Fluorimetric detection of DNA methylation by cerium oxide nanoparticles for early cancer diagnosis.

In this study, a very sensitive fluorescence nano-biosensor was developed using CeO2 nanoparticles for the rapid detection of DNA methylation. The characteristics of CeO2 nanoparticles were determined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD) spectroscopy, UV-visible spectroscopy, and fluorescence spectroscopy. The CeO2 nanoparticles were reacted with a single-stranded DNA (ssDNA) probe, and then methylated and unmethylated target DNAs hybridized with an ssDNA probe, and the fluorescence emission was measured. Upon adding the target unmethylated and methylated ssDNA, the fluorescence intensity increased in the linear range of concentration from 2 × 10-13 - 10-18 M. The limit of detection (LOD) was 1.597 × 10-6 M for methylated DNA and 1.043 × 10-6 M for unmethylated DNA. The fluorescence emission intensity of methylated sequences was higher than of that unmethylated sequences. The fabricated DNA nanobiosensor showed a fluorescence emission at 420 nm with an excitation wavelength of 280 nm. The impact of CeO2 binding on methylated and unmethylated DNA was further demonstrated by agarose gel electrophoresis. Finally, the actual sample analysis suggested that the nanobiosensor could have practical applications for detecting methylation in the human plasma samples.

Synthesis of Cu3Fe4V6O24 Nanoparticles to Produce 1,2,3-Triazoles by Azide-Alkyne Cycloaddition Reactions.

This paper presents the fabrication of novel Cu3Fe4V6O24 nanoparticles (NPs) via a facile sol-gel method as efficient nanocatalysts (NCs) to produce azide-alkyne 1,3-dipolar cycloaddition compounds. The effect of the calcination time on the formation of NPs was investigated. The as-prepared NPs were characterized using X-ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM), electron-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), and Brunauer-Emmett-Teller (BET) analyses. Cu3Fe4V6O24 NCs were applied to azide-alkyne 1,3-dipolar cycloaddition reactions. The effect of the catalyst loading, temperature, and time of reaction was optimized to improve the efficiency of the NC function by the response surface methodology-central composite design (RSM-CCD) method. In optimal conditions, the yield of the reaction was 96%. In addition, the effect of different solvents on the yield of the reaction was investigated. Moreover, Cu3Fe4V6O24 NPs efficiently catalyze different 1,2,3-triazoles in excellent yields.

Synthesis and characterization of a new copper-based polyoxomolybdate and its catalytic activity for azide-alkyne cycloaddition reaction under UV light irradiation.

A new organic-functionalized Cu-based Anderson-type polyoxomolybdate, namely (C7H15N4)2[Na(H2O)4]2[C6H12CuMo6N2O24]·2(H2O) (CuII-POM), was synthesized via a simple one-pot reaction and subsequently characterized using a range of analytical and spectral techniques. Structural investigation by single crystal X-ray diffraction analysis revealed that the polyanion component of the synthesized compound (i.e. [C6H12CuMo6N2O24]4-) possesses a δ-isomer Anderson-type structure, which is surrounded by four lattice water molecules and four [C7H15N4-NaH15(H2O)8]4+ cations in the crystal packing arrangement. The resulting double-sided tris-functionalized Anderson-type compound can function as highly effective heterogeneous photocatalysts for the copper(I)-catalyzed Huisgen azide-alkyne cycloaddition (Cu-AAC) reaction of terminal alkyne, benzyl halides, and sodium azide (acts as the azidonation and reducing agent) in aqueous media. Ultraviolet light irradiation enhances the catalytic activity of CuII-POM ~ 4.4 times of the "off" situation under reaction conditions of 0.00239 mmol cat., 80 °C, 8 h, 2 mL H2O, So that the isolated yields for the AAC reaction involving a variety of terminal alkynes and benzyl halides using the CuII-POM catalyst ranged between 19-97%. The current study is the first report about using an efficient and economical Cu(II)-POM/UV/NaN3 catalytic system in the Cu-AAC reaction and reveals its significant potential for applying to other Cu(I)-catalyzed reactions.

Stromal localization of inactive CD8+ T cells in metastatic mismatch repair deficient colorectal cancer.

BACKGROUND: The determinants of metastasis in mismatch repair deficiency with high levels of microsatellite instability (MSI-H) in colorectal cancer (CRC) are poorly understood. Here, we hypothesized that distinct immune and stromal microenvironments in primary tumors may discriminate between non-metastatic MSI-H CRC and metastatic MSI-H CRC. METHODS: We profiled 46,727 single cells using high-plex imaging mass cytometry and analyzed both differential cell type abundance, and spatial distribution of fibroblasts and immune cells in primary CRC tumors with or without metastatic capacity. We validated our findings in a second independent cohort using immunohistochemistry. RESULTS: High-plex imaging mass cytometry and hierarchical clustering based on microenvironmental markers separated primary MSI-H CRC tumors with and without metastatic capacity. Primary tumors with metastatic capacity displayed a high stromal content and low influx of CD8+ T cells, which expressed significantly lower levels of markers reflecting proliferation (Ki67) and antigen-experience (CD45RO) compared to CD8+ T cells in non-metastatic tumors. CD8+ T cells showed intra-epithelial localization in non-metastatic tumors, but stromal localization in metastatic tumors, which was validated in a second cohort. CONCLUSION: We conclude that localization of phenotypically distinct CD8+ T cells within stroma may predict metastasis formation in MSI-H CRC.

High-Performance Novel Polyoxometalate-LDH Nanocomposite-Modified Thin-Film Nanocomposite Forward Osmosis Membranes: A Study of Desalination and Antifouling Performance.

Numerous investigations have focused on creating effective membranes for desalination in order to alleviate the water scarcity crisis. In this study, first, LDH nanoplates were synthesized and utilized to alter the surface of thin-film composite (TFC) membranes in the course of this investigation. Following that, a simple technique was used to produce a novel nanocomposite incorporating LDH layers and Na14(P2W18Co4O70)·28H2O polyoxometalate nanoparticles, resulting in the creation of a fresh variety of thin-film nanocomposite (TFN). The performance of all of the membranes acquired was examined in the process of forward osmosis (FO). The impact of the compounds that were prepared was assessed on the hydrophilicity, topology, chemical structure, and morphology of the active layer of polyamide (PA) through analysis methods such as atomic force microscopy (AFM), energy-dispersive X-ray (EDX), FTIR spectroscopy, powder X-ray diffraction (XRD), scanning electron microscopy (SEM), and water contact angle (WCA) goniometry. After evaluating the outcomes of both modified membrane types, it was observed that the membrane equipped with the nanocomposite modifier at a concentration of 0.01 wt % exhibited the highest water flux, measuring 46.6 LMH and selectivity of 0.23 g/L. This membrane was thus considered the best option. Furthermore, the membrane's ability to prevent fouling was examined, and the findings revealed an enhancement in its resistance to fouling in comparison to the filler-free membrane.

Highly multiplexed spatial analysis identifies tissue-resident memory T cells as drivers of ulcerative and immune checkpoint inhibitor colitis.

Colitis is a prevalent adverse event associated with immune checkpoint inhibitor (ICI) therapy with similarities to inflammatory bowel disease. Incomplete mechanistic understanding of ICI colitis curtails evidence-based treatment. Given the often-overlooked connection between tissue architecture and mucosal immune cell function, we here applied imaging mass cytometry (IMC) to gain spatial proteomic insight in ICI colitis in comparison to ulcerative colitis (UC). Using a cell segmentation pipeline that simultaneously utilizes high-resolution nuclear imaging and high-multiplexity IMC, we show that intra-epithelial CD8+ T cells are significantly more abundant (and numerically dominant) in anti-PD-1 ± anti-CTLA-4-induced colitis compared to anti-CTLA-4-induced colitis and UC. We identified activated, cycling CD8+ tissue-resident memory T(RM) cells at the lamina propria-epithelial interface as drivers of cytotoxicity in ICI colitis and UC. Moreover, we found that combined ICI-induced colitis featured highest granzyme B levels both in tissue and serum. Together, these data reinforce CD8+ TRM cells as potentially targetable drivers of ICI colitis.

High-Performance Novel MoS2@Zeolite X Nanocomposite-Modified Thin-Film Nanocomposite Forward Osmosis Membranes: A Study of Desalination and Antifouling Performance.

Novel thin-film nanocomposite (TFN) membranes modified by the MoS2@Zeolite X nanocomposite were made and studied for desalination by the forward osmosis (FO) method. Herein, MoS2@Zeolite X nanocomposite (MoS2@Z) and zeolite X particles are integrated into the polyamide (PA) selective layer of the TFN membranes, separately. The aim of this study is the synthesis of nanocomposites containing hydrophilic zeolite X particles with a modified surface and pore and improvement of their effective properties on desalination and antifouling performance. For this purpose, MoS2 nanosheets with a high hydrophilicity were selected. The existence of polymer-matrix-compatible MoS2@Z inside the PA active layer caused the formation of a defect-free smooth surface with further channels within this layer that could increase the water flux and fouling resistance of the TFN membranes. The TFN-MZ2 membrane (containing 0.01 wt % MoS2@Z) showed the top desalination performance in the FO process. In contrast to the pristine thin-film composite (TFC) and TFN-Z2 membrane (containing 0.025 wt % zeolite X, the most optimal membrane among the zeolite-modified membranes), its water flux has increased by 2.6 and 1.8 times, respectively. Furthermore, in the fouling test, this optimal TFN-MZ2 membrane with a flux decrement of 19.6% revealed an ∼2.2- and 1.8-fold enhancement in antifouling tendency compared to the TFC and TFN-Z2, respectively. Also, based on the antibiofouling test, the water flux drop of 48.6% for the TFC membrane has reached 36.9% for the optimal membrane. Hence, this high-performance TFN-MZ2 membrane shows good capability for commercial employment in FO desalination application.

A new binuclear Ni(II) complex, an effective A3-coupling catalyst in solvent-free condition.

A newly binuclear nickel(II) complex, [Ni2(en)4(ox)](ClO4)2 (1) (where en = ethylenediamine, and ox = oxalate), has been isolated from a reaction of NiCl2·6H2O, ethylenediamine, ammonium oxalate and sodium perchlorate in water and its crystal structure has been determined by X-ray crystallography and infra-red techniques. Compound 1 was successfully employed to promote the one-pot reaction of aldehydes, amines and acetylenes for the construction of corresponding propargylamines under solvent-free media with fine yields. Further studies reveal this catalytic system can be refreshed and used again in five runs.

Chitosan-based Schiff base-metal (Fe, Cu, and Zn) complexes mitigate the negative consequences of drought stress on pomegranate fruits.

Drought is one of the major environmental stresses that impairs fruit productivity and quality. The proper management of minerals can, however, assist plant to maintain their growth even under drought incidents, and is considered one of the encouraging approaches to refine the drought tolerance of plants. The beneficial effects of chitosan (CH)-based Schiff base-metal complexes (e.g., CH-Fe, CH-Cu and CH-Zn) in reducing the harmful impacts of different levels of drought stress on the growth and productivity of 'Malase Saveh' pomegranate cultivar were examined. All CH-metal complexes displayed favorable effects on the yield- and growth-related attributes of pomegranate trees cultivated under well-watered and different drought situations, with the best effects were observed with CH-Fe application. Specifically, leaves of CH-Fe-treated pomegranate plants showed higher concentrations of photosynthetic pigments [chlorophyll a (Chl a), Chl b, Chl a+b, and carotenoids by 28.0, 29.5, 28.6 and 85.7%, respectively] and microelements (Fe by 27.3%), along with increased levels of superoxide dismutase (by 35.3%) and ascorbate peroxidase (by 56.0%) enzymatic activities relative to those of CH-Fe-non-treated pomegranate plants under intense drought stress. CH-Fe-treated drought-stressed pomegranate leaves showed high increment of abscisic acid (by 25.1%) and indole-3-acetic acid (by 40.5%) relative to CH-Fe-non-treated pomegranates. The increased contents of total phenolics, ascorbic acid, total anthocyanins, and titratable acidity (by 24.3, 25.8, 9.3 and 30.9%, respectively) in the fruits of CH-Fe-treated drought-stressed pomegranates indicated the advantageousness of CH-Fe on the enhancement of fruit nutritional qualities. Collectively, our results prove the explicit functions of these complexes, particularly CH-Fe, in the control of drought-induced negative effects on pomegranate trees grown in semi-arid and dry areas.

Comparing the toxicity of tungsten and vanadium oxide nanoparticles on Spirulina platensis.

The production and release of nanoparticles and their impacts on living organisms are among the most important concerns in the world. Spirulina platensis was chosen because of its ability to absorb more elements than other algae. Therefore, an experiment was conducted to improve the product quality of spirulina exposed to new type of nanoparticles. In this experiment, vanadium oxide nanoparticles (VNPs) and tungsten oxide nanoparticles (WNPs) were used at concentrations of 0, 0.001, 0.017, and 0.05 g/l. The measured indices such as protein percentage and concentrations of phycobiliproteins and carbohydrates were the most important parameters of spirulina. Results showed that the concentration of 0.001 g/l of VNPs significantly affected the amounts of protein and phycocyanin. It has also been observed that 0.001 g/l of WNPs significantly influenced the amounts of protein (5.3%) and phycocyanin (90%); however, WNPs at all concentrations increased the concentrations of protein and phycocyanin. A concentration of 0.05 g/l of WNPs increased phycocyanin content by 83% over the control. The examination of nanoparticles by spirulina showed that VNPs were more adsorbed by spirulina than WNPs. In general, VNPs were toxic to algae at concentrations of 0.017 and 0.05 g/l, but WNPs did not show any fatal toxicity.

Synthesis of rod-like CeO2 nanoparticles and their application to catalyze the luminal-O2 chemiluminescence reaction used in the determination of oxcarbazepine and ascorbic acid.

Rod-like CeO2 nanoparticles (NPs) were produced by the quick precipitation approach and employed as a catalyzer to increase the chemiluminescence (CL) intensity of the luminol-O2 reaction. The transmission electron microscopy (TEM) images of the CeO2 NPs showed that rod-like particles with the length and diameter about 15 nm and 5 nm, respectively, were produced. Furthermore, pharmaceuticals including oxcarbazepine (OXP) and ascorbic acid (AA) showed an inhibitory effect against the CL intensity such that the more concentration of the pharmaceuticals, the less was the CL intensity. Therefore, the new CeO2 NPs-luminol-O2 CL reaction was developed to determine OXP and AA in the pharmaceutical formulations. It is the first CL method established for the quantification of OXP. The linear dynamic range of this method for OXP was from 6.0 × 10-7 to 6.0 × 10-5 mol L-1 and for AA from 1.0 × 10-6 to 1.0 × 10-4 mol L-1.

A sensitive colori/fluorimetric nanoprobe for detection of polyphenols using peroxidase-mimic plasma-modified MoO3 nanoparticles.

Herein, MoO3 nanoparticles were synthesized and modified using Argon cold plasma treatment (Ar-MoO3NPs) for the first time. Various characterization studies were performed using various methods, including SEM, XRD, and FTIR techniques. The catalytic activity of MoO3NPs before and after modification was investigated using fluorometric and colorimetric experiments. The results indicated that the enzyme-mimic activity of MoO3NPs increased after plasma-surface modification (1.5 fold). Also, a fluorometric method based on the oxidation of a non-fluorescent terephthalic acid by Ar-MoO3NPs in the presence of H2O2 and the production of a compound with a high emission was designed for polyphenols detection. Quercetin was used as a polyphenol standard for the optimization of the proposed system. Under the optimum conditions, the dynamic ranges of the calibration graphs and the detection limits were calculated for different polyphenols (µmol/L): quercetin (2-232, 12.22), resveratrol (2-270, 61.89), curcumin (39-400, 38.89), gallic acid (2-309, 21.5) and ellagic acid (39-309, 16.25). Also, the precision of the method, which was expressed as RSD%, was in the range of 0.286-1.19%. The proposed system could detect individual polyphenols and total polyphenols in three different fruit extracts (apple, orange, and grapes) with high sensitivity. The obtained total concentrations of polyphenols in real samples were comparable to those calculated by the spectrophotometric method. So, a novel and sensitive optical nanosensor for the detection of polyphenols was reported as an alternative to the routine Folin-Ciocalteu spectrophotometric technique.

MATISSE: An analysis protocol for combining imaging mass cytometry with fluorescence microscopy to generate single-cell data.

Exploring tissue heterogeneity on a single-cell level by imaging mass cytometry (IMC) remains challenging because of its limiting resolution. We previously demonstrated that combining higher resolution fluorescence with IMC data in the analysis pipeline resulted in high-quality single-cell segmentation. Here, we provide a step-by-step workflow of this MATISSE pipeline, including instructions regarding the staining procedure, and the analysis route to generate single-cell data. For complete details on the use and execution of this protocol, please refer to Baars et al., 2021.

Dual enzymes-mimic activity of nanolayered manganese-calcium oxide for fluorometric determination of metformin.

There are different analytical methods available for the determination of metformin, as an oral hypoglycemic and antidiabetic drug, in biological samples. However, most of these methods suffer from some drawbacks, including high-priced materials and equipment, damaging chemical reagents, time-consuming nature, and tedious operation procedures. So, in this work a new, sensitive and simple method was reported for the detection of metformine. In this regard, nanolayered manganese-calcium oxide (NL-MnCaO2) were synthesized and characterized using scanning electron microscopy (SEM), fourier transform infrared (FTIR) spectroscopy, and X-ray powder diffraction (XRD) techniques. Also, we studied the enzyme-like activity of synthesized particles and reported a bifunctional nanozyme, which performs the dual roles for peroxidase and catalase-mimicking. The results demonstrated the hindering effect of metformin on the peroxidase-mimic activity of NL-MnCaO2 and this effect was increased by raising metformin concentration. So, a sensitive fluorometric detection system was designed for the analytical assay of metformin, based on the terephthalic acid (TA)-H2O2 reaction with NL-MnCaO2. An acceptable linearity was observed between the metformin concentration and fluorescence quenching of the system in the range of 0.07-0.77 mM. Limit of detection (LOD) and limit of quantification (LOQ) were 0.17 µM and 0.96 µM, respectively. The proposed system was applied for the estimation of metformin concentration in serum samples by recoveries of 86.68-106%. So, the proposed fluorometric method provides some main advantages such as wide linear range, low detection limit, rapid detections, high sensitivity, and good practicability for the determination of metformin in biological samples.

Protective effects of cerium oxide nanoparticles in grapevine (Vitis vinifera L.) cv. Flame Seedless under salt stress conditions.

High levels of soil salinity can cause substantial decline in growth and productivity of crops worldwide, thus representing a major threat to global agriculture. In recent years, engineered nanoparticles (NPs) have been deemed as a promising alternative in combating abiotic stress factors, such as salinity. In this context, the present study was designed to explore the potential of cerium oxide nanoparticles (CeO2NPs) in alleviating salt stress in grapevine (Vitis vinifera L. cv. Flame Seedless) cuttings. Specifically, the interaction between CeO2 NPs (25, 50 and 100 mg L-1) and salinity (25 and 75 mM NaCl) was evaluated by assaying an array of agronomic, physiological, analytical and biochemical parameters. Treatments with CeO2 NPs, in general, alleviated the adverse impacts of salt stress (75 mM NaCl) significantly improving relevant agronomic traits of grapevine. CeO2 NPs significantly ameliorated chlorophyll damage under high levels of salinity. Furthermore, the presence of CeO2 NPs attenuated salinity-induced damages in grapevine as indicated by lower levels of proline, MDA and EL; however, H2O2 content was not ameliorated by the presence of CeO2 NPs under salt stress. Additionally, salinity caused substantial increases in enzymatic activities of GP, APX and SOD, compared with control plants. Similar to stress conditions, all concentrations of CeO2 NPs triggered APX activity, while the highest concentration of CeO2 NPs significantly increased GP activity. However, CeO2 NPs did not significantly modify SOD activity. Considering mineral nutrient profile, salinity increased Na and Cl content as well as Na/K ratio, while it decreased K, P and Ca contents. Nevertheless, the presence of CeO2 NPs did not lead to significant alterations in Na, K and P content of salt-stressed plants. Taken together, current findings suggest that CeO2 NPs could be employed as promising salt-stress alleviating agents in grapevine.

MATISSE: a method for improved single cell segmentation in imaging mass cytometry.

BACKGROUND: Visualizing and quantifying cellular heterogeneity is of central importance to study tissue complexity, development, and physiology and has a vital role in understanding pathologies. Mass spectrometry-based methods including imaging mass cytometry (IMC) have in recent years emerged as powerful approaches for assessing cellular heterogeneity in tissues. IMC is an innovative multiplex imaging method that combines imaging using up to 40 metal conjugated antibodies and provides distributions of protein markers in tissues with a resolution of 1 µm2 area. However, resolving the output signals of individual cells within the tissue sample, i.e., single cell segmentation, remains challenging. To address this problem, we developed MATISSE (iMaging mAss cyTometry mIcroscopy Single cell SegmEntation), a method that combines high-resolution fluorescence microscopy with the multiplex capability of IMC into a single workflow to achieve improved segmentation over the current state-of-the-art. RESULTS: MATISSE results in improved quality and quantity of segmented cells when compared to IMC-only segmentation in sections of heterogeneous tissues. Additionally, MATISSE enables more complete and accurate identification of epithelial cells, fibroblasts, and infiltrating immune cells in densely packed cellular areas in tissue sections. MATISSE has been designed based on commonly used open-access tools and regular fluorescence microscopy, allowing easy implementation by labs using multiplex IMC into their analysis methods. CONCLUSION: MATISSE allows segmentation of densely packed cellular areas and provides a qualitative and quantitative improvement when compared to IMC-based segmentation. We expect that implementing MATISSE into tissue section analysis pipelines will yield improved cell segmentation and enable more accurate analysis of the tissue microenvironment in epithelial tissue pathologies, such as autoimmunity and cancer.

Ultra-small and highly dispersive iron oxide hydroxide as an efficient catalyst for oxidation reactions: a Swiss-army-knife catalyst.

Ultra-small and highly dispersive (< 10 nm) iron oxide hydroxide is characterized by some methods. The compound is an efficient and stable catalyst for alcohol oxidation, organic sulfide oxidation, and epoxidation of alkenes in the presence of H2O2. The electrochemical oxygen-evolution reaction of the iron oxide hydroxide is also tested under acidic, neutral, and alkaline conditions. In the presence of the iron oxide hydroxide, excellent conversions (75-100%) and selectivities of substrates (92-97%), depending on the nature of the sulfide, were obtained. Benzylalcohols having electron-donating and-withdrawing substituents in the aromatic ring were oxidized to produce the corresponding aldehydes with excellent conversion (65-89%) and selectivity (96-100%) using this iron oxide hydroxide. The conversion of styrene and cyclooctene toward the epoxidation in the presence of this catalyst are 60 and 53%, respectively. Water oxidation for the catalysts was investigated at pH 2, 6.7, 12, and 14. The onset of OER at pH 14 is observed with a 475 mV overpotential. At 585 mV overpotential, a current density of more than 0.18 mA/cm2 and a turnover frequency of 1.5/h is observed. Operando high-resolution visible spectroscopy at pH 14, similar to previously reported investigations, shows that Fe(IV)=O is an intermediate for water oxidation.

Dysregulated RASGRP1 expression through RUNX1 mediated transcription promotes autoimmunity.

RasGRP1 is a Ras guanine nucleotide exchange factor, and an essential regulator of lymphocyte receptor signaling. In mice, Rasgrp1 deletion results in defective T lymphocyte development. RASGRP1-deficient patients suffer from immune deficiency, and the RASGRP1 gene has been linked to autoimmunity. However, how RasGRP1 levels are regulated, and if RasGRP1 dosage alterations contribute to autoimmunity remains unknown. We demonstrate that diminished Rasgrp1 expression caused defective T lymphocyte selection in C57BL/6 mice, and that the severity of inflammatory disease inversely correlates with Rasgrp1 expression levels. In patients with autoimmunity, active inflammation correlated with decreased RASGRP1 levels in CD4+ T cells. By analyzing H3K27 acetylation profiles in human T cells, we identified a RASGRP1 enhancer that harbors autoimmunity-associated SNPs. CRISPR-Cas9 disruption of this enhancer caused lower RasGRP1 expression, and decreased binding of RUNX1 and CBFB transcription factors. Analyzing patients with autoimmunity, we detected reduced RUNX1 expression in CD4+ T cells. Lastly, we mechanistically link RUNX1 to transcriptional regulation of RASGRP1 to reveal a key circuit regulating RasGRP1 expression, which is vital to prevent inflammatory disease.