Evaluation of Ilex guayusa and Piper marginatum Extract Cytotoxicity on Human Dental Pulp Mesenchymal Stem Cells.

BACKGROUND: Amelogenesis imperfecta is a hereditary disorder affecting dental enamel. Among its phenotypes, hypocalcified AI is characterized by mineral deficiency, leading to tissue wear and, consequently, dental sensitivity. Excessive fluoride intake (through drinking water, fluoride supplements, toothpaste, or by ingesting products such as pesticides or insecticides) can lead to a condition known as dental fluorosis, which manifests as stains and teeth discoloration affecting their structure. Our recent studies have shown that extracts from Colombian native plants, Ilex guayusa and Piper marginatum, deposit mineral ions such as phosphate and orthophosphate into the dental enamel structure; however, it is unknown whether these extracts produce toxic effects on the dental pulp. OBJECTIVE: To assess cytotoxicity effects on human dental pulp stem cells (hDPSCs) exposed to extracts isolated from I. guayusa and P. marginatum and, hence, their safety for clinical use. METHODS: Raman spectroscopy, fluorescence microscopy, and flow cytometry techniques were employed. For Raman spectroscopy, hDPSCs were seeded onto nanobiochips designed to provide surface-enhanced Raman spectroscopy (SERS effect), which enhances their Raman signal by several orders of magnitude. After eight days in culture, I. guayusa and P. marginatum extracts at different concentrations (10, 50, and 100 ppm) were added. Raman measurements were performed at 0, 12, and 24 h following extract application. Fluorescence microscopy was conducted using an OLIMPUS fv1000 microscope, a live-dead assay was performed using a kit employing a BD FACS Canto TM II flow cytometer, and data analysis was determined using a FlowJo program. RESULTS: The Raman spectroscopy results showed spectra consistent with viable cells. These findings were corroborated using fluorescence microscopy and flow cytometry techniques, confirming high cellular viability. CONCLUSIONS: The analyzed extracts exhibited low cytotoxicity, suggesting that they could be safely applied on enamel for remineralization purposes. The use of nanobiochips for SERS effect improved the cell viability assessment.

Probing the cw-Laser-Induced Fluorescence Enhancement in CsPbBr3 Nanocrystal Thin Films: An Interplay between Photo and Thermal Activation.

Perovskite nanocrystals hold significant promise for a wide range of applications, including solar cells, LEDs, photocatalysts, humidity and temperature sensors, memory devices, and low-cost photodetectors. Such technological potential stems from their exceptional quantum efficiency and charge carrier conduction capability. Nevertheless, the underlying mechanisms of photoexcitation, such as phase segregation, annealing, and ionic diffusion, remain insufficiently understood. In this context, we harnessed hyperspectral fluorescence microspectroscopy to advance our comprehension of fluorescence enhancement triggered by UV continuous-wave (cw) laser irradiation of CsPbBr3 colloidal nanocrystal thin films. Initially, we explored the kinetics of fluorescence enhancement and observed that its efficiency (φph) correlates with the laser power (P), following the relationship φph = 7.7⟨P⟩0.47±0.02. Subsequently, we estimated the local temperature induced by the laser, utilizing the finite-difference method framework, and calculated the activation energy (Ea) required for fluorescence enhancement to occur. Our findings revealed a very low activation energy, Ea ∼ 9 kJ/mol. Moreover, we mapped the fluorescence photoenhancement by spatial scanning and real-time static mode to determine its microscale length. Below a laser power of 60 µW, the photothermal diffusion length exhibited nearly constant values of approximately (22 ± 5) µm, while a significant increase was observed at higher laser power levels. These results were ascribed to the formation of nanocrystal superclusters within the film, which involves the interparticle spacing reduction, creating the so-called quantum dot solid configuration along with laser-induced annealing for higher laser powers.

Interaction Between Macrophages and Cryptococcus neoformans: Distinguishing Phagocytosed Versus External Fungi.

The interaction between macrophages and Cryptococcus neoformans is crucial in the pathogenesis of cryptococcosis. These phagocytes are important immune effectors, but also a niche in which facultative intracellular parasites, such as C. neoformans, thrive. Consequently, phagocytosis of cryptococcal cells and its outcomes are very frequently studied. One major issue with several of the tests used for this, however, is that macrophage-C. neoformans interaction does not always result in phagocytosis, as fungi may be attached to the external surface of the phagocyte. The most used methodologies to study phagocytosis of cryptococcal cells have varying degrees of precision in separating fungi that are truly internalized from those that are outside macrophages. Here we describe two assays to measure phagocytosis that can differentiate internal from external C. neoformans cells.

Study of the Membrane Activity of the Synthetic Peptide ∆M3 Against Extended-Spectrum ß-lactamase Escherichia coli Isolates.

Escherichia coli is the most common microorganism causing nosocomial or community-acquired bacteremia, and extended-spectrum ß-lactamase-producing Escherichia coli isolates are identified worldwide with increasing frequency. For this reason, it is necessary to evaluate potential new molecules like antimicrobial peptides. They are recognized for their biological potential which makes them promising candidates in the fight against infections. The goal of this research was to evaluate the potential of the synthetic peptide ΔM3 on several extended-spectrum ß-lactamase producing E. coli isolates. The antimicrobial and cytotoxic activity of the peptide was spectrophotometrically determined. Additionally, the capacity of the peptide to interact with the bacterial membrane was monitored by fluorescence microscopy and infrared spectroscopy. The results demonstrated that the synthetic peptide is active against Escherichia coli isolates at concentrations similar to Meropenem. On the other hand, no cytotoxic effect was observed in HaCaT keratinocyte cells even at 10 times the minimal inhibitory concentration. Microscopy results showed a permeabilizing effect of the peptide on the bacteria. The infrared results showed that ΔM3 showed affinity for the lipids of the microorganism's membrane. The results suggest that the ∆M3 interacts with the negatively charged lipids from the E. coli by a disturbing effect on membrane. Finally, the secondary structure experiments of the peptide showed a random structure in solution that did not change during the interaction with the membranes.

A bird's-eye view of the endoplasmic reticulum in filamentous fungi.

SUMMARYThe endoplasmic reticulum (ER) is one of the most extensive organelles in eukaryotic cells. It performs crucial roles in protein and lipid synthesis and Ca2+ homeostasis. Most information on ER types, functions, organization, and domains comes from studies in uninucleate animal, plant, and yeast cells. In contrast, there is limited information on the multinucleate cells of filamentous fungi, i.e., hyphae. We provide an analytical review of existing literature to categorize different types of ER described in filamentous fungi while emphasizing the research techniques and markers used. Additionally, we identify the knowledge gaps that need to be resolved better to understand the structure-function correlation of ER in filamentous fungi. Finally, advanced technologies that can provide breakthroughs in understanding the ER in filamentous fungi are discussed.

The Virus-Induced Cytopathic Effect.

The cytopathic effect comprises the set of cellular alterations produced by a viral infection. It is of great relevance since it constitutes a direct marker of infection. Likewise, these alterations are often virus-specific which makes them a phenotypic marker for many viral species. All these characteristics have been used to complement the study of the dynamics of virus-cell interactions through the kinetic study of the progression of damage produced by the infection. Various approaches have been used to monitor the cytopathic effect, ranging from light microscopy, immunofluorescence assays, and direct labeling with fluorescent dyes, to plaque assay for the characterization of the infection over time. Here we address the relevance of the study of cytopathic effect and describe different experimental alternatives for its application.

The Role of Trypan Blue as a Conventional and Fluorescent Dye for the Diagnosis of Superficial Mycoses by Potassium Hydroxide (KOH) Testing.

Direct microscopic examination of samples using potassium hydroxide (KOH) is a fast, simple, and inexpensive method to confirm clinical suspicion of superficial mycosis. However, the lack of color contrast in this test makes it difficult to separate any fungal structures from artifacts. The sensitivity of the KOH mount technique may be enhanced using both fluorochromes and conventional stains that highlight the fungal structures when observed under fluorescence microscopy and bright-field, respectively. Here we study the possibility of using Trypan Blue (TB), an azo dye which is often used as a live/dead marker, in the diagnosis of superficial mycoses by KOH testing. TB at 0.01% displayed a fluorescent staining pattern similar to that of Calcofluor White (CFW), the conventional cell wall fluorophore. Furthermore, by adjusting the TB concentration to 0.1-0.3%, in addition to maintaining the fluorescent staining pattern, the fungal elements were stained in blue under bright-field microscopy. Thus, we demonstrate for the first time that TB has the unique property as a fungal stain that can be used in both bright-field and fluorescence microscopy for diagnosis of superficial mycoses by direct microscopic examination.

rcell2: Microscopy-Based Cytometry in R.

This article describes a method for quantifying various cellular features (e.g., volume, curvature, total and sub-cellular fluorescence localization) of individual cells from sets of microscope images, and for tracking them over time-course microscopy experiments. One purposely defocused transmission image (sometimes referred to as bright-field or BF) is used to segment the image and locate each cell. Fluorescence images (one for each of the color channels or z-stacks to be analyzed) may be acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses a set of R packages called rcell2. Relative to the original release of Rcell (Bush et al., 2012), the updated version bundles, into a single software suite, the image-processing capabilities of Cell-ID, offers new data analysis tools for cytometry, and relies on the widely used data analysis and visualization tools of the statistical programming framework R. © 2023 Wiley Periodicals LLC. Basic Protocol: Extracting quantitative information from single cells Support Protocol 1: Obtaining and installing Cell-ID and R Support Protocol 2: Preparing cells for imaging.

In Vitro Characterization of Protein:Nucleic Acid Liquid-Liquid Phase Separation by Microscopy Methods and Nanoparticle Tracking Analysis.

Uncontrolled assembly/disassembly of physiologically formed liquid condensates is linked to irreversible aggregation. Hence, the quest for understanding protein-misfolding disease mechanism might lie in the studies of protein:nucleic acid coacervation. Several proteins with intrinsically disordered regions as well as nucleic acids undergo phase separation in the cellular context, and this process is key to physiological signaling and is related to pathologies. Phase separation is reproducible in vitro by mixing the target recombinant protein with specific nucleic acids at various stoichiometric ratios and then examined by microscopy and nanotracking methods presented herein. We describe protocols to qualitatively assess hallmarks of protein-rich condensates, characterize their structure using intrinsic and extrinsic dyes, quantify them, and analyze their morphology over time. Analysis by nanoparticle tracking provides information on the concentration and diameter of high-order protein oligomers formed in the presence of nucleic acid. Using the model protein (globular domain of recombinant murine PrP) and DNA aptamers (high-affinity oligonucleotides with 25 nucleotides in length), we provide examples of a systematic screening of liquid-liquid phase separation in vitro.

The Contribution of Cell Imaging to the Study of Anterior Pituitary Function and Its Regulation.

Advances in the knowledge of the neuroendocrine system are closely related to the development of cellular imaging and labeling techniques. This synergy ranges from the staining techniques that allowed the first characterizations of the anterior pituitary gland, its relationship with the hypothalamus, and the birth of neuroendocrinology; through the development of fluorescence microscopy applications, specific labeling strategies, transgenic systems, and intracellular calcium sensors that enabled the study of processes and dynamics at the cellular and tissue level; until the advent of super-resolution microscopy, miniscopes, optogenetics, fiber photometry, and other imaging methods that allowed high spatiotemporal resolution and long-term three-dimensional cellular activity recordings in living systems in a conscious and freely moving condition. In this review, we briefly summarize the main contributions of cellular imaging techniques that have allowed relevant advances in the field of neuroendocrinology and paradigm shifts that have improved our understanding of the function of the hypothalamic-pituitary axes. The development of these methods and equipment is the result of the integration of knowledge achieved by the integration of several disciplines and effort to solve scientific questions and problems of high impact on health and society that this system entails.

Quantitative melanoma diagnosis using spectral phasor analysis of hyperspectral imaging from label-free slices.

Introduction: Melanoma diagnosis traditionally relies on microscopic examination of hematoxylin and eosin (H&E) slides by dermatopathologists to search for specific architectural and cytological features. Unfortunately, no single molecular marker exists to reliably differentiate melanoma from benign lesions such as nevi. This study explored the potential of autofluorescent molecules within tissues to provide molecular fingerprints indicative of degenerated melanocytes in melanoma. Methods: Using hyperspectral imaging (HSI) and spectral phasor analysis, we investigated autofluorescence patterns in melanoma compared to intradermal nevi. Using UV excitation and a commercial spectral confocal microscope, we acquired label-free HSI data from the whole-slice samples. Results: Our findings revealed distinct spectral phasor distributions between melanoma and intradermal nevi, with melanoma displaying a broader phasor phase distribution, signifying a more heterogeneous autofluorescence pattern. Notably, longer wavelengths associated with larger phases correlated with regions identified as melanoma by expert dermatopathologists using H&E staining. Quantitative analysis of phase and modulation histograms within the phasor clusters of five melanomas (with Breslow thicknesses ranging from 0.5 mm to 6 mm) and five intradermal nevi consistently highlighted differences between the two groups. We further demonstrated the potential for the discrimination of several melanocytic lesions using center-of-mass comparisons of phase and modulation variables. Remarkably, modulation versus phase center of mass comparisons revealed strong statistical significance among the groups. Additionally, we identified the molecular endogenous markers responsible for tissue autofluorescence, including collagen, elastin, NADH, FAD, and melanin. In melanoma, autofluorescence is characterized by a higher phase contribution, indicating an increase in FAD and melanin in melanocyte nests. In contrast, NADH, elastin, and collagen dominate the autofluorescence of the nevus. Discussion: This work underscores the potential of autofluorescence and HSI-phasor analysis as valuable tools for quantifying tissue molecular fingerprints, thereby supporting more effective and quantitative melanoma diagnosis.

Study of resveratrol against bone loss by using in-silico and in-vitro methods / Estudo do resveratrol contra a perda óssea usando métodos in silico e in vitro

By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa-Β [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de −6,9, −7,6, −7,1, −6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 μg / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 μg / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

Study of resveratrol against bone loss by using in-silico and in-vitro methods

Abstract By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa- ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of 6.9, 7.6, 7.1, 6.9, 6.7, and 7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 g / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 g / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Resumo Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa- [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de 6,9, 7,6, 7,1, 6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow-derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 g / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 g / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

Study of resveratrol against bone loss by using in-silico and in-vitro methods / Estudo do resveratrol contra a perda óssea usando métodos in silico e in vitro

Abstract By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Resumo Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis ​​pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa-Β [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de −6,9, −7,6, −7,1, −6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow-derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 μg / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 μg / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

Study of resveratrol against bone loss by using in-silico and in-vitro methods / Estudo do resveratrol contra a perda óssea usando métodos in silico e in vitro

By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.(AU)

Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa-Β [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de −6,9, −7,6, −7,1, −6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 μg / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 μg / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.(AU)

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu.

Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200 nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4 nm-10 nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

Cellulose in Secondary Xylem of Cactaceae: Crystalline Composition and Anatomical Distribution.

Cellulose is the main polymer that gives strength to the cell wall and is located in the primary and secondary cell walls of plants. In Cactaceae, there are no studies on the composition of cellulose. The objective of this work was to analyze the crystallinity composition and anatomical distribution of cellulose in Cactaceae vascular tissue. Twenty-five species of Cactaceae were collected, dried, and milled. Cellulose was purified and analyzed with Fourier transform infrared spectroscopy, the crystallinity indexes were calculated, and statistical analyzes were performed. Stem sections were fixed, cut, and stained with safranin O/fast green, for observation with epifluorescence microscopy. The crystalline cellulose ratios had statistical differences between Echinocereus pectinatus and Coryphantha pallida. All cacti species presented a higher proportion of crystalline cellulose. The fluorescence emission of the cellulose was red in color and distributed in the primary wall of non-fibrous species; while in the fibrous species, the distribution was in the pits. The high percentages of crystalline cellulose may be related to its distribution in the non-lignified parenchyma and primary walls of tracheary elements with helical or annular thickenings of non-fibrous species, possibly offering structural rigidity and forming part of the defense system against pathogens.

A Smartphone-Based Low-Cost Inverted Laser Fluorescence Microscope for Disease Diagnosis.

Fluorescence microscopy is an important tool for disease diagnosis, often requiring costly optical components, such as fluorescence filter cubes and high-power light sources. Due to its high cost, conventional fluorescence microscopy cannot be fully exploited in low-income settings. Smartphone-based fluorescence microscopy becomes an interesting low-cost alternative, but raises challenges in the optical system. We present the development of a low-cost inverted laser fluorescence microscope that uses a smartphone to visualize the fluorescence image of biological samples. Our fluorescence microscope uses a laser-based simplified optical filter system that provides analog optical filtering capabilities of a fluorescence filter cube. Firstly, we validated our inverted optical filtering by visualizing microbeads labeled with three different fluorescent compounds or fluorophores commonly used for disease diagnosis. Secondly, we validated the disease diagnosis capabilities by comparing the results of our device with those of a commercial fluorescence microscope. We successfully detected and visualized Trypanosoma cruzi parasites, responsible for the Chagas infectious disease and the presence of Antineutrophil cytoplasmic antibodies of the ANCA non-communicable autoimmune disease. The samples were labeled with the fluorescein isothiocyanate (FITC) fluorophore, one of the most commonly used fluorophores for disease diagnosis. Our device provides a 400× magnification and is at least one order of magnitude cheaper than conventional commercial fluorescence microscopes.

Assessment of Intracellular Amyloid Formation in Fixed and Live Bacteria Using Fluorescence Microscopy.

Although amyloid aggregation has been generally associated with protein misfolding and neurodegenerative diseases in mammals, bacteria and other organisms have harnessed amyloidogenesis to perform diverse biological processes. These functional amyloids, some of them secreted and others intracellular, require that the producing cells keep aggregation under control in the cytoplasm upon protein translation, preventing their inherent toxicity. Thus, it is highly relevant to understand how intracellular amyloid formation occurs and is regulated, its metabolic consequences, and the formation dynamics and fate of the amyloid inclusions upon cell division. This chapter describes methods leveraging fluorescence microscopy and fixed- or live-cell imaging to monitor intracellular amyloid formation in bacterial cells.

Fluorescence fluctuation-based super-resolution microscopy: Basic concepts for an easy start.

Due to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of approximately half of the wavelength of visible light, that is, within the range of 200 to 350 nm. Fluorescence fluctuation-based super-resolution microscopy (FF-SRM) is a term used to encompass a collection of image analysis techniques that rely on the statistical processing of temporal variations of the fluorescence signal. FF-SRM aims to reduce the uncertainty of the location of fluorophores within an image, often improving spatial resolution by several tens of nanometers. FF-SRM is suitable for live-cell imaging due to its compatibility with most fluorescent probes and relatively simple instrumental and experimental requirements, which are mostly camera-based epifluorescence instruments. Each FF-SRM approach has strengths and weaknesses, which depend directly on the underlying statistical principles through which enhanced spatial resolution is achieved. In this review, the basic concepts and principles behind a range of FF-SRM methods published to date are described. Their operational parameters are explained and guidance for their selection is provided.

Due to light's wave nature, an optical microscope's resolution range is 200 to 350 nanometers. Several techniques enhance resolution; this work encompasses several fluorescence fluctuation super-resolution (FF-SMR) methods capable of achieving nanoscopic scales. FF-SRM is known to be suitable for fixed or live-cell imaging and compatible with most conventional microscope setups found in a laboratory. However, each FF-SRM approach has its strengths and weaknesses, which depend directly on the underlying principles through which enhanced spatial resolution is achieved. Therefore, the basic concepts and principles behind diverse FF-SRM methods are revisited in this review. In addition, their operational parameters are explained, and guidance for their selection is provided for microscopists interested in FF-SRM.