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Objective: This study aims to evaluate the risk stratification among elderly Nasopharyngeal carcinoma (NPC) patients (≥60 years old) and select the beneficiaries from concurrent chemotherapy (CCRT) combined with induction chemotherapy (IC). Materials and Methods: A total of 909 elderly non‐metastatic NPC patients treated with cisplatin‐based CCRT or IC + CCRT between January 2007 and December 2016 were included. Prognostic nomograms were generated according to clinical characteristics and serum biomarkers. The survival outcomes of patients treated with CCRT versus IC + CCRT were compared in three well‐matched risk groups (high, medium, and low risk) after PSM analysis. Benefit of IC in people older or younger than 70 years and effect of different IC regimens and cycles on prognosis were analyzed. Results: Nomograms of overall survival (OS) (C‐index: 0.64, 95% CI, 0.61–0.89) and disease special survival (DSS) (C‐index: 0.65, 95% CI, 0.62–0.71) showed good prognostic accuracy. The nomogram for DSS included variables of age, gender, ACE, EBV DNA, N stage, and T stage. OS included variables of age, smoking history, ACE, ALB, EBV DNA, N stage, and T stage. The corresponding 5‐year OS rates of high, medium and low risk groups were 87.4%, 82.2%, and 60.9%, respectively (p < 0.001), while the 5‐year DSS rates were 92.2%, 84.3%, and 69.0%, respectively (p < 0.001). In the high risk group, IC + CCRT led to significantly higher 5‐year OS and DSS rate compared with CCRT (5‐year OS rate, 73.5% versus 51.8%, p = 0.006; 5‐year DSS rate, 81.4% versus 61.3%, p = 0.002). While in the medium and low risk groups, OS and DSS were not significantly different (OS: p = 0.259, 0.186; DSS: p = 0.29, 0.094). Subgroup analysis showed in the high risk group, only people younger than 70 years old could benefit from IC. TPF and IC cycles of three could lead to the best survival results. Conclusion: Compared with CCRT, OS, and DSS among high risk elderly patients were significantly improved by the addition of IC in patients younger than 70 years old. TPF and three IC cycles were recommended. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that p53 plays a suppressive role in cancer metastasis, yet the underlying mechanism remains largely unclear. Regulation of actin dynamics is essential for the control of cell migration, which is an important step in metastasis. The Arp2/3 complex is a major nucleation factor to initiate branched actin polymerization that drives cell migration. However, it is unknown whether p53 could suppress metastasis through modulating Arp2/3 function. Here, we report that WDR63 is transcriptionally upregulated by p53. We show with migration assays and mouse xenograft models that WDR63 negatively regulates cell migration, invasion, and metastasis downstream of p53. Mechanistically, WDR63 interacts with the Arp2/3 complex and inhibits Arp2/3‐mediated actin polymerization. Furthermore, WDR63 overexpression is sufficient to dampen the increase in cell migration, invasion, and metastasis induced by p53 depletion. Together, these findings suggest that WDR63 is an important player in the regulation of Arp2/3 function and also implicate WDR63 as a critical mediator of p53 in suppressing metastasis. Synopsis: As a direct transcriptional target of p53, WDR63 interacts with the Arp2/3 complex and inhibits Arp2/3‐dependent actin polymerization, thereby mediating the inhibitory role of p53 on metastasis. WDR63 is a direct transcriptional target of p53.WDR63 negatively regulates cell migration, invasion, and metastasis.WDR63 interacts with the Arp2/3 complex and inhibits Arp2/3‐dependent actin polymerization.WDR63 overexpression dampens the increase in cell migration, invasion and metastasis induced by p53 depletion. [ABSTRACT FROM AUTHOR]

The Nit (nitrilase-like) protein subfamily constitutes branch 10 of the nitrilase superfamily. Nit proteins are widely distributed in nature. Mammals possess two members of the Nit subfamily, namely Nit1 and Nit2. Based on sequence similarity, yeast Nit2 (yNit2) is a homologue of mouse Nit1, a tumour-suppressor protein whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2 (also a putative tumour suppressor) is identical to ω-amidase, an enzyme that catalyzes the hydrolysis of α-ketoglutaramate (α-KGM) and α-ketosuccinamate (α-KSM) to α-ketoglutarate (α-KG) and oxaloacetate (OA), respectively. In the present study, crystal structures of wild-type (WT) yNit2 and of WT yNit2 in complex with α-KG and with OA were determined. In addition, the crystal structure of the C169S mutant of yNit2 (yNit2-C169S) in complex with an endogenous molecule of unknown structure was also solved. Analysis of the structures revealed that α-KG and OA are covalently bound to Cys169 by the formation of a thioester bond between the sulfhydryl group of the cysteine residue and the γ-carboxyl group of α-KG or the β-carboxyl group of OA, reflecting the presumed reaction intermediates. However, an enzymatic assay suggests that α-KGM is a relatively poor substrate of yNit2. Finally, a ligand was found in the active site of yNit2-C169S that may be a natural substrate of yNit2 or an endogenous regulator of enzyme activity. These crystallographic analyses provide information on the mode of substrate/ligand binding at the active site of yNit2 and insights into the catalytic mechanism. These findings suggest that yNit2 may have broad biological roles in yeast, especially in regard to nitrogen homeostasis, and provide a framework for the elucidation of the substrate specificity and biological role of mammalian Nit1. [ABSTRACT FROM AUTHOR]

Phosphorylated derivatives of phosphatidylinositol (PtdIns), also called phosphoinositides (PIPs), are basic components of membrane-associated signalling systems. A family of PtdIns-transfer proteins (PITPs) called the Sec14 family have been predicted to form a set of functional modules that can sense different types of lipid metabolism and transmit the information to the PIP signalling system. In eukaryotic cells, the Sec14 family exhibits a wide diversity of activity, but the structural basis of this diversity remains unclear. In the present study, the dimeric structure of Sfh3 (Sec14 family homologue 3 in yeast) is reported for the first time and differs from the Sec14 proteins reported to date, all of which are monomeric. Some variations in the binding pocket of Sfh3 were observed and the dimer interface was identified and proposed to provide a link between dimer-monomer state changes and PtdIns binding. Together, these structural changes and the oligomeric state transformation of Sfh3 support ideas of diversity within the Sec14 family and provide some new clues to function. [ABSTRACT FROM AUTHOR]

The yeast Paf1 complex (Paf1C), which is composed of the proteins Paf1, Cdc73, Ctr9, Leo1 and Rtf1, accompanies RNA polymerase II from the promoter to the 3′-end formation site of mRNA- and snoRNA-encoding genes. As one of the first identified subunits of Paf1C, yeast Cdc73 (yCdc73) takes part in many transcription-related processes, including binding to RNA polymerase II, recruitment and activation of histone-modification factors and communication with other transcriptional activators. The human homologue of yCdc73, parafibromin, has been identified as a tumour suppressor linked to breast, renal and gastric cancers. However, the functional mechanism of yCdc73 has until recently been unclear. Here, a 2.2 Å resolution crystal structure of the highly conserved C-terminal region of yCdc73 is reported. It revealed that yCdc73 appears to have a GTPase-like fold. However, no GTPase activity was observed. The crystal structure of yCdc73 will shed new light on the modes of function of Cdc73 and Paf1C. [ABSTRACT FROM AUTHOR]

Agkisacucetin is a snake C-type lectin isolated from the venom of Agkistrodon acutus (A. acutus). It binds specifically to the platelet glycoprotein (GP) Ib and prevents the von Willebrand factor (VWF) accessing it. We determined the crystal structure of agkisacucetin to 1.9Å resolution. The structure of agkisacucetin has an (αβ) fold similar to another GPIb-binding protein, flavocetin-A, but lacks the C-terminal cysteine in the β-subunit, does not form (βα)4 tetramers, and does not cluster GPIbs, like flavocetin-A. Proteins 2012;. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In bacteria and eukaryotes, the last two steps of de novo purine biosynthesis are catalyzed by bifunctional purine-biosynthesis protein (PurH), which is composed of two functionally independent domains linked by a flexible region. The N-terminal domain possesses IMP cyclohydrolase activity and the C-terminal domain possesses aminoimidazole-4-carboxamide ribonucleotide transformylase activity. This study reports the expression, purification, crystallization and preliminary X-ray crystallographic analysis of PurH from Escherichia coli with an N-terminal His6 tag. The crystals diffracted to a maximum resolution of 3.05 Å and belonged to the monoclinic space group P21, with unit-cell parameters a = 76.37, b = 132.15, c = 82.64 Å, β = 111.86°. [ABSTRACT FROM AUTHOR]

Snake-venom thrombin-like enzymes (SVTLEs) are serine proteases that are widely distributed in snakes from the Crotalinae subfamily of the Viperidae. In contrast to other snake-venom serine proteases, they have a biochemical activity similar to that of thrombin and play an important role in the process of blood coagulation. However, SVTLEs cannot activate factor VIII, which is essential in blood-clot stabilization. Consequently, blood clots produced by SVTLEs are not stable and are cleared rapidly. This characteristic makes SVTLEs attractive as potential candidates for antithrombotic therapy. Saxthrombin, an SVTLE from Gloydius saxatilis, was purified and crystallized to obtain a high-quality crystal, from which data were acquired to 1.43 Å resolution. Preliminary X-ray diffraction analysis showed that the crystal belonged to space group C2, with unit-cell parameters a = 94.2, b = 52.2, c = 50.1 Å, β = 96.7°. The crystal structure was determined by molecular replacement and the final R factor was 18.69%; the Rfree was 20.01%. This is the first report of a crystal structure of an SVTLE. Saxthrombin belongs to the typical α/β-hydrolase fold of serine proteases. Its structure was compared with those of thrombin and other snake-venom serine proteases. The observed differences in the amino-acid composition of the loops surrounding the active site appear to contribute to different surface-charge distributions and thus alter the shape of the active-site cleft, which may explain the differences in substrate affinity. [ABSTRACT FROM AUTHOR]

Fungal laccases are oxidoreductases that belong to the multinuclear copper-containing oxidases. They are able to oxidize a wide range of substrates, preferably phenolic compounds, which makes them suitable for employment in the bioremediation of soil and water as well as in other biotechnological applications. Here, the structural analysis of natural laccase B (LacB) from Trametes sp. AH28-2 is presented. This structure provides the opportunity to study the natural post-translational modifications of the enzyme. The overall fold shows a high homology to those of previously analyzed laccases with known three-dimensional structure. However, LacB contains a new structural element, a protruding loop near the substrate-binding site, compared with the previously reported laccase structures. This unique structural feature may be involved in modulation of the substrate recognition of LacB. [ABSTRACT FROM AUTHOR]

Transcription in archaea employs a eukaryotic-type transcription apparatus but uses bacterial-type transcription factors. NusG is one of the few archaeal transcription factors whose orthologs are essential in both bacteria and eukaryotes. Archaeal NusG is composed of only an NusG N-terminal (NGN) domain and a KOW domain, which is similar to bacterial NusG but not to the eukaryotic ortholog, Spt5. However, archaeal NusG was confirmed recently to form a complex with rpoE″ that was similar to the Spt5-Spt4 complex. Thus, archaeal NusG presents hybrid features of Spt5 and bacterial NusG. Here we report the crystal structure of NGN from the archaea Methanocaldococcus jannaschii ( MjNGN). MjNGN folds to an α-β-α sandwich without the appendant domain of bacterial NGNs, and forms a unique homodimer in crystal and solution. MjNGN alone was found to be sufficient for rpoE″ binding and an MjNGN-rpoE″ model has been constructed by rigid docking. Proteins 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The Gos1 protein (Golgi SNAP receptor complex member 1) is involved in the SNARE complex, which is the core machinery that drives membrane fusion between cargo-carrying vesicles and their target membranes in the secretory and endocytic pathways in yeast. Truncated versions of the Gos1 protein from Saccharomyces cerevisiae were cloned, expressed, purified and crystallized. The crystal belonged to space group P212121, with unit-cell parameters a = 39.67, b = 43.58, c = 81.94 Å, α = β = γ = 90°. An X-ray diffraction data set was collected at 100 K to 1.63 Å resolution. Matthews coefficient ( VM) calculations suggest that one molecule is present in the asymmetric unit, corresponding to a solvent content of ∼55%. [ABSTRACT FROM AUTHOR]

Adenylate kinase plays a very important role in regulating adenylate species in the cell. Methanocaldococcus jannaschii is a rich resource of unique enzymes. Here, MJ0458, an adenylate kinase from M. jannaschii, was crystallized. A set of X-ray diffraction data to 2.70 Å resolution was collected on beamline BL-17U of the Shanghai Synchrotron Radiation Facility (SSRF). The crystal belonged to space group P41212 or P43212. The unit-cell parameters were a = b = 76.18, c = 238.70 Å, α = β = γ = 90°. [ABSTRACT FROM AUTHOR]

Synaptotagmin acts as the Ca2+ sensor for neural and endocrine exocytosis. Synaptotagmin 5 has been demonstrated to play a key role in the acquisition of cathepsin D and the vesicular proton ATPase and in Ca2+-dependent insulin exocytosis. The C2 domains modulate the interaction of synaptotagmin with the phospholipid bilayer of the presynaptic terminus and effector proteins such as the SNARE complex. This study reports the cloning, expression in Escherichia coli, purification, crystallization and preliminary X-ray analysis of the C2A domain of human synaptotagmin 5 with an N-terminal His6 tag. The crystals diffracted to 1.90 Å resolution and belonged to the hexagonal space group P65, with unit-cell parameters a = b = 93.97, c = 28.05 Å. A preliminary model of the protein structure has been built and refinement of the model is ongoing. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, TRM6 and TRM61 compose a tRNA methyltransferase which catalyzes the methylation of the N1 of adenine at position 58 in tRNAs, especially initiator methionine tRNA. TRM61 is the subunit that binds S-adenosyl- l-methionine and both subunits contribute to target tRNA binding. In order to elucidate the catalytic mechanism of TRM6-TRM61 and the mode of interaction between the two subunits, expression, purification, crystallization and X-ray diffraction analysis of the TRM6-TRM61 complex were performed in this study. The crystals diffracted to 2.80 Å resolution and belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 139.14, c = 101.62 Å. [ABSTRACT FROM AUTHOR]

Nitrogen fixation is catalyzed by the nitrogenase complex in Azotobacter, which is composed of dinitrogenase and dinitrogenase reductase. Dinitrogenase is an α2β2 heterotetramer of the proteins NifD and NifK. Dinitrogenase reductase is a homodimer of the protein NifH. The expression of NifD/K and NifH nitrogenase homologues (named NflD/K and NflH for Nif-like D and H, respectively) has been detected in the non-nitrogen-fixing hyperthermophilic methanogen Methanocaldococcus jannaschii. Solving the structure of MjNifH1 may help in better understanding its function and may supply some clues to understanding the evolution of nitrogenase. The full-length protein with an additional His6 tag at the C-terminus was expressed, purified and crystallized by the hanging-drop vapour-diffusion method at 287 K. An X-ray diffraction data set was collected to a resolution of 3.3 Å. The crystal belonged to space group P4132, with unit-cell parameters a = b = c = 139.45 Å, and was estimated to contain one protein molecule per asymmetric unit. [ABSTRACT FROM AUTHOR]

Nitrogenases are protein complexes that are only found in Azotobacter and are required for biological nitrogen fixation. They are made up of a nitrogenase, which is a NifD2/NifK2 heterotetramer, and a nitrogenase reductase, which is a homodimer of NifH. Many homologues of nitrogenase have been found in various non-nitrogen-fixing prokaryotes; in particular, they are found in all known methanogens. This indicates that these homologues may play a role in methane production. Here, the cloning of NifH2, a homologue of the NifH nitrogenase component, from Methanocaldococcus jannaschii ( MjNifH2) and its expression in Escherichia coli with a polyhistidine tag, purification and crystallization are described. MjNifH2 crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 2.85 Å. The crystals belonged to space group P2, with unit-cell parameters a = 64.01, b = 94.38, c = 98.08 Å, α = γ = 90, β = 98.85°. [ABSTRACT FROM AUTHOR]

5-Aminolaevulinic acid dehydratase (ALAD), a crucial enzyme in the biosynthesis of tetrapyrrole, catalyses the condensation of two 5-aminolaevulinic acid (ALA) molecules to form porphobilinogen (PBG). The gene encoding ALAD was amplified from genomic DNA of Bacillus subtilis and the protein was overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified and crystallized with an additional MGSSHHHHHHSSGLVPRGSH- tag at the N-terminus of the target protein. Diffraction-quality single crystals were obtained by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at a resolution of 2.7 Å. [ABSTRACT FROM AUTHOR]

Escherichia coli YeaD (ecYeaD) is suggested to be a member of the galactose mutarotase-like superfamily. Galactose mutarotase is an enzyme that converts α-galactose to β-galactose. The known structures of these galactose mutarotase-like proteins are similar to those of galactose mutarotases, with the catalytic residues being conserved, but there are some differences between them in the substrate-binding pocket. In order to reveal the specificity of ecYeaD, a three-dimensional structure is essential. Full-length ecYeaD with an additional 6×His tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant at 283 K. An X-ray diffraction data set was collected to a resolution of 1.9 Å from a single flash-cooled crystal that belonged to space group P212121. [ABSTRACT FROM AUTHOR]

The histone chaperone SET encoded by the SET gene, which is also known as template-activating factor Iβ (TAF-Iβ), is a multifunctional molecule that is involved in many biological phenomena such as histone binding, nucleosome assembly, chromatin remodelling, replication, transcription and apoptosis. A truncated SET/TAF-IβΔN protein that lacked the first 22 residues of the N-terminus but contained the C-terminal acidic domain and an additional His6 tag at the C-terminus was overexpressed in Escherichia coli and crystallized by the hanging-drop vapour-diffusion method using sodium acetate as precipitant at 283 K. The crystals diffracted to 2.7 Å resolution and belonged to space group P43212. [ABSTRACT FROM AUTHOR]

Multiphase flow hydrodynamics in a novel gas–liquid–solid jet‐loop reactor (JLR) were experimentally investigated at the macroscales and mesoscales. The chord length distribution was measured by an optical fiber probe and transformed for bubble size distribution through the maximum entropy method. The impacts of key operating conditions (superficial gas and liquid velocity, solid loading) on hydrodynamics at different axial and radial locations were comprehensively investigated. JLR was found to have good solid suspension ability owing to the internal circulation of bubbles and liquid flow. The gas holdup, axial liquid velocity, and bubble velocity increase with gas velocity, while liquid velocity has little influence on them. Compared with the gas–liquid JLRs, solids decrease the gas holdup and liquid circulation, reduces the bubble velocity and delays the flow development due to the enhanced interaction between bubbles and particles (Stokes number >1). This work also provides a benchmark data for computational fluid dynamics (CFD) model validation. © 2019 American Institute of Chemical Engineers AIChE J, 65: e16537, 2019 [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, TRM6 and TRM61 compose a tRNA methyltransferase which catalyzes the methylation of the N1 of adenine at position 58 in tRNAs, especially initiator methionine tRNA. TRM61 is the subunit that binds S-adenosyl-L-methionine and both subunits contribute to target tRNA binding. In order to elucidate the catalytic mechanism of TRM6-TRM61 and the mode of interaction between the two subunits, expression, purification, crystallization and X-ray diffraction analysis of the TRM6-TRM61 complex were performed in this study. The crystals diffracted to 2.80 Å resolution and belonged to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 139.14, c = 101.62 Å., (© 2011 International Union of Crystallography. All rights reserved.)

The histone chaperone SET encoded by the SET gene, which is also known as template-activating factor Iß (TAF-Iß), is a multifunctional molecule that is involved in many biological phenomena such as histone binding, nucleosome assembly, chromatin remodelling, replication, transcription and apoptosis. A truncated SET/TAF-Iß ΔN protein that lacked the first 22 residues of the N-terminus but contained the C-terminal acidic domain and an additional His6 tag at the C-terminus was overexpressed in Escherichia coli and crystallized by the hanging-drop vapour-diffusion method using sodium acetate as precipitant at 283 K. The crystals diffracted to 2.7 A resolution and belonged to space group P4(3)2(1)2.

ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody-antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1-192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45 A resolution from a single flash-cooled crystal; the crystal belonged to space group P2(1)2(1)2(1).

Transfer RNA (tRNA) (m(7)G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-L-methionine (SAM) as the methyl-group donor to catalyze the formation of N(7)-methylguanosine (m(7)G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His(6) tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2(1).

The fifth HMG-box domain in human upstream binding factor (hUBF) contributes to the synthesis of rRNA by RNA polymerase I (Pol I). The 2.0 A resolution crystal structure of this protein has been solved using the single-wavelength anomalous dispersion method (SAD). The crystal structure and the reported NMR structure have r.m.s. deviations of 2.18-3.03 A for the C(alpha) atoms. However, there are significant differences between the two structures, with displacements of up to 9.0 A. Compared with other HMG-box structures, the r.m.s. deviations for C(alpha) atoms between hUBF HMG box 5 and HMG domains from Drosophila melanogaster protein D and Rattus norvegicus HMG1 are 1.5 and 1.6 A, respectively. This indicates that the differences between the crystal and NMR structures of hUBF HMG box 5 are larger than those with its homologous structures. The differences between the two structures potentially reflect two states with different structures. The specific interactions between the hUBF HMG box 5 and the first bromodomain of TBP-associated factor 1 (TAF1) were studied by ultrasensitive differential scanning calorimetry and chemical shift perturbation. Based on these experimental data, possible sites in hUBF HMG box 5 that may interact with the first bromodomain of TAF1 were proposed.

5-Aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole-biosynthesis pathway. Plants, algae and many other bacteria synthesize ALA from glutamate by a C5 pathway in which the carbon skeleton of glutamate is converted into ALA by a series of enzymes. Glutamate-1-semialdehyde aminotransferase (GSAT) is the last enzyme in this pathway. The gene that codes for GSAT was amplified from the cDNA library of Bacillus subtilis and overexpressed in Escherichia coli strain BL21(DE3). The protein was purified and crystallized. Well diffracting single crystals were obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray diffraction studies yielded excellent diffraction data to a resolution of 2.0 angstroms.