Relationship between meat quality and intramuscular collagen characteristics of muscles from calf-fed, yearling-fed and mature crossbred beef cattle.

Intramuscular Ehrlich Chromogen (EC) and pyridinoline (Pyr) concentrations in the gluteus medius (GM) and semitendinosus (ST) from crossbred Angus calf- (n = 14) and yearling-fed (n = 14) steer and mature cow (MC, n = 12) carcasses were related to collagen and intramuscular connective tissue (IMCT) thermal stability and peak Warner-Bratzler shear force (WBSF). In both muscles, Pyr density was greater in MC, while EC concentrations were comparable in calf- and yearling-fed steer muscles and lowest in MC muscles. Thermal denaturation temperature and enthalpy of IMCT were highest in both muscles when from MC, although only total collagen was correlated with WBSF in calf fed-yearling fed steer data. Results confirmed that EC concentration contributed to collagen thermal stability in steer muscles, but decreased it in MC muscles, while Pyr was consistently associated with collagen thermal stability.

Colorimetric Polymerase Chain Reaction Enabled by a Fast Light-Activated Substrate Chromogenic Detection Platform.

Miniaturization of nucleic acid tests (NATs) into portable, inexpensive detection platforms may aid disease diagnosis in point-of-care (POC) settings. Colorimetric signals are ideal readouts for portable NATs, and it remains of high demand to develop color readouts that are simple, quantitative, and versatile. Thus motivated, we report a fast light-activated substrate chromogenic polymerase chain reaction (FLASH PCR) that uses DNA intercalating dyes (DIDs) to enable colorimetric nucleic acid detection and quantification. The FLASH system is established on our finding that DID-DNA intercalation can promote the rapid photooxidation of chromogenic substrates through light-induced production of singlet oxygen. Using this principle, we have successfully converted DID-based fluorescent PCR assays into colorimetric FLASH PCR. To demonstrate the practical applicability of FLASH PCR to POC diagnosis, we also fabricated two readout platforms, including a portable electronic FLASH reader and a paper-based FLASH strip. Using the FLASH reader, we were able to detect as low as 60 copies of DNA standards, a limit of detection (LOD) comparable with commercial quantitative PCR. The FLASH strip further enables the reader-free detection of PCR amplicons by converting the colorimetric signal into the visual measurement of distance as a readout. Finally, the practical applicability of the FLASH PCR was demonstrated by the detection and/or quantification of nucleic acid markers in diverse clinical and biological samples.

Chromogenic Tissue-Based Methods for Detection of Gene Amplifications (or Rearrangements) Combined with Protein Overexpression in Clinical Samples.

Immunohistochemistry (IHC) is a well-established, tissue-based assay for the visualization of target proteins. For analysis of DNA targets, chromogenic in situ hybridization (CISH) applications have significant advantages over traditional fluorescence in situ hybridization (FISH). CISH slides can be analyzed using a regular light microscope, while FISH slides require the use of a specialized fluorescence microscope in a dark room. CISH slides allow observers to correlate the gene status (gene amplifications, gene rearrangements, and gene deletions) in the context of tissue morphology better than FISH slides. Recently, a combination of IHC and CISH assays (gene-protein assay, GPA) was developed to study the relationship between gene status and protein expression on the same tissue section. CISH and GPA applications can be optimized using an automated tissue slide processing system to generate reproducible results for a long and complex assay protocol. GPA applications are an ideal approach for tumor status and heterogeneity analyses for research and clinical investigations.

Chromogenic culture media or rapid immunochromatographic test: Which is better for detecting Klebsiella pneumoniae that produce OXA-48 and can they be used in blood and urine specimens.

Our goal was to compare a rapid test (OXA-48K-SeT) and four different chromogenic media (CHROMagar KPC, CHROMagar mSuperCARBA, ChromID Carba and ChromID OXA-48) for the detection of OXA-48 producing Klebsiella pneumoniae isolates and spiked urine/blood samples with these bacteria. In total 100 K.pneumoniae isolates, including 60 OXA-48 positive, 15 other carbapenemase producing, 15 Extended spectrum betalactamases (ESBL) positive and 10 carbapenem sensitive K.pneumoniae were included in the study. After all samples were inoculated into all chromogenic media, temocillin discs were placed onto the media. OXA-48K-SeT was studied according to the manufacturer's instructions and the lower detection limit was determined. Sensitivities and specificities of all chromogenic media and rapid test were detected as 100%. All of the OXA-48 producers were found resistant to temocillin on all chromogenic media. The lower detection limit of the rapid assay was determined as 106 in both direct bacterial samples and in spiked urine/blood samples. As a result, four chromogenic culture media and OXA-48 K-SeT can be used safely for detection of OXA-48 positive K.pneumoniae isolates. Although direct clinical specimens were not used, our study suggests that this media and OXA-48 K-SeT may be used in patient samples like blood and urine. Further studies are needed to assess this suggestion.

DNAzyme-aptamer or aptamer-DNAzyme paradigm: Biochemical approach for aflatoxin analysis.

DNAzyme and aptamer conjugations have already been used for sensitive and accurate detection of several molecules. In this study, we tested the relationship between conjugation orientation of DNAzyme and aflatoxin B1 aptamer and their subsequent peroxidase activity. Circular dichroism (CD) spectroscopy and biochemical analysis were used here to differentiate between these two conjugation patterns. Results showed that DNAzyme-aptamer has more catalytic activity and efficiency than aptamer-DNAzyme. Thereby, DNAzyme-aptamer with its superior efficiency can be used for design and development of more sensitive aflatoxin B1 DNA based biosensors.

Chromogenic detection procedure for the multidrug-resistant, neonatal sepsis-associated clone Staphylococcus capitis NRCS-A.

The multiresistant Staphylococcus capitis clone NRCS-A is a major pathogen in neonates worldwide. We show that NRCS-A grows as mauve colonies with a cream-color halo after a 5-day incubation on MRSA Brilliance 2 agar (Oxoid®). This innovative protocol will ease the screening of clinical and environmental niches of this clone.

CHROMagar mSuperCARBA and RAPIDEC® Carba NP test for detection of carbapenemase-producing Enterobacteriaceae.

A novel chromogenic medium CHROMagar mSuperCARBA was evaluated to detect carbapenem-resistant Gram-negatives. This medium is as sensitive and as specific as the SUPERCARBA medium for detecting KPC, MBL and OXA-48-type producers (100% and 100%, respectively) and is compatible with subsequent testing of carbapenemase activity using the RAPIDEC® CARBA NP.

Chromogenic Factor VIII Assays for Improved Diagnosis of Hemophilia A.

Hemophilia A is an inherited bleeding disorder caused by a reduced level of factor VIII coagulant activity (FVIII:C) in blood. Bleeding episodes may occur spontaneously in the severe form of hemophilia or after trauma in the milder forms. It is important that patients are diagnosed correctly, which includes placing them into the correct severity category of the disorder so that appropriate treatment can be given. Diagnosis is made by determination of the amount of FVIII:C in the blood, usually using a one-stage factor VIII:C assay. However, approximately one third of patients with mild or moderate hemophilia will have much lower results by the chromogenic assay, with some of them having normal results by the one-stage assay. The chromogenic factor VIII assay is used in some specialized hemophilia reference centers and is recommended for the diagnosis of mild hemophilia A, as this assay is considered to better reflect the severity status of hemophilia patients than the one-stage assay.

Antithrombin Activity of Erythrocyte Microvesicles.

Coagulation and optical (based on chromogenic substrate) methods were employed to examine antithrombin activity of erythrocytes and erythrocyte-derived microvesicles isolated days 7, 14, 21, and 28 on erythrocyte storage. The erythrocyte-derived microvesicles decelerated fibrin clot formation from fibrinogen in the presence of exogenous thrombin both with and without heparin. Microvesicles reduced optical density of chromogenic substrate. These data suggest that erythrocyte-derived microvesicles display a prominent antithrombin activity, which significantly increases during erythrocyte storage.

Feasibility of rapid measurement of Rivaroxaban plasma levels in patients with acute stroke.

Plasma levels of Rivaroxaban (RivLev) might be useful to guide therapeutic decisions in patients with acute stroke under Rivaroxaban. A prerequisite for the potential clinical usefulness is their rapid availability in emergency situations. Single-center explorative analysis from the Novel-Oral-Anticoagulants-in-Stroke-Patients-registry (NOACISP, cinicaltrials.gov:NCT02353585). We included consecutive patients with acute ischemic or hemorrhagic stroke under Rivaroxaban (last intake <48 h) in which RivLev determined by an automated anti-factor Xa-based chromogenic assay (Hyphen-Biomed, France) are available. Primary endpoint was the turnaround time (TAT), defined as time from registration of the blood sample in the lab to first result published. Furthermore, we studied, whether TAT is influenced by (1) on- and off-hour-measurements and (2) early versus later patient arrival (cut-off: 270 min after symptom onset). Thirty-eight patients met the eligibility criteria (mean age 77 years, 44 % female). TAT was 34 min (IQR 29-65 min). TATs were similar for on- (n = 14; median 34 min; IQR 30-56 min) and off-hours-TATs (n = 24; median 35 min; IQR 29-75 min) as well as for early (n = 16; median 33 min; IQR 30-40 min) and late patient arrival (n = 22, median 34 min, IQR 28-58 min; all nonsignificant.). Taking into account RivLev in the decision process about the use of intravenous thrombolysis, three patients received intravenous thrombolysis on an individualized basis, none of them with bleeding complications. Emergency measurement of RivLev among patients with acute stroke is available within a median of 34 min and therefore feasible for ED use. Due to the rapid availability, further research to evaluate the role of RivLev in order to guide acute treatment decisions is warranted.

Two-color fluorescence in situ hybridization using chromogenic substrates in Xenopus.

Xenopus embryo yolk proteins present a serious barrier to fluorescence microscopy. Previously, in situ assays of gene transcripts in whole embryos was limited to the use of chromogenic alkaline phosphatase substrates, which restricted researchers' ability to gauge coexpression of transcripts. Here, we describe a modified in situ hybridization (ISH) protocol that uses fluorescent substrates and a novel yolk-clearing technique for simultaneous visualization of the expression of two genes in whole Xenopus embryos with high resolution. This protocol employs two well-known fluorescent substrates, nitro blue tetrazolium/5-bromo- 4-chloro-3-indolyl-phosphate (NBT/BCIP) and Vector Red, in a sequential dual in situ hybridization procedure. Subsequent clearing of the samples with refractive-index-matching solution (RIMS) renders the samples amenable to confocal microscopy, allowing imaging at sufficiently high resolution to discern coexpression of transcripts within individual cells.

Automated measurement of estrogen receptor in breast cancer: a comparison of fluorescent and chromogenic methods of measurement.

Whereas FDA-approved methods of assessment of estrogen receptor (ER) are 'fit for purpose', they represent a 30-year-old technology. New quantitative methods, both chromogenic and fluorescent, have been developed and studies have shown that these methods increase the accuracy of assessment of ER. Here, we compare three methods of ER detection and assessment on two retrospective tissue microarray (TMA) cohorts of breast cancer patients: estimates of percent nuclei positive by pathologists and by Aperio's nuclear algorithm (standard chromogenic immunostaining), and immunofluorescence as quantified with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). Reproducibility was excellent (R(2)>0.95) between users for both automated analysis methods, and the Aperio and QIF scoring results were also highly correlated, despite the different detection systems. The subjective readings show lower levels of reproducibility and a discontinuous, bimodal distribution of scores not seen by either mechanized method. Kaplan-Meier analysis of 10-year disease-free survival was significant for each method (Pathologist, P=0.0019; Aperio, P=0.0053, AQUA, P=0.0026); however, there were discrepancies in patient classification in 19 out of 233 cases analyzed. Out of these, 11 were visually positive by both chromogenic and fluorescent detection. In 10 cases, the Aperio nuclear algorithm labeled the nuclei as negative; in 1 case, the AQUA score was just under the cutoff for positivity (determined by an Index TMA). In contrast, 8 out of 19 discrepant cases had clear nuclear positivity by fluorescence that was unable to be visualized by chromogenic detection, perhaps because of low positivity masked by the hematoxylin counterstain. These results demonstrate that automated systems enable objective, precise quantification of ER. Furthermore, immunofluorescence detection offers the additional advantage of a signal that cannot be masked by a counterstaining agent. These data support the usage of automated methods for measurement of this and other biomarkers that may be used in companion diagnostic tests.

Rapid Photodegradation of Methyl Orange (MO) Assisted with Cu(II) and Tartaric Acid.

Cu(II) and organic carboxylic acids, existing extensively in soil and aquatic environments, can form complexes that may play an important role in the photodegradation of organic contaminants. In this paper, the catalytic role of Cu(II) in the removal of methyl orange (MO) in the presence of tartaric acid with light was investigated through batch experiments. The results demonstrate that the introduction of Cu(II) could markedly enhance the photodegradation of MO. In addition, high initial concentrations of Cu(II) and tartaric acid benefited the decomposition of MO. The most rapid removal of MO assisted by Cu(II) was achieved at pH 3. The formation of Cu(II)-tartaric acid complexes was assumed to be the key factor, generating hydroxyl radicals (•OH) and other oxidizing free radicals under irradiation through a ligand-to-metal charge-transfer pathway that was responsible for the efficient degradation of MO. Some intermediates in the reaction system were also detected to support this reaction mechanism.

Specific and global coagulation tests in patients with mild haemophilia A with a double mutation (Glu113Asp, Arg593Cys).

BACKGROUND: Heterogeneous bleeding phenotypes are observed in haemophilia A patients with the same mutation in the F8 gene. Specific mutations in the A2 domain of factor VIII are associated with mild haemophilia and a higher risk of inhibitor development. Double mutations in mild haemophilia A are rarely reported. In this study, we investigated the in vitro function of factor VIII, performing different specific and global coagulation assays, observed clinical characteristics and assessed the possible predictive diagnostic value of the differences. MATERIALS AND METHODS: The clinical features of haemophiliacs with a mild phenotype were reviewed. Blood samples were obtained and analysed for mutations and coagulation assays: activated partial thromboplastin time, one-stage and chromogenic factor VIII activity, factor VIII antigen and rotational thromboelastometry. RESULTS: We report on a cohort of 22 patients with double Glu113Asp, Arg593Cys mutations. All our patients have a quantitative defect of factor VIII and preserved similar functional activity. Factor VIII activities measured by the one-stage or chromogenic method were not discrepant, although the chromogenic assay resulted in 20% lower factor VIII activities. Waveform analysis showed a lower maximum value of the second derivative curve (Max2) of APTT with curve shape alternation, while thromboelastometry (INTEM) showed low sensitivity in comparison to results in a normal population. DISCUSSION: In genotyping, the coexistence of a second mutation should never be excluded, especially in cases of discordant clinical presentation. Waveform analysis correlates better with factor VIII activity than thromboelastometry and the Max2 parameter could provide additional information in managing haemophilia patients. The utility of specific factor activity and global haemostatic assays in general practice still needs to be investigated.

Low-Cost, Disposable, Flexible and Highly Reproducible Screen Printed SERS Substrates for the Detection of Various Chemicals.

Ideal SERS substrates for sensing applications should exhibit strong signal enhancement, generate a reproducible and uniform response, and should be able to fabricate in large-scale and low-cost. Herein, we demonstrate low-cost, highly sensitive, disposable and reproducible SERS substrates by means of screen printing Ag nanoparticles (NPs) on a plastic PET (Polyethylene terephthalate) substrates. While there are many complex methods for the fabrication of SERS substrates, screen printing is suitable for large-area fabrication and overcomes the uneven radial distribution. Using as-printed Ag substrates as the SERS platform, detection of various commonly known chemicals have been done. The SERS detection limit of Rhodamine 6G (R6G) is higher than the concentration of 1 × 10(-10) M. The relative standard deviation (RSD) value for 784 points on the detection of R6G and Malachite green (MG) is less than 20% revealing a homogeneous SERS distribution and high reproducibility. Moreover, melamine (MA) is detected in fresh liquid-milk without additional pretreatment, which may accelerate the application of rapid on-line detection of MA in liquid milk. Our screen printing method highlights the use of large-scale printing strategies for the fabrication of well-defined functional nanostructures with applications well beyond the field of SERS sensing.

Evaluation of New bioMérieux Chromogenic CPS Media for Detection of Urinary Tract Pathogens.

Four chromogenic media were compared for their ability to detect urinary tract pathogens in 299 urine specimens, of which 175 were found positive, allowing the growth of 279 microorganisms. After 18 to 24 h of incubation, the CPS ID4, CPSE, CPSO (bioMérieux), and UriSelect4 (Bio-Rad) media showed sensitivities of 97.1%, 99.3%, 99.6%, and 99.6%, respectively.

Femtogram detection of horseradish peroxidase by a common desktop scanner.

We report an image-based detection of horseradish peroxidase (HRP) by different color spaces. The results show excellent correlation between color saturation and absorbance (Pearson correlation coefficient; 0.9868) with respect to HRP. The present method can detect 185 and 46.45 fg/ml of HRP using o-phenylenediamine dihydrochloride and 3,3',5,5'-tetramethylbenzidine as chromogenic substrates respectively.

Two-color fluorescent in situ hybridization using chromogenic substrates in zebrafish.

Two-color fluorescent in situ hybridization (FISH) is a widely used technique for comparing relative gene expression patterns. Current two-color FISH protocols are not ideal for detecting weakly expressed transcripts or monitoring signal strength and background levels during the course of the reaction. Here we describe an improved FISH protocol using the conventional highly sensitive chromogenic substrates nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Vector Red in zebrafish embryos. This protocol substantially improves on existing FISH techniques by combining the advantages of long reactivity of alkaline phosphatase, chromogenic monitoring of both developing reactions, and the ability to perform subsequent high-resolution fluorescent imaging. Although tested in zebrafish, a similar approach is expected to be applicable to ISH in any model organism.

Comparative evaluation of two chromogenic tests for rapid detection of carbapenemase in Enterobacteriaceae and in Pseudomonas aeruginosa isolates.

We compared the performance of the Carba NP test and the Rosco Rapid CARB screen kit for detecting carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Both tests are rapid and highly sensitive; however, the Carba NP test showed superior specificity, and several uninterpretable results were observed with the Rapid CARB screen.