The ReCoDe addiction research consortium: Losing and regaining control over drug intake-Findings and future perspectives.

Substance use disorders (SUDs) are seen as a continuum ranging from goal-directed and hedonic drug use to loss of control over drug intake with aversive consequences for mental and physical health and social functioning. The main goals of our interdisciplinary German collaborative research centre on Losing and Regaining Control over Drug Intake (ReCoDe) are (i) to study triggers (drug cues, stressors, drug priming) and modifying factors (age, gender, physical activity, cognitive functions, childhood adversity, social factors, such as loneliness and social contact/interaction) that longitudinally modulate the trajectories of losing and regaining control over drug consumption under real-life conditions. (ii) To study underlying behavioural, cognitive and neurobiological mechanisms of disease trajectories and drug-related behaviours and (iii) to provide non-invasive mechanism-based interventions. These goals are achieved by: (A) using innovative mHealth (mobile health) tools to longitudinally monitor the effects of triggers and modifying factors on drug consumption patterns in real life in a cohort of 900 patients with alcohol use disorder. This approach will be complemented by animal models of addiction with 24/7 automated behavioural monitoring across an entire disease trajectory; i.e. from a naïve state to a drug-taking state to an addiction or resilience-like state. (B) The identification and, if applicable, computational modelling of key molecular, neurobiological and psychological mechanisms (e.g., reduced cognitive flexibility) mediating the effects of such triggers and modifying factors on disease trajectories. (C) Developing and testing non-invasive interventions (e.g., Just-In-Time-Adaptive-Interventions (JITAIs), various non-invasive brain stimulations (NIBS), individualized physical activity) that specifically target the underlying mechanisms for regaining control over drug intake. Here, we will report on the most important results of the first funding period and outline our future research strategy.

Recommendations for robust and reproducible preclinical research in personalised medicine.

BACKGROUND: Personalised medicine is a medical model that aims to provide tailor-made prevention and treatment strategies for defined groups of individuals. The concept brings new challenges to the translational step, both in clinical relevance and validity of models. We have developed a set of recommendations aimed at improving the robustness of preclinical methods in translational research for personalised medicine. METHODS: These recommendations have been developed following four main steps: (1) a scoping review of the literature with a gap analysis, (2) working sessions with a wide range of experts in the field, (3) a consensus workshop, and (4) preparation of the final set of recommendations. RESULTS: Despite the progress in developing innovative and complex preclinical model systems, to date there are fundamental deficits in translational methods that prevent the further development of personalised medicine. The literature review highlighted five main gaps, relating to the relevance of experimental models, quality assessment practices, reporting, regulation, and a gap between preclinical and clinical research. We identified five points of focus for the recommendations, based on the consensus reached during the consultation meetings: (1) clinically relevant translational research, (2) robust model development, (3) transparency and education, (4) revised regulation, and (5) interaction with clinical research and patient engagement. Here, we present a set of 15 recommendations aimed at improving the robustness of preclinical methods in translational research for personalised medicine. CONCLUSIONS: Appropriate preclinical models should be an integral contributor to interventional clinical trial success rates, and predictive translational models are a fundamental requirement to realise the dream of personalised medicine. The implementation of these guidelines is ambitious, and it is only through the active involvement of all relevant stakeholders in this field that we will be able to make an impact and effectuate a change which will facilitate improved translation of personalised medicine in the future.

Towards best practices in research: Role of academic core facilities.

Academic Core Facilities are optimally situated to improve the quality of preclinical research by implementing quality control measures and offering these to their users.

Electronic Lab Notebooks and Experimental Design Assistants.

Documentation of experiments is essential for best research practice and ensures scientific transparency and data integrity. Traditionally, the paper lab notebook (pLN) has been employed for documentation of experimental procedures, but over the course of the last decades, the introduction of electronic tools has changed the research landscape and the way that work is performed. Nowadays, almost all data acquisition, analysis, presentation and archiving are done with electronic tools. The use of electronic tools provides many new possibilities, as well as challenges, particularly with respect to documentation and data quality. One of the biggest hurdles is the management of data on different devices with a substantial amount of metadata. Transparency and integrity have to be ensured and must be reflected in documentation within LNs. With this in mind, electronic LNs (eLN) were introduced to make documentation of experiments more straightforward, with the development of enhanced functionality leading gradually to their more widespread use. This chapter gives a general overview of eLNs in the scientific environment with a focus on the advantages of supporting quality and transparency of the research. It provides guidance on adopting an eLN and gives an example on how to set up unique Study-IDs in labs in order to maintain and enhance best practices. Overall, the chapter highlights the central role of eLNs in supporting the documentation and reproducibility of experiments.

Linear ubiquitination prevents inflammation and regulates immune signalling.

Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpin(cpdm/cpdm)) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1ß was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.

Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction.

TNF is a key inflammatory cytokine. Using a modified tandem affinity purification approach, we identified HOIL-1 and HOIP as functional components of the native TNF-R1 signaling complex (TNF-RSC). Together, they were shown to form a linear ubiquitin chain assembly complex (LUBAC) and to ubiquitylate NEMO. We show that LUBAC binds to ubiquitin chains of different linkage types and that its recruitment to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2- but not in RIP1- or NEMO-deficient MEFs. Furthermore, the E3 ligase activity of cIAPs, but not TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. LUBAC enhances NEMO interaction with the TNF-RSC, stabilizes this protein complex, and is required for efficient TNF-induced activation of NF-kappaB and JNK, resulting in apoptosis inhibition. Finally, we demonstrate that sustained stability of the TNF-RSC requires LUBAC's enzymatic activity, thereby adding a third form of ubiquitin linkage to the triggering of TNF signaling by the TNF-RSC.

Implementation of the EQIPD Quality System.

The EQIPD Quality System (QS) was conceptualized and established by an international consortium consisting of academic and industrial partners to ensure that non-regulated biomedical research will be conducted according to Good Research Practice expectations. The QS supports researchers to reflect on and improve internal practices by providing a systematic framework and guidance for implementing the EQIPD QS in a time and cost effective manner. This report describes the content of the EQIPD QS with its key features and 18 Core Requirements (CR) in more detail. It gives a short background on each CR and hands on examples on how they were addressed by two different research labs in their respective laboratory environments. Thereby, this article provides examples and direction for other research labs who aim to implement the QS as well. The final paragraphs discuss the potential benefits of the QS in respect to different user groups and stakeholders within the scientific community and summarize the overall governance structure of the EQIPD framework.

Assessing and Improving Research Quality in Core Facilities.

Core facilities allow scientists to perform experiments needing specialized technologies in a time- and cost-efficient way. They became increasingly important and now produce a significant amount of research data. Experiments carried out in core facilities are typically shared between the facility staff and the users. However, sharing experiments brings additional challenges to ensure data rigor and reproducibility-for example, in communication, trust, and accountability. We present here an interactive website developed especially for core facilities that offers tools to help them assess, improve, and ensure research quality.

eLabFTW as an Open Science tool to improve the quality and translation of preclinical research.

Reports of non-replicable research demand new methods of research data management. Electronic laboratory notebooks (ELNs) are suggested as tools to improve the documentation of research data and make them universally accessible. In a self-guided approach, we introduced the open-source ELN eLabFTW into our life-science lab group and, after using it for a while, think it is a useful tool to overcome hurdles in ELN introduction by providing a combination of properties making it suitable for small life-science labs, like ours. We set up our instance of eLabFTW, without any further programming needed. Our efforts to embrace open data approach by introducing an ELN fits well with other institutional organized ELN initiatives in academic research and our goals towards data quality management.

Improving target assessment in biomedical research: the GOT-IT recommendations.

Academic research plays a key role in identifying new drug targets, including understanding target biology and links between targets and disease states. To lead to new drugs, however, research must progress from purely academic exploration to the initiation of efforts to identify and test a drug candidate in clinical trials, which are typically conducted by the biopharma industry. This transition can be facilitated by a timely focus on target assessment aspects such as target-related safety issues, druggability and assayability, as well as the potential for target modulation to achieve differentiation from established therapies. Here, we present recommendations from the GOT-IT working group, which have been designed to support academic scientists and funders of translational research in identifying and prioritizing target assessment activities and in defining a critical path to reach scientific goals as well as goals related to licensing, partnering with industry or initiating clinical development programmes. Based on sets of guiding questions for different areas of target assessment, the GOT-IT framework is intended to stimulate academic scientists' awareness of factors that make translational research more robust and efficient, and to facilitate academia-industry collaboration.

Introduction to the EQIPD quality system.

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Supported through the European Union's Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers, and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e. performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.

A survey of research quality in core facilities.

Core facilities are an effective way of making expensive experimental equipment available to a large number of researchers, and are thus well placed to contribute to efforts to promote good research practices. Here we report the results of a survey that asked core facilities in Europe about their approaches to the promotion of good research practices, and about their interactions with users from the first contact to the publication of the results. Based on 253 responses we identified four ways that good research practices could be encouraged: (i) motivating users to follow the advice and procedures for best research practice; (ii) providing clear guidance on data-management practices; (iii) improving communication along the whole research process; and (iv) clearly defining the responsibilities of each party.

TRAIL signalling: decisions between life and death.

The TNF-related apoptosis-inducing ligand, TRAIL, has been shown to selectively kill tumour cells. This property has made TRAIL and agonistic antibodies against its death inducing receptors (TRAIL-R1 and TRAIL-R2) to some of the most promising novel biotherapeutic agents for cancer therapy. Here we review the signalling pathways initiated by the apoptosis- as well as the non-apoptosis-inducing receptors, TRAIL-R3 and TRAIL-R4. The TRAIL "death-inducing signalling complex" (DISC) transmits the apoptotic signal. DISC formation leads to activation of a protease cascade, finally resulting in cell death. The TRAIL death receptor-mediated "extrinsic" pathway and the "intrinsic" pathway, which is controlled by the interaction of members of the Bcl-2 family, interact with each other in the decision about life or death of a cell. Apoptotic and non-apoptotic signalling is influenced by the NF-kappaB, PKB/Akt and the MAPK signalling pathways. In this review we intend to summarise the most important findings on the TRAIL signalling network and the interplay in the decisions between life and death of a tumor cell.

Targeting repulsive guidance molecule A to promote regeneration and neuroprotection in multiple sclerosis.

Repulsive guidance molecule A (RGMa) is a potent inhibitor of neuronal regeneration and a regulator of cell death, and it plays a role in multiple sclerosis (MS). In autopsy material from progressive MS patients, RGMa was found in active and chronic lesions, as well as in normal-appearing gray and white matter, and was expressed by cellular meningeal infiltrates. Levels of soluble RGMa in the cerebrospinal fluid were decreased in progressive MS patients successfully treated with intrathecal corticosteroid triamcinolone acetonide (TCA), showing functional improvements. In vitro, RGMa monoclonal antibodies (mAbs) reversed RGMa-mediated neurite outgrowth inhibition and chemorepulsion. In animal models of CNS damage and MS, RGMa antibody stimulated regeneration and remyelination of damaged nerve fibers, accelerated functional recovery, and protected the retinal nerve fiber layer as measured by clinically relevant optic coherence tomography. These data suggest that targeting RGMa is a promising strategy to improve functional recovery in MS patients.

C-terminal domains deliver the DNA replication factor Ciz1 to the nuclear matrix.

Cip1-interacting zinc finger protein 1 (Ciz1) stimulates DNA replication in vitro and is required for mammalian cells to enter S phase. Here, we show that a significant proportion of Ciz1 is retained in nuclear foci following extraction with nuclease and high salt. This suggests that Ciz1 is normally immobilized by interaction with non-chromatin nuclear structures, consistent with the nuclear matrix. Furthermore, matrix-associated Ciz1 foci strikingly colocalize with sites of newly synthesized DNA in S phase nuclei, suggesting that Ciz1 is present in DNA replication factories. Analysis of green fluorescent protein-tagged fragments indicates that nuclear immobilization of Ciz1 is mediated by sequences in its C-terminal third, encoded within amino acids 708-830. Immobilization occurs in a cell-cycle-dependent manner, most probably during late G1 or early S phase, to coincide with its reported point of action. Although C-terminal domains are sufficient for immobilization, N-terminal domains are also required to specify focal organization. Combined with previous work, which showed that the DNA replication activity of Ciz1 is encoded by N-terminal sequences, we suggest that Ciz1 is composed of two functionally distinct domains: an N-terminal replication domain and a C-terminal nuclear matrix anchor. This could contribute to the formation or function of DNA replication factories in mammalian cells.