XomAnnotate: Analysis of Heterogeneous and Complex Exome- A Step towards Translational Medicine

In translational cancer medicine, implicated pathways and the relevant master genes are of focus. Exome''s specificity, processing-time, and cost advantage makes it a compelling tool for this purpose. However, analysis of exome lacks reliable combinatory analysis tools and techniques. In this paper we present XomAnnotate – a meta- and functional-analysis software for exome. We compared UnifiedGenotyper, Freebayes, Delly, and Lumpy algorithms that were designed for whole-genome and combined their strengths in XomAnnotate for exome data through meta-analysis to identify comprehensive mutation profile (SNPs/SNVs, short inserts/deletes, and SVs) of patients. The mutation profile is annotated followed by functional analysis through pathway enrichment and network analysis to identify most critical genes and pathways implicated in the disease genesis. The efficacy of the software is verified through MDS and clustering and tested with available 11 familial non-BRCA1/BRCA2 breast cancer exome data. The results showed that the most significantly affected pathways across all samples are cell communication and antigen processing and presentation. ESCO1, HYAL1, RAF1 and PRKCA emerged as the key genes. Network analysis further showed the purine and propanotate metabolism pathways along with RAF1 and PRKCA genes to be master regulators in these patients. Therefore, XomAnnotate is able to use exome data to identify entire mutation landscape, pathways, and the master genes accurately with wide concordance from earlier microarray and whole-genome studies -- making it a suitable biomedical software for using exome in next-generation translational medicine.

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http://www.iomics.in/research/XomAnnotate     

Polyamines and jasmonic acid induce plasma membrane potential variations in lima bean

Exogenous polyamines [cadaverine (Cad), putrescine (Put), spermidine (Spd) and spermine (Spm)] elicit the production of volatiles in Lima bean (Phaseolus lunatus). Among the tested PAs, Spm induces the production of some volatile terpenoids that are known to be induced by the spider mite Tetranychus urticae. Spm treatment elicits the biosynthesis of Jasmonic acid (JA), a phytohormone known to regulate the production of the volatile terpenoids. The treatment with JA together with Spm resulted in the increased volatile emission, and predatory mites Phytoseiulus persimilis preferred JA and Spm-treated leaves over those treated with JA alone.⁵ JA and Spm treatment has no effects on polyamine oxidase (PAO) and Cu-amine oxidase (CuAO) but has a significant induction of calcium influx, ROS production, enzyme activities for NADPH-oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase, and gene expressions except for NADPH-oxidase complex.⁵ Here, we report that a plasma membrane potential (V_m) depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd. JA perfusion did not alter V_m but the perfusion of JA and the polyamines significantly increased Cad and Put V_m depolarization. When JA was perfused with polyamines, a negative correlation was found between V_m depolarization and the number of amino group of the polyamines tested.Key words: polyamines, lima bean, herbivore-induced volatile organic compounds, calcium and ROS signalling, jasmonic acid, quantitative gene expression, transmembrane potentialPolyamines are involved in plants’ stress responses and growth. By activating biosynthesis of nucleic acids, polyamines concern the plant growth and differentiation.^1–3 Furthermore, it has been reported that polyamines are involved in the response against environmental stress and plant disease.^1–4 We recently reported that exogenously applied polyamines ∼diamines [cadaverine (Cad), putrescine (Put)], triamine [spermidine (Spd)] and tetraamine ]spermine (Spm)]∽ induce volatile emission in Lima bean leaves.⁵ Membrane potentials (V_m) and intracellular calcium variations were also studied in Lima bean leaves after perfusion with the polyamines and with these addition of JA and here we report on these additional results.The primary candidate for intercellular signaling in higher plants is the stimulus-induced change in V_m.⁶ The plasma membrane potential (V_m), which lies in the range of −50 to −200 mV in Lima bean leaves,⁷ may be shifted either to more negative (hyperpolarization) or to more positive values (depolarization) in response to various biotic or abiotic stresses.Measurement of V_m were performed and data statistically treated as previously described (ANOVA and Tukey-Kramer’s HSD test).⁷ Perfusion with the polyamines (Fig. 1 single arrow) shows a specific response of the leaf tissues with a different V_m depolarization, depending on the polyamine. In general, a V_m depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd (Fig. 1). Spm and Spd V_m depolarization values were significantly different (p < 0.05) from all other polyamines, whereas no significant difference was found between Put and Cad V_m depolarization (p = 0.435). In all cases, V_m depolarization was reversed by washing polyamine-treated leaves with a fresh buffer solution (Fig. 1 double arrow); however, a full recovery of the V_m was observed only for Put (Fig. 1). The linearization of the data from Figure 1 allowed to calculate the rate of V_m depolarization after perfusion of the polyamines which was higher for Spd (6.0 mV min⁻¹; R = 0.96), equal for Put and Cad (4.8 mV min⁻¹; Put R = 0.95; Cad R = 0.97) and lower for Spm (3.0 mV min⁻¹; R = 0.96).Open in a separate windowFigure 1Effect of 1 mM polyamines (arrow) on the V_m of Lima bean palisade cells. Spermine (Spm) caused the lowest V_m depolarization, whereas spermidine (Spd) showed the highest values of V_m depolarization. intermediate values were found when putrescine (Put) and cadaverine (cad) were perfused. after washing the tissues with fresh buffer (double arrow) V_m was always hyperpolarized, however the initial potential was recovered only for Put, while for all other polyamines the V_m never reached the initial values. Metric bars indicate standard deviation.Perfusion with JA caused a slight and not significant (p = 0.332) V_m depolarization (Fig. 2) with respect to control. The addition of JA caused a significant increase (p < 0.01) in V_m depolarization when perfused with Cad, with respect to the sole perfusion with Cad (Fig. 1). The same was observed when JA was perfused with Put, whereas not significant differences were observed when Spm (p = 0.513) and Spd (p = 0.107) were perfused with JA (Fig. 2), with respect to the sole perfusion with Spm and Spd (Fig. 1). The linearization of the data from Figure 2 allowed to calculate the rate of V_m depolarization after perfusion of the polyamines + JA, which was higher for Cad (24.40 mV min⁻¹; R = 0.99), almost equal for Put and Spd (Put: 14.21 mV min⁻¹, R = 0.99; Spd: 13.49 mV min⁻¹, R = 0.99) and lower for Spm (1.34 mV min⁻¹; R = 0.93). For JA the rate of V_m depolarization was 0.19 mV min⁻¹ (R = 0.96). With the addition of JA, a negative correlation was found between V_m depolarization and the number of amino group of the polyamines tested.Open in a separate windowFigure 2Effect of 1 mM polyamines + 0.1 mMJA (arrow) on the V_m of Lima bean palisade cells. the perfusion with Ja did not cause any variation in the V_m. addition of JA to Spm and Spd caused the same V_m depolarization observed in the absence of JA, whereas when JA was added to Put and Cad a stronger and significantly different V_m depolarization was observed. even in this case washing the tissues with fresh buffer (double arrow) caused a V_m hyperpolarized, however in this case Spd reached V_m values significantly more negative that the initial V_m. Metric bars indicate standard deviation. For abbreviations see Figure 1.Since ion fluxes through channels directly influence V_m, it seems reasonable to assume that molecules able to act on channel activity might be considered as important factors inducing electrical signals. Among the various channels, calcium and potassium channels are predominantly involved in cell signaling.⁸ In the present study, rapid and reversible V_m depolarization observed upon perfusion of Lima bean mesophyll cells with polyamines was found to be significantly increased when JA was added to Cad and Put. The reversibility of the V_m may be linked to the overall physico-chemical amphiphilic properties of polyamines, probably depending on non covalent interaction with plasma membrane molecules, as polyamines occur in plants in free form, bound electrostatically to negatively charged molecules, and conjugated to small molecules and proteins.⁹ Liu et al.¹⁰ showed that Spm, Spd, Cad and Put strongly inhibited opening and closing of stomata in Vicia faba, suggesting that polyamines target inward potassium channels in guard cells and modulate stomatal movements, so providing a link between abiotic stress, polyamine levels and stomatal regulation. Moreover, the transport of polyamines across the plasma membrane of plant cells is energy-dependent and calcium is involved in the uptake mechanism.^1,11 Both mechanisms can be correlated to the observed V_m depolarization, and the positive correlation between intracellular Ca²⁺ concentration⁵ and V_m depolarizing activity of polyamines confirms the involvement of Ca²⁺ during polyamine uptake.¹¹     

Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana

Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild-type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild-type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues.Key words: LORELEI, glycosylphosphatidylinositol (GPI)-anchored protein, embryogenesis, DD45, seed germination, primary and lateral root growth, seedling developmentDouble fertilization is unique to flowering plants. Upon female gametophyte reception of a pollen tube, the egg and central cells of the female gametophyte fuse with the two released sperm cells to form zygote and endosperm, respectively and initiate seed development.¹ The female gametophyte controls seed development by (1) repressing premature central cell or egg cell proliferation until double fertilization is completed,^1–3 (2) supplying factors that mediate early stages of embryo and endosperm development^1,4,5 and (3) regulating imprinting of genes required for seed development.^1,6The molecular mechanisms underlying female gametophyte control of early seed development are poorly understood. Although much progress has been made in identifying genes and mechanisms by which the female gametophyte represses premature central cell or egg cell proliferation until double fertilization is completed and regulates imprinting of genes required for seed development,^1,6 only a handful of female gametophyte-expressed genes that affect early embryo^7,8 and endosperm⁹ development after fertilization have been characterized. This is particularly important given that a large number of female gametophyte-expressed genes likely regulate early seed development.⁵We recently reported on a mutant screen for plants with reduced fertility and identification of a mutant that contained a large number of undeveloped ovules and very few viable seeds.¹⁰ TAIL-PCR revealed that this mutant is a new allele of LORELEI(LRE) [At4g26466].^10,11 Four lre alleles have been reported;¹¹ so, this mutant was designated lre-5.¹⁰ The Arabidopsis LORELEI protein contains 165 amino acids and possesses sequence features indicative of a glycosylphosphatidylinositol (GPI)-anchor containing cell surface protein. GPI-anchors serve as an alternative to transmembrane domains for anchoring proteins in cell membranes and GPI-anchored proteins participate in many functions including cell-cell signaling.¹²     

Enzymatic bleaching of kraft pulp by xylanase from Aspergillus sydowii SBS 45

Crude xylanase from Aspergillus sydowii SBS 45 was tested for enzymatic bleaching of kraft (Decker) pulp. After optimization of three parameters, consistency of pulp, retention time and enzyme dose, considerable increase in the release of UV and visible absorbance spectra of materials and reducing sugars was observed, which clearly indicated the action of xylanase on pulp. Final brightness of pulp was increased from 29.42 to 70.42% and kappa number was reduced from 15.93 to 1.61, when 25 U of xylanase was given with a retention time of 5 h and at a consistency of 10%. When 10 U g⁻¹ xylanase was given, 14.3% elemental chlorine and 14.3% H₂O₂ could be reduced and when 25 U g⁻¹ xylanase was given 14.3% elemental chlorine and 28.6% H ₂O₂ could be reduced thereby retaining the brightness at control level.     

Direct shoot bud induction and plant regeneration in <Emphasis Type="Italic">Capsicum frutescens</Emphasis> Mill.: influence of polyamines and polarity

Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472–497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28 μM indole-3-acetic acid, 10 μM silver nitrate and either of 13.31–89.77 μM benzyl adenine (BA), 9.29–23.23 μM kinetin, 0.91–9.12 μM zeatin, 2.46–9.84 μM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63 μM) as a cytokinin source and 19.4 ± 4.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24 h photoperiod. These buds were elongated in MS medium containing 2.8 μM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.     

Treatment of paper factory effluent using a phenol degrading Alcaligenes sp. under free and immobilized conditions

Treatment of the paper factory effluent was done with free and immobilized cells of a phenol degrading Alcaligenes sp. d(2). The free cells could bring a maximum of 99% reduction in phenol and 40% reduction in chemical oxygen demand (COD) after 32 and 20 h of treatment, respectively. In the case of immobilized cells, a maximum of 99% phenol reduction and 70% COD reduction was attained after 20 h of treatment under batch process. In the continuous mode of operation using packed bed reactor, the strain was able to give 99% phenol removal and 92% COD reduction in 8h of residence time The optimum flow rate was 2.5 ml/h and the half life period was 76 h. Even after the complete removal of phenol, the strain could further enhance reduction in chemical oxygen demand, which clearly indicated that in the paper factory effluent, this strain could also oxidize organic matter other than phenol.     

Assessment of culture medium without commercial ammonium nitrate for in vitro culture of industrially important plant species

Plant Cell, Tissue and Organ Culture (PCTOC) - Ammonium nitrate (AN) is one of the major nitrogen sources of Murashige and Skoog (MS) medium. It is prohibited in various countries, including India...