ABSTRACT
Tender coconut water has been very popular as a natural beverage rich in various electrolytes, amino acids, and vitamins, and hence a large amount of tender coconut kernel is left without efficient utilization. To explore the possibility of making infused tender coconut kernel, we investigated the effects of two osmosis methods, including solid-state osmotic dehydration and liquid-state osmotic dehydration, as well as two osmosis agents such as sorbitol and sucrose, on the mass transfer of coconut kernel under solid-state osmotic dehydration conditions. The results showed that under the conditions of solid-state osmosis using sucrose and liquid-state osmosis using sucrose solution, the water diffusion coefficients were 9.0396 h-1/2 and 2.9940 h-1/2, respectively, with corresponding water mass transfer coefficients of 0.3373 and 0.2452, and the equilibrium water loss rates of 49.04% and 17.31%, respectively, indicating that the mass transfer efficiency of solid-state osmotic dehydration of tender coconut kernel was significantly higher than that of liquid-state osmotic dehydration. Under solid osmosis conditions, the water loss rates using sucrose and sorbitol were 38.64% and 41.95%, respectively, with dry basis yield increments of 61.38% and 71.09%, respectively, demonstrating superior dehydration efficiency of sorbitol over sucrose under solid-state osmosis. This study can provide a reference for the theoretical study of the mass transfer of tender coconut kernel through osmotic dehydration, and also provide technical support for the development and utilization of tender coconut kernel.
ABSTRACT
Alcoholic liver damage is caused by long-term or heavy drinking, and it may further progress into alcoholic liver diseases (ALD). Probiotic supplements have been suggested for the prevention or improvement of liver damage. This study was designed to consider the ameliorative effects of Lactobacillus rhamnosus NKU FL1-8 isolated from infant feces against alcoholic liver damage. The mice were gavaged with a 50% ethanol solution and treated with 109 CFU of L. rhamnosus NKU FL1-8 suspension. The factors for liver function, oxidative stress, inflammation, gut microbiota composition, and intestinal barrier integrity were measured. The results showed that L. rhamnosus NKU FL1-8 could decrease the levels of aspartate aminotransferase (AST) to 61% and alanine aminotransferase (ALT) to 50% compared with ethanol given by gavage. It could inhibit the expression level of malondialdehyde (MDA), increase superoxide dismutase (SOD), glutathione (GSH) to relieve oxidative stress, and down-regulate the cytokines to decrease hepatic inflammation. After treatment, the level of triglycerides was reduced, and the expression levels of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and the peroxisome proliferators-activated receptor-α (PPAR-α) pathway were up-regulated. Additionally, the 16S rRNA sequencing analysis showed that L. rhamnosus NKU FL1-8 increased the relative abundance of Lactobacillus, Ruminococcaceae, etc. At the same time, L. rhamnosus NKU FL1-8 could significantly reduce lipopolysaccharides (LPS) and enhance intestinal tight junction proteins. These results demonstrated that L. rhamnosus NKU FL1-8 could reduce the level of oxidative stress, fat accumulation, and liver inflammation caused by alcohol in the host. The underlying mechanism could be that L. rhamnosus NKU FL1-8 inhibits LPS by regulating the gut microbiota and repairing the intestinal barrier. Thereby, these findings support L. rhamnosus NKU FL1-8 as a potential functional food for the relief of ALD.
Subject(s)
Feces , Gastrointestinal Microbiome , Lacticaseibacillus rhamnosus , Liver Diseases, Alcoholic , Mice, Inbred C57BL , Oxidative Stress , Probiotics , Animals , Gastrointestinal Microbiome/drug effects , Feces/microbiology , Oxidative Stress/drug effects , Liver Diseases, Alcoholic/prevention & control , Probiotics/pharmacology , Mice , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/drug effects , Male , Liver/drug effects , Liver/metabolism , Infant , Ethanol , Disease Models, AnimalABSTRACT
It has been shown that prostaglandin (PG) E2 synthesized in the lateral parabrachial nucleus (LPBN) is involved in lipopolysaccharide-induced fever. But the neural mechanisms of how intra-LPBN PGE2 induces fever remain unclear. In this study, we investigated whether the LPBN-preoptic area (POA) pathway, the thermoafferent pathway for feed-forward thermoregulatory responses, mediates fever induced by intra-LPBN PGE2 in male rats. The core temperature (Tcore) was monitored using a temperature radiotelemetry transponder implanted in rat abdomen. We showed that microinjection of PGE2 (0.28 nmol) into the LPBN significantly enhanced the density of c-Fos-positive neurons in the median preoptic area (MnPO). The chemical lesioning of MnPO with ibotenate or selective genetic lesioning or inhibition of the LPBN-MnPO pathway significantly attenuated fever induced by intra-LPBN injection of PGE2. We demonstrated that EP3 receptor was a pivotal receptor for PGE2-induced fever, since microinjection of EP3 receptor agonist sulprostone (0.2 nmol) or EP3 receptor antagonist L-798106 (2 nmol) into the LPBN mimicked or weakened the pyrogenic action of LPBN PGE2, respectively, but this was not the case for EP4 and EP1 receptors. Whole-cell recording from acute LPBN slices revealed that the majority of MnPO-projecting neurons originating from the external lateral (el) and dorsal (d) LPBN were excited and inhibited, respectively, by PGE2 perfusion, initiating heat-gain and heat-loss mechanisms. The amplitude but not the frequency of spontaneous and miniature glutamatergic excitatory postsynaptic currents (sEPSCs and mEPSCs) in MnPO-projecting LPBel neurons increased after perfusion with PGE2; whereas the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) and the A-type potassium (IA) current density did not change. In MnPO-projecting LPBd neurons, neither sEPSCs nor sIPSCs responded to PGE2; however, the IA current density was significantly increased by PGE2 perfusion. These electrophysiological responses and the thermoeffector reactions to intra-LPBN PGE2 injection, including increased brown adipose tissue thermogenesis, shivering, and decreased heat dissipation, were all abolished by L-798106, and mimicked by sulprostone. These results suggest that the pyrogenic effects of intra-LPBN PGE2 are mediated by both the inhibition of the LPBd-POA pathway through the EP3 receptor-mediated activation of IA currents and the activation of the LPBel-POA pathway through the selective enhancement of glutamatergic synaptic transmission via EP3 receptors.
Subject(s)
Body Temperature Regulation , Dinoprostone , Fever , Parabrachial Nucleus , Preoptic Area , Receptors, Prostaglandin E, EP3 Subtype , Animals , Male , Rats , Body Temperature Regulation/drug effects , Dinoprostone/pharmacology , Fever/chemically induced , Fever/metabolism , Neurons/drug effects , Neurons/metabolism , Parabrachial Nucleus/drug effects , Parabrachial Nucleus/physiology , Preoptic Area/drug effects , Preoptic Area/metabolism , Rats, Sprague-Dawley , Receptors, Prostaglandin E, EP3 Subtype/metabolismABSTRACT
China, with the third largest share of global tuberculosis cases, faces a substantial challenge in its healthcare system as a result of the high burden of multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB). This study employs a genomic epidemiological approach to assess recent tuberculosis transmissions between individuals, identifying potential risk factors and discerning the role of transmitted resistant isolates in the emergence of drug-resistant tuberculosis in China. We conducted a population-based retrospective study on 5052 Mycobacterium tuberculosis (MTB) isolates from 70 surveillance sites using whole genome sequencing (WGS). Minimum spanning tree analysis identified resistance mutations, while epidemiological data analysis pinpointed transmission risk factors. Of the 5052 isolates, 23% (1160) formed 452 genomic clusters, with 85.6% (387) of the transmissions occurring within the same counties. Individuals with younger age, larger family size, new cases, smear positive, and MDR/RR were at higher odds for recent transmission, while higher education (university and above) and occupation as a non-physical workers emerged as protective factors. At least 61.4% (251/409) of MDR/RR-TB were likely a result of recent transmission of MDR/RR isolates, with previous treatment (crude OR = 2.77), smear-positive (cOR = 2.07) and larger family population (cOR = 1.13) established as risk factors. Our findings highlight that local transmission remains the predominant form of TB transmission in China. Correspondingly, drug-resistant tuberculosis is primarily driven by the transmission of resistant tuberculosis isolates. Targeted interventions for high-risk populations to interrupt transmission within the country will likely provide an opportunity to reduce the prevalence of both tuberculosis and drug-resistant tuberculosis.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Whole Genome Sequencing , Humans , China/epidemiology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/classification , Male , Adult , Female , Middle Aged , Tuberculosis, Multidrug-Resistant/epidemiology , Tuberculosis, Multidrug-Resistant/transmission , Tuberculosis, Multidrug-Resistant/microbiology , Cross-Sectional Studies , Retrospective Studies , Young Adult , Risk Factors , Adolescent , Aged , Rifampin/pharmacology , Antitubercular Agents/pharmacology , Genome, Bacterial , Drug Resistance, Multiple, BacterialABSTRACT
Electrochemistry that empowers innovative nanoscopic analysis has long been pursued. Here, the concept of aggregation-enabled electrochemistry (AEE) in a confined nanopore is proposed and devised by reactive oxygen species (ROS)-responsive aggregation of CdS quantum dots (QDs) within a functional nanopipette. Complementary Faradaic and non-Faradaic operations of the CdS QDs aggregate could be conducted to simultaneously induce the signal-on of the photocurrents and the signal-off of the ionic signals. Such a rationale permits the cross-checking of the mutually corroborated signals and thus delivers more reliable results for single-cell ROS analysis. Combined with the rich biomatter-light interplay, the concept of AEE can be extended to other stimuli-responsive aggregations for electrochemical innovations.
ABSTRACT
Peanut is widely used for food supplementation with potential allergic reactions in infants and adults, which prompted the development of reliable and accurate detection of peanut allergens with emphasis on Ara h 1. In this study, a nanobody (Nb)-based micro-total electrochemical immunoassay (Nb-µTEI) was proposed to be generated. Generally, an alpaca was immunized with Ara h 1 to yield a Nb reservoir for selection of four specific Nbs. Nb-mediated immunocapturing allowed the identification of the target as Ara h 1. The Nb-based electrochemical immunoassay was developed by constructing a capturing electrode with cycles of signal enhancement. After construction of the capturing electrode, Nb152 with HA-tag was directly applied to connect immobilized anti-HA IgG for the capture of different concentrations of Ara h 1, which was labeled by biotinylated Nb152 to facilitate signal development with alkaline phosphatase conjugated streptavidin (SA-ALP). A linear range from 4.5 to 55 ng/mL was acquired with LOD and LOQ of 0.86 and 2.10 ng/mL, respectively, with an 11-fold increase of the sensitivity compared with the established sandwich ELISA. The dedicated immunoassay was verified by detecting the antigen spiked in food samples and demonstrated the successful conjugation of Nb with advanced detecting techniques.
Subject(s)
Arachis , Peanut Hypersensitivity , Adult , Humans , Antigens, Plant , Immunoassay/methods , Enzyme-Linked Immunosorbent Assay/methods , Allergens , Plant ProteinsABSTRACT
Milk proteins are widely used for food supplementation, despite the potential risk of food allergy, especially against ß-lactoglobulin (BLG), which makes BLG surveillance critical. Possible interaction of detecting antibodies with BLG-derived peptides will result in unprecise inspection. Thus, in this study, it was proposed to generate nanobodies (Nbs) and validate the immunological detection of intact BLG rather than hydrolytic peptides. Nbs were successfully retrieved and characterized with high stability and target specificity. A competitive enzyme-linked immunosorbent assay (cELISA) was developed with a linear range from 39 to 10,000 ng/mL and a detection limit (LOD) of 4.55 ng/mL, with a recovery of 86.30%-95.09% revealed by analysis of spiked samples. Meanwhile, a sandwich ELISA (sELISA) was established with Nb82 and BLG polyclonal antibody (pAb-BLG) providing a linear range from 29.7 to 1250 ng/mL and an LOD of 13.82 ng/mL with a recovery of 87.82%-103.97%. The interaction of selected Nbs with BLG-derived peptides was investigated by Nb structure modeling and BLG docking. No binding on hydrolytic peptides was revealed, confirming the precision of Nb-mediated immunoassays. In summary, this study successfully identified BLG-specific Nbs for immunoassay development and guaranteed the monitoring of intact BLG without interference of hydrolytic peptides, providing experimental evidence that our Nbs recognize intact food allergen.
Subject(s)
Lactoglobulins , Single-Domain Antibodies , Allergens , Indicators and ReagentsABSTRACT
Serum ferritin (SF) is an iron-rich protein tightly connected with iron homeostasis, and the variations are frequently observed in diseased states, including iron-deficiency anemia, inflammation, liver disease, and tumors, which renders SF level an indicator of potential malignancies in clinical practice. Nanobodies (Nbs) have been widely explored and developed into theranostic reagents. Surprisingly, no reports stated the identification of anti-SF Nbs, nor the potential of such Nbs as a diagnostic tool. In this study, we generated SF-specific Nbs and provided novel clinical diagnostic approaches to develop an immunoassay. An immune library was constructed after immunizing an alpaca with SF, and five Nbs specifically targeting human SF were retrieved. The obtained Nbs exhibited robust properties including high stability, affinity, and specificity. Then, an ELISA-based test using a heterologous Nb-pair was developed. The calibration curve demonstrated a linear range of SF between 9.0 to 1100 ng/mL, and a limit of detection (LOD) of 1.01 ng/mL. The detecting recovery and coefficient variation (CV) were determined by spiking different concentrations of SF into the serum sample, to verify the successful application of our selected Nbs for SF monitoring. In general, this study generated SF-specific Nbs and demonstrated their potential as diagnostic immunoassay tools.
Subject(s)
Single-Domain Antibodies , Enzyme-Linked Immunosorbent Assay , Ferritins , Humans , Immunoassay , IronABSTRACT
A novel sulfate-loaded iron-nitrogen co-doped carbon quantum dots (SO42--CQDs)-based fluorescent method was synthesized by the facile and environmentally friendly pyrolysis of persimmon frost (carbon source) and (NH4)2Fe(SO4)2·6H2O. After SMMC-7721 cells were incubated with the SO42--CQDs for 24 h, more than 95% of the cells remained viable, even at a high concentration of the SO42--CQDs, indicating excellent biocompatibility and low toxicity. In addition, it was able to be taken up by the cells to emit their bright blue fluorescence after excitation at 365 nm, indicating suitable cell permeability. The SO42--CQDs also exhibited a unique response to changes in pH, which was applied in the detection of OPs by relying on the production of acetic acid from the hydrolysis of acetylcholine (ACh) by acetylcholinesterase (AChE), which decreased the pH and engendered an increase in the fluorescence of the SO42--CQDs; however, the inhibition of AChE by glyphosate resulted in little influence on fluorescence intensity due to the lack of acetic acid produced. This mechanism was the basis for the development of a sensitive assay for the detection of glyphosate. The resulting assay had a limit of detection of 0.066 ng/mL. Furthermore, the method was successfully applied for the precise and accurate monitoring of the concentration, distribution, and variation of glyphosate residues in chives and cultivated soil. Therefore, the proposed method was anticipated to provide a promising alternative for other detection methods to enable the reliable analysis of OPs in food products.
Subject(s)
Quantum Dots , Acetylcholinesterase , Carbon/chemistry , Fluorescent Dyes/chemistry , Glycine/analogs & derivatives , Hydrogen-Ion Concentration , Nitrogen/chemistry , Quantum Dots/chemistry , GlyphosateABSTRACT
Trophoblast cell-surface antigen 2 (Trop-2) is a tumor-associated antigen that is connected with the development of various tumors and has been identified as a promising target for tumor immunotherapy. To date, the immunotherapy against Trop-2 mainly relies on the specific targeting by monoclonal antibody in antibody-drug conjugate (ADC). Alternatively, the single domain antibodies of nanobodies (Nbs) possesses unique properties such as smaller size, better tissue penetration, etc., to make them good candidates for tumor targeting. Thus, it was proposed to develop anti-Trop-2 Nbs for tumor targeting in this study. Generally, three consecutive rounds of bio-panning were performed against immobilized recombinant Trop-2, and yielded three Nbs (Nb60, Nb65, and Nb108). The affinity of selected Nbs was determined in the nanomolar range, especially the good properties of Nb60 were verified as a promising candidate for tumor labeling. The binding to native Trop-2 was confirmed by flow cytometry against tumor cells. The inhibitory effects of the selected Nbs on tumor cell proliferation and migration were confirmed by wound healing and Transwell assay. The clear localization of the selected Nbs on the surface of tumor cells verified the potent labeling efficiency. In conclusion, this study provided several Nbs with the potential to be developed as targeting moiety of drug conjugates.
Subject(s)
Immunoconjugates , Neoplasms , Single-Domain Antibodies , Antibodies, Monoclonal , Flow Cytometry , Humans , Immunoconjugates/therapeutic use , Neoplasms/drug therapyABSTRACT
The unique characteristics of camelid heavy-chain only antibody (HCAb) derived nanobodies (Nbs) have facilitated their employment as tools for research and application in extensive fields including food safety inspection, diagnosis and therapy of diseases, etc., to develop immune detecting techniques or alternative candidates of conventional antibodies as diagnostic and therapeutic reagents. The wide application in the fields of food allergen inspection and immunotherapy has not been addressed as not much results published in the literature. The robust properties and straightforward selecting strategy of Nbs impel the advantageous employment compared with monoclonal antibodies (mAbs) to establish immunoassay and serve as blocking antibodies to compete immunoglobulin E (IgE) binding epitopes on food allergens. More and more efforts have been invested to develop specific Nbs against food allergen proteins, such as macadamia allergen of Mac i 1, peanut allergen of Ara h 3, and lupine allergen of Lup an 1, which demonstrated the potential of Nbs for research and application in food allergen surveillance. Meanwhile, the paratopes of Nbs preferably targeting the unique epitopes of food allergens can provide more possibilities to serve as blocking antibodies to shield IgE binding epitopes for food allergy immunotherapy. Regardless, the research and application of Nbs in the field of food allergen and allergic reactions are expected to attract dramatic focus and produce promising research outputs.
Subject(s)
Food Hypersensitivity , Single-Domain Antibodies , Allergens , Antibodies, Blocking , Antigens, Plant , Epitopes , Food Hypersensitivity/therapy , Humans , Immunoglobulin E , Immunologic Factors , Immunotherapy/methodsABSTRACT
A colorimetric gold nanoparticles (AuNPs)-based acetylcholinesterase (AChE) assay was designed for the first time to measure the concentration of parathion-methyl (PM) in lake water samples. In this assay, the analyte PM inhibited the hydrolysis of acetylthiocholine (ATCh) by AChE, preventing the formation of thiocholine (TCh) that would otherwise react with the AuNPs catalyst and deactivate the catalyst. Therefore, in the presence of PM, the AuNPs catalyzed the oxidation of the 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric indicator to oxTMB, inducing a visual color change from colorless to blue. However, in the absence of PM, AChE hydrolyzed ATCh to TCh, which then reacted with the AuNPs, preventing the oxidation of TMB to oxTMB and rendering the solution colorless. Therefore, the change in the color of the analyte solution indicated the presence of PM, and the absorbance of the resulting solution was measured by UV-Vis spectroscopy to calculate the concentration of PM after generation of a calibration curve. This method was then employed using the smartphone app Color Picker, which converted the color information from the photos of the solution into digital red (R), green (G), and blue (B) values. The ratio of green (G) to blue (B) (G/B) was then plotted against the corresponding concentration to calculate the standard curve, whose regression equation was expressed by y = -0.012x + 1.02 (ng/mL), and the coefficient of determination (R2) was 0.97. In addition, this method was also used to determine the amount of PM in real lake water samples with recovery of 90.2-133.3%.
Subject(s)
Metal Nanoparticles , Methyl Parathion , Acetylcholinesterase , Colorimetry , GoldABSTRACT
Objectives: To develop a rapid and low-cost method for 16S rDNA nanopore sequencing. Methods: This was a prospective study on a 16S rDNA nanopore sequencing method. We developed this nanopore barcoding 16S sequencing method by adding barcodes to the 16S primer to reduce the reagent cost and simplify the experimental procedure. Twenty-one common pulmonary bacteria (7 reference strains, 14 clinical isolates) and 94 samples of bronchoalveolar lavage fluid from children with severe pneumonia were tested. Results indicating low-abundance pathogenic bacteria were verified with the polymerase chain reaction (PCR). Further, the results were compared with those of culture or PCR. Results: The turnaround time was shortened to 6~8 hours and the reagent cost of DNA preparation was reduced by employing a single reaction adding barcodes to the 16S primer in advance. The accuracy rate for the 21 common pulmonary pathogens with an abundance ≥ 99% was 100%. Applying the culture or PCR results as the gold standard, 71 (75.5%) of the 94 patients were positive, including 25 positive cultures (26.6%) and 52 positive quantitative PCRs (55.3%). The median abundance in the positive culture and qPCR samples were 29.9% and 6.7%, respectively. With an abundance threshold increase of 1%, 5%, 10%, 15% and 20%, the test sensitivity decreased gradually to 98.6%, 84.9%, 72.6%, 67.1% and 64.4%, respectively, and the test specificity increased gradually to 33.3%, 71.4%, 81.0%, 90.5% and 100.0%, respectively. Conclusions: The nanopore barcoding 16S sequencing method can rapidly identify the pathogens causing bacterial pneumonia in children.
Subject(s)
Nanopore Sequencing , Pneumonia, Bacterial , Humans , Child , DNA, Ribosomal/genetics , Prospective Studies , DNA, Bacterial/genetics , DNA, Bacterial/analysis , Bacteria , Pneumonia, Bacterial/diagnosis , RNA, Ribosomal, 16S/geneticsABSTRACT
The population prehistory of Xinjiang has been a hot topic among geneticists, linguists, and archaeologists. Current ancient DNA studies in Xinjiang exclusively suggest an admixture model for the populations in Xinjiang since the early Bronze Age. However, almost all of these studies focused on the northern and eastern parts of Xinjiang; the prehistoric demographic processes that occurred in western Xinjiang have been seldomly reported. By analyzing complete mitochondrial sequences from the Xiabandi (XBD) cemetery (3,500-3,300 BP), the up-to-date earliest cemetery excavated in western Xinjiang, we show that all the XBD mitochondrial sequences fall within two different West Eurasian mitochondrial DNA (mtDNA) pools, indicating that the migrants into western Xinjiang from west Eurasians were a consequence of the early expansion of the middle and late Bronze Age steppe pastoralists (Steppe_MLBA), admixed with the indigenous populations from Central Asia. Our study provides genetic links for an early existence of the Indo-Iranian language in southwestern Xinjiang and suggests that the existence of Andronovo culture in western Xinjiang involved not only the dispersal of ideas but also population movement.
ABSTRACT
The identity of the earliest inhabitants of Xinjiang, in the heart of Inner Asia, and the languages that they spoke have long been debated and remain contentious1. Here we present genomic data from 5 individuals dating to around 3000-2800 BC from the Dzungarian Basin and 13 individuals dating to around 2100-1700 BC from the Tarim Basin, representing the earliest yet discovered human remains from North and South Xinjiang, respectively. We find that the Early Bronze Age Dzungarian individuals exhibit a predominantly Afanasievo ancestry with an additional local contribution, and the Early-Middle Bronze Age Tarim individuals contain only a local ancestry. The Tarim individuals from the site of Xiaohe further exhibit strong evidence of milk proteins in their dental calculus, indicating a reliance on dairy pastoralism at the site since its founding. Our results do not support previous hypotheses for the origin of the Tarim mummies, who were argued to be Proto-Tocharian-speaking pastoralists descended from the Afanasievo1,2 or to have originated among the Bactria-Margiana Archaeological Complex3 or Inner Asian Mountain Corridor cultures4. Instead, although Tocharian may have been plausibly introduced to the Dzungarian Basin by Afanasievo migrants during the Early Bronze Age, we find that the earliest Tarim Basin cultures appear to have arisen from a genetically isolated local population that adopted neighbouring pastoralist and agriculturalist practices, which allowed them to settle and thrive along the shifting riverine oases of the Taklamakan Desert.
Subject(s)
Archaeology , Genome, Human/genetics , Genomics , Human Migration/history , Mummies/history , Phylogeny , Agriculture/history , Animals , Cattle , China , Cultural Characteristics , Dental Calculus/chemistry , Desert Climate , Diet/history , Europe , Female , Goats , Grassland , History, Ancient , Humans , Male , Milk Proteins/analysis , Phylogeography , Principal Component Analysis , Proteome/analysis , Proteomics , Sheep , Whole Genome SequencingABSTRACT
The declaration of lupine supplements is mandatory to avoid lupine allergy for sensitive individuals. However, reliable detection methods against lupine allergen remain critical to prevent the unintended consumption of allergen contaminated food. In this study, we have immunized an alpaca with lupine protein extracts and retrieved nanobodies (Nbs). Nevertheless, the target antigen has been recognized as Lup an 1, which has been classified as ß-conglutin, and confirmed to connect with lupine allergy. After selection of the best Nb-pair, a sandwich enzyme-linked immunosorbent assay (ELISA) has been developed providing a linear range of 0.036-4.4 µg/mL with detection limit of 1.15 ng/mL. This immunoassay was confirmed by detecting the samples with spiked allergen, and a recovery from 86.25% to 108.45% with coefficient of variation (CV) less than 4.0% has been determined. Generally, this study demonstrated the selection of Nbs against allergen with crude protein content to develop the immunoassay for lupine surveillance in foods.
ABSTRACT
Tea polyphenol of epigallocatechin-3-gallate (EGCG) has been verified to possess multiple biological activities. Interleukin-23 (IL-23) is a heterodimeric cytokine consisting of two subunits of IL-23p19 and IL-12p40, with the functionality in regulating the production of cytokines under physiological or pathological conditions. By serendipity, the raised expression of IL-23 was observed after treating cells with EGCG, whereas the detailed mechanism remains poorly understood. This study was proposed to investigate the signaling related to EGCG-induced IL-23. The raised expression of IL-23 was confirmed primarily by intraperitoneally injecting with different concentrations of EGCG (0, 20, 50, 80 mg/kg) into BALB/c mice, and the raised expression was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Results from enzyme-linked immunosorbent assay (ELISA) revealed the increase of IL-23 in serum from 116.09 to 153.90 pg/mL after treating with EGCG. The same results were also observed in RAW264.7 and peritoneal macrophages after treating with EGCG (0, 1, 5, 10, 25 µM) with the increased tendency of IL-23 in cultural medium (7.98 to 25.38 pg/mL for RAW264.7; 3.64 to 260.93 pg/mL for peritoneal macrophages). After preliminary exploration of the signaling related to the increased IL-23, the classical signaling pathways and key transcription factors, such as nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK) signaling pathways, and interferon regulatory factor 5 (IRF5), were demonstrated with no relevant contribution. A further study revealed the involvement of the key transcription factor of BATF2, which could antagonistically modulate the transcription and translation of IL-23. The signaling of STAT3-BATF2-c-JUN/ATF2-IL-23 has been further verified in RAW264.7 macrophages using the STAT3 inhibitor of AG490 and the activator of Colivelin TFA. The results indicated that EGCG inhibits the phosphorylation of STAT3 to facilitate the decreased level of BATF2, which contributed to the increased level of IL-23 by the enhancing heterodimerization of c-JUN and ATF2.
Subject(s)
Catechin , Interleukin-23 , Activating Transcription Factor 2 , Animals , Basic-Leucine Zipper Transcription Factors , Catechin/analogs & derivatives , Interferon Regulatory Factors , Interleukin-23/genetics , Mice , Mice, Inbred BALB C , NF-kappa B/metabolism , Proto-Oncogene Proteins c-jun , STAT3 Transcription FactorABSTRACT
Macadamia nut contains important food allergens that potentially cause allergic reactions with severe adverse effects in infants and adults. Reliable and accurate detection of macadamia is critical to avoid allergic reactions. However, knowledge on macadamia allergen is scarce and a reliable detection method has not been reported, yet. In this study, an unbiased immunization and selection strategy was employed to select nanobodies (Nbs) recognizing specifically macadamia allergen, as well as to establish a detection method to unveil a macadamia protein contamination. An alpaca was immunized with a crude protein extract of macadamia followed by construction of a Nb library from its lymphocytes. The panning and screening of this immune Nb repertoire resulted in the selection of six target-specific Nbs. Nb-mediated immuno-capturing combined with mass spectrometry allowed us to identify the target as the macadamia vicilin-like antimicrobial peptides 2-3 (MiAMP2), a novel food allergenic protein abbreviated as Mac i 1. Later on, an immunoassay of a heterologous sandwich ELISA method based on the selected Nb-pairs was established, providing a linear response in the range of 0.442-2,800 µg/mL and with a limit of detection of 27.1 ng/mL. The dedicated immunoassay has been verified by detecting the antigen spiked in food samples. Our study provided evidence for the successful application of the unprejudiced strategy to retrieve Nbs against a priori undefined macadamia allergen. These target-specific Nbs were used to design a highly reliable and effective immunoassay.
Subject(s)
Macadamia , Single-Domain Antibodies , Adult , Allergens , Humans , Immunization , Immunoassay , Seed Storage ProteinsABSTRACT
The interaction between conventional immunoglobulins (Igs) and the Ig-binding surface proteins of Staphylococcus aureus (S. aureus) have obstructed the development of immuno-assays to detect these bacteria. The current study aimed to select nanobodies (Nbs) recognizing specifically S. aureus and to establish an immuno-assay to uncover S. aureus contaminations in foods. An alpaca was immunized with an inactivated S. aureus strain followed by the construction of a Nb library from which four target-specific Nbs were retrieved. Subsequently, a sandwich ELISA employing the Nb147 and biotinylated-Nb147 pair to capture and to detect S. aureus, respectively, was established to possess a detection limit of 1.4 × 105 colony forming units (CFU)/mL. The dedicated immuno-assay has been verified by detecting 10 CFU/mL of S. aureus in milk samples after an 8 h-enrichment step. This study provides the basis of an easy, reproducible and effective immuno-assay to screen for S. aureus contaminations in foods.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Milk/microbiology , Single-Domain Antibodies/immunology , Staphylococcus aureus/isolation & purification , Animals , Bacterial Proteins/immunology , Food Contamination/analysis , Limit of Detection , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Staphylococcus aureus/metabolismABSTRACT
In this paper, a novel fluorescence assay has been constructed for the determination of parathion-methyl (PM) by using 4-amino-3-hydroxy-1-naphthalenesulfonic acid (AHNSA) as probe. MnO2 nanosheets (MnO2 NS) could quench the fluorescence of AHNSA, while Mn2+, the reduction product of MnO2 NS, has no influence on it, resulting in fluorescence recovery. This is because that MnO2 NS have oxidized characteristic, and they can react with choline (TCh), which is the product of acetylthiocholine (ATCh) catalyzed by acetylcholinesterase (AChE). In the presence of OPs, the activity of AChE was inhibited, accompanied by the restraint of the redox reaction of MnO2 NS, therefore the fluorescence of AHNSA was quenched. Under the optimized experimental conditions, a linear range of PM was determined to be 0.4-40 ng/mL (R2 = 0.997) by the proposed method with the limit of detection for 0.18 ng/mL (S/N = 3). The assay was successfully applied to the determination of PM in lake water, which average recoveries were between 86.5% and 114.4%.