Characterizing the relationship between peak assistance torque and metabolic cost reduction during running with ankle exoskeletons.

BACKGROUND: Reducing the energy cost of running with exoskeletons could improve enjoyment, reduce fatigue, and encourage participation among novice and ageing runners. Previously, tethered ankle exoskeleton emulators with offboard motors were used to greatly reduce the energy cost of running with powered ankle plantarflexion assistance. Through a process known as "human-in-the-loop optimization", the timing and magnitude of assistance torque was optimized to maximally reduce metabolic cost. However, to achieve the maximum net benefit in energy cost outside of the laboratory environment, it is also necessary to consider the tradeoff between the magnitude of device assistance and the metabolic penalty of carrying a heavier, more powerful exoskeleton. METHODS: In this study, tethered ankle exoskeleton emulators were used to characterize the effect of peak assistance torque on metabolic cost during running. Three recreational runners participated in human-in-the-loop optimization at four fixed peak assistance torque levels to obtain their energetically optimal assistance timing parameters at each level. RESULTS: We found that the relationship between metabolic rate and peak assistance torque was nearly linear but with diminishing returns at higher torque magnitudes, which is well-approximated by an asymptotic exponential function. At the highest assistance torque magnitude of 0.8 Nm/kg, participants' net metabolic rate was 24.8 ± 2.3% (p = 4e-6) lower than running in the unpowered devices. Optimized timing of peak assistance torque was as late as allowed during stance (80% of stance) and optimized timing of torque removal was at toe-off (100% of stance); similar assistance timing was preferred across participants and torque magnitudes. CONCLUSIONS: These results allow exoskeleton designers to predict the energy cost savings for candidate devices with different assistance torque capabilities, thus informing the design of portable ankle exoskeletons that maximize net metabolic benefit.

Proteomic Analysis After Status Epilepticus Identifies UCHL1 as Protective Against Hippocampal Injury.

Brief, non-harmful seizures (preconditioning) can temporarily protect the brain against prolonged, otherwise injurious seizures. Following focal-onset status epilepticus (SE) in preconditioned (tolerance) and sham-preconditioned (injury) mice, we screened for protein changes using a proteomic approach and identified several putative candidates of epileptic tolerance. Among SE-induced changes to both proteomic screens, proteins clustered in key regulatory pathways, including protein trafficking and cytoskeletal regulation. Downregulation of one such protein, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), was unique to injury and not evident in tolerance. UCHL1 inhibition decreased hippocampal ubiquitin, disrupted UPS function, interfered with seizure termination and exacerbated seizure-induced cell death. Though UCHL1 transcription was maintained after SE, we observed downregulation of the pro-translational antisense Uchl1 (AsUchl1) and confirmed that both AsUchl1 and rapamycin can increase UCHL1 expression in vivo. These data indicate that the post-transcriptional loss of UCHL1 following SE is deleterious to neuronal survival and may contribute to hyperexcitability, and are suggestive of a novel modality of rapamycin therapy.

Diversity in growth patterns among strains of the lethal fungal pathogen Batrachochytrium dendrobatidis across extended thermal optima.

The thermal sensitivities of organisms regulate a wide range of ecological interactions, including host-parasite dynamics. The effect of temperature on disease ecology can be remarkably complex in disease systems where the hosts are ectothermic and where thermal conditions constrain pathogen reproductive rates. Amphibian chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis (Bd), is a lethal fungal disease that is influenced by temperature. However, recent temperature studies have produced contradictory findings, suggesting that our current understanding of thermal effects on Bd may be incomplete. We investigated how temperature affects three different Bd strains to evaluate diversity in thermal responses. We quantified growth across the entire thermal range of Bd, and beyond the known thermal limits (T max and T min). Our results show that all Bd strains remained viable and grew following 24 h freeze (-12 °C) and heat shock (28 °C) treatments. Additionally, we found that two Bd strains had higher logistic growth rates (r) and carrying capacities (K) at the upper and lower extremities of the temperature range, and especially in low temperature conditions (2-3 °C). In contrast, a third strain exhibited relatively lower growth rates and carrying capacities at these same thermal extremes. Overall, our results suggest that there is considerable variation among Bd strains in thermal tolerance, and they establish a new thermal sensitivity profile for Bd. More generally, our findings point toward important questions concerning the mechanisms that dictate fungal thermal tolerances and temperature-dependent pathogenesis in other fungal disease systems.

Differential DNA methylation profiles of coding and non-coding genes define hippocampal sclerosis in human temporal lobe epilepsy.

Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain.

Improved Electrosynthesis of Biomass Derived Furanic Compounds via Nitroxyl Radical Redox Mediation.

Biomass is an abundantly available, underutilized feedstock for the production of bulk and fine chemicals, polymers, and sustainable and biodegradable plastics that are traditionally sourced from petrochemicals. Among potential feedstocks, 2,5-furan dicarboxylic acid (FDCA) stands out for its potential to be converted to higher-value polymeric materials such as polyethylene furandicarboxylate (PEF), a bio-based plastic alternative. In this study, the sustainable, electrocatalytic oxidation of stable furan molecule 2,5-bis(hydroxymethyl)furan (BHMF) to FDCA is investigated using a variety of TEMPO derivative electrocatalysts in a mediated electrosynthetic reaction. Three TEMPO catalysts (acetamido-TEMPO, methoxy-TEMPO, and TEMPO) facilitate full conversion to FDCA in basic conditions with >90% yield and >100% Faradaic efficiency. The remaining three TEMPO catalysts (hydroxy-TEMPO, oxo-TEMPO, and amino-TEMPO) all perform intermediate oxidation of BHMF in basic conditions but do not facilitate full conversion to FDCA. On the basis of pH studies completed on all TEMPO derivatives to assess their electrochemical reversibility and response to substrate, pH and reversibility play significant roles in the catalytic ability of each catalyst, which directly influences catalyst turnover and product formation. More broadly, this study also highlights the importance of an effective and rapid electroanalytical workflow in mediated electrosynthetic reactions, demonstrating how voltammetric catalyst screening can serve as a useful tool for predicting the reactivity and efficacy of a catalyst-substrate electrochemical system.

Differential DNA methylation patterns define status epilepticus and epileptic tolerance.

Prolonged seizures (status epilepticus) produce pathophysiological changes in the hippocampus that are associated with large-scale, wide-ranging changes in gene expression. Epileptic tolerance is an endogenous program of cell protection that can be activated in the brain by previous exposure to a non-harmful seizure episode before status epilepticus. A major transcriptional feature of tolerance is gene downregulation. Here, through methylation analysis of 34,143 discrete loci representing all annotated CpG islands and promoter regions in the mouse genome, we report the genome-wide DNA methylation changes in the hippocampus after status epilepticus and epileptic tolerance in adult mice. A total of 321 genes showed altered DNA methylation after status epilepticus alone or status epilepticus that followed seizure preconditioning, with >90% of the promoters of these genes undergoing hypomethylation. These profiles included genes not previously associated with epilepsy, such as the polycomb gene Phc2. Differential methylation events generally occurred throughout the genome without bias for a particular chromosomal region, with the exception of a small region of chromosome 4, which was significantly overrepresented with genes hypomethylated after status epilepticus. Surprisingly, only few genes displayed differential hypermethylation in epileptic tolerance. Nevertheless, gene ontology analysis emphasized the majority of differential methylation events between the groups occurred in genes associated with nuclear functions, such as DNA binding and transcriptional regulation. The present study reports select, genome-wide DNA methylation changes after status epilepticus and in epileptic tolerance, which may contribute to regulating the gene expression environment of the seizure-damaged hippocampus.

Draft Genome Sequence of an Aspergillus Strain Isolated from a Honey Bee Pupa.

Insect-associated fungi play an important role in wild and agricultural communities. We present a draft genome sequence of an entomopathogenic strain from the fungal genus Aspergillus, isolated from a honey bee pupa.

A Bacterial Symbiont Protects Honey Bees from Fungal Disease.

Fungal pathogens, among other stressors, negatively impact the productivity and population size of honey bees, one of our most important pollinators (1, 2), in particular their brood (larvae and pupae) (3, 4). Understanding the factors that influence disease incidence and prevalence in brood may help us improve colony health and productivity. Here, we examined the capacity of a honey bee-associated bacterium, Bombella apis, to suppress the growth of fungal pathogens and ultimately protect bee brood from infection. Our results showed that strains of B. apis inhibit the growth of two insect fungal pathogens, Beauveria bassiana and Aspergillus flavus, in vitro. This phenotype was recapitulated in vivo; bee broods supplemented with B. apis were significantly less likely to be infected by A. flavus. Additionally, the presence of B. apis reduced sporulation of A. flavus in the few bees that were infected. Analyses of biosynthetic gene clusters across B. apis strains suggest antifungal candidates, including a type 1 polyketide, terpene, and aryl polyene. Secreted metabolites from B. apis alone were sufficient to suppress fungal growth, supporting the hypothesis that fungal inhibition is mediated by an antifungal metabolite. Together, these data suggest that B. apis can suppress fungal infections in bee brood via secretion of an antifungal metabolite. IMPORTANCE Fungi can play critical roles in host microbiomes (5-7), yet bacterial-fungal interactions are understudied. For insects, fungi are the leading cause of disease (5, 8). In particular, populations of the European honey bee (Apis mellifera), an agriculturally and economically critical species, have declined in part due to fungal pathogens. The presence and prevalence of fungal pathogens in honey bees have far-reaching consequences, endangering other species and threatening food security (1, 2, 9). Our research highlights how a bacterial symbiont protects bee brood from fungal infection. Further mechanistic work could lead to the development of new antifungal treatments.

Draft Genome Sequences of Four Saccharibacter sp. Strains Isolated from Native Bees.

The genus Saccharibacter is currently understudied, with only one described species, Saccharibacter floricola, isolated from a flower. In an effort to better understand the microbes that come in contact with native bee pollinators, we isolated and sequenced four additional strains of Saccharibacter from native bees in the genera Melissodes and Anthophora These genomes range in size from 2,104,494 to 2,316,791 bp (mean, 2,246,664 bp) and contain between 1,860 and 2,167 (mean, 2,060) protein-coding genes.

Transitions and transmission: behavior and physiology as drivers of honey bee-associated microbial communities.

Microbial communities have considerable impacts on animal health. However, only in recent years have the host factors impacting microbiome composition been explored. An increasing wealth of microbiome data in combination with decades of research on behavior, physiology, and development have resulted in the European honey bee (Apis mellifera) as a burgeoning model system for studying the influence of host behavior on the microbiota. Honey bees are eusocial insects which exhibit striking behavioral and physiological differences between castes and life stages. These include changes in social contact, environmental exposure, diet, and physiology: all factors which can affect microbial composition and function. The honey bee system offers an opportunity to tease apart the interactive effects of all these factors on microbiota composition, abundance, and diversity.

Draft Genome Sequence of a Bombella apis Strain Isolated from Honey Bees.

Bombella apis occupies a variety of distinct niches within a honey bee hive, including queen guts, royal jelly, and larval food. In an effort to better understand its evolution and identify signatures of honey bee association, we sequenced a strain isolated from hive honey stores. This genome is 2,086,308 bp long and contains 1,975 protein-coding genes.

A physiologically relevant 3D collagen-based scaffold-neuroblastoma cell system exhibits chemosensitivity similar to orthotopic xenograft models.

3D scaffold-based in vitro cell culturing is a recent technological advancement in cancer research bridging the gap between conventional 2D culture and in vivo tumours. The main challenge in treating neuroblastoma, a paediatric cancer of the sympathetic nervous system, is to combat tumour metastasis and resistance to multiple chemotherapeutic drugs. The aim of this study was to establish a physiologically relevant 3D neuroblastoma tissue-engineered system and explore its therapeutic relevance. Two neuroblastoma cell lines, chemotherapeutic sensitive Kelly and chemotherapeutic resistant KellyCis83 were cultured in a 3D in vitro model on two collagen-based scaffolds containing either glycosaminoglycan (Coll-GAG) or nanohydroxyapatite (Coll-nHA) and compared to 2D cell culture and an orthotopic murine model. Both neuroblastoma cell lines actively infiltrated the scaffolds and proliferated displaying >100-fold increased resistance to cisplatin treatment when compared to 2D cultures, exhibiting chemosensitivity similar to orthotopic xenograft in vivo models. This model demonstrated its applicability to validate miRNA-based gene delivery. The efficacy of liposomes bearing miRNA mimics uptake and gene knockdown was similar in both 2D and 3D in vitro culturing models highlighting the proof-of-principle for the applicability of 3D collagen-based scaffolds cell system for validation of miRNA function. Collectively, this data shows the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. While neuroblastoma is the specific disease being focused upon, the platform may have multi-functionality beyond this tumour type. STATEMENT OF SIGNIFICANCE: Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.

Transcriptional Response of Polycomb Group Genes to Status Epilepticus in Mice is Modified by Prior Exposure to Epileptic Preconditioning.

Exposure of the brain to brief, non-harmful seizures can activate protective mechanisms that temporarily generate a damage-refractory state. This process, termed epileptic tolerance, is associated with large-scale down-regulation of gene expression. Polycomb group (PcG) proteins are master controllers of gene silencing during development that are re-activated by injury to the brain. Here, we explored the transcriptional response of genes associated with polycomb repressive complex (PRC) 1 (Ring1A, Ring1B, and Bmi1) and PRC2 (Ezh1, Ezh2, and Suz12), as well as additional transcriptional regulators Sirt1, Yy1, and Yy2, in a mouse model of status epilepticus (SE). Findings were contrasted to changes after SE in mice previously given brief seizures to evoke tolerance. Real-time quantitative PCR showed SE prompted an early (1 h) increase in expression of several genes in PRC1 and PRC2 in the hippocampus, followed by down-regulation of many of the same genes at later times points (4, 8, and 24 h). Spatio-temporal differences were found among PRC2 genes in epileptic tolerance, including increased expression of Ezh2, Suz12, and Yy2 relative to the normal injury response to SE. In contrast, PRC1 complex genes including Ring 1B and Bmi1 displayed differential down-regulation in epileptic tolerance. The present study characterizes PcG gene expression following SE and shows prior seizure exposure produces select changes to PRC1 and PRC2 composition that may influence differential gene expression in epileptic tolerance.

Investigating gene promoter methylation in a mouse model of status epilepticus.

Epigenetic modification of DNA by methylation of the cytosine present in CG dinucleotides constitutes a key regulatory mechanism in the control of gene expression in neurological diseases. In this chapter, we describe an in-depth methodology of methylated DNA immunoprecipitation used in combination with tiling microarrays (MeDIP-chip) in order to analyze genome-wide gene promoter methylation in the hippocampus of mice following status epilepticus (prolonged seizure). While a specific mouse model and array format are described, the method can be applied to DNA from many tissues to analyze the methylation status of promoter regions across whole genomes, using a wide range of available array formats (both custom designed and commercially catalogued). We conclude the chapter with the description of bisulfite sequencing validation of MeDIP-chip results.

Spatio-temporally restricted blood-brain barrier disruption after intra-amygdala kainic acid-induced status epilepticus in mice.

Mesial temporal lobe epilepsy (MTLE) is the most common, intractable seizure disorder in adults. Blood-brain barrier (BBB) disruption, including interruption of endothelial tight cell junctions and serum protein and immunoglobulin G (IgG) extravasation into brain parenchyma, has been reported in experimental and human MTLE and implicated in disease pathogenesis. Triggering status epilepticus in mice by intra-amygdala microinjection of kainic acid produces damage mainly within the CA3 subfield of the ipsilateral hippocampus, and recurrent spontaneous seizures emerge during the following week. To investigate whether BBB impairment is a feature of this model, we characterized endothelial tight cell junction proteins and IgG and albumin in the hippocampus up to three weeks after status epilepticus. Hippocampal microvessels displayed a reduction in continuous staining for zonula occludens 1 (ZO-1), the main tight junction protein, after status epilepticus and in epileptic mice, although western blotting found ZO-1 protein levels in the hippocampal subfields were not different from controls at any time. Increased IgG and albumin were detected in damaged and non-damaged ipsilateral hippocampal subfields, mainly 4-24h after status epilepticus, although increased serum protein extravasation was also found in the CA3 subfield in epileptic mice. Thus, BBB opening or damage occurs mainly in the period shortly after status epilepticus but may also persist within the CA3 subfield as a feature of the pathophysiology of chronic epilepsy in this model.

Plxdc2 is a mitogen for neural progenitors.

The development of different brain regions involves the coordinated control of proliferation and cell fate specification along and across the neuraxis. Here, we identify Plxdc2 as a novel regulator of these processes, using in ovo electroporation and in vitro cultures of mammalian cells. Plxdc2 is a type I transmembrane protein with some homology to nidogen and to plexins. It is expressed in a highly discrete and dynamic pattern in the developing nervous system, with prominent expression in various patterning centres. In the chick neural tube, where Plxdc2 expression parallels that seen in the mouse, misexpression of Plxdc2 increases proliferation and alters patterns of neurogenesis, resulting in neural tube thickening at early stages. Expression of the Plxdc2 extracellular domain alone, which can be cleaved and shed in vivo, is sufficient for this activity, demonstrating a cell non-autonomous function. Induction of proliferation is also observed in cultured embryonic neuroepithelial cells (ENCs) derived from E9.5 mouse neural tube, which express a Plxdc2-binding activity. These experiments uncover a direct molecular activity of Plxdc2 in the control of proliferation, of relevance in understanding the role of this protein in various cancers, where its expression has been shown to be altered. They also implicate Plxdc2 as a novel component of the network of signalling molecules known to coordinate proliferation and differentiation in the developing nervous system.