SARM1 regulates NAD+-linked metabolism and select immune genes in macrophages.

Sterile alpha and HEAT/armadillo motif-containing protein (SARM1) was recently described as a NAD+-consuming enzyme and has previously been shown to regulate immune responses in macrophages. Neuronal SARM1 is known to contribute to axon degeneration due to its NADase activity. However, how SARM1 affects macrophage metabolism has not been explored. Here, we show that macrophages from Sarm1-/- mice display elevated NAD+ concentrations and lower cyclic ADP-ribose, a known product of SARM1-dependent NAD+ catabolism. Further, SARM1-deficient macrophages showed an increase in the reserve capacity of oxidative phosphorylation and glycolysis compared to WT cells. Stimulation of macrophages to a proinflammatory state by lipopolysaccharide (LPS) revealed that SARM1 restricts the ability of macrophages to upregulate glycolysis and limits the expression of the proinflammatory gene interleukin (Il) 1b, but boosts expression of anti-inflammatory Il10. In contrast, we show macrophages lacking SARM1 induced to an anti-inflammatory state by IL-4 stimulation display increased oxidative phosphorylation and glycolysis, and reduced expression of the anti-inflammatory gene, Fizz1. Overall, these data show that SARM1 fine-tunes immune gene transcription in macrophages via consumption of NAD+ and altered macrophage metabolism.

Implementation of Ireland's first state-funded day-case total hip arthroplasty programme.

BACKGROUND: Over the last two decades, many elective procedures have transitioned to day-case surgery thanks to the introduction of 'enhanced recovery' protocols. Only recently has total hip arthroplasty been considered a candidate for day-case surgery, as it was once associated with significant pain, mobility impairment and prolonged postoperative recovery. The National Orthopaedic Hospital Cappagh became the first public hospital in Ireland to set up a day-case total hip arthroplasty service in June 2018, and since then has performed over 109 such cases. AIMS: We outline our day-case total hip arthroplasty pathway, with specific focus on anaesthetic considerations. We report rates of failed discharge and readmission. RESULTS: We achieved successful same-day discharge in 90.8% of our first 109 cases. Readmission rate was 4.6%. CONCLUSION: Our experience of implementing a day-case total hip arthroplasty pathway was highly positive and congruent with expectations from the literature. With appropriate patient selection and education, day-case total hip arthroplasty is not just safe, but of benefit to both patients and healthcare systems.

Investigation of the input-output relationship of engineered neural networks using high-density microelectrode arrays.

Bottom-up neuroscience utilizes small, engineered biological neural networks to study neuronal activity in systems of reduced complexity. We present a platform that establishes up to six independent networks formed by primary rat neurons on planar complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs). We introduce an approach that allows repetitive stimulation and recording of network activity at any of the over 700 electrodes underlying a network. We demonstrate that the continuous application of a repetitive super-threshold stimulus yields a reproducible network answer within a 15 ms post-stimulus window. This response can be tracked with high spatiotemporal resolution across the whole extent of the network. Moreover, we show that the location of the stimulation plays a significant role in the networks' early response to the stimulus. By applying a stimulation pattern to all network-underlying electrodes in sequence, the sensitivity of the whole network to the stimulus can be visualized. We demonstrate that microchannels reduce the voltage stimulation threshold and induce the strongest network response. By varying the stimulation amplitude and frequency we reveal discrete network transition points. Finally, we introduce vector fields to follow stimulation-induced spike propagation pathways within the network. Overall we show that our defined neural networks on CMOS MEAs enable us to elicit highly reproducible activity patterns that can be precisely modulated by stimulation amplitude, stimulation frequency and the site of stimulation.

Can teleconsent improve patient recall of surgical risks in knee arthroplasty? A randomised controlled trial.

OBJECTIVES: Informed consent plays a vital role in managing patients undergoing knee arthroplasty (KA). Unfortunately, patient recall of informed consent remains poor. Evidence has suggested that telemedicine and teleconsent can be safe, cost-effective, and well-received by patients. The primary aim of this study was to evaluate the effect of an additional preoperative teleconsent session on patient recall of surgical risks 1 month after knee arthroplasty. The secondary aim was to assess its impact on patient satisfaction. METHODS: Sixty adult patients awaiting knee arthroplasty were randomly allocated to receive an additional preoperative teleconsent consultation (intervention group) or not (control group), along with the standard informed consent on the day of surgery. Participants were contacted 1 month after surgery to assess recall of surgical risks and satisfaction with the process. Demographics and education levels were recorded for each patient. RESULTS: The mean recall rates were 16% and 12% in the study and control groups, respectively, with no significant difference (p = 0.42). There was a significant difference between the mean satisfaction scores in the intervention group and the control group (9.8/10 vs 9/10, p = 0.0004). Lastly, there was a significant positive correlation between the education level and the number of risks recalled in the study (p = 0.05) and control groups (p = 0.04). CONCLUSION: The additional preoperative teleconsent session had no significant effect on the risk recall rate but improved patient satisfaction. Our findings suggest education level may play a role in information recall. We can advocate for the increased use of teleconsent and telemedicine in patients undergoing KA or any elective orthopaedic procedure due to its perceived positive effects on patient satisfaction rates.

CRISPR/Cas9-mediated SARM1 knockout and epitope-tagged mice reveal that SARM1 does not regulate nuclear transcription, but is expressed in macrophages.

SARM1 is a toll/interleukin-1 receptor -domain containing protein, with roles proposed in both innate immunity and neuronal degeneration. Murine SARM1 has been reported to regulate the transcription of chemokines in both neurons and macrophages; however, the extent to which SARM1 contributes to transcription regulation remains to be fully understood. Here, we identify differential gene expression in bone-marrow-derived macrophages (BMDMs) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared with wild type (WT). However, we found that passenger genes, which are derived from the 129 donor strain of mice that flank the Sarm1 locus, confound interpretation of the results, since many of the identified differentially regulated genes come from this region. To re-examine the transcriptional role of SARM1 in the absence of passenger genes, here we generated three Sarm1-/- mice using CRISPR/Cas9. Treatment of neurons from these mice with vincristine, a chemotherapeutic drug causing axonal degeneration, confirmed SARM1's function in that process; however, these mice also showed that lack of SARM1 has no impact on transcription of genes previously shown to be affected such as chemokines. To gain further insight into SARM1 function, we generated an epitope-tagged SARM1 mouse. In these mice, we observed high SARM1 protein expression in the brain and brainstem and lower but detectable levels in macrophages. Overall, the generation of these SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages yet has no general role in macrophage transcriptional regulation and has provided important new models to further explore SARM1 function.

Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Batch release testing for human and veterinary tetanus vaccines still relies heavily on methods that involve animals, particularly for potency testing. The quantity and quality of tetanus antigen present in these products is of utmost importance for product safety and clinical effect. Immunochemical methods that measure consistency of antigen content and quality, potentially as an indicator of potency, could be a better choice and negate the need for an in vivo potency test. These immunochemical methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against tetanus with a view to select antibodies that can be used for development of an in vitro potency immunoassay. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined using an in-house cell-based neutralisation assay to support prior in vivo potency data that was available for some, but not all, of the antibodies. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Double-Binding Botulinum Molecule with Reduced Muscle Paralysis: Evaluation in In Vitro and In Vivo Models of Migraine.

With a prevalence of 15%, migraine is the most common neurological disorder and among the most disabling diseases, taking into account years lived with disability. Current oral medications for migraine show variable effects and are frequently associated with intolerable side effects, leading to the dissatisfaction of both patients and doctors. Injectable therapeutics, which include calcitonin gene-related peptide-targeting monoclonal antibodies and botulinum neurotoxin A (BoNT/A), provide a new paradigm for treatment of chronic migraine but are effective only in approximately 50% of subjects. Here, we investigated a novel engineered botulinum molecule with markedly reduced muscle paralyzing properties which could be beneficial for the treatment of migraine. This stapled botulinum molecule with duplicated binding domain-binary toxin-AA (BiTox/AA)-cleaves synaptosomal-associated protein 25 with a similar efficacy to BoNT/A in neurons; however, the paralyzing effect of BiTox/AA was 100 times less when compared to native BoNT/A following muscle injection. The performance of BiTox/AA was evaluated in cellular and animal models of migraine. BiTox/AA inhibited electrical nerve fiber activity in rat meningeal preparations while, in the trigeminovascular model, BiTox/AA raised electrical and mechanical stimulation thresholds in Aδ- and C-fiber nociceptors. In the rat glyceryl trinitrate (GTN) model, BiTox/AA proved effective in inhibiting GTN-induced hyperalgesia in the orofacial formalin test. We conclude that the engineered botulinum molecule provides a useful prototype for designing advanced future therapeutics for an improved efficacy in the treatment of migraine.

Duplication of clostridial binding domains for enhanced macromolecular delivery into neurons.

Neurological diseases constitute a quarter of global disease burden and are expected to rise worldwide with the ageing of human populations. There is an increasing need to develop new molecular systems which can deliver drugs specifically into neurons, non-dividing cells meant to last a human lifetime. Neuronal drug delivery must rely on agents which can recognise neurons with high specificity and affinity. Here we used a recently introduced 'stapling' system to prepare macromolecules carrying duplicated binding domains from the clostridial family of neurotoxins. We engineered individual parts of clostridial neurotoxins separately and combined them using a strong alpha-helical bundle. We show that combining two identical binding domains of tetanus and botulinum type D neurotoxins, in a sterically defined way by protein stapling, allows enhanced intracellular delivery of molecules into neurons. We also engineered a botulinum neurotoxin type C variant with a duplicated binding domain which increased enzymatic delivery compared to the native type C toxin. We conclude that duplication of the binding parts of tetanus or botulinum neurotoxins will allow production of high avidity agents which could deliver imaging reagents and large therapeutic enzymes into neurons with superior efficiency.

Cell Survival and Cytokine Release after Inflammasome Activation Is Regulated by the Toll-IL-1R Protein SARM.

Assembly of inflammasomes after infection or injury leads to the release of interleukin-1ß (IL-1ß) and to pyroptosis. After inflammasome activation, cells either pyroptose or enter a hyperactivated state defined by IL-1ß secretion without cell death, but what controls these different outcomes is unknown. Here, we show that removal of the Toll-IL-1R protein SARM from macrophages uncouples inflammasome-dependent cytokine release and pyroptosis, whereby cells displayed increased IL-1ß production but reduced pyroptosis. Correspondingly, increasing SARM in cells caused less IL-1ß release and more pyroptosis. SARM suppressed IL-1ß by directly restraining the NLRP3 inflammasome and, hence, caspase-1 activation. Consistent with a role for SARM in pyroptosis, Sarm1-/- mice were protected from lipopolysaccharide (LPS)-stimulated sepsis. Pyroptosis-inducing, but not hyperactivating, NLRP3 stimulants caused SARM-dependent mitochondrial depolarization. Thus, SARM-dependent mitochondrial depolarization distinguishes NLRP3 activators that cause pyroptosis from those that do not, and SARM modulation represents a cell-intrinsic mechanism to regulate cell fate after inflammasome activation.

Analysis of the Effectiveness of Sub-sensory Electrical Noise Stimulation During Visuomotor Adaptations in Different Visual Feedback Conditions.

Sub-sensory electrical noise stimulation has been shown to improve motor performance in tasks that mainly rely on proprioceptive feedback. During the execution of movements such as reaching, proprioceptive feedback combines dynamically with visual feedback. It is still unclear whether boosting proprioceptive information in tasks where proprioception mixes with vision can influence motor performance. To better understand this point, we tested the effect of electrical noise stimulation applied superficially to the muscle spindles during four different experiments consisting of isometric reaching tasks under different visual feedback conditions. The first experiment (n = 40) consisted of a reach-and-hold task where subjects had to hold a cursor on a target for 30 s and had visual feedback removed 10 s into the task. Subjects performed 30 repetitions of this task with different stimulation levels, including no stimulation. We observed that trials in which the stimulation was present displayed smaller movement variability. Moreover, we observed a positive correlation between the level of stimulation and task performance. The other three experiments consisted of three versions of an isometric visuomotor adaptation task where subjects were asked to reach to random targets in <1.5 s (otherwise incurring in negative feedback) while overcoming a 45° clockwise rotation in the mapping between the force exerted and the movement of the cursor. The three experiments differed in the visual feedback presented to the subjects, with one group (n = 20) performing the experiment with full visual feedback, one (n = 10) with visual feedback restricted only to the beginning of the trajectory, and one (n = 10) without visual feedback of the trajectory. All subjects performed their experiment twice, with and without stimulation. We did not observe substantial effects of the stimulation when visual feedback was present (either completely or partially). We observed a limited effect of the stimulation in the absence of visual feedback consisting in a significant smaller number of negative-feedback trials and a significant smaller movement time in the first block of the adaptation phase. Our results suggest that sub-sensory stimulation can be beneficial when proprioception is the main feedback modality but mostly ineffective in tasks where visual feedback is actively employed.

Dendritic Cell RIPK1 Maintains Immune Homeostasis by Preventing Inflammation and Autoimmunity.

Necroptosis is a form of cell death associated with inflammation; however, the biological consequences of chronic necroptosis are unknown. Necroptosis is mediated by RIPK1, RIPK3, and MLKL kinases but in hematopoietic cells RIPK1 has anti-inflammatory roles and functions to prevent necroptosis. Here we interrogate the consequences of chronic necroptosis on immune homeostasis by deleting Ripk1 in mouse dendritic cells. We demonstrate that deregulated necroptosis results in systemic inflammation, tissue fibrosis, and autoimmunity. We show that inflammation and autoimmunity are prevented upon expression of kinase inactive RIPK1 or deletion of RIPK3 or MLKL. We provide evidence that the inflammation is not driven by microbial ligands, but depends on the release of danger-associated molecular patterns and MyD88-dependent signaling. Importantly, although the inflammation is independent of type I IFN and the nucleic acid sensing TLRs, blocking these pathways rescues the autoimmunity. These mouse genetic studies reveal that chronic necroptosis may underlie human fibrotic and autoimmune disorders.

A Cell Line for Detection of Botulinum Neurotoxin Type B.

Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products. Refined assays for botulinum product testing are urgently needed. Specifically, in vitro replacement assays which can account for all steps of BoNT intoxication are in high demand. Here, we describe a novel SiMa cell-based approach where re-engineering of the VAMP molecule allows detection of all BoNT/B intoxication steps using a luminescent enzymatic reaction with sensitivity comparable to mouse LD50 bioassay. The presented one-step enzyme-linked immunosorbent assay meets 3Rs (replacement, reduction, and refinement of the use of animals) objectives, is user-friendly and will accelerate development of new botulinum drugs. The sensitive enzymatic reporter cell line could also be adapted for the detection of toxin activity during the manufacture of botulinum and tetanus vaccines.

Veratridine produces distinct calcium response profiles in mouse Dorsal Root Ganglia neurons.

Nociceptors are a subpopulation of dorsal root ganglia (DRG) neurons that detect noxious stimuli and signal pain. Veratridine (VTD) is a voltage-gated sodium channel (VGSC) modifier that is used as an "agonist" in functional screens for VGSC blockers. However, there is very little information on VTD response profiles in DRG neurons and how they relate to neuronal subtypes. Here we characterised VTD-induced calcium responses in cultured mouse DRG neurons. Our data shows that the heterogeneity of VTD responses reflects distinct subpopulations of sensory neurons. About 70% of DRG neurons respond to 30-100 µM VTD. We classified VTD responses into four profiles based upon their response shape. VTD response profiles differed in their frequency of occurrence and correlated with neuronal size. Furthermore, VTD response profiles correlated with responses to the algesic markers capsaicin, AITC and α, ß-methylene ATP. Since VTD response profiles integrate the action of several classes of ion channels and exchangers, they could act as functional "reporters" for the constellation of ion channels/exchangers expressed in each sensory neuron. Therefore our findings are relevant to studies and screens using VTD to activate DRG neurons.

Mouse DRG Cell Line with Properties of Nociceptors.

In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.