Optical device for parallel online measurement of dissolved oxygen and pH in shake flask cultures

We describe a new device with parallel optical measurement of dissolved oxygen (DO) and pH in up to nine shake flasks applicable in any conventional shaking incubator. Measurement ranges are 0–500% of air saturation for oxygen and 5.5–8.5 for pH. It was used to characterize growth profiles of different l-lysine producing strains of Corynebacterium glutamicum, of Saccharomyces cerevisiae and of Escherichia coli. Cultures in unbaffled flasks were highly reproducible. Oxygen limitation was indicated online which is particularly important when cultivating fast growing cells as E. coli. C. glutamicum strains showed distinct characteristic patterns of DO and pH indicating biological events. During the cultivation of S. cerevisiae on glucose, fructose and galactose, oxygen uptake rate was determined using the predetermined value of k _L a. pH measurement was used to determine the minimum buffer requirement for a culture of C. glutamicum.     

Development of alcoholic and malolactic fermentations in highly acidic and phenolic apple musts

This work reports the influence of the high acidity and high phenolic content in apple musts on the development of alcoholic and malolactic fermentations and on the final chemical and microbiological composition of the ciders. Four different musts were obtained by pressing several varieties and proportions of cider apples from the Basque Country (Northern Spain). Specially acidic and phenolic varieties were selected. Three musts were obtained in experimental stations and the fourth one, in a cider factory following usual procedures. The evolution of these musts was monitored during five months by measuring 18 parameters throughout eight samplings. In the most acidic of the three experimental musts, yeasts were added to complete the alcoholic fermentation. In the rest of the musts, alcoholic and malolactic fermentations took place spontaneously due to natural microflora and no chemical was added to control these processes. Malolactic fermentation (MLF) finished before alcoholic fermentation in the three tanks obtained in experimental stations, even in the most acidic and phenolic one (pH 3.18, 1.78 g tannic acid/l). After four months, these ciders maintained low levels of lactic acid bacteria (10(4)CFU/ml) and low content of acetic acid (<0.60 g/l). Both fermentations began simultaneously in the must obtained in the cider factory, but MLF finished 10 days after alcoholic fermentation. Subsequently, this must maintained a high population of lactic acid bacteria (>10(6)CFU/ml), causing a higher production of acetic acid (>1.00 g/l) than in the other ciders. These results show the possible advantages of MLF finishing before alcoholic fermentation.     

Elicitation and in situ adsorption enhanced secondary metabolites production of Tripterygium wilfordii Hook. f. adventitious root fragment liquid cultures in shake flask and a modified bubble column bioreactor

The experiments of elicitation and in situ adsorption were conducted in shake flasks and then tested in a modified bubble column bioreactor for enhancing the productions of three active metabolites in Tripterygium wilfordii Hook. f., triptolide, wilforgine and wilforine. Methyl jasmonate was screened out as the elicitor and the non-ionic polymeric ion-exchange resin of Amberlite^® XAD-7 was used for in situ product removal and protecting the alkaloids from degradation in the medium. In shake flask experiments, 3.55-fold, 49.11-fold, and 10.40-fold of triptolide, wilforgine, and wilforine, respectively, could be recovered from the medium and XAD-7 resin by elicitation and in situ product removal, compared with the control. The modified 10 L bubble column bioreactor had similar productions of the three active metabolites but needed a further optimization of parameters for better growth of adventitious roots.     

A method for the accurate measurement of opsonic activity in uterine secretions of the mare

This study was undertaken to develop a method for accurately measuring opsonic activity in the uterine secretions of the mare. Ten mares were used in the study. They ranged in ages from 6 to 19 years and were of various genital health status. Undiluted uterine secretions were collected by inserting a tampon into the uterus during estrus; serum samples were collected simultaneously Opsonic activity in the secretions and serum was analyzed in a chemiluminescence assay, in which zymosan particles were opsonized. Opsonic activity was determined as peak chemiluminescence, time to peak chemiluminescence, and total chemiluminescence (area under curve). The peak chemiluminescence was 16 to 17 times higher when uterine secretions were used for opsonization rather than when buffer was used. Compared to the opsonic activity in serum, the peak chemiluminescence was 21% (P相似文献   

Development of a Lentivirus Vector-Based Assay for Non-Destructive Monitoring of Cell Fusion Activity

Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPe^NLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPe^NLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPe^NLS+ or an NLS-less version of FLPe (FLPe^NLS−) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPe^NLS+ and FLPe^NLS− both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPe^NLS+ generally yielded slightly higher signals than FLPe^NLS− while at low acceptor-to-donor cell ratios FLPe^NLS− was usually superior. The ability of FLPe^NLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for quantifying cell-to-cell fusion progression based on GpLuc has been developed.     

The use of fluorometry for on-line measurement of mixing time and hold-up in fermentations

Summary Mixing times and gas hold-ups in a 250L bioreactor containing a phosphate buffer or an active fermentation were determined on-line using fluorometric (MEFS and NADH) probes as functions of agitation and aeration rates. Both mixing time and hold-up of a fermentaion can be determined using a MEFS probe. Hold-up may also be measured with a presently available commercial NADH probe.     

Design and characterization of a modified T‐flask bioreactor for continuous monitoring of engineered tissue stiffness

Controlling environmental conditions, such as mechanical stimuli, is critical for directing cells into functional tissue. This study reports on the development of a bioreactor capable of controlling the mechanical environment and continuously measuring force‐displacement in engineered tissue. The bioreactor was built from off the shelf components, modified off the shelf components, and easily reproducible custom built parts to facilitate ease of setup, reproducibility and experimental flexibility. A T‐flask was modified to allow for four tissue samples, mechanical actuation via a LabView controlled stepper motor and transduction of force from inside the T‐flask to an external sensor. In vitro bench top testing with instrumentation springs and tissue culture experiments were performed to validate system performance. Force sensors were highly linear (R² > 0.998) and able to maintain force readings for extended periods of time. Tissue culture experiments involved cyclic loading of polyurethane scaffolds seeded with and without (control) human foreskin fibroblasts for 8 h/day for 14 days. After supplementation with TGF‐β, tissue constructs showed an increase in stiffness between consecutive days and from the acellular controls. These experiments confirmed the ability of the bioreactor to distinguish experimental groups and monitor tissue stiffness during tissue development. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Development of catalase activity in yeast in relation to growth and respiration

1. 1) When yeast cells grown anaerobically were adapted to aerobicculture in a normal medium, catalase formation was markedlyenhanced after the earlier stage of exponential growth of thecells. The same thing occurred with cells transferred from ananaerobic culture into a nitrogen deficient medium.
2. 2) Thecatalase activity of aerobically grown cells declinedprogressivelyuntil glucose, which had been added to the mediumwas profoundlyexhausted. This decline was followed by a progressiverecoveryof activity to a normal level with the growth of thecells.Similar behavior of catalase was also seen at low concentrationsof glucose, except that an abrupt rise in activity was observedat the beginning of incubation. Even when cells which had declinedto a minimum of catalase activity were aerated in phosphatebuffer, they continued to synthesize catalase.
3. 3) The patternof alteration of catalase activity during cellgrowth was accompaniedby a comparable pattern of alterationin respiratory capacity.On the basis of this finding, togetherwith the fact that antimycinA causes intensive depression incatalase formation, it maybe inferred that the formation ofthe respiratory chain conductsthe formation of catalase.
4. 4) In the presence of ethyl alcoholas the carbon source inplace of glucose, a rise in both catalaseactivity and respiratorycapacity occurred from initiation ofincubation. This fact canbe interpreted to mean that the repressiveeffect of glucoseon catalase formation depends on the aerobiccharacter of thecells.

(Received February 26, 1968; )     

A simple and modified manometric method for measuring oxygen uptake rate of plant cells in flask cultures

Summary A simple and convenient technique was developed based on the principle of Warburg manometric method to measure O₂ uptake rate (OUR) and CO₂ evolution rate (CER) of suspended cells in a shake flask culture. It was successfully applied to suspension cultures of rice (Oryza sativa) and Panax notoginseng cells, and some important bioprocess parameters, such as OUR, CER, respiratory quotient (RQ), specific OUR (SOUR) and specific CER (SCER), were quantitatively obtained. The measuring system is easy to operate, able to treat many samples simultaneously and is economical.     

Development of a coupled enzyme assay for the measurement of alternanase activity**, ***

Alternanase, an endoglucanase that hydrolyzes the bacterial exopolysaccharide alternan, will also hydrolyze the trisaccharide, panose, to produce glucose and a disaccharide that can be formed into a novel, cyclic tetrasaccharide. The glucose can then be selectively and quantitatively measured by enzyme-based reaction which forms the basis of a coupled enzyme assay to quantitate alternanase activity. By this method a preparation of alternanase purified by affinity chromatography on immobilized isomaltose had a maximum reaction rate (V _max) of 0.75 mol glucose min^–1 and a K _m of 34 mM for panose. Two competitive inhibitors of alternanase activity were also evaluated using this coupled enzyme assay: isomaltose had a K _i of 94 mM while the cyclic tetrasaccharide had a K _i of 66 mM.     

Development of a highly sensitive fluorescence probe for peptidyl arginine deiminase (PAD) activity

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of arginine residues to citrulline residues. Aberrant levels of PAD activity are associated with various diseases, such as rheumatoid arthritis, Alzheimer’s disease, and multiple sclerosis, so there is a need for simple and convenient high-throughput screening systems to discover PAD inhibitors as candidate therapeutic agents. Here, we report a highly sensitive off/on-type fluorescence probe for PAD activity based on the donor-excited photoinduced electron transfer (d-PeT) mechanism, utilizing the specific cycloaddition reaction between the benzil group of the probe and the ureido group of the PAD product, citrulline, under acidic conditions. We synthesized and functionally evaluated a series of probes bearing substituents on the benzil phenyl group, and found that 4MEBz-FluME could successfully detect citrulline with higher sensitivity and broader dynamic range than our previously reported fluorescence probe, FGME. Moreover, we succeeded in establishing multiple assay systems for PAD subtypes activities, including PAD2 and PAD4, with 4MeBz-FluME thanks to its high sensitivity. We expect that our fluorescence probes will become a powerful tool for discovering PAD inhibitors of several subtypes. Thus, it should be suitable for high-throughput screening of chemical libraries for inhibitors of PADs.     

Cartesian-diver microrespirometry: a technique for the accurate measurement of respiratory flux in plant cells

Abstract. A Cartesian-diver microrespirometer system is described which can be used to measure respiratory fluxes of oxygen accurately for cells of higher plants in a liquid phase. This microrespirometry technique has been adapted from protozoological and microfaunal studies to plant physiology. The Cartesian-diver has considerable scope for investigation of oxygen flux in plant cells and has several advantages compared to the oxygen electrode in terms of sensitivity to changing oxygen levels in respiring material. Because the volumes of liquid are small in the Cartesian-divers, diffusional distances arc measured in micrometres and there is no need for stirring to overcome diffusional problems, thus minimizing the risk of mechanical damage to the experimental material. In addition, only very small quantities of experimental material are required for the Cartesian-diver which is invaluable where only limited amounts of tissue or numbers of cells can be obtained. Examples of respiratory oxygen consumption by protoplasts from intercalary meristematic regions of light-grown barley ( Hordeum vulgare L.c.v. Patty) seedlings, in response to abscisic and gibberellic acids, are presented. The advantages and disadvantages of Cartesian-diver microrespirometry compared to oxygen electrodes are also discussed.     

Simplified model and measurement of specific absorption rate distribution in a culture flask within a transverse electromagnetic mode exposure system

In vitro experiments in bioelectromagnetics frequently require the determination of specific absorption rate (SAR) within a layer of cells on the bottom of a culture flask when the SAR has rapid spatial variation both horizontally within the cell layer and vertically in the medium bathing the cells. This problem has only recently been treated in the literature; and it is here approached differently for another irradiation system. It is shown that a simple two-dimensional frequency-domain guided-wave treatment yields results qualitatively comparable to those of more computationally intensive three-dimensional time-domain free-field scattering treatments. The problem of inferring local SARs from temperature-vs.-time curves is shown to be seriously confounded by thermal diffusion; and specific analytic and numerical results are presented to aid in understanding this effect. A novel experimental technique is introduced for measuring millikelvin temperature offsets with subsecond resolution, and illustrative experimental data are presented. Finally, present experimental and theoretical uncertainties are considered; and it is pessimistically asserted that, in a culture flask where spatial SAR variation is rapid, point SAR measurements by thermal methods may be in error by as much as +/- 3 dB. More reliable thermal determinations will require extreme care, challenging technological innovations, or both.     

An accurate colorimetric method for measurement of sulfaminohexose in heparins and heparan sulfates

A modification of a method for hexosamine analysis is presented which adapts it to measurement of sulfaminohexose in heparins and heparan sulfates. Unlike methods of sulfaminohexose analysis based upon coupling with indole, the absorptivity of polymeric and monomeric hexosamines is identical. N-Sulfated hexosamines are specifically deaminated in 33% acetic acid to yield free 2,5-anhydromannose residues which are then coupled to the color reagent 3-methyl-2-benzothiazolinone hydrazone hydrochloride. The sulfaminohexose content of a variety of heparins and heparan sulfates was determined with this methodology and compared with the indole-coupling method. Interferences by amino acids, proteins, and neutral sugar were evaluated in the sulfaminohexose assay and in the originally reported procedure for total hexosamine analysis.     

Development of a highly sensitive in vitro assay method for biological activity of endotoxin contamination in biological products.

The pyrogen test in rabbits has been replaced by the bacterial endotoxin test. The endotoxin test, however, showed a considerable discrepancy with pyrogenicity and was, therefore, assumed to have an efficacy limitation in directly predicting harmful biological effects of endotoxin. We developed a sensitive in vitro assay method by making use of tumour necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells, which showed a fine correlation with pyrogenicity in rabbits. RAW264.7 cells maintained by serial subculture under an endotoxin-free condition have gained the similar level of sensitivity as the endotoxin test to allow extensive dilutions of a drug for eliminating adverse effects on the cells. The in vitro TNF-alpha induction assay was shown to be capable to detect quantitatively a synergistic effect of a drug and endotoxin. The synergy is assumed necessary to be taken into consideration to define the limit value for the endotoxin test for guaranteeing the similar level of safety as by the pyrogen test.     

Purification of a highly modified RNA-aptamer: Effect of complete denaturation during chromatography on product recovery and specific activity

To evaluate RNA-aptamers as potential drug candidates, efficient and scaleable purification protocols are needed. Because aptamers are highly structured and rigid molecules, denaturation during the purification process is a critical aspect to obtain a pure and active product. A two-step chromatographic procedure was developed to purify a synthetic anti-VEGF aptamer at the preparative scale. A reversed-phase chromatographic step was optimized with a highly hydrophobic ion pairing reagent, followed by ion-exchange chromatography in which heat and a chaotropic salt were used. Because of the presence of 2′-modified ribose, denaturation conditions had to be optimized in both chromatographic steps to achieve a fully active molecule.     

Evaluation of Voltage-Sensitive Fluorescence Dyes for Monitoring Neuronal Activity in the Embryonic Central Nervous System

Using an optical imaging technique with voltage-sensitive dyes (VSDs), we investigated the functional organization and architecture of the central nervous system (CNS) during embryogenesis. In the embryonic nervous system, a merocyanine-rhodanine dye, NK2761, has proved to be the most useful absorption dye for detecting neuronal activity because of its high signal-to-noise ratio (S/N), low toxicity and small dye bleaching. In the present study, we evaluated the suitability of fluorescence VSDs for optical recording in the embryonic CNS. We screened eight styryl (hemicyanine) dyes in isolated brainstem–spinal cord preparations from 7-day-old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recording system. The signal size, S/N, photobleaching, effects of perfusion and recovery of neural responses after staining were compared. We also evaluated optical responses with various magnifications. Although the S/N was lower than with the absorption dye, clear optical responses were detected with several fluorescence dyes, including di-2-ANEPEQ, di-4-ANEPPS, di-3-ANEPPDHQ, di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA and di-2-ANEPPTEA. Di-2-ANEPEQ showed the largest S/N, whereas its photobleaching was faster and the recovery of neural responses after staining was slower. Di-4-ANEPPS and di-3-ANEPPDHQ also exhibited a large S/N but required a relatively long time for recovery of neural activity. Di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA and di-2-ANEPPTEA showed smaller S/Ns than di-2-ANEPEQ, di-4-ANEPPS and di-3-ANEPPDHQ; but the recovery of neural responses after staining was faster. This study demonstrates the potential utility of these styryl dyes in optical monitoring of voltage changes in the embryonic CNS.     

Design and performance of a modified buckle transducer for the measurement of ligament tension

The basic features of a modified buckle transducer for the direct in-situ measurement of tension in ligamentous or tendonous tissues are described. The slender shape of the modified design allows measurement in ligaments located in confined spaces with reduced risk of physical interference between the transducer and adjacent bony structures. In addition, simultaneous measurements in different fiber groups of the same ligament are made convenient. The design procedure of the proposed transducer and its performance characteristics are described.     

Evaluation of a fiberoptic-based system for measurement of optical properties in highly attenuating turbid media

Background  

Accurate measurements of the optical properties of biological tissue in the ultraviolet A and short visible wavelengths are needed to achieve a quantitative understanding of novel optical diagnostic devices. Currently, there is minimal information on optical property measurement approaches that are appropriate for in vivo measurements in highly absorbing and scattering tissues. We describe a novel fiberoptic-based reflectance system for measurement of optical properties in highly attenuating turbid media and provide an extensive in vitro evaluation of its accuracy. The influence of collecting reflectance at the illumination fiber on estimation accuracy is also investigated.     

Development of an Intelligent Mobile Health Monitoring System for the Health Surveillance System in Indonesia

Mobile phone applications have been widely used in various fields, including health care. Generally, this technology is used to overcome problems in health care by utilising mobile phone features for facilitating basic needs in health services. This study proposes an intelligent mobile health monitoring system that can be used in rural and remote areas where health services are still lacking. The system was made based on client/server architecture. Nine symptoms of typhoid, cough and diarrhoea from 30 patients were gathered from a hospital. Based on this data, a machine learning model using Support Vector Machine (SVM) was performed to distinguish these diseases. To find the best model parameters of the SVM, three different kernels (linear, polynomial, and Radial Basis Function (RBF)) were analysed. The result showed that RBF with degree 2 provided the best result in this particular application. The system was designed to receive input from patients about symptoms of the disease they have. The mobile phone application sends the data of the symptoms using Short Message Service (SMS) to the server. Furthermore, a machine algorithm module in the server identifies to which disease it belongs to based on the machine learning model created before. The prediction result is accessible to the doctor and the nearest Community Health Center (CHC). Based on the result, the doctor proposes a treatment plan for the patient to be recorded and sent to the patient by CHC. The proposed mobile health monitoring system has run properly and is ready to be evaluated in a real situation.