Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies

The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×10 3 cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×10 3 cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×10 3 and 11×10 3 and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages

Comparison of Different Procedures for Separating Microplastics from Sediments

The separation of microplastics from environmental matrices is still challenging, especially for sediments where microplastics can accumulate affecting benthic organisms. Many authors have adopted different procedures, but their effectiveness has been rarely compared. The present study aims to compare the recovery rate of three different methodologies for the separation of dense microplastics from fine sediments and provide insights about contamination processes occurring in microplastic separation techniques. The protocols tested are a density separation method with NaCl and NaI, a density separation with NaI followed by a centrifugation step, and a digestion method with 10%KOH (m/v). The recovery yields of two high-density polymers of three different dimensional classes were tested. The highest recovery rate was reported for the first protocol. However, this method proved to be expensive, and unsatisfactory results were found when using merely NaCl. The digestion method was the one that was proven to be simple, reproducible, and affordable. The contamination tests highlighted as multiple filtration steps can increase the number of fibers deriving from airborne contamination. Since a unified approach for microplastic separation from sediments is still not selected, this study is of paramount importance as it provides data about the reliability of different methods widely adopted.

Enrichment of leukocytes in peripheral blood using 3D printed tubes

Leukocytes have an essential role in patient clinical trajectories and progression. Traditional methods of leukocyte enrichment have many significant limitations for current applications. It is demonstrated a novel 3D printing leukocyte sorting accumulator that combines with centrifugation to ensure label-free initial leukocyte enrichment based on cell density and size. The internal structure of leukocyte sorting accumulator (revealed here in a new design, leukocyte sorting accumulator-3, upgraded from earlier models), optimizes localization of the buffy coat fraction and the length of the period allocated for a second centrifugation step to deliver a higher recovery of buffy coats than earlier models. Established methodological parameters were evaluated for reliability by calculating leukocyte recovery rates and erythrocyte depletion rates by both pushing and pulling methods of cell displacement. Results indicate that leukocyte sorting accumulator-3 achieves a mean leukocytes recovery fraction of 96.2 ± 2.38% by the pushing method of layer displacement. By the pulling method, the leukocyte sorting accumulator-3 yield a mean leukocytes recovery fraction of 94.4 ± 0.8%. New procedures for preliminary enrichment of leukocytes from peripheral blood that avoid cellular damage, as well as avert metabolic and phase cycle intervention, are required as the first step in many modern clinical and basic research assays.

Assessing Brazilian food establishments' hygienic handling of leafy vegetables and their microbiological quality

In this study, the hygienic handling of leafy vegetables was evaluated in ten selected food establishments located in south-eastern Brazil. A 14-question survey was applied in these establishments, addressing the origin of the vegetables, washing procedures, and temperature conditions during storage and exposure. In addition, 30 samples of leafy vegetables were collected and submitted to the enumeration of total coliforms, coliforms at 45 °C and tested for Salmonella. Based on the responses to the survey, the establishments could be clustered into three groups. Group 1 included four establishments, where the vegetables originated from farms exclusively and were displayed to consumers at room temperature, and where most of the food handlers had participated in courses on good handling practices. Group 2 included two establishments that performed a centrifugation step and where vegetables were not available to consumers in a self-service. Finally, group 3 included four establishments, where vegetables originated from suppliers exclusively, were stored under refrigeration, and displayed to consumers on refrigerated tables. The mean count of total coliforms was 2.5 ± 0.7 log MPN/g, while coliforms at 45 °C were detected in only one sample (0.9 log MPN/g). Salmonella was not found in any sample. Microbiological results obtained for leafy vegetables from establishments clustered in the three groups did not differ significantly. These data contribute to a better understanding of the hygienic handling of leafy vegetables in the surveyed Brazilian food establishments.

Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols

Microvesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma membrane. They are involved in a variety of physiological and pathological conditions and represent promising biomarkers for diseases. MV heterogeneity together with the lack of specific markers had strongly hampered the development of effective methods for MV isolation and differential centrifugation remains the most used method to purify MVs. In this study, we analysed the capacity of the differential centrifugation method to isolate MVs from cell-conditioned medium using flow cytometry and TEM/AFM microscopy. We found that the loss of MVs (general population and/or specific subpopulations) represents a major and underestimate drawback of the differential centrifugation protocol. We demonstrate that the choice of the appropriate rotor type (fixed-angle vs swinging-bucket) and the implementation of an additional washing procedure to the first low-speed centrifugation step of the protocol allow to overcome this problem increasing the total amount of isolated vesicles and avoiding the selective loss of MV subpopulations. These parameters/procedures should be routinely employed into optimized differential centrifugation protocols to ensure isolation of the high-quantity/quality MVs for the downstream analysis/applications.

A retrospective study of Uncinaria stenocephala in domestic dogs: Age, sex distribution, and risk factors

Background and Aim: Uncinaria infection often appears in domestic dogs. In the present study, parasitological examination of fecal samples from 782 dogs were analyzed for the presence of Uncinaria stenocephala. Materials and Methods: Fecal samples were analyzed by means of a standardized flotation method using a saturated salt solution containing NaNO3 (specific gravity 1.38), with a centrifugation step. Results: The highest prevalence rates were found among young adult dogs (8.3%), followed by puppies (5.4%); the lowest prevalence rates were found in dogs older than 3 years (4.3%). The prevalence was 5.8% among female dogs and 7.2% in male dogs. Coinfections with roundworms and protozoan parasites were frequently observed in U. stenocephala-positive dogs (15%). In total, three types of coinfections were registered. Coinfection of U. stenocephala + Sarcocystids oocysts was recorded in 19.1% of the dogs (n=10). This may relate to higher prevalence of S. oocysts in dogs (n=153; 19.5%). There were two cases of coinfection of U. stenocephala + Toxocara canis (3.9%), which may relate to low prevalence of T. canis (3.9 %). One case of coinfection of Dipylidium caninum + U. stenocephala (0.1%) also appeared. Conclusion: The present study showed that male dogs and young dogs were most susceptible to U. stenocephala infection.

Therapeutic and diagnostic use of pyrantel embonate for kittens with toxocariasis

Toxocara cati are widespread in cats and are the causative agent of zoonosis. Prevention of the spread of infective Toxocara eggs in the environment is a priority and includes timely treatment of toxocariasis in cats. The aim of study. To study the effectiveness of the use of the pyrantel embonate in domestic and homeless kittens with confirmed intestinal toxocariasis and with a negative result from the study of feces. Materials and methods. In the experimental groups took 44 kittens under the age of 10 months. A coproscopic examination was performed by flotation method with a solution of sodium nitrate (density 1.34 g/cm3) with the inclusion of a centrifugation step. Kittens of all groups were treated with an anthelmintic containing a pyrantel embonate. Repeated coprovoscopy was performed on the 10th and 30th day after deworming. Results. The pyrantel embonate showed 100% effectiveness in groups of domestic and homeless kittens infected T. cati. Conclusion. We recommend to carry out the deworming in young homeless kittens up to a year and with a negative result from the study of feces. This will prevent the spread of pathogen eggs in the environment and the accumulation of larvae in animal tissues.

Properties Of An Analogue Cheese Obtained From Raw Peanut

ABSTRACT The focus is on a peanut suspension in which starch is added and that exhibits specific mechanical characteristics relevant for food products. The mixture is composed of water, lipids, starch, and proteins. The process consists of blending together the different constituents, and the study changes the experimental conditions to tune the mechanical behavior of the mixture. The rheological properties (viscosity, indentation) and physical parameters such as color, dry extract, and particle size distribution were measured. The matrix behavior was studied after a centrifugation step necessary to determine stability of the emulsion, and for varying shearing durations. Short shearing duration induce a maximum of firmness, observed by measuring indentation resistance, and a maximum of spreadability, evaluated by shear rheometry. On the contrary, long shearing durations destabilize the matrix emulsion by increasing the oil separation capacity. This study observes structural changes in the rheological behavior of this analogue artificial cheese that correlates with the extent of shearing.

Modern approaches for the analytical speciation of chromium at very low concentration in waters and leachates

<p>The speciation of chromium in waters and leachates obtained from soils and sediments has aroused interest in the last years. The element may be present in two oxidation degrees that have quite different toxicity. While chromium (III) is even essential for human beings due to its role in the metabolism of glucose and lipids, Cr(VI) is toxic due to its oxidant properties. The concentration of chromium in waters is usually of a few micrograms per liter, and the difficulty of carrying out the measurement at such low levels is further increased due to the distribution of the total element in the two mentioned forms. The technique commonly used nowadays for the purpose is inductively coupled plasma mass spectrometry (ICP-MS), a powerful analytical tool, but expensive both in acquisition and maintenance. Speciation, in addition, requires some type of previous separation or suitable strategy since the signal obtained by ICP-MS depends on the total amount of the metal present.</p><p>Recent advances in microextraction techniques have demonstrated that the determination and speciation is also possible by using electrothermal atomization atomic absorption spectrometry (ETAAS), an analytical technique which is of lesser cost than ICP-MS and is present in most laboratories worldwide. This communication summarizes some recent studies carried out in our laboratory based on the use of dispersive solid-phase microextraction to concentrate chromium. The small volume of liquid extract finally obtained can be injected into the electrothermal atomizer, and a very sensitive chromium determination is achieved. The extreme sensitivity in this way obtained is the consequence of combining the efficient preconcentration step with the sensitivity inherent to ETAAS measurement. Selectivity is also guaranteed by the characteristics of ETAAS. Speciation can be carried out by means of simple previous redox treatments without the need for a chromatographic separation. Three procedures are compared, one of them using graphene oxide as the active micro-solid phase, other based in the use of cellulose, an inexpensive reagent. Both procedures require a centrifugation step to separate the micro-solid phase from the supernatant. The third procedure uses freshly prepared ferrite particles and avoids the centrifugation step since the magnetic characteristics of the solid material permit an easy separation of phases with an external magnet. In all cases, chromium is measured after desorption from the micro-solid phase by a small volume of a suitable reagent. The limits of detection are close to 0.01 micrograms/L. The reliability of the three procedures is checked by using several reference samples with a certified chromium content. Data for the speciation of the metal are also given, a point that may be of practical interest for those involved in risk assessment or toxicity studies, since the dealers of the reference materials only provide the total chromium content.</p>

Lossless Immunocytochemistry using Photo-polymerized Hydrogel Thin-films

Immunocytochemistry (ICC), or immunofluorescence microscopy, is an essential biological technique for phenotyping cells in both research and diagnostic applications. Standard ICC methods often do not work well when the cell sample contains a small number of cells (<10,000) because of the significant cell loss that occurs during washing, staining, and centrifugation steps. Cell loss is particularly relevant when working with rare cells, such as circulating tumor cells, where such losses could significantly bias experimental outcomes. In order to eliminate cell loss in ICC protocols, we present a method to encapsulate the cell sample in a photo-polymerized hydrogel thin-film. The hydrogel thin-film is permeable to antibodies and other ICC reagents, thereby allowing the use of standard ICC protocols without modification. The cell sample is physically constrained by the hydrogel at the bottom surface of a standard (unmodified) imaging microtiter plate, thereby enabling the acquisition of high-quality micrographs regardless of the properties of the cell sample or staining reagents. Furthermore, while standard ICC requires several centrifugation steps during staining and washing, our hydrogel encapsulation method requires only a single centrifugation step. This property greatly reduces the time required to perform ICC protocols and is more compatible with robotic platforms. In this study, we show that standard ICC and Cytospin protocols are extremely lossy (>70% loss) when the sample contains less than 10,000 cells, while encapsulating the cells using a permeable hydrogel thin-film results in a lossless ICC process.