Glycosylation status of bone sialoprotein and its role in mineralization.

The highly glycosylated bone sialoprotein (BSP) is an abundant non-collagenous phosphoprotein in bone which enhances osteoblast differentiation and new bone deposition in vitro and in vivo. However, the structural details of its different glycosylation linkages have not been well studied and their functions in bone homeostasis are not clear. Previous studies suggested that the O-glycans, but not the N-glycans on BSP, are highly sialylated. Herein, we employed tandem mass spectrometry (MS/MS) to demonstrate that the N-glycanson the recombinant human integrin binding sialoprotein (rhiBSP) are also enriched in sialic acids (SAs) at their termini. We also identified multiple novel sites of N-glycan modification. Treatment of rhiBSP enhances osteoblast differentiation and mineralization of MC3T3-E1 cells and this effect could be partially reversed by efficient enzymatic removal of its N-glycans. Removal of all terminal SAs has a greater effect in reversing the effect of rhiBSP on osteogenesis, especially on mineralization, suggesting that sialylation at the termini of both N-glycans and O-glycans plays an important role in this regulation. Moreover, BSP-conjugated SAs may affect mineralization via ERK activation of VDR expression. Collectively, our results identified novel N-glycans enriched in SAs on the rhiBSP and demonstrated that SAs at both N- and O-glycans are important for BSP regulation of osteoblast differentiation and mineralization in vitro.

Profiling analysis of low molecular weight heparins by multiple heart-cutting two dimensional chromatography with quadruple time-of-flight mass spectrometry.

Low molecular weight heparins (LMWHs) are polydisperse and microheterogenous mixtures of polysaccharides used as anticoagulant drugs. Profiling analysis is important for obtaining deeper insights into the structure of LMWHs. Previous oligosaccharide mapping methods are relatively low resolution and are unable to show an entire picture of the structural complexity of LMWHs. In the current study a profiling method was developed relying on multiple heart-cutting, two-dimensional, ultrahigh performance liquid chromatography with quadruple time-of-flight mass spectrometry. This represents an efficient, automated, and robust approach for profiling LMWHs. Using size-exclusion chromatography and ion-pairing reversed-phase chromatography in a two-dimensional separation, LMW components of different sizes and LMW components of the same size but with different charges and polarities can be resolved, providing a more complete picture of a LMWH. Structural information on each component was then obtained with quadrupole time-of-flight mass spectrometry. More than 80 and 120 oligosaccharides were observed and unambiguously assigned from the LMWHs, nadroparin and enoxaparin, respectively. This method might be useful for quality control of LMWHs and as a powerful tool for heparin-related glycomics.

Role of Fas-associated death domain-containing protein (FADD) phosphorylation in regulating glucose homeostasis: from proteomic discovery to physiological validation.

Fas-associated death domain-containing protein (FADD), a classical apoptotic signaling adaptor, participates in different nonapoptotic processes regulated by its phosphorylation. However, the influence of FADD on metabolism, especially glucose homeostasis, has not been evaluated to date. Here, using both two-dimensional electrophoresis and liquid chromatography linked to tandem mass spectrometry (LC/MS/MS), we found that glycogen synthesis, glycolysis, and gluconeogenesis were dysregulated because of FADD phosphorylation, both in MEFs and liver tissue of the mice bearing phosphorylation-mimicking mutation form of FADD (FADD-D). Further physiological studies showed that FADD-D mice exhibited lower blood glucose, enhanced glucose tolerance, and increased liver glycogen content without alterations in insulin sensitivity. Moreover, investigations on the molecular mechanisms revealed that, under basal conditions, FADD-D mice had elevated phosphorylation of Akt with alterations in its downstream signaling, leading to increased glycogen synthesis and decreased gluconeogenesis. Thus, we uncover a novel role of FADD in the regulation of glucose homeostasis by proteomic discovery and physiological validation.

Cumulative effects of the ApoE genotype and gender on the synaptic proteome and oxidative stress in the mouse brain.

Elderly females, particularly those carrying the apolipoprotein E (ApoE)-ε4 allele, have a higher risk of developing Alzheimer's disease (AD). However, the underlying mechanism for this increased susceptibility remains unclear. In this study, we investigated the effects of the ApoE genotype and gender on the proteome of synaptosomes. We isolated synaptosomes and used label-free quantitative proteomics, to report, for the first time, that the synaptosomal proteomic profiles in the cortex of female human-ApoE4 mice exhibited significantly reduced expression of proteins related to energy metabolism, which was accompanied by increased levels of oxidative stress. In addition, we also first demonstrated that the proteomic response in synaptic termini was more susceptible than that in the soma to the adverse effects induced by genders and genotypes. This suggests that synaptic mitochondria might be 'older' than mitochondria in the soma of neurons; therefore, they might contain increased cumulative damage from oxidative stress. Furthermore, female human-ApoE4 mice had much lower oestrogen levels in the cortex and treatment with oestrogen protected ApoE3 stable transfected C6 neurons from oxidative stress. Overall, this study reveals complex ApoE- and gender-dependent effects on synaptic function and also provides a basis for future studies of candidates based on specific pathways involved in the pathogenesis of AD. The lack of oestrogen-mediated protection regulated by the ApoE genotype led to synaptic mitochondrial dysfunction and increased oxidative stress, which might make older females more susceptible to AD.

Gastrointestinal manifestations of critical ill heatstroke patients and their associations with outcomes: A multicentre, retrospective, observational study.

BACKGROUND: Extreme heat exposure is a growing health problem, and the effects of heat on the gastrointestinal (GI) tract is unknown. This study aimed to assess the incidence of GI symptoms associated with heatstroke and its impact on outcomes. AIM: To assess the incidence of GI symptoms associated with heatstroke and its impact on outcomes. METHODS: Patients admitted to the intensive care unit (ICU) due to heatstroke were included from 83 centres. Patient history, laboratory results, and clinically relevant outcomes were recorded at ICU admission and daily until up to day 15, ICU discharge, or death. GI symptoms, including nausea/vomiting, diarrhoea, flatulence, and bloody stools, were recorded. The characteristics of patients with heatstroke concomitant with GI symptoms were described. Multivariable regression analyses were performed to determine significant predictors of GI symptoms. RESULTS: A total of 713 patients were included in the final analysis, of whom 132 (18.5%) patients had at least one GI symptom during their ICU stay, while 26 (3.6%) suffered from more than one symptom. Patients with GI symptoms had a significantly higher ICU stay compared with those without. The mortality of patients who had two or more GI symptoms simultaneously was significantly higher than that in those with one GI symptom. Multivariable logistic regression analysis revealed that older patients with a lower GCS score on admission were more likely to experience GI symptoms. CONCLUSION: The GI manifestations of heatstroke are common and appear to impact clinically relevant hospitalization outcomes.

Increased hippocampal tau phosphorylation and axonal mitochondrial transport in a mouse model of chronic stress.

Corticotropin-releasing hormone (CRH) is considered the driving force of the hypothalamo-pituitary-adrenal (HPA) axis and plays an important role in mood regulation. The HPA axis is reported to be closely related to acute stress-induced tau phosphorylation in the rodent hippocampus. However, the relationship between the hyperactive HPA axis and tau phosphorylation in the hippocampus and hence the functional implications for chronic stress are not fully understood. In this study, we aimed to examine tau phosphorylation and the effect on axonal transport of mitochondria in the hippocampus of a chronic stress model. A mouse model was created by neonatal isolation before weaning, followed by chronic mild stress by social isolation after weaning. Behavioural tests showed that the model had a typical depression/anxiety-like behaviour accompanied by increased plasma corticosterone level and hypothalamic CRH mRNA expression. Phosphorylated tau increased significantly, accompanied by increased synaptosomal mitochondrial levels in hippocampus of the chronic stress model. CRH receptor 1 antagonist (CP154,526) treatment, not glucocorticoid receptor antagonist (RU486) treatment, decreased tau phosphorylation and synaptosomal mitochondrial levels in the hippocampus of the mouse model. Consistent with an in-vivo model, when hyperphosphorylated tau was inhibited by lithium in cultured primary hippocampal neurons, mitochondrial transport monitored by live imaging was also decreased. We show here for the first time that phosphorylated tau in the hippocampus of a chronic stress model, accompanied by increased mitochondrial transport, was mediated by CRH receptor 1, not by glucocorticoid receptors, which suggests that centrally derived CRH may be involved in the process of mitochondrial axon transport and hence play an important role in hippocampus of a chronic stress model.

Pharmacokinetics, distribution, and excretion of sodium oligomannate, a recently approved anti-Alzheimer's disease drug in China.

The National Medical Products Administration has authorized sodium oligomannate for treating mild-to-moderate Alzheimer's disease. In this study, an LC-MS/MS method was developed and validated to quantitate sodium oligomannate in different biomatrices. The plasma pharmacokinetics, tissue distribution, and excretion of sodium oligomannate in Sprague-Dawley rats and beagle dogs were systematically investigated. Despite its complicated structural composition, the absorption, distribution, metabolism, and excretion profiles of the oligosaccharides in sodium oligomannate of different sizes and terminal derivatives were indiscriminate. Sodium oligomannate mainly crossed the gastrointestinal epithelium through paracellular transport following oral administration, with very low oral bioavailability in rats (0.6%-1.6%) and dogs (4.5%-9.3%). Absorbed sodium oligomannate mainly resided in circulating body fluids in free form with minimal distribution into erythrocytes and major tissues. Sodium oligomannate could penetrate the blood-cerebrospinal fluid (CSF) barrier of rats, showing a constant area under the concentration-time curve ratio (CSF/plasma) of approximately 5%. The cumulative urinary excretion of sodium oligomannate was commensurate with its oral bioavailability, supporting that excretion was predominantly renal, whereas no obvious biliary secretion was observed following a single oral dose to bile duct-cannulated rats. Moreover, only 33.7% (male) and 26.3% (female) of the oral dose were recovered in the rat excreta within 96 h following a single oral administration, suggesting that the intestinal flora may have ingested a portion of unabsorbed sodium oligomannate as a nutrient.

Electrochemiluminescence of terbium (III)-two fluoroquinolones-sodium sulfite system in aqueous solution.

The electrochemiluminescence (ECL) of Tb3+-enoxacin-Na2SO3 system (ENX system) and Tb3+-ofloxacin-Na2SO3 system (OFLX system) in aqueous solution is reported. ECL is generated by the oxidation of Na2SO3, which is enhanced by Tb3+-fluoroquinolone (FQ) complex. The ECL intensity peak versus potential corresponds to oxidation of Na2SO3, and the ECL emission spectra (the peaks are at 490, 545, 585 and 620 nm) match the characteristic emission spectrum of Tb3+, indicating that the emission is from the excited state of Tb3+. The mechanism of ECL is proposed and the difference of ECL intensity between ENX system and OFLX system is explained. Conditions for ECL emission were optimized. The linear range of ECL intensity versus concentrations of pharmaceuticals is 2.0 x 10(-10) -8.0 x 10(-7)mol l(-1) for ENX and 6.0 x 10(-10) -6.0 x 10(-7)mol l(-1) for OFLX, respectively. A theoretical limit of detection is 5.4 x 10(-11)mol l(-1) for ENX and 1.6 x 10(-10)mol l(-1) for OFLX, respectively. The ECL was satisfactorily applied to the determination of the two FQs in dosage form and urine sample.

Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients.

Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.

Analysis of magnetic elements in otoliths of the macula lagena in homing pigeons with inductively coupled plasma mass spectrometry.

OBJECTIVE: The macula lagena in birds is located at the apical end of the cochlea and contains many tiny otoliths. The macula lagena is innervated and has neural projections to the brainstem, but its physiological function is still unclear. It remains disputable that it is because otoliths in the lagena are rich in elements Fe and Zn that birds can obtain geomagnetic information for homing. To clarify this issue, we carried out a study to determine whether or not otoliths in the lagena of homing pigeons are richer in magnetic elements than those in the saccule and the utricle. METHODS: The contents of ferromagnetic elements (Fe, Co, Ni) and other metal elements in lagenal otoliths of adult homing pigeons were precisely analyzed with inductively coupled plasma mass spectrometry (ICP-MS) of high sensitivity, and then they were compared with those in saccular and utricular otoliths (all the contents were normalized to Ca). RESULTS: In adult homing pigeons, the contents of ferromagnetic elements (Fe, Co, Ni) in lagenal otoliths were less than 0.7% (normalized to Ca element) and were the same order in magnitude as those in saccular and utricular otoliths. The content of Fe in lagenal otoliths was not significantly different from that in utricular otoliths and was even lower than that in saccular otoliths. The content of Co in lagenal otoliths was lower than that in saccular otoliths and higher than that in utricular otoliths. The content of Ni in lagenal otoliths was not significantly different from that in saccular otoliths and was higher than that in utricular otoliths. The contents of other metal elements Na, Mg, K, Al, Mn and Pb in lagenal otoliths were not significantly different from those in utricular and saccular otoliths. The contents of metal elements Zn, Ba and Cu in lagenal otoliths were lower than those in saccular otoliths. CONCLUSION: The contents of magnetic elements in lagenal otoliths of homing pigeons are not much higher than those in utricular and saccular otoliths, which does not support the hypothesis that birds depend on high contents of Fe and Zn in lagenal otoliths for sensation of geomagnetic information. Similarities in morphology, element ingredient and element content between lagenal otoliths and utricular otoliths suggest that the two types of otolithic organs may play similar roles in sensing gravitational and acceleration signals.

[Introduction on a forecasting model for infectious disease incidence rate based on radial basis function network].

It is important to forecast incidence rates of infectious disease for the development of a better program on its prevention and control. Since the incidence rate of infectious disease is influenced by multiple factors, and the action mechanisms of these factors are usually unable to be described with accurate mathematical linguistic forms, the radial basis function (RBF) neural network is introduced to solve the nonlinear approximation issues and to predict incidence rates of infectious disease. The forecasting model is constructed under data from hepatitis B monthly incidence rate reports from 1991-2002. After learning and training on the basic concepts of the network, simulation experiments are completed, and then the incidence rates from Jan. 2003-Jun. 2003 forecasted by the established model. Through comparing with the actual incidence rate, the reliability of the model is evaluated. When comparing with ARIMA model, RBF network model seems to be more effective and feasible for predicting the incidence rates of infectious disease, observed in the short term.

Insights into photodissociation dynamics of propionyl chloride from ab initio calculations and molecular dynamics simulations.

The potential energy surfaces of isomerization, dissociation, and elimination reactions for CH3CH2COCl in the S0 and S1 states have been mapped with the different ab initio calculations. Mechanistic photodissociation of CH3CH2COCl at 266 nm has been characterized through the computed potential energy surfaces, the optimized surface crossing structure, intrinsic reaction coordinate, and ab initio molecular dynamics calculations. Photoexcitation at 266 nm leads to the CH3CH2COCl molecules in the S1 state. From this state, the C-Cl bond cleavage proceeds in a time scale of picosecond in the gas phase. The barrier to the C-Cl bond cleavage on the S1 surface is significantly increased by effects of the matrix and the internal conversion to the ground state prevails in the condensed phase. The HCl eliminations as a result of internal conversion to the ground state become the dominant channel upon photodissociation of CH3CH2COCl in the argon matrix at 10 K.

[Study on the factors influencing the quality of life among patients with malignant lymphoma].

OBJECTIVE: To measure and assess the quality of life (QOL) and to explore the influencing factors on patients with malignant lymphoma. METHODS: QOL of 110 patients with malignant lymphoma were marked using EORTC QLQ-C30 short form, and multiple linear regression models were used to study the main factors influencing the QOL of patients with malignant lymphoma on five functional scales (physical, role, cognitive, emotional, and social) and the total scores. RESULTS: The influencing factors of quality of life on patients with malignant lymphoma appeared to be: history of relapse, refraining from smoking, older age, educational level, space for living, exercises, medical care system, and available health care programs. Relapse (beta = 5.997, P= 0.020) and refraining from smoking (beta = -6.526, P= 0.006) were associated with total QOL scores, educational level (beta = -2.144, P= 0.057), History of relapse (beta = 5.857, P = 0.003) was associated with total functional scales while exercises (beta= -0.771, P = 0.097) and refraining from smoking (beta= -4.106, P = 0.005) were with physical scales, refraining from smoking (beta = -4.644,P = 0.008) and older age (beta = 0.989, P= 0.029) were with role scales, relapse (beta = 14.035, P= 0.001) and older age (beta = 2.230, P= 0.023) were with cognitive scales, relapse (beta = 8.500, P= 0.031) and living space (beta = - 3.054, P= 0.0901) were with emotional scales and medical care system and available health care programs (beta = -6.577, P= 0.018) were with social scales respectively. CONCLUSION: Factors as prevention of relapse, correct cognition on malignant lymphoma, reasonable exercise, refrain from bad habits, improving medical care system could all increase the functions of malignant lymphoma patient, and to improve their quality of life.

Determination of norfloxacin using a terbium-sensitized electrogenerated chemiluminescence method.

A simple electrogenerated chemiluminescence (ECL) analysis method for the determination of norfloxacin (NFLX) is reported. It is based on ECL produced by Na(2)SO(3), which is sensitized by the Tb-NFLX complex. The relative ECL intensity of the Tb(3+)-NFLX-Na(2)SO(3) system is proportional to the amount of NFLX. The optimized experimental conditions were investigated. The linear range and detection limit for NFLX were 1.0 x 10(-10)-8.0 x 10(-7) mol/L and 2.8 x 10(-11) mol/L, respectively. This method was successfully applied to the determination of NFLX in a capsule. NFLX in urine can be directly detected without pretreatment or separation.

[Study on the relations between serum insulin-like growth factor-1, insulin-like growth factor binding protein-3 and colorectal cancer: a meta-analysis].

OBJECTIVE: To evaluate the relationship between circulating levels of insulin-like growth factor-1 (IGF-1), IGF-binding protein-3 (IGFBP-3) and colorectal cancer. METHODS: A meta-analysis of 6 epidemiological studies on insulin-like growth factors and risk of colorectal cancer were performed. RESULTS: The pooled odds ratio (OR) of IGF-1 and IGFBP-3 were 1.56 (95% CI: 1.14-2.13) and 0.78 (95% CI: 0.43-1.44) respectively. According to the results from different measurements (enzyme-linked immunoabsorbent assay and immunoradiometric assay), the pooled OR were 1.92 and 1.23 for IGF-1, 0.46 and 1.44 for IGFBP-3 respectively. CONCLUSION: High serum levels of IGF-1 were independent risk factors of colorectal cancer but the OR of IGFBP-3 was not statistically significant. The heterogeneity between studies on IGFBP-3 and colorectal cancer was caused by different measurements used, but there was still a need to conduct simultaneous large size study under 2 different measurements for further conclusion.

[Study on the application of artificial neural network on diabetes mellitus/insulin-glucose tolerance classification].

OBJECTIVE: To discuss the potential application of artificial neural network (ANN) on the epidemiological classification of disease. METHODS: Learning vector quantification neural network (LVQNN) and discriminate analysis were applied to data from epidemiological survey in a mine in 1996. RESULTS: The structure of LVQNN was 25-->13-->3. The total veracity rates was 96.98%, and 92.45% among the abnormal blood glucose individuals. Through stepwise discriminate analysis, the discriminate equations were established including 11 variables with a total veracity rate of 87.34%, but was 85.53% in the abnormal blood glucose individuals. Further analysis on 30 cases with missing values showed that the disagreement ratio of LVQ was 1/30, lower than that of discriminate analysis of 7/30. CONCLUSIONS: Compared to the conventional statistics method, LVQ not only showed better prediction precision, but could treat data with missing values satisfactorily plus it had no limit to the type or distribution of relevant data, thus provided a new powerful method to epidemiologic prediction.

Proteomic analysis of SARS associated coronavirus using two-dimensional liquid chromatography mass spectrometry and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by mass spectroemtric analysis.

The proteomes of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and its infected Vero E6 cells were detected in the present study. The cytosol and nucleus fractions of virus-infected cells as well as the crude virions were analyzed either by one-dimensional electrophoresis followed by ESI-MS/MS identification or by shotgun strategy with two-dimensional liquid chromatography-ESI-MS/MS. For the first time, all of the four predicted structural proteins of SARS-CoV were identified, including S (Spike), M (Membrane), N (Nucleocapsid), and E (Envolope) proteins. In addition, a novel phosphorylated site of M protein was observed. The combination of these gel-base and non-gel methods provides fast and complimentary approaches to SARS-CoV proteome and can be widely used in the analysis of other viruses.