CBD Inhibits In Vivo Development of Human Breast Cancer Tumors.

Inflammation is a critical component of cancer development. Previously, we showed in vitro that IL-1ß treatment of non-invasive human breast cancer MCF-7 cells promoted their transition to a malignant phenotype (6D cells). This epithelial-mesenchymal transition was reverted by exposure to cannabidiol (CBD). We show in a murine model that subcutaneous inoculation of 6D cells induced formation and development of tumors, the cells of which keep traits of malignancy. These processes were interrupted by administration of CBD under two schemes: therapeutic and prophylactic. In the therapeutic scheme, 6D cells inoculated mice developed tumors that reached a mean volume of 540 mm3 at 45 days, while 50% of CBD-treated mice showed gradual resorption of tumors. In the prophylactic scheme, mice were pre-treated for 15 days with CBD before cells inoculation. The tumors formed remained small and were eliminated under continuous CBD treatment in 66% of the animals. Histological and molecular characterization of tumors, from both schemes, revealed that CBD-treated cells decreased the expression of malignancy markers and show traits related with apoptosis. These results confirm that in vivo CBD blocks development of breast cancer tumors formed by cells induced to malignancy by IL-1ß, endorsing its therapeutic potential for cancer treatment.

Differential Expression of Proteins in an Atypical Presentation of Autoimmune Lymphoproliferative Syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare disease defined as a defect in the lymphocyte apoptotic pathway. Currently, the diagnosis of ALPS is based on clinical aspects, defective lymphocyte apoptosis and mutations in Fas, FasL and Casp 10 genes. Despite this, ALPS has been misdiagnosed. The aim of this work was to go one step further in the knowledge of the disease, through a molecular and proteomic analysis of peripheral blood mononuclear cells (PBMCs) from two children, a 13-year-old girl and a 6-year-old boy, called patient 1 and patient 2, respectively, with clinical data supporting the diagnosis of ALPS. Fas, FasL and Casp10 genes from both patients were sequenced, and a sample of the total proteins from patient 1 was analyzed by label-free proteomics. Pathway analysis of deregulated proteins from PBMCs was performed on the STRING and PANTHER bioinformatics databases. A mutation resulting in an in-frame premature stop codon and protein truncation was detected in the Fas gene from patient 2. From patient 1, the proteomic analysis showed differences in the level of expression of proteins involved in, among other processes, cell cycle, regulation of cell cycle arrest and immune response. Noticeably, the most down-regulated protein is an important regulator of the cell cycle process. This could be an explanation of the disease in patient 1.

Giardia intestinalis coiled-coil cytolinker protein 259 interacts with actin and tubulin.

Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic.

CBD Reverts the Mesenchymal Invasive Phenotype of Breast Cancer Cells Induced by the Inflammatory Cytokine IL-1ß.

Cannabidiol (CBD) has been used to treat a variety of cancers and inflammatory conditions with controversial results. In previous work, we have shown that breast cancer MCF-7 cells, selected by their response to inflammatory IL-1ß cytokine, acquire a malignant phenotype (6D cells) through an epithelial-mesenchymal transition (EMT). We evaluated CBD as a potential inhibitor of this transition and inducer of reversion to a non-invasive phenotype. It decreased 6D cell viability, downregulating expression of receptor CB1. The CBD blocked migration and progression of the IL-1ß-induced signaling pathway IL-1ß/IL-1RI/ß-catenin, the driver of EMT. Cannabidiol reestablished the epithelial organization lost by dispersion of the cells and re-localized E-cadherin and ß-catenin at the adherens junctions. It also prevented ß-catenin nuclear translocation and decreased over-expression of genes for ∆Np63α, BIRC3, and ID1 proteins, induced by IL-1ß for acquisition of malignant features. Cannabidiol inhibited the protein kinase B (AKT) activation, a crucial effector in the IL-1ß/IL-1RI/ß-catenin pathway, indicating that at this point there is crosstalk between IL-1ß and CBD signaling which results in phenotype reversion. Our 6D cell system allowed step-by-step analysis of the phenotype transition and better understanding of mechanisms by which CBD blocks and reverts the effects of inflammatory IL-1ß in the EMT.

Synthesis and toxicity assessment of Coffea arabica extract-derived gold nanoparticles loaded with doxorubicin in lung cancer cell cultures.

Cancer is the second leading cause of death worldwide, despite the many treatments available, cancer patients face side effects that reduce their quality of life. Therefore, there is a need to develop novel strategies to increase the efficacy of treatments. In this study, gold nanoparticles obtained by green synthesis with Coffea arabica green bean extract were loaded with Doxorubicin, (a highly effective but non-specific drug) by direct interaction and using commercial organic ligands that allow colloidal dispersion at physiological and tumor pH. Conjugation of these components resulted in stable nanohybrids at physiological pH and a tumor pH release dependent, with a particle size less than 40 nm despite having the ligands and Doxorubicin loaded on their surface, which gave them greater specificity and cytotoxicity in H69 tumor cells.

Identification of Transferrin Receptor 1 (TfR1) Overexpressed in Lung Cancer Cells, and Internalization of Magnetic Au-CoFe2O4 Core-Shell Nanoparticles Functionalized with Its Ligand in a Cellular Model of Small Cell Lung Cancer (SCLC).

Lung cancer is, currently, one of the main malignancies causing deaths worldwide. To date, early prognostic and diagnostic markers for small cell lung cancer (SCLC) have not been systematically and clearly identified, so most patients receive standard treatment. In the present study, we combine quantitative proteomics studies and the use of magnetic core-shell nanoparticles (mCSNP's), first to identify a marker for lung cancer, and second to functionalize the nanoparticles and their possible application for early and timely diagnosis of this and other types of cancer. In the present study, we used label-free mass spectrometry in combination with an ion-mobility approach to identify 220 proteins with increased abundance in small cell lung cancer (SCLC) cell lines. Our attention was focused on cell receptors for their potential application as mCSNP's targets; in this work, we report the overexpression of Transferrin Receptor (TfR1) protein, also known as Cluster of Differentiation 71 (CD71) up to a 30-fold increase with respect to the control cell. The kinetics of endocytosis, evaluated by a flow cytometry methodology based on fluorescence quantification, demonstrated that receptors were properly activated with the transferrin supported on the magnetic core-shell nanoparticles. Our results are important in obtaining essential information for monitoring the disease and/or choosing better treatments, and this finding will pave the way for future synthesis of nanoparticles including chemotherapeutic drugs for lung cancer treatments.

Quantitative Proteomic Analysis of MARC-145 Cells Infected with a Mexican Porcine Reproductive and Respiratory Syndrome Virus Strain Using a Label-Free Based DIA approach.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease characterized by severe reproductive failure in sows, acute respiratory disorders in growing pigs, and high mortality in piglets. The causative agent of this syndrome is the PRRS virus (PRRSV), an RNA virus belonging to the Arteriviridae family. To date, several quantitative approaches of proteomics have been applied to analyze the gene expression profiles during PRRSV infection in PAMs and MARC-145 cells, and few proteins have been consistent among independent studies, probably due to the differences in the levels of virulence of different PRRSV strains used and/or due to analytical conditions. In this study, total proteins isolated from noninfected and infected MARC-145 cells with a Mexican PRRSV strain were relatively quantified using label-free based DIA approach in combination with ion-mobility separation. As a result, 1456 quantified proteins were found to be shared between the control and infected samples. Afterward, these proteins were filtered, and 699 of them were considered without change. Also, 17 proteins were up-regulated and 19 proteins were down-regulated during the PRSSV infection. Bioinformatic analysis revealed that many of the differentially expressed proteins are involved in processes like antigen processing, presentation of antigens, response to viruses, response to IFNs, and innate immune response, among others. The present work is the first one which provides a detailed proteomic analysis through label-free based DIA approach in MARC-145 cells during the infection with a Mexican PRRSV strain.

The interaction between RE1-silencing transcription factor (REST) and heat shock protein 90 as new therapeutic target against Huntington's disease.

The wild type huntingtin protein (Htt), supports the production of brain-derived neurotrophic factor (BDNF), a survival factor for striatal neurons, through cytoplasmic sequestering of RE-1silencing transcription factor (REST). In Huntington´s Disease an inherited degenerative disease, caused by a CAG expansion in the 5´coding region of the gene, the mutant huntingtin protein (mHtt), causes that REST enters pathologically into the nucleus of cells, resulting in the repression of neuronal genes including BDNF, resulting in the progressive neuronal death. It has been reported that Htt associates with Hsp90 and this interaction is involved in regulation of huntingtin aggregation. Discovering mechanisms to reduce the cellular levels of mutant huntingtin and REST provide promising strategies for treating Huntington disease. Here, we use the yeast two-hybrid system to show that N-terminus or REST interacts with the heat shock protein 90 (Hsp90) and identifies REST as an Hsp90 Client Protein. To assess the effects of Hsp90 we used antisense oligonucleotide, and evaluated the levels mHtt and REST levels. Our results show that direct knockdown of endogenous Hsp90 significantly reduces the levels of REST and mutant Huntingtin, decreased the percentage of cells with mHtt in nucleus and rescued cells from mHtt-induced cellular cytotoxicity. Additionally Hsp90-specific inhibitors geldanamicyn and PUH71 dramatically reduced mHtt and REST levels, thereby providing neuroprotective activity. Our data show that Hsp90 is necessary to maintain the levels of REST and mHtt, which suggests that the interactions between Hsp90-REST and Hsp90-Huntingtin could be potential therapeutic targets in Huntington's disease.

Electricity generation from Nopal biogas effluent using a surface modified clay cup (cantarito) microbial fuel cell.

A modified clay cup (cantarito) microbial fuel cell (C-MFCs) was designed to digest the biomass effluent from a nopal biogas (NBE). To improve the process, commercial acrylic varnish (AV) was applied to the C-MFCs. The experiment was performed as:Both-C-MFCs, painting of AV on both sides of the clay cup; In-C-MFCs, painting of AV on the internal side, and Out-C-MFCs painting of AV on the external side. The order for the maximum volumetric power densities were Both-C-MFCs (1841.99 mW/m3)>Out-C-MFCs (1023.74 mW/m3) >In-C-MFCs (448.90 mW/m3). The control experiment without applied varnish did not show a stable potential, supporting the idea that the acryloyl group in varnish could favor the performance. Finally, a 4-digits clock was powered with two, Both-C-MFCs connected in series; the microbial diversity in this format was explored and a well-defined bacterial community including members of the phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Synergistetes and candidate division TM7 was found.

Differential sensitivity to interferon influences the replication and transcription of Urabe AM9 mumps virus variants in nerve cells.

Urabe AM9 mumps virus vaccine causes post-vaccination meningitis. Two variants of Urabe AM9 virus differ in their replication efficiency in human nerve cells, HN-A(1081) variant being more neurotropic than HN-G(1081). The effect of interferon (IFN) on viral replication and transcription was analyzed. Priming of nerve cells with IFN reduced more significantly the replication of HN-G(1081) variant (from 10(2.5) to 10(1.3) TCID(50)) than that of HN-A(1081) (from 10(3.5) to 10(2.6) TCID(50)). IFN-priming also reduced the transcription of HN-G(1081) genes, but not of HN-A(1081). The effect of viral infection on the transcription of cellular IFN responsive genes was analyzed. HN-A(1081) virus reduced the transcription of STAT1, STAT2, p48 and MxA in both unprimed and IFN-primed cells; whereas HN-G(1081) virus just reduced MxA transcription. Since rubulavirus V protein inhibits IFN signaling, the V mRNA was cloned and sequenced, finding that HN-G(1081) but not HN-A(1081) presented three extra G in the P/V edition site, producing the insertion of Gly156 in the V protein. Our results suggest that the replication efficiency of Urabe AM9 mumps virus variants is influenced by their sensitivity to interferon and their capacity to reduce the antiviral response.

Amino acid change 335 E to K affects the sialic-acid-binding and neuraminidase activities of Urabe AM9 mumps virus hemagglutinin-neuraminidase glycoprotein.

A mutation coding for the amino acid change E335 to K is frequently found in the hemagglutinin-neuraminidase (HN) gene of Urabe AM9 mumps viruses isolated during post-vaccination meningitis cases. To identify if this mutation modifies the biological activities of the HN glycoprotein, two variants of Urabe AM9 vaccine differing at amino acid 335 (HN-E335 and HN-K335) were isolated and their receptor-binding specificity was determined by means of competence assays. Pre-incubation of the viruses with sialic acids inhibited both syncytia formation in Vero cells and replication in SH-SY5Y cells. Thus, HN-K335 showed higher affinity towards sialylalpha2,6lactose, whereas HN-G335 preferred sialylalpha2,3lactose. These results are relevant because a high expression of sialylalpha2,6lactose in nerve cells was confirmed by means of Sambucus nigra lectin-cytochemistry. In addition, kinetics assays showed that HN-K335 and HN-E335 also differ in their hydrolysis rate (Vmax values of 37.5 vs. 3.5 nmol min-1mg-1, respectively). Therefore, HN-K335 variant presented a neuraminidase activity level 11-fold higher than that of HN-E335 variant. In conclusion, the mutation affects the receptor-binding and neuraminidase activities of Urabe AM9 mumps virus variants.

Two clones obtained from Urabe AM9 mumps virus vaccine differ in their replicative efficiency in neuroblastoma cells.

A high rate of post-vaccinal aseptic meningitis for Urabe AM9 mumps virus strain is well documented. This strain is composed of two virus variants differing at the nt 1081 (A/G) region in the hemagglutinin-neuraminidase (HN) gene. An association of HN-A(1081) variant with neurovirulence has been proposed. In order to test for neurotropism we isolated the HN-A(1081) and HN-G(1081) virus variants from Urabe AM9 mumps virus vaccine. Sequential passages were performed in monkey kidney Vero cells and human neuroblastoma SH-SY5Y cells. Viral replication was determined by conventional and real-time RT-PCR. The results show that clone HN-A(1081) can replicate efficiently in both cell types. However, a defective replication of clone HN-G(1081), lacking its genetic marker, was observed after the third passage in neuroblastoma cells. Kinetics assays showed that clone HN-A(1081) replicates faster than clone HN-G(1081). Viral clones were also inoculated into the brains of newborn rats. Clone HN-A(1081) replicated 14 times, while clone HN-G(1081) merely duplicated its level over the initial inoculum. These results suggest that there is a selective replication of HN-A(1081) mumps virus variants in cells of nervous origin.

Identification of repressor element 1 in cytochrome P450 genes and their negative regulation by RE1 silencing transcription factor/neuron-restrictive silencer factor.

RE1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) mediates transcriptional repression in many neuron-specific genes by interaction with the repressor element 1/neuron-restrictive silencing element (RE1/NRSE). This element has been identified at least in 20 neuron specific genes. REST/NRSF is highly expressed in non-neuronal tissues, where it is thought to repress gene transcription. We performed a BLAST search to look for the presence of RE1/NRSE elements in the rat cytochrome P450 genes. We identified the presence of RE1/NRSE element in the cytochrome P450 genes CYP1A1, 2A2, 2E1 and 3A2. Electrophoretic mobility shift assay and supershift assays were carried out to prove functionality of these sites and detect the interaction of REST/NRSF with this sequence. Cotransfection studies in PC12 cells with a plasmid containing the RE1 element of the CYP genes, cloned upstream of the minimal type II sodium channel promoter, in the presence of REST/NRSF, showed a marked expression inhibition of the CAT reporter gene. These data suggest that the RE1 elements that exist in these four CYP genes might be a target for the REST/NRSF transcription factor and such an interaction might play a role in the negative regulation of these genes.

Isolation of human mesenchymal stem cells and their cultivation on the porous bone matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Pathological-like assembly of tau induced by a paired helical filament core expressed at the plasma membrane.

The hallmark of Alzheimer's disease is the pathological aggregation of tau proteins into paired helical filaments and neurofibrillary tangles. This paper evaluates the abnormal expression and localization of chimeric tau molecules at the plasma membrane of COS-7 cells and its relationship with tau polymerization. Overexpression of these proteins, in combination with either tau441 or tau391, induces tau to assemble into beta-pleated sheets that are recognized by Thiazin red. Immunoelectromicroscopy analysis revealed the presence of filaments close to the plasma membrane resembling those found in Alzheimer's disease. The capacity of plasma membrane-associated chimeric tau proteins to capture full length tau was increased in the presence of H(2)O(2) or okadaic acid treatments. This suggests that hyperphosphorylation or an oxidative environment could both influence the biochemical properties of the cell that lead to assembly of paired helical filaments. The altered localization of tau protein at the plasma membrane could play a key role in the assembly of pathological tau.

Structure-function analysis of two variants of mumps virus hemagglutinin-neuraminidase protein.

A point mutation from guanine (G) to adenine (A) at nucleotide position 1081 in the hemagglutinin-neuraminidase (HN) gene has been associated with neurovirulence of Urabe AM9 mumps virus vaccine. This mutation corresponds to a glutamic acid (E) to lysine (K) change at position 335 in the HN glycoprotein. We have experimentally demonstrated that two variants of Urabe AM9 strain (HN-A1081 and HN-G1081) differ in neurotropism, sialic acidbinding affinity and neuraminidase activity. In the present study, we performed a structure-function analysis of that amino acid substitution; the structures of HN protein of both Urabe AM9 strain variants were predicted. Based on our analysis, the E/K mutation changes the protein surface properties and to a lesser extent their conformations, which in turn reflects in activity changes. Our modeling results suggest that this E/K interchange does not affect the structure of the sialic acid binding motif; however, the electrostatic surface differs drastically due to an exposed short alpha helix. Consequently, this mutation may affect the accessibility of HN to substrates and membrane receptors of the host cells. Our findings appear to explain the observed differences in neurotropism of these vaccine strains.

Structure-function analysis of two variants of mumps virus hemagglutinin-neuraminidase protein

A point mutation from guanine (G) to adenine (A) at nucleotide position 1081 in the hemagglutinin-neuraminidase (HN) gene has been associated with neurovirulence of Urabe AM9 mumps virus vaccine. This mutation corresponds to a glutamic acid (E) to lysine (K) change at position 335 in the HN glycoprotein. We have experimentally demonstrated that two variants of Urabe AM9 strain (HN-A1081 and HN-G1081) differ in neurotropism, sialic acidbinding affinity and neuraminidase activity. In the present study, we performed a structure-function analysis of that amino acid substitution; the structures of HN protein of both Urabe AM9 strain variants were predicted. Based on our analysis, the E/K mutation changes the protein surface properties and to a lesser extent their conformations, which in turn reflects in activity changes. Our modeling results suggest that this E/K interchange does not affect the structure of the sialic acid binding motif; however, the electrostatic surface differs drastically due to an exposed short alpha helix. Consequently, this mutation may affect the accessibility of HN to substrates and membrane receptors of the host cells. Our findings appear to explain the observed differences in neurotropism of these vaccine strains.

Diagnóstico molecular de la neoplasia endocrina múltiple tipo 2B en un caso de novo con la mutación 918 en el oncogen ret / Molecular diagnosis of de novo 918 mutation of ret oncogne in a case of multiple endocrine neoplasia type 2B

Introducción. La neoplasia endocrina múltiple tipo 2B (NEM2B) es un síndrome con carácter dominante hereditario que se caracteriza por el desarrollo de diversas neoplasias de origen neuroendocrino en distintos órganos, tales como carcinoma medular de tiroides (CMT), feocromocitomas, neuromas mucosales, ganglioneuromas del aparato gastrointestinal, también se observan anormalidades esqueléticas y oftálmicas. En más de 95 por ciento de los casos, este padecimiento se asocia con una mutación puntual específica en el dominio tirosina cinasa del proto-oncogen ret, en el codón 918 (METÕTHR), la cual surge de novo en 50 por ciento de los pacientes. Material y métodos. El probando fue un paciente masculino de 19 años de edad sin antecedentes de importancia para la enfermedad y que inició su padecimiento a los 5 años con neuromas submucosos en lengua y labios, así como habitus marfanoide que se acentuó a los 19 años. Determinándose la presencia de la mutación mencionada anteriormente en el DNA de leucocitos de sangre periférica y de carcinoma medular de tiroides de este paciente afectado por NEM2B y se realizó la búsqueda de la misma en leucocitos de sus familiares. Resultados. Los elevados niveles séricos de calcitonina basal (600 pg/mL) sugirieron, además del aspecto clínico y evolución, que el paciente era portador de NEM2B. El estudio histopatológico de tiroides reveló la presencia de CTM clásico. Al estudio del DNA de células de sangre periférica se observó una banda extra sugiriendo que contenía una mutación. Se confirmó la presencia de la mutación ATGÕACG en el codón 918. Conclusión. Al no encontrarse la mutación en los familiares del paciente sugiere que ésta surgió de novo en etapas tempranas del desarrollo embrionario.

Dengue virus: detection and pathogeny

Sequences from a cDNA of dengue virus type 4 were cloned into transcription vectors. These sequences included the E, NS1, NS2A, NS2B, NS3 genes. RNA transcipts produced in vitro from these plasmids were used in hybridization assays to detect dengue viral sequences. With these RNA-probes we have been able to detect molecules of serotype-specific dengue 4 viral RNA. Moreover, the riboprobes detected viral sequences of other serotypes in the following order of sensitivity 4 > 2 > 3 > 1, and might be useful to differentiate serotypes