Design, preparation and testing of suitable probe-receptors for RNA biosensing

Absolute measurements of a given RNA in a cheap, easy, rapid and reproducible manner using biosensors technology could overcome many of the operative and analytical limits of conventional molecular biology methods. To this end, an integrated approach for the design, synthesis, and connection of RNA probes to the transducing surface of a microgravimetric biosensor has been developed. Suitable probes to be used as the bioreceptors in RNA biosensor were successfully designed by using a purposely developed computational method whose selection criteria are based on the accessibility of target region to probe, on pairing stability of probe-target duplex and on the uniqueness of selected targets over all known expressed sequences from a genome data base. Automated chemical synthesis of selected probes was performed and the oligonucleotides produced were covalently conjugated to the sensing surface of a quartz microbalance. The microgravimetric sensor was tested in a flow chamber by measuring the variation of resonance frequency due to the binding of synthetic target substrates. Specific dose dependent binding was observed. Furthermore, the binding of a transcribed full-length mRNA substrate was successfully monitored under similar conditions.     

Occurence of subtelomeric rearrangements in the genome of the microsporidian parasite Encephalitozoon cuniculi, as revealed by a new fingerprinting procedure based on two-dimensional pulsed field gel electrophoresis

In Microsporidia, mitochondria-lacking eukaryotic intracellular parasites, genomic comparisons were so far based on molecular karyotyping. The mammal-infecting species Encephalitozoon cuniculi is characterized by a very low haploid genome size (approximately 2.8 Mbp) and rather high karyotype variability. Recently, we developed a two-dimensional pulsed field gel electrophoresis (2-D PFGE) fingerprinting technique useful for constructing a restriction map fo the genome of a mouse E. cuniculi isolate (karyotype variant A). The so-called karyotype and restriction display 2-D PFGE (KARD-PFGE) protocol involved 1-D chromosome separation, digestion with a rare cutter, Klenow radiolabeling of genomic DNA and 2-D separation of restriction fragments followed by autoradiography. In order to assess its suitability for detecting polymorphic loci in E. cuniculi, we applied KARD-PFGE with either BssHII or Mlul digestion to genome analysis of two rabbit isolates representative of two different karyotype variants (A and C). The 2-D spot pattern of the rabbit isolate variant A is identical to the reference mouse isolate but differs greatly from the rabbit isolate variant C. Chromosomal restriction fragment length polymorphisms (RFLPs) provide strong evidence for homologous chromosomes and frequent DNA rearrangements within subtelomeric regions just upstream of the dispersed rDNA units closely associated with each chromosomal end.     

Direct CI method in restricted configuration spaces

A configuration interaction (CI) method in restricted CI (RCI) space obtained by imposing inequality-type restrictions on the occupancies of groups of molecular orbitals (MOs) was studied. The direct CI approach in such space was analyzed, and some recommendations concerning practical implementation of the RCI method are given. The corresponding program has been written in FORTRAN 77 for an IBM 486 DX personal computer and has been used for electronic structure calculations on transition metal complexes using a valence MO basis with the INDO approximation. © 1996 by John Wiley & Sons, Inc.     

Determining the global DNA methylation status of rat according to the identifier repetitive elements

A large portion of the genome represents repetitive elements. Identifier (ID) elements, the major elements of short interspersed repetitive elements, are widespread with about 150 000 copies in the rat genome. Each ID element contains six CpG dinucleotides, which might account for the global methylation status of rat. We validated the CpG methylation of the ID elements by various methods. The methylation of one CpG site (CpG-3) of the ID element was investigated by performing pyrosequencing. The methylation percentage of the CpG-3 site was 53.6% (SD = 2.2) on average from six rat tissues with blood, but 24.6% (SD = 1.0) in rat pheochromocytoma, PC-12, cell line. This CpG-3 methylation was further verified by whole genome amplification (WGA), 5-azacytidine treatment, and proportional mixing of rat WGA genomic DNA (gDNA) with liver gDNA. Methylation-sensitive restriction enzyme PCR method showed that three other CpG sites (CpG-1, CpG-4, and CpG-5) within the ID element were also methylated (about 60%) in rat gDNA, but not in WGA gDNA. The ID elements may be good candidates for routine analysis of the global DNA methylation changes of rat for pharmaceutical treatment and their use can make basic epigenetic research possible with high accuracy.     

G-rich sequence-specific recognition and scission of human genome by PNA/DNA hybrid G-quadruplex formation

Hole in one: A single peptide nucleic acid (PNA) effectively targets the G-rich region in double-stranded DNA through formation of a PNA/DNA hybrid G-quadruplex. Only one target site in the whole human genome was selectively cleaved by the hybrid G-quadruplex. Such site-selective scission of DNA is central to gene manipulation for molecular biology, biotechnology, and therapy.     

One‐Minute Wear‐Rate Measurement

Summary: Single‐asperity wear measurements were performed on small quantities of polyethylene with different molecular weights. Experiments could be performed on a surface area of 25 × 3000 μm². A quantitative value of wear rate could be obtained that was proportional to macroscopic results relating the wear rate to the molecular weight. Material transfer limited the maximum sliding velocity to 1 μm · s⁻¹. Possible solutions for this problem have been identified and will be a subject of further investigation.

Indentation profile for high‐molecular‐weight polyethylene (PE‐H).     

Chip-based nano-LC-MS/MS identification of proteins in complex biological samples using a novel polymer microfluidic device

Although the proteome of each organism is unambiguously coded in its genome, the proteome shows the real biology in action in each particular organism. New powerful tools are being developed for biochemists and biologists to analyze complex biological samples for studying the complete protein supplement of the genome, i. e., the proteome. There are several methods available for proteome analysis including 2-DE and several forms of MS. In recent years, technologies such as microfluidics and array-based systems have appeared in the field of analysis, identification, and quantification of proteins. These novel approaches might help in solving current technical challenges in proteomics. This paper presents a practical application of the first commercially available microfluidic nano-ESI device coupled with nano-LC (i. e., HPLC-chip) for the analysis of samples of some biological protein mixtures.     

Gel and polymer-solution mediated separation of biopolymers by capillary electrophoresis

In the age of genomics and proteomics, high-resolution separation techniques are routinely utilized in an integrated and automated fashion to solve formidable separation problems and provide the means for large-scale analysis of biological samples with excellent resolution. By automating the current manual procedures, capillary gel (CGE) and polymer-solution mediated electrophoresis greatly enhance the productivity of biopolymer analysis while also reducing both analysis time and the human intervention necessary from sample loading to data processing. The advent of this novel and high-performance bioseparation technique has made it possible to sequence the human genome and revealed global changes in the genome and proteome level, bringing about a revolutionary transition in our views of living systems on the molecular basis. CGE and polymer-solution mediated electrophoresis and related microseparation methods (e.g., electrophoresis microchips) are quickly becoming important separation and characterization tools in analytical biochemistry and molecular biology. This review gives an overview of the key application areas of DNA, protein, and complex carbohydrate analysis, and summarizes the latest developments on CGE column technology, including capillary coatings and sieving polymer matrices. Micropreparative aspects and related microseparation techniques are also discussed.     

Modification of a commercial automatic alpha counter for 2-π alpha counting

A 2-π proportional counting chamber is substituted for the end-window chamber in an automatic α/β counter, to achieve automated α-counting without sacrificing the efficiency and accuracy offered by manually operated 2-π proportional counters. A 2-π chamber was fitted with a thin metal base plate with a hole in the center. Appropriate sample inserts were made, and a mechanism was fabricated for raising the sample insert flush with the hole. A manifold and solenoids mounted in a double-wide NIM module are used to control counter gas purging of the chamber and normal gas flow during counting. The counter operations are controlled by modified logic circuits, also located in the module. During ca. 8 months of operation, the system has performed well.     

Structural effect of synthetic zwitterionic cosolutes on the stability of DNA duplexes

The molecular design of useful cosolutes for polymerase chain reaction (PCR), which is one of the most important techniques in molecular biology, plays a significant role in amplification of highly stable genome sequences because during PCR, strand dissociation sometimes fails due to high melting temperature. Here, we designed and synthesized eight new zwitterionic cosolutes derived from glycine betaine, a destabilizing reagent for GC-rich DNA duplexes, and systematically compared their ability to destabilize DNA duplexes and to amplify genome DNA by PCR. We found that introduction of n-butyl groups rather than methyl groups into the ammonium group reduced the melting temperature of DNA duplexes 11-fold more than what was observed for the scaffold cosolute, glycine betaine, and furthermore, the cosolute can amplify the stable genome sequence by PCR.     

Coupling Pervaporation-Gas Chromatography for Speciation of Volatile Forms of Selenium in Sediments

Abstract

A method for speciation of dimethylselenide (DMeSe), dimethyldiselenide (DMeDSe) and diethylselenide (DEtSe) in sediments based on a coupling between a pervaporation module, a preconcentration sorptive trap and a gas chromatograph-mass spectrometer is reported. The coupling is performed through a high pressure injection valve which allows two different operational modes: (a) analysis without preconcentration, in which analytes are directly driven from the pervaporation chamber to the injection port of the chromatograph, and (b) analysis with preconcentration in a trap, in which the analytes from the pervaporation chamber are first trapped on a Tenax minicolumn and then thermally desorbed and driven to the GC. This second approach improves the sensitivity compared to the direct coupling, reaching estimated absolute detection limits lower than 0.6 ng Se for each tested species. The method is applied to the determination of volatile organic selenium species in several sediments collected from different areas in the Southwest of Spain.     

DNA hybridization enhancement using piezoelectric microagitation through a liquid coupling medium

In conventional DNA microarray hybridization, delivery of target cDNAs to surface-bounded probes depends solely on diffusion, which is notoriously slow, and thus typically requires 6-20 h to complete. In this study, piezoelectric microagitation through a liquid coupling medium is employed to enhance DNA hybridization efficiency and the results are compared with the standard static hybridization method. DNA hybridization was performed in a sealed aluminium chamber containing DNA microarray glass chip, coupling medium and piezoelectric transducers. 3×SSC (Saline Sodium Citrate) was used as a coupling medium to prevent overheating of the piezoelectric transducers and to effectively transmit ultrasonic wave to the glass chip. Flow visualization using fluidic dye and velocimetry (PTV) technique was applied to observe fluid transport in the hybridization chamber. It was revealed that the dye solution was homogeneously distributed within 10 min under dynamic agitation while it took over 1 h to reach the same level of homogeneity in static condition. Plasmodium falciparum DNA microarrays and total RNA extracted from parasite cells were used as a model for DNA microarray experiments. It was found that the required hybridization time may be substantially reduced from 16 h to 4 h by the use of dynamic hybridization scheme. With the same hybridization time of 16 h, dynamic hybridization resulted in higher fluorescent signals of ～33% and ～24% compared to static hybridization in Cy3 and Cy5 channels, respectively. Additionally, good/effective spots, some of which were not formed by static method, were enhanced and distributed more uniformly over the microarray. Therefore, the developed dynamic hybridization with integrated piezoelectric microagitation platform is highly promising for DNA analysis in molecular biology and medical applications.     

A flexible correlation group table (CGT) method for the relativistic configuration interaction wavefunctions

A generalized correlation group table (CGT) method is described for the relativistic configuration interaction (RCI) wavefunctions of molecules containing heavy atoms. In this method first four keywords are defined and two properties are discussed in terms of spectroscopic states and double group theory. These definitions and properties are then used to summarize six principles to stipulate the relationship among relativistic states, nonrelativistic states, as well as RCI configurations. The definitions, properties, and principles comprise the generalized CGT method, which facilitates the classification and assignment of the RCI wavefunctions, and thus, provide a general technique for complex systems containing several open shells. Finally, the techniques are exemplified with a few computational models.     

Effect of polymer molecular weight on the polymer/surfactant interaction

A thermodynamic analysis of the interaction between fourteen different molar mass poly(ethylene oxide)s (PEO) and sodium dodecyl sulfate (SDS) based on the measured surfactant-binding isotherms is given. The surfactant-binding isotherms were determined by the potentiometric method in the presence of 0.1 M inert electrolyte (NaBr). It was found that there is no PEO/SDS complex formation if M(PEO) < 1000. In the molecular weight range 1000 < M(PEO) < 8000, the critical aggregation concentration (cac) and the surfactant aggregation number are decreasing as the polymer molecular weight increases. The saturated bound surfactant amount is proportional to the number concentration of the polymer in this molecular weight range. If M(PEO) exceeds approximately 8000, the cac does not depend on the polymer molar mass, and the saturated bound amount of the surfactant becomes proportional to the mass concentration of the polymer. It was also observed that independently of the polymer molecular weight the surfactant aggregation number increases as the equilibrium surfactant monomer concentration increases from the cac to the critical micellar concentration (cmc). Finally, it was demonstrated that only one polymer molecule is involved in the complex formation independently of the polymer molecular weight.     

PHYSICAL AND CHEMICAL PROPERTIES OF CHLOROPHYLL RCI EXTRACTED FROM PHOTOSYSTEM I OF SPINACH LEAVES AND FROM GREEN ALGAE

Abstract— A new chlorophyll, designated chlorophyll RCI (Chi RCI), with absorption and fluorescence properties different to other known chlorophylls, has been extracted from photosystem I (PSI) sub-chloroplast particles of the green alga Scenedesmus obliquus; it was suggested that this chlorophyll is either the chromophore ofP–700 or the chromophore of another holochrome associated in a 1:1 molar ratio withP–700. We now report the extraction and isolation of a chlorophyll from PSI particle preparations from spinach leaves with properties identical to those of Chi RCI from Scenedesmus. Its molar ratio toP–700 measured in vivo is again approximately 1:1. Chlorophyll RCI is further characterized by its fluorescence characteristics and redox behaviour. Molecular weight determinations show that Chi RCI has a mol wt 35 units higher than that of chlorophyll a (Chi a).     

一种可绝对定量核酸的数字PCR微流控芯片

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.     

Design,synthesis, and characterization of a combined main‐chain/side‐chain liquid crystalline polymer based on mesogen‐jacketed liquid crystal polymer via atom transfer radical polymerization

A novel combined main‐chain/side‐chain liquid crystalline polymer based on mesogen‐jacketed liquid crystal polymers (MJLCPs) containing two biphenyls per mesogenic core of MJLCPs main chain, poly(2,5‐bis{[6‐(4‐butoxy‐4′‐oxy‐biphenyl)hexyl]oxycarbonyl}styrene) (P1–P8) was successfully synthesized via atom transfer radical polymerization (ATRP). The chemical structure of the monomer was confirmed by elemental analysis, ¹H NMR, and ¹³C NMR. The molecular characterizations of the polymer with different molecular weights (P1–P8) were performed with ¹H NMR, gel permeation chromatography (GPC), and thermogravimetric analysis (TGA). Their phase transitions and liquid‐crystalline behaviors of the polymers were investigated by differential scanning calorimetry (DSC) and polarized optical microscope (POM). We found that the polymers P1–P8 exhibited similar behavior with three different liquid crystalline phases upon heating to or cooling in addition to isotropic state, which should be related to the complex liquid crystal property of the side‐chain and the main‐chain. Moreover, the transition temperatures of liquid crystalline phases of P1–P8 are found to be dependent on the molecular weight. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 7310–7320, 2008     

Construction of L‐Lysine Sensor by Layer‐by‐Layer Adsorption of L‐Lysine 6‐Dehydrogenase and Ferrocene‐Labeled High Molecular Weight Coenzyme Derivative on Gold Electrode

A ferrocene‐labeled high molecular weight coenzyme derivative (PEI‐Fc‐NAD) and a thermostable NAD‐dependent L ‐lysine 6‐dehydrogenase (LysDH) from thermophile Geobacillus stearothermophilus were used to fabricate a reagentless L ‐lysine sensor. Both LysDH and PEI‐Fc‐NAD were immobilized on the surface of a gold electrode by consecutive layer‐by‐layer adsorption (LBL) technique. By the simple LBL method, the reagentless L ‐lysine sensor, with co‐immobilization of the mediator, coenzyme, and enzyme was obtained, which exhibited current response to L ‐lysine without the addition of native coenzyme to the analysis system. The amperometric response of the sensor was dependent on the applied potential, bilayer number of PEI‐Fc‐NAD/LysDH, and substrate concentration. A linear current response, proportional to L ‐lysine concentration in the range of 1–120 mM was observed. The response of the sensor to L ‐lysine was decreased by 30% from the original activity after one month storage.