Backbone cyclization of Salmonella typhimurium diaminopropionate ammonia-lyase to enhance the activity and stability.

Diaminopropionate ammonia-lyase transforms D and L isomers of 2,3-diaminopropionate to pyruvate and ammonia. It catalyzes D- and l-serine less effectively. L-2,3-diaminopropionate is a precursor in the biosynthesis of oxalyl diaminopropionate as a neurotoxin in certain legume species. In this work, we cyclized the diaminopropionate ammonia-lyase from Salmonella typhimurium in vitro using the redox-responsive split intein, and identified that backbone cyclization afforded the enzyme with the improved activity, thermal stability and resistance to the exopeptidase proteolysis, different from effects of the incorporated sequence recognized by tobacco vein mottling virus protease at C-terminus. Using analyses of three fluorescent dyes including 8-anilino-1-naphthalenesulfonic acid, N-phenyl-1-naphthylamine, and thioflavin T, the same amounts of the cyclic protein displayed less fluorescence than those of the linear protein upon the heat treatment. The cyclic enzyme displayed the enhanced activity in Escherichia coli cells using the designed novel reporter. In this system, d-serine was added to the culture and transported into the cytoplasm. It was transformed by pre-overexpression of the diaminopropionate ammonia-lyase, and untransformed d-serine was oxidized by the coproduced human d-amino acid oxidase to generate hydrogen peroxide. This oxidant is monitored by the HyPer indicator. The current results presented that the cyclized enzyme could be applied as a better candidate to block the neurotoxin biosynthesis in certain plant species.

Fluorescent Probes Based on Charge and Proton Transfer for Probing Biomolecular Environment.

Fluorescent probes for sensing fundamental properties of biomolecular environment, such as polarity and hydration, help to study assembly of lipids into biomembranes, sensing interactions of biomolecules and imaging physiological state of the cells. Here, we summarize major efforts in the development of probes based on two photophysical mechanisms: (i) an excited-state intramolecular charge transfer (ICT), which is represented by fluorescent solvatochromic dyes that shift their emission band maximum as a function of environment polarity and hydration; (ii) excited-state intramolecular proton transfer (ESIPT), with particular focus on 5-membered cyclic systems, represented by 3-hydroxyflavones, because they exhibit dual emission sensitive to the environment. For both ICT and ESIPT dyes, the design of the probes and their biological applications are summarized. Thus, dyes bearing amphiphilic anchors target lipid membranes and report their lipid organization, while targeting ligands direct them to specific organelles for sensing their local environment. The labels, amino acid and nucleic acid analogues inserted into biomolecules enable monitoring their interactions with membranes, proteins and nucleic acids. While ICT probes are relatively simple and robust environment-sensitive probes, ESIPT probes feature high information content due their dual emission. They constitute a powerful toolbox for addressing multitude of biological questions.

Synthesis of New D-π-A Phenothiazine-Based Fluorescent Dyes: Aggregation Induced Emission and Antibacterial Activity.

Highly solid-state fluorescent dyes based on phenothiazine bearing sulfa-drug derivatives were successfully prepared and fully characterized by NMR, mass spectra, and elemental analysis. The prepared phenothiazine dyes bearing sulfadiazine and sulfathiazole 4-(((10-hexyl-10 H-phenothiazin-3-yl)methylene)amino)-N-(pyrimidin-2yl) benzenesulfonamide (PTZ-1) and 4-(((10-hexyl-10 H-phenothiazin-3-yl) methylene) amino)-N-(thiazol-2-yl)benzenesulfonamide (PTZ-2), showed strong emission in polycrystalline form, and significant emission in solution was observed. The quantum yield of the prepared dyes varied and decreased by increasing the solvent polarity, with the maximum recorded value being 0.63 and 0.6 in dioxane. Aggregation-induced emission (AIE) and the effect of the solvent polarity on absorption and emission spectra were investigated. The dyeing application of polyester fabrics using the prepared phenothiazine-based dyes was studied, showing very good affinity to dyed fabrics. The antibacterial affinity against gram-positive and gram-negative bacteria for the dye powder as well as the dyed PET fabric was investigated, with PTZ-2 showing better affinity against bacteria compared to PTZ-1. This multifunctional property highlights the potential uses of PTZ-1 and PTZ-2 for advanced applications in biomedicine and optoelectronics.

Sodium fluorescein-guided resection of brain metastases: A needed approach or an option? A systematic review and meta-analysis.

PURPOSE: Brain metastases (BM) often leave residual tumors despite having visible margins, which increases the risk of local tumor recurrence and can impact overall patient survival rates. Fluorescence-guided surgery (FGS) utilizing sodium fluorescein (FL) has been reported as an effective technique in recent studies. This study aimed to evaluate the efficacy of FL FGS in improving the extent of resection of brain metastases and its impact on overall survival. METHODS: We conducted a systematic search following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Our primary focus was on gross total resection (GTR). Additionally, we extracted survival data and evaluated the risk of bias using a modified version of the Joanna Briggs Institute critical appraisal tool. RESULTS: The study comprised 970 patients with brain metastases through eight different studies. The study found that patients who underwent FL-guided resection had a significantly higher rate of GTR (OR: 2.02, 95% CI: 1.14-3.56, p = 0.0156, I2 = 41.5%). Additionally, the study concluded that FL-guided resection is associated with better overall survival rates (HR: 0.61, 95%CI: 0.47 0.80, p = 0.0003, I2 = 41.5%). CONCLUSION: Our research suggests that the use of FL is associated with a higher rate of GTR and improved overall patient survival. None of the studies we reviewed reported significant complications associated with the use of FL in patients.

Luminescent photon-upconversion nanoparticles with advanced functionalization for smart sensing and imaging.

Photon-upconversion nanoparticles (UCNP) have already been established as labels for affinity assays in analog and digital formats. Here, advanced, or smart, systems based on UCNPs coated with active shells, fluorescent dyes, and metal and semiconductor nanoparticles participating in energy transfer reactions are reviewed. In addition, switching elements can be embedded in such assemblies and provide temporal and spatial control of action, which is important for intracellular imaging and monitoring activities. Demonstration and critical comments on representative approaches demonstrating the progress in the use of such UCNPs in bioanalytical assays, imaging, and monitoring of target molecules in cells are reported, including particular examples in the field of cancer theranostics.

ß-Galactosidase- and Photo-Activatable Fluorescent Probes for Protein Labeling and Super-Resolution STED Microscopy in Living Cells.

We report on the synthesis of two fluorescent probes which can be activated by ß-Galactosidase (ß-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with ß-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin-Halo fusion protein in live cells with overexpressed ß-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science.

Preparation and Characterization of Bilayer Polymer-Dispersed Liquid Crystals Doped with Gd2O3 Nanoparticles and Rhodamine B Base Fluorescent Dye.

Using the polymerization-induced phase separation (PIPS) method, bilayer polymer-dispersed liquid crystal (PDLC) films with a PDLC-PVA-PDLC structure were prepared in this work. It was found that all PDLC performance indexes were affected by polymer mesh size after comparing the microscopic morphology and electro-optical properties of samples with different monomer ratios. Gd2O3 nanoparticles and rhodamine B base fluorescent dyes introduced into the bilayer PDLC optimized the samples' electro-optical properties and developed new functionalities. In addition, the bilayer PDLC doped with Gd2O3 and rhodamine B base held excellent progressive driving functions as well as stable durability properties. Samples doped with Gd2O3 nanoparticles and rhodamine B base also produced excellent anti-counterfeiting effects under UV irradiation at different angles, further exploiting the application potential of PDLC.

Substituted Maleimides: Self-Reportable Linkers and Tags in Bioconjugation, Material Science, and Nanotechnology.

Substituted maleimides are customisable fluorescent linkers and probes with adaptable reactivity and optical properties. Their compact nature and tunability has led to their use in various applications. For example, their solvatochromic properties offer real-time feedback on linking chemistry and environmental changes, essential for applications in material labelling, drug delivery, and nanoparticle functionalization. This review focuses on developing, synthesising, and modifying substituted maleimides as environment-sensitive fluorescent linkers and probes. It delves into their photophysical dynamics and strategic applications, highlighting their significant contributions to macromolecule conjugation and the development of self-reporting materials.

Prediction of the minimum amount of anti-adhesive agent required for entire intra-abdominal cavity using fluorescent dye.

PURPOSE: Studies on the appropriate amount of anti-adhesive agents for preventing postoperative adhesion are lacking. This animal study aimed to investigate the distribution of an anti-adhesive agent in the abdominal cavity and estimate the necessary amount to cover the entire cavity. METHODS: Fluorescent dye Flamma-552 was conjugated to Guardix-sol to create Guardix-Flamma, which was laparoscopically applied to the abdominal cavity of two 10-kg pigs in different amounts: 15 mL for G1 and 35 mL for G2. After 24 hours, the distribution of Guardix-Flamma was examined under the near-infrared mode of the laparoscope, and the thickness was measured in tissues from the omentum, small, and large intestine by immunohistochemistry. RESULTS: The average area of the abdominal cavity in 10 kg pigs was 2,755 cm2. Guardix-Flamma fluorescence was detected in the greater omentum, ascites in the pelvis, and right quadrant area in G1, whereas in G2, it was detected everywhere. On average, the total thickness of G1 and G2 were 12.68 ± 9.80 µm and 18.16 ± 15.57 µm, respectively. Guardix-Flamma thickness applied to the omentum, small, and large intestines of G2 were 1.31-, 1.45-, and 1.49-times thicker than those of G1, respectively, and were all statistically significant (P < 0.05). CONCLUSION: The entire abdominal cavity of the 10 kg pig was not evenly covered with 15 mL of Guardix. Although 35 mL of Guardix is sufficient to cover the same area with an average thickness of 18 µm, further studies should evaluate the minimum thickness required for an effective anti-adhesive function.

What is the diagnostic accuracy of fluorescence-guided surgery for margin assessment in appendicular bone and soft tissue tumors? - A systematic review of clinical studies.

BACKGROUND: Fluorescence-guided surgery (FGS) is a novel technique to successfully assess surgical margins intraoperatively. Investigation and adoption of this technique in orthopaedic oncology remains limited. METHODS: The PRISMA guidelines were followed for this manuscript. Our study was registered on PROSPERO (380520). Studies describing the use of FGS for resection of bone and soft tissue sarcomas (STS) on humans were included. Diagnostic performance metrics (sensitivity, specificity, positive predictive value [PPV], negative predictive value [NPV] and accuracy) and margin positivity rate were the outcomes assessed. RESULTS: Critical appraisal using the Joanna Brigs Institute checklists showed significant concerns for study quality. Sensitivity of FGS ranged from 22.2 % to 100 % in three of the four studies assessing his metrics; one study in appendicular tumors in the pediatric population reported 0 % sensitivity in the three cases included. Specificity ranged from 9.38 % to 100 %. PPV ranged from 14.6 % to 70 % while NPV was between 53.3 % and 100 %. The diagnostic accuracy ranged from 21.62 % to 92.31 %. Margin positivity rate ranged from 2 % to 50 %, with six of the seven studies reporting values between 20 % and 50 %. CONCLUSIONS: FSG is a feasible technique to assess tumor margins in bone and STS. Reported performance metrics and margin positivity rates vary widely between studies due to low study quality and high heterogeneity in dying protocols. LEVEL OF EVIDENCE: Level III, diagnostic study.

Make It Shine: Labelling the ER for Light and Fluorescence Microscopy.

The ER is a highly dynamic network of tubules and membrane cisternae. Hence, imaging this organelle in its native and mobile state is of great importance. Here we describe methods of labelling the native plant ER using fluorescent proteins and lipid dyes as well as methods for immunolabelling on plant tissue.

Near-infrared fluorescent indolizine-dicyanomethylene-4H-pyran hybrids for viscosity imaging in living cells.

The development of near-infrared organic fluorescent dyes with tunable emission profiles is highly required in the field of biological sensing and imaging. In this paper, we designed and synthesized two organic fluorescent dyes, DCM-1 and DCM-2, through the hybridization of indolizine and dicyanomethylene-4H-pyran skeleton. These two compounds show near-infrared fluorescence with emission maximum approximately at 640 and 680 nm, respectively. Notably, both DCM-1 and DCM-2 have specific responses to viscosity without being interfered by biological relevant species. Cell experiments demonstrate that DCM-1 and DCM-2 can detect dynamic changes in viscosity within living cells, suggesting their potential applications in chemical biology research.

Monitoring Xylem Transport in Arabidopsis thaliana Seedlings Using Fluorescent Dyes.

Fluorescent dyes are often used to observe transport mechanisms in plant vascular tissues. However, it has been technically challenging to apply fluorescent dyes on roots to monitor xylem transport in vivo. Here, we present a fast, noninvasive, and high-throughput protocol to monitor xylem transport in seedlings. Using the fluorescent dyes 5(6)-carboxyfluorescein diacetate (CFDA) and Rhodamine WT, we were able to observe xylem transport on a cellular level in Arabidopsis thaliana roots. We describe how to apply these dyes on primary roots of young seedlings, how to monitor root-to-shoot xylem transport, and how to measure xylem transport velocity in roots. Moreover, we show that our protocol can also be applied to lateral roots and grafted seedlings to assess xylem (re)connection. Altogether, these techniques are useful for investigating xylem functionality in diverse experimental setups.

Fluorescent Solvatochromic Probes for Long-Term Imaging of Lipid Order in Living Cells.

High-resolution spatio-temporal monitoring of the cell membrane lipid order provides visual insights into the complex and sophisticated systems that control cellular physiological functions. Solvatochromic fluorescent probes are highly promising noninvasive visualization tools for identifying the ordering of the microenvironment of plasma membrane microdomains. However, conventional probes, although capable of structural analysis, lack the necessary long-term photostability required for live imaging at the cellular level. Here, an ultra-high-light-resistant solvatochromic fluorescence probe, 2-N,N-diethylamino-7-(4-methoxycarbonylphenyl)-9,9-dimethylfluorene (FπCM) is reported, which enables live lipid order imaging of cell division. This probe and its derivatives exhibit sufficient fluorescence wavelengths, brightness, polarity responsiveness, low phototoxicity, and remarkable photostability under physiological conditions compared to conventional solvatochromic probes. Therefore, these probes have the potential to overcome the limitations of fluorescence microscopy, particularly those associated with photobleaching. FπCM probes can serve as valuable tools for elucidating mechanisms of cellular processes at the bio-membrane level.

Activation of stress reactions in the dinophyte microalga Prorocentrum cordatum as a consequence of the toxic effect of ZnO nanoparticles and zinc sulfate.

According to the results of the experimental study, the main regularities of changes in morphological, structural-functional and fluorescent indices of P. cordatum were established when zinc oxide nanoparticles ZnO NPs (0.3-6.4 mg L-1) and Zn in form of salt (0.09-0.4 mg L-1) were added to the medium. The studied pollutants have cytotoxic (growth inhibition, development of oxidative stress, destruction of cytoplasmic organelles, disorganization of mitochondria) and genotoxic (changes in the morphology of nuclei, chromatin condensation) effects on microalgae, affecting almost all aspects of cell functioning. Despite the similar mechanism of action of zinc sulfate and ZnO NPs on P. cordatum cells, the negative effect of ZnO NPs is also due to the inhibition of photosynthetic activity of cells (significant decrease in the maximum quantum yield of photosynthesis and electron transport rate), reduction of chlorophyll concentration from 3.5 to 1.8 pg cell-1, as well as mechanical effect on cells: deformation and damage of cell membranes, aggregation of NPs on the cell surface. Apoptosis-like signs of cell death upon exposure to zinc sulfate and ZnO NPs were identified by flow cytometry and laser scanning confocal microscopy methods: changes in cell morphology, cytoplasm retraction, development of oxidative stress, deformation of nuclei, and disorganization of mitochondria. It was shown that the first signs of cell apoptosis appear at 0.02 mg L-1 Zn and 0.6 mg L-1 ZnO NPs after 72 h of exposure. At higher concentrations of pollutants, a dose-dependent decrease in algal enzymatic activity (up to 5 times relative to control) and mitochondrial membrane potential (up to 4 times relative to control), and an increase in the production of reactive oxygen species (up to 4-5 times relative to control) were observed. The results of the presented study contribute to the disclosure of fundamental mechanisms of toxic effects of pollutants and prediction of ways of phototrophic microorganisms reaction to this impact.

Conformational Dynamic Studies of Prokaryotic Potassium Channels Explored by Homo-FRET Methodologies.

Fluorescence techniques have been widely used to shed light over the structure-function relationship of potassium channels for the last 40-50 years. In this chapter, we describe how a Förster resonance energy transfer between identical fluorophores (homo-FRET) approach can be applied to study the gating behavior of the prokaryotic channel KcsA. Two different gates have been described to control the K+ flux across the channel's pore, the helix-bundle crossing and the selectivity filter, located at the opposite sides of the channel transmembrane section. Both gates can be studied individually or by using a double-reporter system. Due to its homotetrameric structural arrangement, KcsA presents a high degree of symmetry that fulfills the first requisite to calculate intersubunit distances through this technique. The results obtained through this work have helped to uncover the conformational plasticity of the selectivity filter under different experimental conditions and the importance of its allosteric coupling to the opening of the activation (inner) gate. This biophysical approach usually requires low protein concentration and presents high sensitivity and reproducibility, complementing the high-resolution structural information provided by X-ray crystallography, cryo-EM, and NMR studies.

Tinea capitis: dermoscopy and calcium fluorescent microscopy as highly efficient and precise diagnostic tools

Abstract Tinea capitis comprising of tinea favosa and kerion is mostly seen in school-aged children. Some tinea capitis often presented with insignificant findings under the naked eyes are easily overlooked. The authors describe an unusual case of tinea capitis caused by Trichophyton violaceum. The patient was an 8-year-old girl, with a history of pruritus on the scalp for more than one year. A diagnosis of tinea capitis was confirmed by clinical examination aided by dermoscopy, calcium fluorescent microscopy and culture. Comma and corkscrew hairs are two specific dermoscopic patterns of tinea capitis. The patient was treated with systemic itraconazole, topical application with 1% naftifine 0.25% ketoconazole cream followed after daily hair wash with 2% ketoconazole shampoo for 8 weeks.

Facial nerve identification with fluorescent dye in rats

PURPOSE The parotidectomy technique still has an elevated paresis and paralysis index, lowering patient life's quality. The correct identification of the facial nerve can prevent nerve damage. Fluorescent dye identifies nerves in experimental studies but only few articles focused its use on facial nerve study in parotidectomies. We aimed to stain the rat facial nerve with fluorescent dye to facilitate visualization and dissection in order to prevent injuries. METHODS Forty adult male Wistar rats were submitted to facial injection of saline solution (Gsf-control group, 10) or fluorescent dye solution (Gdye group, 30) followed by parotidectomy preserving the facial nerve, measuring the time for localization and facility of localization (LocTime and LFN). Nerve function was assessed using the Vibrissae Movements (PMV) and Eyelid Closure Motion (PFP) scores. RESULTS Nerve localization was faster in Gdye group, with 83% Easy LFN rate. The Gdye group presented with low nerve injury degree and better PMV and PFP scores, with high sensitivity and accuracy. CONCLUSIONS This experimental method of facial nerve fluorescence was effective for intraoperative nerve visualization, identification and preservation. The technique may be used in future facial nerve studies, translated to humans, contributing to the optimization of parotid surgery in the near future.

Water interaction and bond strength to dentin of dye-labelled adhesive as a function of the addition of rhodamine B

ABSTRACT Objective This study investigated the effect of the fluorescent dye rhodamine B (RB) for interfacial micromorphology analysis of dental composite restorations on water sorption/solubility (WS/WSL) and microtensile bond strength to dentin (µTBS) of a 3-step total etch and a 2-step self-etch adhesive system. Material and Methods The adhesives Adper Scotchbond Multi-Purpose (MP) and Clearfil SE Bond (SE) were mixed with 0.1 mg/mL of RB. For the WS/WSL tests, cured resin disks (5.0 mm in diameter x 0.8 mm thick) were prepared and assigned into four groups (n=10): MP, MP-RB, SE, and SE-RB. For µTBS assessment, extracted human third molars (n=40) had the flat occlusal dentin prepared and assigned into the same experimental groups (n=10). After the bonding and restoration procedures, specimens were sectioned in rectangular beams, stored in water and tested after seven days or after 12 months. The failure mode of fractured specimens was qualitatively evaluated under optical microscope (x40). Data from WS/WSL and µTBS were assessed by one-way and three-way ANOVA, respectively, and Tukey’s test (α=5%). Results RB increased the WSL of MP and SE. On the other hand, WS of both MP and SE was not affected by the addition of RB. No significance in µTBS between MP and MP-RB for seven days or one year was observed, whereas for SE a decrease in the µTBS means occurred in both storage times. Conclusions RB should be incorporated into non-simplified DBSs with caution, as it can interfere with their physical-mechanical properties, leading to a possible misinterpretation of bonded interface.

Diseño de un panel multicolor para evaluar moléculas intracelulares y de superficie mediante citometría de flujo / Design of a multicolor panel to assess intracellular and surface molecules by flow cytometry

Introducción. La citometría de flujo permite detectar la presencia de moléculas intracelulares y de superficie, de forma simultánea sobre cada célula. Objetivo. Describir un método para la construcción armónica de un panel multicolor con 11 parámetros para el análisis fenotípico y funcional de linfocitos T (LT) CD8 + por citometría de flujo. Materiales y métodos. Para la construcción del panel multicolor, se seleccionaron las moléculas y se titularon los conjugados con fluorocromos para la determinación de CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 y CD127, en células mononucleares de sangre periférica. Para la evaluación del panel, se hizo la construcción progresiva adicionando uno a uno los conjugados y la fluorescencia menos uno (FMO). Este método fue aplicado para células ex vivo y para evaluar la producción de IFN ? , IL-2 y TNFa frente al estímulo con la enterotoxina B de Staphylococcus aureus (SEB) y al antígeno crudo de Trypanosoma cruzi . Finalmente, se procedió al análisis de las subpoblaciones de LT CD8 + ex vivo en individuos sanos. Resultados. La evaluación de las moléculas con los conjugados no mostró interferencia en las señales de fluorescencia. Las frecuencias de las subpoblaciones de LT CD8 + evaluadas fueron cercanas a los valores reportados en otros estudios. Además, se observó que la frecuencia de LT CD8 + productores de IFN ? , IL-2 y TNFa fue mayor a las seis horas de cultivo con SEB y con el antígeno crudo de T. cruzi . Conclusiones. El método aplicado para la construcción del panel multicolor permite obtener frecuencias de las subpoblaciones de LT CD8 + que corresponden a lo reportado en la literatura científica.

Introduction: Flow cytometry allows simultaneous detection of surface and intracellular molecules on each cell. Objective: To describe a method for building up a harmonic multicolor panel with 11 flow cytometry parameters for phenotypic and functional analysis on CD8 + T lymphocytes. Materials and methods: For the multicolor panel construction, we selected the molecules and titred conjugated antibodies with fluorochromes for CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 and CD127 determination in peripheral blood mononuclear cells (PBMC). To evaluate the panel, the conjugated antibodies were gradually added one by one and fluorescence minus one (FMO) test was performed. This method was applied to assess ex vivo subpopulations of T cells and the production of intracellular IFN ? , IL-2 and TNF a using polyclonal stimulation with enterotoxin B from Staphylococcus aureus (SEB) and antigen-specific cells with crude Trypanosoma cruzi antigen. Finally, the ex vivo CD8 + T lymphocyte subpopulations frequencies were analyzed in healthy individuals. Results: The evaluation of the selected molecules and conjugates did not show interference in the fluorescence signals and detection. The frequencies of CD8 + T cells evaluated were similar to the values reported in other studies. Additionally, we observed that the frequency of CD8 + T lymphocytes producing IFN ? , IL-2 and TNF a was higher 6 hours after culture with SEB and crude T. cruzi lysate. Conclusions: The method used for the construction of a multicolor panel allows obtaining frequencies of CD8 + T lymphocyte subpopulations corresponding to those reported in the literature.