Morphological and molecular screening of rice germplasm lines for low soil P tolerance

Phosphorus (P) is an essential macronutrient to all crops including rice and it plays a key role in various plant activities and development. Low availability of P in the soils negatively, influences rice crop growth and causes significant yield loss. In the present study, we characterized a set of 56 germplasm lines for their tolerance to low soil P by screening them at low soil P and optimum soil P levels along with low soil P tolerant and sensitive check varieties. These lines were genotyped for the presence/absence of tolerant allele with respect to the major low soil P tolerance QTL, Pup1, using a set of locus specific PCR-based markers, viz., K46-1, K46-2, K52 and K46CG-1. High genetic variability was observed for various traits associated with low soil P tolerance. The yield parameters from normal and low soil P conditions were used to calculate stress tolerance indices and classify the genotypes according to their tolerance level. Out of the total germplasm lines screened, 15 lines were found to be tolerant to low soil P condition based on the yield reduction in comparison to the tolerant check, but most of them harbored the complete or partial Pup1 locus. Interestingly, two tolerant germplasm lines, IC216831 and IC216903 were observed to be completely devoid of Pup1 and hence they can be explored for new loci underlying low soil P tolerance.

     

Combination of AT-101/cisplatin overcomes chemoresistance by inducing apoptosis and modulating epigenetics in human ovarian cancer cells

We investigated the effects of AT-101/cisplatin combination treatment on the expression levels of apoptotic proteins and epigenetic events such as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) enzyme activities in OVCAR-3 and MDAH-2774 ovarian cancer cells. XTT cell viability assay was used to evaluate cytotoxicity. For showing apoptosis, both DNA Fragmentation and caspase 3/7 activity measurements were performed. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array. DNMT and HDAC activities were evaluated by ELISA assay and mRNA levels of DNMT1 and HDAC1 genes were quantified by qRT-PCR. Combination of AT-101/cisplatin resulted in strong synergistic cytotoxicity and apoptosis in human ovarian cancer cells. Combination treatment reduced some pivotal anti-apoptotic proteins such as Bcl-2, HIF-1A, cIAP-1, XIAP in OVCAR-3 cells, whereas p21, Bcl-2, cIAP-1, HSP27, Clusterin and XIAP in MDAH-2774 cells. Among the pro-apoptotic proteins, Bad, Bax, Fas, phospho-p53 (S46), Cleaved caspase-3, SMAC/Diablo, TNFR1 and Cytochrome c were induced in OVCAR-3 cells, whereas, Bax, TRAILR2, FADD, p27, phospho-p53 (S46), Cleaved caspase-3, Cytochrome c, SMAC/Diablo and TNFR1 were induced in MDAH-2774 cells. Combination treatment also inhibited both DNMT and HDAC activities and also mRNA levels in both ovarian cancer cells. AT-101 exhibits great potential in sensitization of human ovarian cancer cells to cisplatin treatment in vitro, suggesting that the combination of AT-101 with cisplatin may hold great promise for development as a novel chemotherapeutic approach to overcome platinum-resistance in human ovarian cancer.     

A liquid chromatography–tandem mass spectrometry assay for d-Ala-d-Lac: A key intermediate for vancomycin resistance in vancomycin-resistant enterococci

Vancomycin exerts its antibacterial activity by binding to d-Ala-d-Ala in bacterial cell wall precursors. Vancomycin resistance in vancomycin-resistant enterococci (VRE) is due to an alternative cell wall biosynthesis pathway in which d-Ala-d-Ala is replaced, most commonly by d-Ala-d-Lac. In this study, we extend our recently developed Marfey’s derivatization-based liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for l-Ala, d-Ala, and d-Ala-d-Ala to d-Ala-d-Lac and apply it to the quantitation of these metabolites in VRE. The first step in this effort was the development of an effective washing method for removing medium components from VRE cells. Mar-d-Ala-d-Lac was well resolved chromatographically from Mar-d-Ala-d-Ala, a prerequisite for MS/MS quantitation of d-Ala-d-Ala and d-Ala-d-Lac. Mar-d-Ala-d-Lac gave similar detection parameters, sensitivity, and linearity as Mar-d-Ala-d-Ala. l-Ala, d-Ala, d-Ala-d-Ala, and d-Ala-d-Lac levels in VRE were then determined in the presence of variable vancomycin levels. Exposure to vancomycin resulted in a dramatic reduction of d-Ala-d-Ala, with a response midpoint at approximately 0.06 μg/ml vancomycin and with a broad response profile up to 128 μg/ml vancomycin. In contrast, d-Ala-d-Lac was present in the absence of vancomycin, with its level constant up to 128 μg/ml vancomycin. This method will be useful for the discovery, characterization, and refinement of new agents targeting vancomycin resistance in VRE.     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

Exploring membrane permeability of Tomatidine to enhance lipid mediated nucleic acid transfections

Intracellular delivery of nucleic acids is one of the critical steps in the transfections. Prior findings demonstrated various strategies including membrane fusion, endosomal escape for the efficient cytoplasmic delivery. In our continuing efforts to improve the nucleic acids transfections, we harnessed cell permeable properties of Tomatidine (T), a steroidal alkaloid abundantly found in green tomatoes for maximizing intracellular delivery of lipoplexes. We doped Tomatidine into liposomes of cationic lipid with amide linker (A) from our lipid library. Six liposomal formulations (AT) of Lipid A (1?mM) with varying concentrations of Tomatidine (0–1?mM) were prepared and evaluated for their transfection efficacies. Owing to its signature characteristic of cell membrane permeability, Tomatidine modulated endocytosis process, enhanced the intracellular delivery of the lipoplexes, and in turn increased the transfection efficacy of cationic liposomes. Our findings provide ‘proof of concept’ for enhancing transfections in gene delivery applications with Tomatidine in cationic liposomal formulations. These findings can be further applied in lipid mediated gene therapy and drug delivery applications.     

A Novel Approach to Flurbiprofen Pulsatile Colonic Release: Formulation and Pharmacokinetics of Double-Compression-Coated Mini-Tablets

A significant plan is executed in the present study to study the effect of double-compression coating on flurbiprofen core mini-tablets to achieve the pulsatile colonic delivery to deliver the drug at a specific time as per the patho-physiological need of the disease that results in improved therapeutic efficacy. In this study, pulsatile double-compression-coated tablets were prepared based on time-controlled hydroxypropyl methylcellulose K100M inner compression coat and pH-sensitive Eudragit S100 outer compression coat. Then, the tablets were evaluated for both physical evaluation and drug-release studies, and to prove these results, in vivo pharmacokinetic studies in human volunteers were conducted. From the in vitro drug-release studies, F6 tablets were considered as the best formulation, which retarded the drug release in the stomach and small intestine (3.42 ± 0.12% in 5 h) and progressively released to the colon (99.78 ± 0.74% in 24 h). The release process followed zero-order release kinetics, and from the stability studies, similarity factor between dissolution data before and after storage was found to be 88.86. From the pharmacokinetic evaluation, core mini-tablets producing peak plasma concentration (C_max) was 14,677.51 ± 12.16 ng/ml at 3 h T_max and pulsatile colonic tablets showed C_max = 12,374.67 ± 16.72 ng/ml at 12 h T_max. The area under the curve for the mini and pulsatile tablets was 41,238.52 and 72,369.24 ng-h/ml, and the mean resident time was 3.43 and 10.61 h, respectively. In conclusion, development of double-compression-coated tablets is a promising way to achieve the pulsatile colonic release of flurbiprofen.KEY WORDS: core mini-tablets, double-compression coating, inner compression coat, outer compression coat, similarity factor     

Ion-pairing liquid chromatography–tandem mass spectrometry-based quantification of uridine diphosphate-linked intermediates in the Staphylococcus aureus cell wall biosynthesis pathway

Bacterial cell wall biosynthesis is the target of several antibiotics and is of interest as a target for new inhibitor development. The cytoplasmic steps of this pathway involve a series of uridine diphosphate (UDP)-linked peptidoglycan intermediates. Quantification of these intermediates is essential for studies of current agents targeting this pathway and for the development of new agents targeting this pathway. In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for quantification of these intermediates in Staphylococcus aureus. To address the problem of poor retention of UDP-linked intermediates on reverse phase media, an ion-pairing (IP) approach using N,N-dimethylhexylamine was developed. MS/MS detection in negative mode was optimized for UDP-GlcNAc, UDP-MurNAc, UDP-MurNAc-l-Ala, UDP-MurNAc-l-Ala-d-Glu, UDP-MurNAc-l-Ala-d-Glu-l-Lys, and UDP-MurNAc-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. The lower limits of quantification (LLOQs) for these analytes were 1.8, 1.0, 0.8, 2.2, 0.6, and 0.5 pmol, respectively, which correspond to LLOQs of 6, 3, 3, 7, 2, and 2 nmol/g bacteria, respectively. This method was demonstrated for quantification of in vivo levels of these intermediates from S. aureus (0.3 mg dry weight analyzed) treated with fosfomycin, d-boroAla, d-cycloserine, and vancomycin. Metabolite accumulation is consistent with the known targets of these antibiotics and indicates potential regulatory loops within this pathway.     

Preparation, characterization, and in vitro and in vivo evaluation of lovastatin solid lipid nanoparticles

The purpose of this research was to study whether the bioavailability of lovastatin could be improved by administering lovastatin solid lipid nanoparticles (SLN) duodenally to rats. Lovastatin SLN were developed using triglycerides by hot homogenization followed by ultrasonication. Particle size and zeta potential were measured by photon correlation spectroscopy. The solid state of the drug in the SLN and lipid modification were characterized. Bioavailability studies were conducted in male Wistar rats after intraduodenal administration of lovastatin suspension and SLN. Stable lovastatin SLN having a mean size range of 60 to 119 nm and a zeta potential range of −16 to −21 mV were developed. More than 99% of the lovastatin was entrapped in the SLN. Lovastatin was dispersed in an amorphous state, and triglycerides were in {ieE162-1} form in the SLN. In vitro stability studies showed the slow release and stability of lovastatin SLN. The relative bioavailabilities of lovastatin and lovastatin hydroxy acid of SLN were increased by ∼173% and 324%, respectively, compared with the reference lovastatin suspension. Published: March 23, 2007