近红外光谱测定灵芝子实体多糖含量

灵芝具有多种药理活性,多糖是其主要活性成分之一.目前灵芝多糖的定量常采用的比色法,使用比较繁琐,也缺乏一定的安全环保性.应用近红外光谱对灵芝进行多糖定量分析,发现对子实体直接分析存在定量不准的问题.为了快速准确地评估灵芝子实体多糖含量,本研究采用对灵芝子实体水提物进行近红外光谱检测与分析,由此建立了较好的定量模型.该模...     

Chemometric analysis of in-line multi-wavelength fluorescence measurements obtained during cultivations with a lipase producing Aspergillus oryzae strain

The filamentous fungus, Aspergillus oryzae, was cultivated in batch and fed-batch cultivations in order to investigate the use of multi-wavelength fluorescence for monitoring course of events during filamentous fungi cultivations. The A. oryzae strain applied expressed a fungal lipase from Thermomyces lanuginosus. Spectra of multi-wavelength fluorescence were collected every 5 min with the BioView system (DELTA, Denmark) and both explorative and predictive models, correlating the fluorescence data with cell mass and lipase activity, were built. During the cultivations, A. oryzae displayed dispersed hyphal growth and under these conditions no fouling of the multi-wavelength fluorescence sensor was observed. The scores of a parallel factor analysis (PARAFAC) model, based on the fluorescence spectra, gave clear evidence of, for example, the on-set of the feeding phase. The predictive models, estimating the cell mass, showed correlations between 0.73 and 0.97 with root mean square error of cross validation (RMSECV) values between 1.48 and 0.77 g . kg(-1). A model estimating the lipase activity was also constructed for the fed-batch cultivations with a correlation of 0.93. The results presented here clearly show that multi-wavelength fluorescence is a useful tool for monitoring fed-batch cultivations of filamentous fungi.     

生物组织的折射率对漫反射光分布的影响

采用杂合算法,讨论了折射率对不同吸收系数的生物组织漫反射光分布的影响。计算结果表明,在近源处生物组织的折射率越小,漫反射光模拟计算的相对误差越小;而在远离源处,其相对误差随折射率的增大而减小。同时也说明杂合模型不太适合模拟吸收生物组织的漫反射光分布。     

Mathematical modeling of an aqueous film coating process in a Bohle Lab-Coater, Part 1: Development of the model

The purpose of this study was to develop a model to predict (1) air and product temperatures, (2) product moisture, and (3) air humidity during an aqueous coating process using a Bohle Lab-Coater. Because of the geometrical properties and the airflow, the drum of the Bohle Lab-Coater can in principle be divided into 2 zones of equal size—the drying and the spraying zones. For each zone, 4 balance equations could be set up describing the change of the air humidity, the product moisture, the enthalpy of the air, and the enthalpy of the product in each zone. For this purpose, knowledge regarding heat and mass transfer and also the motion of the tablets in drums was used. Based on the considerations of the heat and mass transfer, a set of first-order coupled ordinary differential equations (ODEs) was developed. This set of ODEs can be solved numerically. In this part, the development of the model is described in detail, whereas the application of the model can be found in part 2.     

Mathematical modeling of an aqueous film coating process in a Bohle Lab-Coater: Part 2: Application of the model

For the prediction of the air and product temperatures, the product moisture, and the air humidity during a coating process in a Bohle Lab-Coater, a model was developed. The purpose of this work was to determine the limit moisture, the critical moisture, and the constant for the exchange rate between both zones and to use these values for other sets of experiments to test the model. The adaptation of the 3 parameters (limit moisture, critical moisture, and exchange rate constant), was done by calculation of the product temperature in both zones for several sets of parameters in order to minimize the sum of square deviation between the calculated and the measured product temperatures. This set of parameters was used to test the validity of the model. By applying the model, the product temperature could be predicted based on the product, process, and equipment-related parameters. Hence, the model can be used to theoretically investigate the influence of different process paramaters. The mean difference between the predicted, and measured product temperatures in the steady state is ≈2 up to 3 K using the determined parameter set for the limit moisture, the critical moisture, and the exchange rate constant. The model is useful for the prediction of the air and product temperatures, the product moisture, and air humidity during a coating process in the Bohle Lab-Coater using round, biconvex tablets.     

Quantitative analysis of epithelial morphogenesis in Drosophila oogenesis: New insights based on morphometric analysis and mechanical modeling

The process of epithelial morphogenesis is ubiquitous in animal development, but much remains to be learned about the mechanisms that shape epithelial tissues. The follicle cell (FC) epithelium encapsulating the growing germline of Drosophila is an excellent system to study fundamental elements of epithelial development. During stages 8 to 10 of oogenesis, the FC epithelium transitions between simple geometries-cuboidal, columnar and squamous-and redistributes cell populations in processes described as posterior migration, squamous cell flattening and main body cell columnarization. Here we have carried out a quantitative morphometric analysis of these poorly understood events in order to establish the parameters of and delimit the potential processes that regulate the transitions. Our results compel a striking revision of accepted views of these phenomena, by showing that posterior migration does not involve FC movements, that there is no role for columnar cell apical constriction in FC morphogenesis, and that squamous cell flattening may be a compliant response to germline growth. We utilize mechanical modeling involving finite element computational technologies to demonstrate that time-varying viscoelastic properties and growth are sufficient to account for the bulk of the FC morphogenetic changes.     

Construction of hypothetical three-dimensional structure of P2Y1 receptor based on Fourier transform analysis

G protein-coupled receptors constitute a large family of homologous transmembrane proteins that represents one of the most important classes of confirmed drug targets. For novel drug discovery, the 3D structure of target protein is indispensable. To construct hypothetical 3D structures of G protein-coupled receptors, several prediction methods have been proposed. But none of the them has confirmed a correct ligand binding site. In this study we constructed the 3D structure of bovine rhodopsin using the prediction method proposed by Donnelly et al., with some modification. We found that our 3D model showed a good agreement with the reported retinal binding site. Using the similar method, we constructed the 3D structure of the P2Y₁ receptor; one of the G protein-coupled receptors, and showed a binding site of an endogenous ligand, ADP, on the basis of the 3D model and in vitro experimental data. These results should be valuable for design of a specific antagonist for P2Y₁ receptor.     

Quantitative analysis and prediction of curvature in leucine‐rich repeat proteins

Leucine‐rich repeat (LRR) proteins form a large and diverse family. They have a wide range of functions most of which involve the formation of protein–protein interactions. All known LRR structures form curved solenoids, although there is large variation in their curvature. It is this curvature that determines the shape and dimensions of the inner space available for ligand binding. Unfortunately, large‐scale parameters such as the overall curvature of a protein domain are extremely difficult to predict. Here, we present a quantitative analysis of determinants of curvature of this family. Individual repeats typically range in length between 20 and 30 residues and have a variety of secondary structures on their convex side. The observed curvature of the LRR domains correlates poorly with the lengths of their individual repeats. We have, therefore, developed a scoring function based on the secondary structure of the convex side of the protein that allows prediction of the overall curvature with a high degree of accuracy. We also demonstrate the effectiveness of this method in selecting a suitable template for comparative modeling. We have developed an automated, quantitative protocol that can be used to predict accurately the curvature of leucine‐rich repeat proteins of unknown structure from sequence alone. This protocol is available as an online resource at http://www.bioinf.manchester.ac.uk/curlrr/ . Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Soft sensor based on 2D‐fluorescence and process data enabling real‐time estimation of biomass in Escherichia coli cultivations

In bioprocesses, specific process responses such as the biomass cannot typically be measured directly on‐line, since analytical sampling is associated with unavoidable time delays. Accessing those responses in real‐time is essential for Quality by Design and process analytical technology concepts. Soft sensors overcome these limitations by indirectly measuring the variables of interest using a previously derived model and actual process data in real time. In this study, a biomass soft sensor based on 2D‐fluorescence data and process data, was developed for a comprehensive study with a 20‐L experimental design, for Escherichia coli fed‐batch cultivations. A multivariate adaptive regression splines algorithm was applied to 2D‐fluorescence spectra and process data, to estimate the biomass concentration at any time during the process. Prediction errors of 4.9% (0.99 g/L) for validation and 3.8% (0.69 g/L) for new data (external validation), were obtained. Using principal component and parallel factor analyses on the 2D‐fluorescence data, two potential chemical compounds were identified and directly linked to cell metabolism. The same wavelength pairs were also important predictors for the regression‐model performance. Overall, the proposed soft sensor is a valuable tool for monitoring the process performance on‐line, enabling Quality by Design.     

Functionalized micro-capillary film for the rapid at-line analysis of IgG aggregates in a cell culture bioreactor

A micro-capillary film has been developed that offers the potential for an at-line analytical tool for rapid aggregate analysis during biopharmaceutical antibody production. A non-porous walled micro-capillary film (NMCF) with cation exchange functionality was demonstrated to act as a chromatography medium that could be operated with high linear fluid velocities and was highly resistant to blockage by entrained particulates, including cells. The NMCF containing 19 parallel microcapillaries was prepared using a melt extrusion process from poly(ethylene-vinyl alcohol) copolymer (EVOH). The NMCF-EVOH was modified to have cation-exchange functionality (NMCF-EVOH-SP) and shown to differentially bind monomer and aggregated species of IgG antibody directly from a bioreactor. The use of NMCF-EVOH-SP to quantify aggregate concentrations in monoclonal antibody preparations in less than 20 minutes was demonstrated.     

Characterization and application of PBA fiber optic chemical film sensor based on fluorescence multiple quenching

The three types of structure of the pyrenebutyric acid of fiber optic chemical film sensor were stud-ied by fluorescence multiple quenching. They are, for different test samples and purposes, respectively general, three-way and combined. A tri-cup method was designed to demonstrate the multiple quenching of response mechanism, and a relationship formula of mathematical approach was established. The response mechanism was shown to include the dynamic quenching , inner-filter effects and/or resonance energy transfer. To show the response characterization in a series of organic and inorganic quenchers, a new concept of apparent quenching coefficient Kq was advanced. This kind of sensor has been used in continuous and in situ monitoring of the dissolution rate of drug tablets, on line and in situ monitoring of some organic therapeutic drugs in biological fluid and Cr( VI ) in industrial waste water. The measured data were examined and compared with HPLC or HPTLCS. Test results show that the sensors and appa     

An analysis of interspecific relationships ofSaguinus based on cranial measurements

The interspecific relationships of the following nine forms ofSaguinus were analyzed applying the multivariate analysis to the cranial measurements;S. oedipus, S. geoffroyi, S. leucopus, S. nigricollis, S. fuscicollis, S. labiatus, S. mystax, S. midas midas, andS. midas niger. Penrose's size distance was used to express the size factor among the nine forms; and for the shape factor, Q-mode correlation coefficients were utilized. The shape distance betweenS. oedipus andS. geoffroyi was almost equal to that betweenS. nigricollis andS. fuscicollis which are recognized as different species based on biogeographical evidence. Furthermore, the Penrose's size distance betweenS. oedipus andS. geoffroyi was quite large. Therefore, the results of this study support the hypothesis thatS. oedipus andS. geoffroyi are valid species. The analysis of the phylogenetic relationships among the nine forms was based on the shape factor only. The forms were divided into two main clusters: (1)S. oedipus, S. geoffroyi, andS. leucopus; (2)S. nigricollis, S. fuscicollis, S. labiatus, S. mystax, S. midas midas, andS. midas niger. In the former cluster,S. oedipus was more closely related toS. geoffroyi than either was toS. leucopus. The latter cluster was subdivided in two subclusters based on the degree of their affinity: (2a)S. nigricollis, S. fuscicollis, S. labiatus, andS. mystax; and (2b)S. midas midas andS. midas niger. In the former subcluster, [S. nigricollis, S. fuscicollis] and [S. labiatus, S. mystax] were classified into clusters, respectively. The ancestor of theS. nigricollis group differentiated intoS. oedipus, S. geoffroyi, andS. leucopus with the narrowing of the maxilla in the facial region, andS. midas midas andS. midas niger with the downward movement of rhinion.     

散射辐射测量及其对陆地生态系统生产力影响的研究进展

全球变化,特别是大气成分变化引起的散射辐射变化已经并将继续影响陆地生态系统的生产力与碳收支。该文综述了散射辐射的影响因子及其估算方法,分析了散射辐射对植被光能利用率(light-use efficiency,LUE)、陆地生态系统生产力及其碳收支的影响过程与控制机理,在此基础上提出了未来拟加强研究的方面:1)散射辐射对植物光合作用影响的机理及其在不同时空尺度的反应;2)散射辐射及其与其他环境因子的相互作用对植物与冠层光合作用影响的定量描述;3)散射辐射及其与其他环境因子的相互作用对土壤呼吸作用的影响过程与控制机理;4)植物对散射辐射及其与其他环境因子相互作用的适应性研究;5)散射辐射及其与其他环境因子的相互作用对陆地生态系统生产力及其碳收支的影响过程与调控对策。     

A novel hydrogen peroxide sensor based on immobilization of haemoglobin on nano-TiO2/DTAB composite film

Abstract

The direct electron transfer of immobilized haemoglobin (Hb) on nano-TiO₂ and dodecyltrimethylammonium bromide (DTAB) film modified carbon paste electrode (CPE) and its application as a hydrogen peroxide (H₂O₂) biosensor were investigated. On nano-TiO₂/DTAB/Hb/CPE, Hb displayed a rapid electron transfer process with participation of one proton and with an electron transfer rate constant which estimated as 0.29 s^??1. Thus, the proposed biosensor exhibited a high sensitivity and excellent electrocatalytic activity for the reduction of H₂O₂. The catalytic reduction current of H₂O₂ was proportional to H₂O₂ concentration in the range of 0.2–4.0 mM with a detection limit of 0.07 mM. The apparent Michaelis–Menten constant (K_m^app) of the biosensor was calculated to be 0.127 mM, exhibiting a high enzymatic activity and affinity. This sensor for H₂O₂ can potentially be applied in determination of other reactive oxygen species as well.     

Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli

Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by-product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions.     

Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye

Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((gamma-(32)P)ATP(gamma-(33)P)ATP labeling) or phosphoamino acid-selective antibodies. Both of these methods suffer from relatively well-known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro-Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro-Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid-selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312-625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro-Q Diamond dye readily permits differentiation between specific and non-specific peptide labeling at picogram to subpicogram levels of detection sensitivity.     

Vegetation‐mediated impacts of trends in global radiation on land hydrology: a global sensitivity study

Incident solar radiation has changed in the last 50 years, as an initial dimming trend from 1960 to approximately 1990 was followed by an ongoing brightening period, with concomitant changes in the partitioning between direct and diffuse fractions. Such radiation changes are expected to affect the global water cycle. In this study, we use the Community Land Model (CLM) to perform global offline simulations for the period 1948–2004 and study the effects of solar forcing changes on trends in evapotranspiration and runoff. The modeled components of the hydrologic cycle respond strongly to the imposed radiation changes in several regions, especially in the tropics. Exceptions are regions with soil moisture‐limited evapotranspiration regime, such as the U.S. Great Plains. In Europe and the Eastern US, the imposed 7 W m^?2 solar dimming for 1960–1990 leads to an evapotranspiration reduction of 1.5 W m^?2 or approximately 5% of the mean and an enhancement of runoff by equal percentage. In these regions, the imposed 6 W m^?2 solar brightening leads to a 3 W m^?2 increase of evapotranspiration in 1990–2004, and a runoff reduction of between 7 and 10% of the mean. Additional simulations investigating the impact of higher diffuse radiation fraction during 1960–1990 suggest mostly an increase of evapotranspiration in the tropics of 2.5 W m^?2 (3% of mean) due to increased photosynthesis from shaded leaves, but with smaller opposite effects elsewhere because of lower ground evaporation. The runoff trend resulting from the imposed radiation/aerosols effect is of the same sign and approximate relative magnitude (but larger absolute magnitude) as those calculated, in various studies, for other potential drivers of runoff change such as climate, CO₂, or land use. These results thus strengthen the claim that radiation effects on runoff are not to be neglected. Understanding the impacts of radiation on the water cycle will affect projections of river flow and freshwater availability for human consumption.     

Real-time in situ monitoring of freely suspended and immobilized cell cultures based on mid-infrared spectroscopic measurements.

Glucose and lactate profiles in Chinese hamster ovary cell cultures were accurately monitored in real time and in situ during three bioreactor batch cultures lasting 11,15, and 15 days performed within a 60-day period. Monitoring was accomplished using in situ-collected mid-infrared spectra analyzed with a priori one-time established partial least-squares regression models. The robustness of the technique was demonstrated by application of these models without modification after 2.3 years. Neither recalibration nor instrument maintenance was required during the 2.3-year period, except for the daily filling of liquid nitrogen for detector cooling during operation. The lactate calibration model yielded accurate absolute concentration estimations during each of the batch cultures with standard errors of estimate from 1 to 3 mM. The a priori-established glucose calibration model yielded concentration estimations with an off-set, which was constant throughout a culture. Adjustment of the off-set before inoculation resulted in accurate concentration estimations with Standard errors of estimate of approximately 1 mM for each of the bioreactor cultures. Sensitivity in detecting differences of 0.5 mM and selectivity against variation of one metabolite while the other was kept constant was demonstrated during standard additions of either glucose or lactate. The sensor system proved to be reliable, simple, accurate, sterile, and capable of long-term automatic operation and is considered to be mature enough to be routinely applied for in situ (on-line) cell culture monitoring.     

Quantitative analysis of C-bands based on optical density profiles in human chromosomes

Summary A method of quantitative analysis of C segments in human chromosomes 1, 9, and 16 based on longitudinal densitometry has been developed. The way of fitting apparatus data to visual estimates is represented. Density curve parameters not dependent on stain intensity were used. The general C band length error is approx. 0.05 m. Heteromorphic chromosome 16 pairs have been investigated with this method. A significant difference (about 0.18 m) between C bands of the homologues has been detected in chromosome 16. It has been calculated that C bands can be distinguished if the mean difference between the length of the homologues is more than 15%.