Neurodevelopment and early pharmacological interventions in Fragile X Syndrome.

Fragile X Syndrome (FXS) is a neurodevelopmental disorder and the leading monogenic cause of autism and intellectual disability. For years, several efforts have been made to develop an effective therapeutic approach to phenotypically rescue patients from the disorder, with some even advancing to late phases of clinical trials. Unfortunately, none of these attempts have completely succeeded, bringing urgency to further expand and refocus research on FXS therapeutics. FXS arises at early stages of postnatal development due to the mutation and transcriptional silencing of the Fragile X Messenger Ribonucleoprotein 1 gene (FMR1) and consequent loss of the Fragile X Messenger Ribonucleoprotein (FMRP) expression. Importantly, FMRP expression is critical for the normal adult nervous system function, particularly during specific windows of embryogenic and early postnatal development. Cellular proliferation, migration, morphology, axonal guidance, synapse formation, and in general, neuronal network establishment and maturation are abnormally regulated in FXS, underlying the cognitive and behavioral phenotypes of the disorder. In this review, we highlight the relevance of therapeutically intervening during critical time points of development, such as early postnatal periods in infants and young children and discuss past and current clinical trials in FXS and their potential to specifically target those periods. We also discuss potential benefits, limitations, and disadvantages of these pharmacological tools based on preclinical and clinical research.

An ecological study on reinfection rates using a large dataset of RT-qPCR tests for SARS-CoV-2 in Santiago of Chile.

Introduction: As the SARS-CoV-2 continues to evolve, new variants pose a significant threat by potentially overriding the immunity conferred by vaccination and natural infection. This scenario can lead to an upswing in reinfections, amplified baseline epidemic activity, and localized outbreaks. In various global regions, estimates of breakthrough cases associated with the currently circulating viral variants, such as Omicron, have been reported. Nonetheless, specific data on the reinfection rate in Chile still needs to be included. Methods: Our study has focused on estimating COVID-19 reinfections per wave based on a sample of 578,670 RT-qPCR tests conducted at the University of Santiago of Chile (USACH) from April 2020 to July 2022, encompassing 345,997 individuals. Results: The analysis reveals that the highest rate of reinfections transpired during the fourth and fifth COVID-19 waves, primarily driven by the Omicron variant. These findings hold despite 80% of the Chilean population receiving complete vaccination under the primary scheme and 60% receiving at least one booster dose. On average, the interval between initial infection and reinfection was found to be 372 days. Interestingly, reinfection incidence was higher in women aged between 30 and 55. Additionally, the viral load during the second infection episode was lower, likely attributed to Chile's high vaccination rate. Discussion: This study demonstrates that the Omicron variant is behind Chile's highest number of reinfection cases, underscoring its potential for immune evasion. This vital epidemiological information contributes to developing and implementing effective public health policies.

Molecules involved in the sperm interaction in the human uterine tube: a histochemical and immunohistochemical approach.

In humans, even where millions of spermatozoa are deposited upon ejaculation in the vagina, only a few thousand enter the uterine tube (UT). Sperm transiently adhere to the epithelial cells lining the isthmus reservoir, and this interaction is essential in coordinating the availability of functional spermatozoa for fertilization. The binding of spermatozoa to the UT epithelium (mucosa) occurs due to interactions between cell-adhesion molecules on the cell surfaces of both the sperm and the epithelial cell. However, in humans, there is little information about the molecules involved. The aim of this study was to perform a histological characterization of the UT focused on determining the tissue distribution and deposition of some molecules associated with cell adhesion (F-spondin, galectin-9, osteopontin, integrin αV/ß3) and UT's contractile activity (TNFα-R1, TNFα-R2) in the follicular and luteal phases. Our results showed the presence of galectin-9, F-spondin, osteopontin, integrin αV/ß3, TNFα-R1, and TNFα-R2 in the epithelial cells in ampullar and isthmic segments during the menstrual cycle. Our results suggest that these molecules could form part of the sperm-UT interactions. Future studies will shed light on the specific role of each of the identified molecules.

P2X7 receptor is essential for cross-dressing of bone marrow-derived dendritic cells.

T cell activation requires the processing and presentation of antigenic peptides in the context of a major histocompatibility complex (MHC complex). Cross-dressing is a non-conventional antigen presentation mechanism, involving the transfer of preformed peptide/MHC complexes from whole cells, such as apoptotic cells (ACs) to the cell membrane of professional antigen-presenting cells (APCs), such as dendritic cells (DCs). This is an essential mechanism for the induction of immune response against viral antigens, tumors, and graft rejection, which until now has not been clarified. Here we show for first time that the P2X7 receptor (P2X7R) is crucial to induce cross-dressing between ACs and Bone-Marrow DCs (BMDCs). In controlled ex vivo assays, we found that the P2X7R in both ACs and BMDCs is required to induce membrane and fully functional peptide/MHC complex transfer to BMDCs. These findings show that acquisition of ACs-derived preformed antigen/MHC-I complexes by BMDCs requires P2X7R expression.

Anthocyanins from Aristotelia chilensis Prevent Olanzapine-Induced Hepatic-Lipid Accumulation but Not Insulin Resistance in Skeletal Muscle Cells.

Type 2 diabetes and obesity are major problems worldwide and dietary polyphenols have shown efficacy to ameliorate signs of these diseases. Anthocyanins from berries display potent antioxidants and protect against weight gain and insulin resistance in different models of diet-induced metabolic syndrome. Olanzapine is known to induce an accelerated form of metabolic syndrome. Due to the aforementioned, we evaluated whether delphinidin-3,5-O-diglucoside (DG) and delphinidin-3-O-sambubioside-5-O-glucoside (DS), two potent antidiabetic anthocyanins isolated from Aristotelia chilensis fruit, could prevent olanzapine-induced steatosis and insulin resistance in liver and skeletal muscle cells, respectively. HepG2 liver cells and L6 skeletal muscle cells were co-incubated with DG 50 µg/mL or DS 50 µg/mL plus olanzapine 50 µg/mL. Lipid accumulation was determined in HepG2 cells while the expression of p-Akt as a key regulator of the insulin-activated signaling pathways, mitochondrial function, and glucose uptake was assessed in L6 cells. DS and DG prevented olanzapine-induced lipid accumulation in liver cells. However, insulin signaling impairment induced by olanzapine in L6 cells was not rescued by DS and DG. Thus, anthocyanins modulate lipid metabolism, which is a relevant factor in hepatic tissue, but do not significantly influence skeletal muscle, where a potent antioxidant effect of olanzapine was found.

In Vivo Antitumor Effect against Murine Cells of CT26 Colon Cancer and EL4 Lymphoma by Autologous Whole Tumor Dead Cells.

Active immunotherapy against cancer is based on immune system stimulation, triggering efficient and long-lasting antigen-specific immune responses. Immunization strategies using whole dead cells from tumor tissue, containing specific antigens inside, have become a promising approach, providing efficient lymphocyte activation through dendritic cells (DCs). In this work, we generate whole dead tumor cells from CT26, E.G7, and EL4 live tumor cells as antigen sources, which termed immunogenic cell bodies (ICBs), generated by a simple and cost-efficient starvation-protocol, in order to determine whether are capable of inducing a transversal anticancer response regardless of the tumor type, in a similar way to what we describe previously with B16 melanoma. We evaluated the anticancer effects of immunization with doses of ICBs in syngeneic murine tumor models. Our results showed that mice's immunization with ICBs-E.G7 and ICBs-CT26 generate 18% and 25% of tumor-free animals, respectively. On the other hand, all carrying tumor-animals and immunized with ICBs, including ICBs-EL4, showed a significant delay in their growth compared to not immunized animals. These effects relate to DCs maturation, cytokine production, increase in CD4+T-bet+ and CD4+ROR-γt+ population, and decrease of T regulatory lymphocytes in the spleen. Altogether, our data suggest that whole dead tumor cell-based cancer immunotherapy generated by a simple starvation protocol is a promising way to develop complementary, innovative, and affordable antitumor therapies in a broad spectrum of tumors.

Adenosine triphosphate, polymyxin B and B16 cell-derived immunization induce anticancer response.

Aim: Whole dead tumor cells can be used as antigen source and the induction of protective immune response could be enhanced by damage-associated molecular patterns. Materials & methods: We generated whole dead tumor cells called B16-immunogenic cell bodies (ICBs) from B16 melanoma cells by nutrient starvation and evaluated the in vivo antitumor effect of B16-ICBs plus ATP and polymyxin B (PMB). Results: The subcutaneous immunization with B16-ICBs + PMB + ATP a 50% of tumor-free animals and induced a significant delay in tumor growth in a prophylactic approach. These results correlated with maturation of bone marrow-derived dendritic cells and activation of T CD8+ lymphocytes in vitro. Conclusion: Altogether, ICB + ATP + PMB is efficient in inducing the antitumor efficacy of the whole dead tumor cells vaccine.

Cancer disease progression and death during the COVID-19 pandemic: a multidisciplinary analysis for the Peruvian setting.

Since the COVID-19 pandemic began in China in late 2019, infection from the SARS-CoV-2 virus has spread virtually worldwide. This infection has adversely affected several countries; governments have outlined a series of political measures aimed to preserve the health and safety of their populations. In Peru, most actions have prioritised COVID-19 attention, with a subsequent gap in the healthcare facilities needed for other diseases. Cancer, one principal cause of death in the country, is usually diagnosed late. Moreover, in the pandemic context, the prevention and control of cancer have been negatively affected. Therefore, we carried out a multidisciplinary analysis using the Ishikawa diagram to identify the probable factors that contribute to cancer progression and deaths in Peru.

Chitosan-Based Delivery of Avian Reovirus Fusogenic Protein p10 Gene: In Vitro and In Vivo Studies towards a New Vaccine against Melanoma.

Reovirus is known to have an anticancer effect in both the preclinical and clinical assays. Current evidence suggests that the reovirus-mediated impact on tumor growth depends on the activation of specific antitumor immune responses. A feasible explanation for the oncolytic effects and immune system activation is through the expression of the fusogenic reovirus protein. In this work, we evaluated the in vivo antitumor effects of the expression of fusogenic protein p10 of avian reovirus (ARV-p10). We used chitosan nanoparticles (CH-NPs) as a vehicle for the ARV-p10 DNA in murine B16 melanoma models both in vitro and in vivo. We confirmed that ARV-p10 delivery through a chitosan-based formulation (ARV-p10 CH-NPs) was capable of inducing cell fusion in cultured melanoma cells, showing a mild cytotoxic effect. Interestingly, intratumor injection of ARV-p10 CH-NPs delayed tumor growth, without changing lymphoid populations in the tumor tissue and spleen. The injection of chitosan nanoparticles (CH-NPs) also delayed tumor growth, suggesting the nanoparticle itself would attack tumor cells. In conclusion, we proved that in vitro ARV-p10 protein expression using CH-NPs in murine melanoma cells induces a cytotoxic effect associated with its cell fusion. Further studies are necessary for establishing a protocol for efficient in vivo DNA delivery of fusion proteins to produce an antitumoral effect.

Chitosan-Based Nanoparticles for Intracellular Delivery of ISAV Fusion Protein cDNA into Melanoma Cells: A Path to Develop Oncolytic Anticancer Therapies.

Oncolytic virus therapy has been tested against cancer in preclinical models and clinical assays. Current evidence shows that viruses induce cytopathic effects associated with fusogenic protein-mediated syncytium formation and immunogenic cell death of eukaryotic cells. We have previously demonstrated that tumor cell bodies generated from cells expressing the fusogenic protein of the infectious salmon anemia virus (ISAV-F) enhance crosspriming and display prophylactic antitumor activity against melanoma tumors. In this work, we evaluated the effects of the expression of ISAV-F on the B16 melanoma model, both in vitro and in vivo, using chitosan nanoparticles as transfection vehicle. We confirmed that the transfection of B16 tumor cells with chitosan nanoparticles (NP-ISAV) allows the expression of a fusogenically active ISAV-F protein and decreases cell viability because of syncytium formation in vitro. However, the in vivo transfection induces a delay in tumor growth, without inducing changes on the lymphoid populations in the tumor and the spleen. Altogether, our observations show that expression of ISAV fusion protein using chitosan nanoparticles induces cell fusion in melanoma cells and slight antitumor response.

Early postnatal environmental enrichment restores neurochemical and functional plasticities of the cerebral cortex and improves learning performance in hidden-prenatally-malnourished young-adult rats.

Moderate reduction of dietary protein (from 25% to 8% casein) in pregnant rats, calorically compensated by carbohydrates, gives rise to 'hidden prenatal malnutrition' (HPM) in the offspring since it does not alter body and brain weights of pups at birth. However, this dietary treatment leads to decreased ß-adrenoceptor signaling and brain derived neurotrophic factor (BDNF) levels in the pup' brain, altogether with defective cortical long-term potentiation (LTP) and lowered visuospatial memory performance. Since early postnatal environmental enrichment (EE) has been shown to exert plastic effects on the developing brain and neuroprotection both on cognition and on structural properties of the neocortex, in the present study we addressed the question of whether early postnatal EE during the lactation period could exert compensatory changes in the expression of ®-adrenergic receptors and BDNF in the neocortex of HPM rats, and if these effects are associated with an improvement or even a restore of both neocortical LTP in vivo and cognitive performance induced by HPM. The results obtained show that EE restored ß-adrenoceptor density, BDNF expression and the ability to support LTP at prefrontal and occipital cortices of HPM rats. Besides, EE improved learning performance in visuospatial and operant conditioning tasks. The latter support the notion that adequate maternal protein nutrition during pregnancy is required for proper brain development and function. Further, the results highlight the role of environmental enrichment during early postnatal life in increasing later brain plasticity and exerting neuroprotection against brain deficits induced by prenatal malnutrition.

Epilepsia en niños atendidos en el Hospital Nacional Cayetano Heredia de Lima, Perú, 2010- 2016: Lima, Peru, 2010-2016 / Clinical characteristics of epileptic children seen at Cayetano Heredia Hospital

La epilepsia es la principal causa de consulta neurológica en niños peruanos. Sin embargo, hay escasa información sobre sus características clínicas y epidemiológicas. Objetivos: Presentar las características clínicas de los niños epilépticos atendidos en el Hospital Cayetano Heredia, entre los años 2010 y 2016. Material y métodos: Se realizó un estudio observacional descriptivo, retrospectivo, tipo serie de casos, basado en las historias clìnicas de en niños menores de 14 años con diagnóstico de epilepsia, seguimiento clínico, registro electroencefalogràfico y neuroimagenes. Resultados: Se incluyeron 193 pacientes. El 47,2% (91/193) presentaron su primera crisis epiléptica antes del año de edad, cuya etiología fue secundaria en el 59,3% (54/91), destacando los factores perinatales y las malformaciones cerebrales. La etiología primaria fue más frecuente en los niños que iniciaron las crisis después del año de edad. Las crisis generalizadas se presentaron en 64,2% (124/193) sin diferencias por grupos etarios. El 69,9% (135/193) fueron controlados con un solo fármaco, en tanto que el 15% (29/193) fueron refractarios al tratamiento. La comorbilidad ocurrió en el 68,4% (132/193), siendo frecuentes el retraso del desarrollo psicomotor, el retardo mental y la parálisis cerebral. Conclusiones: La mayoría de los niños presentaron su primera crisis antes del año de edad, generalmente de causa secundaria. En los niños mayores fue prevalente la etiología primaria. La mayoría fueron controlados con monoterapia, pero se registró un porcentaje de refractariedad al tratamiento. La comorbilidad fue muy frecuente.

Epilepsy is the main cause of neurological clinical visits in Peruvian children. However, in our country, there is little information on its clinical and epidemiological characterizes. Objectives: To describe the clinical characteristics of children with epilepsy seen at the Cayetano Heredia Hospital, between 2010 and 2016. Material and Methods: A descriptive retrospective observational study was carried out, based on the clinical records of children under 14 years old with diagnosis of epilepsy, with clinical follow-up, electroencephalography and neuroimaging. Results: One hundred ninety-three patients were included. In 47.2% (91/193), the first seizure occurred before the one year of age; the etiology was secondary in 59.3% (54/91), with the main causes being perinatal events and cerebral malformations. Primary etiologies were more frequent in the children who started haviing seizures after one year of age. Generalized seizures ocurred in 64.2% (124/193) without differences by age groups. In 69.9% (135/193) seizures were controlled with a single drug while 15% (29/193) were refractory to treatment. Comorbidity occurred in 68,4% (132/193), with delays in psychomotor development, intellectual disability and cerebral palsy being frequent. Conclusions: Most children had their first seizures before the first year of age, usually with a secondary cause. In older children, the primary epilepsies were more prevalent. Monotherapy controlled the majority of children, but a percentage of refractoriness to the treatment was registered. Comorbidities were very frequent.

The Netrin-4/ Neogenin-1 axis promotes neuroblastoma cell survival and migration.

Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration and cell death, during both embryonic development and adult homeostasis. It has been described as a dependence receptor, because it promotes cell death in the absence of its ligands (Netrin and Repulsive Guidance Molecule (RGM) families) and cell survival when they are present. Although NEO1 and its ligands are involved in tumor progression, their precise role in tumor cell survival and migration remain unclear. Public databases contain extensive information regarding the expression of NEO1 and its ligands Netrin-1 (NTN1) and Netrin-4 (NTN4) in primary neuroblastoma (NB) tumors. Analysis of this data revealed that patients with high expression levels of both NEO1 and NTN4 have a poor survival rate. Accordingly, our analyses in NB cell lines with different genetic backgrounds revealed that knocking-down NEO1 reduces cell migration, whereas silencing of endogenous NTN4 induced cell death. Conversely, overexpression of NEO1 resulted in higher cell migration in the presence of NTN4, and increased apoptosis in the absence of ligand. Increased apoptosis was prevented when utilizing physiological concentrations of exogenous Netrin-4. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced, thus revealing an important role for NEO1 in NB cell survival. In vivo analysis, using the chicken embryo chorioallantoic membrane (CAM) model, showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling.

Astrocitoma pilocítico en niños: Reporte de un caso / Pilocytic astrocytoma in children. Case Report

Se reporta el caso de un niño de 10 años que se presentó con dos semanas de enfermedad caracterizada por cefalea, vómitos, marcha tambaleante y somnolencia. El examen clínico reveló bradilalia, ataxia, hiperreflexia, nistagmo horizontal y vertical, parálisis del VI nervio craneal izquierdo y signos de frontalización. En la tomografía y resonancia cerebrales se encontró hidrocefalia obstructiva por un tumor de la fosa posterior. La anatomía patológica reveló Astrocitoma Pilocítico grado I (WHO)...

We report a 10 years old boy with 2 weeks of symptoms characterized by headache, vomiting, staggering gait and drowsiness. The initial clinical examination showed bradilalia, ataxia, hyperreflexia, horizontal and vertical nystagmus, VI left cranial nerve paresis and signs of frontalization. The brain CT and MRI showed a posterior fossa tumor causing hydrocephaly. The histhopatology revealed grade I pilocytic astrocytoma (WHO)...

Tres formas clínico-radiológicas de compromiso neurológico por toxoplasmosis congénita / Three clinic-radiological forms of neurological compromise by congenital toxoplasmosis

La toxoplasmosis congénita es una enfermedad prevalente entre las infecciones intrauterinas. Describimos tres casos clínicos - radiológicos con diferentes formas de compromiso neurológico. Caso 1: feto con hidrocefalia detectada a las 36 semanas, cuya madre presentó anticuerpos positivos (Ig M: 2,26 UI/ml), nació por cesárea y al examen presentó macrocefalia, uveítis, hepatoesplenomegalia, hipotonía, Moro incompleto y succión débil, en suero IgM 0,95 UI/ml, en LCR se observaron taquizoítos viables, y proteínas en 1204 mg/dl. Caso 2: neonato a término, letárgico desde el nacimiento, con coriorretinitis, hemiparesia izquierda, hipotonía, hiporreflexia y midriasis derecha (síndrome de Weber). LCR con pleocitosis ( leucocitos 52 mm³), glucosa en 63mg/dl y proteínas de 300 mg/dl. IgM en sangre de 1,9 UI/ml, y múltiples lesiones parenquimales. Caso 3: lactante de 3 meses, desnutrido, con fontanela grande, hipotonía axial, hipertonía apendicular e hiperreflexia. IgM 2,1 UI/ml. Hidrocefalia por estenosis del Acueducto de Silvio y multiples lesiones parenquimales.En los tres casos el esquema de tratamiento fue sulfadiazina, pirimetamina, ácido folínico y prednisona. Se colocó drenaje ventricular a los niños con hidrocefalia. En la evolución se observó sordera, perdida de la agudeza visual y retardo del desarrollo.Se concluye que la Toxoplasmosis congénita puede tener diferentes formas de presentación neurológica dependiendo del periodo de infestación, inoculo y de la respuesta inmune del huésped.

Congenital toxoplasmosis is a prevalent disease among intrauterine infections. We describe three cases with different forms of neurological compromise. Case 1: fetus with hydrocephalus detected at 36 weeks , whose mother had positive antibodies (Ig M: 2.26 IU / ml), the patient was born by caesarean section and physical examination showed macrocephaly, uveitis, hepatosplenomegaly , hypotonia, incomplete Moro reflex and weak suction, IgM 0.95 IU / ml , CSF with viable tachyzoites were observed and 1204 mg protein / dl. Case 2: newborn lethargic at birth, with chorioretinitis, left hemiparesis, hypotonia, hyporeflexia and right mydriasis (Weber syndrome), CSF leukocytes showed 52 mm3, glucose 63mg/dl and protein 300 mg/dl, IgM 1.9 IU/ml and multiple parenchymal lesions. Case 3: patient 3 months old, malnourished , with large fontanelle, axial hypotonia, appendicular hypertonia and hyperreflexia, IgM 2.1 IU/ml, hydrocephalus due to aqueductal stenosis and diffuse parenchymal lesions . In all of them the treatment schedule was sulfadiazine, pyrimethamine, folinic acid and prednisone. Ventricular drainage was placed for children with hydrocephalus. Deafness, visual acuity loss and developmental delay was observed at follow up. We conclude that congenital toxoplasmosis can have different forms of neurological presentation depending to infestation period, inoculum and host immune response.

Insight into insulin secretion from transcriptome and genetic analysis of insulin-producing cells of Drosophila.

Insulin-producing cells (IPCs) in the Drosophila brain produce and release insulin-like peptides (ILPs) to the hemolymph. ILPs are crucial for growth and regulation of metabolic activity in flies, functions analogous to those of mammalian insulin and insulin-like growth factors (IGFs). To identify components functioning in IPCs to control ILP production, we employed genomic and candidate gene approaches. We used laser microdissection and messenger RNA sequencing to characterize the transcriptome of larval IPCs. IPCs highly express many genes homologous to genes active in insulin-producing ß-cells of the mammalian pancreas. The genes in common encode ILPs and proteins that control insulin metabolism, storage, secretion, ß-cell proliferation, and some not previously linked to insulin production or ß-cell function. Among these novelties is unc-104, a kinesin 3 family gene, which is more highly expressed in IPCs compared to most other neurons. Knockdown of unc-104 in IPCs impaired ILP secretion and reduced peripheral insulin signaling. Unc-104 appears to transport ILPs along axons. As a complementary approach, we tested dominant-negative Rab genes to find Rab proteins required in IPCs for ILP production or secretion. Rab1 was identified as crucial for ILP trafficking in IPCs. Inhibition of Rab1 in IPCs increased circulating sugar levels, delayed development, and lowered weight and body size. Immunofluorescence labeling of Rab1 showed its tight association with ILP2 in the Golgi of IPCs. Unc-104 and Rab1 join other proteins required for ILP transport in IPCs.

Neogenin1 is a Sonic Hedgehog target in medulloblastoma and is necessary for cell cycle progression.

The canonical Sonic Hedgehog (Shh)/Gli pathway plays multiples roles during central nervous system (CNS) development. To elucidate the molecular repertoire of Shh mediators, we have recently described novel transcriptional targets in response to Shh pathway modulation. Among them, we were able to identify Neogenin1 (Neo1), a death dependence receptor, as a new direct Shh downstream regulator in neural precursor proliferation. As appropriate Shh signaling is required for cerebellar growth and alterations cause Shh-driven medulloblastoma (MB), here we have addressed the role of the Shh/Neogenin1 interaction in the context of cerebellar development and cancer. We demonstrate that the Shh pathway regulates Neogenin1 expression in mouse models that recapitulate the Shh MB subtype. We show that the canonical Shh pathway directly regulates the Neo1 gene acting through an upstream sequence in its promoter both in vitro and in vivo in granule neuron precursor cells. We also identified and characterized a functional Gli-binding site in the first intron of the human NEO1 gene. Gene expression profiling of more than 300 MB shows that NEO1 is indeed upregulated in SHH tumors compared to the other MB subgroups. Finally, we provide evidence that NEO1 is necessary for cell cycle progression in a human MB cell line, because a loss of function of NEO1 arrests cells in the G2/M phase. Taken together, these results highlight Neogenin1 as a novel downstream effector of the Shh pathway in MB and a possible therapeutic target.

Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development.

BACKGROUND: The increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh) pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1) and novel Hh-regulated genes in zebrafish embryos. RESULTS: The combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo. CONCLUSION: A recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in different contexts of vertebrate embryonic development.

Sonic Hedgehog in cancer stem cells: a novel link with autophagy.

The Sonic Hegdehog/GLI (SHH/GLI) pathway has been extensively studied for its role in developmental and cancer biology. During early embryonic development the SHH pathway is involved mainly in pattern formation, while in latter stages its function in stem cell and progenitor proliferation becomes increasingly relevant. During postnatal development and in adult tissues, SHH/GLI promotes cell homeostasis by actively regulating gene transcription, recapitulating the function observed during normal tissue growth. In this review, we will briefly discuss the fundamental importance of SHH/GLI in tumor growth and cancer evolution and we will then provide insights into a possible novel mechanism of SHH action in cancer through autophagy modulation in cancer stem cells. Autophagy is a homeostatic mechanism that when disrupted can promote and accelerate tumor progression in both cancer cells and the stroma that harbors tumorigenesis. Understanding possible new targets for SHH signaling and its contribution to cancer through modulation of autophagy might provide better strategies in order to design combined treatments and perform clinical trials.