First systematic plant proteomics workshop in Botany Department, University of Delhi: transferring proteomics knowledge to next-generation researchers and students.

International Plant Proteomics Organization (INPPO) outlined ten initiatives to promote plant proteomics in each and every country. With greater emphasis in developing countries, one of those was to "organize workshops at national and international levels to train manpower and exchange information". This third INPPO highlights covers the workshop organized for the very first time in a developing country, India, at the Department of Botany in University of Delhi on December 26-30, 2013 titled - "1(st) Plant Proteomics Workshop / Training Program" under the umbrella of INPPO India-Nepal chapter. Selected 20 participants received on-hand training mainly on gel-based proteomics approach along with manual booklet and parallel lectures on this and associated topics. In house, as well as invited experts drawn from other Universities and Institutes (national and international), delivered talks on different aspects of gel-based and gel-free proteomics. Importance of gel-free proteomics approach, translational proteomics, and INPPO roles were presented and interactively discussed by a group of three invited speakers Drs. Ganesh Kumar Agrawal (Nepal), Randeep Rakwal (Japan), and Antonio Masi (Italy). Given the output of this systematic workshop, it was proposed and thereafter decided to be organized every alternate year; the next workshop will be held in 2015. Furthermore, possibilities on providing advanced training to those students / researchers / teachers with basic knowledge in proteomics theory and experiments at national and international levels were discussed. INPPO is committed to generating next-generation trained manpower in proteomics, and it would only happen by the firm determination of scientists to come forward and do it.

S-nitrosylation analysis in Brassica juncea apoplast highlights the importance of nitric oxide in cold-stress signaling.

Reactive nitrogen species (RNS) including nitric oxide (NO) are important components of stress signaling. However, RNS-mediated signaling in the apoplast remains largely unknown. NO production measured in the shoot apoplast of Brassica juncea seedlings showed nonenzymatic nitrite reduction to NO. Thiol pool quantification showed cold-induced increase in the protein (including S-nitrosothiols) as well as non protein thiols. Proteins from the apoplast were resolved as 109 spots on the 2-D gel, while S-nitrosoglutathione-treated (a NO donor), neutravidin-agarose affinity chromatography-purified S-nitrosylated proteins were resolved as 52 spots. Functional categorization after MALDI-TOF/TOF identification showed 41 and 38% targets to be metabolic/cell-wall-modifying and stress-related, respectively, suggesting the potential role(s) of S-nitrosylation in regulating these responses. Additionally, identification of cold-stress-modulated putative S-nitrosylated proteins by nLC-MS/MS showed that only 38.4% targets with increased S-nitrosylation were secreted by classical pathway, while the majority (61.6%) of these were secreted by unknown/nonclassical pathways. Cold-stress-increased dehydroascorbate reductase and glutathione S-transferase activity via S-nitrosylation and promoted ROS detoxification by ascorbate regeneration and hydrogen peroxide detoxification. Taken together, cold-mediated NO production, thiol pool enrichment, and identification of the 48 putative S-nitrosylated proteins, including 25 novel targets, provided the preview of RNS-mediated cold-stress signaling in the apoplast.

Sub-proteome S-nitrosylation analysis in Brassica juncea hints at the regulation of Brassicaceae specific as well as other vital metabolic pathway(s) by nitric oxide and suggests post-translational modifications cross-talk.

Abiotic stress affects the normal physiology of the plants and results in crop loss. Brassica juncea is an oil yielding crop affected by abiotic stress. In future, over 30% yield loss by abiotic stress is predicted in India. Understanding the mechanism of plant response to stress would help in developing stress tolerant crops. Nitric oxide (NO) is now viewed as a remarkably important signaling molecule, involved in regulating stress responses. S-Nitrosylation is a NO based post-translational modification (PTM), linked with the regulation of many physiologically relevant targets. In the last decade, over 700 functionally varied S-nitrosylated proteins were identified, which suggested broad-spectrum regulation. To understand the physiological significance of S-nitrosylation, it was analyzed in cold stress. Functional categorization and validation of some of the B. juncea S-nitrosylated targets, suggested that NO produced during stress regulates cellular detoxification by modulating enzymes of ascorbate glutathione cycle, superoxide dismutase, glutathione S-transferase and glyoxalase I by S-nitrosylation in crude, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) depleted and apoplastic fractions. Interestingly, S-nitrosylation of enzymes associated with glucosinolate hydrolysis pathway, suggests a novel regulation of this Brassicaceae specific pathway by NO. Moreover, identification of enzymes of Glycolysis and Calvin cycle in crude and RuBisCO depleted fractions showed the regulation of metabolic as well as photosynthetic pathways by S-nitrosylation. S-Nitrosylation of cell wall modifying and proteolytic enzymes in the apoplast suggested differential and spatial regulation by S-nitrosylation. To have an overview of physiological role(s) of NO, collective information on NO based signaling (mainly by S-nitrosylation) is presented in this review.

Expanding roles of cross-talk between hydrogen sulfide and nitric oxide under abiotic stress in plants.

Abiotic stress such as salt, heavy metals, drought, temperature, and others can affect plants from seed germination to seedling growth to reproductive maturity. Abiotic stress increases reactive oxygen species and lowers antioxidant enzymes in plants resulted the plant tolerance ability against stress conditions decrease. Hydrogen sulfide (H2S) and nitric oxide (NO) are important gasotransmitters involved in seed germination, photosynthesis, growth and development, metabolism, different physiological processes and functions in plants. In plants, various enzymes are responsible for the biosynthesis of both H2S and NO via both enzymatic and non-enzymatic pathways. They also mediate post-translation modification, such as persulfidation, and nitrosylation, which are protective mechanisms against oxidative damage. They also regulate some cellular signalling pathways in response to various abiotic stress. H2S and NO also stimulate biochemical reactions in plants, including cytosolic osmoprotectant accumulation, reactive oxygen species regulation, antioxidant system activation, K+ uptake, and Na+ cell extrusion or vacuolar compartmentation. In this review, we summarize how H2S and NO interact with each other, the function of both H2S and NO, the mechanism of biosynthesis, and post-translational modification under different abiotic stress. Our main emphasis was to find the cross-talk between NO and H2S and how they regulate genes in plants under abiotic stress.

Nitric oxide-cold stress signalling cross-talk, evolution of a novel regulatory mechanism.

Plants enhance their cold stress tolerance by cold acclimation, a process which results in vast reprogramming of transcriptome, proteome and metabolome. Evidence suggests nitric oxide (NO) production during cold stress which regulates genes (especially the C-repeat binding factor (CBF) cold stress signalling pathway), diverse proteins including transcription factors (TFs) and phosphosphingolipids. About 59% (redox), 50% (defence/stress) and 30% (signalling) cold responsive proteins are modulated by NO-based post translational modifications (PTMs) namely S-nitrosylation, tyrosine nitration and S-glutathionylation, suggesting a cross-talk between NO and cold. Analysis of cold stress responsive deep proteome in apoplast, mitochondria, chloroplast and nucleus suggested continuation of this cross-talk in sub-cellular systems. Modulation of cold responsive proteins by these PTMs right from cytoskeletal elements in plasma membrane to TFs in nucleus suggests a novel regulation of cold stress signalling. NO-mediated altered protein transport in nucleus seems an important stress regulatory mechanism. This review addresses the NO and cold stress signalling cross-talk to present the overview of this novel regulatory mechanism.

Evaluation of relationship between odontogenic infections and maxillary sinus changes: A Cone Beam Computed Tomography-based study.

Introduction: Odontogenic infections affects the maxillary sinus mucosa. Cone Beam Computed Tomography (CBCT) is helpful in diagnosis of maxillary sinusitis of odontogenic origin. This cross-sectional study was planned with the aim to assess maxillary sinus changes associated with odontogenic infection by evaluating CBCT images. Material and methods: In cross-sectional analytical study, total 213 patients (404 sinuses) were evaluated on CBCT after thorough clinical examination of the individuals and taking into consideration the history of rhino or allergic rhinitis. Based upon CBCT images and clinical examination, patients were divided into, study group (odontogenic infection) and control group (no associated odontogenic infection). Effect of the size of periapical lesion, spatial relationship of lesion to the sinus floor and periodontal bone loss on maxillary sinus changes were evaluated. Results: Mucosal changes found in 200 sinuses (49.5%), mucosal thickening being the most prevalent and more commonly associated with odontogenic infections (p = 0.004). A significantly increased risk of mucosal thickening was observed with severe periodontal bone loss (p = 0.008). Size and spatial relationship of lesion to the maxillary sinus had no impact on the prevalence of mucosal thickening (p = 0.6, p = 0.4 respectively). Periodontal bone loss was 2.2 more likely to be associated with mucosal thickening than periapical or combined lesion. Conclusions: Most prevalent sinus change was mucosal thickening. Periodontal bone loss was significantly associated with mucosal thickening. CBCT is an appropriate method for sinus evaluation.

Evaluation of transcutaneous electrical nerve stimulation as an adjunct therapy in trigeminal neuralgia - a randomized double-blind placebo-controlled clinical study.

BACKGROUND: Trigeminal neuralgia (TN) is a severe form of pain that affects the daily activities of a patient. Transcutaneous electrical nerve stimulation (TENS) therapy is an emerging option for the treatment of acute and chronic pain. The aim of this study was to evaluate the effect of TENS therapy as an adjunct to drug therapy for the treatment of TN. METHODS: A total of 52 patients diagnosed with TN according to the International Classification of Headache Disorders (version 3) were included. Each patient was randomized to either the TENS or placebo TENS groups. Intervention was given in continuous mode and 100-Hz frequency for 20 mins biweekly for 6 weeks. Parameters were measured at baseline, TENS completion and 3 months, 6 months, and 1 year of follow up. The parameters observed were mean carbamazepine dose, mean visual analog scale (VAS) score, mean present pain intensity (PPI) score, and functional outcome. Non-parametric analyses, one-way ANOVA and the Kruskal-Wallis test were applied for intragroup comparisons, while the Mann-Whitney U test and independent t-test were used for intergroup comparisons of variables. The chi-square test was applied to analyze categorical data. RESULTS: Compared to the placebo TENS group, the mean dose of carbamazepine in the TENS group was significantly reduced at TENS completion, as well as at 6 months and 1 year follow up. Changes in mean VAS score, mean PPI score, and functional outcome did not show significant differences between the groups (P>0.05). CONCLUSION: TENS therapy does not lead to any changes in pain levels but it may reduce the mean dose of carbamazepine when used as an adjunct treatment in patients with TN.

Asada-Halliwell pathway maintains redox status in Dioscorea alata tuber which helps in germination.

Reactive Oxygen Species (ROS) are important regulatory molecules governing physiological processes. In the present study a biochemical and proteome level comparison of two contrasting growth stages of Dioscorea alata tuber namely germinating and mature tuber was performed in order to understand the tuber physiology and biochemistry. Existence of all the component enzymes [APx (ascorbate peroxidase), GR (glutathione reductase), DHAR (dehydroascorbate reductase), MDHAR (mono-dehydroascorbate reductase)] and major products [ascorbate (ASC) and glutathione (GSH)] of the cycle showed an operational Asada-Halliwell cycle in the tuber. A 2.65 fold increase in ASC content & a 3.8 fold increase in GR activity fortified the redox milieu during germination. In contrast a 5 fold higher H2O2 content (due to 3.08 fold lower APx activity) and accumulation of reactive nitrogen species (RNS) such as nitric oxide (NO, 2.4-fold) and S-nitrosothiol (SNO, 2.08 fold) contributed to overall oxidative conditions in the mature tuber. The carbonic anhydrase (CA, 7.5 fold), DHAR (5.31 fold) and MDHAR (7 fold) activities were higher in the germinating tuber in comparison with the mature tuber. GSNO negatively regulated the CA (3.6 & 3.95 fold), MDHAR (7.5 & 1.5 fold) and APx (2.3 & 1.81 fold) while another NO donor, CysNO negatively regulated the DHAR (2.24 & 1.32 fold) activity in the mature and germinating stages respectively indicating again that the lesser inhibition by NO (via nitrosylation) may be because of overall reducing environment in the germinating tuber. Increased SNO leading to S-nitrosylation of dioscorin was confirmed by Biotin switch assay. This is the first report showing dioscorin nitrosylation. The present analysis showed differential redox regulation and also suggests the physiological relevance of CA, DHAR, MDHAR, APx & GR in tuber germination for the first time. These enzymes may be used as potential markers of tuber germination in future.

Nitric oxide modulates Lycopersicon esculentum C-repeat binding factor 1 (LeCBF1) transcriptionally as well as post-translationally by nitrosylation.

Nitric oxide (NO) production increases in the cold stress. This cold enhanced NO manifests its effect either by regulating the gene expression or by modulating proteins by NO based post-translational modifications (PTMs) including S-nitrosylation. CBF (C-repeat binding factor) dependent cold stress signaling is most studied cold stress-signaling pathway in plants. SNP (sodium nitroprusside, a NO donor) treatment to tomato seedlings showed four fold induction of LeCBF1 (a cold inducible CBF) transcript in cold stress. S-nitrosylation as PTM of CBF has not been analyzed till date. In silico analysis using GPS-SNO 1.0 software predicted Cys 68 as the probable site for nitrosylation in LeCBF1. The 3D structure and motif prediction showed it to be present in the beta hairpin loop and hence available for S-nitrosylation. LeCBF1 was cloned and expressed in Escherichia coli. LeCBF1 accumulated in the inclusion bodies, which were solubilized under denaturing conditions and purified after on column refolding by Ni-NTA His tag affinity chromatography. Purified LeCBF1 resolved as a 34 kDa spot with a slightly basic pI (8.3) on a 2-D gel. MALDI-TOF mass spectrometry identified it as LeCBF1 and western blotting using anti-LeCBF1 antibodies confirmed its purification. Biotin switch assay and neutravidin affinity chromatography showed LeCBF1 to be S-nitrosylated in presence of GSNO (NO donor) as well as endogenously (without donor) in cold stress treated tomato seedlings. Dual regulation of LeCBF1 by NO at both transcriptional as well as post-translational level (by S-nitrosylation) is shown for the first time.

RuBisCO depletion improved proteome coverage of cold responsive S-nitrosylated targets in Brassica juncea.

Although in the last few years good number of S-nitrosylated proteins are identified but information on endogenous targets is still limiting. Therefore, an attempt is made to decipher NO signaling in cold treated Brassica juncea seedlings. Treatment of seedlings with substrate, cofactor and inhibitor of Nitric-oxide synthase and nitrate reductase (NR), indicated NR mediated NO biosynthesis in cold. Analysis of the in vivo thiols showed depletion of low molecular weight thiols and enhancement of available protein thiols, suggesting redox changes. To have a detailed view, S-nitrosylation analysis was done using biotin switch technique (BST) and avidin-affinity chromatography. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is S-nitrosylated and therefore, is identified as target repeatedly due to its abundance. It also competes out low abundant proteins which are important NO signaling components. Therefore, RuBisCO was removed (over 80%) using immunoaffinity purification. Purified S-nitrosylated RuBisCO depleted proteins were resolved on 2-D gel as 110 spots, including 13 new, which were absent in the crude S-nitrosoproteome. These were identified by nLC-MS/MS as thioredoxin, fructose biphosphate aldolase class I, myrosinase, salt responsive proteins, peptidyl-prolyl cis-trans isomerase and malate dehydrogenase. Cold showed differential S-nitrosylation of 15 spots, enhanced superoxide dismutase activity (via S-nitrosylation) and promoted the detoxification of superoxide radicals. Increased S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase sedoheptulose-biphosphatase, and fructose biphosphate aldolase, indicated regulation of Calvin cycle by S-nitrosylation. The results showed that RuBisCO depletion improved proteome coverage and provided clues for NO signaling in cold.