Inhibition of Nonsense-Mediated Decay Induces Nociceptive Sensitization through Activation of the Integrated Stress Response.

RNA stability is meticulously controlled. Here, we sought to determine whether an essential post-transcriptional regulatory mechanism plays a role in pain. Nonsense-mediated decay (NMD) safeguards against translation of mRNAs that harbor premature termination codons and controls the stability of ∼10% of typical protein-coding mRNAs. It hinges on the activity of the conserved kinase SMG1. Both SMG1 and its target, UPF1, are expressed in murine DRG sensory neurons. SMG1 protein is present in both the DRG and sciatic nerve. Using high-throughput sequencing, we examined changes in mRNA abundance following inhibition of SMG1. We confirmed multiple NMD stability targets in sensory neurons, including ATF4. ATF4 is preferentially translated during the integrated stress response (ISR). This led us to ask whether suspension of NMD induces the ISR. Inhibition of NMD increased eIF2-α phosphorylation and reduced the abundance of the eIF2-α phosphatase constitutive repressor of eIF2-α phosphorylation. Finally, we examined the effects of SMG1 inhibition on pain-associated behaviors. Peripheral inhibition of SMG1 results in mechanical hypersensitivity in males and females that persists for several days and priming to a subthreshold dose of PGE2. Priming was fully rescued by a small-molecule inhibitor of the ISR. Collectively, our results indicate that suspension of NMD promotes pain through stimulation of the ISR.SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in their plasticity which may contribute to chronic pain. Translational regulation has emerged as a dominant mechanism in pain. Here, we investigate the role of a major pathway of RNA surveillance called nonsense-mediated decay (NMD). Modulation of NMD is potentially beneficial for a broad array of diseases caused by frameshift or nonsense mutations. Our results suggest that inhibition of the rate-limiting step of NMD drives behaviors associated with pain through activation of the ISR. This work reveals complex interconnectivity between RNA stability and translational regulation and suggests an important consideration in harnessing the salubrious benefits of NMD disruption.

The RNA-Binding Protein HuR Is Integral to the Function of Nociceptors in Mice and Humans.

HuR is an RNA-binding protein implicated in RNA processing, stability, and translation. Previously, we examined protein synthesis in dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We found that the HuR consensus binding element was enriched in transcripts with elevated translation. HuR is expressed in the soma of nociceptors and their axons. Pharmacologic inhibition of HuR with the small molecule CMLD-2 reduced the activity of mouse and human sensory neurons. Peripheral administration of CMLD-2 in the paw or genetic elimination of HuR from sensory neurons diminished behavioral responses associated with NGF- and IL-6-induced allodynia in male and female mice. Genetic disruption of HuR altered the proximity of mRNA decay factors near a key neurotrophic factor (TrkA). Collectively, the data suggest that HuR is required for local control of mRNA stability and reveals a new biological function for a broadly conserved post-transcriptional regulatory factor.SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in excitability, which may contribute to chronic pain. Noxious cues that promote pain lead to rapid induction of protein synthesis. The underlying mechanisms that confer specificity to mRNA control in nociceptors are unclear. Here, we identify a conserved RNA-binding protein called HuR as a key regulatory factor in sensory neurons. Using a combination of genetics and pharmacology, we demonstrate that HuR is required for signaling in nociceptors. In doing so, we report an important mechanism of mRNA control in sensory neurons that ensures appropriate nociceptive responses to inflammatory mediators.

Global analyses of mRNA expression in human sensory neurons reveal eIF5A as a conserved target for inflammatory pain.

Nociceptors are a type of sensory neuron that are integral to most forms of pain. Targeted disruption of nociceptor sensitization affords unique opportunities to prevent pain. An emerging model for nociceptors are sensory neurons derived from human stem cells. Here, we subjected five groups to high-throughput sequencing: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC-derived sensory neurons, mature co-cultures containing hiPSC-derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. Co-culture of nociceptors and astrocytes promotes expression of transcripts enriched in DRG tissues. Comparisons of the hiPSC models to tissue samples reveal that many key transcripts linked to pain are present. Markers indicative of a range of neuronal subtypes present in the DRG were detected in mature hiPSCs. Intriguingly, translation factors were maintained at consistently high expression levels across species and culture systems. As a proof of concept for the utility of this resource, we validated expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissues and hiPSC samples. eIF5A is subject to a unique posttranslational hypusine modification required for its activity. Inhibition of hypusine biosynthesis prevented hyperalgesic priming by inflammatory mediators in vivo and diminished hiPSC activity in vitro. Collectively, our results illuminate the transcriptomes of hiPSC sensory neuron models. We provide a demonstration for this resource through our investigation of eIF5A. Our findings reveal hypusine as a potential target for inflammation associated pain in males.

More evidence that less is better: Sub-optimal choice in dogs.

The less-is-better effect is a preference for the lesser of two alternatives sometimes observed when they are evaluated separately. For example, a dinner service of 24 intact pieces might be judged to be more valuable than a 40-piece dinner service containing nine broken pieces. Pattison and Zentall (Animal Cognition, 17: 1019-1022, 2014) reported similar sub-optimal choice behavior in dogs using a simultaneous choice procedure. Given a choice between a single high-value food item (cheese) or an equivalent high-value item plus a lower-value food item (carrot), their dogs chose the individual item. In a subsequent test, the dogs preferred two high-value items to a single high-value item, suggesting that avoidance of multiple items did not cause the sub-optimal choice behavior. In two experiments, we replicated Pattison and Zentall's procedure while including additional controls. In Experiment 1, habituation of neophobia for multiple items was controlled for by intermixing the two types of test trial within a single experimental session. In Experiment 2, we controlled for avoidance of heterogeneous rewards by including test trials in which a choice was offered between the combination of items and a single low-value item. In both experiments we observed sub-optimal choice behavior which could not be explained by either of these putative mechanisms. Our results, as well as those of Pattison and Zentall, are consistent with the suggestion that dogs' assessment of the total value of multiple items is based, at least partly, on their average quality.

Journey From a Digital Innovation to a Sustainable Health Worker Capacity-Building App in India: Experiences, Challenges, and Lessons Learned.

Health workers, especially auxiliary nurse midwives (ANMs), are among the most critical resources in improving the quality of immunization services and reducing vaccine hesitancy under the Universal Immunization Programme (UIP) in India. To improve health worker immunization skills, UIP trainings in India are primarily conducted through instructor-led classroom, cascade trainings. However, a 2018 capacity-building need assessment revealed several challenges involved in traditional classroom training, such as a single-time exposure to new guidelines, complicated logistics arrangements, a lack of refresher training, and varying quality of training. These complexities make it difficult to meet the timely knowledge and skill needs of every health worker effectively and uniformly in a rapidly changing scenario of UIP. To meet health worker capacity-building needs and address these challenges, Rapid Immunization Skill Enhancement (RISE), a learning management system (LMS) application, was conceptualized. The RISE LMS application was developed as a human-centered, interactive, continuous, and adaptable knowledge and skill-building platform for health workers engaged in the UIP. RISE complements existing classroom-based cascade training for health workers by leveraging digital technologies for faster, easier, and more effective knowledge transfer to accommodate the fast-changing needs of a dynamic health program like UIP. In this article, we share the challenges and strategic solutions to digital training applications, lessons learned, sustainability of the application, and the impact RISE has made in India, all of which stemmed from leadership, coordinated efforts from a team of skilled professionals, government acceptance, detailed planning, and continued stakeholder engagement.

Relationship between sex biases in gene expression and sex biases in autism and Alzheimer's disease.

BACKGROUND: Sex differences in the brain may play an important role in sex-differential prevalence of neuropsychiatric conditions. METHODS: In order to understand the transcriptional basis of sex differences, we analyzed multiple, large-scale, human postmortem brain RNA-Seq datasets using both within-region and pan-regional frameworks. RESULTS: We find evidence of sex-biased transcription in many autosomal genes, some of which provide evidence for pathways and cell population differences between chromosomally male and female individuals. These analyses also highlight regional differences in the extent of sex-differential gene expression. We observe an increase in specific neuronal transcripts in male brains and an increase in immune and glial function-related transcripts in female brains. Integration with single-nucleus data suggests this corresponds to sex differences in cellular states rather than cell abundance. Integration with case-control gene expression studies suggests a female molecular predisposition towards Alzheimer's disease, a female-biased disease. Autism, a male-biased diagnosis, does not exhibit a male predisposition pattern in our analysis. CONCLUSION: Overall, these analyses highlight mechanisms by which sex differences may interact with sex-biased conditions in the brain. Furthermore, we provide region-specific analyses of sex differences in brain gene expression to enable additional studies at the interface of gene expression and diagnostic differences.

We sought to understand why females have higher rates of Alzheimer's disease, and males have higher rates of autism. One idea was that the female brain at baseline may be more similar to an Alzheimer's brain, so it is easier for them to shift into that state (likewise, males may be more similar to autism). To test this, we examined gene expression differences between brains of biological males and biological females. While all people have the same ~ 25,000 genes, each gene can be on or off ('expressed') to different extents. Overall, we found that there were differences in gene expression between males and females in all brain regions tested but more differences in some brain regions than others. By looking at the role of these genes we estimate that female immune system processes might be more active in the brain. We also found female brain gene expression looked slightly more like people with Alzheimer's compared to people without Alzheimer's, which may explain why females get Alzheimer's disease more easily. However, the male brain gene expression did not look more like autism, suggesting that the reason males have higher rates of autism is complex and needs further investigation.

A Massively Parallel Screen of 5'UTR Mutations Identifies Variants Impacting Translation and Protein Production in Neurodevelopmental Disorder Genes.

De novo mutations cause a variety of neurodevelopmental disorders including autism. Recent whole genome sequencing from individuals with autism has shown that many de novo mutations also occur in untranslated regions (UTRs) of genes, but it is difficult to predict from sequence alone which mutations are functional, let alone causal. Therefore, we developed a high throughput assay to screen the transcriptional and translational effects of 997 variants from 5'UTR patient mutations. This assay successfully enriched for elements that alter reporter translation, identifying over 100 potentially functional mutations from probands. Studies in patient-derived cell lines further confirmed that these mutations can alter protein production in individuals with autism, and some variants fall in genes known to cause syndromic forms of autism, suggesting a diagnosis for these individual patients. Since UTR function varies by cell type, we further optimized this high throughput assay to enable assessment of mutations in neurons in vivo. First, comparing in cellulo to in vivo results, we demonstrate neurons have different principles of regulation by 5'UTRs, consistent with a more robust mechanism for reducing the impact of RNA secondary structure. Finally, we discovered patient mutations specifically altering the translational activity of additional known syndromic genes LRRC4 and ZNF644 in neurons of the brain. Overall our results highlight a new approach for assessing the impact of 5'UTR mutations across cell types and suggest that some cases of neurodevelopmental disorder may be caused by such variants.

A compendium of validated pain genes.

The development of novel pain therapeutics hinges on the identification and rigorous validation of potential targets. Model organisms provide a means to test the involvement of specific genes and regulatory elements in pain. Here we provide a list of genes linked to pain-associated behaviors. We capitalize on results spanning over three decades to identify a set of 242 genes. They support a remarkable diversity of functions spanning action potential propagation, immune response, GPCR signaling, enzymatic catalysis, nucleic acid regulation, and intercellular signaling. Making use of existing tissue and single-cell high-throughput RNA sequencing datasets, we examine their patterns of expression. For each gene class, we discuss archetypal members, with an emphasis on opportunities for additional experimentation. Finally, we discuss how powerful and increasingly ubiquitous forward genetic screening approaches could be used to improve our ability to identify pain genes. This article is categorized under: Neurological Diseases > Genetics/Genomics/Epigenetics Neurological Diseases > Molecular and Cellular Physiology.

Code status discussions between attending hospitalist physicians and medical patients at hospital admission.

BACKGROUND: Bioethicists and professional associations give specific recommendations for discussing cardiopulmonary resuscitation (CPR). OBJECTIVE: To determine whether attending hospitalist physicians' discussions meet these recommendations. DESIGN: Cross-sectional observational study on the medical services at two hospitals within a university system between August 2008 and March 2009. PARTICIPANTS: Attending hospitalist physicians and patients who were able to communicate verbally about their medical care. MAIN MEASURES: We identified code status discussions in audio-recorded admission encounters via physician survey and review of encounter transcripts. A quantitative content analysis was performed to determine whether discussions included elements recommended by bioethicists and professional associations. Two coders independently coded all discussions; Cohen's kappa was 0.64-1 for all reported elements. KEY RESULTS: Audio-recordings of 80 patients' admission encounters with 27 physicians were obtained. Eleven physicians discussed code status in 19 encounters. Discussions were more frequent in seriously ill patients (OR 4, 95% CI 1.2-14.6), yet 66% of seriously ill patients had no discussion. The median length of the code status discussions was 1 min (range 0.2-8.2). Prognosis was discussed with code status in only one of the encounters. Discussions of patients' preferences focused on the use of life-sustaining interventions as opposed to larger life goals. Descriptions of CPR as an intervention used medical jargon, and the indication for CPR was framed in general, as opposed to patient-specific scenarios. No physician quantitatively estimated the outcome of or provided a recommendation about the use of CPR. CONCLUSIONS: Code status was not discussed with many seriously ill patients. Discussions were brief, and did not include elements that bioethicists and professional associations recommend to promote patient autonomy. Local and national guidelines, research, and clinical practice changes are needed to clarify and systematize with whom and how CPR is discussed at hospital admission.

A role for translational regulation by S6 kinase and a downstream target in inflammatory pain.

BACKGROUND AND PURPOSE: Translational controls pervade neurobiology. Nociceptors play an integral role in the detection and propagation of pain signals. Nociceptors can undergo persistent changes in their intrinsic excitability. Pharmacological disruption of nascent protein synthesis diminishes acute and chronic forms of pain-associated behaviours. However, the targets of translational controls that facilitate plasticity in nociceptors are unclear. EXPERIMENTAL APPROACH: We used ribosome profiling to probe the translational landscape in dorsal root ganglion (DRG) neurons from male Swiss-Webster mice, after treatment with nerve growth factor and IL-6. Expression dynamics of c-Fos were followed with immunoblotting and immunohistochemistry. The involvement of ribosomal protein S6 kinase 1 (S6K1), a downstream component of mTOR signalling, in the control of c-Fos levels was assessed with low MW inhibitors of S6K1 (DG2) or c-Fos (T-5224), studying their effects on nociceptor activity in vitro using multielectrode arrays (MEAs) and pain behaviour in vivo in Swiss-Webster mice using the hyperalgesic priming model. KEY RESULTS: c-Fos was expressed in sensory neurons. Inflammatory mediators that promote pain in both humans and rodents promote c-Fos translation. The mTOR effector S6K1 is essential for c-Fos biosynthesis. Inhibition of S6K1 or c-Fos with low MW compounds diminished mechanical and thermal hypersensitivity in response to inflammatory cues. Additionally, both inhibitors reduced evoked nociceptor activity. CONCLUSION AND IMPLICATIONS: Our data show a novel role of S6K1 in modulating the rapid response to inflammatory mediators, with c-Fos being one key downstream target. Targeting the S6 kinase pathway or c-Fos is an exciting new avenue for pain-modulating compounds.

Management of long bone fractures in patients with cerebral fat embolism syndrome.

Fat embolism syndrome (FES) is a rare complication associated with long bone fractures. Intramedullary nailing is the gold standard for treating patients with these injuries and early surgical intervention can prevent FES. However, there is a paucity of data on managing these patients once FES has developed. The purpose of this study is to present 3 unique cases of polytrauma patients with long bone fractures who underwent fixation with Taylor Spatial Frame, open reduction and internal fixation, or submuscular plating for treatment of these injuries. All 3 patients had complete cognitive and physical recovery.

Arthroscopic-Assisted Reduction of Tibial Plateau Fractures.

Arthroscopic reduction of tibial plateau fractures have been gaining in popularity. Advantages include accurate diagnosis and treatment of joint pathology, minimally invasive soft tissue dissection, quicker recovery of joint motion, and anatomic reduction of joint surface. Success depends on accurate fracture selection. With arthroscopic-assisted reduction of tibial plateau fractures, patient set-up is similar to standard knee arthroscopy, but the C-arm is used to aid with fracture reduction and fixation. Outcomes are comparable or even improved when compared with standard procedures, and morbidity with arthroscopic reduction can often be lower with decreased rates of infection, wound complications, and thromboembolism.

First-line antihypertensive treatment for severe hypertension in pregnancy: A systematic review and network meta-analysis.

BACKGROUND: Hydralazine, labetalol, and nifedipine are the recommended first-line treatments for severe hypertension in pregnancy. While all three are effective, there is a lack of sufficient evidence regarding their comparative safety and efficacy. OBJECTIVE: To determine the comparative safety and efficacy of the first-line treatment options for severe hypertension in pregnancy. METHODS: A systematic search of Medline, Embase, and Cochrane Central Register of Controlled Trials up to May 31, 2018 was conducted. RCTs in pregnancy comparing a first-line antihypertensive agent to another first-line agent for the treatment of severe hypertension in pregnancy. Screening, data abstraction, and quality assessment were done by two independent reviewers. To estimate relative effects from all available evidence, a Bayesian network meta-analysis with vague priors was conducted. MAIN RESULTS: Of the 1330 publications identified, 17 RCTs comprised of a total of 1591 women met our selection criteria. For successful treatment of severe hypertension, nifedipine was found to be superior to hydralazine (OR 4.13 [95% CrI 1.01-20.75]) but not labetalol (OR 3.43 [95% CrI 0.94-19.95]). This was not associated with an increased risk for caesarean delivery or maternal side effects. There was no significant difference between labetalol and hydralazine. CONCLUSIONS: Given the results of this systematic review and network meta-analysis, maternity care providers should feel comfortable initiating management of severe hypertension in pregnancy using oral nifedipine.