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1.  Novel Strategy for Typing Mycoplasma pneumoniae Isolates by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Coupled with ClinProTools 
Journal of Clinical Microbiology  2014;52(8):3038-3043.
The typing of Mycoplasma pneumoniae mainly relies on the detection of nucleic acid, which is limited by the use of a single gene target, complex operation procedures, and a lengthy assay time. Here, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) coupled to ClinProTools was used to discover MALDI-TOF MS biomarker peaks and to generate a classification model based on a genetic algorithm (GA) to differentiate between type 1 and type 2 M. pneumoniae isolates. Twenty-five M. pneumoniae strains were used to construct an analysis model, and 43 Mycoplasma strains were used for validation. For the GA typing model, the cross-validation values, which reflect the ability of the model to handle variability among the test spectra and the recognition capability value, which reflects the model's ability to correctly identify its component spectra, were all 100%. This model contained 7 biomarker peaks (m/z 3,318.8, 3,215.0, 5,091.8, 5,766.8, 6,337.1, 6,431.1, and 6,979.9) used to correctly identify 31 type 1 and 7 type 2 M. pneumoniae isolates from 43 Mycoplasma strains with a sensitivity and specificity of 100%. The strain distribution map and principle component analysis based on the GA classification model also clearly showed that the type 1 and type 2 M. pneumoniae isolates can be divided into two categories based on their peptide mass fingerprints. With the obvious advantages of being rapid, highly accurate, and highly sensitive and having a low cost and high throughput, MALDI-TOF MS ClinProTools is a powerful and reliable tool for M. pneumoniae typing.
PMCID: PMC4136174  PMID: 24920781
2.  Classifying human operator functional state based on electrophysiological and performance measures and fuzzy clustering method 
Cognitive Neurodynamics  2013;7(6):477-494.
The human operator’s ability to perform their tasks can fluctuate over time. Because the cognitive demands of the task can also vary it is possible that the capabilities of the operator are not sufficient to satisfy the job demands. This can lead to serious errors when the operator is overwhelmed by the task demands. Psychophysiological measures, such as heart rate and brain activity, can be used to monitor operator cognitive workload. In this paper, the most influential psychophysiological measures are extracted to characterize Operator Functional State (OFS) in automated tasks under a complex form of human–automation interaction. The fuzzy c-mean (FCM) algorithm is used and tested for its OFS classification performance. The results obtained have shown the feasibility and effectiveness of the FCM algorithm as well as the utility of the selected input features for OFS classification. Besides being able to cope with nonlinearity and fuzzy uncertainty in the psychophysiological data it can provide information about the relative importance of the input features as well as the confidence estimate of the classification results. The OFS pattern classification method developed can be incorporated into an adaptive aiding system in order to enhance the overall performance of a large class of safety–critical human–machine cooperative systems.
PMCID: PMC3825145  PMID: 24427221
Operator functional state; Fuzzy c-means algorithm; Psychophysiological measures; Feature extraction; Pattern classification
3.  The Construction and Evaluation of Reference Spectra for the Identification of Human Pathogenic Microorganisms by MALDI-TOF MS 
PLoS ONE  2014;9(9):e106312.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique for the rapid and high-throughput identification of microorganisms. There remains a dearth of studies in which a large number of pathogenic microorganisms from a particular country or region are utilized for systematic analyses. In this study, peptide mass reference spectra (PMRS) were constructed and evaluated from numerous human pathogens (a total of 1019 strains from 94 species), including enteric (46 species), respiratory (21 species), zoonotic (17 species), and nosocomial pathogens (10 species), using a MALDI-TOF MS Biotyper system (MBS). The PMRS for 380 strains of 52 species were new contributions to the original reference database (ORD). Compared with the ORD, the new reference database (NRD) allowed for 28.2% (from 71.5% to 99.7%) and 42.3% (from 51.3% to 93.6%) improvements in identification at the genus and species levels, respectively. Misidentification rates were 91.7% and 57.1% lower with the NRD than with the ORD for genus and species identification, respectively. Eight genera and 25 species were misidentified. For genera and species that are challenging to accurately identify, identification results must be manually determined and adjusted in accordance with the database parameters. Through augmentation, the MBS demonstrated a high identification accuracy and specificity for human pathogenic microorganisms. This study sought to provide theoretical guidance for using PMRS databases in various fields, such as clinical diagnosis and treatment, disease control, quality assurance, and food safety inspection.
PMCID: PMC4152241  PMID: 25181391
4.  Clinical features and outcome of acute hepatitis B in pregnancy 
BMC Infectious Diseases  2014;14:368.
The impact of pregnancy on the clinical course of acute hepatitis B (AHB) is still largely unclear, mainly because most studies have not included matched controls. This study was conducted to investigate the clinical features and outcome of AHB in pregnancy using matched controls.
Consecutive AHB inpatients who were admitted to Jinan Infectious Disease Hospital, Jinan, between January 2006 and December 2010 were evaluated and followed. Demographic data, clinical manifestations, and results of laboratory tests were compared between pregnant patients and age and sex matched non-pregnant patients at admission, discharge, and final follow-up.
A total of 618 AHB inpatients were identified during the study period. 22 pregnant patients and 87 age and sex matched non-pregnant patients were enrolled in this study. Prodromal fever was less common (0% vs. 20.7%, P = 0.02), serum alanine aminotransferase levels were significantly lower, and HBsAg > 250 IU/mL rate and serum bilirubin levels were significantly higher in pregnant patients than in non-pregnant patients. After a mean (range) of 7(5.2-8.3) months follow-up, 18.2% pregnant patients and 4.6% non-pregnant patients were still HBsAg positive (P = 0.03). For pregnant patients, the relative risk (95% confidence interval) of HBsAg positive at the end of follow-up was 4.6 (1.1-20.2). The median (95% confidence interval) days of HBsAg seroclearance form disease onset in pregnant and non-pregnant patients were 145.0 (110.5-179.5) and 80.0 (62.6-97.4), respectively.
The HBsAg loss and seroconversion were delayed and lower in pregnant patients. Pregnancy might be a possible risk of chronicity following acute HBV infection.
PMCID: PMC4096733  PMID: 24993389
Acute hepatitis B; Pregnancy; Clinical features; Outcome; Hepatitis B surface antigen; Chronicity
5.  Mesothelin promotes cell proliferation in the remodeling of neonatal rat pancreas 
AIM: To investigate the effect of mesothelin in the remodeling of the endocrine pancreas in neonatal rats.
METHODS: Overexpression or downregulation of mesothelin expression in INS-1 cells was carried out to investigate the effect of mesothelin during cell proliferation and cell apoptosis in vitro. Adenovirus-mediated RNA interference was performed to block mesothelin in vivo to directly assess the role of mesothelin in the remodeling of the endocrine pancreas in neonatal rats.
RESULTS: Exogenous overexpression of mesothelin promoted cell proliferation, cell colony formation and enhanced cell resistance to apoptosis of INS-1 cells. Down-regulation of mesothelin made no difference in cell proliferation and apoptosis compared with that in the control group. After an injection of adenovirus-mesothelin, a significantly increased number of small islets appeared, and the expression of PCNA was decreased on day 7 and day 14 compared with the Ad-EGFP group.
CONCLUSION: Mesothelin was able to promote β cell proliferation in the remodeling stage of neonatal rats. Mesothelin may have an important role in the remodeling of the endocrine pancreas in neonatal rats.
PMCID: PMC4051928  PMID: 24944479
Mesothelin; Pancreatic remodeling; Cell proliferation; Pancreatic development; Small islets
6.  The Core Proteome and Pan Proteome of Salmonella Paratyphi A Epidemic Strains 
PLoS ONE  2014;9(2):e89197.
Comparative proteomics of the multiple strains within the same species can reveal the genetic variation and relationships among strains without the need to assess the genomic data. Similar to comparative genomics, core proteome and pan proteome can also be obtained within multiple strains under the same culture conditions. In this study we present the core proteome and pan proteome of four epidemic Salmonella Paratyphi A strains cultured under laboratory culture conditions. The proteomic information was obtained using a Two-dimensional gel electrophoresis (2-DE) technique. The expression profiles of these strains were conservative, similar to the monomorphic genome of S. Paratyphi A. Few strain-specific proteins were found in these strains. Interestingly, non-core proteins were found in similar categories as core proteins. However, significant fluctuations in the abundance of some core proteins were also observed, suggesting that there is elaborate regulation of core proteins in the different strains even when they are cultured in the same environment. Therefore, core proteome and pan proteome analysis of the multiple strains can demonstrate the core pathways of metabolism of the species under specific culture conditions, and further the specific responses and adaptations of the strains to the growth environment.
PMCID: PMC3933413  PMID: 24586590
7.  Overexpression of insulin-like growth factor-I receptor as a pertinent biomarker for hepatocytes malignant transformation 
AIM: To investigate the dynamic features of insulin-like growth factor-I receptor (IGF-IR) expression in rat hepatocarcinogenesis, and the relationship between IGF-IR and hepatocytes malignant transformation at mRNA or protein level.
METHODS: Hepatoma models were made by inducing with 2-fluorenylacetamide (2-FAA) on male Sprague-Dawley rats. Morphological changes of hepatocytes were observed by pathological Hematoxylin and eosin staining, the dynamic expressions of liver and serum IGF-IR were quantitatively analyzed by an enzyme-linked immunosorbent assay. The distribution of hepatic IGF-IR was located by immunohistochemistry. The fragments of IGF-IR gene were amplified by reverse transcription-polymerase chain reaction, and confirmed by sequencing.
RESULTS: Rat hepatocytes after induced by 2-FAA were changed dynamically from granule-like degeneration, precancerous to hepatoma formation with the progressing increasing of hepatic mRNA or IGF-IR expression. The incidences of liver IGF-IR, IGF-IR mRNA, specific IGF-IR concentration (ng/mg wet liver), and serum IGF-IR level (ng/mL) were 0.0%, 0.0%, 0.63 ± 0.17, and 1.33 ± 0.47 in the control; 50.0%, 61.1%, 0.65 ± 0.2, and 1.51 ± 0.46 in the degeneration; 88.9%, 100%, 0.66 ± 0.14, and 1.92 ± 0.29 in the precancerosis; and 100%, 100%, 0.96 ± 0.09, and 2.43 ± 0.57 in the cancerous group, respectively. IGF-IR expression in the cancerous group was significantly higher (P < 0.01) than that in any of other groups at mRNA or protein level. The closely positive IGF-IR relationship was found between livers and sera (r = 0.91, t = 14.222, P < 0.01), respectively.
CONCLUSION: IGF-IR expression may participate in rat hepatocarcinogenesis and its abnormality should be an early marker for hepatocytes malignant transformation.
PMCID: PMC3785631  PMID: 24106410
Hepatoma; Insulin-like growth factor-I receptor; Immunohistochemistry; Gene amplification; Sequencing; Rat hepatoma model
8.  Outcomes of neuropsychological interventions of stroke 
The reported prevalence of cognitive deficits within the first month of stroke ranges widely from 10% to 82%, depending primarily on the criteria used to define cognitive impairment and on the selected patient population. These cognitive defects progress toward impairment over a course of time if left untreated. Among the most common cognitive deficits are the attentional, the visuoperceptual, the memory and executive function deficits. As these impairments are being increasingly recognized in the scientific communities, more and more studies are being devoted to the outcomes of various therapies for these disorders. In this review, we focus on the outcomes of various therapies for these cognitive disorders over time. We reviewed all the possible medical databases using key words for individual cognitive deficit treatment outcomes. All the possible studies including randomized controlled trials, pre-post design studies, case series and single case reports were included in this study. On the basis of present literature review, we conclude that the evidence is definitively positive only for outcomes of attentional and visuoperceptive skill deficits. On the other hand, there have been very few studies to conclude for effectiveness of various therapies for memory and executive function outcomes.
PMCID: PMC3788273  PMID: 24101809
Cognition; interventions; neuropsychological outcome; stroke
9.  Comparative Proteomics-Based Identification of Genes Associated with Glycopeptide Resistance in Clinically Derived Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Strains 
PLoS ONE  2013;8(6):e66880.
Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is associated with clinical treatment failure. However, the resistance mechanism of hVISA has not been fully clarified. In the present study, comparative proteomics analysis of two pairs of isogenic vancomycin-susceptible S. aureus (VSSA) and hVISA strains isolated from two patients identified five differentially expressed proteins, IsaA, MsrA2, Asp23, GpmA, and AhpC, present in both isolate pairs. All the proteins were up-regulated in the hVISA strains. These proteins were analyzed in six pairs of isogenic VSSA and hVISA strains, and unrelated VSSA (n = 30) and hVISA (n = 24) by real-time quantitative reverse transcriptase–PCR (qRT–PCR). Of the six pairs of isogenic strains, isaA, msrA2 and ahpC were up-regulated in all six hVISA strains; whereas asp23 and gpmA were up-regulated in five hVISA strains compared with the VSSA parental strains. In the unrelated strains, statistical analyses showed that only isaA was significantly up-regulated in the hVISA strains. Analysis of the five differentially expressed proteins in 15 pairs of persistent VSSA strains by qRT–PCR showed no differences in the expression of the five genes among the persistent strains, suggesting that these genes are not associated with persistence infection. Our results indicate that increased expression of isaA may be related to hVISA resistance.
PMCID: PMC3696005  PMID: 23840544
10.  Annexin A2 silencing inhibits invasion, migration, and tumorigenic potential of hepatoma cells 
AIM: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells.
METHODS: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a novel human hepatocellular carcinoma (HCC) cell line with high metastasis potential] and a normal hepatocyte cell line (LO2) were used in this study. The protein and mRNA expression levels of ANXA2 were analysed by western blotting and real-time polymerase chain reaction, respectively. The intracellular distribution profile of ANXA2 expression was determined by immunofluorescence and immunohistochemistry. Short hairpin RNA targeting ANXA2 was designed and stably transfected into MHCC97-H cells. Cells were cultured for in vitro analyses or subcutaneously injected as xenografts in mice for in vivo analyses. Effects of ANXA2 silencing on cell growth were assessed by cell counting kit-8 (CCK-8) assay (in vitro) and tumour-growth assay (in vivo), on cell cycling was assessed by flow cytometry and propidium iodide staining (in vitro), and on invasion and migration potential were assessed by transwell assay and wound-healing assay, respectively (both in vitro).
RESULTS: The MHCC97-H cells, which are known to have high metastasis potential, showed the highest level of ANXA2 expression among the four HCC cell types examined; compared to the LO2 cells, the MHCC97-H expression level was 8-times higher. The ANXA2 expression was effectively inhibited (about 80%) by ANXA2-specific small hairpin RNA (shRNA). ANXA2 expression in the MHCC97-H cells was mainly localized to the cellular membrane and cytoplasm, and some localization was detected in the nucleus. Moreover, the proliferation of MHCC97-H cells was obviously suppressed by shRNA-mediated ANXA2 silencing in vitro, and the tumour growth inhibition rate was 38.24% in vivo. The percentage of MHCC97-H cells in S phase dramatically decreased (to 27.76%) under ANXA2-silenced conditions. Furthermore, ANXA2-silenced MHCC97-H cells showed lower invasiveness (percentage of invading cells decreased to 52.16%) and suppressed migratory capacity (migration distance decreased to 63.49%). It is also worth noting that shRNA-mediated silencing of ANXA2 in the MHCC97-H cells led to abnormal apoptosis.
CONCLUSION: shRNA-mediated silencing of ANXA2 suppresses the invasion, migration, and tumorigenic potential of hepatoma cells, and may represent a useful target of future molecular therapies.
PMCID: PMC3699036  PMID: 23840117
Annexin A2; Small hairpin RNA; Hepatocellular carcinoma; Invasion; Migration; Tumorigenic potential
11.  Identification of tetrodotoxin-producing Shewanella spp. from feces of food poisoning patients and food samples 
Gut Pathogens  2013;5:15.
Shewanella spp. is infrequently recovered from clinical specimens. Following two outbreaks of food poisoning, eight Shewanella spp. strains were obtained from the fecal specimens of patients, food and food processing-related materials. Tetrodotoxin (TTX) was identified in the culture supernatants of these strains, and the toxin’s biological activity was detected using a mouse bioassay. This study suggested that Shewanella strains can colonize and survive in human intestines. The study also raises the issues of the accumulation of TTX produced by Shewanella in food and the possible role of TTX-producing Shewanella in food poisoning.
PMCID: PMC3734009  PMID: 23782739
Tetrodotoxin; Shewanella; Food poisoning
12.  Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells 
BioMed Research International  2013;2013:135189.
Background. Genetic modification, such as the addition of exogenous genes to the MSC genome, is crucial to their use as cellular vehicles. Due to the risks associated with viral vectors such as insertional mutagenesis, the safer nonviral vectors have drawn a great deal of attention. Methods. VEGF, bFGF, vitamin C, and insulin-transferrin-selenium-X were supplemented in the MSC culture medium. The cells' proliferation and survival capacity was measured by MTT, determination of the cumulative number of cells, and a colony-forming efficiency assay. The plasmid pHr2-NL was constructed and nucleofected into MSCs. The recombinants were selected using G418 and characterized using PCR and Southern blotting. Results. BFGF is critical to MSC growth and it acted synergistically with vitamin C, VEGF, and ITS-X, causing the cells to expand significantly. The neomycin gene was targeted to the rDNA locus of human MSCs using a nonviral human ribosomal targeting vector. The recombinant MSCs retained multipotential differentiation capacity, typical levels of hMSC surface marker expression, and a normal karyotype, and none were tumorigenic in nude mice. Conclusions. Exogenous genes can be targeted to the rDNA locus of human MSCs while maintaining the characteristics of MSCs. This is the first nonviral gene targeting of hMSCs.
PMCID: PMC3666425  PMID: 23762822
13.  Draft Genome Sequences of Two Streptococcus pyogenes Strains Involved in Abnormal Sharp Raised Scarlet Fever in China, 2011 
Journal of Bacteriology  2012;194(21):5983-5984.
A scarlet fever outbreak caused by Streptococcus pyogenes occurred in China in 2011. To determine the genomic features of the outbreak strains, we deciphered genomes of two strains isolated from the regions with the highest incidence rates. The sequences will provide valuable information for comprehensive study of mechanisms related to this outbreak.
PMCID: PMC3486120  PMID: 23045496
14.  MFG-E8 and HMGB1 Are Involved in the Mechanism Underlying Alcohol-Induced Impairment of Macrophage Efferocytosis 
Molecular Medicine  2013;19(1):170-182.
Efferocytosis is a unique phagocytic process for macrophages to remove apoptotic cells in inflammatory loci. This event is maintained by milk fat globule-EGF factor 8 (MFG-E8), but attenuated by high mobility group box 1 (HMGB1). Alcohol abuse causes injury and inflammation in multiple tissues. It alters efferocytosis, but precise molecular mechanisms for this effect remain largely unknown. Here, we showed that acute exposure of macrophages to alcohol (25 mmol/L) inhibited MFG-E8 gene expression and impaired efferocytosis. The effect was mimicked by hydrogen peroxide. Moreover, N-acetylcysteine (NAC), a potent antioxidant, blocked acute alcohol effect on inhibition of macrophage MFG-E8 gene expression and efferocytosis. In addition, recombinant MFG-E8 rescued the activity of alcohol-treated macrophages in efferocytosis. Together, the data suggest that acute alcohol exposure impairs macrophage efferocytosis via inhibition of MFG-E8 gene expression through a reactive oxygen species dependent mechanism. Alcohol has been found to suppress or exacerbate immune cell activities depending on the length of alcohol exposure. Thus, we further examined the role of chronic alcohol exposure on macrophage efferocytosis. Interestingly, treatment of macrophages with alcohol for seven days in vitro enhanced MFG-E8 gene expression and efferocytosis. However, chronic feeding of mice with alcohol caused increase in HMGB1 levels in serum. Furthermore, HMGB1 diminished efferocytosis by macrophages that were treated chronically with alcohol, suggesting that HMGB1 might attenuate the direct effect of chronic alcohol on macrophage efferocytosis in vivo. Therefore, we speculated that the balance between MFG-E8 and HMGB1 levels determines pathophysiological effects of chronic alcohol exposure on macrophage efferocytosis in vivo.
PMCID: PMC3745596  PMID: 23552724
15.  Multicenter case-control study of the risk factors for ulcerative colitis in China 
AIM: To evaluate potential risk factors in the development of ulcerative colitis (UC) in China.
METHODS: A total of 1308 patients with UC and 1308 age-matched and sex-matched controls were prospectively studied in China. The UC cases were collected from 17 hospitals in China from April 2007 to April 2010. Uniform questionnaires were designed to investigate risk factors including smoking, appendectomy, stress, socio-economic conditions, nonsteroidal anti-inflammatory drugs (NSAIDs), oral contraceptives, diet, breastfeeding, infections and family sanitary conditions. Group comparisons by each factor were done using simple logistic regression analysis. Conditional logistic regression was used for multivariate analysis.
RESULTS: By univariate analysis, the variables predictive of UC included feeling stress, light and heavy alcoholic drinking, spicy food, sugar consumption and infectious diarrhea, while heavy tea intake and tap water consumption were protective against UC. On multivariate analysis, the protective factor for UC was tap water consumption [odds ratios (OR) = 0.424, 95%CI: 0.302-0.594, P < 0.001]; while the potential risk factors for UC were heavy sugar consumption (OR = 1.632, 95%CI: 1.156-2.305, P < 0.001), spicy food (light intake: OR = 3.329, 95%CI: 2.282-4.857, P < 0.001; heavy intake: OR = 3.979, 95%CI: 2.700-5.863, P < 0.001), and often feeling stress (OR = 1.981, 95%CI: 1.447-2.711, P < 0.001). Other factors, such as smoking habit, appendectomy, breastfeeding, a history of measles, rural or urban residence, education, oral contraceptives, and NSAID use have not been found to have a significant association with the development of UC in the present study.
CONCLUSION: Our study showed tap water consumption was a protective factor for UC, while spicy food, heavy sugar consumption and often feeling stress were risk factors for UC in this Chinese population.
PMCID: PMC3607760  PMID: 23555172
Ulcerative colitis; Risk factors; Case-control study
16.  A New Oxidative Stress Model, 2,2-Azobis(2-Amidinopropane) Dihydrochloride Induces Cardiovascular Damages in Chicken Embryo 
PLoS ONE  2013;8(3):e57732.
It is now well established that the developing embryo is very sensitive to oxidative stress, which is a contributing factor to pregnancy-related disorders. However, little is known about the effects of reactive oxygen species (ROS) on the embryonic cardiovascular system due to a lack of appropriate ROS control method in the placenta. In this study, a small molecule called 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), a free radicals generator, was used to study the effects of oxidative stress on the cardiovascular system during chick embryo development. When nine-day-old (stage HH 35) chick embryos were treated with different concentrations of AAPH inside the air chamber, it was established that the LD50 value for AAPH was 10 µmol/egg. At this concentration, AAPH was found to significantly reduce the density of blood vessel plexus that was developed in the chorioallantoic membrane (CAM) of HH 35 chick embryos. Impacts of AAPH on younger embryos were also examined and discovered that it inhibited the development of vascular plexus on yolk sac in HH 18 embryos. AAPH also dramatically repressed the development of blood islands in HH 3+ embryos. These results implied that AAPH-induced oxidative stress could impair the whole developmental processes associated with vasculogenesis and angiogenesis. Furthermore, we observed heart enlargement in the HH 40 embryo following AAPH treatment, where the left ventricle and interventricular septum were found to be thickened in a dose-dependent manner due to myocardiac cell hypertrophy. In conclusion, oxidative stress, induced by AAPH, could lead to damage of the cardiovascular system in the developing chick embryo. The current study also provided a new developmental model, as an alternative for animal and cell models, for testing small molecules and drugs that have anti-oxidative activities.
PMCID: PMC3585800  PMID: 23469224
17.  MALDI-TOF MS Distinctly Differentiates Nontypable Haemophilus influenzae from Haemophilus haemolyticus 
PLoS ONE  2013;8(2):e56139.
Nontypable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus exhibit different pathogenicities, but to date, there remains no definitive and reliable strategy for differentiating these strains. In this study, we evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a potential method for differentiating NTHi and H. haemolyticus. The phylogenetic analysis of concatenated 16S rRNA and recombinase A (recA) gene sequences, outer membrane protein P6 gene sequencing and single-gene PCR were used as reference methods. The original reference database (ORD, provided with the Biotyper software) and new reference database (NRD, extended with Chinese strains) were compared for the evaluation of MALDI-TOF MS. Through a search of the ORD, 76.9% of the NTHi (40/52) and none of the H. haemolyticus (0/20) strains were identified at the species level. However, all NTHi and H. haemolyticus strains used for identification were accurately recognized at the species level when searching the NRD. From the dendrogram clustering of the main spectra projections, the Chinese and foreign H. influenzae reference strains were categorized into two distinct groups, and H. influenzae and H. haemolyticus were also separated into two categories. Compared to the existing methods, MALDI-TOF MS has the advantage of integrating high throughput, accuracy and speed. In conclusion, MALDI-TOF MS is an excellent method for differentiating NTHi and H. haemolyticus. This method can be recommended for use in appropriately equipped laboratories.
PMCID: PMC3573053  PMID: 23457514
18.  Comparative peptidome analyses of the profiles of the peptides ranging from 1–10 KD in CSF samples pooled from probable sporadic CJD and non-CJD patients 
Prion  2012;6(1):46-51.
The shotgun strategy applying tandem mass spectrometry has been widely used to identify the proteins that are differentially distributed among diseases for its high reliability and efficiency. To find out the potential difference of protein profiles in cerebrospinal fluids (CSF) between Creutzfeldt-Jakob disease (CJD) and non-CJD patients, especially in the fraction ranging from 1–10 KD, the CSF samples of 40 probable sporadic CJD (sCJD) patients, 32 non-CJD cases with dementia and 17 non-CJD cases without dementia were separately pooled and enriched by the magnetic beads based weak cation exchange chromatography (MB-WCX). After trypsin digestion, each enriched CSF was separated and identified by RP-HPLC-ESI-QTOF MS/MS. In total, 42, 53 and 47 signals of proteins were identified in the pooled CSF fraction less than 10 KD of probable sCJD, non-CJD with dementia and non-CJD without dementia, respectively. Compared with that of probable sCJD, the similarity of CSF protein profiles of non-CJD with dementia (76.2%) were higher than that of non-CJD without dementia (57.1%). Nine CSF proteins were found to be specially observed in probable sCJD group. Those data may help to select the potential biomarkers for diagnosis of CJD. Additionally, further studies of the small segments of cellular proteins in CSF of CJD patients may also provide scientific clues for understanding the neuropathogenesis of TSEs.
PMCID: PMC3338965  PMID: 22453178
Creutzfeldt-Jakob disease; CSF; MB-HPLC-ESI-QTOF; 1–10 KD; comparative peptidome
19.  Expression characteristics and diagnostic value of annexin A2 in hepatocellular carcinoma 
AIM: To investigate the characteristics and diagnostic value of annexin A2 (ANXA2) expression in cancerous tissues and sera of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC).
METHODS: Levels of liver ANXA2 gene transcription or protein expression were analyzed in HCC-, their self-controlled precancerous-, and distant cancerous- tissues from 30 HCC. Serum levels of ANXA2 expression in 115 patients with HCC, 25 with metastatic liver cancer, 35 with chronic hepatitis, 28 with acute hepatitis, 38 with cirrhosis, and 30 healthy controls were determined. Clinicopathological characteristics of circulating ANXA2 expression were analyzed, and its diagnostic efficiency and clinical values in HCC were evaluated.
RESULTS: ANXA2 expression was localized in both cell membrane and cytoplasm in HCC tissue, mainly in the cytoplasm of matched adjacent cancerous tissue, and there was almost no positive staining in matched distant cancerous tissue. Abnormal expression of liver ANXA2 was present in HCC tissues compared with self-controlled adjacent- and distant-cancerous tissues at protein or mRNA level. Circulating ANXA2 in HCC patients was significantly higher than that of other liver diseases (P < 0.01) except metastatic liver cancer. If the diagnostic cutoff value of ANXA2 level was more than 18 ng/mL, the incidence of serum ANXA2 was 86.96% in the HCC group, 80% in the metastatic liver cancer group, 31.58% in the liver cirrhosis group, none in the chronic hepatitis or acute hepatitis or normal control group, respectively. Serum ANXA2 expression in HCC patients was correlated with HBV infection (27.38 ± 5.67 ng/mL vs 18.58 ± 7.83 ng/mL, P < 0.01), extrahepatic metastasis (26.11 ± 5.43 ng/mL vs 22.79 ± 5.64 ng/mL, P < 0.01), and portal vein thrombus (26.03 ± 5.99 ng/mL vs 23.06 ± 5.03 ng/mL, P < 0.01), and was significantly higher (P < 0.01) in the moderately- (26.19 ± 5.34 ng/mL) or the poorly- differentiated group (27.05 ± 5.13 ng/mL) than in the well differentiated group (20.43 ± 4.97 ng/mL), and in the tumor node metastasis stages III-IV (P < 0.01) than in stages I-II. ANXA2 was not correlated with patient sex, age, size or α-fetoprotein (AFP) level. Area under the receiver operating characteristic curve for the whole range of sensitivities and specificities was 0.796 for ANXA2 and 0.782 for AFP. Combining detection of serum ANXA2 and AFP substantially improved the diagnostic efficiency (96.52%) and the negative predictive value (96.61%) for HCC.
CONCLUSION: The characteristics and distribution of ANXA2 expression has good diagnostic potential for HCC diagnosis.
PMCID: PMC3491596  PMID: 23139605
Hepatocellular carcinoma; Annexin A2; Liver; Upregulation; Clinicopathological characteristics; Diagnosis; Expression; Biomarker
20.  Retinal image registration and comparison for clinical decision support 
The Australasian Medical Journal  2012;5(9):507-512.
For eye diseases, such as glaucoma and age-related macular degeneration (ARMD), involved in long-term degeneration procedure, longitudinal comparison of retinal images is a common step for reliable diagnosis of these kinds of diseases.
To provide a retinal image registration approach for longitudinal retinal image alignment and comparison.
Two image registration solutions were proposed for facing different image qualities of retinal images to make the registration methods more robust and feasible in a clinical application system.
Thirty pairs of longitudinal retinal images were used for the registration test. The experiments showed both solutions provided good performance for the accurate image registrations with efficiency.
We proposed a set of retinal image registration solutions for longitudinal retinal image observation and comparison targeting a clinical application environment.
PMCID: PMC3477780  PMID: 23115586
Retinal image registration; Glaucoma; ARMD; clinical decision support
21.  Correction: Exploring the Caffeine-Induced Teratogenicity on Neurodevelopment Using Early Chick Embryo 
PLoS ONE  2012;7(8):10.1371/annotation/0dc7c4d1-2601-4267-b56d-a315d89a6310.
PMCID: PMC3415384
22.  Anti-Stress Effects of Carnosine on Restraint-Evoked Immunocompromise in Mice through Spleen Lymphocyte Number Maintenance 
PLoS ONE  2012;7(4):e33190.
Carnosine (β-alanyl-L-histidine), a naturally occurring dipeptide, has been characterized as a putative neurotransmitter and serves as a reservoir for brain histamine, which could act on histaminergic neurons system to relieve stress-induced damages. However, understanding of the role of carnosine in stress-evoked immunocompromise is limited. In this study, results showed that when mice were subjected to restraint stress, spleen index and the number of spleen lymphocytes including Natural Killer (NK) cells were obviously decreased. Results also demonstrated that restraint stress decreased the cytotoxic activity of NK cells per spleen (LU10/spleen) while the activity of a single NK cell (LU10/106 cells) was not changed. However, oral administration of carnosine (150 and 300 mg/kg) increased spleen index and number of spleen lymphocytes (including NK cells), and elevated the cytotoxic activity of NK cells per spleen in restraint-stressed mice. These results indicated that carnosine ameliorated stress-evoked immunocompromise through spleen lymphocyte number maintenance. Carnosine was further found to reduce stress-induced elevation of plasma corticosterone level. On the other hand, results showed that carnosine and RU486 (a glucocorticoids receptor antagonist) treatment prevented the reduction in mitochondrion membrane potential and the release of mitochondrial cytochrome c into cytoplasm, increased Bcl-2/Bax mRNA ratio, as well as decreased terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells in spleen lymphocytes of stressed mice. The results above suggested that the maintenance of spleen lymphocyte number by carnosine was related with the inhibition of lymphocytes apoptosis caused by glucocorticoids overflow. The stimulation of lymphocyte proliferation by carnosine also contributed to the maintenance of spleen lymphocyte number in stressed mice. In view of the elevated histamine level, the anti-stress effects of carnosine on restraint-evoked immunocompromise might be via carnosine-histamine metabolic pathway. Taken together, carnosine maintained spleen lymphocyte number by inhibiting lymphocyte apoptosis and stimulating lymphocyte proliferation, thus prevented immunocompromise in restraint-stressed mice.
PMCID: PMC3325237  PMID: 22511917
23.  Exploring the Caffeine-Induced Teratogenicity on Neurodevelopment Using Early Chick Embryo 
PLoS ONE  2012;7(3):e34278.
Caffeine consumption is worldwide. It has been part of our diet for many centuries; indwelled in our foods, drinks, and medicines. It is often perceived as a “legal drug”, and though it is known to have detrimental effects on our health, more specifically, disrupt the normal fetal development following excessive maternal intake, much ambiguity still surrounds the precise mechanisms and consequences of caffeine-induced toxicity. Here, we employed early chick embryos as a developmental model to assess the effects of caffeine on the development of the fetal nervous system. We found that administration of caffeine led to defective neural tube closures and expression of several abnormal morphological phenotypes, which included thickening of the cephalic mesenchymal tissues and scattering of somites. Immunocytochemistry of caffeine-treated embryos using neural crest cell markers also demonstrated uncharacteristic features; HNK1 labeled migratory crest cells exhibited an incontinuous dorsal-ventral migration trajectory, though Pax7 positive cells of the caffeine-treated groups were comparatively similar to the control. Furthermore, the number of neurons expressing neurofilament and the degree of neuronal branching were both significantly reduced following caffeine administration. The extent of these effects was dose-dependent. In conclusion, caffeine exposure can result in malformations of the neural tube and induce other teratogenic effects on neurodevelopment, although the exact mechanism of these effects requires further investigation.
PMCID: PMC3314624  PMID: 22470550
24.  Bacterial flora concurrent with Helicobacter pylori in the stomach of patients with upper gastrointestinal diseases 
AIM: To investigate the non-Helicobacter pylori (H. pylori) bacterial flora concurrent with H. pylori infection.
METHODS: A total of 103 gastric biopsy specimens from H. pylori positive patients were selected for bacterial culture. All the non-H. pylori bacterial isolates were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
RESULTS: A total of 201 non-H. pylori bacterial isolates were cultivated from 67 (65.0%) of the 103 gastric samples, including 153 isolates identified successfully at species level and 48 at genus level by MALDI-TOF MS. The dominant species were Streptococcus, Neisseria, Rothia and Staphylococcus, which differed from the predominantly acid resistant species reported previously in healthy volunteers. The prevalence of non-H. pylori bacteria was higher in non-ulcer dyspepsia group than in gastric ulcer group (100% vs 42.9%, P < 0.001). Six bacterial species with urease activity (Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus capitis, Staphylococcus aureus, Brevibacterium spp. and Klebsiella pneumoniae) were also isolated.
CONCLUSION: There is a high prevalence of the non-H. pylori bacteria concurrent with H. pylori infection, and the non-H. pylori bacteria may also play important as-yet-undiscovered roles in the pathogenesis of stomach disorders.
PMCID: PMC3309916  PMID: 22468090
Non-Helicobacter pylori; Bacterial flora; Gastrointestinal diseases; Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
25.  Luminal-Applied Flagellin Is Internalized by Polarized Intestinal Epithelial Cells and Elicits Immune Responses via the TLR5 Dependent Mechanism 
PLoS ONE  2011;6(9):e24869.
Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn’s lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.
PMCID: PMC3174220  PMID: 21949773

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