable diagnosis, as well be used to predict treatment efficacy or prognosis for the cancer evolution. Our work goal was to discover genes that have deregulated expression, which could be caused by methylation or SNPs. Then, this gene panel could be used as diagnosis or prognosis approach for CC patients. The in silico analysis was performed with Metacore® software database (Thomson Reuters, USA), with the follow word combinations: cervical cancer and methylation, cervical cancer and polymorphisms and cervical cancer and gene expression. The three sets of results were combined so that only genes that were affected in all aspects were considered. For methylation there were 54 genes, while 3003 genes were linked to expression deregulation and 4095 linked to SNPs. The intersection of these genes resulted in 16 genes. This gene list included genes such as CDKN2A (ciclindependent kinase inhibitor 2A), CDKN1A (ciclin-dependent kinase inhibitor), TP53 (tumor protein p53) and TP73 (Tumor protein p73) are related to cell cycle. Both TP73 and TP53 are tumor suppressor proteins, having their expression reduced when hyper-methylated, which is consistent with cancer development. Several other tumor suppressor proteins were found, for instance: FHIT (Fragile histidine triad), PTEN (Phosphatase and tensin homolog) and RB1 (Retinoblastoma 1), RASSF1 (Ras Association domain-containing protein 1). This indicates that CC development requires extensive damage to cells mechanisms of regulation. APC (adenomatous polyposis coli) also appears in the gene panel, once it was found hypermethylated and downregulated in CC, meaning that it has its activity reduced. APC is antagonist of the WNT signaling pathway. In colorectal cancer, loss of this function is associated with cancer progression. It is interesting to notice this gene presence in cervix samples probably due to epithelial cells similarity between cervical and colotectal regions. Further in vitro studies are needed in order to confirm these in silico results, but the use of genetic markers looks promising for a more reliable and accurate diagnosis of CC

Influence of trace elements supplementation on the production of recombinant frutalin by Pichia pastoris KM71H in fed-batch process

Frutalin, a galactose-specific lectin used to detect specific tumour markers, is a protein with low expression level in breadfruit. In the present study, fed-batch fermentation in a stirred tank bioreactor was used as a strategy to enhance protein production by a recombinant Pichia pastoris KM71H. By using this process, the production of recombinant frutalin was 4-fold higher than the value obtained in shaker flasks batch assays. Supplementation of the fermentation medium with trace elements (Pichia trace minerals, PTM) was also evaluated in order to stimulate production of the recombinant protein. The addition of PTM to the minimum medium afforded a recombinant protein production of 13.4 mg L−1, which was 2.5-fold higher than that achieved from the culture medium without PTM supplementation. These results are significant as the development of strategies to improve the production of recombinant frutalin may broaden its application in cancer diagnosis.The authors are grateful for the financial support received from Erasmus Mundus External Cooperation Window - ISAC programme, and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPQ) - Brazil