Automatic knowledge graph population with model-complete text comprehension for pre-clinical outcomes in the field of spinal cord injury.

The paradigm of evidence-based medicine requires that medical decisions are made on the basis of the best available knowledge published in the literature. Existing evidence is often summarized in the form of systematic reviews and/or meta-reviews and is rarely available in a structured form. Manual compilation and aggregation is costly, and conducting a systematic review represents a high effort. The need to aggregate evidence arises not only in the context of clinical trials, but is also important in the context of pre-clinical animal studies. In this context, evidence extraction is important to support translation of the most promising pre-clinical therapies into clinical trials or to optimize clinical trial design. Aiming at developing methods that facilitate the task of aggregating evidence published in pre-clinical studies, in this paper a new system is presented that automatically extracts structured knowledge from such publications and stores it in a so-called domain knowledge graph. The approach follows the paradigm of model-complete text comprehension by relying on guidance from a domain ontology creating a deep relational data-structure that reflects the main concepts, protocol, and key findings of studies. Focusing on the domain of spinal cord injuries, a single outcome of a pre-clinical study is described by up to 103 outcome parameters. Since the problem of extracting all these variables together is intractable, we propose a hierarchical architecture that incrementally predicts semantic sub-structures according to a given data model in a bottom-up fashion. At the heart of our approach is a statistical inference method that relies on conditional random fields to infer the most likely instance of the domain model given the text of a scientific publication as input. This approach allows modeling dependencies between the different variables describing a study in a semi-joint fashion. We present a comprehensive evaluation of our system to understand the extent to which our system can capture a study in the depth required to enable the generation of new knowledge. We conclude the article with a brief description of some applications of the populated knowledge graph and show the potential implications of our work for supporting evidence-based medicine.

Aromatase mutation in men as a rare cause of osteoporosis: a case report and review of the literature.

OBJECTIVES: Oestrogen deficiency is a rare disease and leads inter alia to arthralgia and osteoporosis in men. The clinical relevance of aromatase to a functioning male metabolism has become evident since 1991, when cases of patients with oestrogen deficiency caused by aromatase mutation were first described. Only few cases are known so far, which will now be presented in a case report and review of the literature. METHODS: All available publications since the first description in 1991 dealing with loss-of-function aromatase mutation in men were summarised and our case report was added. RESULTS: The mutations that cause the aromatase protein to lose function leads to a rather heterogeneous clinical picture. It is, however, clear that oestrogens play a central role in male patients, especially in bone metabolism. Most frequently, tall stature, unclosed epiphyseal joints, and osteoporosis are detected in affected individuals as a consequence of the change in hormonal status. CONCLUSIONS: As low oestrogen is associated with arthralgia, patients with aromatase mutation may be referred to a rheumatologist. Despite aromatase deficiency being a rare disease, the study of the effects of oestrogen on male bone development provides important insights for endocrine bone regulation. It has been demonstrated that androgens alone are not sufficient for adequate skeletal development in males. The described effects of loss of oestrogens are known from the aromatase inhibitor therapy in breast cancer treatment. This work highlights the important role of oestrogens in individual health and disease in men. Molecular effects of oestrogens on bone metabolism are summarised.

Aqueous Solutions of Associating Poly(acrylamide-co-styrene): A Path to Improve Drag Reduction?

Hydrophobically modified associating polymers could be effective drag-reducing agents containing weak "links" which after degradation can reform, protecting the polymer backbone from fast scission. Previous studies using hydrophobically modified polymers in drag reduction applications used polymers with M w ≥ 1000 kg/mol. Homopolymers of this high M w already show significant drag reduction (DR), and the contribution of macromolecular associations on DR remained unclear. We synthesized associating poly(acrylamide-co-styrene) copolymers with M w ≤ 1000 kg/mol and various hydrophobic moiety content. Their DR effectiveness in turbulent flow was studied using a pilot-scale pipe flow facility and a rotating "disc" apparatus. We show that hydrophobically modified copolymers with M w ≈ 1000 kg/mol increase DR in pipe flow by a factor of ∼2 compared to the unmodified polyacrylamide of similar M w albeit at low DR level. Moreover, we discuss challenges encountered when using hydrophobically modified polymers synthesized via micellar polymerization.

Mapping the Long Rocky Road to Effective Spinal Cord Injury Therapy: A Meta-Review of Pre-Clinical and Clinical Research.

Spinal cord injury (SCI) is a rare condition, which even after decades of research, to date still presents an incurable condition with a complex symptomatology. An SCI can result in paralysis, pain, loss of sensation, bladder and sexual dysfunction, and muscle degeneration, to name but a few. The large number of publications makes it difficult to keep track of current progress in the field and of the many treatment options that have been suggested and are being proposed with increasing frequency. Scientific databases with user-oriented search options will offer possible solutions, but they are still mostly in the development phase. In this meta-analysis, we summarize and narrow down SCI therapeutic approaches applied in pre-clinical and clinical research. Statistical analyses of treatment clusters-assorted after counting annual publication numbers in PubMed and ClinicalTrials.gov databases-were performed to allow the comparison of research foci and of their translation efficacy into clinical therapy. Using the example of SCI research, our findings demonstrate the challenges that come with the accelerating research progress-an issue that many research fields are faced with today. The analyses point out similarities and differences in the prioritization of SCI research in pre-clinical versus clinical therapy strategies. Moreover, the results demonstrate the rapidly growing importance of modern (bio-)engineering technologies.

AAV-mediated inhibition of ULK1 promotes axonal regeneration in the central nervous system in vitro and in vivo.

Axonal damage is an early step in traumatic and neurodegenerative disorders of the central nervous system (CNS). Damaged axons are not able to regenerate sufficiently in the adult mammalian CNS, leading to permanent neurological deficits. Recently, we showed that inhibition of the autophagic protein ULK1 promotes neuroprotection in different models of neurodegeneration. Moreover, we demonstrated previously that axonal protection improves regeneration of lesioned axons. However, whether axonal protection mediated by ULK1 inhibition could also improve axonal regeneration is unknown. Here, we used an adeno-associated viral (AAV) vector to express a dominant-negative form of ULK1 (AAV.ULK1.DN) and investigated its effects on axonal regeneration in the CNS. We show that AAV.ULK1.DN fosters axonal regeneration and enhances neurite outgrowth in vitro. In addition, AAV.ULK1.DN increases neuronal survival and enhances axonal regeneration after optic nerve lesion, and promotes long-term axonal protection after spinal cord injury (SCI) in vivo. Interestingly, AAV.ULK1.DN also increases serotonergic and dopaminergic axon sprouting after SCI. Mechanistically, AAV.ULK1.DN leads to increased ERK1 activation and reduced expression of RhoA and ROCK2. Our findings outline ULK1 as a key regulator of axonal degeneration and regeneration, and define ULK1 as a promising target to promote neuroprotection and regeneration in the CNS.

Secretome Analysis of Mesenchymal Stem Cell Factors Fostering Oligodendroglial Differentiation of Neural Stem Cells In Vivo.

Mesenchymal stem cell (MSC)-secreted factors have been shown to significantly promote oligodendrogenesis from cultured primary adult neural stem cells (aNSCs) and oligodendroglial precursor cells (OPCs). Revealing underlying mechanisms of how aNSCs can be fostered to differentiate into a specific cell lineage could provide important insights for the establishment of novel neuroregenerative treatment approaches aiming at myelin repair. However, the nature of MSC-derived differentiation and maturation factors acting on the oligodendroglial lineage has not been identified thus far. In addition to missing information on active ingredients, the degree to which MSC-dependent lineage instruction is functional in vivo also remains to be established. We here demonstrate that MSC-derived factors can indeed stimulate oligodendrogenesis and myelin sheath generation of aNSCs transplanted into different rodent central nervous system (CNS) regions, and furthermore, we provide insights into the underlying mechanism on the basis of a comparative mass spectrometry secretome analysis. We identified a number of secreted proteins known to act on oligodendroglia lineage differentiation. Among them, the tissue inhibitor of metalloproteinase type 1 (TIMP-1) was revealed to be an active component of the MSC-conditioned medium, thus validating our chosen secretome approach.

Heterogeneous fate choice of genetically modulated adult neural stem cells in gray and white matter of the central nervous system.

Apart from dedicated oligodendroglial progenitor cells, adult neural stem cells (aNSCs) can also give rise to new oligodendrocytes in the adult central nervous system (CNS). This process mainly confers myelinating glial cell replacement in pathological situations and can hence contribute to glial heterogeneity. Our previous studies demonstrated that the p57kip2 gene encodes an intrinsic regulator of glial fate acquisition and we here investigated to what degree its modulation can affect stem cell-dependent oligodendrogenesis in different CNS environments. We therefore transplanted p57kip2 knockdown aNSCs into white and gray matter (WM and GM) regions of the mouse brain, into uninjured spinal cords as well as in the vicinity of spinal cord injuries and evaluated integration and differentiation in vivo. Our experiments revealed that under healthy conditions intrinsic suppression of p57kip2 as well as WM localization promote differentiation toward myelinating oligodendrocytes at the expense of astrocyte generation. Moreover, p57kip2 knockdown conferred a strong benefit on cell survival augmenting net oligodendrocyte generation. In the vicinity of hemisectioned spinal cords, the gene knockdown led to a similar induction of oligodendroglial features; however, newly generated oligodendrocytes appeared to suffer more from the hostile environment. This study contributes to our understanding of mechanisms of adult oligodendrogenesis and glial heterogeneity and further reveals critical factors when considering aNSC mediated cell replacement in injury and disease.

Low-pressure micro-mechanical re-adaptation device sustainably and effectively improves locomotor recovery from complete spinal cord injury.

Traumatic spinal cord injuries result in impairment or even complete loss of motor, sensory and autonomic functions. Recovery after complete spinal cord injury is very limited even in animal models receiving elaborate combinatorial treatments. Recently, we described an implantable microsystem (microconnector) for low-pressure re-adaption of severed spinal stumps in rat. Here we investigate the long-term structural and functional outcome following microconnector implantation after complete spinal cord transection. Re-adaptation of spinal stumps supports formation of a tissue bridge, glial and vascular cell invasion, motor axon regeneration and myelination, resulting in partial recovery of motor-evoked potentials and a thus far unmet improvement of locomotor behaviour. The recovery lasts for at least 5 months. Despite a late partial decline, motor recovery remains significantly superior to controls. Our findings demonstrate that microsystem technology can foster long-lasting functional improvement after complete spinal injury, providing a new and effective tool for combinatorial therapies.

HSF1-deficiency affects gait coordination and cerebellar calbindin levels.

Heat shock proteins (HSPs) play an important role in cell homeostasis and protect against cell damage. They were previously identified as key players in different ataxia models. HSF1 is the main transcription factor for HSP activation. HSF1-deficient mice (HSF1-/-) are known to have deficiencies in motor control test. However, little is known about effects of HSF1-deficiency on locomotor, especially gait, coordination. Therefore, we compared HSF-deficient (HSF1-/-) mice and wildtype littermates using an automated gait analysis system for objective assessment of gait coordination. We found significant changes in gait parameters of HSF1-/- mice reminiscent of cerebellar ataxia. Immunohistochemical analyses of a cerebellum revealed co-localization of HSF1 and calbindin in Purkinje cells. Therefore, we tested the hypothesis of a potential interconnection between HSF1 and calbindin in Purkinje cells. Calbindin levels were analyzed qualitatively and quantitatively by immunohistochemistry and immunoblotting, respectively. While quantitative PCR revealed no differences in calbindin mRNA levels between HSF1+/+ and HSF1-/- mice, calbindin protein levels, however, were significantly decreased in a cerebellum of HSF1-/- mice. A pathway analysis supports the hypothesis of an interconnection between HSF1 and calbindin. In summary, the targeted deletion of HSF1 results in changes of locomotor function associated with changes in cerebellar calbindin protein levels. These findings suggest a role of HSF1 in regular Purkinje cell calcium homeostasis.

The pruritus- and TH2-associated cytokine IL-31 promotes growth of sensory nerves.

BACKGROUND: Pruritus is a cardinal symptom of atopic dermatitis, and an increased cutaneous sensory network is thought to contribute to pruritus. Although the immune cell-IL-31-neuron axis has been implicated in severe pruritus during atopic skin inflammation, IL-31's neuropoietic potential remains elusive. OBJECTIVE: We sought to analyze the IL-31-related transcriptome in sensory neurons and to investigate whether IL-31 promotes sensory nerve fiber outgrowth. METHODS: In vitro primary sensory neuron culture systems were subjected to whole-transcriptome sequencing, ingenuity pathway analysis, immunofluorescence, and nerve elongation, as well as branching assays after IL-31 stimulation. In vivo we investigated the cutaneous sensory neuronal network in wild-type, Il31-transgenic, and IL-31 pump-equipped mice. RESULTS: Transgenic Il31 overexpression and subcutaneously delivered IL-31 induced an increase in the cutaneous nerve fiber density in lesional skin in vivo. Transcriptional profiling of IL-31-activated dorsal root ganglia neurons revealed enrichment for genes promoting nervous system development and neuronal outgrowth and negatively regulating cell death. Moreover, the growth cones of primary small-diameter dorsal root ganglia neurons showed abundant IL-31 receptor α expression. Indeed, IL-31 selectively promoted nerve fiber extension only in small-diameter neurons. Signal transducer and activator of transcription 3 phosphorylation mediated IL-31-induced neuronal outgrowth, and pharmacologic inhibition of signal transducer and activator of transcription 3 completely abolished this effect. In contrast, transient receptor potential cation channel vanilloid subtype 1 channels were dispensable for IL-31-induced neuronal sprouting. CONCLUSIONS: The pruritus- and TH2-associated novel cytokine IL-31 induces a distinct transcriptional program in sensory neurons, leading to nerve elongation and branching both in vitro and in vivo. This finding might help us understand the clinical observation that patients with atopic dermatitis experience increased sensitivity to minimal stimuli inducing sustained itch.

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion.

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury. The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion. In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.

Pharmacological Suppression of CNS Scarring by Deferoxamine Reduces Lesion Volume and Increases Regeneration in an In Vitro Model for Astroglial-Fibrotic Scarring and in Rat Spinal Cord Injury In Vivo.

Lesion-induced scarring is a major impediment for regeneration of injured axons in the central nervous system (CNS). The collagen-rich glial-fibrous scar contains numerous axon growth inhibitory factors forming a regeneration-barrier for axons. We demonstrated previously that the combination of the iron chelator 2,2'-bipyridine-5,5'-decarboxylic acid (BPY-DCA) and 8-Br-cyclic AMP (cAMP) inhibits scar formation and collagen deposition, leading to enhanced axon regeneration and partial functional recovery after spinal cord injury. While BPY-DCA is not a clinical drug, the clinically approved iron chelator deferoxamine mesylate (DFO) may be a suitable alternative for anti-scarring treatment (AST). In order to prove the scar-suppressing efficacy of DFO we modified a recently published in vitro model for CNS scarring. The model comprises a co-culture system of cerebral astrocytes and meningeal fibroblasts, which form scar-like clusters when stimulated with transforming growth factor-ß (TGF-ß). We studied the mechanisms of TGF-ß-induced CNS scarring and compared the efficiency of different putative pharmacological scar-reducing treatments, including BPY-DCA, DFO and cAMP as well as combinations thereof. We observed modulation of TGF-ß-induced scarring at the level of fibroblast proliferation and contraction as well as specific changes in the expression of extracellular matrix molecules and axon growth inhibitory proteins. The individual and combinatorial pharmacological treatments had distinct effects on the cellular and molecular aspects of in vitro scarring. DFO could be identified as a putative anti-scarring treatment for CNS trauma. We subsequently validated this by local application of DFO to a dorsal hemisection in the rat thoracic spinal cord. DFO treatment led to significant reduction of scarring, slightly increased regeneration of corticospinal tract as well as ascending CGRP-positive axons and moderately improved locomotion. We conclude that the in vitro model for CNS scarring is suitable for efficient pre-screening and identification of putative scar-suppressing agents prior to in vivo application and validation, thus saving costs, time and laboratory animals.

Characterization of Regenerative Phenotype of Unrestricted Somatic Stem Cells (USSC) from Human Umbilical Cord Blood (hUCB) by Functional Secretome Analysis.

Stem cell transplantation is a promising therapeutic strategy to enhance axonal regeneration after spinal cord injury. Unrestricted somatic stem cells (USSC) isolated from human umbilical cord blood is an attractive stem cell population available at GMP grade without any ethical concerns. It has been shown that USSC transplantation into acute injured rat spinal cords leads to axonal regrowth and significant locomotor recovery, yet lacking cell replacement. Instead, USSC secrete trophic factors enhancing neurite growth of primary cortical neurons in vitro. Here, we applied a functional secretome approach characterizing proteins secreted by USSC for the first time and validated candidate neurite growth promoting factors using primary cortical neurons in vitro. By mass spectrometric analysis and exhaustive bioinformatic interrogation we identified 1156 proteins representing the secretome of USSC. Using Gene Ontology we revealed that USSC secretome contains proteins involved in a number of relevant biological processes of nerve regeneration such as cell adhesion, cell motion, blood vessel formation, cytoskeleton organization and extracellular matrix organization. We found for instance that 31 well-known neurite growth promoting factors like, e.g. neuronal growth regulator 1, NDNF, SPARC, and PEDF span the whole abundance range of USSC secretome. By the means of primary cortical neurons in vitro assays we verified SPARC and PEDF as significantly involved in USSC mediated neurite growth and therewith underline their role in improved locomotor recovery after transplantation. From our data we are convinced that USSC are a valuable tool in regenerative medicine as USSC's secretome contains a comprehensive network of trophic factors supporting nerve regeneration not only by a single process but also maintained its regenerative phenotype by a multitude of relevant biological processes.

Growth/differentiation factor-15 and its role in peripheral nervous system lesion and regeneration.

Growth/differentiation factor-15 (GDF-15) is a distant member of the transforming growth factor beta (TGF-ß) superfamily. It is widely distributed in the nervous system, where it has been shown to play an important role in neuronal maintenance. The present study investigates the role of endogenous GDF-15 in sciatic nerve (SN) lesions using wild-type (WT) and GDF-15 knock-out (KO) mice. SN of 5-6-month-old mice were crushed or transected. Dorsal root ganglia (DRG) and nerve tissue were analyzed at different time points from 6 h to 9 weeks post-lesion. Both crush and transection induced GDF-15 mRNA and protein in the distal portion of the nerve, with a peak at day 7. DRG neuron death did not significantly differ between the genotypes; similarly, remyelination of regenerating axons was not affected by the genotype. Alternative macrophage activation and macrophage recruitment were more pronounced in the KO nerve. Protrusion speed of axons was similar in the two genotypes but WT axons showed better maturation, as indicated by larger caliber at 9 weeks. Furthermore, the regenerated WT nerve showed better performance in the electromyography test, indicating better functional recovery. We conclude that endogenous GDF-15 is beneficial for axon regeneration following SN crush.

AAV-mediated expression of BAG1 and ROCK2-shRNA promote neuronal survival and axonal sprouting in a rat model of rubrospinal tract injury.

A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback, and locomotor deficits. In this study, we used adeno-associated virus (AAV)-mediated over-expression of BAG1 and ROCK2-shRNA in the red nucleus to trace [by co-expression of enhanced green fluorescent protein (EGFP)] and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract, and motor recovery 12 weeks after unilateral dorsal hemisection at Th8 in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV-mediated protein over-expression versus AAV.shRNA-mediated down-regulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2-shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion.

Neural ECM mimetics.

The consequence of numerous neurological disorders is the significant loss of neural cells, which further results in multilevel dysfunction or severe functional deficits. The extracellular matrix (ECM) is of tremendous importance for neural regeneration mediating ambivalent functions: ECM serves as a growth-promoting substrate for neurons but, on the other hand, is a major constituent of the inhibitory scar, which results from traumatic injuries of the central nervous system. Therefore, cell and tissue replacement strategies on the basis of ECM mimetics are very promising therapeutic interventions. Numerous synthetic and natural materials have proven effective both in vitro and in vivo. The closer a material's physicochemical and molecular properties are to the original extracellular matrix, the more promising its effectiveness may be. Relevant factors that need to be taken into account when designing such materials for neural repair relate to receptor-mediated cell-matrix interactions, which are dependent on chemical and mechanical sensing. This chapter outlines important characteristics of natural and synthetic ECM materials (scaffolds) and provides an overview of recent advances in design and application of ECM materials for neural regeneration, both in therapeutic applications and in basic biological research.

Spinal cord injury - there is not just one way of treating it.

In the last century, research in the field of spinal cord trauma has brought insightful knowledge which has led to a detailed understanding of mechanisms that are involved in injury- and recovery-related processes. The quest for a cure for the yet generally incurable condition as well as the exponential rise in gained information has brought about the development of numerous treatment approaches while at the same time the abundance of data has become quite unmanageable. Owing to an enormous amount of preclinical therapeutic approaches, this report highlights important trends rather than specific treatment strategies. We focus on current advances in the treatment of spinal cord injury and want to further draw attention to arising problems in spinal cord injury (SCI) research and discuss possible solutions.