Inflammatory Caspases: Toward a Unified Model for Caspase Activation by Inflammasomes.

Inflammasomes are inflammatory signaling complexes that provide molecular platforms to activate the protease function of inflammatory caspases. Caspases-1, -4, -5, and -11 are inflammatory caspases activated by inflammasomes to drive lytic cell death and inflammatory mediator production, thereby activating host-protective and pathological immune responses. Here, we comprehensively review the mechanisms that govern the activity of inflammatory caspases. We discuss inflammatory caspase activation and deactivation mechanisms, alongside the physiological importance of caspase activity kinetics. We also examine mechanisms of caspase substrate selection and how inflammasome and cell identities influence caspase activity and resultant inflammatory and pyroptotic cellular programs. Understanding how inflammatory caspases are regulated may offer new strategies for treating infection and inflammasome-driven disease.

Uncovering the magnitude of African pangolin poaching with extensive nanopore DNA genotyping of seized scales.

Trade in pangolins is illegal, and yet tons of their scales and products are seized at various ports. These large seizures are challenging to process and comprehensively genotype for upstream provenance tracing and species identification for prosecution. We implemented a scalable DNA barcoding pipeline in which rapid DNA extraction and MinION sequencing were used to genotype a substantial proportion of pangolin scales subsampled from 2 record shipments seized in Singapore in 2019 (37.5 t). We used reference sequences to match the scales to phylogeographical regions of origin. In total, we identified 2346 cytochrome b (cytb) barcodes of white-bellied (Phataginus tricuspis) (from 1091 scales), black-bellied (Phataginus tetradactyla) (227 scales), and giant (Smutsia gigantea) (1028 scales) pangolins. Haplotype diversity was higher for P. tricuspis scales (121 haplotypes, 66 novel) than that for P. tetradactyla (22 haplotypes, 15 novel) and S. gigantea (25 haplotypes, 21 novel) scales. Of the novel haplotypes, 74.2% were likely from western and west-central Africa, suggesting potential resurgence of poaching and newly exploited populations in these regions. Our results illustrate the utility of extensively subsampling large seizures and outline an efficient molecular approach for rapid genetic screening that should be accessible to most forensic laboratories and enforcement agencies.

Revelación de la magnitud de la caza furtiva del pangolín africano mediante el genotipo extenso de nanoporos de ADN de escamas incautadas Resumen Aunque el mercado de pangolines es ilegal, se incautan toneladas de sus escamas y productos derivados en varios puertos comerciales. Es un reto procesar estas magnas incautaciones y obtener el genotipo completo para usarlo en la trazabilidad logística ascendente e identificación de la especie y así imponer sanciones. Implementamos una canalización escalable del código de barras de ADN en el cual usamos la extracción rápida de ADN y la secuenciación MinION para obtener el genotipo de una proporción sustancial de las escamas de pangolín submuestreadas en dos cargamentos incautados en 2019 en Singapur (37.5 t). Usamos secuencias referenciales para emparejar las escamas con las regiones filogeográficas de origen. Identificamos en total 2,346 códigos de citocromo b (cytb) del pangolín de vientre blanco (Phataginus tricuspis) (de 1,091 escamas), de vientre negro (P. tetradactyla) (227 escamas) y del pangolín gigante (Smutsia gigantea) (1,028 escamas). La diversidad de haplotipos fue mayor en las escamas de P. tricuspis (121 haplotipos, 66 nuevos) que en las de P. tetradactyla (22 haplotipos, 15 nuevos) y S. gigantea (25 haplotipos, 21 nuevos). De los haplotipos nuevos, el 74.2% probablemente provenía del occidente y centro­occidente de África, lo que sugiere un resurgimiento potencial de la caza furtiva y poblaciones recién explotadas en estas regiones. Nuestros resultados demuestran la utilidad de submuestrear extensivamente las grandes incautaciones y esboza una estrategia molecular eficiente para un análisis genético rápido que debería ser accesible para la mayoría de los laboratorios forenses y las autoridades de aplicación.

Exploring medicines information needs of mental health service users at a New Zealand hospital.

Providing effective medicines information to individuals can improve outcomes, yet little is known about what information mental health service users need and want about their medicines, and how best to deliver this. The aim of this study was to explore the medicines information needs of mental health service users. Adults (n=30) under the care of acute mental health services in a New Zealand hospital were invited to take part in semi-structured interviews or a focus group to explore preferences for medicines information. Interview data were analysed using an inductive thematic approach. Six key themes were identified: 1) personalisation of information, 2) adverse and beneficial effects, 3) relationships and trust, 4) informed choice, 5) use of reliable internet resources and 6) involvement of family and support people. Preferences on content, timing, provider and format of information delivery were highly individualised indicating the need for information to be personalised to the needs of the service user. A trusted relationship with their healthcare provider was essential . Making informed decisions reduced confusion or fear about medicines. Understanding medicines information needs of patients can help improve the education health professionals provide on medicines, thus potentially improving patient engagement and outcomes.

Visualisation and detection of latent DNA deposited by pangolin scales onto plastic packaging materials.

We report on the detection and visualisation of latent DNA from pangolin scales deposited onto a plastic packaging material through the use of a nucleic acid staining dye. This latent DNA deposited by pangolin scales was subsequently isolated and analysed using DNA barcoding method. Pangolins are the most illegally traded mammalian species due to the demand for their scales and meat. The demand for their scales were mostly fuelled by its use in traditional medicines. The scales are usually packed into bags and transported globally via sea routes. This is the first report detailing the detection of trace latent DNA from processed wildlife products, on surfaces of bags that they were packaged in. Prior to this report, it was not known if the dried pangolin scales contained transferable quantities of biological material for DNA analyses. To address this, scales were removed from a roadkill Sunda pangolin (Manis javanica), processed by drying and packaged into one of five plastic bags. The presence of pangolin latent DNA was detected on the surface of the plastic bags and visualised using Diamond™ nucleic acid dye. Swabs were then used to recover the stained biological material from various locations in the five bags. The DNA was isolated and quantified using a newly designed quantitative PCR (qPCR) specific to M. javanica to amplify a fragment of the mitochondrial DNA cytochrome b gene. There was a positive correlation between the number of stained particles and DNA quantity, and a greater number of stained particles were found at the bottom of the bag than were found at the top. Conventional PCR targeting part of the cyt b gene amplified a product from all 30 samples taken from the bags and in all cases, sequence data generated matched that of the Sunda pangolin, as expected. All negative controls yielded no results. The method described here is the very first use of a nucleic acid staining dye to detect latent DNA from a mammalian species, other than humans, and highlights the opportunity for further use of Diamond™ nucleic acid dye in wildlife forensic science. It is anticipated that this method will be invaluable in retrieving latent DNA deposited by wildlife products from the environment in which they were contained, to determine the presence of these illegal wildlife products even when previously hidden, inaccessible, or no longer present physically. Further research is required to understand if the use on non-human mammalian wildlife species is feasible.

Beliefs about antibiotics, perceptions of antimicrobial resistance, and antibiotic use: initial findings from a multi-country survey.

OBJECTIVES: To examine public beliefs about antibiotics, AMR, and knowledge of antibiotic use, and how these relate to self-reported antibiotic use. METHODS: Two hundred and fifty participants from 23 countries completed a cross-sectional, online survey assessing beliefs about antibiotics and AMR, knowledge of antibiotics, and antibiotic use. Descriptive statistics, Mann-Whitney U tests and Spearman's ρ correlations were used to understand relationships between outcomes. KEY FINDINGS: Respondents generally viewed antibiotics positively, with particularly strong beliefs regarding their benefit (M = 16.48 out of 20, SD = 2.62) and few concerns regarding their harm (M = 3.98 out of 10, SD = 1.82). Greater benefit beliefs about antibiotics were associated with fewer concerns about their overuse (P < .0001) and harm (P < .0001). Stronger perceived importance of AMR was associated with greater beliefs about the benefits of antibiotics (P = .006), greater concerns about their overuse (P = .009), and increased knowledge of appropriate use (P = .006). Those who reported inappropriately using their last antibiotics had greater concerns about overuse (P = .12) and less knowledge regarding appropriate use (P = .015), compared to those who did not. CONCLUSIONS: Generally, the public tends to view antibiotics as having strong benefits and have few concerns about their harm, which may have implications for inappropriate use. These initial findings highlight beliefs that could be targeted in messages to reduce inappropriate demand for antibiotics.

Using interRAI Assessment for Research: Developing a National Research Agenda in Aotearoa New Zealand.

interRAI provides a suite of standardized, validated instruments used to assess health and psychosocial well-being, and to inform person-centered care planning. Data obtained from these standardized tools can also be used at a population level for research and to inform policy, and interRAI is currently used in more than 40 countries globally. We present a brief overview of the use of interRAI internationally within research and policy settings, and then introduce how interRAI is used within the universal public health system in Aotearoa New Zealand (NZ), including considerations relating to Maori, the Indigenous people of NZ. In NZ, improvement in interRAI data utilization for research purposes was called for from aged care, health providers, and researchers, to better use these data for quality improvement and health advancement for New Zealanders. A national research network has been established, providing a medium for researchers to form relationships and collaborate on interRAI research with a goal of translating routinely collected interRAI data to improve clinical care, patient experience, service development, and quality improvement. In 2023, the network members met (hybrid: in-person and online) and identified research priorities. These were collated and developed into a national interRAI research agenda by the NZ interRAI Research Network Working Group. Research priorities included reviewing the interRAI assessment processes, improving methods for data linkage to national data sets, exploring how Indigenous Data Sovereignty can be upheld, as well as a variety of clinically focused research topics. Implications for Practice, Policy, and Research: This appears to be the first time national interRAI research priorities have been formally identified. Priorities identified have the potential to inform quality and clinical improvement activities and are likely of international relevance. The methodology described to cocreate the research priorities will also be of wider significance for those looking to do so in other countries.

Caspase-4 dimerisation and D289 auto-processing elicit an interleukin-1ß-converting enzyme.

The noncanonical inflammasome is a signalling complex critical for cell defence against cytosolic Gram-negative bacteria. A key step in the human noncanonical inflammasome pathway involves unleashing the proteolytic activity of caspase-4 within this complex. Caspase-4 induces inflammatory responses by cleaving gasdermin-D (GSDMD) to initiate pyroptosis; however, the molecular mechanisms that activate caspase-4 and govern its capacity to cleave substrates remain poorly defined. Caspase-11, the murine counterpart of caspase-4, acquires protease activity within the noncanonical inflammasome by forming a dimer that self-cleaves at D285 to cleave GSDMD. These cleavage events trigger signalling via the NLRP3-ASC-caspase-1 axis, leading to downstream cleavage of the pro-IL-1ß cytokine precursor. Here, we show that caspase-4 first dimerises then self-cleaves at two sites-D270 and D289-in the interdomain linker to acquire full proteolytic activity, cleave GSDMD, and induce cell death. Surprisingly, caspase-4 dimerisation and self-cleavage at D289 generate a caspase-4 p34/p9 protease species that directly cleaves pro-IL-1ß, resulting in its maturation and secretion independently of the NLRP3 inflammasome in primary human myeloid and epithelial cells. Our study thus elucidates the key molecular events that underpin signalling by the caspase-4 inflammasome and identifies IL-1ß as a natural substrate of caspase-4.

Detection of a novel Babesia sp. in Amblyomma javanense, an ectoparasite of Sunda pangolins.

BACKGROUND: Babesia is a protozoal, tick-borne parasite that can cause life-threatening disease in humans, wildlife and domestic animals worldwide. However, in Southeast Asia, little is known about the prevalence and diversity of Babesia species present in wildlife and the tick vectors responsible for its transmission. Recently, a novel Babesia species was reported in confiscated Sunda pangolins (Manis javanica) in Thailand. To investigate the presence of this parasite in Singapore, we conducted a molecular survey of Babesia spp. in free-roaming Sunda pangolins and their main ectoparasite, the Amblyomma javanense tick. METHODS: Ticks and tissue samples were opportunistically collected from live and dead Sunda pangolins and screened using a PCR assay targeting the 18S rRNA gene of Babesia spp. DNA barcoding of the cytochrome oxidase subunit I (COI) mitochondrial gene was used to confirm the species of ticks that were Babesia positive. RESULTS: A total of 296 ticks and 40 tissue samples were obtained from 21 Sunda pangolins throughout the 1-year study period. Babesia DNA was detected in five A. javanense ticks (minimum infection rate = 1.7%) and in nine different pangolins (52.9%) located across the country. Phylogenetic analysis revealed that the Babesia 18S sequences obtained from these samples grouped into a single monophyletic clade together with those derived from Sunda pangolins in Thailand and that this evolutionarily distinct species is basal to the Babesia sensu stricto clade, which encompasses a range of Babesia species that infect both domestic and wildlife vertebrate hosts. CONCLUSIONS: This is the first report documenting the detection of a Babesia species in A. javanense ticks, the main ectoparasite of Sunda pangolins. While our results showed that A. javanense can carry this novel Babesia sp., additional confirmatory studies are required to demonstrate vector competency. Further studies are also necessary to investigate the role of other transmission pathways given the low infection rate of ticks in relation to the high infection rate of Sunda pangolins. Although it appears that this novel Babesia sp. is of little to no pathogenicity to Sunda pangolins, its potential to cause disease in other animals or humans cannot be ruled out.

Rapid lamellipodial responses by neighbor cells drive epithelial sealing in response to pyroptotic cell death.

Cell injury poses a substantial challenge for epithelia homeostasis. Several cellular processes preserve epithelial barriers in response to apoptosis, but less is known about other forms of cell death, such as pyroptosis. Here we use an inducible caspase-1 system to analyze how colon epithelial monolayers respond to pyroptosis. We confirm that sporadic pyroptotic cells are physically eliminated from confluent monolayers by apical extrusion. This is accompanied by a transient defect in barrier function at the site of the pyroptotic cells. By visualizing cell shape changes and traction patterns in combination with cytoskeletal inhibitors, we show that rapid lamellipodial responses in the neighbor cells are responsible for correcting the leakage and resealing the barrier. Cell contractility is not required for this resealing response, in contrast to the response to apoptosis. Therefore, pyroptosis elicits a distinct homeostatic response from the epithelium that is driven by the stimulation of lamellipodia in neighbor cells.

Features of successful interventions to improve adherence to inhaled corticosteroids in children with asthma: A narrative systematic review.

INTRODUCTION: Nonadherence to inhaled corticosteroids (ICSs) in children with asthma leads to significant morbidity and mortality. Few adherence interventions have been effective and little is known about what contributes to intervention effectiveness. This systematic review summarizes the efficacy and the characteristics of effective interventions. METHODS: Six databases were systematically searched on October 3, 2020 for randomized control trials measuring adherence to ICS in children with asthma. A narrative synthesis was conducted focusing on intervention efficacy and study reliability. Intervention content was coded based on the National Institute for Health and Care Excellence guidelines for medicines adherence (the Perceptions and Practicalities Approach, PAPA) and behavior change techniques (BCTs), to determine the effective aspects of the intervention. RESULTS: Of 240 studies identified, 25 were eligible for inclusion. Thirteen of the 25 studies were categorized as being highly reliable. Nine of the 13 interventions were effective at increasing adherence and 6 of those met the criteria for a PAPA intervention. Techniques targeting perceptions and practicalities in successful interventions included rewards, reminders, feedback and monitoring of adherence, pharmacological support, instruction on how to take their ICS/adhere, and information about triggers for symptoms and nonadherence. CONCLUSION: Adherence interventions in children with asthma have mixed effectiveness. Effective intervention studies were more frequently of higher quality, were tailored to individuals' perceptual and practical adherence barriers, and used multiple BCTs. However, due to the small number of included studies and varying study design quality, conclusions drawn here are preliminary. Future research is needed to test a PAPA-based intervention with a rigorous study design.

Digital Inhalers for Asthma or Chronic Obstructive Pulmonary Disease: A Scientific Perspective.

Impressive advances in inhalation therapy for patients with asthma and chronic obstructive pulmonary disease (COPD) have occurred in recent years. However, important gaps in care remain, particularly relating to poor adherence to inhaled therapies. Digital inhaler health platforms which incorporate digital inhalers to monitor time and date of dosing are an effective disease and medication management tool, promoting collaborative care between clinicians and patients, and providing more in-depth understanding of actual inhaler use. With advances in technology, nearly all inhalers can be digitalized with add-on or embedded sensors to record and transmit data quantitating inhaler actuations, and some have additional capabilities to evaluate inhaler technique. In addition to providing an objective and readily available measure of adherence, they allow patients to interact with the device directly or through their self-management smartphone application such as via alerts and recording of health status. Clinicians can access these data remotely and during patient encounters, to better inform them about disease status and medication adherence and inhaler technique. The ability for remote patient monitoring is accelerating interest in and the use of these devices in clinical practice and research settings. More than 20 clinical studies of digital inhalers in asthma or COPD collectively show improvement in medication adherence, exacerbation risk, and patient outcomes with digital inhalers. These studies support previous findings about patient inhaler use and behaviors, but with greater granularity, and reveal some new findings about patient medication-taking behaviors. Digital devices that record inspiratory flows with inhaler use can guide proper inhaler technique and may prove to be a clinically useful lung function measure. Adoption of digital inhalers into practice is still early, and additional research is needed to determine patient and clinician acceptability, the appropriate place of these devices in the therapeutic regimen, and their cost effectiveness. Video: Digital Inhalers for Asthma or Chronic Obstructive Pulmonary Disease: A Scientific Perspective (MP4 74535 kb).

Inflammasome signaling and regulation of interleukin-1 family cytokines.

Specific IL-1 family cytokines are expressed by cells as cytosolic pro-forms that require cleavage for their activity and cellular release. IL-1ß, IL-18, and IL-37 maturation and secretion is governed by inflammatory caspases within signaling platforms called inflammasomes. By inducing pyroptosis, inflammasomes can also drive the release of the alarmin IL-1α. Recent advances have transformed our mechanistic understanding of inflammasome signaling, cell death decisions, and cytokine activation and secretion. Here, we provide an updated view of inflammasome signaling; mechanisms underpinning IL-1α, IL-1ß, IL-18, and IL-37 maturation and release; and the functions of these cytokines in protective and pathological inflammation.

SABA Reliance Questionnaire (SRQ): Identifying Patient Beliefs Underpinning Reliever Overreliance in Asthma.

BACKGROUND: Patient overreliance on short-acting beta2 agonists (SABA), with concomitant underuse of inhaled corticosteroids (ICS), is associated with poor asthma control and increased risk of asthma attacks. OBJECTIVE: To develop and validate a brief questionnaire to elicit patients' perceptions of SABA (eg, belief that asthma is best managed by SABA alone) that could lead them to be overly reliant on SABA. METHODS: The 5-item SABA Reliance Questionnaire (SRQ) was adapted from the well-validated Beliefs about Medicines Questionnaire assessing patient perceptions of the importance of, and necessity for, SABA in managing their asthma. The psychometric properties of the questionnaire were studied using Amazon Mechanical Turk, an online survey platform, in 446 people with self-reported asthma. Internal reliability and criterion-related validity were assessed on the basis of relationships between SRQ scores and other variables, including self-reported adherence to ICSs and perceived importance of reliever inhalers. RESULTS: Internal reliability was good with Cronbach α = 0.74. Criterion-related validity was demonstrated by an inverse correlation between SRQ scores and self-reported adherence to ICSs (r = -0.291; P < .0001), and significant correlation between SRQ scores and perceived reliever importance (r = 0.216; P < .0001), as well as by significant differences in SRQ scores between those with high and those with low self-reported ICS adherence (adherence to ICS t = 4.825; P < .0001). CONCLUSIONS: The SRQ demonstrated acceptable internal reliability, and criterion validity, supporting its potential use as a pragmatic tool for identifying patients whose beliefs are indicative of overreliance on SABA for asthma.

Dimerization and auto-processing induce caspase-11 protease activation within the non-canonical inflammasome.

Caspase-11 is a cytosolic sensor and protease that drives innate immune responses to the bacterial cell wall component, LPS. Caspase-11 provides defence against cytosolic Gram-negative bacteria; however, excessive caspase-11 responses contribute to murine endotoxic shock. Upon sensing LPS, caspase-11 assembles a higher order structure called the non-canonical inflammasome that enables the activation of caspase-11 protease function, leading to gasdermin D cleavage and cell death. The mechanism by which caspase-11 acquires protease function is, however, poorly defined. Here, we show that caspase-11 dimerization is necessary and sufficient for eliciting basal caspase-11 protease function, such as the ability to auto-cleave. We further show that during non-canonical inflammasome signalling, caspase-11 self-cleaves at site (D285) within the linker connecting the large and small enzymatic subunits. Self-cleavage at the D285 site is required to generate the fully active caspase-11 protease (proposed here to be p32/p10) that mediates gasdermin D cleavage, macrophage death, and NLRP3-dependent IL-1ß production. This study provides a detailed molecular mechanism by which LPS induces caspase-11-driven inflammation and cell death to provide host defence against cytosolic bacterial infection.

Factors associated with medication adherence in school-aged children with asthma.

Adherence to preventive asthma treatment is poor, particularly in children, yet the factors associated with adherence in this age group are not well understood. Adherence was monitored electronically over 6 months in school-aged children who attended a regional emergency department in New Zealand for an asthma exacerbation and were prescribed twice-daily inhaled corticosteroids. Participants completed questionnaires including assessment of family demographics, asthma responsibility and learning style. Multivariable analysis of factors associated with adherence was conducted. 101 children (mean (range) age 8.9 (6-15) years, 51% male) participated. Median (interquartile range) preventer adherence was 30% (17-48%) of prescribed. Four explanatory factors were identified: female sex (+12% adherence), Asian ethnicity (+19% adherence), living in a smaller household (-3.0% adherence per person in the household), and younger age at diagnosis (+2.7% for every younger year of diagnosis) (all p<0.02). In school-aged children attending the emergency department for asthma, males and non-Asian ethnic groups were at high risk for poor inhaled corticosteroid adherence and may benefit most from intervention. Four factors explained a small proportion of adherence behaviour indicating the difficulty in identifying adherence barriers. Further research is recommended in other similar populations.

The effect of an electronic monitoring device with audiovisual reminder function on adherence to inhaled corticosteroids and school attendance in children with asthma: a randomised controlled trial.

BACKGROUND: Suboptimum adherence to preventive asthma treatment is associated with substantial morbidity and mortality, yet adherence often remains poor. We aimed to investigate whether use of an inhaler with audiovisual reminders leads to improved adherence and asthma outcomes in school-aged children who presented to the emergency department with an asthma exacerbation. METHODS: We did a randomised controlled trial in patients aged 6-15 years who attended the regional emergency department in Auckland, New Zealand with an asthma exacerbation and were on regular inhaled corticosteroids. Using a simple, unrestricted block randomisation with block sizes of 200, we randomly assigned patients to receive an electronic monitoring device for use with their preventer inhaler with the audiovisual reminder functions either enabled to support adherence to inhaled corticosteroids (intervention group) or disabled (control group). Participants were followed up every 2 months for 6 months. The primary outcomes were adherence to preventive inhaled corticosteroids and number of days absent from school for any reason. Asthma control was assessed as a secondary outcome. All analyses were done in the intention-to-treat population. This trial is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12613001353785. FINDINGS: The study took place between May 10, 2010, and Feb 26, 2012. We randomly assigned 220 patients, 110 to the intervention group and 110 to the control group. Median percentage adherence was 84% (10th percentile 54%, 90th percentile 96%) in the intervention group, compared with 30% (8%, 68%) in the control group (p<0·0001). The proportion of days absent from school for any reason was 1·9% (10th percentile 0·0%, 90th percentile 7·9%) in the intervention group and 1·7% (0·0%, 8·6%) in the control group. The change in asthma morbidity score from baseline to 6 months was significantly greater in the intervention group than in the control group (p=0·008), with a reduction of 2·0 points from a mean baseline score of 9·3 (SD 2·2) to 7·3 (2·1) in the intervention group, compared with a reduction of 1·2 points from a baseline of 9·2 (2·5) to 8·0 (2·2) in the control group. INTERPRETATION: Use of an electronic monitoring device with an audiovisual reminder led to significant improvements in adherence to inhaled corticosteroids in school-aged children with asthma. This intervention could be beneficial for the improvement of asthma control in patients for whom poor asthma control is related to poor adherence. FUNDING: Health Research Council of New Zealand and Cure Kids.