Contextual Memory Research Articles

Brain imaging and machine learning reveal uncoupled functional network for contextual threat memory in long sepsis.

Positron emission tomography (PET) utilizes radiotracers like [18F]fluorodeoxyglucose (FDG) to measure brain activity in health and disease. Performing behavioral tasks between the FDG injection and the PET scan allows the FDG signal to reflect task-related brain networks. Building on this principle, we introduce an approach called behavioral task-associated PET (beta-PET) consisting of two scans: the first after a mouse is familiarized with a conditioning chamber, and the second upon recall of contextual threat. Associative threat conditioning occurs between scans. Beta-PET focuses on brain regions encoding threat memory (e.g., amygdala, prefrontal cortex) and contextual aspects (e.g., hippocampus, subiculum, entorhinal cortex). Our results show that beta-PET identifies a biologically defined network encoding contextual threat memory and its uncoupling in a mouse model of long sepsis. Moreover, machine learning algorithms (linear logistic regression) and ordinal trends analysis demonstrate that beta-PET robustly predicts the behavioral defense response and its breakdown during long sepsis.

Comparative Analysis of Excitatory and Inhibitory Hippocampal Neurons on Retrieval of Associative Contextual Memory

Comparative Analysis of Excitatory and Inhibitory Hippocampal Neurons on Retrieval of Associative Contextual Memory

Adverse Effects of Aβ1-42 Oligomers: Impaired Contextual Memory and Altered Intrinsic Properties of CA1 Pyramidal Neurons

Aβ1-42 (amyloid beta) oligomers, the major neurotoxic culprits in Alzheimer’s disease, initiate early pathophysiological events, including neuronal hyperactivity, that underlie aberrant network activity and cognitive impairment. Although several synaptotoxic effects have been extensively studied, neuronal hyperexcitability, which may also contribute to cognitive deficits, is not fully understood. Here, we found several adverse effects of in vivo injection of Aβ1-42 oligomers on contextual memory and intrinsic properties of CA1 pyramidal neurons. Male rats underwent behavioral and electrophysiological studies 1 week after microinjections into the dorsal CA1 region, followed by histological analysis. After 1 week, Aβ1-42 oligomers impaired contextual learning without affecting basic physiological functions and triggered training-induced neuronal excitability. Furthermore, riluzole, a persistent sodium current (INaP) blocker, dose-dependently reduced Aβ1-42 oligomer-induced hyperexcitability. Congo red staining, which detects insoluble amyloid deposits, further identified labeling of CA1 pyramidal neurons while immunohistochemistry with lecanemab, which detects soluble Aβ oligomers, revealed immunoreactivity of both pyramidal and non-pyramidal cells in the target area. Therefore, our study suggests that a single injection of Aβ1-42 oligomers resulted in contextual memory deficits along with concomitant neuronal hyperexcitability and amyloid deposition in the CA1 region after 1 week.

Hippocampal contextualization of social rewards in mice

Acquiring and exploiting memories of rewarding experiences is critical for survival. The spatial environment in which a rewarding stimulus is encountered regulates memory retrieval. The ventral hippocampus (vH) has been implicated in contextual memories involving rewarding stimuli such as food, social cues or drugs. Yet, the neuronal representations and circuits underlying contextual memories of socially rewarding stimuli are poorly understood. Here, using in vivo electrophysiological recordings, in vivo one-photon calcium imaging, and optogenetics during a social reward contextual conditioning paradigm in male mice, we show that vH neurons discriminate between contexts with neutral or acquired social reward value. The formation of context-discriminating vH neurons following learning was contingent upon the presence of unconditioned stimuli. Moreover, vH neurons showed distinct contextual representations during the retrieval of social reward compared to fear contextual memories. Finally, optogenetic inhibition of locus coeruleus (LC) projections in the vH selectively disrupted social reward contextual memory by impairing vH contextual representations. Collectively, our findings reveal that the vH integrates contextual and social reward information, with memory encoding of these representations supported by input from the LC.

Tat-Beclin-1 Ameliorates Memory by Improving Neuronal Cytoarchitecture and Mitigating Mitochondrial Dysfunction in Scopolamine-Induced Amnesic Male Mice.

Mitophagy, the targeted breakdown of damaged mitochondria, plays a vital role in maintaining cellular homeostasis. As impairment of mitophagy leads to neurodegeneration and memory decline, the current study explores the therapeutic potential of an autophagy inducer Tat-Beclin-1 during scopolamine-induced amnesia. Tat-Beclin-1 improved contextual and recognition memory and also mitochondrial ultrastructure by restoring mitochondrial length and area and reducing the number of fragmented mitochondria. Tat-Beclin-1 upregulated the expression of genes associated with mitophagy (PTEN-induced kinase 1, Parkin, Lamp2, and LC3), mitochondrial fusion (Mfn1, Mfn2, and optic atrophy1), and fission (dynamin-related protein 1 and Fis1) in amnesic mice. Subsequently, these results were supported by a decreased level of p-Drp1 (S616) and Drp 1 ratios and an increased level of Mfn2, LC3BI, and BII in Tat-Beclin-1-treated mice. Moreover, Tat-Beclin-1 maintained mitochondrial membrane potential and complex I/V activity in amnesic mice. Tat-Beclin-1 enhanced myelination and diminished the activity of acetylcholinesterase and caspase-3 activity. Sholl analysis revealed augmented dendritic branching and length, elevated dendritic spine density, and upregulated the expression of synaptophysin and PSD95 proteins, indicating neuronal plasticity enhancement by Tat-Beclin-1. Thus, these findings provide valuable insights into the therapeutic potential of Tat-Beclin-1, addressing mitochondrial dysfunction to mitigate cognitive impairment associated with amnesic conditions.

Spatial mRNA expression patterns of orexin receptors in the dorsal hippocampus.

Orexins are wake-promoting neuropeptides that originate from hypothalamic neurons projecting to widespread brain areas throughout the central nervous system. They modulate various physiological functions via their orexin 1 (OXR1) and 2 (OXR2) receptors, including sleep-wake rhythm but also cognitive functions such as memory formation. Here, we provide a detailed analysis of OXR1 and OXR2 mRNA expression profiles in the dorsal hippocampus as a key region for memory formation, using RNAscope multiplex in situ hybridization. Interconnected subareas relevant for cognition and memory such as the medial prefrontal cortex and the nucleus reuniens of the thalamus were assessed as well. Both receptor types display distinct profiles, with the highest percentage of OXR1 mRNA-positive cells in the hilus of the dentate gyrus. Here, the content of OXR1 mRNA per cell was slightly modulated at selected time points over a 12h light/ 12 dark light phase. Using RNAScope and quantitative polymerase chain reaction approaches, we began to address a cell-type specific expression of OXR1 in hilar GABAergic interneurons. The distinct expression profiles of both receptor subtypes within hippocampal subareas and circuits provide an interesting basis for future interventional studies on orexin receptor function in spatial and contextual memory.

Contextual memory bias in emotional events: Neurobiological correlates and depression risk

BackgroundContextual memory loss of emotional events plays a critical role in depression psychopathology. Individuals with depression, clinical or subclinical, exhibit enhanced and impaired memory for emotionally negative stimuli and context in an event, respectively. This suggests that contextual encoding may fail because of attentional interference caused by concurrent negative stimuli, possibly leading to contextual memory loss as a depression risk. Amygdala–prefrontal connectivity and cortisol may underlie the mechanism; however, the relationships remain unknown. MethodsOne hundred twenty participants, including 34 with subclinical depression, underwent behavioral tasks, functional magnetic resonance imaging (fMRI) scans, and saliva collection. Encoding and 24 h later recollection performance of visuoperceptual/spatial/temporal context in a series of events, where fearful (vs. neutral) faces appeared, were measured via contextual memory tasks. Overgeneral autobiographical memory (OGM), a more remote form of contextual memory loss, was also assessed via the Autobiographical Memory Test. Amygdala connectivity was measured by fMRI during attentional interference by fearful (vs. neutral) faces to differentiate selective attention from encoding. Basal cortisol levels were assayed through saliva collected at encoding during the visit day and across 2 consecutive days in the following week (12 time points in total). We explored whether contextual memory encoding failure would explain depressive symptoms through OGM under possible moderation of amygdala connectivity and cortisol. ResultsIn individuals with subclinical depression compared to those without, fearful faces disturbed memory encoding of the visuoperceptual context rather than 24 h later recollection, while neutral faces in their temporal proximity contrastingly augmented it. The more the contextual memory encoding bias (fearful vs. neutral) intensified, the more the amygdala’s functional connectivity with the ventromedial prefrontal cortex (vmPFC) weakened. Higher total cortisol output tended to be correlated with poorer 24-h later recollection of the temporal context. Moderated mediation effects of the amygdala-vmPFC connectivity and cortisol were not significant; however, contextual encoding bias explained depressive symptoms through negatively valenced OGM. ConclusionsNegative stimuli appearing in an event might impair memory encoding of the visuoperceptual context under attentional interference, represented as weakened amygdala-vmPFC connectivity implicated in emotion-related attentional dysregulation. Conversely, negative stimuli might enhance temporally proximal visuoperceptual encoding after their disappearance. Contextual encoding bias could explain the overgeneralization (or lower coherence) of autobiographical memory and increase the risk of depression. The possible role of cortisol in recollecting the context of emotional events over time warrants further investigation.

Ventral hippocampal interneurons govern extinction and relapse of contextual associations

Contextual memories are critical for survival but must be extinguished when new conditions render them nonproductive. By most accounts, extinction forms a new memory that competes with the original association for control over behavior, but the underlying circuit mechanisms remain largely enigmatic. Here, we demonstrate that extinction of contextual fear conditioning recruits somatostatin interneurons (SST-INs) in the ventral hippocampus. Correspondingly, real-time activity of SST-INs correlates with transitions between immobility and movement, signaling exit from defensive freezing bouts. Optogenetic manipulation of SST-INs but not parvalbumin interneurons (PV-INs) elicits bidirectional changes in freezing that are specific to the context in which extinction was acquired. Finally, similar effects were obtained following extinction of sucrose-based appetitive conditioning, in which SST-IN inhibition triggers relapse to reward seeking. These data suggest that ventral hippocampal SST-INs play a fundamental role in extinction that is independent of affective valence and may be related to their disruption of spontaneous emotional responses.

Brain imaging and machine learning reveal uncoupled functional network for contextual threat memory in long sepsis.

Positron emission tomography (PET) is a highly sensitive tool for studying physiology and metabolism through positron-emitting radionuclides that label molecular targets in the body with unparalleled specificity, without disturbing their biological function. Here, we introduce a small-animal technique called behavioral task-associated PET (beta-PET) consisting of two scans: the first after a mouse is familiarized with a conditioning chamber, and the second upon recall of contextual threat. Associative threat conditioning occurs between the scans. Beta-PET focuses on brain regions encoding threat memory (e.g., amygdala, prefrontal cortex) and contextual aspects (e.g., hippocampus, subiculum, entorhinal cortex). Our results show that beta-PET identifies a biologically defined functional network encoding contextual threat memory and its uncoupling in a mouse model of long sepsis. Moreover, machine learning algorithms (linear logistic regression) and ordinal trends analysis demonstrate that beta-PET robustly predicts the behavioral defense response and its breakdown during long sepsis.

A Visualized Exploration of Educational Robotics Over Five Decades

With the swift development of information technologies, educational robotics has been widely used in education, generally leading to improved educational outcomes. While educational robotics has gained popularity and achieved success in education, few researchers have conducted bibliographic analyses in this field. This study complements this missing link by visualizing the top ten authors, organizations, countries, keywords, authors, and sources via VOSviewer. The clustering techniques in CitNetExplorer were used to cluster the publications, where citation networks and qualitative analyses were developed to discuss the topics including the learning outcomes, social and interactive skills, working memory, and higher-order skills in the educational robotics-based context. This study concludes that effective educational robotics can exert a positive influence on the learning outcomes and robotics can improve the development of proximal zones in human brains, conducive to the learning outcomes. Future researchers may focus their attention on robotic competitions, affective features, and evaluative and analytical frameworks to improve educational robotics-based educational effectiveness.

The Phenomenon of Collective Memory in the Semantic Contexts of Post-Modernity

The article is devoted to the study of the phenomenon of collective memory in the value contexts of post-modernity. During the 20th century, the culture of memory is revealed through the individual-collective polarity, and in the postmodern era attention is focused on the boundary between them, on the culture of memory in the dimension of the modern media, on the contexts of the global information war. While individual memory loses touch with the past in the dimension of simulated identity, collective memory is usually associated with tradition as a reservoir of memory of the past. Collective memory in the age of modern media is in certain danger of targeted negative external influence, falsification, and inflation. The deepest cultural fears of the 20th century are the deformation or loss of memory, as well as the fear of memory substitution. The main value is linking memory with authenticity in the reproduction of the basic narrative in the cultural practices of today, in which the individual intersects with the collective. That is why the article pays special attention to the border between individual and collective memory, the culture of recall in communicative projects, and the reflection of moral dilemmas in different models of historical memory, the confrontation of different memories.

Colonialismo italiano e questione meridionale: amnesie, memorie competitive e alleanze intersezionali ai margini di una comunità immaginata bianca1

This article examines the intertwined histories of “internal colonialism” in Southern Italy and Italian colonialism in Africa, addressing the question of Italy’s national amnesia in relation to these topics. Drawing on Michael Rothberg’s “multidirectional memory” framework, the essay explores how the two historical issues mentioned above risk becoming competitive memories in contemporary public history debates. To develop this argument, the essay presents two case studies. The first is Pino Aprile’s Contro l’oblio. Giorno della Memoria per le vittime meridionali dell’Unità d’Italia. The book exemplifies competitive memory in the broader context of the neo-Bourbon approach to the Southern Question. The literal use of concepts such as colonialism and racism as metaphors for the Southern Italian condition risks creating self-apologetic narratives driven by “white anxiety,” absolving Southern Italians from their historical involvement in colonialism and contemporary racial privileges. The second case study examines the Museo Cesare Lombroso in Turin as an archive of Italian antiblackness. This section proposes the museum as a potential site for multidirectional memories, focusing on the interplay between the racialized representation of Southern Italians and Italian colonial racism. The goal is to articulate these two memories without overlapping them, emphasizing different historical responsibilities and contemporary privileges. The article underscores why intersectionality and positionality are critical to analyze the complexities of racial and colonial histories and their relevance to contemporary social justice struggles.

Associative memory in alcohol-related contexts: An fMRI study with young binge drinkers.

Alcohol-related cues are known to influence craving levels, a hallmark of alcohol misuse. Binge drinking (BD), a pattern of heavy alcohol use, has been associated with cognitive and neurofunctional alterations, including alcohol attentional bias, memory impairments, as well as disrupted activity in prefrontal- and reward-related regions. However, literature is yet to explore how memories associated with alcohol-related cues are processed by BDs, and how the recall of this information may influence their reward processing. The present functional magnetic resonance imaging (fMRI) study aimed to investigate the neurofunctional signatures of BD during an associative memory task. In all, 36 university students, 20 BDs and 16 alcohol abstainers, were asked to memorize neutral objects paired with either alcohol or non-alcohol-related contexts. Subsequently, neutral stimuli were presented, and participants were asked to classify them as being previously paired with alcohol- or non-alcohol-related contexts. While behavioral performance was similar in both groups, during the recall of alcohol-related cues, BDs showed increased brain activation in two clusters including the thalamus, globus pallidus and dorsal striatum, and cerebellum and occipital fusiform gyrus, respectively. These findings suggest that BDs display augmented brain activity in areas responsible for mental imagery and reward processing when trying to recall alcohol-related cues, which might ultimately contribute to alcohol craving, even without being directly exposed to an alcohol-related context. These results highlight the importance of considering how alcohol-related contexts may influence alcohol-seeking behavior and, consequently, the maintenance or increase in alcohol use.

Using a conditioned place preference assay in fruit flies to examine effects of insecticidal compounds on contextual memory

Insecticides are vital for safeguarding agricultural crops against pests, albeit many lack selectivity towards pest species and are poorly bio-degradable. This leads to targeting of beneficial organisms like pollinators and widespread environmental contamination of soil and water. Exposure to insecticides such as neonicotinoids causes insect paralysis and mortality at higher doses, while sublethal doses can disrupt other functions that are crucial for survival such as learning and memory performance. Potent and selective arachnid venom peptides affecting a variety of molecular targets are being explored as bioinsecticide candidates. However, their effect on insect learning is poorly understood. We therefore established a sucrose-induced conditioned place preference (CPP) assay using Drosophila melanogaster fruit flies to provide a means of evaluating how various classes of insecticidal compounds interact with insect memory to assess their broader ecological consequences. Our results confirmed the adverse effect of a sublethal dose of the neonicotinoid insecticide imidacloprid (20 pg/fly) on fly CPP formation upon daily injection during the conditioning phase. However, imidacloprid did not affect CPP retrieval when applied after the conditioning phase. Sublethal doses of the two insecticidal spider venom peptides μ-DGTX-Dc1a (Dc1a; 70 pg/fly) and U1-AGTX-Ta1a (Ta1a; 125 pg/fly) had no effect on either CPP formation or retrieval, underlining their potential as novel and safe bioinsecticide candidates.

Archaeology of Historic Football Grounds: A Case Study of the 19 May Stadium in Turkey

In Turkey, being young and healthy was idealized as the politics of the state that had successfully ruptured the link with the Ottoman Empire, thereby making sport a significant component of culture and modern life in Turkey. One of the most important football grounds was the 19 May Stadium, which was the first football ground in Turkey and reflected the political and sports culture of the time. Yet, 19 May Stadium is not perceived to be part of the country’s heritage equation; it has meaning for individuals of all ages, and it contributes significantly to the overall quality of life. Accordingly, this study makes an archaeological examination of 19 May Stadium as a form of material culture. It examines the evolving field of community archaeology projects and how they contribute to the importance of place, memory, and meaning in the historical context of the site, thereby making them more visible on the historical environment agenda in Turkey.

P2X7 receptors modulate acquisition of cue fear extinction and contextual background memory generalization in male mice

The purinergic P2X7 receptors (P2X7R) are activated by adenosine triphosphate (ATP) in several brain regions, particularly those involved with emotional control and the regulation of fear-related memories. Here, we investigate the role of P2X7R in fear learning memory, specifically in the acquisition and consolidation phases of the cued fear conditioning paradigm. C57Bl/6 wildtype (WT) male mice that received a single i.p. injection of the selective P2X7R antagonist A438079 prior the conditioning session showed generalization of cued fear memory and impaired fear extinction recall in the test session, while those treated prior the extinction session exhibited a similar behavior profile accompanied by resistance in the extinction learning. However, no effects were observed when this drug was administered immediately after the conditioning, extinction, or before the test session. Our results with P2X7R knockout (P2X7 KO) mice showed a behavioral profile that mirrored the collective effects observed across all pharmacological treatment conditions. This suggests that the P2X7R KO model effectively replicates the behavioral changes induced by the pharmacological interventions, demonstrating that we have successfully isolated the role of P2X7R in the fear and extinction phases of memory. These findings highlight the role of P2X7R in the acquisition and recall of extinction memory and supports P2X7R as a promising candidate for controlling abnormal fear processing, with potential applications for stress exposure-related disorders such as post-traumatic stress disorder (PTSD).

Memory compromise at extended delays in people with new-onset epilepsy.

Memory is one of the most sensitive markers of cognitive compromise in people with new-onset epilepsy. Nonetheless, around half of these cases score within the normal range on standard memory testing. Here we explore whether memory retention at a 1-week delay reveals otherwise undetected memory compromise in such individuals, and how it relates to subjective memory complaints and mood. Using a prospective design, 38 adults with new-onset epilepsy underwent baseline memory screening via telephone using an abbreviated Rey Auditory Verbal Learning Test (RAVLT). Psychological screening occurred via online questionnaires. One week later, without forewarning, participants completed three follow-up memory tasks. Of particular focus, we explored longer-term memory performances and forgetting trajectories in those individuals (n = 23) who demonstrated normal memory performances (scores >10th percentile) at baseline (30-min delay). Outcomes were compared to 32 healthy controls, matched for age, sex, and education. As a group, people with epilepsy performed worse than controls on all memory measures, with 44 percent impaired at baseline testing. Of those unimpaired at baseline, the rate and volume of information loss over 1 week was significantly greater than for controls. Contextual memory performance at 1 week was also significantly poorer for people with epilepsy. At the individual level, the prevalence of impaired forgetting was not significantly different between patients and controls. Subjective memory complaints were not related to any objective tests but were strongly related to self-reported mood and anxiety symptoms. People with new-onset epilepsy show reduced memory at short and extended intervals. For those showing normal memory at baseline, information does appear to be forgotten more quickly than in healthy controls, though the effect is not large. The findings indicate that while extended delay memory testing is not essential in all new epilepsy cases, it could provide useful information for particular individuals. Memory problems are common in people with epilepsy shortly after seizure onset, however, many individuals still show normal memory performances on standard neuropsychological testing. Through testing memory at an extended timepoint (1 week), our study found that on average, these individuals showed a slightly quicker rate of forgetting over a 1-week period than people without a brain condition. Self-reported memory complaints in people with new epilepsy were unrelated to their actual memory skills on testing at short and long timepoints but were rather linked to lower mood and quality of life.

Memory benefits of daily-living-related contextual cueing for individuals with subjective cognitive decline and mild cognitive impairment.

We aimed to assess how daily-living-related contextual cueing (DLCC) affects memory performance in three groups: healthy older adults (HA), those with subjective cognitive decline (SCD), and mild cognitive impairment (MCI), while accounting for age and education. After gathering demographic information, participants underwent neuropsychological assessments including the Montreal Cognitive Assessment (MoCA), Everyday Cognition Scale (ECog-12), Mini Mental State Examination (MMSE), Geriatric Depression Scale (GDS), and Trail Making Test (TMT) to establish baseline characteristics. Participants were categorized into HA (N = 47), SCD (N = 54), and MCI (N = 43) groups based on MoCA and ECog-12 scores. Memory performance was evaluated through two components of the Contextual Memory Test (CMT): one with non-contextual cues and the other with daily-living-related contextual cues. Interaction effects between contextual cueing and group for immediate recall (p < .001), delayed recall (p < .001), and total recall (p < .001) were found. All recall scores were lower in the MCI group than in the other two groups in the contextual cueing, not non-contextual. The post-hoc results revealed that scores on immediate recall, delayed recall, and total recall were lower in the MCI group than in the other two groups in the contextual cueing condition but not in the non-contextual cueing one. Daily-living-related contextual cueing benefited HA, SCD, and younger-adult MCI groups more than older-adult MCI group, particularly enhancing delayed and total memory performance.

Hippocampal γCaMKII dopaminylation promotes synaptic-to-nuclear signaling and memory formation.

Protein monoaminylation is a class of posttranslational modification (PTM) that contributes to transcription, physiology and behavior. While recent analyses have focused on histones as critical substrates of monoaminylation, the broader repertoire of monoaminylated proteins in brain remains unclear. Here, we report the development/implementation of a chemical probe for the bioorthogonal labeling, enrichment and proteomics-based detection of dopaminylated proteins in brain. We identified 1,557 dopaminylated proteins - many synaptic - including γCaMKII, which mediates Ca2+-dependent cellular signaling and hippocampal-dependent memory. We found that γCaMKII dopaminylation is largely synaptic and mediates synaptic-to-nuclear signaling, neuronal gene expression and intrinsic excitability, and contextual memory. These results indicate a critical role for synaptic dopaminylation in adaptive brain plasticity, and may suggest roles for these phenomena in pathologies associated with altered monoaminergic signaling.

Face naming and recollection represent key memory deficits in developmental prosopagnosia

Previous studies have found that face perception deficits do not fully account for the severity of face recognition deficits in developmental prosopagnosia (DP). Researchers have begun identifying deficient memory mechanisms such as impaired face recollection, but these findings require replication, and further characterization of additional memory deficits is necessary. Our goals were to replicate prior findings of face recollection impairment in DP and extend these findings to assess different types of face associative memory. We had 69 DPs and 99 controls perform a face perception battery as well as three face memory tasks: 1) Old/New task with confidence ratings to calculate recollection and familiarity using ROC analysis, 2) Face/Scene task to examine remember-know judgments and contextual memory for faces, and 3) Face-Name/Occupation task to assess the ability to learn semantic associations with faces. Compared to controls, DPs showed poorer recollection and familiarity across both Old/New and Face/Scene tasks as well as reduced scene accuracy for correct faces. Of these differences, only Old/New recollection remained significant after controlling for group differences in face perception abilities. In the Face-Name/Occupation task, after controlling for face perception, DPs showed poorer recall of names than controls but performed similarly in recalling occupations. Finally, we found that DPs with major, mild, and no face perception deficits showed consistent impairments in Old/New recollection and face-naming, and larger perceptual deficits were associated with larger memory deficits. Together, these results provide several mechanistic insights into the nature of memory deficits in DPs and have diagnostic and treatment implications.