Dye-based Methods Research Articles

Whether Dye-Based Methods Are Ready for Microalbuminuria Detection: A Review of Research in the Field

Whether Dye-Based Methods Are Ready for Microalbuminuria Detection: A Review of Research in the Field

Assessment of Endothelial Barrier Functions in Extra Embryonic Vasculature of Chick Embryo as an Alternative Model.

Vascular permeability, a tightly regulated process, is a direct measure of angiogenic and immune responses in the endothelium altered in several acute and chronic diseases such as sepsis, high-altitude pulmonary edema (HAPE), high-altitude cerebral edema (HACE), ischemia, and coronavirus disease 2019 (COVID-19) endotheliitis. Both endogenous and exogenous factors such as cytokines, chemokines, and hormones may affect vascular permeability. The conventional tools available for the measurement of vascular permeability in vitro and in vivo based on collagen-coated Transwell and dye-based spectrophotometric methods are indirect measures of permeability. In this chapter, we present our live in ovo protocols based on dextran-Texas red and avian chorioallantoic membrane assay developed using custom-made equipment to assess leakiness of endothelial cell barrier both in vitro and in vivo. Further, we validate this assay using different stressors such as ischemia and hypoxia known to affect endothelial barrier properties by potentiating actin stress fiber disorganization and disrupting the cell-cell junctions.

Hexagonal boron nitride nanodots inhibit cell proliferation of HUVECs and the underlying mechanism

Boron nitride (BN)-based nanomaterials have immense potential in nano-biomedicine, such as drug carriers and anti-bacterial agents. However, their biocompatibility remains a crucial concern. Here, we investigated the cytotoxicity of hexagonal BN (h-BN) nanodots (BNNDs) on HUVEC cells, assessing their effects on viability, morphology, proliferation, cell cycle, genes, and protein expression. BNNDs had limited impact on HUVECs viability, even at high concentrations (200 μg/mL, 48 h). Yet, they hindered cell proliferation and caused cell cycle arrest in the S phase in a dose−/time-dependent manner. Cytotoxicity primarily resulted from disturbances in cell proliferation and DNA replication-related genes (e.g., GADD45A) and proteins (e.g., GADD45A). BNNDs also induced oxidative stress, enhancing cytotoxic effects. Therefore, cautious evaluation of long-term cytotoxicity is crucial before employing BNNDs in biological systems. Additionally, certain dye-based cytotoxicity assessment methods may not accurately reflect the cytotoxicity of BNNDs. Understanding these new cytotoxic mechanisms can aid in designing safer BN-based nano-medicines/devices.

Meta-analysis of microbial source tracking for the identification of fecal contamination in aquatic environments based on data-mining

Microbial source tracking (MST) technology represents an innovative approach employed to trace fecal contamination in environmental water systems. The performance of primers may be affected by amplification techniques, target primer categories, and regional differences. To investigate the influence of these factors on primer recognition performance, a meta-analysis was conducted on the application of MST in water environments using three databases: Web of Science, Scopus, and PubMed (n = 2291). After data screening, 46 studies were included in the final analysis. The investigation encompassed Polymerase Chain Reaction (PCR)/quantitative PCR (qPCR) methodologies, dye-based (SYBR)/probe-based (TaqMan) techniques, and geographical differences of a human host-specific (HF183) primer and other 21 additional primers. The results indicated that the primers analyzed were capable of differentiating host specificity to a certain degree. Nonetheless, by comparing sensitivity and specificity outcomes, it was observed that virus-based primers exhibited superior specificity and recognition capacity, as well as a stronger correlation with human pathogenicity in water environments compared to bacteria-based primers. This finding highlights an important direction for future advancements. Moreover, within the same category, qPCR did not demonstrate significant benefits over conventional PCR amplification methods. In comparing dye-based and probe-based techniques, it was revealed that the probe-based method's advantage lay primarily in specificity, which may be associated with the increased propensity of dye-based methods to produce false positives. Furthermore, the heterogeneity of the HF183 primer was not detected in China, Canada, and Singapore respectively, indicating a low likelihood of regional differences. The variation among the 21 other primers may be attributable to regional differences, sample sources, detection techniques, or alternative factors. Finally, we identified that economic factors, climatic conditions, and geographical distribution significantly influence primer performance.

Description of lipase producing novel yeast species Debaryomyces apis f.a., sp. nov. and a modified pH indicator dye-based method for the screening of lipase producing microorganisms

Four yeast strains were isolated from the gut of stingless bee, collected in Churdhar, Himachal Pradesh, India. Physiological characterization, morphological examination, and sequence analysis of small subunit ribosomal RNA (18S rRNA) genes, internal transcribed spacer (ITS) region, and D1/D2 domain of the large subunit rRNA gene revealed that the four strains isolated from the gut of stingless bee belonged to the Debaryomyces clade. Strain CIG-23HT showed sequence divergence of 7.5% from Debaryomyces nepalensis JCM 2095T, 7.8% from Debaryomyces udenii JCM 7855T, and Debaryomyces coudertii JCM 2387T in the D1/D2 domain. In the ITS region sequences, strain CIG-23HT showed a 15% sequence divergence from Debaryomyces nepalensis JCM 2095T and Debaryomyces coudertii JCM 2387T. In 18S rRNA gene sequence, the strain CIG-23HT showed 1.14% sequence divergence from Debaryomyces nepalensis JCM 2095 and and Debaryomyces coudertii JCM 2387, and 0.83% sequence divergence from Debaryomyces hansenii NRRL Y-7426. Strain CIG-23HT can utilize more carbon sources than closely related species. The findings suggest that strain CIG-23HT is a novel species of the genus Debaryomyces, and we propose to name it as Debaryomyces apis f.a., sp. nov. The holotype is CBS 16297T, and the isotypes are MTCC 12914T and KCTC 37024T. The MycoBank number of Debaryomyces apis f.a., sp. nov. is MB836065. Additionally, a method using cresol red and Bromothymol blue pH indicator dyes was developed to screen for lipase producers, which is more sensitive and efficient than the currently used phenol red and rhodamine B dye-based screening methods, and avoids the problem of less differentiable zone of hydrolysis.

Field-portable, rapid, and low-cost RT-LAMP assay for the detection of tomato chlorotic spot virus.

Tomato chlorotic spot virus (TCSV) is a highly destructive, thrips-transmitted, emerging orthotospovirus in various vegetable and ornamental crops. It is important to reduce the risk of spreading this virus by limiting the movement of infected plant materials to other geographic areas by utilizing point-of-care diagnostics. Current diagnostic assays for TCSV require costly lab equipment, skilled personnel, and electricity. Here we report the development of a simple rechargeable battery-operated handwarmer-assisted reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay and demonstrate a step-by-step protocol to achieve in-field detection of TCSV. Under field conditions, handwarmer-assisted RT-LAMP can detect as little as 0.9 pg/μl of total RNA from TCSV-infected tomato plants in <35 minutes. When fully charged, the field-portable device can be used in six consecutive RT-LAMP detection assays, yielding test results for 96 individual samples. Dye-based colorimetric methods, including pH and metal ion indicators, were evaluated to eliminate laboratory-dependent LAMP visualization. Phenol-red combined with hydroxynaphthol blue was adopted in the handwarmer-assisted RT-LAMP detection method to obtain a more robust color difference distinguishable by the naked eye. Overall, handwarmer-assisted RT-LAMP is a rapid, highly sensitive, and cost-effective diagnostic technique that can be used by non-specialist personnel in the field, particularly in rural production areas lacking access to a diagnostic lab or constant electricity.

A case study of optical methods for measuring thickness of liquid sheets

We demonstrate that the usual optical methods can be applied in a simple way to quickly estimate thickness of liquid sheets on sub-millimetre scales. It is shown that an approximate linear relation can be obtained between gap thickness and several of the usual RBG and HSV variables (red and green pixel values in the RBG case and saturation in the HSV case). The method we use is viable as long as the sheet does not become too thin and is much simpler than other dye-based optical methods for determining the thickness.

Fluorimetric method for specific detection of human serum albumin in urine using its pseudoesterase property

Detection of microalbuminuria is an analytical challenge. There are dye-based methods and immunochemical methods. However, these methods are less specific and sensitive respectively. So, people are trying new approaches for microalbuminuria detection. In this context, we have developed a fluorescent spectroscopic method to detect human serum albumin using its pseudoesterase property. Recently, we had discovered that neostigmine does not inhibit Human serum albumin pseudoesterase activity. Using such a phenomenon, we have devised a specific fluorimetric detection method of HSA using 2NA as a substrate for the pseudoesterase activity. The developed method can sense as low as 0.1 μM of HSA in the urine matrix without dye or antibody. We have proposed a scheme of automation of the proposed method.

Effects of endoscopic sinus surgery on nasal spray deposition using dye-based methods for humans and a human silicone sinonasal cavity model

ObjectiveWe have evaluated that the deposition patterns of corticosteroid nasal spray in the sinonasal cavity of both post-operated human cases, which were further compared with a computed tomography-based sinonasal airway model. MethodsFifty-one patients with chronic rhinosinusitis following an endoscopic sinus surgery were enrolled in this study. Nasal spray mometasone furoate hydrate (Nasonex®) containing 0.1% indigocarmine was applied to the patients' nasal cavities and the sinonasal cavity was observed by endoscopy and video documentation. A single plaster sinonasal model was used to quantify the sinonasal deposition of nasal sprays containing 10% red ink solution using 12 round paper strips. ResultsThe predominant areas of the spray deposition of the operated sinonasal cavities were recognized in the ethmoid sinus and the olfactory cleft in the human study. The droplets were mainly deposited in the inferior turbinate followed by the posterior part of the ethmoid sinus, the olfactory cleft, and anterior part of the ethmoid sinus in a sinonasal model. ConclusionThe corticosteroid nasal spray efficiently reached the olfactory cleft and the ethmoid sinus in post-operative conditions, which was demonstrated by post-operated human cases and a computed tomography-based sinonasal airway model.

Comparison of dye-based proteinuria estimation methods with immunoturbidimetric microalbuminuria estimation, in screening for diabetic

We compared the performance of Coomassie Brilliant Blue (CBB) and Ponceau S dyes based proteinuria estimation methods with immunoturbidimetry microalbumin estimation to develop new cut-offs for proteinuria, which would help us detect diabetic nephropathy patients with better sensitivity using the above mentioned dye based methods. These proteinuria testing methods are easy to adapt in smaller laboratories typically associated with peripheral government health centers. Urine sample was collected from 158 diabetic individuals and tested simultaneously for Urine Albumin Creatinine ratio (UACR) using immunoturbidimetry and Urine Protein Creatinine Ratio (UPCR) using Ponceau S method and CBB method. The Ponceau S method gives good correlation with immunoturbidimetry, for screening of diabetic nephropathy, at UPCR cut-off of 50mg/g creatinine. The CBB method gives higher false positives. The overall agreement of Ponceau S and CBB with immunotubidimetry is maximum at cut off 50mg/g, i.e. 91% and 84% respectively. In conclusion, proteinuria estimation with Ponceau S method with UPCR >50mg/g creatinine, may be used for routine screening of diabetic patients to diagnose microalbuminuria. Keywords: Diabetic nephropathy screening, Proteinuria, Microalbuminuria, Ponceau S.

Assessing Chondrocyte Status by Immunofluorescence-Mediated Localization of Parkin Relative to Mitochondria.

Immunofluorescence staining is a widely used and powerful tool for the visualization and colocalization of two or more proteins and/or cellular organelles. For colocalization studies in fixed cells, one target protein/organelle is immunostained and visualized by one fluorophore and the other target protein/organelle is immunostained and visualized by a different fluorophore whose excitation emission spectra does not overlap with the first fluorophore. Parkin (PARK2) is an E3 ubiquitin ligase which performs ubiquitination of surface proteins of dysfunctional mitochondria to mark them for autolysosomal degradation.Here we describe the immunofluorescence staining of parkin protein and immunofluorescence or dye-based methods to visualize mitochondria and study the colocalization of parkin and mitochondria in primary human or mouse chondrocytes or cell lines.

Quantifying and Storing RNA.

Quantifying RNA is an important and necessary step before most RNA analysis techniques. Methods for quantifying RNA can be classified into two categories: ultraviolet (UV) spectrophotometric methods, which are based on the absorption spectra of the purine and pyrimidine bases; and fluorescent dye-based methods, which measure the fluorescence intensity of dyes that selectively fluoresce when bound to nucleic acids. If the RNA sample is pure (i.e., without significant amounts of contaminants such as proteins, phenol, agarose, or other nucleic acids), UV spectrophotometric measurement of the amount of UV irradiation absorbed by the bases is simple and accurate. However, if the sample contains significant quantities of impurities or if the concentration of RNA is very low, it is better to use fluorescent dye-based methods. An overview of spectrophotometric and fluorescent dye-based RNA quantification methods is given here, as are several options for storing purified RNA preparations. Proper storage of RNA samples is important; it can help minimize RNase contamination and consequent sample degradation.

A High-Throughput Method for Directed Evolution of NAD(P)+-Dependent Dehydrogenases for the Reduction of Biomimetic Nicotinamide Analogues.

Engineering flavin-free NAD(P)+-dependent dehydrogenases to reduce biomimetic nicotinamide analogues (mNAD+s) is of importance for eliminating the need for costly NAD(P)+ in coenzyme regeneration systems. Current redox dye-based screening methods for engineering the mNAD+ specificity of dehydrogenases are frequently encumbered by a background signal from endogenous NAD(P) and intracellular reducing compounds, making the detection of low mNAD+-based activities a limiting factor for directed evolution. Here, we develop a high-throughput screening method, NAD(P)-eliminated solid-phase assay (NESPA), which can reliably identify mNAD+-active mutants of dehydrogenases with a minimal background signal. This method involves (1) heat lysis of colonies to permeabilize the cell membrane, (2) colony transfer onto filter paper, (3) washing to remove endogenous NAD(P) and reducing compounds, (4) enzyme-coupled assay for mNADH-dependent color production, and (5) digital imaging of colonies to identify mNAD+-active mutants. This method was used to improve the activity of 6-phosphogluconate dehydrogenase on nicotinamide mononucleotide (NMN+). The best mutant obtained after six rounds of directed evolution exhibits a 50-fold enhancement in catalytic efficiency (k cat/K M) and a specific activity of 17.7 U/mg on NMN+, which is comparable to the wild-type enzyme on its natural coenzyme, NADP+. The engineered dehydrogenase was then used to construct an NMNH regeneration system to drive an ene-reductase catalysis. A comparable level of turnover frequency and product yield was observed using the engineered system relative to NADPH regeneration by using the wild-type dehydrogenase. NESPA provides a simple and accurate readout of mNAD+-based activities and the screening at high-throughput levels (approximately tens of thousands per round), thus opening up an avenue for the evolution of dehydrogenases with specific activities on mNAD+s similar to the levels of natural enzyme/coenzyme pairs.

Spatial Distribution of Noninvasive Break Up Times and Clinical Relevance in Healthy Participants and Mild Dry Eye.

PurposeNoninvasive keratograph break up times (NIKBUTs) are preferred to dye-based methods to evaluate tear stability in translational medicine. We analyzed the NIKBUTs in different regions of the precorneal tear by using a common imaging technology and explored potential correlations with clinical parameters.MethodsWe tested NIKBUTs of 120 participants (62.5% females, aged 61.0 ± 13.8 years) with the Keratograph 5M, with standardized symptoms, ocular surface evaluation, and tear lipid layer interferometry. NIKBUTs were obtained from color maps in up to 165 spatial zones corresponding to 7 concentric rings.ResultsThe lowest NIKBUT of tested zones averaged 7.8 ± 7.4 seconds (median, 4.5; range, 1.5–24 seconds), with the lowest NIKBUT measuring <2 seconds in many inferior zones. A mean of 5 zones had broken up by 2 seconds compared to a mean of about 50 zones by 10 seconds. NIKBUTs in specific inferior peripheral zones were significantly directly correlated to tear lipid thicknesses. The receiver operating characteristics for detecting reduced tear lipid thickness were better than overall NIKBUTs for participants with readings in these zones. Weaker correlations of NIKBUTs with symptoms were observed in two other zones. Overall, the NIKBUT displayed by keratograph was not significantly associated with any clinical parameters.ConclusionsDecreased NIKBUTs in specific peripheral locations may be associated with lower lipid thicknesses. Future measurements of NIKBUTs should ideally be determined in smaller defined zones than current maps.Translational RelevanceAn understanding of how to evaluate tear stability allows a more robust clinical evaluation of new drugs and medical devices for dry eye.

Evaluation of non-reducing end pathologic glycosaminoglycan detection method for monitoring therapeutic response to enzyme replacement therapy in human mucopolysaccharidosis I

Therapeutic development and monitoring require demonstration of effects on disease phenotype. However, due to the complexity of measuring clinically-relevant effects in rare multisystem diseases, robust biomarkers are essential. For the mucopolysaccharidoses (MPS), the measurement of glycosaminoglycan levels is relevant as glycosaminoglycan accumulation is the primary event that occurs due to reduced lysosomal enzyme activity. Traditional dye-based assays that measure total glycosaminoglycan levels have a high background, due to a normal, baseline glycosaminoglycan content in unaffected individuals. An assay that selectively detects the disease-specific non-reducing ends of heparan sulfate glycosaminoglycans that remain undegraded due to deficiency of a specific enzyme in the catabolic pathway avoids the normal background, increasing sensitivity and specificity. We evaluated glycosaminoglycan content by dye-based and non-reducing end methods using urine, serum, and cerebrospinal fluid from MPS I human samples before and after treatment with intravenous recombinant human alpha-l-iduronidase. We found that both urine total glycosaminoglycans and serum heparan sulfate derived non-reducing end levels were markedly decreased compared to baseline after 26 weeks and 52 weeks of therapy, with a significantly greater percentage reduction in serum non-reducing end (89.8% at 26 weeks and 81.3% at 52 weeks) compared to urine total glycosaminoglycans (68.3% at 26 weeks and 62.4% at 52 weeks, p < 0.001). Unexpectedly, we also observed a decrease in non-reducing end levels in cerebrospinal fluid in all five subjects for whom samples were collected (mean 41.8% reduction, p = 0.01). The non-reducing ends in cerebrospinal fluid showed a positive correlation with serum non-reducing end levels in the subjects (r2 = 0.65, p = 0.005). Results suggest utility of the non-reducing end assay in evaluating a therapeutic response in MPS I.

Standalone interferometry-based calibration of convex lens-induced confinement microscopy with nanoscale accuracy.

Strongly confined environments (confined dimensions between 1-100 nm) represent unique challenges and opportunities for understanding and manipulating molecular behavior due to the significant effects of electric double layers, high surface-area to volume ratios, and other phenomena at the nanoscale. Convex Lens-induced Confinement (CLiC) can be used to analyze the dynamics of individual molecules or particles confined in a planar slit geometry with continuously varying gap thickness. We describe an interferometry-based method for precise measurement of the slit pore geometry. Specifically, this approach permitted accurate characterization of separation distances as small as 5 nm, with 1 nm precision, without a priori knowledge or assumptions about the contact geometry, as well as a greatly simplified experimental setup that required only a lens, coverslip, and inverted microscope. The interferometry-based measurement of gap height offered a distinct advantage over conventional fluorescent dye-based methods; e.g., accurate interferometric height measurements were made at low gap heights regardless of solution conditions, while the concentration of fluorescent dye was significantly impacted by solution conditions such as ionic strength or pH. The accuracy of the interferometric measurements was demonstrated by comparing the experimentally measured concentration of a charged fluorescent dye as a function of gap thickness with dye concentration profiles calculated using Debye-Hückel theory. Accurate characterization of nanoscale gap thickness will enable researchers to study a variety of practical and biologically relevant systems within the CLiC geometry.

Lysyl oxidase activates cancer stromal cells and promotes gastric cancer progression: quantum dot-based identification of biomarkers in cancer stromal cells.

PurposeSemiconductor quantum dots (QDs) are a promising alternative to organic fluorescent dyes for multiplexed molecular imaging of cancer stroma, which have great advantages in holistically analyzing the complex interactions among cancer stromal components in situ.Patients and methodsA QD probe-based multiplexed spectral molecular imaging method was established for simultaneous imaging. Three tissue microarrays (TMAs) including 184 gastric cancer (GC) tissues were constructed for the study. Multispectral analyses were performed for quantifying stromal biomarkers, such as lysyl oxidase (LOX). The stromal status including infiltrating of immune cells (high density of macrophages), angiogenesis (high density of microvessel density [MVD], low neovessel maturation) and extracellular matrix (ECM) remodeling (low density of type IV collagen, intense expression of matrix metalloproteinase 9 [MMP-9]) was evaluated.ResultsThis study compared the imaging features of the QD probe-based single molecular imaging method, immunohistochemistry, and organic dye-based immunofluorescent methods, and showed the advantages of the QD probe-based multiple molecular imaging method for simultaneously visualizing complex components of cancer stroma. The risk of macrophages in high density, high MVD, low neomicrovessel maturation, MMP-9 expression and low type IV collagen was significantly increased for the expression of LOX. With the advantages of the established QD probe-based multiplexed molecular imaging method, the spatial relationship between LOX and stromal essential events could be simultaneously evaluated histologically. Stromal activation was defined and then evaluated. Survival analysis showed that the stromal activation was correlated with overall survival and disease-free survival (P<0.001 for all). The expression of LOX was significantly increased in the intense activation subgroup (P<0.001).ConclusionQuantifying assessment of the stroma indicates that the LOX may be a stromal marker for GC and stromal activation, which is not only responsible for the ECM remodeling morphologically, but also for the formation of invasive properties and recurrence. These results support the possibility to integrate morphological and molecular biomarker information for cancer research by the biomedical application of QDs.

Library Quantification Using PicoGreen Fluorometry

This protocol describes the quantification of DNA using PicoGreen and a fluorometer to determine the concentration of DNA sample for downstream processing. Because dye-based methods will not detect degraded or short DNA fragments, PicoGreen requires DNAs ≥50 bp, but can less reliably detect fragments as small as 20 bp.

Agreement of noninvasive tear break-up time measurement between Tomey RT-7000 Auto Refractor-Keratometer and Oculus Keratograph 5M.

BackgroundIt is difficult to standardize assessment of dry eye in different clinical settings. Increasingly, tear stability is recognized to be important for the definition and assessment of patients with dry eye. Recently, two commercially available instruments have been made available for objectively measuring noninvasive tear break-up time (NIBUT), as an indicator of tear stability: the Tomey RT-7000 Auto Refractor-Keratometer and Oculus Keratograph (K)5M. We aim to assess the agreement of NIBUT measurements using these modalities.MethodsThis prospective cross-sectional study was carried out in a tertiary referral eye center and involved 126 consecutive dry eye patients. NIBUT assessment was performed on the right eyes of participants with both the RT-7000 and the K5M techniques, with the order of assessment randomized. The Standardized Patient Evaluation of Eye Dryness (SPEED) questionnaires were administered to assess dry eye symptoms in the 2 weeks before assessment.ResultsThe age of the participants was 56.0±14.3 years (69.84% females). Measurements for both modalities were non-normally distributed (right-skewed). The median RT-7000 and K5M readings were 4.2 (range 0.1–10.0) and 6.4 (0.1–24.9) seconds, respectively. RT-7000 and K5M readings were poorly correlated (ρ=0.061, P=0.495). Intraclass correlation coefficient between the modalities was 0.187 (95% confidence interval −0.097 to 0.406). The Bland–Altman plot showed no systematic differences between the readings with these machines. The agreement between machines was not different in different SPEED categories.ConclusionWhile there are theoretical and practical benefits of NIBUT for assessment of tear stability over dye-based methods, the agreement between the two modalities was poor. Hence, studies and trials assessing NIBUT should avoid using these modalities interchangeably for NIBUT assessment. More research is needed to improve consensus on how to determine NIBUT.

A Simple, Versatile Antibody-Based Barcoding Method for Flow Cytometry.

Barcoding of biological samples is a commonly used strategy to mark or identify individuals within a complex mixture. However, cell barcoding has not yet found wide use in flow cytometry that would benefit greatly from the ability to analyze pooled experimental samples simultaneously. This is due, in part, to technical and practical limitations of current fluorescent dye-based methods. In this study, we describe a simple, versatile barcoding strategy that relies on combinations of a single Ab conjugated to different fluorochromes and thus in principle can be integrated into any flow cytometry application. To demonstrate the efficacy of the approach, we describe the results of a variety of experiments using live cells as well as fixed and permeabilized cells. The results of these studies show that Ab-based barcoding provides a simple, practical method for identifying cells from individual samples pooled for analysis by flow cytometry that has broad applications in immunological research.