Regeneration of plants from alginate-encapsulated shoot tips of Withania somnifera (L.) Dunal, a medicinally important plant species

A protocol was developed for plant regeneration from encapsulated shoot tips collected from in vitro proliferated shoots of Withania somnifera. The best gel composition was achieved using 3% sodium alginate and 75 mM CaCl2.2H2O. The maximum percentage response (87%) for conversion of encapsulated shoot tips into plantlets was achieved on MS medium supplemented with 0.5 mg/l IBA after 5 weeks of culture. The conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate beads having entrapped propagules were directly sown in autoclaved soilrite moistened with 14-MS salts.     

Catch-bond model derived from allostery explains force-activated bacterial adhesion

High shear enhances the adhesion of Escherichia coli bacteria binding to mannose coated surfaces via the adhesin FimH, raising the question as to whether FimH forms catch bonds that are stronger under tensile mechanical force. Here, we study the length of time that E. coli pause on mannosylated surfaces and report a double exponential decay in the duration of the pauses. This double exponential decay is unlike previous single molecule or whole cell data for other catch bonds, and indicates the existence of two distinct conformational states. We present a mathematical model, derived from the common notion of chemical allostery, which describes the lifetime of a catch bond in which mechanical force regulates the transitions between two conformational states that have different unbinding rates. The model explains these characteristics of the data: a double exponential decay, an increase in both the likelihood and lifetime of the high-binding state with shear stress, and a biphasic effect of force on detachment rates. The model parameters estimated from the data are consistent with the force-induced structural changes shown earlier in FimH. This strongly suggests that FimH forms allosteric catch bonds. The model advances our understanding of both catch bonds and the role of allostery in regulating protein activity.     

Emerging medicinal roles for lysophospholipid signaling

The two lysophospholipids (LPs) lysophosphatidic acid and sphingosine 1-phosphate (S1P) regulate diverse biological processes. Over the past decade, it has become clear that medically relevant LP activities are mediated by specific G protein-coupled receptors, implicating them in the etiology of a growing number of disorders. A new class of LP agonists shows promise for drug therapy: the experimental drug FTY720 is phosphorylated in vivo to produce a potent S1P receptor agonist (FTY720-P) and is currently in Phase III clinical trials for kidney transplantation and Phase II for multiple sclerosis. Recent genetic and pharmacological studies on LP signaling in animal disease models have identified new areas in which interventions in LP signaling might provide novel therapeutic approaches for the treatment of human diseases.     

Large-scale analysis of plasmid relationships through gene-sharing networks

Plasmids are vessels of genetic exchange in microbial communities. They are known to transfer between different host organisms and acquire diverse genetic elements from chromosomes and/or other plasmids. Therefore, they constitute an important element in microbial evolution by rapidly disseminating various genetic properties among different communities. A paradigmatic example of this is the dissemination of antibiotic resistance (AR) genes that has resulted in the emergence of multiresistant pathogenic bacterial strains. To globally analyze the evolutionary dynamics of plasmids, we built a large graph in which 2,343 plasmids (nodes) are connected according to the proteins shared by each other. The analysis of this gene-sharing network revealed an overall coherence between network clustering and the phylogenetic classes of the corresponding microorganisms, likely resulting from genetic barriers to horizontal gene transfer between distant phylogenetic groups. Habitat was not a crucial factor in clustering as plasmids from organisms inhabiting different environments were often found embedded in the same cluster. Analyses of network metrics revealed a statistically significant correlation between plasmid mobility and their centrality within the network, providing support to the observation that mobile plasmids are particularly important in spreading genes in microbial communities. Finally, our study reveals an extensive (and previously undescribed) sharing of AR genes between Actinobacteria and Gammaproteobacteria, suggesting that the former might represent an important reservoir of AR genes for the latter.     

Isorhamnetin Inhibits Proliferation and Invasion and Induces Apoptosis through the Modulation of Peroxisome Proliferator-activated Receptor γ Activation Pathway in Gastric Cancer

Gastric cancer (GC) is a lethal malignancy and the second most common cause of cancer-related deaths. Although treatment options such as chemotherapy, radiotherapy, and surgery have led to a decline in the mortality rate due to GC, chemoresistance remains as one of the major causes for poor prognosis and high recurrence rate. In this study, we investigated the potential effects of isorhamnetin (IH), a 3′-O-methylated metabolite of quercetin on the peroxisome proliferator-activated receptor γ (PPAR-γ) signaling cascade using proteomics technology platform, GC cell lines, and xenograft mice model. We observed that IH exerted a strong antiproliferative effect and increased cytotoxicity in combination with chemotherapeutic drugs. IH also inhibited the migratory/invasive properties of GC cells, which could be reversed in the presence of PPAR-γ inhibitor. We found that IH increased PPAR-γ activity and modulated the expression of PPAR-γ regulated genes in GC cells. Also, the increase in PPAR-γ activity was reversed in the presence of PPAR-γ-specific inhibitor and a mutated PPAR-γ dominant negative plasmid, supporting our hypothesis that IH can act as a ligand of PPAR-γ. Using molecular docking analysis, we demonstrate that IH formed interactions with seven polar residues and six nonpolar residues within the ligand-binding pocket of PPAR-γ that are reported to be critical for its activity and could competitively bind to PPAR-γ. IH significantly increased the expression of PPAR-γ in tumor tissues obtained from xenograft model of GC. Overall, our findings clearly indicate that antitumor effects of IH may be mediated through modulation of the PPAR-γ activation pathway in GC.     

Cooperative stabilization of microtubule dynamics by EB1 and CLIP-170 involves displacement of stably bound P(i) at microtubule ends

End binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in the form of comet tails and regulate microtubule dynamics. However, the mechanism by which they regulate microtubule dynamics is not well understood. Using full-length EB1 and a minimal functional fragment of CLIP-170 (ClipCG12), we found that EB1 and CLIP-170 cooperatively regulate microtubule dynamic instability at concentrations below which neither protein is effective. By use of small-angle X-ray scattering and analytical ultracentrifugation, we found that ClipCG12 adopts a largely extended conformation with two noninteracting CAP-Gly domains and that it formed a complex in solution with EB1. Using a reconstituted steady-state mammalian microtubule system, we found that at a low concentration of 250 nM, neither EB1 nor ClipCG12 individually modulated plus-end dynamic instability. Higher concentrations (up to 2 μM) of the two proteins individually did modulate dynamic instability, perhaps by a combination of effects at the tips and along the microtubule lengths. However, when low concentrations (250 nM) of EB1 and ClipCG12 were present together, the mixture modulated dynamic instability considerably. Using a pulsing strategy with [γ(32)P]GTP, we further found that unlike EB1 or ClipCG12 alone, the EB1-ClipCG12 mixture partially depleted the microtubule ends of stably bound (32)P(i). Together, our results suggest that EB1 and ClipCG12 act cooperatively to regulate microtubule dynamics. They further indicate that stabilization of microtubule plus ends by the EB1-ClipCG12 mixture may involve modification of an aspect of the stabilizing cap.     

Treatment of Visceral Leishmaniasis: Model-Based Analyses on the Spread of Antimony-Resistant L. donovani in Bihar,India

Background

Pentavalent antimonials have been the mainstay of antileishmanial therapy for decades, but increasing failure rates under antimonial treatment have challenged further use of these drugs in the Indian subcontinent. Experimental evidence has suggested that parasites which are resistant against antimonials have superior survival skills than sensitive ones even in the absence of antimonial treatment.

Methods and Findings

We use simulation studies based on a mathematical L. donovani transmission model to identify parameters which can explain why treatment failure rates under antimonial treatment increased up to 65% in Bihar between 1980 and 1997. Model analyses suggest that resistance to treatment alone cannot explain the observed treatment failure rates. We explore two hypotheses referring to an increased fitness of antimony-resistant parasites: the additional fitness is (i) disease-related, by causing more clinical cases (higher pathogenicity) or more severe disease (higher virulence), or (ii) is transmission-related, by increasing the transmissibility from sand flies to humans or vice versa.

Conclusions

Both hypotheses can potentially explain the Bihar observations. However, increased transmissibility as an explanation appears more plausible because it can occur in the background of asymptomatically transmitted infection whereas disease-related factors would most probably be observable. Irrespective of the cause of fitness, parasites with a higher fitness will finally replace sensitive parasites, even if antimonials are replaced by another drug.     

Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives

A new series of betulinic acid derivatives have been synthesized by introducing heterocyclic ring between C-2 and C-3 positions of betulinic acid. Further modifications were also carried out by reduction of C-20(29) unsaturated bond and substitution of C-28 carboxyl group by ester and amide linkage to enhance the selectivity. Compound 11 resulted in IC(50) of 2.44, 2.5, and 2.7 microg/ml on MIAPaCa, PA-1, and SW620 cancer cell lines, respectively. Compound 38 resulted in IC(50) of 0.67 microg/ml on MIAPaCa cell line.