Estimating the accuracy of polymerase chain reaction-based tests using endpoint dilution

Polymerase chain reaction (PCR)-based tests for various microorganisms or target DNA sequences are generally acknowledged to be highly "sensitive," yet the concept of sensitivity is ill-defined in the literature on these tests. We propose that sensitivity should be expressed as a function of the number of target DNA molecules in the sample (or specificity, when the target number is 0). However, estimating this "sensitivity curve" is problematic, since it is difficult to construct samples with a fixed number of targets. Nonetheless, using serially diluted replicate aliquots of a known concentration of the target DNA sequence, we show that it is possible to disentangle random variations in the number of target DNA molecules from the underlying test sensitivity. We develop parametric, nonparametric, and semiparametric (spline-based) models for the sensitivity curve. The methods are compared on a new test for M. genitalium.     

Maillard reaction-like lysine modification by a lipid peroxidation product: immunochemical detection of protein-bound 2-hydroxyheptanal in vivo

2-Hydroxyheptanal (2-HH) is one of the reactive aldehyde species generated during the peroxidation of n-6 polyunsaturated fatty acids, such as linoleic and arachidonic acids. Analogous to the Maillard reaction of reducing sugars, 2-HH readily reacts with lysine epsilon-amino groups. In the present study, to define the occurrence of the Maillard reaction-like lysine modification by 2-HH in vivo, we raised a monoclonal antibody directed to a trihydropyridinone (THPO) structure, 1-alkyl-4-butyl-5-pentyl-1,2,6-trihydropyridin-3-one, formed from 2-HH and lysine, and examined the presence of the antigenic structure in the human atherosclerotic aorta. Mice were immunized with the 2-HH-modified keyhole limpet hemocyanin (KLH) as the immunogen. Using a THPO-carrier protein conjugate, we screened the hybridomas and finally obtained a clone that produced the monoclonal antibody 3C8 (mAb3C8). The antibody strongly recognized bovine serum albumin (BSA) treated with 2-HH, but showed no cross-reactivity with BSAs modified with other related aldehydes. By using this antibody, it was revealed that the antigenic structure was indeed present in atherosclerotic lesions of the human aorta.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)     

A mathematical design of vector vaccine against autoimmune disease

Viruses have been implicated in the initiation, progression, and exacerbation of several human autoimmune diseases. Evidence also exists that viruses can protect against autoimmune disease. Several proposed mechanisms explain the viral effects. One mechanism is “molecular mimicry” which represents a shared immunologic epitope with a microbe and the host. We consider, using a simple mathematical model, whether and how a viral infection with molecular mimicry can be beneficial or detrimental for autoimmune disease. Furthermore, we consider the possibility of development of a vector therapeutic vaccine that can relieve autoimmune disease symptoms. Our findings demonstrate that vaccine therapy success necessitates (i) appropriate immune response function, (ii) appropriate affinities with self and non-self antigen, and (iii) a replicative vector vaccine. Moreover, the model shows that the viral infection can cause autoimmune relapses.     

Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.     

Trypanosoma cruzi (Kinetoplastida Trypanosomatidae): ecology of the transmission cycle in the wild environment of the Andean valley of Cochabamba, Bolivia

An active Trypanosoma cruzi transmission cycle maintained by wild rodents in the Andean valleys of Cochabamba Bolivia is described. Wild and domestic Triatoma infestans with 60% infection with T. cruzi were found and was evidenced in 47.5% (rodents) and 26.7% (marsupial) by parasitological and/or serologycal methods. Phyllotis ocilae and the marsupial species Thylamys elegans, are the most important reservoirs followed by Bolomys lactens and Akodon boliviensis. In spite of both genotypes (TCI and TCII) being prevalent in Bolivia, in our study area only T. cruzi I is being transmitted. Our data suggest that wild T. infestans and wild small mammals play an important role in the maintenance of the transmission cycle of T. cruzi. Furthermore, the finding of high prevalence of T. cruzi infection in wild T. infestans point to the risk of the dispersion of Chagas' disease.     

草原沙漠化过程中植物的耐胁迫类型研究

以内蒙古锡林郭勒盟多伦县草原沙漠化过程中不同梯度共有种群为研究对象 ,探讨植物的受损机理 ,综合 2 0 0 1～ 2 0 0 3年的研究结果表明 :随着沙漠化的进展 ,共有种群的 1叶片含水量及叶绿素 a、b和总叶绿素含量均呈降低趋势。叶片含水量高低顺序为扁蓿豆 (Melilotoides ruthenica) >冷蒿 (Artemisia frigida) >羊草 (L eymus chinensis) >糙隐子草 (Cleistogenessquarrosa) ,反映了共有种群水分状况的不同。2质膜相对透性、游离脯氨酸含量均呈上升趋势 ,其中羊草、糙隐子草和冷蒿变化表现出先升后降再升的规律。3MDA含量变化均表现出先升后降再升的规律 ,不同植物不同阶段 MDA积累程度 (积累率大小顺序为沙漠化初期 :冷蒿 2 9.5 9% >羊草 18.14 % >扁蓿豆 13.6 8% >糙隐子草 3.77% ;沙漠化后期 :糙隐子草 80 .11% >冷蒿 77.2 9% >羊草 39.31% >扁蓿豆 8.5 6 % )的不同反映了不同阶段细胞受伤害程度的差异。4 SOD、CAT活性变化总体上均呈上升趋势 ,其中羊草 CAT活性呈降低趋势 ;酶活性均梯度间差异显著 (P<0 .0 0 1)。 5内源激素 ABA含量的变化因不同种群抗逆性强弱而异。糙隐子草、冷蒿和扁蓿豆 ABA增幅小 ,而羊草 ABA增幅大。综合各种生理特征及其对沙漠化环境的响应 ,对共有种群分类 :羊草为敏感型     

芽胞抗力快速弱化技术研究

以L-丙氨酸缓冲液为发芽剂,结合芬顿反应原理,观察发芽-氧化损伤效应对芽胞的杀灭效果,以期为新型炭疽疫源地净化方法的深入研究奠定基础。以腊样芽胞为试验菌,采用透射电镜、激光扫描共聚焦显微镜、活菌计数等方法观察芽胞发芽过程的超微结构、核酸含量变化,以及在芬顿反应的联合作用下发芽体的活性变化。在20~30 min的发芽过程中,芽胞核心密度降低,核心与皮质、皮质与外壁之间界限模糊,芽胞外壁和芽胞衣有破裂,通透性增加,进一步有皮质消失、细胞核与细胞质融合、细胞膜基本形成的现象;发芽体荧光强度不断增加,显示菌体中核酸的活性和含量不断增加;发芽体对化学因子的抗力明显下降,H2O2浓度为0.20 mol/L的Fenton反应系统作用60 min时,发芽体灭活可达到3.016个对数级。诱导发芽和反应的联合处理程序可显著提高芽胞的灭活水平。