The amplification of the highly conserved chromo-helicase-DNA binding region found in both the Z and W chromosome was evaluated with three sets of primers (P8/P2, 1237L/1272H and 2550F/2718R). DNA extracted from feathers through a simple boiling method was used to address its reliability in generating the sex-linked bands. All the bird samples, including the seven bird families that have not been reported previously, were successfully amplified with the primer set 2550F/2718R. The resulting polymerase chain reaction products showed clearly resolved fragments on a conventional agarose gel electrophoresis with size differences ranging from 80 to 540 bp between the two respective ZW gene copies. Although the P8/P2 primer was not as effective under the same conditions, it was able to produce well-resolved Z and W bands from bird species under the Antidea family, whereas the 2250F/2718R primer set only produced a single amplified fragment of a different size between the male and the female. Zoo Biol 27:62-69, 2008. (c) 2007 Wiley-Liss, Inc.

Background: Lung cancer contributes to high cancer mortality worldwide with 80% of total cases diagnosed as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain serves as a druggable target in NSCLC patients with exon 19 deletion and L858R mutation. However, patients eventually succumbed to resistance to first- and second-generation EGFR-TK inhibitors through activation of T790M mutation. Third-generation EGFR-TKI, Osimertinib exhibits high efficacy in patients with exon 19 deletion/L858R/T790M mutation but they experienced acquired resistance thereafter. Available treatment options in NSCLC patients remains a challenge due to unknown molecular heterogeneity responsible for acquired resistance to EGFR-TKI. In this study, we aim to generate Osimertinib-resistant (OR) cells from H1975 carrying L858R/T790M double mutation which can be used as a model to elucidate mechanism of resistance. Methods: OR cells were established via stepwise-dose escalation and limiting single-cell dilution method. We then evaluated Osimertinib resistance potential via cell viability assay. Proteins expression related to EGFR-signalling, epithelial to mesenchymal transition (EMT), and autophagy were analyzed via western blot. Results: OR cell lines exhibited increased drug resistance potential compared to H1975. Distinguishable mesenchymal-like features were observed in OR cells. Protein expression analysis revealed EGFR-independent signaling involved in the derived OR cells as well as EMT and autophagy activity. Conclusion: We generated OR cell lines in-vitro as evidenced by increased drug resistance potential, increased mesenchymal features, and enhanced autophagy activity. Development of Osimertinib resistance cells may serve as in-vitro model facilitating discovery of molecular aberration present during acquired mechanism of resistance.

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(2E,6E)-2,6-bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC) is a synthetic curcumin analogue, which has been reported to possess anti-tumor, anti-metastatic, and anti-invasion properties on estrogen receptor (ER) negative breast cancer cells in vitro and in vivo. However, the cytotoxic effects of BHMC on ER positive breast cancer cells were not widely reported. This study was aimed to investigate the cytotoxic potential of BHMC on MCF-7 cells using cell viability, cell cycle, and apoptotic assays. Besides, microarray and quantitative polymerase chain reaction (qPCR) were performed to identify the list of miRNAs and genes, which could be dysregulated following BHMC treatment. The current study discovered that BHMC exhibits selective cytotoxic effects on ER positive MCF-7 cells as compared to ER negative MDA-MB-231 cells and normal breast cells, MCF-10A. BHMC was shown to promote G2/M cell cycle arrest and apoptosis in MCF-7 cells. Microarray and qPCR analysis demonstrated that BHMC treatment would upregulate several miRNAs like miR-3195 and miR-30a-3p and downregulate miRNAs such as miR-6813-5p and miR-6132 in MCF-7 cells. Besides, BHMC administration was also found to downregulate few tumor-promoting genes like VEGF and SNAIL in MCF-7. In conclusion, BHMC induced apoptosis in the MCF-7 cells by altering the expressions of apoptotic-regulating miRNAs and associated genes.

BackgroundHuman hair follicles are important for the renewal of new hairs and their development. The generation of induced pluripotent stem cells (iPSCs) from hair follicles is easy due to its accessibility and availability. The pluripotent cells derived from hair follicles not only have a higher tendency to re-differentiate into hair follicles, but are also more suited for growth in hair scalp tissue microenvironment.MethodsIn this study, human hair follicular keratinocytes were used to generate iPSCs, which were then further differentiated in vitro into keratinocytes. The derived iPSCs were characterised by using immunofluorescence staining, flow cytometry, and reverse-transcription PCR to check for its pluripotency markers expression.ResultsThe iPSC clones expressed pluripotency markers such as TRA-1-60, TRA-1-81, SSEA4, OCT4, SOX2, NANOG, LEFTY, and GABRB. The well-formed three germ layers were observed during differentiation using iPSCs derived from hair follicles. The successful formation of keratioctyes from iPSCs was confirmed by the expression of cytokeratin 14 marker.DiscussionHair follicles represent a valuable keratinocytes source for in vitro hair cloning for use in treating hair balding or grafting in burn patients. Our significant findings in this report proved that hair follicles could be used to produce pluripotent stem cells and suggested that the genetic and micro-environmental elements of hair follicles might trigger higher and more efficient hair follicles re-differentiation.