Expression of Intracellular Interferon-Alpha Confers Antiviral Properties in Transfected Bovine Fetal Fibroblasts and Does Not Affect the Full Development of SCNT Embryos

Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α) (without secretory signal sequence) gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT). Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9%) became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR) and the 2′-5′-oligoadenylate synthetase gene (2′-5′ OAS), which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2′-5′ OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.     

利用泌乳乳腺鉴定真核基因表达的研究

为了找到一种新的可用于基因及其表达调控元件鉴定的方法,本试验以蚓激酶(LK)作为目的标志基因,分别构建了在巨细胞病毒(CMV)立即早期启动子、逆转录病毒长末端重复序列(LTR)和beta-酪蛋白启动子调控下的三种真核表达载体pICLK、pLLKSN和pIbLK。制备各重组质粒后,以泌乳期羊乳腺作为支持系统,在处于稳定泌乳阶段分别注射400~800μg重组质粒于山羊乳腺组织中。在注射后不同时间采集奶汁,检测纤溶活性。结果显示,蚓激酶的不同重组质粒在注射到乳腺组织之后迅速得到表达,注射后6~9h蚓激酶表达量达到高峰,并可持续至72h以上;不同表达载体间表达量略有变化,但无显著差异。这些结果表明,利用泌乳乳腺组织表达真核蛋白是鉴定真核基因表达快速有效的手段之一。     

Utility of Physiologically Based Modeling and Preclinical In Vitro/In Vivo Data to Mitigate Positive Food Effect in a BCS Class 2 Compound

Physiologically based pharmacokinetic (PBPK) modeling has become a useful tool to estimate the performance of orally administrated drugs. Here, we described multiple in silico/in vitro/in vivo tools to support formulation development toward mitigating the positive food effect of NVS123, a weak base with a pH-dependent and limited solubility. Administered orally with high-fat meal, NVS123 formulated as dry filled capsules displayed a positive food effects in humans. Three alternative formulations were developed and assessed in in vitro and in vivo preclinical and/or clinical studies. By integrating preclinical in vitro and in vivo data, the PBPK model successfully estimated the magnitude of food effects and the predicted values were within ±30% of the observed results. A model-guided parameter sensitivity analysis illustrated that enhanced solubility and longer precipitation times under fed condition were the main reason for enhanced NVS123's exposure in presence of food. Eventually, exposure after an amorphous formulation was found to be not significantly altered because of remarkably enhanced intestinal solubility and reduced precipitation. Gastroplus population simulations also suggested that the amorphous formulation is promising in mitigating a clinically significant food effect. Overall, these efforts supported the rationale of clinical investigation of the new formulation, and more importantly, highlighted a practical application of PBPK modeling solving issues of undesirable food effects in weakly basic compounds based on preclinical in vitro/in vivo data.     

An Estimation of the Manganese Requirement for Broilers from 1 to 21 Days of Age

A dose–response experiment was conducted to find the sensitive and consistent biomarker for the estimation of dietary manganese (Mn) requirement and establish the optimal Mn level for broilers fed a practical corn–soybean meal diet from 1 to 21 days of age post-hatching. A total of 480 1-day-old Arbor Acres male chicks were randomly allotted to one of eight treatments with five replicates of 12 birds each and fed diets supplemented with 0, 20, 40, 60, 80, 100, 120, or 140 mg Mn/kg from reagent grade Mn sulfate. Tissue Mn concentrations, manganese-containing superoxide dismutase (MnSOD) activity, and MnSOD mRNA concentration within heart tissue were analyzed at 7, 14, and 21 days of age. Tissue Mn concentrations and heart MnSOD activity showed significant quadratic responses, and heart MnSOD mRNA concentration showed an asymptotic response to dietary supplemental Mn level, respectively. The estimate of dietary Mn for chicks from 1 to 21 days of age was 122–128 for heart Mn concentration, 141–159 for pancreas Mn concentration, 127–138 for liver Mn concentration, and 135–156 mg/kg for heart MnSOD activity, respectively. Heart MnSOD mRNA concentration was a consistent index for the estimation of the Mn requirement of broilers. Based on this index, the estimate of dietary Mn requirement for broilers from 1 to 21 days of age post-hatching was about 130 mg/kg, which was a little more than two times of the current NRC (1994) requirement.     

Label-free electrochemical detection of DNA using ferrocene-containing cationic polythiophene and PNA probes on nanogold modified electrodes

A label-free electrochemical method for the detection of DNA-PNA hybridization using a water-soluble, ferrocene-functionalized polythiophene transducer and single-stranded PNA probes on the nanogold modified electrode is investigated. Nanogold modified electrodes can largely increase the immobilization amount of ss-PNA capture probe and lead to an increase of the electrical signal. The ferrocene-containing cationic polythiophene do not interact electrostatically with the PNA probes due to the absence of the anionic phosphate groups on the PNA probes. But after DNA-PNA hybridization, cationic polythiophene is adsorbed on the DNA backbone, giving a clear hybridization detection signal in differential pulse voltammetry (DPV). Very good discrimination against non-complementary DNA and four-base mismatch DNA is observed. These studies show that the proposed method can provide an alternative for expanding the range of detection methods available for DNA hybridization.     

白色链霉菌DSM 41398中洋橄榄菌素和放线吡喃酮的发现及其生物合成途径分析

【目的】从菌株Streptomyces albus DSM 41398的发酵产物中发掘结构多样的由I型聚酮合酶催化形成的化合物,以期找到具有新颖结构或强生物活性的化合物。在结构鉴定的基础上,对其生物合成途径进行分析。【方法】利用HPLC分析方法,通过系统比较野生型菌株S.albus DSM 41398与I型聚酮合酶编码基因簇失活突变株的发酵产物差异,实现目标化合物的定向分离。然后,利用~1H-和~(13)C-NMR以及HR-ESI-MS进行化合物的结构鉴定。最后,利用生物信息学等方法对化合物的生物合成途径进行推测和分析。【结果】从5 L的S.albus DSM 41398发酵产物中,分离得到了2个具有抗肿瘤活性的聚酮类化合物放线吡喃酮和洋橄榄菌素,分别定位了它们的生物合成基因簇,并分别对其生物合成途径进行了推导。其中,放线吡喃酮的生物合成基因簇为首次报道。【结论】本研究一方面为基因组发掘S.albus DSM 41398中其他由I型聚酮合酶催化形成的化合物提供参考,另一方面也为相关化合物的结构修饰改造奠定了良好的基础。