Prevalence of Clostridium botulinum in Retail Peanut Butters from a 2007 Survey in Ottawa, Canada.

The spore-forming, anaerobic bacterium, Clostridium botulinum, can cause intestinal toxemia (colonization) botulism in adults and infants by colonizing the gut and producing botulinum neurotoxin in situ. In 2006, peanut butter was identified as a lab-confirmed source of C. botulinum spores for an adult colonization botulism case in Canada. It is recommended for infants to be exposed to peanut butter at an early age to help prevent the development of a peanut allergy, yet the prevalence of C. botulinum in retail peanut butters is currently unknown. This report details a survey that was conducted in 2007 for the presence of viable C. botulinum spores in 92 peanut butters and 12 other nut butter spreads obtained from retail grocery stores in Ottawa, Canada. Samples were tested for viable C. botulinum spores by detecting botulinum neurotoxin in enrichment cultures by mouse bioassay. Three of the peanut butters from the entire survey of nut butter spreads (3/104, 3%) produced cultures containing botulinum neurotoxin. Whole genome sequencing performed on one isolate from this survey, as well as a clinical isolate and peanut butter isolates associated with the 2006 adult colonization case revealed that all C. botulinum isolates contained a full-length chromosomal bont/A1 gene within an ha-orf + cassette. This study identifies retail peanut butters as a potential source of viable C. botulinum spores at the time of sampling. Whether peanut butter represents a food category that may be contributing to the incidence of infant botulism has yet to be determined.

Infant Botulism: In Search of Clostridium botulinum Spores.

Infant botulism is now the most common form of human botulism in Canada and the United States. Infant botulism is a severe neuroparalytic disease caused by ingestion of the spore-forming neurotoxic clostridia, including Clostridium botulinum that colonize the large intestine and subsequently produce botulinum neurotoxin in situ. It has been over a century since the first surveys documenting the ubiquitous prevalence of C. botulinum in soils around the world. Since then, honey has been identified as the only well-known risk factor for infant botulism despite a multitude of international environmental surveys isolating C. botulinum spores from ground soil, aquatic sediments, and commonly available infant foods. Associations of infant botulism cases with confirmed sources of C. botulinum exposure have primarily implicated outdoor soil and indoor dust, as well as commonly ingested foods including honey, dry cereals, and even powdered infant formula. Yet the origin of infection remains unknown for most infant botulism cases. This review summarizes the various surveys from around the world for C. botulinum in environmental soils and sediments, honey, and other infant foods, as well as laboratory-confirmed associations with documented infant botulism cases. Additional factors are also discussed, including the composition of infant gut microbiota and the practice of breastfeeding. We make several recommendations to better identify sources of exposure to C. botulinum spores that could lead to effective preventive measures and help reduce the incidence of this rare but life-threatening disease.

Temporal Summation but Not Expectations of Pain Relief Predict Response to Acupuncture Treatment in Fibromyalgia.

Fibromyalgia (FM) is a common chronic pain condition for which acupuncture treatment is increasingly utilized. However, there is no universally accepted measure to predict whether a specific patient will benefit from acupuncture. This is a single-center, single-blind, sham-controlled, randomized, noncrossover, longitudinal trial of 76 subjects with FM, assigned to either electroacupuncture (EA) or a placebo control, mock laser (ML) acupuncture. Outcome measures included clinical pain severity (Brief Pain Inventory [BPI]), degree of nociplastic pain (Fibromyalgia Survey Questionnaire), and pressure pain tolerance (PPtol). Baseline measures of temporal summation of pain and expectations for treatment relief were used as predictors. Individuals in both treatment groups experienced significant reductions in BPI (EA: P < .001, ML: P = .018) and Fibromyalgia Survey Questionnaire (EA: P = .032, ML: P = .002) after treatment; however, neither group showed a significant increase in PPtol. Lower temporal summation at baseline was correlated with greater post-treatment improvement in BPI in the EA group (rho = .389, P = .025) but not in the ML group (rho = -.272, P = .109). Lower-baseline temporal summation was correlated with greater decreases in PPtol following EA (rho = .400, P = .040), whereas the opposite was seen for ML (rho = -.562, P = .001). Treatment expectancy at baseline was not correlated with any outcome after EA or ML treatments. Our results support using a quantitative sensory testing metric, temporal summation of pain, but not expectations, to predict analgesia following acupuncture treatment for pain. PERSPECTIVE: A randomized study of acupuncture in FM found baseline temporal summation, but not expectations of pain relief, to be predictive of treatment response. CLINICAL TRIAL REGISTRATION: Registered under ClinicalTrials.gov identifier NCT02064296.

Stem-like T cells are associated with the pathogenesis of ulcerative colitis in humans.

To understand the role of T cells in the pathogenesis of ulcerative colitis (UC), we analyzed colonic T cells isolated from patients with UC and controls. Here we identified colonic CD4+ and CD8+ T lymphocyte subsets with gene expression profiles resembling stem-like progenitors, previously reported in several mouse models of autoimmune disease. Stem-like T cells were increased in inflamed areas compared to non-inflamed regions from the same patients. Furthermore, TCR sequence analysis indicated stem-like T cells were clonally related to proinflammatory T cells, suggesting their involvement in sustaining effectors that drive inflammation. Using an adoptive transfer colitis model in mice, we demonstrated that CD4+ T cells deficient in either BCL-6 or TCF1, transcription factors that promote T cell stemness, had decreased colon T cells and diminished pathogenicity. Our results establish a strong association between stem-like T cell populations and UC pathogenesis, highlighting the potential of targeting this population to improve clinical outcomes.

Red-on-Yellow Queen: Bio-Layer Interferometry Reveals Functional Diversity Within Micrurus Venoms and Toxin Resistance in Prey Species.

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

A Bayesian spatio-temporal model of COVID-19 spread in England.

Exploring the spatio-temporal variations of COVID-19 transmission and its potential determinants could provide a deeper understanding of the dynamics of disease spread. This study aimed to investigate the spatio-temporal spread of COVID-19 infections in England, and examine its associations with socioeconomic, demographic and environmental risk factors. We obtained weekly reported COVID-19 cases from 7 March 2020 to 26 March 2022 at Middle Layer Super Output Area (MSOA) level in mainland England from publicly available datasets. With these data, we conducted an ecological study to predict the COVID-19 infection risk and identify its associations with socioeconomic, demographic and environmental risk factors using a Bayesian hierarchical spatio-temporal model. The Bayesian model outperformed the ordinary least squares model and geographically weighted regression model in terms of prediction accuracy. The spread of COVID-19 infections over space and time was heterogeneous. Hotspots of infection risk exhibited inconsistent clustering patterns over time. Risk factors found to be positively associated with COVID-19 infection risk were: annual household income [relative risk (RR) = 1.0008, 95% Credible Interval (CI) 1.0005-1.0012], unemployment rate [RR = 1.0027, 95% CI 1.0024-1.0030], population density on the log scale [RR = 1.0146, 95% CI 1.0129-1.0164], percentage of Caribbean population [RR = 1.0022, 95% CI 1.0009-1.0036], percentage of adults aged 45-64 years old [RR = 1.0031, 95% CI 1.0024-1.0039], and particulate matter ( PM 2.5 ) concentrations [RR = 1.0126, 95% CI 1.0083-1.0167]. The study highlights the importance of considering socioeconomic, demographic, and environmental factors in analysing the spatio-temporal variations of COVID-19 infections in England. The findings could assist policymakers in developing tailored public health interventions at a localised level.

The first deep rebreather dive using hydrogen: case report.

Bounce diving with rapid descents to very deep depths may provoke the high-pressure neurological syndrome (HPNS). The strategy of including small fractions of nitrogen in the respired gas to produce an anti-HPNS narcotic effect increases the gas density which may exceed recommended guidelines. In 2020 the 'Wetmules' dive team explored the Pearse Resurgence cave (New Zealand) to 245 m breathing trimix (approximately 4% oxygen, 91% helium and 5% nitrogen). Despite the presence of nitrogen, one diver experienced HPNS tremors beyond 200 m. The use of hydrogen (a light yet slightly narcotic gas) has been suggested as a solution to this problem but there are concerns, including the potential for ignition and explosion of hydrogen-containing gases, and accelerated heat loss. In February 2023 a single dive to 230 m was conducted in the Pearse Resurgence to experience hydrogen as a breathing gas in a deep bounce dive. Using an electronic closed-circuit rebreather, helihydrox (approximately 3% oxygen, 59% helium and 38% hydrogen) was breathed between 200 and 230 m. This was associated with amelioration of HPNS symptoms in the vulnerable diver and no obvious adverse effects. The use of hydrogen is a potential means of progressing deeper with effective HPNS amelioration while maintaining respired gas density within advised guidelines.

Brain network hypersensitivity underlies pain crises in sickle cell disease.

Sickle cell disease (SCD) is a genetic disorder causing painful and unpredictable Vaso-occlusive crises (VOCs) through blood vessel blockages. In this study, we propose explosive synchronization (ES) as a novel approach to comprehend the hypersensitivity and occurrence of VOCs in the SCD brain network. We hypothesized that the accumulated disruptions in the brain network induced by SCD might lead to strengthened ES and hypersensitivity. We explored ES's relationship with patient reported outcome measures (PROMs) as well as VOCs by analyzing EEG data from 25 SCD patients and 18 matched controls. SCD patients exhibited lower alpha frequency than controls. SCD patients showed correlation between frequency disassortativity (FDA), an ES condition, and three important PROMs. Furthermore, stronger FDA was observed in SCD patients with a higher frequency of VOCs and EEG recording near VOC. We also conducted computational modeling on SCD brain network to study FDA's role in network sensitivity. Our model demonstrated that a stronger FDA could be linked to increased sensitivity and frequency of VOCs. This study establishes connections between SCD pain and the universal network mechanism, ES, offering a strong theoretical foundation. This understanding will aid predicting VOCs and refining pain management for SCD patients.

Inflammation and autoimmunity are interrelated in patients with sickle cell disease at a steady-state condition: implications for vaso-occlusive crisis, pain, and sensory sensitivity.

This study aimed to comprehensively analyze inflammatory and autoimmune characteristics of patients with sickle cell disease (SCD) at a steady-state condition (StSt) compared to healthy controls (HCs) to explore the pathogenesis of StSt and its impact on patients' well-being. The study cohort consisted of 40 StSt participants and 23 HCs enrolled between July 2021 and April 2023. StSt participants showed elevated white blood cell (WBC) counts and altered hematological measurements when compared to HCs. A multiplex immunoassay was used to profile 80 inflammatory cytokines/chemokines/growth factors in plasma samples from these SCD participants and HCs. Significantly higher plasma levels of 35 analytes were observed in SCD participants, with HGF, IL-18, IP-10, and MCP-2 being among the most significantly affected analytes. Additionally, autoantibody profiles were also altered, with elevated levels of anti-SSA/Ro60, anti-Ribosomal P, anti-Myeloperoxidase (MPO), and anti-PM/Scl-100 observed in SCD participants. Flow cytometric analysis revealed higher rates of red blood cell (RBC)/reticulocyte-leukocyte aggregation in SCD participants, predominantly involving monocytes. Notably, correlation analysis identified associations between inflammatory mediator levels, autoantibodies, RBC/reticulocyte-leukocyte aggregation, clinical lab test results, and pain crisis/sensitivity, shedding light on the intricate interactions between these factors. The findings underscore the potential significance of specific biomarkers and therapeutic targets that may hold promise for future investigations and clinical interventions tailored to the unique challenges posed by SCD. In addition, the correlations between vaso-occlusive crisis (VOC)/pain/sensory sensitivity and inflammation/immune dysregulation offer valuable insights into the pathogenesis of SCD and may lead to more targeted and effective therapeutic strategies. Clinical Trial Registration: ClinicalTrials.gov, Identifier: NCT05045820.

Identification of a Novel Subtype-Selective α1B-Adrenoceptor Antagonist.

α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α1-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease. Our understanding of the precise physiological roles of α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10-15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.

Complete genomes of Clostridium botulinum type B(F) isolates associated with a 1995 foodborne botulism outbreak from commercial pâté reveals a recombination event disrupting the ntnh gene.

Foodborne botulism is a neuroparalytic disease caused by ingestion of foods contaminated with botulinum neurotoxin (BoNT), produced by Clostridium botulinum. In 1995 a husband and wife from Québec, Canada, were hospitalized for several months with prolonged muscle paralysis after ingesting a commercial pâté de campagne. Examination of faecal samples from both patients and the pâté produced viable Group I (proteolytic) C. botulinum type B from each of the three samples. Whole genome sequencing revealed that all three isolates contain identical bont/B5 and bont/F2 genes encoded on a plasmid. Both faecal isolate genomes were identical in chromosome and plasmid length, as well as gene content. The genome of the pâté isolate was nearly identical to that of the faecal isolates with the notable difference of a missing 13-gene insertion on the bont/B5 cluster disrupting the ntnh gene. Examination of the insertion revealed several mobile genetic elements that participate in recombination.

[ 11 C]-PBR28 positron emission tomography signal as an imaging marker of joint inflammation in knee osteoarthritis.

ABSTRACT: Although inflammation is known to play a role in knee osteoarthritis (KOA), inflammation-specific imaging is not routinely performed. In this article, we evaluate the role of joint inflammation, measured using [ 11 C]-PBR28, a radioligand for the inflammatory marker 18-kDa translocator protein (TSPO), in KOA. Twenty-one KOA patients and 11 healthy controls (HC) underwent positron emission tomography/magnetic resonance imaging (PET/MRI) knee imaging with the TSPO ligand [ 11 C]-PBR28. Standardized uptake values were extracted from regions-of-interest (ROIs) semiautomatically segmented from MRI data, and compared across groups (HC, KOA) and subgroups (unilateral/bilateral KOA symptoms), across knees (most vs least painful), and against clinical variables (eg, pain and Kellgren-Lawrence [KL] grades). Overall, KOA patients demonstrated elevated [ 11 C]-PBR28 binding across all knee ROIs, compared with HC (all P 's < 0.005). Specifically, PET signal was significantly elevated in both knees in patients with bilateral KOA symptoms (both P 's < 0.01), and in the symptomatic knee ( P < 0.05), but not the asymptomatic knee ( P = 0.95) of patients with unilateral KOA symptoms. Positron emission tomography signal was higher in the most vs least painful knee ( P < 0.001), and the difference in pain ratings across knees was proportional to the difference in PET signal ( r = 0.74, P < 0.001). Kellgren-Lawrence grades neither correlated with PET signal (left knee r = 0.32, P = 0.19; right knee r = 0.18, P = 0.45) nor pain ( r = 0.39, P = 0.07). The current results support further exploration of [ 11 C]-PBR28 PET signal as an imaging marker candidate for KOA and a link between joint inflammation and osteoarthritis-related pain severity.

Naturalistic Bladder Filling Reveals Subtypes in Overactive Bladder Syndrome That Differentially Engages Urinary Urgency-Related Brain Circuits: Results From the Symptoms of Lower Urinary Tract Dysfunction Research Network (LURN).

PURPOSE: Overactive bladder (OAB) may be attributed to dysfunction in supraspinal brain circuits. Overactive bladder participants enrolled in the LURN (Symptoms of Lower Urinary Tract Dysfunction Research Network) study reported sensations of urinary urgency during a bladder-filling paradigm while undergoing brain functional MRI to map supraspinal dysfunction. MATERIALS AND METHODS: OAB participants and controls (CONs) completed 2 resting-state functional MRI scans following consumption of 350 mL water. Scans were conducted at fuller and emptier bladder states, interleaved with voiding. Urgency ratings (0-10) were assessed. Patterns of urgency during bladder filling were investigated using latent class trajectory models. Clusters of participants encompassing each pattern (ie, subtype) were derived from aggregated groups of OAB and CON independent of diagnosis. RESULTS: Two distinct patterns of urgency trajectories were revealed: first subtype with OAB and CON who were unresponsive to bladder filling (OAB-1 and CON-1) and second highly responsive subtype predominantly containing OAB (OAB-2). OAB-2 participants scored significantly higher on urinary symptoms but not pain or psychosocial measures. Neuroimaging analyses showed change in urgency due to both bladder filling and voided volume related to multiple loci of brain network connectivity in OAB-2, and in some cases, different than OAB-1 and/or CON-1. Sensorimotor to dorsomedial/dorsolateral prefrontal connectivity mediated the relationship between stimulus (voided volume) and percept (urgency) in OAB-2. CONCLUSIONS: Our results reveal different OAB subtypes with latent class trajectory models of urgency ratings during natural bladder filling. Functional MRI revealed differences in pathophysiology between subtypes, namely sensorimotor-prefrontal connectivity is a key locus in OAB patients with higher urinary symptoms.

Relationship Between Nociplastic Pain Involvement and Medication Use, Symptom Relief, and Adverse Effects Among People Using Medical Cannabis for Chronic Pain.

OBJECTIVES: Cannabis is increasingly being used for chronic pain management, but cannabis' effects remain poorly characterized in chronic nociplastic pain (NPP), which is posited to be caused by disturbances in nervous system pain processing. In this cross-sectional study (n=1213), we used the 2011 Fibromyalgia (FM) Survey Criteria as a surrogate measure for degree of NPP among individuals using medical cannabis for chronic pain. METHODS: Using a quartile-split, we investigated associations between the degree of NPP and medication use, cannabis use characteristics, and symptom relief. Continuous variables were assessed using one-way analysis of variance and categorical variables with Pearson χ 2 test and binomial logistic regression for calculation of odds ratios. RESULTS: Participants were predominately female (59%), with a mean ± SD age of 49.4±13.6 years. Higher FM scores were associated with less self-reported improvement in pain and health since initiating medical cannabis use, as well as more cannabis-related side effects. Paradoxically, higher FM scores were also associated with higher usage of concomitant medication use (including opioids and benzodiazepines) but also with substituting cannabis for significantly more medication classes, including opioids and benzodiazepines. DISCUSSION: This article presents evidence that individuals in higher NPP quartiles have higher analgesic intake, higher odds of substituting cannabis for medications, higher side effect burden, and lower therapeutic effect from cannabis. These seemingly contradictory findings may reflect higher symptom burden, polypharmacy at baseline, or that NPP may be challenging to treat with cannabis. Further research is necessary to further explain cannabinoid effects in NPP.

Psychedelic concentrations of nitrous oxide reduce functional differentiation in frontoparietal and somatomotor cortical networks.

Despite the longstanding use of nitrous oxide and descriptions of its psychological effects more than a century ago, there is a paucity of neurobiological investigation of associated psychedelic experiences. We measure the brain's functional geometry (through analysis of cortical gradients) and temporal dynamics (through analysis of co-activation patterns) using human resting-state functional magnetic resonance imaging data acquired before and during administration of 35% nitrous oxide. Both analyses demonstrate that nitrous oxide reduces functional differentiation in frontoparietal and somatomotor networks. Importantly, the subjective psychedelic experience induced by nitrous oxide is inversely correlated with the degree of functional differentiation. Thus, like classical psychedelics acting on serotonin receptors, nitrous oxide flattens the functional geometry of the cortex and disrupts temporal dynamics in association with psychoactive effects.

Differential clinical characteristics across traditional Chinese medicine (TCM) syndromes in patients with sickle cell disease.

Background: Pain is a common, debilitating, and poorly understood complication of sickle cell disease (SCD). The need for clinical pain management of SCD is largely unmet and relies on opioids as the main therapeutic option, which leads to a decreased quality of life (QoL). According to the literature, acupuncture has shown certain therapeutic effects for pain management in SCD. However, these clinical studies lack the guidance of Traditional Chinese Medicine (TCM) Syndrome Differentiation principles for treatment. Aim: To characterize differences in clinical presentation amongst TCM-diagnosed syndromes in SCD patients. Method: 52 patients with SCD and 28 age- and sex-matched healthy controls (HCs) were enrolled in an ongoing trial of acupuncture. Each participant completed a series of questionnaires on pain, physical function, fatigue, sleep, anxiety, depression, and QoL and underwent cold- and pressure-based quantitative sensory testing at baseline. Data on prescription opioid use over the 12 months prior to study enrollment was used to calculate mean daily morphine milligram equivalents (MME). Differences among the three TCM syndromes were analyzed by one-way ANOVA followed by Tukey post hoc testing. Two-sample t-tests were used to compare SCD and HC groups. Results: TCM diagnosis criteria classified SCD patients into one of three TCM syndromes: a) Equal; b) Deficiency; and c) Stagnation. The Stagnation group exhibited higher pain interference, physical dysfunction, nociplastic pain, fatigue, anxiety, depression, MME consumption, and lower sleep quality and QoL compared to the Equal group. Few differences were observed between HCs and the Equal SCD group across outcomes. Deficiency and Stagnation groups were differentiated with observed- and patient-reported clinical manifestations. Conclusion: These findings suggest that TCM-diagnosed syndromes in SCD can be differentially characterized using validated objective and patient-reported outcomes. Because characteristics of pain and co-morbidities in each SCD patient are unique, targeting specific TCM "syndromes" may facilitate treatment effectiveness with a syndrome-based personalized treatment plan that conforms to TCM principles. These findings lay the foundation for the development of tailored acupuncture interventions based on TCM syndromes for managing pain in SCD. Larger samples are required to further refine and validate TCM diagnostic criteria for SCD.

Prevalence and Mortality of Moose (Alces alces) Infected with Elaeophora schneideri in Montana, USA.

Elaeophora schneideri is a filarial nematode of North America that occasionally infects aberrant ruminant hosts such as moose (Alces alces). The role E. schneideri plays in clinical morbidity or mortality of moose remains uncertain. We sampled predominantly hunter-killed adult moose (n=127) to characterize the spatial patterns of prevalence and intensity of worms in carotid arteries of moose in Montana. We compared prevalence and intensity of E. schneideri within these moose to a separate sample of adult moose that died of health-related causes (n=34). We found lower prevalence in northwest Montana (0.06) than in the remainder of the state (0.42). We also found both higher prevalence of E. schneideri and higher intensity to be correlated with increased probability of health-related mortality. Our results suggest presence and intensity of E. schneideri correlate with mortality of moose, although the mechanisms of mortality remain uncertain.