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Find video protocols related to scientific articles indexed in Pubmed.
808 nm driven Nd(3+)-sensitized upconversion nanostructures for photodynamic therapy and simultaneous fluorescence imaging.
Nanoscale
PUBLISHED: 11-20-2014
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The in vivo biological applications of upconversion nanoparticles (UCNPs) prefer excitation at 700-850 nm, instead of 980 nm, due to the absorption of water. Recent approaches in constructing robust Nd(3+) doped UCNPs with 808 nm excitation properties rely on a thick Nd(3+) sensitized shell. However, for the very important and popular Förster resonance energy transfer (FRET)-based applications, such as photodynamic therapy (PDT) or switchable biosensors, this type of structure has restrictions resulting in a poor energy transfer. In this work, we have designed a NaYF4:Yb/Ho@NaYF4:Nd@NaYF4 core-shell-shell nanostructure. We have proven that this optimal structure balances the robustness of the upconversion emission and the FRET efficiency for FRET-based bioapplications. A proof of the concept was demonstrated for photodynamic therapy and simultaneous fluorescence imaging of HeLa cells triggered by 808 nm light, where low heating and a high PDT efficacy were achieved.
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Plasmid-encoding extended-spectrum ?-lactamase CTX-M-55 in a clinical Shigella sonnei strain, China.
Future Microbiol
PUBLISHED: 11-19-2014
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ABSTRACT? Aims: To characterize a clinical Shigella sonnei strain harboring a conjugatable bla CTX-M-55-borne plasmid. Materials & methods: S. sonnei strain #1081 was isolated from a dysentery patient in China. A CTX-M-55-encoding plasmid harbored in this strain was transformed to Escherichia coli, and then its complete nucleotide sequence was determined by next generation sequencing. The MIC values of bacterial strains were tested by using Vitec(®) 2 (Biomerieux, Marcy l'Etoile, France). Results: Strain #1081 conferred the resistance to multiple beta-lactam antibiotics. bla CTX-M-55 was the only known antibiotic resistance gene and located in a 3090-bp ISEcp1-bla CTX-M-55-orf477 transposition unit carried by a conjugatable plasmid p1081-CTXM in #1081. The ISEcp1-mediated transposition provided a sole promoter, which was located adjacently upstream of the inverted repeat right element of ISEcp1, to drive the expression of CTX-M-55. Conclusion: Plasmid p1081-CTXM was a close variant of the IncI2-type plasmid pHN1122-1 that was harbored in a faecal E. coli strain recovered from a dog in China, indicating the potential transfer of CTX-M-55-encoding plasmids from faecal flora E. coli to human pathogen S. sonnei.
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Fabrication of ultraviolet-curable adhesive bottle-like microresonators by wetting and photocuring.
Appl Opt
PUBLISHED: 11-18-2014
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This work presents a remarkably simple method for the fabrication of ultraviolet (UV)-curable adhesive bottle-like microresonators (BLMRs). The main fabrication process involves two steps: (1) creating liquid bottle-like microcavities along the taper waist of an optical fiber taper under interfacial tension and (2) curing the liquids into solids by UV light irradiation. The shape of the BLMRs can be fitted with a truncated harmonic-oscillator profile. Whispering gallery mode resonances of the bottle-like microcavity were excited via a tapered fiber at different positions along its axis. A cleaner spectrum with identifiable and traceable features over a broad wavelength range at the center excitation position and the estimated Q factors close to 105 around 1.55 ?m are observed. The shifts of resonance frequency by the input light power change demonstrate the potential applications of thermo-optic sensing and frequency tuning.
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Passively Q-switched Nd:YAlO3 nanosecond laser using MoS2 as saturable absorber.
Opt Express
PUBLISHED: 11-18-2014
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We report on the first passively Q-switched Nd:YAlO3 laser at ~1079.5 nm using MoS2 as saturable absorber. The MoS2 saturable absorber is fabricated by transferring the liquid-phase-exfoliated MoS2 nanosheets onto a BK7 glass substrate. By inserting the glass MoS2 saturable absorber into a plano-concave Nd:YAlO3 laser cavity, we obtain a stable Q-switched laser operation with a maximum average output power of 0.26 W corresponding to a pulse repetition rate of 232.5 kHz, a pulse width of 227 ns and a pulse energy of about 1.11 ?J. The results experimentally confirm the promising application of the new kind of 2D material, few-layer MoS2, in solid state lasers.
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Widely-tunable, passively Q-switched erbium-doped fiber laser with few-layer MoS2 saturable absorber.
Opt Express
PUBLISHED: 11-18-2014
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We propose and demonstrate a MoS2-based passively Q-switched Er-doped fiber laser with a wide tuning range of 1519.6-1567.7 nm. The few-layer MoS2 nano-platelets are prepared by the liquid-phase exfoliation method, and are then made into polymer-composite film to construct the fiber-compatible MoS2 saturable absorber (SA). It is measured at 1560 nm wavelength, that such MoS2 SA has the modulation depth of ?2% and the saturable optical intensity of ?10 MW/cm2. By further inserting the filmy MoS2-SA into an Er-doped fiber laser, stable Q-switching operation with a 48.1 nm continuous tuning from S- to C-waveband is successfully achieved. The shortest pulse duration and the maximum pulse energy are 3.3 ?s and 160 nJ, respectively. The repetition rate and the pulse duration under different operation conditions have been also characterized. To the best of our knowledge, it is the first demonstration of MoS2 Q-switched, widely-tunable fiber laser.
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Distinct outcomes in patients with different molecular subtypes of inflammatory breast cancer.
Saudi Med J
PUBLISHED: 11-17-2014
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To determine the outcome of patients with luminal A, luminal B, human epidermal growth factor receptor-2 (HER-2) positive, and triple negative molecular subtypes of inflammatory breast cancer (IBC) using a retrospective analysis.
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Efficient laser-diode end-pumped Nd:GGG lasers at 1054 and 1067??nm.
Appl Opt
PUBLISHED: 10-17-2014
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Efficient and compact laser-diode end-pumped Nd:GGG simultaneous multiwavelength continuous-wave lasers at ?1059, ?1060 and ?1062??nm were first demonstrated in a free-running 30 mm plano-concave laser cavity. The maximum output power was up to 3.92 W with a slope efficiency of about 53.6% with respect to the absorbed pump power. By inserting a 0.1 mm optical glass plate acting as a Fabry-Pérot etalon, a single-wavelength laser at ?1067??nm with a maximum output power of 1.95 W and a slope efficiency of 28.5% can be obtained. Multiwavelength lasers, including those at ?1054 or ?1067??nm, were also achievable by suitably tilting the glass etalon. These simultaneous multiwavelength lasers provide a potential source for terahertz wave generation.
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Key Mechanistic Features of Ni-Catalyzed C-H/C-O Biaryl Coupling of Azoles and Naphthalen-2-yl Pivalates.
J. Am. Chem. Soc.
PUBLISHED: 10-10-2014
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The mechanism of the Ni-dcype-catalyzed C-H/C-O coupling of benzoxazole and naphthalen-2-yl pivalate was studied. Special attention was devoted to the base effect in the C-O oxidative addition and C-H activation steps as well as the C-H substrate effect in the C-H activation step. No base effect in the C(aryl)-O oxidative addition to Ni-dcype was found, but the nature of the base and C-H substrate plays a crucial role in the following C-H activation. In the absence of base, the azole C-H activation initiated by the C-O oxidative addition product Ni(dcype)(Naph)(PivO), 1B, proceeds via ?G = 34.7 kcal/mol barrier. Addition of Cs2CO3 base to the reaction mixture forms the Ni(dcype)(Naph)[PivOCs·CsCO3], 3_Cs_clus, cluster complex rather than undergoing PivO(-) ? CsCO3(-) ligand exchange. Coordination of azole to the resulting 3_Cs_clus complex forms intermediate with a weak Cs-heteroatom(azole) bond, the existence of which increases acidity of the activated C-H bond and reduces C-H activation barrier. This conclusion from computation is consistent with experiments showing that the addition of Cs2CO3 to the reaction mixture of 1B and benzoxazole increases yield of C-H/C-O coupling from 32% to 67% and makes the reaction faster by 3-fold. This emerging mechanistic knowledge was validated by further exploring base and C-H substrate effects via replacing Cs2CO3 with K2CO3 and benzoxazole (1a) with 1H-benzo[d]imidazole (1b) or quinazoline (1c). We proposed the modified catalytic cycle for the Ni(cod)(dcype)-catalyzed C-H/C-O coupling of benzoxazole and naphthalen-2-yl pivalate.
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Mechanistic Investigation of Dirhodium-Catalyzed Intramolecular Allylic C-H Amination versus Alkene Aziridination.
J. Org. Chem.
PUBLISHED: 10-07-2014
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The reaction mechanisms and chemoselectivity on the intramolecular allylic C-H amination versus alkene aziridination of 4-pentenylsulfamate promoted by four elaborately selected dirhodium paddlewheel complexes are investigated by a DFT approach. A predominant singlet concerted, highly asynchronous pathway and an alternative triplet stepwise pathway are obtained in either C-H amination or alkene aziridination reactions when mediated by weak electron-donating catalysts. A singlet stepwise C-H amination pathway is obtained under strongly donating catalysts. The rate-determining step in the C-H amination is the H-abstraction process. The subsequent diradical-rebound C-N formation in the triplet pathway or the combination of the allylic carbocation and the negative changed N center in the singlet pathway require an identical energy barrier. A mixed singlet-triplet pathway is preferred in either the C-H insertion or alkene aziridination in the Rh2(NCH3CHO)4 entry that the triplet pathway is initially favorable in the rate-determining steps, and the resultant triplet intermediates would convert to a singlet reaction coordinate. The nature of C-H amination or alkene aziridination is estimated to be a stepwise process. The theoretical observations presented in the paper are consistent with the experimental results and, more importantly, provide a thorough understanding of the nature of the reaction mechanisms and the minimum-energy crossing points.
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Molecular characterization of a CpTRIM35-like protein and its splice variants from whitespotted bamboo shark (Chiloscyllium plagiosum).
Biochem. Biophys. Res. Commun.
PUBLISHED: 09-18-2014
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The tripartite motif (TRIM) proteins play important roles in a broad range of biological processes, including apoptosis, cell proliferation and innate immunity response. In this study, a TRIM gene and its three splice variants were cloned from an elasmobranch fish-whitespotted bamboo shark (Chiloscyllium plagiosum Bennett). Phylogenetic analysis indicated that the gene was closely related to TRIM35 homologs, thus termed CpTRIM35-like. Deduced CpTRIM35 has a RBCC-PRY/SPRY structure typical of TRIM proteins, and its splice variants (CpTRIM35-1-3) have different truncations at the C-terminus. The gene products were constitutively expressed in adult sharks with the highest levels in spleen and kidney. The different subcellular locations, upregulation upon LPS and poly I:C stimulation, and significant E3 ubiquitin ligase activities suggested their different roles in immune responses as an E3 ubiquitin ligase. This is the first TRIM protein ever characterized in elasmobranch fish.
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Yersinia Protein Kinase A Phosphorylates Vasodilator-Stimulated Phosphoprotein to Modify the Host Cytoskeleton.
Cell. Microbiol.
PUBLISHED: 09-13-2014
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Pathogenic Yersinia species evolved a type III secretion system that injects a set of effectors into the host cell cytosol to promote infection. One of these effectors, Yersinia protein kinase A (YpkA), is a multidomain effector that harbors a Ser/Thr kinase domain and a guanine dissociation inhibitor (GDI) domain. The intercellular targets of the kinase and GDI domains of YpkA were identified to be G?q and the small GTPases RhoA and Rac1, respectively, which synergistically induce cytotoxic effects on infected cells. In this study, we demonstrate that vasodilator-stimulated phosphoprotein (VASP), which is critical for regulation of actin assembly, cell adhesion, and motility is a direct substrate of YpkA kinase activity. Ectopic coexpression of YpkA and VASP in HEK293T cells lead to the phosphorylation of VASP at S157, and YpkA kinase activity is essential for VASP phosphorylation at this site. Moreover, YpkA directly phosphorylates VASP in in vitro kinase assay. YpkA-mediated VASP phosphorylation significantly inhibits actin polymerization and promotes the disruption of actin cytoskeleton, which inhibits the phagocytosis. Taken together, our study found a novel molecular mechanism used by YpkA to disrupt cytoskeleton dynamics, thereby promoting the anti-phagocytosis ability of pathogenic Yersiniae.
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Lipid rafts participate in aberrant degradative autophagic-lysosomal pathway of amyloid-beta peptide in Alzheimer's disease.
Neural Regen Res
PUBLISHED: 09-11-2014
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Amyloid-beta peptide is the main component of amyloid plaques, which are found in Alzheimer's disease. The generation and deposition of amyloid-beta is one of the crucial factors for the onset and progression of Alzheimer's disease. Lipid rafts are glycolipid-rich liquid domains of the plasma membrane, where certain types of protein tend to aggregate and intercalate. Lipid rafts are involved in the generation of amyloid-beta oligomers and the formation of amyloid-beta peptides. In this paper, we review the mechanism by which lipid rafts disturb the aberrant degradative autophagic-lysosomal pathway of amyloid-beta, which plays an important role in the pathological process of Alzheimer's disease. Moreover, we describe this mechanism from the view of the Two-system Theory of fasciology and thus, suggest that lipid rafts may be a new target of Alzheimer's disease treatment.
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Structural perspectives of an engineered ?-1,4-xylanase with enhanced thermostability.
J. Biotechnol.
PUBLISHED: 09-03-2014
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The glycoside hydrolase 10 (GH10) xylanase from Streptomyces sp. 9 (XynAS9) can operate in a broad range of pH and temperature, and thus is a potential candidate for commercial applications. Recently, we engineered XynAS9 via mutating several residues in accordance with the consensus sequences of GH10 thermophilic xylanases in an attempt to improve the enzyme thermostability and thermotolerance. The most promising effects were observed in the double mutant V81P/G82E. In order to investigate the molecular mechanism of the improved thermal profile of XynAS9, complex crystal structures of the wild type (WT) and mutant (MT) enzyme were solved at 1.88-2.05? resolution. The structures reveal a classical GH10 (?/?)8 TIM-barrel fold. In MT XynAS9, E82 forms several interactions to its neighboring residues, which might aid in stabilizing the local structure. Furthermore, the MT structure showed lower B factors for individual residues compared to the WT structure, reflecting the increased MT protein rigidity. Analyses of the XynAS9 structures also delineate the detailed enzyme-substrate interaction network. More importantly, possible explanations for the enhanced thermal profiles of MT XynAS9 are proposed, which may be a useful strategy for enzyme engineering in the future.
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Angiogenesis and airway reactivity in asthmatic Brown Norway rats.
Angiogenesis
PUBLISHED: 08-23-2014
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Expanded and aberrant bronchial vascularity, a prominent feature of the chronic asthmatic airway, might explain persistent airway wall edema and sustained leukocyte recruitment. Since it is well established that there are causal relationships between exposure to house dust mite (HDM) and the development of asthma, determining the effects of HDM in rats, mammals with a bronchial vasculature similar to humans, provides an opportunity to study the effects of bronchial angiogenesis on airway function directly. We studied rats exposed bi-weekly to HDM (Der p 1; 50 ?g/challenge by intranasal aspiration, 1, 2, 3 weeks) and measured the time course of appearance of increased blood vessels within the airway wall. Results demonstrated that within 3 weeks of HDM exposure, the number of vessels counted within airway walls of bronchial airways (0.5-3 mm perimeter) increased significantly. These vascular changes were accompanied by increased airway responsiveness to methacholine. A shorter exposure regimen (2 weeks of bi-weekly exposure) was insufficient to cause a significant increase in functional vessels or reactivity. Yet, (19)F/(1)H MR imaging at 3T following ?v?3-targeted perfluorocarbon nanoparticle infusion revealed a significant increase in (19)F signal in rat airways after 2 weeks of bi-weekly HDM, suggesting earlier activation of the process of neovascularization. Although many antigen-induced mouse models exist, mice lack a bronchial vasculature and consequently lack the requisite human parallels to study bronchial edema. Overall, our results provide an important new model to study the impact of bronchial angiogenesis on chronic inflammation and airways hyperreactivity.
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Identification of a Novel HOG1 Homologue from an Industrial Glycerol Producer Candida glycerinogenes.
Curr. Microbiol.
PUBLISHED: 08-14-2014
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Candida glycerinogenes, a glycerol production industrial strain with hyperosmo-adaptation can grow well in 15 % (w/v) NaCl or 55 % (w/v) glucose. To understand the osmo-adaptation mechanism in C. glycerinogenes, the mitogen-activated protein kinase HOG1 gene (CgHOG1), which plays an essential role in the yeast hyperosmotic response, was isolated by degenerate PCR and SEFA-Formed Adaptor PCR. The CgHOG1 gene was then transformed in Saccharomyces cerevisiae hog1? null mutant, which restored the recombination S. cerevisiae to the wild-type phenotype with osmo-adaptation. To further clarify the function of CgHOG1, the phosphorylation of CgHOG1 and transcription of the glycerol-3-phosphate dehydrogenase gene (GPD1) of the CgHOG1-harbouring S. cerevisiae mutant was detected, and found to be similar to that of wild-type S. cerevisiae. In addition, the recombination S. cerevisiae with CgHOG1 gene significantly accumulated intracellular glycerol when stressed with NaCl.
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Biochemical characterization of a thermophilic ?-mannanase from Talaromyces leycettanus JCM12802 with high specific activity.
Appl. Microbiol. Biotechnol.
PUBLISHED: 08-08-2014
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Thermophilic ?-mannanases are of increasing importance for wide industrial applications. In the current study, gene cloning, functional expression in Pichia pastoris, and characterization of a thermophilic ?-mannanase (Man5A) from thermophilic Talaromyces leycettanus JCM12802 are reported. Deduced Man5A exhibits the highest identity with a putative ?-mannanase from Talaromyces stipitatus ATCC10500 (70.3 %) and is composed of an N-terminal signal peptide, a fungal-type carbohydrate-binding module (CBM) of family 1, and a catalytic domain of glycosyl hydrolase (GH) family 5 at the C-terminus. Two recombinant proteins with different glycosylation levels, termed Man5A1 (72 kDa) and Man5A2 (60 kDa), were identified after purification. Both enzymes were thermophilic, exhibiting optimal activity at 85-90 °C, and were highly stable at 70 °C. Man5A1 and Man5A2 had a pH optimum of 4.5 and 4.0, respectively, and were highly stable over the broad pH range of 3.0-10.0. Most metal ions and sodium dodecyl sulfate (SDS) had no effect on the enzymatic activities. Man5A1 and Man5A2 exhibited high specific activity (2,160 and 1,800 U/mg, respectively) when using locust bean gum as the substrate. The CBM1 and two key residues D191 and R286 were found to affect Man5A thermostability. Man5A displays a classical four-site-binding mode, hydrolyzing mannooligosaccharides into smaller units, galactomannan into mannose and mannobiose, and glucomanman into mannose, mannobiose, and mannopentaose, respectively. All these properties make Man5A a good candidate for extensive applications in the bioconversion, pulp bleaching, textile, food, and feed industries.
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Large-energy, wavelength-tunable, all-fiber passively Q-switched Er:Yb-codoped double-clad fiber laser with mono-layer chemical vapor deposition graphene.
Appl Opt
PUBLISHED: 08-05-2014
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We demonstrate a large-energy, wavelength-tunable, all-fiber passively Q-switched Er:Yb-codoped laser using a mono-layer chemical vapor deposition (CVD) graphene saturable absorber (SA). By exploiting the large laser gain of Er:Yb double-clad fiber and optimizing the coupling ratio of the output coupler, not only can the mono-layer CVD graphene SA be protected from oversaturation and thermal damage, but also a large pulse energy up to 1.05 ?J (corresponding to the average output power of 25.6 mW) is thus achieved. Using a tunable fiber Fabry-Perot filter, stable Q-switched pulses can operate with a tunable range from 1530.97 to 1546.92 nm, covering a wavelength range of ?16??nm. The Q-switching states at the different lasing wavelengths have been observed and recorded. The Q-switched repetition rate and the pulse duration (with the minimum one of 2.6 ?s) have been characterized as well. This is, to the best of our knowledge, the largest pulse energy from an all-fiber graphene Q-switched laser.
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Integrative expression vectors for overexpression of xylitol dehydrogenase (XYL2) in Osmotolerant yeast, Candida glycerinogenes WL2002-5.
J. Ind. Microbiol. Biotechnol.
PUBLISHED: 07-18-2014
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Yeasts are excellent hosts for the production of recombinant proteins. Candida glycerinogenes WL2002-5, an osmotolerant yeast with extremely high glycerol productivity, provides an attractive eukaryotic expression platform. The integrative vectors PURGAP-gfp and PURGPD-gfp harbouring phleomycin-resistance coding sequence and GFP coding sequence with PCgGAP, PCgGPD promoter, respectively, were constructed. The recombinant plasmid PURPpGAP-gfp with the promoter PPpGAP based on the sequence of Pichia pastoris GAPDH gene and the plasmid PURScGAP-gfp with the promoter PScGAP from Saccharomyces cerevisiae were constructed. After transformation, the copy number of gfp gene, which determined using fluorescent quantitative real-time polymerase chain reaction (FQ-RTPCR) in genome of C. glycerinogenes is 1. Expressions of gfp at different levels were conducted using different promoters by osmotic stress containing NaCl or glucose for the recombinant strains. In this study, C. glycerinogenes WL2002-5, expressing xylitol dehydrogenase (XYL2) gene from Pichia stipitis, has the ability to produce glycerol from xylose entered into pentose phosphate pathway. Two recombinant strains of PURGAPX, PURGPDX with XYL2 overexpression were constructed to ferment a mixture of glucose and xylose simultaneously in batch fermentation. Compared to C. glycerinogenes WL2002-5 strain, glycerol production from xylose in strains PURGAPX, PURGPDX were increased by 95.9 and 121.1 %, respectively.
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[Monitoring and control of pan-drug resistant Acinetobacter baumannii colonization and infection in a medical intensive care unit].
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue
PUBLISHED: 07-17-2014
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To describe the monitoring and control of pan-drug resistant Acinetobacter baumannii (XDRABA) colonization and infection in a medical intensive care unit (ICU), and to summarize the effective measures of surveillance of nosocomial infection and control.
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[A method for primary culture of pulmonary microvascular endothelial cells].
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi
PUBLISHED: 07-09-2014
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To explore a simple and practical method of primarily culturing rat pulmonary microvascular endothelial cells (PMECs) in vitro, and observe the cell growth status and identify the PMECs.
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Whole-cell biotransformation systems for reduction of prochiral carbonyl compounds to chiral alcohol in Escherichia coli.
Sci Rep
PUBLISHED: 07-07-2014
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Lactobacillus brevis alcohol dehydrogenase (Lb-ADH) catalyzes reduction of prochiral carbonyl compounds to chiral alcohol and meanwhile consumes its cofactor NADH into NAD(+), while the cofactor regeneration can be catalyzed by Candida boidinii formate dehydrogenase (Cb-FDH). This work presents three different Escherichia coli whole-cell biocatalyst systems expressing recombinant ADH/FDH, FDH-LIN1-ADH and FDH-LIN2-ADH, respectively, all of which display very high efficacies of prochiral carbonyl conversion with respect to conversion rates and enantiomeric excess values. ADH/FDH represents co-expression of Lb-ADH and Cb-FDH under different promoters in a single vector. Fusion of Lb-ADH and Cb-FDH by a linker peptide LIN1 (GGGGS)2 or LIN2 (EAAAK)2 generates the two bifunctional enzymes FDH-LIN1-ADH and FDH-LIN2-ADH, which enable efficient asymmetric reduction of prochiral ketones in whole-cell biotransformation.
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Isolation of a Novel Cold-Active Family 11 Xylanase from the Filamentous Fungus Bispora antennata and Deletion of its N-Terminal Amino Acids on Thermostability.
Appl. Biochem. Biotechnol.
PUBLISHED: 06-29-2014
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In the present study, we first reported a cold-active xylanase of glycosyl hydrolase family 11, Xyn11, from the filamentous fungus Bispora antennata. The coding gene (xyn11) was cloned and successfully expressed in Pichia pastoris. Deduced Xyn11 exhibited the highest identity of 65 % with a family 11 endo-?-1,4-xylanase from Alternaria sp. HB186. Recombinant Xyn11 exhibited maximal activity at 35 °C and remained 21 % of the activity at 0 °C. Sequence alignment showed that the N-terminal sequence of Xyn11 is distinct from those of thermophilic xylanases of family 11. To determine its effect on enzyme properties, the Xyn11 mutant without the N-terminal sequence, t-Xyn11, was then constructed, expressed in P. pastoris, and compared with Xyn11. Both enzymes showed optimal activities at 35 °C and pH 5.5 and were stable at pH 2.0-12.0. Compared with truncated mutant t-Xyn11, Xyn11 retained more activity after 20-min incubation at 40 °C (Xyn11:28 %  vs. t-Xyn11:4 %) and degraded xylan substrates more completely. Thus, a new factor affecting the thermostability of cold-active xylanase of family 11 was identified.
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Role of Mitochondrial Calcium Uniporter in Early Brain Injury After Experimental Subarachnoid Hemorrhage.
Mol. Neurobiol.
PUBLISHED: 06-25-2014
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Previous studies have shown that mitochondrial Ca(2+) is undertaken by mitochondrial calcium uniporter (MCU), and its accumulation is associated with the development of many diseases. However, little was known about the role of MCU in early brain injury (EBI) after subarachnoid hemorrhage (SAH). MCU can be opened by spermine under a physiological condition and inhibited by ruthenium red (RR). Herein, we investigated the effects of RR and spermine to reveal the role of MCU in SAH animal model. The data obtained with biochemical and histological assays showed that mitochondrial Ca(2+) concentration was significantly increased in the temporal cortex of rats 1, 2, and 3 days after SAH, consistent with constant high levels of cellular Ca(2+) concentration. In agreement with the observation in the acute phase, SAH rats showed an obvious increase of reactive oxygen species (ROS) level and decrease of ATP production. Blockage of MCU prevented Ca(2+) accumulation, abated the level of oxidative stress, and improved the energy supply. Translocation of cytochrome c, increased cleaved caspase-3, and a large amount of apoptotic cells after SAH were reversed by RR administration. Surprisingly, exogenous spermine did not increase cellular Ca(2+) concentration, but lessened the Ca(2+) accumulation after SAH to benefit the rats. Taken together, our results demonstrated that blockage of MCU or prevention of Ca(2+) accumulation after SAH is essential in EBI after SAH. These findings suggest that MCU is considered to be a therapeutic target for patients suffering from SAH.
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Diagnostic and therapeutic advancements for aerobic vaginitis.
Arch. Gynecol. Obstet.
PUBLISHED: 05-01-2014
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Aerobic vaginitis (AV) is a newly defined clinical entity that is distinct from candidiasis, trichomoniasis and bacterial vaginosis (BV). Because of the poor recognition of AV, this condition can lead to treatment failures and is associated with severe complications, such as pelvic inflammatory disease, infertility, preterm birth and foetal infections.
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Transcriptomic response to Yersinia pestis: RIG-I like receptor signaling response is detrimental to the host against plague.
J Genet Genomics
PUBLISHED: 04-24-2014
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Bacterial pathogens have evolved various mechanisms to modulate host immune responses for successful infection. In this study, RNA-sequencing technology was used to analyze the responses of human monocytes THP1 to Yersinia pestis infection. Over 6000 genes were differentially expressed over the 12 h infection. Kinetic responses of pathogen recognition receptor signaling pathways, apoptosis, antigen processing, and presentation pathway and coagulation system were analyzed in detail. Among them, RIG-I-like receptor (RLR) signaling pathway, which was established for antiviral defense, was significantly affected. Mice lacking MAVS, the adaptor of the RLR signaling pathway, were less sensitive to infection and exhibited lower IFN-? production, higher Th1-type cytokines IFN-? and IL-12 production, and lower Th2-type cytokines IL-4 and IL-13 production in the serum compared with wild-type mice. Moreover, infection of pathogenic bacteria other than Y. pestis also altered the expression of the RLR pathway, suggesting that the response of RLR pathway to bacterial infection is a universal mechanism.
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Specific dephosphorylation of janus kinase 2 by protein tyrosine phosphatases.
Proteomics
PUBLISHED: 04-17-2014
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Many protein kinases are activated through phosphorylation of an activation loop thereby turning on downstream signaling pathways. Activation of JAK2, a non-receptor tyrosine kinase with an important role in growth factor and cytokine signaling, requires phosphorylation of the 1007 and 1008 tyrosyl residues. Dephosphorylation of these two sites by phosphatases presumably inactivates the enzyme, but the underlying mechanism is not known. In this study, we employed MALDI-TOF/TOF and triple quadrupole mass spectrometers to analyze qualitatively and quantitatively the dephosphorylation process by using synthetic peptides derived from the tandem autophosphorylation sites (Y1007 and Y1008) of human JAK2. We found that tyrosine phosphatases catalyzed the dephosphorylation reaction sequentially, but different enzymes exhibited different selectivity. PTP1B caused rapid dephosphorylation of Y1008 followed by Y1007, while SHP-1 and SHP-2 selectively dephosphorylated Y1008 only, and yet HePTP randomly removed a single phosphate from either Y1007 or Y1008, leaving behind mono-phosphorylated peptides. The specificity of dephosphorylation was further confirmed by molecular modeling. The data reveal multiple modes of JAK2 regulation by tyrosine phosphatases, reflecting a complex and intricate interplay between protein phosphorylation and dephosphorylation. This article is protected by copyright. All rights reserved.
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Glucose-regulated protein 78 may play a crucial role in promoting the pulmonary microvascular remodeling in a rat model of hepatopulmonary syndrome.
Gene
PUBLISHED: 04-16-2014
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This study is to investigate the role of glucose-regulated protein 78 (GRP78) in the pulmonary microvascular remodeling during hepatopulmonary syndrome (HPS) development.
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Self-mode-locked 2 ?m Tm(3+)-doped double-clad fiber laser with a simple linear cavity.
Appl Opt
PUBLISHED: 03-26-2014
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We demonstrate the self-mode-locking operation of a thulium (Tm)-doped fiber laser (TDFL) with a simple linear cavity. Since the laser cavity does not include any specific mode-locker, we experimentally investigate and analyze the self-mode-locking mechanism. The mode-locking operation is attributed to the combination of the self-phase modulation effect and the weak saturable absorption of the high-concentration Tm-doped fiber. The mode-locked TDFL operates at a central wavelength of 1985.5 nm with the 3 dB spectral linewidth of 0.18 nm. The self-mode-locking generates a large pulse energy of 32.7 nJ with a pulsed repetition rate of 2.05 MHz and is stable with a radio-frequency signal-to-noise ratio of more than 54 dB. To the best of our knowledge, it is the first demonstration of a 2 ?m Tm-doped fiber laser mode-locked by such technique.
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A C-terminal proline-rich sequence simultaneously broadens the optimal temperature and pH ranges and improves the catalytic efficiency of glycosyl hydrolase family 10 ruminal xylanases.
Appl. Environ. Microbiol.
PUBLISHED: 03-21-2014
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Efficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed in Escherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more ? helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.
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A new ?-galactosidase from thermoacidophilic Alicyclobacillus sp. A4 with wide acceptor specificity for transglycosylation.
Appl. Biochem. Biotechnol.
PUBLISHED: 03-14-2014
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An ?-galactosidase gene (gal36A4) of glycosyl hydrolase family 36 was identified in the genome of Alicyclobacillus sp. A4. It contains an ORF of 2,187 bp and encodes a polypeptide of 728 amino acids with a calculated molecular mass of 82.6 kDa. Deduced Gal36A4 shows the typical GH36 organization of three domains--the N-terminal ?-sheets, the catalytic (?/?)8-barrels, and the C-terminal antiparallel ?-sheet. The gene product was produced in Escherichia coli and showed both hydrolysis and transglycosylation activities. The optimal pH for hydrolysis activity was 6.0, and a stable pH range of 5.0-11.0 was found. The enzyme had a temperature optimum of 60 °C. It is specific for ?-1,6-glycosidic linkages and had a K m value of 1.45 mM toward pNPGal. When using melibiose as both donor and acceptor of galactose, Gal36A4 showed the transfer ratio of 23.25 % at 96 h. With respect to acceptor specificity, all tested monosaccharides, disaccharides, and oligosaccharides except for D-xylose and L-arabinose were good acceptors for transglycosylation. Thus, Gal36A4 may find diverse applications in industrial fields, especially in the food industry.
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SPOT-Seq-RNA: predicting protein-RNA complex structure and RNA-binding function by fold recognition and binding affinity prediction.
Methods Mol. Biol.
PUBLISHED: 02-28-2014
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RNA-binding proteins (RBPs) play key roles in RNA metabolism and post-transcriptional regulation. Computational methods have been developed separately for prediction of RBPs and RNA-binding residues by machine-learning techniques and prediction of protein-RNA complex structures by rigid or semiflexible structure-to-structure docking. Here, we describe a template-based technique called SPOT-Seq-RNA that integrates prediction of RBPs, RNA-binding residues, and protein-RNA complex structures into a single package. This integration is achieved by combining template-based structure-prediction software, SPARKS X, with binding affinity prediction software, DRNA. This tool yields reasonable sensitivity (46 %) and high precision (84 %) for an independent test set of 215 RBPs and 5,766 non-RBPs. SPOT-Seq-RNA is computationally efficient for genome-scale prediction of RBPs and protein-RNA complex structures. Its application to human genome study has revealed a similar sensitivity and ability to uncover hundreds of novel RBPs beyond simple homology. The online server and downloadable version of SPOT-Seq-RNA are available at http://sparks-lab.org/server/SPOT-Seq-RNA/.
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Carbohydrate-binding protein identification by coupling structural similarity searching with binding affinity prediction.
J Comput Chem
PUBLISHED: 02-27-2014
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Carbohydrate-binding proteins (CBPs) are potential biomarkers and drug targets. However, the interactions between carbohydrates and proteins are challenging to study experimentally and computationally because of their low binding affinity, high flexibility, and the lack of a linear sequence in carbohydrates as exists in RNA, DNA, and proteins. Here, we describe a structure-based function-prediction technique called SPOT-Struc that identifies carbohydrate-recognizing proteins and their binding amino acid residues by structural alignment program SPalign and binding affinity scoring according to a knowledge-based statistical potential based on the distance-scaled finite-ideal gas reference state (DFIRE). The leave-one-out cross-validation of the method on 113 carbohydrate-binding domains and 3442 noncarbohydrate binding proteins yields a Matthews correlation coefficient of 0.56 for SPalign alone and 0.63 for SPOT-Struc (SPalign?+?binding affinity scoring) for CBP prediction. SPOT-Struc is a technique with high positive predictive value (79% correct predictions in all positive CBP predictions) with a reasonable sensitivity (52% positive predictions in all CBPs). The sensitivity of the method was changed slightly when applied to 31 APO (unbound) structures found in the protein databank (14/31 for APO versus 15/31 for HOLO). The result of SPOT-Struc will not change significantly if highly homologous templates were used. SPOT-Struc predicted 19 out of 2076 structural genome targets as CBPs. In particular, one uncharacterized protein in Bacillus subtilis (1oq1A) was matched to galectin-9 from Mus musculus. Thus, SPOT-Struc is useful for uncovering novel carbohydrate-binding proteins. SPOT-Struc is available at http://sparks-lab.org. © 2014 Wiley Periodicals, Inc.
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TLR4 inhibitor resatorvid provides neuroprotection in experimental traumatic brain injury: implication in the treatment of human brain injury.
Neurochem. Int.
PUBLISHED: 02-16-2014
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Toll-like receptor 4 (TLR4) is considered to play an important role in neuronal death in animal models and could be an important therapeutic target following traumatic brain injury (TBI). Resatorvid is a small molecule, commonly accepted to inhibit TLR4-mediated pathway. The purpose of this study was to investigate the neuroprotective effect of resatorvid after TBI. Our data revealed that inhibition of TLR4 by resatorvid attenuated the development of TBI in mouse model. And we found that resatorvid administration dramatically reduced neuronal apoptosis. To investigate the cellular mechanism, we evaluated the expression of transforming growth factor-?-activated kinase 1 (TAK1), which plays a crucial role in TLR4 signal transduction pathway and is activated by phosphorylation in response to TBI. In addition, enzyme-linked immunosorbent assay was used to determine the expression of tumor necrosis factor-? (TNF-?) and interlukin-1? (IL-1?) at 24h after injury. Our results showed that resatorvid treatment significantly reduced the protein levels of TAK1, p-TAK1, TNF-?, and IL-1? compared with vehicle treatment. Importantly, the delayed therapy (4h post injury) once daily consecutively for 5days ameliorated brain damage and improved neurological recovery, suggesting that this drug has a wide therapeutic time window. Clinically, we observed that TLR4 and TAK1 expression was significantly increased in human contusion specimens after TBI. These data provide an experimental rationale for the evaluation of TLR4 as a clinical target and therapeutic implication of resatorvid in human traumatic brain injury.
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The hydrophobic contacts between the center of the ?I domain and the ?1/?7 helices are crucial for the low-affinity state of integrin ?4 ?7.
FEBS J.
PUBLISHED: 02-11-2014
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Integrin ?4 ?7 mediates both rolling and firm adhesion of lymphocytes by modulating its affinity to the ligand: mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Integrin activation is associated with allosteric reshaping in the ? subunit I (?I) domain. A prominently conformational change comprises displacement of the ?1 and ?7 helices in the ?I domain, suggesting that the location of these helices is important for the change in integrin affinity. In the present study, we report that the hydrophobic contacts between the center of the ?7 I domain and the ?1/?7 helices play critical roles in keeping ?4 ?7 in a low-affinity state. Using molecular dynamics simulation, we identified nine hydrophobic residues that might be involved in the critical hydrophobic contacts maintaining integrin in a low-affinity state. Integrin ?7 I domain exhibited a lower binding free energy for ligand after disrupting these hydrophobic contacts by substituting the hydrophobic residues with Ala. Moreover, these ?4 ?7 mutants not only showed high-affinity binding to soluble MAdCAM-1, but also demonstrated firm cell adhesion to immobilized MAdCAM-1 in shear flow and enhanced the strength of the ?4 ?7 -MAdCAM-1 interaction. Disruption of the hydrophobic contacts also induced the active conformation of ?4 ?7 . Thus, the findings obtained in the present study reveal an important structural basis for the low-affinity state of integrin.
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Pure nongestational uterine choriocarcinoma in a postmenopausal Chinese woman confirmed with short tandem repeat analysis.
Am. J. Obstet. Gynecol.
PUBLISHED: 02-10-2014
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Nongestational choriocarcinomas have been observed in the ovaries but rarely the uterus in postmenopausal women. Choriocarcinomas of gestational and nongestational origin have distinct prognoses but cannot be distinguished with routine histologic examination. We report a case of nongestational uterine choriocarcinoma in a 62-year-old Chinese woman that was confirmed with short tandem repeat analysis.
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Mechanistic investigation into the cleavage of a phosphomonoester mediated by a symmetrical oxyimine-based macrocyclic zinc(II) complex.
Chemphyschem
PUBLISHED: 01-24-2014
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Density functional calculations are utilized to explore the hydrolysis mechanisms of the phosphomonoester 4-nitrophenyl phosphate catalyzed by a symmetrical zinc(II) complex. The formation process and properties of the active catalyst are verified. Eight plausible mechanisms are proposed and categorized into three groups. All of the proposed mechanisms, except for Mechanism 7 (see text), are S(N)2-type addition-substitution reaction pathways. Nucleophilic attack at the ortho position occurs in Mechanism 7 with a relatively high reaction barrier. Mechanisms 1 and 2 in the monocatalyst model, Mechanisms 5 to 7 in the sandwich-dual-catalyst model, as well as the nucleophilic addition-substitution step in Mechanism 8 are concerted reaction pathways, whereas the rest appear to occur in a stepwise manner. Meanwhile, the explicit solvent model is utilized to consider direct hydrogen bonds and solvation interactions and these results indicate that the added water molecule is involved in the hydrolysis process, but does not change the mechanisms significantly. Mechanism 8, with the lowest reaction barrier, is the most favored reaction pathway of the eight proposed mechanisms, although Mechanisms 1, 4, and 6 are in competition with Mechanism 8. In consideration of the zinc(II) complex concentration, Mechanism 1 is only the predominant reaction pathway at a low zinc(II) complex concentration; Mechanisms 4 and 6 tend to be more competitive with increasing concentration of the zinc(II) complexes, and Mechanism 8 is favored at high zinc(II) complex concentrations. Our calculated results are consistent with, and can be used to systematically interpret, experimental observations. More importantly, insightful suggestions are made regarding the catalyst design and selection of the reaction environment.
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Thermostability improvement of a streptomyces xylanase by introducing proline and glutamic acid residues.
Appl. Environ. Microbiol.
PUBLISHED: 01-24-2014
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Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner ?-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.
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A thermophilic endo-1,4-?-glucanase from Talaromyces emersonii CBS394.64 with broad substrate specificity and great application potentials.
Appl. Microbiol. Biotechnol.
PUBLISHED: 01-20-2014
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Thermophilic cellulases are of significant interest to the efficient conversion of plant cell wall polysaccharides into simple sugars. In this study, a thermophilic and thermostable endo-1,4-?-glucanase, TeEgl5A, was identified in the thermophilic fungus Talaromyces emersonii CBS394.64 and functionally expressed in Pichia pastoris. Purified recombinant TeEgl5A exhibits optimal activity at pH 4.5 and 90 °C. It is highly stable at 70 °C and over a broad pH range of 1.0-10.0, and shows strong resistance to most metal ions, sodium dodecyl sulfate (SDS), and proteases. TeEgl5A has broad substrate specificity and exhibits high activity on substrates containing ?-1,4-glycosidic bonds and ?-1,3-glycosidic bonds (barley ?-glucan, laminarin, lichenan, CMC-Na, carob bean gum, and birchwood xylan). Under simulated mashing conditions, addition of 60 U TeEgl5A reduced more viscosity (10.0 vs.7.6 %) than 80 U of Ultraflo XL from Novozymes. These properties make TeEgl5A a good candidate for extensive application in the detergent, textile, feed, and food industries.
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Impact of human pathogenic micro-insertions and micro-deletions on post-transcriptional regulation.
Hum. Mol. Genet.
PUBLISHED: 01-16-2014
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Small insertions/deletions (INDELs) of ?21 bp comprise 18% of all recorded mutations causing human inherited disease and are evident in 24% of documented Mendelian diseases. INDELs affect gene function in multiple ways: for example, by introducing premature stop codons that either lead to the production of truncated proteins or affect transcriptional efficiency. However, the means by which they impact post-transcriptional regulation, including alternative splicing, have not been fully evaluated. In this study, we collate disease-causing INDELs from the Human Gene Mutation Database (HGMD) and neutral INDELs from the 1000 Genomes Project. The potential of these two types of INDELs to affect binding-site affinity of RNA-binding proteins (RBPs) was then evaluated. We identified several sequence features that can distinguish disease-causing INDELs from neutral INDELs. Moreover, we built a machine-learning predictor called PinPor (predicting pathogenic small insertions and deletions affecting post-transcriptional regulation, http://watson.compbio.iupui.edu/pinpor/) to ascertain which newly observed INDELs are likely to be pathogenic. Our results show that disease-causing INDELs are more likely to ablate RBP-binding sites and tend to affect more RBP-binding sites than neutral INDELs. Additionally, disease-causing INDELs give rise to greater deviations in binding affinity than neutral INDELs. We also demonstrated that disease-causing INDELs may be distinguished from neutral INDELs by several sequence features, such as their proximity to splice sites and their potential effects on RNA secondary structure. This predictor showed satisfactory performance in identifying numerous pathogenic INDELs, with a Matthews correlation coefficient (MCC) value of 0.51 and an accuracy of 0.75.
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Preliminary X-ray diffraction analysis of thermostable ?-1,4-xylanase from Streptomyces sp. S9.
Acta Crystallogr F Struct Biol Commun
PUBLISHED: 01-15-2014
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Xylanase, which catalyzes the random hydrolysis of internal xylosidic linkages, is a critical enzyme participating in xylan decomposition and has been widely applied in industrial utilizations. Xylanase isolated from the extremophilic Streptomyces sp. S9 (XynAS9) possesses broad adaptability to temperature and pH and thus is an attractive candidate in industrial applications. In particular, the major products of XynAS9 are xylose and xylobiose, which enable the subsequent bioconversion to be carried out with higher efficiency. Therefore, the three-dimensional structure of XynAS9 and its catalytic machinery are of great interest. Here, recombinant XynAS9 protein was expressed in Pichia pastoris, purified and crystallized. Crystals belonging to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 80.9, c = 289.3?Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.08?Å resolution. Initial phase determination using molecular replacement indicated that the crystal contains one molecule in an asymmetric unit. Further model building and structural refinement are in progress.
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An upconversion nanoparticle--Zinc phthalocyanine based nanophotosensitizer for photodynamic therapy.
Biomaterials
PUBLISHED: 01-14-2014
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Recent advances in NIR triggering upconversion-based photodynamic therapy have led to substantial improvements in upconversion-based nanophotosensitizers. How to obtain the high efficiency of singlet oxygen generation under low 980 nm radiation dosage still remains a challenge. A highly efficient nanophotosensitizer, denoted as UCNPs-ZnPc, was constructed for photodynamic therapy, which is based on near infrared (NIR) light upconversion nanoparticle (UCNP) and Zn(II)-phthalocyanine (ZnPc) photosensitizer (PS). The high (1)O2 production efficiency came from the enhancement of the 660 nm upconversion emission of NaYF4:Yb(3+), Er(3+) UCNP with 25% Yb(3+) doping, covalent assemblage of UCNP and ZnPc which significantly shortened the distance and enhanced the energy transfer between the two. The high (1)O2 production led to a secure and efficient PDT treatment, as evidenced by the in vivo test where UCNPs-ZnPc of 50 mg per kg body weight was locally injected into the liver tumor in mice, a low 980 nm radiation dose of 351 J/cm(2) (0.39 W/cm(2)) and short irradiation duration of 15 min were sufficient to perform image-guided PDT and caused the liver tumor inhibitory ratio of approximately 80.1%. Histological analysis revealed no pathological changes and inflammatory response in heart, lung, kidney, liver or spleen.
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A novel low-temperature-active pectin methylesterase from Penicillium chrysogenum F46 with high efficiency in fruit firming.
Food Chem
PUBLISHED: 01-12-2014
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A pectin methylesterase gene (pe8F46) was cloned from Penicillium chrysogenum F46 and successfully expressed in Pichia pastoris. The full-length cDNA consists of 969 bp and encodes a 322-residue polypeptide with the calculated molecular weight of 34.1 kDa. Deduced PE8F46 belongs to family 8 of carbohydrate esterases and shares 54% identity with a functionally characterised counterpart from Myceliophthora thermophile. Purified recombinant PE8F46 showed the optimal activity at pH 5.0 and 40°C, and remained 52% maximum activity even at 10°C. An orthogonal experiment was employed to determine the best conditions for firming pineapple dices. After incubation with 0.75% (w/v) PE8F46 and 0.4% calcium lactate (w/v) for 20 min, the firmness of pineapple dices was improved by 47.6%, 13.7% higher than that of a commercial pectinase complex. These results suggest that PE8F46 has application potential in the food industry.
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Characterization of a novel cytochrome P450 from Amycolatopsis sp. CGMCC1149 for hydroxylation of lovastatin.
Biotechnol. Appl. Biochem.
PUBLISHED: 01-12-2014
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Wuxistatin, a novel and potent statin, is converted from lovastatin by Amycolatopsis sp. CGMCC1149. In the bioconversion, lovastatin is firstly hydroxylated to 3-hydroxymethyl lovastatin (product I) by a hydroxylase. In the current study, a novel hydroxylase gene p450lov was isolated from Amycolatopsis sp. CGMCC1149 by degenerate PCR and self-formed adaptor PCR and expressed in Escherichia coli. The gene encodes a 403-amino-acid protein with a molecular weight of 44.8 kDa and was designated as a new member of cytochrome P450 (CYP) 105 family, CYP105A44. Meanwhile, a lovastatin catalytic in vitro system was established, and an optimal hydroxylation reaction system contained 30 µM lovastatin, 600 µM NADH, 120 µM ferredoxin, 0.04 U ferredoxin-nicotinamide adenine dinucleotide phosphate reductase, and 100 µM CYP105A44 in a final volume of 200 µL Tris HCl buffer (50 mM, pH 7.4). These would be helpful for further studies on the hydroxylation of statins.
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A novel bifunctional pectinase from Penicillium oxalicum SX6 with separate pectin methylesterase and polygalacturonase catalytic domains.
Appl. Microbiol. Biotechnol.
PUBLISHED: 01-12-2014
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A multimodular pectinase of glycoside hydrolase family 28, S6A, was identified in Penicillium oxalicum SX6 that consists of an N-terminal catalytic domain of pectin methylesterase, a Thr/Ser-rich linker region, and a C-terminal catalytic domain of polygalacturonase. Recombinant S6A and its two derivatives, S6PE (the catalytic domain of pectin methylesterase) and S6PG (the catalytic domain of polygalacturonase), were produced in Pichia pastoris. S6A was a bifunctional protein and had both pectin methylesterase and polygalacturonase activities. Three enzymes showed similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 50 °C) and excellent stability at pH 3.5-6.0 and 40 °C. Most metal ions tested (Na(+), K(+), Ca(2+), Li(+), Co(2+), Cr(3+), Ni(2+), Cu(2+), Mn(2+),Mg(2+), Fe(3+), Zn(2+), and Pb(2+)) enhanced the pectin methylesterase activities of S6PE and S6A, but had little or inhibitory effects on the polygalacturonase activities of S6A and S6PG. In comparison with most fungal pectin methylesterases, S6A had higher specific activity (271.1 U/mg) towards 70 % DM citrus pectin. When S6PE and S6PG were combined at the activity ratio of 1:4, the most significant synergistic effect was observed in citrus pectin degradation and degumming of sisal fiber, which is comparable with the performance of S6A (95 v.s. 100 % and 16.9 v.s. 17.2 %, respectively). To the best of our knowledge, this work represents the first report of gene cloning, heterologous expression, and biochemical characterization of a bifunctional pectinase with separate catalytic domains.
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Diabetes Attitude Scale: validation in type-2 diabetes patients in multiple centers in China.
PLoS ONE
PUBLISHED: 01-01-2014
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The aim of the paper is to report the development and psychometric testing of Diabetes Attitude Scale.
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Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.
PLoS ONE
PUBLISHED: 01-01-2014
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As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions). A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC) of 0.77 with high precision (94%) and high sensitivity (65%). We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA)] is available as an on-line server at http://sparks-lab.org.
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Anti-angiogenesis therapy in the Vx2 rabbit cancer model with a lipase-cleavable Sn 2 taxane phospholipid prodrug using ?(v)??-targeted theranostic nanoparticles.
Theranostics
PUBLISHED: 01-01-2014
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In nanomedicine, the hydrophobic nature of paclitaxel has favored its incorporation into many nanoparticle formulations for anti-cancer chemotherapy. At lower doses taxanes are reported to elicit anti-angiogenic responses. In the present study, the facile synthesis, development and characterization of a new lipase-labile docetaxel prodrug is reported and shown to be an effective anti-angiogenic agent in vitro and in vivo. The Sn 2 phosphatidylcholine prodrug was stably incorporated into the lipid membrane of ?(v)??-integrin targeted perfluorocarbon (PFC) nanoparticles (?(v)??-Dxtl-PD NP) and did not appreciably release during dissolution against PBS buffer or plasma over three days. Overnight exposure of ?(v)??-Dxtl-PD NP to plasma spiked with phospholipase enzyme failed to liberate the taxane from the membrane until the nanoparticle integrity was compromised with alcohol. The bioactivity and efficacy of ?(v)??-Dxtl-PD NP in endothelial cell culture was as effective as Taxol(®) or free docetaxel in methanol at equimolar doses over 96 hours. The anti-angiogenesis effectiveness of ?(v)??-Dxtl-PD NP was demonstrated in the Vx2 rabbit model using MR imaging of angiogenesis with the same ?(v)??-PFC nanoparticle platform. Nontargeted Dxtl-PD NP had a similar MR anti-angiogenesis response as the integrin-targeted agent, but microscopically measured decreases in tumor cell proliferation and increased apoptosis were detected only for the targeted drug. Equivalent dosages of Abraxane(®) given over the same treatment schedule had no effect on angiogenesis when compared to control rabbits receiving saline only. These data demonstrate that ?(v)??-Dxtl-PD NP can reduce MR detectable angiogenesis and slow tumor progression in the Vx2 model, whereas equivalent systemic treatment with free taxane had no benefit.
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IL-17A Produced by Neutrophils Protects against Pneumonic Plague through Orchestrating IFN-?-Activated Macrophage Programming.
J. Immunol.
PUBLISHED: 12-13-2013
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Innate immune cells, including neutrophils and macrophages, are critically involved in host antimicrobial defense responses. Intrinsic regulatory mechanisms controlling neutrophil and macrophage activities are poorly defined. In this study, we found that IL-17A, a natural signal factor, could provide protection against early pneumonic plague inflammation by coordinating the functions of neutrophils and programming of macrophages. The IL-17A level is promptly increased during the initial infection. Importantly, abrogation of IL-17A or IL-17AR significantly aggravated the infection, but mIL-17A treatment could significantly alleviate inflammatory injury, revealing that IL-17A is a critical requirement for early protection of infection. We also demonstrated that IL-17A was predominantly produced by CD11b(+)Ly6G(+) neutrophils. Although IL-17A could not significantly affect the antimicrobial responses of neutrophils, it could target the proinflammatory macrophage (M1) programming and potentiate the M1s defense against pneumonic plague. Mechanistically, IFN-? treatment or IFN-?-activated M1 macrophage transfer could significantly mitigate the aggravated infection of IL-17A(-/-) mice. Finally, we showed that IL-17A and IFN-? could synergistically promote macrophage anti-infection immunity. Thus, our findings identify a previously unrecognized function of IL-17A as an intrinsic regulator in coordinating neutrophil and macrophage antimicrobial activity to provide protection against acute pneumonic plague.
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Mechanism and enantioselectivity of dirhodium-catalyzed intramolecular C-h amination of sulfamate.
J. Org. Chem.
PUBLISHED: 12-05-2013
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The mechanisms and enantioselectivities of the dirhodium (Rh2L4, L = formate, N-methylformamide, S-nap)-catalyzed intramolecular C-H aminations of 3-phenylpropylsulfamate ester have been investigated in detail with BPW91 density functional theory computations. The reactions catalyzed by the Rh2(II,II) catalysts start from the oxidation of the Rh2(II,II) dimer to a triplet mixed-valent Rh2(II,III)-nitrene radical, which should facilitate radical H-atom abstraction. However, in the Rh2(formate)4-promoted reaction, as a result of a minimum-energy crossing point (MECP) between the singlet and triplet profiles, a direct C-H bond insertion is postulated. The Rh2(N-methylformamide)4 reaction exhibits quite different mechanistic characteristics, taking place via a two-step process involving (i) intramolecular H-abstraction on the triplet profile to generate a diradical intermediate and (ii) C-N formation by intersystem crossing from the triplet state to the open-shell singlet state. The stepwise mechanism was found to hold also in the reaction of 3-phenylpropylsulfamate ester catalyzed by Rh2(S-nap)4. Furthermore, the diradical intermediate also constitutes the starting point for competition steps involving enantioselectivity, which is determined by the C-N formation open-shell singlet transition state. This mechanistic proposal is supported by the calculated enantiomeric excess (94.2% ee) with the absolute stereochemistry of the product as R, in good agreement with the experimental results (92.0% ee).
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Comparison of fecundity and offspring immunity in zebrafish fed Lactobacillus rhamnosus CICC 6141 and Lactobacillus casei BL23.
Reproduction
PUBLISHED: 10-17-2013
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To increase the knowledge of probiotic effects on zebrafish (Danio rerio), we compare the effects of two probiotic strains, Lactobacillus rhamnosus CICC 6141 (a highly adhesive strain) and Lactobacillus casei BL23 (a weakly adhesive strain), on zebrafish reproduction and their offsprings innate level of immunity to water-borne pathogens. During probiotics treatments from 7 to 28 days, both the Lactobacillus strains, and especially L. casei BL23, significantly increased fecundity in zebrafish: higher rates of egg ovulation, fertilization, and hatching were observed. Increased densities of both small and large vitellogenic follicles, seen in specimens fed either Lactobacillus strain, demonstrated accelerated oocyte maturation. Feeding either strain of Lactobacillus upregulated gene expression of leptin, kiss2, gnrh3, fsh, lh, lhcgr, and paqr8, which were regarded to enhance fecundity and encourage oocyte maturation. Concomitantly, the gene expression of bmp15 and tgfb1 was inhibited, which code for local factors that prevent oocyte maturation. The beneficial effects of the Lactobacillus strains on fecundity diminished after feeding of the probiotics was discontinued, even for the highly adhesive gut Lactobacillus strain. Administering L. rhamnosus CICC 6141 for 28 days was found to affect the innate immunity of offspring derived from their parents, as evinced by a lower level of alkaline phosphatase activity in early larval stages. This study highlights the effects of probiotics both upon the reproductive process and upon the offsprings immunity during early developmental stages.
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A thermophilic ?-galactosidase from Neosartorya fischeri P1 with high specific activity, broad substrate specificity and significant hydrolysis ability of soymilk.
Bioresour. Technol.
PUBLISHED: 10-05-2013
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An extracellular ?-galactosidase (Gal27A) with high specific activity of 423Umg(-1) was identified in thermophilic Neosartorya fischeri P1. Its coding gene (1680bp) was cloned and functionally expressed in Pichia pastoris. Sequence analysis indicated that deduced Gal27A contains a catalytic domain of glycoside hydrolase family 27. The native and recombinant enzymes shared some similar properties, such as pH optima at 4.5, temperature optima at 60-70°C, resistance to most chemicals and saccharides, and great abilities to degrade raffinose and stachyose in soymilk. Considering the high yield (3.1gL(-1)) in P. pastoris, recombinant rGal27A is more favorable for industrial applications. This is the first report on purification and gene cloning of Neosartorya ?-galactosidase.
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Encapsulation of liver microsomes into a thermosensitive hydrogel for characterization of drug metabolism and toxicity.
Biomaterials
PUBLISHED: 09-04-2013
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This study reported the encapsulation of liver microsomes into a thermosensitive hydrogel to characterize drug metabolism and predict drug effects. Pluronic(®)F-127 (F127) and acrylamide-bisacrylamide (Acr-Bis) were utilized as the two precursors. After chemical crosslinking catalyzed by ammonium persulfate (APS) and N,N,N,N-tetramethylethylenediamine (TEMED), the resulting Pluronic F127-acrylamide-bisacrylamide (FAB) hydrogel could encapsulate microsomes at 4 °C and facilitate metabolic reactions at 37 °C. The gel morphology at different Acr-Bis concentrations was characterized using field emission scanning electron microscopy (FE-SEM). Higher concentrations of Acr-Bis could lead to higher degrees of cross-linking of the gel. A fluorescent staining assay was subsequently used to demonstrate successful encapsulation of microsomes into the gel as well as the free diffusion process of micromolecular substrates. The thermosensitivity of the FAB gel was studied using swelling ratio and protein release assay to verify its ability to encapsulate microsomes. The metabolic activity of microsomes encapsulated in gels was investigated by detecting the metabolites of FDA-approved substrates, including dextromethorphan, chlorzoxazone and testosterone. Compared with the traditional method of microsomal incubation, the FAB gel maintained 60%-70% of microsome activity. Lastly, the classic anticancer prodrug cyclophosphamide (CTX) was chosen as a model drug for the study of drug metabolism and the prediction of drug effects. When the microsomes encapsulated in the FAB gel were used in the cell culture system, CTX induced a higher level of apoptosis in MCF-7 cells compared with traditional microsomes.
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Outer membrane proteins ail and OmpF of Yersinia pestis are involved in the adsorption of T7-related bacteriophage Yep-phi.
J. Virol.
PUBLISHED: 09-04-2013
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Yep-phi is a T7-related bacteriophage specific to Yersinia pestis, and it is routinely used in the identification of Y. pestis in China. Yep-phi infects Y. pestis grown at both 20°C and 37°C. It is inactive in other Yersinia species irrespective of the growth temperature. Based on phage adsorption, phage plaque formation, affinity chromatography, and Western blot assays, the outer membrane proteins of Y. pestis Ail and OmpF were identified to be involved, in addition to the rough lipopolysaccharide, in the adsorption of Yep-phi. The phage tail fiber protein specifically interacts with Ail and OmpF proteins, and residues 518N, 519N, and 523S of the phage tail fiber protein are essential for the interaction with OmpF, whereas residues 518N, 519N, 522C, and 523S are essential for the interaction with Ail. This is the first report to demonstrate that membrane-bound proteins are involved in the adsorption of a T7-related bacteriophage. The observations highlight the importance of the tail fiber protein in the evolution and function of various complex phage systems and provide insights into phage-bacterium interactions.
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The Role of Semidisorder in Temperature Adaptation of Bacterial FlgM Proteins.
Biophys. J.
PUBLISHED: 09-02-2013
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Probabilities of disorder for FlgM proteins of 39 species whose optimal growth temperature ranges from 273 K (0°C) to 368 K (95°C) were predicted by a newly developed method called Sequence-based Prediction with Integrated NEural networks for Disorder (SPINE-D). We showed that the temperature-dependent behavior of FlgM proteins could be separated into two subgroups according to their sequence lengths. Only shorter sequences evolved to adapt to high temperatures (>318 K or 45°C). Their ability to adapt to high temperatures was achieved through a transition from a fully disordered state with little secondary structure to a semidisordered state with high predicted helical probability at the N-terminal region. The predicted results are consistent with available experimental data. An analysis of all orthologous protein families in 39 species suggests that such a transition from a fully disordered state to semidisordered and/or ordered states is one of the strategies employed by nature for adaptation to high temperatures.
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Promoter RNA links transcriptional regulation of inflammatory pathway genes.
Nucleic Acids Res.
PUBLISHED: 09-02-2013
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Although many long non-coding RNAs (lncRNAs) have been discovered, their function and their association with RNAi factors in the nucleus have remained obscure. Here, we identify RNA transcripts that overlap the cyclooxygenase-2 (COX-2) promoter and contain two adjacent binding sites for an endogenous miRNA, miR-589. We find that miR-589 binds the promoter RNA and activates COX-2 transcription. In addition to miR-589, fully complementary duplex RNAs that target the COX-2 promoter transcript activate COX-2 transcription. Activation by small RNA requires RNAi factors argonaute-2 (AGO2) and GW182, but does not require AGO2-mediated cleavage of the promoter RNA. Instead, the promoter RNA functions as a scaffold. Binding of AGO2 protein/small RNA complexes to the promoter RNA triggers gene activation. Gene looping allows interactions between the promoters of COX-2 and phospholipase A2 (PLA2G4A), an adjacent pro-inflammatory pathway gene that produces arachidonic acid, the substrate for COX-2 protein. miR-589 and fully complementary small RNAs regulate both COX-2 and PLA2G4A gene expression, revealing an unexpected connection between key steps of the eicosanoid signaling pathway. The work demonstrates the potential for RNA to coordinate locus-dependent assembly of related genes to form functional operons through cis-looping.
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Bioluminescent tracking of colonization and clearance dynamics of plasmid-deficient Yersinia pestis strains in a mouse model of septicemic plague.
Microbes Infect.
PUBLISHED: 08-30-2013
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Yersinia pestis 201 contains 4 plasmids pPCP1, pMT1, pCD1 and pCRY, but little is known about the effects of these plasmids on the dissemination of Y. pestis. We developed a plasmid-based luxCDABE bioreporter in Y. pestis 201, Y. pestis 201-pCD1(+), Y. pestis 201-pMT1(+), Y. pestis 201-pPCP1(+), Y. pestis 201-pCRY(+), Y. pestis 201-p(-) and Yersinia pseudotuberculosis Pa36060 strains, and investigated their dissemination by bioluminescence imaging during primary septicemic plague in a mouse model. These strains mainly colonized the livers and spleens shortly after intravenous inoculation. Y. pestis 201-pMT1(+) appeared to have a stronger ability to survive in the livers, spleens and blood, and to be more virulent than other plasmid-deficient strains. Y. pestis 201-pPCP1(+) appeared to have a stronger ability to colonize lungs than other plasmid-deficient strains. Pa36060 has the strongest ability to colonize intestines and lungs. Y. pestis 201 has the strongest ability to survive in blood, and the strongest virulence. These results indicated that the plasmid pMT1 was an important determinent in the colonization of livers, spleens and blood, whereas the plasmid pPCP1 appeared to correlate with the colonization in lungs. The resistance to killing in mouse blood seemed to be the critical factor causing animal death.
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Prediction of RNA binding proteins comes of age from low resolution to high resolution.
Mol Biosyst
PUBLISHED: 07-23-2013
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Networks of protein-RNA interactions is likely to be larger than protein-protein and protein-DNA interaction networks because RNA transcripts are encoded tens of times more than proteins (e.g. only 3% of human genome coded for proteins), have diverse function and localization, and are controlled by proteins from birth (transcription) to death (degradation). This massive network is evidenced by several recent experimental discoveries of large numbers of previously unknown RNA-binding proteins (RBPs). Meanwhile, more than 400 non-redundant protein-RNA complex structures (at 25% sequence identity or less) have been deposited into the protein databank. These sequences and structural resources for RBPs provide ample data for the development of computational techniques dedicated to RBP prediction, as experimentally determining RNA-binding functions is time-consuming and expensive. This review compares traditional machine-learning based approaches with emerging template-based methods at several levels of prediction resolution ranging from two-state binding/non-binding prediction, to binding residue prediction and protein-RNA complex structure prediction. The analysis indicates that the two approaches are complementary and their combinations may lead to further improvements.
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Two family 11 xylanases from Achaetomium sp. Xz-8 with high catalytic efficiency and application potentials in the brewing industry.
J. Agric. Food Chem.
PUBLISHED: 07-03-2013
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This study identified two family-11 xylanase genes (xynC81 and xynC83) in Achaetomium sp. Xz-8, a thermophilic strain from a desert area with substantial xylanase activity, and successfully expressed them in Pichia pastoris . Their deduced amino acid sequences showed the highest identity of ?90% to known fungal xylanases and of ?62% with each other. The purified recombinant xylanases showed optimal activities at pH 5.5 and 60-65 °C and exhibited stability over pH 5.0-10.0 and temperatures at 55 °C and below. XynC81 had high catalytic efficiency (6082 mL/s/mg), and XynC83 was favorable for xylooligosaccharide production. Under simulated mashing conditions, combination of XynC83 and a commercial ?-glucanase improved the filtration rate by 34.76%, which is much better than that of Novozymes Ultraflo (20.71%). XynC81 and XynC83 had a synergistic effect on viscosity reduction (7.08%), which is comparable with that of Ultraflo (8.47%). Thus, XynC81 and XynC83 represent good candidates for application in the brewing industry.
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Prediction and validation of the unexplored RNA-binding protein atlas of the human proteome.
Proteins
PUBLISHED: 06-21-2013
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Detecting protein-RNA interactions is challenging both experimentally and computationally because RNAs are large in number, diverse in cellular location and function, and flexible in structure. As a result, many RNA-binding proteins (RBPs) remain to be identified. Here, a template-based, function-prediction technique SPOT-Seq for RBPs is applied to human proteome and its result is validated by a recent proteomic experimental discovery of 860 mRNA-binding proteins (mRBPs). The coverage (or sensitivity) is 42.6% for 1217 known RBPs annotated in the Gene Ontology and 43.6% for 860 newly discovered human mRBPs. Consistent sensitivity indicates the robust performance of SPOT-Seq for predicting RBPs. More importantly, SPOT-Seq detects 2418 novel RBPs in human proteome, 291 of which were validated by the newly discovered mRBP set. Among 291 validated novel RBPs, 61 are not homologous to any known RBPs. Successful validation of predicted novel RBPs permits us to further analysis of their phenotypic roles in disease pathways. The dataset of 2418 predicted novel RBPs along with confidence levels and complex structures is available at http://sparks-lab.org (in publications) for experimental confirmations and hypothesis generation. Proteins 2013. © 2013 Wiley Periodicals, Inc.
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Molecular MR imaging of neovascular progression in the Vx2 tumor with ?v?3-targeted paramagnetic nanoparticles.
Radiology
PUBLISHED: 06-14-2013
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To assess the dependence of neovascular molecular magnetic resonance (MR) imaging on relaxivity (r1) of ?v?3-targeted paramagnetic perfluorocarbon (PFC) nanoparticles and to delineate the temporal-spatial consistency of angiogenesis assessments for individual animals.
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Two xylose-tolerant GH43 bifunctional ?-xylosidase/?-arabinosidases and one GH11 xylanase from Humicola insolens and their synergy in the degradation of xylan.
Food Chem
PUBLISHED: 06-07-2013
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Two ?-xylosidases of family 43 (Xyl43A and Xyl43B) and one xylanase of family 11 (Xyn11A) were identified from the genome sequence of Humicola insolens Y1, and their gene products were successfully expressed in heterologous hosts. The optimal activities of the purified Xyl43A, Xyl43B, and Xyn11A were found at pH 6.5-7.0 and 50-60°C. They were stable over a pH range of 5.0-10.0 and temperatures of 50°C and below. Xyl43A and Xyl43B had the activities of ?-xylosidase, ?-arabinosidase and xylanase, and showed xylose tolerance up to 79 and 292mM, respectively. Xyn11A and Xyl43A or Xyl43B showed significant synergistic effects on the degradation of various xylans, releasing more reduced sugars (up to 1.29 folds) by simultaneous or sequential addition. This study provides several enzymes for synergistic degradation of xylan and contributes to the formulation of optimised enzyme mixtures for the efficient hydrolysis of plant biomass.
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Theoretical elucidation of the origin for assembly of the DAP12 dimer with only one NKG2C in the lipid membrane.
J Phys Chem B
PUBLISHED: 04-16-2013
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In this work, we have investigated in details the origin of the assembly of the DAP12 dimer with only one NKG2C in the activating immunoreceptor complex from thew two aspects of electronic properties and dynamic structures by performing density functional theory (DFT) calculations and molecular dynamics (MD) simulations. In the DFT calculations, we studied the aggregation ability of the NKG2C(TM) with the DAP12(TM) dimer and the DAP12(TM)-DAP12(TM)-NKG2C(TM) complex by analyzing the electrostatic potentials and frontier molecular orbitals (FMOs), and in the MD simulations we mainly investigated the dynamic structures of the DAP12(TM)-DAP12(TM)-NKG2C(TM) complex and its mutants, as well as the tetramer complex consisting of two DAP12(TM) and two NKG2C(TM) helixes without any restriction. Through the studies of the electrostatic potential, the FMOs, and the dynamic structures, we have provided reasonable explanations to some extent for the experimental observation that only one NKG2C can associate with the DAP12 homodimer. The present theoretical results are expected to give valuable information for further studying the assembly between receptors and signaling subunits.
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20-Hydroxyeicosatetraenoic acid mediates isolated heart ischemia/reperfusion injury by increasing NADPH oxidase-derived reactive oxygen species production.
Circ. J.
PUBLISHED: 04-13-2013
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It has been reported that 20-hydroxyeicosatetraenoic acid (20-HETE) aggravates myocardial ischemia/reperfusion (I/R) injury, but the exact mechanism of action is still unclear.
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Characterization of early neovascular response to acute lung ischemia using simultaneous (19)F/ (1)H MR molecular imaging.
Angiogenesis
PUBLISHED: 04-12-2013
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Angiogenesis is an important constituent of many inflammatory pulmonary diseases, which has been unappreciated until recently. Early neovascular expansion in the lungs in preclinical models and patients is very difficult to assess noninvasively, particularly quantitatively. The present study demonstrated that (19)F/(1)H MR molecular imaging with ?v?3-targeted perfluorocarbon nanoparticles can be used to directly measure neovascularity in a rat left pulmonary artery ligation (LPAL) model, which was employed to create pulmonary ischemia and induce angiogenesis. In rats 3 days after LPAL, simultaneous (19)F/(1)H MR imaging at 3T revealed a marked (19)F signal in animals 2 h following ?v?3-targeted perfluorocarbon nanoparticles [(19)F signal (normalized to background) = 0.80 ± 0.2] that was greater (p = 0.007) than the non-targeted (0.30 ± 0.04) and the sham-operated (0.07 ± 0.09) control groups. Almost no (19)F signal was found in control right lung with any treatment. Competitive blockade of the integrin-targeted particles greatly decreased the (19)F signal (p = 0.002) and was equivalent to the non-targeted control group. Fluorescent and light microscopy illustrated heavy decorating of vessel walls in and around large bronchi and large pulmonary vessels. Focal segmental regions of neovessel expansion were also noted in the lung periphery. Our results demonstrate that (19)F/(1)H MR molecular imaging with ?v?3-targeted perfluorocarbon nanoparticles provides a means to assess the extent of systemic neovascularization in the lung.
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Androgen insensitivity syndrome with gynandroblastoma and vulvar leiomyoma: case report and literature review.
J Low Genit Tract Dis
PUBLISHED: 04-05-2013
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This study aimed to describe a rare case of androgen insensitivity syndrome (AIS) in association with gynandroblastoma and vulvar leiomyomata.
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A novel thermophilic endo-?-1,4-mannanase from Aspergillus nidulans XZ3: functional roles of carbohydrate-binding module and Thr/Ser-rich linker region.
Appl. Microbiol. Biotechnol.
PUBLISHED: 04-01-2013
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The gene man5XZ3 from Aspergillus nidulans XZ3 encodes a multimodular ?-mannanase of glycoside hydrolase family 5 that consists of a family 1 carbohydrate-binding module (CBM1), a Thr/Ser-rich linker region, and a catalytic domain. Recombinant Man5XZ3 and its two truncated derivatives, Man5?CBM (removing the CBM1) and Man5?CL (removing both the CBM1 and linker region), were produced in Pichia pastoris and showed significant variance in the secondary structure. The three enzymes had similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 80 °C) and excellent pH stability at pH 4.0-10.0. Removal of the CBM1 alone could improve the thermostability of Man5XZ3, but further removal of the linker region resulted in worse thermostability. Man5XZ3 retained greater enzyme activity in the presence of an organic solvent (acetone), two detergents (SDS and Triton X-100), and a chaotropic agent (urea) compared with Man5?CBM and Man5?CL. This study provides an excellent ?-mannanase candidate favorable for various industries and primarily demonstrates the relationship between enzyme structure and function.
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Investigation binding patterns of human carboxylesterase I (hCES I) with broad substrates by MD simulations.
Curr Top Med Chem
PUBLISHED: 03-26-2013
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Human carboxylesterase I (hCES 1) plays an important role in the metabolism and activation of prodrugs, such as, the hydrolysis of a variety of drugs of prodrugs featuring an ester, amide or carbamate function. The bindings of the substrates of different lengths and cocaine to hCES1 at two different binding sites, catalytic site and Z-site, were studies through MD simulations. For each case, the correlation analysis has been performed to explore the binding patterns of a broad range of substrates binding to the hCES1.
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