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Genetic linkage maps are used to localize markers on the genome based on the recombination frequency. Most often, these maps are based on the segregation observed within a single biparental population of limited size (n < 300) where relatively few recombination events are sampled and in which some genomic regions are monomorphic because both parents carry the same alleles. Together, these two limitations affect both the resolution and extent of genome coverage of such maps. Consensus genetic maps overcome the limitations of individual genetic maps by merging the information from multiple segregating populations derived from a greater diversity of parental combinations, thus increasing the number of recombination events and reducing the number of monomorphic regions. The aim of this study was to construct a high-density consensus genetic map for single nucleotide polymorphism (SNP) markers obtained through a genotyping-by-sequencing (GBS) approach. Individual genetic maps were generated from six F4:5 mapping populations (n = 278-365), totaling 1857 individuals. The six linkage maps were then merged to produce a consensus map comprising a total of 16 311 mapped SNPs that jointly cover 99.5% of the soybean genome with only two gaps larger than 10 cM. Compared to previous soybean consensus maps, it offers a more extensive and uniform coverage.
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Fabaceae , Genoma de Planta , Polimorfismo de Nucleotídeo Único , Alelos , Consenso , Fabaceae/genética , Ligação Genética , Genótipo , Glycine max/genéticaRESUMO
Background: The genesis of SMAC mimetic drugs is founded on the observation that many cancers amplify IAP proteins to facilitate their survival, and therefore removal of these pathways would re-sensitize the cells towards apoptosis. It has become increasingly clear that SMAC mimetics also interface with the immune system in a modulatory manner. Suppression of IAP function by SMAC mimetics activates the non-canonical NF-κB pathway which can augment T cell function, opening the possibility of using SMAC mimetics to enhance immunotherapeutics. Methods: We have investigated the SMAC mimetic LCL161, which promotes degradation of cIAP-1 and cIAP-2, as an agent for delivering transient costimulation to engineered BMCA-specific human TAC T cells. In doing so we also sought to understand the cellular and molecular effects of LCL161 on T cell biology. Results: LCL161 activated the non-canonical NF-κB pathway and enhanced antigen-driven TAC T cell proliferation and survival. Transcriptional profiling from TAC T cells treated with LCL161 revealed differential expression of costimulatory and apoptosis-related proteins, namely CD30 and FAIM3. We hypothesized that regulation of these genes by LCL161 may influence the drug's effects on T cells. We reversed the differential expression through genetic engineering and observed impaired costimulation by LCL161, particularly when CD30 was deleted. While LCL161 can provide a costimulatory signal to TAC T cells following exposure to isolated antigen, we did not observe a similar pattern when TAC T cells were stimulated with myeloma cells expressing the target antigen. We questioned whether FasL expression by myeloma cells may antagonize the costimulatory effects of LCL161. Fas-KO TAC T cells displayed superior expansion following antigen stimulation in the presence of LCL161, suggesting a role for Fas-related T cell death in limiting the magnitude of the T cell response to antigen in the presence of LCL161. Conclusions: Our results demonstrate that LCL161 provides costimulation to TAC T cells exposed to antigen alone, however LCL161 did not enhance TAC T cell anti-tumor function when challenged with myeloma cells and may be limited due to sensitization of T cells towards Fas-mediated apoptosis.
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Mieloma Múltiplo , NF-kappa B , Humanos , NF-kappa B/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Linhagem Celular Tumoral , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismoRESUMO
The SoyaGen project was a collaborative endeavor involving Canadian soybean researchers and breeders from academia and the private sector as well as international collaborators. Its aims were to develop genomics-derived solutions to real-world challenges faced by breeders. Based on the needs expressed by the stakeholders, the research efforts were focused on maximizing realized yield through optimization of maturity and improved disease resistance. The main deliverables related to molecular breeding in soybean will be reviewed here. These include: (1) SNP datasets capturing the genetic diversity within cultivated soybean (both within a worldwide collection of > 1,000 soybean accessions and a subset of 102 short-season accessions (MG0 and earlier) directly relevant to this group); (2) SNP markers for selecting favorable alleles at key maturity genes as well as loci associated with increased resistance to key pathogens and pests (Phytophthora sojae, Heterodera glycines, Sclerotinia sclerotiorum); (3) diagnostic tools to facilitate the identification and mapping of specific pathotypes of P. sojae; and (4) a genomic prediction approach to identify the most promising combinations of parents. As a result of this fruitful collaboration, breeders have gained new tools and approaches to implement molecular, genomics-informed breeding strategies. We believe these tools and approaches are broadly applicable to soybean breeding efforts around the world.
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Smac mimetic compounds (SMCs) potentiate TNFα-mediated cancer cell death by targeting the inhibitor of apoptosis (IAP) proteins. In addition to TNFα, the tumor microenvironment is exposed to a number of pro-inflammatory cytokines, including IL-1ß. Here, we investigated the potential impact of IL-1ß on SMC-mediated death of cancer cells. Synergy was seen in a subset of a diverse panel of 21 cancer cell lines to the combination of SMC and IL-1ß treatment, which required IL-1ß-induced activation of the NF-κB pathway. Elevated NF-κB activity resulted in the production of TNFα, which led to apoptosis dependent on caspase-8 and RIP1. In addition, concurrent silencing of cIAP1, cIAP2, and X-linked IAP by siRNA was most effective for triggering IL-1ß-mediated cell death. Importantly, SMC-resistant cells that produced TNFα in response to IL-1ß treatment were converted to an SMC-sensitive phenotype by c-FLIP knockdown. Reciprocally, ectopic expression of c-FLIP blocked cell death caused by combined SMC and IL-1ß treatment in sensitive cancer cells. Together, our study indicates that a positive feed-forward loop by pro-inflammatory cytokines can be exploited by SMCs to induce apoptosis in cancer cells.
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Alcinos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Dipeptídeos/farmacologia , Interleucina-1beta/farmacologia , Neoplasias/tratamento farmacológico , Peptidomiméticos/farmacologia , Alcinos/agonistas , Animais , Antineoplásicos/agonistas , Proteína 3 com Repetições IAP de Baculovírus , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Dipeptídeos/agonistas , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Interleucina-1beta/agonistas , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Peptidomiméticos/agonistas , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Grain size is a key agronomic trait that contributes to grain yield in hexaploid wheat. Grain length and width were evaluated in an international collection of 157 wheat accessions. These accessions were genetically characterized using a genotyping-by-sequencing (GBS) protocol that produced 73,784 single nucleotide polymorphism (SNP) markers. GBS-derived genotype calls obtained on Chinese Spring proved extremely accurate when compared to the reference (> 99.9%) and showed > 95% agreement with calls made at SNP loci shared with the 90 K SNP array on a subset of 71 Canadian wheat accessions for which both types of data were available. This indicates that GBS can yield a large amount of highly accurate SNP data in hexaploid wheat. The genetic diversity analysis performed using this set of SNP markers revealed the presence of six distinct groups within this collection. A GWAS was conducted to uncover genomic regions controlling variation for grain length and width. In total, seven SNPs were found to be associated with one or both traits, identifying three quantitative trait loci (QTLs) located on chromosomes 1D, 2D and 4A. In the vicinity of the peak SNP on chromosome 2D, we found a promising candidate gene (TraesCS2D01G331100), whose rice ortholog (D11) had previously been reported to be involved in the regulation of grain size. These markers will be useful in breeding for enhanced wheat productivity.
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Genes de Plantas , Estudo de Associação Genômica Ampla , Oryza/genética , Característica Quantitativa Herdável , Mapeamento Cromossômico , Grão Comestível/genética , Genética Populacional , Genoma de Planta , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Genótipo , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Polyploidy is known to be common in plants and recent work has focused on the rapid changes in genome structure and expression that occur upon polyploidization. In Arabidopsis, much of this work has been done on a synthetic allotetraploid obtained by crossing a tetraploid Arabidopsis thaliana (2n = 4x = 20) with A. arenosa (2n = 4x = 32). To explore an alternative route to polyploidy in this model species, we have developed a synthetic allopolyploid by crossing two diploid species: A. thaliana (2n = 2x = 10) and Arabidopsis lyrata subsp. petraea (2n = 2x = 16). F(1) hybrids were easy to obtain and phenotypically more similar to A. lyrata. Spontaneous chromosome doubling events occurred in about 25% of the F(1)s, thus restoring fertility. The resulting allotetraploids (2n = 26) exhibited many genomic changes typically reported upon polyploidization. Nucleolar dominance was observed as only the A. lyrata rDNA loci were expressed in the F(1) and allotetraploids. Changes in the degree of methylation were observed at almost 25% of the loci examined by MSAP analysis. Finally, structural genomic alterations did occur as a large deletion covering a significant portion of the upper arm of chromosome II was detected but no evidence of increased mobility of transposons was obtained. Such allotetraploids derived from two parents with sequenced (or soon to be sequenced) genomes offer much promise in elucidating the various changes that occur in newly synthesized polyploids.
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Arabidopsis/genética , Modelos Genéticos , Poliploidia , Arabidopsis/classificação , Sequência de Bases , Metilação de DNA , Primers do DNA/genética , Elementos de DNA Transponíveis , DNA de Plantas/genética , Inativação Gênica , Genes de Plantas , Hibridização Genética , RNA de Plantas/genética , RNA Ribossômico/genética , Especificidade da EspécieRESUMO
BACKGROUND: Skeletal muscle atrophy is a pathological condition that contributes to morbidity in a variety of conditions including denervation, cachexia, and aging. Muscle atrophy is characterized as decreased muscle fiber cross-sectional area and protein content due, in part, to the proteolytic activities of two muscle-specific E3 ubiquitin ligases: muscle RING-finger 1 (MuRF1) and muscle atrophy F-box (MAFbx or Atrogin-1). The nuclear factor-kappa B (NF-κB) pathway has emerged as a critical signaling network in skeletal muscle atrophy and has become a prime therapeutic target for the treatment of muscle diseases. Unfortunately, none of the NF-κB targeting drugs are currently being used to treat these diseases, likely because of our limited knowledge and specificity, for muscle biology and disease. The cellular inhibitor of apoptosis 1 (cIAP1) protein is a positive regulator of tumor necrosis factor alpha (TNFα)-mediated classical NF-κB signaling, and cIAP1 loss has been shown to enhance muscle regeneration during acute and chronic injury. METHODS: Sciatic nerve transection in wild-type, cIAP1-null and Smac mimetic compound (SMC)-treated mice was performed to investigate the role of cIAP1 in denervation-induced atrophy. Genetic in vitro models of C2C12 myoblasts and primary myoblasts were also used to examine the role of classical NF-κB activity in cIAP1-induced myotube atrophy. RESULTS: We found that cIAP1 expression was upregulated in denervated muscles compared to non-denervated controls 14 days after denervation. Genetic and pharmacological loss of cIAP1 attenuated denervation-induced muscle atrophy and overexpression of cIAP1 in myotubes was sufficient to induce atrophy. The induction of myotube atrophy by cIAP1 was attenuated when the classical NF-κB signaling pathway was inhibited. CONCLUSIONS: These results demonstrate the cIAP1 is an important mediator of NF-κB/MuRF1 signaling in skeletal muscle atrophy and is a promising therapeutic target for muscle wasting diseases.
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Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Denervação Muscular/efeitos adversos , Atrofia Muscular/etiologia , Animais , Proteínas Reguladoras de Apoptose/farmacologia , Linhagem Celular , Feminino , Marcação de Genes , Humanos , Proteínas Inibidoras de Apoptose/deficiência , Proteínas Inibidoras de Apoptose/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/farmacologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Mioblastos Esqueléticos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Tiazóis/farmacologia , Regulação para CimaRESUMO
The controlled production and downstream signaling of the inflammatory cytokine tumor necrosis factor-α (TNF-α) are important for immunity and its anticancer effects. Although chronic stimulation with TNF-α is detrimental to the health of the host in several autoimmune and inflammatory disorders, TNF-α-contrary to what its name implies-leads to cancer formation by promoting cell proliferation and survival. Smac mimetic compounds (SMCs), small-molecule antagonists of inhibitor of apoptosis proteins (IAPs), switch the TNF-α signal from promoting survival to promoting death in cancer cells. Using a genome-wide siRNA screen to identify factors required for SMC-to-TNF-α-mediated cancer cell death, we identified the transcription factor SP3 as a critical molecule in both basal and SMC-induced production of TNF-α by engaging the nuclear factor κB (NF-κB) transcriptional pathway. Moreover, the promotion of TNF-α expression by SP3 activity confers differential sensitivity of cancer versus normal cells to SMC treatment. The key role of SP3 in TNF-α production and signaling will help us further understand TNF-α biology and provide insight into mechanisms relevant to cancer and inflammatory disease.
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Materiais Biomiméticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição Sp3/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Proteínas Mitocondriais/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias/genética , Neoplasias/patologia , Interferência de RNA , Transdução de Sinais/genética , Fator de Transcrição Sp3/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Smac mimetic compounds (SMCs) are anti-cancer drugs that antagonize Inhibitor of Apoptosis proteins, which consequently sensitize cancer cells to death in the presence of proinflammatory ligands such as tumor necrosis factor alpha (TNF-α). SMCs synergize with the attenuated oncolytic vesicular stomatitis virus (VSVΔ51) by eliciting an innate immune response, which is dependent on the endogenous production of TNF-α and type I interferon. To improve on this SMC-mediated synergistic response, we generated TNF-α-armed VSVΔ51 to produce elevated levels of this death ligand. Due to ectopic expression of TNF-α from infected cells, a lower viral dose of TNF-α-armed VSVΔ51 combined with treatment of the SMC LCL161 was sufficient to improve the survival rate compared to LCL161 and unarmed VSVΔ51 co-therapy. This improved response is attributed to a bystander effect whereby the spread of TNF-α from infected cells leads to the death of uninfected cells in the presence of LCL161. In addition, the treatments induced vascular collapse in solid tumors with a concomitant increase of tumor cell death, revealing another mechanism by which cytokine-armed VSVΔ51 in combination with LCL161 can kill tumor cells. Our studies demonstrate the potential for cytokine-engineered oncolytic virus and SMCs as a new combination immunotherapy for cancer treatment.
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This corrects the article DOI: 10.1038/ncomms14278.
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Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells.
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Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Interferon-alfa/imunologia , Interferon beta/imunologia , Tiazóis/farmacologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Antineoplásicos/síntese química , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/imunologia , Glioblastoma/genética , Glioblastoma/imunologia , Glioblastoma/mortalidade , Humanos , Imunidade Inata/efeitos dos fármacos , Memória Imunológica , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/imunologia , Interferon-alfa/genética , Interferon-alfa/farmacologia , Interferon beta/genética , Interferon beta/farmacologia , Camundongos , Poli I-C/farmacologia , Transdução de Sinais , Análise de Sobrevida , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Tiazóis/síntese química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Vesiculovirus/genética , Vesiculovirus/imunologia , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Educação Continuada em Enfermagem/organização & administração , Capacitação em Serviço/organização & administração , Nefrologia/educação , Comportamento Cooperativo , Currículo , França , Humanos , Comunicação Interdisciplinar , Falência Renal Crônica/enfermagem , Equipe de Assistência ao Paciente/organização & administraçãoRESUMO
Estimation of allelic frequencies is often required in breeding but genotyping many individuals at many loci can be expensive. We have developed a genotyping-by-sequencing (GBS) approach for estimating allelic frequencies on pooled samples (Pool-GBS) and used it to examine segregation distortion in doubled haploid (DH) populations of barley ( L.). In the first phase, we genotyped each line individually and exploited these data to explore a strategy to call single nucleotide polymorphisms (SNPs) on pooled reads. We measured both the number of SNPs called and the variance of the estimated allelic frequencies at various depths of coverage on a subset of reads containing 5 to 25 million reads. We show that allelic frequencies could be cost-effectively and accurately estimated at a depth of 50 reads per SNP using 15 million reads. This Pool-GBS approach yielded 1984 SNPs whose allelic frequency estimates were highly reproducible (CV = 10.4%) and correlated ( = 0.9167) with the "true" frequency derived from analysis of individual lines. In a second phase, we used Pool-GBS to investigate segregation bias throughout androgenesis from microspores to a population of regenerated plants. No strong bias was detected among the microspores resulting from the meiotic divisions, whereas significant biases could be shown to arise during embryo formation and plant regeneration. In summary, this methodology provides an approach to estimate allelic frequencies more efficiently and on materials that are unsuitable for individual analysis. In addition, it allowed us to shed light on the process of androgenesis in barley.
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Técnicas de Genotipagem , Hordeum/genética , Melhoramento Vegetal/métodos , Frequência do Gene , Genótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Stable expression of short-hairpin RNAs (shRNAs) directed against the X-linked inhibitor of apoptosis (XIAP) resulted in the generation of three MDA-MB-231 cell lines (XIAP shRNA cells) with reductions in XIAP mRNA and protein levels > 85% relative to MDA-MB-231 cells stably transfected with the U6 RNA polymerase III promoter alone (U6 cells). This RNA interference (RNAi) approach dramatically sensitized these cells to killing by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, loss of XIAP also sensitized the cells to killing by taxanes but had no additional effects on killing by carboplatin and doxorubicin. The increased sensitivity of the XIAP shRNA cells to killing by TRAIL and taxanes correlated with enhanced caspase cleavage and activation, including caspase-8, and robust processing of poly(ADP-ribose) polymerase and BID compared to U6 cells. Additionally, increasing XIAP levels by adenovirus-mediated expression protected both XIAP shRNA and U6 cells from TRAIL killing in a dose-dependent manner. The effects observed by stable RNAi with respect to TRAIL sensitization were also achieved following downregulation of XIAP in Panc-1 cells treated with a second-generation, mixed-backbone antisense oligonucleotide, AEG 35156/GEM640. These data indicate that reducing XIAP protein expression by either RNAi or antisense approaches increases cancer cell susceptibility to functionally diverse chemotherapeutic agents and supports the notion that downregulation of XIAP in vivo may synergize with disease-relevant chemotherapeutic regimes, including TRAIL and taxanes, to increase the effectiveness of antineoplastic agents.
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Elementos Antissenso (Genética)/farmacologia , Neoplasias/tratamento farmacológico , Proteínas/antagonistas & inibidores , Interferência de RNA , Proteínas Reguladoras de Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Interferon beta/farmacologia , Glicoproteínas de Membrana/farmacologia , Proteínas/genética , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids.
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Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Medicago sativa/genética , Tetraploidia , Alelos , Sequência de Bases , Mapeamento Cromossômico/métodos , Análise Mutacional de DNA/métodos , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Medicago sativa/classificação , Medicago truncatula/genética , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Homologia de Sequência do Ácido NucleicoRESUMO
Cyst nematodes are important agricultural pests responsible for billions of dollars of losses each year. Plant resistance is the most effective management tool, but it requires a close monitoring of population genetics. Current technologies for pathotyping and genotyping cyst nematodes are time-consuming, expensive and imprecise. In this study, we capitalized on the reproduction mode of cyst nematodes to develop a simple population genetic analysis pipeline based on genotyping-by-sequencing and Pool-Seq. This method yielded thousands of SNPs and allowed us to study the relationships between populations of different origins or pathotypes. Validation of the method on well-characterized populations also demonstrated that it was a powerful and accurate tool for population genetics. The genomewide allele frequencies of 23 populations of golden nematode, from nine countries and representing the five known pathotypes, were compared. A clear separation of the pathotypes and fine genetic relationships between and among global populations were obtained using this method. In addition to being powerful, this tool has proven to be very time- and cost-efficient and could be applied to other cyst nematode species.
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Frequência do Gene , Genética Populacional/métodos , Técnicas de Genotipagem/métodos , Nematoides/classificação , Nematoides/genética , Animais , Produtos Agrícolas/parasitologia , Nematoides/isolamento & purificação , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Smac mimetic compounds (SMC), a class of drugs that sensitize cells to apoptosis by counteracting the activity of inhibitor of apoptosis (IAP) proteins, have proven safe in phase 1 clinical trials in cancer patients. However, because SMCs act by enabling transduction of pro-apoptotic signals, SMC monotherapy may be efficacious only in the subset of patients whose tumors produce large quantities of death-inducing proteins such as inflammatory cytokines. Therefore, we reasoned that SMCs would synergize with agents that stimulate a potent yet safe "cytokine storm." Here we show that oncolytic viruses and adjuvants such as poly(I:C) and CpG induce bystander death of cancer cells treated with SMCs that is mediated by interferon beta (IFN-ß), tumor necrosis factor alpha (TNF-α) and/or TNF-related apoptosis-inducing ligand (TRAIL). This combinatorial treatment resulted in tumor regression and extended survival in two mouse models of cancer. As these and other adjuvants have been proven safe in clinical trials, it may be worthwhile to explore their clinical efficacy in combination with SMCs.
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Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Morte Celular/efeitos dos fármacos , Neoplasias Experimentais/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose/uso terapêutico , Citocinas/metabolismo , Sinergismo Farmacológico , Feminino , Células HEK293 , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/farmacologia , Oligodesoxirribonucleotídeos/uso terapêutico , Terapia Viral Oncolítica , Poli I-C/farmacologia , Poli I-C/uso terapêuticoRESUMO
Highly parallel SNP genotyping platforms have been developed for some important crop species, but these platforms typically carry a high cost per sample for first-time or small-scale users. In contrast, recently developed genotyping by sequencing (GBS) approaches offer a highly cost effective alternative for simultaneous SNP discovery and genotyping. In the present investigation, we have explored the use of GBS in soybean. In addition to developing a novel analysis pipeline to call SNPs and indels from the resulting sequence reads, we have devised a modified library preparation protocol to alter the degree of complexity reduction. We used a set of eight diverse soybean genotypes to conduct a pilot scale test of the protocol and pipeline. Using ApeKI for GBS library preparation and sequencing on an Illumina GAIIx machine, we obtained 5.5 M reads and these were processed using our pipeline. A total of 10,120 high quality SNPs were obtained and the distribution of these SNPs mirrored closely the distribution of gene-rich regions in the soybean genome. A total of 39.5% of the SNPs were present in genic regions and 52.5% of these were located in the coding sequence. Validation of over 400 genotypes at a set of randomly selected SNPs using Sanger sequencing showed a 98% success rate. We then explored the use of selective primers to achieve a greater complexity reduction during GBS library preparation. The number of SNP calls could be increased by almost 40% and their depth of coverage was more than doubled, thus opening the door to an increase in the throughput and a significant decrease in the per sample cost. The approach to obtain high quality SNPs developed here will be helpful for marker assisted genomics as well as assessment of available genetic resources for effective utilisation in a wide number of species.
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Técnicas de Genotipagem/métodos , Técnicas de Genotipagem/normas , Sequenciamento de Nucleotídeos em Larga Escala , Polimorfismo de Nucleotídeo Único , Mapeamento Cromossômico , Evolução Molecular , Genoma de Planta , Genômica , Genótipo , Filogenia , Reprodutibilidade dos Testes , Glycine max/classificação , Glycine max/genéticaRESUMO
The eukaryotic DNA mismatch repair (MMR) system contributes to maintaining the fidelity of genetic information by correcting replication errors and preventing illegitimate recombination events. This study aimed to examine the function(s) of the Arabidopsis thaliana PMS1 gene (AtPMS1), one of three homologs of the bacterial MutL gene in plants. Two independent mutant alleles (Atpms1-1 and Atpms1-2) were obtained and one of these (Atpms1-1) was studied in detail. The mutant exhibited a reduction in seed set and a bias against the transmission of the mutant allele. Somatic recombination, both homologous and homeologous, was examined using a set of reporter constructs. Homologous recombination remained unchanged in the mutant while homeologous recombination was between 1.7- and 4.8-fold higher than in the wild type. This increase in homeologous recombination frequency was not correlated with the degree of sequence divergence. In RNAi lines, a range of increases in homeologous recombination were observed with two lines showing a 3.3-fold and a 3.6-fold increase. These results indicate that the AtPMS1 gene contributes to an antirecombination activity aimed at restricting recombination between diverged sequences.