RESUMO
Among arthropod vectors, ticks transmit the most diverse human and animal pathogens, leading to an increasing number of new challenges worldwide. Here we sequenced and assembled high-quality genomes of six ixodid tick species and further resequenced 678 tick specimens to understand three key aspects of ticks: genetic diversity, population structure, and pathogen distribution. We explored the genetic basis common to ticks, including heme and hemoglobin digestion, iron metabolism, and reactive oxygen species, and unveiled for the first time that genetic structure and pathogen composition in different tick species are mainly shaped by ecological and geographic factors. We further identified species-specific determinants associated with different host ranges, life cycles, and distributions. The findings of this study are an invaluable resource for research and control of ticks and tick-borne diseases.
Assuntos
Variação Genética/genética , Doenças Transmitidas por Carrapatos/microbiologia , Carrapatos/genética , Animais , Linhagem Celular , Vetores de Doenças , Especificidade de Hospedeiro/genéticaRESUMO
Small extracellular vesicles (sEVs) are emerging as pivotal players in a wide range of physiological and pathological processes. However, a pressing challenge has been the lack of high-throughput techniques capable of unraveling the intricate heterogeneity of sEVs and decoding the underlying cellular behaviors governing sEV secretion. Here we leverage droplet-based single-cell RNA sequencing (scRNA-seq) and introduce an algorithm, SEVtras, to identify sEV-containing droplets and estimate the sEV secretion activity (ESAI) of individual cells. Through extensive validations on both simulated and real datasets, we demonstrate SEVtras' efficacy in capturing sEV-containing droplets and characterizing the secretion activity of specific cell types. By applying SEVtras to four tumor scRNA-seq datasets, we further illustrate that the ESAI can serve as a potent indicator of tumor progression, particularly in the early stages. With the increasing importance and availability of scRNA-seq datasets, SEVtras holds promise in offering valuable extracellular insights into the cell heterogeneity.
Assuntos
Vesículas Extracelulares , Neoplasias , Humanos , Transcriptoma , AlgoritmosRESUMO
Currently, most paired link based scaffolding algorithms intrinsically mask the sequences between two linked contigs and bypass their direct link information embedded in the original de Bruijn assembly graph. Such disadvantage substantially complicates the scaffolding process and leads to the inability of resolving repetitive contig assembly. Here we present a novel algorithm, inGAP-sf, for effectively generating high-quality and continuous scaffolds. inGAP-sf achieves this by using a new strategy based on the combination of direct link and paired link graphs, in which direct link is used to increase graph connectivity and to decrease graph complexity and paired link is employed to supervise the traversing process on the direct link graph. Such advantage greatly facilitates the assembly of short-repeat enriched regions. Moreover, a new comprehensive decision model is developed to eliminate the noise routes accompanying with the introduced direct link. Through extensive evaluations on both simulated and real datasets, we demonstrated that inGAP-sf outperforms most of the genome scaffolding algorithms by generating more accurate and continuous assembly, especially for short repetitive regions.
Assuntos
Algoritmos , Genômica/métodos , Análise de Sequência de DNA/métodos , DNA/química , Proteínas Associadas a Pancreatite , Sequências Repetitivas de Ácido NucleicoRESUMO
OBJECTIVE: The initial colonisation of the human microbiota and the impact of maternal health on neonatal microbiota at birth remain largely unknown. The aim of our study is to investigate the possible dysbiosis of maternal and neonatal microbiota associated with gestational diabetes mellitus (GDM) and to estimate the potential risks of the microbial shift to neonates. DESIGN: Pregnant women and neonates suffering from GDM were enrolled and 581 maternal (oral, intestinal and vaginal) and 248 neonatal (oral, pharyngeal, meconium and amniotic fluid) samples were collected. To avoid vaginal bacteria contaminations, the included neonates were predominantly delivered by C-section, with their samples collected within seconds of delivery. RESULTS: Numerous and diverse bacterial taxa were identified from the neonatal samples, and the samples from different neonatal body sites were grouped into distinct clusters. The microbiota of pregnant women and neonates was remarkably altered in GDM, with a strong correlation between certain discriminatory bacteria and the oral glucose tolerance test. Microbes varying by the same trend across the maternal and neonatal microbiota were observed, revealing the intergenerational concordance of microbial variation associated with GDM. Furthermore, lower evenness but more depletion of KEGG orthologues and higher abundance of some viruses (eg, herpesvirus and mastadenovirus) were observed in the meconium microbiota of neonates associated with GDM. CONCLUSION: GDM can alter the microbiota of both pregnant women and neonates at birth, which sheds light on another form of inheritance and highlights the importance of understanding the formation of early-life microbiome.
Assuntos
Diabetes Gestacional/diagnóstico , Diabetes Gestacional/metabolismo , Disbiose/metabolismo , Microbiota , Adulto , Glicemia/metabolismo , Cesárea/métodos , Diabetes Gestacional/sangue , Disbiose/complicações , Feminino , Teste de Tolerância a Glucose/métodos , Humanos , Recém-Nascido , GravidezRESUMO
The Amur ide (Leuciscus waleckii) is a cyprinid fish that is widely distributed in Northeast Asia. The Lake Dali Nur population inhabits one of the most extreme aquatic environments on Earth, with an alkalinity up to 50 mmol/L (pH 9.6), thus providing an exceptional model with which to characterize the mechanisms of genomic evolution underlying adaptation to extreme environments. Here, we developed the reference genome assembly for L. waleckii from Lake Dali Nur. Intriguingly, we identified unusual expanded long terminal repeats (LTRs) with higher nucleotide substitution rates than in many other teleosts, suggesting their more recent insertion into the L. waleckii genome. We also identified expansions in genes encoding egg coat proteins and natriuretic peptide receptors, possibly underlying the adaptation to extreme environmental stress. We further sequenced the genomes of 10 additional individuals from freshwater and 18 from Lake Dali Nur populations, and we detected a total of 7.6 million SNPs from both populations. In a genome scan and comparison of these two populations, we identified a set of genomic regions under selective sweeps that harbor genes involved in ion homoeostasis, acid-base regulation, unfolded protein response, reactive oxygen species elimination, and urea excretion. Our findings provide comprehensive insight into the genomic mechanisms of teleost fish that underlie their adaptation to extreme alkaline environments.
Assuntos
Adaptação Fisiológica/genética , Evolução Biológica , Cyprinidae/genética , Animais , Ásia , Evolução Molecular , Ambientes Extremos , Feminino , Perfilação da Expressão Gênica/métodos , Estudos de Associação Genética , Genômica/métodos , Concentração de Íons de Hidrogênio , Lagos , Análise de Sequência de DNA/métodos , Estresse Fisiológico/genética , TranscriptomaRESUMO
16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Microbiota/genética , RNA Ribossômico 16S , Mapeamento de Sequências Contíguas , Escherichia coli/genética , Humanos , Metagenoma , Phaeophyceae/microbiologia , Filogenia , RNA Ribossômico 16S/genética , Saliva/microbiologiaRESUMO
Cocaine and amphetamine-regulated transcript (CART), agouti-related proteins (AgRP) and Melanocortin 4 Receptor (MC4R) involves in the control of appetite. The genes were cloned and characterized, and their regulation was studied in common carp. The CARTI and CARTII genes encode 117- and 120-amino acids, respectively. The AgRP-1 and AgRP-2 genes encode 128- and 136-amino acids, respectively. CARTI was principally expressed in the brain, eye and ovary, while CARTII was highly expressed in the brain. AgRP-1 was strongly expressed in the brain, intestine, testis and eye, while AgRP-2 was highly expressed only in the gill and eye. The MC4R gene, encoding 326-amino acids, was mainly expressed in the brain testis, pituitary and eye. Phylogenetic analysis had been conducted which implied that both CARTI/CARTII and AgRP-1/AgRP-2 might derived from gene duplication events during genome evolution of common carp. CART, AgRP and MC4R gene expression in brain were decreased after fasting treatment and increased sharply after refeeding comparing with normal fed controls, which suggested that CART, AgRP and MC4R are involved in appetite regulation in common carp.
Assuntos
Proteína Relacionada com Agouti/genética , Carpas , Jejum/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/genética , Filogenia , Receptor Tipo 4 de Melanocortina/genética , Proteína Relacionada com Agouti/metabolismo , Animais , Clonagem Molecular , DNA Complementar/genética , Componentes do Gene , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 4 de Melanocortina/metabolismoRESUMO
In order to supply sufficient microsatellite loci for high-density linkage mapping, whole genome shotgun (WGS) sequences of the common carp (Cyprinus carpio) were assembled and surveyed for microsatellite identification. A total of 79,014 microsatellites were collected which were harbored in 68,827 distinct contig sequences. These microsatellites were characterized in the common carp genome. Information of all microsatellites, including previously published BAC-based microsatellites, was then stored in a MySQL database, and a web-based database interface (http://genomics.cafs.ac.cn/ssrdb) was built for public access and download. A total of 3,110 microsatellites, including 1,845 from WGS and 1,265 from BAC end sequences (BES), were tested and genotyped on a mapping family with 192 individuals. A total of 963 microsatellites markers were validated with polymorphism in the mapping family. They will soon be used for high-density linkage mapping with a vast number of polymorphic SNP markers.
Assuntos
Carpas/genética , Genoma/genética , Ensaios de Triagem em Larga Escala , Repetições de Microssatélites/genética , Polimorfismo Genético/genética , Animais , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Biologia Computacional , Análise de Sequência de DNARESUMO
Autism spectrum disorder (ASD), a group of neurodevelopmental disorders characterized by social communication deficits and stereotyped behaviors, may be associated with changes to the gut microbiota. However, how gut commensal bacteria modulate brain function in ASD remains unclear. Here, we used chromodomain helicase DNA-binding protein 8 (CHD8) haploinsufficient mice as a model of ASD to elucidate the pathways through which the host and gut microbiota interact with each other. We found that increased levels of amino acid transporters in the intestines of the mouse model of ASD contribute to the high level of serum glutamine and the increased excitation/inhibition (E/I) ratio in the brain. In addition, elevated α-defensin levels in the haploinsufficient mice resulted in dysregulation of the gut microbiota characterized by a reduced abundance of Bacteroides. Furthermore, supplementation with Bacteroides uniformis improved the ASD-like behaviors and restored the E/I ratio in the brain by decreasing intestinal amino acid transport and the serum glutamine levels. Our study demonstrates associations between changes in the gut microbiota and amino acid transporters, and ASD-like behavioral and electrophysiology phenotypes, in a mouse model.
Assuntos
Transtorno do Espectro Autista , Microbioma Gastrointestinal , Microbiota , Sistemas de Transporte de Aminoácidos/genética , Animais , Transtorno do Espectro Autista/genética , Modelos Animais de Doenças , Microbioma Gastrointestinal/genética , Glutamina , CamundongosRESUMO
A cumulative effect of enterovirus and gluten intake on the risk of celiac disease autoimmunity in infants highlights the significance of viral exposure in early life on the health of children. However, pathogenic viruses may be transmitted to the offspring in an earlier period, raising the possibility that women whose vaginas are inhabited by such viruses may have had their babies infected as early as the time of delivery. A high-resolution intergenerational virome atlas was obtained by metagenomic sequencing and virome analysis on 486 samples from six body sites of 99 mother-neonate pairs. We found that neonates had less diverse oral and enteric viruses than mothers. Vaginally delivered newborns seconds after birth had a more similar oral virome and more viruses of vaginal origin than cesarean-section (C-section) newborns (56.9% vs. 5.8%). Such viruses include both Lactobacillus phage and potentially pathogenic viruses, such as herpesvirus, vaccinia virus, and hepacivirus, illustrating a relatively high variety of the pioneer viral taxa at the time of delivery and a delivery-dependent mother-to-neonate transmission along the vaginal-oral-intestinal route. Neonates are exposed to vaginal viruses as they pass through the reproductive tract, and viruses of vaginal origin may threaten their health. These findings challenge the conventional notion that vaginal delivery is definitely better than cesarean delivery from the perspective of microbial transmission. Screening for vaginal virome before delivery is a worthwhile step to advocate in normal labor to eliminate the risk of intergenerational transmission of pathogenic viruses to offspring.
RESUMO
BACKGROUND: Common carp (Cyprinus carpio), a member of Cyprinidae, is the third most important aquaculture species in the world with an annual global production of 3.4 million metric tons, accounting for nearly 14% of the all freshwater aquaculture production in the world. Apparently genomic resources are needed for this species in order to study its performance and production traits. In spite of much progress, no physical maps have been available for common carp. The objective of this project was to generate a BAC-based physical map using fluorescent restriction fingerprinting. RESULT: The first generation of common carp physical map was constructed using four- color High Information Content Fingerprinting (HICF). A total of 72,158 BAC clones were analyzed that generated 67,493 valid fingerprints (5.5 × genome coverage). These BAC clones were assembled into 3,696 contigs with the average length of 476 kb and a N50 length of 688 kb, representing approximately 1.76 Gb of the common carp genome. The largest contig contained 171 BAC clones with the physical length of 3.12 Mb. There are 761 contigs longer than the N50, and these contigs should be the most useful resource for future integrations with linkage map and whole genome sequence assembly. The common carp physical map is available at http://genomics.cafs.ac.cn/fpc/WebAGCoL/Carp/WebFPC/. CONCLUSION: The reported common carp physical map is the first physical map of the common carp genome. It should be a valuable genome resource facilitating whole genome sequence assembly and characterization of position-based genes important for aquaculture traits.
Assuntos
Carpas/genética , Cromossomos Artificiais Bacterianos , Mapeamento de Sequências Contíguas , Impressões Digitais de DNA , Genoma , Animais , Repetições de Microssatélites , Análise de Sequência de DNARESUMO
Calmodulin (CaM) plays an important role in calcium-dependent signal transduction pathways. In the present study, two alternative splicing isoforms of CaM (named LvCaM-A and LvCaM-B) cDNA were cloned from the Pacific white shrimp, Litopenaeus vannamei. LvCaM-A was of 1101 bp, including a 5'-terminal untranslated region (UTR) of 70 bp, a 3'-terminal UTR of 581 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with a calculated molecular weight (Mw) of 17 kDa and pI of 4.41. LvCaM-B was 689 bp, including a same 5'-UTR of 70 bp, a 3'-terminal UTR of 109 bp and an ORF of 510 bp encoding a polypeptide of 169 amino acids with a calculated Mw of 19 kDa and pI of 4.36. Both LvCaM-A and LvCaM-B contained 4 conservative EF-hand motifs. Quantitative real-time reverse transcription PCR analysis revealed LvCaM-A to be expressed in most shrimp tissues, with the predominant expression in nerve and the weakest expression in heart. However, LvCaM-B expression level was much weaker than those of LvCaM-A in all the tested tissues with main expression in hepatopancreas. The expression of LvCaM-A and LvCaM-B after challenge with Vibrio parahaemolyticus and WSSV were tested in hemocytes, hepatopancreas and nerve. The results indicated that LvCaM-A and LvCaM-B transcripts could be significantly induced in hemocytes and hepatopancreas respectively by injection with V. parahaemolyticus. The highest expression of LvCaM-A was in the hemocytes with 2.3 times (at 48 h) greater expression than in the control (p < 0.05). However, sharp down-regulation of both LvCaM-A and LvCaM-B were detected in nerve after Vibrio- and WSSV injection (p < 0.05). These results suggested that CaM might play an important role in shrimp's defense against pathogenic infection.
Assuntos
Calmodulina/genética , Calmodulina/imunologia , Penaeidae/genética , Penaeidae/imunologia , Vibrio parahaemolyticus , Vírus da Síndrome da Mancha Branca 1 , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Hemócitos/imunologia , Hemócitos/microbiologia , Hemócitos/virologia , Hepatopâncreas/imunologia , Hepatopâncreas/microbiologia , Hepatopâncreas/virologia , Dados de Sequência Molecular , Penaeidae/microbiologia , Penaeidae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
The present study investigated the in vivo hemocytic and hepatopancreatic response to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) injection in shrimp Litopenaeus vannamei. The proliferation of bacteria and virus in shrimp, animal mortality, total hemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst, and gene expression of immune factors associated with immune recognition (lectin), prophenoloxidase (proPO) activation, and the anti-microorganism (lysozyme) and active oxygen defense response (including respiratory burst, cytosolic manganese superoxide dismutase [C-MnSOD], and catalase [CAT]) were quantified. Shrimp death rate increased significantly and was time-dependent after V. parahaemolyticus or WSSV injection. The production of superoxide anion, and the gene expression including lectin in hemocytes, proPO in the hepatopancreas, lysozyme, C-MnSOD and CAT could be induced by injection with V parahaemolyticus and WSSV. The highest value of lysozyme was in the hemocytes with 66.59 times (at 3 h) greater expression than in the control group after WSSV injection and 3.69 times (24 h) greater than in the control group after V parahaemolyticus injection. In the hepatopancreas, CAT expression showed a significant increase, with up to 16 times greater expression than in the control group at 6 h postinjection with WSSV and 7.02 times greater expression than in the control group at 48 h postinjection with V parahaemolyticus (p < 0.05). However, significant decreases in PO activity and proPO transcripts in hemocytes and lectin transcripts in the hepatopancreas were detected after V parahaemolyticus and WSSV injection (p < 0.05). The results suggest that lysozyme, the antioxidase system, and reactive oxygen species might play a crucial role in shrimp defense against bacterial and viral infection.
Assuntos
Penaeidae/imunologia , Espécies Reativas de Oxigênio/imunologia , Vibrio parahaemolyticus/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Animais , Penaeidae/microbiologia , Penaeidae/virologia , Fatores de TempoRESUMO
Reconstructing the sequence of circular RNAs (circRNAs) from short RNA sequencing reads has proved challenging given the similarity of circRNAs and their corresponding linear messenger RNAs. Previous sequencing methods were unable to achieve high-throughput detection of full-length circRNAs. Here we describe a protocol for enrichment and full-length sequencing of circRNA isoforms using nanopore technology. Circular reverse transcription and size selection achieves a 20-fold higher enrichment of circRNAs from total RNA compared to previous methods. We developed an algorithm, called circRNA identifier using long-read sequencing data (CIRI-long), to reconstruct the sequence of circRNAs. The workflow was validated with simulated data and by comparison to Illumina sequencing as well as quantitative real-time RT-PCR. We used CIRI-long to analyze adult mouse brain samples and systematically profile circRNAs, including mitochondria-derived and transcriptional read-through circRNAs. We identified a new type of intronic self-ligated circRNA that exhibits special splicing and expression patterns. Our method takes advantage of nanopore long reads and enables unbiased reconstruction of full-length circRNA sequences.
Assuntos
Sequenciamento por Nanoporos , RNA Circular/genética , Animais , Sequência de Bases , Simulação por Computador , Regulação da Expressão Gênica , Humanos , Camundongos , Isoformas de RNA , Reprodutibilidade dos TestesRESUMO
The importance of the nitric oxide synthase (NOS) gene family is demonstrated by many studies in recent years. However, the lack of sequence information and clones of shrimp NOS cDNA limits further study on its characterization and function in this species. In this report, the cDNA of NOS contained full-length ORF was cloned from the Pacific white shrimp, Litopenaeus vannamei. It was of 4680 bp, including a 5'-terminal untranslated region (UTR) of 278 bp, a 3'-terminal UTR of 862 bp, which contained 5 ATTTA repeats, and an open reading frame (ORF) of 3540 bp encoding a polypeptide of 1179 amino acids. It contained a typical NO synthase domain at the N-terminal, next to a flavodoxin 1 domain, a flavin adenine dinucleotide (FAD) binding domain, respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domain structure at the C-terminal. Quantitative real-time reverse transcription PCR analysis revealed L. vannamei NOS (LvNOS) to be expressed in most shrimp tissues, with highest expression in the hepatopancreas and weakest expression in skin. The expression of LvNOS after challenge with LPS and poly I:C was tested in hemocytes, hepatopancreas and nerve. The results indicated that the NOS transcript level could be induced in hemocytes by injection with LPS. The highest expression was in the hemocyte, with 8.8 times (at 3 h) as much as that in the control (p < 0.05). However, sharp down-regulation of NOS was found in hepatopancreas and nerve after LPS and poly I:C injection (p < 0.05). These results suggested that NOS might play an important role in shrimp's defense against pathogenic infection.
Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Penaeidae/enzimologia , Penaeidae/genética , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Óxido Nítrico Sintase Tipo I/química , Penaeidae/classificação , Penaeidae/imunologia , Filogenia , Poli I-C/farmacologia , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Existing circular RNA (circRNA) databases have become essential for transcriptomics. However, most are unsuitable for mining in-depth information for candidate circRNA prioritization. To address this, we integrate circular transcript collections to develop the circAtlas database based on 1070 RNA-seq samples collected from 19 normal tissues across six vertebrate species. This database contains 1,007,087 highly reliable circRNAs, of which over 81.3% have been assembled into full-length sequences. We profile their expression pattern, conservation, and functional annotation. We describe a novel multiple conservation score, co-expression, and regulatory networks for circRNA annotation and prioritization. CircAtlas can be accessed at http://circatlas.biols.ac.cn/.
Assuntos
Bases de Dados de Ácidos Nucleicos , RNA Circular/metabolismo , Transcriptoma , Animais , Sequência Conservada , Humanos , Camundongos , Anotação de Sequência Molecular , RNA Circular/química , RNA Circular/genética , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Ratos , Vertebrados/genética , Vertebrados/metabolismoRESUMO
The effects of some pathogen-associated molecular patterns (PAMPs) (laminarin, LPS and poly I:C) on total hemocyte counts (THC), phenoloxidase (PO) activity, superoxide anion production and lectin, prophenoloxidase, lysozyme, cytosolic manganese superoxide dismutase (C-MnSOD) and catalase (CAT) gene expression were studied. The results showed that the production or activity of most tested immune factors and the expression of most tested genes were up-regulated after stimulation with PAMPs, among which the highest value of lectin with 4.4 times as much as that of the control group appeared at 6 h in hemocytes, of CAT with 47 times as much as that of the control group appeared at 12 h in hepatopancreas, and with 2.7 times higher than that of the control group at 24 h of C-MnSOD in hepatopancreas after laminarin injection. The peak value of proPO, lysozyme and C-MnSOD appeared at 6 h in hepatopancreas, 24 h in hepatopancreas and 24 h in hemocytes after LPS injection, respectively. The highest expression level of lysozyme appeared at 12 h in hemocytes after poly I:C injection. However, significant decreases of PO activity in hemocytes and lectin expression in hepatopancreas were found after poly I:C injection, and a dramatic down-regulation of proPO expression from 3 h to 48 h was found in hemocytes after injection with laminarin, LPS and poly I:C. The results suggest that the shrimp immune response could be activated or inhibited by different PAMPs, and that the hepatopancreas also plays a key role by synthesizing immune factors.
Assuntos
Regulação da Expressão Gênica/imunologia , Hemócitos/metabolismo , Hepatopâncreas/metabolismo , Penaeidae/citologia , Animais , Perfilação da Expressão GênicaRESUMO
Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. In this paper, the full-length cDNA of AK was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Escherichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps.
Assuntos
Arginina Quinase/genética , Arginina Quinase/metabolismo , Penaeidae/enzimologia , Penaeidae/genética , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Arginina Quinase/química , Catálise , Clonagem Molecular , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hemócitos/enzimologia , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Músculos/enzimologia , Proteínas Recombinantes/metabolismoRESUMO
Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor and Toll-like receptor-induced activation of nuclear factor-kappaB (NF-kappaB). In this report, the full-length cDNA of MyD88 was cloned from the large yellow croaker, Pseudosciaena crocea. It was of 1574 bp, including a 5'-terminal untranslated region (UTR) of 89 bp, a 3'-terminal UTR of 621bp and an open reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids. It contained a typical death domain at the N-terminal and a conservative Toll/IL-1R (TIR) domain structure at the C-terminal. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of MyD88 with the highest expression in the spleen and the weakest expression in the muscle. The expression of MyD88 after challenge with formalin-inactivated Gram-negative bacterium Vibrio parahaemolyticus was tested in blood, spleen and liver. It suggested that the highest expression was in the spleen (p<0.05) with 1.9 times (at 48 h) as much as that in the control and the lowest expression of MyD88 was in the liver (p<0.05) with 0.29 times (at 3h) of that in the control. These results indicated that as a universal key adaptor in the Toll-like receptor pathway in mammals, MyD88 might play an important role in large yellow croaker defense against pathogenic infection.
Assuntos
Regulação da Expressão Gênica , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Fígado/imunologia , Dados de Sequência Molecular , Fator 88 de Diferenciação Mieloide/sangue , Fator 88 de Diferenciação Mieloide/química , Perciformes/classificação , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Baço/imunologia , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio parahaemolyticus/imunologiaRESUMO
Currently, circRNA studies are shifting from the identification of circular transcripts to understanding their biological functions. However, such endeavors have been limited by large-scale determination of their full-length sequences and also by the inability of accurate quantification at the isoform level. Here, we propose a new feature, reverse overlap (RO), for circRNA detection, which outperforms back-splice junction (BSJ)-based methods in identifying low-abundance circRNAs. By combining RO and BSJ features, we present a novel approach for effective reconstruction of full-length circRNAs and isoform-level quantification from the transcriptome. We systematically compared the difference between the BSJ-level and isoform-level differential expression analyses using human liver tumor and normal tissues and highlight the necessity of deepening circRNA studies to the isoform-level resolution. The CIRI-full software can be accessed at https://sourceforge.net/projects/ciri .