RESUMO
We present a method to detect copy number variants (CNVs) that are differentially present between two groups of sequenced samples. We use a finite-state transducer where the emitted read depth is conditioned on the mappability and GC-content of all reads that occur at a given base position. In this model, the read depth within a region is a mixture of binomials, which in simulations matches the read depth more closely than the often-used negative binomial distribution. The method analyzes all samples simultaneously, preserving uncertainty as to the breakpoints and magnitude of CNVs present in an individual when it identifies CNVs differentially present between the two groups. We apply this method to identify CNVs that are recurrently associated with postglacial adaptation of marine threespine stickleback (Gasterosteus aculeatus) to freshwater. We identify 6664 regions of the stickleback genome, totaling 1.7 Mbp, which show consistent copy number differences between marine and freshwater populations. These deletions and duplications affect both protein-coding genes and cis-regulatory elements, including a noncoding intronic telencephalon enhancer of DCHS1 The functions of the genes near or included within the 6664 CNVs are enriched for immunity and muscle development, as well as head and limb morphology. Although freshwater stickleback have repeatedly evolved from marine populations, we show that freshwater stickleback also act as reservoirs for ancient ancestral sequences that are highly conserved among distantly related teleosts, but largely missing from marine stickleback due to recent selective sweeps in marine populations.
Assuntos
Adaptação Fisiológica/genética , Variações do Número de Cópias de DNA/genética , Seleção Genética , Smegmamorpha/genética , Animais , Água Doce , Genoma/genética , Polimorfismo de Nucleotídeo Único/genética , Estudos de AmostragemRESUMO
Marine stickleback fish have colonized and adapted to thousands of streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high-quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of twenty additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine-freshwater divergence. Our results indicate that reuse of globally shared standing genetic variation, including chromosomal inversions, has an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine-freshwater evolution, but regulatory changes appear to predominate in this well known example of repeated adaptive evolution in nature.
Assuntos
Adaptação Fisiológica/genética , Evolução Biológica , Genoma/genética , Smegmamorpha/genética , Alaska , Animais , Organismos Aquáticos/genética , Inversão Cromossômica/genética , Cromossomos/genética , Sequência Conservada/genética , Ecótipo , Feminino , Água Doce , Variação Genética/genética , Genômica , Dados de Sequência Molecular , Água do Mar , Análise de Sequência de DNARESUMO
BACKGROUND: Dorsal spine reduction in threespine sticklebacks (Gasterosteus aculeatus) is a classic example of recurrent skeletal evolution in nature. Sticklebacks in marine environments typically have long spines that form part of their skeletal armor. Many derived freshwater populations have evolved shorter spines. Changes in spine length are controlled in part by a quantitative trait locus (QTL) previously mapped to chromosome 4, but the causative gene and mutations underlying the repeated evolution of this interesting skeletal trait have not been identified. RESULTS: Refined mapping of the spine length QTL shows that it lies near the MSX2A transcription factor gene. MSX2A is expressed in developing spines. In F1 marine × freshwater fish, the marine allele is preferentially expressed. Differences in expression can be attributed to splicing regulation. Due to the use of an alternative 5 ' splice site within the first exon, the freshwater allele produces greater amounts of a shortened, non-functional transcript and makes less of the full-length transcript. Sequence changes in the MSX2A region are shared by many freshwater fish, suggesting that repeated evolution occurs by reuse of a spine-reduction variant. To demonstrate the effect of full-length MSX2A on spine length, we produced transgenic freshwater fish expressing a copy of marine MSX2A. The spines of the transgenic fish were significantly longer on average than those of their non-transgenic siblings, partially reversing the reduced spine lengths that have evolved in freshwater populations. CONCLUSIONS: MSX2A is a major gene underlying dorsal spine reduction in freshwater sticklebacks. The gene is linked to a separate gene controlling bony plate loss, helping explain the concerted effects of chromosome 4 on multiple armor-reduction traits. The nature of the molecular changes provides an interesting example of morphological evolution occurring not through a simple amino acid change, nor through a change only in gene expression levels, but through a change in the ratio of splice products encoding both normal and truncated proteins.
Assuntos
Evolução Biológica , Proteínas de Peixes/genética , Splicing de RNA , Smegmamorpha/anatomia & histologia , Smegmamorpha/genética , Coluna Vertebral/anatomia & histologia , Fatores de Transcrição/genética , Alelos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/anatomia & histologia , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Água Doce , Fenótipo , Locos de Características Quantitativas , Alinhamento de Sequência , Smegmamorpha/metabolismo , Coluna Vertebral/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication.
Assuntos
Quebras de DNA de Cadeia Simples , DNA Ligases/química , DNA Ligases/metabolismo , Replicação do DNA/fisiologia , DNA/química , DNA/metabolismo , Motivos de Aminoácidos , Animais , DNA/genética , DNA Ligase Dependente de ATP , DNA Ligases/genética , Humanos , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteína de Replicação C/química , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismoRESUMO
Immune checkpoint inhibitors (ICIs), including CTLA-4- and PD-1-blocking antibodies, can have profound effects on tumor immune cell infiltration that have not been consistent in biopsy series reported to date. Here, we analyze seven molecular datasets of samples from patients with advanced melanoma (N = 514) treated with ICI agents to investigate clinical, genomic, and transcriptomic features of anti-PD-1 response in cutaneous melanoma. We find that prior anti-CTLA-4 therapy is associated with differences in genomic, individual gene, and gene signatures in anti-PD-1 responders. Anti-CTLA-4-experienced melanoma tumors that respond to PD-1 blockade exhibit increased tumor mutational burden, inflammatory signatures, and altered cell cycle processes compared with anti-CTLA-4-naive tumors or anti-CTLA-4-experienced, anti-PD-1-nonresponsive melanoma tumors. We report a harmonized, aggregate resource and suggest that prior CTLA-4 blockade therapy is associated with marked differences in the tumor microenvironment that impact the predictive features of PD-1 blockade therapy response.
Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Antígeno CTLA-4/genética , Biomarcadores Tumorais , Imunoterapia , Microambiente TumoralRESUMO
Understanding the mechanisms leading to new traits or additional features in organisms is a fundamental goal of evolutionary biology. We show that HOXDB regulatory changes have been used repeatedly in different fish genera to alter the length and number of the prominent dorsal spines used to classify stickleback species. In Gasterosteus aculeatus (typically 'three-spine sticklebacks'), a variant HOXDB allele is genetically linked to shortening an existing spine and adding an additional spine. In Apeltes quadracus (typically 'four-spine sticklebacks'), a variant HOXDB allele is associated with lengthening a spine and adding an additional spine in natural populations. The variant alleles alter the same non-coding enhancer region in the HOXDB locus but do so by diverse mechanisms, including single-nucleotide polymorphisms, deletions and transposable element insertions. The independent regulatory changes are linked to anterior expansion or contraction of HOXDB expression. We propose that associated changes in spine lengths and numbers are partial identity transformations in a repeating skeletal series that forms major defensive structures in fish. Our findings support the long-standing hypothesis that natural Hox gene variation underlies key patterning changes in wild populations and illustrate how different mutational mechanisms affecting the same region may produce opposite gene expression changes with similar phenotypic outcomes.
Assuntos
Genes Homeobox , Smegmamorpha , Animais , Elementos de DNA Transponíveis , Fenótipo , Smegmamorpha/genéticaRESUMO
Immune and molecular profiling of CD8 T cells of patients receiving DC vaccines expressing three full-length melanoma antigens (MAs) was performed. Antigen expression levels in DCs had no significant impact on T cell or clinical responses. Patients who received checkpoint blockade before DC vaccination had higher baseline MA-specific CD8 T cell responses but no evidence for improved functional responses to the vaccine. Patients who showed the best clinical responses had low PD-1 expression on MA-specific T cells before and after DC vaccination; however, blockade of PD-1 during antigen presentation by DC had minimal functional impact on PD-1high MA-specific T cells. Gene and protein expression analyses in lymphocytes and tumor samples identified critical immunoregulatory pathways, including CTLA-4 and PD-1. High immune checkpoint gene expression networks correlated with inferior clinical outcomes. Soluble serum PD-L2 showed suggestive positive association with improved outcome. These findings show that checkpoint molecular pathways are critical for vaccine outcomes and suggest specific sequencing of vaccine combinations.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , Ativação Linfocitária/imunologia , Antígenos de Neoplasias/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Antígeno CTLA-4/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Intervalo Livre de Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Antígeno MART-1/metabolismo , Melanoma/sangue , Melanoma/genética , Melanoma/imunologia , Melanoma/patologia , Receptor de Morte Celular Programada 1/metabolismo , VacinaçãoRESUMO
In human cells, there are three genes that encode DNA ligase polypeptides with distinct but overlapping functions. Previously small molecule inhibitors of human DNA ligases were identified using a structure-based approach. Three of these inhibitors, L82, a DNA ligase I (LigI)-selective inhibitor, and L67, an inhibitor of LigI and DNA ligases III (LigIII), and L189, an inhibitor of all three human DNA ligases, have related structures that are composed of two 6-member aromatic rings separated by different linkers. Here we have performed a structure-activity analysis to identify determinants of activity and selectivity. The majority of the LigI-selective inhibitors had a pyridazine ring whereas the LigI/III- and LigIII-selective inhibitors did not. In addition, the aromatic rings in LigI-selective inhibitors had either arylhydrazone or acylhydrazone, but not vinyl linkers. Among the LigI-selective inhibitors, L82-G17 exhibited increased activity against and selectivity for LigI compared with L82. Notably. L82-G17 is an uncompetitive inhibitor of the third step of the ligation reaction, phosphodiester bond formation. Cells expressing LigI were more sensitive to L82-G17 than isogenic LIG1 null cells. Furthermore, cells lacking nuclear LigIIIα, which can substitute for LigI in DNA replication, were also more sensitive to L82-G17 than isogenic parental cells. Together, our results demonstrate that L82-G17 is a LigI-selective inhibitor with utility as a probe of the catalytic activity and cellular functions of LigI and provide a framework for the future design of DNA ligase inhibitors.
Assuntos
DNA Ligase Dependente de ATP/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Piridazinas/farmacologia , DNA Ligase Dependente de ATP/metabolismo , Replicação do DNA , Humanos , Cinética , Piridazinas/síntese química , Relação Estrutura-AtividadeAssuntos
Anestesia Local/métodos , Toracoscopia/métodos , Anestesia Local/efeitos adversos , Competência Clínica , Humanos , Derrame Pleural Maligno/diagnóstico , Derrame Pleural Maligno/terapia , Pleurodese/métodos , Pneumotórax/terapia , Toracoscopia/efeitos adversos , Tuberculose Pleural/diagnósticoRESUMO
During DNA replication, DNA joining events link Okazaki fragments on the lagging strand. In addition, they are required to repair DNA single- and double-strand breaks and to complete repair events initiated by the excision of mismatched and damaged bases. In human cells, there are three genes encoding DNA ligases. These enzymes are ATP-dependent and contain a conserved catalytic region. Biophysical studies have shown that the catalytic region contains three domains that, in the absence of DNA, are in an extended conformation. When the catalytic region engages a DNA nick, it adopts a compact, ring structure around the DNA nick with each of the three domains contacting the DNA. Protein-protein interactions involving the regions flanking the conserved catalytic regions of human DNA ligases play a major role in directing these enzymes to participate in specific DNA transactions. Among the human LIG genes, the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different N- and C-termini. One of these polypeptides is targeted to mitochondria where it plays an essential role in the maintenance of the mitochondrial genome. In the nucleus, DNA ligases I, III and IV have distinct but overlapping functions in DNA replication and repair. Small molecule inhibitors of human DNA ligases have been identified using structure-based approaches. As expected, these inhibitors are cytotoxic and also potentiate the cytotoxicity of DNA damaging agents. The results of preclinical studies with human cancer cell lines and mouse models of human cancer suggest that DNA ligase inhibitors may have utility as anti-cancer agents.
RESUMO
Estrogens are known to be important in the growth of breast cancers in both pre and postmenopausal women. As the number of breast cancer patients increases with age, the majority of breast cancer patients are postmenopausal women. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. As aromatase catalyzes the final and rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme are effective targeted therapy for breast cancer. Three aromatase inhibitors (AIs) are now FDA approved and have been shown to be more effective than the antiestrogen tamoxifen and are well tolerated. AIs are now a standard treatment for postmenopausal patients. AIs are effective in adjuvant and first-line metastatic setting. This review describes the development of AIs and their current use in breast cancer. Recent research focuses on elucidating mechanisms of acquired resistance that may develop in some patients with long term AI treatment and also in innate resistance. Preclinical data in resistance models demonstrated that the crosstalk between ER and other signaling pathways particularly MAPK and PI3K/Akt is an important resistant mechanism. Blockade of these other signaling pathways is an attractive strategy to circumvent the resistance to AI therapy in breast cancer. Several clinical trials are ongoing to evaluate the role of these novel targeted therapies to reverse resistance to AIs. Article from the special issue on 'Targeted Inhibitors'.
Assuntos
Inibidores da Aromatase/uso terapêutico , Aromatase/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/uso terapêutico , Inibidores da Aromatase/metabolismo , Estrogênios/metabolismo , Feminino , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais/fisiologia , TrastuzumabRESUMO
Aromatase inhibitors (AIs) have become the front-line choice for treatment of ER+ breast cancer. Nevertheless, although patients are responsive initially, they may acquire resistance and become unresponsive to further treatment. In addition, approximately 25% of breast cancers do not express the estrogen receptor (ERα) and consequently, are innately resistant to endocrine therapy. We have investigated the mechanisms associated with this lack of treatment response using xenograft models. We found that in cells and tumors that acquired resistance to the AI letrozole therapy, expression of the ER was reduced whereas growth factor signally was enhanced, including a marked increase in HER2 expression. Treatment with trastuzumab (HER2 antibody) resulted in a significant down-regulation of HER2 and p-MAPK as well as restoration of ERα expression. Thus, when trastuzumab was added to letrozole treatment at the time of tumor progression, there was significantly prolonged tumor suppression compared to trastuzumab or letrozole alone. This suggests that inhibition of both HER2 and ERα signaling pathways are required for overcoming resistance and restoring treatment sensitivity. ER negative tumors are innately resistant to endocrine therapy. Repression of the ERα has been found to be due to epigenetic modifications such as increased methylation and histone deacetylation. We found that entinostat (ENT), a histone deacetylase inhibitor (HDACi), activated not only expression of ERα but also aromatase in MDA-MB-231 ER-negative breast cancer cells, resulting in their ability to respond to estrogen and letrozole. Treatment with ENT in combination with letrozole significantly reduced tumor growth rate in xenografts compared to control tumors (p<0.001). ENT plus letrozole treatment also prevented the colonization and growth of MDA-MB-231 cells in the lung with a significant reduction (p<0.03) in both visible and microscopic foci. These results provide a strong indication for possible use of AIs in combination with HDAC inhibitors for the treatment of ER-negative breast cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Inibidores da Aromatase/uso terapêutico , Aromatase/metabolismo , Neoplasias da Mama/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Aromatase/genética , Benzamidas/uso terapêutico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Letrozol , Camundongos , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Nitrilas/uso terapêutico , Fosforilação , Piridinas/uso terapêutico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Trastuzumab , Triazóis/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
STUDY OBJECTIVES: To look for an association between the vertical position of the hyoid, as measured by the sella-hyoid (S-H) distance and the severity of obstructive sleep apnea (OSA). A previously published study on this subject showed a significant correlation between S-H distances more than 120 mm and severe OSA. DESIGN AND SETTING: A retrospective study of patients who were treated with a mandibular advancement splint (MAS) for sleep disordered breathing during June 2000 to May 2005 at the orthodontic department of Colchester University Hospital. PATIENTS AND PARTICIPANTS: One hundred and six consecutive patients who were treated with MAS during the period. MEASUREMENTS AND RESULTS: Lateral cephalograms were taken and traced prior to the provision of MAS therapy, and the distance S-H was measured. The mean S-H distance was 125.5 mm (103-148 mm) with a median of 126 mm. Four out of 8 patients (50%) with severe OSA had a S-H distance more than 120 mm, as did 38 out of 50 (76%) with mild to moderate OSA (AHI 5-30), and 17 out of 22 patients (77.27%) with an AHI of 0 to 4. No significant correlations between the S-H distance and the severity of OSA were found (Pearson correlation coefficient -0.034 and 95% confidence interval: -0.25, 0.18). CONCLUSION: There was no correlation between patients' OSA severity and their cephalometric S-H distance. The utility of the latter as a screening test cannot therefore be recommended as a substitute for existing diagnostic tests.