Fast and sensitive detection of an anthrax biomarker using SERS-based solenoid microfluidic sensor.

We report the application of a fully automated surface-enhanced Raman scattering (SERS)-based solenoid-embedded microfluidic device to the quantitative and sensitive detection of anthrax biomarker poly-γ-D-glutamic acid (PGA) in solution. Analysis is based on the competitive reaction between PGA and PGA-conjugated gold nanoparticles with anti-PGA-immobilized magnetic beads within a microfluidic environment. Magnetic immunocomplexes are trapped by yoke-type solenoids embedded within the device, and their SERS signals were directly measured and analyzed. To improve the accuracy of measurement process, external standard values for PGA-free serum were also measured through use of a control channel. This additional measurement greatly improves the reliability of the assay by minimizing the influence of extraneous experimental variables. The limit of detection (LOD) of PGA in serum, determined by our SERS-based microfluidic sensor, is estimated to be 100 pg/mL. We believe that the defined method represents a valuable analytical tool for the detection of anthrax-related aqueous samples.

Conversion of agroindustrial residues for high poly(γ-glutamic acid) production by Bacillus subtilis NX-2 via solid-state fermentation.

Poly(γ-glutamic acid) (γ-PGA) production by Bacillus subtilis NX-2 was carried out through solid-state fermentation with dry mushroom residues (DMR) and monosodium glutamate production residues (MGPR; a substitute of glutamate) for the first time. Dry shiitake mushroom residue (DSMR) was found to be the most suitable solid substrate among these DMRs; the optimal DSMR-to-MGPR ratio was optimized as 12:8. To increase γ-PGA production, industrial waste glycerol was added as a carbon source supplement to the solid-state medium. As a result, γ-PGA production increased by 34.8%. The batch fermentation obtained an outcome of 115.6 g kg(-1) γ-PGA and 39.5×10(8) colony forming units g(-1) cells. Furthermore, a satisfactory yield of 107.7 g kg(-1) γ-PGA was achieved by compost experiment on a scale of 50 kg in open air, indicating that economically large-scale γ-PGA production was feasible. Therefore, this study provided a novel method to produce γ-PGA from abundant and low-cost agroindustrial residues.

Assessment of the relationship between methotrexate polyglutamates in red blood cells and clinical response in patients commencing methotrexate for rheumatoid arthritis.

BACKGROUND AND OBJECTIVES: Therapeutic drug monitoring in patients with rheumatoid arthritis (RA) receiving methotrexate (MTX, MTXGlu1) has not been established. In this study, we aim to explore the relationship between red blood cell (RBC) concentrations of MTX and its polyglutamate metabolites (MTXGlu(n); n = 2, 3, 4, 5) and clinical response in RA patients commencing MTX. METHODS: The binding activity of MTXGlu(n) to three putative enzymes involved in the MTX mechanism of action­dihydrofolate reductase, thymidylate synthase, and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase­was simulated. RBC MTXGlu(n) concentrations that gave the highest inhibition activity across all three enzymes were linked with the disease activity score DAS28-3v (C-reactive protein [CRP]). A population pharmacokinetic-pharmacodynamic model was developed to describe the relationship between RBC MTX polyglutamate concentrations and clinical response in 12 RA patients commencing MTX. RESULTS: The highest inhibition activity was with RBC MTXGlu(3-5). These polyglutamates were further evaluated for their relationship with DAS28-3v (CRP). Three of the 12 patients had a high DAS28-3v (CRP) at baseline (mean = 6.1) and showed a delayed response to MTX treatment. The remaining nine patients with a lower DAS28-3v (CRP) baseline (mean = 3.6) showed an immediate response. The developed MTX pharmacokinetic-pharmacodynamic model provided an acceptable description of the observed DAS28-3v (CRP) across all patients. CONCLUSIONS: The developed model describes a longitudinal relationship between RBC MTXGlu(3-5) concentrations and DAS28-3v (CRP) in patients with RA commencing MTX. Further work is required to determine whether measurement of RBC MTX polyglutamates might be useful for dose individualisation in patients with RA.

Role of methotrexate polyglutamation and reduced folate carrier 1 (RFC1) gene polymorphisms in clinical assessment indexes.

The aims of the present study were to define inter-individual differences in response to methotrexate (MTX) through MTX polyglutamate (MTX-PG) levels in red blood cells (RBC) and MTX-related gene polymorphisms. A total of 145 rheumatoid arthritis patients were recruited. MTX-PG1-5 concentrations in RBC were measured, and 11 single nucleotide polymorphisms, all in MTX-related genes involved in the folate pathway, were analyzed. Disease activity was also assessed. There was no direct relationship between any MTX-PG concentration and the patient's disease condition, but detectability of MTX-PG5 was extracted as a candidate marker for response to MTX. When disease activity was compared between patients in which MTX-PG5 was detectable and undetectable, all indexes except the visual analog scale (VAS) and C-reactive protein (CRP) were found to be significantly lower in the former patients. Reduced folate carrier 1 (RFC1) 80G>A was significantly associated with the detectability of MTX-PG5; detectability of MTX-PG5 was lower in patients with the A mutant allele. The present study suggests that detectability of MTX-PG5 in RBC is a possible biomarker for response to MTX, and the RFC1 80G>A mutation is associated with low detectability of MTX-PG5. Prospective studies with a sufficient number of patients are needed to confirm the present findings.

Economical production of poly(γ-glutamic acid) using untreated cane molasses and monosodium glutamate waste liquor by Bacillus subtilis NX-2.

The production of poly(γ-glutamic acid) by Bacillus subtilis NX-2 from cane molasses and monosodium glutamate waste liquor (MGWL) was studied for the first time in this work. When batch fermentation was carried out with untreated molasses, 33.6±0.37 g L(-1) PGA was obtained with a productivity of 0.46±0.006 g L(-1) h(-1). In order to minimize the substrate inhibition, fed-batch fermentation was performed with untreated or hydrolyzed molasses in 7.5 L bioreactor, giving 50.2±0.53 and 51.1±0.51 g L(-1) of PGA at 96 h, respectively. Further studies were carried out by using MGWL as another carbon source, resulting in a PGA concentration of 52.1±0.52 g L(-1) with a productivity of 0.54±0.003 g L(-1) h(-1). These results suggest that the low-cost cane molasses and MGWL can be used for the environmental-friendly and economical production of PGA by B. subtilis NX-2.

Assessment of intracellular methotrexate and methotrexate-polyglutamate metabolite concentrations in erythrocytes by ultrafast matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry.

A new ultrafast quantitative and high-throughput mass spectrometric method using matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry has been developed and validated for determination of intracellular erythrocyte concentrations of the antifolate drug methotrexate (MTX) and its polyglutamate metabolites. The method consists of a solid-phase extraction of MTX and MTX-polyglutamate metabolites from deproteinized erythrocyte lysates spiked with aminopterin as internal standard. The newly developed method was validated according to the most recent FDA guidelines on linearity, recovery, within-run and between-run accuracy and precision and stability of the analytes. The low limit of quantification (LLOQ) was 10 nmol/L for all analytes while the limit of detection (LOD) determined at a signal-to-noise (S/N) ratio = 3:1 in drug- free erythrocyte lysate was on average 0.3 nmol/L. After validation, the new method was used in the measurement of intracellular erythrocyte concentrations of MTX and MTX-polyglutamate metabolites (MTXPG2 to MTXPG7) in packed human erythrocyte samples collected from patients with rheumatoid arthritis receiving low-dose oral methotrexate therapy. Mean (SD) intracellular erythrocyte concentrations observed in patient samples were 12.8 (12.6), 12.4 (9.4), 44.4 (30.0), 33.6 (35.9) and 9.4 (8.2) nmol/L for MTX to MTXPG5, respectively, in 10(6) erythrocytes. The highest observed glutamylation degree of MTX was MTXPG5, the very long chain MTX-polyglutamate metabolites MTXPG6 and MTXPG7 were not detected in the packed erythrocyte pellets collected from rheumatoid arthritis patients.