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The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 Å in diameter, connected via a three-section neck to an 840-Å-long contractile tail, ending with a three-module baseplate composed of only six protein components. This simplified baseplate consists of a central hub-spike surrounded by six wedge heterotriplexes, to which twelve tail fibers are covalently attached via disulfide bonds in alternating upward and downward configurations. In vitro reduction assays revealed a putative redox-dependent mechanism of baseplate assembly and tail sheath contraction. These findings establish a minimal myophage that might become a user-friendly chassis phage in synthetic biology.

The folding and assembly of RuBisCO, the most abundant enzyme in nature, needs a series of chaperones, including the RuBisCO accumulation factor Raf1, which is highly conserved in cyanobacteria and plants. Here, we report the crystal structures of Raf1 from cyanobacteria Anabaena sp. PCC 7120 and its complex with RuBisCO large subunit RbcL. Structural analyses and biochemical assays reveal that each Raf1 dimer captures an RbcL dimer, with the C-terminal tail inserting into the catalytic pocket, and further mediates the assembly of RbcL dimers to form the octameric core of RuBisCO. Furthermore, the cryo-electron microscopy structures of the RbcL-Raf1-RbcS assembly intermediates enable us to see a dynamic assembly process from RbcL8Raf18 to the holoenzyme RbcL8RbcS8. In vitro assays also indicate that Raf1 can attenuate and reverse CcmM-mediated cyanobacterial RuBisCO condensation. Combined with previous findings, we propose a putative model for the assembly of cyanobacterial RuBisCO coordinated by the chaperone Raf1.

Lake Chaohu, one of the five largest freshwater lakes in China, has been suffering from severe cyanobacterial blooms in the summer for many years. Cyanophages, the viruses that specifically infect cyanobacteria, play a key role in modulating cyanobacterial population, and thus regulate the emergence and decline of cyanobacterial blooms. Here we report a long-tailed cyanophage isolated from Lake Chaohu, termed Mic1, which specifically infects the cyanobacterium Microcystis aeruginosa. Mic1 has an icosahedral head of 88 nm in diameter and a long flexible tail of 400 nm. It possesses a circular genome of 92,627 bp, which contains 98 putative open reading frames. Genome sequence analysis enabled us to define a novel terminase large subunit that consists of two types of intein, indicating that the genome packaging of Mic1 is under fine control via posttranslational maturation of the terminase. Moreover, phylogenetic analysis suggested Mic1 and mitochondria share a common evolutionary origin of DNA polymerase γ gene. All together, these findings provided a start-point for investigating the co-evolution of cyanophages and its cyanobacterial hosts.

Summary Despite previous structural analyses of bacteriophages, quite little is known about the structures and assembly patterns of cyanophages. Using cryo-EM combined with crystallography, we solve the near-atomic-resolution structure of a freshwater short-tailed cyanophage, Pam1, which comprises a 400-A-long tail and an icosahedral capsid of 650 A in diameter. The outer capsid surface is reinforced by trimeric cement proteins with a β-sandwich fold, which structurally resemble the distal motif of Pam1's tailspike, suggesting its potential role in host recognition. At the portal vertex, the dodecameric portal and connected adaptor, followed by a hexameric needle head, form a DNA ejection channel, which is sealed by a trimeric needle. Moreover, we identify a right-handed rifling pattern that might help DNA to revolve along the wall of the ejection channel. Our study reveals the precise assembly pattern of a cyanophage and lays the foundation to support its practical biotechnological and environmental applications.

The coordination of carbon and nitrogen metabolism is essential for bacteria to adapt to nutritional variations in the environment, but the underlying mechanism remains poorly understood. In autotrophic cyanobacteria, high CO2 levels favor the carboxylase activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) to produce 3-phosphoglycerate, whereas low CO2 levels promote the oxygenase activity of RuBisCO, leading to 2-phosphoglycolate (2-PG) production. Thus, the 2-PG level is reversely correlated with that of 2-oxoglutarate (2-OG), which accumulates under a high carbon/nitrogen ratio and acts as a nitrogen-starvation signal. The LysR-type transcriptional repressor NAD(P)H dehydrogenase regulator (NdhR) controls the expression of genes related to carbon metabolism. Based on genetic and biochemical studies, we report here that 2-PG is an inducer of NdhR, while 2-OG is a corepressor, as found previously. Furthermore, structural analyses indicate that binding of 2-OG at the interface between the two regulatory domains (RD) allows the NdhR tetramer to adopt a repressor conformation, whereas 2-PG binding to an intradomain cleft of each RD triggers drastic conformational changes leading to the dissociation of NdhR from its target DNA. We further confirmed the effect of 2-PG or 2-OG levels on the transcription of the NdhR regulon. Together with previous findings, we propose that NdhR can sense 2-OG from the Krebs cycle and 2-PG from photorespiration, two key metabolites that function together as indicators of intracellular carbon/nitrogen status, thus representing a fine sensor for the coordination of carbon and nitrogen metabolism in cyanobacteria.

Background Renal ischemia/reperfusion (I/R)-induced acute kidney injury remains to be a troublesome condition in clinical practice. Although the exact molecular mechanisms underlying renal I/R injury are incompletely understood, the deleterious progress of renal I/R injury involves inflammation, apoptosis, and oxidative stress. Diosmetin is a member of the flavonoid glycosides family, which suppresses the inflammatory response and cellular apoptosis and enhances antioxidant activity. The purpose of this study was to investigate the protective effect of diosmetin on I/R-induced renal injury in mice. Methods Thirty BALB/c mice were randomly divided into five groups. Four groups of mice received diosmetin (0.25, 0.5, and 1 mg/kg) or vehicle (I/R group) before ischemia. Another group received vehicle without ischemia to serve as a negative control (sham-operated group). Twenty-four hours after reperfusion, serum and renal tissues were harvested to evaluate renal function and histopathologic features. In addition, the expression of inflammation-related proteins, apoptotic molecules, and antioxidant enzymes was analyzed. Results Compared with sham mice, the I/R group significantly exacerbated renal function and renal tube architecture and increased the inflammatory response and renal tubule apoptosis. Nevertheless, pretreatment with diosmetin reversed these changes. In addition, diosmetin treatment resulted in a marked increase in antioxidant protein expression compared with I/R mice. Conclusions The renoprotective effects of diosmetin involved suppression of the nuclear factor-κB and mitochondrial apoptosis pathways, as well as activation of the nuclear factor erythroid 2–related factor 2/heme oxygenase-1 pathway. Diosmetin has significant potential as a therapeutic intervention to ameliorate renal injury after renal I/R.

Cyanophages, widespread in aquatic systems, are a class of viruses that specifically infect cyanobacteria. Though they play important roles in modulating the homeostasis of cyanobacterial populations, little is known about the freshwater cyanophages, especially those hypothetical proteins of unknown function. Mic1 is a freshwater siphocyanophage isolated from the Lake Chaohu. It encodes three hypothetical proteins Gp65, Gp66, and Gp72, which share an identity of 61.6% to 83%. However, we find these three homologous proteins differ from each other in oligomeric state. Moreover, we solve the crystal structure of Gp72 at 2.3 A, which represents a novel fold in the α + β class. Structural analyses combined with redox assays enable us to propose a model of disulfide bond mediated oligomerization for Gp72. Altogether, these findings provide structural and biochemical basis for further investigations on the freshwater cyanophage Mic1.

The folding and assembly of RuBisCO, the most abundant enzyme in nature, needs a series of chaperones, including the RuBisCO accumulation factor Raf1, which is highly conserved in cyanobacteria and plants. Here, we report the crystal structures of Raf1 from cyanobacteria Anabaena sp. PCC 7120 and its complex with RuBisCO large subunit RbcL. Structural analyses and biochemical assays reveal that each Raf1 dimer captures an RbcL dimer, with the C-terminal tail inserting into the catalytic pocket, and further mediates the assembly of RbcL dimers to form the octameric core of RuBisCO. Furthermore, the cryo-electron microscopy structures of the RbcL-Raf1-RbcS assembly intermediates enable us to see a dynamic assembly process from RbcL

English course plays a significant part in graduate education. It is advocated in this paper that applying the Approach of Length Writing to graduate English teaching is not only effective in improving the teaching efficiency but also helpful in developing students’ metacognition. By reviewing and summarizing the findings of recent studies and teaching experiments in ESL, some teaching strategies have been put forward in this paper about how to adopt Length Writing Approach in graduate English course to develop students’ metacognitive strategies, reducing their learning anxiety and increasing their comprehensive ability in English using simultaneously. It is claimed by the author in this paper that such a teaching approach will produce prolonged positive effects in graduate students’ English learning and therefore deserves more attention, discussion as well as application.

Despite over 3300 protein-DNA complex structures have been reported in the past decades, there remain some unknown recognition patterns between protein and target DNA. The silkgland-specific transcription factor FMBP-1 from the silkworm Bombyx mori contains a unique DNA-binding domain of four tandem STPRs, namely the score and three amino acid peptide repeats. Here we report three structures of this STPR domain (termed BmSTPR) in complex with DNA of various lengths. In the presence of target DNA, BmSTPR adopts a zig-zag structure of three or four tandem α-helices that run along the major groove of DNA. Structural analyses combined with binding assays indicate BmSTPR prefers the AT-rich sequences, with each α-helix covering a DNA sequence of 4 bp. The successive AT-rich DNAs adopt a wider major groove, which is in complementary in shape and size to the tandem α-helices of BmSTPR. Substitutions of DNA sequences and affinity comparison further prove that BmSTPR recognizes the major groove mainly via shape readout. Multiple-sequence alignment suggests this unique DNA-binding pattern should be highly conserved for the STPR domain containing proteins which are widespread in animals. Together, our findings provide structural insights into the specific interactions between a novel DNA-binding protein and a unique deformed B-DNA.

The soilborne fungus Verticillium dahliae is the major pathogen that causes the verticillium wilt disease of plants, which leads to huge economic loss worldwide. At the early stage of infection, growth of the pathogen is subject to the nutrition stress of limited nitrogen. To investigate the secreted pathogenic proteins that play indispensable roles during invasion at this stage, we compared the profiles of secreted proteins of V. dahliae under nitrogen starvation and normal conditions by using in-gel and in-solution digestion combined with liquid chromatography–nano-electrospray ionization tandem mass spectrometry (LC–nanoESI-MS). In total, we identified 212 proteins from the supernatant of liquid medium, including 109 putative secreted proteins. Comparative analysis indicated that the expression of 76 proteins was induced, whereas that of 9 proteins was suppressed under nitrogen starvation. Notably, 24 proteins are constitutively expressed. Further bioinformatic exploration enabled us to classify the stress-induced proteins into seven functional groups: cell wall degradation (10.5%), reactive oxygen species (ROS) scavenging and stress response (11.8%), lipid effectors (5.3%), protein metabolism (21.1%), carbohydrate metabolism (15.8%), electron–proton transport and energy metabolism (14.5%), and other (21.0%). In addition, most stress-suppressed proteins are involved in the cell-wall remodeling. Taken together, our analyses provide insights into the pathogenesis of V. dahliae and might give hints for the development of novel strategy against the verticillium wilt disease.

Following the Aeromonas hydrophila aerolysin, various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. We have recently reported the mechanism of receptor recognition and in vitro pore-formation of a zebrafish aerolysin-like protein Dln1/Aep1. However, the physiological function of Aep1 remains unknown. Here we detected that aep1 gene is constitutively expressed in various immune-related tissues of adult zebrafish; and moreover, its expression is significantly up-regulated upon bacterial challenge, indicating its involvement in antimicrobial infection. Pre-injection of recombinant Aep1 into the infected zebrafish greatly accelerated the clearance of bacteria, resulting in significantly increased survival rate. Meanwhile, the induced expression of cytokines such as interleukin IL-1β and tumor necrosis factor TNF-α in zebrafish upon injection of recombinant Aep1 suggested that Aep1 may be a pro-inflammatory protein that triggers the antimicrobial immune responses. However, compared to the overproduction of these cytokines in the infected zebrafish, pre-injection of Aep1 could significantly reduce the expression level of these cytokines, accompanying with a reduced bacterial load. Moreover, the expression profiles through the developmental stages of zebrafish demonstrated that aep1 is activated at the very early stage prior to the maturation of adaptive immune system. Altogether, our findings proved that Aep1 is an innate immune molecule that prevents the bacterial infection.

With the trend of globalization, especially cultural interaction and integration, the development of Chinese music has welcomed unprecedented chances and opportunities. However, under the increasingly aggressive import and infusion of western cultures, the problem of sustainable development without losing one’s national traits cannot be underestimated. Therefore, with an attempt to promote the stable and scientific development of Chinese music, the author of this paper suggests that it is necessary to construct the cultural subjectivity of Chinese music. It is argued in this paper that by strengthening the conception of cultural identity, deconstructing the misbelief of West-centralism, highlighting the cultural nationality and perfecting the theoretic system building, the scientific development of Chinese music characterized by its own national uniqueness will be enhanced.

To examine the effects of formononetin (FMN) on Acetaminophen (APAP)-induced liver injury in vitro and in vivo. Human non-tumor hepatic cells LO2 were pretreated with either vehicle or FMN (20, 40 μM), for 6 h, followed by incubation with or without APAP (10 mM) for 24 h. In an in vivo assay, male BALB/c mice were randomly divided into four groups: (1) control group; (2) APAP group; (3) APAP + FMN (50 mg/Kg); (4) APAP + FMN (100 mg/Kg). The mice in the control and APAP groups were pre-treated with vehicle; the other two groups were pretreated daily with FMN (50, 100 mg/Kg) orally for 7 consecutive days. After the final treatment, acute liver injury was induced in all groups, except the control group, by intraperitoneal (i.p.) injection of 300 mg/Kg APAP. In LO2 cells, APAP exposure decreased the cell viability and glutathione (GSH) content, which were both greatly restored by FMN pretreatment. Overdose of APAP increased hepatic malondialdehyde (MDA) content, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activity in experimental mice. Supplementation with 100 mg/Kg FMN significantly reduced APAP-induced elevated levels of MDA (1.97 ± 0.27 vs 0.55 ± 0.14 nmol/mg protein, p < 0.001), ALT (955.80 ± 209.40 vs 46.90 ± 20.40 IU/L, p < 0.001) and AST (1533.80 ± 244.80 vs 56.70 ± 28.80 IU/L, p < 0.001), and hepatic GSH level (5.54 ± 0.93 vs 8.91 ± 1.11 μmol/mg protein, p < 0.001) was significantly increased. These results were further validated by histopathology and TdT-mediated biotin-dUTP nick-endlabeling (TUNEL) staining, pretreatment with 100 mg/Kg FMN significant decreased APAP-induced hepatocellular damage and cell apoptosis (36.55 ± 3.82 vs 2.58 ± 1.80%, p < 0.001). Concomitantly, FMN stimulated the expression of Nrf2 and antioxidant gene expression in the presence of APAP. These data provide an experimental basis for the use of FMN in the treatment of patients with APAP-induced hepatotoxicity.

The promotion of the green buildings is the necessary way to expand Chinese urbanization. The idea of green energy-saving building and green life-styles should be a kind of behavioral patterns. In the paper, the idea of green and energy-saving buildings is put forward. The management systems about green and energy conservation buildings are combed. Some Problems existing in the promotion of Chinese green energy-saving building at present are pointed out. From the perspective of implementation, some countermeasures are also pointed out in the paper, including improving supervision about green energy-saving buildings, perfecting technology system of intelligent and green energy conservation buildings, based on optimizing the design, setting up scientific assessment system, promoting the use of new technologies, new materials, and so on.

The juvenile hormone (JH) is a kind of epoxidecontaining sesquiterpene ester secreted by a pair of corpora allatum behind the brain of insects.1 It controls the metamorphosis development of insects together with the ecdysone.2,3 Thus the synthesis and degradation of JH are tightly regulated in different development stages.4 The degradation of JH is catalyzed by two hydrolases, juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone esterase. JHEH is responsible for opening the epoxide ring of JH to produce JH diol, whereas JHE catalyzes the removal of the methyl ester moiety of JH to form JH acid.5,6 JHEH belongs to the microsomal epoxide hydrolase (mEH) (EC 3.3.2.9) family, which is one of the most widely distributed families of epoxide hydrolases (EHs). EHs can transform epoxides to compounds with decreased chemical reactivity, increased water solubility, and altered biological activity.7,8 In addition to participating in the catabolism of JH in insects, mEHs also play important roles in cytoprotection, steroid metabolism, bile acid transport, and xenobiotic metabolism.9 To date, the only structure of the mEH from the fungus Aspergillus niger (termed AnEH, PDB 1QO7) revealed a typical a/b-hydrolase core composed of a twisted eightstranded b-sheet packing on both sides with several a-helices.10,11 Structural analyses suggested a bimolecular nucleophilic substitution (SN2) reaction mechanism involving a standard nucleophile–histidine–acid catalytic triad of Asp–His–Glu/Asp.11 However, the mechanism of substrate recognition and catalysis of mEHs remains unclear. Here we report the crystal structure of Bombyx mori JHEH (BmJHEH) at 2.30 A resolution. Structural analyses together with molecular simulation reveal insights into the specific binding of JH in the active-site pocket. These findings increase our understanding of the substrate recognition and catalysis of mEHs and might help the design of JH-derived pesticides.

The EPC mode is one kind of total engineering management mode. In EPC engineering, the contractor commitments the project design, equipment procurement and construction, etc, which does not mean that general contractor must complete personally the entire construction project. The general contractor of EPC engineering can subcontract a part of the works to some subcontractors. So it is very important for the general contractor to tighten up the management of subcontractors through rules on contract. In the paper, some existing problems in signing stage and performance stage are analyzed. To address the problems, some solutions are put forward, including paying attention to the relevant qualification of the subcontractors audit, setting up warning systems of sub-contract, conducting risk assessment of sub-contract variations, etc.

It is of great significance for the measurement of low concentration of VOCs that the sample pretreatment preconcentration system. We design a gas sample pre-treatment preconcentration system based on LabVIEW. It’s in this system, of whose PC uses LabVIEW software and of whose following part adopts data acquisition card USB4711A; intelligent temperature control instrument FB400 of RKC and mass flow controller of burkert (MFC8711). The standardized drive mode of OPC and DataSocket of LabVIEW realize the PC real-time communication with lower computer .This system has a good man-machine interface and it is a reliable control system. This system can realize the real-time display and control of parameters such as temperature, flow, and also realize the running condition of all equipments control and alarm function.

Cyanobacteria are the most abundant photosynthetic microorganisms, the global distribution of which is mainly regulated by the corresponding cyanophages. A systematic screening of water samples in the Lake Chaohu enabled us to isolate a freshwater siphocyanophage that infects Microcystis wesenbergii, thus termed Mic1. Using cryoelectron microscopy, we solved the 3.5-Å structure of Mic1 capsid. The major capsid protein gp40 of an HK97-like fold forms two types of capsomers, hexons and pentons. The capsomers interact with each other via the interweaved N-terminal arms of gp40 in addition to a tail-in-mouth joint along the three-fold symmetric axis, resulting in the assembly of capsid in a mortise-and-tenon pattern. The novel-fold cement protein gp47 sticks at the two-fold symmetric axis and further fixes the capsid. These findings provide structural insights into the assembly of cyanophages, and set up a platform to explore the mechanism of specific interactions and co-evolution with cyanobacteria.

Highway engineering construction due to a long development cycle, complex and large scale, is filled with risks. The research of the risk and countermeasures of highway engineering construction is of great significance. In the paper, risk factors analysis of highway engineering construction contract in signing stage and implement stage are discussed, and some countermeasures are proposed, including risk aversion and risk transfer.

Invertases catalyze the hydrolysis of sucrose to glucose and fructose, thereby playing a key role in primary metabolism and plant development. According to the optimum pH, invertases are classified into acid invertases (Ac-Invs) and alkaline/neutral invertases (A/N-Invs), which share no sequence homology. Compared with Ac-Invs that have been extensively studied, the structure and catalytic mechanism of A/N-Invs remain unknown. Here we report the crystal structures of Anabaena alkaline invertase InvA, which was proposed to be the ancestor of modern plant A/N-Invs. These structures are the first in the GH100 family. InvA exists as a hexamer in both crystal and solution. Each subunit consists of an (α/α)6 barrel core structure in addition to an insertion of three helices. A couple of structures in complex with the substrate or products enabled us to assign the subsites -1 and +1 specifically binding glucose and fructose, respectively. Structural comparison combined with enzymatic assays indicated that Asp-188 and Glu-414 are putative catalytic residues. Further analysis of the substrate binding pocket demonstrated that InvA possesses a stringent substrate specificity toward the α1,2-glycosidic bond of sucrose. Together, we suggest that InvA and homologs represent a novel family of glucosidases.

Iron homeostasis plays a crucial role in growth and division of cells in all kingdoms of life. Although yeast iron metabolism has been extensively studied, little is known about the molecular mechanism of response to surplus iron. In this study, expression profiling of Saccharomyces cerevisiae in the presence of surplus iron revealed a dual effect at 1 and 4 h. A cluster of stress-responsive genes was upregulated via activation of the stress-resistance transcription factor Msn4, which indicated the stress effect of surplus iron on yeast metabolism. Genes involved in aerobic metabolism and several anabolic pathways are also upregulated in iron-surplus conditions, which could significantly accelerate yeast growth. This dual effect suggested that surplus iron might participate in a more complex metabolic network, in addition to serving as a stress inducer. These findings contribute to our understanding of the global response of yeast to the fluctuating availability of iron in the environment.

Fibroins serve as the major building blocks of silk fiber. As the major component of fibroin, the fibroin heavy chain is a considerably large protein comprising N-terminal and C-terminal hydrophilic domains and 12 highly repetitive Gly-Ala-rich regions flanked by internal hydrophilic blocks. Here, we show the crystal structure of the fibroin N-terminal domain (FibNT) at pH 4.7, revealing a remarkable double-layered anti-parallel β-sheet with each layer comprising two FibNT molecules entangled together. We also show that FibNT undergoes a pH-responsive conformational transition from random coil to β-sheets at around pH 6.0. Dynamic light scattering demonstrates that FibNT tends to oligomerize as pH decreases to 6.0, and electron microscopy reveals micelle-like oligomers. Our results are consistent with the micelle assembly model of silk fibroin and, more importantly, show that the N-terminal domain in itself has the capacity to form micelle-like structures in response to pH decrease. Structural and mutagenesis analyses further reveal the important role of conserved acidic residues clustered in FibNT, such as Glu56 and Asp100, in preventing premature β-sheet formation at neutral pH. Collectively, we suggest that FibNT functions as a pH-responsive self-assembly module that could prevent premature β-sheet formation at neutral pH yet could initiate fibroin assembly as pH decreases along the lumen of the posterior silk gland to the anterior silk gland.

The hemolymph of the fifth instar larvae of the silkworm Bombyx mori contains a group of homologous proteins with a molecular weight of approximately 30 kDa, termed B. mori low molecular weight lipoproteins (Bmlps), which account for about 5% of the total plasma proteins. These so-called "30 K proteins" have been reported to be involved in the innate immune response and transportation of lipid and/or sugar. To elucidate their molecular functions, we determined the crystal structure of a 30 K protein, Bmlp7, at 1.91Å. It has two distinct domains: an all-α N-terminal domain (NTD) and an all-β C-terminal domain (CTD) of the β-trefoil fold. Comparative structural analysis indicates that Bmlp7 represents a new family, adding to the 14 families currently identified, of the β-trefoil superfamily. Structural comparison and simulation suggest that the NTD has a putative lipid-binding cavity, whereas the CTD has a potential sugar-binding site. However, we were unable to detect the binding of either lipid or sugar. Therefore, further investigations are needed to characterize the molecular function of this protein.

Saccharomyces cerevisiae Hsp33/YOR391Cp is a member of the ThiI/DJ-1/PfpI superfamily. Hsp33 was overexpressed in Escherichia coli and its crystal structure was determined at 2.40 Å resolution. Structural comparison revealed that Hsp33 adopts an α/β-hydrolase fold and possesses the putative Cys-His-Glu catalytic triad common to the Hsp31 family, suggesting that Hsp33 and Hsp31 share similar aminopeptidase activity, while structural deviations in helices α2-α3 of the core domain might be responsible for the access of different peptide substrates.

Cu/Zn superoxide dismutases (Cu/Zn-SODs) are a large family of cytosolic antioxidant proteins involved in responses to oxidative stress.1 They catalyze the disproportionation of superoxide radicals into less toxic hydrogen peroxide and dioxygen via cyclic reduction and reoxidation of its bound copper ion as shown below.2 SOD CuðIIÞ þ O2 ! SOD CuðIÞ þ O2 SOD CuðIÞ þO2 þ 2Hþ ! SOD CuðIIÞ þH2O2

During prepupal stage, the genes expression in silkgland is considered as a model for gene expression and regulation of eukaryotes. Aiming to comprehensively interpret gene expression profile in the silkgland, we collected all currently available EST, complete cDNA and protein expression information and other gene expression testing data published before, and explored their roles in their function pathways level. With the analysis of interaction between the known proteins and putative bio-macromolecules partners in silico, we list our prediction results in the form of pathway classification and test some of their expressions by experiments.

For the complexity and concealment of the construction, the bolt anchoring system has great difficulty in quality detection. Due to various reasons the traditional detection methods of quality are difficult to obtain effective results. In recent years, stress wave reflection has been the main method in quality nondestructive testing of rock bolts, but the reflection signal contains noise which brings great inconvenience for the signal analysis. Harmonic wavelet has a strict "box" spectrum characteristics, and by using the harmonic wavelet packet, signal can be decomposed into different frequency bands and the unwanted ingredients can be effectively eliminated, so the denoised signal could be obtained. In this paper, a reflection signal is decomposed by using the harmonic wavelet packet, and the appropriate decomposition layers and the frequency band width are chosen, the effective frequency band is extracted and reconstructed. Compared with the classical wavelet transform, experiment results show that the bolt parameters accuracy using harmonic wavelet packet is improved.

Chitinases (EC 3.2.1.14), as one kind of glycosyl hydrolase, hydrolyze the β-(1,4) linkages of chitin. According to the sequence similarity, chitinases can be divided into glycoside hydrolase family 18 and family 19. Here, a chitinase from Nosema bombycis (NbchiA) was cloned and purified by metal affinity chromatography and molecular exclusion chromatography. Sequence analysis indicated that NbchiA belongs to glycoside hydrolase family 19 class IV chitinase. The optimal pH and temperature of NbchiA are 7.0 and 40 °C, respectively. This purified chitinase showed high activity toward soluble substrates such as ethylene glycol chitin and soluble chitosan. The degradation of chitin oligosaccharides (GlcNAc)(2-5) detected by high-performance liquid chromatography showed that NbchiA hydrolyzed mainly the second glycosidic linkage from the reducing end of (GlcNAc)(3-5). On the basis of structure-based multiple-sequence alignment, Glu51 and Glu60 are believed to be the key catalytic residues. The site-directed mutation analysis revealed that the enzymatic activity was decreased upon mutation of Glu60, whereas mutation of Glu51 totally abolished the enzymatic activity. This is the first report of a GH19 chitinase in fungi and in Microsporidia.

ACFI is an anticoagulant C-type lectin-like protein (CLP) isolated from Agkistrodon acutus venom. To investigate the function of ACFI and its subunits, the cDNAs of two subunits were transformed and expressed in Pichia pastoris separately or together by a novel strategy using two vectors with different selectable markers. The results showed that recombinant homodimers were secreted when the subunits were expressed alone, while heterodimers (rACFI) were secreted when two subunits were co-expressed. The secreted proteins were purified from culture supernatants in one step by metal-chelating affinity chromatography with the yields of 1–4 mg/L. PAGE and ELISA showed that rACFI competed the binding of native ACFI for human factor X and IX with affinities of 1.6 and 30 nM, respectively. In addition, rACFI prolonged the activated partial thromboplastin time (APTT) in a concentration dependent manner as same as native ACFI. However, recombinant α or β homodimers completely lost these activities, indicating the heterodimerization of two subunits is required for its function. It also suggests that P. pastoris is a promising system for structure-function studies of snake CLPs.

Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested withBamHI andXhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested withBamHI andXhoI, then the NOR1, gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1, was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunoblot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography. The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1, is obtained in JM 105, and its antiserum can be prepared successfully.

Objective: To identify the relation between nasopharyngeal carcinoma and the human novel gene UBAP1, which is located in the region of minimal heterozygosity deletion at 9pl3.2 and down-expressed in NPC. Methods: Five single nucleotide polymorphisms (SNPs) within UBAP1 gene were analysed by sequencing in 105 NPC patients and 183 control subjects which matched to the NPC cases on age, sex and residence. Results: Significant association was found between NPC with one SNP mark (rslO49557), which is located at 3′ non-region of UBAP1 gene; the relative risk of this SNP mark is 1.64 (genotype GG) and 1.31 (genotype CG). Conclusion: The result has proved again that UBAP1 gene may play a certain role in the occurrence and development of nasopharyngeal carcinoma. The SNP mark rslO49557, considering its location, may influence the expression of UBAP1 gene.

Objective: To identify the differential expression profile of human novel gene UBAP1, a putative nasopharyngeal neoplasms (NPC) relate gene, in multiple cancers. Methods: We first present an EST approach for electronic Northern in silico to analyse expression patterns of UBAP1 in tumor and normal tissues. Full length cDNA of UBAP1 gene was taken as a “probe” sequence, and a blastn search was performed against human EST Database. The Blastn report can be used to determine the fold differences between the pedigree ESTs in different libraries. Especially, the ESTs corresponding to UBAP1 present in fifteen tumor-derived libraries were compared against their normal counterpart to produce an electronic differential expression profile. Second, the distinct down-regulation of UBAP1 in meningioma, glioma, and colorectal tumor was confirmed by differentially RT-PCR analysis. Results: Database surveys indicated that UBAP1 gene was not only ubiquitously expressed in many normal tissues with various levels but also differentially expressed in different tumor tissues, especially down-regulated in multiple neoplastic tissues such as brain, breast, skin, colon, testis and uterus tumor tissues. Furthermore, differential RT-PCR analysis demonstrated that expression of UBAP1 was down-regulated or absent in 7 of 12 (58%) meningioma samples, 6 of 9 (66%) glioma and 7 of 11 (63%) colorectal tumor tissues respectively. Conclusion: we described a data mining procedure in silico that proved to be useful for the identification of differential expression patterns of UBAP1. These findings could be valuable for the investigation of the mechanism the differential expression of UBAP1 gene and its significance in the progression of multiple cancers.

Renal ischemia/reperfusion (I/R) injury continues to be a complicated situation in clinical practice. Genistein, the main isoflavone found in soy products, is known to possess a wide spectrum of biochemical and pharmacological activities. However, the protective effect of genistein on renal I/R injury has not been well investigated. In the current study, we explore whether genistein exhibits its renal-protective effects through SIRT1 (Sirtuin 1) in I/R-induced mice model. We found the treatment of genistein significantly reduced renal I/R-induced cell death, simultaneously stimulating renal cell proliferation. Meanwhile, SIRT1 expression was up-regulated following the administration of genistein in renal region. Furthermore, pharmacological inhibition or shRNA-mediated depletion of SIRT1 significantly reversed the protective effect of genistein on renal dysfunction, cellular damage, apoptosis, and proliferation following I/R injury, suggesting an indispensible role of the increased SIRT1 expression and activity in this process. Meanwhile, the reduced p53 and p21 expression and increased PCNA (Proliferating Cell Nuclear Antigen) expression were blocked after the depletion of SIRT1 compared with the genistein treatment group in the renal I/R process. Hence, our results provided further experimental basis for the potential use of genistein for the treatment of kidney disease with deficiency of SIRT1 activity.

Saccharomyces cerevisiae Gre2 (EC1.1.1.283) serves as a versatile enzyme that catalyzes the stereoselective reduction of a broad range of substrates including aliphatic and aromatic ketones, diketones, as well as aldehydes, using NADPH as the cofactor. Here we present the crystal structures of Gre2 from S. cerevisiae in an apo-form at 2.00A and NADPH-complexed form at 2.40A resolution. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain and a variable C-terminal domain, which participates in substrate recognition. The induced fit upon binding to the cofactor NADPH makes the two domains shift toward each other, producing an interdomain cleft that better fits the substrate. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis enabled us to define a potential substrate-binding pocket that determines the stringent substrate stereoselectivity for catalysis.

To study the pollution characteristics of volatile organic compounds (VOCs) in Tianjin downtown ambient air, 26 sampling points were distributed in Tianjin and systematic sampling was carried out in Spring, Summer, Autumn and Winter, respectively. More than 80 kinds of VOCs were detected which mainly included alkanes, BETX and halohydrocarbon with detection rates of over 80%. The total concentration of VOCs changed with the seasons, in the order of Spring (110.43 x 10(-9))Autumn (93.73 x 10(-9))Winter (73.37 x 10(-9))Summer (60.43 x 10(-9)). The result of concentration statistics indicates that the total concentration of VOCs in urban areas was in the range of 30 x 10(-9)-90 x 10(-9) and the number of samples in every season in this concentration range accounted for over 50%. There were also differences in the composition of VOCs with the change of seasons in Tianjin. Oxygenated organic compounds and hydrocarbons were the main substances of VOCs and the concentration percentage of them was over 50% in every season. Two main pollutants, BETX and halohydrocarbon, were analyzed for seasonal variation in this paper.

The manganese-specific superoxide dismutase SOD2 from the yeast Saccharo­myces cerevisiae is a protein that resides in the mitochondrion and protects it against attack by superoxide radicals. However, a high iron concentration in the mitochondria results in iron misincorporation at the active site, with subsequent inactivation of SOD2. Here, the crystal structures of SOD2 bound with the native metal manganese and with the ‘wrong’ metal iron are presented at 2.05 and 1.79 A resolution, respectively. Structural comparison of the two structures shows no significant conformational alteration in the overall structure or in the active site upon binding the non-native metal iron. Moreover, residues Asp163 and Lys80 are proposed to potentially be responsible for the metal specificity of the Mn-specific SOD. Additionally, the surface-potential distribution of SOD2 revealed a conserved positively charged electrostatic zone in the proximity of the active site that probably functions in the same way as in Cu/Zn-SODs by facilitating the diffusion of the superoxide anion to the metal ion.

Glutathione transferases (GSTs) are ubiquitous detoxification enzymes that conjugate hydrophobic xenobiotics with reduced glutathione. The silkworm Bombyx mori encodes four isoforms of GST Omega (GSTO), featured with a catalytic cysteine, except that bmGSTO3-3 has an asparagine substitution of this catalytic residue. Here, we determined the 2.20-A crystal structure of bmGSTO3-3, which shares a typical GST overall structure. However, the extended C-terminal segment that exists in all the four bmGSTOs occupies the G-site of bmGSTO3-3 and makes it unworkable, as shown by the activity assays. Upon mutation of Asn29 to Cys and truncation of the C-terminal segment, the in vitro GST activity of bmGSTO3-3 could be restored. These findings provided structural insights into the activity regulation of GSTOs.