A Novel Method for In Situ RT-PCR Based on Capsules from Centrifuge Tubes, Ideal for Transcripts Detection in Plant Tissues.

In situ RT-PCR presents advantages over other expression analysis methods due to its rapid processing and low-cost equipment. However, this technique is not without its challenges. A protocol based on a capsule made from centrifuge tubes that offers advantages over slides is presented. This capsule protects histological sections from drying out, and its easy assembly reduces time pauses between incubations. In addition, the container size where the sample is deposited allows the addition and withdrawal of the different solutions. The capsule does not need previous sealing after each incubation, and, above all, it is a low-cost and accessible material. A guideline for tissue sectioning using a cryostat that offers advantages over other sectioning methods is also described.

Glass wool column filtration for stallion sperm selection: A comparative analysis with the single-layer colloid centrifugation.

Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome-intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF-50 showed an improvement (p < .05) in PM and HOS+ sperm after selection. With GWCF-75, an increase (p < .05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll-E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF-75 (GWCF-50 = 60.0; GWCF-75 = 51.0; Androcoll-E™ = 76.0 million sperm; median; p = .013), but results on total progressive sperm count were similar (GWCF-50 = 23.0; GWCF-75 = 27.0; Androcoll-E™ = 24.0 million sperm; median; p = .3850). Using frozen-thawed semen samples (n = 16), an improvement (p < .05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF-75 filtrates. Results were comparable to Androcoll-E™ centrifugation, except HOS+ that increased (p < .05) only after GWCF-75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low-cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll-E™.

Effects of platelet-rich fibrin produced by three centrifugation protocols on bone neoformation in defects created in rat calvaria.

This study evaluated the potential of Leukocyte-platelet-rich fibrin (L-PRF; fixed angle centrifugation protocol), Advanced-platelet-rich fibrin (A-PRF; low-speed fixed angle centrifugation protocol), and Horizontal-platelet-rich fibrin (H-PRF; horizontal centrifugation protocol) in bone neoformation in critical size defects (CSDs) in rat calvaria. Thirty-two rats were divided into groups: Control (C), L-PRF, A-PRF, and H-PRF. 5 mm diameter CSDs were created in the animals' calvaria. Defects from group Control (C) were filled with blood clots, while defects from groups L-PRF, A-PRF, and H-PRF were filled with respective platelet-rich fibrin (PRF) membranes. L-PRF, A-PRF, and H-PRF were prepared from animal blood collection and specific centrifugation protocols. At 14 and 30 days, calcein (CA) and alizarin (AL) injections were performed, respectively. Animals were euthanized at 35 days. Microtomographic, laser confocal microscopy, and histomorphometric analyzes were performed. Data were statistically analyzed (ANOVA, Tukey, p < .05). L-PRF, A-PRF, and H-PRF groups showed higher values of bone volume (BV), newly formed bone area (NFBA), and precipitation of CA and AL than the C group (p < .05). The H-PRF group showed higher values of BV, number of trabeculae (Tb. N), NFBA, and higher precipitation of AL than the A-PRF and L-PRF groups (p < .05). Therefore, it can be concluded that: i) L-PRF, A-PRF, and H-PRF potentiate bone neoformation in CSDs in rat calvaria; ii) H-PRF demonstrated more biological potential for bone healing.

After tooth loss, the alveolar bone (which supports the teeth) undergoes a natural process called bone remodeling, which can lead to significant decreases in bone height and thickness over time. Faced with the need to replace missing teeth, especially when it comes to dental implants, the lack of supporting tissues can compromise their correct positioning, leading to negative impacts on the success and longevity of the treatment. Therefore, over the years, several materials and procedures have been proposed to preserve and regenerate oral tissues. Leukocyte-platelet-rich fibrin (L-PRF) consists of a membrane obtained by centrifuging the patient's blood in a fixed-angle centrifuge, allowing cells to be available to stimulate tissue regeneration directly at the place of action. Several reports demonstrate high potential in stimulating the formation of new tissues using L-PRF. In recent years, new protocols have been proposed to increase cell concentration and improve the regenerative potential of these membranes, changing the speed and time of centrifugation and introducing horizontal centrifugation. However, there still needs to be concrete evidence of the superiority of the new protocols in relation to the original protocol. In this study, we evaluated the healing of defects created in rat calvaria using platelet aggregates obtained through different centrifugation protocols. Within the limits of this study, it can be concluded that platelet aggregates improve bone healing, and horizontal centrifugation promotes more satisfactory results compared to fixed-angle protocols.

Potential effect of platelet rich fibrin prepared under differen centrifugation protocols on stem cells from the apical papila / O potencial efeito da fibrina rica em plaquetas preparada sob diferentes protocolos de centrifugação nas células tronco da papila apical

Objetivo: Este estudo objetivou avaliar o potencial proliferativo e de diferenciação das células tronco da papila cultivadas conjuntamente com fibrina rica em plaquetas (PRF) preparados sob dois protocolos de centrifugação distintos. Material e Métodos: Protocolos padrão e avançado de PRF foram utilizados. As células foram divididas em 4 grupos: controle negativo, controle positivo, padrão (L-PRF) e avançado (A-PRF). A contagem de células e ensaio de viabilidade foram realizados para verificar a capacidade proliferativa. Coloração vermelho de alizarina S, atividade de fosfatase alcalina e imunofluorescência para o receptor ativador do fator nuclear kappa-B (RANKL) foram utilizados para avaliar o potencial osteogênico e de diferenciação celular. Resultados: Ambos os tipos de PRF aumentaram o número de células, viabilidade celular sem toxicidade o que refletiu no aumento da proliferação e diferenciação de acordo com os testes realizados. Conclusão: O grupo A-PRF aumentou significativamente a proliferação e diferenciação comparado com o grupo L-PRF.(AU)

Objectives: The present work was designed to evaluate the proliferation and differentiation potential of stem cells from the apical papilla (SCAP) seeded along with platelet rich fibrin (PRF) scaffolds prepared under two different centrifugation protocols. Materials and Methods: Standard and advanced PRF protocols were used. Cells were divided into 4 groups: negative control, positive control, standard (L-PRF) and advanced (A-PRF) groups. Cell count and cell viability assays were carried out to assess the proliferation capacity. Alizarin red S (ARS) stain, Alkaline phosphatase (ALP) activity and Receptor activator of nuclear factor-kappa B ligand (RANKL) immunofluorescence staining were used to evaluate the osteogenic potential in the differentiated cells. Results:Both types of platelet rich fibrin increased the cell count, cell viability with no cytotoxicity that was reflected on increased proliferation and differentiation in terms of the performed tests. Conclusion: A-PRF group showed significant increase in proliferation and differentiation potentials compared to L-PRF group

Block freeze concentration by centrifugation and vacuum increases the content of lactose-free milk macronutrients.

Lactose-free milk is rising in popularity among consumers due to its claim to be a better digestible product compared to regular fluid milk. For that reason, concentrating on this food is a good alternative for increasing its versatility and usability in different dairy industry segments. Block freeze concentration (BFC) is a simple technology used to concentrate liquid foods through ice crystal formation and subsequent removal of water. Thus, this work aimed to test two variants of the BFC technique on lactose-free milk concentration. In the first approach, it was investigated the centrifugation-assisted BFC of skim lactose-free milk by applying a factorial experimental design. Temperature, time, and rotation speed were the factors, and the response variables included the concentrate yield, concentration index, and efficiency of the process. Concentrate yield and concentration index were mainly affected by the centrifugation temperature. On the other hand, individual factors did not have a significant effect on the efficiency, only their interactions. In the case of centrifugation-assisted BFC in a single step, the condition at 40°C, 70 min, and 4500 rpm was considered the best, given the highest values of efficiency and concentrate yield (80.87 and 67.02, respectively), and still an excellent value for concentration index (2.05). Conversely, the condition at 30°C, 45 min, and 3500 rpm was chosen to integrate a freeze concentration process in two stage. Then, the ice obtained from the first cycle was subjected to the vacuum-assisted BFC, which consisted in the second cycle. The concentrate obtained from the vacuum-assisted BFC presented contents of total solids, carbohydrates, and protein 2.95, 3.00, and 2.91 times more than the initial lactose-free milk, respectively. Therefore, we believe that the concentrates obtained can be used for the development of innovative lactose-free dairy products. PRACTICAL APPLICATION: Using concentration processes in the dairy industry can significantly contribute to enhancing the overall efficiency of milk processing since huge quantities of water from milk can be reduced, increasing the total solids content. In turn, dairy products that provide a high amount of solids (especially protein) are gaining in popularity among consumers, with consequent interest from researchers. In addition, milk concentration shows advantages in terms of processing, packaging, transportation, and handling. Since most changes occur in an aqueous environment, the removal of some parts of water results in the preservation of milk. It is noteworthy that dairy industries are concerned principally with food preservation, green technologies, and the production of high-quality products. Thus, concentration processes could favor the development of milk products rich in proteins to meet certain demands on functional and nutritional properties, for example in beverages and formulated food.

Comparison of the effects of platelet concentrates produced by high and low-speed centrifugation protocols on the healing of critical-size defects in rat calvaria: a microtomographic and histomorphometric study.

The current study evaluated the healing of critical-size defects (CSD) created in rat calvaria treated with platelet concentrates produced by high-speed (Leukocyte- and Platelet-Rich Fibrin - L-PRF) and low-speed (Advanced Platelet-Rich Fibrin - A-PRF) protocols of centrifugation. Twenty-four rats were distributed into three groups: Control, L-PRF, and A-PRF. Five mm diameter CSD were created on the animals' calvaria. The defects of the L-PRF and A-PRF groups were filled with 0.01 ml of L-PRF and A-PRF, respectively. The control group defects were filled with a blood clot only. All animals were euthanized on the 35th postoperative day. Histomorphometric and microtomographic analyses were then performed. The L-PRF and A-PRF groups had significantly higher bone volume and neoformed bone area than those of the control group and lowered bone porosity values (p < .05). No significant differences were observed between A-PRF and L-PRF groups for the analyzed parameters. Therefore, it can be concluded that i) L-PRF and A-PRF potentiated the healing of CSD in rat calvaria; ii) high and low-speed centrifugation protocols did not produce PRF matrices with different biological impacts on the amount of bone neoformation.

Cellular components and TGF-beta1 content of a closed Tube system for Platelet Rich Plasma acquisition in horse] / [Componentes celulares e TGF-beta1 do plasma rico em plaquetas obtido por sistema fechado em equinos]

Platelet-rich plasma (PRP) has been proposed as an agent to accelerate the healing process and stimulate the regenerative capacity of tissues due to its abundance of growth factors. A large variety of kits and protocols are available to obtain PRP by different cell-separation systems. However, the lack of standardization may lead to inconsistent results. The aim of this study was to characterize cellular composition, platelet parameters using the ADVIA 120 flow cytometer, and TGF-ß1 concentration from the PRP product obtained through a closed system, using simple centrifugation. Six clinically healthy horses were used in this study. The protocol in the closed system resulted in approximately 1.6-fold higher platelet and approximately 2.0-fold lower white blood cell concentrations in comparison with whole blood values. The evaluated system was efficient in concentrating platelets and in retrieving a small number of leukocytes, using a protocol of single centrifugation at low speed.

O plasma rico em plaquetas (PRP) tem sido proposto como um agente para acelerar o processo de cicatrização e estimular a capacidade regenerativa dos tecidos, devido à sua abundância de fatores de crescimento. Uma grande variedade de kits e de protocolos está disponível para obtenção de PRP, utilizando-se diferentes sistemas de separação de células. No entanto, a falta de padronização pode levar a resultados inconsistentes. O objetivo deste estudo foi caracterizar a composição celular, os parâmetros plaquetários pelo citômetro de fluxo ADVIA 120 e a concentração de TGF-ß1 do PRP obtido em sistema fechado, por centrifugação simples. Seis cavalos clinicamente saudáveis ​​foram usados ​​neste estudo. O protocolo no sistema fechado resultou em concentrações de plaquetas aproximadamente 1,6 vez maior e concentrações de leucócitos aproximadamente 2,0 vezes menores em comparação com os valores do sangue total. O sistema avaliado foi eficiente na concentração de plaquetas e na recuperação de um pequeno número de leucócitos, utilizando um protocolo de centrifugação única em baixa velocidade.

Harvesting of Chlorella sorokiniana BR001 cultivated in a low-nitrogen medium using different techniques / Colheita de Chlorella sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio utilizando diferentes técnicas

The harvesting process is a current challenge for the commercial production of microalgae because the biomass is diluted in the culture medium. Several methods have been proposed to harvest microalgae cells, but there is not a consensus about the optimum method for such application. Herein, the methods based on sedimentation, flocculation, and centrifugation were evaluated on the recovery of Chlorella sorokiniana BR001 cultivated in a low-nitrogen medium. C. sorokiniana BR001 was cultivated using a low-nitrogen medium to trigger the accumulation of neutral lipids and neutral carbohydrates. The biomass of C. sorokiniana BR001 cultivated in a low-nitrogen medium showed a total lipid content of 1.9 times higher (23.8 ± 4.5%) when compared to the biomass produced in a high-nitrogen medium (12.3 ± 1.2%). In addition, the biomass of the BR001 strain cultivated in a low-nitrogen medium showed a high content of neutral carbohydrates (52.1 ± 1.5%). The natural sedimentation-based process was evaluated using a sedimentation column, and it was concluded that C. sorokiniana BR001 is a non-flocculent strain. Therefore, it was evaluated the effect of different concentrations of ferric sulfate (0.005 to 1 g L-1) or aluminum sulfate (0.025 to 0.83 g L-1) on the flocculation process of C. sorokiniana BR001, but high doses of flocculant agents were required for an efficient harvest of biomass. It was evaluated the centrifugation at low speed (300 to 3,000 g) as well, and it was possible to conclude that this process was the most adequate to harvest the non-flocculent strain C. sorokiniana BR001.(AU)

O processo de colheita é um desafio atual para a produção comercial de microalgas porque a biomassa é diluída no meio de cultivo. Diversos métodos têm sido propostos para coletar células de microalgas, porém não existe um consenso sobre um método ótimo para tal aplicação. Neste estudo, métodos baseados em sedimentação, floculação e centrifugação foram avaliados na recuperação de Chlorella sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio. C. sorokiniana BR001 foi cultivada em um meio com baixo teor de nitrogênio para induzir ao acúmulo de lipídeos e carboidratos neutros. A biomassa de C. sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio apresentou um teor de lipídeos 1,9 vezes superior (23,8 ± 4,5%), quando comparada à biomassa produzida em um meio com alto teor de nitrogênio (12,3 ± 1,2%). Adicionalmente, a biomassa da linhagem BR001 cultivada em um meio com baixo teor de nitrogênio apresentou alto teor de carboidratos neutros (52,1 ± 1,5%). O processo baseado em sedimentação natural foi avaliado utilizando uma coluna de sedimentação e concluiu-se que C. sorokiniana BR001 é uma linhagem não floculante. Portanto, o efeito de diferentes concentrações de sulfato férrico (0,005 a 1 g L-1) ou sulfato de alumínio (0,025 a 0,83 g L-1) foram avaliados no processo de floculação de C. sorokiniana BR001, mas altas doses de floculantes foram necessárias para uma colheita de biomassa eficiente. Também foi avaliada a centrifugação em baixa velocidade (300 a 3.000 g), e foi possível concluir que este processo constituiu o mais adequado para a colheita da linhagem não floculante C. sorokiniana BR001.(AU)

Harvesting of Chlorella sorokiniana BR001 cultivated in a low-nitrogen medium using different techniques / Colheita de Chlorella sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio utilizando diferentes técnicas

The harvesting process is a current challenge for the commercial production of microalgae because the biomass is diluted in the culture medium. Several methods have been proposed to harvest microalgae cells, but there is not a consensus about the optimum method for such application. Herein, the methods based on sedimentation, flocculation, and centrifugation were evaluated on the recovery of Chlorella sorokiniana BR001 cultivated in a low-nitrogen medium. C. sorokiniana BR001 was cultivated using a low-nitrogen medium to trigger the accumulation of neutral lipids and neutral carbohydrates. The biomass of C. sorokiniana BR001 cultivated in a low-nitrogen medium showed a total lipid content of 1.9 times higher (23.8 ± 4.5%) when compared to the biomass produced in a high-nitrogen medium (12.3 ± 1.2%). In addition, the biomass of the BR001 strain cultivated in a low-nitrogen medium showed a high content of neutral carbohydrates (52.1 ± 1.5%). The natural sedimentation-based process was evaluated using a sedimentation column, and it was concluded that C. sorokiniana BR001 is a non-flocculent strain. Therefore, it was evaluated the effect of different concentrations of ferric sulfate (0.005 to 1 g L-1) or aluminum sulfate (0.025 to 0.83 g L-1) on the flocculation process of C. sorokiniana BR001, but high doses of flocculant agents were required for an efficient harvest of biomass. It was evaluated the centrifugation at low speed (300 to 3,000 g) as well, and it was possible to conclude that this process was the most adequate to harvest the non-flocculent strain C. sorokiniana BR001.

O processo de colheita é um desafio atual para a produção comercial de microalgas porque a biomassa é diluída no meio de cultivo. Diversos métodos têm sido propostos para coletar células de microalgas, porém não existe um consenso sobre um método ótimo para tal aplicação. Neste estudo, métodos baseados em sedimentação, floculação e centrifugação foram avaliados na recuperação de Chlorella sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio. C. sorokiniana BR001 foi cultivada em um meio com baixo teor de nitrogênio para induzir ao acúmulo de lipídeos e carboidratos neutros. A biomassa de C. sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio apresentou um teor de lipídeos 1,9 vezes superior (23,8 ± 4,5%), quando comparada à biomassa produzida em um meio com alto teor de nitrogênio (12,3 ± 1,2%). Adicionalmente, a biomassa da linhagem BR001 cultivada em um meio com baixo teor de nitrogênio apresentou alto teor de carboidratos neutros (52,1 ± 1,5%). O processo baseado em sedimentação natural foi avaliado utilizando uma coluna de sedimentação e concluiu-se que C. sorokiniana BR001 é uma linhagem não floculante. Portanto, o efeito de diferentes concentrações de sulfato férrico (0,005 a 1 g L-1) ou sulfato de alumínio (0,025 a 0,83 g L-1) foram avaliados no processo de floculação de C. sorokiniana BR001, mas altas doses de floculantes foram necessárias para uma colheita de biomassa eficiente. Também foi avaliada a centrifugação em baixa velocidade (300 a 3.000 g), e foi possível concluir que este processo constituiu o mais adequado para a colheita da linhagem não floculante C. sorokiniana BR001.

Method for the physical characterization of substrates using centrifugation

The characterization of substrate properties supports the correct choice of components and directs management decisions in container cultivation. There are several systems of analysis, however, the routine procedures to determine the water retention curve (WRC) are time consuming and inaccurate. This study proposed a reproducible, less time-consuming, and easy to perform method for physical characterization of substrates. We used Sphagnum peat, coconut powder, carbonized rice husk, expanded vermiculite, and expanded clay in isolation. The WRC was obtained at tensions 0, 10, 50, and 100 hPa using the method of Büchner funnel, the European standard (CEN-EN13041), and by the centrifugation method of samples. In centrifugation, tensions were simulated by combining the angular velocity, sample height, and radius measured from the sample outermost point to the central point of the centrifuge. Three rotations were applied (230; 520 and 730 rpm), testing the centrifugation time for each tension and then the volumetric water content was compared between the methods. The results showed a positive correlation (p < 0.001) between the centrifugation and standard methods, considering that the centrifugation time required for moisture stabilization varied according to the material analyzed and the tension applied. However, the time required to obtain a report was reduced by half. Sample centrifugation is a promising method, ensuring that the tensile force actually acts on the entire sample to determine the water retention curve of the substrate also reducing the duration of the analyses.

OPTIMIZING A PLATELET-RICH PLASMA CONCENTRATION PROTOCOL FOR SOUTH AMERICAN SEA LIONS (OTARIA FLAVESCENS).

Accelerated healing in wild or captive South American sea lions (Otaria flavescens) is a key tool to help minimize infection and complications associated with open wounds, dental disease, and ocular pathology. Platelet-rich plasma (PRP) is an autogenous source for growth factors based on platelet concentration, which can be obtained by centrifuging whole blood collected in sodium citrate anticoagulant. Currently, there are well-defined PRP concentration protocols for humans and most domestic companion animal species. However, there is no clear centrifugation protocol for obtaining PRP in most marine mammal species. This study aimed to optimize the platelet concentration protocol based on whole blood centrifugation using speeds ranging from 500 to 5,000 rpm and times ranging from 3 to 6 min. Blood was drawn from seven adult South American sea lions, placed into 1-ml sodium citrate tubes, and centrifuged following 12 different centrifugation protocols. PRP was designated as the lower third fraction of the centrifuged plasma. Platelet counts were performed using flow cytometry and statistical analysis was carried out to establish a well-defined protocol for efficient PRP production. Transmission electron microscopy (TEM) analysis was performed to evaluate possible platelet degranulation during the different centrifugation protocols and measure platelet areas. Maximum concentration of platelets in PRP was 4.73-fold higher than the number of platelets in equal volume of whole blood, and significant differences in the concentrations obtained were found between the 12 centrifugation protocols evaluated using different speed and time combinations. The best one-step centrifugation protocol resulted from using 900-rpm speed for 3 min. The highest-fold increase was achieved using a two-step centrifugation protocol, which combined the most efficient one-step centrifugation protocol (900 rpm, 3 min) with a second centrifugation using 2,000-rpm speed for 6 min. TEM analysis confirmed that platelets were complete and maintained integrity after the proposed protocol.

Commercial blood cell separation systems versus tube centrifugation methods for the preparation of platelet-rich plasma: a preliminary cross-sectional study.

OBJECTIVE: Clinical studies claim that platelet-rich plasma (PRP) accelerates tissue healing due to its high concentration of growth factors and that the combination with leukocytes improves the antimicrobial effect of the concentrate. Most of these studies obtained PRP using different separation systems, and few analyzed the content of the PRP used for treatment. This study characterized the composition of PRP and white blood cells (WBC) from a single donor produced by three commercially available PRP separation systems and two anticoagulated general analytical tubes. METHODS: Five patients donated 50 mL of blood, which was processed to produce PRP and WBC using three PRP concentration systems (i.e., Easy PRP Kit, GloPRP, and Wego) and two tubes for general analysis anticoagulated with ethylenediaminetetraacetic acid (EDTA) and citrate. Platelets and WBC in combination with their concentrates were analyzed by automated systems in a clinical laboratory. RESULTS: There were no significant differences in the average concentrations of PRP platelets and WBC between GloPRP and the tubes for general analysis with EDTA and citrate; however, the Easy PRP Kit gave results much superior to the rest of the methods, especially comparing it with the Wego Kit, whose concentrates were especially low, even nonexistent for WBC. CONCLUSIONS: The Easy PRP Kit concentrates WBC-rich PRP, resulting in increased WBC concentrations, compared with low WBC-low PRP of GloPRP and general tube methods for EDTA and citrate analysis and the even lower concentration of PRP from the Wego Kit, with the absence of leukocytes.

Laboratory diagnosis of mange-causing mites in dogs using a modified centrifugation-flotation technique in sucrose solution.

Among the skin disorders of dogs, scabies is notable for its high occurrence rate and the need for veterinary interventions. There are two obstacles to making this diagnosis through direct investigation under a microscope (DIM): the continual need to make new slides when the results are negative and the long time needed for reading these slides. Thus, the objective of the present study was to compare efficacy between DIM and the technique of centrifugation-flotation in sucrose solution (CFSS) in samples from dogs in the semiarid region of the state of Paraíba, Brazil. Samples from 136 dogs were used, and three slides were made for each examination (DIM and CFSS). The readings were halted in cases of positivity. Positive samples were obtained from 56.6% of the dogs (77/136), of which 76.6% (59/77) were positive through both techniques, 13% (10/77) only through DIM and 10.4% (8/77) only through CFSS. The positivity rate did not differ statistically between the techniques. CFSS showed higher quality of readings, due to the considerably fewer artifacts on the slides, thereby optimizing the reading time. Sensitivity (85.6%), specificity (88.1%), accuracy (86.8%), positive predictive value (88.1%) and negative predictive value (85.1%) were obtained and the kappa coefficient (0.73) was considered substantial. It was concluded that CFSS showed high diagnostic capacity for scabies, similar to that of DIM, with optimized reading time, fewer artifacts and better display of mites.

Therapeutic plasma exchange in critically ill children: experience of the pediatric intensive care unit of two centers in Chile.

INTRODUCTION: Therapeutic plasma exchange (TPE) is an extracorporeal blood purification technique used in a wide spectrum of diseases. We aim to review the indications, complications, and outcomes of critically ill children who received TPE and to compare a membrane versus centrifugal method in this cohort. METHODS: A retrospective observational study in two pediatric intensive care units in Chile during eight years (2011-2019) Results: A total of 36 patients underwent 167 TPE sessions (20 centrifugation and 16 membrane-based). The more frequent indications for TPE were autoimmune neurological diseases in 14 cases, renal diseases (9), and rheumatological disorders (5). 58 % of children received other immunomodulatory therapy. According to ASFA, 45 % of cases were I-II category, 50 % to III, and 5% not classified. Response to treatment was complete in 64 % (23/36) and partial in 33 % (12/36). Complications occurred in 17.4 % of sessions, and the most frequent was transient hypotension during the procedure. Overall survival at discharge from the PICU was 92 %. Patients who received TPE as a single therapy (n = 26) survived 96 %. The clinical outcomes between the two apheresis methods were similar. Survivors had a significantly lower PELOD score on admission (14.5 vs. 6.5, p = 0.004). CONCLUSIONS: TPE is mainly indicated as a rescue treatment in neurological autoimmune diseases refractory to conventional immunomodulatory treatment. Complications in critically ill children are mild and low. The outcome in children requiring TPE as a single therapy is good, and no differences were observed with centrifugation or membrane method.

Alcance de las células madre derivadas de tejido adiposo / Scope of stem cells derived from adipose tissue

Introducción: En el tejido adiposo se han identificado células madre mesenquimales con capacidad autorrenovadora y multipotencial. Mediante digestión enzimática y centrifugado del lipoaspirado se libera una población heterogénea de células denominada fracción vascular estromal, con innumerables potencialidades terapéuticas en el campo de la medicina regenerativa. Objetivo: Actualizar el alcance de las células madre derivadas de tejido adiposo en la terapia regenerativa. Método: Se revisaron 38 artículos entre los años 2000 y 2019 en las bases de datos Scielo, ScienceDirect, Medline y Pubmed. Desarrollo: Las células de la fracción vascular estromal se caracterizan por su capacidad de generar tejido adiposo y vasos sanguíneos, y por la producción de factores de crecimiento que ayudan en la supervivencia de los adipocitos y la formación de una red vascular. El principal mecanismo de acción de las células madre derivadas de tejido adiposo parece deberse a su acción paracrina y a la sinergia con células endoteliales. En el campo de la medicina regenerativa se han utilizado en el tratamiento de cicatrices patológicas y de fibrosis deformantes con impotencia funcional, en las reconstrucciones de secuelas por cáncer y en el cierre precoz de zonas cruentas. Conclusiones: La lipotransferencia es un procedimiento con un mínimo de complicaciones que constituye una de las opciones terapéuticas más empleadas para corregir defectos en los tejidos, debido a que no solo es un medio de relleno, sino que también permite la regeneración y restauración tisular. La presencia de células madre en el tejido adiposo, unido a su accesibilidad, disponibilidad e histocompatibilidad, ha motivado su aplicación cada vez más expandida en la medicina estética, reconstructiva y regenerativa(AU)

Introduction: Mesenchymal stem cells with self-renewing and multipotential capacity have been identified in adipose tissue. By means of enzymatic digestion and centrifugation of the lipoaspirate a heterogeneous population of cells called vascular stromal fraction is released. It has innumerable therapeutic potentialities in the field of regenerative medicine. Objective: To update the scope of stem cells derived from adipose tissue in regenerative therapy. Method: 38 articles published between 2000 and 2019 in the Scielo, ScienceDirect, Medline and Pubmed databases were reviewed. Development: The cells of the vascular stromal fraction are characterized by generating adipose tissue and blood vessels and by the production of growth factors that help in the survival of adipocytes and the formation of a vascular network. The main mechanism of action of stem cells derived from adipose tissue appears to be due to their paracrine action and synergy with endothelial cells. Stem cells derived from adipose tissue have been used in regenerative medicine for the treatment of pathological scars and deforming fibrosis with functional impotence, in the reconstruction of cancer sequelae and in the early closure of bloody areas. Conclusions: Lipotransfer is a procedure with a minimum of complications that constitutes one of the most widely used therapeutic options to correct tissue defects, since it is not only a filling medium, but also allows tissue regeneration and restoration. The presence of stem cells in adipose tissue, together with their accessibility, availability and histocompatibility, has motivated their increasingly widespread application in aesthetic, reconstructive and regenerative medicine(AU)

Polyphenolic Compounds Extracted and Purified from Buddleja Globosa Hope (Buddlejaceae) Leaves Using Natural Deep Eutectic Solvents and Centrifugal Partition Chromatography.

Chemical profiling of Buddleja globosa was performed by high-performance liquid chromatography coupled to electrospray ionization (HPLC-DAD-ESI-IT/MS) and quadrupole time-of-flight high-resolution mass spectrometry (HPLC-ESI-QTOF/MS). The identification of 17 main phenolic compounds in B. globosa leaf extracts was achieved. Along with caffeoyl glucoside isomers, caffeoylshikimic acid and several verbascoside derivatives (ß-hydroxyverbascoside and ß-hydroxyisoverbascoside) were identified. Among flavonoid compounds, the presence of 6-hydroxyluteolin-7-O-glucoside, quercetin-3-O-glucoside, luteolin 7-O-glucoside, apigenin 7-O-glucoside was confirmed. Campneoside I, forsythoside B, lipedoside A and forsythoside A were identified along with verbascoside, isoverbascoside, eukovoside and martynoside. The isolation of two bioactive phenolic compounds verbascoside and forsythoside B from Buddleja globosa (Buddlejaceae) was successfully achieved by centrifugal partition chromatography (CPC). Both compounds were obtained in one-step using optimized CPC methodology with the two-phase solvent system comprising ethyl acetate-n-butanol-ethanol-water (0.25:0.75:0.1:1, v/v). Additionally, eight Natural Deep Eutectic Solvents (NADESs) were tested for the extraction of polyphenols and compared with 80% methanol. The contents of verbascoside and luteolin 7-O-glucoside after extraction with 80% methanol were 26.165 and 3.206 mg/g, respectively. Among the NADESs tested in this study, proline- citric acid (1:1) and choline chloride-1, 2- propanediol (1:2) were the most promising solvents. With these NADES, extraction yields for verbascoside and luteolin 7-O-glucoside were 51.045 and 4.387 mg/g, respectively. Taken together, the results of this study confirm that CPC enabled the fast isolation of bioactive polyphenols from B. globosa. NADESs displayed higher extraction efficiency of phenolic and therefore could be used as an ecofriendly alternative to classic organic solvents.

Cytocentrifugation as an additional method to study echinoderm coelomocytes: a comparative approach combining living cells, stained preparations, and energy-dispersive x-ray spectroscopy / Citocentrifugación como un método adicional para estudiar celomitos de equinodermos: un enfoque comparativo que combina células vivas, preparaciones teñidas y espectroscopía de rayos-x de dispersión de energía

Introduction: Echinoderm coelomocytes have traditionally been investigated through a morphological approach using light microscopy, which relies on the idea of constant cell shape as a stable character. However, this can be affected by biotic or abiotic conditions. Objective: To analyze if the consistency in cell morphology offered by the cytocentrifugation method, might be used as a convenient tool to study echinoderm coelomocytes. Methods: Cells of Echinaster (Othilia) brasiliensis (Asteroidea), Holothuria (Holothuria) tubulosa (Holothuroidea), Eucidaris tribuloides, Arbacia lixula, Lytechinus variegatus, and Echinometra lucunter (Echinoidea) were spread on microscope slides by cytocentrifugation, stained, and analyzed through light microscopy. Additionally, fluorescence microscopy, scanning electron microscopy, and energy-dispersive x-ray spectroscopy were applied to cytospin preparations, to complement the analysis of granular and colorless spherulocytes of Eucidaris tribuloides. Results: Altogether, 11 cell types, including phagocytes, spherulocytes, vibratile cells, and progenitor cells were identified in the samples analyzed. The granular spherulocyte, a newly-described cell type, was observed in all Echinoidea and was very similar to the acidophilic spherulocytes of Holothuria (Holothuria) tubulosa. Conclusions: Cytocentrifugation proved to be versatile, either as the main method of investigation in stained preparations, or as a framework on which other procedures may be performed. Its ability to maintain a constant morphology allowed accurate correspondence between live and fixed/stained cells, differentiation among similar spherulocytes as well as comparisons between similar cells of Holothuroidea and Echinoidea.

Introducción: Los celomocitos de equinodermos se han investigado tradicionalmente a través de un enfoque morfológico utilizando microscopía óptica, que se basa en la idea de la forma celular constante como un carácter estable. Sin embargo, esto puede verse afectado por condiciones bióticas o abióticas. Objetivo: Analizar si la consistencia en la morfología celular que ofrece el método de citocentrifugación podría utilizarse como una herramienta conveniente para estudiar los celomocitos de equinodermos. Métodos: Células de Echinaster (Othilia) brasiliensis (Asteroidea), Holothuria (Holothuria) tubulosa (Holothuroidea), Eucidaris tribuloides, Arbacia lixula, Lytechinus variegatus y Echinometra lucunter (Echinoidea) se esparcieron en portaobjetos de microscopio por citocentrifugación, se tiñeron y analizaron mediante microscopía óptica. Adicionalmente, se aplicó microscopía de fluorescencia, microscopía electrónica de barrido y espectroscopía de rayos X con dispersión de energía a las preparaciones de citoespina, para complementar el análisis de los esferulocitos granulares e incoloros de Eucidaris tribuloides. Resultados: En total, se identificaron en las muestras analizadas 11 tipos de células, incluidos fagocitos, esferulocitos, células vibrátiles y células progenitoras. El esferulocito granular, un tipo de célula recién descrito, se observó en todos los Echinoidea y fue muy similar a los esferulocitos acidófilos de Holothuria (Holothuria) tubulosa. Conclusiones: La citocentrifugación demostró ser un método bastante versátil, ya sea como el método principal de investigación en preparaciones teñidas o como un marco en el que se pueden realizar otros procedimientos. Su capacidad para mantener una morfología constante permitió una correspondencia precisa entre las células vivas y las células fijas/teñidas, la diferenciación entre esferulocitos similares, así como comparaciones entre células similares de Holothuroidea y Echinoidea.

High-speed centrifugation of extender of freeze-thaw boar semen.

In semen cryopreservation, egg yolk is still widely used as a non-penetrating cryoprotectant. Much has been developed in the search for alternatives for this biological product. This work aimed to evaluate the processed egg yolk through ultracentrifugation and/or sonication in the cryopreservation of swine semen. Twenty-seven semen doses were purchased from a commercial boar stud and processed for cryopreservation using egg yolk lactose 11% (control) extender, processed using two different methods: high-speed centrifugation and sonication. Then, they were submitted to freeze-thawing protocol and were assessed for kinematic and cell structural parameters. Samples in which extenders underwent centrifugation had better results in velocity parameters, meanwhile those that only sonication was performed had poorest results in this parameter. The preservation of the membrane and mitochondria structure had better results when the diluent was only centrifuged in comparison with the other treatments. Therefore, centrifugation of extender containing egg yolk is important for better cryopreservation of swine semen.

Rapid molecular diagnostics of COVID-19 by RT-LAMP in a centrifugal polystyrene-toner based microdevice with end-point visual detection.

Infection caused by the new coronavirus (SARS-CoV-2) has become a serious worldwide public health problem, and one of the most important strategies for its control is mass testing. Loop-mediated isothermal amplification (LAMP) has emerged as an important alternative to simplify the diagnostics of infectious diseases. In addition, an advantage of LAMP is that it allows for easy reading of the final result through visual detection. However, this step must be performed with caution to avoid contamination and false-positive results. LAMP performed on microfluidic platforms can minimize false-positive results, in addition to having potential for point-of-care applications. Here, we describe a polystyrene-toner (PS-T) centrifugal microfluidic device manually controlled by a fidget spinner for molecular diagnosis of COVID-19 by RT-LAMP, with integrated and automated colorimetric detection. The amplification was carried out in a microchamber with 5 µL capacity, and the reaction was thermally controlled with a thermoblock at 72 °C for 10 min. At the end of the incubation time, the detection of amplified RT-LAMP fragments was performed directly on the chip by automated visual detection. Our results demonstrate that it is possible to detect COVID-19 in reactions initiated with approximately 10-3 copies of SARS-CoV-2 RNA. Clinical samples were tested using our RT-LAMP protocol as well as by conventional RT-qPCR, demonstrating comparable performance to the CDC SARS-CoV-2 RT-qPCR assay. The methodology described in this study represents a simple, rapid, and accurate method for rapid molecular diagnostics of COVID-19 in a disposable microdevice, ideal for point-of-care testing (POCT) systems.

Effects of rotor angle and time after centrifugation on the biological in vitro properties of platelet rich fibrin membranes.

This study evaluated the impact of rotor angle and time of storage after centrifugation on the in vitro biological properties of platelet-rich fibrin (PRF) membranes. Blood samples (n = 9) were processed with a vertical fixed-angle (V) or a swing-out horizontal (H) centrifuge, with 20-60 min of sample storage after centrifugation. Leukocytes, platelets, and red blood cells were counted, and fibrin architecture was observed by scanning electron microscopy (SEM). The release of FGF2, PDGFbb, VEGF, IL-6, and IL-1ß was measured after incubation on culture media for 7-21 days. Cell content was equivalent in all experimental groups (p > .05). The fibrin matrix was similar for fixed-angle and horizontal centrifugation. Horizontal centrifugation induced a twofold increase in PDGF and 1.7× increase on FGF release as compared to V samples, while IL-1ß was significantly reduced (p < .05). No significant difference was observed on the release of growth factors and cytokines at different times after centrifugation (p < .05). These data suggest that both angles of centrifugation produce PRF membranes with similar structure and cellularity, but horizontal centrifugation induces a higher release of growth factors. Higher times of storage after centrifugation did not impact on cell content and the release of growth factors.