FUNCTIONAL GENOMICS & BIOMEDICINE

The technological advances in genotyping, expression profiling and proteomics, coupled with systems biology, offer exciting and promising advances in biomedicine, both in understanding the basis of common diseases as well as in improved diagnosis and therapy. The combination of genetic and genomic information will allow early and more accurate prediction and diagnosis of disease and disease progression; the redefinition of disease subtypes through functional genomics is likely to provide many examples of differential response to therapy; and understanding of individual responses to drugs will have implications for their use and development by the pharmaceutical industry. In this programme, we will focus initially on the currently emerging areas of epigenomics and neurogenomics and the insights they provide into the mechanisms of cancer and neurological disorders respectively; and on metabolomics, pharmacogenomics and predictive, preventive and personalised medicine for assessment of disease susceptibility and response to therapy.

Epigenetics, epigenomics and cancer
Epigenetics is a key area of current and future research that can elucidate how genomes function. It combines genetics and the environment to address complex biological systems such as genome plasticity. Much of the control of gene expression is governed by epigenetic changes, such as differential DNA methylation and histone modification. The involvement of DNA methylation in genetic imprinting, gene regulation, chromatin structure, genome stability and diseases, especially cancer, is now well established. In the wake of the human genome project, epigenetic phenomena can be studied on a genome-wide scale, giving rise to the new field of epigenomics. There are far-reaching implications of epigenomic research for areas of functional genomics and disease, including stem cells, cancer and ageing.

While epigenetic modifications of the DNA do not alter the sequence code, they are heritable and regulate gene transcription. Genomic imprinting is a form of epigenetic regulation in mammals which results in the silencing of one allele of specific genes according to parental origin. Characteristic of imprinted genes are differences in DNA methylation of maternal and paternal alleles. While some 70 imprinted genes have been identified in the mouse, most of which are similarly imprinted in humans, it is not clear how many imprinted loci exist, creating a need for systematic approaches to imprinted gene discovery. Screens have been based on methylation analysis, since many imprinted genes and all imprinting control elements exhibit differences in DNA methylation between the parental alleles. Congenital abnormalities may occur when both copies of imprinted genes are active, due to a failure in the establishment of the normal methylation pattern, as in Prader-Willi syndrome, Angelman's syndrome and Beckwith-Wiedemann syndrome. Diseases can also arise when two copies of an imprinted gene are inactive.

Understanding the many complex roles of DNA methylation is a very active area. Methylation occurs on cytosine bases at CpG sequences and is involved in controlling correct gene expression; methylated cytosines in promotor regions generally lead to gene inactivation, acting as an expression switch. Mechanisms through which they have this effect include inhibition of binding of transcription factors, or recruitment of repressor complexes that include histone deacetylases. Methylation is the only flexible genomic parameter that can change genome function under exogenous influence. Hence it constitutes a link between genetics, disease and the environment that is widely thought to play a decisive role in the aetiology of many human pathologies. DNA methylation is essential for normal development - deletion of DNA methyltransferases (DNMTs) is lethal in homozygous knockout mice. Problems with the methylation machinery give rise to human developmental diseases, eg mutations in the DNMt3b enzyme cause the ICF immunodeficiency syndrome, while abnormalities in one of the proteins (MeCP2) recognising and binding methyl cytosine leads to Rett syndrome, a form of mental retardation affecting young girls. Abnormalities of DNA methylation, either increased or decreased, also develop with ageing and are found in many human cancers. Whether epigenetics plays a role in common diseases such as heart disease, diabetes and obesity is now ripe for investigation.

Large-scale methylation studies became possible with the introduction of bisulfite treatment of genomic DNA that converts unmethylated cytosines into uracils but leaves methylcytosines unconverted. PCR assays and array-based detection methods can discriminate methylated and unmethylated sequences, allowing rapid high throughput screening of multiple samples. Differentially methylated cytosines give rise to distinct patterns specific for tissue type and disease state. Methylation variable positions (MVPs) are common epigenetic markers which, like SNPs, promise to significantly advance our ability to understand and diagnose human disease.

Histone modifications Considerable attention is also being given to histone modifications. Characteristic patterns of site-specific acetylation and methylation on histone tails (the ‘histone code') are involved in gene regulation through changes in chromatin structure and condensation. The histone code is recognised by effector proteins that bind to nucleosomes and recognise specific patterns of histone modification, eg hypoacetylation of histone H3 and H4 is associated with heterochromatic, transcriptionally inactive regions in the genome.

Epigenomics and cancer Epigenetic modifications are frequently involved in transcriptional changes in tumour suppressor genes (TSGs) and oncogenes. The interplay of chromatin modifications, histones, DNA methylation and gene regulation in cancer is beginning to be understood. In particular, gene-associated CpG islands are targets of hypermethylation, which plays a critical role in gene silencing. Methylated TSGs have been found in virtually every tumour type and promoter methylation has been established as a key mechanism in TSG inactivation. The cause of aberrant methylation in cancers is the major question in cancer epigenomics. The assessment of methylation status in hundreds of CpG island sequences in a single hybridization experiment on oligonucleotide microarrays has demonstrated that methylation events discriminate normal versus tumour tissue and holds great potential as a diagnostic tool for tumour subclassification. DNA methylation events occur early in tumorigenesis, making them ideal targets for early detection of malignant cells, while the fact that epigenetic modifications are reversible also makes them candidates for therapeutic interventions. Clinical trials are in progress to study the effects of demethylating agents in malignancies.

European initiatives in epigenomics The Epigenome Network of Excellence is a recently funded EC FP6 consortium formed with 25 partners to promote a coherent European Research Area and prioritise research into molecular mechanisms of epigenetic control. The research programme of the NoE is organised into 8 subprogrammes, focusing on central questions, such as the existence of a histone code in addition to the genetic code, the molecular mechanisms of epigenetic plasticity, and how epigenetic dysfunction affects disease.

Neurogenomics and disease
Neurogenomics is the study of how the genome as a whole contributes to the evolution, development, structure and function of the nervous system. Using the new technologies for probing polymorphisms and gene expression, neurogenomics aims to understand the molecular basis of nervous system function and dysfunction. It includes the application of high throughput transcriptomics and proteomics to existing model systems in order to obtain global views of gene expression in the brain. A wealth of data is also available for comparative genomics studies, eg human and mouse; the latter is a key model for molecular studies of the brain and the recently completed mouse genome provides access to informative models of human somatic and psychological diseases. Large-scale analysis of gene expression in human postmortem brain has the potential to elucidate molecular changes that occur in disease and may lead to identification of genes and proteins specifically associated with psychotic conditions, eg schizophrenia, bipolar disorder, and neurodevelopmental/neurodegenerative disorders, eg Down's syndrome, Alzheimer's.

Transcriptional profiling It is thought that up to half of all genes are largely or exclusively dedicated to directing the development, maintenance and functioning of the brain. Although it has a limited number of primary cell types, they show immense phenotypic diversity, with thousands of different classes defined by morphology, connectivity, neurotransmitters, receptors, etc. Subpopulations are increasingly distinguishable on their pattern of gene expression, which reflects the complexity of neuronal cell types and circuits. A molecular map of gene expression in the brain would revolutionise the study of both normal brain function and development, as well as neurological and psychiatric disease. Approaches to mapping expression can be gene-based (eg in situ hybridisation) or cell-based (eg expression analysis). A major obstacle is the difficulty of obtaining mRNA from just the cells of interest. In experimental animals, specific neuronal populations can often be isolated and purified as a source of mRNA, but this is of limited utility in the human brain. Laser capture microdissection is applicable to human tissue. Detailed anatomical atlases of gene expression in the developing and adult brain are being developed, eg the US Gensat project (Gene Expression Nervous System Atlas), a fully public, searchable database of gene expression in all CNS cell types.

Proteomics Cell type-specific expression patterns of protein products must also be defined, with their subcellular localization on particular portions of neurons or in specific intracellular compartments. Proteomics is providing insights into general biological structures, as well as synapses, receptor complexes and other neuronal and glial features, including neurotransmitter and adhesion protein complexes, synaptic preparations and axo-glial junctions. Programmes are now underway to map the entire proteome of the human brain (Human Brain Proteome Project) and detailed 2D electrophoresis and mass spectrometry analyses have been carried out on the mouse brain and human foetal brain, CSF and various cultured cell lines. Such global datasets are essential for a systems biology approach. Comparative proteomics has also been used to study neurological and psychiatric disorders, including neurodegeneration, psychiatry, trauma, stroke and tumours.

Mutation analysis Phenotype-driven approaches in model systems are important in bridging the gap between gene identification and understanding gene function. A number of centres are conducting genome-wide, phenotype-driven mutagenesis (eg ENU) screens in the mouse, to detect and characterise neurological and behavioural phenotypes. Phenotypic screens include neuroendocrine and behavioural responses to stress, learning and memory, psychostimulant response, vision, and circadian rhythm.

Challenges There are major difficulties for microarray and proteomics studies in using brain in general and postmortem brains in particular. They result from heterogeneity of the starting material, coupled with problems of standardisation due to the anatomical and physiological complexity and variability of clinical phenotypes. Availability of postmortem material is limited, and there is extreme diversity with respect to age, race, postmortem interval, medication history, etc. DNA microarray analysis of brain tissue is highly complex and expression changes may affect only subpopulations of cells; as a result, their magnitude is often modest and hard to separate from noise. Moreover, as many neurons project to remote areas, transcript and protein changes may occur in different compartments. The transcriptome is also shaped by medication, so that distinguishing effects of disease from those of treatment is a challenging aspect.

Benefits for diagnosis and therapy Although the diagnostic specificity of the observed gene expression changes in brain microarray studies is often unknown, major advances in diagnosis and treatment may result if they can be developed into quantitative traits correlated with genetic variation and neurobehavioural phenotypes. The identification of genes expressed in particular classes of neurons linked to specific diseases may provide new drug targets for the treatment of stroke, spinal cord injury, neurodegenerations, tumours, schizophrenia, depression, anxiety disorders and addiction. It is particularly exciting to consider that human psychiatric disorders could be accurately diagnosed by these means and broken down into subphenotypes with different contributing genes, with enormous implications for the more effective use of therapeutics and identification of new drug targets.

Standardisation One of the areas which will be of particular interest to the programme is the development of standards for bioinformatics and databases, which is especially relevant to neurogenomics. The data generated by high throughput approaches must be organized into freely available, central repositories that are easily accessed by the community. Data must be mapped onto a digital brain atlas, accessible through graphical interfaces, which can integrate quantitative gene expression analysis with histochemical data from immunostaining and in situ hybridization. Such a database will require standards for data organisation and display, as well as for data collection that are compatible with the digital atlas framework. The current absence of such standards is severely limiting the utility of data that are already being generated by existing large-scale efforts.

Systems biology & neurogenomics A second area of particular focus in this programme is the application of the systems biology approach, which is now poised to tackle neurobiological questions. In a general neurosystems strategy, integrating all functional genomics approaches, behaviour and brain are broken down into a hierarchy with genome, transcriptome and proteome studies at the base and electrophysiological, anatomical and behavioural analysis at higher levels. The general goal is to obtain large scale data from multiple levels of analysis and then integrate and model it.

Metabolomics
Metabolomics is the study of the metabolome, the entire metabolic content of a cell or organism at a given moment. Its novelty is the attempt to tabulate and quantify all the small molecules within a sample, to find new markers for disease or metabolite patterns as indicators of nutritional status. Metabolomics has been studied in microorganisms and in plants, though less systematic work has so far been performed on animals or humans, where it has generally concentrated on biofluids and a greater attention to cells is needed. Examining metabolomics, or changes in metabolic profiles, will be an important part of an integrative approach for assessing gene function and relationships to phenotypes, as well as having potential implications in biomedicine.

Size of the metabolome Several general questions arise about the size, nature, structure and organisation of metabolic networks. Annotated genomic data alone can provide the baseline of reactions and although these are available in the general metabolic databases such as KEGG (Kyoto encyclopaedia of genes and genomes), it is necessary to produce organism-specific ones. It is clear that we have only just begun to recognise how many metabolites a typical cell can contain or produce. Studies of Arabidopsis thaliana indicated the presence of some 326 metabolites, but better deconvolution of the data has raised this to over 1000. The latest E. coli model has 931 unique biochemical reactions. The yeast metabolic reaction scheme is especially useful, as in addition to genomics data it also exploits biochemical and physiological knowledge, sequence matching and reaction guessing. These models give numbers of metabolites in the hundreds, which are comparatively easy to handle, and while these will be underestimates due to imperfect knowledge, the lack of specificity among enzymes, and the production of substances at very low concentrations that are not routinely detected, they form a good starting point. A candidate mouse metabolome is already available, and one for humans must be imminent.

Methods The first requirement is to have available techniques that are as comprehensive as possible for metabolic analyses. As the chemistry of different metabolites is very heterogeneous, isolating and measuring them all together is problematic, and most metabolic studies are really ‘metabolic profiling' of subsets of chemical classes. Recent advances incorporate NMR spectroscopy, mass spectrometry (MS), chromatographic analysis, and metabolic network analysis models to estimate cellular metabolic fluxes. As well as increasingly refined gas chromatography (GC)-MS methods, multidimensional separation methods are coming to the fore as they can routinely separate more than 1000 compounds. Although it will probably never get close to matching MS for sensitivity, NMR continues to improve in resolution and sensitivity. Its chief virtue is arguably its non-invasive nature, which can allow one to obtain spatially resolved metabolic profiles and to investigate metabolomics in vivo.

Applications in biomedicine Several significant findings of basic or applied biomedical interest have emerged from recent metabolomic studies. In nutritional studies, metabolomics has excellent potential for evaluating subtle differences in individiuals in the metabolic response to diet. It is also vital to gain an understanding of the normal human serum metabolome in health, where interesting diet-dependent or disease-related changes could be observed. Metabolic changes during tumour cell proliferation has been studied utilising metabolomic approaches. The tumour metabolome is characterized by high glycolytic and glutaminolytic capacities, high phosphometabolite levels and channeling of glucose carbons to synthetic processes, which allows tumour cells to proliferate over broad variations in oxygen and glucose supply. This type of research will allow for a better understanding of metabolism in tumour cells and thus approaches that might be effective in altering their rates of proliferation. A major driver in metabolomics is discovering biomarkers of disease status, eg to distinguish various forms of coronary heart disease via blood samples. There is much interest in determining the mode or site of action of compounds, in functional genomics, in target discovery and toxicity assessment, which are thereby interrelated. Since the methods are generic, metabolomics will increase in importance in toxicology, mode of action analysis and functional genomics.

Metabolomics & systems biology There is a huge interest in understanding complex biological systems from the systems point of view, combining quantitative experimentation and mathematical simulation/modelling in an iterative fashion. Metabolomic data is used for the large-scale reconstruction of biological systems and for the generation of both testable hypotheses and the predictive models that lie at the heart of systems biology. As part of a systems analysis, metabolic information is useful for determining the route and function of metabolic pathways. Measurement and comparison of metabolite concentration and flux under different conditions can also provide information about the active regulatory mechanisms and metabolic networks and the role of unknown genes. One strategy combines the networks that are reconstructed qualitatively from the genomic data with the constraints imposed by quantitative determinations. This allows one to make some successful predictions of whole cell behaviour at the metabolic and physiological levels from such in silico analysis alone. It also highlights the importance of the topological structure of metabolic networks (independently of their kinetic properties), to their effective functioning. An especially helpful analysis of metabolic networks is in terms of ‘network motifs' which, by loose analogy with protein structural motifs, are arrangements of reactions, including feedback structures, which regularly occur in biology and are therefore assumed to have functional use. Of all the possible feedback arrangements between separate elements, a very restricted subset is found to occur regularly in nature (ie to have been selected by evolution). Although metabolomics measurements have a major role to play in metabolic network reconstruction, true systems biology will require the integration of metabolomic measurements with measurements of the time-dependent concentrations of other types of components, ie mRNA and proteins. It will be of particular interest to integrate metabolic models in genomically characterised organisms with their experimentally determined metabolomes to allow an iterative improvement of our understanding of the latter. This can be seen as the hallmark and purpose of the systems biology agenda.

Pharmacogenomics
The term pharmacogenomics describes a polygenic or genome-wide approach to identifying genetic determinants of drug response, utilising both information from the human genome project and technologies such as high throughput sequencing, DNA and protein microarrays, and bioinformatics. Improving therapeutic efficacy and reducing drug toxicity are two of the most important goals of genomics and genetics in clinical practice. While there is a growing list of polymorphisms in genes encoding drug-metabolising enzymes, drug transporters and drug targets, as well as disease-modifying genes, most drug effects and treatment outcomes are determined by the interplay of multiple genes. Thus, while early studies identifed highly penetrant, single-gene traits, future advances hinge on the more difficult challenge of elucidating multigene determinants of drug response. The integration of genomic tests with extensive phenotypic characterisation of uniformly treated patients is essential in order to define the inherited nature of most drug effects. Genome information may in the future be used widely to target drugs more accurately and to improve their therapeutic usefulness. This intersection of genomics and medicine also has the potential to yield a new set of molecular diagnostic tools that can be used to individualise and optimise drug therapy. There are many challenges to overcome in order to understand fully the contribution of polymorphisms to individual differences in drug effects and to translate it into clinical practice.

Methods To make an informed prediction of the genes in which polymorphisms might affect the disposition or response to a given drug, the 'candidate gene' or 'candidate pathway' approach has often been used. In many cases, known phenotypic variability in the gene product has guided the search for a genetic basis for such differences. Functionally important polymorphisms can include amino acid substitutions, promoter or enhancer polymorphisms, gene duplications, synonymous coding SNPs that affect transcript stability, or intronic SNPs that cause splice variants that create early stop codons. The candidate gene approach can fail for several reasons, eg other mechanisms alter protein function (for example, post-translational modifications). Genome-wide approaches, such as expression arrays, genome-wide scans or proteomic assays, can identify previously unrecognized candidates, eg by detecting genes or proteins whose expression differentiates drug responders from non-responders. Transcriptome array and proteomics have the advantage that the level of the signal may directly reflect functional variation. There are pitfalls, eg such experiments are highly influenced by choice of tissue, which may or may not reflect that of concern for toxicity and response, so that false negatives are possible, while the large number of transcripts or proteins that can be interrogated may give false-positives. Once a gene or its product has been implicated in a drug response, large-scale molecular epidemiological association studies (in vivo or in vitro with human tissues), biochemical functional studies and pre-clinical animal models of candidate gene polymorphisms can be used to further establish genetic variability as a determinant of specific drug effects.

Applications Pharmacogenomics may enhance drug discovery and development by the identification of new targets, eg through the discovery of genes that are under- or overexpressed in cancer cells that are sensitive to anticancer agents compared with those that are resistant. The products of such overexpressed genes represent plausible targets for inhibitors that could reverse the drug-resistance phenotype. Gene expression in target tissues can be used to ascertain the effects of chemotherapy and how cells respond to treatment with single drugs or combinations.

A much discussed application is the development and use of drugs in specific, identifiable subpopulations. Pharmacogenomic approaches to prescribing drugs bear the twofold promise of improved treatment outcomes by allowing the a priori selection of individuals who are likely to respond to a given medicine and of reducing the incidence of adverse events by similarly excluding patients who are likely to suffer such events. Indeed, establishing individual profiles of likely drug responses is already changing the practice and economics of medicine. Decreasing the frequency of adverse drug effects and increasing the probability of successful therapy will probably lower the cost of health care. Pharmacogenomics has the potential to facilitate this process by translating knowledge of human genome variability into better therapeutics. Few health care systems will allow patients to receive drugs that they are unlikely to benefit from, and new information will help to drive genomic diagnostics into routine practice.

Moreover, it is important for the pharmaceutical industry to be able to identify polymorphisms that predispose patients to adverse drug effects which, although they may occur in only a small subset of people treated with a new medication, are sufficiently toxic to jeopardize further development of the drug for all patients. One approach is to obtain genomic DNA from patients entered on large phase III clinical trials of a new agent, and then retrospectively search for polymorphisms that predispose a small subset to toxicities. Where patients prone to adverse effect could be prospectively identified based on genotype, an otherwise efficacious new drug might be 'saved' from abandonment during development, or from withdrawal after approval and widespread use. Given the potential value of knowing all the possible factors that influence the effects of new agents, it is likely that pharmacogenomics will have an increasingly important role in drug discovery and development.

Predictive, Preventive and Personalised Medicine

Predictive medicine The Human Genome Project and associated technology developments have accelerated the process of analysing entire genomes. In turn this has catalysed the major development of predictive, preventive and personalised medicine which will impact profoundly on clinical practice. In particular, it has provided access to the extensive human genome variability in the form of SNPs, some of which predispose to disease. This knowledge introduces the prospect of clinical prognosis based on identification of susceptibility genes. It is likely that a predictive medicine will gradually emerge, capable of determining a probabilistic ‘future health history' for each individual. This will require ability to analyse the relevant DNA sequences at lower cost and higher throughput, a declared aim being to sequence a human genome for less than $1000 in a fraction of an hour. This would make it realistic to examine the polymorphisms of the 30,000 or so genes for each individual and make statements about disease likelihood. Disease-related SNPs could then be used, in combination with other factors, to define populations and individuals at risk, so the potential may be realised of informing an individual that, with some probability or with near certainty, he or she will suffer from a disease of greater or lesser severity. In addition, genomic tools will define disease subtypes more precisely on the basis of their individual pathophysiology and responsiveness to therapy.

An important problem is to understand the molecular basis of polygenic or multifactorial disorders. The genetic dissection of common diseases involves association studies performed on large, tiered population samples and family-based controls, and there is still much to learn about the complex path from genotype to phenotype, providing a major challenge for functional genomics in the area of molecular medicine. Thus, the main limitation on the deployment of broad DNA-based tests to assess individual susceptibilities to diseases like cancers, cardiovascular disease and stroke is the sparse knowledge of how different combinations of genetic variations might affect predisposition to any particular condition. At present, too little is known about which SNPs to type for non-Mendelian disease, while the clinical application of the HapMap will be limited, at least in the short term, because varying linkage disequilibrium patterns in different populations make it unsuitable for unsorted patient samples. Therefore it will probably be some time before we see widespread use of human genetic diagnostics to assess disease susceptibility and future risk, other than for some inherited cancers (eg breast cancers caused by mutations in BRCA1 and BRCA2) and simple genetic diseases.

As far as diagnosis is concerned, through use of mRNA and protein expression profiling, we can expect data to be generated on multiple biomarkers that vary quantitatively with disease onset, disease progression or therapeutic response, leading to the ability to define disease subtypes and predict response to a drug or adverse reactions (see also pharmacogenomics, above). Simultaneous systematic, multiparametric analysis of biomarkers (mRNA, protein or small molecules, particularly in plasma) will be required for disease prediction. Currently these approaches have not reached routine diagnostics and clear evidence of the predictive strength of these markers in clinical settings remains essential for their implementation.

Preventive and personalised medicine Along with the ability to predict susceptibility, eg to complex and late-onset diseases, will come a need to adopt preventive strategies based on dietary measures, control of environmental factors, early therapy or targeted screening. Inevitably, medicine will become oriented towards disease prevention rather than efforts to cure people at later stages of illness. Because individuals will have different potential disease combinations, medicine will also become highly personalised, with the knowledge of individual genetic makeup and lifestyle influences. Improved disease understanding and definition will make treatments available for individuals who cannot be helped today because their clinical syndromes do not fit neatly into a traditional category. As noted above (pharmacogenomics), DNA-based screens will provide information to aid in dosing individual medications, avoid side effects and to predict drug non-responders on the basis of polymorphisms in drug targets or pathways. While the opportunities for clinical genomics to become a mainstream component of clinical medicine are now apparent and the move to the clinic inevitable, it will nevertheless take time to implement.

Predictive and personalised medicine & systems biology The effectiveness of predictive and personalised medicine will hinge on characterisation of biological systems in their normal states and definition of the molecular basis for their pathology, tasks that will require an integrative, systems biology approach. Systems biology in medicine will manifest itself in at least two major forms. First, it will continually improve the capacity to understand and model biological systems on a more global and in-depth scale than hitherto. Secondly, the development of new technologies will enhance the efficiency, scale and precision with which cellular measurements are made. This latter influence will facilitate all aspects of healthcare, including detection and monitoring of diseases, drug discovery, and treatment evaluation. To fully realise the potential of these technologies and insights, however, a number of major challenges remain. First systems biology must be more widely utilised. This will require developing new global technologies for genomics, proteomics, metabolomics and phenotyping, as well as software that can capture, store, analyse, graphically display, integrate, model and disperse global data sets across the dynamic transitions of development or physiological responses. The natures of protein and gene regulatory networks and their integrations must be determined. Finally, there must be access to biological samples from large numbers of normal and diseased patients to begin the global correlative studies that will establish the foundation of predictive medicine and pave the way for preventive medicine (see Biobanking). In the long run functional genomics will create a new approach to clinical practice with many benefits for patients.