Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)_n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.     

The Effect of TCM-Induced HAMP on Key Enzymes in the Hydrolysis of AD Model Cells

Neurochemical Research - Alzheimer’s disease (AD) process is characterized classically by two hallmark pathologies: β-amyloid (Aβ) plaque deposition and neurofibrillary tangles of...     

Deliberately infecting healthy volunteers with malaria parasites: Perceptions and experiences of participants and other stakeholders in a Kenyan-based malaria infection study

Controlled human malaria infection (CHMI) studies involve the deliberate infection of healthy volunteers with malaria parasites under controlled conditions to study immune responses and/or test drug or vaccine efficacy. An empirical ethics study was embedded in a CHMI study at a Kenyan research programme to explore stakeholders’ perceptions and experiences of deliberate infection and moral implications of these. Data for this qualitative study were collected through focus group discussions, in-depth interviews and non-participant observation. Sixty-nine participants were involved, including CHMI study volunteers, community representatives and research staff. Data were managed using QSR Nvivo 10 and analysed using an inductive-deductive approach, guided by ethics literature. CHMI volunteers had reasonable understanding of the study procedures. Decisions to join were influenced by study incentives, trust in the research institution, their assessment of associated burdens and motivation to support malaria vaccine development. However, deliberate malaria infection was a highly unusual research strategy for volunteers, community representatives and some study staff. Volunteers’ experiences of physical, emotional and social burdens or harms were often greater than anticipated initially, and fluctuated over time, related to specific procedures and events. Although unlikely to deter volunteers' participation in similar studies in furture, we argue that the dissonance between level of understanding of the burdens involved and actual experiences are morally relevant in relation to community engagement, informed consent processes, and ongoing support for volunteers and research staff. We further argue that ethics oversight of CHMI studies should take account of these issues in deciding whether consent, engagement and the balance of benefits and harms are reasonable in a given context.     

濒危植物永瓣藤叶片功能性状对环境因子的响应

植物叶片功能性状能够响应环境条件的变化，反应了植物对环境的适应策略。当前，针对藤本植物叶片功能性状地理格局及其环境驱动力的研究较少。以国家重点保护植物永瓣藤（Monimopetalum chinense）为研究对象，对其分布区内11个种群的15个叶片功能性状进行测量，并结合气候、土壤因子来解释叶性状变异。比较叶片性状在局域和区域尺度上的种内变异程度，利用多元逐步回归分析环境因子对叶性状的影响。结果表明，在局域尺度上，永瓣藤叶功能性状变异系数介于3.0%-22.5%，其中，叶面积变异程度最大，叶片碳含量变异最小。永瓣藤叶片形状随纬度上升而变得宽且圆。叶片磷含量相对较低，永瓣藤的生长可能受到了磷限制。土壤与气候因子是叶片性状的重要驱动因素，解释了25%-97%的叶片性状变异。在温度和水分充足的情况下，永瓣藤叶片趋向于的慢速生长的保守策略。总体来说，永瓣藤叶片功能性状通过一定的种内变异和性状组合，并与气候、土壤因子相互作用，适应当前的环境条件。     

Ontogeny of head and caudal fin shape of an apex marine predator: The tiger shark (Galeocerdo cuvier)

How morphology changes with size can have profound effects on the life history and ecology of an animal. For apex predators that can impact higher level ecosystem processes, such changes may have consequences for other species. Tiger sharks (Galeocerdo cuvier) are an apex predator in tropical seas, and, as adults, are highly migratory. However, little is known about ontogenetic changes in their body form, especially in relation to two aspects of shape that influence locomotion (caudal fin) and feeding (head shape). We captured digital images of the heads and caudal fins of live tiger sharks from Southern Florida and the Bahamas ranging in body size (hence age), and quantified shape of each using elliptical Fourier analysis. This revealed changes in the shape of the head and caudal fin of tiger sharks across ontogeny. Smaller juvenile tiger sharks show an asymmetrical tail with the dorsal (upper) lobe being substantially larger than the ventral (lower) lobe, and transition to more symmetrical tail in larger adults, although the upper lobe remains relatively larger in adults. The heads of juvenile tiger sharks are more conical, which transition to relatively broader heads over ontogeny. We interpret these changes as a result of two ecological transitions. First, adult tiger sharks can undertake extensive migrations and a more symmetrical tail could be more efficient for swimming longer distances, although we did not test this possibility. Second, adult tiger sharks expand their diet to consume larger and more diverse prey with age (turtles, mammals, and elasmobranchs), which requires substantially greater bite area and force to process. In contrast, juvenile tiger sharks consume smaller prey, such as fishes, crustaceans, and invertebrates. Our data reveal significant morphological shifts in an apex predator, which could have effects for other species that tiger sharks consume and interact with. J. Morphol. 277:556–564, 2016. © 2016 Wiley Periodicals, Inc.     

Rapid improvement of rice eating and cooking quality through gene editing toward glutelin as target

Rice eating and cooking quality (ECQ) is a major concern of breeders and consumers, determining market competitiveness worldwide. Rice grain protein content (GPC) is negatively related to ECQ, making it possible to improve ECQ by manipulating GPC. However, GPC is genetically complex and sensitive to environmental conditions; therefore, little progress has been made in traditional breeding for ECQ. Here, we report that CRISPR/Cas9-mediated knockout of genes encoding the grain storage protein glutelin rapidly produced lines with downregulated GPC and improved ECQ. Our finding provides a new strategy for improving rice ECQ.     

酸敏感离子通道与脑梗死

急性脑梗死约占全部脑卒中的70%,病死率和致残率高,且极易复发。但目前针对急性脑梗死在时间窗内溶栓、抗凝等治疗手段不能从根本上切实有效地修复受损脑组织,且伴有出血等风险。寻找脑梗死形成发展的原因并予以治疗迫在眉睫。酸中毒是引起缺血性脑损伤的重要机制。大量实验研究表明,酸中毒能加重神经元的缺血性损伤,且其梗死面积与酸中毒的程度直接相关。但缺血产生的酸中毒如何引起神经元损伤的确切机制尚不明确。最近研究发现酸中毒能激活一种在中枢及周围神经中广泛存在的膜通道,即酸敏感离子通道,它对Ca2+通透,能引起细胞内Ca2+超载,同时能激活胞内酶引起细胞内蛋白质、脂类及核酸的降解,加重缺血后脑损伤。本文就酸敏感离子通道1a与脑梗死做一综述。     

Analyzing the normal mode dynamics of macromolecules by the component synthesis method: Residue clustering and multiple-component approach

A study of the component synthesis method (CSM) for analyzing the normal mode dynamics of macromolecules is reported. The procedure involves a reduction of the dimensions of the normal mode problems for large molecular systems and the accurate extraction of the low-frequency modes. A macromolecule is divided into small components based on a hierarchical clustering of the residues in the structure. Interactions between coupled components are treated by the method of static correlation. The normal modes of the components are obtained first, and a fraction of the low-frequency normal modes of the components under mutual correlations are then used as a reduced basis for solving for the normal modes of the whole molecule. Multiple components are introduced for large macromolecules so that the dimensions of the eigenvalue problems at the component level are small. The method is applied to the protein crambin. In test calculations in which the dimensions of the eigenvalue equations are reduced to 1/6 of their natural size, the errors in the normal mode frequencies calculated by the CSM procedure are only about 1–2% when compared with the exact values. The rms fluctuations of all atoms in crambin calculated by the CSM procedure are basically identical to the exact results. The CSM procedure is shown to be accurate for calculating the normal modes of large macromolecules with a significant reduction of the size of the problem. © 1994 John Wiley & Sons, Inc.