The Development of an Isotope Dilution Mass Spectrometry Method for Interleukin-6 Quantification.

Inflammatory responses and tumor developments are closely related, with interleukin-6 (IL-6) playing important roles in both processes. IL-6 has been extensively identified as a potential tumor biomarker. This study developed an isotope dilution mass spectrometry (IDMS) method for quantifying IL-6 based on signature peptides. These peptides were screened by excluding those with missed cleavage or post-translational modification. The method's accuracy was verified using amino acid-based IDMS, in which purified IL-6 protein samples were quantified after hydrolyzing them into amino acids, and no significant difference was observed (p-value < 0.05). The method demonstrated good linearity and sensitivity upon testing. The specificity and matrix effect of the method were verified, and a precision study showed that the coefficient of variation was less than 5% for both the intra-day and inter-day tests. Compared to immunoassays, this method offers distinct advantages, such as the facilitation of multi-target analysis. Furthermore, the peptides used in this study are much more convenient for storage and operation than the antibodies or purified proteins typically used in immunoassays.

Development of a Phage-Displayed Nanobody-Based Competitive Immunoassay for the Sensitive Detection of Soybean Agglutinin.

Soybean agglutinin (SBA) is a primary antinutritional factor in soybeans that can inhibit the growth of humans and mammals, disrupt the intestinal environment, and cause pathological changes. Therefore, detecting and monitoring SBA in foods is essential for safeguarding human health. In this paper, M13 phage-displayed nanobodies against SBA were isolated from a naive nanobody library. An M13 phage-displayed nanobody-based competitive enzyme-linked immunosorbent assay (P-cELISA) was then established for SBA analysis using biotinylated anti-M13 phage antibody (biotin-anti-M13) and streptavidin poly-HRP conjugate (SA-poly-HRP). The biotin-anti-M13@SA-poly-HRP probe can easily amplify the detection signal without the chemical modifications of phage-displayed nanobodies. The established P-cELISA presented a linear detection range of 0.56-250.23 ng/mL and a limit of detection (LOD) of 0.20 ng/mL, which was 12.6-fold more sensitive than the traditional phage-ELISA. Moreover, the developed method showed good specificity for SBA and acceptable recoveries (78.21-121.11%) in spiked wheat flour, albumen powder, and whole milk powder. This study proposes that P-cELISA based on biotin-anti-M13@SA-poly-HRP may provide a convenient and effective strategy for the sensitive detection of SBA.

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/µL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

Establishment of Indirect Competitive Enzyme-linked Immunosorbent Assay (ic-ELISA) for Copper ion (Cu2+) in Raw Meat Products.

Adding an appropriate amount of copper to feed can promote the growth and development of livestock; however, a large amount of heavy metal copper can accumulate in livestock through the enrichment effect, which poses a serious threat to human health. Traditional Cu2+ detection relies heavily on complex and expensive instruments, such as inductively coupled plasma-optical emission spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS); thus, convenient and simple rapid detection technologies are urgently needed. In this paper, synthesized copper antigens were used to immunize mice and highly specific anticopper monoclonal antibodies were obtained, which were verified to exhibit high affinity and specificity. Based on the above antibodies, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established for the rapid detection of copper content in pork. The standard inhibition curve of the method was obtained by antigen-antibody working concentration screening, in which the half inhibitory concentration (IC50) was 11.888 ng/mL, the limit of detection (LOD) was 0.841 ng/mL and the correlation coefficient R2 of the curve was 0.998. In the additive recovery experiment, the recovery rate ranged from 90% to 110%, and the coefficient of variation (CV) was less than 10%, indicating that the method achieved high accuracy and precision. Finally, the results of ic-ELISA combined with Bland-Altman analysis showed a high correlation with ICP-MS, and the correlation coefficient (R2) reached 0.990 when the copper concentration was less than 200 ng/mL. Thus, the ic-ELISA method exhibits high reliability.

Development of an isotope dilution mass spectrometry assay for the quantification of insulin based on signature peptide analysis.

An isotope dilution mass spectrometry (IDMS) method that involves peptide-based protein analysis was developed to accurately quantify insulin. In this study, a signature peptide (GFFYTPK) obtained from tryptic digestion of insulin was selected as a surrogate for insulin. Then, the optimal conditions for signature peptide analysis through mass spectrometry detection and enzymatic digestion were determined. The analytical performance of this method was assessed and validated using porcine insulin-certified reference material. The linear range of the insulin calibration curve ranged from 0.05 ~ 2 mass ratios, with recoveries ranging from 96.15 to approximately 101.15%. The limit of detection was 0.19 ng/mL, and the limit of quantification was 0.63 ng/mL. The quantitative results corresponded well with a certified value that was obtained from measuring a porcine insulin reference material with amino acid-based IDMS. In addition, the target peptide GFFYTPK can be found in other species of insulin. This method was also applied for the quantification of human insulin-certified reference material. Finally, we applied the method to quantify the concentrations of simulated serum insulin. These findings suggested that this signature peptide-based IDMS approach can accurately quantify insulin levels, can assign a certified value to insulin reference materials, and has the potential to quantify serum insulin with traceable measurements.

Carboxymethyl chitosan-modified UiO-66 for the rapid detection of fenpropathrin in grains.

Fenpropathrin residues in grain are potentially harmful to humans. Therefore, a fluorimetric lateral flow immunoassay using a zirconium-based organic skeleton (UiO-66) as a signal marker was developed for detecting fenpropathrin. Herein, carboxymethyl chitosan (CMCS) was used to modify UiO-66 and improve its water solubility to facilitate stable binding with sodium fluorescein (NaFL). This resulted in formation of a new fluorescent probe that is more suitable for lateral flow immunoassay (LFIA). The materials were characterized via electron microscopy, Fourier-transform infrared spectroscopy, and powder X-ray diffraction. CMCS and NaFL were successfully bound to UiO-66. Under optimized conditions, the constructed NaFL/UiO-66@CMCS-LFIA exhibited a good linear relationship within the range of 0.98-62.5 µg/L, with a detection limit of 3.91 µg/L. This probe was fourfold more sensitive than traditional colloidal gold nanoparticle-based LFIA. Finally, NaFL/UiO-66@CMCS-LFIA was successfully applied to detect fenpropathrin in wheat and maize samples. The detection limit was 1.56 µg/kg and recoveries ranged from 96.58 % to 118.56 %. This study provides a sensitive, stable, and convenient method for the rapid detection of pesticide residues.

Traceability of Microplastic Fragments from Waste Plastic Express Packages Using Near-Infrared Spectroscopy Combined with Chemometrics.

The pollution from waste plastic express packages (WPEPs), especially microplastic (MP) fragments, caused by the blowout development of the express delivery industry has attracted widespread attention. On account of the variety of additives, strong complexity, and high diversity of plastic express packages (PEPs), the multi-class classification of WPEPs is a typical large-class-number classification (LCNC). The traceability and identification of microplastic fragments from WPEPs is very challenging. An effective chemometric method for large-class-number classification would be very beneficial for the comprehensive treatment of WPEP pollution through the recycling and reuse of waste plastic express packages, including microplastic fragments and plastic debris. Rather than using the traditional one-against-one (OAO) and one-against-all (OAA) dichotomies, an exhaustive and parallel half-against-half (EPHAH) decomposition, which overcomes the defects of the OAO's classifier learning limitations and the OAA's data proportion imbalance, is proposed for feature selection. EPHAH analysis, combined with partial least squares discriminant analysis (PLS-DA) for large-class-number classification, was performed on 750 microplastic fragments of polyethylene WPEPs from 10 major courier companies using near-infrared (NIR) spectroscopy. After the removal of abnormal samples through robust principal component analysis (RPCA), the root mean square error of cross-validation (RMSECV) value for the model was reduced to 0.01, which was 21.5% lower than that including the abnormal samples. The best models of PLS-DA were obtained using SNV combined with SG-17 smoothing and 2D (SNV+SG-17+2D); the latent variables (LVs), the error rates of Monte Carlo cross-validation (ERMCCVs), and the final classification accuracies were 6.35, 0.155, and 88.67% for OAO-PLSDA; 5.37, 0.103, and 87.33% for OAA-PLSDA; and 3.12, 0.054, and 96.00% for EPHAH-PLSDA. The results showed that the EPHAH strategy can completely learn the complex LCNC decision boundaries for 10 classes, effectively break the tie problem, and greatly improve the voting resolution, thereby demonstrating significant superiority to both the OAO and OAA strategies in terms of classification accuracy. Meanwhile, PLS-DA further maximized the covariance and data interpretation abilities between the potential variables and categories of microplastic debris, thereby establishing an ideal performance identification model with a recognition rate of 96.00%.

Structural insights into IL-6 signaling inhibition by therapeutic antibodies.

Antibody inhibitors of the interleukin-6 (IL-6) signaling pathway, such as tocilizumab and sarilumab, have been used to treat rheumatoid arthritis, chimeric antigen receptor T cell-induced cytokine storm, and severe COVID-19 pneumonia. Here, we solve the cryogenic electron microscopy structures of sarilumab and tocilizumab in complex with IL-6R to resolutions of 3.2 and 3.3 Å, respectively. These structures reveal that both tocilizumab and sarilumab bind to the D3 domain of IL-6R. The binding surfaces of the two antibodies largely overlap, but the detailed interactions are different. Functional studies of various mutants show results consistent with our structural analysis of the antibodies and IL-6R interactions. Structural comparisons with the IL-6/IL-6R/gp130 complex indicate that sarilumab and tocilizumab probably inhibit IL-6/IL-6R signaling by competing for the IL-6 binding site. In summary, this work reveals the antibody-blocking mechanism of the IL-6 signaling pathway and paves the way for future antibody discovery.

Targeting autophagy drug discovery: Targets, indications and development trends.

Autophagy plays a vital role in sustaining cellular homeostasis and its alterations have been implicated in the etiology of many diseases. Drugs development targeting autophagy began decades ago and hundreds of agents were developed, some of which are licensed for the clinical usage. However, no existing intervention specifically aimed at modulating autophagy is available. The obstacles that prevent drug developments come from the complexity of the actual impact of autophagy regulators in disease scenarios. With the development and application of new technologies, several promising categories of compounds for autophagy-based therapy have emerged in recent years. In this paper, the autophagy-targeted drugs based on their targets at various hierarchical sites of the autophagic signaling network, e.g., the upstream and downstream of the autophagosome and the autophagic components with enzyme activities are reviewed and analyzed respectively, with special attention paid to those at preclinical or clinical trials. The drugs tailored to specific autophagy alone and combination with drugs/adjuvant therapies widely used in clinical for various diseases treatments are also emphasized. The emerging drug design and development targeting selective autophagy receptors (SARs) and their related proteins, which would be expected to arrest or reverse the progression of disease in various cancers, inflammation, neurodegeneration, and metabolic disorders, are critically reviewed. And the challenges and perspective in clinically developing autophagy-targeted drugs and possible combinations with other medicine are considered in the review.

Dosage-sensitive and simultaneous detection of multiple small-molecule pollutants in environmental water and agriproducts using portable SERS-based lateral flow immunosensor.

The co-contamination of pesticide residues and mycotoxins in agricultural products is a global concern, with the potential for cumulative and synergistic damaging effects, imposing substantial health and economic burdens to the public. The dosage-sensitive and simultaneous detection of multiple pollutants, with a heightened sensitivity in real samples, poses a significant demand and challenge. Herein, we propose a portable detection method integrating surface-enhanced Raman scattering (SERS)-with lateral flow immunoassay (LFIA), offering high sensitivity and multiplex analysis capabilities. This approach enables the simultaneous detection of imidacloprid (IMI), pyraclostrobin (PYR) and aflatoxin B1 (AFB1) through a single test strip. Utilizing the immune-specific binding between antigen and antibodies, we immobilised antibody- conjugated SERS nanotags on three test lines of the strips to generate Raman signal amplification in the proposed biosensor. Accurate quantitative analysis was performed by measuring the SERS signal intensity on the test lines. The limits of detection were 8.6 pg/mL for IMI, 97.4 pg/mL for PYR and 8.9 pg/mL for AFB1, exhibiting sensitivities 12-fold, 102-fold and11-fold higher than the colorimetric signals, respectively. Importantly, the SERS-LFIA immunosensor demonstrated robust performance when applied to real samples, yielding recoveries ranging from 86.16 % to 115.0 %, with relative standard deviation values below 8.67 %. These results underscore the excellent stability, high selectivity and reliability the proposed SERS-LFIA immunosensor. Consequently, it holds promise for the detection of multiple pesticides and mycotoxins in both environmental and agricultural samples.

Advances in Detection Techniques for the H5N1 Avian Influenza Virus.

Avian influenza is caused by avian influenza virus infection; the H5N1 avian influenza virus is a highly pathogenic subtype, affecting poultry and human health. Since the discovery of the highly pathogenic subtype of the H5N1 avian influenza virus, it has caused enormous losses to the poultry farming industry. It was recently found that the H5N1 avian influenza virus tends to spread among mammals. Therefore, early rapid detection methods are highly significant for effectively preventing the spread of H5N1. This paper discusses the detection technologies used in the detection of the H5N1 avian influenza virus, including serological detection technology, immunological detection technology, molecular biology detection technology, genetic detection technology, and biosensors. Comparisons of these detection technologies were analyzed, aiming to provide some recommendations for the detection of the H5N1 avian influenza virus.

Recent developments in ionization techniques for single-cell mass spectrometry.

The variation among individual cells plays a significant role in many biological functions. Single-cell analysis is advantageous for gaining insight into intricate biochemical mechanisms rarely accessible when studying tissues as a whole. However, measurement on a unicellular scale is still challenging due to unicellular complex composition, minute substance quantities, and considerable differences in compound concentrations. Mass spectrometry has recently gained extensive attention in unicellular analytical fields due to its exceptional sensitivity, throughput, and compound identification abilities. At present, single-cell mass spectrometry primarily concentrates on the enhancement of ionization methods. The principal ionization approaches encompass nanoelectrospray ionization (nano-ESI), laser desorption ionization (LDI), secondary ion mass spectrometry (SIMS), and inductively coupled plasma (ICP). This article summarizes the most recent advancements in ionization techniques and explores their potential directions within the field of single-cell mass spectrometry.

Recent Advances in Non-Targeted Screening of Compounds in Plastic-Based/Paper-Based Food Contact Materials.

Ensuring the safety of food contact materials has become a pressing concern in recent times. However, detecting hazardous compounds in such materials can be a complex task, and traditional screening methods may not be sufficient. Non-targeted screening technologies can provide comprehensive information on all detectable compounds, thereby supporting the identification, detection, and risk assessment of food contact materials. Nonetheless, the non-targeted screening of food contact materials remains a challenging issue. This paper presents a detailed review of non-targeted screening technologies relying on high-resolution mass spectrometry for plastic-based and paper-based food contact materials over the past five years. Methods of extracting, separating, concentrating, and enriching compounds, as well as migration experiments related to non-targeted screening, are examined in detail. Furthermore, instruments and devices of high-resolution mass spectrometry used in non-targeted screening technologies for food contact materials are discussed and summarized. The research findings aim to provide a theoretical basis and practical reference for the risk management of food contact materials and the development of relevant regulations and standards.

Prokaryotic Argonaute Proteins: A New Frontier in Point-of-Care Viral Diagnostics.

The recent pandemic of SARS-CoV-2 has underscored the critical need for rapid and precise viral detection technologies. Point-of-care (POC) technologies, which offer immediate and accurate testing at or near the site of patient care, have become a cornerstone of modern medicine. Prokaryotic Argonaute proteins (pAgo), proficient in recognizing target RNA or DNA with complementary sequences, have emerged as potential game-changers. pAgo present several advantages over the currently popular CRISPR/Cas systems-based POC diagnostics, including the absence of a PAM sequence requirement, the use of shorter nucleic acid molecules as guides, and a smaller protein size. This review provides a comprehensive overview of pAgo protein detection platforms and critically assesses their potential in the field of viral POC diagnostics. The objective is to catalyze further research and innovation in pAgo nucleic acid detection and diagnostics, ultimately facilitating the creation of enhanced diagnostic tools for clinic viral infections in POC settings.

Biosynthetic Pathways and Functions of Indole-3-Acetic Acid in Microorganisms.

Indole-3-acetic acid (IAA) belongs to the family of auxin indole derivatives. IAA regulates almost all aspects of plant growth and development, and is one of the most important plant hormones. In microorganisms too, IAA plays an important role in growth, development, and even plant interaction. Therefore, mechanism studies on the biosynthesis and functions of IAA in microorganisms can promote the production and utilization of IAA in agriculture. This mini-review mainly summarizes the biosynthesis pathways that have been reported in microorganisms, including the indole-3-acetamide pathway, indole-3-pyruvate pathway, tryptamine pathway, indole-3-acetonitrile pathway, tryptophan side chain oxidase pathway, and non-tryptophan dependent pathway. Some pathways interact with each other through common key genes to constitute a network of IAA biosynthesis. In addition, functional studies of IAA in microorganisms, divided into three categories, have also been summarized: the effects on microorganisms, the virulence on plants, and the beneficial impacts on plants.

Diploid mycelia of Ustilago esculenta fails to maintain sustainable proliferation in host plant.

Smut fungi display a uniform life cycle including two phases: a saprophytic phase in vitro and a parasitic phase in host plants. Several apathogenic smut fungi are found, lacking suitable hosts in their habitat. Interestingly, MT-type Ustilago esculenta was found to maintain a parasitic life, lacking the saprophytic phase. Its long period of asexual proliferation in plant tissue results in severe defects in certain functions. In this study, the growth dynamics of U. esculenta in plant tissues were carefully observed. The mycelia of T- and MT-type U. esculenta exhibit rapid growth after karyogamy and aggregate between cells. While T-type U. esculenta successfully forms teliospores after aggregation, the aggregated mycelia of MT-type U. esculenta gradually disappeared after a short period of massive proliferation. It may be resulted by the lack of nutrition such as glucose and sucrose. After overwintering, infected Zizania latifolia plants no longer contained diploid mycelia resulting from karyogamy. This indicated that diploid mycelia failed to survive in plant tissues. It seems that diploid mycelium only serves to generate teliospores. Notably, MT-type U. esculenta keeps the normal function of karyogamy, though it is not necessary for its asexual life in plant tissue. Further investigations are required to uncover the underlying mechanism, which would improve our understanding of the life cycle of smut fungi and help the breeding of Z. latifolia.

Domestication, breeding, omics research, and important genes of Zizania latifolia and Zizania palustris.

Wild rice (Zizania spp.), an aquatic grass belonging to the subfamily Gramineae, has a high economic value. Zizania provides food (such as grains and vegetables), a habitat for wild animals, and paper-making pulps, possesses certain medicinal values, and helps control water eutrophication. Zizania is an ideal resource for expanding and enriching a rice breeding gene bank to naturally preserve valuable characteristics lost during domestication. With the Z. latifolia and Z. palustris genomes completely sequenced, fundamental achievements have been made toward understanding the origin and domestication, as well as the genetic basis of important agronomic traits of this genus, substantially accelerating the domestication of this wild plant. The present review summarizes the research results on the edible history, economic value, domestication, breeding, omics research, and important genes of Z. latifolia and Z. palustris over the past decades. These findings broaden the collective understanding of Zizania domestication and breeding, furthering human domestication, improvement, and long-term sustainability of wild plant cultivation.

Characterization and transcriptomic analysis of a native fungal pathogen against the rice pest Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is one of the most destructive pests of rice. Given the threats posed by insecticide resistance to its control, eco-friendly strategies based on microbial pathogens emerged as a promising biocontrol alternative. In the present study, we isolated a native fungal pathogen against BPH from infected BPH cadavers and preliminarily identified as a strain of Aspergillus fumigatus based on morphological and molecular methods. Laboratory bioassay revealed that this fungal strain was highly virulent to BPH both at nymphal and adult stages, with the median lethal times (LT50) of 7.5 and 5.8 days under high conidial concentration of 1 × 109 conidia mL-1. A genome-wide view of gene expressions in BPH against fungal attack was analyzed by transcriptomic sequencing and consequently a large number of differentially expressed genes that mainly involved in host immune defense and cell detoxification were found. RNAi-mediated knockdown of an upregulated gene encoding a serine protease (NlSPN) could cause a significant decrease in BPH survival. Combination of dsRNA injection and fungal infection showed an additive effect on BPH mortality, which provided clues to develop new pest management strategies against BPH.

The MAP Kinase PvMK1 Regulates Hyphal Development, Autophagy, and Pathogenesis in the Bayberry Twig Blight Fungus Pestalotiopsis versicolor.

Bayberry twig blight caused by the ascomycete fungus Pestalotiopsis versicolor is a devastating disease threatening worldwide bayberry production. However, the molecular basis underlying the pathogenesis of P. versicolor is largely unknown. Here, we identified and functionally characterized the MAP kinase PvMk1 in P. versicolor through genetic and cellular biochemical approaches. Our analysis reveals a central role of PvMk1 in regulating P. versicolor virulence on bayberry. We demonstrate that PvMk1 is involved in hyphal development, conidiation, melanin biosynthesis, and cell wall stress responses. Notably, PvMk1 regulates P. versicolor autophagy and is essential for hyphal growth under nitrogen-depleting conditions. These findings suggest the multifaceted role of PvMk1 in regulating P. versicolor development and virulence. More remarkably, this evidence of virulence-involved cellular processes regulated by PvMk1 has paved a fundamental way for further understanding the impact of P. versicolor pathogenesis on bayberry.

The Novel Effector Ue943 Is Essential for Host Plant Colonization by Ustilago esculenta.

The smut fungus Ustilago esculenta obligately parasitizes Zizania latifolia and induces smut galls at the stem tips of host plants. Previous research identified a putative secreted protein, Ue943, which is required for the biotrophic phase of U. esculenta but not for the saprophytic phase. Here, we studied the role of Ue943 during the infection process. Conserved homologs of Ue943 were found in smut fungi. Ue943 can be secreted by U. esculenta and localized to the biotrophic interface between fungi and plants. It is required at the early stage of colonization. The Ue943 deletion mutant caused reactive oxygen species (ROS) production and callose deposition in the host plant at 1 and 5 days post inoculation, which led to failed colonization. The virulence deficiency was restored by overexpressing gene Ue943 or Ue943:GFP. Transcriptome analysis further showed a series of changes in plant hormones following ROS production when the host plant was exposed to ΔUe943. We hypothesize that Ue943 might be responsible for ROS suppression or avoidance of recognition by the plant immune system. The mechanism underlying Ue943 requires further study to provide more insights into the virulence of smut fungi.