Brain imaging and neuropsychological assessment of individuals recovered from a mild to moderate SARS-CoV-2 infection.

As severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infections have been shown to affect the central nervous system, the investigation of associated alterations of brain structure and neuropsychological sequelae is crucial to help address future health care needs. Therefore, we performed a comprehensive neuroimaging and neuropsychological assessment of 223 nonvaccinated individuals recovered from a mild to moderate SARS-CoV-2 infection (100 female/123 male, age [years], mean ± SD, 55.54 ± 7.07; median 9.7 mo after infection) in comparison with 223 matched controls (93 female/130 male, 55.74 ± 6.60) within the framework of the Hamburg City Health Study. Primary study outcomes were advanced diffusion MRI measures of white matter microstructure, cortical thickness, white matter hyperintensity load, and neuropsychological test scores. Among all 11 MRI markers tested, significant differences were found in global measures of mean diffusivity (MD) and extracellular free water which were elevated in the white matter of post-SARS-CoV-2 individuals compared to matched controls (free water: 0.148 ± 0.018 vs. 0.142 ± 0.017, P < 0.001; MD [10-3 mm2/s]: 0.747 ± 0.021 vs. 0.740 ± 0.020, P < 0.001). Group classification accuracy based on diffusion imaging markers was up to 80%. Neuropsychological test scores did not significantly differ between groups. Collectively, our findings suggest that subtle changes in white matter extracellular water content last beyond the acute infection with SARS-CoV-2. However, in our sample, a mild to moderate SARS-CoV-2 infection was not associated with neuropsychological deficits, significant changes in cortical structure, or vascular lesions several months after recovery. External validation of our findings and longitudinal follow-up investigations are needed.

Linking Cognitive Integrity to Working Memory Dynamics in the Aging Human Brain.

Aging is accompanied by a decline of working memory, an important cognitive capacity that involves stimulus-selective neural activity that persists after stimulus presentation. Here, we unraveled working memory dynamics in older human adults (male and female) including those diagnosed with mild cognitive impairment (MCI) using a combination of behavioral modeling, neuropsychological assessment, and MEG recordings of brain activity. Younger adults (male and female) were studied with behavioral modeling only. Participants performed a visuospatial delayed match-to-sample task under systematic manipulation of the delay and distance between sample and test stimuli. Their behavior (match/nonmatch decisions) was fit with a computational model permitting the dissociation of noise in the internal operations underlying the working memory performance from a strategic decision threshold. Task accuracy decreased with delay duration and sample/test proximity. When sample/test distances were small, older adults committed more false alarms than younger adults. The computational model explained the participants' behavior well. The model parameters reflecting internal noise (not decision threshold) correlated with the precision of stimulus-selective cortical activity measured with MEG during the delay interval. The model uncovered an increase specifically in working memory noise in older compared with younger participants. Furthermore, in the MCI group, but not in the older healthy controls, internal noise correlated with the participants' clinically assessed cognitive integrity. Our results are consistent with the idea that the stability of working memory contents deteriorates in aging, in a manner that is specifically linked to the overall cognitive integrity of individuals diagnosed with MCI.

Neural correlates of an illusionary sense of agency caused by virtual reality.

Sense of agency (SoA) is the sensation that self-actions lead to ensuing perceptual consequences. The prospective mechanism emphasizes that SoA arises from motor prediction and its comparison with actual action outcomes, while the reconstructive mechanism stresses that SoA emerges from retrospective causal processing about the action outcomes. Consistent with the prospective mechanism, motor planning regions were identified by neuroimaging studies using the temporal binding (TB) effect, a behavioral measure often linked to implicit SoA. Yet, TB also occurs during passive observation of another's action, lending support to the reconstructive mechanism, but its neural correlates remain unexplored. Here, we employed virtual reality (VR) to modulate such observation-based SoA and examined it with functional magnetic resonance imaging (fMRI). After manipulating an avatar hand in VR, participants passively observed an avatar's "action" and showed a significant increase in TB. The binding effect was associated with the right angular gyrus and inferior parietal lobule, which are critical nodes for inferential and agency processing. These results suggest that the experience of controlling an avatar may potentiate inferential processing within the right inferior parietal cortex and give rise to the illusionary SoA without voluntary action.

How emotional contexts modulate item memory in individuals with high and low negative affect and worry.

Emotional stimuli are usually remembered with high confidence. Yet, it remains unknown whether-in addition to memory for the emotional stimulus itself-memory for a neutral stimulus encountered just after an emotional one can be enhanced. Further, little is known about the interplay between emotion elicited by a stimulus and emotion relating to affective dispositions. To address these questions, we examined (1) how emotional valence and arousal of a context image preceding a neutral item image affect memory of the item, and (2) how such memory modulation is affected by two hallmark features of emotional disorders: trait negative affect and tendency to worry. In two experiments, participants encoded a series of trials in which an emotional (negative, neutral, or positive) context image was followed by a neutral item image. In experiment 1 (n = 42), items presented seconds after negative context images were remembered better and with greater confidence compared to those presented after neutral and positive ones. Arousal ratings of negative context images were higher compared to neutral and positive ones and the likelihood of correctly recognizing an item image was related to higher arousal of the context image. In experiment 2 (n = 59), better item memory was related to lower trait negative affect. Participants with lower trait negative affect or tendency to worry displayed higher confidence compared to those with high negative affect or tendency to worry. Our findings describe an emotional "carry-over" effect elicited by a context image that enhances subsequent item memory on a trial-by-trial basis, however, not in individuals with high trait negative affect who seem to have a general memory disadvantage.

Is Short Sleep Bad for the Brain? Brain Structure and Cognitive Function in Short Sleepers.

Many sleep less than recommended without experiencing daytime sleepiness. According to prevailing views, short sleep increases risk of lower brain health and cognitive function. Chronic mild sleep deprivation could cause undetected sleep debt, negatively affecting cognitive function and brain health. However, it is possible that some have less sleep need and are more resistant to negative effects of sleep loss. We investigated this using a cross-sectional and longitudinal sample of 47,029 participants of both sexes (20-89 years) from the Lifebrain consortium, Human Connectome project (HCP) and UK Biobank (UKB), with measures of self-reported sleep, including 51,295 MRIs of the brain and cognitive tests. A total of 740 participants who reported to sleep <6 h did not experience daytime sleepiness or sleep problems/disturbances interfering with falling or staying asleep. These short sleepers showed significantly larger regional brain volumes than both short sleepers with daytime sleepiness and sleep problems (n = 1742) and participants sleeping the recommended 7-8 h (n = 3886). However, both groups of short sleepers showed slightly lower general cognitive function (GCA), 0.16 and 0.19 SDs, respectively. Analyses using accelerometer-estimated sleep duration confirmed the findings, and the associations remained after controlling for body mass index, depression symptoms, income, and education. The results suggest that some people can cope with less sleep without obvious negative associations with brain morphometry and that sleepiness and sleep problems may be more related to brain structural differences than duration. However, the slightly lower performance on tests of general cognitive abilities warrants closer examination in natural settings.SIGNIFICANCE STATEMENT Short habitual sleep is prevalent, with unknown consequences for brain health and cognitive performance. Here, we show that daytime sleepiness and sleep problems are more strongly related to regional brain volumes than sleep duration. However, participants sleeping ≤6 h had slightly lower scores on tests of general cognitive function (GCA). This indicates that sleep need is individual and that sleep duration per se is very weakly if at all related brain health, while daytime sleepiness and sleep problems may show somewhat stronger associations. The association between habitual short sleep and lower scores on tests of general cognitive abilities must be further scrutinized in natural settings.

Digital nature: Unveiling the impact and safety of FlowVR group intervention for depression in a feasibility trial.

OBJECTIVE: This study addresses the limitations of existing interventions for depression, such as a deficit-oriented focus, overlooking the utilization of positive elements such as nature, and neglecting the incorporation of group effects. The present feasibility study examines FlowVR, a resource-oriented, nature-inspired virtual reality (VR)-based group therapy. Previously tested individually in a pilot study for non-clinical participants, FlowVR has demonstrated positive effects on depressive symptoms. This study assesses the impact and safety of FlowVR in a group setting within a clinical sample using a one-armed study design. METHOD: Forty-two inpatients and day patients with depression were recruited. Before and after the FlowVR intervention period of 4 weeks (two sessions per week), depressive symptoms were assessed (Beck Depression Inventory-II; BDI). Symptomatology (i.e., depressive symptoms), depression-associated variables (i.e., self-efficacy), intervention-specific variables (feeling of flow), and VR-specific variables (simulator sickness) were assessed before and after each session. RESULTS: Linear mixed effect models showed that symptomatology (depression, negative affect, current anxiety), depression-associated constructs (self-efficacy, motivation), and intervention-specific variables (flow) improved over the course of the sessions. No variable deteriorated more in one session compared to any other session. The lasso regression identified five potential predictors for the change in depression (BDI-II), yet these could not be validated in a subsequent linear regression analysis. CONCLUSION: To conclude, FlowVR had the hypothesized positive impact over the course of the sessions, showing, for example, improvements in symptomatology. The sessions have demonstrated safety with no notable deteriorations. Therefore, FlowVR is deemed safe for clinical patients and group settings. However, further research is needed to explore predictors for the change in depression.

Individual differences in brain aging: heterogeneity in cortico-hippocampal but not caudate atrophy rates.

It is well documented that some brain regions, such as association cortices, caudate, and hippocampus, are particularly prone to age-related atrophy, but it has been hypothesized that there are individual differences in atrophy profiles. Here, we document heterogeneity in regional-atrophy patterns using latent-profile analysis of 1,482 longitudinal magnetic resonance imaging observations. The results supported a 2-group solution reflecting differences in atrophy rates in cortical regions and hippocampus along with comparable caudate atrophy. The higher-atrophy group had the most marked atrophy in hippocampus and also lower episodic memory, and their normal caudate atrophy rate was accompanied by larger baseline volumes. Our findings support and refine models of heterogeneity in brain aging and suggest distinct mechanisms of atrophy in striatal versus hippocampal-cortical systems.

Can a nature walk change your brain? Investigating hippocampal brain plasticity after one hour in a forest.

In cities, the incidence of mental disorders is higher, while visits to nature have been reported to benefit mental health and brain function. However, there is a lack of knowledge about how exposure to natural and urban environments affects brain structure. To explore the causal relationship between exposure to these environments and the hippocampal formation, 60 participants were sent on a one hour walk in either a natural (forest) or an urban environment (busy street), and high-resolution hippocampal imaging was performed before and after the walks. We found that the participants who walked in the forest had an increase in subiculum volume, a hippocampal subfield involved in stress response inhibition, while no change was observed after the urban walk. However, this result did not withstand Bonferroni correction for multiple comparisons. Furthermore, the increase in subiculum volume after the forest walk was associated with a decrease in self-reported rumination. These results indicate that visits to nature can lead to observable alterations in brain structure, with potential benefits for mental health and implications for public health and urban planning policies.

Blue skies: Does the visual composition of sky guide subjective judgments of naturalness in the environment?

Expanding on previous findings, that highlighted the significance of sky in environmental perception, our analysis investigated whether the visual composition of the sky shapes perceptions of environmental naturalness. The study employed a novel, free-selection task in which participants viewed a series of environmental images with varying levels of natural and urban elements, as well as different sky visibility conditions, and were asked to identify "nature" within these images. The task procedure also involved subjective ratings of each scene. Using previously gathered data, we reassessed 105 participants' selection of the sky as "nature" across 96 photos of diverse outdoor scenes to understand which visuospatial features influence these perceptions. Utilizing the Boruta feature selection algorithm, we identified key characteristics-fractal dimensions, brightness, and entropy in brightness, hue, and saturation-that significantly predicted the selection of sky as "nature", irrespective of the environment type (urban or natural). Results indicated that lower fractal dimensions are preferred for sky selected as "nature", inversely affecting the naturalness judgment of scenes with the additional effect of brightness. These findings enhance our understanding of how visuospatial features influence environmental perception, offering implications for future research directions and theoretical advancements in understanding environmental perception.

Impact of exposure to natural versus urban soundscapes on brain functional connectivity, BOLD entropy and behavior.

BACKGROUND: Humans have been moving from rural to urban environments for decades. This process may have important consequences for our health and well-being. Most previous studies have focused on visual input, and the auditory domain has been understudied so far. Therefore, we set out to investigate the influence of exposure to natural vs urban soundscapes on brain activity and behavior. METHODS: Resting-state fMRI data was acquired while participants (N = 35) listened to natural and urban soundscapes. Two affective questionnaires (the Positive and Negative Affect Schedule (PANAS) and the Perceived Stress Scale) and two cognitive tasks (dual n-back (DNB) and the backward digit-span (BDS)) were assessed before and after each soundscape condition. To quantify brain function we used complexity and network measures, namely brain entropy (BEN) and whole brain functional connectivity (FC). To study the link between brain and behavior, changes in BEN and whole brain FC were correlated to changes in cognitive performance and self-reported affect. RESULTS: We found higher BEN when listening to urban sounds in posterior cingulate gyrus, cuneus and precuneus, occipital lobe/calcarine as compared to nature sounds, which was negatively correlated to (post-pre) differences in positive affect (PANAS) in the urban soundscape condition. In addition, we found higher FC between areas in the auditory, cinguloopercular, somatomotor hand and mouth networks when listening to nature as compared to urban sounds which was positively correlated to (post-pre) differences of the of the composite score of Digit span and N-back for nature soundscape. CONCLUSIONS: This study provides a framework for the neural underpinnings of how natural versus urban soundscapes affect both whole brain FC and BEN and bear implications for the understanding of how the physical auditory environment affects brain function and subsequently observed behavior. Moreover, correlations with cognition and affect reveal the meaning that exposure to soundscapes may have on the human brain. To the best of our knowledge this is the first study to analyze BEN and whole brain FC at rest during exposure to nature and urban soundscapes and to explore their relationship to behavior.

Are we afraid of the woods? - An investigation of the implicit and explicit fear reactions to forests.

The beneficial effects of nature exposure have been repeatedly documented and encourage frequent and regular contact with nature and especially highlight forests. However, in human history, forests have also been associated with negative emotions such as fear and were seen as dangerous environments. While existing literature could demonstrate that natural environments can evoke fear, the focus was on the explicit perception. Given that research has shown the significance of additional implicit processes in fear-related behaviour, we aim to explore the presence of an implicit fear response to forests. Therefore, in an online study, we investigated the explicit and implicit fear reactions to forests by a Northern German sample of N = 256. Using three explicit measurements, we investigated fear and danger perception on a semantic and visual level of the stimulus category "forest" compared to the human-made urban green space "park" and the urban setting "house". Additionally, we assessed the unconscious response tendencies towards the forest within three implicit tasks: Subliminal Priming Procedure (SPP), Affect Misattribution Procedure (AMP) and Approach-Avoidance Task (AAT). Within the analyzed sample, the subliminally presented word forest evoked a stronger positive valence response compared to park (SPP). In contrast to houses, the forest showed a stronger approach and weaker avoidance tendency (AAT). At the same time, both the three explicit and one implicit measurement (AMP) showed a stronger fear perception of forests compared to parks or houses. Considering the increasingly utilised beneficial effects of nature in interventions, these findings should be acknowledged when implementing nature exposure in interventions and treatments.

Deficits in memory metacognitive efficiency in late adulthood are related to distinct brain profile.

The tendency of falsely remembering events that did not happen in the past increases with age. This is particularly evident in cases in which features presented at study are re-presented at test in a recombined constellation (termed rearranged pairs). Interestingly, older adults also express high confidence in such false memories, a tendency that may indicate reduced metacognitive efficiency. Within an existing cohort study, we aimed at investigating age-related differences in memory metacognitive efficiency (as measured by meta d' ratio) in a sample of 1522 older adults and 397 young adults. The analysis showed an age-related deficit in metacognition which was more pronounced for rearranged pairs than for new pairs. We then explored associations between cortical thickness and memory metacognitive efficiency for rearranged pairs in a subsample of 231 older adults. By using partial least square analysis, we found that a multivariate profile composed by ventromedial prefrontal cortex, insula, and parahippocampal cortex was uniquely associated with between-person differences in memory metacognitive efficiency. These results suggest that the impairment in memory metacognitive efficiency for false alarms is a distinct age-related deficit, above and beyond a general age-related decline in memory discrimination, and that it is associated with brain regions involved in metacognitive processes.

Educational attainment does not influence brain aging.

Education has been related to various advantageous lifetime outcomes. Here, using longitudinal structural MRI data (4,422 observations), we tested the influential hypothesis that higher education translates into slower rates of brain aging. Cross-sectionally, education was modestly associated with regional cortical volume. However, despite marked mean atrophy in the cortex and hippocampus, education did not influence rates of change. The results were replicated across two independent samples. Our findings challenge the view that higher education slows brain aging.

Subjective evaluation of home environment and levels of self-reported depression in middle to old age: Results from the HCHS study.

OBJECTIVES: The immediate living environment might, like other lifestyle factors, be significantly related to mental well-being. The current study addresses the question whether five relevant subjective home environment variables (i.e., protection from disturbing nightlight, daylight entering the home, safety at home, quality of window views, and noise disturbance) are associated with levels of self-reported depression over and above well-known sociodemographic and common lifestyle variables. METHODS: Data from the Hamburg City Health Study (HCHS) were analyzed. In N = 8757 with available PHQ-9 depression data, multiple linear regression models were computed, with demographic data, lifestyle variables, and variables describing the subjective evaluation of the home environment. RESULTS: The model explained 15% of variance in depression levels, with ratings for the subjective evaluation of home environment accounting for 6%. Better protection from disturbing light at night, more daylight entering the home, feeling safer, and perceived quality of the window views, were all significantly associated with lower, while more annoyance by noise was associated with higher levels of self-reported depression. Results did not differ if examining a sample of the youngest (middle-aged participants: 46-50 years) versus oldest (70-78 years) participants within HCHS. CONCLUSION: Beyond studying the role of lifestyle factors related to self-reported depression, people's homes may be important for subclinical levels of depression in middle and older age, albeit the direction of effects or causality cannot be inferred from the present study. The development of a consensus and tools for a standardized home environment assessment is needed.

Do we really need ligands in Ir-catalyzed C-H borylation?

Direct borylation of C-H bonds is a privileged strategy to access versatile building blocks and valuable derivatives of complex molecules (late-stage functionalization, metabolite synthesis). This perspective aims to provide an overview and classification of the catalytic systems developed in this fast-growing area of research. Unexpected selectivity differences between two established directed-borylation systems have been discovered using high-throughput experimentation highlighting the importance of classical control experiments in catalysis research.

Randomized Controlled Trial on Imaginal Retraining for Problematic Alcohol Use: A Dismantling Study.

Imaginal retraining (IR) is an approach-avoidance procedure that has shown promising results in previous studies. The aim of the present study was to dismantle the efficacy of IR's components in a randomized controlled trial (RCT). We conducted a RCT with nine conditions comprising eight intervention groups and a waitlist control group (WLC). Alcohol craving (primary outcome), consumption, depressive symptoms, quality of life, subjective appraisal, and side effects were assessed online at baseline, post intervention (6 weeks), and follow-up (12 weeks). The sample consisted of 426 participants (age: M = 47.22, SD = 11.82, women: 50.5%). The intervention groups received instructions for four different components of IR (mood induction, mental avoidance of unhealthy stimuli, motor avoidance of unhealthy stimuli, approach to healthy stimuli) that were each conveyed with or without prior psychoeducation (PE). The intervention was delivered online. At total of 163 individuals (42.9%) used the intervention at least once. No group differences were found for any primary or secondary outcome after Sidák correction. Uncorrected statistics showed effects of significantly decreased alcohol consumption for the approach + PE group in the intention-to-treat and the merged motor avoidance group in the per-protocol analyses at post assessment compared with the WLC. Exploratory moderation analyses revealed that individuals with high visualization skills benefited most. The authors conclude that visualization training and motivational components may increase the efficacy and adherence of IR.

How nature nurtures: Amygdala activity decreases as the result of a one-hour walk in nature.

Since living in cities is associated with an increased risk for mental disorders such as anxiety disorders, depression, and schizophrenia, it is essential to understand how exposure to urban and natural environments affects mental health and the brain. It has been shown that the amygdala is more activated during a stress task in urban compared to rural dwellers. However, no study so far has examined the causal effects of natural and urban environments on stress-related brain mechanisms. To address this question, we conducted an intervention study to investigate changes in stress-related brain regions as an effect of a one-hour walk in an urban (busy street) vs. natural environment (forest). Brain activation was measured in 63 healthy participants, before and after the walk, using a fearful faces task and a social stress task. Our findings reveal that amygdala activation decreases after the walk in nature, whereas it remains stable after the walk in an urban environment. These results suggest that going for a walk in nature can have salutogenic effects on stress-related brain regions, and consequently, it may act as a preventive measure against mental strain and potentially disease. Given rapidly increasing urbanization, the present results may influence urban planning to create more accessible green areas and to adapt urban environments in a way that will be beneficial for citizens' mental health.

Can you trust this source? Advice taking in borderline personality disorder.

Research suggests that patients with borderline personality disorder (BPD) share a range of cognitive biases with patients with psychosis. As the disorder often manifests in dysfunctional social interactions, we assumed associated reasoning styles would be exaggerated in a social setting. For the present study, we applied the Judge-Advisor System by asking participants to provide initial estimates of a person's age and presumed hostility based on a portrait photo. Afterwards, we presented additional cues/advice in the form of responses by anonymous previous respondents. Participants could revise their estimate, seek additional advice, or make a decision. Contrary to our preregistered hypothesis, patients with BPD (n = 38) performed similarly to healthy controls (n = 30). Patients sought the same number of pieces of advice, were equally confident, and used advice in similar ways to revise their estimates. Thus, patients with BPD did trust advice. However, patients gave higher hostility ratings to the portrayed persons. In conclusion, patients with BPD showed no cognitive biases in seeking, evaluating, and integrating socially provided information. While the study implies emotional rather than cognitive biases in the disorder, cognitive biases may still prove to be useful treatment targets in order to encourage delaying and reflecting on extreme emotional responses in social interactions.

Association between periodontal disease and microstructural brain alterations in the Hamburg City Health Study.

AIM: The aim of the PAROBRAIN study was to examine the association of periodontal health with microstructural white matter integrity and cerebral small vessel disease (CSVD) in the Hamburg City Health Study, a large population-based cohort with dental examination and brain magnetic resonance imaging (MRI). MATERIALS AND METHODS: Periodontal health was determined by measuring clinical attachment loss (CAL) and plaque index. Additionally, the decayed/missing/filled teeth (DMFT) index was quantified. 3D-FLAIR and 3D-T1-weighted images were used for white matter hyperintensity (WMH) segmentation. Diffusion-weighted MRI was used to quantify peak width of skeletonized mean diffusivity (PSMD). RESULTS: Data from 2030 participants were included in the analysis. Median age was 65 years, with 43% female participants. After adjusting for age and sex, an increase in WMH load was significantly associated with more CAL, higher plaque index and higher DMFT index. PSMD was significantly associated with the plaque index and DMFT. Additional adjustment for education and cardiovascular risk factors revealed a significant association of PSMD with plaque index (p < .001) and DMFT (p < .01), whereas effects of WMH load were attenuated (p > .05). CONCLUSIONS: These findings suggest an adverse effect of periodontal health on CSVD and white matter integrity. Further research is necessary to examine whether early treatment of periodontal disease can prevent microstructural brain damage.

Combination of Two Behavioral Techniques Reduces Craving in Problematic Alcohol Consumption by One Third: A Randomized Controlled Trial.

INTRODUCTION: Craving alcohol is a core symptom of alcohol use disorder (AUD) and an important target for treatment. A new line of treatment for AUD aims at overriding the urge to consume alcohol by changing implicit cognitions via approach bias modification (ApBM). In a prior study, we tested a variant of ApBM called imaginal retraining, which reduced craving. As addiction and body-focused repetitive behaviors (BFRBs) share important symptoms (e.g., inability to resist urges), for the present study we merged imaginal retraining with a technique aimed at BFRB, called decoupling, to augment treatment effects. We hypothesized that the new technique, which is called 3P, would lead to a greater reduction in craving relative to (active) control conditions. METHODS: The study was conducted online. Data from 227 participants were considered. Participants were randomized to 1 out of 5 conditions. Craving for alcohol before and after the brief intervention was the primary outcome. RESULTS: Only the 3P condition lessened craving by approximately one third at an almost medium effect size (improvement: 34.5%, p = 0.003, d = 0.458). Effects were significantly larger relative to the wait-list control and two active control conditions (p's < 0.02; greater reduction than imaginal retraining at a small but nonsignificant effect size). DISCUSSION/CONCLUSION: If replicated, the combination of imaginal retraining and decoupling (3P) represents a promising, easy-to-implement self-help technique to reduce immediate craving. Long-term effects in participants with formally diagnosed AUD have not yet been investigated.