Search

5-Fluorouracil (5-FU), an anti-cancer drug, has been used for hepatoblastoma (HB) chemotherapy in children, who may have impaired ovarian follicle pool reserve with lasting effects to reproduction. Therefore, this study aimed to investigate 5-FU effects on survival, growth, and morphology of ovarian preantral follicles from C57BL6J young mice. Experiments were carried-out both in vivo and in vitro. Mice were treated with 5-FU injection (450 mg/kg i.p) or saline and sacrificed 3 days after to obtain ovaries for histology and molecular biology. Ovaries for in vitro studies were obtained from unchallenged mice and cultured under basic culture medium (BCM) or BCM plus 5-FU (9.2, 46.1, 92.2 mM). Preantral follicles were classified according to developmental stages, and as normal or degenerated. To assess cell viability, caspase-3 immunostaining was performed. Transcriptional levels for apoptosis (Bax, Bcl2, p53, Bax/Bcl2) and Wnt pathway genes (Wnt2 and Wnt4) were also analyzed. Ultrastructural analyses were carried-out on non-cultured ovaries. In addition, β-catenin immunofluorescence was assessed in mouse ovaries. The percentage of all-types normal follicles was significantly lower after 5-FU challenge. A total loss of secondary normal follicles was found in the 5-FU group. The highest 5-FU concentrations reduced the percentage of cultured normal primordial follicles. Large vacuoles were seen in granulosa cells and ooplasm of preantral follicles by electron microscopy. A significantly higher gene expression for Bax and Bax/Bcl2 ratio was seen after 5-FU treatment. A marked reduction in β-catenin immunolabeling was seen in 5-FU-challenged preantral follicles. In the in vitro experiments, apoptotic and Wnt gene transcriptions were significantly altered. Altogether, our findings suggest that 5-FU can deleteriously affect the ovarian follicle reserve by reducing preantral follicles survival.

This review highlights the work that my research group has been developing, together with international collaborators, during the last decade. Since we were able to establish the Xenopus laevis experimental model in Brazil, we have been focused on understanding early embryonic patterns regarding neural induction and axes establishment. In this context, the Wnt pathway appears as a major player and has been much explored by us and other research groups. Here, we chose to review three published works which we consider to be landmarks within the course of our research and also within the history of modern findings regarding neural induction and patterning. We intend to show how our series of discoveries, when painted together, tells a story that covers crucial developmental windows of early differentiation paths of anterior neural tissue: 1. establishing the head organizer in contrast to the trunk organizer in the early gastrula; 2. deciding between neural ectoderm and epidermis ectoderm at the blastula/gastrula stages, and 3. the gathering of prechordal unique properties in the late gastrula/early neurula.

Objetivou-se avaliar as características produtivas, nutricionais e econômicas de vacas de leite submetidas a níveis de ureia com dietas à base de silagem de sorgo. Foram utilizadas 05 vacas lactantes ¾ Holandês x ¼ Gir Leiteiro, no terço médio de lactação, com idade média de 60 meses e peso corporal médio de 518 kg ± 52 kg, que foram distribuídas em cinco tratamentos, em delineamento experimental de quadrado latino 5x5. Os tratamentos (U-50, U-75, U-100, U-125 e U-150) foram constituídos de diferentes níveis de ureia no concentrado (5,7; 8,6; 11,4; 14,3 e 17,1 g de ureia/kg de MS do concentrado, respectivamente). Adotou-se como nível de significância 0,05. Não houve efeito dos níveis de ureia no consumo alimentar, digestibilidade e parâmetros metabólicos de vacas de leite. Não foram observadas diferenças significativas na produção de leite corrigida para 3,5% de gordura. A composição do leite não sofreu efeitos significativos. O custo total com volumoso, concentrado e alimentação não sofreu efeitos significativos. A receita bruta com a venda de leite não sofreu efeito significativo, apresentando média de BRL 28,65. A taxa de retorno marginal não apresentou diferenças significativas. Os diferentes níveis de ureia não afetaram a produção de vacas de leite. Conclui-se que na dieta de vacas de leite à base de silagem de sorgo pode-se realizar a inclusão de ureia no concentrado com níveis de 5,7 a 17,1 g/kg do concentrado, sem redução do desempenho produtivo dos animais.

Background In Latin America, there is scarce information about severe asthma (SA) according to the ERS/ATS 2014 criteria. This study aimed to compare the demographic, socio, clinical characteristics, treatment, and use of healthcare resources between SA and non-severe asthma (NSA) patients in Argentina, Colombia, Chile and Mexico. Methods A cross-sectional study was conducted including 594 asthma patients from outpatient specialized sites. A descriptive analysis was performed comparing SA patients and NSA. Chi-square and Mann Whitney tests were used to assess associations between asthma severity and outcome variables. Results Using ERS/ATS 2014 criteria, 31.0% of the patients were identified as SA. SA patients were older at diagnosis (mean age 31.64 years vs 24.71 years, p

Expression of estrus near timed artificial insemination (TAI) is associated with greater fertility, and estrus detection could improve TAI fertility or direct TAI management, although accurate estrus detection can be difficult and time-consuming using traditional methods. The aim of this study is to evaluate influence of estrus on pregnancy (artificial insemination pregnancy rates (P/AI)) and to validate an alternative method to classify estrus/heat expression using tail chalking (HEATSC) in postpartum Bos indicus cows subjected to TAI in progesterone–estrogen-based protocols. In experiment 1 (Exp. 1), cows (5491) were subjected to visual observation of estrus after progesterone device removal, before TAI, and P/AI was evaluated according to estrus and body condition score (BCS). Cows received a progesterone device and 2 mg estradiol benzoate (EB). After 8 days, the device was removed and 150 μg of d-cloprostenol and 300 IU equine chorionic gonadotrophin was given. Later, animals in Exp. 1 received 1 mg EB and TAI 44 to 48 h. In the Exp. 2 – 3830 cows using similar protocol, received different ovulation inducers: 1 mg EB (n=1624) or 1 mg estradiol cypionate (EC; n=2206) on day 8 (D8). Cows were then marked with chalk, and HEATSC evaluated at TAI on D10 (HEATSC1 – no chalk removal=no estrus expression; HEATSC2 – partial chalk removal=low estrus expression; HEATSC3 – near complete/complete chalk removal=high estrus expression). In Exp. 1, cows showing estrus presented greater P/AI (48.4% v. 40.2%, P

The work aims were to describe the histological and histochemical structure of the gastroesophageal tube of Iguana iguana and verify the occurrence and distribution of immunoreactive serotonin (5-HT) and somatostatin (SS) cells. Fragments of the gastrointestinal tract (GIT) of five iguanas were which underwent standard histological and immunohistochemistry technique. Immunoreactive cells for 5-HT and SS were quantified using the STEPanizer. The oesophagus has ciliated columnar pseudostratified epithelium with staining Alcian blue (AB) + and goblet cells highly reactive to periodic acid Schiff (PAS). In the cervical oesophagus, the numerical density of 5-HT cells per unit area (QA [5-HT cells]/µm2) was 4.6x10-2 ± 2.0 and celomatic oesophagus presented QA = 4.0x10-2 ± 1.0. The epithelium of the stomach is simple columnar, PAS and AB +. The cranial and middle regions of the stomach presented (QA [5-HT cells]/µm2) = 6.18x10-2 ± 3.2 and the caudal region, QA = 0.6x10-2 ± 0.2. The SS cells were only observed in the caudal stomach, with numerical density (QA [SS cells]/µm2) = 1.4x10-2 ± 0.9 In I. iguana, variation was observed in terms of the distribution of mucus secretions and the pattern of occurrence of serotonin and somatostatin-secreting enteroendocrine cells in the TGI, which possibly will result in an interspecific adaptive response. Resumo Os objetivos do trabalho foram descrever a estrutura histológica e histoquímica do tubo gastroesofágico da Iguana iguana e verificar a ocorrência e distribuição de células serotonina (5-HT) e somatostatina (SS) imunorreativas. Fragmentos do trato gastrointestinal (TGI) de cinco iguanas foram submetidos à técnica histológica e imunohistoquímica padrão. As células imunorreativas para 5-HT e SS foram quantificadas usando o STEPanizer. O esôfago apresenta epitélio pseudoestratificado colunar ciliado Alcian blue (AB) positivo, com células caliciformes altamente reativas ao ácido periódico de Schiff (PAS). No esôfago cervical, a densidade numérica de células 5-HT por unidade de área (QA [células 5-HT] / µm2) foi de 4.6x10-2 ± 2.0 e o esôfago celomático apresentou QA = 4.0x10-2 ± 1.0. O epitélio do estômago é colunar simples, PAS e AB positivo. As regiões cranial e média do estômago apresentaram (QA [células 5-HT] / µm2) = 6.18x10-2 ± 3.2 e a região caudal, QA = 0.6x10-2 ± 0.2. As células SS foram observadas apenas no estômago caudal, com densidade numérica (QA [células SS] / µm2) = 1.4x10-2 ± 0.9. Em I. iguana, foi observada variações em termos da distribuição das secreções de muco e padrão de ocorrência das células enteroendócrinas secretoras de serotonina e somatostatina no TGI, o que possivelmente reflete uma resposta adaptativa interespecifica.

Wnt signaling through the Frizzled (FZD) family of serpentine receptors is essential for embryogenesis and homeostasis, and stringent control of the FZD protein level is critical for stem cell regulation. Through CRISPR/Cas9 genome-wide screening in human cells, we identified TMEM79/MATTRIN, an orphan multi-span transmembrane protein, as a specific inhibitor of Wnt/FZD signaling. TMEM79 interacts with FZD during biogenesis and promotes FZD degradation independent of ZNRF3/RNF43 ubiquitin ligases (R-spondin receptors). TMEM79 interacts with ubiquitin-specific protease 8 (USP8), whose activating mutations underlie human tumorigenesis. TMEM79 specifically inhibits USP8 deubiquitination of FZD, thereby governing USP8 substrate specificity and promoting FZD degradation. Tmem79 and Usp8 genes have a pre-bilaterian origin, and Tmem79 inhibition of Usp8 and Wnt signaling is required for anterior neural development and gastrulation in Xenopus embryos. TMEM79 is a predisposition gene for Atopic dermatitis, suggesting deregulation of Wnt/FZD signaling a possible cause for this most common yet enigmatic inflammatory skin disease.

More than 94% of colorectal cancer cases have mutations in one or more Wnt/β-catenin signaling pathway components. Inactivating mutations in APC or activating mutations in β-catenin (CTNNB1) lead to signaling overactivation and subsequent intestinal hyperplasia. Numerous classes of medicines derived from synthetic or natural small molecules, including alkaloids, have benefited the treatment of different diseases, including cancer, Piperine is a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper (Piper nigrum) and long pepper (Piper longum). Studies have shown that piperine has a wide range of pharmacological properties; however, piperine molecular mechanisms of action are still not fully understood. By using Wnt/β-catenin pathway epistasis experiment we show that piperine inhibits the canonical Wnt pathway induced by overexpression of β-catenin, β-catenin S33A or dnTCF4 VP16, while also suppressing β-catenin nuclear localization in HCT116 cell line. Additionally, piperine impairs cell proliferation and migration in HCT116, SW480 and DLD-1 colorectal tumor cell lines, while not affecting the non-tumoral cell line IEC-6. In summary, piperine inhibits the canonical Wnt signaling pathway and displays anti-cancer effects on colorectal cancer cell lines.

In the present work we have focused on the Histone Deacetylase (HDAC) control of myeloid cells behavior during Xenopus tail regeneration. Here we show that myeloid differentiation is crucial to modulate the regenerative ability of Xenopus tadpoles in a HDAC activity-dependent fashion. HDAC activity inhibition during the first wave of myeloid differentiation disrupted myeloid cells dynamics in the regenerative bud as well the mRNA expression pattern of myeloid markers, such as LURP, MPOX, Spib and mmp7. We also functionally bridge the spatial and temporal dynamics of lipid droplets, the main platform of lipid mediators synthesis in myeloid cells during the inflammatory response, and the regenerative ability of Xenopus tadpoles. In addition, we showed that 15-LOX activity is necessary during tail regeneration. Taken together our results support a role for the epigenetic control of myeloid behavior during tissue and organ regeneration, which may positively impact translational approaches for regenerative medicine.

This study aims to describe the eligibility for biologic therapies for severe asthma (SA) in a cohort of patients attending the Program for Control of Asthma (ProAR) in Bahia, Brazil.Data from SA patients (≥18 years old) attending the ProAR, that were included in a case-control study conducted from 2013 to 2015, were used to reassess patients according to a modified ERS/ATS 2014 SA criteria. Patients were then classified according to the eligibility for SA biological therapy based on current prescription labels.From 544 patients in the cohort, 531 (97.6%) were included and 172 (32.4%) were identified as SA patients according to the ERS/ATS 2014 modified criteria. Of these 172 patients, 69 (40.1%) were ineligible for any of the biologicals approved for asthma (omalizumab, mepolizumab, reslizumab and benralizumab), 60 (34.9%) patients were eligible for one of the biological therapies, and 10 (5.8%) patients were eligible for all biological therapies.More than half of patients with SA were eligible for biologic therapy in our study, but none of them received this form of treatment. Almost half of them were not eligible to any of the approved biologics, however. The variability and overlap in patients' eligibility highlight the importance of evaluating each patient individually for a more personalized treatment approach. While there is a need to increase access for some of those eligible that may really need a biologic treatment, continuous efforts are required to develop alternatives to those who are not eligible.

Escherichia coli is an important pathogen responsible for a variety of diseases. We have recently shown that Pic, a serine protease secreted by E. coli, mediates immune evasion by the direct cleavage of complement molecules. The aim of this study was to investigate the action of a Pic-producing bacteria in a murine model of sepsis. Mice were infected with Pic-producing E. coli (F5) or F5∆pic mutant. Animal survival was monitored for five days, and a subset of mice was euthanized after 12 h for sample acquisition. The inoculation of Pic-producing bacteria induced 100% death within 24 h. The colony forming units count in the organs was significantly higher in F5. Hematological analysis showed a decrease of total leukocytes. Nitric oxide and cytokines were detected in serum, as well as on peritoneal lavage of the F5 group in higher levels than those detected in the other groups. In addition, immunophenotyping showed a decrease of activated lymphocytes and macrophages in the F5 group. Therefore, Pic represents an important virulence factor, allowing the survival of the bacterium in the bloodstream and several organs, as well as inducing a high production of proinflammatory mediators by the host, and concomitantly a cellular immunosuppression, leading to sepsis and death.

In the present work we propose to shed light on the epigenetic control of immune mechanisms involved duringXenopustail regeneration. Here we show that the first 24 hour post amputation (hpa), which exclusively encompasses the first wave of myeloid differentiation, are crucial to epigenetically modulate the regenerative ability ofXenopustadpoles. During this developmental window, HDAC activity was shown to be necessary for the proper establishment of myeloid cells dynamics in the regenerative bud, mainly contributing to modulate the behavior of monocytes/macrophages and neutrophils as well the mRNA expression pattern of the main myeloid markers, such as LURP, MPOX, Spib and mmp7. In addition, we functionally bridge the spatial and temporal dynamics of lipid droplets, the main platform of lipid mediators synthesis in myeloid cells during the inflammatory response, and the regenerative ability ofXenopustadpoles showing that 15-LOX activity is a key player during tail regeneration. Taken together our results support a role for the epigenetic control of inflammatory response during tissue and organ regeneration, which may positively impact translational approaches for regenerative medicine.Summary statementWe propose that Epigenetic mechanisms HDAC-dependent can control myeloid cells behavior upon tissue injury and that HDAC inhibitors may be used for tissue regeneration in translational studies.

Multi-wall lipid-core nanocapsule (MLNC) functionalized with captopril and nanoencapsulating furosemide within the core was developed as a liquid formulation for oral administration. The nanocapsules had mean particle size below 200 nm, showing unimodal and narrow size distributions with moderate dispersity (laser diffraction and dynamic light scattering). Zeta potential was inverted from &minus, 14.3 mV [LNC-Fur(0,5)] to +18.3 mV after chitosan coating. Transmission electron microscopy and atomic force microscopy showed spherical structures corroborating the nanometric diameter of the nanocapsules. Regarding the systolic pressure, on the first day, the formulations showed antihypertensive effect and a longer effect than the respective drug solutions. When both drugs were associated, the anti-hypertensive effect was prolonged. On the fifth day, a time effect reduction was observed for all treatments, except for the nanocapsule formulation containing both drugs [Capt(0.5)-Zn(25)-MLNC-Fur(0.45)]. For diastolic pressure, only Capt(0.5)-Zn(25)-MLNC-Fur(0.45) presented a significant difference (p &lt, 0.05) on the first day. On the fifth day, both Capt(0.5)-MLNC-Fur(0.45) and Capt(0.5)-Zn(25)-MLNC-Fur(0.45) had an effect lasting up to 24 h. The analysis of early kidney damage marker showed a potential protection in renal function by Capt(0.5)-Zn(25)-MLNC-Fur(0.45). In conclusion, the formulation Capt(0.5)-Zn(25)-MLNC-Fur(0.45) proved to be suitable for hypertension treatment envisaging an important innovation.

Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular heterogeneity and capability to communicate with microenvironment components, such as microglia. Microglia-glioblastoma interaction contributes to an increase in tumor invasiveness, and Wnt signaling pathway is one of the main cascades related to tumor progression through changes in cell migration and invasion. However, very little is known about the role of canonical Wnt signaling during microglia-glioblastoma crosstalk. Here, we show for the first time that Wnt3a is one of the factors that regulate interactions between microglia and glioblastoma cells. Wnt3a activates the Wnt/β-catenin signaling of both glioblastoma and microglial cells. Glioblastoma-conditioned medium not only induces nuclear translocation of microglial β-catenin but also increases microglia viability and proliferation as well as Wnt3a, cyclin-D1, and c-myc expression. Moreover, glioblastoma-derived Wnt3a increases microglial ARG-1 and STI1 expression, followed by an upregulation of IL-10 mRNA levels, and a decrease in IL1β gene expression. The presence of Wnt3a in microglia-glioblastoma co-cultures increases the formation of membrane nanotubes accompanied by changes in migration capability. In vivo, tumors formed from Wnt3a-stimulated glioblastoma cells presented greater microglial infiltration and more aggressive characteristics such as growth rate than untreated tumors. Thus, we propose that Wnt3a belongs to the arsenal of factors capable of stimulating the induction of M2-like phenotype on microglial cells, which contributes to the poor prognostic of glioblastoma, reinforcing that Wnt/β-catenin pathway can be a potential therapeutic target to attenuate glioblastoma progression.

The aim of this study was to evaluate the linear correlations between feeding behavior and apparent digestibility in feedlot lambs. Fifty castrated Santa Ines ram lambs with an average body weight of 20 ± 4.38 kg and four months of age were used in this experiment. Animals received different feed levels aiming at promoting variations in feed intake and apparent digestibility. A randomized complete design was adopted. Digestibility and feeding behavior were measured. Analyses were performed using Pearson’s linear correlations, applying the t test at 5% probability level. A positive correlation was detected between feeding time and apparent digestibility of ether extract and total digestible nutrients (TDN) (p

Glioblastoma, the most aggressive and fatal type of brain tumor, is capable of interacting with brain immune cells such as microglia, which contributes to the growth of these tumors. Various molecules, including growth factors and cytokines, have been identified as regulators of microglia-glioblastoma interaction. Recent studies suggest that the Wnt family of lipoglycoproteins plays an important role, not only in biological events during development, but also in cancer progression, and can be part of microglia recruitment to glioblastoma as well as of tumor growth and invasion. Here, we discuss recent interesting findings that support a role for Wnt signaling pathways in the microglia-glioblastoma crosstalk.

Summary Objectives The diagnosis of pediatric tuberculosis (TB) presents many challenges, and is further complicated in HIV-infected patients. While many diagnostic systems have been proposed, there is no pediatric TB diagnosis gold standard. The outcomes of four TB diagnostic systems in HIV-infected children were compared in this study. Methods A retrospective cohort study was conducted at a TB/HIV reference hospital in Rio de Janeiro. HIV-infected pediatric patients evaluated for TB from 1998 to 2010 were reassessed using four diagnostic systems: Kenneth Jones, 1969; Tidjani, 1986; Ben Marais, 2006; Brazilian Ministry of Health, 2010. Results were compared to standardized diagnoses made by an expert panel of physicians. Results Of the 121 patients in the study cohort, the expert panel diagnosed 64 as TB and 57 as not TB cases. The Tidjani system showed the highest diagnostic accuracy, with and without the inclusion of microbiological data. The Tidjani and Kenneth Jones systems produced fewer false-positives, and the Ben Marais and Ministry of Health fewer false-negatives. Across systems, there was little agreement between TB diagnoses. Conclusions In HIV-infected pediatric patients, the Ben Marais and Ministry of Health systems are useful for TB diagnostic screening, whereas the Tidjani and Kenneth Jones systems are best used in a reference center setting.

The deregulation of the Wnt/&beta, catenin signaling pathway is a central event in colorectal cancer progression, thus a promising target for drug development. Many natural compounds, such as flavonoids, have been described as Wnt/&beta, catenin inhibitors and consequently modulate important biological processes like inflammation, redox balance, cancer promotion and progress, as well as cancer cell death. In this context, we identified the chalcone lonchocarpin isolated from Lonchocarpus sericeus as a Wnt/&beta, catenin pathway inhibitor, both in vitro and in vivo. Lonchocarpin impairs &beta, catenin nuclear localization and also inhibits the constitutively active form of TCF4, dnTCF4-VP16. Xenopus laevis embryology assays suggest that lonchocarpin acts at the transcriptional level. Additionally, we described lonchocarpin inhibitory effects on cell migration and cell proliferation on HCT116, SW480, and DLD-1 colorectal cancer cell lines, without any detectable effects on the non-tumoral intestinal cell line IEC-6. Moreover, lonchocarpin reduces tumor proliferation on the colorectal cancer AOM/DSS mice model. Taken together, our results support lonchocarpin as a novel Wnt/&beta, catenin inhibitor compound that impairs colorectal cancer cell growth in vitro and in vivo.

Staphylococcus aureus is a notorious human pathogen associated with serious nosocomial and community-acquired infections, such as pneumonia, meningitis, endocarditis, toxic shock syndrome, and sepsis, among others. The objective of this study was to investigate the molecular profile, antimicrobial resistance, and clonal diversity of S. aureus isolated from the bloodstream. The determination of the minimum inhibitory concentration (MIC) of the antimicrobial was performed by an automated method. The presence of several virulence and resistance genes was evaluated by PCR. In addition, multilocus sequence typing (MLST) was used to analyze the clonal diversity of S. aureus. A high resistance to oxacillin (78%), clindamycin (78%), erythromycin (70%), ciprofloxacin (61%), and gentamicin (52%) was observed among the isolates. In most of them, the following virulence genes were detected: hlb (83%), ebpS (61%), icaA (57%), fnbpA (17%), and clfA (13%). Only one isolate carried the pvl gene. MLST analysis identified five new sequence types (STs): 5429, 5430, 5431, 5432, and 5433, as well as another seven&mdash, ST5, ST97, ST398, ST101, ST30, ST461, and ST2779&mdash, among the remaining strains. These seven STs and the four new STs are clustered in four clonal complexes: CC1, CC2, CC7, and CC17. Phylogenetic analysis showed the genetic relationship of the five new ST strains with another 18 strains. Altogether, these analyses indicate the horizontal transfer acquisition of virulence factor genes and multidrug resistance.

Escherichia coli is responsible for cases of diarrhea around the world, and some studies have shown the benefits of cinnamaldehyde in the treatment of bacterial disease. Therefore, the objective of this study was to evaluate the effects of cinnamaldehyde in mice colonized by pathogenic E. coli, as well as to provide more insights into its antimicrobial action mechanism. After determination of minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations, the interference of cinnamaldehyde in macromolecular pathways (synthesis of DNA, RNA, protein, and cell wall) was measured by incorporation of radioisotopes. The anti-adhesive properties of cinnamaldehyde towards E. coli 042 were evaluated using human epithelial type 2 (HEp-2) cells. Intestinal colonization was tested on mice, and the effect of cinnamaldehyde on Tenebrio molitor larvae. Cinnamaldehyde showed MIC and MBC values of 780 μg/mL and 1560 μg/mL, respectively, reduced the adhesion of E. coli 042 on HEp-2 cells, and affected all the synthetic pathways evaluated, suggesting that compost impairs the membrane/cell wall structure leading bacteria to total collapse. No effect on the expression of genes related to the SOS pathway (sulA and dinB1) was observed. The compound did not interfere with cell viability and was not toxic against T. molitor larvae. In addition, cinnamaldehyde-treated mice exhibited lower levels of colonization by E. coli 042 than the untreated group. Therefore, the results show that cinnamaldehyde is effective in treating the pathogenic E. coli strain 042 and confirm it as a promising lead molecule for the development of antimicrobial agents.

Direct development in amphibians is characterized by the loss of aquatic breeding. The anuran Adelophryne maranguapensis is one example of a species with direct development, and it is endemic to the state of Ceara, Brazil. Detailed morphological features of A. maranguapensis embryos and the stages of sequential development have not been described before. Here, we analyzed all available genetic sequence tags in A. maranguapensis (tyr exon 1, pomc and rag1) and compared them with sequences from other species of Adelophryne frogs. We describe the A. maranguapensis reproductive tract and embryonic body development, with a focus on the limbs, tail, ciliated cells of the skin, and the egg tooth, which were analyzed using scanning electron microscopy. Histological analyses revealed ovaries containing oocytes surrounded by follicular cells, displaying large nuclei with nucleoli inside. Early in development, the body is unpigmented, and the neural tube forms dorsally to the yolk vesicle, typical of a direct-developing frog embryo. The hindlimbs develop earlier than the forelimbs. Ciliated cells are abundant during the early stages of skin development and are less common during later stages. The egg tooth appears in the later stages and develops as a keratinized microridge structure. The developmental profile of A. maranguapensis presented here will contribute to our understanding of the direct-development model and may help preserve this endangered native Brazilian frog. genesis 54:257-271, 2016. © 2016 Wiley Periodicals, Inc.

Chronic use of statins may have anti-inflammatory action, promoting immunomodulation and survival in patients with sepsis. This study aimed to analyze the effects of pretreatment with simvastatin in lethal sepsis induced by cecal ligation and puncture (CLP). Male Swiss mice received prophylactic treatment with simvastatin or pyrogen-free water orally in a single daily dose for 30 days. After this period, the CLP was performed. Naïve and Sham groups were performed as non-infected controls. Animal survival was monitored for 60 h after the CLP. Half of mice were euthanized after 12 h to analyze colony-forming units (CFUs); hematological parameters; production of IL-10, IL-12, IL-6, TNF-α, IFN-γ, and MCP-1; cell counts on peritoneum, bronchoalveolar lavage (BAL), bone marrow, spleen, and mesenteric lymph node; immunephenotyping of T cells and antigen presenting cells and production of hydrogen peroxide (H2O2). Simvastatin induced an increase in survival and a decrease in the CFU count on peritoneum and on BAL cells number, especially lymphocytes. There was an increase in the platelets and lymphocytes number in the Simvastatin group when compared to the CLP group. Simvastatin induced a greater activation and proliferation of CD4+ T cells, as well as an increase in IL-6 and MCP-1 production, in chemotaxis to the peritoneum and in H2O2 secretion at this site. These data suggest that simvastatin has an impact on the survival of animals, as well as immunomodulatory effects in sepsis induced by CLP in mice.

Bufadienolides are cardiotonic steroids (CTS) identified in mammals. Besides Na+/K+-ATPase inhibition, they activate signal transduction via protein&ndash, protein interactions. Diversity of endogenous bufadienolides and mechanisms of action may indicate the presence of functional selectivity and unique cellular outcomes. We evaluated whether the bufadienolides telocinobufagin and marinobufagin induce changes in proliferation or viability of pig kidney (LLC-PK1) cells and the mechanisms involved in these changes. In some experiments, ouabain was used as a positive control. CTS exhibited an inhibitory IC50 of 0.20 (telocinobufagin), 0.14 (ouabain), and 3.40 &mu, M (marinobufagin) for pig kidney Na+/K+-ATPase activity and concentrations that barely inhibited it were tested in LLC-PK1 cells. CTS induced rapid ERK1/2 phosphorylation, but corresponding proliferative response was observed for marinobufagin and ouabain instead of telocinobufagin. Telocinobufagin increased Bax:Bcl-2 expression ratio, sub-G0 cell cycle phase and pyknotic nuclei, indicating apoptosis. Src and MEK1/2 inhibitors blunted marinobufagin but not telocinobufagin effect, which was also not mediated by p38, JNK1/2, and PI3K. However, BIO, a GSK-3&beta, inhibitor, reduced proliferation and, as telocinobufagin, phosphorylated GSK-3&beta, at inhibitory Ser9. Combination of both drugs resulted in synergistic antiproliferative effect. Wnt reporter activity assay showed that telocinobufagin impaired Wnt/&beta, catenin pathway by acting upstream to &beta, catenin stabilization. Our findings support that mammalian endogenous bufadienolides may exhibit functional selectivity.

The yellow fever virus (YFV) epidemic that began in Dec 2016 in Brazil is the largest in decades. The recent discovery of YFV in BrazilianAedes sp.vectors highlights the urgent need to monitor the risk of re-establishment of domestic YFV transmission in the Americas. We use a suite of epidemiological, spatial and genomic approaches to characterize YFV transmission. We show that the age- and sex-distribution of human cases in Brazil is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally using a new protocol reveals an early phase of sylvatic YFV transmission restricted to Minas Gerais, followed in late 2016 by a rise in viral spillover to humans, and the southwards spatial expansion of the epidemic towards previously YFV-free areas. Our results establish a framework for monitoring YFV transmission in real-time, contributing to the global strategy of eliminating future yellow fever epidemics.

Human adipose-derived stromal cells (hADSC) are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1) regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

Summary Secreted Wnt morphogens are essential for embryogenesis and homeostasis and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that belongs to the α/β hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. Here, we report that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus, Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naive ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that Notum is a prerequisite for the "default" neural fate and that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development.

Chenopodium ambrosioides L. (Amaranthaceae) is often used in different kinds of vegetal preparations for medicinal purposes in many clinical situations. Some studies have demonstrated its anti-inflammatory and immunomodulatory properties. The aim of this work was to investigate the effect of prophylactic treatment with the hydroalcoholic extract of C. ambrosioides (HCE) and its hexanic fraction (HEX) on the control of bacterial growth, the activation of phagocytes and the control of the systemic inflammatory response in a sepsis experimental model. Animals were divided into three groups (n = 5/group): Control, which received only NaCl 0.9% solution; HCE, which received the crude extract; and HEX, which received the hexane fraction of the extract. The animals received saline, HCE or HEX (5 mg/kg), subcutaneously (SC), 6 h before cecal ligation and puncture (CLP). Twelve hours after the CLP, the blood was collected to measure the serum cytokines, and the animals were killed for the evaluation of colony-forming units (CFUs), cellular influx and activation of phagocytes in the peritoneal cavity, measured by the secretion of hydrogen peroxide and nitric oxide production. The results showed that only HEX treatment inhibited bacterial growth in the peritoneum and inflammatory cellular influx, especially influx of macrophages and neutrophils. However, HCE and HEX treatments increased ex vivo hydrogen peroxide secretion and nitric oxide production by phagocytes and decreased the pro-inflammatory cytokines in the serum, indicating a systemic anti-inflammatory effect of both. In conclusion, C. ambrosioides treatment decreases bacterial growth likely by activation of phagocytes and, in parallel, ameliorates the general state of mice by reducing the systemic inflammatory response usually observed in sepsis.

It is now well documented that natural products have played an important role in anticancer therapy. Many studies focus on the ability of these natural compounds to modulate tumor-related signaling pathways and the relationship of these properties to an anticancer effect. According to the World Health Organization (WHO), colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death among men and women. Therefore, finding strategies to fight against CRC is an emergent health problem. CRC has a strong association with deregulation of Wnt/β-catenin signaling pathway. As some types of natural compounds are capable of modulating the Wnt/β-catenin signaling, one important question is whether they could counteract CRC. In this review, we discuss the role of flavonoids, a class of natural compounds, on Wnt/β-catenin regulation and its possible potential for therapeutic usage on colorectal cancer.

Background: Cuba is a unique country, and despite limited economic development, has an excellent health system. However, the prevalence of asthma symptoms in children in Havana, Cuba, is unusually high. Aim: As early life exposures are critical to the aetiology of asthma, we have studied environmental influences on the risk of wheezing in Cuban infants. Design: Cross-sectional study. Methods: A random sample of 2032 children aged 12–15 months living in Havana was selected for inclusion in the cohort. Data were collected using questionnaires administered by researchers. Results: Of 2032 infants invited to participate, 1956 (96%) infants provided data. The prevalence of any wheeze was 45%, severe wheeze requiring use of emergency services was 30% and recurrent wheeze on three or more occasions was 20%. The largest adjusted risk factors for any wheeze were presence of eczema [odds ratio (OR) 2.09; 95% confidence interval (CI) 1.48–2.94], family history of asthma (OR 2.05; 95% CI 1.60–2.62), poor ventilation in the house (OR 1.99; 95% CI 1.48–2.67), attendance at nursery (OR 1.78; 95% CI 1.24–2.57), male sex (OR1.52; 95% CI 1.19–1.96) and the number of smokers in the house (P < 0.03 for trend), OR 1.64 (95% CI 1.17–2.31) for three or more smokers in the house compared to no smokers in the household. Conclusion: We have identified several risk factors for any wheeze in young infants living in modern day Cuba. As the prevalence of smoking in the house is high (51%), intervention studies are required to determine effective strategies to improve infant health.

Hospital-acquired infections caused by antibiotic-resistant bacteria threaten the lives of many citizens all over the world. Discovery of new agents to hinder bacterial development would have a significant impact on the treatment of infections. Here, the purification and characterization of Rc-2S-Alb, a protein that belongs to the 2S albumin family, from Ricinus communis seed cake, are reported. Rc-2S-Alb was purified after protein extraction with Tris-HCl buffer, pH 7.5, fractionation by ammonium sulfate (50-75%), and chromatography on Phenyl-Sepharose and DEAE-Sepharose. Rc-2S-Alb, a 75 kDa peptide, displays trypsin inhibitory activity and has high in vitro antibacterial activity against Bacillus subtilis, Klebsiella pneumonia, and Pseudomonas aeruginosa, which are important human pathogenic bacteria. Atomic force microscopy studies indicated that Rc-2S-Alb disrupts the bacterial membrane with loss of the cytoplasm content and ultimately bacterial death. Therefore, Rc-2S-Alb is a powerful candidate for the development of an alternative drug that may help reduce hospital-acquired infections.

National Institutes of Health Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Provost's Office for Research of the University of São Paulo Programa de Incentivo a Pesquisa NAPNA-USP, Brazil International Rett Syndrome Foundation Emerald Foundation California Institute for Regenerative Medicine Grant Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. in the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific beta 3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin- induced receptor activity contributes to neurogenesis. in agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less beta 3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression. Univ São Paulo, Inst Quim, Dept Bioquim, BR-05508000 São Paulo, Brazil Univ Calif San Diego, Dept Pediat, San Diego, CA 92093 USA Univ Calif San Diego, Dept Cellular & Mol Med, San Diego, CA 92093 USA Universidade Federal de São Paulo, Dept Biofis, BR-04023062 São Paulo, Brazil Univ Fed Rio de Janeiro, Inst Ciencias Biomed, BR-21941902 Rio de Janeiro, Brazil Universidade Federal de São Paulo, Dept Biofis, BR-04023062 São Paulo, Brazil National Institutes of Health: 1-DP2-OD006495-01 FAPESP: 2006/61285-9 Provost's Office for Research of the University of São Paulo Programa de Incentivo a Pesquisa: 2011.1.9333.1.3 International Rett Syndrome Foundation: 2517 California Institute for Regenerative Medicine Grant: TR2-01814 Web of Science

Objetivou-se, com este estudo, avaliar as correlações entre a digestibilidade aparente e o comportamento ingestivo de novilhas mestiças a pasto. O experimento foi conduzido na fazenda Princesa do Mateiro, município de Ribeirão do Largo, Bahia. Foram utilizadas 20 novilhas com grau de sangue 5/8 Guzerá linhagem leiteira e 3/8 Holandesa, com média de 18 meses de idade e peso corporal médio de 187±13,1 kg. O experimento teve duração de 224 dias e os animais criados em pastejo rotacionado de Brachiaria brizantha "Marandú". Verificou-se uma correlação positiva entre o tempo de pastejo e o coeficiente de digestibilidade da fibra em detergente neutro (CDFDN). Correlações negativas foram verificadas entre o tempo de outras atividades e CDFDN. O tempo de alimentação no cocho apresentou correlações negativas com coeficiente de digestibilidade da matéria seca (CDMS), matéria orgânica (CDMO), carboidratos não fibrosos (CDCNF), carboidratos totais (CDCHOT). Ob-servou-se uma correlação positiva entre o tempo de alimentação total e mastigação total com o CDFDN. O número de períodos de pastejo e ruminação apresentaram correlações negativas com CDMS, CDMO, CDCNF, CDCHOT. Também, verificou-se correlação acima de 0,90 para os nutrientes digestíveis totais (NDT), podendo também ser incluída em equações de predição de consumo. O número de período de alimentação no cocho apresentou correlação negativa com CDMO e coeficiente de digestibilidade de proteína bruta (CDPB). O tempo por período de ruminação apresentou correlação positiva com CDMO, CDCNF, CDCHOT, NDT e CDPB. O tempo por período de outras atividades apresentou correlação positiva com CDCNF e NDT. Correlações negativas foram verificadas entre a taxa de bocados e CDMS, CDMO, CDCNF, CDCHOT e CDPB. O tempo por deglutição correlacionou positivamente com CDMS, CDMO, CDCNF, CDCHOT, NDT, CDPB. A massa do bocado e bocados por deglutição não apre-sentaram correlações com nenhuma das variáveis da digestibilidade. O número de mastigação merícica por dia apresentou correlações negativas com NDT. O tempo de pastejo e ruminação não apresentaram relação com os demais coeficientes de digestibilidade aparente de novilhas mestiças a pasto. O coeficiente da digestibilidade da fibra em detergente neutro provoca variações no tempo de pastejo.

Connective tissue growth factor (CTGF/CCN2) is a protein of the CCN family that modulates cell–ECM interactions in a variety of cell types. In this study, we investigated the chemotactic and adhesive properties of CCN2 protein in embryonic teratocarcinoma P19 cells. Initially, P19 cells were attracted to CCN2-coated agarose beads. In Boyden chamber experiments, CCN2-containing medium induced a threefold greater migration of P19 cells. CCN2 adhesion properties were studied by using optical tweezers. The specific adhesion times of P19 cells to polystyrene beads coated with laminin, fibronectin, CCN2 and bovine serum albumin were 1.8 ± 0.5s, 2.7 ± 0.4s, 10 ± 2s and 13 ± 2s, respectively, revealing an unexpectedly low adhesive capacity of CCN2 protein for P19 cells. In conclusion, our findings support the chemoattractive role of CCN2 for P19 cells, but not its adhesive role when compared to laminin or fibronectin.

Objective High-fat diets have been shown to be a risk factor for ulcerative colitis (UC). Omega-6 polyunsaturated fatty acids are considered to increase lipid peroxidation, while the omega-3 polyunsaturated fatty acid exerts a chemopreventative effect. We evaluated the effect of high-fat diets (20%) enriched with fish or soybean oil on colonic inflammation and DNA damage in dextran sulfate sodium-induced colitis. Methods Male Wistar rats (28–30 days) were fed an American Institute of Nutrition (AIN)-93 diet for 47 days and divided into five groups: control normal fat non-colitic (C) or control colitis (CC), high soybean fat group (HS) colitis, high fish fat group colitis, or high-fat soybean plus fish oil colitis. UC was induced from day 35 until day 41 by 3% dextran sulfate sodium. On day 47, the rats were anesthetized; blood samples collected for corticosterone determination, and the distal colon was excised to quantify interleukin-4 (IL-4), IL-10, and interferon-gamma levels, myeloperoxidase activity, histological analyses, and DNA damage. The disease activity index was recorded daily. Results The disease activity index, histological analysis, myeloperoxidase activity, IL-4, interferon-gamma, and corticosterone levels did not differ among the colitic groups. IL-10 was significantly increased by the high fish fat group diet in relation to HS, but only the high soybean-fish fat diet increased the IL-10/IL-4 ratio (anti-inflammatory/pro-inflammatory) to levels closer to the C group and reduced DNA damage compared to the HS group ( P Conclusion The data show that high-fat diets did not exacerbate UC and suggest that the soybean and fish oil mixture, more than the fish oil alone, could be a complementary therapy to achieve a cytokine balance in UC.

Background: Asynchronous electrical activation induced by right ventricular (RV) pacing can cause several abnormalities in left ventricular (LV) function. However, the effect of ventricular pacing on RV function has not been well established. We evaluated RV function in patients undergoing long-term RV pacing. Methods: Eighty-five patients and 24 healthy controls were included. After pacemaker implantation, conventional echocardiography and strain imaging were used to analyze RV function. Strain imaging measurements included peak systolic strain and strain rate. LV function and ventricular dyssynchrony by tissue Doppler imaging (TDI) were assessed. Intra- and interobserver variabilities of TDI parameters were tested on 15 randomly selected cases. Results: All patients were in New York Heart Association functional class I or II and percentage of ventricular pacing was 96 ± 4%. RV apical induced interventricular dyssynchrony in 49 patients (60%). LV dyssynchrony was found in 51 patients (60%), when the parameter examined was the standard deviation of the time to peak myocardial systolic velocity of all 12 segments greater than 34 ms. Likewise, septal-to-lateral delay ≥65 ms was found in 31 patients (36%). All echocardiographic indexes of RV function were similar between patients and controls (strain: −22.8 ± 5.8% vs −22.1 ± 5.6%, P = 0.630; strain rate: −1.47 ± 0.91 s−1 vs −1.42 ± 0.39 s−1, P = 0.702). Intra- and interobserver variability for RV strain was 3.1% and 5.3%, and strain rate was 1.3% and 2.1%, respectively. Conclusions: In patients with standard pacing indications, RV apical pacing did not seem to affect RV systolic function, despite induction of electromechanical dyssynchrony. (PACE 2011; 34:155–162)

Objetivou-se descrever, por meio de tomografia computadorizada, o trajeto do canal mandibular (CM) em 20 gatos sem raça definida, com ausência de alterações na cavidade oral, provenientes do Centro de Controle de Zoonoses do Distrito Federal. Foram realizados cortes tomográficos com 2mm de espessura, acompanhando todo o trajeto do CM, tendo como referência a região do forame mandibular, as raízes distais e mesiais dos dentes pré-molares e molares e o forame mentoniano, obtendo-se medidas desde o CM até as faces vestibular, lingual, ventral e alveolar (profundidade) do corpo da mandíbula, bem como seu diâmetro. Pôde constatar que o CM manteve-se no aspecto lingual do corpo da mandíbula desde o forame mandibular até a raiz mesial do 1º pré-molar, onde se deslocou para a face vestibular, emergindo no forame mentoniano. Com relação à profundidade, seu trajeto sofreu declive a partir do forame mandibular até a região da raiz mesial do 1º molar, onde alcançou seu ponto mais profundo para prosseguir em suave ascensão até o forame mentoniano. Os dados apresentados contribuem para o estudo anatômico da mandíbula de gatos, bem como auxiliam no melhor planejamento e execução de procedimentos cirúrgicos na mandíbula dessa espécie. ABSTRACT This study aimed to describe the path of the mandibular canal (MC), using computerized tomography, in twenty mongrel cats, with no changes in the oral cavity, from the Zoonosis Control Center of the Federal District. 2mm thick tomographic sections were taken following the entire path of the mandibular canal, considering as reference the region of the mandibular foramen, the distal and mesial roots of premolar and molar teeth, and mental foramen, obtaining measurements from the MC until the buccal, lingual, ventral and alveolar (depth) surfaces of the mandibular body as well as its diameter. MC remained on the lingual aspect of the mandibular body from the mandibular foramen to the mesial root of the first premolar, where it displaced to the buccal surface, emerging from the mental foramen. Regarding the depth, we observed a downward path from the mandibular foramen to the mesial root of the first premolar, where it reached its deepest point, the path continued in gentle ascent until to the mental foramen. Our data contribute to the anatomical study of the feline jaw and allow a better planning and execution of surgical procedures in the mandible of this species.

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.

The harmonious development of the central nervous system depends on the interactions of the neuronal and glial cells. Extracellular matrix elements play important roles in these interactions, especially laminin produced by astrocytes, which has been shown to be a good substrate for neuron growth and axonal guidance. Glioblastomas are the most common subtypes of primary brain tumors and may be astrocytes in origin. As normal laminin-producing glial cells are the preferential substrate for neurons, and glial tumors have been shown to produce laminin, we questioned whether glioblastoma retained the same normal glial–neuron interactive properties with respect to neuronal growth and differentiation. Then, rat neurons were co-cultured onto rat normal astrocytes or onto three human glioblastoma cell lines obtained from neurosurgery. The co-culture confirmed that human glioblastoma cells as well as astrocytes maintained the ability to support neuritogenesis, but non-neural normal or tumoral cells failed to do so. However, glioblastoma cells did not distinguish embryonic from post-natal neurons in relation to neurite pattern in the co-cultures, as normal astrocytes did. Further, the laminin organization on both normal and tumoral glial cells was altered from a filamentous arrangement to a mixed punctuate/filamentous pattern when in co-culture with neurons. Together, these results suggest that glioblastoma cells could identify neuronal cells as partners, to support their growth and induce complex neurites, but they lost the normal glia property to distinguish neuronal age. In addition, our results show for the first time that neurons modulate the organization of astrocytes and glioblastoma laminin on the extracellular matrix.

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative, anti-inflammatory, anti-viral, and anti-tumoral effects. These effects are related to the ability of flavonoids to modulate signaling pathways, such as the canonical Wnt signaling pathway. This pathway controls many aspects of embryonic development and tissue maintenance and has been found to be deregulated in a range of human cancers. We performed several in vivo assays in Xenopus embryos, a functional model of canonical Wnt signaling studies, and also used in vitro models, to investigate whether isoquercitrin affects Wnt/β-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/β-catenin, where the flavonoid acts downstream of β-catenin translocation to the nuclei. Isoquercitrin affects Xenopus axis establishment, reverses double axes and the LiCl hyperdorsalization phenotype, and reduces Xnr3 expression. In addition, this flavonoid shows anti-tumoral effects on colon cancer cells (SW480, DLD-1, and HCT116), whereas exerting no significant effect on non-tumor colon cell (IEC-18), suggesting a specific effect in tumor cells in vitro. Taken together, our data indicate that isoquercitrin is an inhibitor of Wnt/β-catenin and should be further investigated as a potential novel anti-tumoral agent.