[Establishment of clinical features and prognostic scoring model in early-stage hepatitis B-related acute-on-chronic liver failure].

Objective: To explore the clinical characteristics and establish a corresponding prognostic scoring model in patients with early-stage clinical features of hepatitis B-induced acute-on-chronic liver failure (HBV-ACLF). Methods: Clinical characteristics of 725 cases with hepatitis B-related acute-on-chronic hepatic dysfunction (HBV-ACHD) were retrospectively analyzed using Chinese group on the study of severe hepatitis B (COSSH). The independent risk factors associated with 90-day prognosis to establish a prognostic scoring model was analyzed by multivariate Cox regression, and was validated by 500 internal and 390 external HBV-ACHD patients. Results: Among 725 cases with HBV-ACHD, 76.8% were male, 96.8% had cirrhosis base,66.5% had complications of ascites, 4.1% had coagulation failure in respect to organ failure, and 9.2% had 90-day mortality rate. Multivariate Cox regression analysis showed that TBil, WBC and ALP were the best predictors of 90-day mortality rate in HBV-ACHD patients. The established scoring model was COSS-HACHADs = 0.75 × ln(WBC) + 0.57 × ln(TBil)-0.94 × ln(ALP) +10. The area under the receiver operating characteristic curve (AUROC) of subjects was significantly higher than MELD, MELD-Na, CTP and CLIF-C ADs(P < 0.05). An analysis of 500 and 390 cases of internal random selection group and external group had similar verified results. Conclusion: HBV-ACHD patients are a group of people with decompensated cirrhosis combined with small number of organ failure, and the 90-day mortality rate is 9.2%. COSSH-ACHDs have a higher predictive effect on HBV-ACHD patients' 90-day prognosis, and thus provide evidence-based medicine for early clinical diagnosis and treatment.

A multiplexed, indirect enzyme-linked immunoassay for the detection and differentiation of E. coli from other Enterobacteriaceae and P. aeruginosa from other glucose non-fermenters.

Gram-negative bacteria (GNB) are important causes of community (CA) and hospital (HA)- associated infections. Here we describe the development of an indirect ELISA (I-ELISA), which can be used to detect and differentiate the Enterobacteriaceae Escherichia coli, and glucose non-fermenter Pseudomonas aeruginosa from other GNB species. The I-ELISA utilizes six antibodies for bacterial speciation, which were grouped according to their bacterial targets; Enterobacteriaceae (SL-EntA and CH1810 mAb), Escherichia coli (SL-EcA and 6103-46 mAb), Pseudomonas aeruginosa (SL-PaA and SL-PaB). The six, anti-GNB antibodies were first screened against a panel of well-characterized clinical GNB isolates to optimize assay conditions and to determine individual antibody sensitivity and specificity. When tested against a diverse, blinded panel of 94 GNB clinical isolates, the I-ELISA exhibited the following sensitivity/specificity for each target: Enterobacteriaceae (94.4%/95%), E. coli (82.6%/88.7%), P. aeruginosa (83.3%/96%). An I-ELISA to detect and differentiate the most common GNB pathogens offers advantage in terms of simplicity over diagnostic tests currently used in most clinical settings.

Exploring the recognized bio-mimicry materials for gas sensing.

This study was undertaken to synthesize peptides that are partially similar to the binding sites of human olfactory receptor protein. First, a putative 3-D model structure of human olfactory receptor protein (P30953) was modeled using a molecular simulation method. The computer docking simulation was then performed to determine the most plausible binding sites between the model structure and target gases, trimethylamine, ammonia, acetic acid, and o-xylene. According to the simulation result, a series of polypeptide sequences, horp61 for TMA, horp103 for o-xylene, horp109 for ammonia, and horp193 for acetic acid as recognized molecules were designed for gas sensing purposes. Preparing these peptides as corresponding gas sensing probes, the results showed a high relative sensitivity response of 6.7 for TMA (probe horp61), 5.1 for o-xylene (probe horp103), 11 for ammonia (probe horp109), and 28 for acetic acid (probe horp193), respectively. These results indicate that peptide mimicking of binding domain on olfactory receptor opens a new window and offers a novel strategy for the further development of recognized materials for gas sensing.

Synthetic peptide mimicking of binding sites on olfactory receptor protein for use in 'electronic nose'.

A putative tertiary structure model of the dog's olfactory receptor (olfd canfa) is established in this study. By using a target odorous compound (trimethylamine), it is possible to locate the most plausible binding sites between the receptor model structure and the target odorous molecules through computer docking simulations. The two short oligo-peptide sequences (orp61 and orp188) for trimethylamine sensing were identified, synthesized, purified and coated onto the surface of the separate piezoelectric gold electrodes. These two peptides show a high binding capability for trimethylamine. To further enhance the sensitivity of the polypeptides towards the target compound, the polarity and the degree of docking were changed by a site-specific modification technique. The orp61 sequence was modified by substituting two amino acids in the binding pocket resulting in 33% increase in sensitivity towards trimethylamine and reduced noises from other non-target chemicals. The techniques used in the present study offer a unique approach for synthesizing peptides in mimicking binding domain of olfactory receptors. The approach can be easily applied to further development of recognized molecules for gas sensing, especially for use in 'electronic noses'.

Expression of capsid [correction of caspid] protein VP1 for use as antigen for the diagnosis of enterovirus 71 infection.

To produce enterovirus 71 antigen for diagnostic purposes, the gene encoding the entire capsid protein VP1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and expressed in Escherichia coli as a poly-histidine fusion protein. Western blotting experiments with sera from patients with enterovirus 71 infection indicated that immunoglobulin G (IgG) and IgM antibodies bound to a single polypeptide VP1. According to these results, IgM anti-VP1 appeared in sera of patients with a symptomatic enterovirus 71 acute infection, whereas IgG anti-VP1 was present in sera of past infection. This finding suggests that detecting IgG and IgM immune responses against linear epitopes of recombinant VP1 is an effective means of determining the different phases of enterovirus 71 infection. In addition, sera containing coxsackie virus 16 (CA16) antibodies did not cross-react with the recombinant VP1 of enterovirus 71, despite the homology between VP1 proteins of both viruses. Comparison with reference PCR and neutralization assays showed these antibody tests to be appropriate for the serodiagnosis of enterovirus 71 infection.

Purification and characterization of neutral sphingomyelinase from Helicobacter pylori.

Phospholipase activities of human gastric bacterium, Helicobacter pylori, are regarded as the pathogenic factors owing to their actions on epithelial cell membranes. In this study, we purified and characterized neutral sphingomyelinase (N-SMase) from the superficial components of H. pylori strains for the first time. N-SMase was purified 2083-fold with an overall recovery of 37%. The purification steps included acid glycine extraction, ammonium sulfate precipitation, CM-Sepharose, Mono-Q, and Sephadex G-75 column chromatography. Approximate molecular mass for the native N-SMase was around 32 kDa. When N-omega-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM) was used as a substrate, the purified enzyme exhibited a K(m) of 6.7 microM and a V(max) of 15.6 nmol of TNPAL-sphingosine/h/mg of protein at 37 degrees C in 50 mM phosphate-buffered saline, pH 7.4. N-SMase reaches optimal activity at pH 7.4 and has a pI of 7.15. The enzyme activity is magnesium dependent and specifically hydrolyzed sphingomyelin and phosphatidylethanolamine. The enzyme also exhibits hemolytic activity on human erythrocytes. According to Western blot analysis, a rabbit antiserum against purified N-SMase from H. pylori cross-reacted with SMase from Bacillus cereus. Sera from individuals with H. pylori infection but not uninfected ones recognizing the purified N-SMase indicated that it was produced in vivo. In enzyme-linked immunosorbent assays, the purified N-SMase used as an antigen was as effective as crude protein antigens in detecting human antibodies to H. pylori.

A piezoelectric biosensor as an olfactory receptor for odour detection: electronic nose.

Humans can detect and differentiate the presence of different odours even at trace levels of these odorous compounds. The odour quantification of any particular samples is normally based on conventional panel decisions. Other analytical instruments could be used to detect trace levels of odorous molecules. This study presents the results of a biological sensor system subject to different odorants. The system consists of a sensor in which the isolated olfactory receptor proteins (ORPs) from bullfrogs (Rana spp.) were coated onto the surface of a piezoelectric (PZ) electrode, similar to the mechanism of human olfaction. The PZ crystal served as a signal transducer. The results indicate rapid (about 400 s), reversible, and longterm (up to 3 months) stable responses to different volatile compounds such as n-caproic acid, isoamyl acetate, n-decyl alcohol, beta-ionone, linalool, and ethyl caporate. The sensitivity of the sensor ranges from 10(-6)-10(-7) g, fully correlated with the olfactory threshold values of human noses. An array of six sensors consisting of five fractionated ORPs and one referenced phospholipid probe is able to respond to different odorants and form a typical fingerprint for each odorant.

Gene probe coated piezoelectric biosensors for biochemical analysis.

A new method has been developed to monitor DNA hybridization by using piezoelectric (PZ) crystal biosensor system. AT-cut crystals, with a basic resonant frequency of 9 MHz, were modified by a series of surface pretreatment as followings: gold plated, anoidical oxidation and gamma-aminopropyltriethoxysilane treatment. Single stranded DNA probe was electro-blotted onto the surface of crystal electrodes. The DNA probe was further fixed on the crystal surface by UV crosslinking. The crystal sensor is dipped into the hybridization solution and the surface mass increase, caused by hybridization, is measured by the decrease in the resonant frequency of the crystal. The potential capability to obtain qualitative as well as quantitative information on a sample through such an DNA hybridization assay makes this technique an attractive alternative to conventional analytical techniques that require use of radio-isotope.