Study on Cloning, Sequencing and Phylogenetic Analysis of Granulocyte Macrophage Colony Stimulating Factor of Indian Cattle (Bos indicus)

The cDNA coding for Granulocyte macrophage colony stimulating factor (GM-CSF) from LPS activated macrophages was amplified using reverse transcriptase polymerase chain reaction (RT-PCR), which was then cloned into the pCNDA vector and sequenced. The amplified bovine GM-CSF cDNA was 755 bp length without the secretory signal peptide sequence. The ORF coded for 143 amino acids with a molecular weight of 16200 Da and was 99 percent identical to Bos taurus and Bubalus bubalis, respectively. The phylogenetic study found that the GM-CSF sequences of swine and horse are the most distinct from those of buffalo, sheep, goat, and deer.

Author (S) Details

Ramya Kalaivanan

Veterinary College and Research Institute, Namakkal-02, India. and Indian Veterinary Research Institute, Hebbal, India.

P. Sankar

Veterinary College and Research Institute, Namakkal-02, India.

Subodh Kishore

Indian Veterinary Research Institute, Hebbal, India.

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Epigenetic Sensitisation of Chemotherapeutic Compounds in Non- Small Cell Lung Cancer

Lung cancer is now the leading cause of cancer-related death worldwide, with 1.7 million fatalities in 2018. The most common kind of lung cancer is non-small cell carcinoma (NSCLC), which accounts for about 80% of all occurrences. In most countries, chemotherapy is still the first-line treatment for NSCLC patients. Chemotherapy is expected to be utilised for at least another two decades, despite the development of novel medicines (targeted chemicals, immunotherapy). Chemotherapy resistance is one of the key variables contributing to lung cancer mortality, and it has been well established in preclinical investigations and clinical trials to date. Currently, there are only a few biomarkers for predicting chemotherapy efficacy, most of which are clinical. Furthermore, clinically, sensitization of cancer cells to such chemicals is largely useless. This research looks into new ways for lung cancer cells to become sensitive to four standard chemotherapeutics (cisplatin, carboplatin, gemcitabine, and vinorelbine), with a focus on epigenetics and the involvement of extracellular vesicles. Cloning, inducible transgene expression, shRNA-based silencing, proliferation and apoptosis assays, the neutral comet assay, RT-qPCR, pyrosequencing-based DNA methylation analysis, and other molecular and cell biology methods (cloning, inducible transgene expression, shRNA-based silencing, proliferation and apoptosis assays, the neutral comet assay, RT-qPCR I examined a wide range of factors in connection to drug resistance in NSCLC cell lines (e.g., western blots, EV isolation and characterization). Resistant cell lines were chosen for sensitization with epigenetic medicines (VPA and DAC), aminomethylphosphonic acid (AMPA), and fendiline after the IC50 of each of the four medications was determined in eight NSCLC cell lines.

VPA showed strong chemotherapeutic sensitization potential for all four chemotherapeutics, which is a first for lung cancer cells. In cells treated with cisplatin or carboplatin, both VPA and DAC caused a considerable increase in apoptotic activity. Gemcitabine produced double strand DNA breaks in the NSCLC cell lines A549, CALU-6, and COR-L23, and pre-treatment with VPA amplified this impact, which was also a novel discovery in this investigation.

I also looked at the role of LANCL1-AS1, a long-noncoding RNA that was shown to be reduced in NSCLC tissues by our research team. Following the establishment of an inducible expression model in the SK-MES-1 cell line, it was discovered that LANCL1-AS1 induced an increase in proliferation rate, migration invasion, sensitivity to gemcitabine and vinorelbine, increased resistance to platin compounds, and, most notably, expression of its coding counterpart gene, LANCL1. The LANCL1 shRNA-based silencing resulted in a reduction in proliferation, migration, and oxidative stress sensitivity. However, sensitivity to all four medications followed the same pattern as overexpression of LANCL1-AS1, raising doubts about whether these two activities are connected or separate, with no way to rule out the possibility of off-target effects from the shRNA method at this time.

The function of extracellular vesicles (EVs) in cancer development and medication resistance has just lately been studied. As a result, I decided to see if the LANCL1-AS1-dependent gemcitabine sensitivity could be transferred to EVs. Increased LANCL1-AS1 expression resulted in a dose-dependent increase in EV release. Unfortunately, due to a variety of technical concerns with the EV isolation reagent’s toxicity, knowledge of how LANCL1-AS1 produced EVs affect gemcitabine resistance when transmitted to a recipient cell line was not possible. Finally, this research has revealed some new insights towards sensitising NSCLC cells in a preclinical setting. More research is needed to determine whether these findings are clinically useful and how they might alter lung cancer treatment.

Author(s) Details

Ghaliah Obaid F. Alnefaie
Medical Genetic Department, Taif University, Taif, Saudi Arabia.

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