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1.
Rinsho Ketsueki ; 60(9): 1236-1242, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31597849

RESUMO

Recent technology advances in genomic analysis have unraveled the genomic complexity and evolutionary process of multiple myeloma (MM). Hyperdiploidy or IgH translocations t (4;14), t (11;14), t (6;14), t (14;16), and t (14;20), leading to ectopic overexpression of MMSET/FGFR3, CCND1, CCND3, MAF, and MAFB, respectively, are initiating events. Subsequent secondary events, such as gene copy number alterations, and gene somatic mutations, participate in tumor progression in a branching pattern consistent with Darwin's evolutionary model. Copy number alterations, such as 1q21 amplification and del (17p), have been associated with adverse outcomes. N/KRAS mutations are most commonly found in around 20% of patients, but numerous gene mutations are infrequent. Pathological and clinical relevance of gene mutations combined with cytogenetic abnormalities are currently under investigation. Additionally, detailed genomic analysis of individual patients using targeted-sequencing panels has been facilitated, and efforts toward personalized therapy based on molecular features have begun. This paper outlines MM molecular pathology and its clinical application in Japanese patients.


Assuntos
Mieloma Múltiplo/genética , Aberrações Cromossômicas , Dosagem de Genes , Humanos , Japão , Translocação Genética
2.
BMC Bioinformatics ; 20(1): 453, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488068

RESUMO

BACKGROUND: Metagenomics caused a quantum leap in microbial ecology. However, the inherent size and complexity of metagenomic data limit its interpretation. The quantification of metagenomic traits in metagenomic analysis workflows has the potential to improve the exploitation of metagenomic data. Metagenomic traits are organisms' characteristics linked to their performance. They are measured at the genomic level taking a random sample of individuals in a community. As such, these traits provide valuable information to uncover microorganisms' ecological patterns. The Average Genome Size (AGS) and the 16S rRNA gene Average Copy Number (ACN) are two highly informative metagenomic traits that reflect microorganisms' ecological strategies as well as the environmental conditions they inhabit. RESULTS: Here, we present the ags.sh and acn.sh tools, which analytically derive the AGS and ACN metagenomic traits. These tools represent an advance on previous approaches to compute the AGS and ACN traits. Benchmarking shows that ags.sh is up to 11 times faster than state-of-the-art tools dedicated to the estimation AGS. Both ags.sh and acn.sh show comparable or higher accuracy than existing tools used to estimate these traits. To exemplify the applicability of both tools, we analyzed the 139 prokaryotic metagenomes of TARA Oceans and revealed the ecological strategies associated with different water layers. CONCLUSION: We took advantage of recent advances in gene annotation to develop the ags.sh and acn.sh tools to combine easy tool usage with fast and accurate performance. Our tools compute the AGS and ACN metagenomic traits on unassembled metagenomes and allow researchers to improve their metagenomic data analysis to gain deeper insights into microorganisms' ecology. The ags.sh and acn.sh tools are publicly available using Docker container technology at https://github.com/pereiramemo/AGS-and-ACN-tools .


Assuntos
Dosagem de Genes , Tamanho do Genoma , Metagenoma/genética , Metagenômica/métodos , RNA Ribossômico 16S/genética , Benchmarking , Variações do Número de Cópias de DNA , Bases de Dados Genéticas , Oceanos e Mares , Fatores de Tempo
3.
BMC Plant Biol ; 19(1): 346, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391002

RESUMO

BACKGROUND: The safety assessment and control of stacked transgenic crops is increasingly important due to continuous crop development and is urgently needed in China. The genetic stability of foreign genes and unintended effects are the primary problems encountered in safety assessment. Omics techniques are useful for addressing these problems. The stacked transgenic maize variety 12-5 × IE034, which has insect-resistant and glyphosate-tolerant traits, was developed via a breeding stack using 12-5 and IE034 as parents. Using 12-5 × IE034, its parents (12-5 and IE034), and different maize varieties as materials, we performed proteomic profiling, molecular characterization and a genetic stability analysis. RESULTS: Our results showed that the copy number of foreign genes in 12-5 × IE034 is identical to that of its parents 12-5 and IE034. Foreign genes can be stably inherited over different generations. Proteomic profiling analysis found no newly expressed proteins in 12-5 × IE034, and the differences in protein expression between 12 and 5 × IE034 and its parents were within the range of variation of conventional maize varieties. The expression levels of key enzymes participating in the shikimic acid pathway which is related to glyphosate tolerance of 12-5 × IE034 were not significantly different from those of its parents or five conventional maize varieties, which indicated that without selective pressure by glyphosate, the introduced EPSPS synthase is not has a pronounced impact on the synthesis of aromatic amino acids in maize. CONCLUSIONS: Stacked-trait development via conventional breeding did not have an impact on the genetic stability of T-DNA, and the impact of stacked breeding on the maize proteome was less significant than that of genotypic differences. The results of this study provide a theoretical basis for the development of a safety assessment approach for stacked-trait transgenic crops in China.


Assuntos
Variação Genética , Melhoramento Vegetal , Plantas Geneticamente Modificadas , Zea mays/genética , China , Inocuidade dos Alimentos , Dosagem de Genes , Instabilidade Genômica , Análise de Perigos e Pontos Críticos de Controle , Proteômica
4.
BMC Plant Biol ; 19(1): 372, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438864

RESUMO

BACKGROUND: Correct timing of flowering is critical for plants to produce enough viable offspring. In Arabidopsis thaliana (Arabidopsis), flowering time is regulated by an intricate network of molecular signaling pathways. Arabidopsis srr1-1 mutants lacking SENSITIVITY TO RED LIGHT REDUCED 1 (SRR1) expression flower early, particularly under short day (SD) conditions (1). SRR1 ensures that plants do not flower prematurely in such non-inductive conditions by controlling repression of the key florigen FT. Here, we have examined the role of SRR1 in the closely related crop species Brassica napus. RESULTS: Arabidopsis SRR1 has five homologs in Brassica napus. They can be divided into two groups, where the A02 and C02 copies show high similarity to AtSRR1 on the protein level. The other group, including the A03, A10 and C09 copies all carry a larger deletion in the amino acid sequence. Three of the homologs are expressed at detectable levels: A02, C02 and C09. Notably, the gene copies show a differential expression pattern between spring and winter type accessions of B. napus. When the three expressed gene copies were introduced into the srr1-1 background, only A02 and C02 were able to complement the srr1-1 early flowering phenotype, while C09 could not. Transcriptional analysis of known SRR1 targets in Bna.SRR1-transformed lines showed that CYCLING DOF FACTOR 1 (CDF1) expression is key for flowering time control via SRR1. CONCLUSIONS: We observed subfunctionalization of the B. napus SRR1 gene copies, with differential expression between early and late flowering accessions of some Bna.SRR1 copies. This suggests involvement of Bna.SRR1 in regulation of seasonal flowering in B. napus. The C09 gene copy was unable to complement srr1-1 plants, but is highly expressed in B. napus, suggesting specialization of a particular function. Furthermore, the C09 protein carries a deletion which may pinpoint a key region of the SRR1 protein potentially important for its molecular function. This is important evidence of functional domain annotation in the highly conserved but unique SRR1 amino acid sequence.


Assuntos
Brassica napus/genética , Flores/genética , Genes de Plantas , Proteínas de Plantas/genética , Flores/crescimento & desenvolvimento , Dosagem de Genes , Expressão Gênica , Filogenia , Proteínas de Plantas/fisiologia
5.
Nat Commun ; 10(1): 3101, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308377

RESUMO

The identification of cancer-promoting genetic alterations is challenging particularly in highly unstable and heterogeneous cancers, such as esophageal adenocarcinoma (EAC). Here we describe a machine learning algorithm to identify cancer genes in individual patients considering all types of damaging alterations simultaneously. Analysing 261 EACs from the OCCAMS Consortium, we discover helper genes that, alongside well-known drivers, promote cancer. We confirm the robustness of our approach in 107 additional EACs. Unlike recurrent alterations of known drivers, these cancer helper genes are rare or patient-specific. However, they converge towards perturbations of well-known cancer processes. Recurrence of the same process perturbations, rather than individual genes, divides EACs into six clusters differing in their molecular and clinical features. Experimentally mimicking the alterations of predicted helper genes in cancer and pre-cancer cells validates their contribution to disease progression, while reverting their alterations reveals EAC acquired dependencies that can be exploited in therapy.


Assuntos
Adenocarcinoma/genética , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias Esofágicas/genética , Perfilação da Expressão Gênica/métodos , Medicina de Precisão/métodos , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Progressão da Doença , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Instabilidade Genômica , Humanos , Aprendizado de Máquina , Modelos Genéticos , Família Multigênica/efeitos dos fármacos , Taxa de Mutação , Polimorfismo de Nucleotídeo Único
6.
BMC Evol Biol ; 19(1): 152, 2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31340752

RESUMO

BACKGROUND: With an ever-growing number of published genomes, many low levels of the Tree of Life now contain several species with enough molecular data to perform shallow-scale phylogenomic studies. Moving away from using just a few universal phylogenetic markers, we can now target thousands of other loci to decipher taxa relationships. Making the best possible selection of informative sequences regarding the taxa studied has emerged as a new issue. Here, we developed a general procedure to mine genomic data, looking for orthologous single-copy loci capable of deciphering phylogenetic relationships below the generic rank. To develop our strategy, we chose the genus Rosa, a rapid-evolving lineage of the Rosaceae family in which several species genomes have recently been sequenced. We also compared our loci to conventional plastid markers, commonly used for phylogenetic inference in this genus. RESULTS: We generated 1856 sequence tags in putative single-copy orthologous nuclear loci. Associated in silico primer pairs can potentially amplify fragments able to resolve a wide range of speciation events within the genus Rosa. Analysis of parsimony-informative site content showed the value of non-coding genomic regions to obtain variable sequences despite the fact that they may be more difficult to target in less related species. Dozens of nuclear loci outperform the conventional plastid phylogenetic markers in terms of phylogenetic informativeness, for both recent and ancient evolutionary divergences. However, conflicting phylogenetic signals were found between nuclear gene tree topologies and the species-tree topology, shedding light on the many patterns of hybridization and/or incomplete lineage sorting that occur in the genus Rosa. CONCLUSIONS: With recently published genome sequence data, we developed a set of single-copy orthologous nuclear loci to resolve species-level phylogenomics in the genus Rosa. This genome-wide scale dataset contains hundreds of highly variable loci which phylogenetic interest was assessed in terms of phylogenetic informativeness and topological conflict. Our target identification procedure can easily be reproduced to identify new highly informative loci for other taxonomic groups and ranks.


Assuntos
Núcleo Celular/genética , Dosagem de Genes , Loci Gênicos , Genômica , Filogenia , Rosa/genética , Primers do DNA/metabolismo , Genoma de Planta , Funções Verossimilhança , Plastídeos/genética
8.
Arch Virol ; 164(9): 2285-2295, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31250104

RESUMO

Examination of lumpy skin disease virus (LSDV) isolates from different geographic regions and times revealed that assays developed in our laboratory for differentiating between virulent Israeli viruses and Neethling vaccine virus (NVV) are generally useful in most, if not all, endemic areas in which NVV-based vaccines are used. Recently it was revealed that the LSDV126 gene of field isolates contains a duplicated region of 27 bp (9 aa), while the vaccine viruses have only one copy. Phylogenetic analysis of a 532-bp segment carrying the LSDV126 gene and whole virus genome sequences revealed that LSDV isolates formed two groups: virulent and vaccine viruses. In this analysis, all of the capripox viruses that lack the ability to efficiently infect cattle were found to carry only one copy of the 27-bp fragment, suggesting that the LSDV126 gene plays an important role in the ability of capripox viruses to infect cattle. In silico analysis of potential antigenic sites in LSDV126 revealed that LSDV126 variants with only one copy of the repeat lack a potentially important antigenic epitope, supporting its possible significance in cattle infection. This study provides new information about the nature of the LSDV126 gene and its possible role in the life cycle of LSDV.


Assuntos
Doença Nodular Cutânea/virologia , Vírus da Doença Nodular Cutânea/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Mapeamento de Epitopos , Dosagem de Genes , Doença Nodular Cutânea/diagnóstico , Vírus da Doença Nodular Cutânea/química , Vírus da Doença Nodular Cutânea/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética
9.
Prep Biochem Biotechnol ; 49(8): 822-829, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156045

RESUMO

Therapeutic monoclonal antibodies (mAbs) have become the dominant products in biopharmaceutical industry. Mammalian cell expression systems including Chinese hamster ovary (CHO) cells are the most commonly used hosts for the production of complex recombinant proteins. However, development of stable, high producing CHO cell lines suffers from the low expression level and instability of the transgene. The increasing efforts in the development of novel therapeutic antibodies and the advent of biosimilars have revealed the necessity for the development of improved platforms for rapid production of products for initial characterization and testing. In line with this premise, vector design and engineering has been applied to improve the expression level and stability of the transgene. This study reports the application of an improved lentiviral vector system containing the human interferon-ß scaffold attachment region (IFN-SAR) for the development of antibody producing stable CHO cells. mAb expressing clones producing 1100 µg/L of IgG1 monoclonal antibody were isolated without extensive screening of a large number of clones. Our results here indicate the positive effects of IFN-SAR on stable mAb expression using lentiviral based expression vectors. We also observed that although IFN-SAR can improve light chain (LC) and heavy chain (HC) gene copy numbers in stable cell pools, mAb expression in single cell clones was not affected by the transgene copy number.


Assuntos
Anticorpos Monoclonais/genética , Clonagem Molecular/métodos , Vetores Genéticos/genética , Lentivirus/genética , Animais , Células CHO , Linhagem Celular , Cricetulus , Dosagem de Genes , Humanos , Proteínas Recombinantes/genética , Transdução Genética
10.
BMC Genomics ; 20(1): 518, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234802

RESUMO

BACKGROUND: The comparison of expression QTL (eQTL) maps obtained in different tissues is an essential step to understand how gene expression is genetically regulated in a context-dependent manner. In the current work, we have compared the transcriptomic and eQTL profiles of two porcine tissues (skeletal muscle and liver) which typically show highly divergent expression profiles, in 103 Duroc pigs genotyped with the Porcine SNP60 BeadChip (Illumina) and with available microarray-based measurements of hepatic and muscle mRNA levels. Since structural variation could have effects on gene expression, we have also investigated the co-localization of cis-eQTLs with copy number variant regions (CNVR) segregating in this Duroc population. RESULTS: The analysis of differential expresssion revealed the existence of 1204 and 1490 probes that were overexpressed and underexpressed in the gluteus medius muscle when compared to liver, respectively (|fold-change| > 1.5, q-value < 0.05). By performing genome scans in 103 Duroc pigs with available expression and genotypic data, we identified 76 and 28 genome-wide significant cis-eQTLs regulating gene expression in the gluteus medius muscle and liver, respectively. Twelve of these cis-eQTLs were shared by both tissues (i.e. 42.8% of the cis-eQTLs identified in the liver were replicated in the gluteus medius muscle). These results are consistent with previous studies performed in humans, where 50% of eQTLs were shared across tissues. Moreover, we have identified 41 CNVRs in a set of 350 pigs from the same Duroc population, which had been genotyped with the Porcine SNP60 BeadChip by using the PennCNV and GADA softwares, but only a small proportion of these CNVRs co-localized with the cis-eQTL signals. CONCLUSION: Despite the fact that there are considerable differences in the gene expression patterns of the porcine liver and skeletal muscle, we have identified a substantial proportion of common cis-eQTLs regulating gene expression in both tissues. Several of these cis-eQTLs influence the mRNA levels of genes with important roles in meat (CTSF) and carcass quality (TAPT1), lipid metabolism (TMEM97) and obesity (MARC2), thus evidencing the practical importance of dissecting the genetic mechanisms involved in their expression.


Assuntos
Regulação da Expressão Gênica , Fígado/metabolismo , Músculo Esquelético/metabolismo , Suínos/genética , Animais , Dosagem de Genes , Perfilação da Expressão Gênica , Masculino , Locos de Características Quantitativas , Transcriptoma
11.
Curr Microbiol ; 76(9): 1045-1054, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31214822

RESUMO

ATP-dependent Lon protease plays important roles in different physiological processes, including cellular differentiation of the bacteria and is a part of an important stress response regulon (HspR/HAIR). In Streptomyces, biosynthesis of secondary metabolites starts with cellular differentiation and stress is one of the factor that affect metabolite production. To clarify the effect of Lon protease on secondary metabolite production, we constructed a recombinant strain of Streptomyces coelicolor A3(2) that has one extra copy of lon gene with its own promoter and transcriptional terminator in its genome. Expression of lon gene in the recombinant strain was determined by quantitative real time (RT-qPCR). Actinorhodin and undecylprodigiosin production of the recombinant cell was measured in liquid R2YE and it was found to produce about 34 times more actinorhodin and 9 times more undecylprodigiosin than the wild-type at 168 h of growth. Development of stable Streptomyces strains capable of producing high amounts of secondary metabolites is valuable for biotechnology industry. One extra copy of lon gene is enough to boost antibiotic production by S. coelicolor A3(2) and this change do not cause any metabolic burden in the cell.


Assuntos
Antibacterianos/biossíntese , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Antraquinonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dosagem de Genes , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Regulon , Streptomyces coelicolor/crescimento & desenvolvimento
12.
Iran J Allergy Asthma Immunol ; 18(2): 143-152, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31066250

RESUMO

Assessment of the number of T-cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) copies has been recently described as biomarkers of newly formed T and B cells respectively. In this study, we aimed to explore the effects of demographic variables including age, gender, weight, height and ethnicity on these two episomal DNA molecules. Second, for the first time in our country, we determined the reference values of TREC and KREC copy numbers in different age groups of Iranian healthy individuals as a threshold for identifying T cell and B cell lymphopenia. The TREC and KREC copy numbers were evaluated in 251 dried blood spot (DBS) samples from healthy volunteers (age range: 0-60 years). Six primary immunodeficiency (PID) patients were included as disease controls. TREC and KREC copies were markedly reduced with increasing age. Although the levels of TREC and KREC were higher in females than males, this difference did not reach statistical significance. These findings suggest that demographic variables including age should be considered for interpretation results of the TREC/KREC assay.


Assuntos
Fatores Etários , Linfócitos B/fisiologia , Linfopenia/diagnóstico , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/fisiologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Dosagem de Genes , Humanos , Lactente , Recém-Nascido , Irã (Geográfico) , Linfopenia/imunologia , Masculino , Pessoa de Meia-Idade , Padrões de Referência , Adulto Jovem
13.
Plant Cell Rep ; 38(9): 1081-1097, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31134349

RESUMO

KEY MESSAGE: Duplicate POT1 genes must rapidly diverge or be inactivated. Protection of telomeres 1 (POT1) encodes a conserved telomere binding protein implicated in both chromosome end protection and telomere length maintenance. Most organisms harbor a single POT1 gene, but in the few lineages where the POT1 family has expanded, the duplicate genes have diversified. Arabidopsis thaliana bears three POT1-like loci, POT1a, POT1b and POT1c. POT1a retains the ancestral function of telomerase regulation, while POT1b is implicated in chromosome end protection. Here we examine the function and evolution of the third POT1 paralog, POT1c. POT1c is a new gene, unique to A. thaliana, and was derived from a duplication event involving the POT1a locus and a neighboring gene encoding ribosomal protein S17. The duplicate S17 locus (dS17) is highly conserved across A. thaliana accessions, while POT1c is highly divergent, harboring multiple deletions within the gene body and two transposable elements within the promoter. The POT1c locus is transcribed at very low to non-detectable levels under standard growth conditions. In addition, no discernable molecular or developmental defects are associated with plants bearing a CRISPR mutation in the POT1c locus. However, forced expression of POT1c leads to decreased telomerase enzyme activity and shortened telomeres. Evolutionary reconstruction indicates that transposons invaded the POT1c promoter soon after the locus was formed, permanently silencing the gene. Altogether, these findings argue that POT1 dosage is critically important for viability and duplicate gene copies are retained only upon functional divergence.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Dosagem de Genes , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/genética , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Elementos de DNA Transponíveis/genética , Evolução Molecular , Duplicação Gênica , Mutação , Regiões Promotoras Genéticas/genética , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/genética
14.
Bioelectrochemistry ; 129: 18-25, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31075535

RESUMO

Periplasmic c-type cytochromes are essential for the electron transport between the cytoplasmic membrane bound menquinol oxidase CymA and the terminal ferric iron reductase MtrABC in the outer membrane of Shewanella oneidensis cells. Either STC or FccA are necessary for periplasmic electron transfer. We followed the hypothesis that the elimination of potential competing reactions in the periplasm and the simultaneous overexpression of STC (cctA) could lead to an accelerated electron transfer to the cell surface. The genes nrfA, ccpA, napB and napA were replaced by cctA. This led to a 1.7-fold increased ferric iron reduction rate and a 23% higher current generation in a bioelectrochemical system. Moreover, the quadruple mutant had a higher periplasmic flavin content. Further deletion of fccA and its replacement by cctA resulted in a strain with ferric iron reduction rates similar to the wild type and a lower concentration of periplasmic flavin compared to the quadruple mutant. A transcriptomic analysis revealed that the quadruple mutant had a 3.7-fold higher cctA expression which could not be further increased by the replacement of fccA. This work indicates that a synthetic adaptation of Shewanella towards extracellular respiration holds potential for increased respiratory rates and consequently higher current densities.


Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Proteínas Periplásmicas/metabolismo , Shewanella/metabolismo , Proteínas de Bactérias/genética , Grupo dos Citocromos c/genética , Transporte de Elétrons , Desenho de Equipamento , Fumaratos/metabolismo , Deleção de Genes , Dosagem de Genes , Engenharia Genética/métodos , Lactatos/metabolismo , Proteínas Periplásmicas/genética , Shewanella/genética , Transcriptoma , Regulação para Cima
15.
Scand J Immunol ; 90(2): e12776, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31069824

RESUMO

The number of the X chromosome-linked genes has been previously suggested to influence immune responses and the development of autoimmune diseases. In the present study, we aimed at evaluating the level of expression of CD40L (an X-linked gene involved in adaptive immunity) and TLR7 (an X-linked gene involved in innate immunity) in a variety of different karyotypes. Those included males, females and patients with X chromosome aneuploidy. Healthy females (46, XX; n = 10) and healthy males (46, XY; n = 10) were compared to females with Turner syndrome (TS) (45, X; n = 11) and males with Klinefelter syndrome (KS) (47, XXY; n = 5). Stimulation of peripheral blood mononuclear cells (PBMCs) with PMA and ionomycin resulted in higher percentage of CD3 + CD40L+ T cells (P < 0.001) and higher level expression of CD40L in T cell (P < 0.001) in female and KS patients compared with male and TS patients. TLR7-mediated IFN-alpha production by HLADR + CD3- CD19- cells was significantly upregulated in healthy women compared with healthy males, TS and KS patients (P < 0.001). TLR7 agonist-stimulated PBMCs from healthy females and KS patients expressed significantly higher levels of TLR7 mRNA than those from male and TS patients (P < 0.05). The increased expression of the X-linked genes TLR7 and CD40L in healthy females and KS patients suggests that the presence of two X chromosomes plays a major role in enhancing both innate and adaptive immune responses. These results may contribute to the explanation of sex-based differences in immune biology and the sex bias in predisposition to autoimmune diseases.


Assuntos
Imunidade Adaptativa/genética , Ligante de CD40/biossíntese , Ligante de CD40/genética , Cromossomos Humanos X/genética , Dosagem de Genes/genética , Imunidade Inata/genética , Receptor 7 Toll-Like/biossíntese , Receptor 7 Toll-Like/genética , Imunidade Adaptativa/imunologia , Antígenos CD19/biossíntese , Complexo CD3/biossíntese , Células Cultivadas , Variações do Número de Cópias de DNA/genética , Feminino , Humanos , Imunidade Inata/imunologia , Interferon-alfa/biossíntese , Ionomicina/farmacologia , Síndrome de Klinefelter/genética , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Ácidos Polimetacrílicos/farmacologia , RNA Mensageiro/biossíntese , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Síndrome de Turner/genética
16.
Mol Immunol ; 111: 87-94, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31048099

RESUMO

Reticuloendotheliosis virus (REV), an avian retrovirus is able to infect a variety of birds and can cause immunosuppression. The aim of this study was to investigate the relationship of thymic lymphocytes apoptosis, proliferation and T cell subtype with immunosuppression. In this study, a hundred and twenty one-day old SPF chickens were randomly divided into control groups (group C) and a REV infection groups (group I). The chickens of group I received intraperitoneal injections of REV with 104.62/0.1 ml TCID50. On day 14, 21, 28 and 35 post-inoculation, the chickens of C group and I group were sacrificed by cardiac puncture blood collection, and the thymic lymphocytes was sterile collected. The proliferation ability of lymphocytes was tested by Cell Counting Kit-8. Flow cytometry was performed to detect apoptosis, cell cycle stage and the change in T cell subtype. The RNA genome copy numbers of REV virus were detected using real-time PCR. Real-time PCR and western blotting were performed to analyze the expression of CyclinD1 and Bcl-2. Our results showed that REV genome copy number steadily declined, the proliferation potential of thymic lymphocytes was inhibited, lymphocytes apoptosed, the ratio of CD4+/CD8+ decreased and the expression of CyclinD1 and Bcl-2 were firstly inhibited, then rapidly recovered. Thus, immunosuppression lead by REV is closely related to the change of T cell subtype, apoptosis, and proliferation of thymic lymphocytes.


Assuntos
Apoptose/imunologia , Proliferação de Células/fisiologia , Galinhas/imunologia , Vírus da Reticuloendoteliose/imunologia , Infecções por Retroviridae/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ciclo Celular/imunologia , Galinhas/virologia , Ciclina D1/imunologia , Dosagem de Genes/imunologia , Genoma Viral/imunologia , Imunossupressão/métodos , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Infecções por Retroviridae/virologia , Subpopulações de Linfócitos T/virologia , Timo/virologia
17.
J Clin Pathol ; 72(9): 642-646, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31123138

RESUMO

AIMS: To elucidate the clinicopathological and molecular features of intravascular NK/T-cell lymphoma (IVNKTCL). METHODS: Two cases of IVNKTCL were retrieved from a single-centre cohort composed of 25 intravascular lymphomas. Whole-exome and RNA sequencing and immunohistochemistry were performed. RESULTS: We identified somatic mutations in the following epigenetic regulators: four histone genes (HIST1H2AN, HIST1H2BE, HIST1H2BN and H3F3A), histone deacetylase (HDAC5), two helicases (WRN and DDX3X), two methylation-related enzymes (TET2 and DNMT1) and the SNI/SWF pathway (ARID1A). Copy number analysis identified driver gene alterations comprising the loss of ARID1B, HACE1 and SMAD4, and the gain of SOX2 and histone clusters. RNA sequencing analysis did not indicate the presence of any fusion gene. Both cases were positive for Epstein-Barr virus (EBV) and showed strong expression of programmed death-ligand 1 (PD-L1). CONCLUSIONS: This study raises the possibility that, at least for some patients, IVNKTCL may be considered an epigenetic disease with EBV infection-associated aetiopathogenesis.


Assuntos
Biomarcadores Tumorais/genética , Variações do Número de Cópias de DNA , Dosagem de Genes , Linfoma Extranodal de Células T-NK/genética , Linfoma Extranodal de Células T-NK/patologia , Mutação , Idoso , Antígeno B7-H1/análise , Biomarcadores Tumorais/análise , Epigênese Genética , Predisposição Genética para Doença , Herpesvirus Humano 4/isolamento & purificação , Humanos , Imuno-Histoquímica , Linfoma Extranodal de Células T-NK/química , Linfoma Extranodal de Células T-NK/virologia , Masculino , Fenótipo , Estudos Retrospectivos , Análise de Sequência de RNA , Sequenciamento Completo do Exoma , Adulto Jovem
18.
PLoS Comput Biol ; 15(5): e1007015, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31095555

RESUMO

MHC genes, which code for proteins responsible for presenting pathogen-derived antigens to the host immune system, show remarkable copy-number variation both between and within species. However, the evolutionary forces driving this variation are poorly understood. Here, we use computer simulations to investigate whether evolution of the number of MHC variants in the genome can be shaped by the number of pathogen species the host population encounters (pathogen richness). Our model assumed that while increasing a range of pathogens recognised, expressing additional MHC variants also incurs costs such as an increased risk of autoimmunity. We found that pathogen richness selected for high MHC copy number only when the costs were low. Furthermore, the shape of the association was modified by the rate of pathogen evolution, with faster pathogen mutation rates selecting for increased host MHC copy number, but only when pathogen richness was low to moderate. Thus, taking into account factors other than pathogen richness may help explain wide variation between vertebrate species in the number of MHC genes. Within population, variation in the number of unique MHC variants carried by individuals (INV) was observed under most parameter combinations, except at low pathogen richness. This variance gave rise to positive correlations between INV and host immunocompetence (proportion of pathogens recognised). However, within-population variation in host immunocompetence declined with pathogen richness. Thus, counterintuitively, pathogens can contribute more to genetic variance for host fitness in species exposed to fewer pathogen species, with consequences to predictions from "Hamilton-Zuk" theory of sexual selection.


Assuntos
Evolução Molecular , Dosagem de Genes , Complexo Principal de Histocompatibilidade , Imunidade Adaptativa/genética , Alelos , Animais , Apresentação do Antígeno/genética , Biologia Computacional , Simulação por Computador , Variação Genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Modelos Genéticos , Modelos Imunológicos , Mutação , Seleção Genética
19.
World J Microbiol Biotechnol ; 35(6): 79, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134410

RESUMO

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Pichia/genética , Plasmídeos/genética , Transformação Genética , Replicação do DNA , Escherichia coli/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Vetores Genéticos , Instabilidade Genômica , Microbiologia Industrial , Kluyveromyces/genética , Regiões Promotoras Genéticas , RNA Guia , Biologia Sintética
20.
World J Microbiol Biotechnol ; 35(6): 84, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134444

RESUMO

Pectin is a type of complex hydrophilic polysaccharide widely distributed in plant resources. Thermal stable pectinase has its advantage in bioapplication in the fields of food processing, brewing, and papermaking, etc. In this study, we enzymatically characterized a putative endo-polygalacturonase TcPG from a Talaromyces cellulolyticus, realized its high-level expression in Pichia pastoris by in vitro constructing of a series of multi-copy expression cassettes and real time quantitative PCR screening. The secretive expression level of TcPG was nonlinear correlated to the gene dosage. Recombinants with five-copy TcPG gene in the host genome showed the highest expression. After cultivation in a bioreactor for about 96 h, the enzyme activity reached 7124.8 U/mL culture. TcPG has its optimal temperature of 70 °C. Under the optimized parameters, the pectin could be efficiently hydrolyzed into oligosaccharides.


Assuntos
Dosagem de Genes , Pectinas/metabolismo , Pichia/genética , Poligalacturonase/biossíntese , Poligalacturonase/genética , Talaromyces/enzimologia , Talaromyces/genética , Reatores Biológicos , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Hidrólise , Pichia/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/genética , Temperatura Ambiente , Fatores de Tempo
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