Dual Affinity Research Articles

Design, Synthesis, and Cytotoxic Assessment of New Haloperidol Analogues as Potential Anticancer Compounds Targeting Sigma Receptors.

Sigma receptors (SRs), including SR1 and SR2 subtypes, have attracted increasing interest in recent years due to their involvement in a wide range of activities, including the modulation of opioid analgesia, neuroprotection, and potential anticancer activity. In this context, haloperidol (HAL), a commonly used antipsychotic drug, also possesses SR activity and cytotoxic effects. Herein, we describe the identification of novel SR ligands, obtained by a chemical hybridization approach. There wereendowed with pan-affinity for both SR subtypes and evaluated their potential anticancer activity against SH-SY5Y and HUH-7 cancer cell lines. Through a chemical hybridization approach, we identified novel compounds (4d, 4e, 4g, and 4j) with dual affinity for SR1 and SR2 receptors. These compounds were subjected to cytotoxicity testing using a resazurin assay. The results revealed potent cytotoxic effects against both cancer cell lines, with IC50 values comparable to HAL. Interestingly, the cytotoxic potency of the novel compounds resembled that of the SR1 antagonist HAL rather than the SR2 agonist siramesine (SRM), indicating the potential role of SR1 antagonism in their mechanism of action. The further exploration of their structure-activity relationships and their evaluation in additional cancer cell lines will elucidate their therapeutic potential and may pave the way for the development of novel anticancer agents that target SRs.

Electrospun Fiber Membrane with the Dual Affinity of Chelation and Covalent Interactions for the Efficient Enrichment of Glycoproteins.

As important biomarkers of many diseases, glycoproteins are of great significance to biomedical science. It is essential to develop efficient glycoprotein enrichment platforms and investigate their adsorption mechanism. In this work, a conspicuous enrichment strategy for glycoproteins was developed by using an electrospun fiber membrane wrapped with polydopamine (PDA) and modified with 3-aminophenylboronic acid and nickel ions, named PAN/DA@PDA@APBA/Ni. The enrichment characteristics of PAN/DA@PDA@APBA/Ni toward glycoproteins were explored through adsorption behavior. Thanks to the existence of two sites of interaction (metal ion chelation and boronate affinity), PAN/DA@PDA@APBA/Ni exhibited significant enrichment capacity for glycoproteins, ovalbumin (604.6 mg/g), and human immunoglobulin G (331.0 mg/g). The adsorption kinetic results of glycoprotein ovalbumin on PAN/DA@PDA@APBA/Ni conform to the pseudo-first-order kinetic model in the first adsorption stage, while the second half adsorption stage is more in line with the pseudo-second-order kinetic model. Moreover, the physical characteristics of PAN/DA@PDA@APBA/Ni and subsequent adsorption experiments on electrospun fiber modified with only phenylboronic acid or nickel ions both confirmed two sites of interaction (metal ion chelation and boronate affinity, respectively). Furthermore, a stepwise elution method with dual-affinity interaction was designed and successfully applied to enrich glycoproteins in real biological samples. This work provides an idea for sample pretreatment, especially for the design of dual-affinity materials in glycoproteins enrichment.

Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.

Bifunctional immunoaffinity magnetic nanoparticles for high-efficiency separation of exosomes based on host-guest interaction

The investigation of exosomes holds substantial importance in the field of disease diagnosis and prognosis, where in the rapid and low-loss isolation of exosomes emerges as a crucial step owing to their exceedingly low concentration in bodily fluids. Herein, bifunctional immunoaffinity magnetic nanoparticles (BI-NPs) were successfully constructed by binding Ti4+ and antibody to magnetic nanoparticles through host-guest interaction. Exosomes in the cell culture supernatant could be effectively captured by the BI-NPs and then gently eluted with α-CD and 10% ammonia solution. Following the elution process, the content of captured exosome protein was determined to be up to 97 μg/mL. A total of 2822 protein groups were identified in the exosomes isolated by BI-NPs, 1060 protein groups were derived from exosomes. At the same time, the mass of exosome protein obtained by BI-NPs was more than 2 times that of UC isolation, and the above results indicated that BI-NPs had high purity enrichment performance. This is attributed to the fact that BI-NPs combine the dual affinity of Ti4+ and antibody to achieve efficient enrichment of exosomes, in addition to the mild elution property of BI-NPs due to the presence of the host-guest system. BI-NPs offer a novel approach for the isolation of exosomes, in order to further promote the application of exosomes in the required fields.

Scalable dual column cation exchange affinity chromatography based platform process for recombinant protein purification

A novel tandem affinity tag is presented that enables the use of cation exchange resins for initial affinity purification, followed by an additional column step for enhanced purity and affinity tag self-removal. In this method, the highly charged heparin-binding tag binds strongly and selectively to either a strong or weak cation exchange resin based on electrostatic interactions, effectively acting as an initial affinity tag. Combining the heparin-binding tag (HB-tag) with the self-removing iCapTag™ provides a means for removing both tags in a subsequent self-cleaving step. The result is a convenient platform for the purification of diverse tagless proteins with a range of isoelectric points and molecular weights. In this work, we demonstrate a dual column process in which the tagged protein of interest is first captured from an E. coli cell lysate using a cation exchange column via a fused heparin-binding affinity tag. The partially purified protein is then diluted and loaded onto an iCapTag™ split-intein column, washed, and then incubated overnight to release the tagless target protein from the bound tag. Case studies are provided for enhanced green fluorescent protein (eGFP), beta galactosidase (βgal), maltose binding protein (MBP) and beta lactamase (βlac), where overall purity and host cell DNA clearance is provided. Overall, the proposed dual column process is shown to be a scalable platform technology capable of accessing both the high dynamic binding capacity of ion exchange resins and the high selectivity of affinity tags for the purification of recombinant proteins.

Ultrasensitive colorimetric detection of creatinine via its dual binding affinity for silver nanoparticles and silver ions.

Creatinine is an important biomarker for the diagnosis of chronic kidney disease (CKD). Recently, it has been reported that the concentration of salivary creatinine correlates well with the concentration of serum creatinine, which makes the former useful for the development of non-invasive and point-of-care (POC) detection for CKD diagnosis. However, there exists a technical challenge in the rapid detection of salivary creatinine at low concentrations of 3-18 μM when using the current kidney function test strips as well as the traditional methods employed in hospitals. Herein, we demonstrate a simple, sensitive colorimetric assay for the detection of creatinine with a limit-of-detection (LOD) down to the nanomolar level. Our approach utilises the dual binding affinity of creatinine for citrate-capped silver nanoparticles (Ag NPs) and Ag(i) ions, which can trigger the aggregation of Ag NPs and thus lead to the colour change of a sample. The quantitative detection of creatinine was achieved using UV-Vis spectroscopy with a LOD of 6.9 nM in artificial saliva and a linear dynamic range of 0.01-0.06 μM. This method holds promise to be further developed into a POC platform for the CKD diagnosis.

Dual Graph Learning Affinity Propagation for Multimodal Remote Sensing Image Clustering

Dual Graph Learning Affinity Propagation for Multimodal Remote Sensing Image Clustering

In-silico Design and Development of Multi-Target Agents Targeting Glycogen Synthase Kinase-3 Beta and Vascular Endothelial Growth Factor Receptor 2 for Acute Myeloid Leukemia

This research focuses on the in-silico design of multi-target agents for acute myeloid leukemia (AML), targeting GSK-3β and VEGFR2. Using TTD data, we selected a promising target pair and considered 20,818 agents. Ligand-based screening in ChEMBL identified compounds with diverse structures and favorable interactions. Protein structures (GSK-3β: PDB ID 1Q5K, VEGFR2: PDB ID 3QTK) underwent rigorous quality assessment, indicating high-quality models. Molecular docking revealed varied affinities, with CHEMBL183504 showing strong affinity for GSK-3β in pocket C1, and CHEMBL185922 for VEGFR2 in pocket 2. CHEMBL181959 exhibited dual affinity. Further experimental validation is needed. In conclusion, this study provides insights for AML therapy, guiding compound selection and structural quality assessment for potential drug development

Highly sensitive quantitative detection of glycans on exosomes in renal disease serums using fluorescence signal amplification strategies

Exosomal glycoproteins play a significant role in many physiological and pathological processes. However, the detection of exosome surface glycans is currently challenged by the complexity of biological samples or the sensitivity of the methods. Herein, we prepared a novel fluorescent probe of biotin-functionalized nanocrystals (denoted as CdTe@cys-biotin) and applied it for the first time for the detection of the expression of exosomal surface glycans using a fluorescence amplification strategy. First, the dual affinity of TiO2 and CD63 aptamers of Fe3O4@TiO2-CD63 was utilized to rapidly and efficiently capture exosomes within 25 min. In this design, interference from other vesicles and soluble impurities can be avoided due to the dual recognition strategy. The chemical oxidation of NaIO4 oxidized the hydroxyl sites of exosomal surface glycans to aldehydes, which were then labeled with aniline-catalyzed biotin hydrazide. Using the high affinity between streptavidin and biotin, streptavidin-FITC and probes were successively anchored to the glycans on the exosomes. The fluorescent probe achieved the dual function of specific recognition and fluorescent labeling by modifying biotin on the surface of nanocrystals. This method showed excellent specificity and sensitivity for exosomes at concentrations ranging from 3.30 × 102 to 3.30 × 106 particles/mL, with a detection limit of 121.48 particles/mL. The fluorescent probe not only quantified exosomal surface glycans but also distinguished with high accuracy between serum exosomes from normal individuals and patients with kidney disease. In general, this method provides a powerful platform for sensitive detection of exosomes in cancer diagnosis.

Dual Affinity Nanoparticles for the Transport of Therapeutics from Carrier Cells to Target Cells under Physiological Flow Conditions.

In this study, a novel two-stage nanoparticle delivery platform was developed based on the dual functionalization of a liposome with moieties that have fundamentally different strengths of adhesion and binding kinetics. The essential concept of this system is that the nanoparticles are designed to loosely bind to the carrier cell until they come into contact with the target cell, to which they bind with greater strength. This allows the nanoparticle to be transferred from one cell to another, circulating for longer periods of time in the blood and delivering the therapeutic agent to the target circulating tumor cell. Liposomes were prepared using the lipid cake and extrusion technique, then functionalized with E-selectin (ES), anti-cell surface vimentin antibody fragments, and TRAIL via click chemistry. The binding of dual affinity (DA) liposomes was confirmed with the neutrophil-like cell line PLB985, the colorectal cancer cell line HCT116, and healthy granulocytes isolated from peripheral whole blood under physiologically relevant fluid shear stress (FSS) in a cone-and-plate viscometer. Transfer of the DA liposomes from PLB985 to HCT116 cells under FSS was greater compared to all of the control liposome formulations. Additionally, DA liposomes demonstrated enhanced apoptotic effects on HCT116 cells in whole blood under FSS, surpassing the efficacy of the ES/TRAIL liposomes previously developed by the King Lab.

Hierarchical structured Ti-doped CeO2 stabilized CoMn2O4 for enhancing the low-temperature NH3-SCR performance within highly H2O and SO2 resistance

Developing effective and stable catalysts for low-temperature selective catalytic reduction (SCR) of NOx remains challenging. Herein, we constructed a hierarchical structure by loading CoMn2O4 onto Ti-doped CeO2, that CoMn2O4/CeTiOx catalyst has shown superior deNOx activity (>95% at 100–225 °C), prominent reaction activation energy (28.8 ± 0.9 kJ mol−1) and outstanding stability (>75% at 100–200 °C within H2O and SO2). The “low-temperature active sites” and “dual anti-poisoning sites” contribute to excellent activity and stability. Firstly, the hierarchical structure boosts generation of active metal-support interface, which is conducive to oxygen migration (including adsorbed oxygen (Oads), lattice oxygen (Olat) and oxygen vacancy (Ov)) and metal charge transfer (Mn2+/3++Ce4+↔Mn3+/4++Ce3+, Ti4++Ce3+↔Ce4++Ti3+). This is the key to breaking through the limits of catalytic activity stability. Secondly, enhanced surface acidity favors NH3 adsorption and activation, which accelerates -NH2/-NH concatenate with NOx through Eley-Rideal mechanism to generate N2 and H2O. Thirdly, the dual strong SO2 affinity sites by Ti-induced CeO2 crystal reconstruction retard the active center affected by the sulfate species, which contributes to striking stability. This work highlights the importance of design of isolated active sites to improve SO2 and H2O endurance.

Synthesis and biological evaluation of novel 3-(5-substituted-1H-indol-3-yl)pyrrolidine-2,5-dione derivatives with a dual affinity for serotonin 5-HT1A receptor and SERT

The serotonin 1A (5-HT1A) receptors and serotonin transporter (SERT) are important biological targets in the treatment of diseases of the central nervous system, especially for depression. In this study, new 3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives linked with the 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole moiety were synthesised and evaluated for their affinity for 5-HT1A receptor and serotonin reuptake inhibition. Selected compounds were then tested for their affinity for D2, 5-HT2A, 5-HT6 and 5-HT7 receptors, and also in in vitro metabolic stability assays in human microsomes. Finally, in vivo assays allowed us to evaluate the agonist–antagonist properties of pre- and postsynaptic 5-HT1A receptors. 3-(1-(4-(3-(5-methoxy-1H-indol-3-yl)-2,5-dioxopyrrolidin-1-yl)butyl)-1,2,3,6-tetrahydropyridin-4-yl)-1H-indole-5-carbonitrile (4f) emerged as the most promising compound from the series, due to its favourable receptor binding profile (Ki(5-HT1A) = 10.0 nM; Ki(SERT) = 2.8 nM), good microsomal stability and 5-HT1A receptor agonistic activity.

Calcium regulates primary nitrate responseassociated gene transcription in a time- and dose-dependent manner.

Nitrate (NO3-) is the primary source of nitrogen preferred by most arable crops, including wheat. The pioneering experiment on primary nitrate response (PNR) was carried out three decades ago. Since then, much research has been carried out to understand the NO3- signaling. Nitrate is sensed by the dual affinity NO3- transceptor NPF6.3, which further relays the information to a master regulator NIN-like protein 7 (NLP7) through calcium-dependent protein kinases (CPK10, CPK30, CPK32), highlighting the importance of calcium ion (Ca2+) as one of the important secondary messengers in relaying the NO3- signaling in Arabidopsis. In a previous study, we found that Ca2+ regulates nitrogen starvation response in wheat. In this study, 10days old NO3--starved wheat seedlings were exposed to various treatments. Our study on time course changes in expression of PNR sentinel genes; NPF6.1, NPF6.2, NRT2.1, NRT2.3, NR, and NIR in wheat manifest the highest level of expression at 30min after NO3- exposure. The use of Ca2+ chelator EGTA confirmed the involvement of Ca2+ in the regulation of transcription of NPFs and NRTs as well the NO3- uptake. We also observed the NO3- dose-dependent and tissue-specificregulation of nitrate reductase activity involving Ca2+ as a mediator. The participation of Ca2+ in the PNR and NO3- signaling in wheat is confirmed by pharmacological analysis, physiological evidences, and protoplast-based Ca2+ localization.

Recombinant protein purification and immobilization strategies based on peptides with dual affinity to iron oxide and silica.

Iron oxide and silica-based materials have emerged as attractive protein purification and immobilization matrices. His6 has been reported as an effective affinity tag for both iron oxide and silica. Here, the silica-binding tags CotB1p and Car9 were shown to work as effectively as iron oxide-binding tags. Using EGFP as a model protein, commercially available bare iron oxide (BIONs) or silicon dioxide (BSiNs) nanoparticles as low-cost purification/immobilization matrices, and non-hazardous and mild binding and elution conditions, adsorption and desorption studies were performed with lysates from Escherichia coli-producing cells to compare the performance of these dual-affinity tags. Under the conditions tested, the His6 tag stood out as the best-performing tag, followed by CotB1p. Our findings concluded the promising combination of these tags, BIONs and BSiNs for one-step purification of recombinant proteins, and two-step purification and immobilization of recombinant proteins without intermediate buffer exchange. This proof of concept work set the ground for future evaluation of these purification and immobilization strategies using other proteins with different properties, which will be of interest to expand their utility and applicability. This article is protected by copyright. All rights reserved.

The Potential Utility of Tirzepatide for the Management of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is the most prevalent endocrinopathy in women of reproductive age. The metabolic dysfunction associated with PCOS increases the probability of developing type 2 diabetes (T2D), endometrial cancer, and cardiovascular disease. Research has shown that the metabolic features of PCOS may be improved by weight loss following treatment with glucagon-like peptide-1 receptor (GLP-1R) agonists. Tirzepatide is a dual GLP-GIP (gastric inhibitory polypeptide) receptor agonist that shares a very similar mechanism of action with GLP-1R agonists, and it is hypothesized that it may be a potential contender in the treatment of PCOS. The success of GLP-1R agonists is usually hindered by their adverse gastrointestinal effects, leading to reduced compliance. The mechanism of action of Tirzepatide partly addresses this issue, as its dual receptor affinity may reduce the intensity of gastrointestinal symptoms. Tirzepatide has been licensed for the treatment of type 2 diabetes and given the metabolic issues and obesity that accompanies PCOS, it may be of value in its management for those PCOS patients who are obese with metabolic syndrome, although it may not benefit those who are of normal weight. This study reviews the current therapies for the treatment of PCOS and evaluates the potential use of Tirzepatide to address the symptoms of PCOS, including reproductive dysfunction, obesity, and insulin resistance.

One-Step Quantification of anti-Covid-19 Antibodies via Dual Affinity Ratiometric Quenching Assays.

The global pandemic caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people and paralyzed healthcare systems worldwide. Developing rapid and accurate tests to detect and quantify anti-SARS-CoV-2 antibodies in complex fluids is critical to (i) track and address the spread of SARS-CoV-2 variants with different virulence and (ii) support the industrial manufacturing and clinical administration of anti-SARS-CoV-2 therapeutic antibodies. Conventional immunoassays, such as lateral flow, ELISA, and surface plasmon resonance (SPR), are either qualitative or, when quantitative, are laborious and expensive and suffer from high variability. Responding to these challenges, this study evaluates the performance of the Dual-Affinity Ratiometric Quenching (DARQ) assay for the quantification of anti-SARS-CoV-2 antibodies in bioprocess harvests and intermediate fractions (i.e., a Chinese hamster ovary (CHO) cell culture supernatant and a purified eluate) and human fluids (i.e., saliva and plasma). Monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid as well as the spike protein of the delta and omicron variants are adopted as model analytes. Additionally, conjugate pads loaded with dried protein were studied as an at-line quantification method that can be used in clinical or manufacturing laboratories. Our results indicate that the DARQ assay is a highly reproducible (coefficient of variation ∼0.5-3%) and rapid (<10 min) test, whose sensitivity (∼0.23-2.5 ng/mL), limit of detection (23-250 ng/mL), and dynamic range (70-1300 ng/mL) are independent of sample complexity, thus representing a valuable tool for monitoring anti-SARS-CoV-2 antibodies.

Engineered dual affinity protein fragments to bind collagen and capture growth factors.

Collagen type I lacks affinity for growth factors (GFs) and yet it is clinically used to deliver bone morphogenic protein 2 (BMP-2), a potent osteogenic growth factor. To mitigate this lack of affinity, supra-physiological concentrations of BMP-2 are loaded in collagen sponges leading to uncontrolled BMP-2 leakage out of the material. This has led to important adverse side effects such as carcinogenesis. Here, we design recombinant dual affinity protein fragments, produced in E.Coli, which contain two regions, one that spontaneously binds to collagen and a second one that binds BMP-2. By adding the fragment to collagen sponges, BMP-2 is sequestered enabling solid phase presentation of BMP-2. We demonstrate osteogenesis in vivo with ultra-low doses of BMP-2. Our protein technology enhances the biological activity of collagen without using complex chemistries or changing the manufacturing of the base material and so opens a pathway to clinical translation.

In Silico Design of New Dual Inhibitors of SARS-CoV-2 MPRO through Ligand- and Structure-Based Methods.

The viral main protease is one of the most attractive targets among all key enzymes involved in the life cycle of SARS-CoV-2. Considering its mechanism of action, both the catalytic and dimerization regions could represent crucial sites for modulating its activity. Dual-binding the SARS-CoV-2 main protease inhibitors could arrest the replication process of the virus by simultaneously preventing dimerization and proteolytic activity. To this aim, in the present work, we identified two series' of small molecules with a significant affinity for SARS-CoV-2 MPRO, by a hybrid virtual screening protocol, combining ligand- and structure-based approaches with multivariate statistical analysis. The Biotarget Predictor Tool was used to filter a large in-house structural database and select a set of benzo[b]thiophene and benzo[b]furan derivatives. ADME properties were investigated, and induced fit docking studies were performed to confirm the DRUDIT prediction. Principal component analysis and docking protocol at the SARS-CoV-2 MPRO dimerization site enable the identification of compounds 1b,c,i,l and 2i,l as promising drug molecules, showing favorable dual binding site affinity on SARS-CoV-2 MPRO.

Membrane dual-targeting probes: A promising strategy for fluorescence-guided prostate cancer surgery and lymph node metastases detection.

Fluorescence-guided surgery (FGS) with tumor-targeted imaging agents, particularly those using the near-infrared wavelength, has emerged as a real-time technique to highlight the tumor location and margins during a surgical procedure. For accurate visualization of prostate cancer (PCa) boundary and lymphatic metastasis, we developed a new approach involving an efficient self-quenched near-infrared fluorescence probe, Cy-KUE-OA, with dual PCa-membrane affinity. Cy-KUE-OA specifically targeted the prostate-specific membrane antigen (PSMA), anchored into the phospholipids of the cell membrane of PCa cells and consequently showed a strong Cy7-de-quenching effect. This dual-membrane-targeting probe allowed us to detect PSMA-expressing PCa cells both invitro and invivo and enabled clear visualization of the tumor boundary during fluorescence-guided laparoscopic surgery in PCa mouse models. Furthermore, the high PCa preference of Cy-KUE-OA was confirmed on surgically resected patient specimens of healthy tissues, PCa, and lymph node metastases. Taken together, our results serve as a bridge between preclinical and clinical research in FGS of PCa and lay a solid foundation for further clinical research.

Dual PTP1B/TC-PTP Inhibitors: Biological Evaluation of 3-(Hydroxymethyl)cinnoline-4(1H)-Ones

Dual inhibitors of protein phosphotyrosine phosphatase 1B (PTP1B)/T-cell protein phosphotyrosine phosphatase (TC-PTP) based on the 3-(hydroxymethyl)-4-oxo-1,4-dihydrocinnoline scaffold have been identified. Their dual affinity to both enzymes has been thoroughly corroborated by in silico modeling experiments. The compounds have been profiled in vivo for their effects on body weight and food intake in obese rats. Likewise, the effects of the compounds on glucose tolerance, insulin resistance, as well as insulin and leptin levels, have been evaluated. In addition, the effects on PTP1B, TC-PTP, and Src homology region 2 domain-containing phosphatase-1 (SHP1), as well as the insulin and leptin receptors gene expressions, have been assessed. In obese male Wistar rats, a five-day administration of all studied compounds led to a decrease in body weight and food intake, improved glucose tolerance, attenuated hyperinsulinemia, hyperleptinemia and insulin resistance, and also compensatory increased expression of the PTP1B and TC-PTP genes in the liver. The highest activity was demonstrated by 6-Chloro-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 3) and 6-Bromo-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 4) with mixed PTP1B/TC-PTP inhibitory activity. Taken together, these data shed light on the pharmacological implications of PTP1B/TC-PTP dual inhibition, and on the promise of using mixed PTP1B/TC-PTP inhibitors to correct metabolic disorders.