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The elevated plus maze test is used to measure anxiety-like behavior in rodents. It can be used to gain insight into conditions such as posttraumatic stress disorder (PTSD) and other conditions marked by anxious behavior. It can also be used as a component in screening of novel compounds for anxiolytic properties. This model is based on aversion to open spaces, which is seen as the animal spending more time in the enclosed arms of the maze. This chapter describes the steps necessary for setting up and conducting the test, along with interpretation of the results.

The open field test is used in studies of the neurobiological basis of anxiety and screening for novel drug targets and anxiolytic compounds. This test uses a camera to measure movement of the test animal in the peripheral and central zones of a 42 × 42 × 42 cm polyvinyl chloride box. This chapter describes a protocol for carrying out the open-field test for assessment of locomotion and anxiety-like behavior in mice.

Psychiatric disorders affect approximately one quarter of people worldwide at some point in their lifetime. This chapter provides a step-by-step guide to conduct behavioral tests in adult mice for investigations of social behavior, without the need for specific equipment. This test should allow the identification of key abnormalities in social interactions that can be followed up by targeted, more complex, behavioral analysis aimed at identification of new biomarkers and potential drug targets.

The forced swim test assesses learned helplessness, which is a feature of depression-like behavior in rodents. This test has also been used in testing the efficacy of existing and novel antidepressant drugs. It is based on the natural tendency of rodents to escape from water. Rodents are placed in a cylinder filled with water and the presumption is that those with a depression-like phenotype give up swimming earlier than those that are not depressed. Furthermore, antidepressant drugs reverse this effect. This chapter describes the basic setup and conduction of the test, along with interpretation of the results. It should be emphasized that this test should be conducted as part of a series of behavioral assessments in order to increase the accuracy of the results.

The Goto-Kakizaki (GK) rat is a model of type 2 diabetes mellitus, produced originally by selective inbreeding for a hyperglycemic trait. These rats are characterized as having insulin resistance and an insulin secretory defect. Pancreatic islets from these mice show abnormal morphology in the distribution of glucagon-secreting α cells and insulin-secreting β cells, which may affect paracrine functions involved in the secretory response. This chapter describes the characterization of GK rat islets using immunofluorescence microscopy and Western blot analyses to demonstrate the effects on islet architecture and how this translates to changes in expression of predominant islet proteins.

The quest for increased human longevity has been a goal of mankind throughout recorded history. Recent molecular studies are now providing potentially useful insights into the aging process which may help to achieve at least some aspects of this quest. This chapter will summarize the main findings of these studies with a focus on long-lived mutant mice and worms, and the longest living natural species including Galapagos giant tortoises, bowhead whales, Greenland sharks, quahog clams and the immortal jellyfish.

This chapter describes the use of lenses obtained from rats as a model of cataractogenesis. At the molecular level, this is visualized as reduced activity of oxidative reductive enzymes such as aldose reductase and increased proteolysis of lens structural proteins including vimentin. In this chapter, protocols for assessment of these two pathways are presented. Specifically, this analysis shows a comparison of aldose reductase activity and vimentin cleavage in male and female rat lenses. This is because female rats are more susceptible to cataract formation compared to males.

Schizophrenia is a severe and debilitating psychiatric disorder believed to have neurodevelopmental origins. Several studies have associated energy metabolism dysfunction with the disorder, mostly related to glycolysis alterations. Glucose is the obligatory energy substrate of the brain and glycolysis is the first step for its metabolism. This takes place predominantly in glial cells, astrocytes and oligodendrocytes, whereas neurons present a predominant oxidative profile. Thus, glial cells generate either lactate or pyruvate to neurons for ATP production. In addition, some aspects of schizophrenia may reflect an advanced aging phenotype with effects on various neural cell types at different stages of the disease. Given the role of glial cells in brain energy metabolism, the association of glycolysis dysfunction and the accelerated aging of neuronal cells in schizophrenia, studies focusing on those aspects can yield important insights into the causes and implications of the disorder. In turn, this may lead to novel therapeutic strategies for improved treatment of individuals suffering with this disorder.

The identification of biomarkers for toxicity is becoming increasingly important for drug discovery and development. This chapter describes the preparation and utilization of primary rat hepatocytes as a cellular model of steatosis. A protocol is presented for dosing the cells with the steatosis-inducing compound amiodarone, along with the conduction of assays for measuring lipid accumulation and mitochondrial function. A differential solubility extraction procedure is also presented, which can be used for proteomic profiling analysis.

This chapter presents a method for assessing general behavior, well-being, and sensorimotor gating. A detailed protocol is given for assessment of nest building performance in female mice using a strict scoring system. The test can be used for assessing moods and behaviors associated with psychiatric disorders such as depression and schizophrenia. It can also be useful for testing movement disorders such as Parkinson disease.

The Y-maze can be used to assess short term memory in mice. Spontaneous alternation, a measure of spatial working memory, can be assessed by allowing mice to explore all three arms of the maze and is driven by an innate curiosity of rodents to explore previously unvisited areas. A mouse with intact working memory, and hence intact prefrontal cortical functions, will remember the arms previously visited and show a tendency to enter a less recently visited arm. Spatial reference memory, which is underlined by the hippocampus, can also be tested by placing the test mice into the Y-maze with one arm closed off during training. After an inter-trial interval of for example 1 h, the mouse should remember which arm it has not explored previously and should visit this arm more often. This chapter describes the pre-test conditions, the materials required and the protocol for conducting and interpreting the results of these two related tests.

Although human life expectancy has increased significantly over the last two centuries, this has not been paralleled by a similar rise in healthy life expectancy. Thus, an important goal of anti-aging research has been to reduce the impact of age-associated diseases as a way of extending the human healthspan. This review will explore some of the potential avenues which have emerged from this research as the most promising strategies and drug targets for therapeutic interventions to promote healthy aging.

This chapter presents characterization of the db/db mouse model of type 2 diabetes. The protocol describes generation of the mice along with measurements of physical characteristics such as body weight and body composition, and behaviours including food and water consumption. Such a procedure can also be used for omics-based analyses of brain tissues to investigate the potential use of db/db mouse model in neuropsychiatric research. In addition, we present measurement of blood glucose and plasma insulin levels which tend to be elevated in this model and can modulated post treatment with anti-diabetic agents. All of these characteristics can be used for studying the pathophysiology and for monitoring response to potential therapeutics.

This chapter describes the propagation and characterization of transplantable insulinoma cells as model of insulin-producing pancreatic islet cells in the rat. Here, the cells are propagated by transplantation into rats followed by harvesting after growth for approximately 1 month. The cells are then purified by Percoll density gradient centrifugation and characterized by pulse-chase radiolabelling and immunoprecipitation of the insulin-related peptides. The results show that the transplantable insulinoma cells produce insulin in a manner similar to that found in normal pancreatic islets.

Obsessive-compulsive disorder (OCD) can occur in several psychiatric illnesses such as autism spectrum disorders (ASD) and it is more prevalent in children. This condition is characterized by repeated and apparently meaningless behaviors such as frequent hand washing, counting, tapping, and rocking. This can disrupt normal socialization and in some cases lead to self-harm. Therefore there is interest in developing more effective therapies for individuals suffering from these conditions. This chapter describes how to conduct the mouse marble burying test as a sensitive measure of compulsive behaviors.

The islets of Langerhans release vital hormones involved in the regulation of blood sugar and other aspects of metabolism. The islets are housed in diffuse clusters of cells within the exocrine pancreas and, therefore, purification of these cells for research or transplant purposes is a difficult undertaking. Here, a detailed protocol is presented for purification of islets from rat pancreas using limited collagenase digestion and step gradient centrifugation techniques. In addition, a method for assessing islet viability is presented using perifusion under both basal and stimulatory glucose conditions, with measurement of the hormone released using an immunoassay for insulin.

Schizophrenia and diabetes have been known to be linked disorders for decades. One reason is due to the fact that a major side effect of antipsychotic medication treatment is metabolic syndrome, which increases the risk of the patients developing type 2 diabetes and cardiovascular disorders. However, signs of metabolic syndrome in schizophrenia patients were identified more than 100 years ago, even before the development of antipsychotic drugs. This suggests that schizophrenia itself predisposes towards diabetes and, in turn, insulin resistance may be a risk factor for the development of schizophrenia. This review summarizes the findings surrounding this issue and places them into context with regards to increasing our understanding of the aetiology of schizophrenia and in support of biomarker and drug discovery efforts.

This chapter reviews the efficacy of the ketogenic diet in a variety of neurodegenerative, neurodevelopmental and metabolic conditions throughout different stages of life. It describes conditions affecting children, metabolic disorders in adults and disorderrs affecting the elderly. We have focused on application of the ketogenic diet in clinical studies and in preclinical models and discuss the benefits and negative aspects of the diet. Finally, we highlight the need for further research in this area with a view of discovering novel mechanistic targets of the ketogenic diet, as a means of maximising the potential benefits/risks ratio.

Interest in the modulation of endocannabinoid signaling has increased since the discovery of receptors for compounds of Cannabis sativa. Endocannabinoids are crucial neuromodulators of many brain functions and changes in the ligands and their receptors have been associated with psychiatric disorders, such as schizophrenia. Genetic, neuroimaging, and behavioral studies have reinforced the role of endocannabinoids in the pathobiology of schizophrenia. However, molecular pathways and biological processes involved in cannabinoid effects are not totally understood. Additionally, the endocannabinoid signaling network with other non-cannabinoid targets, and the effects of phytocannabinoids increase the complexity to understand their role in schizophrenia and homeostasis conditions. Thus, proteomic studies can provide evidence about the involvement of cannabinoid receptors, as well as the metabolic and synthetic enzymes of the endocannabinoids in these disorders. Additionally, quantification of endocannabinoids in the blood serum or cerebrospinal fluid can be a useful approach to identify new biomarkers in schizophrenia, and lipidomic techniques can be used to quantify these compounds. Herein, the authors review proteomic and lipidomic studies that have been used for analysis of the endocannabinoid system in healthy and schizophrenia function. The findings may contribute to understand the involvement of endocannabinoids in the brain and in the neurobiological basis of schizophrenia., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This is the first study to identify lipidomic markers in plasma associated with response of acutely ill schizophrenia patients in response to specific antipsychotic treatments. The study population included 54 schizophrenia patients treated with antipsychotics for 6 weeks. Treatment led to significant improvement in positive and negative symptoms for 34 patients with little or no improvement for 20 patients. In addition, 37 patients showed an increase in body mass index after the 6 week treatment period, consistent with effects on metabolism and the association of such effects with symptom improvement. Profiling of plasma samples taken prior to therapy using liquid chromatography tandem mass spectrometry (LC-MS/MS) resulted in identification of 38, 10, and 52 compounds associated with the olanzapine, risperidone, and quetiapine treatment groups, which could be used to distinguish responders from non-responders. Limitations include the retroactive active nature of the study and the small sample size. Further investigations with larger sample sets could lead to the development of a molecular test that could be used to help psychiatrists determine the best treatment options for each patient.

Oxidative stress and immune dysregulation have been linked to schizophrenia and depression. However, it is unknown whether these factors are related to the pathophysiology or whether they are an epiphenomenon. Inconsistent oxidative stress-related findings in previous studies may have resulted from the use of different biomarkers which show disparate aspects of oxidative stress. Additionally, disease severity, medication, smoking, endocrine stress axis activation and obesity are potential confounders. In order to address some of these shortcomings, we have analyzed a broader set of oxidative stress biomarkers in our exploratory study, including urinary 8-iso-prostaglandin F2α (8-iso-PGF2α), 8-OH-2-deoyxguanosine (8-OH-2-dG), and blood levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione S-transferase (GST) in acutely ill drug-naïve first episode patients with schizophrenia (n = 22), major depression (n = 18), and controls (n = 43). Possible confounding factors were considered, and patients were followed-up after 6 weeks of treatment. No differences were observed regarding 8-OH-2-dG, MDA and GST. At baseline, 8-iso-PGF2α levels were higher in patients with schizophrenia (p = 0.004) and major depression (p = 0.037), with a trend toward higher SOD concentrations in schizophrenia (p = 0.053). After treatment, schizophrenia patients showed a further increase in 8-iso-PGF2α (p = 0.016). These results were not related to age, sex, disease severity, medication or adipose tissue mass. However, 8-iso-PGF2α was associated with smoking, endocrine stress axis activation, C-reactive protein levels and low plasma concentrations of brain-derived neurotrophic factor. This study suggests a role of lipid peroxidation particularly in drug-naïve acutely ill schizophrenia patients and highlights the importance of taking into account other confounding factors in biomarker studies.

In metabolic diseases such as obesity and type 2 diabetes mellitus, the conversion of proinsulin to mature insulin can be impaired. This could mean that insulin molecules with lower activity toward the insulin receptor can be released under conditions of high metabolic demand, resulting in an inadequate glucoregulatory response. The chapter describes a fluorescent monoclonal antibody-based protocol for measurement of human proinsulin and the proinsulin conversion intermediates (split proinsulins). An example assay is presented using serum from non-diabetic, normal body mass index individuals.

Early parental nutritional interventions during prenatal development have been shown to result in neuropsychiatric sequelae in the adult offspring. In order to understand the impact of such nutritional interventions, the behavior of the animal has to be carefully analyzed. This chapter provides a step-by-step guide to conduct behavioral tests in adult mice for investigators without specific expertise or those without the equipment to carry out behavioral studies. We focus on tests tapping into the main behavioral abnormalities that correspond to mental illnesses. We describe the materials required and the detailed methods to conduct global assessment of parameters such as behavioral integrity and general well-being, psychomotor activity, social behavior, repetitive behavior, anxiety-like behavior, depression-like behavior, short-term spatial working memory, and spatial reference memory.

Over the last few decades, evidence has emerged that the pathogenesis of psychiatric disorders such as schizophrenia can involve perturbations of the hypothalamic-pituitary-adrenal (HPA) axis and other neuroendocrine systems. Variations in the manifestation of these effects could be related to differences in clinical symptoms between affected individuals and to differences in treatment response. Such effects can also arise from the complex interaction between genes and environmental factors. Here, we review the effects of maternal stress on abnormalities in HPA axis regulation and the development of psychiatric disorders such as schizophrenia. Studies in this area may prove critical for increasing our understanding of the multidimensional nature of mental disorders and could lead to the development of improved diagnostics and novel therapeutic approaches for treating individuals who suffer from these conditions.

The emergence of next-generation sequencing (NGS) over the last 10 years has increased the efficiency of DNA sequencing in terms of speed, ease, and price. However, the exact quantification of a NGS library is crucial in order to obtain good data on sequencing platforms developed by the current market leader Illumina. Different approaches for DNA quantification are available currently and the most commonly used are based on analysis of the physical properties of the DNA through spectrophotometric or fluorometric methods. Although these methods are technically simple, they do not allow exact quantification as can be achieved using a real-time quantitative PCR (qPCR) approach. A qPCR protocol for DNA quantification with applications in NGS library preparation studies is presented here. This can be applied in various fields of study such as medical disorders resulting from nutritional programming disturbances.

Under- or overfeeding during pregnancy can lead to behavioral deficits in the offspring in later life. Here, we present a protocol for setting up and carrying out the hyperlocomotion test for assessing behavioral symptoms such as psychosis or mania. As an example, we use the acute rat phencyclidine-injection model which exhibits hyperlocomotion and stereotypic behaviors, resembling the positive symptoms of schizophrenia.

Developmental programming resulting from maternal malnutrition can lead to an increased risk of metabolic disorders such as obesity, insulin resistance, type 2 diabetes and cardiovascular disorders in the offspring in later life. Furthermore, many conditions linked with developmental programming are also known to be associated with the aging process. This review summarizes the available evidence about the molecular mechanisms underlying these effects, with the potential to identify novel areas of therapeutic intervention. This could also lead to the discovery of new treatment options for improved patient outcomes.

Multiplex immunoassays are used for rapid profiling of biomarker proteins and small molecules in biological fluids. The advantages over single immunoassays include lower sample consumption, cost, and labor. This chapter details a protocol to develop a 5-plex assay for glucagon-like peptide 1, growth hormone, insulin, leptin, and thyroid-stimulating hormone on the Luminex ® platform. The results of the analysis of insulin in normal control subjects are given due to the important role of this hormone in nutritional programming diseases.

Peripheral blood mononuclear cells (PBMCs) have been used as a surrogate model of immune function in studies of multiple medical areas, such as metabolic diseases and immune dysfunction. This chapter describes a standardized technique for blood draw and preparation of PBMCs from whole blood using density gradient centrifugation, followed by cell culture. The main focus is on collection of the PBMC culture media and extraction of cellular proteins in order to provide the materials for biomarker studies.

Many diseases result from programming effects in utero. This chapter describes recent advances in proteomic studies which have improved our understanding of the underlying pathophysiological pathways in the major psychiatric disorders, resulting in the development of potential novel biomarker tests. Such tests should be based on measurement of blood-based proteins given the ease of accessibility of this medium and the known connections between the periphery and the central nervous system. Most importantly, emerging biomarker tests should be developed on lab-on-a-chip and other handheld devices to enable point-of-care use. This should help to identify individuals with psychiatric disorders much sooner than ever before, which will allow more rapid treatment options for the best possible patient outcomes.

Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labeled proteins can then be immunoprecipitated and analyzed by electrophoresis and gel imaging techniques. This chapter presents a protocol for the biosynthetic labeling and immunoprecipitation of pancreatic islet proteins which are known to be affected in disorders such as diabetes, obesity, and metabolic syndrome.

There is a strong association between a suboptimal maternal environment and increased risk of developing age-associated diseases such as type 2 diabetes, obesity, and cardiovascular disease in the offspring. Blood clotting time may be altered in all of these conditions, and it is also an important factor that requires monitoring in postoperative and cardiovascular disorder patients who are on coagulant medications. This chapter describes patient self-management of blood coagulation activity using a test strip device and the Coagu app. The app can also be used as a reminder of treatment times and for monitoring the effects of treatment over time.

This chapter describes innovations in biomarker testing that can facilitate earlier and better treatment of patients who suffer from metabolic disorders. The use of new microfluidic devices along with miniaturized biosensors and transducers enables analysis of a single drop of a blood within the time frame of a typical visit to a doctor's office. Steps are underway so that these approaches will incorporate both biochemical and clinical data, resulting in unique bioprofiles for each patient. This will allow earlier, personalized, and more effective therapeutic options. In addition, smartphone apps for self-monitoring will be used increasingly for the best possible patient outcomes.

This chapter describes the basics of two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of two distinct proteomes. The example given describes the analysis of male and female rat lens soluble proteins labeled with fluorescent Cy3 and Cy5 dyes in comparison to a pooled standard labeled with Cy2. After labeling the proteomes are mixed together and electrophoresed on the same 2D gels. Scanning the gels at wavelengths specific for each dye allows direct overlay the two different proteomes. Differences in abundance of specific protein spots can be determined through comparison to the pooled standard.

This chapter describes a proteomic analysis of neural progenitor cells using isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry. A detailed procedure is described for the isolation, proliferation, and differentiation of these cells, including a comparative iTRAQ mass spectrometry analysis of the precursor and differentiated states. In total, there were changes in the levels of 55 proteins, many of which are not resolved easily by other proteomic methods. Therefore, this method should be useful for the identification of important regulatory molecules in the study of other precursor cells involved in neuronal or metabolic regulation in nutritional programming diseases.

Many chronic diseases are associated with hypothalamic-pituitary-adrenal axis dysfunction. Therefore, proteomic profiling of the pituitary gland has potential to uncover new information on the underlying pathways affected in these conditions. This could lead to identification of new biomarkers or drug targets for development of novel therapeutics. Here we present a protocol for preparation of pituitary protein extracts and analysis of the major hormones and accessory proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS). The same methods can be applied in the study of other tissues of the diffuse neuroendocrine system.

Lab-on-a-chip assays allow rapid analysis of one or more molecular analytes on an automated user-friendly platform. Here we describe a fully automated assay and readout for measurement of vitamin D levels in less than 15 min using the Fraunhofer in vitro diagnostics platform. Vitamin D (25-hydroxyvitamin D 3 [25(OH)D 3 ]) dilution series in buffer were successfully tested down to 2 ng/mL. This could be applied in the future as an inexpensive point-of-care analysis for patients suffering from a variety of conditions marked by vitamin D deficiencies.

Poor nutrition during pregnancy leads to an increased risk of metabolic disorders and other diseases in the offspring. This can be modelled in animals through manipulation of the maternal diet. One such model is the maternal low-protein rat which gives rise to offspring characterized by insulin resistance. This chapter gives a detailed protocol for generation of the maternal low-protein rat, which has been used in the study of several disorders including diabetes and psychiatric disorders.

The current inability of clinical psychiatry to objectively select the most appropriate treatment is a major factor contributing to the severity and clinical burden of major depressive disorder (MDD). Here, we have attempted to identify plasma protein signatures in 39 MDD patients to predict response over a 6-week treatment period with antidepressants. LC-MS/MS analysis showed that differences in the levels of 29 proteins at baseline were found in the group with a favorable treatment outcome. Most of these proteins were components of metabolism or immune response pathways as well as multiple components of the coagulation cascade. After 6 weeks of treatment, 43 proteins were altered in responders of which 2 (alpha-actinin and nardilysin) had been identified at baseline. In addition, 46 proteins were altered in non-responders and 9 of these (alpha-actinin, alpha-2-macroglobulin, apolipoprotein B-100, attractin, C-reactive protein, fibrinogen alpha chain, fibrinogen beta chain, nardilysin and serine/threonine-protein kinase Chk1) had been identified at baseline. However, it should be stressed that the small sample size precludes generalization of the main results. Further studies to validate these as potential biomarkers of antidepressant treatment response are warranted considering the potential importance to the field of psychiatric disorders. This study provides the groundwork for development of novel objective clinical tests that can help psychiatrists in the clinical management of MDD through improved prediction and monitoring of patient responses to antidepressant treatments.

Deletions on chromosome 22q11.2 are a strong genetic risk factor for development of schizophrenia and cognitive dysfunction. We employed shotgun liquid chromatography-mass spectrometry (LC-MS) proteomic and metabonomic profiling approaches on prefrontal cortex (PFC) and hippocampal (HPC) tissue from Df(16)A +/- mice, a model of the 22q11.2 deletion syndrome. Proteomic results were compared with previous transcriptomic profiling studies of the same brain regions. The aim was to investigate how the combined effect of the 22q11.2 deletion and the corresponding miRNA dysregulation affects the cell biology at the systems level. The proteomic brain profiling analysis revealed PFC and HPC changes in various molecular pathways associated with chromatin remodelling and RNA transcription, indicative of an epigenetic component of the 22q11.2DS. Further, alterations in glycolysis/gluconeogenesis, mitochondrial function and lipid biosynthesis were identified. Metabonomic profiling substantiated the proteomic findings by identifying changes in 22q11.2 deletion syndrome (22q11.2DS)-related pathways, such as changes in ceramide phosphoethanolamines, sphingomyelin, carnitines, tyrosine derivates and panthothenic acid. The proteomic findings were confirmed using selected reaction monitoring mass spectrometry, validating decreased levels of several proteins encoded on 22q11.2, increased levels of the computationally predicted putative miR-185 targets UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 kDa subunit (OGT1) and kinesin heavy chain isoform 5A and alterations in the non-miR-185 targets serine/threonine-protein phosphatase 2B catalytic subunit gamma isoform, neurofilament light chain and vesicular glutamate transporter 1. Furthermore, alterations in the proteins associated with mammalian target of rapamycin signalling were detected in the PFC and with glutamatergic signalling in the hippocampus. Based on the proteomic and metabonomic findings, we were able to develop a schematic model summarizing the most prominent molecular network findings in the Df(16)A +/- mouse. Interestingly, the implicated pathways can be linked to one of the most consistent and strongest proteomic candidates, (OGT1), which is a predicted miR-185 target. Our results provide novel insights into system-biological mechanisms associated with the 22q11DS, which may be linked to cognitive dysfunction and an increased risk to develop schizophrenia. Further investigation of these pathways could help to identify novel drug targets for the treatment of schizophrenia.

Since many aspects of schizophrenia are also manifested at the peripheral level in proliferating cell types, this chapter describes the analysis of skin fibroblasts biopsied from living patients. The method focuses on cell culture and sample preparation for characterization of the model. The resulting cell extracts can be analysed by any number of proteomic techniques for identification of biomarker candidates. This approach could help to elucidate the molecular mechanisms associated with the pathophysiology of schizophrenia and provide a useful model for a new target and drug discovery.

Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Radiolabeled newly synthesized proteins can be analyzed by a number of techniques such as two dimensional gel electrophoresis (2DE). This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with 35 S-methionine in the presence of basal and stimulatory concentrations of glucose, followed by subcellular fractionation to produce a secretory granule fraction and analysis of the granule protein contents by 2DE. This provides a means of determining whether or not the biosynthetic rates of the entire granule constituents are coordinately regulated.

This chapter describes the basics of two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of up to distinct proteomes. The example given describes the analysis of undifferentiated and differentiated neural precursor cells labelled with fluorescent Cy3 and Cy5 dyes in comparison to a pooled standard labelled with Cy2. After labelling, the proteomes are mixed together and electrophoresed on the same 2D gels. Scanning the gels at wavelengths specific for each dye allows direct overlay of the two different proteomes and the differences in abundance of specific protein spots can be determined through comparison to the pooled standard.

Proteomic-based biomarkers are now an integral part of the drug development process. This chapter covers the role of proteomic biomarker tests as useful tools for improving preclinical research and clinical development. One medical area that has been lagging behind this process is the study of psychiatric disorders, and this is most likely due to the complexity of these diseases. The potential of incorporating biomarkers in the clinical pipeline to improve decision-making, accelerate drug development, improve translation and reduce development costs is also discussed, with a focus on psychiatric diseases like schizophrenia. This chapter will also discuss the next steps that must be taken to keep moving this process forwards.

Leptomeningeal metastases (LM) from solid tumours, lymphoma and leukaemia are characterized by multifocal neurological deficits with a high mortality rate. Early diagnosis and initiation of treatment are essential to kerb neurological deterioration. However, this is not always possible as 25% of cerebrospinal fluid samples produce false-negative results at first cytological examination. The identification of biomarkers that allow stratification of individuals according to risk for developing LM would be a major benefit. Proteomic-based approaches are now in increasing use for this purpose, and these are reviewed in this chapter with a focus on cerebrospinal fluid (CSF) analyses. The construction of a CSF proteome disease database would also facilitate analysis of other neurological disorders.

Lab-on-a-chip assays allow rapid identification of multiple parameters on an automated user-friendly platform. Here we describe a fully automated multiplex immunoassay and readout in less than 15 min using the Fraunhofer in vitro diagnostics (ivD) platform to enable inexpensive point-of-care profiling of sera or a single drop of blood from patients with various diseases such as psychiatric disorders.

A major challenge in proteomic biomarker discovery and validation for psychiatric diseases is the inherent biological complexity underlying these conditions. There are also many technical issues which hinder this process such as the lack of standardization in sampling, processing and storage of bio-samples in preclinical and clinical settings. This chapter describes a reproducible procedure for sampling blood serum and plasma that is specifically designed for maximizing data quality output in two-dimensional gel electrophoresis, multiplex immunoassay and mass spectrometry profiling studies.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes that fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures. Here, we describe a method using the SensiFAST and SensiFAST One-Step probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.