Genome-wide copy number analysis of single cells

Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ～3 d from flow cytometry to sequence-ready DNA libraries.     

The late pollen actins are essential for normal male and female development in Arabidopsis

In angiosperms the late pollen actins (LPAs) are strongly expressed in mature pollen and pollen tubes and at much lower levels in ovules. Four Arabidopsis lines with homozygous knockout mutations in the four individual LPA genes displayed normal flowers, pollen, and seed set. However, when all four LPAs were silenced simultaneously with a single RNA interference (RNAi) construct targeting the 3′UTR of each mRNA, obvious reproductive defects were observed. Western analysis of various Late Pollen actin RNA interference (LPRi) epialleles showed total LPA protein and RNA expression levels were knocked down from 0% to 95% compared to wild-type levels. Reciprocal crosses with the RNAi lines demonstrated that lowered LPA expression was associated with defects in both male and female fertility. Strong epialleles showed significant reductions in normal silique and seed production and were nearly sterile. Dissection of the siliques from moderate LPRi epialleles revealed many unfertilized ovules, increased numbers of aborted seeds, and decreased numbers of healthy seeds. Microscopic analysis of LPRi pollen indicated that the pollen shape and size were normal, but pollen germinated poorly. While multiple LPA genes may have some functional redundancy, the combined expression of multiple LPA genes appears essential to normal male and female reproductive development.     

Levan: A Biocompatible Homopolysaccharide Excipient for Stabilization of Peptide Drugs

Stabilisation of protein/peptide drugs against thermal denaturation is a challenging problem, especially for liquid formulations. Various polysaccharides at high concentrations have been reported to improve stability of polypeptides, probably by providing a crowded environment which retards kinetic unfolding and resultant degradation. Levan is a fructose homopolysaccharide which is finding increasing use in pharmaceutical applications, but its use for protein drug stabilization remains meagre. In this study, we used levan for stabilizing a liquid preparation of a peptide antibiotic, bacitracin. We prepared liquid formulations of bacitracin with or without levan and subjected them to storage at 25 °C. The stored samples were then analysed over 120 days for denaturation and antibacterial activity. Differential Scanning Calorimetry, Circular Dichroism and High Performance Liquid Chromatography were used for evaluating the effect of levan on thermal denaturation of bacitracin. We found that levan at 2.5% w/v significantly preserved the antibacterial activity of bacitracin for 120 days as compared to plain buffered bacitracin, even when stored at 25 °C. Also, levan at high concentrations maintained the secondary structure and increased the melting temperature (Tm) of bacitracin in solution. Levan did not form covalent interactions or strong complexation with bacitracin. Based on this study, levan appears as a promising stabilizing agent for preparing liquid formulations of protein/peptide drugs that can be stored at room temperature.     

Molecular identification and phylogenetic analyses of multiple nucleopolyhedrovirus isolated from <Emphasis Type="Italic">Lymantria obfuscata</Emphasis> (Lepidoptera: Lymantriidae) in India

The Lymantria obfuscata Walker (Lyob) multiple (M) nucleopolyhedrovirus (NPV) (LyobMNPV) has been isolated and successfully applied for the management of the Indian gypsy moth, L. obfuscata in Jammu and Kashmir (J&K), India. The present work aimed to investigate the variability of LyobMNPV isolates from six localities of J&K through molecular [amplification of the polyhedrin (polh), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes] and biological (bioassays) characterization. To identify the position of LyobMNPV in the phylogenetic tree of baculoviruses, partial sequences of the polh, lef-8 and lef-9 genes were determined by using the DNA sequences within their coding regions by optimizing the polymerase chain reaction with degenerate primers. The sequence alignment revealed that LyobMNPV isolates exhibited seven, five and eleven single nucleotide polymorphic sites in the case of polh, lef-8 and lef-9, respectively. The phylogenetic analyses supported placing LyobMNPV with the Lymantria dispar L. MNPV (LdMNPV) isolates from different countries, and showed that it was more closely related to LdMNPV than to Lymantria xylina Swinhoe NPV and Lymantria monacha L. NPV. The contaminated diet plug bioassays using 2nd instar larvae indicated that the median lethal dose (LD₅₀) and median survival time (ST₅₀) of different isolates of LyobMNPV against L. obfuscata were lower than those of LdMNPV against L. dispar. LyobMNPV was more closely related to LdMNPV but its LD₅₀ and ST₅₀ were lower than those of LdMNPV. The study provides novel information on the position of LyobMNPV in the phylogenetic tree of baculoviruses and about biological and genetic variation of Lymantria species’ NPV isolates from different parts of the world.     

Stigmatellin Y – An anti-biofilm compound from Bacillus subtilis BR4 possibly interferes in PQS–PqsR mediated quorum sensing system in Pseudomonas aeruginosa

Hitherto this is the first report pertaining to production of biofilm inhibitory compound(s) (BIC) from Bacillus subtilis BR4 against Pseudomonas aeruginosa (ATCC 27853) coupled with production optimization. In order to achieve this, combinations of media components were formulated by employing statistical tools such as Plackett–Burman analysis and central composite rotatable design (CCRD). It was evident that at 35 ml L^?1 glycerol and 3.8 g L^?1 casamino acid, anti-biofilm activity and production of extracellular protein significantly increased by 1.5-fold and 1.2-fold, respectively. These results corroborate that the combination of glycerol and casamino acid plays a key role in the production of BIC. Further, metabolic profiling of BIC was carried out using liquid chromatography/tandem mass spectrometry (LC–MS/MS) based on m/z value. The presence of Stigmatellin Y was predicted with monoisotopic neutral mass of 484.2825 Da. In support of optimization study, higher production of BIC was confirmed in the optimized-media-grown BR4 (OPT-BR4) than in the ideal-media-grown BR4 (ID-BR4) by LC–MS/MS analysis. PqsR in P. aeruginosa is a potential target for anti-virulent therapy. Molecular docking study has revealed that Stigmatellin Y interacts with PqsR in the similar orientation like a cognate signal (PQS) and synthetic inhibitor. In addition, Stigmatellin Y was found to exhibit interaction with four more amino acid residues of PqsR to establish strong affinity. Stigmatellin Y thus might play a role of competitor for PQS to distract PQS–PqsR mediated communication in P. aeruginosa. The present investigation thus paves new avenues to develop anti-Pseudomonas virulent therapy.     

One plant actin isovariant, ACT7, is induced by auxin and required for normal callus formation.

During plant growth and development, the phytohormone auxin induces a wide array of changes that include cell division, cell expansion, cell differentiation, and organ initiation. It has been suggested that the actin cytoskeleton plays an active role in the elaboration of these responses by directing specific changes in cell morphology and cytoarchitecture. Here we demonstrate that the promoter and the protein product of one of the Arabidopsis vegetative actin genes, ACT7, are rapidly and strongly induced in response to exogenous auxin in the cultured tissues of Arabidopsis. Homozygous act7-1 mutant plants were slow to produce callus tissue in response to hormones, and the mutant callus contained at least two to three times lower levels of ACT7 protein than did the wild-type callus. On the other hand, a null mutation in ACT2, another vegetative actin gene, did not significantly affect callus formation from leaf or root tissue. Complementation of the act7-1 mutants with the ACT7 genomic sequence restored their ability to produce callus at rates similar to those of wild-type plants, confirming that the ACT7 gene is required for callus formation. Immunolabeling of callus tissue with actin subclass-specific antibodies revealed that the predominant ACT7 is coexpressed with the other actin proteins. We suggest that the coexpression, and probably the copolymerization, of the abundant ACT7 with the other actin isovariants in cultured cells may facilitate isovariant dynamics well suited for cellular responses to external stimuli such as hormones.     

Eco-friendly approaches for the management of fenugreek root rot

To evolve eco-friendly management of fenugreek root rot caused by Rhizoctonia solani, a field trial was conducted during Kharif 2002 and Rabi seasons of 2002–2003 and 2003–2004. Experiments were conducted with eight treatments and three replications in RBD using the variety CO-2. The pooled analysis of the three season data showed that seed treatment with Trichoderma viride at 4g/kg of seed + soil application of Trichoderma viride at 5 kg/ha + soil application of neem cake at 150 kg/ha (T₃) recorded a percent disease index (PDI) of 23.1 versus 65.5 PDI in the control which accounted for a disease reduction of 64.7%. It was on par with seed treatment with Trichoderma viride at 4g/kg of seed + soil application of T. viride at 5 kg/ha (T₂) which reduced the disease incidence by 62.3% (24.7 PDI). The chemical treatment used for comparison, i.e. seed treatment with carbendazim + soil drenching at 0.1% + soil application of neem cake at 150 kg/ha recorded the lowest PDI of 16.8 with 74.4% disease reduction. Among the various treatments T₃ gave a seed yield of 572.7 kg/ha followed by T₂ (555.7 kg/ha). Treatment T₇ recorded the highest yield of 578.7 kg/ha. In the control plot the recorded yield was only 359.3 kg/ha. Though T₃ was more effective at reducing the disease incidence than T₂, the C:B ratio was higher (1:9.1) in respect of T₂ than T₃, which gave a C:B ratio of only 1:3.9. Hence, seed treatment with T. viride at 4g/kg + soil application of T. viride at 5kg/ha is a cost effective, eco-friendly management strategy for fenugreek root rot.