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Nature Communications Jul 2023The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from...
The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.
Topics: Luminescent Proteins; Biliverdine; Coloring Agents; Engineering; Red Fluorescent Protein
PubMed: 37438348
DOI: 10.1038/s41467-023-39776-9 -
Advanced Science (Weinheim,... Mar 2024Cancer remains a global health challenge, demanding early detection and accurate diagnosis for improved patient outcomes. An intelligent paradigm is introduced that...
Cancer remains a global health challenge, demanding early detection and accurate diagnosis for improved patient outcomes. An intelligent paradigm is introduced that elevates label-free nonlinear optical imaging with contrastive patch-wise learning, yielding stain-free nonlinear optical computational histology (NOCH). NOCH enables swift, precise diagnostic analysis of fresh tissues, reducing patient anxiety and healthcare costs. Nonlinear modalities are evaluated, including stimulated Raman scattering and multiphoton imaging, for their ability to enhance tumor microenvironment sensitivity, pathological analysis, and cancer examination. Quantitative analysis confirmed that NOCH images accurately reproduce nuclear morphometric features across different cancer stages. Key diagnostic features, such as nuclear morphology, size, and nuclear-cytoplasmic contrast, are well preserved. NOCH models also demonstrate promising generalization when applied to other pathological tissues. The study unites label-free nonlinear optical imaging with histopathology using contrastive learning to establish stain-free computational histology. NOCH provides a rapid, non-invasive, and precise approach to surgical pathology, holding immense potential for revolutionizing cancer diagnosis and surgical interventions.
Topics: Humans; Histological Techniques; Coloring Agents; Optical Imaging; Neoplasms; Tumor Microenvironment
PubMed: 38095543
DOI: 10.1002/advs.202308630 -
Molecules (Basel, Switzerland) Jul 2023In this study, pristine kiwi peel (KP) and nitric acid modified kiwi peel (NA-KP) based adsorbents were prepared and evaluated for selective removal of cationic dye. The...
In this study, pristine kiwi peel (KP) and nitric acid modified kiwi peel (NA-KP) based adsorbents were prepared and evaluated for selective removal of cationic dye. The morphology and chemical structure of KP and NA-KP were fully characterized and compared, and results showed nitric acid modification introduced more functional groups. Moreover, the adsorption kinetics and isotherms of malachite green (MG) by KP and NA-KP were investigated and discussed. The results showed that the adsorption process of MG onto KP followed a pseudo-second-order kinetic model and the Langmuir isotherm model, while the adsorption process of MG onto NA-KP followed a pseudo-first-order kinetic model and the Freundlich isotherm model. Notably, the Langmuir maximum adsorption capacity of NA-KP was 580.61 mg g, which was superior to that of KP (297.15 mg g). Furthermore, thermodynamic studies demonstrated the feasible, spontaneous, and endothermic nature of the adsorption process of MG by NA-KP. Importantly, NA-KP showed superior selectivity to KP towards cationic dye MG against anionic dye methyl orange (MO). When the molar ratio of MG/MO was 1:1, the separation factor (α) of NA-KP was 698.10, which was 5.93 times of KP. In addition, hydrogen bonding, π-π interactions, and electrostatic interaction played important roles during the MG adsorption process by NA-KP. This work provided a low-cost, eco-friendly, and efficient option for the selective removal of cationic dye from dyeing wastewater.
Topics: Coloring Agents; Adsorption; Nitric Acid; Rosaniline Dyes; Thermodynamics; Kinetics; Water Pollutants, Chemical; Hydrogen-Ion Concentration
PubMed: 37513184
DOI: 10.3390/molecules28145310 -
Cells Aug 2023Optical mapping is a powerful imaging technique widely adopted to measure membrane potential changes and intracellular Ca variations in excitable tissues using... (Review)
Review
Optical mapping is a powerful imaging technique widely adopted to measure membrane potential changes and intracellular Ca variations in excitable tissues using voltage-sensitive dyes and Ca indicators, respectively. This powerful tool has rapidly become indispensable in the field of cardiac electrophysiology for studying depolarization wave propagation, estimating the conduction velocity of electrical impulses, and measuring Ca dynamics in cardiac cells and tissues. In addition, mapping these electrophysiological parameters is important for understanding cardiac arrhythmia mechanisms. In this review, we delve into the fundamentals of cardiac optical mapping technology and its applications when applied to hiPSC-derived cardiomyocytes and discuss related advantages and challenges. We also provide a detailed description of the processing and analysis of optical mapping data, which is a crucial step in the study of cardiac diseases and arrhythmia mechanisms for extracting and comparing relevant electrophysiological parameters.
Topics: Humans; Myocytes, Cardiac; Induced Pluripotent Stem Cells; Heart Diseases; Cardiac Electrophysiology; Coloring Agents
PubMed: 37681899
DOI: 10.3390/cells12172168 -
Journal of Extracellular Vesicles Aug 2023Although lipophilic membrane dyes (LMDs) or probes (LMPs) are widely used to label extracellular vesicles (EVs) for detection and purification, their labelling...
Although lipophilic membrane dyes (LMDs) or probes (LMPs) are widely used to label extracellular vesicles (EVs) for detection and purification, their labelling performance has not been systematically characterized. Through concurrent side scattering and fluorescence detection of single EVs as small as 40 nm in diameter by a laboratory-built nano-flow cytometer (nFCM), present study identified that (1) PKH67 and PKH26 could maximally label ∼60%-80% of EVs isolated from the conditioned cell culture medium (purity of ∼88%) and ∼40%-70% of PFP-EVs (purity of ∼73%); (2) excessive PKH26 could cause damage to the EV structure; (3) di-8-ANEPPS and high concentration of DiI could achieve efficient and uniform labelling of EVs with nearly 100% labelling efficiency for di-8-ANEPPS and 70%-100% for DiI; (4) all the four tested LMDs can aggregate and form micelles that exhibit comparable side scatter and fluorescence intensity with those of labelled EVs and thus hardly be differentiate from each other; (5) as the LMD concentration went up, the particle number of self-aggregates increased while the fluorescence intensity of aggregates remained constant; (6) PKH67 and PKH26 tend to form more aggregated micelles than di-8-ANEPPS and DiI, and the effect of LMD self-aggregation can be negligible at optimal staining conditions. (7) All the four tested LMDs can label almost all the very-low-density lipoprotein (VLDL) particles, indicating potential confounding factor in plasma-EV labelling. Besides, it was discovered that DSPE-PEG -biotin can only label ∼50% of plasma-EVs. The number of LMP inserted into the membrane of single EVs was measured for the first time and it was confirmed that membrane labelling by lipophilic dyes did not interfere with the immunophenotyping of EVs. nFCM provides a unique perspective for a better understanding of EV labelling by LMD/LMP.
Topics: Extracellular Vesicles; Micelles; Fluorescent Dyes
PubMed: 37525378
DOI: 10.1002/jev2.12351 -
World Journal of Microbiology &... Sep 2023Fungi have been extensively studied for their capacity to biotransform a wide range of natural and xenobiotic compounds. This versatility is a reflection of the broad... (Review)
Review
Fungi have been extensively studied for their capacity to biotransform a wide range of natural and xenobiotic compounds. This versatility is a reflection of the broad substrate specificity of fungal enzymes such as laccases, peroxidases and cytochromes P450, which are involved in these reactions. This review gives an account of recent advances in the understanding of fungal metabolism of drugs and pollutants such as dyes, agrochemicals and per- and poly-fluorinated alkyl substances (PFAS), and describes the key enzymes involved in xenobiotic biotransformation. The potential of fungi and their enzymes in the bioremediation of polluted environments and in the biocatalytic production of important compounds is also discussed.
Topics: Xenobiotics; Substrate Specificity; Biocatalysis; Coloring Agents; Environmental Pollutants
PubMed: 37658215
DOI: 10.1007/s11274-023-03737-7 -
The Journal of Organic Chemistry Jul 2023This report describes cucurbit[7]uril (CB7) complexation of azobenzene dyes that have a 4-(,'-dimethylamino) or 4-amino substituent. Absorption and NMR data show that...
This report describes cucurbit[7]uril (CB7) complexation of azobenzene dyes that have a 4-(,'-dimethylamino) or 4-amino substituent. Absorption and NMR data show that CB7 encapsulates the protonated form of the azobenzene and that the complexed dye exists as its azonium tautomer with a azo conformation and substantial quinoid resonance character. Because CB7 complexation stabilizes the dye conjugate acid, there is an upward shift in its p, and in one specific case, the p of the protonated azobenzene is increased from 3.09 to 4.47. Molecular modeling indicates that the CB7/azobenzene complex is stabilized by three major noncovalent factors: (i) ion-dipole interactions between the partially cationic 4-(,'-dimethylamino) or 4-amino group on the encapsulated protonated azobenzene and the electronegative carbonyl oxygens on CB7, (ii) inclusion of the upper aryl ring of the azobenzene within the hydrophobic CB7 cavity, and (iii) a hydrogen bond between the proton on the azo nitrogen and CB7 carbonyls. CB7 complexation enhances azobenzene stability and increases azobenzene hydrophilicity; thus, it is a promising way to improve azobenzene performance as a pigment or prodrug. In addition, the striking yellow/pink color change that accompanies CB7 complexation can be exploited to create azobenzene dye displacement assays with naked eye detection.
Topics: Coloring Agents; Bridged-Ring Compounds; Macrocyclic Compounds
PubMed: 37256736
DOI: 10.1021/acs.joc.3c00423 -
Investigative Ophthalmology & Visual... Dec 2023To investigate the flow pattern in unconventional outflow and its correlation with conventional outflow in mouse eyes.
PURPOSE
To investigate the flow pattern in unconventional outflow and its correlation with conventional outflow in mouse eyes.
METHODS
Fluorescent microspheres were injected into the anterior chamber of one eye of anesthetized C57BL/6J mice (n = 4), followed by perfused fixation with 4% paraformaldehyde in situ after 45 minutes. Post-euthanasia, the injected eyes were enucleated, further immersion fixed, and dissected into 12 equal radial segments. Both sides of each segment were imaged using a confocal microscope after nuclear counterstaining. Both unconventional and conventional outflow patterns of each eye were analyzed by ImageJ and ZEN 2.3 imaging software.
RESULTS
Segmental outflow patterns were observed in both the ciliary body (CB) and the supraciliary space and suprachoroidal space (SCS). In the CB, the tracer intensity was the lowest at 12 o'clock and highest at 9 o'clock, whereas in the SCS it was the lowest at 2 o'clock and the highest at 10 o'clock. Consequently, a segmental unconventional outflow was observed, with the lowest and highest flow regions in the superior and temporal quadrants, respectively. The overall segmental uveoscleral outflow has no correlation with trabecular outflow (P > 0.05). Four different outflow patterns were observed: (1) low-flow regions in both outflows, (2) primarily a high-flow region in conventional outflow, (3) primarily a high-flow region in unconventional outflow, and (4) high-flow regions in both outflows.
CONCLUSIONS
Uveoscleral outflow is segmental and unrelated to the trabecular segmental outflow. These findings will lead to future studies to identify the best location for the placement of drainage devices and drug delivery.
Topics: Mice; Animals; Mice, Inbred C57BL; Ciliary Body; Anterior Chamber; Coloring Agents; Drainage
PubMed: 38117243
DOI: 10.1167/iovs.64.15.26 -
The Journal of Organic Chemistry Aug 2023Oxoanions such as carboxylates, phosphates, and sulfates play important roles in both chemistry and biology and are abundant on the cell surface. We report on the...
Oxoanions such as carboxylates, phosphates, and sulfates play important roles in both chemistry and biology and are abundant on the cell surface. We report on the synthesis and properties of a rationally designed guanidinium-containing oxoanion binder, 1-guanidino-8-amino-2,7-diazacarbazole (GADAC). GADAC binds to a carboxylate, phosphate, and sulfate in pure water with affinities of 3.6 × 10, 1.1 × 10, and 4.2 × 10 M, respectively. Like 2-azacarbazole, which is a natural product that enables scorpions to fluoresce, GADAC is fluorescent in water (λ = 356 nm, λ = 403 nm, ε = 13,400 M cm). The quantum yield of GADAC is pH-sensitive, increasing from Φ = 0.12 at pH 7.4 to Φ = 0.53 at pH 4.0 as a result of the protonation of the aminopyridine moiety. The uptake of GADAC into live human melanoma cells is detectable in the DAPI channel at low micromolar concentrations. Its properties make GADAC a promising candidate for applications in oxoanion binding and fluorescence labeling in biological (e.g., the delivery of cargo into cells) and other contexts.
Topics: Humans; Guanidine; Water; Phosphates; Carboxylic Acids; Coloring Agents
PubMed: 37530571
DOI: 10.1021/acs.joc.3c00982 -
Science Advances Oct 2023Small extracellular vesicles (sEVs; <200 nm) that contain lipids, nucleic acids, and proteins are considered promising biomarkers for a wide variety of diseases....
Small extracellular vesicles (sEVs; <200 nm) that contain lipids, nucleic acids, and proteins are considered promising biomarkers for a wide variety of diseases. Conventional methods for sEV isolation from blood are incompatible with routine clinical workflows, significantly hampering the utilization of blood-derived sEVs in clinical settings. Here, we present a simple, viscoelastic-based microfluidic platform for label-free isolation of sEVs from human blood. The separation performance of the device is assessed by isolating fluorescent sEVs from whole blood, demonstrating purities and recovery rates of over 97 and 87%, respectively. Significantly, our viscoelastic-based microfluidic method also provides for a remarkable increase in sEV yield compared to gold-standard ultracentrifugation, with proteomic profiles of blood-derived sEVs purified by both methods showing similar protein compositions. To demonstrate the clinical utility of the approach, we isolate sEVs from blood samples of 20 patients with cancer and 20 healthy donors, demonstrating that elevated sEV concentrations can be observed in blood derived from patients with cancer.
Topics: Humans; Microfluidics; Proteomics; Coloring Agents; Extracellular Vesicles; Neoplasms
PubMed: 37801500
DOI: 10.1126/sciadv.adi5296