The Development of Principles for Patient and Public Involvement (PPI) in Preclinical Spinal Cord Research: A Modified Delphi Study.

INTRODUCTION: There is currently limited guidance for researchers on Patient and Public Involvement (PPI) for preclinical spinal cord research, leading to uncertainty about design and implementation. This study aimed to develop evidence-informed principles to support preclinical spinal cord researchers to incorporate PPI into their research. METHODS: This study used a modified Delphi method with the aim of establishing consensus on a set of principles for PPI in spinal cord research. Thirty-eight stakeholders including researchers, clinicians and people living with spinal cord injury took part in the expert panel. Participants were asked to rate their agreement with a series of statements relating to PPI in preclinical spinal cord research over two rounds. As part of Round 2, they were also asked to rate statements as essential or desirable. RESULTS: Thirty-eight statements were included in Round 1, after which five statements were amended and two additional statements were added. After Round 2, consensus (> 75% agreement) was reached for a total of 27 principles, with 13 rated as essential and 14 rated as desirable. The principles with highest agreement related to diversity in representation among PPI contributors, clarity of the purpose of PPI and effective communication. CONCLUSION: This research developed a previously unavailable set of evidence-informed principles to inform PPI in preclinical spinal cord research. These principles provide guidance for researchers seeking to conduct PPI in preclinical spinal cord research and may also inform PPI in other preclinical disciplines. PATIENT AND PUBLIC INVOLVEMENT STATEMENT: This study was conducted as part of a project aiming to develop PPI in preclinical spinal cord injury research associated with an ongoing research collaboration funded by the Irish Rugby Football Union Charitable Trust (IRFU CT) and the Science Foundation Ireland Centre for Advanced Materials and BioEngineering Research (SFI AMBER), with research conducted by the Tissue Engineering Research Group (TERG) at the RCSI University of Medicine and Health Sciences. The project aims to develop an advanced biomaterials platform for spinal cord repair and includes a PPI Advisory Panel comprising researchers, clinicians and seriously injured rugby players to oversee the work of the project. PPI is included in this study through the involvement of members of the PPI Advisory Panel in the conceptualisation of this research, review of findings, identification of key points for discussion and preparation of the study manuscript as co-authors.

The role of Patient and public involvement (PPI) in pre-clinical spinal cord research: An interview study.

BACKGROUND: Patient and public involvement in research (PPI) has many benefits including increasing relevance and impact. While using PPI in clinical research is now an established practice, the involvement of patients and the public in pre-clinical research, which takes place in a laboratory setting, has been less frequently described and presents specific challenges. This study aimed to explore the perspectives of seriously injured rugby players' who live with a spinal cord injury on PPI in pre-clinical research. METHODS: Semi-structured interviews were conducted via telephone with 11 seriously injured rugby players living with spinal cord injury on the island of Ireland. A purposive sampling approach was used to identify participants. Selected individuals were invited to take part via gatekeeper in a charitable organisation that supports seriously injured rugby players. Interviews were transcribed verbatim and analysed thematically. FINDINGS: Six themes were identified during analysis: 'appreciating potential benefits of PPI despite limited knowledge', 'the informed perspectives of people living with spinal cord injury can improve pre-clinical research relevance', 'making pre-clinical research more accessible reduces the potential for misunderstandings to occur', 'barriers to involvement include disinterest, accessibility issues, and fear of losing hope if results are negative', 'personal contact and dialogue helps people feel valued in pre-clinical research, and 'PPI can facilitate effective dissemination of pre-clinical research as desired by people living with spinal cord injury.' CONCLUSION: People affected by spinal cord injury in this study desire further involvement in pre-clinical spinal cord injury research through dialogue and contact with researchers. Sharing experiences of spinal cord injury can form the basis of PPI for pre-clinical spinal cord injury research.

The zebrafish mutant dreammist implicates sodium homeostasis in sleep regulation.

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene, dreammist (dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed dmist gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of either fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since atpa1a3a and dmist mutants have elevated intracellular Na+ levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+ pump function modulates neuronal excitability to maintain normal sleep behaviour.

Longitudinal characterization of TK6 cells sequentially adapted to animal product-free, chemically defined culture medium: considerations for genotoxicity studies.

Introduction: In vitro approaches are an essential tool in screening for toxicity of new chemicals, products and therapeutics. To increase the reproducibility and human relevance of these in vitro assessments, it is advocated to remove animal-derived products such as foetal bovine serum (FBS) from the cell culture system. Currently, FBS is routinely used as a supplement in cell culture medium, but batch-to-batch variability may introduce inconsistency in inter- and intra-lab assessments. Several chemically defined serum replacements (CDSR) have been developed to provide an alternative to FBS, but not every cell line adapts easily and successfully to CDSR-supplemented medium, and the long-term effect on cell characteristics remains uncertain. Aim: The aim of this study was to adapt the TK6 cell line to animal-product free CDSR-supplemented medium and evaluate the long-term effects on cell health, growth, morphology, phenotype, and function. This included a provisional assessment to determine the suitability of the transitioned cell line for standardised genotoxicity testing using the "in vitro mammalian cell micronucleus test" (OECD TG 487). Materials and methods: Gradual adaptation and direct adaptation methodologies were compared by assessing the cell proliferation, size and viability every passage until the cells were fully adapted to animal-free CDSR. The metabolic activity and membrane integrity was assessed every 4-8 passages by PrestoBlue and CytoTox-ONE™ Homogeneous Membrane Integrity Assay respectively. A detailed morphology study by high content imaging was performed and the expression of cell surface markers (CD19 and CD20) was conducted via flow cytometry to assess the potential for phenotypic drift during longer term culture of TK6 in animal-free conditions. Finally, functionality of cells in the OECD TG 487 assay was evaluated. Results: The baseline characteristics of TK6 cells cultured in FBS-supplemented medium were established and variability among passages was used to set up acceptance criteria for CDSR adapted cells. TK6 were adapted to CDSR supplemented medium either via direct or gradual transition reducing from 10% v/v FBS to 0% v/v FBS. The cell growth rate was compromised in the direct adaptation and therefore the gradual adaptation was preferred to investigate the long-term effects of animal-free CDSR on TK6 cells. The new animal cells showed comparable (p > 0.05) viability and cell size as the parent FBS-supplemented cells, with the exception of growth rate. The new animal free cells showed a lag phase double the length of the original cells. Cell morphology (cellular and nuclear area, sphericity) and phenotype (CD19 and CD20 surface markers) were in line (p > 0.05) with the original cells. The new cells cultured in CDSR-supplemented medium performed satisfactory in a pilot OECD TG 487 assay with compounds not requiring metabolic activation. Conclusion: TK6 cells were successfully transitioned to FBS- and animal product-free medium. The new cell cultures were viable and mimicked the characteristics of FBS-cultured cells. The gradual transition methodology utilised in this study can also be applied to other cell lines of interest. Maintaining cells in CDSR-supplemented medium eliminates variability from FBS, which in turn is likely to increase the reproducibility of in vitro experiments. Furthermore, removal of animal derived products from cell culture techniques is likely to increase the human relevance of in vitro methodologies.

Automated Quantification of the Behaviour of Beef Cattle Exposed to Heat Load Conditions.

Cattle change their behaviour in response to hot temperatures, including by engaging in stepping that indicates agitation. The automated recording of these responses would be helpful in the timely diagnosis of animals experiencing heat loading. Behavioural responses of beef cattle to hot environmental conditions were studied to investigate whether it was possible to assess behavioural responses by video-digitised image analysis. Open-source automated behavioural quantification software was used to record pixel changes in 13 beef cattle videorecorded in a climate-controlled chamber during exposure to a simulated typical heat event in Queensland, Australia. Increased digitised movement was observed during the heat event, which was related to stepping and grooming/scratching activities in standing animals. The 13 cattle were exposed in two cohorts, in which the first group of cattle (n = 6) was fed a standard finisher diet based on a high percentage of cereal grains, and the second group of cattle (n = 7) received a substituted diet in which 8% of the grains were replaced by lucerne hay. The second group displayed a smaller increase in digitised movements on exposure to heat than the first, suggesting less discomfort under hot conditions. The results suggest that cattle exposed to heat display increased movement that can be detected automatically by video digitisation software, and that replacing some cereal grain with forage in the diet of feedlot cattle may reduce the measured activity responses to the heat.

The Manufacture and Characterization of Biomimetic, Biomaterial-Based Scaffolds for Studying Physicochemical Interactions of Neural Cells in 3D Environments.

A particular challenge to the field of neuroscience involves translating findings from 2D in vitro systems to 3D in vivo environments. Standardized cell culture environments that adequately reflect the properties of the central nervous system (CNS) such as the stiffness, protein composition, and microarchitecture in which to study 3D cell-cell and cell-matrix interactions are generally lacking for in vitro culture systems. In particular, there remains an unmet need for reproducible, low-cost, high-throughput, and physiologically relevant environments comprised of tissue-native matrix proteins for the study of CNS microenvironments in 3D. Advances in the field of biofabrication over the past number of years have facilitated the production and characterization of biomaterial-based scaffolds. Typically developed for tissue engineering applications, they also provide sophisticated environments in which to study cell-cell and cell-matrix interactions and have been used for 3D modeling for a range of tissues. Here, we describe a simple and scalable protocol for the production of biomimetic, highly porous freeze-dried hyaluronic acid scaffolds with tunable microarchitecture, stiffness, and protein composition. Furthermore, we describe several different approaches that can be used to characterize a range of physicochemical properties and how to employ the scaffolds for the 3D culture of sensitive CNS cells in vitro. Finally, we detail several approaches for the study of key cell responses within the 3D scaffold environments. Overall, this protocol describes the manufacture and testing of a biomimetic and tunable macroporous scaffold system for neuronal cell culture applications. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Scaffold manufacture Basic Protocol 2: Scaffold characterization Basic Protocol 3: Cell culture and analysis of neurons in scaffolds.

Applying Patient and Public Involvement in preclinical research: A co-created scoping review.

BACKGROUND: Patient and Public Involvement (PPI) in research aims to improve the quality, relevance and appropriateness of research. PPI has an established role in clinical research where there is evidence of benefit, and where policymakers and funders place continued emphasis on its inclusion. However, for preclinical research, PPI has not yet achieved the same level of integration. As more researchers, including our team, aim to include PPI in preclinical research, the development of an evidence-based approach is important. Therefore, this scoping review aimed to identify and map studies where PPI has been used in preclinical research and develop principles that can be applied in other projects. METHODS: A scoping review was conducted to search the literature in Medline (PubMed), EMBASE, CINAHL, PsycInfo and Web of Science Core Collection to identify applied examples of preclinical PPI. Two independent reviewers conducted study selection and data extraction separately. Data were extracted relating to PPI in terms of (i) rationale and aims, (ii) approach used, (iii) benefits and challenges, (iv) impact and evaluation and (v) learning opportunities for preclinical PPI. Findings were reviewed collaboratively by PPI contributors and the research team to identify principles that could be applied to other projects. RESULTS: Nine studies were included in the final review with the majority of included studies reporting PPI to improve the relevance of their research, using approaches such as PPI advisory panels and workshops. Researchers report several benefits and challenges, although evidence of formal evaluation is limited. CONCLUSION: Although currently there are few examples of preclinical research studies reporting empirical PPI activity, their findings may support those aiming to use PPI in preclinical research. Through collaborative analysis of the scoping review findings, several principles were developed that may be useful for other preclinical researchers. PATIENT OR PUBLIC CONTRIBUTION: This study was conducted as part of a broader project aiming to develop an evidence base for preclinical PPI that draws on a 5-year preclinical research programme focused on the development of advanced biomaterials for spinal cord repair as a case study. A PPI Advisory Panel comprising seriously injured rugby players, clinicians, preclinical researchers and PPI facilitators collaborated as co-authors on the conceptualization, execution and writing of this review, including refining the findings into the set of principles reported here.

Biomimetic Scaffolds for Spinal Cord Applications Exhibit Stiffness-Dependent Immunomodulatory and Neurotrophic Characteristics.

After spinal cord injury (SCI), tissue engineering scaffolds offer a potential bridge for regeneration across the lesion and support repair through proregenerative signaling. Ideal biomaterial scaffolds that mimic the physicochemical properties of native tissue have the potential to provide innate trophic signaling while also minimizing damaging inflammation. To address this challenge, taking cues from the spinal cord's structure, the proregenerative signaling capabilities of native cord components are compared in vitro. A synergistic mix of collagen-IV and fibronectin (Coll-IV/Fn) is found to optimally enhance axonal extension from neuronal cell lines (SHSY-5Y and NSC-34) and induce morphological features typical of quiescent astrocytes. This optimal composition is incorporated into hyaluronic acid scaffolds with aligned pore architectures but varying stiffnesses (0.8-3 kPa). Scaffolds with biomimetic mechanical properties (<1 kPa), functionalized with Coll-IV/Fn, not only modulate primary astrocyte behavior but also stimulate the production of anti-inflammatory cytokine IL-10 in a stiffness-dependent manner. Seeded SHSY-5Y neurons generate distributed neuronal networks, while softer biomimetic scaffolds promote axonal outgrowth in an ex vivo model of axonal regrowth. These results indicate that the interaction of stiffness and biomaterial composition plays an essential role in vitro in generating repair-critical cellular responses and demonstrates the potential of biomimetic scaffold design.

Functionalization of Electrospun Polycaprolactone Scaffolds with Matrix-Binding Osteocyte-Derived Extracellular Vesicles Promotes Osteoblastic Differentiation and Mineralization.

Synthetic polymeric materials have demonstrated great promise for bone tissue engineering based on their compatibility with a wide array of scaffold-manufacturing techniques, but are limited in terms of the bioactivity when compared to naturally occurring materials. To enhance the regenerative properties of these materials, they are commonly functionalised with bioactive factors to guide growth within the developing tissue. Extracellular matrix vesicles (EVs) play an important role in facilitating endochondral ossification during long bone development and have recently emerged as important mediators of cell-cell communication coordinating bone regeneration, and thus represent an ideal target to enhance the regenerative properties of synthetic scaffolds. Therefore, in this paper we developed tools and protocols to enable the attachment of MLO-Y4 osteocyte-derived EVs onto electrospun polycaprolactone (PCL) scaffolds for bone repair. Initially, we optimize a method for the functionalization of PCL materials with collagen type-1 and fibronectin, inspired by the behaviour of matrix vesicles during endochondral ossification, and demonstrate that this is an effective method for the adhesion of EVs to the material surface. We then used this functionalization process to attach osteogenic EVs, collected from mechanically stimulated MLO-Y4 osteocytes, to collagen-coated electrospun PCL scaffolds. The EV-functionalized scaffold promoted osteogenic differentiation (measured by increased ALP activity) and mineralization of the matrix. In particular, EV-functionalised scaffolds exhibited significant increases in matrix mineralization particularly at earlier time points compared to uncoated and collagen-coated controls. This approach to matrix-based adhesion of EVs provides a mechanism for incorporating vesicle signalling into polyester scaffolds and demonstrates the potential of osteocyte derived EVs to enhance the rate of bone tissue regeneration.

Patient and Public Involvement (PPI) in preclinical research: A scoping review protocol.

Introduction: Patient and public involvement (PPI) aims to improve the quality, relevance, and appropriateness of research and ensure that it meets the needs and expectations of those affected by particular conditions to the greatest possible degree. The evidence base for the positive impact of PPI on clinical research continues to grow, but the role of PPI in preclinical research (an umbrella term encompassing 'basic', 'fundamental', 'translational' or 'lab-based' research) remains limited. As funding bodies and policymakers continue to increase emphasis on the relevance of PPI to preclinical research, it is timely to map the PPI literature to support preclinical researchers involving the public, patients, or other service users in their research. Therefore, the aim of this scoping review is to explore the literature on patient and public involvement in preclinical research from any discipline. Methods: This scoping review will search the literature in Medline (PubMed), Embase, CINAHL, PsycINFO, Web of Science Core Collection, Scopus, and OpenGrey.net to explore the application of PPI in preclinical research. This review will follow the Joanna Briggs Institute (JBI) guidelines for scoping reviews. It will be reported according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR). Two reviewers will independently review articles for inclusion in the final review. Data extraction will be guided by the research questions. The PPI advisory panel will then collaboratively identify themes in the extracted data. Discussion: This scoping review will provide a map of current evidence surrounding preclinical PPI, and identify the body of literature on this topic, which has not been comprehensively reviewed to date. Findings will inform ongoing work of the research team, support the work of other preclinical researchers aiming to include PPI in their own research, and identify knowledge and practice gaps. Areas for future research will be identified.

Fabrication of blood-derived elastogenic vascular grafts using electrospun fibrinogen and polycaprolactone composite scaffolds for paediatric applications.

The development of tissue-engineered vascular grafts (TEVGs) for paediatric applications must consider unique factors associated with this patient cohort. Although the increased elastogenic potential of neonatal cells offers an opportunity to overcome the long-standing challenge of in vitro elastogenesis, neonatal patients have a lower tolerance for autologous tissue harvest and require grafts that exhibit growth potential. The purpose of this study was to apply a multipronged strategy to promote elastogenesis in conjunction with umbilical cord-derived materials in the production of a functional paediatric TEVG. An initial proof-of-concept study was performed to extract fibrinogen from human umbilical cord blood samples and, through electrospinning, to produce a nanofibrous fibrinogen scaffold. This scaffold was seeded with human umbilical artery-derived smooth muscle cells (hUASMCs), and neotissue formation within the scaffold was examined using immunofluorescence microscopy. Subsequently, a polycaprolactone-reinforced porcine blood-derived fibrinogen scaffold (isolated using the same protocol as cord blood fibrinogen) was used to develop a rolled-sheet graft that employed topographical and biochemical guidance cues to promote elastogenesis and cellular orientation. This approach resulted in a TEVG with robust mechanical properties and biomimetic arrangement of extracellular matrix (ECM) with rich expression of elastic fibre-related proteins. The results of this study hold promise for further development of paediatric TEVGs and the exploration of the effects of scaffold microstructure and nanostructure on vascular cell function and ECM production.

Human bone marrow stem/stromal cell osteogenesis is regulated via mechanically activated osteocyte-derived extracellular vesicles.

Bone formation or regeneration requires the recruitment, proliferation, and osteogenic differentiation of stem/stromal progenitor cells. A potent stimulus driving this process is mechanical loading. Osteocytes are mechanosensitive cells that play fundamental roles in coordinating loading-induced bone formation via the secretion of paracrine factors. However, the exact mechanisms by which osteocytes relay mechanical signals to these progenitor cells are poorly understood. Therefore, this study aimed to demonstrate the potency of the mechanically stimulated osteocyte secretome in driving human bone marrow stem/stromal cell (hMSC) recruitment and differentiation, and characterize the secretome to identify potential factors regulating stem cell behavior and bone mechanobiology. We demonstrate that osteocytes subjected to fluid shear secrete a distinct collection of factors that significantly enhance hMSC recruitment and osteogenesis and demonstrate the key role of extracellular vesicles (EVs) in driving these effects. This demonstrates the pro-osteogenic potential of osteocyte-derived mechanically activated extracellular vesicles, which have great potential as a cell-free therapy to enhance bone regeneration and repair in diseases such as osteoporosis.

Rapid, Phase-free Detection of Long Identity-by-Descent Segments Enables Effective Relationship Classification.

Identity-by-descent (IBD) segments are a useful tool for applications ranging from demographic inference to relationship classification, but most detection methods rely on phasing information and therefore require substantial computation time. As genetic datasets grow, methods for inferring IBD segments that scale well will be critical. We developed IBIS, an IBD detector that locates long regions of allele sharing between unphased individuals, and benchmarked it with Refined IBD, GERMLINE, and TRUFFLE on 3,000 simulated individuals. Phasing these with Beagle 5 takes 4.3 CPU days, followed by either Refined IBD or GERMLINE segment detection in 2.9 or 1.1 h, respectively. By comparison, IBIS finishes in 6.8 min or 7.8 min with IBD2 functionality enabled: speedups of 805-946× including phasing time. TRUFFLE takes 2.6 h, corresponding to IBIS speedups of 20.2-23.3×. IBIS is also accurate, inferring ≥7 cM IBD segments at quality comparable to Refined IBD and GERMLINE. With these segments, IBIS classifies first through third degree relatives in real Mexican American samples at rates meeting or exceeding other methods tested and identifies fourth through sixth degree pairs at rates within 0.0%-2.0% of the top method. While allele frequency-based approaches that do not detect segments can infer relationship degrees faster than IBIS, the fastest are biased in admixed samples, with KING inferring 30.8% fewer fifth degree Mexican American relatives correctly compared with IBIS. Finally, we ran IBIS on chromosome 2 of the UK Biobank dataset and estimate its runtime on the autosomes to be 3.3 days parallelized across 128 cores.

Effects of Constitutive and Acute Connexin 36 Deficiency on Brain-Wide Susceptibility to PTZ-Induced Neuronal Hyperactivity.

Connexins are transmembrane proteins that form hemichannels allowing the exchange of molecules between the extracellular space and the cell interior. Two hemichannels from adjacent cells dock and form a continuous gap junction pore, thereby permitting direct intercellular communication. Connexin 36 (Cx36), expressed primarily in neurons, is involved in the synchronous activity of neurons and may play a role in aberrant synchronous firing, as seen in seizures. To understand the reciprocal interactions between Cx36 and seizure-like neural activity, we examined three questions: (a) does Cx36 deficiency affect seizure susceptibility, (b) does seizure-like activity affect Cx36 expression patterns, and (c) does acute blockade of Cx36 conductance increase seizure susceptibility. We utilize the zebrafish pentylenetetrazol [PTZ; a GABA(A) receptor antagonist] induced seizure model, taking advantage of the compact size and optical translucency of the larval zebrafish brain to assess how PTZ affects brain-wide neuronal activity and Cx36 protein expression. We exposed wild-type and genetic Cx36-deficient (cx35.5-/-) zebrafish larvae to PTZ and subsequently mapped neuronal activity across the whole brain, using phosphorylated extracellular-signal-regulated kinase (pERK) as a proxy for neuronal activity. We found that cx35.5-/- fish exhibited region-specific susceptibility and resistance to PTZ-induced hyperactivity compared to wild-type controls, suggesting that genetic Cx36 deficiency may affect seizure susceptibility in a region-specific manner. Regions that showed increased PTZ sensitivity include the dorsal telencephalon, which is implicated in human epilepsy, and the lateral hypothalamus, which has been underexplored. We also found that PTZ-induced neuronal hyperactivity resulted in a rapid reduction of Cx36 protein levels within 30 min. This Cx36 reduction persists after 1-h of recovery but recovered after 3-6 h. This acute downregulation of Cx36 by PTZ is likely maladaptive, as acute pharmacological blockade of Cx36 by mefloquine results in increased susceptibility to PTZ-induced neuronal hyperactivity. Together, these results demonstrate a reciprocal relationship between Cx36 and seizure-associated neuronal hyperactivity: Cx36 deficiency contributes region-specific susceptibility to neuronal hyperactivity, while neuronal hyperactivity-induced downregulation of Cx36 may increase the risk of future epileptic events.

Harnessing topographical & biochemical cues to enhance elastogenesis by paediatric cells for cardiovascular tissue engineering applications.

The development of tissue-engineered vascular grafts (TEVGs) with a biomimetic extracellular matrix (ECM) structure, including a mature elastic network, remains a key challenge for the production of grafts with long-term functionality. The aim of this study was to investigate the influence of aligned nanofiber substrates on ECM protein synthesis by neonatal smooth muscle cells (SMCs), and to examine the combined effects of this topographical cue in conjunction with transforming growth factor beta-1 (TGF-ß1) - a biochemical elastogenic promoter. Glass coverslips were coated in electrospun fibrinogen nanofibers (average diameter < 500 nm) with either a randomly-orientated or aligned topography. Human umbilical artery smooth muscle cells (hUASMCs) were cultured on the electrospun substrates for 7 and 14 days, with or without a 2 ng/ml TGF-ß1 supplement. The ECM structure was analysed using immunohistochemistry and the quantity of secreted elastin in the cell layer was measured using a dye-binding assay. Aligned fiber substrates induced a directed orientation of both the seeded cells and cell-synthesized ECM fibers. Cells cultured on aligned fibers exhibited a significant increase in the expression of phenotypic contractile proteins, as well as increases in the secreted elastin content of the cell layer, compared to cells cultured on randomly-orientated substrates. TGF-ß1 supplementation was shown to synergistically increase secreted elastin from cells cultured on aligned fiber substrates (p < 0.05). Aligned nanofiber scaffolds can be used to direct cellular orientation, elastin-related protein synthesis and cell phenotype, and consequently there is potential for their application in the development of TEVGs as part of a multi-pronged strategy to promote elastic fiber formation.

Self-homodyne system for next generation intra-datacenter optical interconnects.

We propose a self-homodyne system for next generation intra-datacenter networking. The proposed system has a higher spectral efficiency for the modulated signal compared to the intensity-modulation/direct-detection (IM/DD) systems and uses digital signal processing of reduced complexity compared to a conventional coherent system. The concept of the proposed system is to send the modulated signal and a tone originating from the same laser over the full-duplex fiber with the aid of circulators to be used remotely at the receiver for coherent detection. The overall system physical complexity approaches the equivalent IM/DD system giving the same target data rate for 400G systems and beyond. We experimentally demonstrate emulation of the proposed system and report data rates of 530 Gb/s, 448 Gb/s and 320 Gb/s on a single wavelength below the KP4 forward error correcting threshold over 500 m, 2 km and 10 km of single mode fiber, respectively.

100G and 200G single carrier transmission over 2880 and 320 km using an InP IQ modulator and Stokes vector receiver.

We demonstrate experimentally the transmission of single carrier 56 Gbaud 16-QAM, 8-QAM and QPSK optically modulated signals over 320, 960 and 2,880 km, respectively, using a fully packaged InP IQ modulator and a Stokes vector direct detection (SV-DD) receiver realized using discrete optics. Results show that by optimizing the carrier-to-signal-power ratio, the total throughput-times-distance product for 16 QAM and QPSK are 71,680 Gbps.km and 322,560 Gbps.km, respectively, at bit error rate (BER) below the hard decision forward error correcting threshold (HD-FEC) of 4.5 × 10-3.

VideoHacking: Automated Tracking and Quantification of Locomotor Behavior with Open Source Software and Off-the-Shelf Video Equipment.

Differences in nervous system function can result in differences in behavioral output. Measurements of animal locomotion enable the quantification of these differences. Automated tracking of animal movement is less labor-intensive and bias-prone than direct observation, and allows for simultaneous analysis of multiple animals, high spatial and temporal resolution, and data collection over extended periods of time. Here, we present a new video-tracking system built on Python-based software that is free, open source, and cross-platform, and that can analyze video input from widely available video capture devices such as smartphone cameras and webcams. We validated this software through four tests on a variety of animal species, including larval and adult zebrafish (Danio rerio), Siberian dwarf hamsters (Phodopus sungorus), and wild birds. These tests highlight the capacity of our software for long-term data acquisition, parallel analysis of multiple animals, and application to animal species of different sizes and movement patterns. We applied the software to an analysis of the effects of ethanol on thigmotaxis (wall-hugging) behavior on adult zebrafish, and found that acute ethanol treatment decreased thigmotaxis behaviors without affecting overall amounts of motion. The open source nature of our software enables flexibility, customization, and scalability in behavioral analyses. Moreover, our system presents a free alternative to commercial video-tracking systems and is thus broadly applicable to a wide variety of educational settings and research programs.

Evolutionarily conserved regulation of hypocretin neuron specification by Lhx9.

Loss of neurons that express the neuropeptide hypocretin (Hcrt) has been implicated in narcolepsy, a debilitating disorder characterized by excessive daytime sleepiness and cataplexy. Cell replacement therapy, using Hcrt-expressing neurons generated in vitro, is a potentially useful therapeutic approach, but factors sufficient to specify Hcrt neurons are unknown. Using zebrafish as a high-throughput system to screen for factors that can specify Hcrt neurons in vivo, we identified the LIM homeobox transcription factor Lhx9 as necessary and sufficient to specify Hcrt neurons. We found that Lhx9 can directly induce hcrt expression and we identified two potential Lhx9 binding sites in the zebrafish hcrt promoter. Akin to its function in zebrafish, we found that Lhx9 is sufficient to specify Hcrt-expressing neurons in the developing mouse hypothalamus. Our results elucidate an evolutionarily conserved role for Lhx9 in Hcrt neuron specification that improves our understanding of Hcrt neuron development.

Neuropeptidergic control of sleep and wakefulness.

Sleep and wake are fundamental behavioral states whose molecular regulation remains mysterious. Brain states and body functions change dramatically between sleep and wake, are regulated by circadian and homeostatic processes, and depend on the nutritional and emotional condition of the animal. Sleep-wake transitions require the coordination of several brain regions and engage multiple neurochemical systems, including neuropeptides. Neuropeptides serve two main functions in sleep-wake regulation. First, they represent physiological states such as energy level or stress in response to environmental and internal stimuli. Second, neuropeptides excite or inhibit their target neurons to induce, stabilize, or switch between sleep-wake states. Thus, neuropeptides integrate physiological subsystems such as circadian time, previous neuron usage, energy homeostasis, and stress and growth status to generate appropriate sleep-wake behaviors. We review the roles of more than 20 neuropeptides in sleep and wake to lay the foundation for future studies uncovering the mechanisms that underlie the initiation, maintenance, and exit of sleep and wake states.