Summary Interesting optical and photochemical properties make microbial rhodopsin a promising biological material suitable for various applications, but the cost‐prohibitive nature of production has limited its commercialization. The aim of this study was to explore the natural biodiversity of Indian solar salterns to isolate natural bacteriorhodopsin ( BR ) variants that can be functionally expressed in Escherichia coli . In this study, we report the isolation, functional expression and purification of BR s from three pigmented haloarchaea, wsp3 (water sample Pondicherry), wsp5 and K1 T isolated from two Indian solar salterns. The results of the 16S rRNA data analysis suggest that wsp3, wsp5 and K1 T are novel strains belonging to the genera Halogeometricum, Haloferax and Haloarcula respectively. Overall, the results of our study suggest that 17 N‐terminal residues, that were not included in the gene annotation of the close sequence homologues, are essential for functional expression of BR s. The primary sequence, secondary structural content, thermal stability and absorbance spectral properties of these recombinant BR s are similar to those of the previously reported Haloarcula marismortui Hm BRI . This study demonstrates the cost‐effective, functional expression of BR s isolated from haloarchaeal species using E. coli as an expression host and paves the way for feasibility studies for future applications.
Abnormal proliferation and disrupted differentiation of hematopoietic progenitors mark leukemia. Histone cell cycle regulator A (HIRA), a histone chaperone, regulates hemogenic to hematopoietic transition involved in normal hematopoiesis. But, its role remains unexplored in leukemia, a case of dysregulated hematopoiesis. Here, the Cancer Cell Line Encyclopedia database analysis showed enhanced HIRA mRNA expression in cells of hematopoietic and lymphoid origin with maximal expression in the chronic myeloid leukemia (CML) cell line, K562. This observation was further endorsed by the induced expression of HIRA in CML patient samples compared to healthy individuals and Acute Myeloid Leukemia patients. Downregulation of HIRA in K562 cells displayed cell cycle arrest, loss in proliferation, presence of polyploidy with significant increase in CD41+ population thereby limiting proliferation but inducing differentiation of leukemia cells to megakaryocyte fate. Induced megakaryocyte differentiation of mouse Hira‐knockout hematopoietic progenitors in vivo further confirmed the in vitro findings in leukemia cells. Molecular analysis showed the involvement of MKL1/GATA2/H3.3 axis in dictating differentiation of CML cells to megakaryocytes. Thus, HIRA could be exploited for differentiation induction therapy in CML and in chronic pathological conditions involving low platelet counts.
Introduction‐ Pluripotency of stem cells is associated with the dynamicity of the embryo’s developmental stage from which they are derived. Stem cells derived from the pre‐implantation embryo, called embryonic stem cells (ESCs), are naïve in nature, whereas post‐implantation embryo‐derived stem cells, termed epiblast stem cells (EpiSCs), represent the primed state of pluripotency. Maintaining these stem cells in a naïve state is an essential arena of stem cell research since naïve ESCs are capable of clonal propagation and germline transmission, making them a crucial research tool in organogenesis and disease modeling. Inhibition of PKC (PKCi) signaling was found to maintain an undifferentiated stage of pluripotency in both mouse and rat. Interestingly, when used along with a cocktail of inhibitors, the same inhibitor facilitated the derivation of naïve state pluripotency in human ESC. These findings lead us to a hypothesis that PKC signaling inhibition alone has a role in maintaining the naïve state of pluripotency. Hence, it has intrigued us to explore the complex network of mechanisms exerted by PKC signaling in dictating the transition of naïve to primed state of pluripotency. Methodology‐ In that pursuit, we first observed the morphology of the mouse ESCs (mESCs) by phase‐contrast microscopy across passages and performed MTT assay to optimize the concentration used for further assay. Quantitative Real‐time PCR and western blotting were performed to determine the expression of transcription factors associated with naïve and primed states of pluripotency. Other techniques, including FACS, TiO2 phospho‐enrichment followed by Mass‐spectrometric (MS) analysis, Immunofluorescence assay, Immunoprecipitation assay, Nuclear‐cytoplasmic protein extraction and others were exploited to study the mechanism. Results‐ We observed that PKCi could maintain naïve state of pluripotency in mESC even after prolonged culturing by reducing the expression of a critical primed transcription factor. The inhibition of phosphorylation of the PKCζ isoform is responsible for naïve state maintenance. Cell cycle analysis showed that more cells are in the G1 phase in the PKCi maintained condition than in the cells maintained in the presence of Leukemia Inhibitory Factor (LIF). Also, cell proliferation marker CDK1 expression was less in PKCi maintained cells, which probably is negatively regulated by the transcription factor KLF4. Increased nuclear expression of KLF4 was observed in PKCi maintained ESCs than that of ESCs cultured with LIF. Beside these transcription factor‐mediated regulations, PKCi exerts an epigenetic control over the ESCs absent in LIF maintained ESCs. PKCi mediated loss in phosphorylation of the epigenetic factor contributes to naïve state maintenance. Further, our data indicates towards PKCζ mediated NF‐κβ pathway activation, thereby dictating the transition of naïve to primed state of pluripotency. Conclusions‐ PKCi maintained mESCs could maintain the naïve state of pluripotency. We have unraveled a multifaceted functi...
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