Last general practitioner consultation during the final month of life: a national medical record review of suicides in Sweden.

OBJECTIVES: Individuals who die by suicide often consult their general practitioners (GPs) in their final weeks of life. The aim of this study was to gain a deeper knowledge of the clinical characteristics and GP assessments and treatments among individuals who consulted their GPs during the month preceding suicide. Further, we compared these features in those with and without contact with psychiatric services (PC and NPC, respectively) during the two years that preceded the suicide. DESIGN: A nationwide retrospective explorative study investigating medical records. SETTING: Primary care in Sweden. PARTICIPANTS: Individuals who died by suicide in Sweden in 2015 with a GP visit within 30 days of death. RESULTS: The study cohort corresponds to one fifth (n = 238) of all suicides that occurred in Sweden in 2015 (n = 1179), representing all those with available primary care records showing contact with a GP during the final 30 days of life (NPC: n = 125; PC: n = 113). The mean age was 58 years ± 19. Patients in the NPC group were older (NPC: 63 years ± 19 vs. PC: 53 years ± 18, p < 0.0001) and presented psychiatric symptoms less often (NPC: 50% vs. PC: 67%, p < 0.006). Somatic symptoms were as common as psychiatric symptoms for the whole sample, being present in more than half of individuals. Suicide risk was noted in only 6% of all individuals. Referral to psychiatric services occurred in 14%, less commonly for the NPC group (NPC: 6% vs. PC: 22%, p < 0.001). Cardiovascular or respiratory symptoms were noted in 19%, more often in the NPC group (NPC: 30% vs. PC: 6%, p < 0.001), as were diagnoses involving the circulatory system (all 10%, NPC:14% vs. PC: 5%, p < 0.020). CONCLUSION: A high level of somatic symptoms was observed in primary care patients both with and without psychiatric contact, and this might have influenced GPs' management decisions. Psychiatric symptoms were noted in two thirds of those with psychiatric contact but only half of those without. While GPs noted psychiatric symptoms in over half of all individuals included in the study, they seldom noted suicide risk. These findings highlight the need for increased attention to psychiatric symptoms and suicide risk assessment, particularly among middle-aged and older individuals presenting with somatic symptoms. STRENGTHS AND LIMITATIONS OF THIS STUDY: The National Cause of Death Register has excellent coverage of suicide deaths and access to medical records was very good. The medical record review provided detailed information regarding primary care utilization before death by suicide. Because of the lack of statistical power, due to the limited number of persons with GP contact during the last month of life, we chose not to correct for multiple comparisons. Our study approach did not capture the reasons behind GPs' documentation of elevated suicide risk. No systematic inter-rater reliability (IRR) testing was made, however, reviewers received training and continuous support from the research group.

Energy harvesting from fuel cell bicycles for home DC grids using soft switched DC-DC converter.

Fuel cell vehicles (FCVs) are gaining significance due to their potential to reduce greenhouse gas emissions and dependence on fossil fuels. Their efficient fuel cell cycle makes them ideal for last-mile transportation, offering zero emissions and longer range compared to battery electric vehicles. Additionally, the generation of electricity through fuel cell stacks is becoming increasingly popular, providing a clean energy source for various applications. This paper focuses on utilizing the energy from fuel cycle bicycles when it's not in use and feeding it into the home DC grid. To achieve this, a dual-phase DC to DC converter is proposed to boost stack voltage and integrate with the 24 V DC home grid system. The converter design is simulated using the PSIM platform and tested in a hardware-in-the-loop (HIL) environment with real-time simulation capabilities.

Lidocaine-Associated CNS Toxicity at Therapeutic Dosage: A Case Report and Literature Review.

Lidocaine is a commonly used anesthetic. High doses or intravenous administration of lidocaine, as well as other local anesthetics, may result in systemic effects involving the cardiovascular and neurologic systems. Typically, effects are dependent on the serum concentrations of the offending agent. This is a case where a patient presented with symptoms of systemic lidocaine toxicity despite therapeutic dosage and an undetectable serum lidocaine concentration. A 47-year-old Caucasian male received a lumbar medial branch injection of lidocaine and presented with symptoms of perioral numbness, seizure-like activity, and confusion. The patient had a workup, including a CT head without acute findings and an undetectable serum lidocaine level. Due to symptoms consistent with systemic local anesthetic toxicity, intravenous lipid emulsion (ILE) was administered with resolution and without recurrence of symptoms. There should be a low threshold of suspicion for local anesthetic systemic toxicity when patients have neurologic or cardiovascular symptoms following exposure. Toxicity may be present despite therapeutic dosages and low serum concentrations. ILE may be beneficial and should be considered.

Evolutionary trajectory for nuclear functions of ciliary transport complex proteins.

SUMMARYCilia and the nucleus were two defining features of the last eukaryotic common ancestor. In early eukaryotic evolution, these structures evolved through the diversification of a common membrane-coating ancestor, the protocoatomer. While in cilia, the descendants of this protein complex evolved into parts of the intraflagellar transport complexes and BBSome, the nucleus gained its selectivity by recruiting protocoatomer-like proteins to the nuclear envelope to form the selective nuclear pore complexes. Recent studies show a growing number of proteins shared between the proteomes of the respective organelles, and it is currently unknown how ciliary transport proteins could acquire nuclear functions and vice versa. The nuclear functions of ciliary proteins are still observable today and remain relevant for the understanding of the disease mechanisms behind ciliopathies. In this work, we review the evolutionary history of cilia and nucleus and their respective defining proteins and integrate current knowledge into theories for early eukaryotic evolution. We postulate a scenario where both compartments co-evolved and that fits current models of eukaryotic evolution, explaining how ciliary proteins and nucleoporins acquired their dual functions.

Acute Pain Management in a Multi-site Trauma Patient.

Pain management is often difficult in the setting of multi-site trauma such as that caused by motor vehicle accidents (MVA), which is especially compounded in the setting of polysubstance abuse. This often results in patients with poor pain tolerance requiring escalating doses of opioid therapy, which creates a vicious cycle. The use of peripheral nerve blocks (PNB) has been shown to decrease overall opioid consumption and can be used effectively to manage postoperative pain in this patient population. Our case report aims to highlight the importance of PNBs as part of a multimodal approach to pain management in patients with polytrauma in the setting of polysubstance abuse.

Shuffling-type gradient method with bandwidth-based step sizes for finite-sum optimization.

Shuffling-type gradient method is a popular machine learning algorithm that solves finite-sum optimization problems by randomly shuffling samples during iterations. In this paper, we explore the convergence properties of shuffling-type gradient method under mild assumptions. Specifically, we employ the bandwidth-based step size strategy that covers both monotonic and non-monotonic step sizes, thereby providing a unified convergence guarantee in terms of step size. Additionally, we replace the lower bound assumption of the objective function with that of the loss function, thereby eliminating the restrictions on the variance and the second-order moment of stochastic gradient that are difficult to verify in practice. For non-convex objectives, we recover the last iteration convergence of shuffling-type gradient algorithm with a less cumbersome proof. Meanwhile, we also establish the convergence rate for the minimum iteration of gradient norms. Under the Polyak-Lojasiewicz (PL) condition, we prove that the function value of last iteration converges to the lower bound of the objective function. By selecting appropriate boundary functions, we further improve the previous sublinear convergence rate results. Overall, this paper contributes to the understanding of shuffling-type gradient method and its convergence properties, providing insights for optimizing finite-sum problems in machine learning. Finally, numerical experiments demonstrate the efficiency of shuffling-type gradient method with bandwidth-based step size and validate our theoretical results.

Evolution of Cellular Organization Along the First Branches of the Tree of Life.

Current evidence suggests that some form of cellular organization arose well before the time of the last universal common ancestor (LUCA). Standard phylogenetic analyses have shown that several protein families associated with membrane translocation, membrane transport, and membrane bioenergetics were very likely present in the proteome of the LUCA. Despite these cellular systems emerging prior to the LUCA, extant archaea, bacteria, and eukaryotes have significant differences in cellular infrastructure and the molecular functions that support it, leading some researchers to argue that true cellularity did not evolve until after the LUCA. Here, we use recently reconstructed minimal proteomes of the LUCA as well as the last archaeal common ancestor (LACA) and the last bacterial common ancestor (LBCA) to characterize the evolution of cellular systems along the first branches of the tree of life. We find that a broad set of functions associated with cellular organization were already present by the time of the LUCA. The functional repertoires of the LACA and LBCA related to cellular organization nearly doubled along each branch following the divergence of the LUCA. These evolutionary trends created the foundation for similarities and differences in cellular organization between the taxonomic domains that are still observed today.

The association of female reproductive factors with history of cardiovascular disease: a large cross-sectional study.

BACKGROUND: This study aimed to explore the association of female reproductive factors (age at first birth (AFB), age at last birth (ALB), number of pregnancies, and live births) with history of cardiovascular disease (CVD). METHODS: A total of 15,715 women aged 20 years or over from the National Health and Nutrition Examination Surveys from 1999 to 2018 were included in our analysis. Weighted multivariable logistic regression analysis and restricted cubic spline (RCS) model were used to evaluate the association of AFB and ALB with history of CVD in women. Additionally, the relationship between the number of pregnancies, and live births and history of CVD was also explored. RESULTS: After adjusting for potential confounding factors, the RCS plot showed a U-curve relationship between AFB, ALB and history of CVD. Among them, AFB was associated with congestive heart failure (CHF), heart attack, and stroke in a U-shaped curve. Additionally, this U-shaped correlation also exists between ALB and CHF and stroke. However, the number of pregnancies and live births was liner positive associated with history of CVD, including coronary heart disease, CHF, angina pectoris, heart attack, and stroke. CONCLUSIONS: Women with younger or later AFB and ALB have higher odds of CVD in later life. Further study is warranted to verify the underlying mechanisms of this association.

Expanding community case management of malaria to all ages can improve universal access to malaria diagnosis and treatment: results from a cluster randomized trial in Madagascar.

BACKGROUND: Global progress on malaria control has stalled recently, partly due to challenges in universal access to malaria diagnosis and treatment. Community health workers (CHWs) can play a key role in improving access to malaria care for children under 5 years (CU5), but national policies rarely permit them to treat older individuals. We conducted a two-arm cluster randomized trial in rural Madagascar to assess the impact of expanding malaria community case management (mCCM) to all ages on health care access and use. METHODS: Thirty health centers and their associated CHWs in Farafangana District were randomized 1:1 to mCCM for all ages (intervention) or mCCM for CU5 only (control). Both arms were supported with CHW trainings on malaria case management, community sensitization on free malaria care, monthly supervision of CHWs, and reinforcement of the malaria supply chain. Cross-sectional household surveys in approximately 1600 households were conducted at baseline (Nov-Dec 2019) and endline (Nov-Dec 2021). Monthly data were collected from health center and CHW registers for 36 months (2019-2021). Intervention impact was assessed via difference-in-differences analyses for survey data and interrupted time-series analyses for health system data. RESULTS: Rates of care-seeking for fever and malaria diagnosis nearly tripled in both arms (from less than 25% to over 60%), driven mostly by increases in CHW care. Age-expanded mCCM yielded additional improvements for individuals over 5 years in the intervention arm (rate ratio for RDTs done in 6-13-year-olds, RRRDT6-13 years = 1.65; 95% CIs 1.45-1.87), but increases were significant only in health system data analyses. Age-expanded mCCM was associated with larger increases for populations living further from health centers (RRRDT6-13 years = 1.21 per km; 95% CIs 1.19-1.23). CONCLUSIONS: Expanding mCCM to all ages can improve universal access to malaria diagnosis and treatment. In addition, strengthening supply chain systems can achieve significant improvements even in the absence of age-expanded mCCM. TRIAL REGISTRATION: The trial was registered at the Pan-African Clinical Trials Registry (#PACTR202001907367187).

Short-term fire exclusion affects germination and seed traits in tropical savannas.

In fire-prone ecosystems, plant traits are influenced by the fire regime, thus reproduction and establishment can be altered by this disturbance. Changes in fire frequency and history can therefore influence seed and germination traits. We investigated the effects of short-term fire exclusion on seed and germination traits of species from tropical open savannas. Seeds from 27 species were collected from two areas with distinct fire histories: recently and frequently burned (RB) or unburned for 5 to 7 years (E). Seeds from both areas underwent germination trials under optimal conditions for 30 days. Also, 10 species were exposed to high temperature treatments (100 or 200 °C) and seed and germination traits measured. Comparisons were then made for each trait, analysing each species separately, between the two areas. Approximately 85% of species studied had at least one of their germination traits altered in the RB area compared to the E area. Clear differences included lower viability and faster germination in seeds from RB areas. Seed traits of 70% of measured species differed between the two areas. Our results show species-specific trait response to different fire histories. For example, faster germination and lower viability of seeds from RB plots suggest selection for faster maturing individuals and differences in resourcing, respectively, under a regime of frequent fire. This study provides insights into fire effects on regeneration responses of tropical savanna species and also points to the need for more studies evaluating the effects of fire history on seed traits.

Human impacts overwhelmed climate as the dominant factor controlling lacustrine organic matter accumulation in Erhai Lake 2000 years ago, Southwest China.

Climate and human activity are two important factors in regulating organic matter (OM) accumulation in the lake environment. However, when and how anthropogenic impacts have affected lacustrine OM accumulation in southwest China during the late Holocene have not yet been well defined. Here, a 16.3-kyr n-alkane record derived from Erhai Lake was used to trace OM sources and explore their connections to climate and human activity. The n-alkane distributions indicated that the dominant sediment sources shifted from terrestrial and aquatic plants to algae in the late Holocene. OM accumulation was closely related to catchment soil erosion, sediment transport, and deposition processes regulated by climate conditions before 5.0 cal. kyr B.P., following the patterns that stronger monsoon precipitation favoured more terrestrial and less aquatic OM input, and vice versa. From 5.0 to 2.0 cal. kyr B.P., the synchronous downwards trends in terrestrial OM input and precipitation intensity indicated that climate remained a major driving force for OM accumulation. However, sediment sources experienced large-magnitude and centennial-scale oscillations between allochthonous and autochthonous inputs, reflecting early human impacts appeared and lake ecosystems retained the self-regulated ability to recover from the basin-wide early moderate human disturbances. Afterwards, the increased (decreased) OM contributions from terrestrial (aquatic) plants contradicted the weakening monsoon precipitation since 2.0 cal. kyr B.P., indicating a dominant effect of human activities on OM accumulation. This change was accompanied by highly improved algae productivity and gradually elevated lacustrine trophic status, and the lake ecosystem eventually shifted into another state largely deviating from its climate-driven background due to intensified deforestation and agricultural cultivation. Regional comparison indicated that anthropogenic disturbances have temporal differences in southwest China. This study will further improve our understanding of past climate-human-environment interactions in southwest China.

How can we quantify, explain, and apply the uncertainty of complex soil maps predicted with neural networks?

Artificial neural networks (ANNs) have proven to be a useful tool for complex questions that involve large amounts of data. Our use case of predicting soil maps with ANNs is in high demand by government agencies, construction companies, or farmers, given cost and time intensive field work. However, there are two main challenges when applying ANNs. In their most common form, deep learning algorithms do not provide interpretable predictive uncertainty. This means that properties of an ANN such as the certainty and plausibility of the predicted variables, rely on the interpretation by experts rather than being quantified by evaluation metrics validating the ANNs. Further, these algorithms have shown a high confidence in their predictions in areas geographically distant from the training area or areas sparsely covered by training data. To tackle these challenges, we use the Bayesian deep learning approach "last-layer Laplace approximation", which is specifically designed to quantify uncertainty into deep networks, in our explorative study on soil classification. It corrects the overconfident areas without reducing the accuracy of the predictions, giving us a more realistic uncertainty expression of the model's prediction. In our study area in southern Germany, we subdivide the soils into soil regions and as a test case we explicitly exclude two soil regions in the training area but include these regions in the prediction. Our results emphasize the need for uncertainty measurement to obtain more reliable and interpretable results of ANNs, especially for regions far away from the training area. Moreover, the knowledge gained from this research addresses the problem of overconfidence of ANNs and provides valuable information on the predictability of soil types and the identification of knowledge gaps. By analyzing regions where the model has limited data support and, consequently, high uncertainty, stakeholders can recognize the areas that require more data collection efforts.

Does a place of last drink initiative affect the likelihood of alcohol sales to obviously intoxicated patrons?

BACKGROUND: Although the sale of alcohol to obviously intoxicated patrons (i.e., overservice) is illegal in 48 U.S. states, the likelihood of overservice at bars and restaurants has exceeded 80% across multiple studies, states, and decades. Place of last drink (POLD) enforcement is one proposed strategy to address alcohol overservice. When law enforcement agents respond to an alcohol-related incident, they ask the individuals involved where they had their last alcoholic beverage. POLD information is recorded and ideally systematically reviewed to identify locations that are frequently places of last drink. Law enforcement or other agencies may follow up with or penalize the alcohol license holder at these locations. We compared the likelihood of overservice in communities conducting POLD with communities that did not conduct POLD in Minnesota. METHODS: Pseudo-intoxicated patrons acted out signs of obvious intoxication while attempting to purchase alcohol at 396 bars and restaurants in 26 communities conducting POLD and 26 comparison communities. We calculated rates of alcohol sales to the pseudo-intoxicated patrons overall and in POLD communities versus comparison communities. RESULTS: The overall sales rate to the pseudo-intoxicated buyers was 98%. Rates of sales were 99% at establishments in POLD jurisdictions and 97% in comparison jurisdictions. There were no common characteristics, such as perceived gender/age of the server/bartender or crowdedness of the establishment, among the seven establishments that refused alcohol service. CONCLUSIONS: Our study shows that, as currently implemented in Minnesota, POLD does not reduce the overservice of alcohol at a jurisdiction level. More research is needed to identify interventions that yield sustained reductions in the overservice of alcohol.

Environmental and Biogeographic Drivers behind Alpine Plant Thermal Tolerance and Genetic Variation.

In alpine ecosystems, elevation broadly functions as a steep thermal gradient, with plant communities exposed to regular fluctuations in hot and cold temperatures. These conditions lead to selective filtering, potentially contributing to species-level variation in thermal tolerance and population-level genetic divergence. Few studies have explored the breadth of alpine plant thermal tolerances across a thermal gradient or the underlying genetic variation thereof. We measured photosystem heat (Tcrit-hot) and cold (Tcrit-cold) thresholds of ten Australian alpine species across elevation gradients and characterised their neutral genetic variation. To reveal the biogeographical drivers of present-day genetic signatures, we also reconstructed temporal changes in habitat suitability across potential distributional ranges. We found intraspecific variation in thermal thresholds, but this was not associated with elevation, nor underpinned by genetic differentiation on a local scale. Instead, regional population differentiation and considerable homozygosity within populations may, in part, be driven by distributional contractions, long-term persistence, and migrations following habitat suitability. Our habitat suitability models suggest that cool-climate-distributed alpine plants may be threatened by a warming climate. Yet, the observed wide thermal tolerances did not reflect this vulnerability. Conservation efforts should seek to understand variations in species-level thermal tolerance across alpine microclimates.

Local Anesthetics, Local Anesthetic Systemic Toxicity (LAST), and Liposomal Bupivacaine.

Local anesthetics have played a vital role in the multimodal analgesia approach to patient care by decreasing the use of perioperative opioids, enhancing patient satisfaction, decreasing the incidence of postoperative nausea and vomiting, decreasing the length of hospital stay, and reducing the risk of chronic postsurgical pain. The opioid-reduced anesthetic management for perioperative analgesia has been largely successful with the use of local anesthetics during procedures such as peripheral nerve blocks and neuraxial analgesia. It is important that practitioners who use local anesthetics are aware of the risk factors, presentation, and management of local anesthetic systemic toxicity (LAST).

Local Anesthetic Systemic Toxicity (LAST): More Common Than You Think.

The number of anesthetic body procedures in the United States is rapidly increasing, with many being performed on an outpatient basis. These procedures are advertised as being safe, and many times the serious complications may not be discussed. Although local anesthetic systemic toxicity is a rare complication, it is associated with an increase in morbidity. The emergency department staff should be aware of the possibility of this rare complication, as well as the variety of resulting symptoms (from minor to severe), potential sequelae, and appropriate management for patients who have undergone an outpatient anesthetic body procedure. Multiple factors contribute to the development of local anesthetic systemic toxicity, resulting in life-threatening effects on the neurologic and cardiovascular systems. Also, the site of administration, along with the local anesthetic agent used, can impact the risk of the development of local anesthetic systemic toxicity. To minimize the risk and ensure the best possible outcome for these patients, emergency department staff must be highly aware of the mechanisms, risk factors, prevention, and management/treatment of local anesthetic systemic toxicity.

Lipid A in outer membrane vesicles shields bacteria from polymyxins.

The continuous emergence of multidrug-resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae being a prominent threat. We conducted a comprehensive study on K. pneumoniae's antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last-resort antibiotic. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)-stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed Klebsiella. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and Galleria mellonella. In all models, OMVs protected K. pneumoniae from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed K. pneumoniae could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed K. pneumoniae compared to control OMVs. The lipid A-dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected Klebsiella from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross-protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug-resistant bacterial infections.

Distinct and rich assemblages of giant viruses in Arctic and Antarctic lakes.

Giant viruses (GVs) are key players in ecosystem functioning, biogeochemistry, and eukaryotic genome evolution. GV diversity and abundance in aquatic systems can exceed that of prokaryotes, but their diversity and ecology in lakes, especially polar ones, remain poorly understood. We conducted a comprehensive survey and meta-analysis of GV diversity across 20 lakes, spanning polar to temperate regions, combining our extensive lake metagenome database from the Canadian Arctic and subarctic with publicly available datasets. Leveraging a novel GV genome identification tool, we identified 3304 GV metagenome-assembled genomes, revealing lakes as untapped GV reservoirs. Phylogenomic analysis highlighted their dispersion across all Nucleocytoviricota orders. Strong GV population endemism emerged between lakes from similar regions and biomes (Antarctic and Arctic), but a polar/temperate barrier in lacustrine GV populations and differences in their gene content could be observed. Our study establishes a robust genomic reference for future investigations into lacustrine GV ecology in fast changing polar environments.

Superagers Resist Typical Age-Related White Matter Structural Changes.

Superagers are elderly individuals with the memory ability of people 30 years younger and provide evidence that age-related cognitive decline is not inevitable. In a sample of 64 superagers (mean age, 81.9; 59% women) and 55 typical older adults (mean age, 82.4; 64% women) from the Vallecas Project, we studied, cross-sectionally and longitudinally over 5 years with yearly follow-ups, the global cerebral white matter status as well as region-specific white matter microstructure assessment derived from diffusivity measures. Superagers and typical older adults showed no difference in global white matter health (total white matter volume, Fazekas score, and lesions volume) cross-sectionally or longitudinally. However, analyses of diffusion parameters revealed the better white matter microstructure in superagers than in typical older adults. Cross-sectional differences showed higher fractional anisotropy (FA) in superagers mostly in frontal fibers and lower mean diffusivity (MD) in most white matter tracts, expressed as an anteroposterior gradient with greater group differences in anterior tracts. FA decrease over time is slower in superagers than in typical older adults in all white matter tracts assessed, which is mirrored by MD increases over time being slower in superagers than in typical older adults in all white matter tracts except for the corticospinal tract, the uncinate fasciculus, and the forceps minor. The better preservation of white matter microstructure in superagers relative to typical older adults supports resistance to age-related brain structural changes as a mechanism underpinning the remarkable memory capacity of superagers, while their regional aging pattern is in line with the last-in-first-out hypothesis.