fiddle: a tool to combat publication bias by getting research out of the file drawer and into the scientific community.

Statistically significant findings are more likely to be published than non-significant or null findings, leaving scientists and healthcare personnel to make decisions based on distorted scientific evidence. Continuously expanding ´file drawers' of unpublished data from well-designed experiments waste resources creates problems for researchers, the scientific community and the public. There is limited awareness of the negative impact that publication bias and selective reporting have on the scientific literature. Alternative publication formats have recently been introduced that make it easier to publish research that is difficult to publish in traditional peer reviewed journals. These include micropublications, data repositories, data journals, preprints, publishing platforms, and journals focusing on null or neutral results. While these alternative formats have the potential to reduce publication bias, many scientists are unaware that these formats exist and don't know how to use them. Our open source file drawer data liberation effort (fiddle) tool (RRID:SCR_017327 available at: http://s-quest.bihealth.org/fiddle/) is a match-making Shiny app designed to help biomedical researchers to identify the most appropriate publication format for their data. Users can search for a publication format that meets their needs, compare and contrast different publication formats, and find links to publishing platforms. This tool will assist scientists in getting otherwise inaccessible, hidden data out of the file drawer into the scientific community and literature. We briefly highlight essential details that should be included to ensure reporting quality, which will allow others to use and benefit from research published in these new formats.

Quality in Non-GxP Research Environment.

There has been increasing evidence in recent years that research in life sciences is lacking in reproducibility and data quality. This raises the need for effective systems to improve data integrity in the evolving non-GxP research environment. This chapter describes the critical elements that need to be considered to ensure a successful implementation of research quality standards in both industry and academia. The quality standard proposed is founded on data integrity principles and good research practices and contains basic quality system elements, which are common to most laboratories. Here, we propose a pragmatic and risk-based quality system and associated assessment process to ensure reproducibility and data quality of experimental results while making best use of the resources.

Interaction of ARC and Daxx: A Novel Endogenous Target to Preserve Motor Function and Cell Loss after Focal Brain Ischemia in Mice.

UNLABELLED: The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenous ARC protein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD). TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30 ± 8% (mean ± SD; p = 0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1 µg intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.ß-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20 ± 7% (mean ± SD; p < 0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX-ASK1-JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1-JNK activation. Our work identifies for the first time ARC-DAXX binding to block ASK1-JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy. SIGNIFICANCE STATEMENT: Up to now, the only successful pharmacological target of human ischemic stroke is thrombolysis. Neuroprotective pharmacological strategies are needed to accompany therapies aiming to achieve reperfusion. We describe that apoptosis repressor with CARD (ARC) interacts and inhibits DAXX and proximal signals of cell death. In a murine stroke model mimicking human malignant infarction in the territory of the middle cerebral artery, TAT.ARC salvages brain tissue when given during occlusion or 3 h delayed with sustained functional benefits (28 d). This is a promising novel therapeutic approach because it appears to be effective in a model producing severe injury by interfering with an array of proximal signals and effectors of the ischemic cascade, upstream of JNK, caspases, and BIM and BAX activation.

The EQIPD quality system - Assessment and certification procedures.

The EQIPD Quality System was designed with the ultimate mission to provide a framework to ensure the quality and integrity of non-regulated preclinical biomedical research. For research quality to be sustained over time, it is crucial to have continuous improvement mechanisms that routinely monitor the research-related processes and enable solutions for identified issues. The present article is focused on these monitoring and assessment procedures that make the EQIPD Quality System a fully functional 'system' (as opposed to a mere collection of guidelines, work instructions and policies). In this context, a critical instrument are the internal and external assessments of the EQIPD Quality System performance described in detail. The assessment procedures emphasize the unique nature of the EQIPD Quality System being user-friendly, flexible and fit-for-purpose. By undergoing the (voluntary) external EQIPD assessment (leading to the EQIPD certification after all EQIPD core requirements have been implemented), a research unit: (i) secures confidence in the quality of data generated, (ii) ensures continuous improvement of research processes, and (iii) obtains an independent seal of quality communicating commitment to best research practices to the research community.

As Verified with the Aid of Biotinylated Spermine, the Brain Cannot Take up Polyamines from the Bloodstream Leaving It Solely Dependent on Local Biosynthesis.

The importance of polyamines (PAs) for the central nervous system (CNS) is well known. Less clear, however, is where PAs in the brain are derived from. Principally, there are three possibilities: (i) intake by nutrition, release into the bloodstream, and subsequent uptake from CNS capillaries, (ii) production by parenchymatous organs, such as the liver, and again uptake from CNS capillaries, and (iii) uptake of precursors, such as arginine, from the blood and subsequent local biosynthesis of PAs within the CNS. The present investigation aimed to unequivocally answer the question of whether PAs, especially the higher ones like spermidine (SPD) and spermine (SPM), can or cannot be taken up into the brain from the bloodstream. For this purpose, a biotin-labelled analogue of spermine (B-X-SPM) was synthesized, characterized, and used to visualize its uptake into brain cells following application to acute brain slices, to the intraventricular space, or to the bloodstream. In acute brain slices there is strong uptake of B-X-SPM into protoplasmic and none in fibrous-type astrocytes. It is also taken up by neurons but to a lesser degree. Under in vivo conditions, astrocyte uptake of B-X-SPM from the brain interstitial fluid is also intense after intraventricular application. In contrast, following intracardial injection, there is no uptake from the bloodstream, indicating that the brain is completely dependent on the local synthesis of polyamines.

Eleven strategies for making reproducible research and open science training the norm at research institutions.

Reproducible research and open science practices have the potential to accelerate scientific progress by allowing others to reuse research outputs, and by promoting rigorous research that is more likely to yield trustworthy results. However, these practices are uncommon in many fields, so there is a clear need for training that helps and encourages researchers to integrate reproducible research and open science practices into their daily work. Here, we outline eleven strategies for making training in these practices the norm at research institutions. The strategies, which emerged from a virtual brainstorming event organized in collaboration with the German Reproducibility Network, are concentrated in three areas: (i) adapting research assessment criteria and program requirements; (ii) training; (iii) building communities. We provide a brief overview of each strategy, offer tips for implementation, and provide links to resources. We also highlight the importance of allocating resources and monitoring impact. Our goal is to encourage researchers - in their roles as scientists, supervisors, mentors, instructors, and members of curriculum, hiring or evaluation committees - to think creatively about the many ways they can promote reproducible research and open science practices in their institutions.

Is the future of peer review automated?

The rising rate of preprints and publications, combined with persistent inadequate reporting practices and problems with study design and execution, have strained the traditional peer review system. Automated screening tools could potentially enhance peer review by helping authors, journal editors, and reviewers to identify beneficial practices and common problems in preprints or submitted manuscripts. Tools can screen many papers quickly, and may be particularly helpful in assessing compliance with journal policies and with straightforward items in reporting guidelines. However, existing tools cannot understand or interpret the paper in the context of the scientific literature. Tools cannot yet determine whether the methods used are suitable to answer the research question, or whether the data support the authors' conclusions. Editors and peer reviewers are essential for assessing journal fit and the overall quality of a paper, including the experimental design, the soundness of the study's conclusions, potential impact and innovation. Automated screening tools cannot replace peer review, but may aid authors, reviewers, and editors in improving scientific papers. Strategies for responsible use of automated tools in peer review may include setting performance criteria for tools, transparently reporting tool performance and use, and training users to interpret reports.

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.

BKbeta1 subunits contribute to BK channel diversity in rat hypothalamic neurons.

Large conductance Ca(2+)-activated BK channels are important regulators of action potential duration and firing frequency in many neurons. As the pore-forming subunits of BK channels are encoded by a single gene, channel diversity is mainly generated by alternative splicing and interaction with auxiliary beta-subunits (BKbeta1-4). In hypothalamic neurons several BK channel subtypes have been described electrophysiologically; however, the distribution of BKbeta subunits is unknown so far. Therefore, an antibody against the large extracellular loop of the BKbeta1 subunit was raised, freed from cross-reactivity against BKbeta2-4 and affinity-purified. The resulting polyclonal monospecific BKbeta1 antibody was characterized by Western blot analysis, ELISA techniques and immunocytochemical staining of BKbeta1-4-transfected CHO and COS-1 cells. Regional and cellular distribution in the rat hypothalamus was analysed by immunocytochemistry and in situ hybridization experiments. Immunocytochemical staining of rat hypothalamic neurons indicates strong BKbeta1 expression in the supraoptic nucleus and the magno- and parvocellular parts of the paraventricular nucleus. Lower expression was found in periventricular nucleus, the arcuate nucleus and in the median eminence. Immunostaining was predominantly localized to somata. In addition, pericytes and ependymal epithelial cells showed BKbeta1 labelling. In all cases immunocytochemical results were supported by in situ hybridization.

Improving quality of preclinical academic research through auditing: A feasibility study.

How much can we rely on whether what was reported in a study was actually done? Systematic and independent examination of records, documents and processes through audits are a central element of quality management systems. In the context of current concerns about the robustness and reproducibility of experimental biomedical research audits have been suggested as a remedy a number of times. However, audits are resource intense and time consuming, and due to their very nature may be perceived as inquisition. Consequently, there is very little experience or literature on auditing and assessments in the complex preclinical biomedical research environment. To gain some insight into which audit approaches might best suit biomedical research in academia, in this study we have applied a number of them in a typical academic neuroscience environment consisting of twelve research groups with about 100 researchers, students and technicians, utilizing the full gamut of state-of-the-art methodology. Several types of assessments and internal as well as external audits (including the novel format of a peer audit) were systematically explored by a team of quality management specialists. An experimental design template was developed (and is provided here) that takes into account and mitigates difficulties, risks and systematic errors that may occur during the course of a study. All audits were performed according to a pre-defined workflow developed by us. Outcomes were assessed qualitatively. We asked for feedback from participating employees in every final discussion of an audit and documented this in the audit reports. Based on these reports follow-up audits were improved. We conclude that several realistic options for auditing exist which have the potential to improve preclinical biomedical research in academia, and have listed specific recommendations regarding their benefits and provided practical resources for their implementation (e.g. study design and audit templates, audit workflow).

Upregulation of inward rectifier K+ (Kir2) channels in dentate gyrus granule cells in temporal lobe epilepsy.

In humans, temporal lobe epilepsy (TLE) is often associated with Ammon's horn sclerosis (AHS) characterized by hippocampal cell death, gliosis and granule cell dispersion (GCD) in the dentate gyrus. Granule cells surviving TLE have been proposed to be hyperexcitable and to play an important role in seizure generation. However, it is unclear whether this applies to conditions of AHS. We studied granule cells using the intrahippocampal kainate injection mouse model of TLE, brain slice patch-clamp recordings, morphological reconstructions and immunocytochemistry. With progressing AHS and GCD, 'epileptic' granule cells of the injected hippocampus displayed a decreased input resistance, a decreased membrane time constant and an increased rheobase. The resting leak conductance was doubled in epileptic granule cells and roughly 70-80% of this difference were sensitive to K(+) replacement. Of the increased K(+) leak, about 50% were sensitive to 1 mm Ba(2+). Approximately 20-30% of the pathological leak was mediated by a bicuculline-sensitive GABA(A) conductance. Epileptic granule cells had strongly enlarged inwardly rectifying currents with a low micromolar Ba(2+) IC(50), reminiscent of classic inward rectifier K(+) channels (Irk/Kir2). Indeed, protein expression of Kir2 subunits (Kir2.1, Kir2.2, Kir2.3, Kir2.4) was upregulated in epileptic granule cells. Immunolabelling for two-pore weak inward rectifier K(+) channels (Twik1/K2P1.1, Twik2/K2P6.1) was also increased. We conclude that the excitability of granule cells in the sclerotic focus of TLE is reduced due to an increased resting conductance mainly due to upregulated K(+) channel expression. These results point to a local adaptive mechanism that could counterbalance hyperexcitability in epilepsy.

Gene expression profiling of neurochemically defined regions of the human brain by in situ hybridization-guided laser capture microdissection.

Laser capture microdissection (LCM) permits isolation of specific cell types and cell groups based upon morphology, anatomical landmarks and histochemical properties. This powerful technique can be used for region-specific dissection if the target structure is clearly delineated. However, it is difficult to visualize anatomical boundaries in an unstained specimen, while histological staining can complicate the microdissection process and compromise downstream processing and analysis. We now introduce a novel method in which in situ hybridization (ISH) signal is used to guide LCM on adjacent unstained sections to collect tissue from neurochemically defined regions of the human postmortem brain to minimize sample manipulation prior to analysis. This approach was validated in nuclei that provide monoaminergic inputs to the forebrain, and likely contribute to the pathophysiology of mood disorders. This method was used successfully to carry out gene expression profiling and quantitative real-time PCR (qPCR) confirmation from the dissected material. When compared to traditional micropunch dissections, our ISH-guided LCM method provided enhanced signal intensity for mRNAs of specific monoaminergic marker genes as measured by genome-wide gene expression microarrays. Enriched expression of specific monoaminergic genes (as determined by microarrays and qPCR) was detected within appropriate anatomical locations validating the accuracy of microdissection. Together these results support the conclusion that ISH-guided LCM permits acquisition of enriched nucleus-specific RNA that can be successfully used for downstream gene expression investigations. Future studies will utilize this approach for gene expression profiling of neurochemically defined regions of postmortem brains collected from mood disorder patients.

Atlas registration for edema-corrected MRI lesion volume in mouse stroke models.

Lesion volume measurements with magnetic resonance imaging are widely used to assess outcome in rodent models of stroke. In this study, we improved a mathematical framework to correct lesion size for edema which is based on manual delineation of the lesion and hemispheres. Furthermore, a novel MATLAB toolbox to register mouse brain MR images to the Allen brain atlas is presented. Its capability to calculate edema-corrected lesion size was compared to the manual approach. Automated image registration performed equally well in in a mouse middle cerebral artery occlusion model (Pearson r = 0.976, p = 2.265e-11). Information encapsulated in the registration was used to generate maps of edema induced tissue volume changes. These showed discrepancies to simplified tissue models underlying the manual approach. The presented techniques provide biologically more meaningful, voxel-wise biomarkers of vasogenic edema after stroke.

Agmatine modulates spontaneous activity in neurons of the rat medial habenular complex-a relevant mechanism in the pathophysiology and treatment of depression?

The dorsal diencephalic conduction system connects limbic forebrain structures to monaminergic mesencephalic nuclei via a distinct relay station, the habenular complexes. Both habenular nuclei, the lateral as well as the medial nucleus, are considered to play a prominent role in mental disorders like major depression. Herein, we investigate the effect of the polyamine agmatine on the electrical activity of neurons within the medial habenula in rat. We present evidence that agmatine strongly decreases spontaneous action potential firing of medial habenular neurons by activating I1-type imidazoline receptors. Additionally, we compare the expression patterns of agmatinase, an enzyme capable of inactivating agmatine, in rat and human habenula. In the medial habenula of both species, agmatinase is similarly distributed and observed in neurons and, in particular, in distinct neuropil areas. The putative relevance of these findings in the context of depression is discussed. It is concluded that increased activity of the agmatinergic system in the medial habenula may strengthen midbrain dopaminergic activity. Consequently, the habenular-interpeduncular axis may be dysregulated in patients with major depression.

Anterior and posterior parts of the rat ventral tegmental area and the rostromedial tegmental nucleus receive topographically distinct afferents from the lateral habenular complex.

That activation of the reward system involves increased activity of dopaminergic (DA) neurons in the ventral tegmental area (VTA) is widely accepted. In contrast, the lateral habenular complex (LHb), which is known as the center of the anti-reward system, directly and indirectly inhibits DA neurons in the VTA. The VTA, however, is not a homogenous entity. Instead, it displays major functional differences between its anterior (aVTA) and posterior (pVTA) regions. It is not precisely known, whether habenular input to the aVTA, pVTA, and the newly recognized rostromedial tegmental nucleus (RMTg) are similarly or differently organized. Consequently, the present investigation addressed the connections between LHb and aVTA, pVTA, and RMTg using retrograde and anterograde tracing techniques in the rat. Our experiments disclosed strictly reciprocal and conspicuously focal interconnections between LHbM (LHbMPc/LHbMC) and PN, as well as between RLi and LHbLO. In addition, we found that LHb inputs to the aVTA are dorsoventrally ordered. Dorsal parts of the aVTA receive afferents from LHbL and LHbM, whereas ventral parts of the aVTA are preferentially targeted by the LHbM. LHb afferents to the pVTA are distinct from those to the RMTg, given that the RMTg is primarily innervated from the LHbL, whereas pVTA receives afferents from LHbM and LHbL. These data indicate the existence of two separate pathways from the LHb to the VTA, a direct and an indirect one, which may subserve distinct biological functions.

Microarray analysis of transcripts with elevated expressions in the rat medial or lateral habenula suggest fast GABAergic excitation in the medial habenula and habenular involvement in the regulation of feeding and energy balance.

In vertebrates the "anti-reward-system" mainly is represented by the habenula and its medial (MHb) and especially lateral (LHb) complexes. Considerable knowledge has accumulated concerning subnuclear structures and connectivities of MHb and LHb subnuclei. The present investigation aimed to obtain novel information, whether MHb or LHb or their subnuclei display field-characteristic gene products, which may shed light on biological functions of these areas. Unfortunately this was not the case. Microarray analysis of mRNAs in microdissected habenular and thalamic control areas yielded expression values of 17,745 RNAs representing protein-coding genes, to which annotated gene names could be assigned. High relative values of genes with known expression in MHb, LHb or thalamus in the corresponding areas indicated a high precision of the microdissection procedure. Note that the present report emphasizes differences between and not absolute expression values in the selected regions. The present investigation disclosed that the LHb genetically is much closer related to the thalamus as compared to the MHb. The results presented here focuse on gene transcripts related to major transmitter systems, catecholamines and neuropeptides. Quite surprisingly, our data indicate potentially inhibitory effects of acetylcholine and glutamate in the habenula. In addition, the absence of the K-Cl co-transporter 2 supports a largely excitatory role of GABAergic transmission especially in the MHb. Furthermore, several G-protein related receptors (Gpr83, Gpr139, Gpr149, Gpr151, Gpr158) and many neuropeptides related to feeding are differentially expressed in the habenular region, indicating that its involvement in the regulation of food consumption and energy expenditure may have been underestimated so far.

Hypothalamic, thalamic and hippocampal lesions in the mouse MCAO model: Potential involvement of deep cerebral arteries?

Intraluminal monofilament occlusion of the middle cerebral artery (MCAO) in mice is the most used rodent model to study the pathophysiology of stroke. However, this model often shows brain damage in regions not supplied by the MCA such as the hypothalamus, hippocampus and thalamus. Several studies have suggested some explanations on these localized infarcts. We aim to provide an alternative explanation which could allow each experimenter to better grasp the MCAO model. We propose that the MCA occlusion by the monofilament also occludes deep and small cerebral arteries arising directly from the internal carotid artery, proximally to the origin of MCA. Then, drawbacks and pitfalls of the MCAO model must be appreciated and the almost systematic risk of inducing lesions in some unwanted territories for neuroanatomical reasons, i.e. vascular connections between deep arteries and hypothalamic, thalamic and hippocampal areas in rodents has to be integrated.

Hypocretin-1 activates G proteins in arousal-related brainstem nuclei of rat.

The hypocretin (hcrt) ligand-receptor system contributes to the maintenance of wakefulness. Hypocretin receptors are coupled to guanine nucleotide binding (G) proteins and the present study tested the hypothesis that hcrt-1 would activate G proteins in rat brainstem nuclei known to regulate arousal states. In vitro [35S]GTPgammaS autoradiography was performed using coronal brain stem sections from six rats. Hcrt-1 (200 nM) significantly increased [35S]GTPgammaS binding over basal levels in locus coeruleus (24%), dorsal raphe nucleus (12%), pontine reticular nucleus, oral part (28%), and pontine reticular nucleus, caudal part (40%). This is the first study to quantify and localize hcrt-1-induced G protein activation in specific brain stem nuclei. The finding that hcrt-1 stimulated [35S]GTPgammaS binding suggests that some hcrt receptors in pontine brain stem couple to inhibitory G proteins.