Implementation of proteomic biomarkers: making it work.

While large numbers of proteomic biomarkers have been described, they are generally not implemented in medical practice. We have investigated the reasons for this shortcoming, focusing on hurdles downstream of biomarker verification, and describe major obstacles and possible solutions to ease valid biomarker implementation. Some of the problems lie in suboptimal biomarker discovery and validation, especially lack of validated platforms with well-described performance characteristics to support biomarker qualification. These issues have been acknowledged and are being addressed, raising the hope that valid biomarkers may start accumulating in the foreseeable future. However, successful biomarker discovery and qualification alone does not suffice for successful implementation. Additional challenges include, among others, limited access to appropriate specimens and insufficient funding, the need to validate new biomarker utility in interventional trials, and large communication gaps between the parties involved in implementation. To address this problem, we propose an implementation roadmap. The implementation effort needs to involve a wide variety of stakeholders (clinicians, statisticians, health economists, and representatives of patient groups, health insurance, pharmaceutical companies, biobanks, and regulatory agencies). Knowledgeable panels with adequate representation of all these stakeholders may facilitate biomarker evaluation and guide implementation for the specific context of use. This approach may avoid unwarranted delays or failure to implement potentially useful biomarkers, and may expedite meaningful contributions of the biomarker community to healthcare.

A novel cryptic peptide derived from the rat neuropeptide FF precursor reverses antinociception and conditioned place preference induced by morphine.

Neuropeptide FF (NPFF) precursors from different species contain at least three known neuropeptides, i.e. FF (FLFQPQRF-NH(2)), AF (AGEGLSSPFWSLAAPQR-NH(2)) and SF (SLAAPQRF-NH(2)). We demonstrate that the rat NPFF precursor contains another bioactive sequence, NAWGPWSKEQLSPQA, spanning between positions 85 and 99. Synthetic NPFF precursor (85-99) (10 and 20 nmol, i.c.v.) blocked the expression of conditioned place preference induced by morphine (5 mg/kg, s.c.). This peptide alone (10 and 20 nmol, i.c.v.) had no influence on the baseline latency of a nociceptive reaction but reversed the antinociceptive activity of morphine (5 mg/kg, s.c.) in the tail-immersion test in rats. These data suggest the existence of a novel bioactive cryptic peptide within an already known NPFF precursor.

Cryptic peptide derived from the rat neuropeptide FF precursor affects G-proteins linked to opioid receptors in the rat brain.

Recently, we reported the discovery of a novel amino acid sequence derived from the NPFF precursor NAWGPWSKEQLSPQA, which blocked the expression of conditioned place preference induced by morphine and reversed the antinociceptive activity of morphine (5mg/kg, s.c.) in the tail-immersion test in rats. Here, we name it as NPNA (Neuropeptide NA from its flanking amino acid residues). The synthetic peptide influenced the expression of mRNA coding for Galpha(i1), (i2), and (i3) subunits. The results provide further evidence that yet another bioactive sequence might be present within the NPFF precursor.

The European Innovative Medicines Initiative: Progress to Date.

The Innovative Medicines Initiative is a public-private partnership between the European Union and the pharmaceuticals industry that was established in 2008, with an overall budget of €5.3 billion from 2008 until 2024. The objective of the initiative is to boost pharmaceutical innovation in Europe and speed up the development of innovative medicines, vaccines and medical technologies, in particular in areas with high unmet needs. This article discusses the objectives of the initiative, its governance and main results and impact. The initiative has proved to be a unique platform for multi-stakeholder collaborations across Europe. It has contributed to the acceleration of the development process for medicines, from drug discovery to clinical development. The initiative has made important steps towards accessing and using real-world evidence for pharmaceutical research and development, and for healthcare decision-making. Several projects have contributed to a better understanding of the causes of diseases, and some are already delivering results, such as a vaccine against Ebola virus. The initiative has also significantly contributed to building capacity and resources for open use by the broader research and innovation community.

Neuropeptide FF (NPFF) reduces the expression of morphine- but not of ethanol-induced conditioned place preference in rats.

Neuropeptide FF (NPFF) has been described as an anti-opioid peptide. It plays a role in opioid antinociception, dependence and tolerance. Previous study has indicated that 1DMe ([D-Tyr(1), (NMe)Phe(3)]NPFF), a stable analog of NPFF, inhibits acquisition of the rewarding effect of morphine but not of ethanol in mice. The rewarding effects of these drugs were measured in the unbiased paradigm of conditioned place preference (CPP). The present study examines the influence of NPFF on the expression of morphine- and ethanol-induced CPP in the biased procedure in rats. Our experiments showed that NPFF, given intracerebroventricularly (i.c.v.) at the doses of 5, 10 and 20 nmol, inhibited the expression of morphine-induced CPP. NPFF gave itself, neither induced place preference nor aversion, although a tendency to aversive effect was seen at the highest dose of 20 nmol. NPFF did not indicate fear behavior in the elevated plus maze test, and did not disturb locomotor activity of rats. However, NPFF was unable to inhibit the expression of ethanol-induced CPP. Probably this effect is due to the fact that ethanol reward is a more complex process and apart from the role of opioids, there are other neurotransmitters also involved in this mechanism. These results suggest that NPFF is involved in the expression of morphine reward. Moreover, our study supports an anti-opioid character of this peptide.

The role of neuropeptide FF (NPFF) in the expression of sensitization to hyperlocomotor effect of morphine and ethanol.

Neuropeptide FF (NPFF) has been characterized as an endogenous anti-opioid peptide because its intraventricular injection (icv) reversed morphine- and stress-induced analgesia, and precipitates withdrawal syndrome in morphine-dependent rats. The role of NPFF in other aspects of drug dependence is unknown. Therefore, the aim of this study was to determine NPFF influence on the expression of sensitization to the morphine-induced hyperlocomotion. As the opioid system plays a role in ethanol effects, the influence of NPFF on the expression of sensitization to hyperlocomotor effect of ethanol was also investigated. Our study indicated that acute administration of NPFF (5, 10, 20nmol, icv) inhibited the expression of morphine-induced sensitization at doses of 10 (P<0.05) and 20nmol (P<0.01), and also inhibited ethanol-induced sensitization at a dose of 20nmol (P<0.01). Furthermore, NPFF inhibited the acute locomotor effect of morphine (10 and 20nmol) but not that of ethanol. NPFF, given alone, did not change the locomotor activity of mice and did not disturb motor coordination of animals in the rotarod test. In conclusion, our experiments indicated that NPFF attenuated the acute morphine locomotion and the expression of sensitization to locomotion. We anticipate that NPFF may be involved in both of these effects.

Methods for samples preparation in proteomic research.

Sample preparation is one of the most crucial processes in proteomics research. The results of the experiment depend on the condition of the starting material. Therefore, the proper experimental model and careful sample preparation is vital to obtain significant and trustworthy results, particularly in comparative proteomics, where we are usually looking for minor differences between experimental-, and control samples. In this review we discuss problems associated with general strategies of samples preparation, and experimental demands for these processes.

Rat brain proteome in morphine dependence.

The aim of this study was to reveal potential markers associated with drug dependence, using the proteomic approach. Gels containing samples derived from morphine-treated and control animals were compared and analyzed. Inspection of protein profiles, following TCA/acetone precipitation and the use of nano-scale liquid chromatography coupled to tandem mass spectrometry, allowed for identification of eleven potential dependence markers, mainly cytoplasmic and mitochondrial enzymes, e.g. proteins that belong to GTPase and GST superfamilies, ATPase, asparaginase or proteasome subunit p27 families.

CART (85-102)-inhibition of psychostimulant-induced hyperlocomotion: importance of cyclization.

Synthetic derivative of C-terminal fragment of CART (55-102) with reduced thiol groups, [Abu(86,94)]CART (85-102)(red), given together with amphetamine (5 mg/kg, s.c.) or cocaine (15 mg/kg, s.c.), reversed hyperlocomotion induced by these drugs at a dose of 0.1 microg but not at a higher dose. In the cerebral cortex homogenate, [Abu(86,94)]CART (85-102)(red) was nonspecifically cleaved from N- and C-termini. This peptide contains two chemically blocked Cys residues, and two others in reduced form. Concomitant with cleavage, rapid cyclization occurred. The newly formed cyclic peptides were stable. The cyclic peptide [Abu(86,94)]CART (85-102)(ox) failed to inhibit amphetamine- and cocaine-induced locomotor activity. The ability to inhibit the locomotor-stimulant activity of amphetamine was retained in [Abu(86,88,94,101)]CART (85-102), in which all Cys were replaced with 2-aminobutyric acid to prevent their pairing. Disulfide bridge formation may be an interesting mechanism that prevents proteolysis of [Abu(86,94)]CART (85-102)(red) and terminates its ability to reverse amphetamine-induced hyperlocomotion.

The activity of CART peptide fragments.

Cocaine- and amphetamine-regulated transcript (CART) peptides attracted much attention after the discovery that the level of CART mRNA is increased in rat striatum after acute administration of cocaine and amphetamine. The most widely investigated sequence is CART (55-102), whose roles were confirmed in modulation of various physiological processes such as feeding, energy expenditure, stress control, endocrine secretion, and reward. However, peptides other than (55-102) may be generated from the CART precursor as well. This review describes biological activity of peptides derived from the CART precursor in vivo, and of synthetic CART fragments that have not been found in the nature. In particular, the activity of CART (85-102) is described, whose ability to exert behavioral responses was confirmed by the observed attenuation of the expression of sensitization to morphine-induced hyperlocomotion. This fragment also decreased the number of escape jumps evoked by naloxone in morphine-addicted mice after intracerebroventricular administration.

Proteomic analysis of rat cerebral cortex, hippocampus and striatum after exposure to morphine.

Although a series of proteins in the brain have been shown to be qualitatively or quantitatively dysregulated following morphine administration, a systematic proteomic study has not been carried out so far. We therefore aimed to show the effect of morphine on protein levels in the rat brain. For this purpose rats were given a morphine base in subcutaneously placed pellets and subsequently the cerebral cortex, hippocampus and striatum were taken for proteomic studies after three days. Extracted proteins were run on two-dimensional gel electrophoresis, scanned and quantified by specific software. Proteins with significantly different levels were analysed by mass spectrometry (MALDI-TOF-TOF). Twenty-six proteins were found to be differentially expressed and were unambiguously identified. Dysregulated proteins were from several protein pathways and cascades including signaling, metabolic, protein handling, antioxidant and miscellaneous classes. These findings represent an initial approach to the generation of a 'morphinome' and may form the basis for further protein chemical studies as a valuable analytical tool. Moreover, the study reveals morphine-regulated proteins in different brain areas and indicates the pathways involved following morphine administration in the rat, the main species for pharmacological studies in the field.

Non-peptidergic OP4 receptor agonist inhibits morphine antinociception but does not influence morphine dependence.

The non-peptidergic opioid receptor-like 1 (ORL1, OP4) receptor ligand, Ro 64-6198 [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one], is a full agonist of the OP4 receptor. The aim of this study was to evaluate whether this compound influences morphine antinociception and dependence in mice. Ro 64-6198 inhibits the acute analgesic effect of morphine in the tail-immersion test, however, when given chronically during the acquisition of morphine dependence, development of this dependence is not prevented. The acute injection of Ro 64-6198 suppresses withdrawal escape jumps in morphine dependent mice, though this effect may be a result of the loss of locomotor activity induced by this compound and/or its myorelaxant action. The study provides evidence that stimulation of the OP4 receptor suppresses acute morphine antinociception, but is not sufficient to inhibit the development of morphine dependence in mice.

Influence of nociceptin(1-17) fragments and its tyrosine-substituted derivative on morphine-withdrawal signs in rats.

Our previous studies demonstrated that endogenous ligand of nociceptin (NOP) receptor, nociceptin(1-17) (also known as orphanin FQ), inhibits morphine-withdrawal syndrome measured as wet dog shakes in rats [Life Sci. 66 (2000) PL119]. This peptide is metabolized in the spinal cord, both in vitro and in vivo, to shorter fragments, including nociceptin(1-11) and nociceptin(1-6). These fragments, formed after cleavage by endogenous peptidase, are behaviorally active and modulate nociception in a bi-phasic process [Peptides 20 (1999) 239]. As these peptides induced transient naloxone-reversible analgesia in behavioral tests [Peptides 20 (1999) 239], in the present study we tested the influence of nociceptin(1-11) (10 and 20 microg) and nociceptin(1-6) (10, 20 and 40 microg) on the morphine-withdrawal syndrome in rats. Furthermore, the modified fragment of nociceptin(1-6) with an opioid-message domain achieved by replacement of Phe1 with Tyr was tested. Morphine-withdrawal syndrome was precipitated by the i.p. injection of naloxone hydrochloride (2 mg/kg), 72 h after implantation of morphine pellets. The wet-dog shakes were chosen for statistical analyses of the abstinence signs. The results show that nociceptin(1-11) and (1-6) attenuate this morphine-withdrawal symptom. The replacement of Phe1 with Tyr in nociceptin(1-6) fragment did not potentiate the influence of nociceptin(1-6) on wet dog shakes precipitated by naloxone in morphine-dependent rats.

Nociceptin inhibits acquisition of amphetamine-induced place preference and sensitization to stereotypy in rats.

Nociceptin (also called orphanin FQ), a 17-amino-acid peptide, is the natural ligand of the nociceptin opioid peptide (NOP) receptor. This peptide shows similarities, in its structure, to opioid peptides, mainly to dynorphin A. However, unlike opioid peptides, it does not produce a conditioned place preference or aversion but inhibits rewarding effect of drugs of abuse. The present study was designed to examine the ability of nociceptin to block the acquisition of amphetamine-induced place preference, and the development of amphetamine-induced sensitization to stereotypy in rats. Our experiments indicated that repeated administration of nociceptin at increasing doses during conditioning significantly attenuated the reinforcing effect of amphetamine in conditioned place preference paradigm. Nociceptin did not change the acute effect of amphetamine-induced stereotypy but prevented the development of sensitization to stereotypy measured on the challenge day. Our results suggest the involvement of nociceptin in long-lasting neuronal adaptation after repeated amphetamine treatment.

Desorption/ionization on silicon for small molecules:a promising alternative to MALDI TOF.

A method has been developed for laser desorption/ionization of catecholamines from porous silicon. This methodology is particularly attractive for analysis of small molecules. MALDI TOF mass spectrometry, although a very sensitive technique, utilizes matrices that need to be mixed with the sample prior to their analysis. Each matrix produces its own background, particularly in the low-molecular mass region. Therefore, detection and identification of molecules below 400 Da can be difficult. Desorption/ionization of samples deposited on porous silicon does not require addition of a matrix, thus, spectra in the low-molecular mass region can be clearly readable. Here, we describe a method for the analysis of catecholamines. While MALDI TOF is superior for proteomics/peptidomics, desorption/ionization from porous silicon can extend the operating range of a mass spectrometer for studies on metabolomics (small organic molecules and their metabolites, such as chemical neurotransmitters, prostaglandins, steroids, etc.).

Synthesis and evaluation of in vivo activity of diphenylhydantoin basic derivatives.

During the search for antiarrhythmic agents among amide derivatives of phenytoin, compound 7 {3-ethyl-1-[2-hydroxy-3-(4-phenyl-piperazin-1-yl)-propyl]-2,4-dioxo-5,5-diphenyl-imidazolidine} was selected as it showed antiarrhythmic as well as antihypertensive activity. Treating this compound as a lead, new derivatives 8-19 were synthesised, differing in piperazine phenyl ring substitution (2-, 3-, 4-Cl, 2-CH3O) as well as in hydantoin N3 alkyl chain (ethyl, ethyl acetate or ethyl 2-propionate). The obtained compounds in form of hydrochlorides 7a-19a were examined for prophylactic antiarrhythmic and antihypertensive properties. Compounds containing ethyl 2-propionate moiety (17a, 18a) exhibited the highest antihypertensive properties. Water-soluble compounds, containing 2-methoxyphenylpiperazine group (11a, 19a), showed strong antiarrhythmic properties in adrenaline-induced arrhythmia; compound 9a {1-[3-(4-(3-chloro-phenyl)-piperazin-1-yl)- 2-hydroxy-propyl]- 3-ethyl-2,4-dioxo-5,5-diphenyl-imidazolidine hydrochloride} exhibited the highest antiarrhythmic activity in barium chloride arrhythmia model.

"4D Biology for health and disease" workshop report.

The "4D Biology Workshop for Health and Disease", held on 16-17th of March 2010 in Brussels, aimed at finding the best organising principles for large-scale proteomics, interactomics and structural genomics/biology initiatives, and setting the vision for future high-throughput research and large-scale data gathering in biological and medical science. Major conclusions of the workshop include the following. (i) Development of new technologies and approaches to data analysis is crucial. Biophysical methods should be developed that span a broad range of time/spatial resolution and characterise structures and kinetics of interactions. Mathematics, physics, computational and engineering tools need to be used more in biology and new tools need to be developed. (ii) Database efforts need to focus on improved definitions of ontologies and standards so that system-scale data and associated metadata can be understood and shared efficiently. (iii) Research infrastructures should play a key role in fostering multidisciplinary research, maximising knowledge exchange between disciplines and facilitating access to diverse technologies. (iv) Understanding disease on a molecular level is crucial. System approaches may represent a new paradigm in the search for biomarkers and new targets in human disease. (v) Appropriate education and training should be provided to help efficient exchange of knowledge between theoreticians, experimental biologists and clinicians. These conclusions provide a strong basis for creating major possibilities in advancing research and clinical applications towards personalised medicine.

Basic and clinical proteomics from the EU Health Research perspective.

The European Union (EU) is one of the main public funders of research in Europe and its major instrument for funding is the Seventh Framework Programme for research and technological development (FP7). The bulk of funding in FP7 goes to collaborative research, with the objective of establishing excellent research projects and networks. Understanding the functions of proteins is essential for the rational development of disease prevention, diagnosis and treatment, therefore the EU has largely invested in proteomics, in particular for technology development, data standardisation and sharing efforts, and the application of proteomics in the clinic. The scientific community, including both academia and industry, is encouraged to apply for FP7 funding so that the EU can even more efficiently support innovative health research and ultimately, bring better healthcare to patients.

Recommendations from the 2008 International Summit on Proteomics Data Release and Sharing Policy: the Amsterdam principles.

Policies supporting the rapid and open sharing of genomic data have directly fueled the accelerated pace of discovery in large-scale genomics research. The proteomics community is starting to implement analogous policies and infrastructure for making large-scale proteomics data widely available on a precompetitive basis. On August 14, 2008, the National Cancer Institute (NCI) convened the "International Summit on Proteomics Data Release and Sharing Policy" in Amsterdam, The Netherlands, to identify and address potential roadblocks to rapid and open access to data. The six principles agreed upon by key stakeholders at the summit addressed issues surrounding (1) timing, (2) comprehensiveness, (3) format, (4) deposition to repositories, (5) quality metrics, and (6) responsibility for proteomics data release. This summit report explores various approaches to develop a framework of data release and sharing principles that will most effectively fulfill the needs of the funding agencies and the research community.

Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family.

Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties of Staphylococcus epidermidis staphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinant S. epidermidis staphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases from S. aureus cleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue, S. aureus staphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by non-target cysteine proteases. Conversely, S. aureus staphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.