Stable isotope labelled mass spectrometry for quantification of the relative abundances for expressed proteins induced by PeaT1

The protein elicitor from the mycelium of Alternaria tenuissima has been isolated.The elicitor triggered resistance to the tobacco mosaic virus in tobacco by inducing relative oxygen species,but without causing hypersensitive necrosis.The elicitor is reported to impart resistance against Verticillium dahliae and to increase yield in cotton,but its mechanism is not yet clear.In this study,the stable isotope labelled mass spectrometry method was used to quantify the relative abundances of protein expression induced by PeaT1 in Arabidopsis.A significant difference in the relative abundances for the expression of different proteins related to metabolism,modification,regulatory,defense,stress and antioxidation was found in Arabidopsis.

Stable isotope labeling of nanomaterials for biosafety evaluation and drug development

Quantitative information,such as environmental migration,absorption,biodistribution,biotransformation,and elimination,is fundamental and essential for the nanosafety evaluations of nanomaterials.Due to the complexity of biological and environmental systems,it is challenging to develop quantitative approaches and tools that could characterize intrinsic behaviors of nanomaterials in the organisms.The isotopic tracers are ideal candidates to tune the physical properties of nanomaterials while preserving their chemical properties.In this review article,we summarized the stable isotope labeling methods of nanomaterials for evaluating their environmental and biological effects.The skeleton labeling protocols of carbon nanomaterials and metal/metal oxide nanoparticles were introduced.The advantages and disadvantages of stable isotope labeling were discussed in comparison with other quantitative methods for nanomaterials.The quantitative information of nanomaterials in environmental and biological systems was summarized along with the biosafety data.The benefits for drug development of nanomedicine were analyzed based on the targeting effects,persistent accumulation,and safety.Finally,the challenges and future perspectives of stable isotope labeling in nanoscience and nanotechnology were discussed.

Proteomic analysis of energy metabolism and signal transduction in irradiated melanoma cells

AIM: To analyze proteomic and signal transduction alterations in irradiated melanoma cells. METHODS: We combined stable isotope labeling with amino acids in cell culture (SILAC) with highly sensitive shotgun tandem mass spectrometry (MS) to create an efficient approach for protein quantification. Protein protein interaction was used to analyze relationships among proteins. RESULTS: Energy metabolism protein levels were significantly different in glycolysis and not significantly different in oxidative phosphorylation after irradiation. Conversely, tumor suppressor proteins related to cell growth and development were downregulated, and those related to cell death and cell cycle were upregulated in irradiated cells. CONCLUSION: Our results indicate that irradiation induces differential expression of the 29 identified proteins closely related to cell survival, cell cycle arrest, and growth inhibition. The data may provide new insights into the pathogenesis of uveal melanoma and guide appropriate radiotherapy.

Spatial patterns nitrogen transfer models of ectomycorrhizal networks in a Mongolian scotch pine plantation

Ectomycorrhizal(EM)networks provide a variety of services to plants and ecosystems include nutrient uptake and transfer,seedling survival,internal cycling of nutrients,plant competition,and so on.To deeply their structure and function in ecosystems,we investigated the spatial patterns and nitrogen(N)transfer of EM networks usingN labelling technique in a Mongolian scotch pine(Pinus sylvestris var.mongolica Litv.)plantation in Northeastern China.In August 2011,four plots(20 × 20 m)were set up in the plantation.125 ml 5 at.%0.15 mol/LNHNOsolution was injected into soil at the center of each plot.Before and 2,6,30 and 215 days after theN application,needles(current year)of each pine were sampled along four 12 m sampling lines.Needle total N andN concentrations were analyzed.We observed needle N andN concentrations increased significantly over time afterN application,up to 31 and0.42%,respectively.There was no correlation between needle N concentration andN/N ratio(R2=0.40,n=5,P=0.156),while excess needle N concentration and excess needleN/N ratio were positively correlated across different time intervals(R~2=0.89,n=4,P\0.05),but deceased with time interval lengthening.NeedleN/N ratio increased with time,but it was not correlated with distance.NeedleN/N ratio was negative with distance before and 6th day and 30th day,positive with distance at 2nd day,but the trend was considerably weaker,their slop were close to zero.These results demonstrated that EM networks were ubiquitous and uniformly distributed in the Mongolian scotch pine plantation and a random network.We found N transfer efficiency was very high,absorbed N by EM network was transferred as wide as possible,we observed N uptake of plant had strong bias forN andN,namely N fractionation.Understanding the structure and function of EM networks in ecosystems may lead to a deeper understanding of ecological stability and evolution,and thus provide new theoretical approaches to improve conservation practices for the management of the Earth’s ecosystems.

Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis

Ethylene participates in the regulation of numerous cellular events and biological processes, including wa- ter loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a lSN stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eill-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene- regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-O and ein3eill genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up- regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs.

A novel strategy for high-specificity, high-sensitivity, and high-throughput study for gut microbiome metabolism of aromatic carboxylic acids

Aromatic carboxylic acids(ACAs) may be as transformed key metabolites via gut microbiome for playing better pharmacological effects. However, it's rare to achieve high-specificity, high-sensitivity, and highthroughput detection simultaneously, especially, for tracing trace ACAs in gut microbiome. In this work,firstly, a novel dual-template and double-shelled molecularly imprinted 96-well microplates(DDMIPs)was designed and amplified signal for p-hydroxybenzoic acid(PBA) and 3,4,5-trimethoxycinnamic acid(TMA). Additionally, the DDMIPs and a stable isotope labeling derivatization(SILD) method combined with the ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry(UHPLC-TQ MS) was firstly stepwise integrated, achieving high-effective, high-sensitive, and highthroughput study of gut microbiome metabolism. The whole strategy showed lower limits of detections(LODs) up to 1000 folds than the traditional method, and revealed a more real metabolism-time profile of PBA and TMA by 3-step signal amplification. The platform also laid the foundation for fast, simple,high-selective, high-effective, and high-throughput metabolism and pharmacological research.

Transformation of 5-Carboxylcytosine to Cytosine Through C-C Bond Cleavage in Human Cells Constitutes a Novel Pathway for DNA Demethylation

Active demethylation of 5-methylcytosine(5mC)can be realized through ten-eleven translocation(TET)dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC),and 5-carboxylcytosine(5caC),followed by thymine DNA glycosylase(TDG)-initiated base excision repair(BER).The TDG-BER pathwaymay lead to the generation of DNA strand breaks,potentially compromising genome integrity.Alternatively,direct decarboxylation of TET-produced 5caC is highly attractive because this mechanism allows for conversion of 5mC to cytosine without the formation of DNA strand breaks.However,cleavage of the C–C bond in 5caC in human cells remains an open question.We examined this reaction in cell extract and live cells using 5caC-carrying hairpin DNA substrate.After incubation with whole-cell protein extract or transfection into human cells,we monitored the transformation of 5caC to cytosine through direct decarboxylation or BER using liquid chromatography–tandem mass spectrometry(LCMS/MS)analyses at both the mononucleotide and oligodeoxynucleotide levels.Our results clearly showed the direct conversion of 5caC to cytosine in human cells,providing evidence to support a novel pathway for active DNA demethylation.