Use of Electron Microscopy for the Detection of Contaminant Endophytic Bacteria In Vitro.

The success of in vitro cultivation, particularly for micropropagation purposes, depends on the efficient control of contaminants. In this context, the sterilization of plant material constitutes a fundamental step in initiating cultures. Microbial contaminants can be found either on the surface (epiphyte) or inside plant explants (endophyte). However, the latter is generally challenging to detect and may not always be eradicated through surface sterilization alone. Endophyte contaminants, such as bacteria, can persist within plant material over several cultivation cycles, potentially interfering with or inhibiting in vitro establishment, growth, or recovery of cryopreserved materials. Therefore, microscopy techniques, such as electron microscopy, can yield valuable insights into bacterial endophytes' localization, tissue colonization patterns, and functions in in vitro plant culture. This information is essential for adopting effective strategies for eliminating, preventing, or harmonious coexistence with contaminants.

Micromorphological characterization and electron microscopic study of parasitic species of the family Loranthaceae Juss.

The Loranthaceae Juss. family includes parasitic species that invade important trees such as fruit trees. In Saudi Arabia, Loranthaceae comprises four genera, which include six species that grow in the western, southwestern, and northern regions: Tapinanthus globifer (A.Rich.) Tiegh, Oncocalyx glabratus (Engl.) M. G. Gilbert, Loranthella deflersii (Tiegh.) S. Blanco & C. E. Wetze, Phragmanthera austroarabica A. G. Mill. & J. Nyberg, Plicosepalus curviflorus (Benth.ex Oliv.) Tiegh. and Plicosepalus acaciae (Zucc.). The species present in the Kingdom of Saudi Arabia have not been the subject of enough studies. This work aims to screen and evaluate the taxonomic importance of the micromorphological traits of leaves and fruits in Loranthaceae species native to Saudi Arabia (SA) using scanning electron microscope (SEM). In this study, cluster dendrogram (CD), principal component analysis (PCA) and analysis of variance (ANOVA) were used to evaluate the ability to discriminate Loranthaceae species using micromorphological characteristics. Most of the micromorphological characteristics of the leaf and fruit surfaces used reflected significant variation between the species of Loranthaceae. The type of stomata, trichome, lenticels, fine relief of the cell wall and wax form were the most taxonomically important characteristics. In addition, the cluster dendrogram of morphological characteristics showed species distribution within branches based on affiliation to subtribes Tapinanthinae and Emelianthinae. To the best of our knowledge, the fruit and leaves of the species under study have never been described using electron microscopy, and this study is considered the first of its kind. It also contributes to solving the classification problems of the family Loranthaceae in general and confirms the importance of the characteristics and methods used as tools for characterizing parasitic species that infect trees and helps to verify their identities. This will help to improve resistance efforts and put effective control plans in place.

A perspective of the prevalent H9N2 virus with a special focus on molecular and pathological aspects in commercial broiler chicken in Punjab, Pakistan / Perspectiva do vírus H9N2 prevalente com foco especial em aspectos moleculares e patológicos em frangos de corte comerciais em Punjab, Paquistão

Frequent outbreaks of avian influenza H9N2 virus in Pakistan revealed that this subtype has become endemic in the poultry industry and, besides economic losses, poses a threat to public health. The present study describes the molecular characterization and pathological alterations in naturally infected broiler chickens with the current H9N2 field strain and their phylogenomic dynamics. In this study, tissue samples (trachea, lung, kidney and intestine) from 100 commercial chicken flocks were collected from July 2018 to August 2019. Samples were subjected to molecular detection, phylogeny and subsequent pathological examination. The complete length of the HA gene was successfully amplified in five samples. Nucleotide sequencing revealed positive samples placed in a clade belonging to the B2 sub-lineage of the G1 genotype and categorized as LPAIV based on the amino acid sequence of the HA gene at the cleavage site (PAKSSR/G). Genetic analysis of the haemagglutinin (HA) gene revealed nt: 80.5%-99.5%; aa: 83.8%-98.9% homology to H9N2 strains reported previously from Pakistan, neighbouring countries, and (A/Quail/Hong Kong/G1/97). Gross lesions include a slight airsacculitis, mild hemorrhages, diffuse congestion and purulent exudate in tracheal mucosa, fibrinonecrotic cast in the trachea lumen and mild pulmonary congestion. Histopathological alterations include sloughing of epithelial cells and infiltration of inflammatory cells in the trachea, mononuclear cells (MNCs) infiltration, pulmonary congestion and exudate in the lumen of parabronchi, peritubular congestion in the kidneys with degeneration of tubular epithelial cells and degenerative changes in the intestinal villi epithelial cells and goblet cell hyperplasia. Immunohistochemistry analysis confirmed the presence of AIVH9N2 antigen in the trachea, lungs, kidney and intestine. Electron microscopy revealed ultrastructural changes in the trachea, including degenerated cilia, mitochondrial swelling and enlarged endoplasmic reticulum. Based on all essential analysis, the present study revealed the distribution of the H9N2 virus of G1 genotype in Punjab, Pakistan, with mild to moderate pathogenicity.

Surtos frequentes do vírus da gripe aviária H9N2 no Paquistão revelaram que esse subtipo se tornou endêmico na avicultura e, além das perdas econômicas, representa uma ameaça à saúde pública. O presente estudo descreve a caracterização molecular e as alterações patológicas em frangos de corte naturalmente infectados com a atual cepa H9N2 e sua dinâmica filogenômica. Neste estudo, amostras de tecidos (traqueia, pulmões, rim e intestino) de 100 lotes comerciais de frangos foram coletadas de julho de 2018 a agosto de 2019. As amostras foram submetidas à detecção molecular, filogenia e posterior exame patológico. O comprimento completo do gene HA foi amplificado com sucesso em cinco amostras. O sequenciamento de nucleotídeos revelou amostras positivas colocadas em um clado pertencente à sublinhagem B2 do genótipo G1 e categorizado como LPAIV com base na sequência de aminoácidos do gene da hemaglutinina (HA) no local de clivagem (PAKSSR/G). A análise genética do gene da HA revelou: nt = 80,5%-99,5%; aa = 83,8%-98,9% de homologia com cepas de H9N2 relatadas anteriormente no Paquistão e em países vizinhos (A/Quail/Hong Kong/G1/97). As lesões macroscópicas incluíram aerossaculite leve, hemorragias leves, congestão difusa e exsudato purulento na mucosa traqueal, cilindro fibrinonecrótico no lúmen da traqueia e congestão pulmonar leve. As alterações histopatológicas incluíram descamação de células epiteliais, infiltração de células inflamatórias na traqueia, infiltração de células mononucleares (MNCs), congestão pulmonar e exsudato no lúmen dos parabrônquios, congestão peritubular nos rins com degeneração das células epiteliais tubulares, alterações degenerativas nas células epiteliais das vilosidades intestinais e hiperplasia de células caliciformes. A análise imunoistoquímica confirmou a presença do antígeno AIVH9N2 na traqueia, nos pulmões, no rim e no intestino. A microscopia eletrônica revelou alterações ultraestruturais na traqueia, incluindo cílios degenerados, inchaço mitocondrial e retículo endoplasmático aumentado. Com base em todas as análises, o presente estudo revelou a distribuição do vírus H9N2 do genótipo G1 em Punjab, Paquistão, com patogenicidade de leve a moderada.

Herramientas de corte para optimizar parámetros de clasificación de especies maderables con redes neuronales convolucionales / Cutting tools to optimize classification parameters of timber species with convolutional neural networks

Introducción: La gran diversidad de especies maderables tropicales demanda el desarrollo de nuevas tecnologías de identificación con base en sus patrones o características anatómicas. La aplicación de redes neuronales convolucionales (CNN) para el reconocimiento de especies maderables tropicales se ha incrementado en los últimos años por sus resultados prometedores. Objetivo: Evaluamos la calidad de las imágenes macroscópicas con tres herramientas de corte para mejorar la visualización y distinción de las características anatómicas en el entrenamiento del modelo CNN. Métodos: Recolectamos las muestras entre el 2020 y 2021 en áreas de explotación forestal y aserraderos de Selva Central, Perú. Luego, las dimensionamos y, previo a la identificación botánica y anatómica, las cortamos en secciones transversales. Generamos una base de datos de imágenes macroscópicas de la sección transversal de la madera, a través del corte, con tres herramientas para ver su rendimiento en el laboratorio, campo y puesto de control. Resultados: Usamos tres herramientas de corte para obtener una alta calidad de imágenes transversales de la madera; obtuvimos 3 750 imágenes macroscópicas con un microscopio portátil que corresponden a 25 especies maderables. El cuchillo ''Tramontina'' es duradero, pero pierde el filo con facilidad y se necesita una herramienta para afilar, el cúter retráctil ''Pretul'' es adecuado para madera suave y dura en muestras pequeñas de laboratorio; el cuchillo ''Ubermann'' es apropiado para el campo, laboratorio y puesto de control, porque tiene una envoltura duradera y láminas intercambiables en caso de pérdida de filo. Conclusiones: La calidad de las imágenes es decisiva en la clasificación de especies maderables, porque permite una mejor visualización y distinción de las características anatómicas en el entrenamiento con los modelos de red neuronal convolucional EfficientNet B0 y Custom Vision, lo cual se evidenció en las métricas de precisión.

Introduction: The great diversity of tropical timber species demands the development of new technologies capable of identifying them based on their patterns or anatomical characteristics. The application of convolutional neural networks (CNN) for the recognition of tropical timber species has increased in recent years due to the promising results of CNNs. Objective: To evaluate the quality of macroscopic images with three cutting tools to improve the visualization and distinction of anatomical features in the CNN model training. Methods: Samples were collected from 2020 to 2021 in areas of logging and sawmills in the Central Jungle, Peru. They were later sized and, after botanical and anatomical identification, cut in cross sections. A database of macroscopic images of the cross-section of wood was generated through cutting with three different tools and observing its performance in the laboratory, field, and checkpoint. Results: Using three cutting tools, we obtained high quality images of the cross section of wood; 3 750 macroscopic images were obtained with a portable microscope and correspond to 25 timber species. We found the ''Tramontina'' knife to be durable, however, it loses its edge easily and requires a sharpening tool, the ''Pretul'' retractable cutter is suitable for cutting soft and hard wood in small laboratory samples and finally the ''Ubermann'' knife is suitable for use in the field, laboratory, and checkpoint, because it has a durable sheath and interchangeable blades in case of dullness. Conclusion: The quality of the images is decisive in the classification of timber species, because it allows a better visualization and distinction of the anatomical characteristics in training with the EfficientNet B0 and Custom Vision convolutional neural network models, which was evidenced in the precision metrics.

Pathogenesis of an experimental mycobacteriosis in an apple snail.

In this work, we aimed at investigating cell and tissue responses of the apple snail Pomacea canaliculata, following the inoculation of the zoonotic pathogen Mycobacterium marinum. Different doses were tested (10, 20, 65, and 100 M CFU) and the mortality rate was negligible. The histopathogenesis was followed at 4, 9, and 28 days after inoculation. Overt histopathological lesions were consistently observed after the two largest doses only. In the lung, marked hemocyte aggregations, including intravascular nodule formation, were observed within the large blood veins that run along the floor and roof of this organ. Hemocyte aggregations were found occluding many of the radial sinuses supplying the respiratory lamina. Acid-fast bacilli were contained in the different hemocyte aggregations. In addition, hemocytes were observed infiltrating the storage tissue, which makes up most of the lung wall, and the connective tissue of the mantle edge. Additionally, signs of degradation in the storage tissue were observed in the lung wall on day 28. In the kidney, nodules were formed associated with the constitutive hemocyte islets and with the subpallial hemocoelic space, in whose hemocytes the acid-fast structures were found. Electron microscopy analysis revealed the presence of bacteria-containing phagosomes within hemocytes located in the surface zone of the islets. Additionally, electron-dense spheroidal structures, which are likely remnants of digested mycobacteria, were observed in close proximity to the hemocytes' nuclei. The size attained by the hemocyte nodules varied during the observation period, but there was no clear dependence on dose or time after inoculation. Nodules were also formed subpallially. Some of these nodules showed 2-3 layers with different cellular composition, suggesting they may also form through successive waves of circulating cells reaching them. Nodular cores, including those formed intravascularly in the lung, would exhibit signs of hemocyte dedifferentiation, possibly proliferation, and death. Hemocyte congestion was observed in the hemocoelic spaces surrounding the pallial ends of the renal crypts, and the renal crypts themselves showed de-epithelization, particularly on day 28. The diverse cellular responses of P. canaliculata to M. marinum inoculation and the high resilience of this snail to the pathogen make it a suitable species for studying mycobacterial infections and their effects on cellular and physiological processes.

Effect of Clomiphene Citrate on the Ultrastructure of Testis in Albino Rats

SUMMARY: The aim of the present work was to study the closer effect of clomiphene citrate on the ultrastructure of the testis of adult albino rats to provide a basis for optimizing this drug in the treatment of male infertility. The testes were removed from both groups under anesthesia and then prepared for examination by light using hematoxylin and eosin stains and a transmission electron microscope. Semithin sections were cut into 1 µm thick sections, stained with toluidine blue, and examined by light microscopy for a survey. The desired areas were placed in the center, and other areas were trimmed. Primary spermatocytes showed marked nuclear changes (pyknosis), and their nuclear membranes were ill-defined and disrupted. The cytoplasm showed widespread degeneration of mitochondria and lysosomes and focal degeneration of the rough endoplasmic reticulum compared with the control group. The spermatids were pale, and the two phases of spermatogenesis were distinctly identifiable in the control group but were confused in the treated group. Some spermatids had interrupted nuclear membranes, also containing degenerated mitochondria, focal fragmentation of rough endoplasmic reticulum, and free ribosomes. Spermatozoa in the treated group appeared deformed compared to the control, where they had deformed head caps. Leydig cells of the treated group have an irregularly shaped nucleus, with focal chromatin aggregation and peripheral chromatin condensation on the inner surface of the nuclear membrane. The observations of the present work indicate a possible causal relationship between testicular affection and ingestion of clomiphene citrate, which can be avoided by close medical observations using ultrasonography, semen analysis, or testicular biopsy to detect early malignant changes. Furthermore, the drug should not be used for more than three to six cycles and should be stopped for at least three cycles before reuse. When clomiphene citrate is ineffective in the treatment of male infertility, human menopausal gonadotropin (hMG) administration is typically selected. However, high-dose hMG therapy is associated with a variety of adverse effects. In this work, we report the success of a modified clomiphene citrate regimen in increasing sperm count without any hazards to the testicular tissue.

El objetivo del trabajo fue estudiar el efecto del citrato de clomifeno sobre la estructura de los testículos de la rata albina adulta, con la finalidad de determinar la mejor manera de utilizar este fármaco en el tratamiento de la infertilidad masculina. Los testículos se extrajeron bajo anestesia y para su análisis a través de microscopio de luz se tiñeron con HE. Además, las muestras fueron preparadas para su examen con microscopía electrónica de transmisión. Por otra parte, se cortaron secciones semifinas de 1 µm de espesor, se tiñeron con azul de toluidina y se examinaron mediante microscopía óptica. Los espermatocitos primarios mostraron cambios nucleares marcados (picnosis) y sus membranas nucleares estaban mal definidas y alteradas. En el grupo experimental las células presentaban el citoplasma con degeneración generalizada de las mitocondrias y de los lisosomas y una degeneración focal del retículo endoplásmico rugoso en comparación con el grupo control. Las espermátidas estaban pálidas y las dos fases de la espermatogénesis eran claramente identificables en el grupo control, pero se confundían en el grupo tratado. Algunas espermátidas tenían membranas nucleares interrumpidas, y también contenían mitocondrias degeneradas, fragmentación focal del retículo endoplásmico rugoso y ribosomas libres. Los espermatozoides del grupo tratado se presentaban deformados en comparación con el control. Las células de Leydig del grupo tratado presentaban un núcleo de forma irregular, con agregación focal de cromatina y condensación de cromatina periférica en la superficie interna de la membrana nuclear. Las observaciones del presente trabajo indican una posible relación causal entre la afección testicular y la ingestión de citrato de clomifeno, que puede evitarse mediante observaciones médicas minuciosas a través de ecografía, análisis de semen o biopsia testicular para detectar cambios malignos tempranos. Además, el medicamento no debiera ser usado durante más de tres a seis ciclos y debe suspenderse durante al menos tres ciclos antes de volver a usarlo. Cuando el citrato de clomifeno es ineficaz en el tratamiento de la infertilidad masculina, normalmente se selecciona la administración de gonadotropina menopáusica humana (hMG). Sin embargo, la terapia con hMG en dosis altas se asocia con una variedad de efectos adversos. En este trabajo, informamos el éxito de un régimen modificado con citrato de clomifeno para aumentar el recuento de espermatozoides sin riesgo para el tejido testicular.

Alternativas de procesos para la búsqueda de casos de tuberculosis bajo condiciones económicas limitadas. Cuba 2022-2025 / Process Alternatives for the Tuberculosis Case Finding under Limited Economic Conditions. Cuba 2022-2025

Ejecutar procesos efectivos de búsqueda de casos de tuberculosis es crucial para acele-rar el paso hacia su eliminación. El empeoramiento de las condiciones económicas mun-diales y nacionales no nos permite aplicar extensivamente las tecnologías rápidas mo-leculares idóneas de diagnóstico. Consideramos sensato entonces aplicar algoritmos alternativos que satisfagan las necesidades nacionales presentes hasta que las condi-ciones permitan la cobertura completa de las tecnologías moleculares recomendadas. Sugerimos introducir la radiografía digital para todos los algoritmos, utilizar mejor la microscopía de fluorescencia LED y la óptica convencional ya probadas. En conclusión, es preciso que este enfoque de trabajo, que procura optimizar la efectividad y eficiencia del programa, se introduzca en la práctica cotidiana hasta que lo idóneo sea permisible

Executing effective tuberculosis case-finding processes is crucial to accelerate the path towards elimination of the disease. The worsening of global and national economic conditions do not allow us to extensively apply rapid molecular diagnostic technolo-gies. We consider it sensible and necessary to apply alternative algorithms that meet the current national needs, until conditions allow full coverage of the recommended molecular technologies. We suggest introducing digital X-rays for all algorithms, bet-ter use of LED fluorescence microscopy and conventional optics already appropriate-ly tested. In conclusion, it is necessary that this approach that seeks to optimize the effectiveness and efficiency of the Cuban program be introduced into daily practice until the ideal is permissible

Ayudas diagnósticas y diagnóstico diferencial de la enfermedad de Pompe / Diagnostic aids and differential diagnosis of Pompe disease

Introducción: La enfermedad de Pompe (EP) o glucogenosis tipo II es una enfermedad autosómica recesiva causada por mutaciones en el gen GAA que codifica para la proteína alfa-1,4-glucosidasa. Su deficiencia lleva a un almacenamiento anormal de glucógeno en los lisosomas de varias células, a través de los diferentes tejidos, lo que causa un compromiso musculoesquelético predominante. Contenidos: Los fenotipos de la enfermedad dependen de las variantes genéticas y de los niveles de la actividad enzimática residual. La enfermedad se presenta como EP de inicio infantil, EP de inicio tardío y EP intermedio, por lo que es de suma importancia su diagnóstico temprano, por medio de estudios moleculares como la secuenciación de Sanger y la secuenciación de nueva generación. Conclusiones: Se ha demostrado, mediante diferentes estudios, que las variaciones genéticas pueden diferir entre etnias, y es importante su caracterización molecular para determinar el tratamiento más adecuado, de acuerdo con el estado del material inmunológico de reacción cruzada (CRIM).

Introduction: Pompe disease (PD) or Glycogenosis Type II is a rare autosomal recessive disease caused by mutations in the GAA gene that codes for the alpha-1,4-glucosidase protein. Its deficiency leads to abnormal glycogen storage in the lysosomes of various cells throughout the different tissues causing a predominant musculoskeletal compromise. Contents: The phenotypes of the disease depend on the genetic variants and the levels of residual enzyme activity, presenting as infantile-onset PD, late-onset PD, and intermediate PD; Therefore, early diagnosis of the disease through molecular studies such as Sanger sequencing and new generation sequencing is of utmost importance. Conclusions: It has been shown through different studies that genetic variations can vary between ethnic groups and the molecular characterization of the variants is important to determine the most appropriate treatment depending on the state of the cross-reactive immunological material (CRIM)

A spatially resolved elemental nanodomain organization within acidocalcisomes in Trypanosoma cruzi.

How are ions distributed in the three-dimensional (3D) volume confined in a nanoscale compartment? Regulation of ionic flow in the intracellular milieu has been explained by different theoretical models and experimentally demonstrated for several compartments with microscale dimensions. Most of these models predict a homogeneous distribution of ions seconds or milliseconds after an initial diffusion step formed at the ion translocation site, leaving open questions when it comes to ion/element distribution in spaces/compartments with nanoscale dimensions. Due to the influence of compartment size on the regulation of ionic flow, theoretical variations of classical models have been proposed, suggesting heterogeneous distributions of ions/elements within nanoscale compartments. Nonetheless, such assumptions have not been fully proven for the 3D volume of an organelle. In this work, we used a combination of cutting-edge electron microscopy techniques to map the 3D distribution of diffusible elements within the whole volume of acidocalcisomes in trypanosomes. Cryofixed cells were analyzed by scanning transmission electron microscopy tomography combined with elemental mapping using a high-performance setup of X-ray detectors. Results showed the existence of elemental nanodomains within the acidocalcisomes, where cationic elements display a self-excluding pattern. These were validated by Pearson correlation analysis and in silico molecular dynamic simulations. Formation of element domains within the 3D space of an organelle is demonstrated. Distribution patterns that support the electrodiffusion theory proposed for nanophysiology models have been found. The experimental pipeline shown here can be applied to a variety of models where ion mobilization plays a crucial role in physiological processes.

Morphological Effects of Cigarette Smoking on Respiratory System and Related Functions Based on AQPs Expression

SUMMARY: A great deal of attention of air pollution on respiratory health is increasing, particularly in relation to haze days. It is that exposure to cigarette smoke augments the toxicity of common air contaminants, thereby increasing the complexity of respiratory diseases. Although there are various mechanisms involved to respiratory diseases caused or worsen by cigarette smoking, in which the role of AQPs in the lung with regard to fluid homeostasis still remains elusive. In this paper, we copied the rat models based on smoke generator, and investigated the morphological changes of mucosa and related functions depending on the balance of lining liquid of alveoli via AQPs expression. Compared with normal group, weak labelling of AQP1 and AQP5 protein abundance were clearly detected in the corresponding part of smoke exposure groups compared with normal group. Hence, it is suggested that the contribution of AQPs in the lung is diminished, thereby causing perturbed balancing between resorptive and secretory fluid homeostasis under cigarette smoking.

Cada vez se presta más atención a la contaminación del aire en la salud respiratoria, particularmente, en relación con los días de neblina. En consecuencia la exposición al humo del cigarrillo aumenta la toxicidad de los contaminantes comunes del aire, lo que además aumenta la complejidad de las enfermedades respiratorias. Aunque existen varios mecanismos involucrados en las enfermedades respiratorias causadas o empeoradas por el tabaquismo, en las que el papel de las AQP en el pulmón respecto a la homeostasis de líquidos sigue siendo difícil de alcanzar. En este artículo, copiamos los modelos de rata basados en el generador de humo e investigamos los cambios morfológicos de la mucosa y las funciones relacionadas según el equilibrio del líquido de revestimiento de los alvéolos a través de la expresión de AQP. En comparación con el grupo normal, se detectó claramente un etiquetado débil de la abundancia de proteínas AQP1 y AQP5 en la parte correspondiente de los grupos de exposición al humo en comparación con el grupo control. Por lo tanto, se sugiere que la contribución de las AQP en el pulmón está disminuida, provocando así un equilibrio perturbado entre la homeostasis del líquido secretor y de reabsorción bajo el hábito de fumar cigarrillos.

Diversity of Surface Fibril Patterns in Mimivirus Isolates.

Among the most intriguing structural features in the known virosphere are mimivirus surface fibrils, proteinaceous filaments approximately 150 nm long, covering the mimivirus capsid surface. Fibrils are important to promote particle adhesion to host cells, triggering phagocytosis and cell infection. However, although mimiviruses are one of the most abundant viral entities in a plethora of biomes worldwide, there has been no comparative analysis on fibril organization and abundance among distinct mimivirus isolates. Here, we describe the isolation and characterization of Megavirus caiporensis, a novel lineage C mimivirus with surface fibrils organized as "clumps." This intriguing feature led us to expand our analyses to other mimivirus isolates. By employing a combined approach including electron microscopy, image processing, genomic sequencing, and viral prospection, we obtained evidence of at least three main patterns of surface fibrils that can be found in mimiviruses: (i) isolates containing particles with abundant fibrils, distributed homogeneously on the capsid surface; (ii) isolates with particles almost fibrilless; and (iii) isolates with particles containing fibrils in abundance, but organized as clumps, as observed in Megavirus caiporensis. A total of 15 mimivirus isolates were analyzed by microscopy, and their DNA polymerase subunit B genes were sequenced for phylogenetic analysis. We observed a unique match between evolutionarily-related viruses and their fibril profiles. Biological assays suggested that patterns of fibrils can influence viral entry in host cells. Our data contribute to the knowledge of mimivirus fibril organization and abundance, as well as raising questions on the evolution of those intriguing structures. IMPORTANCE Mimivirus fibrils are intriguing structures that have drawn attention since their discovery. Although still under investigation, the function of fibrils may be related to host cell adhesion. In this work, we isolated and characterized a new mimivirus, called Megavirus caiporensis, and we showed that mimivirus isolates can exhibit at least three different patterns related to fibril organization and abundance. In our study, evolutionarily-related viruses presented similar fibril profiles, and such fibrils may affect how those viruses trigger phagocytosis in amoebas. These data shed light on aspects of mimivirus particle morphology, virus-host interactions, and their evolution.

Intraspecific variation in ultrastructure and secretion of the resin canals in Anacardium humile (Anacardiaceae).

The study combines a range of light and electron microscopy methods to access variation in secretion and ultrastructure in the secretory canals in the above- and belowground stems of Anacardium humile, which here serves as a model system. The aboveground stem canals show epithelial cells with ultrastructural characteristics typical of cells active in secretion, while in the belowground stems, the subcellular characteristics are typical of cells with low rates of metabolism. The secretory canals of the belowground stems show uniformity in size and shape, a large central vacuole, a cytoplasm reduced to a thin layer at the cell periphery, and a reduced population of organelles. The aboveground stem canals had voluminous nuclei with evident nucleoli, a very dense cytoplasm with free ribosomes, polyribosomes, mitochondria with developed cristae, and ellipsoid plastids with electron-opaque droplets surrounded by a periplastid reticulum. The vacuoles were of different sizes and often had membranous contents and the dictyosomes were very developed with dilated ends to the cisternae, rough endoplasmic reticulum, and numerous vesicles. The results show that particularities in above- and belowground environment have significant implications for ultrastructural morphology and functioning of secretory canals in the stems of A. humile.

Acacia senegal inhibits diabetic nephropathy ultrastructure alteration and renal biomarkers changes in rats / Acacia senegal inhibe la alteración de la ultraestructura de la nefropatía diabética y los cambios en los biomarcadores renales en rata

SUMMARY: Diabetic nephropathy (DN) is the most common complication of diabetes. Several studies have been done in a trial to protect against this problem at the ultrastructure level. This study investigates the protective effect of oral administration of Acacia senegal (AS) against the development of DN. Sixty male albino rats were randomly divided into six groups: control, Acacia senegal control, Diabetic untreated, diabetic insulin-treated, Diabetic AS treated, and Diabetic insulin and AS combined treated groups. Plasma glucose, HbA1c, serum Albumin, creatinine, urine creatinine was measured using specific kits. Determinations of creatinine clearance and blood pressure were done. The renal tissues of both kidneys were prepared to investigate under both light (LM) and electron microscope (EM). Ultrastructure examination of renal rats tissue of diabetic untreated rats showed the destruction of the glomerular basement membrane and endothelial cells together with hemorrhage in glomerular capsules (Bowman's capsules). On the other side, both LM and EM revealed improving the endothelial cells and the other glomerular capsules structures, especially with the combined treated group, which confirmed the improvement of the biochemical investigation in the study. In conclusion, from the present study, using the oral AS together with SC insulin could be protected against the development of DN.

RESUMEN: La nefropatía diabética (ND) es la complicación más común de la diabetes. Se han realizado varios estudios de ensayo para abordar esta dificultad a nivel de ultraestructura. Este estudio investiga el efecto protector de la administración oral de Acacia senegal (AS) contra el desarrollo de la ND. Se dividieron sesenta ratas albinas machos aleatoriamente en seis grupos: control, control de Acacia senegal, diabéticos no tratados, diabéticos tratados con insulina, diabéticos tratados con AS y grupos tratados con compuesto de insulina diabética + AS. Se midieron utilizando kits específicos, glucosa plasmática, HbA1c, albúmina sérica, creatinina en sangre y en orina. Se registraron la creatinina y la presión arterial. Los tejidos renales de ambos riñones se prepararon para investigar tanto con microscopio óptico (MO) como electrónico (ME). El examen de la ultraestructura del tejido renal de ratas diabéticas no tratadas mostró la destrucción de la membrana basal glomerular y las células endoteliales junto con hemorragia en las cápsulas glomerulares (cápsulas de Bowman). Por otro lado, tanto MO como ME revelaron una mejora de las células endoteliales y las estructuras capsulares glomerulares, en el grupo tratado con el compuesto, lo que confirmó la mejora de la investigación bioquímica. En conclusión, el uso de AS oral en combinación con insulina podría proteger contra el desarrollo de ND.

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.

Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding.

The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

A comprehensive ultrastructural analysis of the Toxoplasma gondii cytoskeleton.

The invasive nature of Toxoplasma gondii is closely related to the properties of its cytoskeleton, which is constituted by a group of diverse structural and dynamic components that play key roles during the infection. Even if there have been numerous reports about the composition and function of the Toxoplasma cytoskeleton, the ultrastructural organization of some of these components has not yet been fully characterized. This study used a detergent extraction process and several electron microscopy contrast methods that allowed the successful isolation of the cytoskeleton of Toxoplasma tachyzoites. This process allowed for the conservation of the structures known to date and several new structures that had not been characterized at the ultrastructural level. For the first time, characterization was achieved for a group of nanofibers that allow the association between the polar apical ring and the conoid as well as the ultrastructural characterization of the apical cap of the parasite. The ultrastructure and precise location of the peripheral rings were also found, and the annular components of the basal complex were characterized. Finally, through immunoelectron microscopy, the exact spatial location of the subpellicular network inside the internal membrane system that forms the pellicle was found. The findings regarding these new structures contribute to the knowledge concerning the biology of the Toxoplasma gondii cytoskeleton. They also provide new opportunities in the search for therapeutic strategies aimed at these components with the purpose of inhibiting invasion and thus parasitism.

SARS-CoV-2: Ultrastructural Characterization of Morphogenesis in an In Vitro System.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted public health and the world economy and fueled a worldwide race to approve therapeutic and prophylactic agents, but so far there are no specific antiviral drugs. Understanding the biology of the virus is the first step in structuring strategies to combat it, and in this context several studies have been conducted with the aim of understanding the replication mechanism of SARS-CoV-2 in vitro systems. In this work, studies using transmission and scanning electron microscopy and 3D electron microscopy modeling were performed with the goal of characterizing the morphogenesis of SARS-CoV-2 in Vero-E6 cells. Several ultrastructural changes were observed-such as syncytia formation, cytoplasmic membrane projections, lipid droplets accumulation, proliferation of double-membrane vesicles derived from the rough endoplasmic reticulum, and alteration of mitochondria. The entry of the virus into cells occurred through endocytosis. Viral particles were observed attached to the cell membrane and in various cellular compartments, and extrusion of viral progeny took place by exocytosis. These findings allow us to infer that Vero-E6 cells are highly susceptible to SARS-CoV-2 infection as described in the literature and their replication cycle is similar to that described with SARS-CoV and MERS-CoV in vitro models.

Three-dimensional architecture of Cyrilia lignieresi gametocyte-stage development inside red blood cells.

The Haemogregarinidae family (Apicomplexa: Adeleina) comprises hemoprotozoa that infect mammals, birds, amphibians, fish, and reptiles. Some morphological characteristics of the Cyrilia lignieresi have been described previously, but the parasite-erythrocyte relationship is still poorly understood. In order to understand the structural architecture of C. lignieresi-infected red blood cells, electron microscopy-based three-dimensional reconstruction was carried out using TEM as well as FIB-SEM tomography. Results showed that development of the macrogametocyte-stage inside the red blood cell is related to an increase in cleft-like structures in the host cell cytoplasm. Furthermore, other aspects related to parasite intraerythrocytic development were explored by 3D visualization techniques. We observed the invagination of a large extension of the Inner Membrane Complex (IMC) on the parasite body, which results from or induces a folding of the posterior end of the parasite. Small tubular structures were seen associated with areas related to IMC folding. Taken together, results provide new information on the remodeling of erythrocytes induced by the protozoan C. lignieresi.