Chaperone-tip adhesin complex is vital for synergistic activation of CFA/I fimbriae biogenesis.

Colonization factor CFA/I defines the major adhesive fimbriae of enterotoxigenic Escherichia coli and mediates bacterial attachment to host intestinal epithelial cells. The CFA/I fimbria consists of a tip-localized minor adhesive subunit, CfaE, and thousands of copies of the major subunit CfaB polymerized into an ordered helical rod. Biosynthesis of CFA/I fimbriae requires the assistance of the periplasmic chaperone CfaA and outer membrane usher CfaC. Although the CfaE subunit is proposed to initiate the assembly of CFA/I fimbriae, how it performs this function remains elusive. Here, we report the establishment of an in vitro assay for CFA/I fimbria assembly and show that stabilized CfaA-CfaB and CfaA-CfaE binary complexes together with CfaC are sufficient to drive fimbria formation. The presence of both CfaA-CfaE and CfaC accelerates fimbria formation, while the absence of either component leads to linearized CfaB polymers in vitro. We further report the crystal structure of the stabilized CfaA-CfaE complex, revealing features unique for biogenesis of Class 5 fimbriae.

Structural and Functional Insights into PpgL, a Metal-Independent ß-Propeller Gluconolactonase That Contributes to Pseudomonas aeruginosa Virulence.

Biofilm formation is a critical determinant in the pathopoiesis of Pseudomonas aeruginosa It could significantly increase bacterial resistance to drugs and host defense. Thus, inhibition of biofilm matrix production could be regarded as a promising attempt to prevent colonization of P. aeruginosa and the subsequent infection. PpgL, a periplasmic gluconolactonase, has been reported to be involved in P. aeruginosa quorum-sensing (QS) system regulation. However, the detailed function and catalysis mechanism remain elusive. Here, the crystal structure of PpgL is described in the current study, along with biochemical analysis, revealing that PpgL is a typical ß-propeller enzyme with unique metal-independent lactone hydrolysis activity. Consequently, comparative analysis of seven-bladed propeller lactone-catalyzing enzymes and mutagenesis studies identify the critical sites which contribute to the diverse catalytic and substrate recognition functions. In addition, the reduced biofilm formation and attenuated invasion phenotype resulting from deletion of ppgL confirm the importance of PpgL in P. aeruginosa pathogenesis. These results suggest that PpgL is a potential target for developing new agents against the diseases caused by P. aeruginosa.

Mechanistic insights into the allosteric regulation of Pseudomonas aeruginosa aspartate kinase.

In plants and microorganisms, aspartate kinase (AK) catalyzes an initial commitment step of the aspartate family amino acid biosynthesis. Owing to various structural organizations, AKs from different species show tremendous diversity and complex allosteric controls. We report the crystal structure of AK from Pseudomonas aeruginosa (PaAK), a typical α2ß2 hetero-tetrameric enzyme, in complex with inhibitory effectors. Distinctive features of PaAK are revealed by structural and biochemical analyses. Essentially, the open conformation of Lys-/Thr-bound PaAK structure clarifies the inhibitory mechanism of α2ß2-type AK. Moreover, the various inhibitory effectors of PaAK have been identified and a general amino acid effector motif of AK family is described.

Dietary Patterns Associated Metabolic Syndrome in Chinese Adults.

Dietary pattern has been revealed to be associated with metabolic syndrome. However, the association was not well documented in Chinese due to the complexity of Chinese foods. We mainly assessed the dietary patterns and examined their effects on metabolic syndrome among Chinese adults. Four dietary patterns including 'Refined Grains & Vegetables' Pattern, 'Dairy & Eggs' Pattern, 'Organ Meat & Poultry' Pattern, and 'Coarse Grains & Beans' Pattern were extracted. 'Dairy & Eggs' Pattern was associated with a decreased odds of metabolic syndrome in women, and 'Coarse Grains & Beans' Pattern was associated with a decreased odds of hypertension in men. These results provided a scientific basis for future research and dietary guideline perfection.

[Secular trends of cesarean delivery and cesarean delivery on maternal request among primiparous women with singleton pregnancy in Southern and Northern China during 1993-2010].

OBJECTIVE: To explore the secular trends of cesarean delivery and cesarean delivery on maternal request (CDMR) among primiparous women with singleton pregnancy in Southern and Northern China. METHODS: The Perinatal Healthcare Surveillance System was established in 26 counties/cities of 3 Chinese provinces since 1993. A total of 1 317 774 primiparous women with singleton pregnancy were monitored through the system during the 1993 - 2010 period. Chi-square tests were performed to assess the linear trends in the prevalence of cesarean delivery and CDMR. RESULTS: During the 18-year period, 496 054 cesarean deliveries and 129 219 CDMRs were identified. The prevalence rates of cesarean delivery and CDMR were 37.6% and 10.0% respectively. CDMR accounted for 26.0% of all cesarean births. The prevalence of cesarean delivery increased from 29.4% during the 1993 - 1995 period to 58.7% during the 2006 - 2010 in Southern urban area, from 18.2% to 58.3% in Southern rural area and from 4.3% to 49.5% in Northern rural area. The prevalence of CDMR increased by 34 folds from 0.6% during the 1993 - 1995 period to 21.3% during the 2006 - 2010 period in Southern urban area, by 40 folds from 0.6% to 24.4% in Southern rural area and by 44 folds from 0.6% to 27.3% in Northern rural area. The proportions of CDMR in all cesarean deliveries significantly increased in all three regions. Women aged over 30 years, overweight and well-educated were more likely to experience a cesarean delivery including CDMR. CONCLUSION: The Chinese prevalence rates of cesarean delivery and CDMR increased dramatically during the 1993 - 2010 period and ranked the highest in the world by 2010. And CDMR became a non-negligible component in all cesarean sections.

Nucleotide binding as an allosteric regulatory mechanism for Akkermansia muciniphila ß-N-acetylhexosaminidase Am2136.

ß-N-acetylhexosaminidases (EC3.2.1.52), which belong to the glycosyl hydrolase family GH20, are important enzymes for oligosaccharides modification. Numerous microbial ß-N-acetylhexosaminidases have been investigated for applications in biology, biomedicine and biotechnology. Akkermansia muciniphila is an anaerobic intestinal commensal bacterium which possesses specific ß-N-acetylhexosaminidases for gut mucosal layer colonization and mucin degradation. In this study, we assessed the in vitro mucin glycan cleavage activity of the A. muciniphila ß-N-acetylhexosaminidase Am2136 and demonstrated its ability that hydrolyzing the ß-linkages joining N-acetylglucosamine to a wide variety of aglycone residues, which indicated that Am2136 may be a generalist ß-N-acetylhexosaminidase. Structural and enzyme activity assay experiments allowed us to probe the essential function of the inter-domain interactions in ß23-ß33. Importantly, we revealed that the hydrolysis activity of Am2136 was enhanced by nucleotides. We further speculated that this activation mechanism might be associated with the conformational motions between domain III and IV. To our knowledge, this is the first report of nucleotide effector regulated ß-N-acetylhexosaminidase, to reveal its novel biological functions. These findings contribute to understanding the distinct properties within the GH20 family and lay a certain foundation to develop controllable glycan hydrolyzing catalysts.Abbreviations: OD600 - optical cell densities at 600 nm; LB - Luria-Bertani; IPTG - isopropyl ß-D-1-thiogalactopyranoside; PMSF - phenylmethanesulfonyl fluoride; rmsd - root mean square deviation; GlcNAc - N-acetyl-ß-D-glucosamine; GalNAc - N-acetyl-ß-D-galactosamine; Gal - galactose.

Comparison of the accuracy of dental implant placement using static and dynamic computer-assisted systems: an in vitro study.

OBJECTIVES: The study aimed to compare the accuracy of implant placement between static and dynamic computer-assisted systems (CAS) in a partially edentulous mandible model. MATERIALS & METHODS: A total of 80 implants was placed in mandible models. The implants were placed using either static or dynamic computer-assisted system. Deviations of implant hex, apex and angulation were measured between preoperative planning and postoperative CBCT in planning software. RESULTS: The mean deviations of implant hex, apex and angulation in static CAS group were 1.15 ± 0.34 mm, 1.37 ± 0.38 mm and 2.60 ± 1.11 degree, while in dynamic CAS group were 0.40 ± 0.41 mm, 0.34 ± 0.33 mm and 0.97 ± 1.21 degree, respectively. Implant placement with dynamic CAS showed less deviations of shoulder, apex and angulation than with static CAS significantly. CONCLUSIONS: The implant accuracy using CAS system could be influenced by the guiding technique.

Structural and molecular dynamic studies of Pseudomonas aeruginosa OdaA reveal the regulation role of a C-terminal hinge element.

BACKGROUND: Crotonase superfamily members exhibit great catalytic diversity towards various acyl-CoA substrates. A common CoA moiety binding pattern is usually observed in this family, understanding the substrate-binding mechanism would facilitate the rational engineering of crotonases for improved properties. METHODS: We applied X-ray crystallography to investigate a putative enoyl-CoA hydratase/isomerase OdaA in Pseudomonas aeruginosa. Thermal shift assay (TSA) were performed to explore the binding of OdaA with CoA thioester substrates. Furthermore, we performed molecular dynamics (MD) simulations to elucidate the dynamics of its CoA-binding site. RESULTS: We solved the crystal structures of the apo and CoA-bound OdaA. Thermal shift assay (TSA) showed that CoA thioester substrates bind to OdaA with a different degree. MD simulations demonstrated that the C-terminal alpha helix underwent a structural transition and a hinge region would associate with this conformational change. CONCLUSIONS: TSA in combination with MD simulations elucidate that the dynamics of C-terminal alpha helix in CoA-binding, and a hinge region play an important role in conformational change. GENERAL SIGNIFICANCE: Those results help to extend our knowledge about the nature of crotonases and would be informative for future mechanistic studies and industry applications.

Structural analysis of the sulfatase AmAS from Akkermansia muciniphila.

Akkermansia muciniphila, an anaerobic Gram-negative bacterium, is a major intestinal commensal bacterium that can modulate the host immune response. It colonizes the mucosal layer and produces nutrients for the gut mucosa and other commensal bacteria. It is believed that mucin desulfation is the rate-limiting step in the mucin-degradation process, and bacterial sulfatases that carry out mucin desulfation have been well studied. However, little is known about the structural characteristics of A. muciniphila sulfatases. Here, the crystal structure of the premature form of the A. muciniphila sulfatase AmAS was determined. Structural analysis combined with docking experiments defined the critical active-site residues that are responsible for catalysis. The loop regions I-V were proposed to be essential for substrate binding. Structure-based sequence alignment and structural superposition allow further elucidation of how different subclasses of formylglycine-dependent sulfatases (FGly sulfatases) adopt the same catalytic mechanism but exhibit diverse substrate specificities. These results advance the understanding of the substrate-recognition mechanisms of A. muciniphila FGly-type sulfatases. Structural variations around the active sites account for the different substrate-binding properties. These results will enhance the understanding of the roles of bacterial sulfatases in the metabolism of glycans and host-microbe interactions in the human gut environment.

Selection of Reference Genes for Normalization of Gene Expression in Thermobia domestica (Insecta: Zygentoma: Lepismatidae).

Zygentoma occupies a key evolutionary position for understanding the evolution of insect metamorphosis but has received little attention in terms of genetic analysis. To develop functional genomic studies in this insect, we evaluated five candidate internal reference genes for quantitative RT-PCR (qPCR) studies from Thermobia domestica, a representative species of Zygentoma, including Actin 5C (Actin5C), Elongation factor-1 alpha (EF1A), Ribosome protein S26 (RPS26), Ribosome protein L32 (RPL32), and Superoxide dismutase 2 (SOD2), at different developmental stages, in various body parts, and under dsRNA microinjection and starvation stresses, using four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) and a comparative algorithm (RefFinder). Specific suitable reference genes were recommended across specific experimental conditions, and the combination of RPS26 and RPL32 was appropriate for all tested samples. Employing our selected reference gene combination, we investigated the gene expression pattern of Myoglianin (Myo), a crucial gene-regulating insect metamorphosis, in ametabolous T. domestica, and demonstrated the efficiency of RNA interference (RNAi) in firebrat nymphs. This study provides a basis for reliable quantitative studies of genes and greatly benefits evolutionary and functional genomics studies in Zygentoma.

Structural and biochemical analysis of 1-Cys peroxiredoxin ScPrx1 from Saccharomyces cerevisiae mitochondria.

BACKGROUND: ScPrx1 is a yeast mitochondrial 1-Cys peroxiredoxins (Prx), a type of Prx enzyme which require thiol-containing reducing agents to resolve its peroxidatic cysteine. ScPrx1 plays important role in protection against oxidative stress. Mitochondrial thioredoxin ScTrx3 and glutathione have been reported to be the physiological electron donor for ScPrx1. However, the mechanism underlying their actions, especially the substrate recognition of ScPrx1 requires additional elucidation. METHODS: The structure of ScPrx1 was obtained through crystallization experiments. The oligomeric state of ScPrx1 was monitored by Blue-Native PAGE. Mutations were generated by the QuikChange PCR-based method. The ScPrx1 activity assay was carried out by measuring the change of 340 nm absorption of the NADPH oxidation. RESULTS: ScPrx1 exist as a homodimer in solution. The structure adopts a typical Prx-fold core which is preceded by an N-terminal ß-hairpin and has a C-terminal extension. Mutations (Glu94Ala, Arg198Ala and Trp126) close to the active site could enhance the catalytic efficiency of ScPrx1 while His83Ala and mutations on α4-ß6 region exhibited reduced activity. The biochemical data also show that the deletion or mutations on ScPrx1 C-terminal have 2-4.56 fold increased activity. CONCLUSION: We inferred that conformational changes of ScPrx1 C-terminal segment were important for its reaction, and the α4-ß6 loop regions around the ScPrx1 active sites were important for the catalytic function of ScPrx1. Collectively, these structural features provides a basis for understanding the diverse reductant species usage in different 1-Cys Prxs.

Structural characterization of a Δ3, Δ2-enoyl-CoA isomerase from Pseudomonas aeruginosa: implications for its involvement in unsaturated fatty acid metabolism.

Gene PA4980 from Pseudomonas aeruginosa encodes a putative enoyl-coenzyme A hydratase/isomerase that is associated with the function of the biofilm dispersion-inducing signal molecule cis-2-decenoic acid. To elucidate the role of PA4980 in cis-2-decenoic acid biosynthesis, we reported the crystal structure of its protein product at 2.39 Å. The structural analysis and substrate binding prediction suggest that it acts as a monofunctional enoyl-coenzyme A isomerase, implicating an alternative pathway of the cis-2-decenoic acid synthesis.

Biosynthesis of (R)-2-hydroxy-3-phenylpropionic acid using whole recombinant Escherichia coli cells in an aqueous/n-octane biphasic system.

(R)-2-hydroxy-3-phenylpropionic acid (PLA) is an ideal antimicrobial compound with broad-spectrum activity against a wide range of Gram-positive bacteria, some Gram-negative bacteria, and fungi. We studied the bioconversion of phenylpyruvate (PPA) to PLA using whole recombinant Escherichia coli cells in a series of buffer/organic solvent systems. Octane was found to be the best organic solvent. The optimum volume ratio of the water phase to the n-octane phase, conversion temperature, substrate concentration, and cell concentration were 6:4, 40 °C, 12.5 g/L, and 30 g/L wet cells, respectively. Under the optimized conditions, the average PLA productivity in the aqueous/ n-octane system was 30.69% higher than that in the aqueous system, and 32.31 g/L PLA was obtained with the use of a stirred reactor (2-L scale). Taken together, our findings indicated that PLA biosynthesis was more efficient in an aqueous/n-octane biphasic system than in a monophasic aqueous system. The proposed biphasic system is an effective strategy for enhancing PLA yield and the biosynthesis of its analogues.

Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

DspI, a putative enoyl-coenzyme A (CoA) hydratase/isomerase, was proposed to be involved in the synthesis of cis-2-decenoic acid (CDA), a quorum sensing (QS) signal molecule in the pathogen Pseudomonas aeruginosa (P. aeruginosa). The present study provided a structural basis for the dehydration reaction mechanism of DspI during CDA synthesis. Structural analysis reveals that Glu126, Glu146, Cys127, Cys131 and Cys154 are important for its enzymatic function. Moreover, we show that the deletion of dspI results in a remarkable decreased in the pyoverdine production, flagella-dependent swarming motility, and biofilm dispersion as well as attenuated virulence in P. aeruginosa PA14. This study thus unravels the mechanism of DspI in diffusible signal factor (DSF) CDA biosynthesis, providing vital information for developing inhibitors that interfere with DSF associated pathogenicity in P. aeruginosa.

Author Correction: Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

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Structure-Function Relationship of Aminopeptidase P from Pseudomonas aeruginosa.

PepP is a virulence-associated gene in Pseudomonas aeruginosa, making it an attractive target for anti-P. aeruginosa drug development. The encoded protein, aminopeptidases P (Pa-PepP), is a type of X-prolyl peptidase that possesses diverse biological functions. The crystal structure verified its canonical pita-bread fold and functional tetrameric assembly, and the functional studies measured the influences of different metal ions on the activity. A trimetal manganese cluster was observed at the active site, elucidating the mechanism of inhibition by metal ions. Additionally, a loop extending from the active site appeared to be important for specific large-substrate binding. Based on the structural comparison and bacterial invasion assays, we showed that this non-conserved surface loop was critical for P. aeruginosa virulence. Taken together, these findings can extend our understanding of the catalytic mechanism and virulence-related functions of Pa-PepP and provide a solid foundation for the design of specific inhibitors against pathogenic-bacterial infections.

[An animal study of peripheral neurophysiologic mechanism in osseoperception phenomena of dental implant].

OBJECTIVE: To investigate the existence of functional neuroreceptors in peri-implant bone tissue and to test the peri-implant neural feedback pathway reconstruction and its influential factors. METHODS: Three beagle dogs were involved in this study. After the extraction of 3 premolars and 1 molar of the bilateral mandible, 27 implants were placed immediately or delayed. The implants were loaded for 3 - 6 months, then sensory nerve action potential (SNAP) tests were performed. The stimulating electrode was connected to the implant or natural tooth. The recording electrode was fixed near the inferior alveolar nerve (IAN) to record the latency period and amplitude of SNAP. The specimens with peri-implant bone tissues were taken and examined by immunohistochemistry. RESULTS: SNAP of IAN could be recorded following stimulations to natural teeth and implants. The differences in SNAP amplitudes between natural teeth [(44.04 +/- 6.23) microV] and all implant groups were statistically significant (P < 0.01). Free nerve endings and nerve tract were found in the peri-implant bone tissue, but much less than those in periodontal membrane. Some nerve fibers were directly connected with implant surface. CONCLUSIONS: Functional neuroreceptors, though much less than that of natural tooth, exist in peri-implant bone tissue. Surgical methods and loading time do not have obvious influences on peri-implant neural feedback pathway reconstruction.

[Clinical features of cleft palate with micrognathia and the risk of early palatoplasty].

This review focus on the characters of patients with cleft palate and micrognathia, the pathologic and physiological changes of this malformation and their influence on the patients, the risk factors of cleft palate repair, the indication and the outcome of palatoplasty. It is aimed to give a guideline for the optimal timing of cleft palate repair and the reduction of the risk factors,for these unique patients.