High Specificity (high + specificity)

Distribution by Scientific Domains
Distribution within Medical Sciences

Selected Abstracts

Characterization and Application of a New Monoclonal Antibody with High Specificity for Helicobacter hepaticus

HELICOBACTER, Issue 1 2009
Yoshihiro Fukuda
Abstract Background and Aims:, Infection with Helicobacter hepaticus is suggested to play a role in the pathogenesis of chronic liver disease in humans. However, reactive antigens among Helicobacter species make the development of an H. hepaticus ELISA test with high specificity difficult. A new monoclonal antibody from a hybridoma clone (HRII-51) showed high specificity to H. hepaticus without cross-reaction to other gastrointestinal bacteria. Methods:, The molecular weight of HRII-51 immunoreactive antigen was examined by Western blot of H. hepaticus probed with the monoclonal antibody HRII-51. A HRII-51-immunoreactive antigen capture ELISA was prepared in which the specific antigen was anchored by HRII-51-immobilized ELISA plate. Accuracy of HRII-51 antigen capture ELISA was examined using sera obtained from mice inoculated with Helicobacter species. Specificity of HRII-51 antigen capture ELISA was compared to that of H. hepaticus antigen-based ELISA using human sera with absorption by H. pylori cell lysate. Results:, HRII-51 immunoreactive antigen had a molecular weight of 15 kDa. Sensitivity and specificity of HRII-51 antigen capture ELISA were 87.0% and 97.6% in mice inoculated with Helicobacter species. In human sera, modification of the results by absorption with H. pylori lysate was smaller in HRII-51 antigen capture ELISA comparing with H. hepaticus -antigen-based ELISA. Conclusion:, Use of the HRII-51 antigen capture ELISA would be a useful approach for the serodiagnosis of H. hepaticus infection in both experimental animals and humans. [source]

Diversity of rice glutelin polypeptides in wild species assessed by the higher-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subunit-specific antibodies

Nadar Khan
Abstract In efforts to find genetic resources with high nutritional value of rice seed, we assessed the diversity of the major storage protein glutelin in 13 wild and 2 cultivated rice species by a unique SDS-PAGE method and subunit-specific antibodies. Maximum separation of microheterogeneous glutelin ,-polypeptides, which is a prerequisite for the diversity evaluation, could be attained by SDS-PAGE performed at higher temperature (45C) than the generally employed temperatures (4,25C). Seven antipeptide antibodies were raised against subunit-specific epitope sequences designed at five sites from four variable regions spanning the glutelin ,-polypeptides. High specificity of each antibody was confirmed using rice glutelin mutants, and demonstrated considerable variation in amino acid sequence and accumulation level of glutelin subunit in wild species, in combination with the higher-temperature SDS-PAGE. The degree of the variation was, however, changed according to the site of variable regions and the type of subunit. Some wild species accumulated nutritious GluB subunits more than cultivated rice. The wild species Oryza longiglumis and O. brachyantha had glutelin with low reactivity against most antibodies examined in this study, reflecting the significant divergence. Such wild species may hopefully serve as important genetic resources for nutritional improvement of cultivated rice. [source]

High specificity of V3 serotyping among human immunodeficiency virus type-1 subtype C infected patients with varying disease status and viral phenotype

Polly R. Walker
Abstract V3 serotyping is a technique for determining HIV-1 genetic subtype based on the binding of antibodies from patient sera or plasma to synthetic V3 peptides derived from subtype consensus sequences. Variation in the performance of this assay has been attributed to V3 sequence heterogeneity, the degree of which varies with patient disease progression, virus co-receptor usage, and genetic subtype. This study assessed the performance of a competitive peptide enzyme immunoassay (cPEIA) in samples from HIV-1 subtype C infected patients with varying disease profiles, including those with syncytium (SI) and non-syncytium-inducing (NSI) viruses. Out of 90 sera tested, 94.4% reacted strongly against the subtype C peptide. There was no significant difference in assay sensitivity among samples from advanced AIDS patients in which humoral immune response may be lower, nor among SI viruses which carry changes in the V3 sequence. Four samples were found to be cross-reactive with other subtypes and one acutely infected patient sample was non-reactive due to low anti-gp120 antibody titers. A significantly higher number of samples showed secondary reactivity to subtype A, compared to other subtypes (P,<,0.005). In conclusion, the assay was able to identify HIV-1 subtype C infection with a high level of sensitivity (94%) irrespective of the stage of disease and therefore provides a valuable resource for the large-scale epidemiological monitoring of the spread of HIV-1 subtypes in South Africa. J. Med. Virol. 78:1262,1268, 2006. 2006 Wiley-Liss, Inc. [source]

Ficus racemosa is pollinated by a single population of a single agaonid wasp species in continental South-East Asia

Abstract High specificity in the Ficus -agaonid wasp mutualism has lead to the assumption of a mostly ,one-to-one' relationship, albeit with some exceptions. This view has been challenged by new molecular data in recent years, but surprisingly little is known about local and spatial genetic structuring of agaonid wasp populations. Using microsatellite markers, we analysed genetic structuring of Ceratosolen fusciceps, the fig wasp pollinating Ficus racemosa, a fig tree species widely distributed from India to Australia. In sampling stretching from the south of China to the south of Thailand we found evidence for only a single pollinating wasp species in continental South-East Asian mainland. We found no evidence for the co-occurrence of cryptic species within our subcontinent sampling zone. We observed no spatial genetic structure within sites and only limited structuring over the whole sampling zone, suggesting that F. racemosa is pollinated by a single population of a single agaonid wasp species all over continental South-East Asia. An additional sample of wasps collected on F. racemosa in Australia showed clear-cut genetic differentiation from the Asian continent, suggesting allopatric divergence into subspecies or species. We propose that the frequent local co-occurrence of sister species found in the literature mainly stems from contact zones between biogeographic regions, and that a single pollinator species over wide areas might be the more common situation everywhere else. [source]

High specificity generally characterizes mycorrhizal association in rare lady's slipper orchids, genus Cypripedium

Abstract Lady's slipper orchids (Cypripedium spp.) are rare terrestrial plants that grow throughout the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for germination and seedling nutrition. The nutritional relationships of adult Cypripedium mycorrhizae are unclear; however, Cypripedium distribution may be limited by mycorrhizal specificity, whether this specificity occurs only during the seedling stage or carries on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100 Cypripedium plants, and successfully did so with two Cypripedium calceolus, 10 Cypripedium californicum, six Cypripedium candidum, 16 Cypripedium fasciculatum, two Cypripedium guttatum, 12 Cypripedium montanum, and 11 Cypripedium parviflorum plants from a total of 44 populations in Europe and North America, yielding fungal nuclear large subunit and mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism) data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting structures, we assessed fungal identity through direct PCR (polymerase chain reaction) amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic analysis revealed that the great majority of Cypripedium mycorrhizal fungi are members of narrow clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus, Phialophora. C. californicum was the only orchid species with apparently low specificity, as it associated with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our results add support to the growing literature showing that high specificity is not limited to nonphotosynthetic plants, but also occurs in photosynthetic ones. [source]

Validity of medication-based co-morbidity indices in the Australian elderly population

Agnes Vitry
Abstract Objectives: To determine the validity of two medication-based co-morbidity indices, the Medicines Disease Burden Index (MDBI) and Rx-Risk-V in the Australian elderly population. Methods: In Phase I, the sensitivity and specificity of both indices were determined in 767 respondents from wave 6 of the Australian Longitudinal Study of Ageing (ALSA). Medication-defined index disease categories were compared to self-reported medical conditions. Correlation with self-rated health was examined and Cox proportional hazards models were used to assess the predictive validity for mortality. Phase II verified the predictive ability of Rx-Risk-V in a sample of 213,191 veterans from Australian Department of Veterans' Affairs (DVA) database. Results: MDBI and Rx-Risk-V scores could be calculated for 28% and 73% of the ALSA sample respectively. Both indices had high specificities and low to moderate sensitivities compared to self-reported medical conditions. Total weighted scores were significantly related to self-rated health (p<0.001). Both indices were predictive of mortality (Hazard Ratio (HR) =3.690 (95% CI 2.264-6.015) for MDBI and HR 1.079 (95% CI 1.045-1.114) for Rx-Risk-V. The predictive validity for mortality of Rx-Risk-V was confirmed using DVA data (HR= 1.090, 95% CI 1.088-1.092). Conclusions: Medication-based co-morbidity indices Rx-Risk-V and MDBI are valid measures of co-morbidity. However, Rx-Risk-V detects more comorbidity in the Australian elderly population and is likely to be a more suitable index to use in administrative datasets, particularly where studies include large numbers of outpatients. [source]

Evaluation of 2 self-administered questionnaires to ascertain dermatitis among metal workers and its relation with exposure to metalworking fluids

Berna Van Wendel de Joode
We performed an exploratory study to evaluate 2 self-administered questionnaires assessing hand dermatitis and investigate a possible exposure,response relationship between dermal exposure to semi-synthetic metalworking fluids (SMWF) and dermatitis. In a cross-sectional survey on dermatitis, a symptom-based questionnaire and a picture-based skin screening list were applied in 80 SMWF-exposed workers and 67 referents. To evaluate the accuracy of the questionnaires, 47 subjects were examined by a dermatologist. Dermal exposure levels to SMWF were assessed on hands, forearms, and face with a observational method that was validated using a fluorescent tracer method. The symptom-based questionnaire had a relatively high sensitivity (0.86) but moderate specificity (0.64), and the skin screening list had a low sensitivity (0.36) and a relatively high specificity (0.84). The skin screening list seemed to represent the more severe cases of dermatitis and showed a significant relation with exposure for dermatitis on hands, forearms, or face. In epidemiological surveys where workers are not seen by a dermatologist, the skin screening list seems to be more appropriate to detect cases of dermatitis, as its higher specificity results in less false positives. Alternatively, it would be preferable applying the symptom-based questionnaire; workers with symptoms should be seen by a dermatologist to identify false positives. [source]

The Brset violence checklist (BVC)

Phil Woods
Objective:, The Brset violence checklist (BVC) is a short-term violence prediction instrument assessing confusion, irritability, boisterousness, verbal threats, physical threats and attacks on objects as either present or absent. The aim of this paper is to describe the evolution and usefulness of the BVC. Method:, This paper reviews studies on the BVC and discusses implications for further research. Results:, Empirical research has shown that it has moderate sensitivity and high specificity with an adequate inter-rater reliability. Conclusion:, The BVC is a useful instrument for predicting inpatient violence within the next 24-h period. The psychometric properties of the instrument are satisfactory. Results from ongoing studies will give important information on cultural differences, the validity of the BVC in less well staffed wards, the clinical use of the checklist and its ability to predict violence throughout all the hospital stay. [source]

Nipple aspirate fluid and ductoscopy to detect breast cancer

Edward R. Sauter M.D., Ph.D.
Abstract We prospectively performed cytologic assessment and image analysis (IA) on matched nipple aspirate fluid (NAF) and mammary ductoscopy (MD) specimens to determine (1) the accuracy of these methods in cancer detection and (2) whether the two collection methods provide complementary information. NAF and MD specimens were collected from 84 breasts from 75 women (nine bilateral samples) who underwent breast surgery. Cytologic evaluation was performed on all samples. IA was performed on slides with sufficient epithelial cells. Cytologic evaluation proved more accurate in patients without pathologic spontaneous nipple discharge (PND) than those with PND, mainly because of the potential false positive diagnosis in the latter. While the sensitivity of NAF and MD cytology was low (10% and 14%, respectively), both were 100% specific in cancer detection in the non-PND cohort. Combining NAF and MD cytology information improved sensitivity (24%) without sacrificing specificity. Similar to cytology, IA was more accurate in patients without PND having high specificity (100% for aneuploid IA), but relatively low sensitivity (36%). Combining NAF and MD cytology with aneuploid IA improved the sensitivity (45%) while maintaining high specificity (100%). The best predictive model was positive NAF cytology and/or MD cytology combined with IA aneuploidy, which resulted in 55% sensitivity and 100% specificity in breast cancer detection. Cytologic evaluation and IA of NAF and MD specimens are complementary. The presence of atypical cells arising from an intraductal papilloma in ductoscopic specimens is a potential source of false positive diagnosis in patients with nipple discharge. Diagn. Cytopathol. 2010 2009 Wiley-Liss, Inc. [source]

The diagnostic utility of D2-40 for malignant mesothelioma versus pulmonary carcinoma with pleural involvement

Ph.D., Reda S. Saad M.D.
Abstract Differentiating malignant mesothelioma (MM) from pulmonary carcinoma in pleural fluid cytology can be challenging. Recent studies have suggested that D2-40, a novel lymphatic marker, may be a useful marker for mesothelial differentiation in surgical specimens. However, there are no available data regarding its utility in effusion cytology specimens. We investigated the utility of D2-40 in pleural fluid cytology in differentiating MM from pulmonary carcinomas. Twenty cases of pleural effusion smears of surgically confirmed MM with their corresponding cell blocks were retrieved from the database of the hospital computer system. We also included 10 cases of metastatic pulmonary adenocarcinoma (PA) and 10 cases metastatic pulmonary squamous cell carcinoma (PSCC) involving the pleural fluid. Cell blocks were formalin-fixed, paraffin embedded, and immunostained for TTF1, p63, calretinin, CK5/6, WT-1, and D2-40. Cases were scored as negative (<5% positivity) or positive (>5% moderate/strong positivity). The positive rates for TTF1, p63, calretinin, CK5/6, WT-1, and D2-40 were as follows: MM (0/20), (0/20), (17/20), (18/20), (19/20), (17/20), for PA (8/10), (0/10), (3/10), (0/10), (0/10), (0/10), and for PSCC (1/10), (10/10), (6/10), (10/10), (0/15), (0/10). The staining pattern for D2-40 was characterized by thick membranous staining. Diffuse cytoplasmic staining by D2-40 was seen in 2 cases of pulmonary carcinoma, counted as negative. Our study showed that in differentiating MM from PA, CK5/6, WT-1, and D2-40 have high specificity and sensitivity for MM. Although calretinin is a sensitive IHC marker for MM, it is not specific since it stained 30% of PA. Conversely, to differentiate between MM and PSCC, p63 and WT-1 are the best available markers. We recommend a panel of CK5/6, p63, D2-40, and WT-1 to differentiate MM from pulmonary carcinomas in effusion cytology specimens. Diagn. Cytopathol. 2006; 34:801,806. 2006 Wiley-Liss, Inc. [source]

Primary sclerosing cholangitis as a cause of false positive bile duct brushing cytology: Report of two cases.

Lester J. Layfield M.D.
Abstract Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown etiology characterized by ongoing inflammation, destruction, and fibrosis of intrahepatic and extrahepatic bile ducts. Irregular narrowing and dilation of the biliary duct system produces the characteristic beaded pattern seen on cholangiogram. Malignant degeneration resulting in cholangiocarcinoma is a well-recognized sequela of PSC. Bile duct brushing cytology is the primary screening technique for cholangiocarcinoma. It is associated with a relatively low sensitivity but high specificity. Few false positive bile duct brushings have been reported in the literature, with the majority of these having occurred in a background of PSC. We report two patients with PSC in whom bile duct brush cytologies were falsely positive for carcinoma. Diagn. Cytopathol. 2005;32:119,124. 2005 Wiley-Liss, Inc. [source]

A tethered ascorbate-norepinephrine compound, 4-UT, displays long-acting adrenergic activity on rabbit aortic smooth muscle

Robert Root-Bernstein
Abstract We previously demonstrated that adrenergic and histaminergic receptors have an ascorbic acid (vitamin C) binding site on the first extracellular loop, immediately adjacent to the aminergic binding site. Binding of ascorbate to this site strongly potentiates any sub-maximal dose of an adrenergic or histaminergic compound, significantly increasing its duration of activity. We report here the successful synthesis of a tethered compound that mimics the combined effects of a mixture of ascorbate with norepinephrine. The tethered compound uses a four-unit polyethylene linker to tether ascorbate to norepinephrine. The tethered compound is about tenfold less effective than norepinephrine in stimulating rabbit aortic smooth muscle, but has a very significantly enhanced duration of activity compared with norepinephrine alone and comparable to a mixture of norepinephrine and ascorbate. Additional ascorbate does not enhance the tethered compound's effects and we demonstrate that the compound binds to a synthetic peptide spanning the ascorbate binding site of the receptor. These experiments strongly suggest that the compound binds to both the adrenergic binding site and the ascorbate binding site simultaneously. Tethered compounds with linkers of other lengths did not have these properties. We believe that the synthesis of enhanced adrenergic and histaminergic drugs by tethering them to potentiators such as ascorbate will permit a new class of potential drugs to be created with high specificity and long duration of activity. Drug Dev Res 69:242,250, 2008. 2008 Wiley-Liss, Inc. [source]

Brief screening questionnaires to identify problem drinking during pregnancy: a systematic review

ADDICTION, Issue 4 2010
Ethel Burns
ABSTRACT Aims Although prenatal screening for problem drinking during pregnancy has been recommended, guidance on screening instruments is lacking. We investigated the sensitivity, specificity and predictive value of brief alcohol screening questionnaires to identify problem drinking in pregnant women. Methods Electronic databases from their inception to June 2008 were searched, as well as reference lists of eligible papers and related review papers. We sought cohort or cross-sectional studies that compared one or more brief alcohol screening questionnaire(s) with reference criteria obtained using structured interviews to detect ,at-risk' drinking, alcohol abuse or dependency in pregnant women receiving prenatal care. Results Five studies (6724 participants) were included. In total, seven instruments were evaluated: TWEAK (Tolerance, Worried, Eye-opener, Amnesia, Kut down), T-ACE [Take (number of drinks), Annoyed, Cut down, Eye-opener], CAGE (Cut down, Annoyed, Guilt, Eye-opener], NET (Normal drinker, Eye-opener, Tolerance), AUDIT (Alcohol Use Disorder Identification Test), AUDIT-C (AUDIT-consumption) and SMAST (Short Michigan Alcohol Screening Test). Study quality was generally good, but lack of blinding was a common weakness. For risk drinking sensitivity was highest for T-ACE (69-88%), TWEAK (71,91%) and AUDIT-C (95%), with high specificity (71,89%, 73,83% and 85%, respectively). CAGE and SMAST performed poorly. Sensitivity of AUDIT-C at score ,3 was high for past year alcohol dependence (100%) or alcohol use disorder (96%) with moderate specificity (71% each). For life-time alcohol dependency the AUDIT at score ,8 performed poorly. Conclusion T-ACE, TWEAK and AUDIT-C show promise for screening for risk drinking, and AUDIT-C may also be useful for identifying alcohol dependency or abuse. However, their performance as stand-alone tools is uncertain, and further evaluation of questionnaires for prenatal alcohol use is warranted. [source]

Detection of denitrification genes by in situ rolling circle amplification-fluorescence in situ hybridization to link metabolic potential with identity inside bacterial cells

Tatsuhiko Hoshino
Summary A target-primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single-copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene to target both DNA strands; the target DNA was cut by a restriction endonuclease close to the probe binding sites, which subsequently were made accessible by 5,-3, exonucleolysis. After hybridization, the padlock probe was circularized by ligation and served as template for in situ RCA, primed by the probe target site. Finally, the RCA product inside the cells was detected by standard fluorescence in situ hybridization (FISH). The optimized protocol showed high specificity and signal-to-noise ratio but low detection frequency (up to 15% for single-copy genes and up to 43% for the multi-copy 16S rRNA gene). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA-FISH was demonstrated on activated sludge by the differential detection of two types of nirS -defined denitrifiers; one of them was identified as Candidatus Accumulibacter phosphatis by combining in situ RCA-FISH with 16S rRNA-targeted FISH. While not suitable for quantification because of its low detection frequency, in situ RCA-FISH will allow to link metabolic potential with 16S rRNA (gene)-based identification of single microbial cells. [source]

Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics

Catalina Arevalo-Ferro
Summary The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N -acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type. These results add haemoglobin utilization to the list of phenotypes controlled through QS in P. aeruginosa. The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms. To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem. [source]

Evaluation of decision criteria for detection of spinal cord compression based on cervical myelography in horses: 38 cases (1981,2001)

J. Van Biervliet
Summary Reasons for performing study: Different criteria have been described based on height reduction of the total myelographic contrast column and components of it as tests for compression of the spinal cord due to cervical stenotic myelopathy (CSM). Fifty percent height reduction of the dorsal myelographic column (DMC), <2 mm empiric height of the DMC and a 40% reduction of the ratio of stenosis calculated based on the height reduction of the entire dural diameter (DD) have been described as decision criteria for considering the test result positive. The reasons for selecting these decision criteria or their accuracies have rarely been reported. Objectives: To evaluate the accuracy of diagnostic criteria based on reduced height of the total myelographic column and components of it for diagnosing extradural spinal cord compression using different decision criteria, and make recommendations for consistent myelographic interpretation in horses suspected of having CSM. Methods: Four measurements were obtained by 2 readers in a retrospective sample population of 38 horses in which both cervical myelography and histopathological examination of the cervical spinal cord were performed. The prevalence of CSM in the sample was 50%. At intervertebral sites, the minimum heights of the DD and DMC were measured. At intravertebral sites, the maximum heights of the entire DD and DMC were obtained. Percent height reductions of the DMC and DD were determined as the ratio of minimum intervertebral height to maximum intravertebral height within the next cranial vertebra. Histological examination was used as the gold standard for determining the actual site of spinal cord compression. Sensitivity and specificity for the diagnostic criteria were estimated at each site in neutral and flexed neck positions using several different decision criteria. Conclusions: At C6-C7, in neutral or flexed neck position and using 20% reduction of DD, the test was highly sensitive and specific for CSM. At other sites, reduced height of the myelographic column generally was not accurate for diagnosing extradural spinal cord compression. Using 20% reduction of DD in neutral position at the mid-cervical sites, the test had only low sensitivity and high specificity. Flexion of the neck appeared to increase detection of spinal cord compression in the mid-cervical region, but also substantially increased the frequency of false-positive diagnoses. Potential relevance: By using the reported sensitivity and specificity estimates, readers may decide on a decision criterion for diagnosis of extradural spinal cord compression due to CSM. However, in planning a surgical correction, it is difficult to define a decision criterion that combines acceptable sensitivity and specificity, especially at the mid-cervical sites. [source]

Clinical importance of antibodies against platelet activating factor in antiphospholipid syndrome manifestations

Background We assessed whether antibodies against platelet activating factor (PAF) are related to the presence of antiphospholipid syndrome (APS) clinical manifestations, in particular thrombosis, in patients with connective tissue diseases. Materials and methods Anti-PAF, anticardiolipin (aCL), anti,2 glycoprotein I (anti,2GPI) and antiphosphatidylcholine (anti-PC) antibodies were determined in 52 patients with APS, 29 patients with systemic lupus erythematosus (SLE) aCL but without APS, 30 patients with SLE without aCL, and 30 patients with scleroderma. A new enzyme-linked immunosorbent assay (ELISA) was developed for determining anti-PAF antibodies in a bovine serum-free fashion. Results The ELISA showed high specificity. Homologous inhibition experiments showed 60,70% inhibition. Anti-PAF antibodies were found in 18/52 APS patients, 10/29 SLE/aCL+ patients, 9/30 SLE/aCL, patients and 3/30 scleroderma patients. Anti-PAF antibodies were significantly associated with anti-PC antibodies (odds ratio [OR] 12.7, P < 0.01), and there was a modest association with immunoglobulin G (IgG) aCL (OR 3.1, P > 0.10), but not with IgM aCL or anti,2GPI. Three SLE/aCL+ patients and five SLE/aCL, patients had clinical manifestations characteristic of APS. All these patients had anti-PAF antibodies, while none had high titres of aCL or anti,2GPI antibodies and only one had anti-PC antibodies. Among the combined APS and SLE groups, the presence of anti-PAF antibodies was significantly associated with clinical manifestations which are characteristic of APS (OR 2.6, P = 0.02). The effect was independent of IgG aCL and anti,2GPI antibodies. Conclusions Anti-PAF antibodies are common in APS and SLE and comprise an independent factor for the development of thrombosis. Several patients experiencing thromboses have anti-PAF antibodies without other antiphospholipid specificities. [source]

Kinetic mechanism for p38 MAP kinase ,

FEBS JOURNAL, Issue 18 2005
A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate
p38 Mitogen-activated protein kinase alpha (p38 MAPK,) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPK,, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1,10) that at pH 7.5, 10 mm Mg2+ and 27 C p38 MAPK, phosphorylates ATF2,115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (,,,-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPK, with dissociation constants of KA = 360 m, KI = 240 m, and KI > 2000 m, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPK,,ATF2,115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd , 70 m. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2,115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a ,-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPK, inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations >,100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor. [source]

Oxidation of phenols by laccase and laccase-mediator systems

FEBS JOURNAL, Issue 21 2002
Solubility, steric issues
To investigate how solubility and steric issues affect the laccase-catalysed oxidation of phenols, a series of oligomeric polyphenol compounds, having increasing size and decreasing solubility in water, was incubated with laccase. The extent of substrate conversion, and the nature of the products formed in buffered aqueous solutions, were compared to those obtained in the presence of an organic cosolvent, and also in the presence of two mediating species, i.e. N -hydroxyphthalimide (HPI) and 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO). This approach showed not only an obvious role of solubility, but also a significant role of the dimension of the substrate upon the enzymatic reactivity. In fact, reactivity decreases as substrate size increases even when solubility is enhanced by a cosolvent. This effect may be ascribed to limited accessibility of encumbered substrates to the enzyme active site, and can be compensated through the use of the appropriate mediator. While TEMPO was highly efficient at enhancing the reactivity of large, less soluble substrates, HPI proved less effective. In addition, whereas the laccase/HPI system afforded the same products as laccase alone, the use of TEMPO provided a different product with high specificity. These results offer the first evidence of the role of ,oxidation shuttles' that the mediators of laccase may have, but also suggest two promising routes towards an environmentally friendly process for kraft pulp bleaching: (a) the identification of mediators which, once oxidized by laccase, are able to target strategic functional groups present in lignin, and (b) the introduction of those strategic functional groups in an appropriate pretreatment. [source]

Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae

Wang Yang
Abstract Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae. Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (,2 test, P<0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection. [source]

Affinity-Based Protein Surface Pattern Formation by Ligand Self-Selection from Mixed Protein Solutions

Manish Dubey
Abstract Photolithographically prepared surface patterns of two affinity ligands (biotin and chloroalkane) specific for two proteins (streptavidin and HaloTag, respectively) are used to spontaneously form high-fidelity surface patterns of the two proteins from their mixed solution. High affinity protein-surface self-selection onto patterned ligands on surfaces exhibiting low non-specific adsorption rapidly yields the patterned protein surfaces. Fluorescence images after protein immobilization show high specificity of the target proteins to their respective surface patterned ligands. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging further supports the chemical specificity of streptavidin and HaloTag for their surface patterned ligands from mixed protein solutions. However, ToF-SIMS did detect some non-specific adsorption of bovine serum albumin, a masking protein present in excess in the adsorbing solutions, on the patterned surfaces. Protein amino acid composition, surface coverage, density, and orientation are important parameters that determine the relative ToF-SIMS fragmentation pattern yields. ToF-SIMS amino acid-derived ion fragment yields summed to produce surface images can reliably determine which patterned surface regions contain bound proteins, but do not readily discriminate between different co-planar protein regions. Principal component analysis (PCA) of these ToF-SIMS data, however, improves discrimination of ions specific to each protein, facilitating surface protein pattern identification and image contrast. [source]

Characterization and Application of a New Monoclonal Antibody with High Specificity for Helicobacter hepaticus

HELICOBACTER, Issue 1 2009
Yoshihiro Fukuda
Abstract Background and Aims:, Infection with Helicobacter hepaticus is suggested to play a role in the pathogenesis of chronic liver disease in humans. However, reactive antigens among Helicobacter species make the development of an H. hepaticus ELISA test with high specificity difficult. A new monoclonal antibody from a hybridoma clone (HRII-51) showed high specificity to H. hepaticus without cross-reaction to other gastrointestinal bacteria. Methods:, The molecular weight of HRII-51 immunoreactive antigen was examined by Western blot of H. hepaticus probed with the monoclonal antibody HRII-51. A HRII-51-immunoreactive antigen capture ELISA was prepared in which the specific antigen was anchored by HRII-51-immobilized ELISA plate. Accuracy of HRII-51 antigen capture ELISA was examined using sera obtained from mice inoculated with Helicobacter species. Specificity of HRII-51 antigen capture ELISA was compared to that of H. hepaticus antigen-based ELISA using human sera with absorption by H. pylori cell lysate. Results:, HRII-51 immunoreactive antigen had a molecular weight of 15 kDa. Sensitivity and specificity of HRII-51 antigen capture ELISA were 87.0% and 97.6% in mice inoculated with Helicobacter species. In human sera, modification of the results by absorption with H. pylori lysate was smaller in HRII-51 antigen capture ELISA comparing with H. hepaticus -antigen-based ELISA. Conclusion:, Use of the HRII-51 antigen capture ELISA would be a useful approach for the serodiagnosis of H. hepaticus infection in both experimental animals and humans. [source]

Oral contrast-enhanced sonography for the diagnosis and grading of postsurgical recurrence of Crohn's disease

Fabiana Castiglione MD
Abstract Background: Postsurgical recurrence (PSR) is very common in patients with Crohn's disease (CD) and previous surgery. Endoscopy is crucial for the diagnosis of PSR, also showing high prognostic value. Bowel sonography (BS) with or without oral contrast enhancement (OCBS) is accurate for CD diagnosis but its role in PSR detection and grading is poorly investigated. The aim was to evaluate the diagnostic accuracy of BS and OCBS for PSR compared to the endoscopical Rutgeerts's grading system. Methods: We prospectively performed endoscopy, BS, and OCBS in 40 CD patients with previous bowel resection to provide evidence of possible PSR. Endoscopy, BS, and OCBS were executed 1 year after surgery, with PSR diagnosis and grading made in accordance with Rutgeerts. BS and OCBS were considered suggestive for PSR in the presence of bowel wall thickness (BWT) >3 mm. OCBS was performed after ingestion of 750 mL of polyethylene glycol (PEG). Also, a receiver operating characteristic (ROC) curve was constructed in order to define the best cutoff of BWT to discriminate mild from severe PSR (grade 0,2 versus 3,4 of Rutgeerts) for both BS and OCBS. Results: In all, 22 out of the 40 CD showed an endoscopic evidence of PSR (55%). A severe PSR was present in 14 patients (64%). Sensitivity, specificity, and positive and negative predictive values were 77%, 94%, 93%, and 80% for BS, and 82%, 94%, 93%, and 84% for OCBS. On the ROC curve a BWT >5 mm showed sensitivity, specificity, and positive and negative predictive values of 93%, 96%, 88%, and 97% for the diagnosis of severe PSR at BS, while a BWT >4 mm was the best cutoff differentiating the mild from the severe CD recurrence for OCBS, with a sensitivity, specificity, and positive and negative predictive values of 86%, 96%, 97%, and 79%, respectively. Conclusions: Both BS and OCBS show good sensitivity and high specificity for the diagnosis of PSR in CD, with a BWT >5 mm for BS and BWT >4 mm for OCBS strongly indicative of severe endoscopic PSR. Accordingly, these techniques could replace endoscopy for the diagnosis and grading of PSR in many cases. (Inflamm Bowel Dis 2008) [source]

Directed Assembly of Polymer Blends Using Nanopatterned Templates

Ming Wei
The direct assembly of polymer blends on chemically functionalized surfaces is shown to produce a variety of nonuniform complex patterns. This method provides a powerful tool for easily producing nonuniform patterns in a rapid (30 s), one-step process with high specificity and selectivity for a variety of applications, such as nanolithography, polymeric optoelectronic devices, integrated circuits, and biosensors. [source]

Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of ochratoxin A

Won-Bo Shim
Summary A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the determination of ochratoxin A (OTA) was developed. Fluorescein-labelled OTA derivative (tracer) was synthesized and purified by thin-layer chromatography. The optimized OTA FPIA had a dynamic range from 5 to 200 ng mL,1 with IC50 value of 30 ng mL,1 and a detection limit of 3 ng mL,1. The method developed was characterized by high specificity and reproducibility. Cross-reactivity with other mycotoxins (zearalenone, aflatoxins, patulin and T-2 toxin) was negligible (<0.1%). Methanol extracts of barley samples were used for the analysis. The results of OTA determination in barley were compared with those determined by indirect competitive enzyme-linked immunosorbent assay (ELISA). Recoveries for the samples spiked at 50, 100 and 500 ng g,1 levels were 91, 90 and 97%, respectively, for FPIA, and 98, 98 and 102%, for ELISA. Naturally contaminated barley samples were analysed by these methods but some disagreement was observed between the results. The FPIA method can be applied for screening of food samples for OTA residues without a complicated clean-up. [source]

Accuracy of Spirometry in Diagnosing Pulmonary Restriction in Elderly People

Simone Scarlata MD
OBJECTIVES: To compare the accuracy of a diagnosis of pulmonary restriction made using forced vital capacity (FVC) less than the lower limit of normal (LLN) with the criterion standard diagnosis made using total lung capacity (TLC) less than the LLN in an elderly population. DESIGN: Retrospective analysis. SETTING: A teaching hospital. PARTICIPANTS: Five hundred sixty-four ambulatory and acute care hospital patients aged 65 to 96 underwent complete pulmonary function evaluation. MEASUREMENTS: Sensitivity, specificity, positive and negative predictive values (PPV, NPV) of diagnosis of pulmonary restriction defined as FVC less than the LLN were calculated in the overall sample and after stratification according to bronchial obstruction. Expected PPV and NPV at different background prevalence of true pulmonary restriction (5% and 15%) were calculated using the Bayes theorem. RESULTS: Low sensitivity (0.32) and high specificity (0.95) were found, with an area under the receiver operating characteristic curve (AUC) of 0.89. In participants without bronchial obstruction, specificity was even higher, although sensitivity decreased to 0.28 (AUC=0.92). The PPV was good (0.81), whereas with a low to moderate a priori probability (prevalence from 5% to 15%) the NPV was fair (,0.89). CONCLUSION: A reduction in FVC below LLN cannot reliably identify true pulmonary restriction in elderly people, confirming previous findings in the adult population. Normal FVC, instead, can effectively exclude pulmonary restriction regardless of the presence of bronchial obstruction when the a priori probability is low or moderately high. [source]

Development and evaluation of a one-step loop-mediated isothermal amplification for detection of spring viraemia of carp virus

Z. Liu
Abstract Aim:, Spring viraemia of carp virus (SVCV) is the causative agent of SVC disease. The main aim of our study was to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid, sensitive and effective detection of SVCV. Methods and Results:, A set of four specific primers, two outer and two inner primers were designed based on the SVCV M gene for RT-LAMP assay. The sensitivity and specificity of RT-LAMP were determined and clinical test was performed under optimized amplification conditions (64C, 60 min). The results showed that the assay has a high specificity and the detection limit was 80 copies using 10-fold series dilutions of SVCV RNA, 10 times more sensitive than nest reverse transcription-polymerase chain reaction. In the detection of 472 fish samples, this assay showed excellent agreement with the standard virus isolation method (, = 0807). Conclusions:, A sensitive and specific RT-LAMP assay was successfully developed to monitor and detect SVCV. Significance and Impact of the Study:, This work provides a robust method for evaluating the risk of SVCV. Given the advantages of LAMP in the detection of SVCV, this method can be applied to diagnose other viruses, which pose serious threats to the aquaculture industry. [source]

The application of chromogenic media in clinical microbiology

J.D. Perry
Summary Since 1990, a wide range of chromogenic culture media has been made commercially available providing useful tools for diagnostic clinical microbiology. By the inclusion of chromogenic enzyme substrates targeting microbial enzymes, such media are able to target pathogens with high specificity. Examples of target pathogens include Staphylococcus aureus, Streptococcus agalactiae, Salmonella spp. and Candida spp. The inclusion of multiple chromogenic substrates into culture media facilitates the differentiation of polymicrobial cultures, thus allowing for the development of improved media for diagnosis of urinary tract infections and media for the enhanced discrimination of yeasts. The purpose of this review is to provide some insight into how such media work and appraise their utility in routine clinical diagnostics, in comparison with conventional media. [source]

In vitro inhibition of oral streptococci binding to the acquired pellicle by algal lectins

E.H. Teixeira
Abstract Aims:, The initial colonization of the tooth by streptococci involves their attachment to adsorbed components of the acquired pellicle. Avoiding this adhesion may be successful in preventing caries at early stages. Salivary mucins are glycoproteins that when absorbed onto hydroxyapatite may provide binding sites for certain bacteria. Algal lectins may be especially interesting for oral antiadhesion trials because of their great stability and high specificity for mucins. This work aimed to evaluate the potential of two algal lectins to inhibit the adherence of five streptococci species to the acquired pellicle in vitro. Methods and Results:, The lectins used were extracted from Bryothamnion triquetrum (BTL) and Bryothamnion seaforthii (BSL). Fluorescence microscopy was applied to visualize the ability of fluorescein isothiocyanate-labelled lectins to attach to the pellicle and revealed a similar capability for both lectins. Streptococcal adherence assays were performed using saliva-coated microtitre plates. BSL inhibited more than 75% of Streptococcus sanguis, Streptococcus mitis, Streptococcus sobrinus and Streptococcus mutans adherence, achieving 92% to the latter. BTL only obtained statistically significant results on S. mitis and S. sobrinus, whose adherence was decreased by 325% and 544%, respectively. Conclusion:, Algal lectins are able to inhibit streptococcal adherence. Significance and Impact of the Study:, Our results support the proposed application of lectins in antiadhesion therapeutics. [source]

Identification of Escherichia coli O172 O-antigen gene cluster and development of a serogroup-specific PCR assay

H. Guo
Abstract Aim:, To characterize the locus for O-antigen biosynthesis from Escherichia coli O172 type strain and to develop a rapid, specific and sensitive PCR-based method for identification and detection of E. coli O172. Methods and Results:, DNA of O-antigen gene cluster of E. coli O172 was amplified by long-range PCR method using primers based on housekeeping genes galF and gnd Shot gun bank was constructed and high quality sequencing was performed. The putative genes for synthesis of UDP-FucNAc, O-unit flippase, O-antigen polymerase and glycosyltransferases were assigned by the homology search. The evolutionary relationship between O-antigen gene clusters of E. coli O172 and E. coli O26 is shown by sequence comparison. Genes specific to E. coli O172 strains were identified by PCR assays using primers based on genes for O-unit flippase, O-antigen polymerase and glycosyltransferases. The specificity of PCR assays was tested using all E. coli and Shigella O-antigen type strains, as well as 24 clinical E. coli isolates. The sensitivity of PCR assays was determined, and the detection limits were 1 pg ,l,1 chromosomal DNA, 02 CFU g,1 pork and 02 CFU ml,1 water. The total time required from beginning to end of the procedure was within 16 h. Conclusion:, The O-antigen gene cluster of E. coli O172 was identified and PCR assays based on O-antigen specific genes showed high specificity and sensitivity. Significance and Impact of the Study:, An O-antigen gene cluster was identified by sequencing. The specific genes were determined for E. coli O172. The sensitivity of O-antigen specific PCR assay was tested. Although Shiga toxin-producing O172 strains were not yet isolated from clinical specimens, they may emerge as pathogens. [source]