Preparation of chicken IgY against recombinant E2 protein of bovine viral diarrhea virus (BVDV) and development of ELISA and ICA for BVDV detection

Bovine viral diarrhea virus (BVDV) infects cattle and may lead to persistent infection (PI). The PI animals harbor BVDV throughout their life and become immune tolerant against BVDV. Thus, diagnosis of this virus in herd is highly important. Recombinant E2 protein expression (using pET-32a in Escherichia coli) was confirmed by SDS-PAGE and Western blotting; then purified by Ni⁺ affinity chromatography. Chickens were immunized with BVDV-E2 protein, and IgY antibodies were extracted from egg yolk by PEG-6000. The peak titer of anti-BVDV-E2-IgY was 1:128,000 after the fifth immunization. IgY-based enzyme-linked immuno sorbent assay (ELISA) and immunochromatographic assay (ICA) were further developed. Coincidence of ELISA and ICA test with RT-PCR was 95.45 and 90.91%, respectively. The anti-BVDV-E2 IgY could be used in routine screening of BVDV infection. Besides, it can also be applicable while licensing and/or using live vaccines; screening of imported products containing bovine serum and strong surveillance of BVDV outbreaks.     

Comparison between principal component analysis and independent component analysis in electroencephalograms modelling

Principal Component Analysis (PCA) is a classical technique in statistical data analysis, feature extraction and data reduction, aiming at explaining observed signals as a linear combination of orthogonal principal components. Independent Component Analysis (ICA) is a technique of array processing and data analysis, aiming at recovering unobserved signals or 'sources' from observed mixtures, exploiting only the assumption of mutual independence between the signals. The separation of the sources by ICA has great potential in applications such as the separation of sound signals (like voices mixed in simultaneous multiple records, for example), in telecommunication or in the treatment of medical signals. However, ICA is not yet often used by statisticians. In this paper, we shall present ICA in a statistical framework and compare this method with PCA for electroencephalograms (EEG) analysis.We shall see that ICA provides a more useful data representation than PCA, for instance, for the representation of a particular characteristic of the EEG named event-related potential (ERP).     

Correlative GC-TOF-MS-based metabolite profiling and LC-MS-based protein profiling reveal time-related systemic regulation of metabolite–protein networks and improve pattern recognition for multiple biomarker selection

A novel approach is presented combining quantitative metabolite and protein data and multivariate statistics for the analysis of time-related regulatory effects of plant metabolism at a systems level. For the analysis of metabolites, gas chromatography coupled to a time-of-flight mass analyzer (GC-TOF-MS) was used. Proteins were identified and quantified using a novel procedure based on shotgun sequencing as described recently (Weckwerth etal., 2004b, Proteomics 4, 78–83). For comparison, leaves of Arabidopsis thaliana wild type plants and starchless mutant plants deficient in phosphoglucomutase activity (PGM) were sampled at intervals throughout the day/night cycle. Using principal and independent components analysis, each dataset (metabolites and proteins) displayed discrete characteristics. Compared to the analysis of only metabolites or only proteins, independent components analysis (ICA) of the integrated metabolite/protein dataset resulted in an improved ability to distinguish between WT and PGM plants (first independent component) and, in parallel, to see diurnal variations in both plants (second independent component). Interestingly, levels of photorespiratory intermediates such as glycerate and glycine best characterized phases of diurnal rhythm, and were not influenced by high sugar accumulation in PGM plants. In contrast to WT plants, PGM plants showed an inversely regulated cluster of N-rich amino acid metabolites and carbohydrates, indicating a shift in C/N partitioning. This observation corresponds to altered utilization of urea cycle intermediates in PGM plants suggesting enhanced protein degradation and carbon utilization due to growth inhibition. Among the proteins chloroplastidic GAPDH (At3g26650) was the best discriminator between WT and PGM plants in contrast to the cytosolic isoform (At1g13440) according to the primary effect of mutation located in the chloroplast. The described method is applicable to all kinds of biological systems and enables the unbiased identification of biomarkers embedded in correlative metabolite–protein networks.     

Delta-opioid augments cardiac contraction through β-adrenergic and CGRP-receptor co-signaling

Cardiac epinephrine and calcitonin gene-related peptide (CGRP) are produced by intrinsic cardiac adrenergic cells (ICA cells) residing in human and animal hearts. ICA cells are neuroparicine cells expressing δ-opioid receptors (DOR). We hypothesized that δ-opioid stimulation of ICA cells enhances epinephrine and CGRP release, which results in the augmentation of heart contraction. Rats were injected with DOR-agonist DPDPE (100 μg/kg) with or without 10-min pretreatment with either β-adrenergic receptor (β-AR) blocker propranolol (2mg/kg) or CGRP-receptor (CGRPR) blocker CGRP(8-37) (300 μg/kg), or their combination. Hemodynamics were monitored with echocardiogram and systolic blood pressure (SBP) was monitored via a tail arterial catheter. Changes in left ventricular fraction-shortening (LVFS) and heart rate (HR) were observed at 5-min after DPDPE infusion. At 5-min DPDPE induced a 36 ± 18% (p<0.001) increase of the LVFS, which continues to increase to 51 ± 24% (p<0.0001) by 10 min, and 68 ± 19% (p<0.001) by 20 min. The increase in LVFS was accompanied by the decrease of HR by 9±5% (p<0.01) by 5 min and 11 ± 6% (p<0.001) by 15 min post DPDPE infusion. This magnitude of HR reduction was observed for the remainder of the 20 min. Despite the HR-reduction, cardiac output was increased by 17 ± 8% (p<0.05) and 28±5% (p<0.001) by 5- and 20-min post DPDPE administration, respectively. There was a modest (9 ± 9%, p=0.03) decrease in SBP that was not apparent until 20 min post DPDPE infusion. The positive inotropism of DPDPE was abrogated in animals pretreated with propranolol, CGRP(8-37), or combined propranolol+CGRP(8-37). Furthermore, in whole animal and cardiomyocyte cell culture preparations, DPDPE induced myocardial protein-kinase A (PKA) activation which was abrogated in the animals pretreated with propranolol+CGRP(8-37). DOR agonists augment myocardial contraction through enhanced β-AR and CGRPR co-signaling.     

胰岛细胞抗体分型与胰岛细胞凋亡的实验研究

研究通过大量临床糖尿病病人胰岛细胞抗体（Islet Cell Antibody,ICA)检测,发现ICA阳性反应有两种完全不同的形态学表现;弥漫型ICA和边缘型ICA,经免疫组织化学双标技术鉴别。弥漫型ICA可同时有着染α细胞和β细胞,边缘型ICA则仅着染α细胞。这种只着染α细胞的ICA国内外尚未见有报道,为探讨其在糖尿病发病中所起的作用。选择1型糖尿病（1-DM）的弥漫型ICA和边缘型ICA各3例。另选正常3例作对照,用患血清分别以2、4、8小时三个时相与分离并贴壁生长的正常人胰岛细胞孵育后,进行原位细胞凋亡检测。结果发现,弥漫型ICA,边缘型ICA均可导致胰岛细胞产生凋亡,其中弥漫型ICA使β细胞及α细胞出现凋亡;边缘型ICA使α细胞产生凋亡,这一结果提示;糖尿病发病机制除与分泌胰岛素的β细胞有密切关系的经典途径之外,可能还与分泌胰高血糖素的α细胞存在某种关系。     

用猪、家兔、大鼠、小鼠和豚鼠胰腺做抗原免疫组织化学检测ICA的比较研究

免疫组织化学检测Ⅰ型糖尿病（ＩＤＤＭ）病人血清中的胰岛细胞抗体（ＩＣＡ）需用“Ｏ”型血人胰腺做抗原。因为人胰腺来源困难,所以,本研究选用猪、家兔、大鼠、小鼠和豚鼠以及“Ｏ”型血正常人胰腺作抗原,对１１例ＩＤＤＭ患者和１０例正常人血清,进行ＩＣＡ免疫组织化学检测,以期筛选寻找ＩＣＡ免疫组织化学检测的最佳抗原替代品。选用已确诊为ＩＤＤＭ并用“Ｏ”型血正常人胰腺作抗原ＩＣＡ检测为阳性患者的血清１１份,正常人血清１０份,同步用ＰＯＰＡ免疫组织化学技术进行检测。结果发现：在１１份ＩＤＤＭ患者血清中,用猪胰腺与用“Ｏ”型血正常人胰腺作抗原,１１例均为阳性,而用家兔和大鼠胰腺作抗原各有１０例为阳性。但用小鼠和豚鼠胰腺作抗原,则分别只有２例和１例呈阳性。在１０例正常人血清中,人和各种动物胰腺均呈阴性反应。本研究结果表明猪胰腺是ＩＣＡ免疫组织化学最佳替代品,其次是家兔和大鼠的胰腺。小鼠和豚鼠胰腺作抗原检测ＩＣＡ结果差异较大     

Numerical simulations of the blood flow in the patient-specific arterial cerebral circle region

The Cerebral Circle Region, also known as the Circle of Willis (CoW), is a loop of arteries that form arterial connections between supply arteries to distribute blood throughout the cerebral mass. Among the population, only 25% to 50% have a complete system of arteries forming the CoW. 3D time-varying simulations for three different patient-specific artery anatomies of CoW were performed in order to gain a better insight into the phenomena existing in the cerebral blood flow. The models reconstructed on the basis of computer tomography images start from the aorta and include the largest arteries that supply the CoW and the arteries of CoW. Velocity values measured during the ultrasound examination have been compared with the results of simulations. It is shown that the flow in the right anterior artery in some cases may be supplied from the left internal carotid artery via the anterior communicating artery. The investigations conducted show that the computational fluid dynamic tool, which provides high resolution in both time and space domains, can be used to support physicians in diagnosing patients of different ages and various anatomical arterial structures.     

Skatole metabolites in urine as a biological marker of pigs with enhanced hepatic metabolism

Boar taint is a quality defect in meat, related to accumulation of skatole and androstenone in male pigs. The levels of skatole and its main metabolites in plasma and urine samples were measured with a validated liquid chromatography-MS method and related to activity of hepatic cytochrome P450 (CYP450) in order to identify ‘fast metabolizing’ pigs. Urine (n=46), blood (n=12), liver (n=25) and adipose tissue (n=46) were sampled from a total of 46 entire male pigs. Skatole levels in fat were negatively correlated to CYP2E1 activity and positively to 3-hydroxy-3-methyloxindole (HMOI), indole-3-carboxylic acid (ICA) and 2-aminoacetophenone in urine. HMOI and ICA levels in urine were the best predictors of high skatole levels in fat. In summary, the present study provided further evidence for the key role of CYP2E1 in skatole metabolism and suggested that measurement of HMOI and/or ICA in urine might provide information about skatole levels in live pigs.