Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)

Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants.Phosphorylation plays a pivotal role in the regulation of a majority of cellular processes via signaling transduction pathways. During the last decade, quantitative phosphoproteomics has become a powerful and versatile platform to profile signaling pathways at a system-wide scale. Multiple signaling networks in different organisms have been characterized through global phosphorylation profiling (1–3), which has evolved over the years with highly optimized procedures for sample preparation and phosphopeptide enrichment, high resolution mass spectrometry, and data analysis algorithms to identify and quantify thousands of phosphorylation events (4–8).Quantitative phosphoproteomics can be achieved mainly by two major techniques, stable isotope labeling and label-free quantitation. Isotope labeling prior to liquid chromatography-mass spectrometry (LC-MS)¹ has been widely used, including metabolic labeling such as stable isotope labeling by amino acids in cell culture (SILAC), chemical labeling such as multiplexed isobaric tags for relative and absolute quantification (iTRAQ) and isotope-coded affinity tags (ICAT) (9–12). On the other hand, label-free quantitation has gained momentum in recent years (13–15), as no additional chemistry or sample preparation steps are required. Compared with stable isotope labeling, label-free quantitation has higher compatibility with the source of the samples, the number of samples for comparison, and the instrument choice.Many label-free approaches, in particular to phosphoproteomics, are based on ion intensity (16, 17), but they are relatively error-prone because of run-to-run variations in LC/MS performance (18). In theory, such systematic errors can be corrected by spiking an internal standard into every sample to be compared. Several methods based on internal standard spiking have been reported so far. Absolute quantification peptide technology (AQUA) (19), for example, uses synthetic peptides with isotope labeling for absolute quantitation. For a global quantitative proteomics study, it is unrealistic to spike-in all reference peptides. Another labeling reference method, spike-in SILAC appears to be a promising technique to quantify the proteome in vivo with multiplex capability and it can be extended to clinical samples (20). One solution to large-scale quantitation without any isotope labeling is pseudo internal standard approach (21), which selects endogenous house-keeping proteins as the internal standard so that no spike-in reagent is required. However, finding a good pseudo internal standard remains a challenge for phosphoproteome samples. Spike-in experiments are an alternative way to improve normalization profile. Some internal standard peptides such as MassPREP^TM (Waters) were already widely used. Other groups spiked an identical amount of standard protein into samples prior to protein digestion (22–24). There are two major normalization procedures. In one approach, sample peptides were normalized to the total peak intensity of spike-in peptides (25). Alternatively, the digested peptides were compared at first and the normalization factor was determined in different ways such as the median (26) or average of ratios (27). However, spiking an identical amount of standard proteins into phosphoproteomic samples before protein digestion may not be compatible with phosphoproteomic analyses which typically require a phosphopeptide enrichment step. Spectral counting has been extensively applied in large sets of proteomic samples because of its simplicity but the method is often not reliable for the quantitation of phosphoproteins, which are typically identified by single phosphopeptides with few spectra (28–30). Many software packages have been implemented to support the wide variety of those quantitation techniques, including commercial platforms such as Progenesis LC-MS^TM, Mascot Distiller^TM, PEAKS Q^TM, etc., as well as open-source software packages including MaxQuant (31), PEPPeR (32), Skyline (33), etc.In this study, we have devised a novel label-free quantitation strategy termed Library Assisted eXtracted Ion Chromatogram (LAXIC) for plant phosphoproteomic analyses with high accuracy and consistency (Fig. 1). The approach employs synthetic peptide libraries as the internal standard. These peptides were prepared to have proper properties for quality control assessments and mass spectrometric measurements. In particular, peptides were designed to elute sequentially over an entire LC gradient and to have suitable ionization efficiency and m/z values within the normally scanned mass range. Local normalization of peak intensity is performed using Loess Procedure, a data treatment adopted from cDNA microarray data analysis (34). To monitor the diverse ion suppression effect across retention time, the local normalization factors (LNFs) are determined by internal standard pairs in individual time windows. Finally, samples will be quantified with LNFs in order to correct variance of LC-MS conditions. This quantification occurs in a small time frame centered to each target peptide.Open in a separate windowFig. 1.Work flow for the LAXIC strategy to quantify the phosphorylation change in response to osmotic stress. A, Schematic representation of the LAXIC algorithm. First, all the chromatographic peaks were aligned and the ratios were calculated. Second, the normalization factors which equal to ratios of library peptide peaks between MS runs were chosen to construct normalization curve. Third, sample peptide peak ratios were normalized against predicted normalization factor corresponding to certain retention time. B, Schematic representation of quantitative phosphoproteomics. Plants either treated with mannitol or PBS were lysed and mixed proportionally at first. Following peptide digestion and enrichment, phosphopeptides were identified and further quantified through LAXIC incorporated with self-validating process using thelinear regression model to analyze the fold change (fold), linearity (R²) and accuracy (%Acc).Water deficit and salinity causes osmotic stress, which is a major environmental factor limiting plant agricultural productivity. Osmotic stress rapidly changes the metabolism, gene expression and development of plant cells by activating several signaling pathways. Several protein kinases have been characterized as key components in osmotic stress signaling. Arabidopsis histidine kinase AHK1 can complement the histidine kinase mutant yeast, which can act as the osmosensor in yeast (35). Overexpression of AHK1 gene increases the drought tolerance of transgenic plants in Arabidopsis (36). Similar to yeast, the MAPK kinase cascade is also involved in osmotic stress response in plants. It is reported that AtMPK3, AtMPK6, and tobacco SIPK can be activated by NaCl or mannitol, and play positive roles in osmotic signaling (37, 38). MKK7 and MKKK20 may act as the up-stream kinase in the kinase cascade (39). Involvement of some calcium-dependent protein kinases, such as AtCPK21, AtCPK6, and OsCPK7 (CDPK) in osmotic stress signaling has also been reported (40–42). Another kinase family, SNF1-related protein kinase (SnRK) 2, also participates in osmotic stress response. In Arabidopsis, there are ten members in the SnRK2 family. Five from the ten SnRK2s, SnRK2, 3, 6, 7, and 8, can be activated by abscisic acid (ABA) and play central roles in ABA-receptor coupled signaling (43, 44). Furthermore, all SnRK2s except SnRK2.9 can be activated by NaCl or mannitol treatment (43). The decuple mutant of SnRK2 showed a strong osmotic hypersensitive phenotype (45). It is proposed that protein kinases including MAPK and SnRK2s have a critical function in osmotic stress (46), but the detailed mechanism and downstream substrates or target signal components are not yet clarified. We applied, therefore, the LAXIC approach with a self-validating method (47) to profile the osmotic stress-dependent phosphoproteome in Arabidopsis by quantifying phosphorylation events before and after mannitol treatment. Among a total of over 2000 phosphopeptides, more than 400 of them are dependent on osmotic stress. Those phosphoproteins are present on enzymes participating in signaling networks that are involved in many processes such as signal transduction, cytoskeleton development, and apoptosis. Overall, LAXIC represents a powerful tool for label-free quantitative phosphoproteomics.     

PDEA-coated magnetic nanoparticles for gene delivery to Hep G2 cells

Poly(2-(diethylamino)ethyl methacrylate) coated magnetic nanoparticles (PDEA-MNPs) were synthesized as a new gene nanocarrier to delivery plasmids (pEGFPN1 and pRL-TK) into human hepatoma (Hep G2) cells. The PDEA-MNPs shows the pH-sensitive property. These nanoparticles are positively charged at acidic pH and negatively charged at neutral or alkaline pH. The PDEAMNPs exhibited a low cytotoxicity in Hep G2 cells. PDEA-MNPs could bind and protect DNA from DNase I degradation. The transfection study demonstrated that the PDEA-MNPs could carry plasmid into Hep G2 cells and exhibited a high gene transfection efficiency. These results indicated that the novel magnetic nanoparticles could enhance gene transfection in vitro and hold the potential to be a promising non-viral nanodevice.     

Lactobacillus paracasei subsp. paracasei LC01 positively modulates intestinal microflora in healthy young adults

Lactobacillus paracasei subsp. paracasei LC01 (LC01) can tolerate intestinal stresses and has antioxidant activity. To evaluate the effect of the bacterium on human intestinal microflora, a randomized, double-blind, placebo-controlled human trial was carried out. Fifty-two healthy adult volunteers were randomized equally to two groups. One group consumed 12% (wt/vol) skimmed milk supplemented with 10¹⁰ CFU of LC01 each day for the 4-week treatment period, and then consumed placebo in the next treatment period, separated by a 2-week washout. The other group followed the reverse order. Group-specific real-time PCR and biochemical analyses was used to determine the intestinal bacterial composition of fecal samples collected at the end of every period, and the concentration of short-chain fatty acids and ammonia. A significant inhibition in fecal Escherichia coli and increase in Lactobacillus, Bifidobacterium, and Roseburia intestinalis were observed after consumption of LC01. Acetic acid and butyric acid were significantly higher in the probiotic stage and fecal ammonia was significantly lower. The results indicated a modulation effect of LC01 on the intestinal microflora of young adults, suggesting a beneficial effect on bowel health. LC01 may have potential value as a probiotic.     

Phylogenetic position of Jaoa,a green algal genus endemic to China

Jaoa prasina, a freshwater green alga endemic to China, was collected from a stream in Hubei province, China. Unialgal cultivation, morphological observation, and phylogenetic analyses of small subunit ribosomal DNA and RuBisCO large subunit sequences were performed. When cultured on agar medium, the alga was irregularly filamentous, similar to marine species of Acrochaete. Aplanospores were observed on solid medium. A vesicular‐like thallus without rhizoids developed in liquid medium, similar to specimen development in natural habitats. Molecular phylogenetic analyses revealed that Jaoa was closely related to the marine genera Acrochaete Pringsheim and Ulvella Crouan & Crouan. The results suggested the genus Jaoa is a member of the family Ulvellaceae (Ulvophyceae), which contains mostly marine algae. The family name Jaoaceae should be abandoned. We speculate that Jaoa may have evolved from a marine Ulvellaceae ancestor.     

Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs

Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed “toolkits” utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.     

Improvements in the primary culture of neonate rat myocardial cells by study of the mechanism of endoplasmic reticulum stress

We previously found that endoplasmic reticulum stress (ERS) might be exhibited in the conventional protocol of the primary culture of neonate rat myocardial cells (NRMCs) and that the high glucose concentration (25 mmol/L) in the culture medium might be the cause. Here, we investigated if the high concentration of glucose might influence ERS in myocardial cells during culture. GRP78 expression (ERS marker) was similar in groups with tunicamycin (TM) and without TM in high glucose cultured cells (p?>?0.01). Different glucose concentrations elicited different GRP78 expressions according to analyses of protein and RNA levels, which showed ERS in H/H groups. Finally, we found that GRP78 expression was higher in TM groups compared with M/M groups (p?<?0.01). The conventional high-glucose culture media during primary culture of NRMCs induced ERS. We propose that medium-glucose culture media should be used and describe an improved protocol for the primary culture of NRMCs.     

hPNAS-4 inhibits proliferation through S phase arrest and apoptosis: underlying action mechanism in ovarian cancer cells

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Previous studies have shown that hPNAS-4 can inhibit tumor growth when over-expressed in ovarian cancer cells. However, the underlying action mechanism remains elusive. In this work, we found that hPNAS-4 expression was significantly increased in SKOV3 cells when exposed to cisplatin, methyl methanesulfonate or mitomycin C, and that its overexpression could induce proliferation inhibition, S phase arrest and apoptosis in A2780s and SKOV3 ovarian cancer cells. The S phase arrest caused by hPNAS-4 was associated with up-regulation of p21. p21 is p53-dispensable and correlates with activation of ERK, and activation of the Cdc25A-Cdk2-Cyclin E/Cyclin A pathway, while the pro-apoptotic effects of hPNAS-4 were mediated by activation of caspase-9 and -3 other than caspase-8, and accompanied by release of AIF, Smac and cytochrome c into the cytosol. Taken together, these data suggest a new mechanism by which hPNAS-4 inhibits proliferation of ovarian cancer cells by inducing S phase arrest and apoptosis via activation of Cdc25A-Cdk2-Cyclin E/Cyclin A axis and mitochondrial dysfunction-mediated caspase-dependent and -independent apoptotic pathways. To our knowledge, we provide the first molecular evidence for the potential application of hPNAS-4 as a novel target in ovarian cancer gene therapy.     

Th1/Th2/Th17/Treg cytokines in Guillain–Barré syndrome and experimental autoimmune neuritis

Guillain–Barré syndrome (GBS) is an immune-mediated acute inflammatory disorder in the peripheral nervous system (PNS) of humans characterized by inflammatory infiltration and damage to myelin and axon. Experimental autoimmune neuritis (EAN) is a useful animal model for GBS. Although GBS and EAN have been widely studied, the pathophysiological basis of GBS/EAN remains largely unknown. Immunocompetent cells together with cytokines produced by various cells contribute to the inflammatory process of EAN by acting as mediators or effectors. Both GBS and EAN have hitherto been attributed to T helper (Th)1 cells-mediated disorders, however, some changes in GBS and EAN could not be explained by the pathogenic role of Th1 cells and a disturbance of the Th1/Th2 balance, which has previously been considered to be important for the homeostatic maintenance of the immune responses and to explain the adaptive immunity and autoimmune diseases. The Th1/Th2 paradigm in autoimmune diseases has been greatly challenged in recent years, with the identification of a particular T cell subset Th17 cells. Studies on the associations between Th17 cells/cytokines and GBS/EAN are reviewed. But some of them occasionally yield conflicting results, indicating an intricate network of cytokines in immune response.