Mn2+-Mediated Modulation of PfAgo Activity for Biosensing.

Argonaute (Ago) as a powerful enzyme has provided new insights into biosensing due to its programmability, high sensitivity, and user-friendly operation. However, current strategies mainly rely on phosphorylated guide DNA to modulate the cleavage activity of Ago, which is limited in versatility and simplicity. Herein, the authors report the Mn2+-enhanced cleavage activity of Ago and employ Mn-ions with variable valence to regulate the activity of Pyrococcus furiosus Ago (PfAgo) for biosensing applications. The conversion of Mn ions with different valence states through MnO2 nanoflowers enables the sensitive detection of ascorbic acid, alkaline phosphatase, and arsenic with limits of detection of 2.5 nmol L-1, 0.009 U L-1, and 0.4 ng mL-1, respectively. A PfAgo-based immunoassay is further developed that allows for the detection of diverse targets, thus providing a promising toolbox to broaden PfAgo-based sensors into versatile bioanalytical and biomedical applications.

Simultaneous detection of seven bacterial pathogens transmitted by flies using the reverse line blot hybridization assay.

BACKGROUND: Traditional methods for detecting insect-borne bacterial pathogens are time-consuming and require specialized laboratory facilities, limiting their applicability in areas without access to such resources. Consequently, rapid and efficient detection methods for insect-borne bacterial diseases have become a pressing need in disease prevention and control. METHODS: We aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/µl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China. RESULTS: The established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/µl for S. aureus, 103 copies/µl for S. flexneri, 105 copies/µl for A. caviae, 105 copies/µl for V. vulnificus, 100 copies/µl for S. enterica, 105 copies/µl for P. vulgaris, and 100 copies/µl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species. CONCLUSIONS: Overall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.

"Four-In-One" Multifunctional Dandelion-Like Gold@platinum Nanoparticles-Driven Multimodal Lateral Flow Immunoassay.

The traditional lateral flow immunoassay (LFIA) with a single signal output mode may encounter challenges such as low sensitivity, poor detection range, and susceptibility to external interferences. These limitations hinder its ability to meet the growing demand for advanced LFIA. To address these issues, the rational development of multifunctional labels for multimodal LFIA emerges as a promising strategy. Herein, this study reports a multimodal LFIA using "four-in-one" multifunctional dandelion-like gold@platinum nanoparticles (MDGP). The inherent properties of MDGP, such as the broad absorption spectrum, porous dandelion-like nanostructure, and bimetallic composition with gold and platinum, endow them with capacities in dual spectral-overlapped fluorescence quenching, optical readout, catalytic activity, and photothermal effect. Benefiting from their multifunctional properties, the MDGP-LFIA enables multimodal outputs including fluorescent, colorimetric, and photothermal signals. This multimodal MDGP-LFIA allows for the detection of acetamiprid at a range of 0.01-50 ng mL-1 , with the lowest qualitative and quantitative detection results of 0.5 and 0.008 ng mL-1 , respectively, significantly better than the traditional gold nanoparticles-based LFIA. The diversity, complementarity, and synergistic effect of integrated output signals in this multimodal MDGP-LFIA improve the flexibility, practicability, and accuracy of detection, holding great promise as a point-of-care testing platform in versatile application scenarios.

Global Proteome-Wide Analysis of Cysteine S-Nitrosylation in Toxoplasma gondii.

Toxoplasma gondii transmits through various routes, rapidly proliferates during acute infection and causes toxoplasmosis, which is an important zoonotic disease in human and veterinary medicine. T. gondii can produce nitric oxide and derivatives, and S-nitrosylation contributes to their signaling transduction and post-translation regulation. To date, the S-nitrosylation proteome of T. gondii remains mystery. In this study, we reported the first S-nitrosylated proteome of T. gondii using mass spectrometry in combination with resin-assisted enrichment. We found that 637 proteins were S-nitrosylated, more than half of which were localized in the nucleus or cytoplasm. Motif analysis identified seven motifs. Of these motifs, five and two contained lysine and isoleucine, respectively. Gene Ontology enrichment revealed that S-nitrosylated proteins were primarily located in the inner membrane of mitochondria and other organelles. These S-nitrosylated proteins participated in diverse biological and metabolic processes, including organic acid binding, carboxylic acid binding ribose and phosphate biosynthesis. T. gondii S-nitrosylated proteins significantly contributed to glycolysis/gluconeogenesis and aminoacyl-tRNA biosynthesis. Moreover, 27 ribosomal proteins and 11 microneme proteins were identified as S-nitrosylated proteins, suggesting that proteins in the ribosome and microneme were predominantly S-nitrosylated. Protein-protein interaction analysis identified three subnetworks with high-relevancy ribosome, RNA transport and chaperonin complex components. These results imply that S-nitrosylated proteins of T. gondii are associated with protein translation in the ribosome, gene transcription, invasion and proliferation of T. gondii. Our research is the first to identify the S-nitrosylated proteomic profile of T. gondii and will provide direction to the ongoing investigation of the functions of S-nitrosylated proteins in T. gondii.

Point-of-Care Detection of Antioxidant in Agarose-Based Test Strip through Antietching of Au@Ag Nanostars.

Antioxidants are crucial for human health, and the detection of antioxidants can provide valuable information for disease diagnosis and health management. In this work, we report a plasmonic sensing approach for the determination of antioxidants based on their antietching capacity toward plasmonic nanoparticles. The Ag shell of core-shell Au@Ag nanostars can be etched by chloroauric acid (HAuCl4), whereas antioxidants can interact with HAuCl4, which prevents the surface etching of Au@Ag nanostars. We modulate the thickness of the Ag shell and morphology of the nanostructures, showing that the core-shell nanostars with the smallest thickness of Ag shell have the best etching sensitivity. Owing to the extraordinary surface plasmon resonance (SPR) property of Au@Ag nanostars, the antietching effect of antioxidants can induce a significant change in both the SPR spectrum and the color of solution, facilitating both the quantitative detection and naked-eye readout. This antietching strategy enables the determination of antioxidants such as cystine and gallic acid with a linear range of 0.1-10 µM. The core-shell Au@Ag nanostars are further immobilized in agarose gels to fabricate test strips, which can display different color changes in the presence of HAuCl4 from 0 to 1000 µM. The agarose-based test strip is also capable of detecting antioxidants in real samples, which allows naked-eye readout and quantitative detection by a smartphone.

Preparation and photocatalytic performance study of dual Z-scheme Bi2Zr2O7/g-C3N4/Ag3PO4 for removal of antibiotics by visible-light.

At present, the high re-combination rate of photogenerated carriers and the low redox capability of the photocatalyst are two factors that severely limit the improvement of photocatalytic performance. Herein, a dual Z-scheme photocatalyst bismuthzirconate/graphitic carbon nitride/silver phosphate (Bi2Zr2O7/g-C3N4/Ag3PO4 (BCA)) was synthesized using a co-precipitation method, and a dual Z-scheme heterojunction photocatalytic system was established to decrease the high re-combination rate of photogenerated carriers and consequently improve the photocatalytic performance. The re-combination of electron-hole pairs (e- and h+) in the valence band (VB) of g-C3N4 increases the redox potential of e- and h+, leading to significant improvements in the redox capability of the photocatalyst and the efficiency of e--h+ separation. As a photosensitizer, Ag3PO4 can enhance the visible light absorption capacity of the photocatalyst. The prepared photocatalyst showed strong stability, which was attributed to the efficient suppression of photo-corrosion of Ag3PO4 by transferring the e- to the VB of g-C3N4. Tetracycline was degraded efficiently by BCA-10% (the BCA with 10 wt.% of AgPO4) under visible light, and the degradation efficiency was up to 86.2%. This study experimentally suggested that the BCA photocatalyst has broad application prospects in removing antibiotic pollution.

Comparative Phosphoproteomic Analysis of Sporulated Oocysts and Tachyzoites of Toxoplasma gondii Reveals Stage-Specific Patterns.

Toxoplasma gondii is an obligate intracellular protozoan of severe threat to humans and livestock, whose life history harbors both gamic and apogamic stages. Chinese 1 (ToxoDB#9) was a preponderant genotype epidemic in food-derived animals and humans in China, with a different pathogenesis from the strains from the other nations of the world. Posttranslational modifications (PTMs) of proteins were critical mediators of the biology, developmental transforms, and pathogenesis of protozoan parasites. The phosphoprotein profiling and the difference between the developmental phases of T. gondii, contributing to development and infectivity, remain unknown. A quantitative phosphoproteomic approach using IBT integrated with TiO2 affinity chromatography was applied to identify and analyze the difference in the phosphoproteomes between the sporulated oocysts and the tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii. A total of 4058 differential phosphopeptides, consisting of 2597 upregulated and 1461 downregulated phosphopeptides, were characterized between sporulated the oocysts and tachyzoites. Twenty-one motifs extracted from the upregulated phosphopeptides contained 19 serine motifs and 2 threonine motifs (GxxTP and TP), whereas 16 motifs identified from downregulated phosphopeptides included 13 serine motifs and 3 threonine motifs (KxxT, RxxT, and TP). Beyond the traditional kinases, some infrequent classes of kinases, including Ab1, EGFR, INSR, Jak, Src and Syk, were found to be corresponding to motifs from the upregulated and downregulated phosphopeptides. Remarkable functional properties of the differentially expressed phosphoproteins were discovered by GO analysis, KEGG pathway analysis, and STRING analysis. S8GFS8 (DNMT1-RFD domain-containing protein) and S8F5G5 (Histone kinase SNF1) were the two most connected peptides in the kinase-associated network. Out of these, phosphorylated modifications in histone kinase SNF1 have functioned in mitosis and interphase of T. gondii, as well as in the regulation of gene expression relevant to differentiation. Our study discovered a remarkable difference in the abundance of phosphopeptides between the sporulated oocysts and tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii, which may provide a new resource for understanding stage-specific differences in PTMs and may enhance the illustration of the regulatory mechanisms contributing to the development and infectivity of T. gondii.

Lateral flow immunoassay based on dual spectral-overlapped fluorescence quenching of polydopamine nanospheres for sensitive detection of sulfamethazine.

Herein, we propose a lateral flow immunoassay (LFIA) based on the dual spectral-overlapped fluorescence quenching of polydopamine nanospheres (PDANs) caused by the inner filter effect to sensitively detect sulfamethazine (SMZ). The fluorescence quenching LFIA device consists of four parts: absorbent pad, polyvinyl chloride pad, sample pad, and nitrocellulose membrane. Compared with traditional quenchers such as gold nanoparticles (AuNPs) with single spectral-overlapped quenching ability, PDANs can quench the excitation light and emission light of three fluorescence donors (aggregation-induced emission fluorescent microsphere, AIEFM; fluorescent microsphere, FM; and quantum dot bead, QB). The fluorescence intensity changes (ΔF) are numerically larger for PDANs-LFIA (ΔFAIEFM = 2315, ΔFFM = 979, ΔFQB = 910) than those for AuNPs-LFIA (ΔFAIEFM = 1722, ΔFFM = 833, ΔFQB =;520). AIEFM-based PDANs-LFIA exhibits a large ΔF (2315) in response to the changes in the SMZ concentration, and produces a high signal-to-noise ratio. The limit of detection (LOD) and visual LOD of LFIA based on PDANs quenching AIEFM for the detection of SMZ in chicken are 0.043 and 0.5 ng/mL, respectively. The results confirm that the proposed method can be used for the detection of hazardous materials in practical applications.

Global phosphoproteome analysis reveals significant differences between sporulated oocysts of virulent and avirulent strains of Toxoplasma gondii.

In this study, the differences in the phosphoproteomic landscape of sporulated oocysts between virulent and avirulent strains of Toxoplasma gondii were examined using a global phosphoproteomics approach. Phosphopeptides from sporulated oocysts of the virulent PYS strain (Chinese ToxoDB#9) and the avirulent PRU strain (type II) were enriched by titanium dioxide (TiO2) affinity chromatography and quantified using IBT approach. A total of 10,645 unique phosphopeptides, 8181 nonredundant phosphorylation sites and 2792 phosphoproteins were identified. We also detected 4129 differentially expressed phosphopeptides (DEPs) between sporulated oocysts of PYS strain and PRU strain (|log1.5 fold change| > 1 and p < 0.05), including 2485 upregulated and 1644 downregulated phosphopeptides. Motif analysis identified 24 motifs from the upregulated phosphorylated peptides including 22 serine motifs and two threonine motifs (TPE and TP), and 15 motifs from the downregulated phosphorylated peptides including 12 serine motifs and three threonine motifs (TP, RxxT and KxxT) in PYS strain when comparing PYS strain to PRU strain. Several kinases were consistent with motifs of overrepresented phosphopeptides, such as PKA, PKG, CKII, IKK, MAPK, EGFR, INSR, Jak, Syk, Src, Ab1. GO enrichment, KEGG pathway analysis and STRING analysis revealed DEPs significantly enriched in many biological processes and pathways. Kinase related network analysis showed that AGC kinase was the most connected kinase peptide. Our findings reveal significant difference in phosphopeptide profiles of sporulated oocysts between virulent and avirulent T. gondii strains, providing new resources for further elucidation of the mechanisms underpinning the virulence of T. gondii.

Quantum Dot Nanobeads Based Fluorescence Immunoassay for the Quantitative Detection of Sulfamethazine in Chicken and Milk.

Sulfamethazine (SMZ) as a broad antibiotic is widely used in livestock and poultry. However, the abuse of SMZ in livestock feed can lead to SMZ residues in food and the resistance of bacteria to drugs. Thus, a method for the detection of SMZ in food is urgently needed. In this study, quantum dot (QD) nanobeads (QBs) were synthesized by encapsulating CdSe/ZnS QDs using a microemulsion technique. The prepared QBs as signal probes were applied in lateral flow immunoassay (LFIA) for the detection of SMZ in chicken and milk. Our proposed method had limits of detection of 0.1138-0.0955 ng/mL and corresponding linear ranges of 0.2-12.5, 0.1-15 ng/mL in chicken and milk samples, respectively. The recovery of LFIA for the detection of SMZ was 80.9-109.4% and 84-101.6% in chicken and milk samples, respectively. Overall, the developed QBs-LFIA had high reliability and excellent potential for rapid and sensitive screening of SMZ in food.

Effective mineralization of p-nitrophenol by catalytic ozonation using Ce-substituted La1‒xCexFeO3 catalyst.

In this study, cerium-doped lanthanum ferrite perovskite oxides (La1‒xCexFeO3) with different A-site were synthesized using a sol-gel method and they were used as ozonation catalyst for p-nitrophenol (PNP) mineralization for the first time. Catalytic activity in terms of total organic carbon (TOC) removal followed the order of La0.8Ce0.2FeO3 > La0.4Ce0.6FeO3 > La0.6Ce0.4FeO3 > La0.2Ce0.8FeO3 > LaFeO3 with 77, 66, 61, 60 and 56% respectively. The synthesized catalysts were characterized by diffraction of X-ray (XRD), Raman spectroscopy, Brunauer-Emmett-Teller (BET) and scanning electronic microscopy (SEM). Moreover, electron spin resonance (ESR) and radicals quenching experiments showed that the active oxygen species in the ozone decomposition process are mainly hydroxyl radical (·OH), and also include superoxide radical (O2-) and singlet oxygen (1O2). Furthermore, the superior activity of La0.8Ce0.2FeO3 could be attributed to the higher surface area, the richer lattice oxygen, richer surface -OH groups and the facilitated redox Ce3+/Ce4+ and Fe2+/Fe3+ cycling. In addition, this study provides an insight to use metal-doped perovskite catalysts for catalytic ozonation.

Generation and application of a monoclonal antibody against the 18-kDa oncosphere antigen of Taenia pisiformis.

Taenia pisiformis is a parasite that causes cysticercosis pisiformis, which has acquired economic relevance because of its effects on animal welfare and production. A useful assay for the detection of T. pisiformis is needed for the prevention of cysticercosis pisiformis and control of the parasite. The 18-kDa oncosphere antigen is expressed in the oncosphere of several cysticerci in species of the genus Taenia, including T. pisiformis. This protein plays an important role in tissue invasion and has extensive applications in diagnosis. In this study, the T. pisiformis 18-kDa oncosphere antigen (TPO18) was expressed in soluble form and successfully purified for use in the production of monoclonal antibodies (MAbs) against TPO18. Twenty hybridomas were obtained using ELISA, and the subcloning process identified three positive hybridoma cell lines, which were designated as 4E8, 5G5, and 7E8. MAb 7E8 exhibited the highest titer and had an IgG2b heavy chain and a kappa light chain. Western blot analysis demonstrated that MAb 7E8 reacted with GST-TPO18. Immunohistochemistry showed that TPO18 was widely distributed in the drape and wall of uteri in adults of T. pisiformis adults and in the fibrous layer of the sucker and cyst cavity of T. pisiformis cysticerci. This research will provide a foundation for the development of diagnostic tools and will contribute to a better understanding of the functions of TPO18.

Immunochromatographic assay based on time-resolved fluorescent nanobeads for the rapid detection of sulfamethazine in egg, honey, and pork.

BACKGROUND: Sulfamethazine (SMZ), a veterinary drug widely used in animal husbandry, is harmful to human health when excess residues are present in food. In this study, a fast, reliable, and sensitive immunochromatographic assay (ICA) was developed on the basis of the competitive format by using time-resolved fluorescent nanobeads (TRFN) as label for the detection of SMZ in egg, honey, and pork samples. RESULTS: Under optimized working conditions, this method had limits of detection of 0.016, 0.049, and 0.029 ng mL-1 and corresponding linear ranges of 0.05 to 1.00, 0.05 to 5.00, and 0.05 to 1.00 ng mL-1 in egg, honey, and pork samples, respectively. The recovery experiments showed that the average recoveries ranged from 90.5% to 113.9%, 82.4% to 112.0%, and 79.8% to 93.4% with corresponding coefficients of variation of 4.1% to 11.7%, 7.5% to 11.5%, and 4.8% to 8.7% for egg, honey, and pork samples, respectively. The developed TRFN-ICA was also systematically compared with high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) by analyzing 45 actual samples from egg, honey, and pork. CONCLUSION: Overall, the developed TRFN-ICA had high reliability and excellent potential for the ultrasensitive detection of SMZ for food safety monitoring, also providing a universal platform for the on-site detection of other targets. © 2020 Society of Chemical Industry.

Activation of peroxymonosulfate using metal-free in situ N-doped carbonized polypyrrole: A non-radical process.

Metal-free catalysts are widely considered as promising alternatives to traditional metal-based catalysts, which can effectively activate peroxymonosulfate (PMS). In this study, a novel metal-free catalyst, carbonized polypyrrole (CPPy) was synthesized through high-temperature carbonization of PPy, easily achieving the in situ N doping without the addition of nitrogen sources. Tetracycline (TC) was selected as the target contaminant to assess the catalytic activity of the CPPy/PMS system. Enhanced catalytic activity was observed in CPPy/PMS over a wide pH range (3.0-9.0), and the removal rate of TC by CPPy-3/PMS reached 91.3% after 10 min. After regenerating the used catalyst, the catalytic activity was refreshed, implying its stability and recyclability. The catalytic degradation of TC by CPPy/PMS was mainly attributed to a non-radical process. CPPy, as an intermediary, grabbed electrons from the electron-donating groups of TC and transferred them to the PMS molecule. Thereafter, TC that lost electrons was oxidized and degraded, and the O-O bonds of PMS were destroyed by the transferred electrons to form SO42- and OH-. Moreover, O⋅2- and 1O2 were involved in TC degradation. TC degradation pathway was investigated through HPLC-MS analysis. These findings provide a promising strategy for the construction of catalysts for PMS and environmental remediation.

Reliable performance of aggregation-induced emission nanoparticle-based lateral flow assay for norfloxacin detection in nine types of animal-derived food.

The wide application of lateral flow assay (LFA) was limited by its low sensitivity and poor matric tolerance. Aggregation induced emission (AIE) materials show superior luminescence and good stability under close packing state, which may accelerate the development of LFA. However, the detection performance of the AIE-based LFA in different real samples was unclear. In this work, an AIE-LFA was established for norfloxacin (NOR) detection in nine types of animal-derived food. Results indicated that AIE-LFA had the average recovery range of 75.6%-95.1%, 78.6%-94.6%, 71.4%-112.7%, 81.7%-121.8%, 72.7%-93.5%, 79.8%-108.5%, 79.2%-109.4%, 76.3%-103.6%, and 80.6%-108.3% in pork, pig liver, fish, lamb, beef, milk, chicken, egg, and honey, respectively. The detection results of AIE-LFA were compatible with HPLC-MS/MS in detecting NOR in 135 real samples from nine types of animal-derived food. The developed AIE-LFA was sensitive and reliable for NOR detection in those real samples.

Acetylome analysis of the feline small intestine following Toxoplasma gondii infection.

Toxoplasma gondii is a protozoan parasite capable of infecting a large number of warm-blooded animals and causes serious health complications in immunocompromised patients. T. gondii infection of the feline small intestine is critical for the completion of the life cycle and transmission of T. gondii. Protein acetylation is an important posttranslational modification, which plays roles in the regulation of various cellular processes. Therefore, understanding of how T. gondii reprograms the protein acetylation status of feline definitive host can help to thwart the production and spread of T. gondii. Here, we used affinity enrichment and high-resolution liquid chromatography with tandem mass spectrometry to profile the alterations of the acetylome in cat small intestine 10 days after infection by T. gondii Prugniuad (Pru) strain. Our analysis showed that T. gondii induced significant changes in the acetylation of proteins in the cat intestine. We identified 2606 unique lysine acetylation sites in 1357 acetylated proteins. The levels of 334 acetylated peptides were downregulated, while the levels of 82 acetylated peptides were increased in the infected small intestine. The proteins with differentially acetylated peptides were particularly enriched in the bioenergetics-related processes, such as tricarboxylic acid cycle, oxidative phosphorylation, and oxidation-reduction. These results provide the first baseline of the global acetylome of feline small intestine following T. gondii infection and should facilitate further analysis of the role of acetylated protein in the pathogenesis of T. gondii infection in its definitive host.

Dual-mode immunoassay system based on glucose oxidase-triggered Fenton reaction for qualitative and quantitative detection of danofloxacin in milk.

In this study, a novel colorimetric and fluorescent dual-mode ELISA based on glucose oxidase (GOx)-triggered Fenton reaction was developed for the qualitative and quantitative detection of danofloxacin (DAN). In this system, streptavidin-linked biotinylated anti-DAN-monoclonal antibody (SA-Bio-mAb) and biotinylated GOx (Bio-GOx) form the immune complex mAb-Bio-SA-Bio-GOx. In the absence of DAN, the mAb-Bio-SA-Bio-GOx would be immobilized by combining with coated DAN-BSA and catalyzed glucose to generate H2O2. The Fenton reaction between H2O2 and Fe2+ generated hydroxyl radicals, which oxidized the o-phenylenediamine to 2,3-diamino-phenazine. A dual-signal immunoassay with colorimetry and fluorescence as the signal readout was established. In the presence of DAN, DAN and DAN-BSA competed with Bio-mAb, decreasing the connection between immune complexes and DAN-BSA and finally resulting in lower signal of colorimetry and fluorescence. Under optimal conditions, the limit of detection of the fluorescence immunoassay was 0.337 ng/mL and was 5.24-fold lower than that of traditional ELISA. The colorimetric immunoassay cut-off value was 30 ng/mL in milk. The average recoveries of the method for milk samples that are spiked with different concentrations of DAN were 91.1 to 128.3%, with a coefficient of variation of 0.7 to 8.2%. These results of the method exhibited good agreement with those of liquid chromatography-tandem mass spectrometry system (LC-MS/MS) method. In brief, this work provides an improved screening strategy with high sensitivity and accuracy for the qualitative or quantitative detection of DAN in milk monitoring.

Calcium formate assisted catalytic pyrolysis of pine for enhanced production of monocyclic aromatic hydrocarbons over bimetal-modified HZSM-5.

An efficient process was developed to selectively produce monocyclic aromatic hydrocarbons (MAHs) from ex-situ catalytic fast pyrolysis (CFP) of pine assisted with calcium formate (CF) over bimetal-modified HZSM-5. Mo and another metal (Mg, Ga or Zn) were used to modify the HZSM-5, and the as-synthesized bimetal-modified HZSM-5 catalysts were utilized for both pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and lab-scale CFP tests with CF as a hydrogen donor to selectively obtain MAHs. The results revealed that the presence of CF and Mg-Mo modified HZSM-5 (0.5Mg1Mo/HZ) exhibited excellent capability for MAHs production with tiny generation of polycyclic aromatic hydrocarbons (PAHs). The maximum MAHs yield attained 12.79 wt% at 650 °C from Py-GC/MS with the CF-to-pine (CF-to-PN) ratio of 3 and catalyst-to-pine (CA-to-PN) ratio of 11, and became 9.67 wt% from lab-scale device with CF-to-PN and CA-to-PN ratios of 0.5 and 4, respectively. In addition, compared with HZSM-5, 0.5Mg1Mo/HZ possessed better anti-deactivation ability.

Label-Free Quantitative Acetylome Analysis Reveals Toxoplasma gondii Genotype-Specific Acetylomic Signatures.

Distinct genotypic and pathogenic differences exist between Toxoplasma gondii genotypes. For example, genotype I is highly virulent, whereas genotype II and genotype III are less virulent. Moreover, Chinese 1 genotype (ToxoDB#9) is also virulent. Here, we compare the acetylomes of genotype 1 (RH strain) and Chinese 1 genotype (ToxoDB#9, PYS strain) of T. gondii. Using mass spectrometry enriched for acetylated peptides, we found a relationship between the levels of protein acetylation and parasite genotype-specific virulence. Notably, lysine acetylation was the largest (458 acetylated proteins) in RH strain, followed by PYS strain (188 acetylated proteins), whereas only 115 acetylated proteins were detected in PRU strain. Our analysis revealed four, three, and four motifs in RH strain, PRU strain and PYS strain, respectively. Three conserved sequences around acetylation sites, namely, xxxxxKAcHxxxx, xxxxxKAcFxxxx, and xxxxGKAcSxxxx, were detected in the acetylome of the three strains. However, xxxxxKAcNxxxx (asparagine) was found in RH and PYS strains but was absent in PRU strain. Our analysis also identified 15, 3, and 26 differentially expressed acetylated proteins in RH strain vs. PRU strain, PRU strain vs. PYS strain and PYS strain vs. RH strain, respectively. KEGG pathway analysis showed that a large proportion of the acetylated proteins are involved in metabolic processes. Pathways for the biosynthesis of secondary metabolites, biosynthesis of antibiotics and microbial metabolism in diverse environments were featured in the top five enriched pathways in all three strains. However, acetylated proteins from the virulent strains (RH and PYS) were more enriched in the pyruvate metabolism pathway compared to acetylated proteins from PRU strain. Increased levels of histone-acetyl-transferase and glycyl-tRNA synthase were detected in RH strain compared to PRU strain and PYS strain. Both enzymes play roles in stress tolerance and proliferation, key features in the parasite virulence. These findings reveal novel insight into the acetylomic profiles of major T. gondii genotypes and provide a new important resource for further investigations of the roles of the acetylated parasite proteins in the modulation of the host cell response to the infection of T. gondii.

iTRAQ-Based Global Phosphoproteomics Reveals Novel Molecular Differences Between Toxoplasma gondii Strains of Different Genotypes.

To gain insights into differences in the virulence among T. gondii strains at the post-translational level, we conducted a quantitative analysis of the phosphoproteome profile of T. gondii strains belonging to three different genotypes. Phosphopeptides from three strains, type I (RH strain), type II (PRU strain) and ToxoDB#9 (PYS strain), were enriched by titanium dioxide (TiO2) affinity chromatography and quantified using iTRAQ technology. A total of 1,441 phosphopeptides, 1,250 phosphorylation sites and 759 phosphoproteins were detected. In addition, 392, 298, and 436 differentially expressed phosphoproteins (DEPs) were identified in RH strain when comparing RH/PRU strains, in PRU strain when comparing PRU/PYS strains, and in PYS strain when comparing PYS/RH strains, respectively. Functional characterization of the DEPs using GO, KEGG, and STRING analyses revealed marked differences between the three strains. In silico kinase substrate motif analysis of the DEPs revealed three (RxxS, SxxE, and SxxxE), three (RxxS, SxxE, and SP), and five (SxxE, SP, SxE, LxRxxS, and RxxS) motifs in RH strain when comparing RH/PRU strains, in PRU strain when comparing PRU/PYS, and in PYS strain when comparing PYS/RH strains, respectively. This suggests that multiple overrepresented protein kinases including PKA, PKG, CKII, IKK, and MAPK could be involved in such a difference between T. gondii strains. Kinase associated network analysis showed that ROP5, ROP16, and cell-cycle-associated protein kinase CDK were the most connected kinase peptides. Our data reveal significant changes in the abundance of phosphoproteins between T. gondii genotypes, which explain some of the mechanisms that contribute to the virulence heterogeneity of this parasite.