Epitope selection and their placement for increased virus neutralization in a novel vaccination strategy for porcine epidemic diarrhea virus utilizing the Hepatitis B virus core antigen

Porcine epidemic diarrhea virus (PEDV) is a member of the Alphacoronaviridae genus within the Coronaviridae family. It is the causative agent of porcine epidemic diarrhea, a disease that can have mortality rates as high as 100% in suckling piglets. PEDV causes severe economic loss, and has been in existence for decades. A panzootic starting in 2010 renewed interest in the development of a universal vaccine toward PEDV. This report details several design changes made to a Hepatitis B virus core antigen (HBcAg)-based recombinant vaccine strategy, and their effect in vivo. Initially, several multi-antigen vaccine candidates were able to elicit antibodies specific to three out of four B-cell epitopes inserted into the chimeric proteins. However, a lack of virus neutralization led to a redesign of the vaccines. The focus of the newly redesigned vaccines was to elicit a strong immune response to the YSNIGVCK amino acid motif from PEDV. Genetically modified new vaccine candidates were able to elicit a strong antibody (Ab) response to the YSNIGVCK epitope, which correlated with an increased ability to neutralize the CO strain of PEDV. Additionally, the location of the inserted PEDV epitopes within the vector protein was shown to affect the immune recognition toward the native HBcAg during vaccination.     

Reference antigen-free and antibody-free LTD-IDMS assay for influenza H7N9 vaccine in vitro potency determination

Influenza vaccines are the most effective intervention to prevent the substantial public health burden of seasonal and pandemic influenza. Hemagglutinin (HA), as the main antigen in inactivated influenza vaccines (IIVs), elicits functional neutralizing antibodies and largely determines IIV effectiveness. HA potency has been evaluated by single-radial immunodiffusion (SRID), the standard in vitro potency assay for IIVs, to predict vaccine immunogenicity with a correlation to protective efficacy. We previously reported that limited trypsin digestion (LTD) selectively degraded stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique could specifically quantify trypsin-resistant, immunologically active HA. Here, we demonstrate that isotope dilution mass spectrometry (IDMS), a method capable of quantifying the absolute HA concentration without reference antigen use, can be further expanded by adding LTD followed with precipitation to selectively quantify the active HA. We test the LTD-IDMS assay on H7N9 vaccines stressed by low pH, raised temperature, or freeze/thaw cycles. This method, unlike SRID, has no requirement for strain-specific reference antigens or antibodies and can generate potency values that correlate with SRID. Thus, LTD-IDMS is a promising alternative in vitro potency assay for influenza vaccines to complement and potentially replace SRID in a pandemic when strain specific reagents may not be readily available.     

成年大鼠心肌细胞培养方法的建立和形态学观察

为探索稳定的成年大鼠心肌细胞的分离和培养方法 ,应用生物酶 ( 5g/L胰蛋白酶及 1 g/L胶原酶 )灌注法分离成年大鼠心肌细胞 ,用形态学及台盼蓝染色法鉴定分离的心肌细胞 ,并在光镜和电镜下观察和摄像记录。结果 :心肌细胞的存活率为 91 .7% ;存活的心肌细胞静止地贴在培养板底上 ,细胞完整 ,呈杆状 ,长宽比约为 4～ 6∶1。结果提示 :该方法是比较理想的心肌细胞培养法。     

体外循环期间乌司他丁对炎性因子变化的影响

目的　观察乌司他丁 (UTI)对体外循环 (CPB)心脏手术期间体液因子变化的影响 ,探讨其手术期间的保护作用。方法　 30例择期CPB心脏手术病人 ,随机分为两组 (每组 15例 ) ,乌司他丁组 (U组 )给予乌司他丁 2万U kg ,对照组 (C组 )给予等量的生理盐水。分别于麻醉诱导后 (t1 )、主动脉开放后 10min(t2 )、CPB停机后 10min(t3)、CPB停机后 1h(t4 )测定血浆TNF -α、ET - 1、TXA2 的水平及血浆丙二醛 (MDA)的浓度和过氧化物歧化酶 (SOD)的活性。结果　两组的TNF -α在t2 升高 ,U组在t3开始下降 ,C组持续至t4 与基础值相比仍明显升高。ET - 1在t2 升高 ,并逐渐下降 ,两组比较差异无显著性意义。两组MDA在t2 、t3升高 ,但U组明显低于C组 ,且U组MDA在t4 恢复至基础水平 ,而C组仍明显高于基础值 (P <0 .0 1)。两组的SOD活性在主动脉开放后至CPB停机后 1h持续升高 ,在t2 、t3U组SOD活性显著高于C组 (P <0 .0 1)。两组的TXA2 在t2 、t3较t1 明显升高 ,且C组与U组相比差异有显著性意义 ,U组的TXA2 在t4 已恢复至基础值 ,C组仍明显高于基础值。结论　乌司他丁可以抑制体外循环时TNF -α、TXA2 等炎性因子的产生 ,并能提高SOD的活性 ,在行体外循环的心脏手术中应用前景广阔。     

Structure analysis of trypsin inhibitors isolated from Cucurbitacae seeds. Circular dichroism studies

Structure analysis jointly based on circular dichroism and Chou and Fasman analysis has been performed on a homologous group of 12 squash proteinase inhibitors. The analysis favours a structure incorporating 40–45% helix which is tightly crosslinked by three disulphide bonds. The proposed structure is consistent with the previously published CD and ¹H n.m.r. spectra. The amino acid which forms the centre of the interaction between trypsin and the inhibitor, arginine⁵ or lysine⁵, is located on the N-terminus of one of the helical segments. The basic residue is orientated away from the rest of the structure and thus is ideally situated for interaction with the confined space of the trypsin active site.     

比较四种消化方法对神经干细胞传代增殖及活性的影响

目的：观察4种消化方法对体外培养的神经干细胞（N SC s）增殖及活性的影响。方法：原代培养新生大鼠海马N SC s,采用机械消化、0.25%单纯胰酶、0.25%胰酶＋0.02%EDTA和0.02%EDTA 4种不同消化方法对N SC s球进行传代培养,经四唑盐比色法（M TT法）法检测不同方法对N SC s活性及增殖能力的变化。结果：与机械消化相比,0.25%单纯胰酶组,0.25%胰酶＋EDTA组消化后的大鼠海马N SC s球长时间不聚集,细胞增殖停顿,而单纯EDTA组则明显提高了N SC s的增殖及活性,其余各组差异无统计学意义。结论：随培养时间延长,单纯0.02%EDTA组能够显著提高体外培养的大鼠海马N SC s的增殖水平及活力,且对细胞的后续损伤相对较小。     

一种有效分离人尿激肽释放酶和人尿胰蛋白酶抑制剂的方法

人尿激肽释放酶和人尿胰蛋白酶抑制剂是存在于尿中的酸性蛋白.本文通过ZnCl2胶体沉淀和乙醇沉淀相结合的方法可有效分离两者,得率高、活性保留好.     

乌司他丁对法洛氏四联症根治患者体外循环期间全身炎症反应的影响

目的观察乌司他丁对法洛氏四联症根治术患者体外循环期间全身炎性反应的影响。方法拟施法洛氏四联症根治术患者40例,性别不限,体重12—47kg,年龄2-18岁,ASA II—III级,用随机、双盲方法分为2组（n=20）：对照组（c组）和乌司他丁组（u组）。u组于麻醉后静脉恒速注射乌司他丁8000U／kg,CPB开始前输完,然后以4000u／（kg．h）的速率持续静脉输注至CPB结束,c组以等容量生理盐水替代乌司他丁。于CPB前5min（T1）、CPB开始后10min（T2）、CPB结束后30min（T3）、60min（T4）时测定血浆白细胞介素6（IL-6）、IL-8、IL-10及肿瘤坏死因子a（TNFa）的浓度。结果与C组比较,u组CPB期间和CPB结束后血浆IL-6、IL8和TNF-a的浓度降低,IL-10浓度升高（P〈0．05或0．01）;与T1比较T2．3时两组血浆IL-6、IL-8、IL-10、TNF—a的浓度升高（P〈0．01）。结论乌司他丁可减低法洛氏四联症根治术患者CPB期间促炎．抗炎反应失衡,减轻全身炎性反应。     

A method for in vitro enzymatic dissociation of nerve roots and peripheral nerves from adult mammals

Preparations yielding a high percentage of undamaged axons from fresh peripheral nerve or nerve root were made using an enzymatic dissociation regimen. The nerve was placed in a temperature-controlled chamber mounted over an inverted phase-contrast microscope. An oxygenated solution (Brimijoins) or modified Hank's solution was pumped through the chamber, first in a calcium-free form and then containing enzymes. The enzymes for dissociation were collagenase and trypsin, alternated. Enzymatic dissociation of the epineurium, perineurium and extracellular matrix was achieved. We supplemented the gentle agitation of a 10-roller peristaltic pump by periodically raising and lowering the fluid level in the chamber to provide a controlled mechanical agitation that promoted dissociation. A large percentage of the axons can be dissociated from the nerve, varying from approximately one-quarter to occasional complete dissociation. Action potentials were still conducted through dissociated axons, and axon transport was also still present, as documented by direct visualization using an AVEC-DIC type of microscope system. The axons had a better morphological appearance and displayed better transport than comparison preparations prepared by the usual mechanical teasing method, in our hands. The enzymatic method allows study of axons in an adult or developing mammal with regard to their electrical conduction and axon transport mechanisms. It should help to avoid a selection process for more hardy axons which may be imposed by traditional mechanical teasing methods. Mechanical stress was observed to cause widened Schmidt-Lanterman clefts, widened nodes, myelin bubbles, and other abnormal morphology as evidence of damage.