A clinical and histopathological study of malformations observed in fetuses infected by the Zika virus.

BACKGROUND: The recent outbreak of Zika virus (ZIKV) infection and the associated increased prevalence of microcephaly in Brazil underline the impact of viral infections on embryo fetal development. The aim of the present study is to provide a detailed clinical and histopathological study of the fetal disruption caused by the ZIKV, with a special focus on the associated neuropathological findings. METHODS: A detailed feto-placental examination, as well as neuropathological and neurobiological studies were performed on three fetuses collected after pregnancy termination between 22 and 25 weeks of gestation (WG), because brain malformations associated with a maternal and fetal ZIKV infection was diagnosed. RESULTS: In all three cases, the maternal infection occurred during the first trimester of pregnancy. A small head was observed on the ultrasound examination of the second trimester of pregnancy and led to the diagnosis of ZIKV fetopathy and pregnancy termination. The fetal histopathological examination was unremarkable on the viscera but showed on the testis an interstitial lymphocytic infiltrate. The placenta contained a Hofbauer cells hyperplasia with signs of inflammation. Neuropathological findings included a meningoencephalitis and an ex vacuo hydrocephalus. Immunohistochemical studies showed the presence of T lymphocytic and histiocytic meningitis associated with an abundant cerebral astroglial and macrophagic reaction. In situ hybridization demonstrated, abundant ZIKV particles within the cerebral parenchyma mainly in the ventricular/subventricular zone and in the cortical plate. In addition massive cells death and endoplasmic reticulum damage were present. CONCLUSION: The present study reports on the clinical and histopathological findings observed in three fetuses infected by the ZIKV. It emphasizes the severity of brain damages and the minimal visceral and placental changes observed upon ZIKV infection. This confirms the selective neurotropism of ZIKV. Finally, it allows us to describe the cascade of multifactorial developmental defects leading to microcephaly.

Unveiling the effects of berenil, a DNA-binding drug, on Trypanosoma cruzi: implications for kDNA ultrastructure and replication.

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a single mitochondrion with an enlarged portion termed kinetoplast. This unique structure harbors the mitochondrial DNA (kDNA), composed of interlocked molecules: minicircles and maxicircles. kDNA is a hallmark of kinetoplastids and for this reason constitutes a valuable target in chemotherapeutic and cell biology studies. In the present work, we analyzed the effects of berenil, a minor-groove-binding agent that acts preferentially at the kDNA, thereby affecting cell proliferation, ultrastructure, and mitochondrial activity of T. cruzi epimastigote form. Our results showed that berenil promoted a reduction on parasite growth when high concentrations were used; however, cell viability was not affected. This compound caused significant changes in kDNA arrangement, including the appearance of membrane profiles in the network and electron-lucent areas in the kinetoplast matrix, but nuclear ultrastructure was not modified. The use of the TdT technique, which specifically labels DNA, conjugated to atomic force microscopy analysis indicates that berenil prevents the minicircle decatenation of the network, thus impairing DNA replication and culminating in the appearance of dyskinetoplastic cells. Alterations in the kinetoplast network may be associated with kDNA lesions, as suggested by the quantitative PCR (qPCR) technique. Furthermore, parasites treated with berenil presented higher levels of reactive oxygen species and a slight decrease in the mitochondrial membrane potential and oxygen consumption. Taken together, our results reveal that this DNA-binding drug mainly affects kDNA topology and replication, reinforcing the idea that the kinetoplast represents a potential target for chemotherapy against trypanosomatids.

The kinetoplast ultrastructural organization of endosymbiont-bearing trypanosomatids as revealed by deep-etching, cytochemical and immunocytochemical analysis.

The endosymbiont-bearing trypanosomatids present a typical kDNA arrangement, which is not well characterized. In the majority of trypanosomatids, the kinetoplast forms a bar-like structure containing tightly packed kDNA fibers. On the contrary, in trypanosomatids that harbor an endosymbiotic bacterium, the kDNA fibers are disposed in a looser arrangement that fills the kinetoplast matrix. In order to shed light on the kinetoplast structural organization in these protozoa, we used cytochemical and immunocytological approaches. Our results showed that in endosymbiont-containing species, DNA and basic proteins are distributed not only in the kDNA network, but also in the kinetoflagellar zone (KFZ), which corresponds to the region between the kDNA and the inner mitochondrial membrane nearest the flagellum. The presence of DNA in the KFZ is in accordance with the actual model of kDNA replication, whereas the detection of basic proteins in this region may be related to the basic character of the intramitochondrial filaments found in this area, which are part of the complex that connects the kDNA to the basal body. The kinetoplast structural organization of Bodo sp. was also analyzed, since this protozoan lacks the highly ordered kDNA-packaging characteristic of trypanosomatid and represents an evolutionary ancestral of the Trypanosomatidae family.

The nuclear phenotypic plasticity observed in fish during rRNA regulation entails Cajal bodies dynamics.

Cajal bodies (CBs) are small mobile organelles found throughout the nucleoplasm of animal and plant cells. The dynamics of these organelles involves interactions with the nucleolus. The later has been found to play a substantial role in the compensatory response that evolved in eurythermal fish to adapt to the cyclic seasonal habitat changes, i.e., temperature and photoperiod. Contrary to being constitutive, rRNA synthesis is dramatically regulated between summer and winter, thus affecting ribosomal biogenesis which plays a central role in the acclimatization process. To examine whether CBs, up to now, never described in fish, were also sustaining the phenotypic plasticity observed in nuclei of fish undergoing seasonal acclimatization, we identified these organelles both, by transmission electronic microscopy and immunodetection with the marker protein p80-coilin. We found transcripts in all tissues analyzed. Furthermore we assessed that p80-coilin gene expression was always higher in summer-acclimatized fish when compared to that adapted to the cold season, indicating that p80-coilin expression is modulated upon seasonal acclimatization. Concurrently, CBs were more frequently found in summer-acclimatized carp which suggests that the organization of CBs is involved in adaptive processes and contribute to the phenotypic plasticity of fish cell nuclei observed concomitantly with profound reprogramming of nucleolar components and regulation of ribosomal rRNAs.

Ultrastructural changes of the carp (Cyprinus carpio) hepatocyte nucleolus during seasonal acclimatization.

BACKGROUND INFORMATION: The eurythermal fish carp (Cyprinus carpio) adjusts to the seasonal changes in the temperature and photoperiod of its habitat through diverse cellular and molecular mechanisms. We have observed that ribosomal biogenesis is modulated during the acclimatization process and correlates with profound phenotypic changes, reflecting a seasonal-dependent ultrastructural appearance of the nucleolar components. Previous studies using classical techniques showed that in winter-adapted carp the nucleolus appears to be segregated. In the present work, we have reassessed the nucleolar ultrastructural organization of the carp in summer- and winter-adapted fish by using more specific cytochemical and immunocytological techniques. RESULTS: The acetylation method provided evidence that the nucleolar organization is different between winter- and summer-adapted carp. In winter-adapted fish the fibrillar component appears as a unique mass surrounded by several granular caps, whereas in summer-adapted carp the fibrillar component forms few cordons surrounded by granular masses. The nucleolar structure and distribution of the condensed chromatin observed varies upon seasonal acclimatization. In winter the nucleolar chromatin is densely packed in masses that surround the nucleolus, whereas during summer it displays a rather looser organization formed by filaments that not only surround the nucleolus, but also go through the nucleolar body. Using the TdT (terminal deoxynucleotidyl transferase)-immunogold labelling technique, we detected condensed and decondensed nucleolar chromatin, and found some labelling of fibrillar components in both seasons. When liver tissue from summer-adapted carp was treated with AMD (actinomycin D), we observed that the rearrangement of the nucleolar components and condensed chromatin were similar to that found in winter-adapted fish, with differences in the distribution of the perinucleolar chromatin. CONCLUSIONS: The acetylation and TdT-immunogold labelling experiments indicated that the rearrangement of the nucleolar components of winter-adapted carp is very similar to the AMD-treated summer-adapted carp nucleolus, with the latter representing the repression of the ribosomal biogenesis that occurs during the cold season. Nevertheless, the distribution of the condensed perinucleolar chromatin in winter-adapted carp compared with AMD-treated cells suggests that the transcription of rRNA genes in winter-adapted fish is less strongly inhibited and does not lead to the classical segregation of the nucleolus of that described after AMD treatment. In addition, we have confirmed that carp hepatocyte nucleoli comprise only two main structural compartments: a fibrillar component and a granular component. Fibrillar centres were not observed.

Immunocytochemical detection of DNA and RNA in endosymbiont-bearing trypanosomatids.

Research about the kinetoplast of trypanosomatids has yielded valuable information about the organization of extranuclear structure. However, the ultrastructural localization of nucleic acids within these protozoa remains uncertain. We have applied cytochemical and immunocytochemical approaches to precisely identify DNA and RNA in lower endosymbiont-bearing trypanosomatids. Using the Terminal deoxynucleotidyl Transferase (TdT) immunogold technique, we showed that nuclear DNA is seen associated with the nuclear envelope during the trypanosomatid cell cycle. By combining the TdT technique with the acetylation method, which improves the contrast between structures containing fibrils and granules, we have demonstrated that the nucleolus of endosymbiont-bearing trypanosomatids is composed of two constituents: a granular component and a DNA-positive fibrillar zone. Moreover, we revealed that DNA of endosymbiotic bacteria consisted of electron-dense filaments which are usually in close contact with the prokaryote envelope. Using a Lowicryl post-embedding immunogold labeling procedure with anti-RNA antibodies, we showed the presence of RNA not only over the cytoplasm, the interchromatin spaces and the nucleolus, but also over the kinetoplast and virus-like particles present in Crithidia desouzai.

Chromosome localization changes in the Trypanosoma cruzi nucleus.

Chromosome localization in the interphase nuclei of eukaryotes depends on gene replication and transcription. Little is known about chromosome localization in protozoan parasites such as trypanosomes, which have unique mechanisms for the control of gene expression, with most genes being posttranscriptionally regulated. In the present study, we examined where the chromosomes are replicated in Trypanosoma cruzi, the agent of Chagas' disease. The replication sites, identified by the incorporation of 5-bromodeoxyuridine, are located at the nuclear periphery in proliferating epimastigote forms in the early S phase of the cell cycle. When the S phase ends and cells progress through the cell cycle, 5-bromodeoxyuridine labeling is observed in the nuclear interior, suggesting that chromosomes move. We next monitored chromosome locations in different stages of the cell cycle by using a satellite DNA sequence as a probe in a fluorescence in situ hybridization assay. We found two distinct labeling patterns according to the cell cycle stage. The first one is seen in the G(1) phase, in hydroxyurea-arrested epimastigotes or in trypomastigotes, which are differentiated nondividing forms. In all of these forms the satellite DNA is found in dots randomly dispersed in the nucleus. The other pattern is found in cells from the S phase to the G(2) phase. In these cells, the satellite DNA is found preferentially at the nuclear periphery. The labeling at the nuclear periphery disappears only after mitosis. Also, DNA detected with terminal deoxynucleotidyl transferase is found distributed throughout the nuclear space in the G(1) phase but concentrated at the nuclear periphery in the S phase to the G(2) phase. These results strongly suggest that T. cruzi chromosomes move and, after entering the S phase, become constrained at the nuclear periphery, where replication occurs.