In Vivo Monitoring of Obligate Biotrophic Pathogen Growth by Kinetic PCR

The plant kingdom is constantly challenged by a battery of evolving pathogens. New species or races of pathogens are discovered on crops that were initially bred for disease resistance, and globalization is facilitating the movement of exotic pests. Among these pests, obligate biotrophic parasites make up some of the most damaging groups and have been particularly challenging to study. Here we demonstrate the utility of kinetic PCR (kPCR) (real-time PCR, quantitative PCR) to assess the growth of poplar rust, caused by Melampsora species, by quantification of pathogen DNA. kPCR allowed the construction of reliable growth curves from inoculation through the final stages of uredinial maturation, as well as pathogen monitoring before symptoms become visible. Growth parameters, such as latency period, generation time in logarithmic growth, and the increase in DNA mass at saturation, were compared in compatible, incompatible, and nonhost interactions. Pathogen growth was monitored in different applications dealing with plant pathology, such as host and pathogen diversity and transgenic crop improvement. Finally, the capacity of kPCR to differentiate pathogens in the same sample has broad molecular ecology applications for dynamically monitoring the growth of fungi in their environments or in mixed populations or to measure the efficacy of pest control strategies.     

Supersaturation-Driven Optical Tuning of Ag Nanocomposite Glasses for Photonics: An In Situ Optical Microspectroscopy Study

Silver nanoparticle (NP) precipitation in a melt-quenched aluminophosphate glass matrix has been studied and compared for 8 mol% and 4 mol% concentrations of both Ag₂O and SnO dopants. The assessment is carried out by monitoring the plasmonic evolution of glass-embedded Ag NPs in real time during thermal treatments by in situ optical microspectroscopy and complemented by transmission electron microscopy and X-ray diffraction characterization. The time variation in the surface plasmon resonance of Ag NPs is analyzed in the framework of Mie extinction theory in connection with nanocrystal precipitation in the supersaturated solid solutions. For the higher concentration of silver and tin, nucleation and growth processes were distinguished, which appeared to be temperature- and time-dependent. Hence, favorable conditions were induced for the precipitation of a large amount of small NPs in the system. On the other hand, the nucleation and growth stages were not well separated in time for the lower concentration of dopants, resulting in Ag NPs of a broad size range. However, such less-concentrated nanocomposite allowed for the precipitation of NPs much larger than those observed for the 8% doped glass. Varying the degree of supersaturation in the system has been established as an important means for the tuning of material optical properties for photonic (nanoplasmonic) applications.     

Discrimination Between Paraffin-Embedded and Frozen Skin Sections Using Synchrotron Infrared Microspectroscopy

The difference between paraffin-embedded and frozen skin sections is always questionable. Ten patients of early stage mycosis fungoides, ten patients with psoriasis and ten normal controls were included in this study. Aim of this study is to differentiate between paraffin-embedded and frozen skin sections in inflammatory and malignant dermatoses using synchrotron infrared microspectroscopy (SIRM). It was found that epidermal beta sheets in paraffin-embedded sections were higher in a highly significant manner than frozen sections (P < 0.001). Also, epidermal nucleic acids in paraffin-embedded sections were lower in a highly significant manner than frozen sections (P < 0.001). However, when various skin diseases were compared with the control. It was found that the difference between paraffin-embedded and frozen skin sections were almost similar. In conclusion SIRM is a unique promising diagnostic technique and it seems that frozen processing preserve skin tissue more, this was represented by less apoptosis (beta sheets) and more nucleic acids than paraffin processing. However, there are still many advantages of both approaches over the other depending on the goal of the study.     

Optical lock-in detection of FRET using synthetic and genetically encoded optical switches

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.     

橄榄绿链霉菌细胞膜中几丁单、寡糖结合蛋白质的分离、纯化和结合动力学

橄榄绿链霉菌细胞膜中存在能与N 乙酰葡糖胺特异性结合的蛋白质。采用超声波破碎、超速离心等方法 ,分离得到细胞膜后用TritonX 10 0抽提 ,经连续的阴离子层析后 ,可分别得到能与N 乙酰葡糖胺特异性结合的 46 .0kD和 47.5kD蛋白质。底物竞争结合实验证实 ,这两种蛋白质还能与其他几丁质降解产物 (几丁二糖至几丁六糖 )特异性结合而不与葡萄糖、纤维二糖等结合。采用介质表面感应技术 (surfaceplasmonresonance)检测出46kD蛋白质与几丁单糖至几丁六糖的解离常数 (Kd)在 8.2 9× 10 -9～ 1.95× 10 -5mol/L之间 ,单糖最高而三糖最低。决定其差异的主要因子是底物结合速率 (Kon)而非解离速率 (Koff)。N末端氨基酸序列分析结果推测 ,该蛋白质属于一个潜在的ATP结合转运系统     

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera’s collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.     

Melting of DNA Oligomers: Dynamical Models and Comparison with Experimental Results

We compare experimental melting curves of short heterogeneous DNA oligomers with theoretical curves derived from statistical mechanics. Partition functions are computed with the one-dimensional Peyrard-Bishop (PB) Hamiltonian, already used in the study of the melting of long DNA chains. Working with short chains we take into account, in the computations, not only the breaking of the interstrand hydrogen bonds, but also the complete dissociation of the double helix into separate single strands. Since this dissociation equilibrium is of general relevance, independent of the particular microscopic model, we give some details of its treatment. We discuss how the non bonded three-dimensional interactions, not explicitly considered in the one-dimensional PB model, are taken into account through the treatment of the dissociation equilibrium. We also evaluate the relevance of the dissociation as a function of the chain length.     

APPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy

Background

Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.

Methodology

Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.

Conclusions

All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.     

Kinematic Analysis and Experimental Verification on the Locomotion of Gecko

This paper presents a kinematic analysis of the locomotion of a gecko,and experimental verification of the kinematicmodel.Kinematic analysis is important for parameter design,dynamic analysis,and optimization in biomimetic robot research.The proposed kinematic analysis can simulate,without iteration,the locomotion of gecko satisfying the constraint conditionsthat maintain the position of the contacted feet on the surface.So the method has an advantage for analyzing the climbing motionof the quadruped mechanism in a real time application.The kinematic model of a gecko consists of four legs based on 7-degreesof freedom spherical-revolute-spherical joints and two revolute joints in the waist.The motion of the kinematic model issimulated based on measurement data of each joint.The motion of the kinematic model simulates the investigated real gecko’smotion by using the experimental results.The analysis solves the forward kinematics by considering the model as a combinationof closed and open serial mechanisms under the condition that maintains the contact positions of the attached feet on the ground.The motions of each joint are validated by comparing with the experimental results.In addition to the measured gait,three othergaits are simulated based on the kinematic model.The maximum strides of each gait are calculated by workspace analysis.Theresult can be used in biomimetic robot design and motion planning.     

A New Approach to Determine the Effect of Mismatches on Kinetic Parameters in DNA Hybridization Using an Optical Biosensor

We have demonstrated a simple yet direct method for determiningthe kinetic parameters in DNA-DNA interactions using biosensortechnology based on the surface plasmon resonance phenomenon;a technique that does not require complex DNA labeling. To determinethe effect of mismatches on the kinetics involved in DNA-DNAinteractions, DNA hybridization kinetics were monitored in realtime using synthetic oligonucleotides less than 20 bases inlength which contained either a complementary sequence or mismatchedbases. Upon analysis of the kinetic parameters obtained in oligonucleotidehybridization, we found that they were significantly affectedby the presence of mismatches as well as by their number andlocation in a DNA duplex. In addition, the presented biosensormethod is sensitive enough to detect kinetic effects causedby the presence of a single-mismatched base pair. Our findingsstrongly suggest that analysis of kinetic parameters involvedin DNA-DNA interactions is advantageous for detecting the presenceof mismatch base pairs in a DNA duplex.     

Estimating Orientation Factors in the FRET Theory of Fluorescent Proteins: The TagRFP-KFP Pair and Beyond

The orientation factor κ², one of the key parameters defining Förster resonance energy transfer efficiency, is determined by the transition dipole moment orientations of the donor and acceptor species. Using the results of quantum chemical and quantum mechanical/molecular mechanical calculations for the chromophore-containing pockets in selected colored proteins of the green fluorescent protein family, we derived transition dipole moments corresponding to the S_0,min → S₁ excitation for green fluorescent protein, red fluorescent protein (TagRFP), and kindling fluorescent protein, and the S_1,min → S₀ emission for TagRFP. These data allowed us to estimate κ² values for the TagRFP-linker-kindling fluorescent protein tetrameric complex required for constructing novel sensors.     

Micro- and Macro-ecological Factors in the Speciation of Obligate Parasites

The role of ecological factors in the speciation of obligateparasites is studied in the light of an example, the genusAnthracoidea,Ustilaginales. The factors may be divided into two groups inrelation to the life cycle of the parasite. The macro-ecologicalfactors are those which affect the parasite directly, i.e. mainlyduring that part of its life which it spends outside the hostplant. The micro-ecological factors exert their effect whenthe parasite is within the tissues of the host, or they areenvironmental factors in which the host is involved. For theparasite, the most critical point in its life cycle is the timewhen it infects its host, for at that time the micro-and macro-ecologicalfactors may even operate simultaneously. On morphological groundsthe genus Anthracoidea is divided into a number of highly specializedspecies. There also exist cytological grounds for this specialization:pseudo-homothallism and homothallism are postulated to limitthe possibilities of genetic recombination. However, ecological,and especially micro-ecological, factors also operate in theform of barriers limiting the exchange of genetic material betweenpopulations, and thus augmenting the effects of the cytologicalpeculiarities.