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The increased environmental presence of micro-/nanoplastics (MNPLs) and the potential health risks associated with their exposure classify them as environmental pollutants with special environmental and health concerns. Consequently, there is an urgent need to investigate the potential risks associated with secondary MNPLs. In this context, using "true-to-life" MNPLs, resulting from the laboratory degradation of plastic goods, may be a sound approach. These non-commercial secondary MNPLs must be labeled to track their presence/journeys inside cells or organisms. Because the cell internalization of MNPLs is commonly analyzed using fluorescence techniques, the use of fluorescent dyes may be a sound method to label them. Five different compounds comprising two chemical dyes (Nile Red and Rhodamine-B), one optical brightener (Opticol), and two industrial dyes (Amarillo Luminoso and iDye PolyPink) were tested to determine their potential for such applications. Using commercial standards of polystyrene nanoplastics (PSNPLs) with an average size of 170 nm, different characteristics of the selected dyes such as the absence of impact on cell viability, specificity for plastic staining, no leaching, and lack of interference with other fluorochromes were analyzed. Based on the overall data obtained in the wide battery of assays performed, iDye PolyPink exhibited the most advantages, with respect to the other compounds, and was selected to effectively label "true-to-life" MNPLs. These advantages were confirmed using a proposed protocol, and labeling titanium-doped PETNPLs (obtained from the degradation of milk PET plastic bottles), as an example of "true-to-life" secondary NPLs. These results confirmed the usefulness of iDye PolyPink for labeling MNPLs and detecting cell internalization.

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BACKGROUND: There is a little information about of expression of C4d (complement fragment) in Focal segmental glomerulosclerosis (FSGS) subtypes. Our aim was to determine the expression of C4d in FSGS subtypes in percutaneous native renal biopsies in a second-level hospital and its correlation with clinical, biochemical and histological variables. MATERIAL AND METHODS: A retrospective study in paraffin blocks of patients with biopsy with FSGS aged 16-65 years, indistinct sex, not diabetic or obese. Immunohistochemistry was performed for C4d and their expression was analyzing in non-sclerosed glomerular capillaries (GC) and sclerosis areas (SA). Clinical and biochemical variables were recorded. The cases were divided into C4d positive and C4d negative groups and compared. The correlation between C4d staining scores in CG and SA with clinical and biochemical variables were analyzed. RESULTS: Twenty samples were analyzed, 4 for each subtype. At the time of biopsy average age 38.8 ±â€¯18.6 years, 65% male, 8.7% were hypertension. The percentage of positivity for C4d was 40% in GC, 30% SA and 35% in mesangium. The highest expression was for cellular and collapsing subtypes. C4d positivity cases had increased proteinuria (p = 0.035). A significant correlation was found between percentage of C4d expression in CG with SA (p = 0.012) and SA with tubular atrophy and interstitial fibrosis (p < 0.05). CONCLUSIONS: C4d expression in FSGS predominated in the cellular and collapsing subtypes, which translates complement activation. C4d is a possible surrogate marker in GSFS.

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INTRODUCTION: Membranous nephropathy (MN) is the most common cause of primary nephrotic syndrome in adults (20-30%). Light microscopy shows thickening of glomerular basement membrane with appearance of spikes. These histological findings are not evident in early forms, in which case the granular deposition pattern of IgG and/or C3 in the basement membrane by immunofluorescence (IF) constitutes the diagnostic tool that allows to differentiate it from minimal change disease (MCD). Complement system plays a key role in the pathophysiology of MN. C4d is a degradation product and a marker of the complement system activation. C4d labelling by immunohistochemical (HI) technique can help in the differential diagnosis between both glomerulopathies NM and MCD when the material for IF is insufficient and light microscopy is normal. Our objective was to explore the discrimination power of C4d to differentiate between MN and MCD in renal biopsy material. METHODS: Paraffin-embedded samples were recovered from renal biopsies with a diagnosis of MN and MCD performed between 1/1/2008 and 4/1/2019. IH staining was performed by immunoperoxidase technique using a rabbit anti-human C4d polyclonal antibody. RESULTS: In all cases with MN (n = 27, 15 males) with a median age of 63 (range: 18-87) years, C4d deposits were detected. In 21 cases with MCD (12 males) with a median age of 51 (range: 18-87) years, the C4d marking was negative in every samples. CONCLUSION: The results indicate that the marking of the renal biopsy with C4d is a useful tool for the differential diagnosis between NM and MCD.

Introducción: La nefropatía membranosa (NM) es la causa más frecuente de síndrome nefrótico primario en adultos (20-30%). En la microscopia óptica se observa engrosamiento de membrana basal glomerular con aparición de espigas. Estos hallazgos histológicos no son evidentes en formas tempranas, en cuyo caso el patrón de depósito granular de IgG y/o C3 en la membrana basal por inmunofluorescencia (IF) permite diferenciarla de enfermedad por cambios mínimos (ECM). El sistema del complemento juega un papel central en la fisiopatología de la NM. C4d es producto de degradación y un marcador de la activación del complemento. La marcación con C4d en muestras de biopsias renales, por técnica de inmunohistoquímica (IH) puede colaborar en el diagnóstico diferencial entre ambas glomerulopatías. Nuestro objetivo fue explorar el poder de discriminación del C4d para diferenciar NM de ECM en material de biopsias renales. Métodos: Se recuperaron muestras en parafina de biopsias renales con diagnóstico de NM y ECM realizados entre 1/1/2008 y 1/4/2019. Se realizaron tinciones de IH por técnica de inmunoperoxidasa con C4d usando un anticuerpo policlonal antihumano de conejo. Resultados: En todos los casos con NM (n = 27, 15 hombres) con mediana de edad de 63 (rango: 18-86) años se detectaron depósitos de C4d. En los 21 casos con ECM (12 hombres) con mediana de edad de 51 (rango: 18-87) años la marcación de C4d fue negativa. Conclusión: Los resultados indican que la marcación de la biopsia renal con C4d es una herramienta útil para el diagnóstico diferencial entre NM y ECM.

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The selection of oleaginous bacteria, potentially applicable to biotechnological approaches, is usually carried out by different expensive and time-consuming techniques. In this study, we used Oil Red O (ORO) as an useful dye for staining of neutral lipids (triacylglycerols and wax esters) on thin-layer chromatography plates. ORO could detect minimal quantities of both compounds (detection limit, 0.0025 mg of tripalmitin or 0.005 mg of cetylpalmitate). In addition, we developed a specific, rapid, and inexpensive screening methodology to detect triacylglycerol-accumulating microorganisms grown on the agar plate. This staining methodology detected 9/13 strains with a triacylglycerol content higher than 20% by cellular dry weight. ORO did not stain polyhydroxyalkanoates-producing bacteria. The four oleaginous strains not detected by this screening methodology exhibited a mucoid morphology of their colonies. Apparently, an extracellular polymeric substance produced by these strains hampered the entry of the lipophilic dye into cells. The utilization of the developed screening methodology would allow selecting of oleaginous bacteria in a simpler and faster way than techniques usually used nowadays, based on unspecific staining protocols and spectrophotometric or chromatographic methods. Furthermore, the use of ORO as a staining reagent would easily characterize the neutral lipids accumulated by microorganisms as reserve compounds. KEY POINTS: • Oil Red O staining is specific for triacylglycerols • Oil Red O staining is useful to detect oleaginous bacteria • Fast and inexpensive staining to isolate oleaginous bacteria from the environment.

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The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry.

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SUMMARY: Over time, Goldner's trichrome staining has been essential in paraffin soft tissue research. However, its classic application involves prior decalcification, generating disadvantages in the integrity of the samples and the interpretation of results. This study seeks to overcome the limitations associated with decalcification when applying Goldner's trichrome stain with plastic resins. It focuses on detailed visualization of non-decalcified bone and dental samples in animal models. Samples of jaw and tooth from a dog (Canis familiaris) were used, as well as tibia from a rabbit (Oryctolagus cuniculus) with a titanium dental implant and bone graft substitute. Adjustments were made to the original protocol, including a surface treatment prior to staining. Plastination and inclusion in specific plastic resins were part of the process. The microplastinated and stained samples showed optimal quality for optical microscopy. Those from dogs allowed detailed observation of the tooth-periodontal tissue relationship, while those from rabbits revealed a clear differentiation between mineralized and osteoid bone tissue. The staining made it easy to examine the precise interface between soft tissues, bone graft, and implant. The successful adaptation of Goldner's trichrome stain to specimens in plastic resins represents a significant advance in histological investigation of hard tissues. This methodology stands out as an effective tool to evaluate implants and biomaterials in animal models, providing detailed visualization without compromising the integrity of the samples. The combination of histochemistry and plastic resins offers a valuable alternative for microanatomical studies, opening new possibilities in hard tissue research and evaluation of bone structures.

A lo largo del tiempo, la tinción tricrómica de Goldner ha sido esencial en la investigación de tejidos blandos en parafina. Sin embargo, su aplicación clásica conlleva la descalcificación previa, generando desventajas en la integridad de las muestras y la interpretación de resultados. Este estudio busca superar las limitaciones asociadas con la descalcificación al aplicar la tinción tricrómica de Goldner con resinas plásticas. Se enfoca en visualizar detalladamente muestras óseas y dentales no descalcificadas en modelos animales. Se emplearon muestras de mandíbula y diente de perro (Canis familiaris), así como tibia de conejo (Oryctolagus cuniculus) con implante dental de titanio y substituto de injerto óseo. Se realizaron ajustes al protocolo original, incluyendo un tratamiento superficial previo a la tinción. La plastinación y la inclusión en resinas plásticas específicas fueron parte del proceso. Las muestras microplastinadas y teñidas mostraron una calidad óptima para microscopía óptica. Las de perro permitieron la observación detallada de la relación diente-tejido periodontal, mientras que las de conejo revelaron una clara diferenciación entre tejido óseo mineralizado y osteoide. La tinción facilitó examinar la interface precisa entre tejidos blandos, injerto óseo e implante. La adaptación exitosa de la tinción tricrómica de Goldner a muestras en resinas plásticas representa un avance significativo en la investigación histológica de tejidos duros. Esta metodología destaca como una herramienta eficaz para evaluar implantes y biomateriales en modelos animales, brindando una visualización detallada sin comprometer la integridad de las muestras. La combinación de histoquímica y resinas plásticas ofrece una alternativa valiosa para estudios microanatómicos, abriendo nuevas posibilidades en la investigación de tejidos duros y evaluación de estructuras óseas.

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OBJECTIVE: Evaluate the color and translucency of a nanoceramic resin subjected to hygiene practices and immersion in coffee over time. MATERIALS AND METHODS: Specimens (n = 80, HT and LT, 0.5-mm thick) of a nanoceramic resin were obtained and were divided in groups according to the simulated oral hygiene: no intervention (NT), brushing with an electric brush and water (BN), brushing with an electric brush and toothpaste for 10 s (BT), and immersion in mouthwash for 30 s (MW). Oral hygiene practices were repeated after staining in coffee solution for 15, 48, 336, 1440, 8640, and 35,040 min. Spectral wavelengths were recorded with a spectrophotometer at each time. ∆E00, ∆L', ∆C', ∆H', and TP00 were calculated by CIEDE2000 and were evaluated by 2-way ANOVA, repeated measures ANOVA, and Tukey's HSD test (α = 0.05). RESULTS: ANOVA showed significance among factors for ΔE00 (p < 0.05), with higher ΔE00 for NT-HT (11.7) and NT-LT (11.2). After T6 (2-year simulation), the lowest values were found for BT-HT (5.3) and BT-LT (4.9). All groups showed a reduction in lightness and translucency and increased chroma and hue. However, the smallest variations were found in the BT groups (p < 0.05). CONCLUSIONS: Brushing with toothpaste effectively minimized the color change of NCRs caused by coffee staining. CLINICAL SIGNIFICANCE: Oral hygiene practices, such as brushing with toothpaste, might be important to minimize staining of nanoceramic resin restorations, especially in patients who frequently consume coffee.

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Las técnicas de doble tinción y transparentación se han usado desde 1897, pero su utilidad ha sido poco explorada en los estudios anatómicos de micromamíferos adultos. No obstante, la combinación de estas técnicas con el análisis alométrico mutivariado posibilita el estudio de esqueletos poscraneales articulados de tales grupos de micromamíferos como los roedores, los cuales son muy limitados ya que casi siempre se enfocan en los cráneos. En este estudio, analizamos y comparamos la morfometría del esqueleto de Neotomodon alstoni con la de Meriones unguiculatus, Phodopus campbelli y Rattus norvegicus. Usamos la técnica de doble tinción y transparentación para analizar las relaciones morfométricas entre estos roedores utilizando sesenta caracteres esqueléticos. Se encontró que tres especies comparten dos correlaciones comunes y compartieron el mismo tipo de crecimiento isométrico en una de ellas; además se encontraron similitudes aparentes entre los patrones de la morfometría de P campbelli con el patrón de osificación descrito para la especie relacionada Mesocricetus auratus. Las diferencias en el crecimiento alométrico pueden representar también diferencias en el ritmo de desarrollo de acuerdo con el tipo de historia de vida de cada especie. Aquí demostramos que tanto la técnica de preparación como el método de análisis morfométricos son herramientas poderosas pero simples, para realizar estudios anatómicos y morfológicos en el laboratorio. Nuestros resultados reflejan las condiciones del desarrollo ontogenético derivados delpropio patrón de heterocronía para cada especie, y además representan la historia evolutiva de este grupo analizado. Sin embargo, consideramos que es deseable más investigación.

SUMMARY: Clearing and staining techniques have been present since 1897, However, their use in anatomical studies of adult micromammals has been limited. When using such techniques in combination with allometric method, it is possible to study articulated skeletons of micromammals, instead of relying only on the skulls, which is important in morphologically complicated groups as the rodents. Research involving multivariate allometric analysis of postcranial skeleton of rodents has been limited and confined to specific items. In this study, we analyzed and compared the morphometry of the skeleton of Neotomodon alstoni with that of Meriones unguiculatus, Phodopus campbelli and Rattus norvegicus. We applied the double staining and clearing technique in order to determine the morphometric relation between these rodents using sixty skeletal characters. We found that three species share two common correlations and one isometric, with apparent similarities between the morphometry patterns of P campbelli with the ossification pattern described for the related species Mesocricetus auratus. The differences in allometric growth could represent differences in the development stages according to the type of life history for each species. In this analysis we confirmed that both the preparation technique and morphometric analysis method, are simple yet verifiable tools for anatomical and morphological studies. Our results reflect the conditions of ontogenetic development derived from the heterochrony pattern for each species, representing the evolutionary history for this group. Therefore, as this approach continues to be discussed, ongoing research is warranted.

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Intracellular cytokine staining is a versatile technique used to analyze cytokine production in individual cells by flow cytometry. This methodology has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation, and functional parameters pertaining to responding T cells. This methodology applied after short-term culture of cells, followed by fixation and permeabilization make this technique ideal for the assessment of T-cell immune responses induced by different challenges. Here we describe an intracellular staining method followed by flow cytometry after cell stimulation with immune-relevant antigens for Lyme disease.

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Pre-clinical trials are an essential step that underpins the drug discovery, development, and safety process. During this process, animal testing is performed to determine the safety of new compounds and any potential adverse effects. Developmental toxicity tests are carried out to verify whether the drug has potential to cause congenital anomalies to the developing embryo/fetus. Chicken embryos are very useful for these purposes and present several advantages, such as low cost of production and housing, easy handling and manipulation, and rapid development in addition to sharing similarities to the human embryo at molecular, cellular, and anatomical levels. In this chapter, we bring methods for using the chicken embryo model for testing the teratogenic effects of drugs and assessing the main outcomes of them.

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Iron is an essential micronutrient for life. During the development of the seed, iron accumulates during embryo maturation. In Arabidopsis thaliana, iron mainly accumulates in the vacuoles of only one cell type, the cell layer that surrounds provasculature in hypocotyl and cotyledons. Iron accumulation pattern in Arabidopsis is an exception in plant phylogeny, most part of the dicot embryos accumulate iron in several cell layers including cortex and, in some cases, even in protodermis. It remains unknown how does iron reach the internal cell layers of the embryo, and in particular, the molecular mechanisms responsible of this process. Here, we use transgenic approaches to modify the iron accumulation pattern in an Arabidopsis model. Using the SDH2-3 embryo-specific promoter, we were able to express VIT1 ectopically in both a wild type background and a mutant vit1 background lacking expression of this vacuolar iron transporter. These manipulations modify the iron distribution pattern in Arabidopsis from one cell layer to several cell layers, including protodermis, cortex cells, and the endodermis. Interestingly, total seed iron content was not modified compared with the wild type, suggesting that iron distribution in embryos is not involved in the control of the total iron amount accumulated in seeds. This experimental model can be used to study the processes involved in iron distribution patterning during embryo maturation and its evolution in dicot plants.

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Trend of biofuel production from microalgal triacylglycerols is enhancing, because this substrate is a good sustainable and advantageous alternative to oil and gas fuel. In the present study, indigenous micro algal isolates were screened from water (n=30) and soil (n=30) samples collected from three districts of Punjab, Pakistan to evaluate their biofuel production potential. The samples were inoculated on BG - 11 agar medium plates by incubating at room temperature of 25°C providing 1000 lux for 16h light cycle followed by 8h of dark cycle for 15 d. Water samples were found to be rich in microalgae and 65.33% microalgae (49 isolates) were isolated from Faisalabad district. On the basis of microscopic morphology microalgal isolates (n=180) were selected and subjected to lipid detection by Nile red staining assay. Nile red positive isolates (n=23) were processed for biochemical (lipid, protein and carbohydrates) characterization. AIN63 isolate showed higher lipids (17.4%) content as detected by micro vanillin assay. Algal isolate AIN128 showed best protein contents (42.91%) detected by Bradford assay and AIN172 isolate showed higher carbohydrate contents (73.83%) as detected by anthrone assay. The selected algal isolates were also analyzed by Fourier transform infrared (FTIR) spectroscopy for confirmation of carbohydrate, protein and lipid analysis. These indigenous algae have the potential for in-vitro biofuel production from agricultural waste.

A tendência de produção de biocombustíveis a partir de triacilgliceróis de microalgas está aumentando, porque esse substrato é uma boa alternativa sustentável e vantajosa ao combustível de petróleo e gás. No presente estudo, isolados de microalgas indígenas foram selecionados de amostras de água (n = 30) e solo (n = 30) coletadas em três distritos de Punjab, Paquistão, para avaliar seu potencial de produção de biocombustíveis. As amostras foram inoculadas em placas de meio BG ­ 11 agar por incubação em temperatura ambiente de 25°C proporcionando 1000 lux por 16h de ciclo claro seguido de 8h de escuro por 15 dias. As amostras de água foram ricas em microalgas e 65.33% de microalgas (49 isolados) foram isolados do distrito de Faisalabad. Com base na morfologia microscópica, isolados de microalgas (n = 180) foram selecionados e submetidos à detecção de lipídios por ensaio de coloração com vermelho do Nilo. Isolados positivos para vermelho do Nilo (n = 23) foram processados ​​para caracterização bioquímica (lipídica, proteica e de carboidratos). O isolado AIN63 apresentou maior teor de lipídios (17.4%) conforme detectado pelo ensaio de microvanilina. O isolado de alga AIN128 apresentou os melhores teores de proteína (42.91%) detectados pelo ensaio de Bradford, e o isolado AIN172 apresentou maiores teores de carboidratos (73.83%), conforme detectado pelo ensaio de anthrone. Os isolados de algas selecionados também foram analisados ​​por espectroscopia de infravermelho com transformada de Fourier (FTIR) para confirmação da análise de carboidratos, proteínas e lipídios. Essas algas nativas têm potencial para a produção de biocombustíveis in vitro a partir de resíduos agrícolas.

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Abstract The study was conducted to evaluate the effect of Moringa olifera on the growth and gut health of Tilapia (Oreochromis niloticus). The feed having 30% crude protein was prepared as an experimental diet with 4%, 8% and 10% M. olifera leaf supplementation, respectively. The control diet was devoid of M. olifera leaves. The 10 weeks feeding trial was carried out on 60 fish in aquaria. Fish was fed @ 3% of body weight twice a day. Diet with the high level of inclusion of M. olifera leaves significantly increased the growth rate, Survival Rate (SR), Specific Growth Rate (SGR) and Feed Conversion Efficiency (FCE) in all treatment groups compared to the control group. Similarly, Feed Conversion Ratio (FCR) gradually decreased and found highly-significant. To check the gut health of the Tilapia, random samples were selected and dissected. Nutrient agar was used as culture media to check the growth of bacteria. Pour Plate Method was used for viable colonies count by colony counter. Through staining method, the different bacteria such as Escherichia coli, Salmonella, Shigella and Pseudomonas aeruginosa were identify abundantly in the intestine of control diet fish but less number present in treatment diets groups. These results showed that M. olifera leaves up to 10% of dietary protein can be used for Nile tilapia for significant growth and healthy gut microbiota of fish.

Resumo O estudo foi conduzido para avaliar o efeito da Moringa olifera no crescimento e saúde intestinal da tilápia (Oreochromis niloticus). A ração com 30% de proteína bruta foi preparada como dieta experimental com 4%, 8% e 10% de suplementação de folhas de M. olifera, respectivamente. A dieta controle foi desprovida de folhas de M. olifera. O ensaio de alimentação de 10 semanas foi realizado em 60 peixes em aquários. O peixe pesava 3% do peso corporal duas vezes ao dia. A dieta com alto nível de inclusão de folhas de M. olifera aumentou significativamente a taxa de crescimento, taxa de sobrevivência (SR), taxa de crescimento de sobrevivência (SGR) e eficiência de conversão alimentar (FCE) em todos os grupos de tratamento em comparação com o grupo de controle. Da mesma forma, a taxa de conversão de alimentação (FCR) diminuiu gradualmente e foi considerada altamente significativa. Para verificar a saúde intestinal da tilápia, amostras aleatórias foram selecionadas e dissecadas. O ágar nutriente foi usado como meio de cultura para verificar o crescimento das bactérias. O método da placa de Verter foi usado para a contagem de colônias viáveis ​​por contador de colônias. Através do método de coloração, diferentes como Escherichia coli, Salmonella, Shigella e Pseudomonas aeruginosa foram identificados abundantemente no intestino de peixes da dieta controle, mas em menor número nos grupos de dieta de tratamento. Esses resultados mostraram que M. olifera deixa até 10% da proteína dietética e pode ser usado para tilápia do Nilo para um crescimento significativo e microbiota intestinal saudável de peixes.

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Abstract The study was conducted to evaluate the effect of Moringa olifera on the growth and gut health of Tilapia (Oreochromis niloticus). The feed having 30% crude protein was prepared as an experimental diet with 4%, 8% and 10% M. olifera leaf supplementation, respectively. The control diet was devoid of M. olifera leaves. The 10 weeks feeding trial was carried out on 60 fish in aquaria. Fish was fed @ 3% of body weight twice a day. Diet with the high level of inclusion of M. olifera leaves significantly increased the growth rate, Survival Rate (SR), Specific Growth Rate (SGR) and Feed Conversion Efficiency (FCE) in all treatment groups compared to the control group. Similarly, Feed Conversion Ratio (FCR) gradually decreased and found highly-significant. To check the gut health of the Tilapia, random samples were selected and dissected. Nutrient agar was used as culture media to check the growth of bacteria. Pour Plate Method was used for viable colonies count by colony counter. Through staining method, the different bacteria such as Escherichia coli, Salmonella, Shigella and Pseudomonas aeruginosa were identify abundantly in the intestine of control diet fish but less number present in treatment diets groups. These results showed that M. olifera leaves up to 10% of dietary protein can be used for Nile tilapia for significant growth and healthy gut microbiota of fish.

Resumo O estudo foi conduzido para avaliar o efeito da Moringa olifera no crescimento e saúde intestinal da tilápia (Oreochromis niloticus). A ração com 30% de proteína bruta foi preparada como dieta experimental com 4%, 8% e 10% de suplementação de folhas de M. olifera, respectivamente. A dieta controle foi desprovida de folhas de M. olifera. O ensaio de alimentação de 10 semanas foi realizado em 60 peixes em aquários. O peixe pesava 3% do peso corporal duas vezes ao dia. A dieta com alto nível de inclusão de folhas de M. olifera aumentou significativamente a taxa de crescimento, taxa de sobrevivência (SR), taxa de crescimento de sobrevivência (SGR) e eficiência de conversão alimentar (FCE) em todos os grupos de tratamento em comparação com o grupo de controle. Da mesma forma, a taxa de conversão de alimentação (FCR) diminuiu gradualmente e foi considerada altamente significativa. Para verificar a saúde intestinal da tilápia, amostras aleatórias foram selecionadas e dissecadas. O ágar nutriente foi usado como meio de cultura para verificar o crescimento das bactérias. O método da placa de Verter foi usado para a contagem de colônias viáveis por contador de colônias. Através do método de coloração, diferentes como Escherichia coli, Salmonella, Shigella e Pseudomonas aeruginosa foram identificados abundantemente no intestino de peixes da dieta controle, mas em menor número nos grupos de dieta de tratamento. Esses resultados mostraram que M. olifera deixa até 10% da proteína dietética e pode ser usado para tilápia do Nilo para um crescimento significativo e microbiota intestinal saudável de peixes.

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Abstract The prevalence of gingivitis is substantial within the general population, necessitating rigorous oral hygiene maintenance. Objective This study assessed a Garcinia indica (GI) fruit extract-based mouthrinse, comparing it to a 0.1% turmeric mouthrinse and a 0.2% Chlorhexidine (CHX) mouthrinse. The evaluation encompassed substantivity, staining potential, antimicrobial efficacy and cytocompatibility. Methodology The study employed 182 tooth sections. For antimicrobial analysis, 64 extracted human teeth coated with a polymicrobial biofilm were divided into four groups, each receiving an experimental mouthrinse or serving as a control group with distilled water. Microbial reduction was assessed through colony forming units (CFU). Substantivity was evaluated on 54 human tooth sections using a UV spectrophotometer, while staining potential was examined on 64 tooth sections. Cytocompatibility was tested using colorimetric assay to determine non-toxic levels of 0.2% GI fruit extract, 0.1% Turmeric, and 0.2% CHX. Results Data were analysed with one-way ANOVA (α=0.05). Cell viability was highly significant (p<0.001) in the 0.2% GI group (64.1±0.29) compared to 0.1% Turmeric (40.2±0.34) and 0.2% CHX (10.95±1.40). For antimicrobial activity, both 0.2% GI (20.18±4.81) and 0.2% CHX (28.22±5.41) exhibited no significant difference (P>0.05) at end of 12 hours. However, 0.1% Turmeric showed minimal CFU reduction (P<0.001). Substantivity results at 360 minutes indicated statistically significant higher mean release rate in 0.1%Turmeric (12.47±5.84 ) when compared to 0.2% GI (5.02±3.04) and 0.2% CHX (4.13±2.25) (p<0.001). The overall discoloration changes (∆E) were more prominent in the 0.2% CHX group (18.65±8.3) compared to 0.2% GI (7.61±2.4) and 0.1% Turmeric (7.32±4.9) (P<0.001). Conclusion This study supports 0.2% GI and 0.1% Turmeric mouth rinses as potential natural alternatives to chemical mouth rinses. These findings highlight viability of these natural supplements in oral healthcare.

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OBJECTIVE: We evaluated the effects of crocin supplementation during culture of intact and half-destroyed four-cell mouse embryos. Outcomes measured included rate of cleavage arrest, blastocyst formation, and blastocyst cell number. METHODS: We used laser to create two zonal holes without blastomere destruction in Groups 1 (n=100) and 2 (n=100), and to destroy two of the four blastomeres in Groups 3 (n=150) and 4 (n=150). Embryos were cultured in groups of ten in drops of medium without (Groups 1 and 3) or with 20 µg/ml of crocin supplementation (Groups 2 and 4). RESULTS: Embryos in Groups 1 and 2 had no difference in the rate of cleavage arrest (6.0% vs. 7.0%, respectively; p=0.774) or blastocyst formation (89.0% vs. 86.0%, respectively; p=0.521). Neither was there a difference in the number of cells in the blastocysts (99.6±23.5 vs. 95.6± 8.2, respectively, p=0.83). Half-destroyed embryos cultured in crocin-supplemented medium (Group 4) had a lower rate of cleavage arrest (14.7% vs. 30.0%, p=0.001), and a higher rate of blastocyst formation (51.3% vs. 37.3%, p=0.015), than those in non-supplemented medium (Group 3). In blastocysts derived from half-destroyed embryos, there was no difference in the number of cells in ICM (14.5±3.9 vs. 13.7±2.9, p=0.285), TE (45.2±12.3 vs. 46.0±13.3, p=0.764), or total cells (59.7±12.2 vs. 59.7±14.8, respectively, p=0.990) among the two groups. CONCLUSIONS: Crocin supplementation during in vitro development of impaired embryos improved their development, but had no effect on intact embryos.

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Macroscopic staining in anatomical samples of the central nervous system is a technique that has been used for decades to achieve better differentiation of multiple gray matter structures, such as the cortex, basal ganglia, and cerebellar nuclei. Staining methods are based on using the different components of the brain, mainly the lipids present in the white matter. These techniques have been progressively forgotten while computer renderings are increasing; however, as a primary exposure to surgical anatomy, stained brain specimens are considered a helpful tool. We aim to summarize different staining techniques, their principles, and their current applications for neuroanatomy learning purposes. In total, four gray matter staining protocol descriptions (Mulligan's, Roberts's, Alston's, and Prussian Blue) were performed, as well as Likert scale surveys of second-year medical students about their perceptions of the stained sections. The results showed that the different macroscopic stains for brain tissue are based on lipid and reactant interactions, intending to increase the white matter (WM) and gray matter (GM) contrast. The search also showed that most staining protocols would take 2 days to develop. Efficient preservation options include submerging the sections in formaldehyde solutions, formaldehyde-free solutions, ethanol, or applying plastination techniques. Based on the student's perspective, the stained slices seem to be a valuable alternative to facilitate the study and identification of the basal ganglia and their relationships with the white matter (from 51.2 to 72% based on the Likert scale) compared with the non-stained sections. In conclusion, macroscopic staining of brain tissue continues to be a valuable tool for comprehensively studying the brain. Further research is needed to determine the efficacy of stained specimens as teaching tools.

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Background: The objective of the current study was comparing the impact of two staining techniques on semen morphological parameters and their influence on patient diagnosis. The ideal staining method should preserve cell integrity while providing detailed information. Methods: Semen samples from fifty men were stained using Diff-Quick or Spermac methods. Morphological parameters were classified based on the Tygerberg criteria, and final diagnosis was according to WHO manual guidelines. Statistical analysis was performed through conducting paired t-tests or Wilcoxon rank-sum tests, with GLIMMIX and Fisher's exact test for determining the significance (p≤0.05). Results: Both staining methods highlighted head and tail regions, with Spermac offering better visualization of the midpiece. Spermac demonstrated fewer normal spermatozoa (2.8±0.3%) compared to Diff-Quick (3.98±0.4%; p=0.0385). Midpiece abnormalities were more evident with Spermac (55.7±2.1%) than Diff-Quick (24.8±2.0%; p<0.0001). No significant difference was found in head and tail abnormalities (p>0.05). Conclusion: Diff-Quick staining resulted in a higher proportion of normal spermatozoa, primarily due to its midpiece evaluation. The choice of staining method significantly impacts the diagnosis of infertile males. These findings have important implications for clinical practice and future research, suggesting the need for further investigations to assess different staining methods and determine optimal diagnostic thresholds.

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Neonatal mortality in dogs reaches up to 40%. Due to the high rates, promptly detecting the causes and preventing newborns from dying are extremely important. Vitality evaluation, blood parameters, and the degree of meconium staining on the skin are valuable resources in canine perinatology. In this study, 435 puppies from 85 bitches close to parturition were recruited and divided into four quartiles according to the puppy's birth weight: Q1 (127-200 g) n = 110 puppies, Q2 (201-269 g) n = 108 puppies, Q3 (270-388 g) n = 108 puppies, and Q4 (389-464 g) n = 109 puppies. This experimental article aimed to report the effect of birth weight on the blood profile variables, the vitality of newborn puppies, and the meconium staining degree, integrating these three aspects. It was concluded that the weight of newborns was correlated with the degree of meconium staining, presenting more cases of severe meconium staining in the puppies of the highest birth weight group. The weight of the newborns was correlated with a higher number of stillbirths and alterations in the blood variables, showing the most severe cases of metabolic acidosis, hypoxia, and hypoglycemia in the puppies of the Q4 quartile. On the contrary, no statistically significant correlations were found between the weight of newborns and vitality. Nevertheless, the analysis of the results showed that the most vigorous puppies were found at Q1; however, at minute 60 after birth (AB), all the puppies in the four quartiles standardized their vitality scores.

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Chromosome banding based on base-specific fluorochromes, mainly double staining with chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI), has been widely used since the 1970s. This technique allows the differential staining of distinct types of heterochromatin. Afterward, the fluorochromes can be easily removed and leave the preparation ready for sequential procedures such as FISH or immunodetection. Interpretations of similar bands obtained with different techniques, however, merit certain caution. Here we present a detailed protocol for CMA/DAPI staining optimized for plant cytogenetics and call attention to the most common sources of misinterpretation of DAPI bands.

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