Sensory involvement in the SOD1-G93A mouse model of amyotrophic lateral sclerosis

A subset of patients of amyotrophic lateral sclerosis (ALS) present with mutation of Cu/Zn superoxide dismutase 1 (SOD1), and such mutants caused an ALS-like disorder when expressed in rodents. These findings implicated SOD1 in ALS pathogenesis and made the transgenic animals a widely used ALS model. However, previous studies of these animals have focused largely on motor neuron damage. We report herein that the spinal cords of mice expressing a human SOD1 mutant (hSOD1-G93A), besides showing typical destruction of motor neurons and axons, exhibit significant damage in the sensory system, including Wallerian-like degeneration in axons of dorsal root and dorsal funiculus, and mitochondrial damage in dorsal root ganglia neurons. Thus, hSOD1-G93A mutation causes both motor and sensory neuropathies, and as such the disease developed in the transgenic mice very closely resembles human ALS.     

Fluorescence assay for monitoring Zn-deficient superoxide dismutase in vitro

A method has been developed for selective detection of the zinc-deficient form of Cu, Zn superoxide dismutase (SOD1) in vitro. Zinc-deficient SOD1 mutants have been implicated in the death of motor neurons leading in amyotrophic lateral sclerosis (ALS or Lou Gerhig's disease). Thus, this method may have applicability for detecting zinc-deficient SOD1 mutants in human ALS patients samples as well as in a transgenic mouse model of ALS and in cultured motor neurons. We determined previously that structural analogs of 1,10 phenanthroline, which react specifically with Cu(I), react with the active Cu(I) of SOD1 when zinc is absent, but not when zinc is also bound, as evidenced by the fact that the reaction is inhibited by pretreatment of the enzyme with zinc. We report herein that bathocuproine, or its water-soluble derivative bathocuproine disulfonate, react with zinc-deficient SOD1 to form a complex which fluoresces at 734 nm when excited at 482 nm. Fluorescent intensity is concentration dependent, thus we propose to use fluorescent confocal microscopy to measure intracellular levels of zinc-deficient SOD1 in situ.     

Separation and characterization of superoxide dismutase 1 (SOD-1) from human erythrocytes by capillary electrophoresis time-of-flight mass spectrometry

Cu, Zn-superoxide dismutase (SOD-1) is a homodimeric metalloenzyme that has been related to ALS (amyotrophic lateral sclerosis). The majority of ALS cases are sporadic while approximately 10% are inherited (familial ALS, FALS). Mutations in the amino acid sequence of human SOD-1 cause only 25% of the FALS cases, while the explanation for the rest is not clear yet. In this way, several authors have suggested the importance of posttranslational modifications or dimer dissociation on formation of the characteristic fatal intraneuronal SOD-1 aggregates. In this paper, we used capillary electrophoresis-electrospray mass spectrometry with an accurate mass and high-resolution time-of-flight mass spectrometer (CE-TOF-MS) for separation and characterization of standard bovine SOD-1 and human SOD-1 purified from erythrocytes. Two background electrolytes (BGEs) were used for CE-TOF-MS experiments in positive ion mode. An acidic BGE allowed detection of apo-monomer SOD-1, because the metal ions were completely released during the electrophoretic separation. The better sensitivity at acidic pH was especially interesting to detect different isoforms of human SOD-1. In contrast, a neutral BGE provided enhanced conditions for detection of the fully metalated dimeric and monomeric enzyme, but selecting an appropriate fragmentor voltage value in the TOF analyzer was critical to obtain reliable quantitative information. Anyway, only the metalated forms involving the main isoform of human SOD-1 were detected due to the lower sensitivity. Hence, the combination of both methodologies resulted necessary to obtain detailed structural information from the enzyme.     

Optimizing separation efficiency of 2-DE procedures for visualization of different superoxide dismutase forms in a cellular model of amyotrophic lateral sclerosis

Neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease (PD) have been associated with increased production of reactive oxygen species. In AD and PD patients, superoxide dismutase (SOD1) was also indicated as a major target of oxidative damage. In particular, in brain tissue of these patients, different SOD1 isoforms have been identified, although their functional role still remains to be elucidated. In the light of the possibility that different SOD1 entities could be expressed also in other neurodegenerative disorders, as a sort of unifying event with AD and PD, we have investigated amyotrophic lateral sclerosis (ALS) using human neuroblastoma SH-SY5Y cells with mutated SOD1 gene H46R as cellular model. 2-DE using a narrow-range IPG 4-7 strips in the first dimension and linear 15% SDS-PAGE in the second allowed to separate different SOD1 spots. MALDI-TOF MS and CapLC-MS/MS have been used for their complete identification. This is the first report in which the presence of SOD1 (iso) forms in a cellular model of ALS has been evidenced.     

Intracellular amyloid beta interacts with SOD1 and impairs the enzymatic activity of SOD1: implications for the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the degeneration of motor neurons. Mutations in Cu/Zn superoxide dismutase (SOD1), including G93A, were reportedly linked to familial ALS. SOD1 is a key antioxidant enzyme, and is also one of the major targets for oxidative damage in the brains of patients suffering from Alzheimer''s disease (AD). Several lines of evidence suggest that intracellular amyloid beta (Aβ) is associated with the pathogenesis of AD. In this report we demonstrate that intracellular Aβ directly interacts with SOD1, and that this interaction decreases the enzymatic activity of the enzyme. We observed Aβ-SOD1 aggregates in the perinuclear region of H4 cells, and mapped the SOD1 binding region to Aβ amino acids 26-42. Interestingly, intracellular Aβ binds to the SOD1 G93A mutant with greater affinity than to wild-type SOD1. This resulted in considerably less mutant enzymatic activity. Our study implicates a potential role for Aβ in the development of ALS by interacting with the SOD1 G93A mutant.     

Growth factor-expressing human neural progenitor cell grafts protect motor neurons but do not ameliorate motor performance and survival in ALS mice

Neural progenitor cells (NPs) have shown several promising benefits for the treatment of neurological disorders. To evaluate the therapeutic potential of human neural progenitor cells (hNPs) in amyotrophic lateral sclerosis (ALS), we transplanted hNPs or growth factor (GF)-expressing hNPs into the central nervous system (CNS) of mutant Cu/Zn superoxide dismutase (SOD1^G93A) transgenic mice. The hNPs were engineered to express brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), VEGF, neurotrophin-3 (NT-3), or glial cell-derived neurotrophic factor (GDNF), respectively, by adenoviral vector and GDNF by lentiviral vector before transplantation. Donor-derived cells engrafted and migrated into the spinal cord or brain of ALS mice and differentiated into neurons, oligodendrocytes, or glutamate transporter-1 (GLT1)-expressing astrocytes while some cells retained immature markers. Transplantation of GDNF- or IGF-1-expressing hNPs attenuated the loss of motor neurons and induced trophic changes in motor neurons of the spinal cord. However, improvement in motor performance and extension of lifespan were not observed in all hNP transplantation groups compared to vehicle-injected controls. Moreover, the lifespan of GDNF-expressing hNP recipient mice by lentiviral vector was shortened compared to controls, which was largely due to the decreased survival times of female animals. These results imply that although implanted hNPs differentiate into GLT1-expressing astrocytes and secrete GFs, which maintain dying motor neurons, inadequate trophic support could be harmful and there is sexual dimorphism in response to GDNF delivery in ALS mice. Therefore, additional therapeutic approaches may be required for full functional recovery.     

Clinical perspectives of high-resolution mass spectrometry-based proteomics in neuroscience: exemplified in amyotrophic lateral sclerosis biomarker discovery research

Biomarker discovery is a central application in today's proteomic research. There is an urgent need for valid biomarkers to improve diagnostic tools and treatment in many disorders, such as the rapidly progressing neurodegenerative disorder amyotrophic lateral sclerosis (ALS) that has a fatal outcome in about 3 years and yet no curative treatment. Screening for clinically relevant biomarkers puts high demands on high-throughput, rapid and precise proteomic techniques. There is a large variety in the methods of choice involving mainly gel-based approaches as well as chromatographic techniques for multi-dimensional protein and peptide separations followed by mass spectrometry (MS) analysis. This special feature article will discuss some important aspects of MS-based clinical proteomics and biomarker discovery in the field of neurodegenerative diseases and ALS research respectively, with the aim to provide a prospective view on current and future research aspects in the field. Furthermore, examples for application of high-resolution MS-based proteomic strategies for ALS biomarker discovery will be demonstrated with two studies previously reported by our group. These studies include among others, utilization of capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) for advanced protein pattern classification in cerebrospinal fluid (CSF) samples of ALS patients as well as highly sensitive protein identification in minimal amounts of postmortem spinal cord tissue and laser micro-dissected motor neurons using FT-ICR-MS in conjunction with nanoflow LC coupled to matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (LC-MALDI-TOF-TOF-MS).     

Parsing disease‐relevant protein modifications from epiphenomena: perspective on the structural basis of SOD1‐mediated ALS

Conformational change and modification of proteins are involved in many cellular functions. However, they can also have adverse effects that are implicated in numerous diseases. How structural change promotes disease is generally not well‐understood. This perspective illustrates how mass spectrometry (MS), followed by toxicological and epidemiological validation, can discover disease‐relevant structural changes and therapeutic strategies. We (with our collaborators) set out to characterize the structural and toxic consequences of disease‐associated mutations and post‐translational modifications (PTMs) of the cytosolic antioxidant protein Cu/Zn‐superoxide dismutase (SOD1). Previous genetic studies discovered >180 different mutations in the SOD1 gene that caused familial (inherited) amyotrophic lateral sclerosis (fALS). Using hydrogen–deuterium exchange with mass spectrometry, we determined that diverse disease‐associated SOD1 mutations cause a common structural defect – perturbation of the SOD1 electrostatic loop. X‐ray crystallographic studies had demonstrated that this leads to protein aggregation through a specific interaction between the electrostatic loop and an exposed beta‐barrel edge strand. Using epidemiology methods, we then determined that decreased SOD1 stability and increased protein aggregation are powerful risk factors for fALS progression, with a combined hazard ratio > 300 (for comparison, a lifetime of smoking is associated with a hazard ratio of ~15 for lung cancer). The resulting structural model of fALS etiology supported the hypothesis that some sporadic ALS (sALS, ~80% of ALS is not associated with a gene defect) could be caused by post‐translational protein modification of wild‐type SOD1. We developed immunocapture antibodies and high sensitivity top‐down MS methods and characterized PTMs of wild‐type SOD1 using human tissue samples. Using global hydrogen–deuterium exchange, X‐ray crystallography and neurotoxicology, we then characterized toxic and protective subsets of SOD1 PTMs. To cap this perspective, we present proof‐of‐concept that post‐translational modification can cause disease. We show that numerous mutations (N➔D; Q➔E), which result in the same chemical structure as the PTM deamidation, cause multiple diseases. Copyright © 2017 John Wiley & Sons, Ltd.     

A Novel Anti-Inflammatory d-Peptide Inhibits Disease Phenotype Progression in an ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterised by selective neuronal death in the brain stem and spinal cord. The cause is unknown, but an increasing amount of evidence has firmly certified that neuroinflammation plays a key role in ALS pathogenesis. Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and has been implicated as driver of disease progression. Here, we describe a treatment study demonstrating the therapeutic potential of a tandem version of the well-known all-d-peptide RD2 (RD2RD2) in a transgenic mouse model of ALS (SOD1*G93A). Mice were treated intraperitoneally for four weeks with RD2RD2 vs. placebo. SOD1*G93A mice were tested longitudinally during treatment in various behavioural and motor coordination tests. Brain and spinal cord samples were investigated immunohistochemically for gliosis and neurodegeneration. RD2RD2 treatment in SOD1*G93A mice resulted not only in a reduction of activated astrocytes and microglia in both the brain stem and lumbar spinal cord, but also in a rescue of neurons in the motor cortex. RD2RD2 treatment was able to slow progression of the disease phenotype, especially the motor deficits, to an extent that during the four weeks treatment duration, no significant progression was observed in any of the motor experiments. Based on the presented results, we conclude that RD2RD2 is a potential therapeutic candidate against ALS.     

Advanced analytical tools in proteomics

Proteomics deals with the study of proteins, their structures, localizations, posttranslational modifications, functions and interactions with other proteins. The mapping of protein structure-function holds the key to a better understanding of cellular functions under both normal and disease states, which is critical for modern drug discovery. However, the study of human proteome presents scientists with a task much more daunting than the human genome project. In fact, the estimated >100,000 different proteins expressed from 30,000 to 40,000 human genes make it extremely challenging, if not impossible with existing protein analysis techniques, to map the entire cellular functions at the translational level. Consequently, there have been rapid advances in the techniques and methods capable of large-scale proteomic studies. Among them, the recently developed high-throughput screening methods have enabled scientists to analyze proteins quickly and efficiently at an organism-wide scale. Herein, we overview some of these emerging tools for high-throughput protein analysis. In particular, we focus on recent advances in the bioassay development, which has provided sensitive and selective tools for high-throughput identification and characterizations of enzymes. Finally, the recently developed bioimaging techniques to visualize and quantify proteins in living cells are also discussed.     

HCV全基因组培养细胞的比较蛋白组学研究

利用比较蛋白质组技术研究了转染丙型肝炎病毒(Hepatitis C virus, HCV)全基因组的人肝癌细胞系Huh7细胞模型中蛋白质表达谱的变化, 建立了Huh7-HCV的双向凝胶电泳蛋白质表达图谱和数据库. 通过双向凝胶电泳分离和图像分析, 对表达差异2倍以上蛋白质点进行了胶内酶解和MALDI-TOF MS鉴定. 得到包括与细胞骨架蛋白、细胞周期、凋亡和信号转导等相关的14个蛋白质, 并且用Western blot验证了热休克蛋白70的蛋白质组研究结果. 利用HCV全基因组培养系统, 采用蛋白质组学技术, 为研究HCV病毒和宿主细胞相互作用提供了新的实验数据, 为深入研究HCV病毒复制和分子致病机理奠定了基础.     

Proteomic and redox‐proteomic study on the role of glutathione reductase in human lung cancer cells

Glutathione reductase (GR), a cytosolic protein, plays a vital role in maintaining a correct redox status in cells. However, comprehensive investigations of GR‐modulated cellular responses, including protein level alteration and redox regulation, have yet to be performed. In this study, we cultured a human lung adenocarcinoma line transfected with empty pLKO.1 vector as a control, CL1‐0shControl, and its GR‐knockdown derivative, CL1‐0shΔGR, to evaluate differential protein level alteration and redox regulation of these two cell lines. We identified 34 spots that exhibited marked changes in intensities, and 13 proteins showing significant changes in thiol reactivity, in response to GR depletion. Several proteins involved in redox regulation, calcium signaling, cytoskeleton regulation, and protein folding showed significant changes in expression, whereas proteins involved in redox regulation, protein folding, and glycolysis displayed changes in thiol reactivity. Interestingly, GR knockdown induces peroxiredoxin‐1 overexpression in the air‐exposed tissue and high oxygen consuming tissue such as cornea and liver, but not in the low oxygen consuming tissues such as breast and uterine. In summary, we used a comprehensive lung adenocarcinoma based proteomic approach for identifying GR‐modulated protein expression alteration and redox modification. Based on our research, this is the first comprehensive proteomic and redox‐proteomic analysis used to investigate the role of GR in a mammalian cell model.     

Calcium-influx increases SOD1 aggregates via nitric oxide in cultured motor neurons

Familial amyotrophic lateral sclerosis (fALS) is caused by mutations in Cu/Zn-superoxide dismutase (SOD1), and SOD1 aggregation and calcium toxicity are involved in neuronal death. However, the effect of altered calcium homeostasis on the SOD1 aggregation is unknown. To investigate whether calcium triggers mutant SOD1 aggregation in vitro, human mutant SOD1 (G93A) was transfected into motor neuronal cell line (VSC 4.1 cells). These cells were then treated with calcium ionophore A23187 or agents that induce intracellular calcium release like cyclic ADP ribose, ryanodine or thapsigargin. A23187 was found to increase mutant SOD1 aggregation and neuronal nitric oxide synthase (nNOS) expression. Moreover, the NOS inhibitor (L-NAME) and a NO-dependent cyclic GMP cascade inhibitor (ODQ) reduced SOD1 aggregation, whereas an exogenous NO donor (GSNO) increased mutant SOD1 aggregation, which was also prevented by NOS or cGMP cascade inhibitor. Our data demonstrate that calcium-influx increases SOD1 aggregation by upregulating NO in cultured motor neuronal cells.     

Proteomic analysis of human cerebral cortex in epileptic patients

Epilepsy affects more than 0.5% of the world population and is known to be associated with a large genetic component eliciting an electrical hyperexcitability in the central nervous system. However, its pathogenic mechanisms remain poorly understood. In order to gain greater molecular incite in the pathogenesis in epilepsy, we analyzed proteomes of human cerebral cortices. Quantitative proteome analysis was used to compare signals corresponding to individual proteins between epileptic cerebral cortices from patients with temporal lobe epilepsy and age-matched non-epileptic subjects. To minimize individual variations, gender and age of the patients were matched. Changes of several spots were consistent among 6 pairs of epileptic patients and nonepileptic subjects. One of the spots was identified as the mitochondrial type Mn-superoxide dismutase (Mn-SOD) confirmed by Western blot analysis with Mn-SOD antibody and enzyme activity assay. Such results were agreeable with chemical and physical parameters given by the 2-dimensional electrophoresis (2-DE) gel. Mn-SOD was consistently down-regulated in epileptic cerebral cortices compared with those of nonepileptic subjects. Our results demonstrate a clear link between pathogenesis of epilepsy and SOD. Additionally, we identified four proteins that were consistently over-expressed in all epileptic temporal neocortices specimens and the other four proteins that were found to be expressed less than non-epileptic control subjects. These proteomic data provide cellular markers in the understanding mechanism of the epilepsy pathogenesis.     

Establishment of an ectopically expressed and functional PRMT1 for proteomic analysis of arginine-methylated proteins

Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), plays crucial roles in a variety of cellular processes. Mammalian PRMT1 exists in a large protein complex in cells, which has been implied in modulating the regulatory and catalytic properties of this enzyme. Establishment of a mammalian comparative approach will help to identify putative substrates of PRMT1 in an authentic condition. Here, we showed that ectopically expressed PRMT1 in mammalian HEK293 cells not only exhibited catalytic properties comparable to the endogenous enzyme but also existed in a functional complex together with endogenous PRMT1 and thus functioned as an endogenous counterpart. In addition, the measured methylation level of cellular proteins using a tritium-labeled methyl donor was accordingly enhanced upon ectopic expression of PRMT1. Subsequent proteomic analysis with such PRMT1-expressing cells allowed us to identify several known and putative methylated proteins. In vitro methylation of selected proteins, eukaryotic translation initiation factor 4A-I and vimentin, by cellular PRMT1 was shown. Together, we have demonstrated the functional equivalence of ectopically expressed PRMT1 in HEK293 cells and its application to systematically identify the substrate proteins in a mammalian cell context.     

Proteomics Analysis of T Lymphocytes Damage Induced by Ionizing Irradiation

Human T lymphocytes were found to be highly radiosensitive and complex cellular responses including apoptosis could be induced upon exposure to X‐ray irradiation. However, the mechanism of apoptosis associated with irradiation was not clear. In this study, a proteomic method was applied to investigation on alteration of proteome of human T‐lymphocyte cells after irradiation. The Jurkat cells were irradiated with 4 Gy X‐ray and the cell lysates were collected at different times after irradiation (6, 12, 18, 24 and 48 h). The whole proteins were separated and quantified by two‐dimensional fluorescence difference gel electrophoresis, and then the differentially expressed proteins were identified by mass spectrometry. 4 proteins exhibited significant irradiation‐induced difference in abundance, including L‐plastin, bifunctional purine biosynthesis protein, tubulin beta chain, beta‐actin. Differentially expressed proteins were reported to be directly or indirectly involved in the function of human T lymphocyte. Thus, this study might provide clues to identify proteins with biological significance related to irradiation.     

Comparative Bioinformatics Analyses and Profiling of Lysosome-Related Organelle Proteomes

Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for 7 lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.     

Reduced net charge and heterogeneity of pI isoforms in familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase

Familial cases of amyotrophic lateral sclerosis (fALS) are related to mutations of copper/zinc superoxide dismutase 1 (SOD1). Aggregation of SOD1 plays a central role in the pathogenesis of fALS and altered metallation of SOD1 mutants could be involved in this process. Using IEF gel electrophoresis under non‐denaturating conditions and particle induced X‐ray emission (PIXE) analysis, we studied the pI distribution and metallation status of fALS SOD1 mutants (A4V, G93A, D125H) compared to human wild‐type (hWT). SOD1 fALS mutants are characterized by a variable number of isoforms and higher pI compared to hWT, reflecting a reduced net charge that might explain their greater propensity to precipitation and aggregation. Cu/Zn ratios were slightly different for the predominant expressed isoforms of A4V, G93A, and D125H mutants compared to hWT. Differences in metallation were observed within each genotype, the more basic isoforms exhibiting lower Cu/Zn ratios. Moreover, we revealed the existence of a pool of fALS mutants SOD1 pI isoforms, slightly expressed (<10%), with a low Cu/Zn ratio and high pI values. Overall, IEF‐PIXE results suggest that the toxicity of SOD1 mutants should be studied at the pI isoform level with a particular attention to the species with the lowest charges.     

Fuzzy adaptive least squares and its use in quantitative structure-activity relationships

Fuzzy adaptive least squares (FALS), a pattern recognition method designed to correlate molecular structure with activity rating, has been developed. A novel feature of FALS is that the degree to which each sample belongs to an activity class is given using a membership function. The algorithm involves an iterative modification of forcing factors to maximize the sum of the membership function values over all samples. This paper first describes the method and calculation procedure of FALS89 (1989 version of FALS), and then shows its application to the correlation of structure with a potency rating of anticarcinogenic mitomycin derivatives and arginine-vasopressin antagonists. FALS89 applied to these samples showed considerably high reliability in both recognition and leave-one-out prediction.     

An integrated map of the murine hippocampal proteome based upon five mouse strains

With the advent of proteomics technologies it is possible to simultaneously demonstrate the expression of hundreds of proteins. The information offered by proteomics provides context-based understanding of cellular protein networks and has been proven to be a valuable approach in neuroscience studies. The mouse hippocampus has been a major target of analysis in the search for molecular correlates to neuronal information storage. Although human and rat hippocampal samples have been successfully subjected to proteomic profiling, no elaborate analysis providing the fundamental experimental basis for protein-expression studies in the mouse hippocampus has been carried out as yet. This led us to construct a master map generated from the individual hippocampal proteomes of five different mouse strains. A proteomic approach, based upon 2-DE coupled to MS (MALDI-TOF/TOF) has been chosen in an attempt to establish a comprehensive reference database of proteins expressed in the mouse hippocampus. 469 individual proteins, represented by 1156 spots displaying various functional states of the respective gene products were identified. Proteomic profiling of the hippocampus, a brain region with a pivotal role for neuronal information processing and storage may provide insight into the characteristics of proteins serving this highly sophisticated function.