Insight into the Stabilization Mechanism of Succinylation Modification on Black Bean Protein Gels: Molecular Conformation, Microstructure, and Gel Properties.

The objective of this work was to investigate the effect of succinylation treatment on the physicochemical properties of black bean proteins (BBPI), and the relationship mechanism between BBPI structure and gel properties was further analyzed. The results demonstrated that the covalent formation of higher-molecular-weight complexes with BBPI could be achieved by succinic anhydride (SA). With the addition of SA at 10% (v/v), the acylation of proteins amounted to 92.53 ± 1.10%, at which point there was a minimized particle size of the system (300.90 ± 9.57 nm). Meanwhile, the protein structure was stretched with an irregular curl content of 34.30% and the greatest processable flexibility (0.381 ± 0.004). The dense three-dimensional mesh structure of the hydrogel as revealed by scanning electron microscopy was the fundamental prerequisite for the ability to resist external extrusion. The thermally induced hydrogels of acylated proteins with 10% (v/v) addition of SA showed excellent gel elastic behavior (1.44 ± 0.002 nm) and support capacity. Correlation analysis showed that the hydrogel strength and stability of hydrogels were closely related to the changes in protein conformation. This study provides theoretical guidance for the discovery of flexible proteins and their application in hydrogels.

Efficient removal of p-nitrophenol from water by imidazolium ionic liquids functionalized cellulose microsphere.

Removing p-nitrophenol (PNP) from water resources is crucial due to its significant threat to the environment and human health. Herein, imidazolium ionic liquids with short/long alkyl chain ([C2VIm]Br and [C8VIm]Br) modified cellulose microspheres (MCC-[C2VIm]Br and MCC-[C8VIm]Br) were synthesized by radiation method. To examine the impact of adsorbent hydrophilicity on adsorption performance, batch and column experiments were conducted for PNP adsorption. The MCC-[C2VIm]Br and MCC-[C8VIm]Br, with an equivalent molar import amount of ionic liquids, exhibited maximum adsorption capacities of 190.84 mg/g and 191.20 mg/g for PNP, respectively, and the adsorption equilibrium was reached within 30 min. Both adsorbents displayed exceptional reusability. Integrating the findings from XPS and FTIR analyses, and AgNO3 identification, the suggested adsorption mechanism posited that the adsorbents engaged with PNP through ion exchange, hydrogen bonds and π-π stacking. Remarkably, the hydrophobic MCC-[C8VIm]Br exhibited superior selectivity for PNP than the hydrophilic MCC-[C2VIm]Br, while had little effect on adsorption capacity and rate. MCC-[C8VIm]Br-2 with high grafting yield increased the adsorption capacity to 327.87 mg/g. Moreover, MCC-[C8VIm]Br-2 demonstrated efficient PNP removal from various real water samples, and column experiments illustrated its selective capture of PNP from groundwater. The promising adsorption performance indicates that MCC-[C8VIm]Br-2 holds potential for PNP removal from wastewater.

Gut microbial metabolism is linked to variations in circulating non-high density lipoprotein cholesterol.

BACKGROUND: Non-high-density lipoprotein cholesterol (non-HDL-c) was a strong risk factor for incident cardiovascular diseases and proved to be a better target of lipid-lowering therapies. Recently, gut microbiota has been implicated in the regulation of host metabolism. However, its causal role in the variation of non-HDL-c remains unclear. METHODS: Microbial species and metabolic capacities were assessed with fecal metagenomics, and their associations with non-HDL-c were evaluated by Spearman correlation, followed by LASSO and linear regression adjusted for established cardiovascular risk factors. Moreover, integrative analysis with plasma metabolomics were performed to determine the key molecules linking microbial metabolism and variation of non-HDL-c. Furthermore, bi-directional mendelian randomization analysis was performed to determine the potential causal associations of selected species and metabolites with non-HDL-c. FINDINGS: Decreased Eubacterium rectale but increased Clostridium sp CAG_299 were causally linked to a higher level of non-HDL-c. A total of 16 microbial capacities were found to be independently associated with non-HDL-c after correcting for age, sex, demographics, lifestyles and comorbidities, with the strongest association observed for tricarboxylic acid (TCA) cycle. Furthermore, decreased 3-indolepropionic acid and N-methyltryptamine, resulting from suppressed capacities for microbial reductive TCA cycle, functioned as major microbial effectors to the elevation of circulating non-HDL-c. INTERPRETATION: Overall, our findings provided insight into the causal effects of gut microbes on non-HDL-c and uncovered a novel link between non-HDL-c and microbial metabolism, highlighting the possibility of regulating non-HDL-c by microbiota-modifying interventions. FUNDING: A full list of funding bodies can be found in the Sources of funding section.

The lethal K18-hACE2 knock-in mouse model mimicking the severe pneumonia of COVID-19 is practicable for antiviral development.

Animal models of COVID-19 facilitate the development of vaccines and antivirals against SARS-CoV-2. The efficacy of antivirals or vaccines may differ in different animal models with varied degrees of disease. Here, we introduce a mouse model expressing human angiotensin-converting enzyme 2 (ACE2). In this model, ACE2 with the human cytokeratin 18 promoter was knocked into the Hipp11 locus of C57BL/6J mouse by CRISPR - Cas9 (K18-hACE2 KI). Upon intranasal inoculation with high (3 × 105 PFU) or low (2.5 × 102 PFU) dose of SARS-CoV-2 wildtype (WT), Delta, Omicron BA.1, or Omicron BA.2 variants, all mice showed obvious infection symptoms, including weight loss, high viral loads in the lung, and interstitial pneumonia. 100% lethality was observed in K18-hACE2 KI mice infected by variants with a delay of endpoint for Delta and BA.1, and a significantly attenuated pathogenicity was observed for BA.2. The pneumonia of infected mice was accompanied by the infiltration of neutrophils and pulmonary fibrosis in the lung. Compared with K18-hACE2 Tg mice and HFH4-hACE2 Tg mice, K18-hACE2 KI mice are more susceptible to SARS-CoV-2. In the antivirals test, REGN10933 and Remdesivir had limited antiviral efficacies in K18-hACE2 KI mice upon the challenge of SARS-CoV-2 infections, while Nirmatrelvir, monoclonal antibody 4G4, and mRNA vaccines potently protected the mice from death. Our results suggest that the K18-hACE2 KI mouse model is lethal and stable for SARS-CoV-2 infection, and is practicable and stringent to antiviral development.

Highly Reactive Graphene Dispersant and Its Effective Reinforcement for Phase Change Coatings.

High efficient dispersant that meanwhile possesses additional functions is highly desirable for the fabrication of graphene-based composite. In this paper, a new reactive dispersant, multi-silanols grafted naphthalenediamine (MSiND), is synthesized, which shows superiority compared with conventional dispersants. It can not only stabilize graphene in water at a high concentration of up to 16 mg mL-1 , but also simultaneously be applicable for ethanol medium, in which the graphene concentration can be as high as 12 mg mL-1 at the weight ratio of 1:1 (MSiND:graphene). The dispersion is compatible with multi-matrixes and affinity to various substrates. In addition, MSiND exhibits excellent reactivity due to the existence of high-density silanol groups. Tough graphene coatings are constructed on glass slides and non-woven fabric simply by direct painting and dip-coating. Moreover, with the assistance of MSiND, graphene-doped phase-change coatings on hydrophobic non-woven fabric (e.g., functional mask) are prepared via the spray method. The composite coatings show enhanced mechanical strength and excellent energy storage performance, exhibiting great potential in heat preservation and thermotherapy.

Alistipes indistinctus-derived hippuric acid promotes intestinal urate excretion to alleviate hyperuricemia.

Hyperuricemia induces inflammatory arthritis and accelerates the progression of renal and cardiovascular diseases. Gut microbiota has been linked to the development of hyperuricemia through unclear mechanisms. Here, we show that the abundance and centrality of Alistipes indistinctus are depleted in subjects with hyperuricemia. Integrative metagenomic and metabolomic analysis identified hippuric acid as the key microbial effector that mediates the uric-acid-lowering effect of A. indistinctus. Mechanistically, A. indistinctus-derived hippuric acid enhances the binding of peroxisome-proliferator-activated receptor γ (PPARγ) to the promoter of ATP-binding cassette subfamily G member 2 (ABCG2), which in turn boosts intestinal urate excretion. To facilitate this enhanced excretion, hippuric acid also promotes ABCG2 localization to the brush border membranes in a PDZ-domain-containing 1 (PDZK1)-dependent manner. These findings indicate that A. indistinctus and hippuric acid promote intestinal urate excretion and offer insights into microbiota-host crosstalk in the maintenance of uric acid homeostasis.

An Environment-Tolerant Ion-Conducting Double-Network Composite Hydrogel for High-Performance Flexible Electronic Devices.

High-performance ion-conducting hydrogels (ICHs) are vital for developing flexible electronic devices. However, the robustness and ion-conducting behavior of ICHs deteriorate at extreme temperatures, hampering their use in soft electronics. To resolve these issues, a method involving freeze-thawing and ionizing radiation technology is reported herein for synthesizing a novel double-network (DN) ICH based on a poly(ionic liquid)/MXene/poly(vinyl alcohol) (PMP DN ICH) system. The well-designed ICH exhibits outstanding ionic conductivity (63.89 mS cm-1 at 25 °C), excellent temperature resistance (- 60-80 °C), prolonged stability (30 d at ambient temperature), high oxidation resistance, remarkable antibacterial activity, decent mechanical performance, and adhesion. Additionally, the ICH performs effectively in a flexible wireless strain sensor, thermal sensor, all-solid-state supercapacitor, and single-electrode triboelectric nanogenerator, thereby highlighting its viability in constructing soft electronic devices. The highly integrated gel structure endows these flexible electronic devices with stable, reliable signal output performance. In particular, the all-solid-state supercapacitor containing the PMP DN ICH electrolyte exhibits a high areal specific capacitance of 253.38 mF cm-2 (current density, 1 mA cm-2) and excellent environmental adaptability. This study paves the way for the design and fabrication of high-performance multifunctional/flexible ICHs for wearable sensing, energy-storage, and energy-harvesting applications.

Progressive acceleration in the genetic algorithm by intragenerational mutation.

The intragenerational mutation of the genetic algorithm (IMGA) is proposed to actively broaden the searching space during the optimization process. The searching space is aggressively increased by expanding the variation of mutation rates of all individuals within each generation, leading to the reduction of the required number of iterations, improving the convergence speed and the enhancement factor.

microRNAs: Key Regulators in Plant Responses to Abiotic and Biotic Stresses via Endogenous and Cross-Kingdom Mechanisms.

Dramatic shifts in global climate have intensified abiotic and biotic stress faced by plants. Plant microRNAs (miRNAs)-20-24 nucleotide non-coding RNA molecules-form a key regulatory system of plant gene expression; playing crucial roles in plant growth; development; and defense against abiotic and biotic stress. Moreover, they participate in cross-kingdom communication. This communication encompasses interactions with other plants, microorganisms, and insect species, collectively exerting a profound influence on the agronomic traits of crops. This article comprehensively reviews the biosynthesis of plant miRNAs and explores their impact on plant growth, development, and stress resistance through endogenous, non-transboundary mechanisms. Furthermore, this review delves into the cross-kingdom regulatory effects of plant miRNAs on plants, microorganisms, and pests. It proceeds to specifically discuss the design and modification strategies for artificial miRNAs (amiRNAs), as well as the protection and transport of miRNAs by exosome-like nanovesicles (ELNVs), expanding the potential applications of plant miRNAs in crop breeding. Finally, the current limitations associated with harnessing plant miRNAs are addressed, and the utilization of synthetic biology is proposed to facilitate the heterologous expression and large-scale production of miRNAs. This novel approach suggests a plant-based solution to address future biosafety concerns in agriculture.

Plant-derived strategies to fight against severe acute respiratory syndrome coronavirus 2.

The coronavirus disease 2019 (COVID-19) pandemic has caused an unprecedented crisis, which has been exacerbated because specific drugs and treatments have not yet been developed. In the post-pandemic era, humans and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will remain in equilibrium for a long time. Therefore, we still need to be vigilant against mutated SARS-CoV-2 variants and other emerging human viruses. Plant-derived products are increasingly important in the fight against the pandemic, but a comprehensive review is lacking. This review describes plant-based strategies centered on key biological processes, such as SARS-CoV-2 transmission, entry, replication, and immune interference. We highlight the mechanisms and effects of these plant-derived products and their feasibility and limitations for the treatment and prevention of COVID-19. The development of emerging technologies is driving plants to become production platforms for various antiviral products, improving their medicinal potential. We believe that plant-based strategies will be an important part of the solutions for future pandemics.

High Plant Protein Diet Ameliorated Hepatic Lipid Accumulation Through the Modulation of Gut Microbiota.

SCOPE: Substituting plant protein for animal protein has emerged as a promising strategy for managing atherogenic lipids. However, the impact of long-term intake of a high plant protein diet (HPD) on hepatic lipid disorder remains unclear. METHODS AND RESULTS: Eight-week-old apolipoprotein E deficient (apoE-/- ) mice are fed with either a normal protein diet (NCD) or HPD for 12 weeks. HPD intervention results in decreased body weight accompanied by increased energy expenditure, with no significant effect on glycemic control. Long-term intake of HPD improves the serum and hepatic lipid and cholesterol accumulation by suppressing hepatic squalene epoxidase (SQLE) expression, a key enzyme in cholesterol biosynthesis. Integrated analysis of 16S rDNA sequencing and metabolomics profiling reveals that HPD intervention increases the abundance of the Lachnospiraece family and serum levels of 12,13-DiHOME. Furthermore, in vivo studies demonstrate that 12,13-DiHOME significantly inhibits lipid accumulation, as well as SQLE expression induced by oleic acid in HepG2 cells. CONCLUSION: Diet rich in plant protein diet alleviates hyperlipidemia via increased microbial production of 12,13-DiHOME.

Efficient and selective capture of Au(III) from PCBs by pentaethylenehexamine-modified chloromethylated polystyrene beads.

Recycling of gold promotes solving the problems of resource waste and environmental pollution. In this work, pentaethylenehexamine (PEHA)-modified chloromethylated polystyrene beads (PEHA-CMPS) was synthesized for the recovery of Au(III) from actual printed circuits boards (PCBs) leaching solution. PEHA-CMPS exhibited excellent adsorption efficiency at a wide pH range. It was discovered that the pseudo-second-order and Langmuir model provided a superior match for the Au(III) adsorption process. The maximum adsorption capacity for Au(III) was 1186 mg/g. Furthermore, PEHA-CMPS was able to selectively capture trace Au(III) with recovery efficiencies of above 80% from the actual PCBs leaching solution. In addition, the column separation approach was utilized to better assess the practical applications for PEHA-CMPS, proving that the prepared adsorbent exhibited great prospects in industrial applications. The adsorption efficiency still maintained 95% after five adsorption-desorption cycles. The FTIR, XRD, and XPS analyses demonstrated that Au(III) uptake on PEHA-CMPS was a collaborative process involving electrostatic interaction, chelation, and oxidation-reduction. The PEHA-CMPS provided a promising strategy in Au(III) recovery and environmental remediation.

Selective recovery of Re(VII) by nucleobases functionalized cellulose microspheres from the simulated uranium ore leaching solution.

From a practical standpoint, it is still challenging to develop adsorbents with high adsorption capacity and outstanding selectivity for rhenium in uranium ore leaching solution. In this study, in order to explore the structure-property relationship, four nucleobases (Adenine, Guanine, Hypoxanthine and Xanthine) were used as functionalization reagents to modify cellulose (MCC-g-GMA-A, MCC-g-GMA-G, MCC-g-GMA-H and MCC-g-GMA-X) via radiation method. The effect of the type of nucleobases on the adsorption performance was evaluated by batch and dynamic experiments. The order of maximum adsorption capacity was MCC-g-GMA-A (194.0 mg g-1) > MCC-g-GMA-G (123.4 mg g-1) > MCC-g-GMA-H (45.59 mg g-1) > MCC-g-GMA-X (23.43 mg g-1), which was associated with the category of nitrogen-functional groups. Different nitrogen-containing functional groups have different degrees of protonation, which leads to differences in the interaction of the adsorbent with Re(VII). Notably, the adsorbents were able to selectively capture trace Re(VII) from the simulated uranium ore leaching solution. The FT-IR, XPS analyses, DFT theoretical calculations exhibited that the adsorption mechanism of nucleobases functionalized cellulose microspheres and Re(VII) was electrostatic interaction. MCC-g-GMA-A and MCC-g-GMA-G exhibited excellent selectivity towards Re(VII), which are potential adsorbents for Re(VII) recovery in uranium ore leaching solutions.

Cardiometabolic benefits of Lacticaseibacillus paracasei 8700:2: A randomized double-blind placebo-controlled trial.

BACKGROUND & AIMS: Modulating microbial metabolism via probiotic supplementation has been proposed as an attractive strategy for the prevention of cardiometabolic diseases. Recently, Lacticaseibacillus paracasei (L. paracasei) was reported to alleviate metabolic disorders in murine models, however, its beneficial effects in humans remain to be determined. This study evaluated whether L. paracasei supplementation could improve endothelial function and cardiometabolic health in subjects with metabolic syndrome (MetS). METHODS: In this randomized, double-blind and placebo-controlled trial among 130 participants with MetS, subjects were randomly assigned to placebo or L. paracasei 8700: 2 (10 billion CFU) daily for 12 weeks. Endothelial function was measured by flow-mediated slowing, and cardiometabolic health was determined by both components and severity of MetS. Ideal compliance was defined as consumption no less than 70% of the capsules. RESULTS: 130 individuals (mean [SD] age, 45.97 [7.11] years; 95 men [73.1%]) were enrolled and randomized to L. paracasei (n = 66) or placebo control (n = 64). Compared to placebo, L. paracasei supplementation led to a greater reduction in remnant cholesterol (-0.16 mmol/L, 95%CI: -0.29 mmol/L to -0.02 mmol/L; P = 0.024). Such a reduction in remnant cholesterol was significantly associated with improvement in endothelial function (r = -0.23, P = 0.027). In subjects with an ideal compliance with trial protocol, L. paracasei treatment additionally lowered triglycerides, alleviated MetS severity and delayed weight gain. On the contrary, no obvious effect on insulin sensitivity or pancreatic beta-cell function was observed after L. paracasei intervention. Moreover, regarding safety and tolerability, no significant between-group difference in protocol-specified adverse events of interest was observed. CONCLUSIONS: L. paracasei supplementation enhanced endothelial function potentially through downregulating remnant cholesterol levels. Our study provides a feasible and safe strategy for the prevention of cardiometabolic diseases in subjects with severe dyslipidemia and endothelial dysfunction. REGISTERED: Under ClinicalTrails.gov identifier NCT05005754.

HDAC3 Knockdown Dysregulates Juvenile Hormone and Apoptosis-Related Genes in Helicoverpa armigera.

Insect development requires genes to be expressed in strict spatiotemporal order. The dynamic regulation of genes involved in insect development is partly orchestrated by the histone acetylation-deacetylation via histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although histone deacetylase 3 (HDAC3) is required for mice during early embryonic development, its functions in Helicoverpa armigera (H. armigera) and its potential to be used as a target of insecticides remain unclear. We treated H. armigera with HDAC3 siRNA and RGFP966, a specific inhibitor, examining how the HDAC3 loss-of-function affects growth and development. HDAC3 siRNA and RGFP966 treatment increased mortality at each growth stage and altered metamorphosis, hampering pupation and causing abnormal wing development, reduced egg production, and reduced hatching rate. We believe that the misregulation of key hormone-related genes leads to abnormal pupa development in HDAC3 knockout insects. RNA-seq analysis identified 2788 differentially expressed genes (≥two-fold change; p ≤ 0.05) between siHDAC3- and siNC-treated larvae. Krüppel homolog 1 (Kr-h1), was differentially expressed in HDAC3 knockdown larvae. Pathway-enrichment analysis revealed the significant enrichment of genes involved in the Hippo, MAPK, and Wnt signaling pathways following HDAC3 knockdown. Histone H3K9 acetylation was increased in H. armigera after siHDAC3 treatment. In conclusion, HDAC3 knockdown dysregulated juvenile hormone (JH)-related and apoptosis-related genes in H. armigera. The results showed that the HDAC3 gene is a potential target for fighting H. armigera.

Gossypol Broadly Inhibits Coronaviruses by Targeting RNA-Dependent RNA Polymerases.

Outbreaks of coronaviruses (CoVs), especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have posed serious threats to humans and animals, which urgently calls for effective broad-spectrum antivirals. RNA-dependent RNA polymerase (RdRp) plays an essential role in viral RNA synthesis and is an ideal pan-coronaviral therapeutic target. Herein, based on cryo-electron microscopy and biochemical approaches, gossypol (GOS) is identified from 881 natural products to directly block SARS-CoV-2 RdRp, thus inhibiting SARS-CoV-2 replication in both cellular and mouse infection models. GOS also acts as a potent inhibitor against the SARS-CoV-2 variant of concern (VOC) and exerts same inhibitory effects toward mutated RdRps of VOCs as the RdRp of the original SARS-CoV-2. Moreover, that the RdRp inhibitor GOS has broad-spectrum anti-coronavirus activity against alphacoronaviruses (porcine epidemic diarrhea virus and swine acute diarrhea syndrome coronavirus), betacoronaviruses (SARS-CoV-2), gammacoronaviruses (avian infectious bronchitis virus), and deltacoronaviruses (porcine deltacoronavirus) is showed. The findings demonstrate that GOS may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and other coronavirus outbreaks.

Cotton flower metabolites inhibit SARS-CoV-2 main protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading globally for over 2 years, causing serious contagious disease and incalculable damage. The introduction of vaccines has slowed the spread of SARS-CoV-2 to some extent, but there remains a need for specific and effective treatment. The high chemical diversity and safety profiles of natural products make them a potential source of effective anti-SARS-CoV-2 drugs. Cotton plant is one of the most important economic and medical crops and is the source of a large number of antiviral phytochemicals. In this work, we used SARS-CoV-2 main protein (Mpro ) as the target to identify potential anti-SARS-CoV-2 natural products in cotton. An in vitro assay showed that of all cotton tissues examined, cotton flower extracts (CFs) exhibited optimal inhibitory effects against Mpro . We proceeded to use the CF metabolite database to screen natural Mpro inhibitors by combining virtual screening and biochemical assays. We identified that several CF natural products, including astragalin, myricitrin, and astilbin, significantly inhibited Mpro with half-maximal inhibitory concentrations (IC50s) of 0.13, 10.73, and 7.92 µm, respectively. These findings may serve as a basis for further studies into the suitability of cotton as a source of potential therapeutics for SARS-CoV-2.

A Broad Antiviral Strategy: Inhibitors of Human DHODH Pave the Way for Host-Targeting Antivirals against Emerging and Re-Emerging Viruses.

New strategies to rapidly develop broad-spectrum antiviral therapies are urgently required for emerging and re-emerging viruses. Host-targeting antivirals (HTAs) that target the universal host factors necessary for viral replication are the most promising approach, with broad-spectrum, foresighted function, and low resistance. We and others recently identified that host dihydroorotate dehydrogenase (DHODH) is one of the universal host factors essential for the replication of many acute-infectious viruses. DHODH is a rate-limiting enzyme catalyzing the fourth step in de novo pyrimidine synthesis. Therefore, it has also been developed as a therapeutic target for many diseases relying on cellular pyrimidine resources, such as cancers, autoimmune diseases, and viral or bacterial infections. Significantly, the successful use of DHODH inhibitors (DHODHi) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection further supports the application prospects. This review focuses on the advantages of HTAs and the antiviral effects of DHODHi with clinical applications. The multiple functions of DHODHi in inhibiting viral replication, stimulating ISGs expression, and suppressing cytokine storms make DHODHi a potent strategy against viral infection.

Featuring few essential Raman spectroscopic signatures between heterogeneous cells.

Here we demonstrate it is instructive to quantify cell Raman spectroscopy by sparse regularization. To be able to extract the specific spectral differences in a heterogeneous cell system with great spectroscopic similarities derived from many common biomolecular components, the maximum information entropy probability was proposed and exemplified by identifying normal lymphocytes from leukemia cells. The essential spectroscopic features were observed to locate at three Raman peaks whose spectral signatures were commensurate. The applicability of the extracted features was acknowledged by that the predicted identification accuracy of up to 93% was still achieved when only two peaks were loaded into decision tree model, which may provide the possibility of a clinically rapid hematological malignancy detection.