e18011 Background: Tobacco consumption accounts for 1.6 million deaths annually in the South East Asia Region (SEAR). Notably, amongst 10-20% of the global population consuming the betel quid and tobacco, about 81% concentration is in SEAR regions, including India and Bangladesh. The prevalence of HNSCC in these regions is rising alarmingly. For example, HNCs account for 23% of total 156775 cancer incidences in Bangladesh. Liquid biopsy tools are unavailable and expensive for most patients in this region. However, early cancer detection using tumor biomarkers, for example, Circulating Tumor Cells (CTCs) is highly implicated. Furthermore, such biomarkers are being validated and have potential for screening of high-risk patients, such as genetic predisposition, tobacco consumption, etc. We report the first observational study in HNSCC patients in Bangladesh correlating the presence of CTCs to chronic tobacco consumption. Methods: The study involved 70 cancer patients and 10 healthy volunteers (no prior cancer history). 87% of the patients had a specified history of chronic tobacco consumption. CTCs were isolated in 1.5 ml of blood using OncoDiscover Liquid Biopsy Test, which is clinically approved by the Drug Controller General of India, that contains an enriching anti-EPCAM antibody immunomagnetic kit. CTCs are qualified as CK18+, DAPI+and CD45-. Subsequently, CTCs were imaged using Zeiss Axio Observer 7 and quantified for Mean Fluorescence Intensity (MFI) for clinicopathological parameters; age/gender, HNSCC sub-population, and CTC distribution. Results: This is the 1st study on Bangladesh phenotype accounting for the presence of CTCs in HNSCC patients. In this population, 34 males (66%) and 10 females (52%) accounted for 91 CTCs. CTC distribution was 0 to 6 with mean and median ̃ 2.02 and 2, respectively. 25 patients (17 males, 8 females) were negative for any CTCs. Interestingly, 02 patients exhibited CTC clusters indicative of aggressive metastasis in which 01 patient had no prior tobacco usage or family cancer history. There was no correlation between CTC presence in males (66 %) and females (52 %). Healthy volunteer samples exhibited no false positives. The MFI values ranged between 23 and 766, with mean and median MFI values were 157 and 96, respectively, indicative of CK overexpression on CTCs of HNSCC patients. Conclusions: HNSCC patients with a history of chronic tobacco consumption in Bangladesh correlated the presence of CTCs in 64 % of the cases. Prospectively, CTCs may be validated as a biomarker for screening chronic tobacco users in Bangladesh to detect early cancers and HNSCC. Clinical trial information: BMRC/Grants/2018/99 (1-100).

Antifreeze proteins or ice-binding proteins (IBPs) facilitate the survival of certain cellular organisms in freezing environment by inhibiting the growth of ice crystals in solution. Present study identifies orthologs of the IBP of Colwellia sp. SLW05, which were obtained from a wide range of taxa. Phylogenetic analysis on the basis of conserved regions (predicted as the 'ice-binding domain' [IBD]) present in all the orthologs, separates the bacterial and archaeal orthologs from that of the eukaryotes'. Correspondence analysis pointed out that the bacterial and archaeal IBDs have relatively higher average hydrophobicity than the eukaryotic members. IBDs belonging to bacterial as well as archaeal AFPs contain comparatively more strands, and therefore are revealed to be under higher evolutionary selection pressure. Molecular docking studies prove that the ice crystals form more stable complex with the bacterial as well as archaeal proteins than the eukaryotic orthologs. Analysis of the docked structures have traced out the ice-binding sites (IBSs) in all the orthologs which continue to facilitate ice-binding activity even after getting mutated with respect to the well-studied IBSs of Typhula ishikariensis and notably, all these mutations performing ice-binding using 'anchored clathrate mechanism' have been found to prefer polar and hydrophilic amino acids. Horizontal gene transfer studies point toward a strong selection pressure favoring independent evolution of the IBPs in some polar organisms including prokaryotes as well as eukaryotes because these proteins facilitate the polar organisms to acclimatize to the adversities in their niche, thus safeguarding their existence.

Our environment is stressed with a load of heavy and toxic metals. Microbes, abundant in our environment, are found to adapt well to this metal-stressed condition. A comparative study among five Cupriavidus/Ralstonia genomes can offer a better perception of their evolutionary mechanisms to adapt to these conditions. We have studied codon usage among 1051 genes common to all these organisms and identified 15 optimal codons frequently used in highly expressed genes present within 1051 genes. We found the core genes of Cupriavidus metallidurans CH34 have a different optimal codon choice for arginine, glycine and alanine in comparison with the other four bacteria. We also found that the synonymous codon usage bias within these 1051 core genes is highly correlated with their gene expression. This supports that translational selection drives synonymous codon usage in the core genes of these genomes. Synonymous codon usage is highly conserved in the core genes of these five genomes. The only exception among them is C. metallidurans CH34. This genomewide shift in synonymous codon choice in C. metallidurans CH34 may have taken place due to the insertion of new genes in its genomes facilitating them to survive in heavy metal containing environment and the co-evolution of the other genes in its genome to achieve a balance in gene expression. Structural studies indicated the presence of a longer N-terminal region containing a copper-binding domain in the cupC proteins of C. metallidurans CH3 that helps it to attain higher binding efficacy with copper in comparison with its orthologs.

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