Comparative analysis of SARS-CoV-2 variants Alpha (B.1.1.7), Gamma (P.1), Zeta (P.2) and Delta (B.1.617.2) in Vero-E6 cells: ultrastructural characterization of cytopathology and replication kinetics

Abstract This study compares the effects of virus-cell interactions among SARS-CoV-2 variants of concern (VOCs) isolated in Brazil in 2021, hypothesizing a correlation between cellular alterations and mortality and between viral load and transmissibility. For this purpose, reference isolates of Alpha, Gamma, Zeta, and Delta variants were inoculated into monolayers of Vero-E6 cells. Viral RNA was quantified in cell supernatants by RT‒PCR, and infected cells were analyzed by Transmission Electron Microscopy (TEM) for qualitative and quantitative evaluation of cellular changes 24, 48, and 72 hours postinfection (hpi). Ultrastructural analyses showed that all variants of SARS-CoV-2 altered the structure and function of mitochondria, nucleus, and rough endoplasmic reticulum of cells. Monolayers infected with the Delta variant showed the highest number of modified cells and the greatest statistically significant differences compared to those of other variants. Viral particles were observed in the cytosol and the cell membrane in 100 % of the cells at 48 hpi. Alpha showed the highest mean particle diameter (79 nm), and Gamma and Delta were the smallest (75 nm). Alpha and Gamma had the highest particle frequency per field at 48 hpi, while the same was observed for Zeta and Delta at 72 hpi and 24 hpi, respectively. The cycle threshold of viral RNA varied among the target protein, VOC, and time of infection. The findings presented here demonstrate that all four VOCs evaluated caused ultrastructural changes in Vero-E6 cells, which were more prominent when infection occured with the Delta variant.

Evaluation of Structural and Compositional Changes on the Root Surface Exposed to Diode Laser (810 nm): An In?vitro Study

Background: The laser therapy has been used as an adjuvant for conventional periodontal disease as they exhibit a bactericidal effect on scaling and root planning by its thermal and photo disruptive effects on the pathogens. This study focuses on the structural and compositional changes induced on the root surfaces of teeth following diode laser (DL) application with increasing quantum of exposure time. Objective: The objective of this study was to evaluate the structural and compositional changes on the root surface of extracted human permanent teeth after application of DLs (810 nm) with varying time interval. Materials and Methods: Twenty samples of single?rooted periodontally compromised extracted teeth were utilized for this study. Root planning was done and the roughness caused by the instrumentation was measured using profilometric analysis. Then, the samples were divided into four groups, with DL application time: Group 1 – laser application for 15 s, Group 2 – laser application for 30 s, Group 3 – laser application for 45 s, and Group 4 – laser application for 60 s. A scanning electron microscope was used to examine the cemental surface and energy?dispersive X?ray analysis software assesses the compositional changes of the teeth in each group. Results: This study reveals that on exposure of DL (810 nm) on the root surface when time of exposure increases, there were relative increases in surface irregularities and charring. There were significant changes in the chemical composition of the tooth surface.

Monkeypox (Mpox) virus isolation and ultrastructural characterisation from a Brazilian human sample case

BACKGROUND According to the last 2023 Monkeypox (Mpox) Outbreak Global Map from the Centres for Disease Control and Prevention (CDC), more than 100 countries with no Mpox infection report cases. Brazil stands out in this group and is the second country with the highest number of cases in the last outbreak. OBJECTIVE To contribute to knowledge of the virus infection effects in a cellular model, which is important for diagnosis infections not yet included in a provider´s differential diagnosis and for developing viral inhibition strategies. METHODS We describe a virus isolation protocol for a human clinical sample from a patient from Brazil, the viral growth in a cell model through plaque forming units (PFU) assay, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM). FINDINGS We follow the viral isolation in Vero cell culture from a Mpox positive clinically diagnosed sample and show the infection effects on cellular structures using a TEM. MAIN CONCLUSIONS Understanding the impact of viral growth on cellular structures and its replication kinetics may offer better strategies for the development of new drugs with antiviral properties.

Vitamin E suppresses aortic ultrastructural alterations induced by toxic doses of monosodium glutamate / La vitamina E suprime las alteraciones ultraestructurales aórticas inducidas por dosis tóxicas de glutamato monosódico

SUMMARY: An association between certain food additives and chronic diseases is reported. Current study determined whether administering toxic doses of the food additive monosodium glutamate (MSG) into rats can induce aortopathy in association with the oxidative stress and inflammatory biomarkers upregulation and whether the effects of MSG overdose can be inhibited by vitamin E. MSG at a dose of (4 mg/kg; orally) that exceeds the average human daily consumption by 1000x was administered daily for 7 days to the rats in the model group. Whereas, rats treated with vitamin E were divided into two groups and given daily doses of MSG plus 100 mg/ kg vitamin E or MSG plus 300 mg/kg vitamin E. On the eighth day, all rats were culled. Using light and electron microscopy examinations, a profound aortic injury in the model group was observed demonstrated by damaged endothelial layer, degenerated smooth muscle cells (SMC) with vacuoles and condensed nuclei, vacuolated cytoplasm, disrupted plasma membrane, interrupted internal elastic lamina, clumped chromatin, and damaged actin and myosin filaments. Vitamin E significantly protected aorta tissue and cells as well as inhibited MSG-induced tissue malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). The highest used vitamin E dosage was more effective. Additionally, a significant correlation was observed between the aortic injury degree and tissue MDA, TNF-α, IL-6, and superoxide dismutase (SOD) levels (p=0.001). Vitamin E effectively protects against aortopathy induced by toxic doses of MSG in rats and inhibits oxidative stress and inflammation.

RESUMEN: Se reporta una asociación entre ciertos aditivos alimentarios y enfermedades crónicas. El objetivo de este estudio fue determinar si la administración de dosis tóxicas del aditivo alimentario glutamato monosódico (MSG) en ratas puede inducir aortopatía en asociación con el estrés oxidativo y la regulación positiva de los biomarcadores inflamatorios y si el efecto de una sobredosis de MSG se puede inhibir con vitamina E. Se administró MSG diariamente durante 7 días una dosis de (4 g/kg; por vía oral) que excede el consumo diario humano promedio, en 1000x a las ratas del grupo modelo. Mientras que las ratas tratadas con vitamina E se dividieron en dos grupos y se administraron dosis diarias de MSG más 100 mg/kg de vitamina E o MSG más 300 mg/kg de vitamina E. Todas las ratas fueron sacrificadas en el octavo día. Usando exámenes de microscopía óptica y electrónica, se observó una lesión aórtica profunda en el grupo modelo demostrada por una capa endotelial dañada, células musculares lisas degeneradas (SMC) con vacuolas y núcleos condensados, citoplasma vacuolado, membrana plasmática rota, lámina elástica interna interrumpida, cromatina agrupada y filamentos de actina y miosina dañados. La vitamina E protegió significativamente el tejido y las células de la aorta, además de inhibir el malondialdehído tisular (MDA) inducido por MSG, la interleucina-6 (IL-6) y el factor de necrosis tumoral alfa (TNF-α). La dosis más alta de vitamina E utilizada fue más efectiva. Además, se observó una correlación significativa entre el grado de lesión aórtica y los niveles tisulares de MDA, TNF-α, IL-6 y superóxido dismutasa (SOD) (p=0,001). La vitamina E efectivamente protege contra la aortopatía inducida por dosis tóxicas de MSG en ratas e inhibe el estrés oxidativo y la inflamación.

Morphometry of bovine blastocysts produced in vitro in culture media with antioxidants cysteamine or oily extract of Lippia origanoides / [Morfometria de blastocistos bovinos produzidos in vitro em meio de cultura com antioxidantes cisteamina ou extrato oleoso de Lippia origanoides]

This study aimed to evaluate the ultrastructural morphometry of bovine embryos produced in vitro grown at different concentrations of antioxidants. After in vitro maturation and fertilization, the presumptive zygotes were assigned into five treatments. T1) without the addition of any antioxidants (negative control); T2) addition of 50µM/mL cysteamine; and T3, T4 and T5) adding 2.5µg/mL, 5.0µg/mL or 10.0µg/mL of the antioxidants derived from the oily extract from Lippia origanoides, respectively. On D7 of culture, the embryos in the blastocyst stage were fixed and prepared for electron transmission microscopy. These were evaluated for the proportion of cytoplasm-to-nucleus, cytoplasm-to-mitochondria, cytoplasm-to-vacuoles, cytoplasm-to-autophagic vacuoles and cytoplasm-to-lipid droplets. Blastocysts cultured in media containing oily extract of Lippia origanoides presented morphological characteristics such as high cell:mitochondria ratio and low cell:vacuoles and cell:autophagic vacuole ratio, possibly been morphological indicators of embryonic quality. Inner cell mass (ICM) from blastocysts cultured in media without any antioxidants had the highest cell:vacuole ratio. Similar results were found in the trophectoderm (TE) cells of blastocysts from treatment 2. Embryo culture media supplemented with antioxidants derived from Lippia origanoides oil produced embryos with a higher cytoplasmic proportion of organelles, such as mitochondria. Also, treatments without any antioxidants or with the addition of cysteamine presented cytoplasmic vacuolization, a characteristic related to production of poor-quality embryos.(AU)

Este estudo teve como objetivo avaliar a morfometria ultraestrutural de embriões bovinos produzidos in vitro e cultivados em diferentes concentrações de antioxidantes. Após a maturação e a fertilização in vitro, os possíveis zigotos foram divididos em cinco tratamentos: T1) sem adição de antioxidantes (controle negativo); T2) adição de 50µM/mL de cisteamina; e T3, T4 e T5) adição de 2,5µg/mL, 5,0µg/mL ou 10,0µg/mL dos antioxidantes derivados do extrato oleoso de Lippia origanoides, respectivamente. No D7 de cultivo, os embriões em estágio de blastocisto foram fixados e preparados para microscopia eletrônica de transmissão. Estes foram avaliados para a proporção entre citoplasma e núcleo, citoplasma e mitocôndria, citoplasma e vacúolos, citoplasma e vacúolos autofágicos e citoplasma e gotículas lipídicas. Blastocistos cultivados em meio contendo extrato oleoso de Lippia origanoides apresentaram características morfológicas como alta relação célula:mitocôndria e baixa relação célula:vacúolos e célula:vacúolo autofágico, possíveis indicadores morfológicos de qualidade embrionária. A massa celular interna (MCI) de blastocistos cultivados em meio sem quaisquer antioxidantes teve a maior razão célula:vacúolo. Resultados semelhantes foram encontrados nas células do trofectoderma (TE) de blastocistos do tratamento 2. Portanto, o meio de cultivo embrionário suplementado com antioxidantes derivados do óleo de Lippia origanoides produziu embriões com maior proporção citoplasmática de organelas, como mitocôndrias. Além disso, tratamentos sem antioxidantes ou com adição de cisteamina apresentaram vacuolização citoplasmática, característica relacionada à produção de embriões de baixa qualidade.(AU)

Progress on electron microscopy diagnosis on Banff classification for renal allograft pathology / 器官移植

With the development of organ transplantation in clinical practice, allograft pathology has been constantly developing and advancing. The convening of Banff conference on allograft pathology and the establishment of Banff classification on allograft pathology (Banff classification) are pivotal milestones in the development of international allograft pathology. Since then, Banff classification on pathological diagnosis of various transplant organs have been continually updated and improved. Ultrastructural pathological observation by electron microscope plays an irreplaceable role in the early diagnosis of antibody-mediated rejection, recurrent disease and de novo disease of renal allograft. Early detection and rational treatment help to maintain the long-term survival of renal allograft and reduce the failure of renal allograft. In this article, the basic definition of electron microscope and the ultrastructural pathological diagnosis, the research history and main progress on electron microscope diagnosis on Banff classification for renal allograft pathology were introduced, and typical pathological changes, specific terminology and diagnostic criteria of electron microscope diagnosis on renal allograft biopsy were summarized, aiming to provide reference for clinical and basic research of organ transplantation.

Age-associated hemogram and ultrastructural leukocyte morphology in Pygocentrus nattereri (Characiformes: Serrasalmidae) from the Brazilian Pantanal

Pygocentrus nattereri is a carnivorous fish widely distributed in the Brazilian wetland and occupies different river strata. Based on the sexual maturity, the standard length (SL) has been used to categorize age and physiological attributes. However, hematological parameters and their relationship with age and growth features are unknow. Here, the aim was to evaluate the hematological profile and leukocyte data of juvenile and adult specimens, associating it with SL. Specimens of both sexes were weighted, and SL were measured for classify as juveniles (SL ≤16.0 cm) and adults (SL ≥16.1 cm). Blood was collected to perform the hemogram, for leukocyte quantification and ultrastructural descriptions. In general, hemogram values and leukocyte, lymphocyte, and monocyte counts were high in juveniles. Hemogram variables were negatively correlated with SL. Thrombocyte and neutrophil counts were higher in adults than in juveniles. SL was correlated with leukogram variables. Ultrastructural analysis showed that granular leukocyte count in P. nattereri was similar another fishes. PAS-positive granular leukocytes are slightly smaller than neutrophils and are characterized by dense elongated and semi-lunar shapes in the cytoplasm. Our results suggest that age-based SL may be useful for the analysis of the relationship between P. nattereri health status and wetland aquatic environments.(AU)

Pygocentrus nattereri é um peixe carnívoro amplamente distribuído no pantanal brasileiro e ocupa diferentes estratos de rios. Com base na maturidade sexual, o comprimento padrão (CP) tem sido usado para categorizar a idade e os atributos fisiológicos. No entanto, os parâmetros hematológicos e sua relação com a idade e características de crescimento são desconhecidos. O objetivo foi avaliar o perfil hematológico e leucocitário de espécimes juvenis e adultos, associando-o ao CP. Espécimes de ambos os sexos foram pesados e os CPs foram medidos para classificação em juvenis (CP ≤16,0 cm) e adultos (CP ≥16,1 cm). Foi coletado sangue para realização do hemograma, perfil e morfologia dos leucócitos. Em geral, os valores do hemograma e as contagens de leucócitos, linfócitos e monócitos foram maiores nos jovens. A contagem de trombócitos e neutrófilos foi maior em adultos do que em jovens. A análise ultraestrutural mostrou que a contagem de leucócitos granulares em P. nattereri foi semelhante a outros peixes. Os leucócitos granulares PAS-positivos são menores do que os neutrófilos, sendo caracterizados por formas alongadas e semilunares densas no citoplasma. Nossos resultados sugerem que CP é útil para a análise da relação entre o estado de saúde de P. nattereri em ambientes aquáticos alagados.(AU)

Collagenofibrotic glomerulopathy – A rare disease diagnosed with the aid of transmission electron microscopy

Collagenofibrotic glomerulopathy (CFG) is a rare idiopathic kidney disease characterized by abnormal deposition of atypical Type III collagen fibers in the glomerulus causing subendothelial and mesangial expansion, manifesting as progressive renal dysfunction accompanied by proteinuria. The majority of CFG cases reported in literature are from Japan where this disease entity was initially recognized. There is an increased awareness and diagnosis of this rare renal disease in India with the recent increase in utilization of electron microscopy (EM) in clinical diagnostic settings. We describe a 28-year-old Bangladeshi woman who presented with hypertension and nephrotic range proteinuria not amenable to treatment with steroids and cyclophosphamide, whose renal biopsy demonstrated diagnostic ultrastructural features of CFG. This illustrative case is presented to highlight the role of EM analysis for diagnostic accuracy in renal biopsy evaluation in addition to demonstrating the unusual renal biopsy findings of this rare entity.

Ginger extract ameliorates renal damage in high fat diet-induced obesity in rats: biochemical and ultrastructural study / El extracto de jengibre mejora el daño renal en la obesidad inducida por la dieta alta en grasas en ratas: estudio bioquímico y ultraestructural

Obesity is a modifiable risk factor for the development and progression of kidney disease. Obesity may harm kidneys in individuals without hypertension, diabetes, or pre-existing renal disease. Ginger, Zingiber officinale, has many beneficial pharmaceutical benefits. This study aimed to evaluate the Zingiber officinale protective effect against obesity complications which induced by high fat diet and caused renal dysfunctions. The study period was two months, and the experimental animals' groups were four, 80 Wistar rats were appropriated similarly 20 animals/group: control group; ginger extract group (GE); high-fat diet (HFD); and GE+HFD group. Body and fat weight, creatinine, leptin, TNF-α, total antioxidants, renal histopathological and ultrastructure were investigated. Rats in group of HFD showed a significant increase (P<0.05) in the body and fat weights, creatinine, leptin and TNF-α, and significant decrease (P<0.05) in total antioxidants (TAS). Ginger administration significantly showed the protective restoring the altered parameters. Furthermore, rats co-treated with ginger extract improved the histopathological and ultrastructural renal injury induced by obesity. The study concluded that the ginger extract used could suppress and decrease the renal damage induced by high-fat diet as it possesses potential medicinal values.

La obesidad es un factor de riesgo modificable para el desarrollo y la progresión de la enfermedad renal. La obesidad puede dañar los riñones en personas sin hipertensión, diabetes o enfermedad renal preexistente. El jengibre, Zingiber officinale, tiene muchos beneficios farmacéuticos. Este estudio tuvo como objetivo evaluar el efecto protector de Zingiber officinale en las complicaciones de la obesidad inducida por una dieta alta en grasas y las enfermedad renal. El período de estudio fue de dos meses, y los grupos de animales experimentales fueron cuatro, se asignaron 80 ratas Wistar de manera similar, 20 animales por grupo: grupo de control; grupo de extracto de jengibre (GE); dieta alta en grasas (DAG); y el grupo GE + DAG. Se evaluó el peso corporal y la grasa, creatinina, leptina, TNF-α, antioxidantes totales, histopatología renal y ultraestructura. Las ratas en el grupo de DAG mostraron un aumento significativo (P<0,05) en el peso corporal y de grasa, creatinina, leptina y TNF-a, y una disminución significativa (P<0,05) en los antioxidantes totales. La administración de jengibre mostró una protección significativa restaurando los parámetros alterados. Además, las ratas tratadas conjuntamente con extracto de jengibre mejoraron la lesión renal histopatológica y ultraestructural inducida por la obesidad. El estudio concluyó que el extracto de jengibre podría suprimir y disminuir el daño renal inducido por la dieta alta en grasas, ya que posee potenciales valores medicinales.

In vivo therapeutic effects of albendazole in combination with IFN-α on cystic echinococcosis in mice / 中国人兽共患病学报

This study aimed to evaluate the in vivo efficacy of combined ABZ-interferon (IFN)-α treatment for CE in mice.After 5 months of secondary infection with protoscoleces,mice were randomly allocated into four groups:ABZ-treated group,IFNαtreated group,ABZ+IFN-α group and untreated control group.Drugs in diverse treated groups were respectively administered for 2 months,of which,sera were respectively collected in 0 d,7 d,14 d,28 d,36 d,48 d,and 60 d.Mice were then euthanized and associated indications were investigated to evaluate the therapeutic efficacy.Results showed that ABZ+IFN-α induced a significant reduction of the number,size as well as weight of cysts,compared with that in ABZ (P＜0.05) or untreated group (P＜0.01) respectively.This effect was associated with ultrastructural modification of the cyst in ABZ+IFN-α group.Interestingly,significant decrease of IL (interleukin)-10 in serum and in vitro production by spleen cells with ABZ+ IFN-α treatment was observed in comparison with untreated control (P＜0.01).Serum IgE,IgG and subsets were respectively decreased in ABZ+IFN-α treatment,compared with that in control group (P＜0.01).Our findings demonstrated that combination of ABZ with IFN-α may contribute to an efficient therapeutic regimen of human and animal CE.

Ameliorative effect of vitamin B12 on seminiferous epithelium of cimetidine-treated rats: a histopathological, immunohistochemical and ultrastructural study / 대한해부학회지

Cimetidine is an H2 receptor antagonist that has an antiandrogenic effect. It intervenes with the conversion of testosterone into estrogen in the Sertoli cells with accompanying testicular structural changes. In the present study, the microscopic and the ultrastructural changes induced by cimetidine and the effect of vitamin B12 as a protective agent on rat testes were studied. Immunoexpression of estrogen receptor β (ERβ) in testes was evaluated. Twenty-four adult male rats were divided into four groups: control, cimetidine-treated, vitamin B12 treated, and combined cimetidine and vitamin B12 treated. The experimental rats were administered with cimetidine and/or vitamin B12 for 52 days. Group II rats showed marked atrophy of the seminiferous tubules with a significant increase in tubular diameter and decrease in the tubular luminal and epithelial areas. Ultrastructure of this group showed irregular Sertoli cells with basal cytoplasmic vacuolation and significantly thickened basement membrane. ERβ immunoexpression was similar to controls. Group III rats showed near normal seminiferous tubular structures with minimal cellular alterations and the immunoreactivity of the testicular sections was very close to normal. However, group IV rats showed markedly immunopositive detached cells, spermatids, and primary spermatocytes. Cimetidine interferes with the control of spermatogenesis as evidenced by microscopic and ultrastructural studies and affection of ERβ receptors and vitamin B12 has a protective action against this harmful effect.

Morpho and Cytological Differentiation of Calli of Eucalyptus grandis x Eucalyptus urophylla During Somatic Embryogenesis

ABSTRACT The aim of this study was to induce and analyze embryogenic calli from two types of explants (leaves and meristems) of the hybrid Eucalyptus grandis x Eucalyptus urophylla. Leaves and meristems of plants kept in a nursery were disinfected and inoculated in Petri dishes containing MS culture medium supplemented with different concentrations of the growth regulator dicamba (1.13, 4.52, and 9.04 µM) and without it. At 60 days of culturing, the calli were analyzed by scanning electron microscopy and at 90 days were evaluated by light microscopy in regard to the embryogenic characteristics of the cells. Different type of calli were induced in leaf explants, designated as Type I with light yellow coloring, Type II with dark yellow coloring, and Type III of brown coloring; however, only Type I had embryogenic characteristics. In the meristematic explants, only one type of callus was induced, and it had embryogenic characteristics. At 90 days of culturing, the formation of somatic embryos in the different embryogenic stages was observed and the formation of procambium, protoderm, and ground meristem tissues. At 150 days of culturing, the concentration of 1.13 µM of dicamba was prominent in the formation of somatic embryos in the different embryogenic stages.

Immunohistochemical, lectin histochemical and ultrastructural studies of canine transmissible venereal tumor in Brazil / Estudos imuno-histoquímico, lectino-histoquímico e ultraestrutural do Tumor Venéreo Transmissível Canino no Brasil

Canine transmissible venereal tumor (CTVT) is a naturally occurring contagious round-cell neoplasia, with poorly understood origin and transmission. This study aims to further investigate the tumor nature through immunohistochemistry, lectin histochemistry and transmission electron microscopy (TEM) analysis, and to provide support for diagnostic and differential diagnoses of CTVT. Immunohistochemistry was performed in 10 genital and six exclusively extragenital tumors, which were previously diagnosed by citology and histopathology. CTVT samples were incubated with biotinylated antibodies to specific membrane and cytoplasmic antigens (anti-lysozyme, anti-macrophage, anti-vimentin, anti-CD18, monoclonal anti-CD117, monoclonal anti-CD3, polyclonal anti-CD117, polyclonal CD3 and anti-CD79a), followed by the avidin-biotin-peroxidase complex technique. The lectins Con A, DBA, SBA, PNA, UEA-1, WGA, sWGA, GSL, JSA, PSA, PHA-L, PHA-E and RCA were additionally tested in four genital CTVTs and TEM was performed in eight genital tumors. The anti-vimentin antibody revealed strong immunoreactivity to neoplastic cells in all the assessed samples (16/16). The polyclonal anti-CD3 antibodies showed moderate to strong immunoreactivity in fourteen (14/16) and the polyclonal anti-CD117 in fifteen cases (15/16). There was no immunoreactivity to anti-lysozyme, anti-macrophage, anti-CD18, monoclonal anti-CD117, monoclonal anti-CD3 and anti-CD79a antibodies. At lectin histochemistry, it was observed strong staining of tumor cells to Con-A, PHA-L and RCA. There was no histopathological and immunoreactivity differences between genital and extragenital CTVTs. These findings do not support the hypothesis of histiocytic origin of CTVT. In contrast, the lectin histochemical results were similar to cells from lymphoid/myeloid origin.(AU)

O Tumor Venéreo Transmissível Canino (CTVT) é uma neoplasia de células células redondas, contagiosa, com origem e transmissão ainda mal compreendidas. Com a finalidade de aprofundar a investigação sobre a natureza (origem) do TVTC, bem como fornecer subsídios para o estabelecimento do diagnóstico e diagnóstico diferencial, realizaram-se avaliações imuno-histoquímica, lectino-histoquímica e ultraestrutural de TVTC(s). A avaliação imuno-histoquímica foi feita em 10 TVTCs genitais e em 6 exclusivamente extragenitais previamente diagnosticados através de citologia e da histopatologia. Os TVTCs foram testados para reagentes específicos de antígenos de membrana e citoplasmáticos (anti-lisozima, anti-macrófago, anti-vimentina, anti-CD18, anti-CD3, anti-CD79, anti-CD117) com utilização da técnica complexo avidina-biotina-peroxidase. Adicionalmente, foram utilizadas as lectinas Con A, DBA, SBA, PNA, UEA-1, WGA, sWGA, GSL, SJA, PSA, PHA-L, PHA-E e RCA em quatro TVTCs genitais. Microscopia eletrônica foi realizada em oito TVTC genitais. Em 100% dos tumores testados (16/16) com anticorpo anti-vimentina (mono e policlonal) houve forte imuno-reatividade. Não houve reatividade para os anticorpos anti-lisozima, anti-macrófago, anti-CD18, anti-CD3, anti-CD79a e anti-CD117 quando empregamos anticorpos monoclonais, entretanto, com a utilização de anticorpos policlonais verificou-se marcação dos tumores com os anticorpos anti-CD3 e anti-CD117. Na avaliação lectino-histoquímica foi verificada forte marcação das células tumorais com Con-A, PHA-L e RCA. Não houve diferença histopatológica e de imuno-reatividade entre os TVTCs genitais e extragenitais. Estes achados não corroboram com a hipótese da origem histiocítica do CTVT (ausência de reatividade dos anticorpos anti-lisozima, anti-macrófago e anti-CD18), entretanto, os resultados da avaliação lectino-histoquímica foram em parte similares aos obtidos quando células de origem linfóide/mielóide (ConA, PHA-L e RCA) foram analisadas (Gimeno et al. 1995).(AU)

Identification of ultrastructural and functional damages in sperm from six-banded armadillos (Euphractus sexcinctus) due to cryopreservation / Identificação de danos ultraestruturais e funcionais em espermatozoides de tatus-peba (Euphractus sexcinctus) oriundos da criopreservação

The aim of the study was to cryopreserve the semen of six-banded armadillos (Euphractus sexcinctus) in Tris-yolk and glycerol diluent, and to determine the damage caused by the freezing-thawing process, using fluorescent markers and ultrastructural analysis. Semen samples (n=11) collected from 4 adult six-banded armadillos by electroejaculation were cryopreserved in Tris diluent plus 20% egg yolk and 3% glycerol, in a fast freezing curve. Classical analysis of samples was performed after dilution, refrigeration and thawing, followed by fluorescence analysis, using a combination of fluorescent probes to assess membrane integrity (propidium iodide - PI and Hoechst - H342), and mitochondrial activity (CMXRos - Mito Tracker Red®). We also used the ultrastructural analysis to verify possible morphological alterations caused by cryoinjuries. When compared with fresh samples, we verified a significant decline in all the armadillos' semen parameters after thawing, in which only 6.1% motile sperm were found. However, the percentage of sperm which remained with viable (13%) and functional (24.7%) membranes after thawing suggests that some cells could be live but immotile. Analysis using fluorescent markers revealed that the mitochondria of armadillos' sperm is highly sensible to the freezing protocol and the findings through ultrastructure analysis proved this statement. Additionally, the images obtained by transmission electron microscopy revealed that frozen-thawed sperm presented damaged plasma membrane, nuclear modifications as changes in chromatin and acrossomal changes relative to sperm capacitation. In conclusion, this study is the first attempt to cryopreserve the semen of an armadillo species, and to help us to identify critical points on the freezing-thawing procedure in order to improve the protocol.(AU)

O objetivo deste estudo foi criopreservar o sêmen de tatus-peba (Euphractus sexcinctus) em diluente Tris-gema e glicerol, e determinar os danos causados pelo processo de congelação-descongelação, utilizando marcadores fluorescentes e análise ultraestrutural. As amostras de sêmen (n=11) coletadas de 4 tatus-peba adultos por eletroejaculação foram criopreservadas em diluente Tris acrescido de 20% de gema de ovo e 3% de glicerol, em curva rápida de congelação. A análise clássica das amostras foi realizada após a diluição, refrigeração e descongelação, seguida por análise de fluorescência, utilizando uma combinação de sondas fluorescentes para avaliar a integridade da membrana (Iodeto de Propídio - PI e Hoechst - H342), e a atividade mitocondrial (CMXRos - Mito Tracker RED®). Foi também utilizada a análise ultraestrutural para verificar possíveis alterações morfológicas causadas pela crioinjúria. Quando comparadas com as amostras a fresco, verificou-se uma queda significativa em todos os parâmetros seminais dos tatus após a descongelação, em que apenas 6,1% de espermatozoides móveis foram encontrados. No entanto, o percentual de espermatozoides que permaneceu com membrana viável (13%) e funcional (24,7%) após a descongelação sugere que algumas células podem estar vivas, mas imóveis. Análises utilizando marcadores fluorescentes revelaram que as mitocôndrias dos espermatozoides de tatus são altamente sensíveis ao protocolo de congelação e os achados através da análise ultraestrutural comprovaram esta afirmação. Além disso, as imagens obtidas por microscopia eletrônica de transmissão revelaram que espermatozoides congelados-descongelados apresentaram membranas plasmáticas danificadas, modificações nucleares como alterações na cromatina, e alterações acrossomais relativas à capacitação espermática. Em conclusão, este estudo é a primeira tentativa de criopreservação de sêmen em uma espécie de tatu, e nos auxiliou a identificar pontos críticos no processo de congelação-descongelação, a fim de melhorar o protocolo.(AU)

Effects of sildenafil on dental pulp: immunohistochemical and ultrastructural evaluation / Efectos de sildenafil en la pulpa dental: evaluación inmunohistoquímica y ultraestructural

Sildenafil is a strong peripheral vasodilator and is used to treat cardiovascular and neurosurgery. The purpose of this study was to investigate the immunohistochemical and ultrastructural effects of sildenafil on dental pulp of rats. The study was performed with adult female Wistar-Albino rats. Control group (n= 7) were fed on standard laboratory diet until surgery. The study group (n= 7) were administered sildenafil orally with orogastric tube 10 mg·kg-1 once a day for 30 days. Each rat was anesthetized and incisor teeth were removed. This study examined the immunohistochemical and ultrastructural effects of sildenafil on the dental pulp in rats. The relaxation from the vessel, endothelial cell hyperplasia, moderate degeneration of collagen fibers were observed to cause degenerative changes in odontoblast with sildenafil. In the pulp tissue long-term use sildenafil is thought to cause degeneration and new vessel formation.

El sildenafil es un vasodilatador periférico importante y se utiliza para tratar enfermedades cardiovasculares y en neurocirugía. El propósito de este estudio fue investigar los efectos inmunohistoquímicos y ultraestructurales del sildenafil sobre la pulpa dental de ratas. El estudio se realizó con ratas Wistar albinas, hembras adultas. El grupo de control (n= 7) fue alimentado con una dieta estándar de laboratorio hasta que se realizó la cirugía. El grupo de estudio (n= 7) fue tratado con sildenafil por vía oral y sonda orogástrica 10 mg·kg-1 una vez al día durante 30 días. Cada rata fue anestesiada y se extrajeron los dientes incisivos. Se examinaron los efectos inmunohistoquímicos y ultraestructurales del sildenafil sobre la pulpa dentaria. Con la administración de sildenafil se observó la relajación de los vasos, la hiperplasia de las células endoteliales y una degeneración moderada de fibras colágenas causando cambios degenerativos en los odontoblastos. En el tejido pulpar, el uso de sildenafil a largo plazo puede causar la degeneración y neoformación de vasos.

Comparative analysis in ultrastructure and histological types for congenital cystic adenomatoid malformation / 中华实用儿科临床杂志

Objective To explore relationship between imaging classification and pathological type,and the electron microscopic ultrastructure by observing the ultrastructural features of congenital cystic adenomatoid malformation (CCAM).Methods Children with CCAM in the Guangdong Women and Children Hospital from May 2014 to November 2014,were analyzed,imaging classification was performed according to enhanced Computed Tomography (CT) scanning,and the surgical specimens were stained by HE and scanning electron microscope was used to observe the tissue structures of various of CCAM.Results Eight cases were enrolled in this research (5 boys and 3 girls),and the mean age of surgery was (5.4 ± 1.1) month old.According to the features of enhanced CT scanning,there were 2 cases of the type-Ⅰ,and the other 3 cases of type-Ⅱ,and 3 cases of type-Ⅲ;According to the characteristics of the pathological types,there was 1 case of type-Ⅰ,4 cases of type-Ⅱ,and the other 3 cases of type-Ⅲ.There was 1 case which classified as type-Ⅰ by CT,however classified as type-Ⅱ by pathological typing.The findings of CT features were in complete correspondence with the pathological types in the rest cases.Electron microscopic findings showed the alveolar spaces were enlarged,the alveolar cells were reduced,the lamellar bodies were decreased and immature and microvilli were also decreased.There was no significant difference between Ⅰ and Ⅱ type under the viewpoint of electron microscope.The common features between type Ⅰ,and type Ⅱ included that the alveolar space was enlarged,the alveolar space had been pressed between enlarged alveolar space,the alveolar cell were decreased and immature,and fibrous hypertrophy was also found.Conclusions There are differences between ultrastructural analysis and clinical pathological typing which suggests the clinical importance of the electron microscopy ultrastructural analysis combined with pathology for typing.There need further clinical and laboratory studies,especially the combination of microscopy-electrical control microscope for typing with related predictive models for follow-up of CCAM.

Asymmetric cell division and its role in cell fate determination in the green alga Tetraselmis indica.

The prasinophytes (early diverging Chlorophyta), consisting of simple unicellular green algae, occupy a critical position at the base of the green algal tree of life, with some of its representatives viewed as the cell form most similar to the first green alga, the ‘ancestral green flagellate’. Relatively large-celled unicellular eukaryotic phytoflagellates (such as Tetraselmis and Scherffelia), traditionally placed in Prasinophyceae but now considered as members of Chlorodendrophyceae (core Chlorophyta), have retained some primitive characteristics of prasinophytes. These organisms share several ultrastructural features with the other core chlorophytes (Trebouxiophyceae, Ulvophyceae and Chlorophyceae). However, the role of Chlorodendrophycean algae as the evolutionary link between cellular individuality and cellular cooperation has been largely unstudied. Here, we show that clonal populations of a unicellular chlorophyte, Tetraselmis indica, consist of morphologically and ultrastructurally variant cells which arise through asymmetric cell division. These cells also differ in their physiological properties. The structural and physiological differences in the clonal cell population correlate to a certain extent with the longevity and function of cells.

Functional anatomical, histological and ultrastructural studies of three chameleon species: Chamaeleo chamaeleon, Chamaeleo africanus, and Chamaeleon vulgaris / Estudios anatómicos, histológicos y ultraestructurales funcionales de tres especies de camaleón: Chamaeleo chamaeleon, Chamaeleo africanus, y Chamaeleon vulgaris

Three chamaeleon species including Chameleon Chamaeleo chamaeleon, Chameleon Chamaeleo africanus, and Chamaeleon vulgaris were collected and their tongue were dissected and examined morphologically and investigated using light and scanning electron microscopy. Both species showed similar histological manifestation of lingual papillae and tubular glands with dense mucous secretion especially in Chamaeleon vulgaris. There is no keratinization of lingual surfaces. Ultrastructurally, filliform represent the only pattern of lingual pappillae and take either cylindrical, conical and leaflet structure.Although the examined chalmaeleon species collected from different habitat, it shows almost similarities in their histological and ultrastructural structures.

Fueron recolectadas tres especies de Camaleón incluyendo Camaleón Chamaeleo chamaeleon, Camaleón Chamaeleo africanus y Chamaeleon vulgaris. Se disecó su lengua y examinó morfológicamente mediante el uso de microscopía de luz y electrónica de barrido. Ambas especies mostraron características histológicas similares en relación a las papilas linguales y glándulas tubulares con secreción mucosa densa, especialmente el Chamaeleon vulgaris. No hubo queratinización de las superficies linguales. Ultraes-tructuralmente, el único patrón de papilas linguales fue el filiforme, tomando una estructura ya sea cilíndrica, cónica y de hoja. Aunque las especies de Camaleón examinadas se recogieron de diferentes hábitat, ellas mostraron similitudes en su estructura histológica y ultraestructural.

Seasonal morphological variation of the vas deferens of scorpion mud turtle (Kinosternon scorpioides) / Variação morfológica sazonal dos ductos deferentes de tartaruga jurará (Kinosternon scorpioides)

This study aimed to characterize the morphology of the vas deferens of Kinosteron scorpioides by macroscopic and microscopic analysis. Were used 20 adult male jurarás collected at regular intervals during the year and divided into four experimental groups in the rainy and dry seasons, being processed for light microscopy, scanning electron microscopy and transmission electron microscopy. Morphometry for tubular and luminal diameters and epithelial height were also performed. On rainy season, vas deferens presented pseudostratified epithelium with cylindrical cells, spermatozoids and milky fluid in the lumen, with cytoplasmic organelles and lipid vesicles. On dry season, epithelium was pseudostratified with cuboid cells, with cellular debris and no spermatozoids. There was significant variation (p<0,05) for morphometry of vas deferens, with lower values of tubular and luminal diameters on rainy season, and higher epithelial height on dry season.

Este estudo teve como objetivo caracterizar a morfologia dos ductos deferentes de Kinosteron scorpioides por meio de análise macroscópica e microscópica. Foram utilizados 20 machos adultos, coletados em intervalos regulares durante o ano, os quais foram divididos em quatro grupos experimentais nas estações chuvosa e seca. Os ductos deferentes foram processados para análise por microscopia de luz, microscopia eletrônica de varredura e microscopia eletrônica de transmissão. Morfometria para diâmetros tubular e luminal e altura epitelial também foram realizadas. Na época das chuvas, o ducto deferente apresentou epitélio pseudoestratificado com células cilíndricas, espermatozoides e líquido leitoso no lúmen, além de com organelas citoplasmáticas e vesículas lipídicas. Na estação seca, o epitélio do ducto deferente foi do tipo pseudoestratificado com células cuboides e debris celulares, sendo que nenhum espermatozoide foi encontrado nesta estação. Houve variação significativa (p <0,05) para a morfometria dos ductos deferentes, com menores valores de diâmetros tubular e luminal na estação chuvosa, e maior altura do epitélio na estação seca.

Association between insulin-like growth factor-I, thyroxine and follicle stimulating hormone on the survival and in vitro development of caprine preantral follicles / Associação entre fator de crescimento semelhante à insulina-I, tiroxina ehormônio folículo estimulante sobre a sobrevivência e o desenvolvimentoin vitro de folículos pré-antrais caprinos

O objetivo deste estudo foi investigar a influência da interação entre o Hormônio Folículo Estimulante obtido da pituitária Humana (hFSH; 10 ng/mL), Fator de Crescimento Semelhante a Insulina Tipo 1 (IGF-I; 100 ng/mL) e Tiroxina (T4 ; 10, 20 ng/mL) na sobrevivência, ativação e crescimento de folículos pré-antrais cultivados in vitro. Fragmentos do córtex ovariano coletados a partir de 17 cabras adultas não gestantes foram submetidos a diferentes tratamentos que incluíam meio α-MEM+, IGF-I, hFSH, T4 e em seguida foram processados para histologia clássica e microscopia eletrônica de transmissão. Os resultados demonstraram uma redução no percentual de folículos normais, após um e sete dias de cultivo nos meios α-MEM+ (controle cultivado), T4 (10)/IGF-I, T4 (20)/IGF-I/hFSH e nos meios α-MEM+, IGF-I/hFSH, T4 (20)/IGF-I/hFSH, T4 (20)/IGF-I/hFSH, quando comparados ao controle não cultivado. Entretanto, o meio contendo T4 (20)/hFSH apresentou taxa de sobrevivência folicular superior aos demais tratamentos, quando cultivado por sete dias. Observou-se, ainda, que o diâmetro folicular quando cultivado neste meio não se alterou, quando comparado ao controle (não cultivado). As análises ultraestruturais confirmaram a integridade de folículos cultivados em meio contendo T4 (20)/hFSH. Em conclusão, o meio com T4 (20)/hFSH apresentou os melhores resultados, quando se avaliou sobrevivência, crescimento e aspectos ultraestruturais de folículos pré-antrais de caprinos cultivados in vitro.

The aim of this study was to investigate the influence of the interaction between follicle stimulating hormone obtained from Human Pituitary (hFSH; 10 ng/mL), Insulin-like Growth Factor Type 1 (IGF-I; 100 ng/mL) and thyroxine (T4 ; 10, 20 ng/mL) on survival, activation and growth of preantral follicles (PAF) cultured in vitro. Fragments of ovarian cortex collected from 17 nonpregnant adult goats underwent different treatments that included α-MEM+ medium, IGF-I, hFSH, T4 and were processed for classic histology and transmission electron microscopy. The results showed a reduction in the percentage of normal follicles, after one and seven days of culture in media α-MEM+ (cultured control), T4 (10)/IGF-I, T4 (20)/IGF-I/hFSH and in media α-MEM+, IGF-I/hFSH, T4 (20)/IGF-I/hFSH, T4 (20)/IGF-I/hFSH, when compared to the non-cultured control. However, the medium containing T4 (20)/hFSH showed a follicular survival rate higher than the other treatments, when cultured for seven days. It was also observed that the follicular diameter did not change when cultured in this medium, when compared to the control (non-cultured). The ultrastructural analysis confirmed the integrity of follicles cultured in a medium containing T4 (20)/hFSH. In conclusion, the medium containing T4 (20)/hFSH showed the best results, when evaluating the survival, growth and ultrastructural aspects of caprine preantral follicles cultured in vitro.