18F-Radiolabeling and Evaluation of an AMD3465 Derivative for PET Imaging of CXCR4 in a Mouse Breast Tumor Model.

The exploration of pharmaceutically active agents and positron emission tomography (PET) tracers targeting CXCR4 has been a focal point in cancer research given its pivotal role in the development and progression of various cancers. While significant strides have been made in PET imaging with radiometal-labeled tracers, the landscape of 18F-labeled small molecule tracers remains relatively limited. Herein, we introduce a novel and promising derivative, [18F]SFB-AMD3465, as a targeted PET tracer for CXCR4. The compound was synthesized by modifying the pyridine ring of AMD3465, which was subsequently labeled with 18F using [18F]SFB. The study provides comprehensive insights into the design, synthesis, and biological evaluation of [18F]SFB-AMD3465. In vitro and in vivo assessments demonstrated the CXCR4-dependent, specific, and sensitive uptake of [18F]SFB-AMD3465 in the CXCR4-overexpressing 4T1 cell line and the corresponding xenograft-bearing mouse model. These findings contribute to bridging the gap in 18F-labeled PET tracers for CXCR4 and underscore the potential of [18F]SFB-AMD3465 as a PET radiotracer for in vivo CXCR4 imaging.

Establishment of fluorescence multi-flow cytometric immunoassay for the simultaneous quantitative detection of six allergen-sIgE antibodies.

BACKGROUND: The in vitro specific IgE (sIgE) assays now commonly used in clinical laboratories are not only time-consuming and expensive but also require many serum samples. To address these limitations, a novel fluorescent microsphere-based multiplex flow cytometric immunoassay was developed. This innovative assay enables rapid and simultaneous quantitative detection of multiple allergen-sIgE antibodies. OBJECTIVE: To establish a new method for the simultaneous quantitative detection of 6 allergen-sIgE antibodies based on fluorescence multiplex flow cytometry. METHODS: Six different encoded fluorescent microspheres were selected to covalently couple 6 allergens, and their antigen-coupling activities were verified. After optimizing the multiplexing procedure and reaction conditions, including the concentration of microspheres encapsulated by allergens, reaction temperature, and reaction time, standard curves were established to quantify the 6 allergen-sIgE, and their performance was evaluated according to clinical guidelines. RESULTS: The chosen analytical mode was optimized for the detection of the 6 allergens-sIgE for 70 minutes. The established coefficients of variation for multiplex flow cytometry reproducibility and intermediate precision were less than 10%. Linear regression analysis showed a highly significant quantitative correlation between the results of the multiple analyses of Dermatophagoides pteronyssinus, Dermatophagoides farinae, Artemisia, and cat hair allergens and ImmunoCAP (Thermo Fisher Scientific): the r2 values ranged from 0.85 to 0.97 (P < .0001). In addition, there was a high correlation between the results of the multiplex analysis of dog hair allergens and the capture enzyme-linked immunosorbent assay (r2 = 0.92, P < .0001). CONCLUSION: A high-throughput system called multiplex flow cytometry has been developed for the simultaneous detection of 6 inhalant allergens. The method has the advantage of being rapid and using less serum. Furthermore, it has the potential to be expanded to include other allergens and biologic agents.

Establishment of a homogeneous chemiluminescence immunoassay for cat dander sIgE antibody detection.

BACKGROUND: In vitro specific IgE (sIgE) testing has become an important tool for the diagnosis of IgE-mediated allergic diseases. Current methods used to detect allergen sIgE are time consuming and/or expensive. Therefore, a new method was developed for rapid quantitative detection of cat dander-sIgE antibody based on homogeneous chemiluminescence immunoassay. METHODS: Selection of chemibeads with different chemical groups, and the best Light-initiated chemiluminescence assay (LiCA) analytical mode for cat dander-sIgE detection. To validate and eliminate the interference of IgE on the detection of cat dander-sIgE, concentration of biotinylated anti-human IgE antibody was optimized. For quantification of cat dander-sIgE, a calibration curve was established, and the performance of the assay was evaluated according to clinical guidelines. RESULTS: Indirect LiCA is the best mode of analysis and biotinylated anti-human IgE antibody at a dilution ratio of 1:250 minimizes IgE interference. The coefficient of variation of the developed LiCA was 1.49% to 4.66%, with an intermediate precision of 6.90% to 8.21%. The LoB, LoD, and LoQ of the assay were 0.023 kUA/L, 0.056 kUA/L and 0.185 kUA/L. The coefficient of correlation (r) between LiCA and ImmounoCAP was 0.9478. CONCLUSIONS: A cat dander-sIgE quantitation assay based on homogeneous chemiluminescence immunoassay was established, which could be a new reliable analytical tool for the determination of cat dander-sIgE.

The EPFL-ERf-SERK signaling controls integument development in Arabidopsis.

As the seed precursor, the ovule produces the female gametophyte (or embryo sac), and the subsequent double fertilization occurs in it. The integuments emerge sequentially from the integument primordia at the early stages of ovule development and finally enwrap the embryo sac gradually during gametogenesis, protecting and nursing the embryo sac. However, the mechanisms regulating integument development are still obscure. In this study, we show that SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES (SERKs) play essential roles during integument development in Arabidopsis thaliana. The serk1/2/3 triple mutant shows arrested integuments and abnormal embryo sacs, similar defects also found in the triple loss-of-function mutants of ERECTA family (ERf) genes. Ovules of serk1/2/3 er erl1/2 show defects similar to er erl1/2 and serk1/2/3. Results of yeast two-hybrid analyses, bimolecular fluorescence complementation (BiFC) analyses, and co-immunoprecipitation assays demonstrated that SERKs interact with ERf, which depends on EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family small peptides. The sextuple mutant epfl1/2/3/4/5/6 shows integument defects similar to both of er erl1/2 and serk1/2/3. Our results demonstrate that ERf-SERK-mediated EPFL signaling orchestrates the development of the female gametophyte and the surrounding sporophytic integuments.

Essential roles of SERKs in the ROOT MERISTEM GROWTH FACTOR-mediated signaling pathway.

ROOT MERISTEM GROWTH FACTORs (RGFs), a group of peptide hormones, play key roles in root apical meristem development. In Arabidopsis (Arabidopsis thaliana), there are 11 members of RGFs, in which at least RGF1, RGF2, and RGF3 are expressed at the root tip and are involved in root stem cell niche maintenance. RGFs are perceived by five functionally redundant receptor-like protein kinases, RGF1 INSENSITIVE 1 (RGI1) to RGI5, to maintain the expression of two downstream APETALA 2 (AP2) transcription factor genes, PLETHORA 1 (PLT1) and PLT2, and to stabilize PLT2. RGI1 to RGI3 were also named RGF RECEPTOR 1 (RGFR1) to RGFR3, respectively. Although previous studies have suggested that BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and its paralogs, SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASEs (SERKs), may act as coreceptors of RGIs, comprehensive genetic and biochemical analyses have not been well documented. Here, we report that single, double, and triple mutants of SERKs show various degrees of short root phenotypes and insensitivity to exogenously applied RGF1. The interaction between RGIs and BAK1 and their mutual phosphorylation are RGF1 dependent. We also found that RGF1-induced MAPK activation relies on both RGIs and SERKs. We demonstrate that RGIs play redundant roles in regulating root apical meristem development. Therefore, we genetically and biochemically substantiated that SERKs, as coreceptors, play essential roles in the RGF1-mediated signaling pathway.

SERKs regulate embryonic cuticle integrity through the TWS1-GSO1/2 signaling pathway in Arabidopsis.

The embryonic cuticle integrity is critical for the embryo to separate from the neighboring endosperm. The sulfated TWISTED SEED1 (TWS1) peptide precursor generated in the embryo diffuses through gaps of the nascent cuticle to the surrounding endosperm, where it is cleaved by ABNORMAL LEAF SHAPE1 (ALE1) and becomes an active mature form. The active TWS1 is perceived by receptor-like protein kinases GASSHO1 (GSO1) and GSO2 in the embryonic epidermal cells to start the downstream signaling and guide the formation of an intact embryonic cuticle. However, the early signaling events after TWS1 is perceived by GSO1/2 are still unknown. Here, we report that serk1/2/3 embryos show cuticle defects similar to ale1, tws1, and gso1/2. Genetic and biochemical analyses were performed to dissect the signaling pathway mediated by SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASEs (SERKs) during cuticle development. SERKs function with GSO1/2 in a common pathway to monitor the integrity of the embryonic cuticle. SERKs interact with GSO1/2, which can be enhanced dramatically by TWS1. The phosphorylation levels of SERKs and GSO1/2 rely on each other and can respond to and be elevated by TWS1. Our results demonstrate that SERKs may function as coreceptors of GSO1/2 to transduce the TWS1 signal and ultimately regulate embryonic cuticle integrity.

Exploring the role of basophil activation test in diagnosis of Dermatophagoides farinae sensitization and evaluation of therapeutic efficacy of subcutaneous immunotherapy in children.

It aims to detect basophil activation ratio (%CD63+ ) in peripheral blood of children with allergic asthma and rhinitis by using flow cytometry (FCM), so as to analyse the application values and clinical relevance of the basophil activation test (BAT) in diagnosis of Dermatophagoides farinae (Derf) sensitization and monitoring therapeutic efficacy of subcutaneous immunotherapy (SCIT). It was a prospective study. From the newly diagnosed children with asthma and rhinitis in our paediatric clinic, 39 patients diagnosed Derf sensitization and 15 patients not allergic to Derf were enrolled; another 4 healthy children were taken as control group. Using Derf extracts in concentration of 1, 10 µg/mL and 100 µg/mL as the stimulus, BAT results were expressed as %CD63+ in diagnosis of Derf sensitization and its correlation with skin prick tests (SPT), serum total IgE (tIgE), specific IgE (sIgE), sIgE/tIgE, specific IgG4 (sIgG4), FEV1%pred in pulmonary ventilation function, exhaled nitric oxide (FeNO), children asthma control test (C-ACT) and visual analogue scale (VAS) were observed. In sensitization group, %CD63+ , sIgG4 and clinical indicators were detected again from patients who had received SCIT to analyse their internal connections. The average levels of %CD63+ in 3 concentrations showed an increasing concentration-dependent trend overall. %CD63+ in sensitization group was significantly higher than that in the other 2 groups. The analysis of ROC for Derf sensitization showed the area under the curve (AUC) for BAT in 3 concentrations was higher than that for sIgE whose AUC is 0.893. %CD63+ was positively correlated with SPT grade, sIgE, sIgE/tIgE and VAS, and negatively correlated with C-ACT. In patients receiving SCIT, %CD63+ became lower and sIgG4 level became higher than pretreatment. There was no obvious change in sIgG4 in those who had not received SCIT. BAT is a reliable and non-invasive tool for diagnosis of Derf sensitization in children with asthma and rhinitis. CD63-based BAT has clinical value to monitor outcome of SCIT, and the change in basophil activation is inherently related to induction of sIgG4.

Alteration of Allergen Fold of Bos d 5 into a Hypoallergenic Vaccine for Immunotherapy of Cow's Milk Allergy.

BACKGROUND: Cow's milk allergy (CMA) is the most common IgE-mediated food allergy and Bos d 5 is the major allergen in cow's milk proteins. More than 60% of the patients with CMA are sensitized to this protein. METHODS AND RESULTS: A recombinant protein, encoded by a synthetic gene and consisting of reassembled Bos d 5 fragments, was expressed in E. coli strain BL21 (DE3) cells and purified to homogeneity. The B5M lacked relevant IgE-reactivity and allergenic activity compared with Bos d 5 in dot-blot and basophil activation assays. T-cell proliferation experiments demonstrated that B5M preserved the main T cell epitopes of Bos d 5. Immunization of rabbits with B5M induced protective IgG antibodies that blocked the binding of patients' IgE antibodies to the wild-type allergen and inhibited the degranulation of basophils induced by Bos d 5. CONCLUSION: Thus, we developed a new strategy, which was based on rational molecular reassembly for allergen-specific immunotherapy (AIT) of CMA and food allergy.

A Simple and Rapid Light-Initiated Chemiluminescence Assay for Quantitation of Artemisia-Specific Immunoglobulin E.

BACKGROUND: Light-initiated chemiluminescence assay (LICA) is a homogeneous assay that has been successfully used for the quantitation of food allergen-specific immunoglobulin E (sIgE), but not inhaled allergen-sIgE. Simultaneously, current assays used to detect allergen-sIgE are serum consuming and/or time consuming. Hence, we established a method for the quantitation of Artemisia-sIgE based on LICA and verified its performance according to the clinical guideline documents, laying a foundation for the quantitation of inhaled and food allergen-sIgE in parallel on LICA. METHODS: The assay was established after optimizing the first incubation time and the dilutions of Artemisia-coated chemibeads, biotinylated goat anti-human IgE, and serum. In order to quantitate Artemisia-sIgE, the calibration curve was established with a high positive serum of known concentration. The assay performance was confirmed per the clinical guideline documents. In addition, the correlation between the results of LICA and capture enzyme-linked immunosorbent assay was evaluated. RESULTS: The developed LICA's coefficients of variation of repeatability and intermediate precision were 3.20%, 2.14%, and 3.85% and 4.30%, 4.00%, and 4.40%, respectively. The limit of detection was 0.10 kUA/L, and the limit of quantitation was 0.11 kUA/L. The range of linearity was from 0.27 kUA/L to 97.53 kUA/L (r = 0.9968). The correlation coefficient (r) for the correlation analysis between results of LICA and capture ELISA was 0.9087. This assay was successfully applied in 64 human serum samples, showing good sensitivity (82.20%) and specificity (100%). CONCLUSION: An Artemisia-sIgE quantitation assay based on LICA was successfully established. Its performance satisfied the clinical requirements and could be widely used in clinical laboratories.

Performance evaluation of a laboratory-developed light-initiated chemiluminescence assay for quantification of egg white-specific IgE.

BACKGROUND: Specific IgE (sIgE) testing has become one of the most important tools for diagnosing IgE-mediated food allergy. Enzyme-linked immunosorbent assay (ELISA) and dot-enzyme-linked immunosorbent assay (Dot-ELISA) have been used to measure sIgE in clinical widely. Light-initiated chemiluminescence assay (LICA) is a new method for measuring allergen-sIgE. We aimed to establish a LICA method for quantitative detection of egg white-sIgE and evaluate its performances. METHODS: The best chemibeads coupling method in detecting egg white-sIgE was selected, and a LICA method for quantitative detection of egg white-sIgE was established. The precision study was performed according to Clinical and Laboratory Standards Institute (CLSI) EP5-A2. Detection capability which contains limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) was evaluated according to National Health Commission of the People's Republic of China (NHC) WS/T 514-2017. Linear range was evaluated according to CLSI EP6-A. All data were analyzed using SPSS software. RESULTS: Precision contains repeatability and intermediate precision. The CV of repeatability ranged from 2.72% to 7.29%, and the CV of intermediate precision ranged from 4.93% to 8.64%. The LoB, LoD, and LoQ of the assay were 0.000 kUA/L, 0.053 kUA/L, and 0.076 kUA/L. The assay linear range was 0.076-34.125 kUA /L (r = 0.9979 ≥ 0.9900). CONCLUSION: This laboratory-developed LICA method can detect egg white-sIgE, and performance meets clinical requirements. This method shows rapid turnaround cycles and high sensitivity. It can be used as an alternative method for clinical detection of egg white-sIgE.

Light-initiated chemiluminescent assay of 17ß-estradiol metrological traceability system established by manufacturer according to ISO17511:2020 and basic performance evaluation performed by clinical end-users.

BACKGROUND: In order to ensure the accuracy of the product, we established 1st model of metrological traceability hierarchy for light-initiated chemiluminescent assay (LICA) of 17ß-estradiol (E2 ) at the manufacturer, based on International Organization for Standardization (ISO) 17511:2020. Moreover, we verified/validated the basic performance (such as matrix effect and long-term stability of end-user IVD MD calibrator, precision, linearity interval, accuracy/ trueness, and detection capability) at the clinical end-user. METHODS: Human serum samples were used in this study. E2 was detected by mass spectrometry (MS) and LICA. The metrological traceability of LICA for E2 was established according to ISO 17511: 2020 standards, and pools of human samples were used as the m.3. secondary calibrator. Precision was validated according to Clinical and Laboratory Standards Institute (CLSI) EP05-A3. The linear interval was verified according to CLSI EP06-ED2. Comparison of accuracy and trueness of E2 with MS and Roche according to CLSI EP09-A3. The detection capability was validated according to EP17-A2. Matrix effect and long-term stability evaluation of end-user IVD MD calibrator were carried out according to CLSI EP14-A2, EP25-A. Statistical software was used for data analyses. RESULTS: The use of pools of human samples and fine adjusting calibrators ensured the accuracy of end-user test results. The metrological traceability of LICA for E2 was established. It showed excellent precision, meeting the requirements of allowable imprecision (7.5%). The allowable deviation from linearity (ADL) of 5% was allowed to show a good linear interval (12.52-4167.25 pg/ml). The accuracy/ trueness was verified, and relative deviation in the medical decision level met the performance specification of 10.03% compared with MS or Roche. The validated limit of blank, limit of detection, and limit of quantitation of E2 were 4.95 pg/ml, 8.93 pg/ml, and 9.88 pg/ml, respectively (the allowed imprecision is 20.00%). The interference rate of E2 ranged from -5.5% to 6.6%. CONCLUSION: LICA showed high sensitivity, high specificity, excellent precision, wide linearity interval, IVD MD calibrator has long-term stability, and no matrix effect. The metrological traceability of E2 established by using pools of human samples as M.3. can deliver accuracy to the end-user IVD MD and show good consistency with MS and Roche.

Establishment of a sandwich light-initiated chemiluminescence assay with double antigen for detecting human cytomegalovirus IgG antibody.

Determination of human cytomegalovirus IgG (HCMV IgG) level is of great importance in the diagnosis of HCMV infections. In this study, a novel, double antigen sandwich homogeneous immunoassay-based light-initiated chemiluminescent assay (LICA) for measuring HCMV IgG serum levels was developed. This sandwich LICA for HCMV IgG was performed by incubating serum samples with HCMV pp150 protein coated with chemibeads, streptavidin-coated sensibeads, and biotinylated HCMV pp150 protein. The working conditions of this assay were optimized and the correlation between the results of the LICA and enzyme-linked immunosorbent assay was evaluated. As a homogeneous immunoassay, this sandwich LICA could accurately and rapidly determine the serum levels of HCMV IgG with a high-throughput. Thus, this newly developed assay could be a useful analytical tool in the clinical diagnosis of HCMV infections.

A New Perspective on the Role of Glutamine Synthetase in Nitrogen Remobilization in Wheat (Triticum aestivum L.).

Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is closely related to nitrogen remobilization. However, how GS isoforms participate in nitrogen remobilization remains unclear. Here, the spatiotemporal expression of the TaGS gene family after anthesis was investigated, and the results showed that TaGS1;1 was mainly encoded by TaGS1;1-6A, while the other isozymes were mainly encoded by TaGS localized on the A and D subgenomes. TaGS1;2-4A/4D had the highest expression level, especially in rachis and peduncle. Furthermore, immunofluorescence showed TaGS1;2 was located in the phloem of rachis and peduncle. GUS (ß-glucuronidase) staining confirmed that ProTaGS1;2-4A/4D::GUS activity was mainly present in the vascular system of leaves, roots, and petal of Arabidopsis. Ureides, an important transport form of nitrogen, were mainly synthesized in flag leaves and transported to grains through the phloem of peduncle and rachis during grain filling. TaAAH, which encodes the enzyme that degrades ureides to release NH4+, had a higher expression in rachis and peduncle and was synchronized with the increase in NH4+ concentration in phloem, indicating that NH4+ in phloem is from ureide degradation. Taking the above into account, TaGS1;2, which is highly expressed in the phloem of peduncle and rachis, may participate in N remobilization by assimilating NH4+ released from ureide degradation.

Detection of Bacterial Alkaline Phosphatase Activity by Enzymatic In Situ Self-Assembly of the AIEgen-Peptide Conjugate.

Abnormal levels of alkaline phosphatase (ALP) activity are associated with various diseases, and many ALP probes have been developed to date. However, the development of ALP-sensitive probes for living cells, especially for the detection of bacterial ALP, remains challenging because of the complex and dynamic context. In this study, we constructed the first fluorescent probe (TPEPy-pY) for sensing bacterial ALP activity. TPEPy-pY is an AIEgen-peptide conjugate with property of aggregation-induced emission (AIE) and could turn on its fluorescence by ALP-catalyzed in situ self-assembly of the probe. The probe shows excellent selectivity and sensitivity for ALP activity, with a detection limit of 6.6 × 10-3 U mL-1. TPEPy-pY performs well in detection and in situ imaging of bacterial ALP activity against E. coli. Also, the detection does not require tedious washing steps and takes approximately 1 h, which is advantageous over commercial ALP kits. Therefore, the proposed strategy paved a new avenue for bacterial ALP detection, and we envision that more self-assembling fluorescent probes will be designed with higher sensitivity in the near future.

Quantitation of estradiol by competitive light-initiated chemiluminescent assay using estriol as competitive antigen.

BACKGROUND: Light-initiated chemiluminescent assays (LICA) are homogeneous assays that are sensitive, specific, and free of separation and washing steps and have high throughput and high precision. METHODS: In this research, we developed a competitive method by LICA to achieve accurate quantification of estradiol (E2) in human serum. E2 competed with estriol (E3) for binding to anti-human E2 antibodies. E3 was linked to biotin via bovine serum albumin as a linker. As this assay used competition between the labeled tracer and the analyte, an increase in E2 concentration will cause a signal decrease. RESULTS: The expected detection range of E2 was 20-5000 pg/mL. The analytical and functional sensitivities were 7.16 and 13.7 pg/mL, respectively. The intra- and inter-assay coefficients of variation were both below 15%, and the recovery rate ranged from 97.5% to 106.8%. The interference rates ranged from -3.6% to 5.4% and met detection requirements for E2 in hyperbilirubinemia, hemolysis, and lipemia in clinical samples. In addition, the cross-reactivity rates between E2 and structural analogs and some reproductive hormones varied from 1.9% to 10.6% which showed that LICA is highly specific for E2. Moreover, our results showed high accordance with the IMMULITE 2000 (y = 0.6695x + 47.92, r2  = .843) and VIDAS systems (y = 1.099x - 821.5, r2  = .9392). CONCLUSION: Our data show that the LICA, which is easy to automate, is a promising technique for quantification of E2 in human serum and could be used for clinical detection.

Competitive light-initiated chemiluminescent assay: using 5-α-dihydrotestosterone-BSA as competitive antigen for quantitation of total testosterone in human sera.

This paper described a homogeneous method, light-initiated chemiluminescent assay (LICA), for quantitation of total testosterone in human sera. The assay was bead based and built on a competitive-binding reaction format, in which 5-α-dihydrotestosterone (5-α-DHT) competed with the testosterone in serum samples in binding with biotinylated anti-testosterone antibody. The more testosterone in the serum sample, the less 5-α-DHT that bonded with biotinylated anti-testosterone antibodies. 5-α-DHT was coupled with emission beads (doped with thioxene derivatives and Eu(III) as a chemiluminescence emitter) via bovine serum albumin as a linker. Once streptavidin-coated sensitizer beads (modified with phthalocyanine as a photosensitizer) were added, the streptavidin/biotin reaction between 5-α-DHT-bound anti-testosterone antibody and sensitizer beads could bring emission and sensitizer beads together, which allowed energy transfer from sensitizer bead to emission bead. As such, an exciting light (680 nm) impinging on the sensitizer beads led to light emission at 520-620 nm by emission beads. The strength of the emitted light was inversely proportional to the testosterone in serum sample. The detection range of this assay was from 13.3 to 1200 ng/dL. The coefficient variation for intra- and inter-assay was lower than 15%. The recovery of this method ranged from 95.5 to 105.9% for different samples. Moreover, the LICA assay was highly specific with low cross-reactivity and interference. The concentration of testosterone from 58 serum samples analyzed by the LICA method significantly correlated (y = 0.97x + 1.87, R2 = 0.970, p < 0.001) with those obtained with the SIEMENS Centaur Xp System. Graphical abstract ᅟ.

Development of a light-initiated chemiluminescent assay for the quantitation of sIgE against egg white allergens based on component-resolved diagnosis.

The determination of specific IgE (sIgE) level is of great importance in IgE-mediated food allergies. Our aim was to develop a homogeneous immunoassay-light-initiated chemiluminescent assay (LICA)-for measuring allergen sIgE of a single component in egg white, thus evaluating the LICA-sIgE assay as a useful tool in the diagnosis of food allergy. The LICA-sIgE assay was performed by incubating serum sample with anti-human IgE antibody coated with chemiluminescer beads, streptavidin-coated sensitizer beads, and biotinylated antigens, which consist of four components in egg white. Serum samples from egg allergic patients (n = 70) and healthy volunteers (n = 30) were collected. For calibration, purified human IgE was used as the calibrator. Working conditions of this homogeneous immunoassay were optimized, analytical performance was determined, and correlation of the results between LICA and ImmunoCAP was evaluated. The assays were performed in 8-well plates with a sample volume diluted to 1:10 of 25 µl. Intra-assay precision (% coefficient of variation) ranged from 1.83 to 4.13%, and inter-assay precision ranged from 2.70 to 8.70%. It exhibited excellent sensitivity, which could distinguish between positive samples and negative samples even at a large dilution level. The sIgE-LICA and ImmunoCAP correlated well in patients allergic to single component (r 2 = 0.929). Also, the components ovomucoid and ovalbumin were best at predicting ImmunoCAP results, with the same area under the ROC curve (AUC) of 0.81, and a specificity of 90.0 and 93.3%, respectively. Our data show effective performance characteristics of LICA to detect sIgE in human serum based on component-resolved diagnostic tests (CRD). The homogeneous sIgE-LICA assay has the following key advantages: requires no washing, simplicity and rapidity, reproducibility, high-throughput, good performance in a liquid phase assay, and good suitability for sIgE diagnosis in food allergy based on CRD. Graphical abstract A light-initiated chemiluminescent assay was developed for the quantitation of sIgE against egg white allergens based on component-resolved diagnosis. Components Gal d 1 and Gal d 2 with the highest AUC values of 0.81 were considered the best at predicting egg allergy.

Shortcut Biological Nitrogen Removal from Coal Gasification Wastewater in Three-Stage MBBRs.

A lab-scale aerobic-anoxic-aerobic (AE1-AN-AE2) MBBR system was tested for the removal of COD, , SCN-, phenols, and nitrogen from coal gasification wastewater, using a shortcut biological nitrogen removal process. Dissolved oxygen concentration in AE1 was maintained at 1.0 to 2.0 mg/L to ensure stable accumulation. Adding methanol wastewater to AN guaranteed denitrification efficiency. AE2 ensured high removal rates of , SCN-, and phenols. The effects of influent pollutant concentration and hydraulic retention time (HRT) on nitrogen removal were studied. Improving the dissolved oxygen concentration in AE1 eliminated the negative effect of increased organic loading on nitrification, but it affected the stability of nitrosation. Shortening the HRT had negative effects on the performance of the system and performance recovered after it was extended. The average total nitrogen removal rate was 82.6% with a CODmethanol/ ratio of 3.5. Biomass and activity of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria were measured to understand the evolution of nitrification.

Frontal metabolic activity contributes to individual differences in vulnerability toward total sleep deprivation-induced changes in cognitive function.

Substantial individual differences characterize the changes induced by total sleep deprivation on cognitive functions. Despite some progress having been achieved, the mechanisms of individual differences in response to total sleep deprivation have not been clearly elucidated. Cerebral metabolism in the resting state is among the key physiological processes supporting the daily function of the brain, and may play an important role in these individual differences. Twenty-two right-handed participants (nine females and 13 males) between 20 and 26 years old completed a mathematical processing task both in resting wakefulness and after 24 h of total sleep deprivation. Fluorine-18 fluorodeoxyglucose positron emission tomography-computed tomography was used to investigate brain metabolism changes. The mathematical task was performed after the positron emission tomography scans were completed. Correlation analysis was used to investigate the correlations between cognitive performance changes and brain metabolism changes. Large inter-individual differences were found in the throughput changes, but these inter-individual differences were not associated with baseline or post-deprivation performance levels. Specifically, deterioration of throughput on the mathematical processing task was significantly correlated with metabolism changes in the superior frontal medial gyrus. These findings suggested that frontal metabolic activity contributes to individual differences in waking-induced impairment of cognitive performance.