Rapid chromatographic method to determine polyamines in urine and whole blood

A procedure is described for the rapid determination of putrescine, spermine and spermidine in urine and whole blood. The samples are hydrolyzed with barium hydroxide and are neutralized with sulfuric acid. The polyamines are concentrated and separated from amino acids on a small bed of ion-exchange resin that then serves to load the samples on a two-channel, automated ion-exchange chromatography apparatus. As many as 100 samples can be analyzed in a 24-h period. The method has been shown to be applicable to the analysis of urine and whole blood samples, but further development is needed for application to serum samples.     

Verfahren und Analysenautomat zur Bestimmung von Nährlösungskomponenten und Stoffwechselprodukten

A continuous flow system with an enzyme electrode and with a new type of measuring instrument, the BIOXY-Meter, is described as an example for the determination of glucose. The BIOXY-Meter combined with various biosensors may be used in biotechnology and in other fields for the solution of a number of analytical problems. Measuring principle is the reduction of oxygen. The peak of first derivation of the current-time-curve is proportional to oxygen consumption and to decrease of substrate concentration. Enzyme fixation with common adhesive and a device for automated saturation of measuring samples with air in the sampler are also described. With the apparatus 60 samples per hour are analyzed for glucose. The linear measuring range is 0.1 mMol to 1.5 mMol with a coefficient of variation of V_k = 0.93 per cent. The described type of enzyme electrode is stable for 2 to 12 weeks or 1,000 to 5,000 measurements.     

Separation and determination of mass and radioactivity of fermentation gases.

A procedure is described for the separation of permanent gases on a gas chromatograph, the determination of each component by means of a thermal conductivity detector and the simultaneous measurement of radioactivity in each peak by means of a proportional counter.Procedures for calibration of the apparatus and for calculation of absolute radioactivities in samples are given.The capabilities of the apparatus are illustrated by some results of experiments with an artificial rumen using ¹⁴C- and ³H-labeled compounds.     

Semiautomated Method for Microbiological Vitamin Assays

A semiautomated method for microbiological vitamin assays is described, which includes separate automated systems for the preparation of the cultures and for the measurement of turbidity. In the dilution and dosage unit based on the continuous-flow principle, vitamin samples were diluted to two different dose levels at a rate of 40 per hr, mixed with the inoculated test broth, and dispensed into culture tubes. After incubation, racks with culture tubes were placed on the sampler of an automatic turbidimeter. This unit, based on the discrete-sample system, measured the turbidity and printed the extinction values at a rate of 300 per hr. Calculations were computerized and the results, including statistical data, are presented in an easily readable form. The automated method is in routine use for the assays of thiamine, riboflavine, pyridoxine, cyanocobalamin, calcium pantothenate, nicotinic acid, pantothenol, and folic acid. Identical vitamin solutions assayed on different days gave variation coefficients for the various vitamin assays of less than 10%.     

Mass spectrometry software for biochemical analysis in electrospray and fast atom bombardment modes.

New mass spectrometry techniques, such as electrospray ionization (ESI), allow the study of large biomolecules and peptide mixtures. The data produced are complex and interpretation can be a long and tedious process. A new suite of data-processing software is described which allows many of these operations to be carried out in a rapid, automated way. Software is described for the deconvolution of the spectra of multiply charged ions, for both pure compounds and mixtures. The rapid peptide mapping of protein digests from h.p.l.c.-m.s. data and peptide sequence confirmation from multiple-stage (MS)-m.s. data using tandem quadrupole m.s. are also described. In addition preliminary results are presented on first principle sequencing of unknown peptides from MS-m.s. experiments.     

USING CONVENTIONAL MICROTITER PLATE TECHNOLOGY FOR THE AUTOMATION OF MICROBIOLOGICAL TESTING OF DRINKING WATER

With the arrival of automated systems for microtiter (MT) plate assays, classical most probable number (MPN) techniques lend themselves to an efficient way to perform quantitative microbiological tests. In contrast to existing automated systems for quantitative determination of microorganisms there is no need for dedicated instruments. Standardized MT equipment available in modern biochemical laboratories, namely a pipetting robot using sterile pipetting tips and a MT reader under computer control are the only prerequisites. The distribution of the media and the samples with optional dilutions, the measurement of growth as well as the statistical calculations of the MPN value are performed automatically. A system was validated for the analysis of the aerobic mesophilic count (AC) of drinking water. The system can cope with low or high bacterial load from 0 to 2 × 10⁴ colonies per mL. The system which takes out the tedium and personnel influence of routine microbiological work is prone to many other determinations like fecal indicator organisms in water as well as other microorganisms of interest in food microbiology.     

Microsequence analysis of peptides and proteins. IX. Manual gas-phase microsequencing of multiple samples

A novel apparatus for performing manual gas-phase Edman chemistry on protein and peptide samples is described. Edman chemistry is performed in 6 to 10 Teflon continuous flow reactors (CFR), previously described by J.E. Shively et al. (1987) Anal. Biochem. 163, 517-529). The CFRs are packed with 10-15 mg of Polybrene-coated spherical silica (Porasil B, Waters Associates). The gas-phase coupling reagent and cleavage reagent are 5% aqueous triethylamine and anhydrous trifluoroacetic acid, respectively, delivered by a stream of argon gas. The delivery of the gas-phase reagents is manually controlled with Hamilton 3-way valves and 2-way valves, and that of the solvents, ethyl acetate and butyl chloride, by syringe pipetting. The average cycle time is 15-20 min for 6 to 10 samples run simultaneously. Conversion of the anilinothiazolinone to phenylthiohydantoin (PTH) amino acid derivatives is accomplished manually with 25% aqueous trifluoroacetic acid. The PTH amino acids are analyzed by reversed-phase HPLC using an autosampler for handling multiple samples. Excellent results were obtained in the 100-200 pmol range. Protein samples can be sequenced from 15-20 cycles, and peptide samples usually to the COOH terminus. Initial yields ranged from 30 to 60% and repetitive yields ranged from 90 to 96%. The sample washout and size of background peaks are significantly reduced, compared to older methods of manual sequence analysis. The yields and background signal to noise are comparable to automated gas-phase Edman chemistry. The improved manual Edman described represents a low cost alternative to automated sequence analysis, and has the advantage being able to process multiple samples simultaneously.     

Rhesus monkey (Macaca mulatta) complex learning skills reassessed

Results from three experiments on basic learning and transfer in rhesus monkeys (Macaca mulatta) are reported in which fully automated testing paradigms, afforded by the Language Research Center's Computerized Test System (LRC-CTS), were employed. Performance levels for discrimination learning set, transfer index, and mediational-learning testing were uniformly higher than was predicted from the literature, in contrast to previous reports of compromised learning under similar conditions (automated apparatus, planimetric stimuli, spatial discontiguity between stimuli and response loci). Analyses reveal relatively advanced learning set performance, transfer-index ratios, and positive transfer of learning even at stringent criterion levels. Moreover, the data suggest that rhesus monkeys tested in these experiments exhibit mediational instead of associative learning strategies, as do great apes and in contrast to previous reports of rhesus learning. We argue that the LRC-CTS enhances learning by nonhuman primate subjects, obviating those factors, reported in the literature from experiments in which manual or other automated systems were employed, that compromise learning.     

Collector device for apparatus hookup connections in microbiological manufacture]

A collector device developed on the basis of a holding tank is discussed. The working principle of the device, its characteristics and advantages as applied to microbiological industry are described. The use of the collector devices in the assembly diagrams of the apparatus provides reduction of the number of the piping fittings, the pipe-line length, the number of the sealing elements, heat evolution into the environment, and time of the apparatus servicing.     

Chip devices for miniaturized biotechnology

Chip devices were introduced in chemistry and molecular biology to improve the read-out of information from molecular systems by efficient analytical procedures and to organize automated experiments. Biochips and chip reactor systems are of interest for cellular processes, too, and can be regarded as components in interfaces for the information exchange between living nature and digital electronic systems. In this minireview, different types of chip reactors for biotechnological applications like nanotiterplates, chip thermocyclers and devices for segmented flow operations are discussed. Finally, an outlook is given on the application of chip reactor systems, which are promising tools for automated experiments with highly parallelized screening procedures, for artificial microcompartmentation, cell analogue systems, micro-ecological studies, investigations on modulated morphogenesis, and for a bioanalogue molecular nanotechnology.     

A system identification approach for developing model predictive controllers of antibody quality attributes in cell culture processes

As the biopharmaceutical industry evolves to include more diverse protein formats and processes, more robust control of Critical Quality Attributes (CQAs) is needed to maintain processing flexibility without compromising quality. Active control of CQAs has been demonstrated using model predictive control techniques, which allow development of processes which are robust against disturbances associated with raw material variability and other potentially flexible operating conditions. Wide adoption of model predictive control in biopharmaceutical cell culture processes has been hampered, however, in part due to the large amount of data and expertise required to make a predictive model of controlled CQAs, a requirement for model predictive control. Here we developed a highly automated, perfusion apparatus to systematically and efficiently generate predictive models using application of system identification approaches. We successfully created a predictive model of %galactosylation using data obtained by manipulating galactose concentration in the perfusion apparatus in serialized step change experiments. We then demonstrated the use of the model in a model predictive controller in a simulated control scenario to successfully achieve a %galactosylation set point in a simulated fed‐batch culture. The automated model identification approach demonstrated here can potentially be generalized to many CQAs, and could be a more efficient, faster, and highly automated alternative to batch experiments for developing predictive models in cell culture processes, and allow the wider adoption of model predictive control in biopharmaceutical processes. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1647–1661, 2017     

THE PREPARATION OF THE GRADED COLLODION MEMBRANES OF ELFORD AND THEIR USE IN THE STUDY OF FILTERABLE VIRUSES

1. The method described by Elford for the preparation of graded collodion membranes suitable for ultrafiltration was found to give excellent results, and his findings are fully confirmed. 2. A formula is given for the preparation of collodion from which satisfactory membranes of graded porosity can be prepared. 3. The technique and apparatus used in the preparation, and standardization of membranes are described in detail. 4. The technique and apparatus required for ultrafiltration experiments are described, and some drawbacks encountered in the experiments are discussed. 5. The results of ultrafiltration experiments show that the pores of the membranes are remarkably uniform in size.     

Simple methods which maintain the barrier status of specific-pathogen-free animals during experimentation

Several simple procedures for experimentation on specific-pathogen-free rats and mice are described which combine the technique of plastic-film-isolator containment and a laminar flow sterile environment. These permit the full range of cellular immunology experiments to be performed without compromising the microbiological barrier.     

A compact multi-channel apparatus for automated real-time monitoring of bioluminescence

We have developed a multi-channel apparatus for automated monitoring of bioluminescence in real time. We designed this apparatus to be compact (230 mm wide, 600 mm deep, and 227.5 mm high) so that it can be operated in a relatively small commercially-available incubator. The apparatus can process 20 samples at maximum in a single run, providing enough processibility in small-scale experiments. We verified the reliability and sensitivity of the apparatus by observing circadian bioluminescence rhythms over one week from a bioluminescent reporter strain (E9) of the cyanobacterium Synechococcus sp. strain PCC 7942 [Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C.R., Tanabe, A., Golden, S.S., Johnson, C.H., Kondo, T., Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria, Science, 281 (1998) 1519–1523]. Our apparatus allows flexible experimental designs and will be effectively used for the studies of gene expression in various purposes.     

A compact multi-channel apparatus for automated real-time monitoring of bioluminescence

We have developed a multi-channel apparatus for automated monitoring of bioluminescence in real time. We designed this apparatus to be compact (230 mm wide, 600 mm deep, and 227.5 mm high) so that it can be operated in a relatively small commercially-available incubator. The apparatus can process 20 samples at maximum in a single run, providing enough processibility in small-scale experiments. We verified the reliability and sensitivity of the apparatus by observing circadian bioluminescence rhythms over one week from a bioluminescent reporter strain (E9) of the cyanobacterium Synechococcus sp. strain PCC 7942 [Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C.R., Tanabe, A., Golden, S.S., Johnson, C.H., Kondo, T., Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria, Science, 281 (1998) 1519-1523]. Our apparatus allows flexible experimental designs and will be effectively used for the studies of gene expression in various purposes.     

Automated Microtransfer Technique for the Assay of Poliovirus- and Mumps Virus-Neutralizing Antibodies

Use of an automated apparatus to quantitate mumps virus- and poliovirus-neutralizing antibody is described. The automated titration equipment affords savings in effort, time, and reagents in conducting large-scale surveys for the determination of mumps- and poliovirus-neutralizing antibodies. This technique has been found to be reproducible and gives results comparable to other antibody assay methods.     

Devices facilitating the preparation of biological specimens for electron microscopy

The construction of a matrix is described which facilitates the process of placing biological objects into polymer media in order to prepare ultrathin sections without the employment of gelatin, starch and polyethylene capsules that can be used only once. The construction of a reactor for cytochemical assays is presented. The apparatus can be used to locate enzymes within the cell, and to identify microorganisms. A modification of the dialysis technique for microbiological objects is proposed which accelerates and simplifies the process.     

A fully automated and computerized system for simultaneous measurements of motility and phototaxis in Chlamydomonas

A fully automated and computerized method for simultaneous measurements of motility and phototaxis of unicellular flagellates is described. Both systems are directly coupled with a homocontinuous culture. The motility measuring apparatus is equipped with a video camera and recorder for simultaneous single cell behaviour studies. First results of studies on the effects of the phototaxis inhibitor sodium azide and the Ca²⁺ conducting ionophore A23 187 on motility and phototaxis of Chlamydomonas are reported and correlated with video observations. These results demonstrate that the described systems give informations of whether phototaxis or motility or both are inhibited by chemicals.     

An automated climbing apparatus to measure chemotherapy-induced neurotoxicity in Drosophila melanogaster

We have developed a novel model system in Drosophila melanogaster to study chemotherapy-induced neurotoxicity in adult flies. Neurological deficits were measured using a manual geotactic climbing assay. The manual assay is commonly used; however, it is laborious, time-consuming, subject to human error and limited to observing one sample at a time. We have designed and built a new automated fly-counting apparatus that uses a “video capture-particle counting technology” to automatically measure 10 samples at a time, with 20 flies per sample. Climbing behavior was assessed manually, as in our previous studies, and with the automated apparatus within the same experiment yielding statistically similar results. Both climbing endpoints as well as the climbing rate can be measured in the apparatus, giving the assay more versatility than the manual assay. Automation of our climbing assay reduces variability, increases productivity and enables high throughput drug screens for neurotoxicity.     

Challenges and opportunities of automated protein-protein docking: HDOCK server vs human predictions in CAPRI Rounds 38-46

Protein-protein docking plays an important role in the computational prediction of the complex structure between two proteins. For years, a variety of docking algorithms have been developed, as witnessed by the critical assessment of prediction interactions (CAPRI) experiments. However, despite their successes, many docking algorithms often require a series of manual operations like modeling structures from sequences, incorporating biological information, and selecting final models. The difficulties in these manual steps have significantly limited the applications of protein-protein docking, as most of the users in the community are nonexperts in docking. Therefore, automated docking like a web server, which can give a comparable performance to human docking protocol, is pressingly needed. As such, we have participated in the blind CAPRI experiments for Rounds 38-45 and CASP13-CAPRI challenge for Round 46 with both our HDOCK automated docking web server and human docking protocol. It was shown that our HDOCK server achieved an “acceptable” or higher CAPRI-rated model in the top 10 submitted predictions for 65.5% and 59.1% of the targets in the docking experiments of CAPRI and CASP13-CAPRI, respectively, which are comparable to 66.7% and 54.5% for human docking protocol. Similar trends can also be observed in the scoring experiments. These results validated our HDOCK server as an efficient automated docking protocol for nonexpert users. Challenges and opportunities of automated docking are also discussed.