DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia,modelling human myeloid leukaemia

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C‐terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs.     

Establishment of Digital RT-PCR Assay for Detection of Swine Influenza Virus H3N2 Subtype

A new assay for the detection of swine influenza virus (SIV) was developed with a novel nucleic acid probe——Molecular beacon in this study. The specific primers and molecular beacon probes were designed according to the conserved region of H3 and N2 genes of SIV H3N2 subtype. A digital RT-PCR assay was developed for detection of SIV H3N2 subtype. The results showed that SIV H3N2 subtype could be identified simultaneously on this microarry with high sensitivity and reproducibility,which could reach to 10⁶ dilute viruse. The conclusion was that the digital RT-PCR method could analyze quantitatively the RNA templates.On the identification of H3N2 SIV,the digital RT-PCR method was much more scientific than Real-time quantitative PCR method.     

Molecular Characterization of Equine Rotavirus Group A Detected in Argentinean Foals During 2009–2014

Equine rotavirus group A (RVA) has been detected in several countries worldwide since its first detection in 1975. Currently, equine RVA is considered the major cause of dehydrating diarrhea in foals younger than 3 months, and the frequency of detection in clinical cases varies from 20% to 77%. The genotypes of epidemiologic relevance found in horses are G3P[12] and G14P[12]. In a survey conducted in Argentina from 1992 to 2008, equine RVA was detected in 21% and 39% of the fecal samples and outbreaks, respectively. Genotype distribution was 51% G3P[12] and 33% G14P[12]. In continuation with the surveillance, the aim of the present study was to characterize the equine RVA detected in Thoroughbred foals in Argentina from 2009 to 2014. A total of 436 stool samples (corresponding to 177 single diarrhea cases or outbreaks) were analyzed. Equine RVA was detected in 31% (135 of 436) of the samples, which corresponded to 42% (74 of 177) of outbreaks. From the positive cases, 42% (57 of 135) were genotyped. Of this, 63% were G3 (36 of 57) and 37% (21 of 57) were G14 genotype. Considering the whole data (1992–2014), equine RVA was detected in 25% (300 of 1,207) of the stool samples and 41% (119 of 293) of the diarrhea outbreaks. The results of this study also show a cyclic pattern of the G3 and G14 prevalence in the horse population with a change in G3:G14 frequencies from year to year. Furthermore, clustering in the phylogenetic tree suggests evolutionary and geographic relationships between the Argentinean strains compared with the strain circulating worldwide.     

3-甲基喹噁啉-2-羧酸的制备及其细胞毒性

通过贝鲁特反应和还原、脱羧反应在体外合成制备3-甲基喹噁啉-2-羧酸,并进行理化鉴定和抗菌活性测试;通过MTT方法研究3-甲基喹噁啉-2-羧酸对多种细胞的生长抑制作用,利用单细胞电泳研究其对细胞DNA的损伤作用,利用流式细胞术研究其对细胞周期的改变。结果显示,体外成功合成制备出3-甲基喹噁啉-2-羧酸,而3-甲基喹噁啉-2-羧酸几乎没有抗菌效果,对多种细胞的生长抑制作用较弱,在剂量检测范围内细胞抑制率不到30%,但在一定剂量下能导致细胞DNA损伤,主要表现为尾长和尾部DNA含量显著升高;并且3-甲基喹噁啉-2-羧酸也能改变Chang细胞的细胞周期,表现为S期阻滞。结果表明,3-甲基喹噁啉-2-羧酸具有一定的细胞毒性。     

Follicular dynamics and changes in oestradiol‐17β, progesterone and LH profiles following PGF2α induced oestrus in early and late ovulating Beetal goats

This study compares the factors associated with variable interval to oestrus and ovulation between early versus late ovulating goats following PGF_2α administration. The time of ovulation in Beetal goats (n = 38) was monitored through transrectal ultrasound at every 6 hr following a single dose of PGF_2α (experiment 1). Variations in oestrus and ovulation times were further explored through the changes in follicular dynamics, endocrine profiles and behaviour in another set of goats (n = 13) following single PGF_2α given randomly during the luteal phase (experiment 2). The ovulation time varied between 60 and 96 hr, and 57% of ovulations occurred by 72 hr following PGF_2α (experiment 1). Accordingly, the goats (n = 13) in the second experiment were retrospectively divided either into early and/or late ovulating, that is, ≤72 and/or ≥84 hr following PGF_2α. The onset of oestrus, peak estradiol‐17β concentration and LH surge after PGF_2α was first observed in early than late ovulating goats (p < 0.05). The goats ovulating early had larger follicle and smaller CL in diameter at the time of PGF_2α administration than those ovulating late (5.4 ± 0.2 vs. 4.3 ± 0.2 mm and 10 ± 0.6 vs. 11.8 ± 0.3 mm, respectively; p < 0.05). Likewise, plasma progesterone concentration tended to be lower (p = 0.087) in early than late ovulating goats. In conclusion, the size of dominant follicle and CL at the time of PGF_2a determines the interval to ovulation following a single dose of PGF_2a during the luteal phase.     

Expression of 14-3-3 σ protein in normal and neoplastic canine mammary gland

14-3-3 σ protein is a negative cell cycle regulator, with both reduced and elevated levels associated with cancer in humans. This study assessed the expression of this protein in canine mammary tissues using immunohistochemistry and Western blotting. 14-3-3 σ was detected in 97% of the mammary tissue samples examined and was found in both myoepithelial (MECs) and epithelial (ECs) cells. Expression levels were elevated and reduced in neoplastic ECs and MECs, respectively (P < 0.001). Intense expression of 14-3-3 σ was detected in neoplastic ECs infiltrating blood vessels and lymph nodes and suggests a possible role for this protein in the malignant transformation of mammary neoplasms. Moreover, double immunostaining for 14-3-3 σ and the MEC – specific marker p63, confirmed that 14-3-3 σ is a highly sensitive marker of MECs since all p63 – positive cells were also positive for 14-3-3 σ. However, this protein is not exclusive to MECs as ECs also labelled positively.     

Low-density lipoprotein-related receptor protein 1 (LRP-1) is not required for insulin-like growth factor binding protein 3 (IGFBP-3) to suppress L6 myogenic cell proliferation

Insulin-like growth factor binding protein-3 (IGFBP-3) suppresses proliferation of numerous cell types, including myogenic cells, via both insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms; however, the mechanism of IGF-independent suppression of proliferation is not clearly defined. In nonmuscle cells, binding of IGFBP-3 to the low-density lipoprotein receptor-related protein-1 (LRP-1)/activated α(2)M receptor is reportedly required for IGFBP-3 to inhibit proliferation. These findings suggest that binding to this receptor also may be required for IGFBP-3 to suppress proliferation of cultured myogenic cells. To investigate the role of the LRP-1 receptor in suppression of myogenic cell proliferation by IGFBP-3, we have examined the effect of receptor-associated protein, an LRP-1 receptor antagonist, on recombinant porcine (rp)IGFBP-3 inhibition of L6 myogenic cell proliferation. Treatment with receptor-associated protein results in a 37% decrease (P < 0.05) in the ability of rpIGFBP-3 to inhibit L6-cell proliferation. In L6 cells subjected to LRP-1 small interfering RNA treatment for 48 h (LRP-1 silenced), LRP-1 mRNA levels were reduced by greater than 80% compared with control cultures treated with nonsense small interfering RNA (mock silenced). In addition, the 85-kDa transmembrane subunit of LRP-1 was undetectable in Western immunoblots of total protein lysates from LRP-1-silenced cells. Even though LRP-1 mRNA and protein levels were dramatically reduced in LRP-1-silenced L6 cells compared with mock-silenced controls, rpIGFPB-3 suppressed proliferation rate to the same extent in both LRP-1-silenced and mock-silenced cultures. Our results strongly suggest that, in contrast to data obtained for nonmuscle cell lines, the LRP-1 receptor is not required for IGFBP-3 to suppress proliferation of L6 myogenic cells.     

人源H3N2亚型猪流感重组病毒的分离鉴定及全基因序列分析

本研究由疑似猪流感病料样品中分离一株病毒,通过HA、HI、电镜观察、生物学特性测定及全基因序列测定表明,分离病毒株为H3N2亚型人流感病毒和H1N1亚型古典猪流感病毒(SIV)的重组体,命名为A/Swine/Fujian/F2/07(H3N2).该分离株含有8个基因片段,共13 442 bp,与GenBank中登录的H3N2亚型人流感病毒、H1N1亚型古典SIV和H3N2亚型SIV进行比较分析显示:分离株的HA、NS、NA基因与H3N2亚型人流感病毒株的同源性分别为84.7 ％～98.1％、94.4 ％～99.5％和88.6 ％～97.6％;与H3N2亚型SIV的核苷酸同源性分别为87.7％～98.5％、82.5 ％～99.9％和87.6 ％～98.4％;M基因与H3N2亚型人流感病毒和SIV的同源性均在90.1％以下,而与H1N1亚型古典SIV的同源性在97.6％以上.基因型分析表明分离株的PB2、PBI、PA、HA、NP、NA和NS基因片段来源于1975年～1982年的人流感病毒,而M基因来源于H1N1亚型古典SIV,充分证明猪作为流感病毒“混合器”的作用.     

3R在免疫功能评价实验中应用的研究

利用实验中有共性的程序和个性特点来进行组合式设计的方法,对某受试的保健食品进行免疫功能的测定。结果用 160 只小鼠就完成了 8 个项目 9 项指标的实验。表明采用组合式设计的方法,既可以完成免疫实验所要求的各项指标,又达到减少和优化的目的。     

H3亚型猪流感病毒荧光定量PCR检测方法的建立

通过RT-PCR方法克隆了H3亚型猪流感病毒HA基因一段靶序列,构建重组质粒作为标准阳性模板.根据GenBank中的H3亚型猪流感病毒HA基因保守序列设计了用于FQ-PCR的1对引物和1条TaqMan探针.通过条件优化,以10倍系列稀释的质粒为标准品进行荧光定量PCR扩增,并制作标准曲线,建立了检测H3亚型猪流感的荧光定量PCR方法.结果表明,该方法检测灵敏度可达1.0×100拷贝/μL,线性范围为109～100,达10个数量级;对起始浓度为1.0×109、1.0×108、1.0×107拷贝/μL的标准品的最终实际测得值(Ct)分别为13.68,18.21和20.57;变异系数分别为0.31%、0.17%和0.12%,均小于5%,说明此方法具有良好的准确性和重现性.对阳性组织病料的检测表明,该方法的检测灵敏度高出常规PCR,与套式PCR具有相近的灵敏度.