RESUMEN
The aim of the study was to investigate the grass carp hemorrhagic infection pathway and its key-related genes. Grass carp reovirus (GCRV) might cause hemorrhagic disease in grass carps. Healthy grass carp fingerlings (N = 60) were divided into control and infected groups. Fish in the control group were intraperitoneally (ip) injected with 0.6% fish physiological saline; the infected group received 5,000,000 50% tissue culture infective doses of GCRV 873 standard strain, a double-stranded RNA (dsRNA) virus strain, ip, in 0.5 mL. Illumina HiSeqTM 2000 was used for transcriptome sequencing, and real-time polymerase chain reaction (PCR) used to detect complement factors II (C2), III (C3), and V (C5); profibrinolysin (PLG); and coagulation factor II (F2) expression. A total of 2,722,223 reads were detected in the control group, and 2,751,111 in the infected group. Among 11,023 unigenes obtained after transcriptome assembly, 10,021 unigenes were significantly differentially expressed. Gene ontology and KEGG analysis, a collection of databases dealing with genomes and biological pathways, were performed to classify unigenes into functional categories, to understand gene function and identify regulatory pathways. Real-time PCR analysis showed that C2, C3, C5, PLG, and F2 expression levels were down-regulated, confirming results of pathway-enrichment analysis. This is the first application of high-throughput sequencing technology to investigate the in vivo effects of GCRV, on genes and pathways involved in the immune response to infection in grass carp.
Asunto(s)
Carpas/genética , Infecciones por Reoviridae/genética , Bazo/metabolismo , Transcriptoma/genética , Animales , Carpas/virología , Proteínas de Peces/biosíntesis , Proteínas de Peces/genética , Regulación de la Expresión Génica , Reoviridae/patogenicidad , Infecciones por Reoviridae/virología , Bazo/patología , Bazo/virologíaRESUMEN
Here, we characterized the structure and function of the coagulation factor II (FII) gene in grass carp and determined its role in coagulation mechanisms. The FII gene EST was obtained using a constructed splenic transcriptome database; the full-length FII gene sequence was obtained by 3' and 5' RACE. The open reading frame (ORF) of FII was cloned and the full-length gene was found to be 1718 bp, with an ORF of 1572 bp; the gene contained a 25 bp 5'-untranslated region (UTR) and 108 bp 3'-UTR. The ORF encoded 524 amino acids, including 74 alkaline amino acids (arginine and lysine) and 69 acidic amino acids (aspartic acid and glutamic acid). The theoretical pI was 6.22. The calculated instability index (II) was 39.81, indicating that FII was a stable protein; the half-life period was predicted to be approximately 30 h. Amino acid sequence comparisons indicated that grass carp FII showed most similarity (71%) to FII of Takifugu rubripes, followed by Oplegnathus fasciatus (48% similarity) and Larimichthys crocea (47% similarity). A real-time reverse transcription PCR analysis showed that under normal circumstances, FII was most highly expressed in the liver, followed by the gill, spleen, thymus, and head-kidney (P < 0.001). After injection of the grass carp reovirus 873 (GCRV873), the pattern of FII expression was significantly altered (P < 0.001); gene expression was high after injection, suggesting a response involving the initiation of the coagulation system and defense of the body in combination with the platelet and complement system.
Asunto(s)
Carpas/genética , Clonación Molecular , Expresión Génica , Protrombina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Evolución Molecular , Modelos Moleculares , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Filogenia , Conformación Proteica , Protrombina/química , Empalme del ARN , ARN Mensajero/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The study examined the clinicopathological characteristics and treatment options in patients with luminal A breast cancer. This retrospective cohort included 1580 patients with luminal A breast cancer treated between January 2005 and June 2007. Patients were divided into four subgroups according to lymph node status. Prognostic factors and 5-year overall survival (OS) and disease-free survival (DFS) of patients were analyzed. The median duration of follow-up was 67 months. Multivariate Cox-regression analysis revealed that patients in the LN2 and LN3 subgroups had a higher risk of recurrence and death than patients in the LN0 subgroup (LN2: HR = 2.2 for DFS and HR = 2.1 for OS; LN3: HR = 4.7 for DFS and HR = 4.7 for OS). In the LN2 subgroup, there was a trend towards reduced risk of recurrence and death for patients receiving adjuvant chemotherapy plus endocrine therapy, although this difference did not reach statistical significance. In the LN0 and LN1 subgroups, there was a trend towards an increased risk of death in patients receiving chemotherapy. Although lymph node status remains one of the most important independent prognostic predictors for luminal A breast cancer, in patients with 0-3 positive lymph nodes endocrine therapy can be considered sufficient. However, patients with ≥4 positive lymph nodes, and especially in those with ≥ 10, should receive chemotherapy.