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1.
Genome Biol ; 21(1): 177, 2020 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-32684159

RESUMO

BACKGROUND: Heteromorphic sex chromosomes have evolved repeatedly across diverse species. Suppression of recombination between X and Y chromosomes leads to degeneration of the Y chromosome. The progression of degeneration is not well understood, as complete sequence assemblies of heteromorphic Y chromosomes have only been generated across a handful of taxa with highly degenerate sex chromosomes. Here, we describe the assembly of the threespine stickleback (Gasterosteus aculeatus) Y chromosome, which is less than 26 million years old and at an intermediate stage of degeneration. Our previous work identified that the non-recombining region between the X and the Y spans approximately 17.5 Mb on the X chromosome. RESULTS: We combine long-read sequencing with a Hi-C-based proximity guided assembly to generate a 15.87 Mb assembly of the Y chromosome. Our assembly is concordant with cytogenetic maps and Sanger sequences of over 90 Y chromosome BAC clones. We find three evolutionary strata on the Y chromosome, consistent with the three inversions identified by our previous cytogenetic analyses. The threespine stickleback Y shows convergence with more degenerate sex chromosomes in the retention of haploinsufficient genes and the accumulation of genes with testis-biased expression, many of which are recent duplicates. However, we find no evidence for large amplicons identified in other sex chromosome systems. We also report an excellent candidate for the master sex-determination gene: a translocated copy of Amh (Amhy). CONCLUSIONS: Together, our work shows that the evolutionary forces shaping sex chromosomes can cause relatively rapid changes in the overall genetic architecture of Y chromosomes.

2.
Neoplasia ; 22(8): 294-310, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32512502

RESUMO

Using a mini-library of 1062 lentiviral shRNAs targeting 40 nuclear hormone receptors and 70 of their co-regulators, we searched for potential therapeutic targets that would be important during in vivo tumor growth using a parallel in vitro and in vivo shRNA screening strategy in the non-small cell lung cancer (NSCLC) line NCI-H1819. We identified 21 genes essential for in vitro growth, and nine genes specifically required for tumor survival in vivo, but not in vitro: NCOR2, FOXA1, HDAC1, RXRA, RORB, RARB, MTA2, ETV4, and NR1H2. We focused on FOXA1, since it lies within the most frequently amplified genomic region in lung adenocarcinomas. We found that 14q-amplification in NSCLC cell lines was a biomarker for FOXA1 dependency for both in vivo xenograft growth and colony formation, but not mass culture growth in vitro. FOXA1 knockdown identified genes involved in electron transport among the most differentially regulated, indicating FOXA1 loss may lead to a decrease in cellular respiration. In support of this, FOXA1 amplification was correlated with increased sensitivity to the complex I inhibitor phenformin. Integrative ChipSeq analyses reveal that FOXA1 functions in this genetic context may be at least partially independent of NKX2-1. Our findings are consistent with a neomorphic function for amplified FOXA1, driving an oncogenic transcriptional program. These data provide new insight into the functional consequences of FOXA1 amplification in lung adenocarcinomas, and identify new transcriptional networks for exploration of therapeutic vulnerabilities in this patient population.

3.
Mol Biol Evol ; 37(6): 1547-1562, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32076722

RESUMO

The Dobzhansky-Muller (DM) model provides a widely accepted mechanism for the evolution of reproductive isolation: incompatible substitutions disrupt interactions between genes. To date, few candidate incompatibility genes have been identified, leaving the genes driving speciation mostly uncharacterized. The importance of interactions in the DM model suggests that gene coexpression networks provide a powerful framework to understand disrupted pathways associated with postzygotic isolation. Here, we perform weighted gene coexpression network analysis to infer gene interactions in hybrids of two recently diverged European house mouse subspecies, Mus mus domesticus and M. m. musculus, which commonly show hybrid male sterility or subfertility. We use genome-wide testis expression data from 467 hybrid mice from two mapping populations: F2s from a laboratory cross between wild-derived pure subspecies strains and offspring of natural hybrids captured in the Central Europe hybrid zone. This large data set enabled us to build a robust consensus network using hybrid males with fertile phenotypes. We identify several expression modules, or groups of coexpressed genes, that are disrupted in subfertile hybrids, including modules functionally enriched for spermatogenesis, cilium and sperm flagellum organization, chromosome organization, and DNA repair, and including genes expressed in spermatogonia, spermatocytes, and spermatids. Our network-based approach enabled us to hone in on specific hub genes likely to be influencing module-wide gene expression and hence potentially driving large-effect DM incompatibilities. A disproportionate number of hub genes lie within sterility loci identified previously in the hybrid zone mapping population and represent promising candidate barrier genes and targets for future functional analysis.

4.
Sci Data ; 7(1): 27, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964871

RESUMO

A gene expression-based siRNA screen was used to evaluate functional similarity between genetic perturbations to identify functionally similar proteins. A siRNA library (siGenome library, Dharmacon) consisting of multiple siRNAs per gene that have been pooled in to one well per gene was arrayed in a 384-well format and used to individually target 14,335 proteins for depletion in HCT116 colon cancer cells. For each protein depletion, the gene expression of eight genes was quantified using the multiplexed Affymetrix Quantigene 2.0 assay in technical triplicate. As a proof of concept, six genes (BNIP3, NDRG1, ALDOC, LOXL2, ACSL5, BNIP3L) whose expression pattern reliably reflect the disruption of the molecular scaffold KSR1 were measured upon each protein depletion. The remaining two genes (PPIB and HPRT) are housekeeping genes used for normalization. The gene expression signatures from this screen can be used to estimate the functional similarity between any two proteins and successfully identified functional relationships for specific proteins such as KSR1 and more generalized processes, such as autophagy.

5.
Mol Cell ; 76(5): 838-851.e5, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31564558

RESUMO

Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes, including signal transduction and gene expression patterns, arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here, we quantified metabolic features, mostly from the 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression, and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral/metabolismo , Metaboloma/fisiologia , Biomarcadores Tumorais/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Regulação Neoplásica da Expressão Gênica/fisiologia , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Redes e Vias Metabólicas/genética , Metabolômica/métodos , Neoplasias/metabolismo
6.
Cell Chem Biol ; 26(10): 1380-1392.e6, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31378711

RESUMO

Gene expression signature-based inference of functional connectivity within and between genetic perturbations, chemical perturbations, and disease status can lead to the development of actionable hypotheses for gene function, chemical modes of action, and disease treatment strategies. Here, we report a FuSiOn-based genome-wide integration of hypomorphic cellular phenotypes that enables functional annotation of gene network topology, assignment of mechanistic hypotheses to genes of unknown function, and detection of cooperativity among cell regulatory systems. Dovetailing genetic perturbation data with chemical perturbation phenotypes allowed simultaneous generation of mechanism of action hypotheses for thousands of uncharacterized natural products fractions (NPFs). The predicted mechanism of actions span a broad spectrum of cellular mechanisms, many of which are not currently recognized as "druggable." To enable use of FuSiOn as a hypothesis generation resource, all associations and analyses are available within an open source web-based GUI (http://fusion.yuhs.ac).

8.
J Bacteriol ; 201(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31182499

RESUMO

Biofilms occur in a broad range of environments under heterogeneous physicochemical conditions, such as in bioremediation plants, on surfaces of biomedical implants, and in the lungs of cystic fibrosis patients. In these scenarios, biofilms are subjected to shear forces, but the mechanical integrity of these aggregates often prevents their disruption or dispersal. Biofilms' physical robustness is the result of the multiple biopolymers secreted by constituent microbial cells which are also responsible for numerous biological functions. A better understanding of the role of these biopolymers and their response to dynamic forces is therefore crucial for understanding the interplay between biofilm structure and function. In this paper, we review experimental techniques in rheology, which help quantify the viscoelasticity of biofilms, and modeling approaches from soft matter physics that can assist our understanding of the rheological properties. We describe how these methods could be combined with synthetic biology approaches to control and investigate the effects of secreted polymers on the physical properties of biofilms. We argue that without an integrated approach of the three disciplines, the links between genetics, composition, and interaction of matrix biopolymers and the viscoelastic properties of biofilms will be much harder to uncover.

9.
Curr Opin Psychol ; 27: 93-97, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30933894

RESUMO

Virtually all human psychological and behavioral traits are at least partially heritable. For nearly a century, classical genetic studies have sought to understand how genetic variation contributes to human variation in these traits. More recently, genome-wide association studies have identified large numbers of specific genetic variants linked with complex traits. Many of these variants fall outside of protein-coding genes, in putative gene regulatory elements. This suggests that some fraction of causal human genetic variation acts through gene regulation. New developments in the field of regulatory genomics offer resources and methods to understand how genetic variants that alter gene expression contribute to human psychology and risk for psychiatric disease.

10.
Genome Biol Evol ; 11(6): 1573-1585, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31028697

RESUMO

Meiotic recombination is a highly conserved process that has profound effects on genome evolution. At a fine-scale, recombination rates can vary drastically across genomes, often localized into small recombination "hotspots" with highly elevated rates, surrounded by regions with little recombination. In most species studied, the location of hotspots within genomes is highly conserved across broad evolutionary timescales. The main exception to this pattern is in mammals, where hotspot location can evolve rapidly among closely related species and even among populations within a species. Hotspot position in mammals is controlled by the gene, Prdm9, whereas in species with conserved hotspots, a functional Prdm9 is typically absent. Due to a limited number of species where recombination rates have been estimated at a fine-scale, it remains unclear whether hotspot conservation is always associated with the absence of a functional Prdm9. Threespine stickleback fish (Gasterosteus aculeatus) are an excellent model to examine the evolution of recombination over short evolutionary timescales. Using a linkage disequilibrium-based approach, we found recombination rates indeed varied at a fine-scale across the genome, with many regions organized into narrow hotspots. Hotspots had highly divergent landscapes between stickleback populations, where only ∼15% of these hotspots were shared. Our results indicate that fine-scale recombination rates may be diverging between closely related populations of threespine stickleback fish. Interestingly, we found only a weak association of a PRDM9 binding motif within hotspots, which suggests that threespine stickleback fish may possess a novel mechanism for targeting recombination hotspots at a fine-scale.


Assuntos
Recombinação Genética , Smegmamorpha/genética , Animais , Feminino , Água Doce , Variação Genética , Genética Populacional , Masculino , Meiose , Água do Mar , Smegmamorpha/classificação , Sítio de Iniciação de Transcrição , Washington , Cromossomo X , Cromossomo Y
11.
Genetics ; 210(4): 1453-1465, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30333190

RESUMO

Hybrid sterility is a common form of reproductive isolation between nascent species. Although hybrid sterility is routinely documented and genetically dissected in speciation studies, its developmental basis is rarely examined, especially in generations beyond the F1 generation. To identify phenotypic and genetic determinants of hybrid male sterility from a developmental perspective, we characterized testis histology in 312 F2 hybrids generated by intercrossing inbred strains of Mus musculus domesticus and M. m. musculus, two subspecies of house mice. Hybrids display a range of histologic abnormalities that indicate defective spermatogenesis. Among these abnormalities, we quantified decreased testis size, reductions in spermatocyte and spermatid number, increased apoptosis of meiosis I spermatocytes, and more multinucleated syncytia. Collectively, our phenotypic data point to defects in meiosis I as a primary barrier to reproduction. We identified seven quantitative trait loci (QTL) controlling five histologic traits. A region of chromosome 17 that contains Prdm9, a gene known to confer F1 hybrid male sterility, affects multinucleated syncytia and round spermatids, potentially extending the phenotypic outcomes of this incompatibility. The X chromosome also plays a key role, with loci affecting multinucleated syncytia, apoptosis of round spermatids, and round spermatid numbers. We detected an epistatic interaction between QTL on chromosomes 17 and X for multinucleated syncytia. Our results refine the developmental basis of a key reproductive barrier in a classic model system for speciation genetics.


Assuntos
Hibridização Genética , Infertilidade Masculina/genética , Meiose/genética , Espermatogênese/genética , Animais , Mapeamento Cromossômico , Epistasia Genética , Especiação Genética , Humanos , Infertilidade Masculina/patologia , Masculino , Camundongos , Locos de Características Quantitativas/genética , Reprodução/genética , Espermatócitos/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Cromossomo X/genética
13.
Gastroenterology ; 155(3): 799-814.e13, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29775598

RESUMO

BACKGROUND & AIMS: Markers of the epithelial-to-mesenchymal transition (EMT) in gastric tumor tissues are associated with poor patient outcomes. We performed a screen to identify pharmacologic compounds that kill gastric cancer cells with EMT-associated gene expression patterns and investigate their mechanisms. METHODS: We identified 29 gastric cancer cell lines with a gene expression signature previously associated with an EMT subtype, based on data from RNA sequence analyses, and confirmed the mesenchymal phenotypes of 7 lines (Hs746T, SNU1750, MKN1, SK4, SNU484, SNU668, and YCC11), based on invasive activity and protein markers. We screened 1,345 compounds for their ability to kill cells with the EMT signature compared with cell lines without this pattern. We tested the effects of identified compounds in BALB/c nude mice bearing GA077 tumors; mice were given intraperitoneal injections of the compound or vehicle (control) twice daily for 24 days and tumor growth was monitored. Proteins associated with the toxicity of the compounds were overexpressed in MKN1 and SNU484 cells or knocked down in MKN45 and SNU719 using small interfering RNAs. We performed immunohistochemical analyses of 942 gastric cancer tissues and investigated associations between EMT markers and protein expression patterns. RESULTS: The nicotinamide phosphoribosyltransferase inhibitor FK866 killed 6 of 7 gastric cancer cell lines with EMT-associated gene expression signatures but not gastric cancer cells without this signature. The 6 EMT-subtype gastric cell lines expressed significantly low levels of nicotinic acid phosphoribosyltransferase (NAPRT), which makes the cells hypersensitive to nicotinamide phosphoribosyltransferase inhibition. Gastric cell lines that expressed higher levels of NAPRT, regardless of EMT markers, were sensitized to FK866 after knockdown of NAPRT, whereas overexpression of NAPRT in deficient EMT cell lines protected them from FK866-mediated toxicity. Administration of FK866 to nude mice with tumors grown from GA077 cells (human gastric cancer tumors of the EMT subtype) led to tumor regression in 2 weeks; FK866 did not affect tumors grown from MKN45 cells without the EMT expression signature. Loss of NAPRT might promote the EMT, because it stabilizes ß-catenin. We correlated the EMT gene expression signature with lower levels of NAPRT in 942 gastric tumors from patients; we also found lower levels of NAPRT mRNA in colorectal, pancreatic, and lung adenocarcinoma tissues with the EMT gene expression signature. CONCLUSIONS: FK866 selectively kills gastric cancer cells with an EMT gene expression signature by inhibiting nicotinamide phosphoribosyltransferase in cells with NAPRT deficiency. Loss of NAPRT expression, frequently through promoter hypermethylation, is observed in many gastric tumors of the EMT subtype. FK866 might be used to treat patients with tumors of this subtype.


Assuntos
Acrilamidas/farmacologia , Antineoplásicos/farmacologia , Citocinas/antagonistas & inibidores , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Piperidinas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Interferente Pequeno/administração & dosagem , Neoplasias Gástricas/genética
14.
Nat Commun ; 9(1): 2050, 2018 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-29784984

RESUMO

The originally published version of this Article contained an error in the spelling of the author Nathaniel W. Oswald, which was incorrectly given as Nathaniel W. Olswald. This has now been corrected in both the PDF and HTML versions of the Article.

15.
Cell ; 173(4): 864-878.e29, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29681454

RESUMO

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Família 4 do Citocromo P450/deficiência , Família 4 do Citocromo P450/genética , Descoberta de Drogas , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Glucocorticoides/farmacologia , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
16.
Sci Rep ; 8(1): 3770, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29491475

RESUMO

AMPK is a serine threonine kinase composed of a heterotrimer of a catalytic, kinase-containing α and regulatory ß and γ subunits. Here we show that individual AMPK subunit expression and requirement for survival varies across colon cancer cell lines. While AMPKα1 expression is relatively consistent across colon cancer cell lines, AMPKα1 depletion does not induce cell death. Conversely, AMPKα2 is expressed at variable levels in colon cancer cells. In high expressing SW480 and moderate expressing HCT116 colon cancer cells, siRNA-mediated depletion induces cell death. These data suggest that AMPK kinase inhibition may be a useful component of future therapeutic strategies. We used Functional Signature Ontology (FUSION) to screen a natural product library to identify compounds that were inhibitors of AMPK to test its potential for detecting small molecules with preferential toxicity toward human colon tumor cells. FUSION identified 5'-hydroxy-staurosporine, which competitively inhibits AMPK. Human colon cancer cell lines are notably more sensitive to 5'-hydroxy-staurosporine than are non-transformed human colon epithelial cells. This study serves as proof-of-concept for unbiased FUSION-based detection of small molecule inhibitors of therapeutic targets and highlights its potential to identify novel compounds for cancer therapy development.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Ontologias Biológicas , Neoplasias do Colo/patologia , Inibidores de Proteínas Quinases/farmacologia , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos
17.
Nat Commun ; 8(1): 2270, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29273768

RESUMO

Drugs that mirror the cellular effects of starvation mimics are considered promising therapeutics for common metabolic disorders, such as obesity, liver steatosis, and for ageing. Starvation, or caloric restriction, is known to activate the transcription factor EB (TFEB), a master regulator of lipid metabolism and lysosomal biogenesis and function. Here, we report a nanotechnology-enabled high-throughput screen to identify small-molecule agonists of TFEB and discover three novel compounds that promote autophagolysosomal activity. The three lead compounds include the clinically approved drug, digoxin; the marine-derived natural product, ikarugamycin; and the synthetic compound, alexidine dihydrochloride, which is known to act on a mitochondrial target. Mode of action studies reveal that these compounds activate TFEB via three distinct Ca2+-dependent mechanisms. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles confers hepatoprotection against diet-induced steatosis in murine models and extends lifespan of Caenorhabditis elegans. These results support the therapeutic potential of small-molecule TFEB activators for the treatment of metabolic and age-related disorders.


Assuntos
Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/agonistas , Biguanidas/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Digoxina/farmacologia , Inibidores Enzimáticos/farmacologia , Lactamas/farmacologia , Longevidade/efeitos dos fármacos , Síndrome Metabólica/metabolismo , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Restrição Calórica , Dieta Hiperlipídica , Fígado Gorduroso/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Inanição
18.
Environ Microbiol ; 19(10): 4326-4348, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28892295

RESUMO

Campylobacter jejuni, the most frequent cause of food-borne bacterial gastroenteritis worldwide, is a microaerophile that has to survive high environmental oxygen tensions, adapt to oxygen limitation in the intestine and resist host oxidative attack. Here, oxygen-dependent changes in C. jejuni physiology were studied at constant growth rate using carbon (serine)-limited continuous chemostat cultures. We show that a perceived aerobiosis scale can be calibrated by the acetate excretion flux, which becomes zero when metabolism is fully aerobic (100% aerobiosis). Transcriptome changes in a downshift experiment from 150% to 40% aerobiosis revealed many novel oxygen-regulated genes and highlighted re-modelling of the electron transport chains. A label-free proteomic analysis showed that at 40% aerobiosis, many proteins involved in host colonisation (e.g., PorA, CadF, FlpA, CjkT) became more abundant. PorA abundance increased steeply below 100% aerobiosis. In contrast, several citric-acid cycle enzymes, the peptide transporter CstA, PEB1 aspartate/glutamate transporter, LutABC lactate dehydrogenase and PutA proline dehydrogenase became more abundant with increasing aerobiosis. We also observed a co-ordinated response of oxidative stress protection enzymes and Fe-S cluster biogenesis proteins above 100% aerobiosis. Our approaches reveal key virulence factors that respond to restricted oxygen availability and specific transporters and catabolic pathways activated with increasing aerobiosis.


Assuntos
Aerobiose/fisiologia , Campylobacter jejuni/metabolismo , Campylobacter jejuni/patogenicidade , Estresse Oxidativo/fisiologia , Oxigênio/metabolismo , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/genética , Humanos , Oxirredução , Proteoma/metabolismo , Proteômica , Transcriptoma/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
19.
J Hered ; 108(6): 693-700, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28821183

RESUMO

Scaffolding genomes into complete chromosome assemblies remains challenging even with the rapidly increasing sequence coverage generated by current next-generation sequence technologies. Even with scaffolding information, many genome assemblies remain incomplete. The genome of the threespine stickleback (Gasterosteus aculeatus), a fish model system in evolutionary genetics and genomics, is not completely assembled despite scaffolding with high-density linkage maps. Here, we first test the ability of a Hi-C based proximity-guided assembly (PGA) to perform a de novo genome assembly from relatively short contigs. Using Hi-C based PGA, we generated complete chromosome assemblies from a distribution of short contigs (20-100 kb). We found that 96.40% of contigs were correctly assigned to linkage groups (LGs), with ordering nearly identical to the previous genome assembly. Using available bacterial artificial chromosome (BAC) end sequences, we provide evidence that some of the few discrepancies between the Hi-C assembly and the existing assembly are due to structural variation between the populations used for the 2 assemblies or errors in the existing assembly. This Hi-C assembly also allowed us to improve the existing assembly, assigning over 60% (13.35 Mb) of the previously unassigned (~21.7 Mb) contigs to LGs. Together, our results highlight the potential of the Hi-C based PGA method to be used in combination with short read data to perform relatively inexpensive de novo genome assemblies. This approach will be particularly useful in organisms in which it is difficult to perform linkage mapping or to obtain high molecular weight DNA required for other scaffolding methods.


Assuntos
Mapeamento Cromossômico/métodos , Smegmamorpha/genética , Alaska , Animais , Cromossomos Artificiais Bacterianos , Mapeamento de Sequências Contíguas , Genômica , Masculino , Análise de Sequência de DNA/métodos
20.
Cancer Res ; 77(18): 5077-5094, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28716898

RESUMO

Emerging observations link dysregulation of TANK-binding kinase 1 (TBK1) to developmental disorders, inflammatory disease, and cancer. Biochemical mechanisms accounting for direct participation of TBK1 in host defense signaling have been well described. However, the molecular underpinnings of the selective participation of TBK1 in a myriad of additional cell biological systems in normal and pathophysiologic contexts remain poorly understood. To elucidate the context-selective role of TBK1 in cancer cell survival, we employed a combination of broad-scale chemogenomic and interactome discovery strategies to generate data-driven mechanism-of-action hypotheses. This approach uncovered evidence that TBK1 supports AKT/mTORC1 pathway activation and function through direct modulation of multiple pathway components acting both upstream and downstream of the mTOR kinase itself. Furthermore, we identified distinct molecular features in which mesenchymal, Ras-mutant lung cancer is acutely dependent on TBK1-mediated support of AKT/mTORC1 pathway activation for survival. Cancer Res; 77(18); 5077-94. ©2017 AACR.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Mesoderma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesoderma/efeitos dos fármacos , Mesoderma/patologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Elementos Reguladores de Transcrição/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Células Tumorais Cultivadas
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