Peptidoglycan-type analysis of the N-acetylmuramic acid auxotrophic oral pathogen Tannerella forsythia and reclassification of the peptidoglycan-type of Porphyromonas gingivalis.

BACKGROUND: Tannerella forsythia is a Gram-negative oral pathogen. Together with Porphyromonas gingivalis and Treponema denticola it constitutes the "red complex" of bacteria, which is crucially associated with periodontitis, an inflammatory disease of the tooth supporting tissues that poses a health burden worldwide. Due to the absence of common peptidoglycan biosynthesis genes, the unique bacterial cell wall sugar N-acetylmuramic acid (MurNAc) is an essential growth factor of T. forsythia to build up its peptidoglycan cell wall. Peptidoglycan is typically composed of a glycan backbone of alternating N-acetylglucosamine (GlcNAc) and MurNAc residues that terminates with anhydroMurNAc (anhMurNAc), and short peptides via which the sugar backbones are cross-linked to build up a bag-shaped network. RESULTS: We investigated T. forsythia's peptidoglycan structure, which is an essential step towards anti-infective strategies against this pathogen. A new sensitive radioassay was developed which verified the presence of MurNAc and anhMurNAc in the cell wall of the bacterium. Upon digest of isolated peptidoglycan with endo-N-acetylmuramidase, exo-N-acetylglucosaminidase and muramyl-L-alanine amidase, respectively, peptidoglycan fragments were obtained. HPLC and mass spectrometry (MS) analyses revealed the presence of GlcNAc-MurNAc-peptides and the cross-linked dimer with retention-times and masses, respectively, equalling those of control digests of Escherichia coli and P. gingivalis peptidoglycan. Data were confirmed by tandem mass spectrometry (MS2) analysis, revealing the GlcNAc-MurNAc-tetra-tetra-MurNAc-GlcNAc dimer to contain the sequence of the amino acids alanine, glutamic acid, diaminopimelic acid (DAP) and alanine, as well as a direct cross-link between DAP on the third and alanine on the fourth position of the two opposite stem peptides. The stereochemistry of DAP was determined by reversed-phase HPLC after dabsylation of hydrolysed peptidoglycan to be of the meso-type. CONCLUSION: T. forsythia peptidoglycan is of the A1γ-type like that of E. coli. Additionally, the classification of P. gingivalis peptidoglycan as A3γ needs to be revised to A1γ, due to the presence of meso-DAP instead of LL-DAP, as reported previously.

In-House Solid-Phase Radioassay for the Detection of Anti-thyroglobulin Autoantibodies in Patients with Differentiated Thyroid Cancer.

Thyroglobulin autoantibodies (TgAb) are estimated to detect potential interferences in thyroglobulin (Tg) immunoassays and also for the diagnosis of autoimmune thyroid disease. A user friendly and robust in-house solid-phase radioassay was standardized and parameters like sensitivity, reproducibility and stability were assessed. Further, it was validated and evaluated for the detection of autoantibodies in differentiated thyroid cancer (DTC) patients. Totally 301 samples received in our laboratory for routine serum Tg estimation were studied. The samples were analyzed for TgAb by the solid-phase radioassay developed in-house and compared with commercial anti-hTg IRMA kit (Immunotech, France). The control group comprised of 37 euthyroid males from our Centre. The intra- and inter-assay CVs for the two quality control samples (Control A = 104 ± 12.6 IU/mL and Control B = 1029 ± 114 IU/mL) were found less than or equal to 6.05 and 13.85 % respectively. Solid-phase radioassay showed a good agreement on comparison with Immunotech IRMA (r = 0.99). Using the proposed cut-off thresholds (in-house solid-phase radioassay 52 IU/mL and Immunotech IRMA 30 IU/mL), 5.4 % of the control subjects were positive for TgAb by both the methods. Prevalence of TgAb in DTC patients was 17.3 and 16.6 % using the Immunotech kit and in-house solid-phase radioassay respectively. The in-house solid-phase radioassay has the requisite sensitivity for the evaluation of TgAb comparable to commercial kit and also suitable for routine use as it is rapid, user friendly and economical.

Analytical applications of MIPs in diagnostic assays: future perspectives.

Many efforts have been made to produce artificial materials with biomimetic properties for applications in binding assays. Among these efforts, the technique of molecular imprinting has received much attention because of the high selectivity obtainable for molecules of interest, robustness of the produced polymers, simple and short synthesis, and excellent cost efficiency. In this review, progress in the field of molecularly imprinted sorbent assays is discussed-with a focus on work conducted from 2005 to date.

A Simple Radioassay to Detect Nanoscale Membrane Disruption.

Understanding the mechanisms and kinetics of membrane damage is of interest to researchers in several overlapping fields of biology. In this study, we describe the development and validation of a simple 32PO43- release radioassay used to track nanometer-scale damage to the bacterial cell membrane. Nanoscale membrane damage will result in the release of small cytoplasmic molecules, such as amino acids, sugars, and osmolytes. Our radioassay tracks the release of these molecules using the release of cytoplasmic 32PO43- as a proxy. Our assay can both detect 32PO43- release and track release kinetics in the order of minutes. We demonstrate the use of our radioassay using A. baumannii treated with colistin and Ω76: two agents known to cause membrane damage. Our assay tracks greater membrane damage in A. baumannii treated with both these agents, compared to an untreated control. Our assay fills a niche that is not covered by traditional 51Cr release radioassays and fluorescent staining techniques. Furthermore, our assay can potentially be used to track membrane damage in other membrane systems such as lipid vesicles, animal cells, and organelles.

Population RBC folate concentrations can be accurately estimated from measured whole blood folate, measured hemoglobin, and predicted serum folate-cross-sectional data from the NHANES 1988-2010.

BACKGROUND: RBC folate (RBF) is an indicator of folate status and risk of neural-tube defects. It is calculated from whole blood folate (WBF), serum folate (SFOL), and hematocrit (Hct). SFOL and/or Hct are sometimes unavailable; hemoglobin (Hb) is generally available in surveys. OBJECTIVES: We assessed the ability of different RBF approximations to generate population data in women aged 12-49 y. METHODS: Using SFOL, RBF, Hct, Hb, and mean corpuscular Hb content (MCHC) from prefortification (1988-1994) and postfortification (1999-2006, 2007-2010) NHANES we applied 6 approaches: #1) assume SFOL = 0; #2) impute SFOL (population median); #3) impute Hct (population median); #4) estimate Hct (Hb/MCHC); #5) assume SFOL = 0 and estimate Hct; and #6) predict SFOL (from WBF) and estimate Hct. For each approach, we calculated the paired percentage difference to the "true" RBF and estimated various statistics. RESULTS: For 2007-2010 (unweighted data), the median relative difference from "true" RBF was lowest for approaches #2 (-0.74%), #4 (-0.96%), and #6 (-1.15%), intermediate for #3 (-3.36%), and highest for #5 (4.96%) and #1 (5.78%). The 95% agreement limits were smallest for approach #1 (2.33%, 13.0%) and largest for #3 (-20.8%, 11.3%). Approach #2 showed concentration-dependence (negative compared with positive differences at low compared with high RBF). Using weighted data, we found similar patterns across approaches for mean relative differences by demographic subgroup for all 3 time periods. CONCLUSIONS: We obtained the best agreement between estimated and "true" RBF when we predicted SFOL using a regression equation obtained from a subset of samples (approach #6). Alternatively, the consistent overestimation of RBF when assuming SFOL = 0 (∼6%) could be addressed by adjusting the data (approach #5). Similar observations for pre- and postfortification periods suggest applicability to low and high folate status situations, but should be confirmed elsewhere. To estimate RBF, at least WBF and Hb are needed.

Poly(amino acid)-based fibrous scaffolds modified with surface-pendant peptides for cartilage tissue engineering.

In this study, fibrous scaffolds based on poly(γ-benzyl-l-glutamate) (PBLG) were investigated in terms of the chondrogenic differentiation potential of human tooth germ stem cells (HTGSCs). Through the solution-assisted bonding of the fibres, fully connected scaffolds with pore sizes in the range 20-400 µm were prepared. Biomimetic modification of the PBLG scaffolds was achieved by a two-step reaction procedure: first, aminolysis of the PBLG fibres' surface layers was performed, which resulted in an increase in the hydrophilicity of the fibrous scaffolds after the introduction of N5 -hydroxyethyl-l-glutamine units; and second, modification with the short peptide sequence azidopentanoyl-GGGRGDSGGGY-NH2 , using the 'click' reaction on the previously modified scaffold with 2-propynyl side-chains, was performed. Radio-assay of the 125 I-labelled peptide was used to evaluate the RGD density in the fibrous scaffolds (which varied in the range 10-3 -10 pm/cm2 ). All the PBLG scaffolds, especially with density 90 ± 20 fm/cm2 and 200 ± 100 fm/cm2 RGD, were found to be potentially suitable for growth and chondrogenic differentiation of HTGSCs. Copyright © 2015 John Wiley & Sons, Ltd.

A Continuously Infused Microfluidic Radioassay System for the Characterization of Cellular Pharmacokinetics.

Measurement of cellular tracer uptake is widely applied to learn the physiologic status of cells and their interactions with imaging agents and pharmaceuticals. In-culture measurements have the advantage of less stress to cells. However, the tracer solution still needs to be loaded, unloaded, and purged from the cell culture during the measurements. Here, we propose a continuously infused microfluidic radioassay (CIMR) system for continuous in-culture measurement of cellular uptake. The system was tested to investigate the influence of the glucose concentration in cell culture media on 18F-FDG uptake kinetics. METHODS: The CIMR system consists of a microfluidic chip integrated with a flow-control unit and a positron camera. Medium diluted with radioactive tracer flows through a cell chamber continuously at low speed. Positrons emitted from the cells and from tracer in the medium are measured with the positron camera. The human cell lines SkBr3 and Capan-1 were incubated with media of 3 different glucose concentrations and then measured with 18F-FDG on the CIMR system. In addition, a conventional uptake experiment was performed. The relative uptake ratios between different medium conditions were compared. A cellular 2-compartment model was applied to estimate the cellular pharmacokinetics on CIMR data. The estimated pharmacokinetic parameters were compared with expressions of glucose transporter-1 (GLUT1) and hexokinase-2 measured by quantitative real-time polymerase chain reaction. RESULTS: The relative uptake ratios obtained from CIMR measurements correlated significantly with those from the conventional uptake experiments. The relative SDs of the relative uptake ratios obtained from the CIMR uptake experiments were significantly lower than those from the conventional uptake experiments. The fit of the cellular 2-compartment model to the 18F-FDG CIMR measurements was of high quality. For SkBr3, the estimated pharmacokinetic parameters k1 and k3 were consistent with the messenger RNA expression of GLUT1 and hexokinase-2: culturing with low glucose concentrations led to higher GLUT1 and hexokinase-2 expression as well as higher estimated k1 and k3 For Capan-1, the estimated k1 and k3 increased as the glucose concentration in the culture medium decreased, and this finding did not match the corresponding messenger RNA expression. CONCLUSION: The CIMR system captures dynamic uptake within the cell culture and enables estimation of the cellular pharmacokinetics.

Competitive chemiluminescent enzyme immunoassay for vitamin B12 analysis in human milk.

Recent discoveries of matrix interferences by haptocorrin (HC) in human milk and serum show that past analyses of vitamin B12 in samples with high HC content might have been inaccurate (Lildballe et al., 2009; Carmel & Agrawal, 2012). We evaluated two competitive enzyme-binding immunoassays for serum/plasma (IMMULITE and SimulTRAC-SNB) for B12 analysis in human milk. B12-recovery rates (United States Environmental Protection Agency, 2007) were determined to be 78.9 ± 9.1% with IMMULITE and 225 ± 108% (range 116-553%) using SimulTRAC-SNB, most likely due to the presence of excess HC. HC-interferences were not observed with the IMMULITE assay, rendering previously reported mandatory HC-removal (Lildballe et al., 2009) unnecessary. Linearity continued at low B12-concentrations (24-193 pM; r(2)>0.985). Milk B12 concentrations from Bangladeshi women (72-959 pM) were significantly lower than those from California (154-933 pM; p<0.0001) showing IMMULITE's robustness against the complex milk matrix and its ability to measure low milk B12 concentrations.

Fast metabolic response to drug intervention through analysis on a miniaturized, highly integrated molecular imaging system.

UNLABELLED: We report on a radiopharmaceutical imaging platform designed to capture the kinetics of cellular responses to drugs. METHODS: A portable in vitro molecular imaging system comprising a microchip and a ß-particle imaging camera permitted routine cell-based radioassays of small numbers of either suspended or adherent cells. We investigated the kinetics of responses of model lymphoma and glioblastoma cancer cell lines to (18)F-FDG uptake after drug exposure. Those responses were correlated with kinetic changes in the cell cycle or with changes in receptor tyrosine kinase signaling. RESULTS: The platform enabled direct radioassays of multiple cell types and yielded results comparable to those from conventional approaches; however, the platform used smaller sample sizes, permitted a higher level of quantitation, and did not require cell lysis. CONCLUSION: The kinetic analysis enabled by the platform provided a rapid (≈ 1 h) drug screening assay.