Multiplexed genotyping with sequence-tagged molecular inversion probes

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.     

Oligolabeling DNA probes to high specific activity with sequenase

In this protocol we present a reproducible method of preparing DNA probes of high specific activity using Sequenase. The probes produced by this method had a specific activity of 2.8×10⁹ cpm/μg with 69% of the total radioactivity incorporated into the TCA-precipitable materials. Probes with 5 to 10-fold lower specific activity were obtained using commercially available kits or using currently empolyed methods.     

Bacteriophage-P22 derivative with high density

Bacteriophage P22Cm21 was differentiated in some characters from the original phage P22. The buoyant density in CsCl solution of phage P22Cm21 was higher than that of phage P22 by as much as 0.007 g per cm³. The possible biological implication involved in this higher density is discussed.     

Novel DNA probes with low background and high hybridization-triggered fluorescence

Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5′-end and a non-fluorescent quencher at the 3′-end of the DNA sequence. This configuration provides surprisingly low background and high hybridization-triggered fluorescence. Here, we comparatively study the performance of such probes, MGB-Eclipse probes, and molecular beacons. Unlike the other two probe formats, the Pleiades probes have low, temperature-independent background fluorescence and excellent signal-to-background ratios. The probes possess good mismatch discrimination ability and high rates of hybridization. Based on the analysis of fluorescence and absorption spectra we propose a mechanism of action for the Pleiades probes. First, hydrophobic interactions between the quencher and the MGB bring the ends of the probe and, therefore, the fluorophore and the quencher in close proximity. Second, the MGB interacts with the fluorophore and independent of the quencher is able to provide a modest (2–4-fold) quenching effect. Joint action of the MGB and the quencher is the basis for the unique quenching mechanism. The fluorescence is efficiently restored upon binding of the probe to target sequence due to a disruption in the MGB–quencher interaction and concealment of the MGB moiety inside the minor groove.     

Coupling of photoactivatable glycolipid probes to apolipoproteins A-I and A-II in human high density lipoproteins 2 and 3

High density lipoprotein (HDL) from human serum was subfractionated into HDL2 and HDL3 by rate-zonal density gradient ultracentrifugation. The orientation of apoproteins (apo) A-I and A-II in these subfractions was investigated by use of the photosensitive glycolipid probes, 2-(4-azido-2-nitrophenoxy)-palmitoyl[1-14C]glucosamine (compound A) and 12-(4-azido-2-nitrophenoxy)-stearoyl[1-14C]glucosamine (compound B). Both probes were added to the HDL-structures in a ratio of two or three probe molecules per particle and were photoactivated by irradiation at a wavelength above 340 nm. After delipidation the probe-apoprotein adducts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the "shallow" probe (compound A) and the "depth" probe (compound B) were coupled for 10-14% (of the label added) to apoA-I and apoA-II from HDL3 and for about 6% to apoA-I and apoA-II from HDL2. By taking into account the relative amounts of apoA-I and apoA-II, it was estimated that the "shallow" probe labeled apoA-I 40% more effectively than apoA-II in both HDL2 and HDL3; the "depth" probe labeled apoA-I and apoA-II equally well in both subfractions. The data suggest that towards the surface HDL2 and HDL3 contain a relatively larger portion of apoA-I than apoA-II, whilst towards the core both subfractions are occupied by an equal portion of apoA-I and apoA-II. Application of these photolabels has failed to point out differences in the structural organization of HDL2 and HDL3.     

Fuzzy neural network for the control of high cell density cultivation of recombinant Escherichia coli

A five-layer fuzzy neural network (FNN) was developed for the control of fed-batch cultivation of recombinant Escherichia coli JM103 harboring plasmid pUR 2921. The FNN was believed to represent the membership functions of the fuzzy subsets and to implement fuzzy inference using previous experimental data. This FNN was then used for compensating the exponential feeding rate determined by the feedforward control element. The control system is therefore a feedforward-feedback type. The change in pH of the culture broth and the specific growth rate were used as the inputs to FNN to calculate the glucose feeding rate. A cell density of 84 g DWC/l in the fed-batch cultivation of the recombinant E. coli was obtained with this control strategy. Two different FNNs were then employed before and after induction to enhance plasmid-encoded β-galactosidase production. Before induction the specific growth rate was set as 0.31 h⁻¹, while it was changed to 0.1 h⁻¹ after induction. Compared to when only one FNN was used, the residual glucose concentration could be tightly controlled at an appropriate level by employing two FNNs, resulting in an increase in relative activity of β-galactosidase which was about four times greater. The present investigation demonstrates that a feedforward-feedback control strategy with FNN is a promising control strategy for the control of high cell density cultivation and high expression of a target gene in fed-batch cultivation of a recombinant strain.     

Evolution of surface electromyography: From muscle electrophysiology towards neural recording and interfacing

Surface electromyography (EMG) comprises a recording of electrical activity from the body surface generated by muscle fibres during muscle contractions. Its characteristics depend on the fibre membrane potentials and the neural activation signal sent from the motor neurons to the muscles. EMG has been classically used as the primary investigation tool in kinesiology studies in a variety of applications. More recently, surface EMG techniques have evolved from single-channel methods to high-density systems with hundreds of electrodes. High-density EMG recordings can be deconvolved to estimate the discharge times of spinal motor neurons innervating the recorded muscles, with algorithms that have been developed and validated in the last two decades. Within limits and with some variability across muscles, these techniques provide a non-invasive method to study relatively large populations of motor neurons in humans. Surface EMG is thus evolving from a peripheral measure of muscle electrical activity towards a neural recording and neural interfacing signal. These advances in technology have had a major impact on our fundamental understanding of the neural control of movement and have exposed new perspectives in neurotechnologies. Here we provide an overview and perspective of modern EMG technology, as derived from past achievements, and its impact in neurophysiology and neural engineering.     

Changes in stem water content influence sap flux density measurements with thermal dissipation probes

Key message

Stem WC may decline during the day. Zero-flow dT _m increases when WC decreases. Use of nighttime dT _m in the calculation of sap flux density during the day might introduce errors.

Abstract

There is increasing evidence of diel variation in water content of stems of living trees as a result of changes in internal water reserves. The interplay between dynamic water storage and sap flow is of current interest, but the accuracy of measurement of both variables has come into question. Fluctuations in stem water content may induce inaccuracy in thermal-based measurements of sap flux density because wood thermal properties are dependent on water content. The most widely used thermal method for measuring sap flux density is the thermal dissipation probe (TDP) with continuous heating, which measures the influence of moving sap on the temperature difference between a heated needle and a reference needle vertically separated in the flow stream. The objective of our study was to investigate how diel fluctuations in water content could influence TDP measurements of sap flux density. We analysed the influence of water content on the zero-flow maximum temperature difference, dT _m, which is used as the reference for calculating sap flux density, and present results of a dehydration experiment on cut branch segments of American sycamore (Platanus occidentalis L.). We demonstrate both theoretically and experimentally that dT _m increases when stem water content declines. Because dT _m is measured at night when water content is high, this phenomenon could result in underestimations of sap flux density during the day when water content is lower. We conclude that diel dynamics in water content should be considered when TDP is used to measure sap flow.     

小球藻高密度培养

<正>小球藻(Chlorella)为绿藻门、小球藻属的单细胞绿藻。小球藻富含蛋白质、多种色素、B族维生素、必需氨基酸、微量元素和一些生物活性代谢产物,而且同时具有抗癌、抗辐射、抗感染、抗氧化、防治高血脂症、防治便秘以及骨髓抑制等一系列生理保健功能,是较为理想的保健食品。传统上小球藻的生产采用光自养培养方式,离不开光照,产量很低,限制了小球藻的开发利用。采用异养培养无需光照,同时能使细胞浓度达到较高水平,大大降低分离成本,小球藻异养培养成为人们研究的重点[1]。     

Hypochlorite-modified high density lipoprotein,a high affinity ligand to scavenger receptor class B,type I,impairs high density lipoprotein-dependent selective lipid uptake and reverse cholesterol transport

Hypochlorous acid/hypochlorite (HOCl/OCl(-)), a potent oxidant generated in vivo by the myeloperoxidase-H(2)O(2)-chloride system of activated phagocytes, alters the physiological properties of high density lipoprotein (HDL) by generating a proatherogenic lipoprotein particle. On endothelial cells lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) and scavenger receptor class B, type I (SR-BI), act in concert by mediating the holoparticle of and selective cholesteryl ester uptake from HOCl-HDL. We therefore investigated the ligand specificity of HOCl-HDL to SR-BI-overexpressing Chinese hamster ovary cells. Binding of HOCl-HDL was saturable, and the degree of HOCl modification was the determining factor for increased binding affinity to SR-BI. Competition experiments further confirmed that HOCl-HDL binds with increased affinity to the same or overlapping domain(s) of SR-BI as does native HDL. Furthermore, SR-BI-mediated selective HDL-cholesteryl ester association as well as time- and concentration-dependent cholesterol efflux from SR-BI overexpressing Chinese hamster ovary cells were, depending on the degree of HOCl modification of HDL, markedly impaired. The most significant findings of this study were that the presence of very low concentrations of HOCl-HDL severely impaired SR-BI-mediated bidirectional cholesterol flux mediated by native HDL. The colocalization of immunoreactive HOCl-modified epitopes with apolipoprotein A-I along with deposits of lipids in serial sections of human atheroma shown here indicates that the myeloperoxidase-H(2)O(2)-halide system contributes to oxidative damage of HDL in vivo.     

DNA probes on beads arrayed in a capillary, 'Bead-array', exhibited high hybridization performance

A DNA analysis platform called 'Bead-array' is presented and its features when used in hybridization detection are shown. In 'Bead-array', beads of 100- micro m diameter are lined in a determined order in a capillary. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1-300 micro l) into the capillary with reciprocal flow. For hybridization detection, as little as 1 amol of fluorescent-labeled oligo DNA was detected. The hybridization reaction was completed in 1 min irrespective of the amount of target DNA. When the number of target molecules was smaller than that of probe molecules on the bead, 10 fmol, almost all targets were captured on the bead. 'Bead-array' enables reliable and reproducible measurement of the target quantity. This rapid and sensitive platform seems very promising for various genetic testing tasks.     

Structural differences in the enteric neural network in murine colon: impact on electrophysiology

The enteric neural network in the proximal murine colon shows a regularly occurring hypoganglionic region, which is here characterized by using anatomical and electrophysiological techniques. Staining with NADPH diaphorase, methylene blue, and cuprolinic blue in standard whole mounts and three-dimensional gut preparations of the murine proximal colon consistently revealed two hypoganglionic areas surrounded by a dense clustering of enteric neurons. This irregularity in the ganglionic plexus was found to be present in mice of three different genetic backgrounds, as well as in rats. The lack of myenteric ganglia in these regions was associated with an absence of the longitudinal muscle layer, as shown in cross sections. Histochemical identification of interstitial cells of Cajal in Kit(W-lacZ/+) transgenic mice showed Kit-positive cells oriented parallel to both muscle layers of the colon. Kit-positive cells oriented parallel to the longitudinal muscle layers were absent in the hypoganglionic area described. Electrical field stimulation elicited TTX-sensitive inhibitory junction potentials (IJPs), which showed region-specific characteristics. The initial partly apamin-sensitive hyperpolarization was present in all parts of the murine colon, whereas a second sustained NG-nitro-L-arginine-sensitive hyperpolarization was absent in the cecum and decreased from the proximal to the distal colon. Dissecting the hypoganglionic area from the surrounding tissue abolished the otherwise normal inhibitory neurotransmission to the circular muscle (1.6 +/- 1.4 and 2.6 +/- 1.7 mV for the fast and slow component of IJP amplitude in the hypoganglionic area vs. 16.5 +/- 1.9 and 23.7 +/- 2.7 mV for the fast and slow component of IJP amplitude in the neuron-rich area, respectively, P < 0.01, n = 6), whereas dissection of an area of identical size with an intact myenteric network showed normal inhibitory neurotransmission, indicating that the hypoganglionic area receives essential functional neural input from the neuron-rich surrounding tissue. In summary, in the murine and rat proximal colon, a constant and distinct hypoganglionic region is described with important concomitant changes in local electrophysiology.     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.     

基于BP神经网络和遗传算法的面包酵母高密度发酵培养基优化

为实现面包酵母的高密度发酵培养,构建一个BP神经网络模型,用于回归面包酵母高密度发酵培养基中显著影响因子与菌体密度之间的非线性关系,并在此基础上结合遗传算法进对此模型进行全局寻优,得到关键因子最佳浓度分别为:葡萄糖52.3 g/L,酵母浸出粉10.4 g/L,(NH4)2SO41.9 g/L.采用此优化配方进行摇瓶培养,所得菌体密度为3.95×108个/mL,比对照提高了61.2％.结果证实了人工神经网络的模拟和预测功能在微生物培养基优化方面有一定应用价值.     

Normalization of boutique two-color microarrays with a high proportion of differentially expressed probes

Normalization is critical for removing systematic variation from microarray data. For two-color microarray platforms, intensity-dependent lowess normalization is commonly used to correct relative gene expression values for biases. Here we outline a normalization method for use when the assumptions of lowess normalization fail. Specifically, this can occur when specialized boutique arrays are constructed that contain a subset of genes selected to test particular biological functions.     

Molecular probes for the development and plasticity of neural crest derivatives

We have isolated cDNA clones for several mRNAs expressed in sympathetic neurons but not in adrenal chromaffin cells, two neural crest derivatives thought to share a common precursor. The tissue specificity, developmental expression, and hormonal regulation of these genes have been characterized using Northern blot and in situ hybridization analysis. We find that these mRNAs are independently regulated in development rather than synchronously induced. Our evidence also implicates Nerve Growth Factor (NGF) in the induction of one of these genes in postmigratory crest cells. Two of these genes become induced in mature chromaffin cells, which express a neuronal morphology in response to NGF. These results support the idea that the phenotypic plasticity of neural crest derivatives reflects a common precursor, the multipotentiality of which is sustained through terminal differentiation.