RESUMEN
A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.
Asunto(s)
Biomarcadores , Proteínas del Ojo/metabolismo , Genómica , Células Progenitoras Linfoides/metabolismo , Análisis de la Célula Individual , Animales , Células Cultivadas , Biología Computacional/métodos , Proteínas del Ojo/genética , Perfilación de la Expresión Génica , Genómica/métodos , Hematopoyesis/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteómica , Análisis de la Célula Individual/métodosRESUMEN
Despite advances in single-cell multi-omics, a single stem or progenitor cell can only be tested once. We developed clonal multi-omics, in which daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel "sister" assays are examined either for gene expression by RNA sequencing (RNA-seq) or for fate in culture. We identified, and then validated using CRISPR, genes that controlled fate bias for different dendritic cell (DC) subtypes. This included Bcor as a suppressor of plasmacytoid DC (pDC) and conventional DC type 2 (cDC2) numbers during Flt3 ligand-mediated emergency DC development. We then developed SIS-skew to examine development of wild-type and Bcor-deficient siblings of the same clone in parallel. We found Bcor restricted clonal expansion, especially for cDC2s, and suppressed clonal fate potential, especially for pDCs. Therefore, SIS-seq and SIS-skew can reveal the molecular and cellular mechanisms governing clonal fate.
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Células Dendríticas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Femenino , Expresión Génica/genética , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Células Madre/metabolismoRESUMEN
The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion surfaces and elicit stronger humoral responses than soluble viral antigens. More recently, bacterial antigens have been presented on self-assembling protein nanoparticles and have elicited protective antibody and effective T-helper responses, further supporting the nanoparticle platform as a universal approach for stimulating potent immunogenicity. Here, we present the rational design, structural analysis, and immunogenicity of self-assembling ferritin nanoparticles displaying eight copies of the Neisseria meningitidis trimeric adhesin NadA. We engineered constructs consisting of two different NadA fragments, head only and head with stalk, that we fused to ferritin and expressed in Escherichia coli. Both fusion constructs self-assembled into the expected nanoparticles as determined by Cryo electron microscopy. In mice, the two nanoparticles elicited comparable NadA antibody levels that were 10- to 100-fold higher than those elicited by the corresponding NadA trimer subunits. Further, the NadAferritin nanoparticles potently induced complement-mediated serum bactericidal activity. These findings confirm the value of self-assembling nanoparticles for optimizing the immunogenicity of bacterial antigens and support the broad applicability of the approach to vaccine programs, especially for the presentation of trimeric antigens.
Asunto(s)
Nanopartículas , Neisseria meningitidis , Ratones , Animales , Ferritinas , Antígenos Bacterianos , Antígenos Virales , Anticuerpos Bloqueadores , Vacunas Combinadas , Nanopartículas/químicaRESUMEN
In addition to its canonical functions, vitamin D has been proposed to be an important mediator of the immune system. Despite ample sunshine, vitamin D deficiency is prevalent (>80%) in the Middle East, resulting in a high rate of supplementation. However, the underlying molecular mechanisms of the specific regimen prescribed and the potential factors affecting an individual's response to vitamin D supplementation are not well characterized. Our objective is to describe the changes in the blood transcriptome and explore the potential mechanisms associated with vitamin D3 supplementation in one hundred vitamin D-deficient women who were given a weekly oral dose (50,000 IU) of vitamin D3 for three months. A high-throughput targeted PCR, composed of 264 genes representing the important blood transcriptomic fingerprints of health and disease states, was performed on pre and post-supplementation blood samples to profile the molecular response to vitamin D3. We identified 54 differentially expressed genes that were strongly modulated by vitamin D3 supplementation. Network analyses showed significant changes in the immune-related pathways such as TLR4/CD14 and IFN receptors, and catabolic processes related to NF-kB, which were subsequently confirmed by gene ontology enrichment analyses. We proposed a model for vitamin D3 response based on the expression changes of molecules involved in the receptor-mediated intra-cellular signaling pathways and the ensuing predicted effects on cytokine production. Overall, vitamin D3 has a strong effect on the immune system, G-coupled protein receptor signaling, and the ubiquitin system. We highlighted the major molecular changes and biological processes induced by vitamin D3, which will help to further investigate the effectiveness of vitamin D3 supplementation among individuals in the Middle East as well as other regions.
Asunto(s)
Colecalciferol/genética , Inmunomodulación/inmunología , Receptores de Lipopolisacáridos/genética , Receptor Toll-Like 4/genética , Vitamina D/genética , Adolescente , Adulto , Colecalciferol/administración & dosificación , Colecalciferol/inmunología , Suplementos Dietéticos , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Inmunomodulación/efectos de los fármacos , Terapia Nutricional , Vitamina D/inmunología , Deficiencia de Vitamina D/dietoterapia , Deficiencia de Vitamina D/genética , Deficiencia de Vitamina D/inmunología , Deficiencia de Vitamina D/patología , Adulto JovenRESUMEN
BACKGROUND: Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation. METHODS: DNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation. RESULTS: Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines. CONCLUSION: Cell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.
Asunto(s)
Melanoma , Proteínas Proto-Oncogénicas B-raf , Línea Celular Tumoral , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Mutación/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Vemurafenib/farmacologíaRESUMEN
While histological analysis represents a powerful tool for the classification of melanocytic lesions as benign or malignant, a clear-cut distinction between a nevus and a melanoma is sometimes a challenging step of the diagnostic process. The immunohistochemical detection of tyrosinase, cardinal melanogenic enzyme during melanocytic maturation, has often been helpful in formulating a differential diagnosis due to the peculiar staining pattern in nevocytes compared with melanoma cells. Tyrosinase distribution in nevi appears to overlap with the cytoarchitectural changes observable within these lesions, that result in epidermal or superficial dermal nevocytes being larger and strongly expressing melanocytic differentiation antigens, such as tyrosinase, compared with deeper dermal nevus cells. Our study aimed to evaluate the immunohistochemical expression pattern of tyrosinase in different histological types of acquired dysplastic melanocytic nevi, including junctional, compound, and intradermal nevi. Moreover, to estimate whether in nevocytes the expression of tyrosinase was associated with their differentiation state, we investigated the expression of two recognized markers of pluripotency, CD34 and nestin. In all examined nevi, our analysis revealed a remarkable immunoreactivity for tyrosinase in junctional and superficial dermal nevocytes and a decreasing gradient of staining in dermal nevocytes, up to become negative in deeper dermis. Meanwhile, junctional and dermal nevocytes were lacking in CD34 protein. Furthermore, nestin immunostaining showed an opposite distribution compared with tyrosinase, leading us to look into the tyrosinase/nestin expression pattern in melanocytic nevus as a tool to better understand the final stages of differentiation of melanocyte precursors toward their ultimate anatomical site into the epidermis.
Asunto(s)
Diferenciación Celular , Melanocitos/química , Melanocitos/patología , Monofenol Monooxigenasa/análisis , Nestina/análisis , Nevo Pigmentado/química , Nevo Pigmentado/patología , Adolescente , Adulto , Anciano , Femenino , Humanos , Inmunohistoquímica , Masculino , Melanocitos/metabolismo , Persona de Mediana Edad , Monofenol Monooxigenasa/biosíntesis , Nestina/biosíntesis , Nevo Pigmentado/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Monoallelic expression (MAE) is a frequent genomic phenomenon in normal tissues, however its role in cancer is yet to be fully understood. MAE is defined as the expression of a gene that is restricted to one allele in the presence of a diploid heterozygous genome. Constitutive MAE occurs for imprinted genes, odorant receptors and random X inactivation. Several studies in normal tissues have showed MAE in approximately 5-20% of the cases. However, little information exists on the MAE rate in cancer. In this study we assessed the presence and rate of MAE in melanoma. The genetic basis of melanoma has been studied in depth over the past decades, leading to the identification of mutations/genetic alterations responsible for melanoma development. METHODS: To examine the role of MAE in melanoma we used 15 melanoma cell lines and compared their RNA-seq data with genotyping data obtained by the parental TIL (tumor infiltrating lymphocytes). Genotyping was performed using the Illumina HumanOmni1 beadchip. The RNA-seq library preparation and sequencing was performed using the Illumina TruSeq Stranded Total RNA Human Kit and subsequently sequenced using a HiSeq 2500 according to manufacturer's guidelines. By comparing genotyping data with RNA-seq data, we identified SNPs in which DNA genotypes were heterozygous and corresponding RNA genotypes were homozygous. All homozygous DNA genotypes were removed prior to the analysis. To confirm the validity to detect MAE, we examined heterozygous DNA genotypes from X chromosome of female samples as well as for imprinted and olfactory receptor genes and confirmed MAE. RESULTS: MAE was detected in all 15 cell lines although to a different rate. When looking at the B-allele frequencies we found a preferential pattern of complete monoallelic expression rather then differential monoallelic expression across the 15 melanoma cell lines. As some samples showed high differences in the homozygous and heterozygous call rate, we looked at the single chromosomes and showed that MAE may be explained by underlying large copy number imbalances in some instances. Interestingly these regions included genes known to play a role in melanoma initiation and progression. Nevertheless, some chromosome regions showed MAE without CN imbalances suggesting that additional mechanisms (including epigenetic silencing) may explain MAE in melanoma. CONCLUSION: The biological implications of MAE are yet to be realized. Nevertheless, our findings suggest that MAE is a common phenomenon in melanoma cell lines. Further analyses are currently being undertaken to evaluate whether MAE is gene/pathway specific and to understand whether MAE can be employed by cancers to achieve a more aggressive phenotype.
Asunto(s)
Impresión Genómica/fisiología , Melanoma/genética , Neoplasias Cutáneas/genética , Alelos , Línea Celular Tumoral , Hibridación Genómica Comparativa , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Frecuencia de los Genes , Genotipo , Heterocigoto , Homocigoto , Humanos , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Melanoma/patología , Análisis por Micromatrices , Polimorfismo de Nucleótido Simple , Neoplasias Cutáneas/patologíaRESUMEN
Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Neisseria meningitidis Serogrupo B/metabolismo , Secuencia de Aminoácidos , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Variación Genética , Humanos , Espectrometría de Masas/métodos , Meningitis Meningocócica/inmunología , Meningitis Meningocócica/microbiología , Vacunas Meningococicas/inmunología , Neisseria meningitidis Serogrupo B/clasificación , Neisseria meningitidis Serogrupo B/genética , Filogenia , Especificidad de la EspecieRESUMEN
BACKGROUND: Differences in the concentrations of circulating 25-hydroxyvitamin D [25(OH)D] are associated with a wide range of health outcomes; however, most studies on genetic variants that impact 25(OH)D levels have been conducted in European populations. Here we aimed to identify common genetic variants that affect vitamin D concentrations in individuals of self-reported Arab ethnicity. METHODS: The study included 1151 Arab subjects living in Kuwait. Common variants of single-nucleotide polymorphisms and genes previously associated with vitamin D levels, such as GC, PDE3B, CYP2R1, and NADSYN1, were genotyped. Raw vitamin D level data were corrected for age, body mass index, and sex and then normalized. Regression tree analyses were performed to identify the impact of genetic variants on vitamin D levels. RESULTS: Compared with other gene variants, the GC gene variants exhibited the greatest impact on vitamin D levels in our study population, of which rs2298850 had the lowest p value (0.003). Individuals homozygous for the derived allele C had lower vitamin D levels. Analyses of the interaction between the number of years for which the subjects had lived in Kuwait and genetic variation in the GC gene showed that those with the CC genotype of rs2298850 who had lived in Kuwait for < 51 years had a mean 25(OH)D level of 10 ng/ml, whereas those who were homozygous for the ancestral allele had a mean 25(OH)D level of 17 ng/ml. Furthermore, subjects who had lived in Kuwait for > 51 years had higher vitamin D levels (mean 28 ng/ml) regardless of the genotype of their GC gene. CONCLUSIONS: The GC gene may play a major role in determining vitamin D levels in Arab populations.
Asunto(s)
Árabes/genética , Variación Genética , Vitamina D/sangre , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis de RegresiónRESUMEN
Epithelial-mesenchymal transition (EMT) has been suggested to have a driving role in the acquisition of a metastatic potential by melanoma cells. Important hallmarks of EMT include both E-cadherin downregulation and increased expression of N-cadherin. This switch in distinct classes of adhesion molecules leads melanoma cells to lose contact with adjacent keratinocytes and interact instead with stromal fibroblasts and endothelial cells, thus promoting dermal and vascular melanoma invasion. Consequently, tumor cells migrate to distant host tissues and establish metastases. A key regulator in the induction of EMT in melanoma is the Notch1 signaling pathway that, when activated, is prompt to upregulate N-cadherin expression. By means of this strategy, melanoma cells gain enhanced survival, proliferation and invasion properties, driving the tumor toward a more aggressive phenotype. On the basis of these statements, the present study aimed to investigate the possible association between N-cadherin and Notch1 presence in primary cutaneous melanomas and lymph node metastases. Our results from immunohistochemical analysis confirmed a positive correlation between N-cadherin and Notch1 presence in the same tumor samples. Moreover, this study highlighted that a concomitant high expression of N-cadherin and Notch1, both in primary lesions and in lymph node metastases, predicts an adverse clinical outcome in melanoma patients. Therefore, N-cadherin and Notch1 co-presence can be monitored as a predictive factor in early- and advanced-stage melanomas and open additional therapeutic targets for the restraint of melanoma metastasis.
Asunto(s)
Cadherinas/análisis , Transición Epitelial-Mesenquimal , Melanoma/química , Receptor Notch1/análisis , Neoplasias Cutáneas/química , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Cadherinas/biosíntesis , Niño , Femenino , Humanos , Inmunohistoquímica , Masculino , Melanoma/diagnóstico , Melanoma/metabolismo , Persona de Mediana Edad , Receptor Notch1/biosíntesis , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Ligase IV syndrome, a hereditary disease associated with compromised DNA damage response mechanisms, and Urofacial syndrome, caused by an impairment of neural cell signaling, are both rare genetic disorders, whose reports in literature are limited. We describe the first case combining both disorders in a specific phenotype. CASE PRESENTATION: We report a case of a 7-year old girl presenting with a complex phenotype characterized by multiple congenital abnormalities and dysmorphic features, microcephaly, short stature, combined immunodeficiency and severe vesicoureteral reflux. Whole Genome Sequencing was performed and a novel ligase IV homozygous missense c.T1312C/p.Y438H mutation was detected, and is believed to be responsible for most of the clinical features of the child, except vesicoureteral reflux which has not been previously described for ligase IV deficiency. However, we observed a second rare damaging (nonsense) homozygous mutation (c.C2125T/p.R709X) in the leucine-rich repeats and immunoglobulin-like domains 2 gene that encodes a protein implicated in neural cell signaling and oncogenesis. Interestingly, this mutation has recently been reported as pathogenic and causing urofacial syndrome, typically displaying vesicoureteral reflux. Thus, this second mutation completes the missing genetic explanation for this intriguing clinical puzzle. We verified that both mutations fit an autosomal recessive inheritance model due to extensive consanguinity. CONCLUSIONS: We successfully identified a novel ligase IV mutation, causing ligase IV syndrome, and an additional rare leucine-rich repeats and immunoglobulin-like domains 2 gene nonsense mutation, in the context of multiple autosomal recessive conditions due to extensive consanguinity. This work demonstrates the utility of Whole Genome Sequencing data in clinical diagnosis in such cases where the combination of multiple rare phenotypes results in very intricate clinical pictures. It also reports a novel causative mutation and a clinical phenotype, which will help in better defining the essential features of both ligase IV and leucine-rich repeats and immunoglobulin-like domains 2 deficiency syndromes.
Asunto(s)
Anomalías Craneofaciales/genética , ADN Ligasa (ATP)/genética , Genoma/genética , Trastornos del Crecimiento/genética , Síndromes de Inmunodeficiencia/genética , Enfermedades Urológicas/genética , Anomalías Múltiples/genética , Encéfalo/diagnóstico por imagen , Niño , Anomalías Craneofaciales/patología , Facies , Femenino , Trastornos del Crecimiento/patología , Homocigoto , Humanos , Síndromes de Inmunodeficiencia/patología , Inmunofenotipificación , Imagen por Resonancia Magnética , Glicoproteínas de Membrana/genética , Mutación Missense , Linaje , Fenotipo , Enfermedades Urológicas/patologíaRESUMEN
BACKGROUND: KCNH1 encodes a voltage-gated potassium channel that is predominantly expressed in the central nervous system. Mutations in this gene were recently found to be responsible for Temple-Baraitser Syndrome (TMBTS) and Zimmermann-Laband syndrome (ZLS). METHODS: Here, we report a new case of TMBTS diagnosed in a Lebanese child. Whole genome sequencing was carried out on DNA samples of the proband and his parents to identify mutations associated with this disease. Sanger sequencing was performed to confirm the presence of detected variants. RESULTS: Whole genome sequencing revealed three missense mutations in TMBTS patient: c.1042G > A in KCNH1, c.2131 T > C in STK36, and c.726C > A in ZNF517. According to all predictors, mutation in KCNH1 is damaging de novo mutation that results in substitution of Glycine by Arginine, i.e., p.(Gly348Arg). This mutation was already reported in a patient with ZLS that could affect the connecting loop between helices S4-S5 of KCNH1 with a gain of function effect. CONCLUSIONS: Our findings demonstrate that KCNH1 mutations cause TMBTS and expand the mutational spectrum of KCNH1 in TMBTS. In addition, all cases of TMBTS were reviewed and compared to ZLS. We suggest that the two syndromes are a continuum and that the variability in the phenotypes is the result of the involvement of genetic modifiers.
Asunto(s)
Anomalías Múltiples/genética , Anomalías Craneofaciales/genética , Fibromatosis Gingival/genética , Hallux/anomalías , Deformidades Congénitas de la Mano/genética , Discapacidad Intelectual/genética , Uñas Malformadas/genética , Pulgar/anomalías , Anomalías Múltiples/diagnóstico , Anomalías Craneofaciales/diagnóstico , ADN/química , ADN/aislamiento & purificación , ADN/metabolismo , Análisis Mutacional de ADN , Canales de Potasio Éter-A-Go-Go/genética , Fibromatosis Gingival/diagnóstico , Deformidades Congénitas de la Mano/diagnóstico , Humanos , Lactante , Discapacidad Intelectual/diagnóstico , Masculino , Mutación Missense , Uñas Malformadas/diagnóstico , Proteínas Serina-Treonina Quinasas/genética , Pulgar/diagnóstico por imagen , Dedos del Pie/diagnóstico por imagenRESUMEN
OBJECTIVES: In Qataris, a population characterized by a small size and a high rate of consanguinity, between two-thirds to three-quarters of adults are overweight or obese. We investigated the relevance of 23 obesity-related loci in the Qatari population. METHODS: Eight-hundred-four individuals assessed to be third generation Qataris were included in the study and assigned to 3 groups according to their body mass index (BMI): 190 lean (BMI < 25 kg/m(2)); 131 overweight (25 kg/m(2) ≤ BMI < 30 kg/m(2)) and 483 obese (BMI ≥ 30 kg/m(2)). Genomic DNA was isolated from peripheral blood and genotyped by TaqMan. RESULTS: Two loci significantly associated with obesity in Qataris: the TFAP2B variation (rs987237) (A allele versus G allele: chi-square = 10.3; P = 0.0013) and GNPDA2 variation (rs10938397) (A allele versus G allele: chi-square = 6.15; P = 0.013). The TFAP2B GG genotype negatively associated with obesity (OR = 0.21; P = 0.0031). Conversely, the GNDPA2 GG homozygous genotype associated with higher risk of obesity in subjects of age < 32 years (P = 0.0358). CONCLUSION: We showed a different genetic profile associated with obesity in the Qatari population compared to Western populations. Studying the genetic background of Qataris is of primary importance as the etiology of a given disease might be population-specific.
Asunto(s)
Árabes/genética , Consanguinidad , Sitios Genéticos , Predisposición Genética a la Enfermedad , Obesidad/genética , Adulto , Índice de Masa Corporal , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Análisis de Componente Principal , Qatar , Grupos Raciales/genética , Delgadez/genéticaRESUMEN
The fourth "Melanoma Bridge Meeting" took place in Naples, December 3-6th, 2014. The four topics discussed at this meeting were: Molecular and Immunological Advances, Combination Therapies, News in Immunotherapy, and Tumor Microenvironment and Biomarkers. Until recently systemic therapy for metastatic melanoma patients was ineffective, but recent advances in tumor biology and immunology have led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS). New therapies, such as mitogen-activated protein kinase (MAPK) pathway inhibitors as well as other signaling pathway inhibitors, are being tested in patients with metastatic melanoma either as monotherapy or in combination, and all have yielded promising results. These include inhibitors of receptor tyrosine kinases (BRAF, MEK, and VEGFR), the phosphatidylinositol 3 kinase (PI3K) pathway [PI3K, AKT, mammalian target of rapamycin (mTOR)], activators of apoptotic pathway, and the cell cycle inhibitors (CDK4/6). Various locoregional interventions including radiotherapy and surgery are still valid approaches in treatment of advanced melanoma that can be integrated with novel therapies. Intrinsic, adaptive and acquired resistance occur with targeted therapy such as BRAF inhibitors, where most responses are short-lived. Given that the reactivation of the MAPK pathway through several distinct mechanisms is responsible for the majority of acquired resistance, it is logical to combine BRAF inhibitors with inhibitors of targets downstream in the MAPK pathway. For example, combination of BRAF/MEK inhibitors (e.g., dabrafenib/trametinib) have been demonstrated to improve survival compared to monotherapy. Application of novel technologies such sequencing have proven useful as a tool for identification of MAPK pathway-alternative resistance mechanism and designing other combinatorial therapies such as those between BRAF and AKT inhibitors. Improved survival rates have also been observed with immune-targeted therapy for patients with metastatic melanoma. Immune-modulating antibodies came to the forefront with anti-CTLA-4, programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) pathway blocking antibodies that result in durable responses in a subset of melanoma patients. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors and other approaches such as adoptive cell transfer demonstrate clinical benefit in patients with melanoma as well. These agents are being studied in combination with targeted therapies in attempt to produce longer-term responses than those more typically seen with targeted therapy. Other combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for this patient population. This meeting's specific focus was on advances in combination of targeted therapy and immunotherapy. Both combination targeted therapy approaches and different immunotherapies were discussed. Similarly to the previous meetings, the importance of biomarkers for clinical application as markers for diagnosis, prognosis and prediction of treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma.
Asunto(s)
Melanoma/patología , Biomarcadores de Tumor , Humanos , Inmunoterapia , Italia , Melanoma/inmunología , Melanoma/terapia , Microambiente TumoralRESUMEN
BACKGROUND: Papillary thyroid cancer is the most common endocrine malignancy. The most sensitive and specific diagnostic tool for thyroid nodule diagnosis is fine-needle aspiration (FNA) biopsy with cytological evaluation. Nevertheless, FNA biopsy is not always decisive leading to "indeterminate" or "suspicious" diagnoses in 10%-30% of cases. BRAF V600E detection is currently used as molecular test to improve the diagnosis of thyroid nodules, yet it lacks sensitivity. The aim of the present study was to identify novel molecular markers/computational models to improve the discrimination between benign and malignant thyroid lesions. METHODS: We collected 118 pre-operative thyroid FNA samples. All 118 FNA samples were characterized for the presence of the BRAF V600E mutation (exon15) by pyrosequencing and further assessed for mRNA expression of four genes (KIT, TC1, miR-222, miR-146b) by quantitative polymerase chain reaction. Computational models (Bayesian Neural Network Classifier, discriminant analysis) were built, and their ability to discriminate benign and malignant tumors were tested. Receiver operating characteristic (ROC) analysis was performed and principal component analysis was used for visualization purposes. RESULTS: In total, 36/70 malignant samples carried the V600E mutation, while all 48 benign samples were wild type for BRAF exon15. The Bayesian neural network (BNN) and discriminant analysis, including the mRNA expression of the four genes (KIT, TC1, miR-222, miR-146b) showed a very strong predictive value (94.12% and 92.16%, respectively) in discriminating malignant from benign patients. The discriminant analysis showed a correct classification of 100% of the samples in the malignant group, and 95% by BNN. KIT and miR-146b showed the highest diagnostic accuracy of the ROC curve, with area under the curve values of 0.973 for KIT and 0.931 for miR-146b. CONCLUSIONS: The four genes model proposed in this study proved to be highly discriminative of the malignant status compared with BRAF assessment alone. Its implementation in clinical practice can help in identifying malignant/benign nodules that would otherwise remain suspicious.
Asunto(s)
Biomarcadores de Tumor/genética , MicroARNs/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-kit/genética , Neoplasias de la Tiroides/diagnóstico , Anciano , Teorema de Bayes , Biomarcadores de Tumor/metabolismo , Biopsia con Aguja Fina , Femenino , Perfilación de la Expresión Génica , Marcadores Genéticos , Humanos , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Mutación , Proteínas de Neoplasias/metabolismo , Análisis de Componente Principal , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN/metabolismo , ARN Mitocondrial , Curva ROC , Sensibilidad y Especificidad , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismoRESUMEN
Due to its non-invasive nature and easy accessibility, urine serves as a convenient bi-ological fluid for research purposes. Furthermore, urine samples are uncomplicated to preserve and relatively inexpensive. MicroRNA (miRNAs), small molecules that regulate gene expression post-transcriptionally, play vital roles in numerous cellular processes, including apoptosis, cell differentiation, development, and proliferation. Their dysregulated expression in urine has been proposed as a potential bi-omarker for various human diseases, including bladder cancer. To draw reliable conclusions about the roles of urinary miRNAs in human diseases, it is essential to have dependable and reproducible methods for miRNA extraction and profiling. In this review, we address the technical challenges associated with studying urinary miRNAs and provide an update on the current technologies used for urinary miRNA isolation, quality control assessment, and miRNA profiling, highlighting both their advantages and limitations.
RESUMEN
Meningococcal (Neisseria meningitidis) serogroup B (MenB) strain antigens are diverse and a limited number of strains can be evaluated using the human serum bactericidal antibody (hSBA) assay. The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict the likelihood of coverage for large numbers of isolates by the 4CMenB vaccine, which includes antigens Neisseria adhesin A (NadA), Neisserial Heparin-Binding Antigen (NHBA), factor H-binding protein (fHbp), and Porin A (PorA). In this study, we characterized by whole-genome analyses 284 invasive MenB isolates collected from 2010 to 2014 by the Argentinian National Laboratories Network (52-61 isolates per year). Strain coverage was estimated by gMATS on all isolates and by hSBA assay on 74 randomly selected isolates, representative of the whole panel. The four most common clonal complexes (CCs), accounting for 81.3% of isolates, were CC-865 (75 isolates, 26.4%), CC-32 (59, 20.8%), CC-35 (59, 20.8%), and CC-41/44 (38, 13.4%). Vaccine antigen genotyping showed diversity. The most prevalent variants/peptides were fHbp variant 2, NHBA peptides 24, 21, and 2, and PorA variable region 2 profiles 16-36 and 14. The nadA gene was present in 66 (23.2%) isolates. Estimated strain coverage by hSBA assay showed 78.4% of isolates were killed by pooled adolescent sera, and 51.4% and 64.9% (based on two different thresholds) were killed by pooled infant sera. Estimated coverage by gMATS (61.3%; prediction interval: 55.5%, 66.7%) was consistent with the infant hSBA assay results. Continued genomic surveillance is needed to evaluate the persistence of major MenB CCs in Argentina.
The most common clinical manifestations of invasive meningococcal disease include meningitis and septicemia, which can be deadly, and many survivors suffer long-term serious after-effects. Most cases of invasive meningococcal disease are caused by six meningococcal serogroups (types), including serogroup B. Although vaccines are available against meningococcal serogroup B infection, these vaccines target antigens that are highly diverse. Consequently, the effectiveness of vaccination may vary from country to country because the meningococcal serogroup B strains circulating in particular regions carry different forms of the target vaccine antigens. This means it is important to test serogroup B strains isolated from specific populations to estimate the percentage of strains that a vaccine is likely to be effective against (known as 'vaccine strain coverage'). The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict strain coverage by the four-component meningococcal serogroup B vaccine, 4CMenB, against large numbers of serogroup B strains. In this study, we analyzed 284 invasive meningococcal serogroup B isolates collected between 2010 and 2014 in Argentina. Genetic analyses showed that the vaccine antigens of the isolates were diverse and some genetic characteristics had not been found in isolates from other countries. However, vaccine strain coverage estimated by gMATS was consistent with that reported in other parts of the world and with strain coverage results obtained for a subset via another method, the human serum bactericidal antibody (hSBA) assay. These results highlight the need for continued monitoring of circulating bacterial strains to assess the estimated strain coverage of meningococcal serogroup B vaccines.
Asunto(s)
Antígenos Bacterianos , Infecciones Meningocócicas , Vacunas Meningococicas , Neisseria meningitidis Serogrupo B , Humanos , Argentina/epidemiología , Vacunas Meningococicas/inmunología , Vacunas Meningococicas/administración & dosificación , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/prevención & control , Infecciones Meningocócicas/epidemiología , Lactante , Adolescente , Niño , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Preescolar , Adulto Joven , Neisseria meningitidis Serogrupo B/genética , Neisseria meningitidis Serogrupo B/aislamiento & purificación , Neisseria meningitidis Serogrupo B/inmunología , Adulto , Femenino , Masculino , Secuenciación Completa del Genoma , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Genotipo , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Persona de Mediana Edad , Porinas/genética , Porinas/inmunología , Determinación de Anticuerpos Séricos Bactericidas , Anciano , Neisseria meningitidis/genética , Neisseria meningitidis/inmunología , Neisseria meningitidis/aislamiento & purificación , Neisseria meningitidis/clasificaciónRESUMEN
BACKGROUND: Recent observations suggest that immune-mediated tissue destruction is dependent upon coordinate activation of immune genes expressed by cells of the innate and adaptive immune systems. METHODS: Here, we performed a retrospective pilot study to investigate whether the coordinate expression of molecular signature mostly associated with NK cells could be used to segregate breast cancer patients into relapse and relapse-free outcomes. RESULTS: By analyzing primary breast cancer specimens derived from patients who experienced either 58-116 months (~5-9 years) relapse-free survival or developed tumor relapse within 9-76 months (~1-6 years) we found that the expression of molecules involved in activating signaling of NK cells and in NK cells: target interaction is increased in patients with favorable prognosis. CONCLUSIONS: The parameters identified in this study, together with the prognostic signature previously reported by our group, highlight the cooperation between the innate and adaptive immune components within the tumor microenvironment.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Perfilación de la Expresión Génica , Células Asesinas Naturales/inmunología , Antígenos CD1d/metabolismo , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Recuento de Células , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Activación de Linfocitos/genética , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Recurrencia , Transducción de Señal/genética , Máquina de Vectores de Soporte , Resultado del TratamientoRESUMEN
Transcriptome profiling of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Recently, finger-stick blood collection systems have allowed a less invasive and quicker collection of peripheral blood. Such non-invasive sampling of small volumes of blood offers practical advantages. The quality of gene expression data is strictly dependent on the steps used for the sample collection, extraction, preparation and sequencing. Here we have: (i) compared the manual and automated RNA extraction of small volumes of blood using the Tempus Spin RNA isolation kit and the MagMAX for Stabilized Blood RNA Isolation kit , respectively; and (ii) assessed the effect of TURBO DNA Free treatment on the transcriptomic data of RNA isolated from small volumes of blood. We have used the QuantSeq 3' FWD mRNA-Seq Library Prep kit to prepare RNA-seq libraries, which were sequenced on the Illumina NextSeq 500 system. The samples isolated manually displayed a higher variability in the transcriptomic data as compared to the other samples. The TURBO DNA Free treatment affected the RNA samples negatively, decreasing the RNA yield and reducing the quality and reproducibility of the transcriptomic data. We conclude that automated extraction systems should be preferred over manual extraction systems for data consistency, and that the TURBO DNA Free treatment should be avoided when working on RNA samples isolated manually from small volumes of blood.