Functional investigation of the BRCA1 Val1714Gly and Asp1733Gly variants by computational tools and yeast transcription activation assay.

Mutations in the BRCA1 gene are known to be a major cause of hereditary breast cancer. However, characterizing the point mutations associated with cancer in BRCA1 is challenging because the functional impact of most of them is still unknown. Nowadays, a variety of methods are employed to identify cancer-associated mutations in BRCA1. This study is aimed to assess the functional effects of two mutations, Asp1733Gly and Val1714Gly, using a combination of in silico tools and yeast functional transcription activator assay. Our computational analysis showed that theVal1714Gly mutation was deleterious, while the other one, Asp1733Gly, predicted as neutral. Also using yeast functional transcription activator assay, we found that the Asp1733Gly mutation displayed similar ability with positive controls. In contrast, the Val1714Gly mutation completely abrogated transcriptional activity in the yeast. These results suggested that Val1714Gly and Asp1733Gly can be classified as pathogenic and benign mutations for the BRCA1, respectively.

Exosomes in Cancer Liquid Biopsy: A Focus on Breast Cancer.

The important challenge about cancer is diagnosis in primary stages and proper treatment. Although classical clinico-pathological features of the tumor have major prognostic value, the advances in diagnosis and treatment are indebted to discovery of molecular biomarkers and control of cancer in the pre-invasive state. Moreover, the efficiency of available therapeutic options is highly diminished, and chemotherapy is still the main treatment due to lack of enough specific targets. Accordingly, finding the new noninvasive biomarkers for cancer is still an important clinical challenge that is not achieved yet. There are current technologies to screen, diagnose, prognose, and treat cancer, but the limitations of these implements and procedures are undeniable. Liquid biopsy as a noninvasive method has a promising future in the field of cancer, and exosomes as one of the recent areas have drawn much attention. In this review, the potential capability of exosomes is summarized in cancer with the special focus on breast cancer as the second cause of cancer mortality in women all around the world. It discusses reasons to choose exosomes for liquid biopsy and the studies related to different potential biomarkers found in the exosomes. Moreover, exosome studies on milk as a specific biofluid are also discussed. At last, because choosing the method for exosome studies is very challenging, a summary of different techniques is provided.

Clinical significance of NDRG3 in patients with breast cancer.

AIM: The expression level of NDRG3 gene is investigated among breast cancer (BC) patients. METHODS: Real-time quantitative PCR was performed. RESULTS:  NDRG3 was downregulated in BC patients particularly in advanced stage of the disease. HER2 status was significantly correlated with the expression of NDRG3. Also, triple-negative BC patients showed low levels of NDRG3 expression in comparison to other subtypes. Lastly, the expression of NDRG3 had significant impact on survival, with NDRG3 downregulated patients having the worst event-free survival rate among others. CONCLUSION: We have presented that NDRG3 might be a tumor suppressor candidate. NDRG3 downregulation might be involved in the tumorigenesis and development of invasive BC in an advanced phase of the disease.

Expression of granulocyte colony stimulating factor (GCSF) in Hansenula polymorpha.

BACKGROUND AND OBJECTIVES: During past decades Hansenula polymorpha has attracted global attention for the expression of recombinant proteins due to its high growth rate, minimal nutritional porequirements and use of methanol as a low cost inducer. MATERIALS AND METHODS: The corresponding nucleotide sequences for the expression of heterologous genes in Hansenula poylmorpha were extracted and assembled in an E. coli vector. The constructed expression cassette included formate dehydrogenase promoter (pFMD), a secretory signal sequence, a multiple cloning site (MCS) and methanol oxidase (MOX) terminator. Zeocin resistance gene fragment and complete cDNA encoding granulocyte colony stimulating factor (GCSF) were cloned downstream of the expression cassette in-frame with signal sequence. Restriction mapping and sequence analysis confirmed the correct cloning procedures. Final vector was transformed into Hansenula and recombinant host was induced for the expression of GCSF protein by adding methanol. SDS-PAGE and immuno-blotting were performed to confirm the identity of r-GCSF. RESULTS: The expression cassette containing gcsf gene (615bp) and zeocin resistance marker (sh-ble, 1200bp) was prepared and successfully transformed into competent Hansenula polymorpha cells via electroporation. Zeocin resistant colonies were selected and GCSF expression was induced in recombinant Hansenula transformants using 0.5% methanol and an approximately 19kDa protein was observed on SDS-PAGE. Western blot analysis using serum isolated from GCSF-treated rabbit confirmed the identity of the protein. CONCLUSIONS: Molecular studies confirmed the designed expression cassette containing gcsf gene along with pFMD and signal sequence. The expressed 19kDa protein also confirmed the ability of designed vector in expressing heterologous genes in Hansenula cells.