Revisiting Biginelli-like reactions: solvent effects, mechanisms, biological applications and correction of several literature reports.

This study critically reevaluates reported Biginelli-like reactions using a Kamlet-Abboud-Taft-based solvent effect model. Surprisingly, structural misassignments were discovered in certain multicomponent reactions, leading to the identification of pseudo three-component derivatives instead of the expected MCR adducts. Attempts to replicate literature conditions failed, prompting reconsideration of the described MCRs and proposed mechanisms. Electrospray ionization (tandem) mass spectrometry, NMR, melting points, elemental analyses and single-crystal X-ray analysis exposed inaccuracies in reported MCRs and allowed for the proposition of a complete catalytic cycle. Biological investigations using both pure and "contaminated" derivatives revealed distinctive features in assessed bioassays. A new cellular action mechanism was unveiled for a one obtained pseudo three-component adduct, suggesting similarity with the known dihydropyrimidinone Monastrol as Eg5 inhibitors, disrupting mitosis by forming monoastral mitotic spindles. Docking studies and RMSD analyses supported this hypothesis. The findings described herein underscore the necessity for a critical reexamination and potential corrections of structural assignments in several reports. This work emphasizes the significance of rigorous characterization and critical evaluation in synthetic chemistry, urging a careful reassessment of reported synthesis and biological activities associated with these compounds.

Sensitive hydrazine detection and quantification with a fluorescent benzothiadiazole sensor: selective lipid droplets and in vivo imaging.

In this work, we describe the design, synthesis, characterization, photophysical evaluation, DFT calculations, and application of two novel fluorescent benzothiadiazole (BTD) sensors for hydrazine detection and quantification at the cellular and multicellular (in vivo) levels. The two probes were fully characterized, and their photophysical properties were evaluated. We tested the designed fluorogenic dye (named BTD-CHO) as a selective sensor for the rapid, sensitive, and selective detection of hydrazine. When treated with N2H4, the probe affords a new derivative named BTD-HZN, releasing water as the only byproduct. BTD-CHO exhibited a preference for lipid droplets (LDs) and accumulated inside these organelles. Hydrazine detection in LDs could be carried out by the in situ formation of BTD-HZN inside live cells. We efficiently visualized the lipids of a challenging cellular model, microalgae (Chlorella sorokiniana), using these sensors. In vivo experiments indicated rapid and efficient detection of the analyte using C. elegans and zebrafish (Danio rerio) as the multicellular models.

Morphological and ultrastructural study of the palpebral conjunctiva of healthy domestic cats.

OBJECTIVE: To describe the morphology of the meibomian glands and goblet cells in the palpebral conjunctiva of healthy cats. ANIMALS STUDIED: Five healthy domestic cats without ocular changes that had died from causes unrelated to the study were evaluated. PROCEDURES: Forty samples were collected from upper and lower palpebral conjunctiva and 20 from palpebral fornix region in the nasal corner. The samples were processed for scanning electron microscopy (SEM), transmission electron microscopy (TEM), and histopathology. RESULTS: In the SEM analysis of the palpebral fornix, numerous points of mucous extrusion between the cell junctions were visualized, along with the presence of microvilli in the apical portions with small secretory vesicles. A homogeneous surface was highlighted, formed by the arrangement of cell contours in the form of hexagons. The grouping of goblet cells and their cytoplasmic vesicles filled with homogeneous content was visualized using TEM. Histopathology showed goblet cells interspersed with stratified epithelium accompanied by well-vascularized connective tissue. In the samples stained with hematoxylin and eosin, the meibomian glands, formed by acinar cells and with the presence of individual openings of the ducts in the eyelid margin, were easily visualized in the eyelid margins. CONCLUSIONS: This study describes the ultrastructural form of goblet cells and the morphology of the palpebral conjunctiva of healthy cats by the histopathology of the meibomian glands. This description can serve as a parameter of normality and aid in the detection of morphological alterations in these structures, as well as a parameter for comparison with other animal species.

Fermentation of Murta (Ugni molinae) Juice: Effect on Antioxidant Activity and Control of Enzymes Associated with Glucose Assimilation.

Berries are rich in bioactive compounds, including antioxidants and especially polyphenols, known inhibitors of starch metabolism enzymes. Lactic acid fermentation of fruits has received considerable attention due to its ability to enhance bioactivity. This study investigated the effect of fermentation with L. mesenteroides of juice from the Chilean berry murta on antioxidant activity, release of polyphenols, and inhibitory activity against α-amylase and α-glucosidase enzymes. Three types of juices (natural fruit, freeze-dried, and commercial) were fermented. Total polyphenol content (Folin-Ciocalteu), antioxidant activity (DPPH and ORAC), and the ability to inhibit α-amylase and α-glucosidase enzymes were determined. Fermented murta juices exhibited increased antioxidant activity, as evidenced by higher levels of polyphenols released during fermentation. Inhibition of α-glucosidase was observed in the three fermented juices, although no inhibition of α-amylase was observed; the juice from freeze-dried murta stood out. These findings highlight the potential health benefits of fermented murta juice, particularly its antioxidant properties and the ability to modulate sugar assimilation by inhibiting α-glucosidase.

Fluorescent Benzothiadiazole Derivatives as Fluorescence Imaging Dyes: A Decade of New Generation Probes.

The current review describes advances in the use of fluorescent 2,1,3-benzothiadiazole (BTD) derivatives after nearly one decade since the first description of bioimaging experiments using this class of fluorogenic dyes. The review describes the use of BTD-containing fluorophores applied as, inter alia, bioprobes for imaging cell nuclei, mitochondria, lipid droplets, sensors, markers for proteins and related events, biological processes and activities, lysosomes, plasma membranes, multicellular models, and animals. A number of physicochemical and photophysical properties commonly observed for BTD fluorogenic structures are also described.

Diverse 3-Methylthio-4-Substituted Maleimides through a Novel Rearrangement Reaction: Synthesis and Selective Cell Imaging.

A transition metal-free protocol for the preparation of fluorescent and non-fluoresent 3-methylthio-4-arylmaleimides in a single step through a new rearrangement from thiazolidine-2,4-diones is described. By employing the optimized reaction conditions, a broad scope of derivatives was prepared in ≤97% yield. The reaction tolerated several substituted aryl groups, including the challenging preparation of pyridyl-containing derivatives. A series of control experiments strongly suggested that the new rearrangement involves a key isocyanate intermediate and a further reaction with in situ-generated methylthiomethyl acetate. The photophysical properties of some of the synthesized derivatives as well as their use in live cell imaging were also investigated, revealing that some of the substituted maleimides are capable of selectively staining different regions of the cells.

Advances in White Wine Protein Stabilization Technologies.

The unstable proteins in white wine cause haze in bottles of white wine, degrading its quality. Thaumatins and chitinases are grape pathogenesis-related (PR) proteins that remain stable during vinification but can precipitate at high temperatures after bottling. The white wine protein stabilization process can prevent haze by removing these unstable proteins. Traditionally, bentonite is used to remove these proteins; however, it is labor-intensive, generates wine losses, affects wine quality, and harms the environment. More efficient protein stabilization technologies should be based on a better understanding of the main factors and mechanisms underlying protein precipitation. This review focuses on recent developments regarding the instability and removal of white wine proteins, which could be helpful to design more economical and environmentally friendly protein stabilization methods that better preserve the products´ quality.

Synthetic enzyme-catalyzed multicomponent reaction for Isoxazol-5(4H)-one Syntheses, their properties and biological application; why should one study mechanisms?

In this work, we describe the application of a synthetic enzyme (synzyme) as the catalyst to promote the multicomponent synthesis of isoxazol-5(4H)-one derivatives. The catalytic system could be used up to 15 times without any notable loss of its activity. Some derivatives showed fluorescence and their photophysical data were evaluated. The mechanism of the reaction was, for the first time, investigated and, among the three reaction pathway possibilities, only one was operating under the developed conditions. ESI-MS(/MS) allowed for both the simultaneous monitoring of the multicomponent reaction (MCR) and the proposition of a kinetic model to explain the transformation. The kinetic model pointed firmly to only one reaction pathway and helped to discard the other two possibilities. The antimicrobial abilities of all synthesized derivatives against Gram-positive and Gram-negative strains were also evaluated. The abilities of functional chromophores (fluorescent compounds) as live cell-imaging probes were verified and one of the multicomponent adducts could stain early endosomes selectively in bioimaging experiments.

Screening test for COVID-19 in dental practice: Best options.

PURPOSE: To review diverse tests that could serve as a screening tool for the dental community to identify patients with potential COVID-19. METHODS: Detailed automated searches of Medline, PubMed and EBM Review, including ACP Journal Club, the Cochrane Controlled Trials Register, and the Cochrane Database of Systematic Reviews of Effectiveness from 2019 to 2020 were conducted. Real-time polymerase chain reaction (RT-PCR) or quantitative (q) RT-PCR and Western blot assays were excluded. RESULTS: Real time loop-mediated isothermal amplification (RT-LAMP) and Real time recombinase polymerase amplification (RT-RAP) are the most promising methodologies developed as screening tools, and could be a screening test option in dental practice to identify patients potentially carrying SARS-CoV-2 and to prevent cross-infection between patients and dental personnel. CLINICAL SIGNIFICANCE: Biological risk management in the dental clinic is primarily focused on the wearing of protective equipment by dental personnel and disinfection/sterilization procedures for surfaces. With the absence of suitable antiviral drugs or vaccines, simple, rapid, and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be critical for preventing the spread of infection in dental practices.

Fluorescent Benzoselenadiazoles: Synthesis, Characterization, and Quantification of Intracellular Lipid Droplets and Multicellular Model Staining.

In this work, we described the synthesis of 10 new fluorescent 2,1,3-benzoselenadiazole small-molecule derivatives and their chemical- and photocharacterizations. The new derivatives could, for the first time, be successfully applied as selective live cell imaging probes (at nanomolar concentrations) and stained lipid-based structures preferentially. Density functional theory (DFT) calculations were used to help in understanding the photophysical data and the intramolecular charge-transfer (ICT) processes of the synthesized dyes. Some derivatives showed impressive cellular responses, allowing them to be tested as probes in a complex multicellular model (i.e., Caenorhabditis elegans). When compared with the commercially available dye, the new fluorescent compounds showed far better results both at the cellular level and inside the live worm. Inside the multicellular complex model, the tested probes also showed selectivity, a feature not observed when the commercial dye was used to carry out the bioimaging experiments.

Deciphering the Dynamics of Organic Nanoaggregates with AIEE Effect and Excited States: Lipophilic Benzothiadiazole Derivatives as Selective Cell Imaging Probes.

An aggregation-induced emission enhancement (AIEE) effect in fluorescent lipophilic 2,1,3-benzothiadiazole (BTD) derivatives and their organic nanoaggregates were studied. A set of techniques such as single-crystal X-ray, dynamic light scattering (DLS), electron paramagnetic resonance (EPR), UV-vis, fluorescence, and density functional theory (DFT) calculations have been used to decipher the formation/break (kinetics), properties, and dynamics of the organic nanoaggregates of three BTD small organic molecules. An in-depth study of the excited-state also revealed the preferential relaxation emissive pathways for the BTD derivatives and the dynamics associated with it. The results described herein, for the first time, explain the formation of fluorescent BTD nanoaggregate derivatives and allow for the understanding of their dynamics in solution as well as the ruling forces of both aggregation and break processes along with the involved equilibrium. One of the developed dyes could be used at a nanomolar concentration to selectively stain lipid droplets emitting an intense and bright fluorescence at the red channel. The other two BTDs could also stain lipid droplets at very low concentrations and were visualized preferentially at the blue channel.

Palladium Catalyst with Task-Specific Ionic Liquid Ligands: Intracellular Reactions and Mitochondrial Imaging with Benzothiadiazole Derivatives.

A water-soluble and charge-tagged palladium complex (PdMAI) was found to function inside breast cancer live cells of the MCF-7 lineage as an efficient catalyst for cross-coupling reaction. PdMAI, bearing two ionophilic task-specific ionic liquids as ligands, efficiently catalyzed both in cellulo Suzuki and Buchwald-Hartwig amination reactions. For the first time, therefore, the Buchwald-Hartwig amination is described to occur inside the highly complex cellular environment. The 2,1,3-benzothiadiazole (BTD) core was used as the base for the syntheses, and two π-extended fluorescent derivatives (BTD-2APy) and (BTD-1AN), which were found to emit in the green and red channels, had impressive mitochondrial affinity. These chromophores allowed for selective mitochondrial imaging and tracking.

Ultrastructural morphology of goblet cells of the conjunctiva of dogs.

OBJECTIVE: To describe the morphology of goblet cells of the eyelid conjunctiva in dogs using transmission and scanning electron microscopy. ANIMAL STUDIED: Ten dogs, both male and female of different breeds, with no ocular changes were examined (20 eyes). PROCEDURES: Ten samples of conjunctiva were collected and processed for scanning and transmission electron microscopy (SEM and TEM), while another 10 samples were stained with Schiff's periodic stain (SPA) and alcian blue, pH 2.5, and analyzed using light microscopy. RESULTS: Scanning electron microscopy revealed several points of mucus extrusion in the free apical portion of the goblet cells as well as a wide distribution of lymphoid follicles and macrophages intermingling with the microvilli of palpebral epithelium cells. TEM revealed normal goblet cells that were predominantly oval with wide cytoplasm of different diameters, and large vesicles with heterogeneous granules and free edges, suggesting the release of mucus content onto the conjunctival surface. Cytoplasmic organelles, such as the Golgi apparatus, endoplasmic reticulum, and a high number of mitochondria were also observed. All the samples were positive for SPA and alcian blue staining. CONCLUSION: This is the first study to evaluate the goblet cells of the eyelid conjunctiva in healthy dogs using electron microscopy techniques. These results are useful for comparing the palpebral conjunctiva of dogs without ocular changes to palpebral conjunctiva of dogs and other species with ocular changes.

Plasma membrane imaging with a fluorescent benzothiadiazole derivative.

This work describes a novel fluorescent 2,1,3-benzothiadiazole derivative designed to act as a water-soluble and selective bioprobe for plasma membrane imaging. The new compound was efficiently synthesized in a two-step procedure with good yields. The photophysical properties were evaluated and the dye proved to have an excellent photostability in several solvents. DFT calculations were found in agreement with the experimental data and helped to understand the stabilizing intramolecular charge-transfer process from the first excited state. The new fluorescent derivative could be applied as selective bioprobe in several cell lines and displayed plasma-membrane affinity during the imaging experiments for all tested models.

Heteropolyacid-Containing Ionic Liquid-Catalyzed Multicomponent Synthesis of Bridgehead Nitrogen Heterocycles: Mechanisms and Mitochondrial Staining.

The current manuscript describes the use of a heteropolyacid-containing task-specific ionic liquid, supported in imidazolium-based ionic liquids, as the catalyst for an efficient multicomponent synthesis of hexahydroimidazo[1,2-α]pyridine derivatives. The reactions conditions were fully optimized, and the bridgehead nitrogen heterocycle derivatives could be obtained in just 1 h exclusively as a single isomer ( trans). Single crystal X-ray analysis confirmed the trans derivative as the only isomer. The mechanism of the reaction was investigated by ESI(+)-MS(/MS), and the use of the elegant charge-tag strategy allowed a catalytic cycle to be proposed for the current transformation and revealed the possibility of at least two reaction pathways. One derivative bearing a coumarin scaffold was synthesized, and its fluorescent properties allowed it to be tested as a probe for live-cell imaging experiments with a preference for mitochondria.

Benzothiadiazole Derivatives as Fluorescence Imaging Probes: Beyond Classical Scaffolds.

This Account describes the origins, features, importance, and trends of the use of fluorescent small-molecule 2,1,3-benzothiadiazole (BTD) derivatives as a new class of bioprobes applied to bioimaging analyses of several (live and fixed) cell types. BTDs have been successfully used as probes for a plethora of biological analyses for only a few years, and the impressive responses obtained by using this important class of heterocycle are fostering the development of new fluorescent BTDs and expanding the biological applications of such derivatives. The first use of a fluorescent small-molecule BTD derivative as a selective cellular probe dates back to 2010, and since then impressive advances have been described by us and others. The well-known limitations of classical scaffolds urged the development of new classes of bioprobes. Although great developments have been achieved by using classical scaffolds such as coumarins, BODIPYs, fluoresceins, rhodamines, cyanines, and phenoxazines, there is still much to be done, and BTDs aim to succeed where these dyes have shown their limitations. Important organelles and cell components such as nuclear DNA, mitochondria, lipid droplets, and others have already been successfully labeled by fluorescent small-molecule BTD derivatives. New technological systems that use BTDs as the fluorophores for bioimaging experiments have been described in recent scientific literature. The successful application of BTDs as selective bioprobes has led some groups to explore their potential for use in studying membrane pores or tumor cells under hypoxic conditions. Finally, BTDs have also been used as fluorescent tags to investigate the action mechanism of some antitumor compounds. The attractive photophysical data typically observed for π-extended BTD derivatives is fostering interest in the use of this new class of bioprobes. Large Stokes shifts, large molar extinction coefficients, high quantum yields, high stability when stored in solution or as pure solids, no fading even after long periods of irradiation, bright emissions with no blinking, good signal-to-noise ratios, efficiency to transpose the cell membrane, and irradiation preferentially in the visible-light region are just some features noted by using BTDs. As the pioneering group in the use of fluorescent small-molecule BTDs for bioimaging purposes, we feel pleased to share our experience, results, advances, and personal perspectives with the readers of this Account. The readers will clearly note the huge advantages of using fluorescent BTDs over classical scaffolds, and hopefully they will be inspired and motivated to further BTD technology in the fields of molecular and cellular biology.

Fluorescent Peptoids as Selective Live Cell Imaging Probes.

This paper describes the synthesis of fluorescent peptoids using the Ugi multicomponent reaction (4CR). The four synthesized structures had their photophysical properties evaluated and their potential as biomarkers established. The peptidomimetics were used at very low concentrations (10 nM) to follow their internalization in breast cancer cells and had their localization precisely determined. One of the new peptoids displayed mitochondrial affinity and stained this important organelle selectively. Co-staining experiments using MitoTracker Red confirmed the localization inside live cells.

Synthesis, Structure, Properties, and Bioimaging of a Fluorescent Nitrogen-Linked Bisbenzothiadiazole.

This paper describes the synthesis, structure, photophysical properties, and bioimaging application of a novel 2,1,3-benzothiadiazole (BTD)-based rationally designed fluorophore. The capability of undergoing efficient stabilizing processes from the excited state allowed the novel BTD derivative to be used as a stable probe for bioimaging applications. No notable photobleaching effect or degradation could be observed during the experimental time period. Before the synthesis, the molecular architecture of the novel BTD derivative was evaluated by means of DFT calculations to validate the chosen design. Single-crystal X-ray analysis revealed the nearly flat characteristics of the structure in a syn conformation. The fluorophore was successfully tested as a live-cell-imaging probe and efficiently stained MCF-7 breast cancer cell lineages.

Carbon dots (C-dots) from cow manure with impressive subcellular selectivity tuned by simple chemical modification.

Improved cellular selectivity for nucleoli staining was achieved by simple chemical modification of carbon dots (C-dots) synthesized from waste carbon sources such as cow manure (or from glucose). The C-dots were characterized and functionalized (amine-passivated) with ethylenediamine, affording amide bonds that resulted in bright green fluorescence. The new modified C-dots were successfully applied as selective live-cell fluorescence imaging probes with impressive subcellular selectivity and the ability to selectively stain nucleoli in breast cancer cell lineages (MCF-7). The C-dots were also tested in four other cellular models and showed the same cellular selection in live-cell imaging experiments.

Impact of kinesin Eg5 inhibition by 3,4-dihydropyrimidin-2(1H)-one derivatives on various breast cancer cell features.

BACKGROUND: Breast cancer is a complex heterogeneous disease and is one of the leading causes of death among women. In addressing the need for treatments of this life-threatening illness, we studied 3,4-dihydropyrimidin-2(1H)-one (or thione) derivatives (DHPMs), a class of inhibitor molecules of the Eg5 motor spindle protein that shows pronounced antitumor activity against several cancer cell lines. METHODS: An in vitro screening was performed for identification of DHPMs with potent antitumor effects on MCF-7 and MDA-MB-231 cells and the selected DHPMs were evaluated for their inhibitory activity on Eg5 both in silico, using Molecular dynamics, and in vitro Eg5 inhibition assays. Analysis of cell death induction, proliferation, cell cycle and cancer stem cells (CSC) profile were performed by flow cytometry to assess the influence of the selected DPHMs on these important tumor features. Finally, the effects of DHPM treatment on tube formation were evaluated in vitro using HUVEC cells, and in vivo using a model on chorioallantoic membrane (CAM) of fertilized eggs. RESULTS: We identified five DHPMs with pronounced inhibitory activity on Eg5 motor protein interfering with the proper mitotic spindle assembly during cell division. These compounds impair the correct conclusion of cell cycle of the breast cancer cells and showed to be selective for tumor cells. Moreover, DHPMs modulate the CD44(+)/CD24(-) phenotype leading to a decrease in the CSC population in MDA-MB-231 cells, an important effect since CSC are resistant to many conventional cancer therapies and play a pivotal role in tumor initiation and maintenance. This observation was confirmed by the results which demonstrated that DHPM treated cells had impaired proliferation and were unable to sustain angiogenesis events. Finally, the DHMP treated cells were induced to apoptosis, which is one of the most pursued goals in drug development. CONCLUSIONS: The results of our study strongly suggest that DHPMs inhibit important tumorigenic features of breast cancer cells leading them to death by apoptosis. These findings firmly point to DHPM molecular architecture as a promising alternative against breast cancer.