Multi-view confocal microscopy enables multiple organ and whole organism live-imaging.

Understanding how development is coordinated in multiple tissues and gives rise to fully functional organs or whole organisms necessitates microscopy tools. Over the last decade numerous advances have been made in live-imaging, enabling high resolution imaging of whole organisms at cellular resolution. Yet, these advances mainly rely on mounting the specimen in agarose or aqueous solutions, precluding imaging of organisms whose oxygen uptake depends on ventilation. Here, we implemented a multi-view multi-scale microscopy strategy based on confocal spinning disk microscopy, called Multi-View confocal microScopy (MuViScopy). MuViScopy enables live-imaging of multiple organs with cellular resolution using sample rotation and confocal imaging without the need of sample embedding. We illustrate the capacity of MuViScopy by live-imaging Drosophila melanogaster pupal development throughout metamorphosis, highlighting how internal organs are formed and multiple organ development is coordinated. We foresee that MuViScopy will open the path to better understand developmental processes at the whole organism scale in living systems that require gas exchange by ventilation.

Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.

Does attentional suppression occur at the level of perception or decision-making? Evidence from Gaspelin et al.'s (2015) probe letter task.

Visual attention is often inadvertently captured by salient stimuli. It was suggested that it is possible to prevent attentional capture in some search tasks by suppressing salient stimuli below baseline. Evidence for attentional suppression comes from a probe task that was interleaved with the main search task. In the probe task of Gaspelin et al. (Psychol Sci 26(11):1740-1750, 2015. https://doi.org/10.1177/0956797615597913 ), letters were shown on the stimuli of the search display and participants had to identify as many letters as possible. Performance was found to be worse for letters shown on the distractor compared to non-salient non-target stimuli, suggesting that distractor processing was suppressed below baseline. However, it is unclear whether suppression occurred at the level of perception or decision-making because participants may have reported letters on the distractor less frequently than letters on nontargets. This decision-level bias may have degraded performance for letters on distractor compared to nontarget stimuli without changing perception. After replicating the original findings, we conducted two experiments where we avoided report bias by cueing only a single letter for report. We found that the difference between distractor and nontarget stimuli was strongly reduced, suggesting that decision-level processes contribute to attentional suppression. In contrast, the difference between target and non-target stimuli was unchanged, suggesting that it reflected perceptual-level enhancement of the target stimuli.

The cluster depth tests: Toward point-wise strong control of the family-wise error rate in massively univariate tests with application to M/EEG.

The cluster mass test has been widely used for massively univariate tests in M/EEG, fMRI and, recently, pupillometry analysis. It is a powerful method for detecting effects while controlling weakly the family-wise error rate (FWER), although its correct interpretation can only be performed at the cluster level without any point-wise conclusion. It implies that the discoveries of a cluster mass test cannot be precisely localized in time or in space. We propose a new multiple comparisons procedure, the cluster depth tests, that both controls the FWER while allowing an interpretation at the time point level. We show the conditions for a strong control of the FWER, and a simulation study shows that the cluster depth tests achieve large power and guarantee the FWER even in the presence of physiologically plausible effects. By having an interpretation at the time point/voxel level, the cluster depth tests make it possible to take full advantage of the high temporal resolution of EEG recording and give a precise timing of the start and end of the significant effects.

Fetal hemoglobin rescues ineffective erythropoiesis in sickle cell disease.

While ineffective erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in the bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for ineffective erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased ineffective erythropoiesis.

Dimerization and phosphorylation of Lutheran/basal cell adhesion molecule are critical for its function in cell migration on laminin.

Tumor cell migration depends on the interactions of adhesion proteins with the extracellular matrix. Lutheran/basal cell adhesion molecule (Lu/BCAM) promotes tumor cell migration by binding to laminin α5 chain, a subunit of laminins 511 and 521. Lu/BCAM is a type I transmembrane protein with a cytoplasmic domain of 59 (Lu) or 19 (Lu(v13)) amino acids. Here, using an array of techniques, including site-directed mutagenesis, immunoblotting, FRET, and proximity-ligation assays, we show that both Lu and Lu(v13) form homodimers at the cell surface of epithelial cancer cells. We mapped two small-XXX-small motifs in the transmembrane domain as potential sites for monomers docking and identified three cysteines in the cytoplasmic domain as being critical for covalently stabilizing dimers. We further found that Lu dimerization and phosphorylation of its cytoplasmic domain were concomitantly needed to promote cell migration. We conclude that Lu is the critical isoform supporting tumor cell migration on laminin 521 and that the Lu:Lu(v13) ratio at the cell surface may control the balance between cellular firm adhesion and migration.

Permutation inference for canonical correlation analysis.

Canonical correlation analysis (CCA) has become a key tool for population neuroimaging, allowing investigation of associations between many imaging and non-imaging measurements. As age, sex and other variables are often a source of variability not of direct interest, previous work has used CCA on residuals from a model that removes these effects, then proceeded directly to permutation inference. We show that a simple permutation test, as typically used to identify significant modes of shared variation on such data adjusted for nuisance variables, produces inflated error rates. The reason is that residualisation introduces dependencies among the observations that violate the exchangeability assumption. Even in the absence of nuisance variables, however, a simple permutation test for CCA also leads to excess error rates for all canonical correlations other than the first. The reason is that a simple permutation scheme does not ignore the variability already explained by previous canonical variables. Here we propose solutions for both problems: in the case of nuisance variables, we show that transforming the residuals to a lower dimensional basis where exchangeability holds results in a valid permutation test; for more general cases, with or without nuisance variables, we propose estimating the canonical correlations in a stepwise manner, removing at each iteration the variance already explained, while dealing with different number of variables in both sides. We also discuss how to address the multiplicity of tests, proposing an admissible test that is not conservative, and provide a complete algorithm for permutation inference for CCA.

Time, frequency, and time-varying Granger-causality measures in neuroscience.

This article proposes a systematic methodological review and an objective criticism of existing methods enabling the derivation of time, frequency, and time-varying Granger-causality statistics in neuroscience. The capacity to describe the causal links between signals recorded at different brain locations during a neuroscience experiment is indeed of primary interest for neuroscientists, who often have very precise prior hypotheses about the relationships between recorded brain signals. The increasing interest and the huge number of publications related to this topic calls for this systematic review, which describes the very complex methodological aspects underlying the derivation of these statistics. In this article, we first present a general framework that allows us to review and compare Granger-causality statistics in the time domain, and the link with transfer entropy. Then, the spectral and the time-varying extensions are exposed and discussed together with their estimation and distributional properties. Although not the focus of this article, partial and conditional Granger causality, dynamical causal modelling, directed transfer function, directed coherence, partial directed coherence, and their variant are also mentioned.

Sex hormones and mental rotation: an intensive longitudinal investigation.

The present study used an intensive longitudinal design to examine whether mental rotation performance varies according to a monthly cycle in both males and females and whether these variations are related to variations in progesterone, estradiol, and testosterone levels. We collected reaction time and accuracy data for 10 males and seven females each workday over eight weeks using 136 pairs of mental rotation stimuli/day, and measured sexual hormones concentrations in the saliva twice a week. A mixed linear model statistical analysis revealed that all females and seven males showed significant cycle effects in mental rotation performance. The female cycle showed an amplitude that was twice as large compared with the amplitude found in males. For males and females, estradiol and testosterone were significantly linearly and quadratically related to interindividual variation in performance at the beginning of the study (progesterone was linearly related to performance for females). The association between testosterone and performance differed across sexes: for males, it had an inverse U-shape, for females it was U-shaped. Towards the end of the study, none of the hormones were significantly related to performance anymore. Thus, the relationship between hormones and mental rotation performance disappeared with repeated testing. Only estradiol levels were significantly elevated at the lowest point of the cycle in mental rotation performance in females. In conclusion, in this intensive longitudinal study spanning two months, a monthly cycle in mental rotation performance was found among both males and females, with a larger cycle's amplitude for females.

Contextual variation in cognitive performance of older adults: Demonstration of an age-of-examiner effect.

Objective: Consistent with research on stereotype threat, when examiners' characteristics make a stereotype of the participant group salient, it can hamper participants' performance. We hypothesized that younger examiners represent a subtle element activating age stereotypes, leading older people to perform worse as examiners' age decreases. Method: We analyzed data from the Survey of Health, Ageing, and Retirement in Europe (SHARE; NParticipants = 32768) and Vivre-Leben-Vivere studies (VLV, Nparticipants = 960), wherein older people were tested at home by examiners of different ages on eight cognitive tasks. Results: Our results indicate that participants' performance on five tasks was positively linked to examiners' age, showing that the older the examiner, the better the participants' performance. Conclusions: These findings could have implications for the current assessment of memory performance among older adults.

Relationship between reward-related brain activity and opportunities to sit.

The present study tested whether energy-minimizing behaviors evoke reward-related brain activity that promotes the repetition of these behaviors via reinforcement learning processes. Fifty-eight healthy young adults in a standing position performed a task where they could earn a reward either by sitting down or squatting while undergoing electroencephalographic (EEG) recording. Reward-prediction errors were quantified as the amplitude of the EEG-derived reward positivity. Results showed that reward positivity was larger on reward versus no reward trials, confirming the validity of our paradigm to measure evoked reward-related brain activity. However, results showed no evidence that sitting (versus standing and squatting) trials led to larger reward positivity. Moreover, we found no evidence suggesting that this effect was moderated by typical physical activity, physical activity on the day of the study, or energy expenditure during the experiment. However, at the behavioral level, results showed that the probability of choosing the stimulus more likely to lead to sitting than standing increased as the number of trials increased. In addition, results revealed that the probability of changing the selected stimulus was higher when the previous trial was a stand trial relative to a sit trial. In sum, neural results showed no evidence supporting the theory that opportunities to minimize energy expenditure are rewarding. However, behavioral findings suggested participants tend to choose the less effortful behavioral alternative and were therefore consistent with the theory of effort minimization (TEMPA).

The effect of d-cycloserine on brain processing of breathlessness over pulmonary rehabilitation: an experimental medicine study.

Research question: Pulmonary rehabilitation is the best treatment for chronic breathlessness in COPD but there remains an unmet need to improve efficacy. Pulmonary rehabilitation has strong parallels with exposure-based cognitive behavioural therapies (CBT), both clinically and in terms of brain activity patterns. The partial N-methyl-d-aspartate (NMDA)-receptor agonist d-cycloserine has shown promising results in enhancing efficacy of CBT, thus we hypothesised that it would similarly augment the effects of pulmonary rehabilitation in the brain. Positive findings would support further development in phase 3 clinical trials. Methods: 72 participants with mild-to-moderate COPD were recruited to a double-blind pre-registered (ClinicalTrials.gov identifier: NCT01985750) experimental medicine study running parallel to a pulmonary rehabilitation course. Participants were randomised to 250 mg d-cycloserine or placebo, administered immediately prior to the first four sessions of pulmonary rehabilitation. Primary outcome measures were differences between d-cycloserine and placebo in brain activity in the anterior insula, posterior insula, anterior cingulate cortices, amygdala and hippocampus following completion of pulmonary rehabilitation. Secondary outcomes included the same measures at an intermediate time point and voxel-wise difference across wider brain regions. An exploratory analysis determined the interaction with breathlessness anxiety. Results: No difference between d-cycloserine and placebo groups was observed across the primary or secondary outcome measures. d-cycloserine was shown instead to interact with changes in breathlessness anxiety to dampen reactivity to breathlessness cues. Questionnaire and measures of respiratory function showed no group difference. This is the first study testing brain-active drugs in pulmonary rehabilitation. Rigorous trial methodology and validated surrogate end-points maximised statistical power. Conclusion: Although increasing evidence supports therapeutic modulation of NMDA pathways to treat symptoms, we conclude that a phase 3 clinical trial of d-cycloserine would not be worthwhile.

Am I really seeing what's around me? An ERP study on social anxiety under speech induction, uncertainty and social feedback.

Cognitive models of social anxiety propose that socially anxious individuals engage in excessive self-focusing attention when entering a social situation. In the present study, speech anxiety was induced to socially anxious and control participants. Event-related potentials were recorded while participants performed a perceptual judgement task using distinct or ambiguous stimuli, before and after social feedback. Disputed feedback led to more revisions and decreased levels of confidence, especially among socially anxious individuals. Prior feedback, greater occipital P1 amplitudes in both groups for ambiguous probes indicated heightened sensory facilitation to ambiguous information, and greater anterior N1 amplitudes for ambiguous stimuli in highly anxious participants suggested anticipation of negative feedback in this group. Post-feedback, P1, N1 and LPP amplitudes were reduced overall among socially anxious individuals indicating a reduction in sensory facilitation of visual information. These results suggest excessive self-focusing among socially anxious individuals, possibly linked to anticipation of an anxiety-provoking social situation.

HIV-1 Nef inhibits ruffles, induces filopodia, and modulates migration of infected lymphocytes.

The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal "axial tomography," this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.

Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance.

Heterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV. SETDB1 coordinates histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.

Self-defining memories after severe traumatic brain injury: A preliminary study.

Autobiographical memory plays a major role in the (re)construction of sense of identity, a recurrent issue after a traumatic brain injury (TBI). Although the recall of specific autobiographical events is frequently impaired in patients with TBI, little is known about how these changes affect their sense of self and identity. Thus, we examined self-defining memories (SDMs), that is, the most significant personal memories supporting one's sense of identity, in 16 patients with severe TBI and in matched controls. To this end, participants recalled three SDMs and rated their emotions in response to memory retrieval. In addition, characteristics of SDMs such as specificity, meaning-making, self-connections that reflect identity stability or identity change, content (theme, presence of tension, and redemption sequences) were analysed by independent raters. The main results showed that patients' SDMs were less specific and contained fewer redemption sequences than did those of controls but did not significantly differ in thematic content, presence of tension, meaning-making, self-connections that reflect identity stability or identity change and affective responses to memory retrieval. Furthermore, among the patients' memories that refer to the TBI-related event itself, only one contained an explicit meaning. Despite the lack of specificity in memories, patients with severe TBI were able to extract meaning from personal memories although they could struggle with integrating the TBI-related event into their sense of identity. These characteristics of SDMs may contribute to disturbances in sense of self and continuity in patients with severe TBI, as well as difficulties in personal or social adjustment. These results also open up relevant prospects for psychological interventions in identity-related issues in patients with TBI.

Shared and Anxiety-Specific Pediatric Psychopathology Dimensions Manifest Distributed Neural Correlates.

BACKGROUND: Imaging research has not yet delivered reliable psychiatric biomarkers. One challenge, particularly among youth, is high comorbidity. This challenge might be met through canonical correlation analysis designed to model mutual dependencies between symptom dimensions and neural measures. We mapped the multivariate associations that intrinsic functional connectivity manifests with pediatric symptoms of anxiety, irritability, and attention-deficit/hyperactivity disorder (ADHD) as common, impactful, co-occurring problems. We evaluate the replicability of such latent dimensions in an independent sample. METHODS: We obtained ratings of anxiety, irritability, and ADHD, and 10 minutes of resting-state functional magnetic resonance imaging data, from two independent cohorts. Both cohorts (discovery: n = 182; replication: n = 326) included treatment-seeking youth with anxiety disorders, with disruptive mood dysregulation disorder, with ADHD, or without psychopathology. Functional connectivity was modeled as partial correlations among 216 brain areas. Using canonical correlation analysis and independent component analysis jointly we sought maximally correlated, maximally interpretable latent dimensions of brain connectivity and clinical symptoms. RESULTS: We identified seven canonical variates in the discovery and five in the replication cohort. Of these canonical variates, three exhibited similarities across datasets: two variates consistently captured shared aspects of irritability, ADHD, and anxiety, while the third was specific to anxiety. Across cohorts, canonical variates did not relate to specific resting-state networks but comprised edges interconnecting established networks within and across both hemispheres. CONCLUSIONS: Findings revealed two replicable types of clinical variates, one related to multiple symptom dimensions and a second relatively specific to anxiety. Both types involved a multitude of broadly distributed, weak brain connections as opposed to strong connections encompassing known resting-state networks.

Cnr2 Is Important for Ribbon Synapse Maturation and Function in Hair Cells and Photoreceptors.

The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva.

Role of the interaction between Lu/BCAM and the spectrin-based membrane skeleton in the increased adhesion of hereditary spherocytosis red cells to laminin.

Lu/BCAM, the unique erythroid receptor for laminin 511/521, interacts with the erythrocyte membrane skeleton through spectrin binding. It has been reported that Hereditary Spherocytosis red blood cells (HS RBC) exhibit increased adhesion to laminin. We investigated the role of Lu/BCAM-spectrin interaction in the RBC adhesion properties of 2 splenectomised HS patients characterized by 40% spectrin deficiency. Under physiological flow conditions, HS RBC exhibited an exaggerated adhesion to laminin that was completely abolished by soluble Lu/BCAM. Triton extraction experiments revealed that a greater fraction of Lu/BCAM was unlinked to the membrane skeleton of HS RBC, as compared to normal RBC. Disruption of the spectrin interaction site in Lu/BCAM expressed in the transfected K562 cell line resulted in a weakened interaction to the skeleton and an enhanced interaction to laminin. These results demonstrated that the adhesion of HS RBC to laminin was mediated by Lu/BCAM and that its interaction with the spectrin-based skeleton negatively regulated cell adhesion to laminin. Finally, the results of this study strongly suggest that the reinforced adhesiveness of spectrin-deficient HS RBC to laminin is partly brought about by an impaired interaction between Lu/BCAM and the membrane skeleton.