Rohitukine inhibits in vitro adipogenesis arresting mitotic clonal expansion and improves dyslipidemia in vivo

We developed a common feature pharmacophore model using known antiadipogenic compounds (CFPMA). We identified rohitukine, a reported chromone anticancer alkaloid as a potential hit through in silico mapping of the in-house natural product library on CFPMA. Studies were designed to assess the antiadipogenic potential of rohitukine. Rohitukine was isolated from Dysoxylum binacteriferum Hook. to ⬧95% purity. As predicted by CFPMA, rohitukine was indeed found to be an antiadipogenic molecule. Rohitukine inhibited lipid accumulation and adipogenic differentiation in a concentration- and exposure-time-dependent manner in 3T3-L1 and C3H10T1/2 cells. Rohitukine downregulated expression of PPARγ, CCAAT/enhancer binding protein α, adipocyte protein 2 (aP2), FAS, and glucose transporter 4. It also suppressed mRNA expression of LPL, sterol-regulatory element binding protein (SREBP) 1c, FAS, and aP2, the downstream targets of PPARγ. Rohitukine arrests cells in S phase during mitotic clonal expansion. Rohitukine was bioavailable, and 25.7% of orally administered compound reached systemic circulation. We evaluated the effect of rohitukine on dyslipidemia induced by high-fat diet in the hamster model. Rohitukine increased hepatic expression of liver X receptor α and decreased expression of SREBP-2 and associated targets. Rohitukine decreased hepatic and gonadal lipid accumulation and ameliorated dyslipidemia significantly. In summary, our strategy to identify a novel antiadipogenic molecule using CFPMA successfully resulted in identification of rohitukine, which confirmed antiadipogenic activity and also exhibited in vivo antidyslipidemic activity.     

Multiple Drugs Compete for Transport via the Plasmodium falciparum Chloroquine Resistance Transporter at Distinct but Interdependent Sites

Mutations in the “chloroquine resistance transporter” (PfCRT) are a major determinant of drug resistance in the malaria parasite Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance.     

Analysis of Genetic Diversity in Pongamia [Pongamia pinnata (L) Pierrre] using AFLP Markers

In recent years, Pongamia has been considered as important renewable source of biodiesel, however not much molecular information is available in this species. Molecular characterization of this legume tree will enhance our understanding in improving the optimal yields of oil through breeding and enable us to meet the future demands for biodiesel. To assess the molecular genetic diversity in 46 Pongamia pinnata accessions collected from six different states of India, amplified fragment length polymorphism (AFLP) marker system was employed. Five AFLP primer combinations produced 520 discernible fragments, of which 502 (96.5%) were polymorphic. AFLP primer informativeness was estimated evaluating four parameters namely polymorphism information content (PIC), effective multiplex ratio (EMR), marker index (MI) and resolving power (RP). In total, 51 unique fragments were detected of which 19 unique fragments were observed with primer combination E-ACG / M-CTA. Although neighbour joining (NJ) method did not group accessions strictly according to their region of collection, a good level of genetic diversity was observed in examined germplasm. However, accessions collected from Karnataka showed comparatively higher diversity than accessions from other states. The diverse accessions identified in this study may be useful in Pongamia pinnata improvement to meet the future demands of biodiesel.     

Pharmacokinetic applicability of a validated liquid chromatography tandem mass spectroscopy method for orally administered curcumin loaded solid lipid nanoparticles to rats

A simple and sensitive validated LC–MS/MS analytical method was used for determination of curcumin in rat plasma, using nimesulide as internal standard. Analyses were performed on an Agilent LC–MS/MS system using a Chromolith rod? and isocratic elution with acetonitrile:10 mM ammonium acetate buffer (pH 3.5) (80:20, v/v) at a flow rate of 0.8 ml/min with a total run time of 3 min and an overall recovery of 77.15%. A triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, operated in the negative mode was used. Calibration curve in plasma spiked with varying concentration of curcumin were linear over the concentration range of 10–2000 ng/ml with determination coefficient >0.99. The lower limit of quantification was 10 ng/ml. Intra and inter-day variability's (RSD) for extraction of curcumin from plasma were less than 10% and 15% respectively and accuracy was 102.43–108.5%. Multiple reaction monitoring was used to monitor the transition for curcumin (m/z; 367/217 [M?H]^?) and IS (m/z; 307/229). The method was applied for determining curcumin concentration in plasma after peroral administration of 50 mg/kg of free curcumin (C-S) or curcumin loaded solid lipid nanoparticles (C-SLNs) to rats. Results established selectivity and suitability of the method for pharmacokinetic studies of curcumin from C-SLNs.     

Cytosolic proteins regulate alpha-synuclein dissociation from presynaptic membranes

Intracellular accumulation of insoluble alpha-synuclein in Lewy bodies is a key neuropathological trait of Parkinson disease (PD). Neither the normal function of alpha-synuclein nor the biochemical mechanisms that cause its deposition are understood, although both are likely influenced by the interaction of alpha-synuclein with vesicular membranes, either for a physiological role in vesicular trafficking or as a pathological seeding mechanism that exacerbates the propensity of alpha-synuclein to self-assemble into fibrils. In addition to the alpha-helical form that is peripherally-attached to vesicles, a substantial portion of alpha-synuclein is freely diffusible in the cytoplasm. The mechanisms controlling alpha-synuclein exchange between these compartments are unknown and the possibility that chronic dysregulation of membrane-bound and soluble alpha-synuclein pools may contribute to Lewy body pathology led us to search for cellular factors that can regulate alpha-synuclein membrane interactions. Here we reveal that dissociation of membrane-bound alpha-synuclein is dependent on brain-specific cytosolic proteins and insensitive to calcium or metabolic energy. Two PD-linked mutations (A30P and A53T) significantly increase the cytosol-dependent alpha-synuclein off-rate but have no effect on cytosol-independent dissociation. These results reveal a novel mechanism by which cytosolic brain proteins modulate alpha-synuclein interactions with intracellular membranes. Importantly, our finding that alpha-synuclein dissociation is up-regulated by both familial PD mutations implicates cytosolic cofactors in disease pathogenesis and as molecular targets to influence alpha-synuclein aggregation.     

Density dependent population growth of the two-spotted spider mite, Tetranychus urticae, on the host plant Leonurus cardiaca

We observed Tetranychus urticae (Koch), a polyphagous spider mite herbivore, on Leonurus cardiaca (L.) at several sites in eastern North America at variable density, ranging from extremely dense to sparse. To understand the nature of T. urticae 's population dynamics we experimentally manipulated population densities on L. cardiaca and assessed per capita growth after 1 to 2 generations in laboratory and field experiments. In particular, we took a 'bottom-up' approach, manipulating both plant size and quality to examine effects on mite dynamics. Per capita growth was strongly dependent on the initial density of the mite population. Spider mite populations grew (1) in a negatively density dependent manner on small plants and (2) unhindered by density dependence on large plants. Mean per capita growth was 59% higher on small plants compared to large plants, irrespective of mite density. We also found evidence for density dependent induced susceptibility to spider mites in small plants and density dependent induced resistance in large plants. Hence, spider mite populations grew at a relatively fast rate on small plants, and this was associated with negative density dependence due to factors that depress population growth, such as food deterioration or limitation. On large plants, spider mite populations grew at a relatively slow rate, apparently resulting in herbivore densities that may not have been high enough to cause intraspecific competition or other forms of negative density dependence.     

Indel‐seq: a fast‐forward genetics approach for identification of trait‐associated putative candidate genomic regions and its application in pigeonpea (Cajanus cajan)

Identification of candidate genomic regions associated with target traits using conventional mapping methods is challenging and time‐consuming. In recent years, a number of single nucleotide polymorphism (SNP)‐based mapping approaches have been developed and used for identification of candidate/putative genomic regions. However, in the majority of these studies, insertion–deletion (Indel) were largely ignored. For efficient use of Indels in mapping target traits, we propose Indel‐seq approach, which is a combination of whole‐genome resequencing (WGRS) and bulked segregant analysis (BSA) and relies on the Indel frequencies in extreme bulks. Deployment of Indel‐seq approach for identification of candidate genomic regions associated with fusarium wilt (FW) and sterility mosaic disease (SMD) resistance in pigeonpea has identified 16 Indels affecting 26 putative candidate genes. Of these 26 affected putative candidate genes, 24 genes showed effect in the upstream/downstream of the genic region and two genes showed effect in the genes. Validation of these 16 candidate Indels in other FW‐ and SMD‐resistant and FW‐ and SMD‐susceptible genotypes revealed a significant association of five Indels (three for FW and two for SMD resistance). Comparative analysis of Indel‐seq with other genetic mapping approaches highlighted the importance of the approach in identification of significant genomic regions associated with target traits. Therefore, the Indel‐seq approach can be used for quick and precise identification of candidate genomic regions for any target traits in any crop species.     

The fire ant social chromosome supergene variant Sb shows low diversity but high divergence from SB

Variation in social behaviour is common, yet little is known about the genetic architectures underpinning its evolution. A rare exception is in the fire ant Solenopsis invicta: Alternative variants of a supergene region determine whether a colony will have exactly one or up to dozens of queens. The two variants of this region are carried by a pair of ‘social chromosomes’, SB and Sb, which resemble a pair of sex chromosomes. Recombination is suppressed between the two chromosomes in the supergene region. While the X‐like SB can recombine with itself in SB/SB queens, recombination is effectively absent in the Y‐like Sb because Sb/Sb queens die before reproducing. Here, we analyse whole‐genome sequences of eight haploid SB males and eight haploid Sb males. We find extensive SB–Sb differentiation throughout the >19‐Mb‐long supergene region. We find no evidence of ‘evolutionary strata’ with different levels of divergence comparable to those reported in several sex chromosomes. A high proportion of substitutions between the SB and Sb haplotypes are nonsynonymous, suggesting inefficacy of purifying selection in Sb sequences, similar to that for Y‐linked sequences in XY systems. Finally, we show that the Sb haplotype of the supergene region has 635‐fold less nucleotide diversity than the rest of the genome. We discuss how this reduction could be due to a recent selective sweep affecting Sb specifically or associated with a population bottleneck during the invasion of North America by the sampled population.