CAPILLARY ISOELECTRIC FOCUSING-BASED MULTIDIMENSIONAL PEPTIDE/PROTEIN SEPARATIONS FOR PROTEOMICS ANALYSIS

With the completion of the human genome project, the proteomics has become the focus of research interest for better understanding the complex biological processes. The mass spectrometry (MS) detection of vast number and broad dynamic range of the proteins requires that sample fractionation and separation be performed prior to MS analysis. Realizing the limitations of gel-based proteomic techniques, capillary-based and gel free separation technologies are presented as attractive alternatives holding the promises of high separation efficiency and resolution, broad dynamic range, easy system automation as well as high throughput. Coupled with laser free microdissection technology, the combination of CIEF with nano-RPLC in an automated and integrated platform was employed for comprehensive and sensitive proteome studies of limited protein quantities obtained from tissue samples. The peptides were first separated and concentrated by CIEF and were sequentially fractionated. All the CIEF fractions were further resolved by nano-RPLC, followed by tandem MS analysis. A total of 6,866 fully tryptic peptides were detected, leading to the identification of 1,820 distinct proteins. Due to limited peptide sequence coverage of identified proteins, the bottom-up approaches provide very limited molecular information about the intact proteins, particularly towards the detection of post-translational modifications. In contrast, top-down methods are advantageous for the detection of protein modifications. To improve separation efficiency and resolution of nano-RPLC separations for intact proteins, various chromatography conditions, including the chain length of the stationary phase, the column temperature, and the ion-pairing agent utilized in the mobile phase, were optimized using model proteins. Building upon the experience in the development of automated and integrated multidimensional peptide separation platform and the optimization of protein chromatography separation, a top-down proteome characterization of yeast cell lysates was further evaluated. An overall system capacity of 4,320-7,200 was achieved and a total of 534 distinct yeast protein masses were measured, yet required a protein loading of only 9.6 ug. This protein loading is two to three orders of magnitude less than those used in current top-down proteome techniques, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples

Overexpression of antioxidant genes in transgenic tomato tolerant against multiple stresses

The effect of increased manganese superoxide dismutase (Mn-SOD) and cytosolic ascorbate peroxidase (cAPX) on various abiotic stresses was studied using transgenic tomato (Lycopersicon esculentum) plants. Transformants were selected in vitro based on kanamycin resistance, confirmed by PCR and northern blot analyses. Northern blots showed an enhanced gene expression of Mn-SOD and cAPX in transgenic Mn-SOD and cAPX plants, respectively. APX native gel assay showed APX activity in transgenic cAPX plants several fold greater than in wild-type (WT) plants in the absence of stress. An additional Mn-SOD enzyme activity band was detected by native PAGE in transformed Mn-SOD plants. Germination of cAPX tomato plant seeds at low temperature (9°C) was greater than of WT seeds. Leaf tissues of the transgenic cAPX plants had lower electrolyte leakage in response to cold temperatures (4°C) than leaf tissues of WT plants. Transgenic Mn-SOD plants also showed resistance to the superoxide- generating herbicide methyl viologen (MV, 10⁻⁴ M). The total SOD activity was 1.5- to 2-fold higher and APX activity was 6- to 7-fold higher in transgenic Mn- SOD plants than in WT plants under MV stress, respectively. Germination of Mn- SOD tomato plant seeds in the presence of 150 mM NaCl was greater than in seeds from WT plants. Transgenic Mn-SOD shoots developed more roots under salt stress (200 mM and 250 mM NaCl, respectively). Both transgenic Mn-SOD and cAPX plant seedlings had enhanced ability to tolerate NaCl stress (200 mM and 250 mM). Total APX activity of transgenic plants was several fold higher than that of WT under salt stress (200 mM NaCl). Leaves and fruits from Mn-SOD or cAPX transgenic tomato plants were more resistant to heat (40°C) and UV-B (2.5 mW/cm² for 5 days) stresses compared to WT plants, respectively. Detached fruits from transgenic plants showed more resistance to sunscald in a field test. APX activity in leaves of the transgenic cAPX plants was several fold higher than that of WT plant leaves when exposed to the heat, drought, and UV-B stresses

Codon optimization, constitutive expression and antimicrobial characterization of hen egg white lysozyme (HEWL) in Pichia pastoris

Fusarium oxysporum (F. oxysporum) and Verticillium dahlia (V. dahlia) causes severe cotton disease in China and other cotton-producing countries. Hen egg white lysozyme (HEWL) has antimicrobial properties. In this study, a codon-optimized HEWL gene was synthesized and cloned into the yeast expression vector, pPIC9K, under the control of the Pichia pastoris (P. pastoris) glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). Results showed that codon-optimized HEWL (oHEWL) was constitutively expressed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the molecular weight of recombinant HEWL (roHEWL) was 14 kDa which corresponds to the standard HEWL. The expression of the roHEWL reached to 54 mg/L. Activity of the roHEWL was 1680 U/mL. The optimum pH for roHEWL was from 6.0 to 7.0, and the optimum temperature was 55°C. In vitro antimicrobial activity assay revealed that roHEWL can lyse cell walls of the gram positive bacteria, Micrococcus lysodeikticus (M. lysodeikticus). In vivo studies showed that it inhibits plant fungi, F. oxysporum and V. dahlia. roHEWL anti-fungal properties might be useful for future genetically engineered cotton plant resistance against pathogenic fungal disease.Keywords: Hen egg white lysozyme (HEWL), antimicrobial activity, glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, codon optimization, constitutive expressio

Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling

The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. © 2016 Hou et al

Linear brain measurement: a new screening method for cognitive impairment in elderly patients with cerebral small vessel disease

BackgroundThe old adults have high incidence of cognitive impairment, especially in patients with cerebral small vessel disease (CSVD). Cognitive impairment is not easy to be detected in such populations. We aimed to develop clinical prediction models for different degrees of cognitive impairments in elderly CSVD patients based on conventional imaging and clinical data to determine the better indicators for assessing cognitive function in the CSVD elderly.Methods210 CSVD patients were screened out by the evaluation of Magnetic Resonance Imaging (MRI). Then, participants were divided into the following three groups according to the cognitive assessment results: control, mild cognitive impairment (MCI), and dementia groups. Clinical data were collected from all patients, including demographic data, biochemical indicators, carotid ultrasound, transcranial Doppler (TCD) indicators, and linear measurement parameters based on MRI.ResultsOur results showed that the brain atrophy and vascular lesions developed progressive worsening with increased degree of cognitive impairment. Crouse score and Interuncal distance/Bitemporal distance (IUD/BTD) were independent risk factors for MCI in CSVD patients, and independent risk factors for dementia in CSVD were Crouse Score, the pulsatility index of the middle cerebral artery (MCAPI), IUD/BTD, and Sylvian fissure ratio (SFR). Overall, the parameters with high performance were the IUD/BTD (OR 2.28; 95% CI 1.26–4.10) and SFR (OR 3.28; 95% CI 1.54–6.91), and the AUC (area under the curve) in distinguishing between CSVD older adults with MCI and with dementia was 0.675 and 0.724, respectively. Linear brain measurement parameters had larger observed effect than other indexes to identify cognitive impairments in CSVD patients.ConclusionThis study shows that IUD/BTD and SFR are good predictors of cognitive impairments in CSVD elderly. Linear brain measurement showed a good predictive power for identifying MCI and dementia in elderly subjects with CSVD. Linear brain measurement could be a more suitable and novel method for screening cognitive impairment in aged CSVD patients in primary healthcare facilities, and worth further promotion among the rural population

Structural Analysis of Alkaline β-Mannanase from Alkaliphilic Bacillus sp. N16-5: Implications for Adaptation to Alkaline Conditions

Significant progress has been made in isolating novel alkaline β-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these β-mannanases. We report the catalytic domain structure of an industrially important β-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (β/α)8-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 β-mannanases. It was revealed that BSP165 MAN and other subfamily 8 β-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to β-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline β-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (β/α)8-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pKa calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 β-mannanases and will help to further understanding of alkaline adaptation mechanism

Geochemistry of soil gas in the seismic fault zone produced by the Wenchuan Ms 8.0 earthquake, southwestern China

The spatio-temporal variations of soil gas in the seismic fault zone produced by the 12 May 2008 Wenchuan Ms 8.0 earthquake were investigated based on the field measurements of soil gas concentrations after the main shock. Concentrations of He, H2, CO2, CH4, O2, N2, Rn, and Hg in soil gas were measured in the field at eight short profiles across the seismic rupture zone in June and December 2008 and July 2009. Soil-gas concentrations of more than 800 sampling sites were obtained. The data showed that the magnitudes of the He and H2 anomalies of three surveys declined significantly with decreasing strength of the aftershocks with time. The maximum concentrations of He and H2 (40 and 279.4 ppm, respectively) were found in three replicates at the south part of the rupture zone close to the epicenter. The spatio-temporal variations of CO2, Rn, and Hg concentrations differed obviously between the north and south parts of the fault zone. The maximum He and H2 concentrations in Jun 2008 occurred near the parts of the rupture zone where vertical displacements were larger. The anomalies of He, H2, CO2, Rn, and Hg concentrations could be related to the variation in the regional stress field and the aftershock activity

Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes

Direct Deamidation Analysis of Intact Adeno-Associated Virus Serotype 9 (AAV9) Capsid Proteins using Reversed-Phase Liquid Chromatography (RPLC)- Tandem Mass Spectrometry (MS) or RPLC-Fluorescence Detector (FLD)

Recombinant adeno-associated viral (AAV) vectors with nonpathogenic nature and ability to provide long-term gene expression have taken center stage as gene delivery vehicles for gene therapy. AAV capsid proteins (VP) are the major components that determine the tissue specificity, immunogenicity, and in vivo transduction performance of the vector. Asparagine deamidation of AAV capsid proteins has been reported to alter vector function, reduce vector stability and potency of AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification (PTM) of proteins that is readily detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS)-based peptide mapping. However, artificial deamidation can be spontaneously induced during sample preparation for peptide mapping prior to LC-MS analysis. We have developed an optimized sample preparation method to reduce and minimize deamidation artifacts induced during sample preparation for peptide mapping, which typically takes several hours to complete. To shorten turnaround time of deamidation results and to avoid artificial deamidation, we developed orthogonal RPLC-MS and RPLC-fluorescence detection (FLD) methods for direct deamidation analysis at the intact AAV9 capsid protein level to routinely support downstream purification, formulation development, and stability testing. Similar trends of increasing deamidation of AAV9 capsid proteins in stability samples were observed at the intact protein level and peptide level, indicating that the developed direct deamidation analysis of intact AAV9 capsid protein is comparable to the peptide mapping-based deamidation analysis and both methods are suitable/alternative for deamidation monitoring of AAV9 capsid proteins