Microfluidic‐Assisted Blade Coating of Compositional Libraries for Combinatorial Applications: The Case of Organic Photovoltaics

Microfluidic technologies are highly adept at generating controllable compositional gradients in fluids, a feature that has accelerated the understanding of the importance of chemical gradients in biological processes. That said, the development of versatile methods to generate controllable compositional gradients in the solid‐state has been far more elusive. The ability to produce such gradients would provide access to extensive compositional libraries, thus enabling the high‐throughput exploration of the parametric landscape of functional solids and devices in a resource‐, time‐, and cost‐efficient manner. Herein, the synergic integration of microfluidic technologies is reported with blade coating to enable the controlled formation of compositional lateral gradients in solution. Subsequently, the transformation of liquid‐based compositional gradients into solid‐state thin films using this method is demonstrated. To demonstrate efficacy of the approach, microfluidic‐assisted blade coating is used to optimize blending ratios in organic solar cells. Importantly, this novel technology can be easily extended to other solution processable systems that require the formation of solid‐state compositional lateral gradients.     

我国自然保护地历史遗留问题的系统解决方案

近年来, 我国自然保护地内存在的大量的人口密集区域、民生设施、矿业活动、开发建设项目、农牧业生产活动等历史遗留问题在中央环保督察中集中暴露出来。为采取针对性政策措施解决这些遗留问题, 本研究首先将其分为三大类: (1)保护越位与保护空缺并存; (2)生态保护和大量原住居民生计改善间的矛盾突出; (3)保护地内存在大量生产经营活动。在此基础上, 深入剖析了各类遗留问题产生的体制机制原因。为合理化解历史遗留问题导致的一系列矛盾, 提高保护地建设和管理质量, 本文从加强保护地体系的顶层设计、推动相关法律法规的制修订、采取多元化手段解决保护地内土地权属和矿业权问题、建立健全平衡保护与发展关系的体制机制等4个方面提出了历史遗留问题的系统性解决方案。     

Optimization and validation of a cost‐effective protocol for biosurveillance of invasive alien species

Environmental DNA (eDNA) metabarcoding has revolutionized biodiversity monitoring and invasive pest biosurveillance programs. The introduction of insect pests considered invasive alien species (IAS) into a non‐native range poses a threat to native plant health. The early detection of IAS can allow for prompt actions by regulating authorities, thereby mitigating their impacts. In the present study, we optimized and validated a fast and cost‐effective eDNA metabarcoding protocol for biosurveillance of IAS and characterization of insect and microorganism diversity. Forty‐eight traps were placed, following the CFIA''s annual forest insect trapping survey, at four locations in southern Ontario that are high risk for forest IAS. We collected insects and eDNA samples using Lindgren funnel traps that contained a saturated salt (NaCl) solution in the collection jar. Using cytochrome c oxidase I (COI) as a molecular marker, a modified Illumina protocol effectively identified 2,535 Barcode Index Numbers (BINs). BINs were distributed among 57 Orders and 304 Families, with the vast majority being arthropods. Two IAS (Agrilus planipennis and Lymantria dispar) are regulated by the Canadian Food Inspection Agency (CFIA) as plant health pests, are known to occur in the study area, and were identified through eDNA in collected traps. Similarly, using 16S ribosomal RNA and nuclear ribosomal internal transcribed spacer (ITS), five bacterial and three fungal genera, which contain species of regulatory concern across several Canadian jurisdictions, were recovered from all sampling locations. Our study results reaffirm the effectiveness and importance of integrating eDNA metabarcoding as part of identification protocols in biosurveillance programs.     

An exact solution of transient equations describing slow axonal transport

An exact analytical solution of equations describing slow axonal transport of cytoskeletal elements (CEs) injected in an axon is presented. The equations modelling slow axonal transport are based on the stop-and-go hypothesis. The simplest model implementing this hypothesis postulates that CEs switch between pausing and running kinetic states, and that the probabilities of CE transition between these two states are described by first-order rate constants. It is assumed that initially CEs are injected such that they form a uniform pulse of a given width. All injected CEs are initially attributed to the pausing state. It is shown that within 30 s kinetic processes redistribute CEs between pausing and running states; after that the process occurs under quasi-equilibrium conditions. The parameter accessible to experiments is the total concentration of CEs (pausing plus running). As the initial rectangular-shaped pulse moves, it changes its shape to become a bell-shaped wave that spreads out as it propagates. The wave's amplitude is decreasing during the wave's propagation. It is also shown that the system forgets its initial condition, meaning that if one starts with pulses of different widths, after sometime they converge to the same bell-shaped wave.     

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.     

Robust numerical solution for the two parameter solute solvent transport model

Prediction of solute and solvent transport in cells is central to developing and testing cryopreservation protocols. As we show here, however, the models used can be difficult to accurately numerically integrate in some key cases, and thus are a challenge to implement when determining the time dependent cell state during cryoprotectant equilibration and cooling. Exact solution techniques exist for overcoming this problem, but their implementation is also challenging: inversion of a nonlinear function is required that negates much of the utility of the approach. This communication describes a simple approach for more robust numerical integration that can be implemented using any numerical differential equation solver, and can facilitate arbitrarily accurate solutions to transport models without the complication of inversion formulae or complicated numerical integration schemes. Further, a simple relevant example of red blood cell equilibration with 40% glycerol is presented with comments on extending the approach to other settings.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

Solution structures of Mycobacterium tuberculosis thioredoxin C and models of intact thioredoxin system suggest new approaches to inhibitor and drug design

Here, we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C‐terminal region. It appears that crystal packing effects have caused an artifactual shift in the α4 helix in the previously reported crystal structure, compared with the solution structure. On the basis of these TrxC structures, chemical shift mapping, a previously reported crystal structure of the M. tuberculosis thioredoxin reductase (not bound to a Trx) and structures for intermediates in the E. coli thioredoxin catalytic cycle, we have modeled the complete M. tuberculosis thioredoxin system for the various steps in the catalytic cycle. These structures and models reveal pockets at the TrxR/TrxC interface in various steps in the catalytic cycle, which can be targeted in the design of uncompetitive inhibitors as potential anti‐mycobacterial agents, or as chemical genetic probes of function. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Temperature‐dependent conformational change affecting Tyr11 and sweetness loops of brazzein

The sweet protein brazzein, a member of the Csβα fold family, contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Nevertheless, a variable temperature study has revealed that the protein undergoes a local, reversible conformational change between 37 and 3°C with a midpoint about 27°C that changes the orientations and side‐chain hydrogen bond partners of Tyr8 and Tyr11. To test the functional significance of this effect, we used NMR saturation transfer to investigate the interaction between brazzein and the amino terminal domain of the sweet receptor subunit T1R2; the results showed a stronger interaction at 7°C than at 37°C. Thus the low temperature conformation, which alters the orientations of two loops known to be critical for the sweetness of brazzein, may represent the bound state of brazzein in the complex with the human sweet receptor. Proteins 2013; © 2012 Wiley Periodicals, Inc.