One-step detection of procollagen type III N-terminal peptide as a fibrosis biomarker using fluorescent immunosensor (quenchbody).

BACKGROUND: Procollagen type III N-terminal peptide (P-III-NP) is a fibrosis biomarker associated with liver and cardiac fibrosis. Despite the value of P-III-NP as a biomarker, its analysis currently relies on enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), which require more than 3 h. To facilitate early diagnosis and treatment through rapid biomarker testing, we developed a one-step immunoassay for P-III-NP using a quenchbody, which is a fluorescence-labeled immunosensor for immediate signal generation. RESULTS: To create quenchbodies, the total mRNA of P-III-NP antibodies was extracted from early-developed hybridoma cells, and genes of variable regions were obtained through cDNA synthesis, inverse PCR, and sequencing. A single-chain variable fragment (scFv) with an N-terminal Cys-tag was expressed in E. coli Shuffle T7, resulting in a final yield of 9.8 mg L-1. The fluorescent dye was labeled on the Cys-tag of the anti-P-III-NP scFv using maleimide-thiol click chemistry, and the spacer arm lengths between the maleimide-fluorescent dyes were compared. Consequently, a TAMRA-C6-labeled quenchbody exhibited antigen-dependent fluorescence signals and demonstrated its ability to detect P-III-NP at concentrations as low as 0.46 ng mL-1 for buffer samples, 1.0 ng mL-1 for 2 % human serum samples. SIGNIFICANCE: This one-step P-III-NP detection method provides both qualitative and quantitative outcomes within a concise 5-min timeframe. Furthermore, its application can be expanded using a 96-well platform and human serum, making it a high-throughput and sensitive method for testing fibrotic biomarkers.

Development of a simultaneous analytical method for the dietary exposure determination of ß-Carboline alkaloids in foods.

As ß-carboline (ßC) alkaloids, posing potential health risks, are present in a wide variety of foods, determining the exposure degrees of food to these alkaloids from dietary activity is key to ensuring food safety. Here, we developed a rapid and sensitive simultaneous analytical method for six ßC alkaloids in food. We optimized the buffered QuEChERS method, which includes a clean-up process through dispersive solid phase extraction, to extract the target compounds from food matrices; then, these compounds were detected via liquid chromatography-tandem mass spectrometry. We established calibration ranges for each target compound and matrix within the range of 0.05-250 µg/kg, and verified linearity (R2 ≥ 0.99) and limit of quantitation (≤1.63 µg/kg). Furthermore, we validated trueness (85.8%-118.8%) and precision (≤18.7%) at three levels within the calibration range, including the lowest and highest concentrations. Finally, we employed the developed method to determine the ßC alkaloid contents in 304 samples of 41 food items and dietary exposure of six ßC alkaloids resulting from daily intake. Although ßC alkaloids were detected in 86.2% of the samples, exposure level to the 41 food items was insufficient to cause toxicity.

Evaluation and Monitoring of the Natural Toxin Ptaquiloside in Bracken Fern, Meat, and Dairy Products.

Ptaquiloside, a naturally occurring cancer-causing substance in bracken fern, has been detected in the meat and milk of cows fed a diet containing bracken fern. A rapid and sensitive method for the quantitative analysis of ptaquiloside in bracken fern, meat, and dairy products was developed using the QuEChERS method and liquid chromatography-tandem mass spectrometry. The method was validated according to the Association of Official Analytical Chemists guidelines and met the criteria. A single matrix-matched calibration method with bracken fern has been proposed, which is a novel strategy that uses one calibration for multiple matrices. The calibration curve ranged from 0.1 to 50 µg/kg and showed good linearity (r2 > 0.99). The limits of detection and quantification were 0.03 and 0.09 µg/kg, respectively. The intraday and interday accuracies were 83.5-98.5%, and the precision was <9.0%. This method was used for the monitoring and exposure assessment of ptaquiloside in all routes of exposure. A total of 0.1 µg/kg of ptaquiloside was detected in free-range beef, and the daily dietary exposure of South Koreans to ptaquiloside was estimated at up to 3.0 × 10-5 µg/kg b.w./day. The significance of this study is to evaluate commercially available products in which ptaquiloside may be present, to monitor consumer safety.

Crystal structure of the Pseudomonas aeruginosa PA0423 protein and its functional implication in antibiotic sequestration.

Pseudomonas aeruginosa is a widely found opportunistic pathogen. The emergence of multidrug-resistant strains and persistent chronic infections have increased. The protein encoded by the pa0423 gene in P. aeruginosa is proposed to be critical for pathogenesis and could be a virulence-promoting protease or a bacterial lipocalin that binds a lipid-like antibiotic for drug resistance. Although two functions of proteolysis and antibiotic resistance are mutually related to bacterial survival in the host, it is very unusual for a single-domain protein to target unrelated ligand molecules such as protein substrates and lipid-like antibiotics. To clearly address the biological role of the PA0423 protein, we performed structural and biochemical studies. We found that PA0423 adopts a single-domain ß-barrel structure and belongs to the lipocalin family. The PA0423 structure houses an internal tubular cavity, which accommodates a ubiquinone-8 molecule. Furthermore, we reveal that PA0423 can directly interact with the polymyxin B antibiotic using the internal cavity, suggesting that PA0423 has a physiological function in the antibiotic resistance of P. aeruginosa.

Alterations in phospholipid profiles of erythrocytes deep-frozen without cryoprotectants.

The erythrocyte membrane is composed of a phospholipid bilayer, which is known to undergo physicochemical changes during storage at low temperatures. This study was conducted to identify marker phospholipids that indicate alteration during deep-frozen storage and to determine the amount of marker phospholipids. Our research suggested a method to detect phospholipids by profiling analysis of thermally injured red blood cells (RBCs) without protecting agents. Human blood was stored at -80°C for 72 days. The RBC membrane phospholipids were extracted through a modified Bligh and Dyer method. Six selected phospholipids were analyzed and quantified using liquid chromatography-tandem mass spectrometry, and an in vitro model system was developed. The intracellular level of N-nervonoyl-D-erythro-sphingosylphosphorylcholine significantly increased in the thermally injured RBCs, and multiple biomarker candidates were evaluated by profiling analysis and mass spectrometry technology for targeted metabolomics.

MPTP-driven NLRP3 inflammasome activation in microglia plays a central role in dopaminergic neurodegeneration.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the reduction of dopamine levels in the striatum. Although details of the molecular mechanisms underlying dopaminergic neuronal death in PD remain unclear, neuroinflammation is also considered a potent mediator in the pathogenesis and progression of PD. In the present study, we present evidences that microglial NLRP3 inflammasome activation is critical for dopaminergic neuronal loss and the subsequent motor deficits in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Specifically, NLRP3 deficiency significantly reduces motor dysfunctions and dopaminergic neurodegeneration of MPTP-treated mice. Furthermore, NLRP3 deficiency abolishes MPTP-induced microglial recruitment, interleukin-1ß production and caspase-1 activation in the SN of mouse brain. In primary microglia and mixed glial cell cultures, MPTP/ATP treatment promotes the robust assembly and activation of the NLRP3 inflammasome via producing mitochondrial reactive oxygen species. Consistently, 1-methyl-4-phenyl-pyridinium (MPP+) induces NLRP3 inflammasome activation in the presence of ATP or nigericin treatment in mouse bone-marrow-derived macrophages. These findings reveal a novel priming role of neurotoxin MPTP or MPP+ for NLRP3 activation. Subsequently, NLRP3 inflammasome-active microglia induces profound neuronal death in a microglia-neuron co-culture model. Furthermore, Cx3Cr1CreER-based microglia-specific expression of an active NLRP3 mutant greatly exacerbates motor deficits and dopaminergic neuronal loss of MPTP-treated mice. Taken together, our results indicate that microglial NLRP3 inflammasome activation plays a pivotal role in the MPTP-induced neurodegeneration in PD.

Bacterial Secretant from Pseudomonas aeruginosa Dampens Inflammasome Activation in a Quorum Sensing-Dependent Manner.

Inflammasome signaling can contribute to host innate immune defense against bacterial pathogens such as Pseudomonas aeruginosa. However, bacterial evasion of host inflammasome activation is still poorly elucidated. Quorum sensing (QS) is a bacterial communication mechanism that promotes coordinated adaptation by triggering expression of a wide range of genes. QS is thought to strongly contribute to the virulence of P. aeruginosa, but the molecular impact of bacterial QS on host inflammasome defense is completely unknown. Here, we present evidence that QS-related factors of the bacterial secretant (BS) from P. aeruginosa can dampen host inflammasome signaling in mouse bone marrow-derived macrophages. We found that BS from QS-defective ΔlasR/rhlR mutant, but not from wild-type (WT) P. aeruginosa, induces robust activation of the NLRC4 inflammasome. P. aeruginosa-released flagellin mediates this inflammasome activation by ΔlasR/rhlR secretant, but QS-regulated bacterial proteases in the WT BS impair extracellular flagellin to attenuate NLRC4 inflammasome activation. P. aeruginosa-secreted proteases also degrade inflammasome components in the extracellular space to inhibit the propagation of inflammasome-mediated responses. Furthermore, QS-regulated virulence factor pyocyanin and QS autoinducer 3-oxo-C12-homoserine lactone directly suppressed NLRC4- and even NLRP3-mediated inflammasome assembly and activation. Taken together, our data indicate that QS system of P. aeruginosa facilitates bacteria to evade host inflammasome-dependent sensing machinery.