A comparison of high-content screening versus manual analysis to assay the effects of mesenchymal stem cell-conditioned medium on neurite outgrowth in vitro

Bone marrow mesenchymal stem cells (MSCs) promote nerve growth and functional recovery in animal models of spinal cord injury (SCI) to varying levels. The authors have tested high-content screening to examine the effects of MSC-conditioned medium (MSC-CM) on neurite outgrowth from the human neuroblastoma cell line SH-SY5Y and from explants of chick dorsal root ganglia (DRG). These analyses were compared to previously published methods that involved hand-tracing individual neurites. Both methods demonstrated that MSC-CM promoted neurite outgrowth. Each showed the proportion of SH-SY5Y cells with neurites increased by ~200% in MSC-CM within 48 h, and the number of neurites/SH-SY5Y cells was significantly increased in MSC-CM compared with control medium. For high-content screening, the analysis was performed within minutes, testing multiple samples of MSC-CM and in each case measuring >15,000 SH-SY5Y cells. In contrast, the manual measurement of neurite outgrowth from >200 SH-SY5Y cells in a single sample of MSC-CM took at least 1 h. High-content analysis provided additional measures of increased neurite branching in MSC-CM compared with control medium. MSC-CM was also found to stimulate neurite outgrowth in DRG explants using either method. The application of the high-content analysis was less well optimized for measuring neurite outgrowth from DRG explants than from SH-SY5Y cells.

A new randomization assay reveals Unexpected elements of sequence bias in model 'randomized' gene libraries:Implications for biopanning

Although techniques such as biopanning rely heavily upon the screening of randomized gene libraries, there is surprisingly little information available on the construction of those libraries. In general, it is based on the cloning of 'randomized' synthetic oligonucleotides, in which given position(s) contain an equal mixture of all four bases. Yet, many supposedly 'randomized' libraries contain significant elements of bias and/or omission. Here, we report the development and validation of a new, PCR-based assay that enables rapid examination of library composition both prior to and after cloning. By using our assay to analyse model libraries, we demonstrate that the cloning of a given distribution of sequences does not necessarily result in a similarly composed library of clones. Thus, while bias in randomized synthetic oligonucleotide mixtures can be virtually eliminated by using unequal ratios of the four phosphoramidites, the use of such mixtures does not ensure retrieval of a truly randomized library. We propose that in the absence of a technique to control cloning frequencies, the ability to analyse the composition of libraries after cloning will enhance significantly the quality of information derived from those libraries. (C) 2000 Published by Elsevier Science B.V. All rights reserved.

The microbiological destruction of thiocyanate in activated sludge plants treating coke oven effluent

The treatment of effluents produced during the manufacture of metallurgical coke is normally carried out using the activated sludge process. The efficiency of activated sludges in purifying coke oven effluent depends largely on the maintenance of species of micro-organisms which destroy thiocyanate. The composition, production, toxicity and treatment of coke oven effluent at Corby steelworks are described. A review is presented which follows the progress made towards identifying and monitoring the species of bacteria which destroy thiocyanate in biological treatment plants purifying coke oven effluents. In the present study a search for bacteria capable of destroying thiocyanate led to the isolation of a species of bacteria, identified as Pseudomonas putida, which destroyed thiocyanate in the presence of succinate; this species had not previously been reported to use thiocyanate. Washed cell suspensions of P. putida destroyed phenol and thiocyanate simultaneously and thiocyanate destruction was not suppressed by pyridine, aniline or catechol at the highest concentrations normally encountered in coke oven effluent. The isolate has been included, as N.C.I.B. 11198, in the National Collection of Industrial Bacteria, Torrey Research Station, Aberdeen. Three other isolates, identified as Achromobacter sp., Thiobacillus thioparus and T. denitrificans, were also confirmed to destroy thi.ocyanate. A technique has been developed for monitoring populations of different species of bacteria in activated sludges. Application of this technique to laboratory scale and full scale treatment plants at Corby showed that thiobacilli were usually not detected; thiobacilli were el~inated during the commissioning period of the full scale plant. However experiments using a laboratory scale plant indicated that during a period of three weeks an increase in the numbers of thiobacilli might have contributed to an improvement in plant performance. Factors which might have facilitated the development of thiobacilli are discussed. Large numbers of fluorescent pseudomonads capable of using thiocyanate were sometimes detected in the laboratory scale plant. The possibility is considered that catechol or other organic compounds in the feed-liquor might have stimulated fluorescent pseudmonads. Experiments using the laboratory scale plant confirmed that deteriorations in the efficiency of thiocyanate destruction were sometimes caused by bulking sludges, due to the excessive growth of fungal floes. Increased dilution of the coke oven effluent was a successful remedy to this difficulty. The optimum operating conditions recommended by the manufacturer of the full scale activated sludge plant at Corby are assessed and the role of bacterial monitoring in a programme of regular monitoring tests is discussed in relation to the operation of activated sludge plants treating coke oven effluents.

Some aspects of the microbiological properties of polymers and composite materials

The interaction of microorganisms with glass-reinforced polyester resins(GRP), both under laboratory and simulated operating conditions, has been examined following reports of severl! fungal biodeterioration. Although GRP was not previously associated with substantial microbial growth, small amounts of microbial activity would pose problems for products associated with comestible materials. The microbiology of the raw materials was investigated, two ingredients were supportive to microbial populations whilst five materials were biostatic or inhibitory in their action. Production laminate was not susceptible to microbial deterioration or inhibitory to microbes. Incorporation of zinc stearate, one of the supportive ingredients, at 300% manufacturing level or drastic undercuring produced laminate capable of supporting microbial growth but only after a non-biotic stage of degradation. Study of the long-term population dynamics of cisterns of GRP and competitive materials under conditions simulating in-service conditions, monitoring microbial numbers within the experimental vessels and comparing with the populations of the supply water, suggests that the performance of GRP cisterns is slightly superior to conventional competitive materials. An investigation of the biological performance of GRP cisterns in an isolated area of known microbiological hazard was conducted. Severe biodeterioration had been experienced with Preform GRP articles moulded using different production techniques, but substitution of current GRP articles resulted in no recurrence of the problem. All attempts to establish the fungal isolate responsible for the phenomena in cisterns under controlled conditions failed. Scanning Electron Microscopy of GRP surfaces showed that although differences exist between current and Preform laminates, these could not satisfactorily explain the differences in service behaviour. These results and the results of the British Plastics Federation Expert Working Group interlaboratory study are discussed in relation to the original report of gross fungal biodeterioration and, to the design of future testing programmes for the products of industrial concerns.

The development of a model system and high throughput assay for the study of interactions between zinc finger proteins and DNA

It has been recognised for some time that a full code of amino acid-based recognition of DNA sequences would be useful. Several approaches, which utilise small DNA binding motifs called zinc fingers, are presently employed. None of the current approaches successfully combine a combinatorial approach to the elucidation of a code with a single stage high throughput screening assay. The work outlined here describes the development of a model system for the study of DNA protein interactions and the development of a high throughput assay for detection of such interactions. A zinc finger protein was designed which will bind with high affinity and specificity to a known DNA sequence. For future work it is possible to mutate the region of the zinc finger responsible for the specificity of binding, in order to observe the effect on the DNA / protein interactions. The zinc finger protein was initially synthesised as a His tagged product. It was not possible however to develop a high throughput assay using the His tagged zinc finger protein. The gene encoding the zinc finger protein was altered and the protein synthesised as a Glutathione S-Transferase (GST) fusion product. A successful assay was developed using the GST protein and Scintillation Proximity Assay technology (Amersham Pharmacia Biotech). The scintillation proximity assay is a dynamic assay that allows the DNA protein interactions to be studied in "real time". This assay not only provides a high throughput method of screening zinc finger proteins for potential ligands but also allows the effect of addition of reagents or competitor ligands to be monitored.

Serodiagnosis of staphylococcal sepsis

The coagulase-negative staphylococci are the most frequent cause of sepsis associated with indwelling intravascular catheters. Current microbiological investigations to support the diagnosis of catheter-related sepsis (CRS) include the culture of blood and catheter tips, however positive results may reflect specimen contamination, or colonisation of the catheter rather than true sepsis. Previous serological approaches to assist in the diagnosis of CRS based on cellular staphylococcal antigens have been of limited value. In this current study, the serodiagnostic potential of an exocellular antigen produced by 7 strains of coagulase-negative staphylococci cultured in brain heart infusion broth was investigated. Antigenic material isolated by gel permeation from liquid culture was characterised by immunological techniques and chemical analysis. Characterisation of the exocellular antigen revealed a novel glycerophosphoglycolipid, termed lipid S. which shared antigenic determinants with lipoteichoic acid, but differed by comprising a glycerophosphate chain length of only 6 units. In addition, lipid S was immunologically distinct from diphosphatidyl glycerol, a constituent cell membrane phospho lipid. An indirect enzyme linked immunosorbent assay (ELISA) based on lipid S was subsequently developed and used to determine serum antibody levels (IgM and IgG) in 67 patients with CRS due to staphylococci, and 67 patients with a central venous catheter (CVC) in situ who exhibited no evidence of sepsis. The sensitivity and specificity of the lipid S IgG ELISA was 75% and 90% respectively whilst the IgM assay had sensitivity and specificity of 52% and 85%. The addition of GullSORereagent to the EL1SA procedure to remove competing serum IgG and rheumatoid factor did not significantly improve the performance of the IgM assay. The serological response in serial serum samples of 13 patients with CRS due to staphylococci was investigated. Elevated levels of antibody were detected at an early stage of infection, prior to the isolation of microorganisms by standard culture methods, and before the clinical presentation of sepsis in 3 patients. The lipid S ELISA was later optimised and a rapid 4-hour assay developed for the serodiagnosis of CRS. Serum IgG levels were determined in 40 patients with CRS due to staphylococci and 40 patients with a CVC in situ who exhibited no evidence of sepsis. The sensitivity and specificity of the rapid IgG assay was 70% and 100% respectively. Elevated serum antibody levels in patients with endocarditis, prosthetic joint infection and pyogenic spondylodiscitis due to Gram-positive cocci were also detected with the lipid S ELISA suggesting that the assay may facilitate the diagnosis of these infections. Unexpected increased levels of anti-lipid S IgG in 31% of control patients with sciatica suggested a possible microbial aetiology of this condition. Further investigation of some of these patients by culture of microdiscectomy tissue removed at operation, revealed the presence of low-virulent microorganisms in 37% of patients of which Propionibacterium aeries accounted for 85% of the positive culture isolates. The results suggested a previously unrecognised association between P. acnes and sciatica, which may have implications for the future management of the condition.

The formulation of artificial reference standards for use within the ELISPOT assay

Whether to assess the functionality of equipment or as a determinate for the accuracy of assays, reference standards are essential for the purposes of standardisation and validation. The ELISPOT assay, developed over thirty years ago, has emerged as a leading immunological assay in the development of novel vaccines for the assessment of efficacy. However, with its widespread use, there is a growing demand for a greater level of standardisation across different laboratories. One of the major difficulties in achieving this goal has been the lack of definitive reference standards. This is partly due to the ex vivo nature of the assay, which relies on cells being placed directly into the wells. Thus, the aim of this thesis was to produce an artificial reference standard using liposomes, for use within the assay. Liposomes are spherical bilayer vesicles with an enclosed aqueous compartment and therefore are models for biological membranes. Initial work examined pre-design considerations in order to produce an optimal formulation that would closely mimic the action of the cells ordinarily placed on the assay. Recognition of the structural differences between liposomes and cells led to the formulation of liposomes with increased density. This was achieved by using a synthesised cholesterol analogue. By incorporating this cholesterol analogue in liposomes, increased sedimentation rates were observed within the first few hours. The optimal liposome formulation from these studies was composed of 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Chol) and brominated cholesterol (Brchol) at a 16:4:12 µMol ratio, based on a significantly higher (p<0.01) sedimentation (as determined by a percentage transmission of 59 ± 5.9 % compared to the control formulation at 29 ± 12 % after four hours). By considering a range of liposome formulations ‘proof of principle’ for using liposomes as ELISPOT reference standards was shown; recombinant IFN? cytokine was successfully entrapped within vesicles of different lipid compositions, which were able to promote spot formation within the ELISPOT assay. Using optimised liposome formulations composed of phosphatidylcholine with or without cholesterol (16 µMol total lipid) further development was undertaken to produce an optimised, scalable protocol for the production of liposomes as reference standards. A linear increase in spot number by the manipulation of cytokine concentration and/or lipid concentrations was not possible, potentially due to the saturation that occurred within the base of wells. Investigations into storage of the formulations demonstrated the feasibility of freezing and lyophilisation with disaccharide cryoprotectants, but also highlighted the need for further protocol optimisation to achieve a robust reference standard upon storage. Finally, the transfer of small-scale production to a medium lab-scale batch (40 mL) demonstrated this was feasible within the laboratory using the optimised protocol.

Development of an enantiomer selective microsampling assay for the quantification of ketorolac suitable for paediatric pharmacokinetic studies

Background: Ketorolac, a potent nonsteroidal anti-inflammatory drug used for pain control in children, exists as a racemate of inactive R (+) and active S (-) enantiomers. Aim: To develop a microsampling assay for the enantioselective analysis of ketorolac in children. Methods: Ketorolac enantiomers were extracted from 50 µl of plasma by liquid–liquid extraction and separated on a ChiralPak AD-RH. Detection was by a TSQ quantum triple quadrupole mass spectrometer with an electrospray ionisation source operating in a positive ion mode. Five children (age 13.8 (1.6) years, weight 52.7 (7.2) kg), were administered intravenous ketorolac 0.5 mg/kg (maximum 10 mg) and blood samples were taken at 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 12 h post administration. CL, VD and t1/2 were calculated based on non-compartmental methods. Results: The standard curves for R (+) and S (-) ketorolac were linear in the range 0–2000 ng/ml. The LLOQs of the method were 0.15 ng on column and 0.31 ng on column for R (+) and S (-) ketorolac, respectively. The median (range) VD and CL of R (+) and S (-) ketorolac were 0.12 l/kg (0.07–0.17), 0.017 l/h/kg (0.12–0.29) and 0.17 (0.09–0.31) l/kg, 0.049 (0.02–0.1) l/h/kg, p = 0.043), respectively. The median (range) elimination half-life (t1/2) of the R (+) and S (-) ketorolac was 5.0 h (2.5–5.8) and 3.1 h (1.8–4.4), p = 0.043), respectively. Conclusion: The development of a simple, rapid and reliable ketorolac assay suitable for paediatric PK studies is reported. Copyright © 2013 John Wiley & Sons, Ltd.

Microarray-formatted clinical biomarker assay development using peptide aptamers to anterior gradient-2

Anterior gradient-2 protein was identified using proteomic technologies as a p53 inhibitor which is overexpressed in human cancers, and this protein presents a novel pro-oncogenic target with which to develop diagnostic assays for biomarker detection in clinical tissue. Combinatorial phage-peptide libraries were used to select 12 amino acid polypeptide aptamers toward anterior gradient-2 to determine whether methods can be developed to affinity purify the protein from clinical biopsies. Selecting phage aptamers through four rounds of screening on recombinant human anterior gradient-2 protein identified two classes of peptide ligand that bind to distinct epitopes on anterior gradient-2 protein in an immunoblot. Synthetic biotinylated peptide aptamers bound in an ELISA format to anterior gradient-2, and substitution mutagenesis further minimized one polypeptide aptamer to a hexapeptide core. Aptamers containing this latter consensus sequence could be used to affinity purify to homogeneity human anterior gradient-2 protein from a single clinical biopsy. The spotting of a panel of peptide aptamers onto a protein microarray matrix could be used to quantify anterior gradient-2 protein from crude clinical biopsy lysates, providing a format for quantitative screening. These data highlight the utility of peptide combinatorial libraries to acquire rapidly a high-affinity ligand that can selectively bind a target protein from a clinical biopsy and provide a technological approach for clinical biomarker assay development in an aptamer microarray format.

A rapid ELISA for the diagnosis of intravascular catheter related sepsis caused by coagulase negative staphylococci

Aim: To develop and evaluate a rapid enzyme linked immunosorbent assay (ELISA) for the diagnosis of intravascular catheter related sepsis caused by coagulase negative staphylococci. Methods: Forty patients with a clinical and microbiological diagnosis of intravascular catheter related sepsis and positive blood cultures, caused by coagulase negative staphylococci, and 40 control patients requiring a central venous catheter as part of their clinical management were recruited into the study. Serum IgG responses to a previously undetected exocellular antigen produced by coagulase negative staphylococci, termed lipid S, were determined in the patient groups by a rapid ELISA. Results: There was a significant difference (p = < 0.0001) in serum IgG to lipid S between patients with catheter related sepsis and controls. The mean antibody titre in patients with sepsis caused by coagulase negative staphylococci was 10 429 (range, no detectable serum IgG antibody to 99 939), whereas serum IgG was not detected in the control group of patients. Conclusions: The rapid ELISA offers a simple, economical, and rapid diagnostic test for suspected intravascular catheter related sepsis caused by coagulase negative staphylococci, which can be difficult to diagnose clinically. This may facilitate treatment with appropriate antimicrobials and may help prevent the unnecessary removal of intravascular catheters.

The enantioselective population pharmacokinetics of intravenous ketorolac in children using a stereoselective assay suitable for microanalysis

Objective: To describe the effect of age and body size on enantiomer selective pharmacokinetic (PK) of intravenous ketorolac in children using a microanalytical assay. Methods: Blood samples were obtained at 0, 15 and 30 min and at 1, 2, 4, 6, 8 and 12 h after a weight-dependent dose of ketorolac. Enantiomer concentration was measured using a liquid chromatography tandem mass spectrometry method. Non-linear mixed-effect modelling was used to assess PK parameters. Key findings: Data from 11 children (1.7–15.6 years, weight 10.7–67.4 kg) were best described by a two-compartment model for R(+), S(−) and racemic ketorolac. Only weight (WT) significantly improved the goodness of fit. The final population models were CL = 1.5 × (WT/46)0.75, V1 = 8.2 × (WT/46), Q = 3.4 × (WT/46)0.75, V2 = 7.9 × (WT/46), CL = 2.98 × (WT/46), V1 = 13.2 × (WT/46), Q = 2.8 × (WT/46)0.75, V2 = 51.5 × (WT/46), and CL = 1.1 × (WT/46)0.75, V1 = 4.9 × (WT/46), Q = 1.7 × (WT/46)0.75 and V2 = 6.3 × (WT/46)for R(+), S(−) and racemic ketorolac. Conclusions: Only body weight influenced the PK parameters for R(+) and S(−) ketorolac. Using allometric size scaling significantly affected the clearances (CL, Q) and volumes of distribution (V1, V2).

Establishment of a PCR and restriction enzyme assay for the detection of the hemochromatosis gene in normal subjects and diabetic patients

The purpose of this study was to establish a polymerase chain reaction (PCR)-restriction enzyme assay for detecting the hereditary hemochromatosis (HHC) mutation, C282Y, in gestational and gestational diabetic subjects in South Florida. DNA samples from 43 gestational subjects were amplified by PCR, digested with RsaI, and analyzed by electrophoresis. An allelic frequency of 2.33%, or 4.65% heterozygosity, was observed. The assay is successful and applicable to future studies on HHC and gestational diabetes. ^