西藏交通警察道路执法工作的风险与防范

西藏自治区自然、地理与气候环境复杂且特殊,道路交通环境相对恶劣,这导致当地交通警察道路执法工作面临诸多风险,包括警员人身安全和执法效能等风险。对此,文章对西藏交通警察道路执法工作风险进行了调查分析,从地区环境、警员安全意识与技能、执法装备、社会宣传和教育等角度探讨了相关风险的成因,针对性相关风险成因,提出从优化警力配置与道路信息推送、加强警员培训、完善警务装备、加强法治化宣传教育等工作来消除相关风险,以期保障警员安全并改善西藏交通环境。

Cloning, Expression Pattern Analysis and Subcellular Localization of Resveratrol Synthase Gene in Peanut (<i>Arachis hypogaea</i>L.)

Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.

Transformation of Arabidopsis by Rice OsWRKY78::GFP Fusion Gene and Subcellular Localization of OsWRKY78 Protein

[Objective] The study was to understand the subcellular localization of OsWRKY78 protein in plants. [Method] Primers specific for OsWRKY78 gene were designed according to the OsWRKY78 full length sequence in Genbank. The gene was cloned by RT-PCR method. The gene was then recombined into a plasmid expression vector carrying green fluorescent protein (GFP) gene, pBinGFP. The recombinant was confirmed by PCR and enzyme digestion. The recombinant plasmid pBinGFP-OsWRKY was transformed into Arabidopsis through Agrobacterium tumefaciens strain GV3101 and transgenic plants were obtained. [Result] Measured by fluorescence microscopy, the expression of OsWRKY78 and GFP fusion protein in root tip cells was localized in the nucleus. [Conclusion] This study laid the foundation for further investigating the function of OsWRKY78 gene and its role in related signal transduction and provided theoretical basis for exploring the relation between OsWRKY78 gene and brown planthoppers.

Expression, purification, and subcellular localization of phospholipase C in Dunaliella salina

Plants possess effective mechanisms to respond quickly to the external environment. Rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs after a stimulus. The PLC in Dunaliella salina plays important roles in growth and stress responses. However, the molecular basis of PLC action in D. salina remains little understood. To gain insight into the potential biological functions of this enzyme, we cloned a phospholipase C gene from D. salina in a previous study, named DsPLC (GenBank No. KF573428). Here, we present the prokaryotic expression, purification, and characterization of the DsPLC gene. The entire coding region of DsPLC was inserted into an expression vector pET32a, and the DsPLC gene was successfully expressed in Escherichia coli. The DsPLC protein was purified and identified using a polyclonal antibody and western blotting. Expressing DsPLC fused with a green fluorescent protein (GFP) in onion showed that DsPLC-GFP was localized to the intracellular membrane. Quantitative real-time PCR analysis revealed that the relative expression of the DsPLC gene was induced significantly by 3.0-mol/L NaCl at 4 h. Our results support the importance of PLC enzymes in plant defense signaling. This study provides a basis for further functional studies of the DsPLC gene and for additional analysis of the potential roles of PLC enzymes in response to abiotic stress.

Computer-Assisted analysis of subcellular localization signals and post-translational modifications of human prion proteins

In the present work, computational analyses were applied to study the subcellular localiza-tion and posttranslational modifications of hu-man prion proteins (PrPs). The tentative location of prion protein was determined to be in the nu-cleolus inside the nucleus by the following bio-informatics tools: Hum-PLoc, Euk-PLoc and Nuc-PLoc. Based on our results signal peptides with average of 22 base pairs in N-terminal were identified in human PrPs. This theoretical study demonstrates that PrP is post-translationally modified by: 1) attachment of two N-linked complex carbohydrate moieties (N181 and N197), 2) attachmet of glycosylphosphatidylinositol (GPI) at serine 230 and 3) formation of two di-sulfide bonds between “6–22” and “179–214” cysteines. Furthermore, ten protein kinase phosphorylation sites were predicted in human PrP. The above-noted phosphorylation was car-ried out by PKC and CK2. By using bioinfor-matics tools, we have shown that computation-ally human PrPs locate particularly into the nu-cleolus.

Semantic Similarity over Gene Ontology for Multi-Label Protein Subcellular Localization

As one of the essential topics in proteomics and molecular biology, protein subcellular localization has been extensively studied in previous decades. However, most of the methods are limited to the prediction of single-location proteins. In many studies, multi-location proteins are either not considered or assumed not existing. This paper proposes a novel multi-label subcellular-localization predictor based on the semantic similarity between Gene Ontology (GO) terms. Given a protein, the accession numbers of its homologs are obtained via BLAST search. Then, the homologous accession numbers of the protein are used as keys to search against the gene ontology annotation database to obtain a set of GO terms. The semantic similarity between GO terms is used to formulate semantic similarity vectors for classification. A support vector machine (SVM) classifier with a new decision scheme is proposed to classify the multi-label GO semantic similarity vectors. Experimental results show that the proposed multi-label predictor significantly outperforms the state-of-the-art predictors such as iLoc-Plant and Plant-mPLoc.

Essential proteins identification method based on four-order distances and subcellular localization information

Essential proteins are inseparable in cell growth and survival. The study of essential proteins is important for understanding cellular functions and biological mechanisms. Therefore, various computable methods have been proposed to identify essential proteins. Unfortunately, most methods based on network topology only consider the interactions between a protein and its neighboring proteins, and not the interactions with its higher-order distance proteins. In this paper, we propose the DSEP algorithm in which we integrated network topology properties and subcellular localization information in protein–protein interaction(PPI) networks based on four-order distances, and then used random walks to identify the essential proteins. We also propose a method to calculate the finite-order distance of the network, which can greatly reduce the time complexity of our algorithm. We conducted a comprehensive comparison of the DSEP algorithm with 11 existing classical algorithms to identify essential proteins with multiple evaluation methods. The results show that DSEP is superior to these 11 methods.

Expression and subcellular localization of a novel gene <i>Bm-X</i>of the silkworm, <i>Bombyx mori</i>

According to the large-scale sequencing of cDNA library from silkworm pupae, the cDNA of a novel gene with blank research background was identified and temporarily named Bm-X. The length of this cDNA is 778 bp. We obtained its ORF for further study by bioinformatics analysis. It is 444 bp and encodes 147 amino acid residues, with a predicted molecular weight (MW) of 16.4 kD and an isoelectric point (pI) of 3.69. In this study, we successfully constructed a recombinant plasmid pET-28a(+)-Bm-X and expressed it in Escherichia coli. We used the fusion protein rBm-X which purified by Niaffinity chromatography to produce polyclonal antibodies against Bm-X for Western blot analysis. The analysis revealed that Bm-X was expressed in the larval midgut, the epidermis and the silk gland. In addition, the subcellular localization analysis of silkworm ovary epithelial cells (BmN cells) showed that Bm-X protein was located both in cytoplasm and nucleus, and the signal was stronger in cytoplasm than in nucleus. Our findings indicate that Bm-X gene is a novel species-specificity gene and its expression product can be detected in tissues of the fifth silkworm instar larvae and BmN cells.

Prediction of Subcellular Localization of Eukaryotic Proteins Using Position-Specific Profiles and Neural Network with Weighted Inputs

Subcellular location is one of the key biological characteristics of proteins. Position-specific profiles （PSP） have been introduced as important characteristics of proteins in this article. In this study, to obtain position-specific profiles, the Position Specific lterative-Basic Local Alignment Search Tool （PSI-BLAST） has been used to search for protein sequences in a database. Position-specific scoring matrices are extracted from the profiles as one class of characteristics. Four-part amino acid compositions and lst-7th order dipeptide compositions have also been calculated as the other two classes of characteristics. Therefore, twelve characteristic vectors are extracted from each of the protein sequences. Next, the characteristic vectors are weighed by a simple weighing function and inputted into a BP neural network predictor named PSP-Weighted Neural Network （PSP-WNN）. The Levenberg-Marquardt algorithm is employed to adjust the weight matrices and thresholds during the network training instead of the error back propagation algorithm. With a jackknife test on the RH2427 dataset, PSP-WNN has achieved a higher overall prediction accuracy of 88.4% rather than the prediction results by the general BP neural network, Markov model, and fuzzy k-nearest neighbors algorithm on this dataset. In addition, the prediction performance of PSP-WNN has been evaluated with a five-fold cross validation test on the PK7579 dataset and the prediction results have been consistently better than those of the previous method on the basis of several support vector machines, using compositions of both amino acids and amino acid pairs. These results indicate that PSP-WNN is a powerful tool for subcellular localization prediction. At the end of the article, influences on prediction accuracy using different weighting proportions among three characteristic vector categories have been discussed. An appropriate proportion is considered by increasing the prediction accuracy.

Influence of RNA interference on the mitochondrial subcellular localization of alpha-synuclein and on the formation of Lewy body-like inclusions in the cytoplasm of human embryonic kidney 293 cells induced by the overexpression of alphasynuclein

The specific and effective a-synuclein RNA interference （RNAi） plasmids, and the a-synuclein-pEGFP recombinant plasmids were co-transfected into human embryonic kidney 293 （HEK293） cells using the lipofectamine method. Using an inverted fluorescence microscope, a-synuclein proteins were observed to aggregate in the cytoplasm and nucleus. Wild-type a-synuclein proteins co-localized with mitochondria. Hematoxylin-eosin staining revealed round eosinophilic bodies （Lewy body-like inclusions） in the cytoplasm of some cells transfected with a-synuclein-pEGFP plasmid. However, the formation of Lewy body-like inclusions was not observed following transfection with the RNAi pSYN-1 plasmid. RNAi blocked Lewy body-like inclusions in the cytoplasm of HEK293 cells induced by wild-type a-synuclein overexpression, but RNAi did not affect the subcellular localization of wild-type a-synuclein in mitochondria.

The Origins of Bosons and Fermions

This paper proposes that all Bosons and all Fermions originate from even more elementary constituents, which called Spin Angular Momentum Vacuum (SAMV). SAMV is filled with Primitive Spin Particles (PSP). The total square spin angular momentum of each PSP is negative, less than zero. Those PSP labeled by index ?of Casimir Operator, are called Vacuum Spin Particle (VSP), which could be contracted into so-called Vacuum Bubbles (VB). VB are identical bubbles, are 'sub-observable physical quantities'. VB are paired up into Vacuum Bubble Pair VBP. VSP ωj(or ω+,ω-) results from Self-identical vacuum bubble interaction ?through the zero order Phase Transition PT. When the 1st, 2nd, 3rd,... order PT of VBP occur, ?then VBP turn into Bosons and Fermions, excited out of sea level of SAMV ocean.

Cd Distribution and Subcellular Localization in Leaf and Its Effects on Growth of Soybean(Glycine max) Seedlings

In order to investigate Cd accumulation, subcellular distribution, and local-ization in soybean seedlings leaves, soybean seedlings were cultivated in solution containing different concentrations of Cd. The results showed that most Cd associ-ated with the cellwal s and soluble fractions, and a minor part of Cd presented in mitochondria fractions, nuclear and chloroplast fractions, especial y exposure to high Cd concentrations. Under 20.00 mg/L Cd stress, Cd subcellular distribution fol owed a sequence as： soluble fractions （55.00%）＆gt;cellwal s （30.0%）＆gt;mitochondria fractions （8.21%）＆gt;nuclear and chloroplast fractions （6.79%）. Deposited Cd black particles were observed in cellwal s, chloroplasts, nuclei, and vacuoles through electrical microscope slice. This fact indicated that the cellwal s of soybean leaves were the first protecting organel es from Cd toxicity, and the cellwal s and soluble fractions were the main place for Cd storage. Due to Cd accumulated in the organel es, the intercellular space was enlarged and the subcellular structure was damaged, especial y for the chloroplasts.

The assessment of time dependent flow of Williamson fluid with radiative blood flow against a wedge

The present pagination reports both Brownian diffusion and thermophoresis aspects subject to magneto hydrodynamic Williamson fluid model.Assuming the flow is unsteady and blood is treated as Williamson fluid over a wedge with radiation.The governing equations are transformed into ordinary differential equations by using similarity variables.The analytical solutions of the transformed governing equations are obtained by using the RK 4th order method along with shooting technique solver.The effects of various physical parameters such as Hartmann number,local Weissenberg number,radiation parameter,unsteadiness parameter,Prandtl number,Lewis number,Brownian diffusion,thermophoresis,wedge angle parameter,moving wedge parameter,on velocity,temperature,concentration,skin friction,heat transfer rate and mass transfer rate have been discussed in detail.The velocity and temperature profile deprives for larger We and an opposite trend is observed for concentration.The radiation parameter is propositional to temperature and a counter behaviour is observed for Pr.

Analysis of Specific Binding and Subcellular Localization of Wheat ERF Transcription Factor W17

The study aims to detect the subcellular localization of ERF （ethylene-responsive element binding factor） transcription factor W17 protein, the interaction between W 17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS 1000/He. Transformed cells were incubated for 24 h at 22℃ in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST （glutathione-S-transferase） in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 （DE3）. IPTG （0.5 mmol L-1） was added to induce the expression of recombinant GST/W17 for 3 h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32p-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2 kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses （e.g., salt-） induced signaling pathway.

An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

Effect of Subcellular Localization of P21 on Proliferation and Apoptosis of HepG2 Cells

This study examined the effect of subcellular localization of P21 on the proliferation and apoptosis of HepG2 cells. The coding genes of the wild and the mutant P21 were amplified by mega primer PCR from the plasmid pCEP-WAF1 which contains human P21 cDNA in the nuclear localizational signal （NLS） sequence, and then inserted into the eukaryotic expression vector pDsRedl-C1. The recombinants were transfected into HepG2 cells. The transcription and expression of P21 were determined by RT-PCR and fluorescence microscopy. The cell proliferation was measured by MTT, and the cell cycle and apoptosis of HepG2 cells by flow cytometry. The results of restriction analysis, DNA sequencing and fluorescence microscopy confirmed the construction of the wild and the mutant P21 in the eukaryotic expression plasmid. The plasmid containing the mutant P21 was found to accelerate cell proliferation and the wild P21 plasmid to inhibit cell proliferation. Cell cycle analysis showed that the cell ratio of GeYG1 in the wild type group was significantly increased as compared with that in the mutant type group, and cell apoptosis analysis revealed that the apoptosis rate in the wild type group was much higher than that in the mutant type group. It was concluded that the subcellular localization of P21 may contribute to the develonment of heoatic cancer.

Activities, Quantitative Changes and Subcellular Localization of α-Amylase During Development of Apple Fruit

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. α_Amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was often shown extrachloroplastic in living cells. The present experiment showed that α_amylase activity was progressively increasing concomitantly with the decreasing starch concentrations during the development of apple ( Malus domestica Borkh cv. Starkrimson) fruit. The apparent amount of α_amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The enzyme subcellular_localization studies via immunogold electron_ microscopy technique showed that α_amylase visualized by gold particles was predominantly located in plastids, but the gold particles were scarcely found in other subcellular compartments. A high density of the enzyme was observed at the periphery of starch granules during the middle and late developmental stages. These data proved that the enzyme is compartmented in its functional sites in the living cells of the fruit. The predominantly plastid_distributed pattern of α_amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (α_amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that α_amylase is involved in starch hydrolysis in plastids of the fruit cells.

Glucose regulates LXRα subcellular localization and function in rat pancreatic β-cells

Liver X receptors(LXRs)are members of the nuclear receptor superfamily,which have been implicated in lipid ho-meostasis and more recently in glucose metabolism.Here,we show that glucose does not change LXRα protein level,but affects its localization in pancreatic β-cells.LXRα is found in the nucleus at 8 mM glucose and in the cytoplasm at4.2 mM.Addition of glucose translocates LXRα from the cytoplasm into the nucleus.Moreover,after the activation ofLXR by its synthetic non-steroidal agonist(T0901317),insulin secretion and glucose uptake are increased at 8 mM anddecreased at 4.2 mM glucose in a dose-dependent manner.Furthermore,at low glucose condition,okadaic acid reversedLXRα effect on insulin secretion,suggesting the involvement of glucose signaling through a phosphorylation-dependentmechanism.

Multiple subcellular localizations and functions of protein kinase Cδ in liver cancer

Protein kinase Cδ(PKCδ)is a member of the PKC family,and its implications have been reported in various biological and cancerous processes,including cell proliferation,cell death,tumor suppression,and tumor progression.In liver cancer cells,accumulating reports show the bi-functional regulation of PKCδin cell death and survival.PKCδfunction is defined by various factors,such as phosphorylation,catalytic domain cleavage,and subcellular localization.PKCδhas multiple intracellular distribution patterns,ranging from the cytosol to the nucleus.We recently found a unique extracellular localization of PKCδin liver cancer and its growth factor-like function in liver cancer cells.In this review,we first discuss the structural features of PKCδand then focus on the functional diversity of PKCδbased on its subcellular localization,such as the nucleus,cell surface,and extracellular space.These findings improve our knowledge of PKCδinvolvement in the progression of liver cancer.

Molecular Determinants Responsible for the Subcellular Localization of HSV-1 UL4 Protein

The function of the herpes simplex virus type 1 （HSV-1） UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study, fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein （EYFP）, the nuclear export signals （NES） of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition, the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattem of UL4. Furthermore, the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1 （CRM1）-dependent manner involving RanGTP hydrolysis