FOXO3 is involved in the tumor necrosis factor-driven inflammatory response in fibroblast-like synoviocytes.

Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Forkhead box O 3 (FOXO3) perturbations in immune cells are increasingly linked to RA pathogenesis. Here, we show that FOXO3 is distinctly inactivated/phosphorylated in the FLS of rheumatoid synovitis. In vitro, stimulation of FLS with tumor necrosis factor-alpha α (TNFα) induced a rapid and sustained inactivation of FOXO3. mRNA profiling revealed that the inactivation of FOXO3 is important for the sustained pro-inflammatory interferon response to TNFα (CXCL9, CXCL10, CXCL11, and TNFSF18). Mechanistically, our studies demonstrate that the inactivation of FOXO3 results from TNF-induced downregulation of phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1). Thus, we identified FOXO3 and its modulator PIK3IP1 as a critical regulatory circuit for the inflammatory response of the resident mesenchymal cells to TNFα and contribute insight into how the synovial tissue brings about chronic inflammation that is driven by TNFα.

Targeted inhibition of Janus kinases abates interfon gamma-induced invasive behaviour of fibroblast-like synoviocytes.

Objectives: The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods: To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results: Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion: Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

Social learning in Cartilaginous fish (stingrays Potamotrygon falkneri).

Social learning is considered one of the hallmarks of cognition. Observers learn from demonstrators that a particular behavior pattern leads to a specific consequence or outcome, which may be either positive or negative. In the last few years, social learning has been studied in a variety of taxa including birds and bony fish. To date, there are few studies demonstrating learning processes in cartilaginous fish. Our study shows that the cartilaginous fish freshwater stingrays (Potamotrygon falkneri) are capable of social learning and isolates the processes involved. Using a task that required animals to learn to remove a food reward from a tube, we found that observers needed significantly (P < 0.01) fewer trials to learn to extract the reward than demonstrators. Furthermore, observers immediately showed a significantly (P < 0.05) higher frequency of the most efficient "suck and undulation" strategy exhibited by the experienced demonstrators, suggesting imitation. Shedding light on social learning processes in cartilaginous fish advances the systematic comparison of cognition between aquatic and terrestrial vertebrates and helps unravel the evolutionary origins of social cognition.

Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor-ligand interactions.

We describe for the first time fluorescent virus-like particles decorated with biologically active mono- and multisubunit immune receptors of choice and the basic application of such fluorosomes (FSs) to visualize and target immune receptor-ligand interactions. For that purpose, human embryonic kidney (HEK)-293 cells were stably transfected with Moloney murine leukemia virus (MoMLV) matrix protein (MA) GFP fusion constructs. To produce FSs, interleukins (ILs), IL-receptors (IL-Rs), and costimulatory molecules were fused to the glycosyl phosphatidyl inositol anchor acceptor sequence of CD16b and coexpressed along with MoMLV group-specific antigen-polymerase (gag-pol) in MA::GFP(+) HEK-293 cells. We show that IL-2 decorated but not control-decorated FSs specifically identify normal and malignant IL-2 receptor-positive (IL-2R(+)) lymphocytes by flow cytometry. In addition to cytokines and costimulatory molecules, FSs were also successfully decorated with the heterotrimeric IL-2Rs, allowing identification of IL-2(+) target cells. Specificity of binding was proven by complete inhibition with nonlabeled, soluble ligands. Moreover, IL-2R FSs efficiently neutralized soluble IL-2 and thus induced unresponsiveness of T cells receiving full activation stimuli via T-cell antigen receptor and CD28. FSs are technically simple, multivalent tools for assessing and blocking mono- and multisubunit immune receptor-ligand interactions with natural constituents in a plasma membrane context.

Establishment of a human three-dimensional chip-based chondro-synovial coculture joint model for reciprocal cross talk studies in arthritis research.

Rheumatoid arthritis is characterised by a progressive, intermittent inflammation at the synovial membrane, which ultimately leads to the destruction of the synovial joint. The synovial membrane as the joint capsule's inner layer is lined with fibroblast-like synoviocytes that are the key player supporting persistent arthritis leading to bone erosion and cartilage destruction. While microfluidic models that model molecular aspects of bone erosion between bone-derived cells and synoviocytes have been established, RA's synovial-chondral axis has not yet been realised using a microfluidic 3D model based on human patient in vitro cultures. Consequently, we established a chip-based three-dimensional tissue coculture model that simulates the reciprocal cross talk between individual synovial and chondral organoids. When co-cultivated with synovial organoids, we could demonstrate that chondral organoids induce a higher degree of cartilage physiology and architecture and show differential cytokine response compared to their respective monocultures highlighting the importance of reciprocal tissue-level cross talk in the modelling of arthritic diseases.

A new method for studying problem solving and tool use in stingrays (Potamotrygon castexi).

Testing the cognitive abilities of cartilaginous fishes is important in understanding the evolutionary origins of cognitive functions in higher vertebrates. We used five South American fresh water stingrays (Potamotrygon castexi) in a learning and problem-solving task. A tube test apparatus was developed to provide a simple but sophisticated procedure for testing cognitive abilities of aquatic animals. All five subjects quickly learned to use water as a tool to extract food from the testing apparatus. The experimental protocol, which gave the animals the opportunity of correcting a wrong visual cue decision, resulted in four out of five subjects correcting an error rather than making an initial right choice. One of five subjects reached 100% correct trials in the visual discrimination task. The ability to use water as an agent to extract food from the testing apparatus is a first indication of tool use in batoid fishes. Performance in the instrumental task of retrieving food from a novel testing apparatus and the rapid learning in the subsequent discrimination/error correction task shows that cartilaginous fish can be used to study the origins of cognitive functions in the vertebrate lineage.

Monitoring tissue-level remodelling during inflammatory arthritis using a three-dimensional synovium-on-a-chip with non-invasive light scattering biosensing.

Rheumatoid arthritis is a chronic, systemic joint disease in which an autoimmune response translates into an inflammatory attack resulting in joint damage, disability and decreased quality of life. Despite recent introduction of therapeutic agents such as anti-TNFα, even the best current therapies fail to achieve disease remission in most arthritis patients. Therefore, research into the mechanisms governing the destructive inflammatory process in rheumatoid arthritis is of great importance and may reveal novel strategies for the therapeutic interventions. To gain deeper insight into its pathogensis, we have developed for the first time a three-dimensional synovium-on-a-chip system in order to monitor the onset and progression of inflammatory synovial tissue responses. In our study, patient-derived primary synovial organoids are cultivated on a single chip platform containing embedded organic-photodetector arrays for over a week in the absence and presence of tumor-necrosis-factor. Using a label-free and non-invasive optical light-scatter biosensing strategy inflammation-induced 3D tissue-level architectural changes were already detected after two days. We demonstrate that the integration of complex human synovial organ cultures in a lab-on-a-chip provides reproducible and reliable information on how systemic stress factors affect synovial tissue architectures.

Peripheral nerve transfers change target muscle structure and function.

Selective nerve transfers surgically rewire motor neurons and are used in extremity reconstruction to restore muscle function or to facilitate intuitive prosthetic control. We investigated the neurophysiological effects of rewiring motor axons originating from spinal motor neuron pools into target muscles with lower innervation ratio in a rat model. Following reinnervation, the target muscle's force regenerated almost completely, with the motor unit population increasing to 116% in functional and 172% in histological assessments with subsequently smaller muscle units. Muscle fiber type populations transformed into the donor nerve's original muscles. We thus demonstrate that axons of alternative spinal origin can hyper-reinnervate target muscles without loss of muscle force regeneration, but with a donor-specific shift in muscle fiber type. These results explain the excellent clinical outcomes following nerve transfers in neuromuscular reconstruction. They indicate that reinnervated muscles can provide an accurate bioscreen to display neural information of lost body parts for high-fidelity prosthetic control.

Octopus arm choice is strongly influenced by eye use.

This study aims to investigate the octopus' eye and arm coordination and raises the question if visual guidance determines choice of arm use. Octopuses possess eight seemingly identical arms but have recently been reported to show a preference as to which arm they use to initiate contact with objects. These animals also exhibit lateralized eye use, therefore, a connection between eye and arm preference seems possible. Seven Octopus vulgaris were observed during approach, contact initiation and exploration of plastic objects that were positioned on three different levels in the water column. The subjects most commonly used an arm to initiate contact with an object that was in a direct line between the eye used to look at the object, and the object itself. This indicates that choice of arm use is spatially rather opportunistic when depending on visual guidance. Additionally, first contact with an object was usually established by the central third of the arm and in arm contact sequences neighboring arms were the most likely to follow an arm already touching the object. Although results point towards strong eye/arm coordination, we did not find lateralized behavior in this experiment. Results are discussed from a neuro-anatomical, behavioral and ecological perspective.

When do octopuses play? Effects of repeated testing, object type, age, and food deprivation on object play in Octopus vulgaris.

Studying play behavior in octopuses is an important step toward understanding the phylogenetic origins and function of play as well as the cognitive abilities of invertebrates. Fourteen Octopus vulgaris (7 subadults and 7 adults) were presented 2 Lego objects and 2 different food items on 7 consecutive days under 2 different levels of food deprivation. Nine subjects showed play-like behavior with the Lego objects. There was no significant difference in play-like behavior corresponding to food deprivation, age, and sex of the octopuses. The sequence of behaviors, from exploration to play-like behavior, had a significant influence on the establishment of play-like behavior, as it occurred mostly on Days 3-6 of the 7-day experiment. The pattern of development of play-like activities after a period of exploration and habituation in this study agrees with the hypothesis that object play follows object exploration. A homologous origin of this behavioral trait in vertebrates and invertebrates is highly unlikely, as the last common ancestor might not have had the cognitive capacity to possess this trait.

Contrasting activity patterns of two related octopus species, Octopus macropus and Octopus vulgaris.

Octopus macropus and Octopus vulgaris have overlapping habitats and are exposed to similar temporal changes. Whereas the former species is described as nocturnal in the field, there are conflicting reports about the activity time of the latter one. To compare activity patterns, the authors tested both species in the laboratory. Octopuses were exposed to a light-dark cycle and held under constant dim light for 7 days each. O. macropus showed nocturnal and light-cued activity. According to casual observations, O. vulgaris started out nocturnal but had switched to mostly diurnal when the experiment began. Individual variation of its activity was found. The different activity patterns of O. macropus and O. vulgaris might reflect their lifestyles, the latter species being more generalist.

Does Octopus vulgaris have preferred arms?

Previous behavioral studies in Octopus vulgaris revealed lateralization of eye use. In this study, the authors expanded the scope to investigate arm preferences. The octopus's generalist hunting lifestyle and the structure of their arms suggest that these animals have no need to designate specific arms for specific tasks. However, octopuses also show behaviors, like exploration, in which only single or small groups of arms are involved. Here the authors show that octopuses had a strong preference for anterior arm use to reach for and explore objects, which points toward a task division between anterior and posterior arms. Four out of 8 subjects also showed a lateral bias. In addition, octopuses had a preference for a specific arm to reach into a T maze to retrieve a food reward. These findings give evidence for limb-specialization in an animal whose 8 arms were believed to be equipotential.

Correlation Between Multimodal Microscopy, Tissue Morphology, and Enzymatic Resistance in Riboflavin-UVA Cross-Linked Human Corneas.

PURPOSE: To explore the utility of multimodal microscopy as a noninvasive tool to assess corneal collagen cross-linking (CXL) efficacy, we investigated the correlation between riboflavin (RF) axial profile, second harmonic generation (SHG) imaging, and histological/biochemical changes of human corneas after RF-ultraviolet A (UVA)-catalyzed CXL. METHODS: De-epithelialized human corneoscleral tissues were imaged by confocal and multiphoton microscopy to study RF tissue diffusion profile and SHG-based roughness index (Rq) after CXL. We installed 0.1% RF for 5, 10, and 20 minutes, respectively, followed by UVA irradiation, while dextran drug vehicle-treated corneas served as controls. Masson's trichrome staining and collagenase digestion assay were employed to assess ultrastructural modifications of collagen lamellae and bioenzymatic strength following RF-UVA CXL. RESULTS: Stromal absorption of RF was significantly higher in 20 minutes compared with 5- and 10-minute drug instillations. The roughness index of SHG images was reduced after RF-UVA CXL at all RF instillation time points compared with dextran controls. Interestingly, correlation between axial profiles of RF dosage and Rq index was only observed in 10- and 20-minute RF instillations (R(2) = 0.13 and 0.28, respectively, all P < 0.05), but not in the 5-minute group. Masson's trichrome staining revealed collagen fibril compaction in cross-linked corneas in an RF dose-dependent manner. Collagenase digestion assay showed significantly increased biochemical strength by higher RF doses in cross-linked corneas. CONCLUSIONS: Intrastromal RF distribution profiles correlated with histological and functional property changes in RF-UVA cross-linked corneas. A riboflavin-defined threshold further determined the sensitivity of SHG imaging as a noninvasive imaging modality to assess the efficacy of RF-UVA CXL.

Octopus senescence: the beginning of the end.

Senescence is a normal stage of an octopus's life cycle that often occurs before death. Some of the following symptoms typify it: lack of feeding, retraction of skin around the eyes, uncoordinated movement, increased undirected activity, and white unhealing lesions on the body. There is inter- and intraspecific variability. Senescence is not a disease or a result of disease, although diseases can also be a symptom of it. Both males and females go through a senescent stage before dying-the males after mating, the females while brooding eggs and after the eggs hatch. There are many aspects of octopus senescence that have not yet been studied. This study discusses the ecological implications of senescence.

Octopus vulgaris uses visual information to determine the location of its arm.

Octopuses are intelligent, soft-bodied animals with keen senses that perform reliably in a variety of visual and tactile learning tasks. However, researchers have found them disappointing in that they consistently fail in operant tasks that require them to combine central nervous system reward information with visual and peripheral knowledge of the location of their arms. Wells claimed that in order to filter and integrate an abundance of multisensory inputs that might inform the animal of the position of a single arm, octopuses would need an exceptional computing mechanism, and "There is no evidence that such a system exists in Octopus, or in any other soft bodied animal." Recent electrophysiological experiments, which found no clear somatotopic organization in the higher motor centers, support this claim. We developed a three-choice maze that required an octopus to use a single arm to reach a visually marked goal compartment. Using this operant task, we show for the first time that Octopus vulgaris is capable of guiding a single arm in a complex movement to a location. Thus, we claim that octopuses can combine peripheral arm location information with visual input to control goal-directed complex movements.