Novel Chromogens for Immunohistochemistry in Spatial Biology.

Spatial relations between tumor cells and host-infiltrating cells are increasingly important in both basic science and clinical research. In this study, we have tested the feasibility of using standard methods of immunohistochemistry (IHC) in a multiplex staining system using a newly developed set of chromogenic substrates for the peroxidase and alkaline phosphatase enzymes. Using this approach, we have developed a set of chromogens characterized by (1) providing fine cellular detail, (2) non-overlapping spectral profiles, (3) an absence of interactions between chromogens, (4) stability when stored, and (5) compatibility with current standard immunohistochemistry practices. When viewed microscopically under brightfield illumination, the chromogens yielded the following colors: red, black, blue, yellow, brown, and green. By selecting compatible color combinations, we have shown feasibility for four-color multiplex staining. Depending on the particular type of analysis being performed, visual analysis, without the aid of computer-assisted image analysis, was sufficient to differentiate up to four different markers.

A functional chromogen gene C from wild rice is involved in a different anthocyanin biosynthesis pathway in indica and japonica.

KEY MESSAGE: we identified a functional chromogen gene C from wild rice, providing a new insight of anthocyanin biosynthesis pathway in indica and japonica. Accumulation of anthocyanin is a desirable trait to be selected in rice domestication, but the molecular mechanism of anthocyanin biosynthesis in rice remains largely unknown. In this study, a novel allele of chromogen gene C, OrC1, from Oryza rufipongon was cloned and identified as a determinant regulator of anthocyanin biosynthesis. Although OrC1 functions in purple apiculus, leaf sheath and stigma in indica background, it only promotes purple apiculus in japonica. Transcriptome analysis revealed that OrC1 regulates flavonoid biosynthesis pathway and activates a few bHLH and WD40 genes of ternary MYB-bHLH-WD40 complex in indica. Differentially expressed genes and metabolites were found in the indica and japonica backgrounds, indicating that OrC1 activated the anthocyanin biosynthetic genes OsCHI, OsF3H and OsANS and produced six metabolites independently. Artificial selection and domestication of C1 gene in rice occurred on the coding region in the two subspecies independently. Our results reveal the regulatory system and domestication of C1, provide new insights into MYB transcript factor involved in anthocyanin biosynthesis, and show the potential of engineering anthocyanin biosynthesis in rice.

Rapid Detection of Escherichia coli in Water Using Sample Concentration and Optimized Enzymatic Hydrolysis of Chromogenic Substrates.

Methods for rapid detection of fecal indicator bacteria in water are important to ensure that water is safe for drinking, bathing, recreation, fishing and shellfish harvesting. In this study, we tested experimental conditions for bacterial hydrolysis of two promising enzymatic substrates, 5-Bromo-4-chloro-3-indolyl ß-D-glucuronide (X-Gluc) and Resorufin ß-D-glucuronide (REG), and optimized parameters such as temperature and pH to determine conditions for rapid reactions. We then innovated a membrane filter-based approach to facilitate more rapid enzyme-based detection of Escherichia coli in water based on the combination of an initial concentration step and optimized test conditions. For this approach, a water sample (10‒100 mL) is filtered through a 0.45-µm pore size filter with a diameter of 4 or 13 mm. After filtration, a newly designed rapid detection broth is added containing the enzymatic inducer Methyl-beta-D-Glucuronide sodium (MetGlu) and the substrate REG or X-Gluc. After a few (1‒7) hours of incubation at 35 °C, the filter shows pink color (for REG-containing broth) or green color (for X-Gluc containing broth) if E. coli is present. The study provides insights and approaches towards developing a simple, fast, and low-cost method to detect fecal indicator bacteria in water.

Specificity of peptidases secreted by filamentous fungi.

Peptidases are enzymes that cleave peptide bonds, yielding proteins and peptides. Enzymes in this class also perform several other functions, regulating the activation or inactivation of target substrates via proteolysis. Owing to these functions, peptidases have been extensively used in industrial and biotechnological applications. Given their potential functions, it is important to optimize the use of these enzymes, which requires determination of the specificity of each peptidase. The peptidase specificity must be taken into account in choosing a peptidase to catalyze the available protein source within the desired application. The specificity of a peptidase defines the profile of enzyme-substrate interactions, and for this the catalytic site and the arrangement of the amino acid residues involved in peptide bond cleavage need to be known. The catalytic sites of peptidases may be composed of several subsites that interact with amino acid residues for proteolysis. Filamentous fungi produce peptidases with varying specificity, and here we provide a review of those reported to date and their potential applications.

Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation.

The metabolic activity of hepatocytes is a central prerequisite for drug activity and a key element in drug-drug interaction. This central role in metabolism largely depends on the activity of the cytochrome P450 (CYP450) enzyme family, which is not only dependent on liver cell maturation but is also controlled in response to drug and chemical exposure. Here, we report the use of VividDye fluorogenic CYP450 substrates to directly measure and continuously monitor metabolic activity in living hepatocytes. We observed time- and dose-dependent correlation in response to established and putative CYP450 inducers acting through the aryl hydrocarbon receptor and drug combinations. Using repetitive addition of VividDye fluorogenic substrate on a daily basis, we demonstrated the new application of VividDye for monitoring the maturation and dedifferentiation of hepatic cells. Despite a lack of high specificity for individual CYP450 isoenzymes, our approach enables continuous monitoring of metabolic activity in living cells with no need to disrupt cultivation. Our assay can be integrated in in vitro liver-mimetic models for on-line monitoring and thus should enhance the reliability of these tissue model systems.

Variable performance of four commercial chromogenic media for detection of methicillin-resistant Staphylococcus aureus isolates harbouring mecC.

In this study, the performances of four methicillin-resistant Staphylococcus aureus (MRSA) chromogenic screening media were compared for detecting mecC-positive MRSA. Using 111 clinical isolates representative of the various mecC-MRSA clones in Europe, chromID® MRSA (bioMérieux) and BrillianceTM MRSA 2 (Oxoid Ltd.) showed higher sensitivity (99.1% and 97.3%, respectively) than BBLTM CHROMagar® MRSA II (BD Diagnostics) and MRSASelectTM (Bio-Rad) (79.3% and 63.1%, respectively) (P <0.0001). These findings have important implications for effective public health diagnostics and epidemiological surveillance of MRSA.

Faster and economical screening for vancomycin-resistant enterococci by sequential use of chromogenic agar and real-time polymerase chain reaction.

BACKGROUND/PURPOSE: Screening for vancomycin-resistant enterococci (VRE) by culture takes days to generate results, while polymerase chain reaction (PCR) testing directly from clinical specimens lacks specificity. The aims of this study were to develop a real-time PCR to detect and identify Enterococcus faecium, Enterococcus faecalis, and vanA and vanB genes, and to evaluate the impact of this PCR on test-reporting times when performing it directly from suspect VRE isolates present on screening chromogenic media. METHODS: The tetraplex PCR primers were designed to amplify E. faecium, E. faecalis, and vanA and vanB genes, with melt-curve analysis of PCR products. Following analytical and clinical validation of the molecular assay, PCR testing was performed for target colonies present on VRE chromogenic media. PCR results were evaluated against conventional phenotypic identification and susceptibility testing, with the time to result being monitored for both modalities. RESULTS: A total of 519 colonies from clinical specimens were tested concurrently by real-time PCR and phenotypic methods. In all, 223 isolates were identified with phenotypic vancomycin resistance (vanA, n = 108; vanB, n = 105; non-vanA/vanB = 10), with complete agreement between PCR and phenotypic testing for vancomycin-resistant E. faecium and E. faecalis. The majority (88.6%) of PCR results were reported, on average, 24.8 hours earlier than those of phenotypic testing, with 68% reduction in total costs. CONCLUSION: The use of culture on selective media, followed by direct colony PCR confirmation allows faster and economical VRE screening.

Automatic Digital Analysis of Chromogenic Media for Vancomycin-Resistant-Enterococcus Screens Using Copan WASPLab.

Vancomycin-resistant enterococci (VRE) are an important cause of health care-acquired infections (HAIs). Studies have shown that active surveillance of high-risk patients for VRE colonization can aid in reducing HAIs; however, these screens generate a significant cost to the laboratory and health care system. Digital imaging capable of differentiating negative and "nonnegative" chromogenic agar can reduce the labor cost of these screens and potentially improve patient care. In this study, we evaluated the performance of the WASPLab Chromogenic Detection Module (CDM) (Copan, Brescia, Italy) software to analyze VRE chromogenic agar and compared the results to technologist plate reading. Specimens collected at 3 laboratories were cultured using the WASPLab CDM and plated to each site's standard-of-care chromogenic media, which included Colorex VRE (BioMed Diagnostics, White City, OR) or Oxoid VRE (Oxoid, Basingstoke, United Kingdom). Digital images were scored using the CDM software after 24 or 40 h of growth, and all manual reading was performed using digital images on a high-definition (HD) monitor. In total, 104,730 specimens were enrolled and automation agreed with manual analysis for 90.1% of all specimens tested, with sensitivity and specificity of 100% and 89.5%, respectively. Automation results were discordant for 10,348 specimens, and all discordant images were reviewed by a laboratory supervisor or director. After a second review, 499 specimens were identified as representing missed positive cultures falsely called negative by the technologist, 1,616 were identified as containing borderline color results (negative result but with no package insert color visible), and 8,234 specimens were identified as containing colorimetric pigmentation due to residual matrix from the specimen or yeast (Candida). Overall, the CDM was accurate at identifying negative VRE plates, which comprised 84% (87,973) of the specimens in this study.

A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli.

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ß-galactosidase chromogenic substrate. The signal produced by cytoplasmic ß-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 µM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 µg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

A novel quantitative method of pten expression assessment in tumor tissue.

Phosphatase and Tensin Homolog deleted on chromosome 10 (PTEN) gene is one of the most important tumor suppressor genes which is involved in the regulation of many signaling cascades (AKT/PKB and MAPK). Subtle changes in its activity lead to cancer susceptibility or aggressive tumor behaviour. Despite the diversity of mechanisms leading to PTEN inactivation, it is frequently associated with a decreased or complete loss of protein expression. About 20% decrease in PTEN expression could lead to the development of cancer. There have been no objective, quantitative methods of PTEN expression assessment that allow to measure the subtle variations of the protein concentration in a tissue-contextual manner. A new quantitative algorithm of immunostaining evaluation based on combination of color deconvolution and relative chromogen signal intensity was used in the study. The proposed algorithm was implemented in the popular ImageJ image analysis software and positively verified in cancer cell lines and tissue models as well as in the tissue samples of colorectal cancer (CRC) patients. The proposed quantitative method of PTEN expression assessment creates an alternative to currently available subjective methods and forms the basis for inter-case and inter-tissue comparisons. Using the algorithm it would be possible to identify three groups of patients with advanced colorectal cancer which could significantly differ in the overall survival. The research should be continued.

Application of HER2 CISH pharmDX for DNA Ploidy Determination.

Products of conception (POC) are encountered daily in general pathology practice. The molar workup is an important part of POC examination. Ploidy analysis, expressed as DNA index (DI), is part of the pathologic workup of molar pregnancy. For the past decade, chromogenic in situ hybridization (CISH) has become a popular way to detect HER2 gene amplification. Current study aims to determine whether HER2 CISH dual-color assay can be used to determine DI in POCs. Twenty-two POC cases were chosen from the departmental archives, including 6 complete hydatidiform mole (CM), 10 partial mole (PM), and 6 hydropic POC (HP). CISH assay was performed using the HER2 CISH PharmDx Kit (SK109; Dako). This kit generates red (HER2) and blue (CEN-17) chromogenic signals on the same tissue section. In the 10 triploid PM cases, CISH generated HER2 signal value of 2.925±0.19. Nine cases (90%) had values within this range, except 1 case (2.5). In diploid cases, CISH generated HER2 signal value of 2.063±0.19. Results from 11 (91.7%) cases fell within this range, except 1 HP case (2.35). Sensitivity is 90%, specificity 91.6%, and overall accuracy 90.9%. The current study is the first one that demonstrates HER2/CEN-17 dual-color CISH can be used for microscopic analysis of cell ploidy. This technique provides a relatively easy and straight way to access DI using regular bright-field microscope. Concurrent CEN-17 signal and ploidy in both placental and maternal tissue can be used as internal control. This assay can be performed in any laboratory that can perform immunohistochemistry.

Prospective Two-Center Comparison of Three Chromogenic Agars for Methicillin-Resistant Staphylococcus aureus Screening in Hospitalized Patients.

Three chromogenic media, chromID MRSA SMART (SMART), chromID MRSA first generation (chromID), and Brilliance MRSA (OX2), were evaluated for methicillin-resistant Staphylococcus aureus (MRSA) screening using 1,220 samples. The sensitivity at 24 h was significantly better with the SMART agar (66.4%) than that with chromID agar (50.5%). Enrichment and incubation until 48 h are still needed for an optimal yield.

Performance of chromID® CARBA medium for carbapenemases-producing Enterobacteriaceae detection during rectal screening.

Chromogenic chromID® CARBA medium was compared with CDC method and MacConkey agar with imipenem for its performance in detecting carbapenemase-producing Enterobacteriaceae (CPE) during a faecal screening surveillance program. Double rectal swabs were collected from patients hospitalized in the ICU. One swab was inoculated onto the solid media chromID® CARBA and MacConkey agar with imipenem, while the other was tested according to CDC protocol. Suspected colonies from all procedures were identified to species level and tested for their susceptibility to carbapenems by phenotypic tests. All carbapenem non-susceptible isolates were tested by the modified Hodge test (MHT) and synergy tests. Positive results were confirmed by PCR testing for carbapenemase gene detection. Performance of all three procedures applied was statistically analyzed as compared to MHT and PCR results for the presence of carbapenemase-encoding genes. Out of 177 rectal samples tested, 86 samples were found to contain one or more CPE verified by molecular detection of carbapenemase encoding genes among isolated Enterobacteriaceae. Sensitivity of chromID® CARBA was 96.5 % in clinical samples. Specificity was 91.2 % at the reading level and 100.0 % after Gram staining. chromID® CARBA performed with high accuracy among the phenotypic methods applied, giving early results.

Three-way comparison of BBL CHROMagar MRSA II, MRSASelect, and spectra MRSA for detection of methicillin-resistant Staphylococcus aureus isolates in nasal surveillance cultures.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired and life-threatening infections. Active surveillance programs for MRSA utilize either molecular or culture-based methods. A prospective study was performed to compare the performance of selective and differential chromogenic media, BBL CHROMagar MRSA II (CMRSA II; BD Diagnostics, Sparks, MD), MRSASelect (Bio-Rad Laboratories, Redmond, WA), and Spectra MRSA (Remel, Lenexa, KS), for the detection of MRSA in nasal swab specimens. A total of 515 compliant remnant nasal swab specimens were sequentially used to inoculate BBL Trypticase soy agar with 5% sheep blood (TSA II) and each chromogenic medium. After 24 h of incubation, colony color reactions and morphology on chromogenic media were compared to suspicious colonies on nonselective TSA II. MRSA on TSA II was confirmed by Gram staining, a coagulase test, and a cefoxitin disk test. The overall prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) on TSA II was 12.4% (64/515) and 9.7% (50/515), respectively. When each chromogenic medium was compared to the standard culture method, the sensitivity and specificity, respectively, were as follows: CMRSA II, 87.7% and 98.6%; MRSASelect, 89.0% and 93.4%; and Spectra MRSA, 83.6% and 92.1%. The positive predictive values were highest for CMRSA II (91.4%), followed by MRSASelect (69.1%) and Spectra MRSA (63.5%). False-positive results on chromogenic media were mainly due to color interpretation. The negative predictive values for all three media were greater than 97%. In conclusion, CMRSA II gave the best overall results for detecting MRSA from nasal specimens.

Performance of three chromogenic VRE screening agars, two Etest(®) vancomycin protocols, and different microdilution methods in detecting vanB genotype Enterococcus faecium with varying vancomycin MICs.

Frequencies of vanB-type Enterococcus faecium increased in Europe during the last years. VanB enterococci show various levels of vancomycin MICs even below the susceptible breakpoint challenging a reliable diagnostics. The performance of 3 chromogenic vancomycin-resistant enterococci (VRE) screening agars, 2 Etest® vancomycin protocols, and different microdilution methods to detect 129 clinical vanB E. faecium strains was investigated. Altogether, 112 (87%) were correctly identified as VanB-type Enterococcus by microdilution MICs. An Etest® macromethod protocol was more sensitive than the standard protocol while keeping sufficient specificity in identifying 15 vanA/vanB-negative strains. Three chromogenic VRE agars performed similarly with 121 (94%), 123 (95%), and 124 (96%) vanB isolates that grew on Brilliance™ VRE Agar, CHROMagar™ VRE, and chromID™ VRE agar, respectively. Using identical media and conditions, we did not identify different growth behaviour on agar and in broth. A few vanB strains showed growth of microcolonies inside the Etest® vancomycin inhibition zones, suggesting a VanB heteroresistance phenotype.

Design, synthesis, and characterization of chromogenic substrates of coagulation factor XIIIa.

A series of Glu(pNA)-containing peptides was designed to determine the activity of the transglutaminase factor XIIIa at 405 nm due to p-nitroaniline release. The most suitable substrate properties were found for peptides containing the Glu(pNA) residue in the second position from the N terminus. For the best substrate 12 (H-Tyr-Glu(pNA)-Val-Lys-Val-Ile-Gly-NH(2)), a k(cat)/K(m) value of 3531 s(-1)M(-1) was found. Although the k(cat)/K(m) values of the Glu(pNA) peptides are more than 100-fold reduced compared with the previously reported cleavage of natural glutamine-containing substrates such as α(2)-antiplasmin and ß-casein, these chromogenic substrates can be useful tools for convenient determination of FXIII-A(2)* activity e.g., for in vitro inhibitor screening. As an example, peptide 12 was used to characterize the inhibition of FXIII-A(2)* by the well-known irreversible inhibitor iodoacetic acid.

Rectal screening for Klebsiella pneumoniae carbapenemases: comparison of real-time PCR and culture using two selective screening agar plates.

Klebsiella pneumoniae carbapenemases (KPCs) have recently been described in Chicago, IL, especially among residents of long-term acute care hospitals (LTACHs). These patients are frequently transferred to local Chicago hospitals for higher acuity of medical care, and rapid detection and isolation of KPC-colonized LTACH residents may interrupt the introduction of KPCs into acute care hospitals. We evaluated the performance of a real-time PCR for bla(KPC) from enrichment broth versus direct plating of rectal surveillance swabs on two selective culture media, CHROMagar extended-spectrum-ß-lactamase (ESBL) and vancomycin, amphotericin B, ceftazidime, and clindamycin (VACC) plates. Rectal surveillance swabs were collected as part of a point prevalence study of KPC carriage rates among 95 residents of two Chicago area LTACHs. Discrepant results between PCR and culture were resolved by subculturing the enrichment broth. Overall, 66 of 95 patients (69.5%) were colonized with KPCs, using the cumulative results of culture as a reference standard. Real-time PCR from enrichment broth was positive in 64 of 66 (97%) colonized patients, including nine surveillance swabs that were missed by both selective culture media. PCR demonstrated higher sensitivity, 97.0%, than culture using either CHROMagar or VACC plates (both with sensitivity of 77.3%). In addition, turnaround time was significantly shorter for the PCR-based method than for culture, with a mean of 24 h versus 64 to 72 h for CHROMagar and VACC plates (P < 0.0001). Overall, PCR for bla(KPC) represents the best screening test for KPCs with significantly higher sensitivity and with less hands-on time, resulting in a shorter time to results.

Experimental application of a synthetic luminescent substrate assay using endotoxin-specific limulus amebocyte lysate to human blood.

A synthetic luminescent substrate method, using a mutant-type luciferase whose luminescence intensity is more than ten times as intense as the wild type, was developed recently. We conducted the first basic studies on clinical application of the novel endotoxin measurement method. We assessed and established measurement conditions, including reagent concentrations and reaction time, so that it would be possible to apply the luminescent synthetic substrate method proposed by Noda et al. to measurements in human blood. When we added lipopolysaccharide (LPS) to water, it was possible to measure LPS at a concentration of 0.1 pg/ml, whereas it was possible to measure LPS in tenfold diluted and heated plasma at a concentration of 1 pg/ml. When plasma was further diluted, inhibiting activity decreased considerably. Thus, it will be necessary to completely eliminate the inhibitor present in plasma. However, the shortest time after collecting the specimen in which it was possible to make measurements was 30-40 min, suggesting that if an assay is established, it will be possible to use the method as a novel blood endotoxin assay.

Usefulness of Chromogenic CromoCen® AGN agar medium for the identification of the genus Aeromonas: Assessment of faecal samples.

Selective screening media for the detection and identification of Aeromonas strains are needed to guide primary isolation procedures in the clinical laboratory. This study compared the selective CromoCen® AGN chromogenic agar medium for the detection and identification of Aeromonas strains that were isolated from various samples against the conventional selective agar media that are commonly used for the isolation of this organism in food, environmental and clinical samples. The Miles and Misra and ecometric methods were used to evaluate the microbiological performance of CromoCen® AGN chromogenic agar medium, which was shown to be satisfactory. A total of 14 reference Aeromonas strains, 44 wild strains and 106 clinical stool specimens were examined using both non-chromogenic selective agars that are commonly used for Aeromonas isolation and CromoCen® AGN agar. The latter exhibited 94.73% sensitivity and 100% specificity for the various samples. On CromoCen® AGN agar medium, Aeromonas formed colonies with light green, greenish and salmon pigments with or without a surrounding wide transparent zone (halo) of 2-3mm in diameter around the entire border. This medium is recommended for the isolation and potential identification of the Aeromonas genus.

Detection of methicillin-resistant Staphylococcus aureus in clinical specimens from cystic fibrosis patients by use of chromogenic selective agar.

We evaluated the use of a chromogenic selective medium (MRSA ID) as a useful tool for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in cystic fibrosis (CF) patient samples. Fifty-four MRSA isolates were detected by MRSA ID, while only 24/54 (44%) (odds ratio [OR], 2.79; 95% confidence interval [CI], 1.63 to 4.76) were detected by conventional methods. A chromogenic selective medium for MRSA detection may improve its surveillance in CF patients.