A Strainer-Based Platform for the Collection and Immunolabeling of Mouse Intestinal Organoids.

Intestinal organoids have emerged as powerful model systems for studying the complex structure and function of the intestine. However, there is a lack of widely applicable methods for the collection, labeling, and imaging of intestinal organoids. In this study, we developed a novel method for loading and labeling intestinal organoids, a method that efficiently collects the organoids and facilitates imaging of their three-dimensional (3D) structure. Based on this strainer platform, mouse intestinal organoids were adequately collected and immobilized, facilitating the immunolabeling workflow to target proteins of the organoids. After evaluation, the strainer size of 40 µm was considered to be more conducive to the collection and labeling of mouse intestinal organoids. More extensive research on organoids of multiple types and species origins will contribute to broadening the applicability of the methodology. Overall, our study proposes an innovative workflow for loading and analyzing intestinal organoids. The combination of a strainer-based collection method, fluorescent labeling, and 3D reconstruction provides valuable insights into the organization and complexity of these tissue models, thereby offering new avenues for investigating intestinal development, disease modeling, and drug discovery.

A Strainer-Based Platform for the Collection and Immunolabeling of Porcine Epidemic Diarrhea Virus-Infected Porcine Intestinal Organoid.

The development of organoid research has raised new requirements for this methodology. In a previous study, we demonstrated that an emerging protocol achieved the collection, loading, and programmed immunolabeling of mouse intestinal organoids based on a strainer platform. To uncover the applied potential of this novel methodology on organoids from other species, the strainer platform was utilized to characterize the porcine epidemic diarrhea virus (PEDV)-infected porcine intestinal organoid model. Based on a previous study, some steps were changed to improve the efficiency of the assay by simplifying the reagent addition procedure. In addition, we redefined the range of strainer sizes on porcine intestinal organoids, showing that strainers with pore sizes of 40 and 70 µm matched the above protocol well. Notably, the strainer platform was successfully used to label viral proteins, laying the foundation for its application in the visualization of viral infection models. In summary, the potential of the strainer platform for organoid technology was explored further. More extensive exploration of this platform will contribute to the development of organoid technology.

Effects and mechanisms of using "clean" smoke particles in the smoking process on the formation of ß-carboline heterocyclic amines (ß-CHAs) in smoked meat patties.

ß-Carboline heterocyclic amines (ß-CHAs), known for their synergistic neurotoxic and carcinogenic effects, are predominantly produced by humans through cigarette smoke and food and are found particularly in meats cooked at high temperatures. Few studies have explored the differences in the mechanisms of accumulation of ß-CHAs in smoked meat and meat processed at high temperatures. In this research, the concentration of ß-CHAs in smoked meats prepared using a variety of wood materials was measured using LCMS/MS. Additionally, key volatile organic compound markers associated with ß-CHAs accumulation in smoke were identified through GCMS and multivariate statistical analysis and subsequently confirmed in a chemical simulation system. Three types of strainers, each with a distinct aperture size, were used to assess the efficacy of particle filtration in reducing ß-CHAs levels in smoked meat. The findings indicated that smoke exposure indeed increases the ß-CHAs content of meat. However, only the strainer capable of filtering PM2.5-sized particles reduced the amount of ß-CHAs present compared to the control group. In contrast, strainers with larger pore sizes facilitated excessive accumulation of ß-CHAs. The presence of aldehydes such as 1 H-pyrrole-2-carboxaldehyde, 5-methylfurfural, benzaldehyde, furfural, and nonanal exhibited a positive correlation with the accumulation of ß-CHAs. Conversely, phenolic compounds, including 2-methoxy-4-vinylphenol, 2-methoxy-5-methylphenol, p-cresol, phenol, 2-methoxy-4-(1-propenyl)-, (Z)-, phenol, 3-ethyl-, and phenol, 4-ethyl-2-methoxy-, showed a negative correlation. Thus, filters made from chelated carbonyl trap materials both chemically and physically disrupt the buildup of ß-CHAs in smoked meats. The use of this approach will not only improve the quality of these products but will also contribute to decreasing the amount of inhalation pollutants released into the environment.

An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy.

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.

Fine details of the choanocyte filter apparatus in asconoid calcareous sponges (Porifera: Calcarea) revealed by ruthenium red fixation.

Sponges (phylum Porifera) are highly specialized filter-feeding metazoans, pumping and filtering water with a network of canals and chambers, the aquiferous system. Most sponges have a leuconoid aquiferous system, characterized by choanocytes organized in small spherical chambers connected with ambient water by a complex net of canals. Such organization requires substantial pressure difference to drive water through an elaborate system of canals, so the choanocytes in leuconoid sponges have several structural features to generate pressure difference. In contrast, it is generally accepted that asconoid and syconoid sponges with long choanocyte tubes or large choanocyte chambers have no similar structures in their choanocytes. The present study is devoted to the detailed ultrastructural analysis of the choanocytes and their filter apparatus in the asconoid calcareous sponge Leucosolenia variabilis. The general structure of L. variabilis choanocytes is similar to that described for other sponge species. However, the fixation with 0.1% ruthenium red allowed us to reveal for the first time a complex of glycocalyx structures (vanes on the flagella, a fine glycocalyx sealing microvilli in the collar, and a glycocalyx strainer, embedding the apical parts of neighboring collars) in the choanocytes of L. variabilis, which are traditionally associated with the pumping and filtration process in leuconoid demosponges. All revealed glycocalyx structures have dimensions and locations similar to those found in the choanocyte chambers of some demosponges. The data suggest that L. variabilis utilizes the principles of water pumping and filtration similar to those in demosponges and revealed glycocalyx structures are potentially crucial for these processes. It seems that sponges from distant phylogenetic lineages and with different body plans rely on common principles of choanoderm organization for effective pumping and filtration of water. However, while some adaptation for effective pumping and filtration of water have possibly arisen before the diversification of Porifera, others have appeared independently in different lineages.

Strainer-Separable TiO2 on Halloysite Nanocomposite-Embedded Alginate Capsules with Enhanced Photocatalytic Activity for Degradation of Organic Dyes.

Photocatalysis driven by natural sunlight is an attractive approach to removing pollutants from wastewater. Although TiO2-based photocatalysts using various support nano-materials with high catalytic activity and reusability have been developed for purifying wastewater, the centrifugal separation methods used for the nanocatalysts limit their use for treating large amounts of water. Here, we prepared a TiO2 nano-catalyst supported on a halloysite nanotube (HNT)-encapsulated alginate capsule (TiO2@HNT/Alcap) to recapture the catalysts rapidly without centrifugation. The structure of TiO2@HNT/Alcap was characterized by X-ray diffraction, SEM, and TGA. In our system, the combination of HNTs and alginate capsules (Alcaps) improved the efficiency of adsorption of organic pollutants to TiO2, and their milli = meter scale structure allowed ultra-fast filtering using a strainer. The TiO2@HNT/Alcaps showed ~1.7 times higher adsorption of rhodamine B compared to empty alginate capsules and also showed ~10 and ~6 times higher degradation rate compared to the HNT/Alcaps and TiO2/Alcaps, respectively.

Application of the BMWP-Costa Rica biotic index in aquatic biomonitoring: sensitivity to collection method and sampling intensity

The use of aquatic macroinvertebrates as bio-indicators in water quality studies has increased considerably over the last decade in Costa Rica, and standard biomonitoring methods have now been formulated at the national level. Nevertheless, questions remain about the effectiveness of different methods of sampling freshwater benthic assemblages, and how sampling intensity may influence biomonitoring results. In this study, we compared the results of qualitative sampling using commonly applied methods with a more intensive quantitative approach at 12 sites in small, lowland streams on the southern Caribbean slope of Costa Rica. Qualitative samples were collected following the official protocol using a strainer during a set time period and macroinvertebrates were field-picked. Quantitative sampling involved collecting ten replicate Surber samples and picking out macroinvertebrates in the laboratory with a stereomicroscope. The strainer sampling method consistently yielded fewer individuals and families than quantitative samples. As a result, site scores calculated using the Biological Monitoring Working Party-Costa Rica (BMWP-CR) biotic index often differed greatly depending on the sampling method. Site water quality classifications using the BMWP-CR index differed between the two sampling methods for 11 of the 12 sites in 2005, and for 9 of the 12 sites in 2006. Sampling intensity clearly had a strong influence on BMWP-CR index scores, as well as perceived differences between reference and impacted sites. Achieving reliable and consistent biomonitoring results for lowland Costa Rican streams may demand intensive sampling and requires careful consideration of sampling methods. Rev. Biol. Trop. 62 (Suppl. 2): 275-289. Epub 2014 April 01.

En Costa Rica el uso de macroinvertebrados acuáticos como bioindicadores de la calidad ambiental del agua ha aumentado considerablemente en la última década, y se han aplicado métodos estandarizados a nivel nacional. Sin embargo, aún existe controversia sobre la efectividad de los métodos de muestreo y su intensidad. En este estudio comparamos una metodología cualitativa y otra cuantitativa, en 12 sitios de nueve ríos localizados en la cuenca baja del río Sixaola, al sur de la vertiente del Caribe de Costa Rica. Muestras cualitativas fueron recolectadas con un colador durante un período de tiempo establecido y los macroinvertebrados fueron separados en el campo. En el muestreo cuantitativo se recolectaron diez muestras utilizando un Surber y los macroinvertebrados fueron separados en el laboratorio utilizando con esteroscopio. El colador produjo un número menor de individuos y familias que las muestras cuantitativas. Como resultado, la puntuación del índice biótico Biological Monitoring Working Party-Costa Rica (BMWP-CR) en cada sitio dependió del método. La categoría de calidad de agua con ese índice también diferió entre los dos métodos en 11 de los 12 sitios en 2005, y en 9 de los 12 sitios en 2006. La intensidad de muestreo claramente tuvo una fuerte influencia en los resultados del índice BMWP-CR, así como diferencias entre sitios de referencia y los sitios afectados. Debido a la coherencia en los resultados es posible aceptar el método cuantitativo pero con una cantidad prudente de repeticiones en estudios de biomonitoreo acuático.