Differential Extraction and Preliminary Identification of Polyphenols from Ugni candollei (White Murta) Berries.

Ugni candollei, commonly known as white murta, is a native Chilean berry with a polyphenol composition that has been underexplored. This study aimed to establish a comprehensive profile of white murta polyphenols using ultra-performance liquid chromatography electrospray ionization Orbitrap mass spectrometry (UPLC-ESI-ORBITRAP MS). Additionally, it compared the efficacy of conventional extraction methods with emerging techniques such as deep eutectic solvent (DES) extraction and hot pressurized water extraction (HPWE). The analysis tentatively identified 107 phenolic compounds (84 of them reported for the first time for this cultivar), including 25 phenolic acids, 37 anthocyanins, and 45 flavonoids. Among the prominent and previously unreported polyphenols are ellagic acid acetyl-xyloside, 3-p-coumaroylquinic acid, cyanidin 3-O-(6'-caffeoyl-glucoside, and phloretin 2'-O-xylosyl-glucoside. The study found HPWE to be a promising alternative to traditional extraction of hydroxybenzoic acids, while DES extraction was less effective across all categories. The findings reveal that white murta possesses diverse phenolic compounds, potentially linked to various biological activities.

Novel protein stabilization in white wine: A study on thermally treated zirconia-alumina composites.

A major concern for wineries is haze formation in white wines due to protein instability. Despite its prevalent use, the conventional bentonite method has shortcomings, including potential alteration of color and aroma, slow processing times, and notable wine wastage. Zirconium oxide (ZrO2) effectively removes proteins without affecting wine characteristics. However, producing cost-effective ZrO2 materials with efficient protein removal capabilities poses a significant challenge. This research aims to assess the viability of designing a porous material impregnated with zirconia to remove turbidity-causing proteins effectively. For this purpose, the support material alone (Al2O3) and the zirconia-impregnated support (ZrO2/Al2O3) were subjected to different calcination temperatures. It was observed that high-temperature treatments (750 °C) enhanced wine stability and protein adsorption capacity. The optimal adsorbent achieved a notable reduction in turbidity, decreasing the ΔNTU from 42 to 18, alongside a significant 44 % reduction in the total protein content, particularly affecting proteins in the molecular weight range of 10 to 70 kDa. This result is attributed to modifying the textural properties of ZrO2/Al2O3, characterized by the reduction of acidic sites, augmented pore diameters from 4.81 to 7.74 nm, and the emergence of zirconia clusters across the surface of the porous support. In summary, this study presents the first application of zirconia on the alumina support surface for protein stabilization in white wine. Combining ZrO2/Al2O3 and a high-temperature treatment emerges as a promising, cost-efficient, and environmentally sustainable strategy for protein removal in white wine.

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.

Specific antibody production using recombinant proteins to elucidate seed transmission and nuclear localization of Coguvirus citrulli and Coguvirus henanense in radicles of watermelon crop.

Watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2, both belonging to the genus Coguvirus (family Phenuiviridae), have been identified in watermelon plants in Brazil. To study tissue tropism and the potential for seed transmission of these viruses, we initially planned to produce specific antibodies. However, difficulties in isolating and propagating the virus in host plants hindered the purified virus preparations. To overcome this problem, the nucleocapsid (N) proteins of WCLaV-1 and -2 were produced using the pepper ringspot virus vector. The N protein genes and the vector backbone were prepared by (RT-)PCR and ligated by Gibson assembly. The constructs were agro-infiltrated in Nicotiana benthamiana plants. The expressed N proteins were purified and used for polyclonal antibody production. The specificity of both antibodies was confirmed by antigen-coating ELISA, tissue-blot immunobinding assay and Western blot. By antigen-coating ELISA demonstrated that WCLaV-1 showed 93.1% of seed-transmission, while WCLaV-2 showed only 17.8%. The N protein of WCLaV-1 was detected in the cytoplasm of the seed tissues. It was also found in the nuclei of the radicle, as confirmed by confocal microscopy. We concluded that the antibodies exhibited both a high titer and sufficient specificity for use in ELISA-based diagnostics and for subcellular localization study.

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.

Cochayuyo (Durvillaea incurvata) Extracts: Their Impact on Starch Breakdown and Antioxidant Activity in Pasta during In Vitro Digestion.

Seaweeds, notably cochayuyo (Durvillaea incurvata), are recognized for their rich macro- and micronutrient content, along with their inhibitory effects on the α-glucosidase enzyme. The present study aims to evaluate the effectiveness of this inhibition in actual starchy food products under in vitro gastrointestinal conditions. This study utilized freeze-dried cochayuyo, extracted using hot pressurized liquid extraction with 50% ethanol at 120 °C and 1500 psi. The inhibition mechanism of α-glucosidase was determined, and the polyphenol composition of the extract was analyzed using Ultra-High-Performance Liquid Chromatography. This study further evaluated the extract's impact on starch digestibility, total phenolic content, and antioxidant capacity in pasta (noodles) as representative starchy food under gastrointestinal conditions. The results indicate that the α-glucosidase inhibition mechanism is of mixed type. Phenolic compounds, primarily tetraphloroethol, could contribute to this anti-enzymatic activity. The extract was observed to decrease starch digestibility, indicated by a lower rate constant (0.0158 vs. 0.0261 min-1) and digested starch at an infinite time (77.4 vs. 80.5 g/100 g). A significant increase (~1200 vs. ~390 µmol TROLOX/100 g) in antioxidant activity was also noted during digestion when the extract was used. Thus, this study suggests that the cochayuyo extract can reduce starch digestion and enhance antioxidant capacity under gastrointestinal conditions.

Enhanced Polyphenols Recovery from Grape Pomace: A Comparison of Pressurized and Atmospheric Extractions with Deep Eutectic Solvent Aqueous Mixtures.

Deep eutectic solvents (DES) are emerging as potent polyphenol extractors under normal atmospheric conditions. Yet, their effectiveness in hot pressurized liquid extraction (HPLE) must be studied more. We explored the ability of various water/DES and water/hydrogen bond donors (HBDs) mixtures in both atmospheric solid liquid extraction (ASLE) and HPLE (50%, 90 °C) for isolating specific polyphenol families from Carménère grape pomace. We assessed extraction yields based on total polyphenols, antioxidant capacity, and recovery of targeted polyphenols. The HBDs ethylene glycol and glycerol outperformed DES in atmospheric and pressurized extractions. Ethylene glycol exhibited a higher affinity for phenolic acids and flavonols, while flavanols preferred glycerol. Quantum chemical computations indicated that a high-water content in DES mixtures led to the formation of new hydrogen bonds, thereby reducing polyphenol-solvent interactions. HPLE was found to be superior to ASLE across all tested solvents. The elevated pressure in HPLE has caused significant improvement in the recovery of flavanols (17-89%), phenolic acids (17-1000%), and flavonols (81-258%). Scanning electron microscopy analysis of post-extraction residues suggested that high pressures collapse the plant matrix, thus easing polyphenol release.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

From Live Cells to Caenorhabditis elegans: Selective Staining and Quantification of Lipid Structures Using a Fluorescent Hybrid Benzothiadiazole Derivative.

The current article describes the synthesis, characterization, and application of a designed hybrid fluorescent BTD-coumarin (2,1,3-benzothiadiazole-coumarin) derivative (named BTD-Lip). The use of BTD-Lip for live-cells staining showed excellent results, and lipid droplets (LDs) could be selectively stained. When compared with the commercially available dye (BODIPY) for LD staining, it was noted that the designed hybrid fluorescence was capable of staining a considerable larger number of LDs in both live and fixed cells (ca. 40% more). The new dye was also tested on live Caenorhabditis elegans (complex model) and showed an impressive selectivity inside the worm, whereas the commercial dye showed no selectivity in the complex model.