抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

基礎講座 電気泳動(Electrophoresis)

PacBio Sequencing and Its Applications.

Micromachining technology was used to prepare chemical analysis systems on glass chips (1 centimeter by 2 centimeters or larger) that utilize electroosmotic pumping to drive fluid flow and electrophoretic separation to distinguish sample components. Capillaries 1 to 10 centimeters long etched in the glass (cross section, 10 micrometers by 30 micrometers) allow for capillary electrophoresis-based separations of amino acids with up to 75,000 theoretical plates in about 15 seconds, and separations of about 600 plates can be effected within 4 seconds. Sample treatment steps within a manifold of intersecting capillaries were demonstrated for a simple sample dilution process. Manipulation of the applied voltages controlled the directions of fluid flow within the manifold. The principles demonstrated in this study can be used to develop a miniaturized system for sample handling and separation with no moving parts.

Micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.

/pdf/extrinsic-fluorescent-dyes-as-tools-for-protein-nmhkb08wui.pdf

Extrinsic fluorescent dyes as tools for protein characterization.

DNA sequencing is in the throes of an enormous technological shift marked by dramatic throughput increases, a precipitously dropping per-base cost of raw sequence, and an accompanying requirement for substantial investment in large capital equipment in order to utilize the technology. Investigations that were, for most, unreachable luxuries just a few years ago (individual genome sequencing, metagenomics studies, and the sequencing of myriad organisms of interest) are being increasingly enabled, at a rapid pace. This Review concentrates on the technology behind the third- and fourth-generation sequencing methods: their challenges, current limitations, and tantalizing promise.

First-generation sequencing encompasses the chain termination method pioneered by Sanger and Coulson1 in 1975 or the chemical method of Maxam and Gilbert in 1976–1977.2 In 1977, Sanger sequenced the first genome, bacteriophage ΦX 174, which is 5375 bases in length.3 These methods and their early history4 have been reviewed in detail previously.5 Four-color fluorescent Sanger sequencing, where each color corresponds to one of the four DNA bases, is the method used by the automated capillary electrophoresis (CE) systems marketed by Applied Biosystems Inc., now integrated into Life Technologies, and by Beckman Coulter Inc. (Table 1).6 The first composite human genome sequence, reported in 2001, was obtained largely using CE, at great cost and with intense human effort over more than a decade.7,8 While the genome reported in 2001 was a work in progress, the availability of an ever-improving “reference” genome is the basis of an ongoing transformation of biological science and remains fundamental to investigations of genotype–phenotype relationships. Considering reports that have appeared (and not appeared) in the literature to date, it could well be that medically meaningful (actionable) insights into complex diseases will require additional types of “personal” genomic data, for instance, tissue-specific mRNA expression profiling and mRNA sequencing, individualized analysis of gene regulatory regions, epigenetic profiling, and high-quality, long-range chromosome mapping to catalog significant deletions, insertions, rearrangements, etc. Correlation of such integrated genomic data sets with comprehensive medical histories for hundreds or thousands of individuals may be what it takes to reach an era of personalized medicine.9–11 Large-scale sequencing centers are now completing the conversion to next-generation sequencers; the Joint Genome Institute (JGI) has retired all of their Sanger sequencing instruments.12 At the other extreme, until small-scale next-generation sequencers can outperform CE on a cost per accurate base called as well as read length, CE systems will likely remain in heavy use for benchtop-scale, targeted sequencing for directed investigations such as quantitative gene expression, biomarker identification, and pathway analysis.



Table 1

First- and Second-Generation Sequencing Technologies

Landscape of Next-Generation Sequencing Technologies

Continuous flow microfluidic reactors that use immobilized components of enzymatic reactions present special challenges in interpretation of kinetic data. This study evaluates the difference between mass-transfer effects and reduced efficiencies of an enzyme reaction. The kinetic properties of immobilized alkaline phosphatase (AP) were measured by the dephosphorylation of 6,8-difluoro-4-methylumbelliferyl/phosphate to a fluorescent 6,8-difluoro-4-methylumbelliferone. A glass microfluidic chip with an in-channel weir was created for the capture of solid silica microbeads functionalized with enzyme. The input substrate concentrations and flow rates across the bed were varied to probe the flow-dependent transport and kinetic properties of the reaction in the microreactor bed. Unlike previous reactors, substrate was titrated directly over the fixed enzyme bed by controlling the air pressure over the chip reservoirs. The reactor explored substrate conversions from near zero to 100%. The average bed porosity, r...

Measurements of kinetic parameters in a microfluidic reactor.

A fluorogenic enzyme assay was conducted on microchips using pressure driven flow. Real-time on-chip dilutions were performed in both low viscosity buffer and an enzyme solution containing high viscosity solution. Flow regulation of mixed viscosity fluids requires a pressure control on each arm of the chip contributing to the overall flow. An 8-channel pressure controller was used to regulate the pressure above all wells, thereby controlling the dilution ratios of buffer, substrate and enzyme with different viscosity. Well pressures maintained a constant concentration of enzyme in the detector channel while adjusting the flow contribution of substrate and buffer.

A fluorogenic assay using pressure-driven flow on a microchip.

We present a systematic study of the electrophoretic migration of 10-200 kDa protein fragments in dilute-polymer solutions using microfluidic chips. The electrophoretic mobility and dispersion of protein samples were measured in a series of monodisperse polydimethylacrylamide (PDMA) polymers of different molecular masses (243, 443, and 764 kDa, polydispersivity index <2) of varying concentration. The polymer solutions were characterized using rheometry. Prior to loading onto the microchip, the polymer solution was mixed with known concentrations of SDS (SDS) surfactant and a staining dye. SDS-denatured protein samples were electrokinetically injected, separated, and detected in the microchip using electric fields ranging from 100 to 300 V/cm. Our results show that the electrophoretic mobility of protein fragments decreases exponentially with the concentration c of the polymer solution. The mobility was found to decrease logarithmically with the molecular weight of the protein fragment. In addition, the mobility was found to be independent of the electric field in the separation channel. The dispersion is relatively independent of polymer concentration and it first increases with protein size and then decreases with a maximum at about 45 kDa. The resolution power of the device decreases with concentration of the PDMA solution but it is always better than 10% of the protein size. The protein migration does not seem to correspond to the Ogston or the reptation models. A semiempirical expression for mobility given by van Winkle fits the data very well.

Electrophoretic migration of proteins in semidilute polymer solutions

This paper presents a microchip-based system for collecting kinetic time-based information on protein refolding and unfolding. Dynamic protein conformational change pathways were studied in microchannel flow using a microfluidic device. We present a protein-conserving approach for quantifying refolding by dynamically varying the concentration of the chemical denaturants, guanidine hydrochloride and urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. To validate this protein assay method, folding transition experiments were performed using two well-characterized proteins, human serum albumin (HSA) and bovine carbonic anhydrase (BCA). Transition events were monitored through fluorescence intensity shifts of the protein dye 8-anilino-1-naphthalenesulfonic acid (ANS) during dilutions of protein from urea or guanidine hydrochloride solutions. The enzymatic activity of refolded BCA was measured by UV absorption through the conversion of p-nitrophenyl acetate (p-NPA). The microchip protein refolding transitions using ANS were well-correlated with conventional plate-based experiments. The microfluidic platform enables refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material.

Matthew B. Kerby

Papers

Landscape of Next-Generation Sequencing Technologies

Measurements of kinetic parameters in a microfluidic reactor.

A fluorogenic assay using pressure-driven flow on a microchip.

Electrophoretic migration of proteins in semidilute polymer solutions

Kinetic measurements of protein conformation in a microchip.