Integrative analysis of molecular pathways and morphological anomalies associated with congenital Zika syndrome.

Congenital Zika syndrome (CZS) comprises a set of clinical manifestations that can be presented by neonates born to mothers infected by the Zika virus (ZIKV). CZS-associated phenotypes include neurological, skeletal, and systemic alterations and long-term developmental sequelae. One of the most frequently reported clinical conditions is microcephaly characterized by a reduction in head circumference and cognitive complications. Nevertheless, the associations among the diverse signaling pathways underlying CZS phenotypes remain to be elucidated. To shed light on CZS, we have extensively reviewed the morphological anomalies resulting from ZIKV infection, as well as genes and proteins of interest obtained from the published literature. With this list of genes or proteins, we performed computational analyses to explore the cellular processes, molecular mechanisms, and molecular pathways related to ZIKV infection. Therefore, in this review, we comprehensively describe the morphological abnormalities caused by congenital ZIKV infection and, through the analysis noted above, propose common molecular pathways altered by ZIKV that could explain both central nervous system and craniofacial skeletal alterations.

Prechordal structures act cooperatively in early trabeculae development of gnathostome skull.

The vertebrate skull is formed by mesoderm and neural crest (NC) cells. The mesoderm contributes to the skull chordal domain, with the notochord playing an essential role in this process. The NC contributes to the skull prechordal domain, prompting investigation into the embryonic structures involved in prechordal neurocranium cartilage formation. The trabeculae cartilage, a structure of the prechordal neurocranium, arises at the convergence of prechordal plate (PCP), ventral midline (VM) cells of the diencephalon, and dorsal oral ectoderm. This study examines the molecular participation of these embryonic structures in gnathostome trabeculae development. PCP-secreted SHH induces its expression in VM cells of the diencephalon, initiating a positive feedback loop involving SIX3 and GLI1. SHH secreted by the VM cells of the diencephalon acts on the dorsal oral ectoderm, stimulating condensation of NC cells to form trabeculae. SHH from the prechordal region affects the expression of SOX9 in NC cells. BMP7 and SHH secreted by PCP induce NKX2.1 expression in VM cells of the diencephalon, but this does not impact trabeculae formation. Molecular cooperation between PCP, VM cells of the diencephalon, and dorsal oral ectoderm is crucial for craniofacial development by NC cells in the prechordal domain.

Anti-cancer Effects of Fucoxanthin on Human Glioblastoma Cell Line.

BACKGROUND/AIM: Glioblastomas (GBMs) are the most malignant primary brain tumor. New treatment strategies against the disease are urgently needed, as therapies are not completely efficient. In this study, we evaluated the antitumorigenic activity of the carotenoid fucoxanthin (Fx) on human GBM cells in vitro. MATERIALS AND METHODS: GBM1 cell viability and proliferation was assessed by MTT reduction, Ki67 and single cell cloning assays. GBM1 migration and invasion were analyzed by wound healing and Transwell assays. Apoptosis and necrosis were analyzed by flow cytometry, and the mitochondrial membrane potential (ΔΨm) by the selective fluorescent dye tetramethylrhodamine ethyl ester. Cell morphology was analyzed through scanning electron microscopy and transmission electron microscopy. Fx anti-angiogenic effect was assessed by the CAM ex ovo assay. RESULTS: Fx decreased cell viability in a concentration-dependent manner (40-100 µ M) in GBM1, A172 and C6 cell lines and was not cytotoxic to murine astrocytes. In addition, Fx inhibited the proliferation and clonogenic potential, and decreased migration and invasion of GBM1 cells. Furthermore, Fx induced apoptosis, loss of ΔΨm and ultrastructural alterations in GBM1. Fx-treated GBM1 cells-conditioned medium reduced the quail yolk membrane vascularity. CONCLUSION: Fx induces cytotoxicity, anti-proliferative, anti-invasive and anti-angiogenic effects on GBM1 cells.

FGF2 Stimulates the Growth and Improves the Melanocytic Commitment of Trunk Neural Crest Cells.

Neural crest cells (NCCs) comprise a population of multipotent progenitors and stem cells at the origin of the peripheral nervous system (PNS) and melanocytes of skin, which are profoundly influenced by microenvironmental factors, among which is basic fibroblast growth factor 2 (FGF2). In this work, we further investigated the role of this growth factor in quail trunk NC morphogenesis and demonstrated its huge effect in NCC growth mainly by stimulating cell proliferation but also reducing cell death, despite that NCC migration from the neural tube explant was not affected. Moreover, following FGF2 treatment, reduced expression of the early NC markers Sox10 and FoxD3 and improved proliferation of HNK1-positive NCC were observed. Since these markers are involved in the regulation of glial and melanocytic fate of NC, the effect of FGF2 on NCC differentiation was investigated. Therefore, in the presence of FGF2, increased proportions of NCCs positives to the melanoblast marker Mitf as well as NCCs double stained to Mitf and BrdU were recorded. In addition, treatment with FGF2, followed by differentiation medium, resulted in increased expression of melanin and improved proportion of melanin-pigmented melanocytes without alteration in the glial marker Schwann myelin protein (SMP). Taken together, these data further reveal the important role of FGF2 in NCC proliferation, survival, and differentiation, particularly in melanocyte development. This is the first demonstration of FGF2 effects in melanocyte commitment of NC and in the proliferation of Mitf-positive melanoblasts. Elucidating the differentiation process of embryonic NCCs brings us a step closer to understanding the development of the PNS and then undertaking the search for advanced technologies to prevent, or treat, injuries caused by NC-related disorders, also known as neurocristopathies.

Human Dental Pulp Stem Cells in Rat Mandibular Bone Defects.

This study aimed to evaluate the use of human dental pulp stem cells (hDPSCs) in non-critical-sized mandibular bone defects in rats. hDPSCs from permanent teeth were isolated and engrafted in mandibular bone defects in rats for 7, 14, and 28 days; bone defects without cells formed the control group. Samples were evaluated by scanning electron microscopy (SEM), light microscopy (hematoxylin and eosin staining), and the regeneration area was measured by the Image J program. Before surgery procedures, the human dental pulp cells were characterized as dental pulp stem cells: fusiform morphology, plastic-adherent; expression of CD105, CD73, and CD90; lack of expression of CD45 and CD34, and differentiated into osteoblasts, adipocytes, and chondroblasts. The results indicated that within 7 days the control group presented a pronounced bone formation when compared with the treated group (p < 0.05). After 14 days, the treated group showed an increase in bone formation, but with no statistical difference among the groups (p > 0.05). In the final evaluated period there was no difference between the control group and the treated group (p > 0.05). There was a significant difference between 7 and 14 days (p < 0.05) and between 7 and 28 days (p < 0.05) in the treated group. In conclusion, there is no evidence that the use of hDPSCs in the conditions of this study could improve bone formation in non-critical-sized mandibular bone defects.

Latin American contributions to the neural crest field.

The neural crest (NC) is one of the most fascinating structures during embryonic development. Unique to vertebrate embryos, these cells give rise to important components of the craniofacial skeleton, such as the jaws and skull, as well as melanocytes and ganglia of the peripheral nervous system. Worldwide, several groups have been studying NC development and specifically in the Latin America (LA) they have been growing in numbers since the 1990s. It is important for the world to recognize the contributions of LA researchers on the knowledge of NC development, as it can stimulate networking and improvement in the field. We developed a database of LA publications on NC development using ORCID and PUBMED as search engines. We thoroughly describe all of the contributions from LA, collected in five major topics on NC development mechanisms: i) induction and specification; ii) migration; iii) differentiation; iv) adult NC; and, v) neurocristopathies. Further analysis was done to correlate each LA country with topics and animal models, and to access collaboration between LA countries. We observed that some LA countries have made important contributions to the comprehension of NC development. Interestingly, some LA countries have a topic and an animal model as their strength; in addition, collaboration between LA countries is almost inexistent. This review will help LA NC research to be acknowledged, and to facilitate networking between students and researchers worldwide.

Novel synthetic chalcones induces apoptosis in human glioblastoma cells.

Glioblastoma multiforme is the main and most frequent tumor in adults' central nervous system. With a survival average of 5% two years after diagnosis, this type of cancer is a main health problem. Substances like the chalcones have been tested in order to develop new treatments. Here, we studied the effects of three synthetic chalcones (A23, C31 and J11) on A172 and surgery obtained-glioma cells. All chalcones showed a decrease in cell viability, mainly C31. An increase in apoptosis levels with no further increase of necrosis was observed. This augmentation may be linked to the high oxidative effect found, caused by the increased presence of reactive oxygen species and nitric oxide production. Cell cycle distribution showed an arrest at G0/G1 and S phases, suggesting that C31 interferes in cell cycle control. Our results shall aid in directing future research with this substance and its antitumor effect.

Temporo-spatial analysis of Osterix, HNK1 and Sox10 during odontogenesis and maxillaries osteogenesis.

Cell differentiation is essential for maxillaries and tooth development. Facial mesenchymal tissue is formed by neural crest cells (NC). These cells are highly migratory, giving rise to various cell types, considered with a high level of plasticity, indicating that they contain progenitor cells with a great power of differentiation. In this study, it was analyzed the presence of NC cell progenitors and mesenchymal stem cells (MSC) during maxillaries osteogenesis and odontogenesis in rats. Histological slides were collected in two phases: embryonic age of 15 and 17 days; 2, 4 and 7 days after birth. Immunohistochemistry for MSC markers (Osterix) and NC cells (Sox10, HNK1) was performed. The results showed positive expression for Osterix and HNK1 in undifferentiated ectomesenchymal cells in early and late stages; Sox10 was present only in early stages in undifferentiated cells. All markers were present in differentiated cells. Although the experiments performed do not allow us to explain a possible role for Osx, HNK1 and Sox10 in both differentiated and undifferentiated cells during osteogenesis and odontogenesis, it had shown important results not yet described: the presence of HNK1 and Sox10 in osteoblasts and odontoblasts in late development stages and in the tooth germ epithelial cells and ameloblasts.

Epidermal growth factor (EGF) promotes the in vitro differentiation of neural crest cells to neurons and melanocytes.

Proliferation of neural crest (NC) stem cells and their subsequent differentiation into different neural cell types are key early events in the development of the peripheral nervous system. Soluble growth factors present at the sites where NC cells migrate are critical to the development of NC derivatives in each part of the body. In the present study, we further investigate the effect of microenvironmental factors on quail trunk NC development. We show for the first time that EGF induces differentiation of NC to the neuronal and melanocytic phenotypes, while fibroblast growth factor 2 (FGF2) promotes NC differentiation to Schwann cells. In the presence of both EGF and FGF2, the neuronal differentiation predominates. Our results suggest that FGF2 stimulates gliogenesis, while EGF promotes melanogenesis and neurogenesis. The combination of both growth factors stimulates neurogenesis. These findings suggest that these two growth factors may play an important role in the fate decision of NC progenitors and in the development of the peripheral nervous system.

Thyroid hormone modulates the extracellular matrix organization and expression in cerebellar astrocyte: effects on astrocyte adhesion.

The effects of thyroid hormone (T(3)) on extracellular matrix (ECM) expression and organization in cerebellar astrocytes were studied. Control astrocytes exhibit laminin immunostaining distributed in a punctate configuration and fibronectin concentrated in focal points at the cell surface. These cells attach to the substratum by membrane points, as shown by scanning microscopy, possibly by focal points stained to fibronectin. In contrast, after T(3) treatment, laminin assumes a fibrillary pattern and fibronectin becomes organized in filaments homogeneously distributed on the cell surface; the cells acquire a very flat and spread morphology. T(3) treatment also modulates astrocyte adhesion. In addition, increased expression of both laminin and fibronectin was detected by Western blot. These alterations in fibronectin and/or laminin production and organization may be involved in the flat and spread morphology and in altered adhesion. We observed that fibroblast growth factor-2 (FGF(2)) added to cultures had similar effects to those described to T(3). Neutralizing antibodies against FGF(2) reversed T(3) effects on fibronectin and laminin distribution. We also observed that cerebellar neurons co-cultured on T(3)-treated astrocytes had an increase in the number of cells and presented longer neurites. Thus, we propose a novel mechanism of the effect of thyroid hormone on cerebellar development mediated by astrocytes: T(3) may induce astrocyte secretion of growth factors, mainly FGF(2), that autocrinally stimulate astrocyte proliferation, reorganization in ECM proteins, and alterations in cell spreading and adhesion. These effects may indirectly influence neuronal development.

Undersulfation of proteoglycans and proteins alter C6 glioma cells proliferation, adhesion and extracellular matrix organization.

Proteoglycans are considered to be important molecule in cell-microenvironment interactions. They are overexpressed in neoplastic cells modifying their growth and migration in hosts. In this work we verified that undersulfation of proteoglycans and other sulfated molecules, induced by sodium chlorate treatment, inhibited C6 glioma cells proliferation in a dose-dependent way. This effect was restored by the addition of exogenous heparin. We could not detect significant cell mortality in our culture condition. The treatment also impaired in a dose-dependent manner, C6 cell adhesion to extracellular matrix (ECM) proteins (collagen IV, laminin and fibronectin). In addition, sodium chlorate treatment altered C6 glioma cell morphology, from the fibroblast-like to a more rounded one. This effect was accompanied by increased synthesis of fibronectin and alterations in its extracellular network organization. However, we could not observe modifications on laminin organization and synthesis. The results suggest an important connection between sulfation degree with important tumor functions, such as proliferation and adhesion. We suggest that proteoglycans may modulate the glioma microenvironment network during tumor cell progression and invasion.