Exploratory development of PCR-fluorescent probes in rapid detection of mutations associated with extensively drug-resistant tuberculosis.

This study aims to evaluate the clinical value of PCR-fluorescent probes for detecting the mutation gene associated with extensively drug-resistant tuberculosis (XDR-TB). The molecular species identification of 900 sputum specimens was performed using polymerase chain reaction (PCR)-fluorescent probe. The mutations of the drug resistance genes rpoB, katG, inhA, embB, rpsL, rrs, and gyrA were detected. The conventional drug susceptibility testing (DST) and PCR-directed sequencing (PCR-DS) were carried out as control. DST demonstrated that there were 501 strains of rifampicin resistance, 451 strains of isoniazid resistance, 293 strains of quinolone resistance, 425 strains of streptomycin resistance, 235 strains of ethambutol resistance, and 204 strains of amikacin resistance. Furthermore, 427 (47.44%) or 146 (16.22%) strains were MDR-TB or XDR-TB, respectively. The mutations of the rpoB, katG, inhA, embB, rpsL, rrs, and gyrA genes were detected in 751 of 900 TB patients by PCR-fluorescent probe method, and the rate of drug resistance was 751/900 (83.44%). No mutant genes were detected in the other 149 patients. Compared with DST, the mutant rates of rpoB, katG/inhA, rpsL, rrs, embB, and gyrA of six drugs were higher than 88%; five of six drugs were higher than 90% except for SM (88.11%). The MDR and XDR mutant gene types were found in 398 (42.22%) and 137 (15.22%) samples. PCR-DS was also employed and confirmed the PCR-fluorescent probe method with the accordance rate of 100%. The PCR-fluorescent probe method is rapid and straightforward in detecting XDR-TB genotypes and is worthy of being applied in hospitals.

Comparison of the Benzanthrone Luminophores: They Are Not Equal for Rapid Examination of Parafasciolopsis fasciolaemorpha (Trematoda: Digenea).

Luminescent derivatives of benzanthrone are becoming more useful based on their light-absorbing and fluorescent-emitting properties. Our previous studies showed that luminescent staining properties of the same benzanthrone dye differ for variable parasite samples. Therefore, two types of benzanthrone dyes were prepared. One has a strongly basic amidine group and a halogen atom, and the other has an amide moiety and a tertiary amine group. Trematoda Parafasciolopsis fasciolaemorpha is a liver fluke of a moose (Alces alces) and has a significant influence on the health and abundance of the moose population. Staining protocols for parasite P. fasciolaemorpha specific organ or organ systems imaging are mostly time-consuming and labor-intensive. The study aimed to compare the fixation technique and the staining protocol by synthesized benzanthrone luminescent dyes to determine detailed morphology, anatomical arrangement of the organ systems and gross organization of the muscle layers of P. fasciolaemorpha using confocal laser scanning microscopy. Luminophores were tested for samples fixed in different fixatives. Developed dyes and staining protocol resulting in imaging of all parts of trematode without additional sample preparation procedures, which usually are required for parasite examination. Obtained results confirmed that the most qualitative results could be reached using 3-N-(2-piperidinylacetamido)benzanthrone dye which has amide moiety and a tertiary amine group. Based on obtained results, 3-N-(2-piperidinylacetamido)benzanthrone gave more qualitative parasite visualization than 2-bromo-3-N-(N',N'-dimethylformamidino)benzanthrone.

Clinical Validation of an Automated Fluorogenic Factor XIII Activity Assay Based on Isopeptidase Activity.

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

Progress and challenges in the use of fluorescence-based flow cytometric assays for anti-malarial drug susceptibility tests.

Drug-resistant Plasmodium is a frequent global threat in malaria eradication programmes, highlighting the need for new anti-malarial drugs and efficient detection of treatment failure. Plasmodium falciparum culture is essential in drug discovery and resistance surveillance. Microscopy of Giemsa-stained erythrocytes is common for determining anti-malarial effects on the intraerythrocytic development of cultured Plasmodium parasites. Giemsa-based microscopy use is conventional but laborious, and its accuracy depends largely on examiner skill. Given the availability of nucleic acid-binding fluorescent dyes and advances in flow cytometry, the use of various fluorochromes has been frequently attempted for the enumeration of parasitaemia and discrimination of P. falciparum growth in drug susceptibility assays. However, fluorochromes do not meet the requirements of being fast, simple, reliable and sensitive. Thus, this review revisits the utility of fluorochromes, notes previously reported hindrances, and highlights the challenges and opportunities for using fluorochromes in flow cytometer-based drug susceptibility tests. It aims to improve drug discovery and support a resistance surveillance system, an essential feature in combatting malaria.

Supramolecular Assembly of TPE-Based Glycoclusters with Dicyanomethylene-4H-pyran (DM) Fluorescent Probes Improve Their Properties for Peroxynitrite Sensing and Cell Imaging.

Two red-emitting dicyanomethylene-4H-pyran (DM) based fluorescent probes were designed and used for peroxynitrite (ONOO- ) detection. Nevertheless, the aggregation-caused quenching effect diminished the fluorescence and restricted their further applications. To overcome this problem, tetraphenylethylene (TPE) based glycoclusters were used to self-assemble with these DM probes to obtain supramolecular water-soluble glyco-dots. This self-assembly strategy enhanced the fluorescence intensity, leading to an enhanced selectivity and activity of the resulting glyco-dot comparing to DM probes alone in PBS buffer. The glyco-dots also exhibited better results during fluorescence sensing of intracellular ONOO- than the probes alone, thereby offering scope for the development of other similar supramolecular glyco-systems for chemical biological studies.

Evaluation of a six-dye multiplex composed of 27 markers for forensic analysis and databasing.

BACKGROUND: Short tandem repeat (STR) markers play a significant role in genetic applications and have proved to be effective for the personal identification in forensic medicine. In this study, a six-dye multiplex composed of 23 autosomal STR loci (TH01, D3S1358, Penta D, D6S1043, D21S11, TPOX, D1S1656, D12S391, Penta E, D10S1248, D22S1045, D19S433, D8S1179, D2S1338, D2S441, D18S51, vWA, FGA, D16S539, CSF1PO, D13S317, D5S818, D7S820), one Y chromosome STR (DYS391), two internal quality control markers (Quality Sensor QS1 and QS2), and Amelogenin was evaluated. METHODS: Evaluation studies, including PCR-based studies, sensitivity studies, species specificity studies, stability studies, DNA mixture studies, concordance studies, and precision evaluations were performed according to the guidelines of "Validation Guidelines for Forensic DNA Analysis Methods (2016)" by the Scientific Working Group on DNA Analysis Methods (SWGDAM). In addition, the forensic characteristics of 357 unrelated male samples from Han and Hui populations in China were investigated using 27 markers. RESULTS: Full STR profiles were obtained from different reaction volumes (5 ~ 25 µl), cycle numbers (28 ~ 34 cycles) and annealing temperatures (58 ~ 62°C). All STR profiles were obtained at humic acid concentration of up to 200 ng/µl and hematin concentration of up to 500 µM. No peaks were observed in most common animal samples except two innovative internal PCR controls (Quality Sensor QS1 and QS2). The six-dye multiplex showed a notably high value for the combined probability of exclusion (CPE), exhibiting values of with 0.99999999977688 in the Han population and 0.999999999583875 in the Hui population. The values of combined probability of discrimination (CPD) were 0.999999999999999999999999999997453 in the Han population and 0. 999999999999999999999999999994398 in the Hui population. In addition, concordance studies showed that there was no difference with the AGCU Express Marker 22 Kit. CONCLUSION: The results indicated that the Investigator® 26plex QS Kit is a robust, reliable, and suitable tool for forensic analysis and databasing.

Recent progress on the organic and metal complex-based fluorescent probes for monitoring nitric oxide in living biological systems.

Nitric oxide (NO) is an important gaseous signaling molecule related to various human diseases. To investigate the biological functions of NO, many strategies have been developed for real-time monitoring the NO levels in biological systems. Among these strategies, fluorescent probes are considered to be one of the most efficient and applicable methods owing to their excellent sensitivity and selectivity, high spatiotemporal resolution, noninvasiveness, and experimental convenience. Therefore, great efforts have been paid to the design, synthesis, and fluorescence investigation of novel NO fluorescent probes in the past several years. However, few of them exhibit practical applications owing to the low concentration, short half-life, and rapid diffusion characteristics of NO in biological systems. Rational design of NO fluorescent probes with excellent selectivity and sensitivity, low cytotoxicity, long-lived fluorescent emission, and low background interference is still a challenge for scientists all over the word. To provide spatial-temporal information, this article focuses on the progress made in the organic and metal complex-based NO fluorescent probes during the past five years. The key structural elements and sensing mechanisms of NO fluorescent probes are discussed. Some novel ratiometric, luminescence, and photoacoustic probes with low background interference and deep tissue penetrating ability are mentioned. All these probes have been used for imaging exogenous and endogenous NO in cells and animal models. More importantly, this article also describes the development of multi-functional NO fluorescent probes, such as organelle targeting probes, dual-analysis probes, and probe-drug conjugates, which will inspire the design of various functional fluorescent probes.

Pharmacological control of inflammation in wound healing.

Wound inflammation is a rapid and highly orchestrated process that significantly impacts the wound healing cascade. Consequent to injury, a series of events set off that include inflammatory, proliferation and maturation phases leading to wound closure and restoration of normal skin integrity. Stimuli causing stress to host immune system or induce inflammatory response include tissue damage and pathogenic microbial infection.Several evidences points towards the positive role of inflammation as it essential to fight against the attack of invading pathogens and to remove dead tissues from the site of injury. Besides its positive role, prolonged inflammation is injurious and may result in deregulated stages of the wound healing which may lead to excessive scarring. Achieving balance in inflammatory cascade is one of the challenging tasks for development of a wound healing drug. This review mainly focuses on the pharmacological control of inflammation by agents which critically balance the inflammatory cascade. However, none of the agent is available in the healthcare market which exclusively plays a role in wound repair. In this review we shall explore different factors or agents affecting inflammation in wound healing. This information might be helpful in designing and development new process, technologies or drugs for better management of wound care. In addition, understanding the effect of inflammation on the outcome of the healing process will serve as a significant milestone in the area of pathological tissue repair.

Comparative study of two near-infrared coumarin-BODIPY dyes for bioimaging and photothermal therapy of cancer.

Near-infrared (NIR) responsive agents for cancer bioimaging and photothermal therapy are available and significant. Herein, we employed two easily available dyes, boron-dipyrromethane and coumarin, to synthesize a pair of coumarin-borondipyrromethane dyes with different conjugate degrees (BDC and BSC). The difference in conjugate degree made their photophysical properties poles apart. After the self-assembling of BDC and BSC, the newly constructed nanoparticles (BDC NPs and BSC NPs) demonstrated good biocompatibility. Moreover, BDC NPs exhibited a good photothermal effect under irradiation of 808 nm laser, which could effectively inhibit the growth of HeLa cells, and BSC NPs could quickly show a conspicuous fluorescence in the HeLa cells. The exploration demonstrates that the two organic dyes prepared with different conjugation degrees could provide new options for photothermal therapy of cancer and rapid bioimaging.

Green-Emitting Rhodamine Dyes for Vital Labeling of Cell Organelles Using STED Super-Resolution Microscopy.

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining.

Yellow-emissive carbon dots with a large Stokes shift are viable fluorescent probes for detection and cellular imaging of silver ions and glutathione.

Yellow-emissive carbon dots (Y-CDs) were prepared by a solvothermal method using anhydrous citric acid and 2,3-phenazinediamine as the starting materials. The Y-CDs display a 24% fluorescence quantum yield, a 188-nm Stokes' shift and excellent stability. They are shown here to be excellent fluorescent probes for the determination of Ag(I) ion and glutathione (GSH). If exposed to Ag(I) ions, they are bound by the carboxy groups of the Y-CDs, and this causes quenching of fluorescence (with excitation/emission maxima at 380/568 nm) via a static quenching mechanism. This effect was used to design a fluorometric assay for Ag(I). The quenched fluorescence of the Y-CDs can be restored by adding GSH due to the high affinity of GSH for Ag(I). The calibration plot for Ag(I) is linear in the 1-4 µM Ag(I) concentration range, and the limit of detection is 31 nM. The respective values for GSH are 5-32 µM, and 76 nM, respectively. The method was applied to the detection of Ag(I) in spiked environmental water samples and gave recoveries ranging from 93 to 107%. It was also applied to the determination of GSH in tomatoes and purple grapes and gave satisfactory recoveries. The Y-CDs display low cytotoxicity and were successfully used to image Ag(I) and GSH in H1299 cells. Graphical abstract Schematic presentation of the mechanism of yellow fluorescent CDs for the detection of Ag+ and glutathione.

A turn-on fluorescent probe for vitamin C based on the use of a silicon/CoOOH nanoparticle system.

The authors describe a fluorometric method for the turn-on determination of vitamin C (ascorbic acid). The blue fluorescence of silicon nanoparticles (SiNPs; with excitation/emission maxima at 350/450 nm) is found to be quenched by CoOOH nanoparticles (NPs). In the presence of vitamin C, the CoOOH NPs are decomposed by a redox reaction between the diol group of vitamin C and CoOOH NPs. As a result, fluorescence recovers. On the basis of this finding, a fluorometric method was designed for the turn-on detection of vitamin C. Under optimal conditions, the method has a low detection limit (0.47 µM) and a linear response in the 0.5 µM to 20 µM a concentration range. It was successfully applied to the determination of vitamin C in spiked red grape and orange juice, and in vitamin C tablets. Graphical abstract A target-triggered dissociation of quencher-based strategy for the fluorescence "turn-on" detection of vitamin C was developed. It is based on surface energy transfer (SET) and an inner filter effect (IFE) between silicon nanoparticles and CoOOH nanoparticles as well as the redox reaction between vitamin C and CoOOH nanoparticles.

Development of a near-infrared ratiometric fluorescent probe for glutathione using an intramolecular charge transfer signaling mechanism and its bioimaging application in living cells.

A novel near-infrared (NIR) ratiometric fluorescent probe HBT-GSH derived from conjugated benzothiazole was developed for the selective detection of glutathione (GSH) over cysteine (Cys) and homocysteine (Hcy). The probe was sophisticatedly designed based on the GSH selectively induced enhancement of intramolecular charge transfer (ICT) fluorescence. It was synthesized by masking the active phenol group of 2,6-bis(2-vinylbenzothiazolyl)-4-fluorophenol through an acetyl group that acts both as a trigger of the ICT fluorescence and as a recognition moiety for GSH. On its own, the probe HBT-GSH exhibited strong blue fluorescence emission at 426 nm and weak NIR fluorescence emission at 665 nm in aqueous solution, whereas the NIR fluorescence was significantly enhanced and the short emission decreased upon the addition of GSH. Thus an NIR ratiometric fluorescent probe for GSH was developed based on the GSH-selective removal of the acetyl group, therefore switching on the ICT in HBT-GSH. The fluorescence intensity ratio (I665 nm/I426 nm) showed a linear relationship with a GSH concentration of 0-100 µM with a detection limit of 0.35 µM. Moreover, the fluorescent probe was successfully used for the ratiometric fluorescence bioimaging of GSH in living cells.

Lot-to-lot stability of antibody reagents for flow cytometry.

The fluorescence detected using fluorochrome-labelled monoclonal antibodies depends not only on the abundance of the target antigen, but amongst many other factors also on the effective fluorochrome-to-antibody ratio. The diagnostic approach of the EuroFlow consortium relies on reproducible fluorescence intensities over time. A capture bead system for mouse immunoglobulin light chains was utilized to compare the mean fluorescence intensity of 1323 consecutive antibody lots to the currently used lot of the same monoclonal antibody. In total, 157 different monoclonal antibodies were assessed over seven years. Median relative difference between consecutive lots was 3.8% (range: 0.01% to 164.7%, interquartile range: 1.3% to 10.1%). The relative difference exceeded 20% in 8.8% of all comparisons. FITC labelled monoclonal antibodies (median relative difference: 2.1%) showed a significantly smaller variation between lots than antibodies conjugated to PE (3.5%), PECy7 (3.9%), PerCPCy5.5 (5.8%), APC (5.8%), APCH7 (7.4%), and APCC750 (14.5%). Reagents labelled with Pacific Blue (1.4%), Pacific Orange (2.4%), HV450 (0.7%), and HV500 (1.7%) demonstrated more consistent results compared to conjugates of BV421 (4.1%) and BV510 (16.2%). Additionally, significant differences in lot-to-lot fluorescence stability amongst antibodies labelled with the same fluorochrome were observed between manufacturers. These observations might guide future quality recommendations for the production and application of fluorescence-labelled monoclonal antibodies in multicolor flow cytometry.

Reconsidering the H&E stain as the gold standard in assessing the depth of burn wounds.

While histological examination is considered by most as the gold standard for burn depth assessment, it has no practical use in the clinical setting. It has, however, been used in the research setting, as a mean for evaluating emerging techniques of depth measurement. Due to the limitations of the H&E stain, other stains have also been explored, such as lactate dehydrogenase (LDH), as presented in this issue, in "Improving the Histologic Characterization of Burn Depth." As the determination of burn depth is not a typical subject in dermatopathology, a summary of selected techniques and the possible role for the LDH stain in future research, is described herein.

An ultrasensitive stain for negative protein detection in SDS-PAGE via 4',5'-Dibromofluorescein.

A highly sensitive method for brief and economical staining of proteins in 1-D and 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by 4',5'-Dibromofluorescein (DBF) was developed in this study. Down to 0.025-0.05ng protein could be detected within 10min (only 2 steps) by DBF stain, which is approximately 10-fold more sensitive than those of Eosin Y (EY) and SYPRO Ruby stains, and 20-fold more sensitive than that of imidazole-zinc (IZ) negative stain. In addition, the LC-MS/MS results indicated that the newly developed staining method is compatible with the downstream protein identification. Moreover, the mechanism of DBF stain was investigated by molecular docking.

Design, installation, and performance evaluation of a custom dye matrix standard for automated capillary electrophoresis.

CE equipment detects and deconvolutes mixtures containing up to six fluorescently labeled DNA fragments. This deconvolution is done by the collection software that requires a spectral calibration file. The calibration file is used to adjust for the overlap that occurs between the emission spectra of fluorescence dyes. All commercial genotyping and sequencing kits require the installation of a corresponding matrix standard to generate a calibration file. Due to the differences in emission spectrum overlap between fluorescent dyes, the application of existing commercial matrix standards to the electrophoretic separation of DNA labeled with other fluorescent dyes can yield undesirable results. Currently, the number of fluorescent dyes available for oligonucleotide labeling surpasses the availability of commercial matrix standards. Therefore, in this study we developed and evaluated a customized matrix standard using ATTO 633, ATTO 565, ATTO 550, ATTO Rho6G, and 6-FAM dyes for which no commercial matrix standard is available. We highlighted the potential genotyping errors of using an incorrect matrix standard by evaluating the relative performance of our custom dye set using six matrix standards. The specific performance of two genotyping kits (UniQTyper™ Y-10 version 1.0 and PowerPlex® Y23 System) was also evaluated using their specific matrix standards. The procedure we followed for the construction of our custom dye matrix standard can be extended to other fluorescent dyes.

Development of real-time PCR for detection and quantitation of Streptococcus parauberis.

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples.

Using the Peggy Simple Western system for fine needle aspirate analysis.

Simple Western™ assays are capillary-based electrophoretic immunoassays, similar in scope to SDS-PAGE (molecular weight separation, "size") and IEF (isoelectric focusing, "charge") immunoblotting. The enhanced sensitivity and automation of the Simple Western makes it better suited to cancer diagnostics and research than the traditional Western platform. Because of its smaller sample volume requirements, primary cells, such as those obtained from fine needle aspirates (FNAs), and solid tumor slices may be used to generate quantitative comparable data. The Peggy™ instrument is capable of performing either size or charge assays on up to 96 samples in a single unattended run.