Molecular characterization of Ascaridia galli from Bangladesh and development of a PCR method for distinguishing A. galli from Heterakis spp.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.     

伪狂犬病病毒在NF-κB家族p65基因敲除细胞系的复制规律研究

试验旨在研究伪狂犬病病毒(PRV)在NF-κB家族p65基因敲除细胞系中的复制规律。利用慢病毒介导的CRISPR/Cas9基因定点修饰技术构建猪肺泡巨噬细胞(3D4/21)p65基因稳定敲除细胞系。通过构建p65-sgRNA重组质粒,转染至HEK293T/17细胞,收取慢病毒,感染3D4/21细胞后利用嘌呤霉素筛选获得多克隆细胞系,T7核酸酶检测敲除效率,再通过有限稀释法获得3D4/21-p65^-/-的稳定细胞系。CCK-8试剂盒检测3D4/21细胞中敲除p65基因后对细胞增殖的影响;流式细胞术检测PRV-GFP感染3D4/21及3D4/21-p65^-/-细胞后病毒增殖的差异;实时定量PCR检测PRV感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、TK基因mRNA表达水平及PRV感染细胞诱导的IL-1β和IL-6基因mRNA水平表达的变化;Western blotting检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、gE蛋白的表达;滴度测定检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后子代病毒滴度。结果表明,sgRNA2和sgRNA3的基因编辑效率较高,对其进行克隆化培养进而获得敲除p65基因的稳定表达细胞系;CCK-8试剂盒检测细胞活力表明,p65基因敲除对细胞活力无影响;流式细胞仪检测表明,同一时间点PRV-GFP在3D4/21-p65^-/-中的增殖显著高于对照细胞;实时荧光定量PCR表明在3D4/21细胞中敲除p65基因促进了PRV gB、TK基因的mRNA表达水平,而抑制了IL-1β、IL-6基因的mRNA表达;Western blotting结果表明,在3D4/21细胞中敲除p65基因促进了PRV gB、gE蛋白的表达;滴度测定结果表明,同一时间点PRV-QXX在3D4/21-p65^-/-细胞中子代病毒的复制显著高于对照细胞。以上结果均表明,p65基因敲除可促进PRV在3D4/21细胞中复制。     

干扰素诱导跨膜蛋白1对猪源冠状病毒吸附宿主细胞的影响

为探索干扰素诱导跨膜蛋白1（interferon-inducible transmembrane protein 1,IFITM1）对猪源冠状病毒复制的影响,本研究选用猪传染性胃肠炎病毒（Transmissible gastroenteritis virus,TGEV）高致病毒株及其宿主细胞PK15作为研究对象。正常PK15细胞接种TGEV后,实时荧光定量PCR检测感染后不同时间点PK15细胞中一些干扰素刺激基因（interferon-stimulated genes,ISGs）的mRNA表达水平;利用慢病毒表达系统构建稳定表达和稳定干扰IFITM1表达的PK15细胞系。将一段靶向干扰猪源IFITM1的shRNA序列及猪源IFITM1全长,分别插入pLKO.1-EGFP-Puro载体及pLVML-Myc-MCS-IRES-Puro载体中,分别构建出pLKO.1-IFITM1shRNA-EGFP-Puro及pLVML-Myc-IFITM1-IRES-Puro重组质粒。将重组质粒与慢病毒包装质粒共转染293FT细胞后获得带有目的基因的重组慢病毒,慢病毒侵染PK15细胞后用嘌呤霉素进行筛选,获得稳定表达及稳定干扰IFITM1表达的PK15细胞系,分别命名为PK15-IFITM1及PK15-IFITM1^-/-,并分别用实时荧光定量PCR、间接免疫荧光试验（IFA）及Western blotting检测IFITM1干扰效率及表达情况;TGEV接种PK15-IFITM1^-/-和PK15-IFITM1,实时荧光定量PCR测定细胞中TGEV的拷贝数。结果显示,PK15细胞接种TGEV后的48 h内,一些ISGs的mRNA水平均有所上升;PK15-IFITM1^-/-细胞系的干扰效率为70%,PK15-IFITM1细胞系表达成功;在PK15-IFITM1^-/-细胞系中,IFITM1的mRNA水平显著下调,促进了TGEV的复制。反之,在PK15-IFITM1细胞系中,TGEV的复制受到了抑制。但IFITM1的表达或缺失却不影响TGEV对PK15的吸附作用。总之,IFITM1对TGEV有显著的抗病毒作用,IFITM1不影响TGEV对PK15细胞的早期吸附,这为后续IFITM1抗冠状病毒机制的研究奠定了基础。     

Impaired female fertility in tubulointerstitial antigen-like 1-deficient mice

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) is a matricellular protein. Previously, we demonstrated that Tinagl1 expression was restricted to extraembryonic regions during the postimplantation period and detected marked expression in mouse Reichert’s membranes. In uteri, Tinagl1 is markedly expressed in the decidual endometrium during the postimplantation period, suggesting that it plays a physical and physiological role in embryo development and/or decidualization of the uterine endometrium during pregnancy. In the present study, in order to determine the role of Tinagl1 during embryonic development and pregnancy, we generated Tinagl1-deficient mice. Although Tinagl1^–/– embryos were not lethal during development to term, homologous matings of Tinagl1^–/– females and Tinagl1^–/– males showed impaired fertility during pregnancy, including failure to carry pregnancy to term and perinatal lethality. To examine ovarian function, ovulation was induced with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); the number of ovulated oocytes did not differ between Tinagl1^–/– and Tinagl1^flox/flox. In vitro fertilization followed by embryo culture also demonstrated the normal developmental potential of Tinagl1-null embryos during the preimplantation period. Our results demonstrate that Tinagl1 deficiency affects female mice and results in subfertility phenotypes, and they suggest that although the potential of Tinagl1^–/– oocytes is normal, Tinagl1 is related to fertility in adult females but is not essential for either fertilization or preimplantation development in vitro.     

松口蘑菌塘土壤理化特性对子实体营养品质影响

以四川松口蘑(Tricholoma matsutake)代表性主产区菌塘土壤及新鲜子实体为材料,分析菌塘土壤理化性质和子实体的营养品质,利用冗余分析(redundancy analysis,RDA)研究前者对后者的影响.结果表明:调查样点菌塘土壤理化性质差异较大,总体以砂粒为主(50.2％～88.5％),质地较为疏松,pH总体呈弱酸性(5.08～7.26),有机质含量54.8～189.6 g/kg;调查样点松口蘑子实体营养品质总体较好,其中谷氨酸含量较高(2.27％～4.10％),但地区间差异较大;钙含量较高(124.4～725.1 mg/kg),锌和锰含量中等,铜含量较低;冗余分析结果表明,子实体中营养品质与菌塘土壤中交换性锰(TMn)含量极显著正相关(P＜0.01).菌塘土壤中TMn是影响松口蘑子实体关键的土壤理化因子.     

Plethysmographic comparison of breathing pattern in heaves (recurrent airway obstruction) versus experimental bronchoconstriction or hyperpnea in horses

BACKGROUND: Horses with recurrent airway obstruction (heaves) are described as exhibiting "increased abdominal effort," but it is unknown whether this translates to an effective contribution to ventilation. HYPOTHESIS: We hypothesized that heaves is characterized by asynchrony between rib cage and abdominal motions, and that the abdominal component is the major contributor to ventilation. ANIMALS: The rib cage versus abdominal motion in naturally occurring heaves (n = 15) was compared to controls at rest (n = 7) and during hyperpnea because of lobeline treatment, and the effects of histamine-induced bronchoconstriction in controls (n = 10). METHODS: Flow patterns, phase angle (theta) between the rib and abdominal compartments, abdominal (Vabd) contribution to tidal volume (VT), and lung mechanics were measured. RESULTS: Findings unique to the heaves group included the loss of biphasic expiratory flow, severely increased theta with the abdomen consistently lagging behind the rib cage, and a reduced contribution of the abdomen to ventilation. A subgroup of heaves (n = 5) with abdominal paradox showed a significant (P < .05) reduction in tidal volume, and increased respiratory rate. Bronchodilation reduced theta in heaves (P = .06), but theta remained significantly higher after bronchodilation than found in controls. CONCLUSIONS AND CLINICAL IMPORTANCE: We conclude that breathing pattern in horses with heaves is characterized by severe rib cage/abdominal asynchrony, with the rib cage motion in synchrony with flow, therefore dominating ventilation. In a subset of heaves, the abdominal compartment (diaphragm, abdominal muscles) was completely out of synchrony with flow ("abdominal paradox") despite the clinical appearance of "increased abdominal effort."     

Effects of 2 concentrations of sodium citrate on coagulation test results, von Willebrand factor concentration, and platelet function in dogs

BACKGROUND: Blood collection tubes containing 3.2% (0.109 M) sodium citrate, instead of 3.8% (0.129 M) sodium citrate, have recently become available in the United States. These tubes are visually indistinguishable from the traditional 3.8% sodium citrate tubes, except for wording on the label. Consequently, samples for hemostatic evaluation are frequently collected in tubes containing the lower concentration of sodium citrate. HYPOTHESIS: Results of hemostasis assays are different in samples collected in 3.2% versus 3.8% sodium citrate. ANIMALS: Twenty healthy dogs. METHODS: This study aimed at determining whether results of standard coagulation tests, von Willebrand factor concentration (vWF:Ag), and platelet function with the platelet function analyzer PFA-100a were affected by the different concentrations of sodium citrate. Blood samples were collected in tubes containing either 3.2% or 3.8% sodium citrate concentrations and processed routinely for coagulation assays (one-stage prothrombin time [OSPT], activated partial thromboplastin time [aPTT], fibrinogen concentration, and platelet count), vWF:Ag, and platelet function assays with a PFA-100. RESULTS: There was no significant difference between samples collected in 3.2% versus those collected in 3.8% sodium citrate for OSPT, aPTT, fibrinogen concentration, platelet count, or vWF:Ag. The closure times with collagen/adenosine diphosphate were significantly shorter (66 +/- 8.1 versus 74.8 +/- 9.7 seconds; P < .0001) with the 3.2% than with 3.8% sodium citrate concentration, and the hematocrit was significantly higher (47.9 +/- 5.6 versus 46.0 +/- 4.7 seconds; P = .03) in samples collected in 3.2% than in those collected in 3.8% sodium citrate. CONCLUSIONS AND CLINICAL IMPORTANCE: There is no clinically relevant effect of collection of blood into 3.2% or 3.8% sodium citrate.     

沉默蜕皮激素受体对雌性中黑盲蝽生殖力的影响

中黑盲蝽Adelphocoris suturalis是一种重要的农业害虫,主要为害棉花、果树和牧草等作物。昆虫保幼激素(juvenile hormone, JH)与蜕皮激素(molting hormone, 20E)是调控昆虫生殖的主要因素。本研究运用基因克隆、基因沉默(RNAi)、实时荧光定量PCR(qPCR)等技术研究蜕皮激素受体(ecdysone receptor, EcR)、超气门蛋白(ultraspiracle protein, USP)在中黑盲蝽生殖调控中的作用。结果表明,中黑盲蝽EcR基因的ORF全长1 413 bp,编码470个氨基酸。预测蛋白质分子量为53.65 kD,pI值为7.79。中黑盲蝽USP基因ORF全长1 209 bp,编码402个氨基酸。预测蛋白质分子量为44.99 kD, pI值为7.94。与对照相比,注射dsEcR、dsUSP后6～18 d的中黑盲蝽体内EcR基因、USP基因在转录水平分别被显著抑制;卵巢的挂卵量分别仅有对照组的31.5%、86.6%;单雌产卵量减少36.0%～80.4%,显著低于对照。EcR和USP基因沉默后,产卵前期与对照相比无显...