Expanding and improving nanobody repertoires using a yeast display method: Targeting SARS-CoV-2.

COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single-chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry-based method, using B-cell complementary DNA from the same immunized animals as a source of VHH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions of antiviral nanobodies. The results suggested a surprising degree of modularity to complementarity-determining region function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the mass spectrometry-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.

ILF3 Is a Negative Transcriptional Regulator of Innate Immune Responses and Myeloid Dendritic Cell Maturation.

APCs such as myeloid dendritic cells (DCs) are key sentinels of the innate immune system. In response to pathogen recognition and innate immune stimulation, DCs transition from an immature to a mature state that is characterized by widespread changes in host gene expression, which include the upregulation of cytokines, chemokines, and costimulatory factors to protect against infection. Several transcription factors are known to drive these gene expression changes, but the mechanisms that negatively regulate DC maturation are less well understood. In this study, we identify the transcription factor IL enhancer binding factor 3 (ILF3) as a negative regulator of innate immune responses and DC maturation. Depletion of ILF3 in primary human monocyte-derived DCs led to increased expression of maturation markers and potentiated innate responses during stimulation with viral mimetics or classic innate agonists. Conversely, overexpression of short or long ILF3 isoforms (NF90 and NF110) suppressed DC maturation and innate immune responses. Through mutagenesis experiments, we found that a nuclear localization sequence in ILF3, and not its dual dsRNA-binding domains, was required for this function. Mutation of the domain associated with zinc finger motif of ILF3's NF110 isoform blocked its ability to suppress DC maturation. Moreover, RNA-sequencing analysis indicated that ILF3 regulates genes associated with cholesterol homeostasis in addition to genes associated with DC maturation. Together, our data establish ILF3 as a transcriptional regulator that restrains DC maturation and limits innate immune responses through a mechanism that may intersect with lipid metabolism.

Molecular mechanisms of organelle inheritance: lessons from peroxisomes in yeast.

Preserving a functional set of cytoplasmic organelles in a eukaryotic cell requires a process of accurate organelle inheritance at cell division. Studies of peroxisome inheritance in yeast have revealed that polarized transport of a subset of peroxisomes to the emergent daughter cell is balanced by retention mechanisms operating in both mother cell and bud to achieve an equitable distribution of peroxisomes between them. It is becoming apparent that some common mechanistic principles apply to the inheritance of all organelles, but at the same time, inheritance factors specific for each organelle type allow the cell to differentially and specifically control the inheritance of its different organelle populations.

EEG Microstate Dynamics Associated with Dream-Like Experiences During the Transition to Sleep.

Consciousness always requires some representational content; that is, one can only be conscious about something. However, the presence of conscious experience (awareness) alone does not determine whether its content is in line with the external and physical world. Dreams, apart from certain forms of hallucinations, typically consist of non-veridical percepts, which are not recognized as false, but rather considered real. This type of experiences have been described as a state of dissociation between phenomenal and reflective awareness. Interestingly, during the transition to sleep, reflective awareness seems to break down before phenomenal awareness as conscious experience does not immediately fade with reduced wakefulness but is rather characterized by the occurrence of uncontrolled thinking and perceptual images, together with a reduced ability to recognize the internal origin of the experience. Relative deactivation of the frontoparietal and preserved activity in parieto-occipital networks has been suggested to account for dream-like experiences during the transition to sleep. We tested this hypothesis by investigating subjective reports of conscious experience and large-scale brain networks using EEG microstates in 45 healthy young subjects during the transition to sleep. We observed an inverse relationship between cognitive effects and physiological activation; dream-like experiences were associated with an increased presence of a microstate with sources in the superior and middle frontal gyrus and precuneus. Additionally, the presence of a microstate associated with higher-order visual areas was decreased. The observed inverse relationship might therefore indicate a disengagement of cognitive control systems that is mediated by specific, inhibitory EEG microstates.

BizarreVR: Dream-like bizarreness in immersive virtual reality induced changes in conscious experience of reality while leaving spatial presence intact.

Differences in conscious experience of reality occur between waking, dreaming, and psychotic states. Between these states, there are systematic differences in the judgment about the reality of the experience when being confronted with bizarre breaks. However, the mechanisms underlying experience of reality in these different states are still unknown. To investigate the effect of bizarre breaks on experience of reality during the wake state, we propose a new paradigm using dream-like bizarreness and immersive virtual reality. Results showed that the realistic non-bizarre virtual environment induced high levels of reality judgment and spatial presence, whereas the confrontation with bizarre breaks induced high levels of experienced bizarreness. Moreover, experienced bizarreness significantly reduced reality judgment in both the bizarre and the realistic condition. Further, there was no effect of bizarre breaks on spatial presence. These results provide proof of concept for the new method to elicit natural bizarre experience within a realistic scenario.

Pictorial low-level features in mental images: evidence from eye fixations.

It is known that eye movements during object imagery reflect areas visited during encoding. But will eye movements also reflect pictorial low-level features of imagined stimuli? In this paper, three experiments are reported in which we investigate whether low-level properties of mental images elicit specific eye movements. Based on the conceptualization of mental images as depictive representations, we expected low-level visual features to influence eye fixations during mental imagery, in the absence of a visual input. In a first experiment, twenty-five participants performed a visual imagery task with high vs. low spatial frequency and high vs. low contrast gratings. We found that both during visual perception and during mental imagery, first fixations were more often allocated to the low spatial frequency-high contrast grating, thus showing that eye fixations were influenced not only by physical properties of visual stimuli but also by its imagined counterpart. In a second experiment, twenty-two participants imagined high contrast and low contrast stimuli that they had not encoded before. Again, participants allocated more fixations to the high contrast mental images than to the low contrast mental images. In a third experiment, we ruled out task difficulty as confounding variable. Our results reveal that low-level visual features are represented in the mind's eye and thus, they contribute to the characterization of mental images in terms of how much perceptual information is re-instantiated during mental imagery.

Usability of Two New Interactive Game Sensor-Based Hand Training Devices in Parkinson's Disease.

This pilot cross-sectional study aimed to evaluate the usability of two new interactive game sensor-based hand devices (GripAble and Smart Sensor Egg) in both healthy adults as well as in persons with Parkinson's Disease (PD). Eight healthy adults and eight persons with PD participated in this study. Besides a standardised usability measure, the state of flow after one training session and the effect of cognitive abilities on flow were evaluated. High system usability scores (SUS) were obtained both in healthy participants (72.5, IQR = 64.375-90, GripAble) as well as persons with PD (77.5, IQR = 70-80.625, GripAble; 77.5, IQR = 75-82.5, Smart Sensor Egg). Similarly, high FSSOT scores were achieved after one training session (42.5, IQR = 39.75-50, GripAble; 50, IQR = 47-50, Smart Sensor Egg; maximum score 55). Across both groups, FSSOT scores correlated significantly with SUS scores (r = 0.52, p = 0.039). Finally, MoCA did not correlate significantly with FSSOT scores (r = 0.02, p = 0.9). The present study shows high usability for both interactive game sensor-based hand training devices, for persons with PD and healthy participants.

The prioritization of visuo-spatial associations during mental imagery.

While previous research has shown that during mental imagery participants look back to areas visited during encoding it is unclear what happens when information presented during encoding is incongruent. To investigate this research question, we presented 30 participants with incongruent audio-visual associations (e.g. the image of a car paired with the sound of a cat) and later asked them to create a congruent mental representation based on the auditory cue (e.g. to create a mental representation of a cat while hearing the sound of a cat). The results revealed that participants spent more time in the areas where they previously saw the object and that incongruent audio-visual information during encoding did not appear to interfere with the generation and maintenance of mental images. This finding suggests that eye movements can be flexibly employed during mental imagery depending on the demands of the task.

Anodal High-definition Transcranial Direct Current Stimulation over the Posterior Parietal Cortex Modulates Approximate Mental Arithmetic.

The representation and processing of numerosity is a crucial cognitive capacity. Converging evidence points to the posterior parietal cortex (PPC) as primary "number" region. However, the exact role of the left and right PPC for different types of numerical and arithmetic tasks remains controversial. In this study, we used high-definition transcranial direct current stimulation (HD-tDCS) to further investigate the causal involvement of the PPC during approximative, nonsymbolic mental arithmetic. Eighteen healthy participants received three sessions of anodal HD-tDCS at 1-week intervals in counterbalanced order: left PPC, right PPC, and sham stimulation. Results showed an improved performance during online parietal HD-tDCS (vs. sham) for subtraction problems. Specifically, the general tendency to underestimate the results of subtraction problems (i.e., the "operational momentum effect") was reduced during online parietal HD-tDCS. There was no difference between left and right stimulation. This study thus provides new evidence for a causal involvement of the left and right PPC for approximate nonsymbolic arithmetic and advances the promising use of noninvasive brain stimulation in increasing cognitive functions.

Vestibular Stimulation Modulates Neural Correlates of Own-body Mental Imagery.

There is growing evidence that vestibular information is not only involved in reflexive eye movements and the control of posture but it also plays an important role in higher order cognitive processes. Previous behavioral research has shown that concomitant vestibular stimuli influence performance in tasks that involve imagined self-rotations. These results suggest that imagined and perceived body rotations share common mechanisms. However, the nature and specificity of these effects remain largely unknown. Here, we investigated the neural mechanisms underlying this vestibulocognitive interaction. Participants (n = 20) solved an imagined self-rotation task during caloric vestibular stimulation. We found robust main effects of caloric vestibular stimulation in the core region of the vestibular network, including the rolandic operculum and insula bilaterally, and of the cognitive task in parietal and frontal regions. Interestingly, we found an interaction of stimulation and task in the left inferior parietal lobe, suggesting that this region represents the modulation of imagined body rotations by vestibular input. This result provides evidence that the inferior parietal lobe plays a crucial role in the neural integration of mental and physical body rotation.

Individual differences in basic numerical skills: The role of executive functions and motor skills.

The aim of the current study was to explore individual differences in basic numerical skills in a normative sample of 151 kindergarteners (mean age = 6.45 years). Whereas previous research claims a substantial link between executive functions and basic numerical skills, motor abilities have been put forward to explain variance in numerical skills. Regarding the current study, these two assumptions have been combined, revealing interesting results. Namely, executive functions (inhibition, switching, and visuospatial working memory) were found to relate to symbolic numerical skills, and motor skills (gross and fine motor skills) showed a significant correlation to nonsymbolic numerical skills. Suggesting that motor skills and executive functions are associated with basic numerical skills could lead to potential avenues for interventions in certain disorders or disabilities such as nonverbal learning disability, developmental dyscalculia, and developmental coordination disorder.

Reinterpretation in visual imagery is possible without visual cues: a validation of previous research.

Is visual reinterpretation of bistable figures (e.g., duck/rabbit figure) in visual imagery possible? Current consensus suggests that it is in principle possible because of converging evidence of quasi-pictorial functioning of visual imagery. Yet, studies that have directly tested and found evidence for reinterpretation in visual imagery, allow for the possibility that reinterpretation was already achieved during memorization of the figure(s). One study resolved this issue, providing evidence for reinterpretation in visual imagery (Mast and Kosslyn, Cognition 86:57-70, 2002). However, participants in that study performed reinterpretations with aid of visual cues. Hence, reinterpretation was not performed with mental imagery alone. Therefore, in this study we assessed the possibility of reinterpretation without visual support. We further explored the possible role of haptic cues to assess the multimodal nature of mental imagery. Fifty-three participants were consecutively presented three to be remembered bistable 2-D figures (reinterpretable when rotated 180°), two of which were visually inspected and one was explored hapticly. After memorization of the figures, a visually bistable exemplar figure was presented to ensure understanding of the concept of visual bistability. During recall, 11 participants (out of 36; 30.6%) who did not spot bistability during memorization successfully performed reinterpretations when instructed to mentally rotate their visual image, but additional haptic cues during mental imagery did not inflate reinterpretation ability. This study validates previous findings that reinterpretation in visual imagery is possible.

Characterization of Peroxisomal Regulation Networks.

Peroxisome proliferation involves signal recognition and computation by molecular networks that direct molecular events of gene expression, metabolism, membrane biogenesis, organelle proliferation, protein import, and organelle inheritance. Peroxisome biogenesis in yeast has served as a model system for exploring the regulatory networks controlling this process. Yeast is an outstanding model system to develop tools and approaches to study molecular networks and cellular responses and because the mechanisms of peroxisome biogenesis and key aspects of the transcriptional regulatory networks are remarkably conserved from yeast to humans. In this chapter, we focus on the complex regulatory networks that respond to environmental cues leading to peroxisome assembly and the molecular events of organelle assembly. Ultimately, understanding the mechanisms of the entire peroxisome biogenesis program holds promise for predictive modeling approaches and for guiding rational intervention strategies that could treat human conditions associated with peroxisome function.

Recurrence quantification analysis of eye movements during mental imagery.

Several studies demonstrated similarities of eye fixations during mental imagery and visual perception but-to our knowledge-the temporal characteristics of eye movements during imagery have not yet been considered in detail. To fill this gap, the same data is analyzed with conventional spatial techniques such as analysis of areas of interest (AOI), ScanMatch, and MultiMatch and with recurrence quantification analysis (RQA), a new way of analyzing gaze data by tracking re-fixations and their temporal dynamics. Participants viewed and afterwards imagined three different kinds of pictures (art, faces, and landscapes) while their eye movements were recorded. While fixation locations during imagery were related to those during perception, participants returned more often to areas they had previously looked at during imagery and their scan paths were more clustered and more repetitive when compared to visual perception. Furthermore, refixations of the same area occurred sooner after initial fixation during mental imagery. The results highlight not only content-driven spatial similarities between imagery and perception but also shed light on the processes of mental imagery maintenance and interindividual differences in these processes.

Motor response specificity in perceptual learning and its release by double training.

Perceptual learning is usually feature-specific. Recently, we showed that perceptual learning is even specific for the motor response type. In a three-line bisection task, participants indicated whether the central line was offset either to the left or right by pressing a left or a right button, respectively. We found no transfer when the same participants adjusted the offset by using a computer mouse. Here, we first show that perceptual learning with mouse adjustments transfers to the untrained hand, but only for the trained adjustment condition. There was no transfer to the button press conditions, neither for the trained nor the untrained hand. Second, we show that a double training procedure enables transfer from the mouse adjustment to the button press condition. Hence, the specificity of perceptual learning to the motor response type can be overcome by double training as it is the case for visual features. Our results suggest that during perceptual learning, perceptuo-decisional signals are encoded together with the corresponding actions.

Subliminal encoding and flexible retrieval of objects in scenes.

Our episodic memory stores what happened when and where in life. Episodic memory requires the rapid formation and flexible retrieval of where things are located in space. Consciousness of the encoding scene is considered crucial for episodic memory formation. Here, we question the necessity of consciousness and hypothesize that humans can form unconscious episodic memories. Participants were presented with subliminal scenes, that is, scenes invisible to the conscious mind. The scenes displayed objects at certain locations for participants to form unconscious object-in-space memories. Later, the same scenes were presented supraliminally, that is, visibly, for retrieval testing. Scenes were presented absent the objects and rotated by 90°-270° in perspective to assess the representational flexibility of unconsciously formed memories. During the test phase, participants performed a forced-choice task that required them to place an object in one of two highlighted scene locations and their eye movements were recorded. Evaluation of the eye tracking data revealed that participants remembered object locations unconsciously, irrespective of changes in viewing perspective. This effect of gaze was related to correct placements of objects in scenes, and an intuitive decision style was necessary for unconscious memories to influence intentional behavior to a significant degree. We conclude that conscious perception is not mandatory for spatial episodic memory formation.

The Influence of Alertness on the Spatial Deployment of Visual Attention is Mediated by the Excitability of the Posterior Parietal Cortices.

With a reduced level of alertness, healthy individuals typically show a rightward shift when deploying visual attention in space. The impact of alertness on the neural networks governing visuospatial attention is, however, poorly understood. By using a transcranial magnetic stimulation twin-coil approach, the present study aimed at investigating the effects of an alertness manipulation on the excitability of the left and the right posterior parietal cortices (PPCs), crucial nodes of the visuospatial attentional network. Participants' visuospatial attentional deployment was assessed with a free visual exploration task and concurrent eye tracking. Their alertness level was manipulated through the time of the day, that is, by testing chronotypically defined evening types both during their circadian on- and off-peak times. The results revealed an increased excitability of the left compared with the right PPC during low alertness. On the horizontal dimension, these results were accompanied by a significant rightward shift in the center and a bilateral narrowing in the periphery of the visual exploration field, as well as a central upward shift on the vertical dimension. The findings show that the manipulation of non-spatial attentional aspects (i.e., alertness) can affect visuospatial attentional deployment and modulate the excitability of areas subtending spatial attentional control.

The Fantasy Questionnaire: A Measure to Assess Creative and Imaginative Fantasy.

This article reports the construction and validation of a comprehensive self-report measure of fantasy. Unlike previous measures of fantasy, which focus on psychopathology, we conceive fantasy as a trait with positive connotation. Principal component analysis (N = 318) and confirmatory factor analyses (N = 345) were conducted using 2 sociodemographically diverse samples. The results provided support for a 2-factor conceptualization of the construct, with the dimensions imaginative fantasy and creative fantasy. Imaginative fantasy refers to vivid imagination and absorption in these images and daydreams. Creative fantasy refers to the activity of using fantasy to create new ideas. The trait measure showed good internal consistency, test-retest reliability, discriminant and convergent construct validity, as well as incremental validity. Moreover, in 3 behavioral studies, we put fantasy scores in relationship with behavioral data to provide further proof of validity. A comprehensive measure of fantasy can contribute to our understanding of individual differences in inner experiences, creative processes, and problem solving.

Peroxins Pex30 and Pex29 Dynamically Associate with Reticulons to Regulate Peroxisome Biogenesis from the Endoplasmic Reticulum.

Peroxisome proliferation occurs by at least two routes, division of existing peroxisomes and de novo biogenesis from the endoplasmic reticulum (ER). The proteins and molecular mechanisms governing peroxisome emergence from the ER are poorly characterized. In this study, we report that two integral membrane peroxins (proteins required for peroxisome biogenesis) in Saccharomyces cerevisiae, Pex29 and Pex30, reside in distinct regions of the ER and associate with Rtn1 and Yop1, reticulon family members that contribute to ER morphology, to govern peroxisome emergence from the ER. In vivo and in vitro analyses reveal that peroxisome proliferation is therefore not restricted to the peroxisome but begins at the level of the ER.

An ancestral role in peroxisome assembly is retained by the divisional peroxin Pex11 in the yeast Yarrowia lipolytica.

The peroxin Pex11 has a recognized role in peroxisome division. Pex11p remodels and elongates peroxisomal membranes prior to the recruitment of dynamin-related GTPases that act in membrane scission to divide peroxisomes. We performed a comprehensive comparative genomics survey to understand the significance of the evolution of the Pex11 protein family in yeast and other eukaryotes. Pex11p is highly conserved and ancestral, and has undergone numerous lineage-specific duplications, whereas other Pex11 protein family members are fungal-specific innovations. Functional characterization of the in-silico-predicted Pex11 protein family members of the yeast Yarrowia lipolytica, i.e. Pex11p, Pex11Cp and Pex11/25p, demonstrated that Pex11Cp and Pex11/25p have a role in the regulation of peroxisome size and number characteristic of Pex11 protein family members. Unexpectedly, deletion of PEX11 in Y. lipolytica produces cells that lack morphologically identifiable peroxisomes, mislocalize peroxisomal matrix proteins and preferentially degrade peroxisomal membrane proteins, i.e. they exhibit the classical pex mutant phenotype, which has not been observed previously in cells deleted for the PEX11 gene. Our results are consistent with an unprecedented role for Pex11p in de novo peroxisome assembly.