[Clinical application and evaluation of an early non-sedation protocol for critically ill respiratory patients].

Objective: To study the value of an early (mechanical ventilation after 24 h) non-sedation protocol for intubated, mechanically ventilated patients in the respiratory intensive care unit (RICU). Methods: Seventy intubated, mechanically ventilated patients were prospectively enrolled and randomly assigned to management with early non-sedation (intervention group; n=35) or with daily interruption of sedation (DIS) (control group; n=35). The duration of mechanical ventilation, length of the RICU and hospital stay, RICU and hospital mortality, drug consumption, RICU and hospitalization expenses, incidence of complications and adverse events and serum levels of vital organ damage and inflammatory markers after mechanical ventilation for 48 h were recorded and compared. Results: Patients in the intervention group had a shorter duration of mechanical ventilation than those in the control group [(7±5) vs (11±9) d, P<0.05] and were discharged from the RICU [(9±7) vs (18±9) d, P<0.05] and hospital earlier [(17±14) vs (29±22) d, P<0.05] than those in the control group. The doses of midazolam were significantly lower in the intervention group than in the control group [(99±104) vs (482±337) mg, P<0.05]. The RICU and hospitalization expenses were both significantly lower in the intervention group than in the control group [53(84) vs 88(173), 72(195) vs 154(234) thousand CHY, P<0.05]. In the intervention group, the occurrence rates of ventilator associated pneumonia (23% vs 46%), tracheotomy (14% vs 37%) and gastrointestinal adverse reactions (17% vs 40%) were significantly lower than those in the control group (P<0.05). No differences were recorded in RICU and hospital mortality (P>0.05). The occurrence rates of unplanned extubation and reintubation and the need for CT brain scans were similar in the 2 groups (P>0.05). The levels of cardiac, liver and renal damage markers, lactic acid and C-reactive protein were the same in both groups (P>0.05). Conclusions: The early non-sedation protocol decreased the duration of mechanical ventilation and the length of stay in the RICU and hospital, and it did not increase the incidence of complications and adverse events.

Food-grade antimicrobials potentiate the antibacterial activity of 1,2-hexanediol.

UNLABELLED: Preservative agents determining the shelf life of cosmetic products must have effective antimicrobial activity while meeting safety requirements for topical use. In this study, we determined the antimicrobial activity of 1,2-hexanediol against several Gram-positive and Gram-negative bacteria. Antimicrobial susceptibility tests have shown that 1,2-hexanediol exhibits broad-spectrum activity against Gram-positive and Gram-negative bacteria with MICs of 0·5-2% (v/v). The bactericidal concentration of 1,2-hexanediol was ranging from 1 to 2 × MIC as demonstrated by time-kill curve assay. A membrane depolarization assay showed that 1,2-hexanediol disrupted the cytoplasmic membrane potential. A checkerboard assay indicated that the effective concentration of 1,2-hexanediol was reduced up to 0·25-0·5 × MIC when combined with macelignan and octyl gallate against Gram-positive bacteria. However, this combination was not effective against Gram-negative bacteria. A turbidity reduction assay demonstrated that the combination of a high concentration of 1,2-hexanediol with food-grade antimicrobial compounds could trigger lytic activity towards Bacillus cereus cells. The remaining cell turbidity was 24·6 and 22·2% when 2% of 1,2-hexanediol was combined with 8 mg l(-1) octyl gallate or with 32 mg l(-1) macelignan respectively. This study showed that food-grade antimicrobial compounds may be used in combination with 1,2-hexanediol to increase its efficacy as a preservative agent in cosmetics. SIGNIFICANCE AND IMPACT OF THE STUDY: The antimicrobial activity of 1,2-hexanediol against Gram-positive and Gram-negative bacteria was potentiated with food-grade antimicrobials including xanthorrhizol, macelignan, panduratin A and octyl gallate, which have already been reported to display anti-inflammatory and other beneficial activities related to cosmetics. Therefore, the combination of 1,2-hexanediol and these food-grade antimicrobial agents would have benefits not only for increasing the antimicrobial activity but also in cosmetics use.

Relationship between lower urinary tract symptoms and metabolic syndrome in a Chinese male population.

OBJECTIVES: To investigate whether the metabolic syndrome (MetS) is a risk factor for lower urinary tract symptoms (LUTS), as defined by the International Prostate Symptom Score in a Chinese male population with benign prostate hyperplasia (BPH). METHODS: We retrospectively analyzed the clinical data obtained from 1,052 Chinese men with BPH. Serum levels of prostate specific antigen, fasting blood glucose (FBG), high-density lipoprotein cholesterol, total cholesterol and triglyceride were determined and recorded. Multiple logistic regression statistical analysis was used to investigate the degree of the association between LUTS and MetS. RESULTS: Of the 1,052 enrolled patients, 648 (61.60 %) had moderate LUTS and 404 (38.40 %) had severe LUTS. A multiple logistic regression analysis showed that age (OR 2.02, 95 % CI 1.04-4.63), FBG (OR 3.65, 95 % CI 1.68-7.98) and presence of MetS (OR 3.64, 95 % CI 1.24-6.14) were significant predictors of severe LUTS. CONCLUSIONS: The results of our study suggest that MetS is associated with an increase risk of total volume and annual growth rate of prostate.

DNA capturing machinery through spore-displayed proteins.

AIMS: The purpose of this study was to develop a general method for the facile development of a new DNA biosensor which utilizes streptavidin-displayed spores as a molecular machinery. METHODS AND RESULTS: Fluorescence spectroscopy was used as a monitoring tool for the streptavidin displayed on the surface of Bacillus thuringiensis spores and as a diagnosis method for DNA detection. As a proof-of-concept, four pathogenic bacteria including Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumonia were used for the detection of pathogenic species. In addition, a set of mutant variants of Wilson's disease were also used for the detection of single nucleotide polymorphism (SNP) in this system. CONCLUSIONS: This strategy, utilizing streptavidin-displayed spores, is capable of capturing DNA targets for the detection of pathogenic bacteria and for mutation analysis in Wilson's disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach could be useful as a simple platform for developing sensitive spore-based biosensors for any desired DNA targets in diagnostic applications.

Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae.

The ice-nucleation protein (Inp) is a glycosyl phosphatidylinositol-anchored outer membrane protein found in some Gram-negative bacteria. Using Pseudomonas syringae inp as an anchoring motif, we investigated the functional display of a foreign protein, Zymomonas mobilis levansucrase (LevU), on the surface of Escherichia coli. The cells expressing Inp-LevU were found to retain both the ice-nucleation and whole-cell levansucrase enzyme activities, indicating the functional expression of Inp-LevU hybrid protein on the cell surface. The surface localization was further verified by immunofluorescence microscopy, fluorescence-activated cell sorting flow cytometry and immunogold electron microscopical examination. No growth inhibition or changes in the outer membrane integrity were observed upon the induction of fusion protein synthesis. Viability of the cells was also maintained over 48 hours in the stationary phase. Surface-displayed levansucrases were found to be resistant to the externally added proteases unless the cells were treated with EDTA. When the levansucrase-displayed cells were used as the enzyme source, levan (44 g/L) was efficiently synthesized from sucrose (130 g/L) with 34% (wt/wt) conversion yield, generating glucose (65 g/L) as a by-product.

Surface-displayed viral antigens on Salmonella carrier vaccine.

We have developed a recombinant live oral vaccine using the ice-nucleation protein (Inp) from Pseudomonas syringae to display viral antigens on the surface of Salmonella spp. Fusion proteins containing viral antigens were expressed in the oral vaccine strain, Salmonella typhi Ty21a. Surface localization was verified by immunoblotting and fluorescence-activated cell sorting. The immunogenicity of surface-displayed viral antigens on the recombinant live vaccine strain was assessed in mice inoculated intranasally and intraperitoneally. Inoculation resulted in significantly higher serum antibody level than those induced by viral antigens expressed intracellularly. Thus, this multivalent mucosal live vaccine may provide an effective means for inducing mucosal or systemic immune responses against multiple viral antigens.

Performance of tPSA and f/tPSA for prostate cancer in Chinese. A systematic review and meta-analysis.

Performing a systematic review and meta-analysis to evaluate the diagnostic performance of the total prostate-specific antigen (tPSA) tests for diagnosis of prostate cancer among Chinese. Literatures related to diagnosis of prostate cancer by tPSA and the ratio of free to total PSA (f/tPSA) being retrieved in five electronic databases. Sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and diagnostic odds ratio (DOR) were pooled using random effects models. Summary receiver operating characteristic curves (SROC) used to summarize overall test performance. In total 10 studies meeting the inclusion criteria, 2256 patients were included. Among them 423 were patients of prostate cancer compared to 1833 were patients of benign prostate hyperplasia (BPH). The pooled sensitivity and specificity of tPSA>4.0 ng/ml, tPSA>10.0 ng/ml and f/tPSA<0.15 as threshold for the diagnosis of prostate cancer were heterogeneity. The areas under SROC (AUC) were 80, 85 and 87%, respectively (P<0.05). The pooled DOR were 8.44(95%CI: 4.45 approximately 16.00), 9.94(95%CI: 4.15 approximately 23.77) and 13.75(95%CI: 6.35 approximately 29.76), respectively (P<0.05). chi(2) test used for testing the heterogeneity. tPSA and f/tPSA made an important performance for diagnosis of prostate cancer in decade among Chinese. The use of f/tPSA<0.15 as threshold maintain a high prognostic accuracy for prostate cancer.

Involvement of iclR and rpoS in the induction of acs, the gene for acetyl coenzyme A synthetase of Escherichia coli K-12.

Two independent pathways in Escherichia coli convert acetate to acetyl CoA: reversal of acetate production by phosphotransacetylase and acetate kinase, and the acetyl-CoA synthetase (Acs) pathway that scavenges acetate. We investigated acs gene expression by using a cat transcriptional fusion. It was observed that acs expression varies depending on the carbon sources used and occurs in the stationary phase of growth even in the absence of acetate. Mutations in iclR for the repressor of the glyoxylate shunt and in rpoS for the stationary phase sigma factor reduced the consumption of acetate mediated by Acs, indicating that both are involved in acs regulation.

Expression of carboxymethylcellulase on the surface of Escherichia coli using Pseudomonas syringae ice nucleation protein.

Ice-nucleation protein (INP), an outer membrane protein from Pseudomonas syringae, is able to catalyze the ice crystal formation of supercooled water. It was exploited for anchoring of Bacillus subtilis carboxymethylcellulase (CMCase) on the surface of Escherichia coli. A surface anchoring vector, pGINP21M, was created that contains the multicloning sites including BamHI, SmaI and EcoRI at the end of the 3' flanking region encoding the C-terminus of INP instead of the stop codon for subcloning the foreign genes. The CMCase gene was in-frame subcloned for making INP-CMCase fusion proteins. The ability of this vector for directing the actual synthesis of INP-CMCase fusion proteins was confirmed by Western blotting analysis. CMCase targeted on the surface of cells was verified by measuring whole cell CMCase activity and ice-nucleation activity. CMCase activity was mainly detected on the cell surface whereas no enzyme activity was detected in the culture supernatant. Ice-nucleation activity was also maintained even if an INP-CMCase hybrid was made. This means that the fusion protein is functionally expressed and has its biological conformation on the surface. INP-CMCase fusion proteins were stable in the stationary phase. INP deleted of the repeating domain, thus producing no ice-nucleation activity, could also direct CMCase on the cell surface. This suggests that it has the secretion and targeting signal to the outer membrane.

[GC-MS analysis of the essential oil from the root of Ligusticum brachylobum Franch].

The constituents of the essential oil of the root of Ligusticum brachylobum have been analysed by gas chromatography-mass spectrometry qualitatively and gas chromatography quantitatively. Forty-five compounds were identified. The main constituent of the oil is alpha-pinene.

[Chemical studies on essential oils from 6 Artemisia species].

The constituents of the essential oils obtained from the leaves of Artemisia argyi, A. argyi cv.qiai, A. lavandulaefolia, A. mongolica, A. princeps and A. argyi var. gracilis were analysed by GC-MS. 96 compounds including alpha-thujene, 1,8-cineole, camphor and artemisia alcohol, etc. were identified. Their percentages in the oils were given.

[Studies on the essential oils of flos Magnoliae].

Essential oils were extracted from nine species of Flos Magnoliae. Their contents were determined. Analysis of chemical constituents in these oils was performed by means of GC-MS. 69 compounds were identified. The percentage of these components in oils were determined by GC.

[GC-MS analysis of essential oils from four Vitex species].

The chemical constituents of the essential oils obtained from the leaves of Vitex negundo var. cannabifolia, V. negundo var. heterophylla, V. negundo and V. trifolia were analysed by GC-MS. Forty compounds including alpha-pinene, linalool, terpinyl acetate, beta-caryophyllene and caryophyllene oxide, etc. were identified. Their percentages in oils were given.

[GC-MS analysis of essential oils from rhizomes of Atractylodes lancea (Thunb.) DC. and A. chinensis (DC.) Koidz].

OBJECTIVE: To analyze the constituents from the rhizomes of Atractylodes lancea and A. chinensis in essential oils. METHOD: GC-MS method was used. RESULT: 32 and 29 compounds were identified respectively. CONCLUSION: The main constituents in the essential oils from the rhizome of A. chinensis are beta-eudesmol or a mixture of beta-eudesmol and atractylone, whereas from that of A. lancea are hinesol, a mixture of beta-eudesmol and atractylone, and atractylone.

Biomass yields and energetic yields of oleaginous yeasts in batch culture.

This article provides the analysis on biomass yields and energetic yields of the oleaginous yeasts. The biomass yields of the oleaginous yeasts are consistently lower than nonoleaginous microorganisms, whereas their energetic yields are higher. Data inconsistencies of literature are explained by the variation of energy contents of oleaginous yeasts.

Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants.

The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.

Cloning and expression of a gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase from Erwinia cypripedii ATCC 29267 in Escherichia coli.

We have cloned the gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase (GADH) from Erwinia cypripedii ATCC 29267 in Escherichia coli by performing a direct-expression assay. The positive clone converted D-gluconate to 2-keto-D-gluconate (2KDG) in the culture medium. Nucleotide sequence analysis of the GADH clone revealed that the cloned fragment contained the complete structural genes for a 68-kDa dehydrogenase subunit, a 47-kDa cytochrome c subunit, and a 24-kDa subunit of unknown function and that the genes were clustered with the same transcriptional polarity. Comparison of the deduced amino acid sequences and the NH2-terminal sequences determined for the purified protein indicated that the dehydrogenase, cytochrome c, and 24-kDa subunits contained typical signal peptides of 22, 19, and 42 amino acids, respectively. The molecular masses of the processed subunits deduced from the nucleotide sequences (65, 45, and 20 kDa) coincided well with the molecular masses of subunits estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In E. cypripedii and recombinant E. coli, the GADH was constitutively formed and the activity of GADH was enhanced more than twofold by addition of D-gluconate to the medium. The holoenzyme glucose dehydrogenase of E. coli was reconstituted by addition of pyrroloquinoline quinone to the culture medium, and the conversion of D-glucose or D-gluconate to 2KDG by recombinant E. coli harboring the cloned GADH gene was attempted in batch culture. The conversion yields for D-glucose were 0.95 mol of 2KDG/mol of D-glucose after 16 h of cultivation, and those for D-gluconate were 0.95 mol of 2KDG/mol of D-gluconate after 12 h of cultivation.

Purification and characterization of the 2-ketoaldonate reductase from Brevibacterium ketosoreductum ATCC21914.

2-Ketoaldonate reductase, which is involved in ketogluconate catabolism, was purified to homogeneity from Brevibacterium ketosoreductum ATCC21914. The enzyme was found to catalyze the reduction of 2,5-diketo-D-gluconate to 5-keto-D-gluconate, and to a lesser extent, 2-keto-D-gluconate to D-gluconate, and 2-keto-L-gluconate to L-idonate. The molecular mass of the reductase was 35 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 72 kDa by gel filtration, indicating that the native enzyme may exist as a dimer. The reductase was optimally active at pH 6.0 with NADPH as a preferred electron donor. The pI of 4.7 was measured for the enzyme. The apparent Km for 2,5-diketo-D-gluconate and NADPH were 5 microM and 10 microM, respectively. The amino-terminal amino acid sequence was NH2-Ala-Ser-Ile-Ser-Val-Ser-Val-Pro-Ser-Ala- Arg-Leu-Ala-Glu-Asp-Leu-Ser-Asp-Ile-Glu.

Identification of the yqhE and yafB genes encoding two 2, 5-diketo-D-gluconate reductases in Escherichia coli.

The identification of a gene (yiaE) encoding 2-ketoaldonate reductase (2KR) in our previous work led to the hypothesis that Escherichia coli has other ketogluconate reductases including 2, 5-diketo-D-gluconate reductase (25DKGR) and to study of the related ketogluconate metabolism. By using the deduced amino acid sequences of 5-diketo-D-gluconate reductase (5KDGR) of Gluconobacter oxydans and 25DKGR of Corynebacterium sp., protein databases were screened to detect homologous proteins. Among the proteins of E. coli, an oxidoreductase encoded by yjgU and having 56% similarity to 5KDGR of G. oxydans and two hypothetical oxidoreductases encoded by yqhE and yafB and having 49.8 and 42% similarity, respectively, to 25DKGR of Corynebacterium sp. were detected. Recently, the yjgU gene was identified as encoding 5KDGR and renamed idnO (C. Bausch, N. Peekhaus, C. Utz, T. Blais, E. Murray, T. Lowary, and T. Conway, J. Bacteriol. 180:3704-3710, 1998). The pathways involved in the metabolism of ketogluconate by E. coli have been predicted by biochemical analysis of purified enzymes and chemical analysis of the pathway intermediates. The gene products of yqhE and yafB were identified as 25DKGR-A, and 25DKGR-B, respectively, catalyzing the reduction of 25KDG to 2-keto-L-gulonate (2KLG). The native 25DKGR-A, 25DKGR-B, and 5KDGR had apparent molecular weights of about 30,000, 30,000, and 54,000, respectively. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, all three enzymes showed protein bands with a molecular weight of about 29,000, which indicated that 25DKGR-A, 25DKGR-B, and 5KDGR may exist as monomeric, monomeric, and dimeric proteins, respectively. The optimum pHs for reduction were 7.5, 7.0, and 8.0, respectively. The 5KDGR was active with NADH, whereas 25DKGR-A and 25DKGR-B were active with NADPH as a preferred electron donor. 25DKG can be converted to 5KDG by 2KR, which is then reduced to D-gluconate by 5KDGR. The pathways were compared with those of Erwinia sp. and Corynebacterium sp. A BLAST search of published and incomplete microbial genome sequences revealed that the ketogluconate reductases and their related metabolism may be widespread in many species.