A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions.

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, are potentially useful in regenerative therapies for heart disease. For medical applications, clinical-grade cardiac cells must be produced from hPSCs in a defined, cost-effective manner. Cell-based screening led to the discovery of KY02111, a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown, results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free, defined medium, devoid of serum and any kind of recombinant cytokines and hormones, such as BMP4, Activin A, or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies.

Identification of Indian pathogenic races of Fusarium oxysporum f. sp. ciceris with gene specific, ITS and random markers.

In this study we demonstrate the synergistic use of gene-specific markers, ITS-RFLP, ISSR and AFLP for distinguishing Indian F. oxysporum f. sp. ciceris races. We also report for the first time that F. oxysporum f. sp. ciceris race 3, a wilt pathogen of chickpea in India, is actually F. proliferatum based on phylogenetic analysis with EF-1alpha sequence data. F. oxysporum f. sp. ciceris races 1, 2 and 4 were easily distinguished from "race 3" (F. proliferatum) by PCR amplification with oligonucleotides designed from conserved regions of Hop78 transposon (Hop 78), cutinase (Cut), desaturase (Dst). F. oxysporum f. sp. ciceris race 4 was distinguished with the xylanase 3 (xyl3) gene by absence of amplification product only in this race. The Xyl3 amplified-DNA fragment isolated and sequenced from F. oxysporum f. sp. ciceris race 1 was similar to the F-xylanase (Xyl3) gene of F. oxysporum f. sp. lycopersici. A TELD motif, which is characteristic of the F-xylanases family, was detected within the deduced amino acid sequence of F. oxysporum f. sp. ciceris. Similarly the F. oxysporum f. sp. ciceris Hop78 DNA fragment, which identified "race 3" (F. proliferatum), was homologous to the Hop78 transposon of F. oxysporum f. sp. melonis, including the 100 amino acid conserved domain and the characteristic CCHC motif. The internal transcribed spacer region-restriction fragment length polymorphism (ITS-RFLP) approach along with intersimple sequence repeat (ISSR) method also differentiated "race 3" (F. proliferatum). Races 1 and 2 were identified by unique AFLP patterns. Sequence characterization of race-specific AFLP products revealed significant homologies of these sequences with metabolically important genes.

NMR comparison of the native energy landscapes of DLC8 dimer and monomer.

Characterization of the low energy excited states on the energy landscape of a protein is one of the exciting and challenging problems in structural biology today. In this context, we present here residue level NMR description of the low energy excited states representing locally different alternative conformations in the dynein light chain protein, in its dimeric as well as monomeric forms. Important differences have been observed between the two cases and these are not necessarily restricted to the dimer interface. Simulations indicate that the low energy excited states are within a free energy of 2-3 kcal/mol above the native state. In both the monomer and the dimer the energy landscape is very sensitive to small pH perturbations. Nearly 25% of the residues (total of residues at pH 3.0 and 3.5 for the monomer, and at pH 7.0 and 6.0 for the dimer) access alternative conformations. The observations have been rationalized on the basis of protonation-deprotonation equilibria in the side chains; histidines in the case of the dimer and aspartates/glutamates in the case of the monomer. The possible relationship of the observed ruggedness of the native energy landscape with the protein structure, and its implications to protein adaptability and unfolding have been discussed.

pH driven conformational dynamics and dimer-to-monomer transition in DLC8.

Dynein light chain protein, a part of the cytoplasmic motor assembly, is a homodimer at physiological pH and dissociates below pH 4.5 to a monomer. The dimer binds to a variety of cargo, whereas the monomer does not bind any of the target proteins. We report here the pH induced stepwise structural and motional changes in the protein, as derived from line broadening and 15N transverse relaxation measurements. At pH 7 and below until 5, partial protonation and consequent interconversion between molecules carrying protonated and neutral histidines, causes conformational dynamics in the dimeric protein and this increases with decreasing pH. Enhanced dynamics in turn leads to partial loosening of the structure. This would have implications for different efficacies of binding by target proteins due to small variations in pH in different parts of the cell, and hence for cargo trafficking from one part to another. Below pH 5, enhanced charge repulsions, partial loss of hydrophobic interactions, and destabilization of H-bonds across the dimer interface cause further loosening of the dimeric structure, leading eventually to the dissociation of the dimer.