The physiological function of different voltage-gated sodium channels in pain.

Evidence from human genetic pain disorders shows that voltage-gated sodium channel α-subtypes Nav1.7, Nav1.8 and Nav1.9 are important in the peripheral signalling of pain. Nav1.7 is of particular interest because individuals with Nav1.7 loss-of-function mutations are congenitally insensitive to acute and chronic pain, and there is considerable hope that phenocopying these effects with a pharmacological antagonist will produce a new class of analgesic drug. However, studies in these rare individuals do not reveal how and where voltage-gated sodium channels contribute to pain signalling, which is of critical importance for drug development. More than a decade of research utilizing rodent genetic models and pharmacological tools to study voltage-gated sodium channels in pain has begun to unravel the role of different subtypes. Here, we review the contribution of individual channel subtypes in three key physiological processes necessary for transmission of sensory information to the CNS: transduction of stimuli at peripheral nerve terminals, axonal transmission of action potentials and neurotransmitter release from central terminals. These data suggest that drugs seeking to recapitulate the analgesic effects of loss of function of Nav1.7 will need to be brain-penetrant - which most of those developed to date are not.

An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity.

The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3-5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7-10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host-pathogen interactions11-13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23.

CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.

Many anti-viral defence systems generate a cyclic nucleotide signal that activates cellular defences in response to infection. Type III CRISPR systems use a specialised polymerase to make cyclic oligoadenylate (cOA) molecules from ATP. These can bind and activate a range of effector proteins that slow down viral replication. In this study, we focussed on the Csx23 effector from the human pathogen Vibrio cholerae ­ a trans-membrane protein that binds a cOA molecule, leading to anti-viral immunity. Structural studies revealed a new class of nucleotide recognition domain, where cOA binding is transmitted to changes in the trans-membrane domain, most likely resulting in membrane depolarisation. This study highlights the diversity of mechanisms for anti-viral defence via nucleotide signalling.

Nerve Growth Factor and Pain Mechanisms.

Nerve growth factor (NGF) antagonism is on the verge of becoming a powerful analgesic treatment for numerous conditions, including osteoarthritis and lower back pain. This review summarizes the historical research, both fundamental and clinical, that led to our current understanding of NGF biology. We also discuss the surprising number of questions that remain about NGF expression patterns and NGF's various functions and interaction partners in relation to persistent pain and the potential side effects of anti-NGF therapy.

Activation of Csm6 ribonuclease by cyclic nucleotide binding: in an emergency, twist to open.

Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6' ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6' in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the 'jaws' of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.

Spinal fMRI demonstrates segmental organisation of functionally connected networks in the cervical spinal cord: A test-retest reliability study.

Resting functional magnetic resonance imaging (fMRI) studies have identified intrinsic spinal cord activity, which forms organised motor (ventral) and sensory (dorsal) resting-state networks. However, to facilitate the use of spinal fMRI in, for example, clinical studies, it is crucial to first assess the reliability of the method, particularly given the unique anatomical, physiological, and methodological challenges associated with acquiring the data. Here, we characterise functional connectivity relationships in the cervical cord and assess their between-session test-retest reliability in 23 young healthy volunteers. Resting-state networks were estimated in two ways (1) by estimating seed-to-voxel connectivity maps and (2) by calculating seed-to-seed correlations. Seed regions corresponded to the four grey matter horns (ventral/dorsal and left/right) of C5-C8 segmental levels. Test-retest reliability was assessed using the intraclass correlation coefficient. Spatial overlap of clusters derived from seed-to-voxel analysis between sessions was examined using Dice coefficients. Following seed-to-voxel analysis, we observed distinct unilateral dorsal and ventral organisation of cervical spinal resting-state networks that was largely confined in the rostro-caudal extent to each spinal segmental level, with more sparse connections observed between segments. Additionally, strongest correlations were observed between within-segment ipsilateral dorsal-ventral connections, followed by within-segment dorso-dorsal and ventro-ventral connections. Test-retest reliability of these networks was mixed. Reliability was poor when assessed on a voxelwise level, with more promising indications of reliability when examining the average signal within clusters. Reliability of correlation strength between seeds was highly variable, with the highest reliability achieved in ipsilateral dorsal-ventral and dorso-dorsal/ventro-ventral connectivity. However, the spatial overlap of networks between sessions was excellent. We demonstrate that while test-retest reliability of cervical spinal resting-state networks is mixed, their spatial extent is similar across sessions, suggesting that these networks are characterised by a consistent spatial representation over time.

RadSigBench: a framework for benchmarking functional genomics signatures of cancer cell radiosensitivity.

Multiple transcriptomic predictors of tumour cell radiosensitivity (RS) have been proposed, but they have not been benchmarked against one another or to control models. To address this, we present RadSigBench, a comprehensive benchmarking framework for RS signatures. The approach compares candidate models to those developed from randomly resampled control signatures and from cellular processes integral to the radiation response. Robust evaluation of signature accuracy, both overall and for individual tissues, is performed. The NCI60 and Cancer Cell Line Encyclopaedia datasets are integrated into our workflow. Prediction of two measures of RS is assessed: survival fraction after 2 Gy and mean inactivation dose. We apply the RadSigBench framework to seven prominent published signatures of radiation sensitivity and test for equivalence to control signatures. The mean out-of-sample R2 for the published models on test data was very poor at 0.01 (range: -0.05 to 0.09) for Cancer Cell Line Encyclopedia and 0.00 (range: -0.19 to 0.19) in the NCI60 data. The accuracy of both published and cellular process signatures investigated was equivalent to the resampled controls, suggesting that these signatures contain limited radiation-specific information. Enhanced modelling strategies are needed for effective prediction of intrinsic RS to inform clinical treatment regimes. We make recommendations for methodological improvements, for example the inclusion of perturbation data, multiomics, advanced machine learning and mechanistic modelling. Our validation framework provides for robust performance assessment of ongoing developments in intrinsic RS prediction.

Structure of the DNA repair helicase XPD.

The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5' to 3' polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 A crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.

The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence.

Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.

Patient-reported impairment following TKA is reduced when a computationally simulated predicted ideal alignment is achieved.

PURPOSE: Joint dynamics following Total Knee Arthroplasty (TKA) may influence patient-reported outcome. Simulations allow many knee alignment approaches to a single patient to be considered prior to surgery. The simulated kinematics can be matched to patient-reported outcome to predict kinematic patterns most likely to give the best outcome. This study aims to validate one such previously developed algorithm based on a simulated deep knee bend (the Dynamic Knee Score, DKS). METHODS: 1074 TKA patients with pre- and post-operative Computerised Tomography (CT) scans and 12-month post-operative Knee Injury and Osteoarthritis Outcomes (KOOS) Scores were identified from the 360 Med Care Joint Registry. Landmarking and registration of implant position was performed on all CT scans, and each of the achieved TKAs was computationally simulated and received a predictive outcome score from the DKS. In addition, a set of potential alternative surgical plans which might have been followed were simulated. Comparison of patient-reported issues and DKS score was evaluated in a counter-factual study design. RESULTS: Patient-reported impairment with the knee catching and squatting was shown to be 30% lower (p = 0.005) and 22% lower (p = 0.026) in patients where the best possible DKS result was the one surgically achieved. Similar findings were found relating attainment of the best tibial slope and posterior femoral resection DKS plans to patient-reported difficulty straightening the knee (40% less likely, p < 0.001) and descending stairs (35% less likely, p = 0.006). CONCLUSION: The DKS has been shown to correlate with presence of patient-reported impairments post-TKA and the resultant algorithm can be applied in a pre-operative planning setting. Outcome optimization in the future may come from patient-specific selection of an alignment strategy and simulations may be a technological enabler of this trend. LEVEL OF EVIDENCE: III (Retrospective Cohort Study).

Characterization of Intrinsic Radiation Sensitivity in a Diverse Panel of Normal, Cancerous and CRISPR-Modified Cell Lines.

Intrinsic radiosensitivity is a major determinant of radiation response. Despite the extensive amount of radiobiological data available, variability among different studies makes it very difficult to produce high-quality radiosensitivity biomarkers or predictive models. Here, we characterize a panel of 27 human cell lines, including those derived from lung cancer, prostate cancer, and normal tissues. In addition, we used CRISPR-Cas9 to generate a panel of lines with known DNA repair defects. These cells were characterised by measuring a range of biological features, including the induction and repair of DNA double-strand breaks (DSBs), cell cycle distribution, ploidy, and clonogenic survival following X-ray irradiation. These results offer a robust dataset without inter-experimental variabilities for model development. In addition, we used these results to explore correlations between potential determinants of radiosensitivity. There was a wide variation in the intrinsic radiosensitivity of cell lines, with cell line Mean Inactivation Doses (MID) ranging from 1.3 to 3.4 Gy for cell lines, and as low as 0.65 Gy in Lig4-/- cells. Similar substantial variability was seen in the other parameters, including baseline DNA damage, plating efficiency, and ploidy. In the CRISPR-modified cell lines, residual DSBs were good predictors of cell survival (R2 = 0.78, p = 0.009), as were induced levels of DSBs (R2 = 0.61, p = 0.01). However, amongst the normal and cancerous cells, none of the measured parameters correlated strongly with MID (R2 < 0.45), and the only metrics with statistically significant associations are plating efficiency (R2 = 0.31, p = 0.01) and percentage of cell in S phase (R2 = 0.37, p = 0.005). While these data provide a valuable dataset for the modelling of radiobiological responses, the differences in the predictive power of residual DSBs between CRISPR-modified and other subgroups suggest that genetic alterations in other pathways, such as proliferation and metabolism, may have a greater impact on cellular radiation response. These pathways are often neglected in response modelling and should be considered in the future.

Chronic muscle recordings reveal recovery of forelimb function in spinal injured female rats after cortical epidural stimulation combined with rehabilitation and chondroitinase ABC.

Cervical level spinal cord injury (SCI) can severely impact upper limb muscle function, which is typically assessed in the clinic using electromyography (EMG). Here, we established novel preclinical methodology for EMG assessments of muscle function after SCI in awake freely moving animals. Adult female rats were implanted with EMG recording electrodes in bicep muscles and received bilateral cervical (C7) contusion injuries. Forelimb muscle activity was assessed by recording maximum voluntary contractions during a grip strength task and cortical motor evoked potentials in the biceps. We demonstrate that longitudinal recordings of muscle activity in the same animal are feasible over a chronic post-injury time course and provide a sensitive method for revealing post-injury changes in muscle activity. This methodology was utilized to investigate recovery of muscle function after a novel combination therapy. Cervical contused animals received intraspinal injections of a neuroplasticity-promoting agent (lentiviral-chondroitinase ABC) plus 11 weeks of cortical epidural electrical stimulation (3 h daily, 5 days/week) and behavioral rehabilitation (15 min daily, 5 days/week). Longitudinal monitoring of voluntary and evoked muscle activity revealed significantly increased muscle activity and upper limb dexterity with the combination treatment, compared to a single treatment or no treatment. Retrograde mapping of motor neurons innervating the biceps showed a predominant distribution across spinal segments C5-C8, indicating that treatment effects were likely due to neuroplastic changes in a mixture of intact and injured motor neurons. Thus, longitudinal assessments of muscle function after SCI correlate with skilled reach and grasp performance and reveal functional benefits of a novel combination therapy.

Granulocyte-Macrophage Colony Stimulating Factor As an Indirect Mediator of Nociceptor Activation and Pain.

The interaction between the immune system and the nervous system has been at the center of multiple research studies in recent years. Whereas the role played by cytokines as neuronal mediators is no longer contested, the mechanisms by which cytokines modulate pain processing remain to be elucidated. In this study, we have analyzed the involvement of granulocyte-macrophage colony stimulating factor (GM-CSF) in nociceptor activation in male and female mice. Previous studies have suggested GM-CSF might directly activate neurons. However, here we established the absence of a functional GM-CSF receptor in murine nociceptors, and suggest an indirect mechanism of action, via immune cells. We report that GM-CSF applied directly to magnetically purified nociceptors does not induce any transcriptional changes in nociceptive genes. In contrast, conditioned medium from GM-CSF-treated murine macrophages was able to drive nociceptor transcription. We also found that conditioned medium from nociceptors treated with the well established pain mediator, nerve growth factor, could also modify macrophage gene transcription, providing further evidence for a bidirectional crosstalk.SIGNIFICANCE STATEMENT The interaction of the immune system and the nervous system is known to play an important role in the development and maintenance of chronic pain disorders. Elucidating the mechanisms of these interactions is an important step toward understanding, and therefore treating, chronic pain disorders. This study provides evidence for a two-way crosstalk between macrophages and nociceptors in the peripheral nervous system, which may contribute to the sensitization of nociceptors by cytokines in pain development.

The association between pain-induced autonomic reactivity and descending pain control is mediated by the periaqueductal grey.

There is a strict interaction between the autonomic nervous system (ANS) and pain, which might involve descending pain modulatory mechanisms. The periaqueductal grey (PAG) is involved both in descending pain modulation and ANS, but its role in mediating this relationship has not yet been explored. Here, we sought to determine brain regions mediating ANS and descending pain control associations. Thirty participants underwent conditioned pain modulation (CPM) assessments, in which they rated painful pressure stimuli applied to their thumbnail, either alone or with a painful cold contralateral stimulation. Differences in pain ratings between 'pressure-only' and 'pressure + cold' stimuli provided a measure of descending pain control. In 18 of the 30 participants, structural scans and two functional MRI assessments, one pain-free and one during cold-pain were acquired. Heart rate variability (HRV) was simultaneously recorded. Normalised low-frequency HRV (LF-HRVnu) and the CPM score were negatively correlated; individuals with higher LF-HRVnu during pain reported reductions in pain during CPM. PAG-ventro-medial prefrontal cortex (vmPFC) and PAG-rostral ventromedial medulla (RVM) functional connectivity correlated negatively with the CPM. Importantly, PAG-vmPFC functional connectivity mediated the strength of the LF-HRVnu-CPM association. CPM response magnitude was also negatively correlated with vmPFC GM volume. Our multi-modal approach, using behavioural, physiological and MRI measures, provides important new evidence of interactions between ANS and descending pain mechanisms. ANS dysregulation and dysfunctional descending pain modulation are characteristics of chronic pain. We suggest that further investigation of body-brain interactions in chronic pain patients may catalyse the development of new treatments. KEY POINTS: Heart rate variability (HRV) is associated with descending pain modulation as measured by the conditioned pain modulation protocol (CPM). There is an association between CPM scores and the functional connectivity between the periaqueductal grey (PAG) and ventro-medial prefrontal cortex (vmPFC). CPM scores are also associated with vmPFC grey matter volume. The strength of functional connectivity between the PAG and vmPFC mediates the association between HRV and CPM. Our data provide new evidence of interactions between the autonomic nervous system and descending pain mechanisms.

Patient Reported vs Claims Based Measures of Health for Modeling Life Expectancy in Men with Prostate Cancer.

PURPOSE: Life expectancy has become a core consideration in prostate cancer care. While multiple prediction tools exist to support decision making, their discriminative ability remains modest, which hampers usage and utility. We examined whether combining patient reported and claims based health measures into prediction models improves performance. MATERIALS AND METHODS: Using SEER (Surveillance, Epidemiology, and End Results)-CAHPS (Consumer Assessment of Healthcare Providers and Systems) we identified men 65 years old or older diagnosed with prostate cancer from 2004 to 2013 and extracted 4 types of data, including demographics, cancer information, claims based health measures and patient reported health measures. Next, we compared the performance of 5 nested competing risk regression models for other cause mortality. Additionally, we assessed whether adding new health measures to established prediction models improved discriminative ability. RESULTS: Among 3,240 cases 246 (7.6%) died of prostate cancer while 631 (19.5%) died of other causes. The National Cancer Institute Comorbidity Index score was associated but weakly correlated with patient reported overall health (p <0.001, r=0.21). For predicting other cause mortality the 10-year area under the receiver operating characteristic curve improved from 0.721 (demographics only) to 0.755 with cancer information and to 0.777 and 0.812 when adding claims based and patient reported health measures, respectively. The full model generated the highest value of 0.820. Models based on existing tools also improved in their performance with the incorporation of new data types as predictor variables (p <0.001). CONCLUSIONS: Prediction models for life expectancy that combine patient reported and claims based health measures outperform models that incorporate these measures separately. However, given the modest degree of improvement, the implementation of life expectancy tools should balance model performance with data availability and fidelity.

Immune Cytokines and Their Receptors in Inflammatory Pain.

There is burgeoning interest in the interaction between the immune and nervous systems. Pain is mediated by primary sensory neurons (nociceptors) that can respond to a variety of thermal, mechanical and chemical signals. Cytokines are now recognized as important mediators of inflammatory pain. They can induce nociceptor sensitization indirectly via mediators, wherein neurons become primed and thus become more responsive to stimulation; alternatively, there is also evidence that cytokines can directly activate neurons via their specific receptors present on the neuronal cells. We review here the evidence for and against these respective mechanisms, focusing on arthritis and inflammatory skin models. A number of striking inconsistencies amongst the conclusions made in the literature are highlighted and discussed.

Do Fixed or Mobile Bearing Implants Have Better Survivorship in Medial Unicompartmental Knee Arthroplasty? A Study From the Australian Orthopaedic Association National Joint Replacement Registry.

BACKGROUND: During the last 5 years, there has been an increase in the use of unicompartmental knee arthroplasty (UKA) to treat knee osteoarthritis in Australia, and these account for almost 6% of annual knee replacement procedures. However, there is debate as to whether a fixed bearing or a mobile bearing design is best for decreasing revision for loosening and disease progression as well as improving survivorship. Small sample sizes and possible confounding in the studies on the topic may have masked differences between fixed and mobile bearing designs. QUESTIONS/PURPOSES: Using data from the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR), we selected the four contemporary designs of medial compartment UKA: mobile bearing, fixed modular, all-polyethylene, and fixed molded metal-backed used for the treatment of osteoarthritis to ask: (1) How do the different designs of unicompartmental knees compare with survivorship as measured by cumulative percentage revision (CPR)? (2) Is there a difference in the revision rate between designs as a function of patient sex or age? (3) Do the reasons for revision differ, and what types of revision procedures are performed when these UKA are revised? METHODS: The AOANJRR longitudinally maintains data on all primary and revision joint arthroplasties, with nearly 100% capture. The study population included all UKA procedures undertaken for osteoarthritis between September 1999 and December 2018. Of 56,628 unicompartmental knees recorded during the study period, 50,380 medial UKA procedures undertaken for osteoarthritis were included in the analysis after exclusion of procedures with unknown bearing types (31 of 56,628), lateral or patellofemoral compartment UKA procedures (5657 of 56,628), and those performed for a primary diagnosis other than osteoarthritis (560 of 56,628). There were 50,380 UKA procedures available for analysis. The study group consisted of 40% (20,208 of 50,380) mobile bearing UKA, 35% (17,822 of 50,380) fixed modular UKA, 23% (11,461 of 50,380) all-polyethylene UKA, and 2% (889 of 50,380) fixed molded metal-backed UKA. There were similar sex proportions and age distributions for each bearing group. The overall mean age of patients was 65 ± 9.4 years, and 55% (27,496 of 50,380) of patients were males. The outcome measure was the CPR, which was defined using Kaplan-Meier estimates of survivorship to describe the time to the first revision. Hazard ratios from Cox proportional hazards models, adjusted for sex and age, were performed to compare the revision rates among groups. The cohort was stratified into age groups of younger than 65 years and 65 years and older to compare revision rates as a function of age. Differences among bearing groups for the major causes and modes of revision were assessed using hazard ratios. RESULTS: At 15 years, fixed modular UKA had a CPR of 16% (95% CI 15% to 17%). In comparison, the CPR was 23% (95% CI 22% to 24%) for mobile bearing UKA, 26% (95% CI 24% to 27%) for all-polyethylene UKA, and 20% (95% CI 16% to 24%) for fixed molded metal-backed UKA. The lower revision rate for fixed modular UKA was seen through the entire period compared with mobile bearing UKA (hazard ratio 1.5 [95% CI 1.4 to 1.6]; p < 0.001) and fixed molded metal-backed UKA (HR 1.3 [95% CI 1.1 to 1.6]; p = 0.003), but it varied with time compared with all-polyethylene UKA. The findings were consistent when stratified by sex or age. Although all-polyethylene UKA had the highest revision rate overall and for patients younger than 65 years, for patients aged 65 years and older, there was no difference between all-polyethylene and mobile bearing UKA. When compared with fixed modular UKA, a higher revision risk for loosening was shown in both mobile bearing UKA (HR 1.7 [95% CI 1.5 to 1.9]; p < 0.001) and all-polyethylene UKA (HR 2.4 [95% CI 2.1 to 2.7]; p < 0.001). The revision risk for disease progression was higher for all-polyethylene UKA at all time points (HR 1.4 [95% CI 1.3 to 1.6]; p < 0.001) and for mobile bearing UKA after 8 years when each were compared with fixed modular UKA (8 to 12 years: HR 1.4 [95% CI 1.2 to 1.7]; p < 0.001; 12 or more years: HR 1.9 [95% CI 1.5 to 2.3]; p < 0.001). The risk of revision to TKA was higher for mobile bearing UKA compared with fixed modular UKA (HR 1.4 [95% CI 1.3 to 1.5]; p < 0.001). CONCLUSION: If UKA is to be considered for the treatment of isolated medial compartment osteoarthritis, the fixed modular UKA bearing has the best survivorship of the current UKA designs. LEVEL OF EVIDENCE: Level III, therapeutic study.

Periplasmic depolymerase provides insight into ABC transporter-dependent secretion of bacterial capsular polysaccharides.

Capsules are surface layers of hydrated capsular polysaccharides (CPSs) produced by many bacteria. The human pathogen Salmonella enterica serovar Typhi produces "Vi antigen" CPS, which contributes to virulence. In a conserved strategy used by bacteria with diverse CPS structures, translocation of Vi antigen to the cell surface is driven by an ATP-binding cassette (ABC) transporter. These transporters are engaged in heterooligomeric complexes proposed to form an enclosed translocation conduit to the cell surface, allowing the transporter to power the entire process. We identified Vi antigen biosynthesis genetic loci in genera of the Burkholderiales, which are paradoxically distinguished from S. Typhi by encoding VexL, a predicted pectate lyase homolog. Biochemical analyses demonstrated that VexL is an unusual metal-independent endolyase with an acidic pH optimum that is specific for O-acetylated Vi antigen. A 1.22-Å crystal structure of the VexL-Vi antigen complex revealed features which distinguish common secreted catabolic pectate lyases from periplasmic VexL, which participates in cell-surface assembly. VexL possesses a right-handed parallel ß-superhelix, of which one face forms an electropositive glycan-binding groove with an extensive hydrogen bonding network that includes Vi antigen acetyl groups and confers substrate specificity. VexL provided a probe to interrogate conserved features of the ABC transporter-dependent export model. When introduced into S Typhi, VexL localized to the periplasm and degraded Vi antigen. In contrast, a cytosolic derivative had no effect unless export was disrupted. These data provide evidence that CPS assembled in ABC transporter-dependent systems is actually exposed to the periplasm during envelope translocation.

Quantitative Proteomic Analysis of the Central Amygdala in Neuropathic Pain Model Rats.

Pain and emotional distress have a reciprocal relation. The amygdala has been implicated in emotional processing. The central nucleus of the amygdala (CeA) receives nociceptive information from the dorsal horn of spinal cord and is responsible for the central plasticity in chronic pain. Neuropathic pain is a type of severe chronic pain and can be strongly influenced by emotional components. Plastic changes in the CeA may play a key role in the development or maintenance or both of neuropathic pain. We studied the expression levels of proteins in the CeA of spinal nerve transection (SNT) model rats. Total tissue lysate proteins were separated by two-dimensional-gel electrophoresis (2D-PAGE). Gels from different time points were compared using Progenesis SameSpot software, and the spots with Fold Change greater than 2 were excised for protein identification by mass spectrometry. We identified more than 50 cytosolic proteins as significantly altered in their expression levels in the CeA of SNT rats, and most of these changes have been validated at mRNA levels by qRT-PCR. We also identified more than 40 membrane proteins as notably up- or down-regulated in the CeA of SNT model rats relative to a control using stable isotope dimethyl labeling nano-LC-MS/MS based proteomics and found that one such protein, doublecortin (DCX), a microtubule-associated protein expressed by neuronal precursor cells during development, is specifically localized in the membrane fraction without changes in total amount of the protein. Immunohistochemistry showed that doublecortin is expressed in processes in the CeA of rats 7 and 21 days after SNT surgery, suggesting that doublecortin is one of the proteins that may contribute to the plastic changes, namely, redevelopment or rewiring of neural networks, in the CeA in the neuropathic pain model. These dysregulated proteins may play roles in reciprocal relationships between pain and psychological distress in the amygdala and contribute to central sensitization. Data are available via ProteomeXchange with identifier PXD017473.

Changes in the transcriptional fingerprint of satellite glial cells following peripheral nerve injury.

Satellite glial cells (SGCs) are homeostatic cells enveloping the somata of peripheral sensory and autonomic neurons. A wide variety of neuronal stressors trigger activation of SGCs, contributing to, for example, neuropathic pain through modulation of neuronal activity. However, compared to neurons and other glial cells of the nervous system, SGCs have received modest scientific attention and very little is known about SGC biology, possibly due to the experimental challenges associated with studying them in vivo and in vitro. Utilizing a recently developed method to obtain SGC RNA from dorsal root ganglia (DRG), we took a systematic approach to characterize the SGC transcriptional fingerprint by using next-generation sequencing and, for the first time, obtain an overview of the SGC injury response. Our RNA sequencing data are easily accessible in supporting information in Excel format. They reveal that SGCs are enriched in genes related to the immune system and cell-to-cell communication. Analysis of SGC transcriptional changes in a nerve injury-paradigm reveal a differential response at 3 days versus 14 days postinjury, suggesting dynamic modulation of SGC function over time. Significant downregulation of several genes linked to cholesterol synthesis was observed at both time points. In contrast, regulation of gene clusters linked to the immune system (MHC protein complex and leukocyte migration) was mainly observed after 14 days. Finally, we demonstrate that, after nerve injury, macrophages are in closer physical proximity to both small and large DRG neurons, and that previously reported injury-induced proliferation of SGCs may, in fact, be proliferating macrophages.