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Adeno-associated viral capsid proteins (AAV VP) are the major components that determine the tissue specificity and immunogenicity, and

Fine-resolution land cover (LC) products are critical for studies of urban planning, global climate change, the Earth’s energy balance, and the geochemical cycle as fundamental geospatial data products. It is important and urgent to evaluate the performance of the updated global land cover maps. In this study, three widely used LC maps with 30 m spatial resolution (FROM-GLC30-2020, GLC_FCS30, and GlobeLand30) published around 2020 were evaluated in terms of their degree of consistency and accuracy metrics. First, we compared their similarities and difference in the area ratio and spatial patterns over different land cover types. Second, the sample and response protocol was proposed and validation samples were collected. Based on this, the overall accuracy, producer’s accuracy, and user’s accuracy were analyzed. The results revealed that: (1) the consistent areas of the three maps accounted for 65.96% of the total area and that two maps exceeded 75% of it. (2) The dominant land cover types, bare land and grassland, were the most consistent land cover types across the three products. In contrast, the spatial inconsistency of the wetland, shrubland, and built-up areas were relatively high, with the disagreement mainly occurring in the heterogeneous regions. (3) The overall accuracy of the GLC_FCS30 map was the highest with a value of 87.07%, which was followed by GlobeLand30 (85.69%) and FROM-GLC30 (83.49%). Overall, all three of the LC maps were found to be consistent and have a good performance in classification in the arid regions, but their ability to accurately classify specific types varied.

Cotton plants are recalcitrant with regards to transformation and induced regeneration. In the present study, 5-enolpyruvylshikimate-3-phosphate (EPSPS), a glyphosate resistant gene from the bacterium Agrobacterium sp. strain CP4, was introduced into an elite Bt transgenic cotton cultivar with a modified technique involving in planta Agrobacterium-mediated transformation of shoot apex. Primary transformants were initially screened using a 0.26% glyphosate spray and subsequently by PCR analysis. Five out of 4 000 transformants from T1 seeds were obtained resulting in an in planta transformation rate of 0.125%. Four homozygous lines were produced by continuous self-fertilization and both PCR-based selection and glyphosate resistance. Transgene insertion was analyzed by Southern blot analysis. Gene transcription and protein expression levels in the transgenic cotton lines were further investigated by RT-PCR, Western blot, and ELISA methods. Transgenic T3 plants were resistant to as much as 0.4% of glyphosate treatments in field trials. Our results indicate that the cotton shoot apex transformation technique which is both tissue-culture and genotype-independent would enable the exploitation of transgene technology in different cotton cultivars. Since this method does not require sterile conditions, the use of specialized growth media or the application of plant hormones, it can be conducted under the greenhouse condition.

In order to effectively and conveniently detect the β-amyloid oligomer (AβO) for earlier diagnosis of Alzheimer’s disease (AD), a disposable aptamer biosensor has been developed with high performance, facile operation, and low cost. Using a nanocomposite by in situ reduction of chloroauric acid to decorate Au nanoparticles (AuNPs) on Fe-MIL-88NH2 material via Au–N bond to effectively enhance the electrochemiluminescence (ECL) of luminol, the functioned basal electrode provides adequate background for sensing response. When the aptamer is linked via Au–S bond on the surface, the sensor gets the ability of specific recognition and coalescence toward the target (AβO). After incubating the sample on the aptasensor, its ECL signal is inhibited owing to the steric hindrance of the AβO macromolecules. The relative inhibition ratio linearly depends to the logarithm of AβO concentration in the range 0.1 pM to 10 pM, with an LOD of 71 fM. The aptasensor has high selectivity to AβO among its analogs. The recoveries in human serum were 98.9–105.4%. This research provides a new approach for sensitive detection of AβO in clinic laboratories for investigation and diagnosis of AD.

Amyloid β-protein (Aβ) aggregation in the brain is a closely related symptom of Alzheimer’s disease (AD), while Aβ in blood might be a more accessible peripheral biomarker to predict AD much earlier due to its abnormal fluctuation. However, when developing an aptasensor for Aβ detection, the specificity of the sensor will be impaired by Aβ aggregates such as Aβ oligomers or fibers because they have the same unit of recognition. Herein, a novel dual-screening sensing strategy based on specific recognition by aptamers and nano-sieving by a mesoporous silica membrane (MSM) with electrochemiluminescence (ECL) as the output signal is reported, in which the MSM achieves a size-screening effect to eliminate the influence of those macromolecules, assisting the specificity screening of the aptamer. The ECL signal is determined by the steric hindrance of the specifically captured Aβ on the aptamer in the nanochannels of the MSM towards the probe (luminol), affording a highly sensitive and specific detection ability for Aβ in complex matrices. In general, this work develops an ECL sensor that integrates aptamer recognition with size-selective separation for blood Aβ detection, which positively helps to monitor the occurrence and development of AD.

Eco-efficiency enhancement is an inherent requirement of green development and an important indicator of high-quality development in general. It aims to achieve the coordinated development of nature, the economy, and society. Therefore, eco-efficiency measurements should focus on not only total factor input, but also process analysis. Based on the &ldquo, full world&rdquo, model in ecological economic theory, this study constructed a theoretical framework for a composite economic-environmental-social system that reflects human welfare and sustainability. To this end, using network data envelopment analysis (DEA), this study established a staged eco-efficiency evaluation model that uses economic, environmental, and social factors to measure the overall and staged eco-efficiency of China&rsquo, s provinces from 2003 to 2016 and analyze its spatiotemporal characteristics. A geographically weighted regression (GWR) model was also used to analyze the influencing factors of eco-efficiency changes and the spatial differentiation in their effect intensity. The findings were as follows: (1) China&rsquo, s overall eco-efficiency is still at a low level. It varies significantly from region to region, and only three regions are at the frontier of production. The eastern region has the highest eco-efficiency, followed by the central region, and the gap between the central and western regions has gradually narrowed. In terms of staged efficiency, the level of eco-efficiency in the production stage is less than in the environmental governance stage, which is less than that in the social input stage. (2) In terms of the efficiency of each stage, the efficiency level of the production stage showed a downward trend throughout the entire process, and the decline in the central and western regions was more obvious. The social input stage and the environmental governance stage both showed upward trends. The social input stage showed a higher level, and the increase was relatively flat during the period of study. Efficiency continued to rise during the environmental governance stage from 2003 to 2010 and rose overall, but with some fluctuations from 2011 to 2016. (3) Geographically weighted regression showed that the effects of the influencing factors on eco-efficiency had obvious spatial heterogeneity. The factors affecting overall, production stage, and social input eco-efficiency were, in order of effect intensity from high to low, economic growth level, marketization level, and social input level. In terms of environmental governance, social input level had the greatest impact, followed by economic growth, marketization level did not show a significant impact.

The recycling of restaurant food waste can bring environmental benefits and improve food safety for urban residents. We here assessed the entire life cycle of the anaerobic digestion&ndash, aerobic composting technique of restaurant food waste recycling using Lanzhou as a case study. We used the CML2001 method provided with the Gabi software and compared the results to those produced using the traditional treatment techniques (landfill and incineration). This work includes a sensitivity analysis of the results. It is here concluded that the anaerobic digestion&ndash, aerobic composting technique had the smallest environmental impact of the methods here examined. The life cycle of anaerobic digestion&ndash, aerobic composting primarily consumes water, clay, coal, crude oil, and natural gas. The pre-processing phase consumes the most resources, and anaerobic digestion showed the greatest environmental impact. Specific environmental impacts in order from the highest to lowest potential to exacerbate global warming were found to be photochemical ozone production, acidification, eutrophication, marine aquatic ecotoxicity, human toxicity, freshwater aquatic ecotoxicity, and terrestrial ecotoxicity. The main factors associated with different environmental impacts and the environmental impacts themselves were found to differ across different phases. Some environmental impacts were shown to be sensitive to electricity, and the eutrophication potential and photochemical ozone creation potential showed the least sensitivity to all variables. To reduce the environmental impact of the anaerobic digestion&ndash, aerobic composting treatment technique, the energy structure and consumption of electricity, water, and diesel need to be optimized.

Brain expressed x-linked gene 1 (Bex1) which is at high levels in several populations of central nervous system (CNS) neurons, belongs to a family of small proteins of unknown function, playing roles as adaptors or modulators of intracellular signaling pathways. But its distribution and function in CNS remains unclear. Neuronal apoptosis is the major pathogenesis in secondary brain injury of intracerebral hemorrhage (ICH). In this study, the roles of Bex1 were explored in the pathophysiology of ICH. Western blot, immunohistochemistry, and immunofluorescence showed that obvious up-regulation of Bex1 in neurons adjacent to the hematoma after ICH. Furthermore, the increase of Bex1 expression was accompanied by the enhanced expression of Bax and active caspase-3, and decreased expression of B-cell lymphoma 2 (Bcl-2) following ICH. The in vitro study using Bex1 siRNA transfection in hemin-exposed PC12 cells suggested that Bex1 exerted anti-apoptotic function. Therefore, Bex1 may play the neuronal anti-apoptosis role following ICH, implying a novel molecular target for the therapy of ICH.

Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239) was produced from a plant system. Both wild-type (WT) and plant codon-optimized (OP)PI239gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco(Nicotiana tabacum)leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product.In vitrotests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.

Cross-city convergence in green space coverage could contribute to our understanding of the law of motion of distribution dynamics of environmental sustainability across cities and provide crucial information for designing policies regarding complex processes relating to human&ndash, environment interactions in an urban system. The relationship between socioeconomic factors and green space coverage has been widely discussed, but research on the distribution dynamics of green space coverage across cities is limited. This paper explored the pattern of convergence in green space coverage of a group of 285 Chinese cities over the period of 2002&ndash, 2012 by using hybrid approaches, including the nonparametric estimation technique, unconditional &beta, convergence model, and conditional &beta, convergence model. The results indicated that the green space coverage level in most cities has been substantially improved, the spatial disparity across Chinese cities has shrunk, and the level of green space coverage has converged to 104% of the average level. The &beta, convergence estimation results demonstrate the existence of unconditional and conditional convergence in green space coverage among Chinese cities, which indicates that cities with a low initial level of green space coverage grow faster than those with an initial high level. Faster sprawling, more compact and wealthier cities are likely to converge to a higher level of green space coverage. Expansion of the population and spatial scale of cities contributes to increasing green space coverage, sprawling of built-up area leads to a decrease of the green space coverage, and economic growth has little impact on green space coverage.

Food waste has become an issue of great public concern because of its environmental impacts and strategic significance of food security worldwide. In the context of ecological civilization construction and circular economy development in China food waste recycling faces critical theoretical and practice obstacles in reality. As China's urban population grows and the restaurant industry expands, the food waste generated by restaurants increases. Therefore restaurant owners’ attitude and behavior towards food waste recycling plays a pivotal role. As a stakeholder, the awareness of restaurant owners affects their attitudes and behavior towards the environment. In this paper, 1,050 restaurant owners from six cities of Gansu province in northwestern China were surveyed to study their awareness of food waste recycling. Using the awareness assessment model, the awareness level of 1,050 restaurant owners were calculated. The results show that 37.33% of respondents has a high awareness and 62.67% of them has a low awareness of food waste recycling. This indicates that the restaurant owners have a low level of overall awareness. Logistic regression model was used to examine factors that affect the awareness level of restaurant owners. Compared with the male restaurant owner, the female one has a lower awareness. The younger restaurant owner has a higher awareness than the older one. In addition, factors, including the educational level of restaurant owner, restaurant scale, amount of daily restaurant food waste, the way of food waste treatment, and the macroeconomic development of city where restaurants located, have positive effects on the awareness.

Plant molecular farming (PMF), defined as the practice of using plants to produce human therapeutic proteins, has received worldwide interest. PMF has grown and advanced considerably over the past two decades. A number of therapeutic proteins have been produced in plants, some of which have been through pre-clinical or clinical trials and are close to commercialization. Plants have the potential to mass-produce pharmaceutical products with less cost than traditional methods. Tobacco-derived antibodies have been tested and used to combat the Ebola outbreak in Africa. Genetically engineered immunoadhesin (DPP4-Fc) produced in green plants has been shown to be able to bind to MERS-CoV (Middle East Respiratory Syndrome), preventing the virus from infecting lung cells. Biosafety concerns (such as pollen contamination and immunogenicity of plant-specific glycans) and costly downstream extraction and purification requirements, however, have hampered PMF production from moving from the laboratory to industrial application. In this review, the challenges and opportunities of PMF are discussed. Topics addressed include; transformation and expression systems, plant bioreactors, safety concerns, and various opportunities to produce topical applications and health supplements.

The N-glycosylation process that occurs in the Pichia pastoris protein expression system can have a significant effect on the yield of heterologous glycoproteins secreted from the yeast. The basis of the effect of N-glycosylation on yield, however, has not been elucidated. In order to investigate the effect of N-glycosylation on heterologous protein production, site-directed mutation was performed on five potential N-glycosylation sites of the tetanus toxin fragment C (TetC). Unaltered TetC (wild-TetC) and eight mutants, in which different numbers and locations of N-glycosylation sites were altered, were expressed in P. pastoris GS115. The recombinant target proteins presented different levels of N-glycosylation. The wild Tet-C and 4 mutations sites of putative N-glycosylation (4Gly mutant: N280Q) had the highest level of secreted protein, while 1 mutation of putative N-glycosylation sites (1Gly mutant: N39/64/85/205Q) had the highest level of intracellular, non-secreted heterologous protein. Reducing the number of native N-glycosylation sites decreased the level of glycosylation, as well as the level of secretion. Introduction of a N-glycosylation site at position 320, however, also reduced the level of expression and secretion of recombinant protein. These results indicate that the number and location of N-glycosylation sites jointly have an effect on the expression and secretion of heterologous glycoproteins in P. pastoris.

Intact protein analysis via top-down mass spectrometry (MS) provides a bird’s eye view over the protein complexes and complex protein mixtures with the unique capability of characterizing protein variants, splice isoforms, and combinatorial post-translational modifications (PTMs). Here we applied capillary electrophoresis (CE) through a sheathless CE–electrospray ionization interface coupled to an LTQ Velos Orbitrap Elite mass spectrometer to analyze the Dam1 complex from Saccharomyces cerevisiae. We achieved a 100-fold increase in sensitivity compared to a reversed-phase liquid chromatography coupled MS analysis of recombinant Dam1 complex with a total loading of 2.5 ng (12 amol). N-terminal processing forms of individual subunits of the Dam1 complex were observed as well as their phosphorylation stoichiometry upon Mps1p kinase treatment.

Baohong Hu,1,2,* Qianli Wang,3,* Yueju Wang,4 Jian Chen,2 Peng Li,2 Mingyong Han1 1Department of Health Care Oncology, East District of Shandong Provincial Hospital of Shandong University, Jinan, 2Department of Medical Oncology, 3Department of Intensive Care Unit, Yantai Yuhuangding Hospital Affiliated to Qingdao University, Yantai, Shandong, 4Department of Geriatrics, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, People’s Republic of China *These authors contributed equally to this work Aim: To investigate the expression and clinical significance of Holliday junction–recognizing protein (HJURP) in hepatocellular carcinoma (HCC). Methods: In this study, we detected the expression of HJURP protein in samples of 164 patients with HCC, and based on this, we divided the patients into two cohorts: high expression of HJURP and low expression of HJURP. We analyzed the correlation between HJURP expression and the clinicopathological factors using chi-square test. Survival significance of HJURP was defined by Kaplan–Meier method and log-rank test, and the independent prognostic factors were identified by Cox regression model. Using function assays of HCC cell lines, we investigated the influence of HJURP on the proliferation of HCC cells. Results: In our study, the proportion of patients with high HJURP expression was 25.6%, which was significantly associated with the tumor size and Barcelona clinic liver cancer stage. Univariate analysis confirmed that high HJURP expression was remarkably associated with poorer overall survival rates (P=0.003), as well as tumor number (P=0.016), tumor differentiation (P=0.047), TNM stage (P=0.005), and Barcelona clinic liver cancer stage (P=0.004). Multivariate analysis confirmed that high HJURP expression (P

Diabetes has become the third largest cause of death in humans worldwide. Therefore, effective treatment for this disease remains a critical issue. Glucagon-like peptide-1 (GLP-1) plays an important role in glucose homeostasis, and therefore represents a promising candidate to use for the treatment of diabetes. Native GLP-1, however, is quickly degraded in in the circulatory system; which limits its clinical application. In the present study, a chemically-synthesized, modified analogue of human GLP-1 (mGLP-1) was designed. Our analyses indicated that, relative to native GLP-1, mGLP-1 is more resistant to trypsin and pancreatin degradation. mGLP-1 promotes mouse pancreatic β-cell proliferation by up-regulating the expression level of cyclin E, CDK2, Bcl-2 and down-regulating Bax, p21, and stimulates insulin secretion. An oral glucose tolerance test indicated that mGLP-1 significantly improved glucose tolerance in mice. Intraperitoneal injections of mGLP-1 into streptozotocin (STZ)-induced type 2 diabetic mice significantly reduced blood sugar levels and stimulated insulin secretion. Oral gavages of mGLP-1 in diabetic mice did not result in significant hypoglycemic activity.

The analysis of carbohydrate moieties of glycoprotein biopharmaceuticals is of high importance, especially because glycosylation changes can impact the biological effect of the drugs. In the case of monoclonal antibody therapeutics, the degree of core fucosylation significantly influences its effector function, thus should be closely monitored during the clone selection, development and manufacturing processes. MS analysis of labile sugar residues such as core fucosylation and terminal silylation may be misleading as such residues can break off during the ionization process or alter the ionization efficiency, both of which affect the results. In the case of monoclonal antibody therapeutics tailored for antibody-dependent cellular cytotoxicity function, core fucosylation should be kept minimal; therefore, close attention should be paid to the prevalence of this particular sugar residue. This paper reports on the core fucosylation analysis of some major immunoglobulin G N-glycans by direct infusion ESI-MS and CE–LIF. In comparison to the industry standard of CE–LIF, a lower degree of core fucosylated structures was found by ESI-MS analysis, emphasizing the need to use orthogonal quantitative separation methods in addition to MS.

Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans , we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.

Nudel/NudE facilitates the binding of Lis1 to dynein, which subsequently enhances the recruitment of dynactin to dynein, and dynactin antagonizes Lis1 to relieve Lis1-induced dynein stall on microtubules., Lis1, Nudel/NudE, and dynactin are regulators of cytoplasmic dynein, a minus end–directed, microtubule (MT)-based motor required for proper spindle assembly and orientation. In vitro studies have shown that dynactin promotes processive movement of dynein on MTs, whereas Lis1 causes dynein to enter a persistent force-generating state (referred to here as dynein stall). Yet how the activities of Lis1, Nudel/NudE, and dynactin are coordinated to regulate dynein remains poorly understood in vivo. Working in Xenopus egg extracts, we show that Nudel/NudE facilitates the binding of Lis1 to dynein, which enhances the recruitment of dynactin to dynein. We further report a novel Lis1-dependent dynein–dynactin interaction that is essential for the organization of mitotic spindle poles. Finally, using assays for MT gliding and spindle assembly, we demonstrate an antagonistic relationship between Lis1 and dynactin that allows dynactin to relieve Lis1-induced dynein stall on MTs. Our findings suggest the interesting possibility that Lis1 and dynactin could alternately engage with dynein to allow the motor to promote spindle assembly.

We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) capillary electrophoresis–tandem mass spectrometry (CE–MS/MS) procedure which employs a high sensitivity porous electrospray ionization (ESI) sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency, we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC)...

Adherens junctions play key roles in mediating cell–cell contacts during tissue development. In Caenorhabditis elegans embryos, the cadherin–catenin complex (CCC), composed of the classical cadherin HMR-1 and members of three catenin families, HMP-1, HMP-2 and JAC-1, is necessary for normal blastomere adhesion, gastrulation, ventral enclosure of the epidermis and embryo elongation. Disruption of CCC assembly or function results in embryonic lethality. Previous work suggests that components of the CCC are subject to phosphorylation. However, the identity of phosphorylated residues in CCC components and their contributions to CCC stability and function in a living organism remain speculative. Using mass spectrometry, we systematically identify phosphorylated residues in the essential CCC subunits HMR-1, HMP-1 and HMP-2 in vivo. We demonstrate that HMR-1/cadherin phosphorylation occurs on three sites within its β-catenin binding domain that each contributes to CCC assembly on lipid bilayers. In contrast, phosphorylation of HMP-2/β-catenin inhibits its association with HMR-1/cadherin in vitro, suggesting a role in CCC disassembly. Although HMP-1/α-catenin is also phosphorylated in vivo, phosphomimetic mutations do not affect its ability to associate with other CCC components or interact with actin in vitro. Collectively, our findings support a model in which distinct phosphorylation events contribute to rapid CCC assembly and disassembly, both of which are essential for morphogenetic rearrangements during development.

Lysozyme is an enzyme that is essential for protection against bacterial infections. In this study, a T4 lysozyme gene was cloned into the yeast expression vector pPIC9K under the control of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). A Hansenula polymorpha-derived ribosomal DNA (rDNA)-targeting element was inserted into the expression vector and was critical for stable DNA integration into the H. polymorpha chromosome. Recombinant T4 lysozyme was successfully expressed in the yeast H. polymorpha A16; 0.49 g L(-1) secreted recombinant T4 lysozyme was obtained 72 h after incubation in culture broth that had an initial pH of 6.0. Recombinant T4 lysozyme showed lytic activity against the cell walls of the gram positive bacteria, Micrococcus lysodeikticus, and the gram negative bacteria Xanthomonas campestris pv. malvacearum and Xanthomonas oryzae pv. oryzae. The zone of inhibition assay was used to evaluate antimicrobial activity. Mass spectrometry showed the N-terminal sequence of recombinant T4 lysozyme was identical to that of the native enzyme. SDS-PAGE indicated that the molecular mass of recombinant T4 lysozyme was 18.7 kD which corresponds to a monomer of the native enzyme. SDS-PAGE without 0.2 mol L(-1) dithiothreitol treatment detected two bands (15 and 31 kD) suggesting that some recombinant T4 lysozyme formed inter- and intra-molecular disulfide bonds which resulted in loss of enzyme activity.

Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart.

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.

We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.

The 20 S proteasome complexes are major contributors to the intracellular protein degradation machinery in mammalian cells. Systematic administration of proteasome inhibitors to combat disease (e.g. cancer) has resulted in positive outcomes as well as adversary effects. The latter was attributed to, at least in part, a lack of understanding in the organ-specific responses to inhibitors and the potential diversity of proteomes of these complexes in different tissues. Accordingly, we conducted a proteomic study to characterize the 20 S proteasome complexes and their postulated organ-specific responses in the heart and liver. The cardiac and hepatic 20 S proteasomes were isolated from the same mouse strain with identical genetic background. We examined the molecular composition, complex assembly, post-translational modifications and associating partners of these proteasome complexes. Our results revealed an organ-specific molecular organization of the 20 S proteasomes with distinguished patterns of post-translational modifications as well as unique complex assembly characteristics. Furthermore, the proteome diversities are concomitant with a functional heterogeneity of the proteolytic patterns exhibited by these two organs. In particular, the heart and liver displayed distinct activity profiles to two proteasome inhibitors, epoxomicin and Z-Pro-Nle-Asp-H. Finally, the heart and liver demonstrated contrasting regulatory mechanisms from the associating partners of these proteasomes. The functional heterogeneity of the mammalian 20 S proteasome complexes underscores the concept of divergent proteomes among organs in the context of an identical genome.

Post-translational modifications (PTMs) serve as key regulatory mechanisms for 20S proteasome functions. Alterations in 20S PTMs have been previously observed with changes in modified protein degradation patterns and altered cellular phenotypes. Despite decades of investigation, our knowledge pertaining to the various PTMs of 20S complexes and their biological significance remain limited. In this investigation, we show that two-dimensional electrophoresis (2-DE) offers an analytical tool with high resolution and reproducibility. Accordingly, it has been applied for the characterization of PTMs including glycosylation, phosphorylation, oxidation, and nitrosylation. The PTMs of murine cardiac 20S proteasomes and their associating proteins were examined. Our 2-DE analyses displayed over 25 spots for the 20S complexes (17 subunits), indicating multiply modified subunits of cardiac proteasomes. The identification of specific PTM sites subsequent to 2-DE were supported by mass spectrometry. These PTMs included phosphorylation and oxidation. Most of the PTMs occurred in low stoichiometry and required enrichment to enhance the detection sensitivity. In conclusion, our studies support 2-DE as a central tool in the analyses of 20S proteasome PTMs. The approaches utilized in this investigation demonstrate their application in mapping the PTMs of the 20S proteasomes in cardiac tissue, which are applicable to other samples and biological conditions.

The 20 S proteasomes play a critical role in intracellular homeostasis and stress response. Their function is tuned by covalent modifications, such as phosphorylation. In this study, we performed a comprehensive characterization of the phosphoproteome for the 20 S proteasome complexes in both the murine heart and liver. A platform combining parallel approaches in differential sample fractionation (SDS-PAGE, IEF, and two-dimensional electrophoresis), enzymatic digestion (trypsin and chymotrypsin), phosphopeptide enrichment (TiO2), and peptide fragmentation (CID and electron transfer dissociation (ETD)) has proven to be essential for identifying low abundance phosphopeptides. As a result, a total of 52 phosphorylation identifications were made in mammalian tissues; 44 of them were novel. These identifications include single (serine, threonine, and tyrosine) and dual phosphorylation peptides. 34 phosphopeptides were identified by CID; 10 phosphopeptides, including a key modification on the catalytically essential β5 subunit, were identified only by ETD; eight phosphopeptides were shared identifications by both CID and ETD. Besides the commonly shared phosphorylation sites, unique sites were detected in the murine heart and liver, documenting variances in phosphorylation between tissues within the proteasome populations. Furthermore the biological significance of these 20 S phosphoproteomes was evaluated. The role of cAMP-dependent protein kinase A (PKA) to modulate these phosphoproteomes was examined. Using a proteomics approach, many of the cardiac and hepatic 20 S subunits were found to be substrate targets of PKA. Incubation of the intact 20 S proteasome complexes with active PKA enhanced phosphorylation in both existing PKA phosphorylation sites as well as novel sites in these 20 S subunits. Furthermore treatment with active PKA significantly elevated all three peptidase activities (β1 caspase-like, β2 trypsin-like, and β5 chymotrypsin-like), demonstrating a functional role of PKA in governing these 20 S phosphoproteomes.

The cardiac proteasome is increasingly recognized as a complex, heterogeneous and dynamic organelle contributing to the modulation of cardiac function in health and diseases. The emerging picture of the proteasome system reveals a highly regulated and organized molecular machine integrated into multiple biological processes of the cell. Full appreciation of its cardiovascular relevance requires an understanding of its proteolytic function as well as its underlying regulatory mechanisms; of which assembly, stoichiometry, post-translational modification and the role of the associating partners are increasingly poignant.

Mitochondria play essential roles in cardiac pathophysiology and the murine model has been extensively used to investigate cardiovascular diseases. In the present study, we characterized murine cardiac mitochondria using an LC/MS/MS approach. We extracted and purified cardiac mitochondria; validated their functionality to ensure the final preparation contains necessary components to sustain their normal function; and subjected these validated organelles to LC/MS/MS-based protein identification. A total of 940 distinct proteins were identified from murine cardiac mitochondria, among which, 480 proteins were not previously identified by major proteomic profiling studies. The 940 proteins consist of functional clusters known to support oxidative phosphorylation, metabolism, and biogenesis. In addition, there are several other clusters, including proteolysis, protein folding, and reduction/oxidation signaling, which ostensibly represent previously under-appreciated tasks of cardiac mitochondria. Moreover, many identified proteins were found to occupy other subcellular locations, including cytoplasm, ER, and golgi, in addition to their presence in the mitochondria. These results provide a comprehensive picture of the murine cardiac mitochondrial proteome and underscore tissue- and species-specification. Moreover, the use of functionally intact mitochondria insures that the proteomic observations in this organelle are relevant to its normal biology and facilitates decoding the interplay between mitochondria and other organelles.

Vampire bat salivary plasminogen activators (DSPAs) are thrombolytic agents that are under clinical investigation for the treatment of acute ischemic stroke. In this study, the synthetic active salivary plasminogen activator alpha2 (DSPAalpha2) gene optimized for the preferred codons of Pichia pastoris was assembled from 48 oligonucleotides, and cloned into the yeast expression vector pPIC9 with a strong enhancer from human cytomegalovirus (HCMV). This system achieved high expression of an active DSPAalpha2 in P. pastoris yeast GS115. Secreted active DSPAalpha2 recombinant protein was purified from broth supernatant by a simple one-step procedure on Sephadex chromatography and was confirmed by SDS-PAGE and Western blot analysis. ELISA showed that 2.5mg of recombinant protein could be obtained from 100-ml culture broth supernatant. The fibrinolytic activity of the recombinant DSPAalpha2 was 1.28 x 10(5)IU/mg.

Production of reactive oxygen species (ROS) is associated with a number of physiological disorders in plants. Superoxide dismutase (SOD) catalyzes the breakdown of Superoxide (O2-) into O2 and H2O2 and provides the first line of defense against the toxic effects of elevated levels of ROS. The effect of increased expression of Mn Superoxide dismutase (Mn-SOD) on salt stress tolerance was studied using transformed tomato (Lycopersicon esculentum cv. Zhongshu No. 5) plants. Northern blots confirmed expression of the heterologous Mn-SOD in transgenic plants. Strong Mn-SOD enzyme activity was detected by native PAGE in transformed plants. Transgenic plants showed resistance to the superoxide-generating herbicide methyl viologen (MV, 10-4 M). The total SOD activity was one and one half- to two-fold higher, and APX (ascorbate peroxidase) activity was six to seven fold higher in transgenic, than in wild-type (WT) plant under MV stress. Germination of transgenic tomato seeds at a NaCl concentration of 150 mM was greater than wild-type seeds. When exposed to salt stress, roots of transgenic plants were less stunted and leaf injury was lower than that observed in WT plants. Also, the total APX activity of transgenic plants was 4 to 5 fold higher than that of WT under NaCl (200 mM) stress.

Reactive oxygen species (ROS), such as hydrogen peroxide, superoxide and hydroxyl radicals, are by-products of biological redox reactions. ROS can denature enzymes and damage important cellular components. Plants develop antioxidant enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase (APX) to scavenge ROS and detoxify them. The effect of increased cytosolic ascorbate peroxidase (cAPX) on heat and UV-B stress tolerance was studied using transformed tomato (Lycopersicon esculentum cv. Zhongshu No. 5) plants. This research demonstrates, in either laboratory or field tests, the potential to enhance tolerance to heat, UV-B, and sunscald stress by gene transfer. Overexpression of cAPX in transgenic tomato enhanced resistance to heat (40°C) and UV-B stress compared to wild-type plants. When leaf disks were placed at 40°C for 13 hours, the electrolyte leakage of disks from wild-type were 93%, whereas two tested transgenic lines (A9, A16) exhibited 24% and 52% leakage respectively. When fruits of wild-type and transgenic plants were exposed to UV-B (2.5 mW cm-2) for five days, the extent of browning was 95%, and 33%, and 37%, respectively. In field tests, the detached fruits from field-grown transgenic plants showed more resistance to exposure to direct sunlight than fruits from wild-type plants. APX activity in leaves of cAPX transgenic plants was several folds higher than in leaves of wild-type plants when exposed to heat, UV-B, and drought stresses.

Intact protein analysis via top-down mass spectrometry (MS) provides the unique capability of fully characterizing protein isoforms and combinatorial post-translational modifications (PTMs) compared to the bottom-up MS approach. Front-end protein separation poses a challenge for analyzing complex mixtures of intact proteins on a proteomic scale. Here we applied capillary electrophoresis (CE) through a sheathless capillary electrophoresis-electrospray ionization (CESI) interface coupled to an Orbitrap Elite mass spectrometer to profile the proteome from Pyrococcus furiosus. CESI-top-down MS analysis of Pyrococcus furiosus cell lysate identified 134 proteins and 291 proteoforms with a total sample consumption of 270 ng in 120 min of total analysis time. Truncations and various PTMs were detected, including acetylation, disulfide bonds, oxidation, glycosylation, and hypusine. This is the largest scale analysis of intact proteins by CE-top-down MS to date.

This study demonstrates the ability to perform sensitive proteome analysis on the limited protein quantities available through tissue microdissection. Capillary isoelectric focusing combined with nano-reversed-phase liquid chromatography in an automated and integrated platform not only provides systematic resolution of complex peptide mixtures based on their differences in isoelectric point and hydrophobicity but also eliminates peptide loss and analyte dilution. In comparison with strong cation exchange chromatography, the significant advantages of electrokinetic focusing-based separations include high resolving power, high concentration and narrow analyte bands, and effective usage of electrospray ionization-tandem MS toward peptide identifications. Through the use of capillary isoelectric focusing-based multidimensional peptide separations, a total of 6866 fully tryptic peptides were detected, leading to the identification of 1820 distinct proteins. Each distinct protein was identified by at least one distinct peptide sequence. These high mass accuracy and high-confidence identifications were generated from three proteome runs of a single glioblastoma multiforme tissue sample, each run consuming only 10 microg of total protein, an amount corresponding to 20,000 selectively isolated cells. Instead of performing multiple runs of multidimensional separations, the overall peak capacity can be greatly enhanced for mining deeper into tissue proteomics by increasing the number of CIEF fractions without an accompanying increase in sample consumption.

For top-down proteomics, nano-reversed phase liquid chromatography (RPLC) plays a major role in both single and multidimensional protein separations in an effort to increase the overall peak capacity for the resolution of complex protein mixtures prior to mass spectrometry analysis. Effects of various chromatography conditions, including alkyl chain length in the stationary phase, capillary column temperature, and ion-pairing agent, on the resolution of intact proteins are studied using nano-RPLC-electrospray ionization-mass spectrometry. Optimal chromatography conditions include the use of C 18 column heated at 60 ° C and the addition of trifluoroacetic acid instead of heptafluorobutyric acid as the ion-paring agent in the mobile phase. Under optimized chromatography conditions, there are no significant differences in the separation performance of yeast cell lysates present in the native versus denatured states. Denatured yeast proteins resolved and eluted from nano-RPLC can be subjected to proteolytic digestion in an on- or off-line approach to provide improved protein sequence coverage toward protein identification in a combined top-down/bottom-up proteome platform.