Silicone-tubing aerated bioreactors for somatic embryo production

Bioreactors equipped with silicone tubings for bubble free oxygen supply are suitable for culture of embryogenic cell suspensions. The advantages of bubble free aeration systems over various devices for dispersion of air bubbles are the lack of foam formation and the possibilities of precise control of the desired oxygen set point. The specification of silicone tubing (length, diameter, wall thickness) has to be adapted according to the amount of embryogenic biomass to be produced in the bioreactor. Cell suspensions of Euphorbia pulcherrima were cultured --2 l bioreactor at 60% pO₂, supplied by a silicone tubing system of 155 cm length, 4.0 mm diameter and 0.4 mm wall thickness. The oxygen concentration decreased when the packed cell volume exceeded 14% (=3.7 g l^-1 cell dry weight), indicating the upper limit of oxygen supply by the silicone tubing. Mathematical considerations for membrane aerated bioreactors are presented with the intention of enabling a more precise definition for the configuration of silicone tube systems in different bioreactor types.     

An alternative method of endotracheal intubation of common marmosets (Callithrix jacchus)

Endotracheal intubation was carried out in 11 common marmosets (Callithrix jacchus). A commercially available tilting stand and a Miller laryngoscope blade were used to visualize the larynx. Anaesthesia was induced with alphaxalone (10.6 ± 1.6 mg/kg intramuscularly, followed by 3.2 ± 1.2 mg/kg intravenously). The diameter of the proximal trachea easily fitted an endotracheal tube made from readily available material (a 12 G 'over the needle' catheter). Once the tip of the endotracheal tube was at the level of the vocal folds, the tube had to be gently rotated through a 180° angle in order to pass through the larynx into the trachea. Assessment of the dimensions of the larynx and trachea, and comparison with external anatomical features of the animals (n = 10) showed that the length of the trachea could be predicted by multiplying the craniosacral length of the marmoset by a factor of 0.42.     

No peptide left behind: the “out of range” recovery in IPG–IEF fractionation

IEF is often used in multidimensional shotgun proteomics and the narrow range of 3.5–4.5 is the recommended pH interval for the fractionation of tryptic peptides. Usually, even if IEF is performed in IPG strip with a narrow range pH, the entire sample must be loaded onto the strip, including the “out of IPG range” peptides. We describe a simple protocol to recover at least a part of these missing peptides and show that this recovery significantly influences the overall fractionation result, increasing the number of the identified proteins and the protein coverage.     

Two dimensional liquid phase separations of proteins using online fractionation and concentration between chromatographic dimensions

Multi-dimensional liquid chromatography is often presented as an alternative to two-dimensional (2-D) gel electrophoresis for separating complex protein mixtures. The vast majority of analytical-scale 2-D LC systems have employed either off-line fractionation or stepped gradients in the first dimension separation. The latter severely restrict flexibility in setting up the first dimension gradient. We propose a novel two-dimensional LC system that employs online fractionation of proteins into a series of small reversed phase trapping columns. These traps effectively decouple the two separation dimensions and avoid problems associated with off-line fraction collection. Flexibility in determining the gradient programs for the two separations is thus enhanced. The reduced diameter of the trapping columns concentrates analyte between chromatographic dimensions. The apparatus is coupled with online electrospray time-of-flight mass spectrometry to characterize ribosomal proteins of Caulobacter crescentus.     

Proteomics analysis of cells in whole saliva from oral cancer patients via value-added three-dimensional peptide fractionation and tandem mass spectrometry

Whole human saliva possesses tremendous potential in clinical diagnostics, particularly for conditions within the oral cavity such as oral cancer. Although many have studied the soluble fraction of whole saliva, few have taken advantage of the diagnostic potential of the cells present in saliva, and none have taken advantage of proteomics capabilities for their study. We report on a novel proteomics method with which we characterized for the first time cells contained in whole saliva from patients diagnosed with oral squamous cell carcinoma. Our method uses three dimensions of peptide fractionation, combining the following steps: preparative IEF using free flow electrophoresis, strong cation exchange step gradient chromatography, and microcapillary reverse-phase liquid chromatography. We determined that the whole saliva samples contained enough cells, mostly exfoliated epithelial cells, providing adequate amounts of total protein for proteomics analysis. From a mixture of four oral cancer patient samples, the analysis resulted in a catalogue of over 1000 human proteins, each identified from at least two peptides, including numerous proteins with a role in oral squamous cell carcinoma signaling and tumorigenesis pathways. Additionally proteins from over 30 different bacteria were identified, some of which putatively contribute to cancer development. The combination of preparative IEF followed by strong cation exchange chromatography effectively fractionated the complex peptide mixtures despite the closely related physiochemical peptide properties of these separations (pI and solution phase charge, respectively). Furthermore compared with our two-step method combining preparative IEF and reverse-phase liquid chromatography, our three-step method identified significantly more cellular proteins while retaining higher confidence protein identification enabled by peptide pI information gained through IEF. Thus, for detecting salivary markers of oral cancer and possibly other conditions of the oral cavity, the results confirm both the potential of analyzing the cells in whole saliva and doing so with our proteomics method.     

Activation of ras genes in human tumors does not affect localization, modification, or nucleotide binding properties of p21

A comparison of proteins encoded by normal human ras genes and by mutant rasH or rasK genes activated in human carcinomas revealed no changes in subcellular localization, posttranslational modification, or guanine nucleotide binding associated with activation. Subcellular fractionation indicated that both normal and activated ras proteins were associated exclusively with the membrane fraction. Furthermore, both normal and activated ras proteins exhibited similar degrees of posttranslational acylation. The KD for dGTP binding was 1.0-2.2 X 10(-8) M, with no consistent differences between normal and activated ras proteins. In addition, a survey of 13 possible competing nucleotides revealed no differences in the specificity of nucleotide binding associated with ras gene activation. These results indicate that structural mutations which activate ras gene transforming activity do not alter the protein's known biochemical parameters and in particular do not affect the protein's intrinsic ability to bind guanine nucleotides.     

NMR spectroscopy of 14-3-3ζ reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3ζ

Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating diverse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3ζ, but has no direct role in chaperone action. Lys(49) is an important functional residue within the ligand-binding groove of 14-3-3ζ with K49E 14-3-3ζ exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3ζ with high affinity and also reduces the interaction of 14-3-3ζ ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3ζ, implying that the C-terminal extension and binding groove of 14-3-3ζ do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3ζ are most likely to be involved, i.e. the protein's chaperone and phosphoserine-binding activities are functionally and structurally separated.     

Identification and characterization of the Sulfolobus solfataricus P2 proteome

Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely: (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC-ESI-MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed.     

Autotrophic denitrification via a novel membrane-attached biofilm reactor

AIMS: A laboratory-scale autotrophic membrane-attached biofilm reactor was developed to remove nitrate from drinking water. METHODS AND RESULTS: Hydrogen and carbon dioxide flowed together into the lumem side of a gas-permeable silicone tube. The gases diffused through the membrane wall to feed Alcaligenes eutrophus that formed a biofilm on the surface of the silicone tube for autotrophic denitrification. Hydrogen provided the energy source, and carbon dioxide, besides serving as the carbon source, was employed to neutralize the alkalinity from denitrification. The optimal carbon dioxide concentration in the silicone tube was between 20% and 50%. CONCLUSION: This study has demonstrated that a gas-permeable silicone tube is a convenient and efficient method to feed A. eutrophus for autotrophic denitrification. Supplying a suitable amount of carbon dioxide together with hydrogen into the silicone tube solved the problem that alkalinity formation caused during denitrification. The pH of the bioreactor was maintained at about 7 to avoid nitrite accumulation, and then the nitrogen removal rate was increased. A high specific nitrogen removal rate (1.6-5.4 g Nm(2)d(-1-1) of surface area of silicone tube) was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to combining the advantages of the hydrogenotrophic denitrification process and a membrane feeding substrate bioreactor (MFSB), this bioreactor achieved a high nitrogen removal rate and is simple to operate. It therefore is highly promising in drinking-water treatment.     

Evaluation of reproducibility of protein identification results after multidimensional human serum protein separation

We developed a gel- and label-free proteomics platform for comparative studies of human serum. The method involves the depletion of the six most abundant proteins, protein fractionation by Off-Gel IEF and RP-HPLC, followed by tryptic digestion, LC-MS/MS, protein identification, and relative quantification using probabilistic peptide match score summation (PMSS). We evaluated performance and reproducibility of the complete platform and the individual dimensions, by using chromatograms of the RP-HPLC runs, PMSS based abundance scores and abundance distributions as objective endpoints. We were interested if a relationship exists between the quantity ratio and the PMSS score ratio. The complete analysis was performed four times with two sets of serum samples containing different concentrations of spiked bovine beta-lactoglobulin (0.1 and 0.3%, w/w). The two concentrations resulted in significantly differing PMSS scores when compared to the variability in PMSS scores of all other protein identifications. We identified 196 proteins, of which 116 were identified four times in corresponding fractions whereof 73 qualified for relative quantification. Finally, we characterized the PMSS based protein abundance distributions with respect to the two dimensions of fractionation and discussed some interesting patterns representing discrete isoforms. We conclude that combination of Off-Gel electrophoresis (OGE) and HPLC is a reproducible protein fractionation technique, that PMSS is applicable for relative quantification, that the number of quantifiable proteins is always smaller than the number of identified proteins and that reproducibility of protein identifications should supplement probabilistic acceptance criteria.     

Parallel isoelectric focusing chip

Fast isoelectric focusing (IEF) is becoming a key method in modern protein analysis. We report here the theory and experimental results of new parallel isoelectric devices (PID) for fast IEF. The main separation tool of any PID is a dielectric membrane with conducting channels filled by immobiline gels of varying pH. The pH value of the surrounding aqueous solution is not equal to the pH of any of the channels. The membrane is held perpendicular to the applied electric field. Proteins are collected (trapped) in the channels whose pH values are equal to the pI of the proteins. The fast particle transport between different channels takes place due to convection in the aqueous solution. We developed a mathematical model for PID. Experiment duration is shown to be proportional to the number of different bands N (the peak capacity in standard IEF) in contrast with N(2) for usual IEF devices. This model was validated with experimental results. Parallel IEF accelerates the fractionation of proteins by their pI values (down to several minutes) allowing a more desirable collection efficiency to be achieved. The main theoretical limitation of PID resolution is the sensitivity of proteins to pH change due to the Coulomb blockade effect. The existence of a minimal pH change deltapH(min) for each type of protein is shown: deltapH(min) approximately r(-1) for globular molecules with radius r.     

Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry

Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.     

High pH reversed‐phase chromatography as a superior fractionation scheme compared to off‐gel isoelectric focusing for complex proteome analysis

MS/MS is the technology of choice for analyzing complex protein mixtures. However, due to the intrinsic complexity and dynamic range present in higher eukaryotic proteomes, prefractionation is an important step to maximize the number of proteins identified. Off‐gel IEF (OG‐IEF) and high pH RP (Hp‐RP) column chromatography have both been successfully utilized as a first‐dimension peptide separation technique in shotgun proteomic experiments. Here, a direct comparison of the two methodologies was performed on ex vivo peripheral blood mononuclear cell lysate. In 12‐fraction replicate analysis, Hp‐RP resulted in more peptides and proteins identified than OG‐IEF fractionation. Distributions of peptide pIs and hydropathy did not reveal any appreciable bias in either technique. Resolution, defined here as the ability to limit a specific peptide to one particular fraction, was significantly better for Hp‐RP. This leads to a more uniform distribution of total and unique peptides for Hp‐RP across all fractions collected. These results suggest that fractionation by Hp‐RP over OG‐IEF is the better choice for typical complex proteome analysis.     

Analysis of birefringence decay profiles for nucleic acid helices possessing bends: the tau-ratio approach.

For nucleic acid helices in the 100-200-bp range, a central bend or point of flexibility increases the rate of rotational diffusion. In a transient electric birefringence (TEB) experiment, this increase is manifest as a reduction in the terminal (slowest) birefringence decay time. Previous experimental and theoretical work has demonstrated that the ratio of the decay times for a bent/flexible molecule and its fully duplex (linear) counterpart represents a sensitive, quantifiable measure of the apparent bend angle (tau-ratio approach). In the current work, we have examined the influence of helix parameters (e.g., persistence length, helix rise, diameter) on the tau-ratio for a given bend. The tau-ratio is found to be remarkably insensitive to variations and/or uncertainties in the helix parameters, provided that one employs bent and control molecules with the same sequence and length (apart from the bend itself). Although a single tau-ratio determination normally does not enable one to distinguish between fixed and flexible bends, such a distinction can be made from a set of tau-ratios for molecules possessing two variably phased bends. A number of additional uncertainties are examined, including errors in the estimation of the dimensions of nonhelix elements that are responsible for bends; such errors can, in principle, be estimated by performing a series of measurements for molecules of varying length.     

Influence of geometric taper on the derivation of the true propagation coefficient using a three point method

We studied the effect of geometric taper on the derivation of the true propagation coefficient from three pressures determined 10 cm apart ('three-point method'). For this purpose the true propagation coefficients of a uniform latex tube (length 50 cm, outer diameter 12.73 mm, Womersley phase velocity 6.23-6.42 ms-1 (1-10 Hz), Womersley damping coefficient 0.05-0.14 m-1 (1-10 Hz) and of a tapered latex tube (length 50 cm, outer diameter varying from 15.88 to 9.45 mm, in the middle section with same properties as the uniform tube) were determined. The real part of the true propagation coefficient (the damping coefficient) was compared with apparent damping, and with the damping coefficient calculated using Womersley's theory. The imaginary part of the true propagation coefficient (the phase coefficient) was expressed in terms of phase velocity. True phase velocity was compared with measurements of apparent phase velocity, foot-to-foot velocity, and calculations of phase velocity parameters Womersley's theory and the Moens-Korteweg equation. The results show that in the uniform tube the three-point propagation coefficient is in agreement with all other estimates. Taper causes an error in the three-point propagation coefficient. At some frequencies the damping is reversed to amplification (values up to -2 m-1) and the phase velocity may be both overestimated or underestimated (up to 50%). The overestimation of true damping as reported in the literature cannot be explained from vessel taper.     

Wave dissipation in flexible tubes in the time domain: in vitro model of arterial waves

Earlier work of wave dissipation in flexible tubes and arteries has been carried out predominantly in the frequency domain and most of the studies used the measured pressure waveform for presenting the results. In this work we investigate the pattern of wave dissipation in the time domain using the separated forward and backward travelling waves in flexible tubes. We tested four sizes of latex tubes of 2m in length each, where a single semi-sinusoidal in shape, pressure wave, was produced at the inlet of each tube. Simultaneous measurements of pressure and flow waveforms were recorded every 5cm along the tubes and wave speed was determined using the pressure-velocity loop method (PU-loop). The measured data and wave speed were used to separate the pressure waveform and wave intensity, into their forward and backward directions, using wave intensity analysis (WIA). Also, the energy carried by the wave was calculated by integrating the relevant area under the wave intensity curve. The peak of the measured pressure waveform increased downstream, however, the peak of the separated forward pressure waveform decreased exponentially along the tube. Wave intensity and energy also dissipated exponentially along the travelling distance. The peaks of the separated pressure and wave intensity decreased in the forward in a similar exponential way to that in the backward direction in all four tube sizes. Also, the smaller the size of the tube the greater wave dissipation it caused. We conclude that wave separation is useful in studying wave dissipation in elastic tubes, and WIA provides a convenient method for determining the dissipation of the energy carried by the wave along the travelled distance. The separated pressure waveform, wave intensity and wave energy dissipate exponentially with the travelling distance, and wave dissipation varies conversely with the diameter of elastic tubes.     

Influence of a downstream narrowing on the flow profile in a tube

The distance over which the upstream flow conditions in a tube are disturbed by a stenosis downstream, i.e. the outlet length, was investigated for Reynolds numbers in the range 210-2900. Two methods were used, the Navier-Stokes equations were solved with a computer and a physical model was constructed and maximal velocities were measured with an ultrasound Doppler system. The computer model showed that Re number does not influence the outlet length, varying the stenosis area from 25% to 90% has an effect. However, the outlet length remained small, below 70% of the diameter of the tube. The physical model confirmed for a 75% stenosis that the outlet length is small, this method set the limit at not more than 1.2 times the tube diameter.     

Sterilization of various diameter dead-ended tubes

Effect of tube diameter on steam-in-place sterilization of dead-ended tubes was studied by examining temperature profiles and rates of kill of Bacillus stearothermophilus spores. Time required for sterilization was determined for 9.4-cm-long tubes with various inside diameters from 0.4 to 1.7 cm. Sterilization time increased with decreasing tube diameter. Experimentally measured kill kinetics in 1.7-cm tubes were in agreement with those predicted if measured temperatures represented saturated steam. A 12-log spore reduction was achieved in 1.7-cm diameter vertical and horizontal tubes in less than 63 minutes. For smaller diameter tubes, entrapped air remained after 2 hours and rates of kill were very dependent on position within the tube, tube diameter, and tube orientation with respect to the gravitational vector. Times to achieve a 1-log drop in spore population in the smaller tubes were as much as 10 times greater than those expected if measured temperatures represented saturated steam. Sterilization was not achieved throughout the 0.4-cm tubes. Recommendations are made for including steam bleeders or using prevaccum cycles for these smaller diameter tubes. (c) 1993 John Wiley & Sons, Inc.     

Engineering and cell attachment properties of human fibronectin–fibrinogen scaffolds for use in tissue engineered blood vessels

Tissue engineered constructs reported to date have been prepared primarily from poly(glycolic) acid or collagen scaffolds onto which cells are grown and matured. In this paper we report experimental data to demonstrate the use of a natural, human protein, as a tubular scaffold for vascular grafting. Using a manual and a scalable dip-coating technique we prepared fibronectin-based tubes up to 12 cm in length and up to 3 mm in diameter. The tubes were flexible and their mechanical properties, measured in terms of tensile strength and burst pressure as a function of humidity, demonstrated their suitability as scaffolds for use in vascular grafting, e.g. coronary artery by pass grafting. In vitro tests involved the attachment of endothelial cells pumped under laminar flow conditions through the tube lumen and the adherence of smooth muscle cells on the outer surface of the tubes. These tests, carried out in multiwells, showed that the scaffolds had excellent cell attachment and guidance characteristics.     

Purification and characterization of extracellular chitinase from Aeromonas schubertii

A locally isolated stain Aeromonas schubertii was cultured and induced by powdered chitin for the production of chitinases. Extracellular proteins were purified by ammonium sulfate precipitation, dialysis to remove salts, and then preparative isoelectric focusing (IEF) to yield several chitinases. The purified enzymes were analyzed by SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) with and without glycol chitin and were found to be SDS-resistant. The chitinase present in the highest abundance was the one with an estimated molecular weight of 75 kDa. The Michaelis constant and turnover number were determined to be 0.29 mM and 1 s⁻¹, respectively, for this enzyme using colloidal chitin azure as the substrate. However, the ethanol treatment of this enzyme could significantly increase its chitinolytic activity. Other chitinases obtained in the same IEF fraction were determined to have molecular weights of ca. 30, 38, and 110 kDa. Since the proteins with highest chitinase activity were collected from IEF fraction tube with pH value of 4.8, those chitinase were believed to be acidic. An activity assay method using colloidal chitin azure as the substrate was recommended since it possessed a broader range of linearity in comparison with conventional reducing sugar equivalent method.