Angélica Silva-Olivares

Ultrastructural study of the midgut and hindgut in eight species of the genus Dendroctonus Erichson (Coleoptera: Scolytidae)

Abstract Chemical communication mediated by pheromones is a crucial aspect in the life cycle of beetles in the genus Dendroctonus. This communication plays an important role in mate location and in the colonization of host conifers. The study of the alimentary canal of these species is of importance not only because this organ is involved in the processes of digestion, detoxification, nutrient absorption, and transport, but also in the production of semiochemical compounds, such as pheromones. To better understand these functions and where they occur, the ultrastructural differences between the anterior and posterior midgut and the hindgut and their different cellular types were characterized. Adult specimens of both sexes from eight species were dissected and the alimentary canal was removed. It was sectioned into three parts: anterior midgut, posterior midgut, and hindgut, and analyzed by transmission electron microscopy. Results show that the epithelial tissue of the midgut possesses ultrastructural characteristics that permit differentiation of the anterior and posterior midgut. There are no ultrastructural differences within sexes of the same species, but differences exist among species. The ultrastructural characteristics of the hindgut do not differ between sexes or among species, but they do differ from those of the midgut.

Corifungin, a New Drug Lead against Naegleria, Identified from a High-Throughput Screen

ABSTRACT Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

Disruption of MDCK cell tight junctions by the free-living amoeba Naegleria fowleri

Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis. This parasite invades its host by penetrating the olfactory mucosa. However, the mechanism of epithelium penetration is not well understood. In the present study, we evaluated the effect of N. fowleri trophozoites and the non-pathogenic Naegleria gruberi on Madin-Darby canine kidney (MDCK) tight junction proteins, including claudin-1, occludin and ZO-1, as well as on the actin cytoskeleton. Trophozoites from each of the free-living amoeba species were co-cultured with MDCK cells in a 1 : 1 ratio for 1, 3, 6 or 10 h. Light microscopy revealed that N. fowleri caused morphological changes as early as 3 h post-infection in an epithelial MDCK monolayer. Confocal microscopy analysis revealed that after 10 h of co-culture, N. fowleri trophozoites induced epithelial cell damage, which was characterized by changes in the actin apical ring and disruption of the ZO-1 and claudin-1 proteins but not occludin. Western blot assays revealed gradual degradation of ZO-1 and claudin-1 as early as 3 h post-infection. Likewise, there was a drop in transepithelial electrical resistance that resulted in increased epithelial permeability and facilitated the invasion of N. fowleri trophozoites by a paracellular route. In contrast, N. gruberi did not induce alterations in MDCK cells even at 10 h post-infection. Based on these results, we suggest that N. fowleri trophozoites disrupt epithelial monolayers, which could enable their penetration of the olfactory epithelium and subsequent invasion of the central nervous system.

Differences between Naegleria fowleri and Naegleria gruberi in expression of mannose and fucose glycoconjugates

Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis, a rapidly fatal parasitic disease of humans. The adherence of Naegleria trophozoites to the host cell is one of the most important steps in the establishment and invasiveness of this infectious disease. Currently, little is known about the surface molecules that may participate in the interaction of N. fowleri with their target cells. In the present study, we investigated the composition of glycoconjugates present on the surface of trophozoites of the pathogenic N. fowleri and the nonpathogenic Naegleria gruberi. With the use of biotinylated lectins in western blot and flow cytometric analysis, we showed that N. fowleri trophozoites present high levels of surface glycoconjugates that contain α-D-mannose, α-D-glucose, and terminal α-L-fucose residues. A significant difference in the expression of these glycoconjugates was observed between N. fowleri and the nonpathogenic N. gruberi. Furthermore, we suggest that glycoconjugates that contain D-mannose and L-fucose residues participate in the adhesion of N. fowleri and subsequent damage to MDCK cells.

Double staining of β-galactosidase with fibrosis and cancer markers reveals the chronological appearance of senescence in liver carcinogenesis induced by diethylnitrosamine

Cellular senescence is characterized by irreversible cell arrest and is associated with the development of chronic diseases, including cancer. Here, we investigated the induction of cellular senescence during liver carcinogenesis. Liver cancer was induced in Fischer 344 rats with a weekly intraperitoneal injection of diethylnitrosamine (50mg/kg body weight) for 16 weeks. Double-detection of β-galactosidase with Ki67 for cell proliferation; a-SMA and Pdgfrb for cell specificity; p53, p21, p16, and cyclin D1, CDK2, and CDK4 for senescence-associated molecular pathways and γ-glutamyltranspeptidase (GGT) for hepatocarcinogenesis was assessed to determine the association of these markers with cellular senescence. DNA damage was measured through senescence-associated heterochromatin foci (SAHF) detection. Progressive cellular senescence was observed in both fibrotic septa and hepatocytes from week 10 to 18. The maximum peak of positive senescent and fibrotic cells was observed at week 16 and decreased at week 18, but cell proliferation remained high. Whereas the increased p16 expression and SAHF were concomitant with that of β-galactosidase, those of p53 and p21 were barely detected. Furthermore, β-galactosidase positive myofibroblast-like cells were mainly surrounding GGT-positive tumors. Our findings showed that in hepatocarcinogenesis by diethylnitrosamine, cellular senescence is associated with p16 pathway activation and is mainly localized in myofibroblast-like cells.

In Vitro Efficacy of Corifungin against Acanthamoeba castellanii Trophozoites and Cysts

ABSTRACT Painful blinding keratitis and fatal granulomatous amebic encephalitis are caused by the free-living amebae Acanthamoeba spp. Several prescription eye medications are used to treat Acanthamoeba keratitis, but the infection can be difficult to control because of recurrence of infection. For the treatment of encephalitis, no single drug was found useful, and in spite of the use of a combination of multiple drugs, the mortality rate remains high. Therefore, efficient, novel drugs are urgently needed for the treatment of amebic keratitis and granulomatous amebic encephalitis. In this study, we identified corifungin, a water-soluble polyene macrolide, as amebicidal. In vitro, it was effective against both the trophozoites and the cysts. Transmission electron microscopy of Acanthamoeba castellanii incubated with corifungin showed the presence of swollen mitochondria, electron-dense granules, degeneration of cytoplasm architecture, and loss of nuclear chromatin structure. These changes were followed by lysis of amebae. Corifungin also induced the encystment process of A. castellanii. There were alterations in the cyst cell wall followed by lysis of the cysts. Corifungin is a promising therapeutic option for keratitis and granulomatous amebic encephalitis.

Entamoeba histolytica and E. dispar Calreticulin: Inhibition of Classical Complement Pathway and Differences in the Level of Expression in Amoebic Liver Abscess

The role of calreticulin (CRT) in host-parasite interactions has recently become an important area of research. Information about the functions of calreticulin and its relevance to the physiology of Entamoeba parasites is limited. The present work demonstrates that CRT of both pathogenic E. histolytica and nonpathogenic E. dispar species specifically interacted with human C1q inhibiting the activation of the classical complement pathway. Using recombinant EhCRT protein, we demonstrate that CRT interaction site and human C1q is located at the N-terminal region of EhCRT. The immunofluorescence and confocal microscopy experiments show that CRT and human C1q colocalize in the cytoplasmic vesicles and near to the surface membrane of previously permeabilized trophozoites or are incubated with normal human serum which is known to destroy trophozoites. In the presence of peripheral mononuclear blood cells, the distribution of EhCRT and C1q is clearly over the surface membrane of trophozoites. Nevertheless, the level of expression of CRT in situ in lesions of amoebic liver abscess (ALA) in the hamster model is different in both Entamoeba species; this molecule is expressed in higher levels in E. histolytica than in E. dispar. This result suggests that EhCRT may modulate some functions during the early moments of the host-parasite relationship.

Histological Description of Oogenesis in Chiton virgulatus (Mollusca: Polyplacophora)

En el presente trabajo se describe la ovogenesis de Chiton virgulatus, utilizando histologia y las tecnicas de microscopia electronica de barrido y de transmision. Se identificaron tres tipos de ovocitos: i) ovocitos previtelogenicos con un diametro promedio de 50±20,5 µm, rodeados por celulas foliculares de forma alargada y un tamano de aproximadamente 5 µm, ii) ovocitos vitelogenicos inmaduros con un diametro promedio de 113±15,3 µm, este tipo de ovocitos presentan pequenas proyecciones citoplasmaticas, que indican el inicio del desarrollo del casco del ovocito. Adyacentes a cada prolongacion se presentan poros con un diametro aproximado de 0,7 µm y iii) ovocitos vitelogenicos maduros con un diametro promedio de 146±24,8 µm, las proyecciones citoplasmaticas del casco del ovocito crecen y en su parte apical adquieren la forma de un tridente, las celulas foliculares, dado el crecimiento de la prolongacion toman el aspecto bulboso y llegan a medir hasta 20 µm de longitud. La morfologia y la ultraestructura de las proyecciones del casco del ovocito vitelogenico maduro, asi como el tamano del poro en la base de las proyecciones son particulares para C. virgulatus, dichas caracteristicas podrian ser utilizadas en trabajos de taxonomia y fertilizacion.

ULTRASTRUCTURE OF SPERMATOGENESIS IN THE WHITE CLAM CHIONE CALIFORNIENSIS (BRODERIP, 1835) (MOLLUSCA: PELECYPODA)

Abstract Although studies of this species have focused on its reproductive cycle, there is no literature available on the ultra-structure of its germ cells. This study consequently aims to describe the latter cells as well as the shape of the acrosome, a character that may be of help in the taxonomic allocation of species of this family or other bivalve families. Results indicate spermatogonia have a central, spherical nucleus as well as scattered heterochromatin granules throughout the nucleoplasm. Rough endoplasmic reticulum is scarce, but the cytoplasm is rich in mitochondria and disperse glycogen granules. In primary spermatocytes, heterochromatin is more highly condensed and there is a reduction in the amount of cytoplasm and number of mitochondria as compared with spermatogonia. In secondary spermatocytes, heterochromatin is present in peripheral areas of the nucleus, forming occasional projections towards its center. In spermatids chromatin is fully condensed, taking up the entire nucleus. Mitochondria increase in size and migrate to the basal pole, giving rise, along with the centriole, to the spermatozoon neck. In spermatozoa, the nucleus is ovoid, the acrosome is round and the centriole is surrounded by four mitochondria, unlike other bivalves, like Anadara tuberculosa, in which the nucleus is reported to be round, the acrosome is pyramidal and there are five mitochondria.

In Vitro Efficacy of Ebselen and BAY 11-7082 Against Naegleria fowleri

Primary amebic meningoencephalitis (PAM) is a fatal infection caused by the free-living ameba Naegleria fowleri, popularly known as the “brain-eating ameba.” The drugs of choice in treating PAM are the antifungal amphotericin B and an antileishmanial miltefosine, but these are not FDA-approved for this indication and use of amphotericin B is associated with severe adverse effects. Moreover, very few patients treated with the combination therapy have survived PAM. Therefore, development of efficient drugs is a critical unmet need to avert future deaths of children. Since N. fowleri causes extensive inflammation in the brain it is important to select compounds that can enter brain to kill ameba. In this study, we identified two central nervous system (CNS) active compounds, ebselen and BAY 11-7082 as amebicidal with EC50 of 6.2 and 1.6 μM, respectively. The closely related BAY 11-7085 was also found active against N. fowleri with EC50 similar to BAY 11-7082. We synthesized a soluble ebselen analog, which had amebicidal activity similar to ebselen. Transmission electron microscopy of N. fowleri trophozoites incubated for 48 h with EC50 concentration of ebselen showed alteration in the cytoplasmic membrane, loss of the nuclear membrane, and appearance of electron-dense granules. Incubation of N. fowleri trophozoites with EC50 concentrations of BAY 11-7082 and BAY 11-7085 for 48 h showed the presence of large lipid droplets in the cytoplasm, disruption of cytoplasmic and nuclear membranes and appearance of several vesicles and chromatin residues. Blood-brain barrier permeable amebicidal compounds have potential as new drug leads for Naegleria infection.