Biology subjects

Das, K.

Publications and source records attributed to Das, K..

5 recordsLinked to original sources

bioRxiv2018

Rat bone marrow derived mesenchymal stem cells differentiate to germ cell like cells

Summary StatementThe in vitro differentiation of rMSCs provides an excellent model for studying cell commitment and their potential in stem cell technology. We have demonstrated that rat bone marrow derived MSCs are able to differentiate into germ-line cells in vitro which has an enormous scope in the advancement of fertility research. In future, this technique can be utilized in different domestic animal species for propagation of elite germ plasm.\n\nAbstractGerm cells undergo distinctive male or female pathways to produce spermatozoa or oocyte respectively essential for sexual reproduction. Mesenchymal stem cells (MSCs) have the capacity of trans-differentiation to form the multiple lineages of cells of mesoderm, endoderm, and ectoderm origin. Herein, MSCs were isolated from rat bone marrow and characterized by their morphological features, expression of surface markers by immunophenotyping and molecular biology tools as well as self renewal and differentiation capability. Thereafter, by inducing these cells with retinoic acid we could able to show that bone marrow derived MSCs are able to trans-differentiate into male germ cell-like cells which were further characterized by the expressions of germ cell specific markers. This in vitro study for the generation of germ-like cells suggests that bone marrow MSCs can be a potential source of germ cells that could be a sustainable source of sperm / oocyte production for potential therapeutic applications in future. Moreover, this technique can be applied in different domestic animal species for propagation of elite germ plasm.

cell biology↗

bioRxiv2018

Structural basis of ECF-sigma-factor-dependent transcription initiation.

Extracytoplasmic (ECF) {sigma} factors, the largest class of alternative {sigma} factors, are related to primary {sigma} factors, but have simpler structures, comprising only two of the six conserved functional modules present in primary {sigma} factors: region 2 ({sigma}R2) and region 4 ({sigma}R4). Here, we report crystal structures of transcription initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF {sigma} factor {sigma}L, and promoter DNA. The structures show that {sigma}R2 and {sigma}R4 of the ECF {sigma} factor occupy the same sites on RNAP as in primary {sigma} factors, show that the connector between {sigma}R2 and {sigma}R4 of the ECF {sigma} factor--although unrelated in sequence--follows the same path through RNAP as in primary {sigma} factors, and show that the ECF {sigma} factor uses the same strategy to bind and unwind promoter DNA as primary {sigma} factors. The results define protein-protein and protein-DNA interactions involved in ECF-{sigma}-factor-dependent transcription initiation.

molecular biology↗

bioRxiv2017

Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

Fidaxomicin is an antibacterial drug in clinical use in treatment of Clostridium difficile diarrhea1-2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1-4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1-2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6-8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9 and inhibits transcription initiation at an earlier step than Rif8-11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12-13 structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5 [A] resolution. The structure shows that Lpm binds at the base of the RNAP \"clamp,\" interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17{degrees} relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14 experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor {sigma} regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

molecular biology↗

bioRxiv2017

Revealing unidentified heterogeneity in different epithelial cancers using heterocellular subtype classification

Cancers are currently diagnosed, categorised, and treated based on their tissue of origin. However, how different cellular compartments of tissues (e.g., epithelial, immune and stem cells) are similar across cancer types is unknown. Here we used colorectal cancer subtypes and their signatures representing different colonic crypt cell types as surrogates to classify different epithelial cancers into five heterotypic cellular (heterocellular) subtypes. The stem-like and inflammatory heterocellular subtypes are ubiquitous across epithelial cancers so capture intrinsic, tissue-independent properties. Conversely, well-differentiated/specialized goblet-like/enterocyte heterocellular subtypes differ across cancer types due to their colorectum-specific genes. The transit-amplifying heterocellular subtype shows a dynamic range of cellular differentiation with shared common pathways (Wnt, FGFR) in certain cancer types. Importantly, this approach revealed previously unrecognised heterogeneity in pancreatic, breast, microsatellite-instability enriched and KRAS mutation-dependent cancers. Immune cell-type differences are common and useful for patient stratification for immunotherapy. This unique approach identifies cell type-dependent but tissue-independent heterogeneity in different cancers for precision medicine.

bioinformatics↗

bioRxiv2017

Structural basis of Mycobacterium tuberculosis transcription and transcription inhibition

One Sentence SummaryStructures of Mycobacterium tuberculosis RNA polymerase reveal taxon-specific properties and binding sites of known and new antituberculosis agents\n\nAbstractMycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report novel non-Rif-related compounds-N-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.

molecular biology↗