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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo.

Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo. Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo. In neocortical neurons, calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetised mice with a high SNR and fast decay time. These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo.

Improving the sensitivity of confocal laser induced fluorescence detection to the sub-picomolar scale for round capillaries by laterally shifting the laser focus point.

This paper describes a simple and efficient approach to reduce the background level of confocal laser induced fluorescence (LIF) detection for round capillaries by laterally shifting the laser focus point. A phenomenon of spontaneous separation of the fluorescence and reflected laser beams at the pinhole of a confocal LIF system when the laser focus point deviates from the center of a capillary channel to the sides was observed for the first time. On the basis of this phenomenon, the reflected laser light from the capillary-air interfaces could be mostly eliminated with a spatial filtering pinhole. A comprehensive study on the phenomenon and optimization of the shift distance was carried out using both experimental and simulation methods. A best shift distance of ±20 µm was obtained, with which background intensity could be significantly reduced by 98.9%, while fluorescence intensity was only reduced by 25.7%, resulting in an improvement of signal-to-noise ratio of 8.3 times, compared with that at a shift distance of 0 µm usually used in most of the confocal LIF systems for round capillaries. A limit of detection of 66 fM was obtained for sodium fluorescein. To demonstrate its potential as an on-column sensitive detector for microscale separation systems, the present system was coupled with a capillary electrophoresis system for separation of four fluorescein isothiocyanate labeled amino acids with concentrations of 100 pM.

[Another discussion of membrane voltage alterations in growing pollen tube].

Here we give a critical analysis of the opinion of Andreev (2011) on membrane potential distribution along the pollen tube plasmalemma. He assumes that a lateral gradient of dipole potential exists, but suggests a lateral gradient of transmembrane potential impossible. We demonstrate by concrete examples that the argumentation of the initiator of discussion is based on inaccurate citation of our experimental data (Breygina et al., 2009) and incomplete analysis of previously published articles. Speaking about transmembrane potential, he doesn't consider numerous facts demonstrating the uneven distribution of transmembrane ion fluxes and ion-transport proteins in the pollen tube plasmalemma, as well as data obtained by modeling of transmembrane potential distribution in objects of different shape. In addition, the assumption on the uneven distribution of dipole potential doesn't have an experimental basis neither in studies of the pollen tube, nor in the practice of using fluorescent voltage-sensitive dyes DiBAC4(3) and Di-4-ANEPPS. We are expecting the author to obtain experimental data in support of his position.

From imaging to understanding: Frontiers in Live Cell Imaging, Bethesda, MD, April 19-21, 2006.

A recent meeting entitled Frontiers in Live Cell Imaging was attended by more than 400 cell biologists, physicists, chemists, mathematicians, and engineers. Unlike typical special topics meetings, which bring together investigators in a defined field primarily to review recent progress, the purpose of this meeting was to promote cross-disciplinary interactions by introducing emerging methods on the one hand and important biological applications on the other. The goal was to turn live cell imaging from a "technique" used in cell biology into a new exploratory science that combines a number of research fields.

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.

Quantification and calibration of images in fluorescence microscopy.

Fluorescence microscopy is a method widely used in life sciences to image biological processes in living and fixed cells or in fixed tissues. Quantification and calibration of images in fluorescence microscopy is notoriously difficult. We have developed a new methodology to prepare tissue "phantoms" that contain known amounts of (i) fluorophore, (ii) DNA, (iii) proteins, and (iv) DNA oligonucleotide standards. The basis of the phantoms is the ability of gelatin to act as a matrix for the conjugation of fluorophores as either a free-flowing liquid or a gelatinous solid depending on temperature (> or = 40 and < or = 4 degrees C).

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.