Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus.

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here, we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast et al., 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.

Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes.

Introduction: Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. Methods: To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 291 kinases are regulating DENV infection. Results: Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of EPHB4, ERBB2, FGFR2, or IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Discussion: Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.

How spotting technique affects dizziness and postural stability after full-body rotations in dancers.

Consecutive longitudinal axis rotations are very common in dance, ranging from head spins in break dance to pirouettes in ballet. They pose a rather formidable perceptuomotor challenge - and hence form an interesting window into human motor behaviour - yet they have been scarcely studied. In the present study, we investigated dancers' dizziness and postural stability after consecutive rotations. Rotations were performed actively or undergone passively, either with or without the use of a spotting technique in such an order that all 24 ordering options were offered at least once and not more than twice. Thirty-four dancers trained in ballet and/or contemporary dance (aged 27.2 ± 5.1 years) with a mean dance experience of 14.2 ± 7.1 years actively performed 14 revolutions in passé or coupé positions with a short gesture leg "foot down" after each revolution. In addition, they were passively turned through 14 revolutions on a motor-driven rotating chair. Participants' centre-of-pressure (COP) displacement was measured on a force-plate before and after the rotations. Moreover, the dancers indicated their subjective feeling of dizziness on a scale from 0 to 20 directly after the rotations. Both the active and passive conditions were completed with and without the dancers spotting. As expected, dizziness was worse after rotations without the adoption of the spotting technique, both in active and passive rotations. However, the pre-post difference in COP area after active rotations was unaffected by spotting, whereas in the passive condition, spotting diminished this difference. Our results thus suggest that adopting the spotting technique is a useful tool for dizziness reduction in dancers who have to perform multiple rotations. Moreover, spotting appears most beneficial for postural stability when it involves less postural control challenges, such as when seated on a chair and occurs in situations with limited somatosensory feedback (e.g., from the cutaneous receptors in the feet). However, the unexpected finding that spotting did not help postural stability after active rotations needs to be investigated further in future studies, for example with a detailed analysis of whole-body kinematics and eye-tracking.

Automated, image-based quantification of peroxisome characteristics with perox-per-cell.

perox-per-cell automates cumbersome, image-based data collection tasks often encountered in peroxisome research. The software processes microscopy images to quantify peroxisome features in yeast cells. It uses off-the-shelf image processing tools to automatically segment cells and peroxisomes and then outputs quantitative metrics including peroxisome counts per cell and spatial areas. In validation tests, we found that perox-per-cell output agrees well with manually-quantified peroxisomal counts and cell instances, thereby enabling high-throughput quantification of peroxisomal characteristics. The software is available at https://github.com/AitchisonLab/perox-per-cell.

SARS-CoV-2 Orf6 is positioned in the nuclear pore complex by Rae1 to inhibit nucleocytoplasmic transport.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accessory protein Orf6 works as an interferon antagonist, in part, by inhibiting the nuclear import activated p-STAT1, an activator of interferon-stimulated genes, and the export of the poly(A) RNA. Insight into the transport regulatory function of Orf6 has come from the observation that Orf6 binds to the nuclear pore complex (NPC) components: Rae1 and Nup98. To gain further insight into the mechanism of Orf6-mediated transport inhibition, we examined the role of Rae1 and Nup98. We show that Rae1 alone is not necessary to support p-STAT1 import or nuclear export of poly(A) RNA. Moreover, the loss of Rae1 suppresses the transport inhibitory activity of Orf6. We propose that the Rae1/Nup98 complex strategically positions Orf6 within the NPC where it alters FG-Nup interactions and their ability to support nuclear transport. In addition, we show that Rae1 is required for normal viral protein production during SARS-CoV-2 infection presumably through its role in supporting Orf6 function.

Executive functions in patients with bilateral and unilateral peripheral vestibular dysfunction.

Previous research suggests that patients with peripheral vestibular dysfunction (PVD) suffer from nonspatial cognitive problems, including executive impairments. However, previous studies that assessed executive functions are conflicting, limited to single executive components, and assessments are confounded by other cognitive functions. We compared performance in a comprehensive executive test battery in a large sample of 83 patients with several conditions of PVD (34 bilateral, 29 chronic unilateral, 20 acute unilateral) to healthy controls who were pairwise matched to patients regarding age, sex, and education. We assessed basic and complex executive functions with validated neuropsychological tests. Patients with bilateral PVD performed worse than controls in verbal initiation and working memory span, while other executive functions were preserved. Patients with chronic unilateral PVD had equal executive performance as controls. Patients with acute unilateral PVD performed worse than controls in the exact same tests as patients with bilateral PVD (verbal initiation, working memory span); however, this effect in patients with acute PVD diminished after correcting for multiple comparisons. Hearing loss and affective disorders did not influence our results. Vestibular related variables (disease duration, symptoms, dizziness handicap, deafferentation degree, and compensation) did not predict verbal initiation or working memory span in patients with bilateral PVD. The results suggest that bilateral PVD not only manifests in difficulties when solving spatial tasks but leads to more general neurocognitive deficits. This understanding is important for multidisciplinary workgroups (e.g., neurotologists, neurologists, audiologists) that are involved in diagnosing and treating patients with PVD. We recommend screening patients with PVD for executive impairments and if indicated providing them with cognitive training or psychoeducational support.

Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus.

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast, Fridy et al. 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.

Human vestibular perceptual thresholds - A systematic review of passive motion perception.

BACKGROUND: The vestibular system detects head accelerations within 6 degrees of freedom. How well this is accomplished is described by vestibular perceptual thresholds. They are a measure of perceptual performance based on the conscious evaluation of sensory information. This review provides an integrative synthesis of the vestibular perceptual thresholds reported in the literature. The focus lies on the estimation of thresholds in healthy participants, used devices and stimulus profiles. The dependence of these thresholds on the participants clinical status and age is also reviewed. Furthermore, thresholds from primate studies are discussed. RESULTS: Thresholds have been measured for frequencies ranging from 0.05 to 5 Hz. They decrease with increasing frequency for five of the six main degrees of freedom (inter-aural, head-vertical, naso-occipital, yaw, pitch). No consistent pattern is evident for roll rotations. For a frequency range beyond 5 Hz, a U-shaped relationship is suggested by a qualitative comparison to primate data. Where enough data is available, increasing thresholds with age and higher thresholds in patients compared to healthy controls can be observed. No effects related to gender or handedness are reported. SIGNIFICANCE: Vestibular thresholds are essential for next generation screening tools in the clinical domain, for the assessment of athletic performance, and workplace safety alike. Knowledge about vestibular perceptual thresholds contributes to basic and applied research in fields such as perception, cognition, learning, and healthy aging. This review provides normative values for vestibular thresholds. Gaps in current knowledge are highlighted and attention is drawn to specific issues for improving the inter-study comparability in the future.

Foundations for future math achievement: Early numeracy, home learning environment, and the absence of math anxiety.

BACKGROUND: Mathematics achievement is pivotal in shaping children's future prospects. Cognitive skills (numeracy), feelings (anxiety), and the social environment (home learning environment) influence early math development. METHOD: A longitudinal study involved 85 children (mean age T1 = 6.4 years; T2 = 7.9) to explore these predictors holistically. Data were collected on early numeracy skills, home learning environment, math anxiety, and their impact on various aspects of math. RESULTS: The study found that early numeracy skills, home learning environment, and math anxiety significantly influenced math school achievement. However, they affected written computation, sequences, and comparisons differently. Early numeracy skills strongly predicted overall achievement and comparison subtest performance. CONCLUSION: These findings underscore the substantial role of math anxiety and home learning environment in children's math achievement. The study emphasizes the need to consider the selective impacts of these factors in future research, shedding light on the multifaceted nature of mathematics achievement determinants.

Gaze Restriction and Reactivation of Place-bound Content Drive Eye Movements During Mental Imagery.

When we imagine a picture, we move our eyes even though the picture is physically not present. These eye movements provide information about the ongoing process of mental imagery. Eye movements unfold over time, and previous research has shown that the temporal gaze dynamics of eye movements in mental imagery have unique properties, which are unrelated to those in perception. In mental imagery, refixations of previously fixated locations happen more often and in a more systematic manner than in perception. The origin of these unique properties remains unclear. We tested how the temporal structure of eye movements is influenced by the complexity of the mental image. Participants briefly saw and then maintained a pattern stimulus, consisting of one (easy condition) to four black segments (most difficult condition). When maintaining a simple pattern in imagery, participants restricted their gaze to a narrow area, and for more complex stimuli, eye movements were more spread out to distant areas. At the same time, fewer refixations were made in imagery when the stimuli were complex. The results show that refixations depend on the imagined content. While fixations of stimulus-related areas reflect the so-called 'looking at nothing' effect, gaze restriction emphasizes differences between mental imagery and perception.

Predicting host-based, synthetic lethal antiviral targets from omics data.

Traditional antiviral therapies often have limited effectiveness due to toxicity and development of drug resistance. Host-based antivirals, while an alternative, may lead to non-specific effects. Recent evidence shows that virus-infected cells can be selectively eliminated by targeting synthetic lethal (SL) partners of proteins disrupted by viral infection. Thus, we hypothesized that genes depleted in CRISPR KO screens of virus-infected cells may be enriched in SL partners of proteins altered by infection. To investigate this, we established a computational pipeline predicting SL drug targets of viral infections. First, we identified SARS-CoV-2-induced changes in gene products via a large compendium of omics data. Second, we identified SL partners for each altered gene product. Last, we screened CRISPR KO data for SL partners required for cell viability in infected cells. Despite differences in virus-induced alterations detected by various omics data, they share many predicted SL targets, with significant enrichment in CRISPR KO-depleted datasets. Comparing data from SARS-CoV-2 and influenza infections, we found possible broad-spectrum, host-based antiviral SL targets. This suggests that CRISPR KO data are replete with common antiviral targets due to their SL relationship with virus-altered states and that such targets can be revealed from analysis of omics datasets and SL predictions.

Probabilistic integration of preceding responses explains response bias in perceptual decision making.

Expectations of sensory information change not only how well but also what we perceive. Even in an unpredictable environment, the brain is by default constantly engaged in computing probabilities between sensory events. These estimates are used to generate predictions about future sensory events. Here, we investigated the predictability of behavioral responses using three different learning models in three different one-interval two-alternative forced choice experiments with either auditory, vestibular, or visual stimuli. Results indicate that recent decisions, instead of the sequence of generative stimuli, cause serial dependence. By bridging the gap between sequence learning and perceptual decision making, we provide a novel perspective on sequential choice effects. We propose that serial biases reflect the tracking of statistical regularities of the decision variable, offering a broader understanding of this phenomenon.

High-current galvanic vestibular stimulation impairs working memory span, but not other executive functions.

Patients with peripheral vestibular dysfunction (PVD) suffer not only from physical problems such as imbalance or vertigo but also from neuropsychological difficulties, including executive deficits. However, it is unclear whether the PVD directly causes executive problems. To examine the causal vestibular influence on executive functions, we induced either high-current (2 mA), low-current (0.8 mA), or sham current (0 mA) galvanic vestibular stimulation (GVS) in 79 healthy participants. Participants solved three tasks, measuring the core executive components (working memory, inhibition, cognitive flexibility) before and during GVS. High-current GVS impaired working memory span, but not inhibition and cognitive flexibility performance. Low-current GVS did not influence executive performance. Results indicate a causal vestibular influence on working memory span. Joint cortical areas of vestibular and working memory processing are discussed. Since high-current GVS in healthy participants serves as a model for an artificial vestibular dysfunction, our results could improve the diagnostics and therapy of patients with PVD.

Nuclear pore complexes mediate subtelomeric gene silencing by regulating PCNA levels on chromatin.

The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The Saccharomyces cerevisiae inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interactions, genetic interactions, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170. The Ctf18-RFC complex is recruited to a subpopulation of NPCs that lack the nuclear basket proteins Mlp1 and Mlp2. In the absence of Nup170, PCNA levels on DNA are reduced, resulting in the loss of silencing of subtelomeric genes. Increasing PCNA levels on DNA by removing Elg1, which is required for PCNA unloading, rescues subtelomeric silencing defects in nup170Δ. The NPC, therefore, mediates subtelomeric gene silencing by regulating PCNA levels on DNA.

Processing of semantic incongruency at the onset of sleep: An auditory N400 evoked potential study.

The ability to organize self-generated thought into coherent, meaningful semantic representations is a central aspect of human cognition and undergoes regular alterations throughout the day. To investigate whether changes in semantic processing might explain the loss of coherence, logic, and voluntary control over thinking typically accompanying the transition to sleep, we recorded N400 evoked potentials from 44 healthy subjects. Auditory word pairs with varying semantic distance were presented while they were allowed to fall asleep. Using semantic distance and wakefulness level as regressors, we found that semantic distance reliably elicited an N400, and lower wakefulness levels were associated with increased frontal negativity within a similar time range. Additionally, and contrary to our initial hypothesis, the results showed an interaction of semantic distance and wakefulness that is best interpreted as an increased N400 effect with decreasing wakefulness. While these results do not rule out a possible role of semantic processes in the generation of diminished logic and thought control during the transition to sleep, we discuss the possibility of additional brain mechanisms that usually constrain the inner stream of consciousness during wakefulness.

Measuring the Influence of Magnetic Vestibular Stimulation on Nystagmus, Self-Motion Perception, and Cognitive Performance in a 7T MRT.

Strong magnetic fields induce dizziness, vertigo, and nystagmus due to Lorentz forces acting on the cupula in the semi-circular canals, an effect called magnetic vestibular stimulation (MVS). In this article, we present an experimental setup in a 7T MRT scanner (MRI scanner) that allows the investigation of the influence of strong magnetic fields on nystagmus as well as perceptual and cognitive responses. The strength of MVS is manipulated by altering the head positions of the participants. The orientation of the participants' semicircular canals with respect to the static magnetic field is assessed by combining a 3D magnetometer and 3D constructive interference in steady-state (3D-CISS) images. This approach allows to account for intra- and inter-individual differences in participants' responses to MVS. In the future, MVS can be useful for clinical research, for example, in the investigation of compensatory processes in vestibular disorders. Furthermore, it could foster insights into the interplay between vestibular information and cognitive processes in terms of spatial cognition and the emergence of self-motion percepts under conflicting sensory information. In fMRI studies, MVS can elicit a possible confounding effect, especially in tasks influenced by vestibular information or in studies comparing vestibular patients with healthy controls.

Training and proficiency level in endoscopic sinus surgery change residents' eye movements.

Nose surgery is challenging and needs a lot of training for safe and efficient treatments. Eye tracking can provide an objective assessment to measure residents' learning curve. The aim of the current study was to assess residents' fixation duration and other dependent variables over the course of a dedicated training in functional endoscopic sinus surgery (FESS). Sixteen residents performed a FESS training over 18 sessions, split into three surgical steps. Eye movements in terms of percent fixation on the screen and average fixation duration were measured, in addition to residents' completion time, cognitive load, and surgical performance. Results indicated performance improvements in terms of completion time and surgical performance. Cognitive load and average fixation duration showed a significant change within the last step of training. Percent fixation on screen increased within the first step, and then stagnated. Results showed that eye movements and cognitive load differed between residents of different proficiency levels. In conclusion, eye tracking is a helpful objective measuring tool in FESS. It provides additional insights of the training level and changes with increasing performance. Expert-like gaze was obtained after half of the training sessions and increased proficiency in FESS was associated with increased fixation duration.

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.

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.

Persistent Nonviral Plasmid Vector in Nasal Tissues Causes False-Positive SARS-CoV-2 Diagnostic Nucleic Acid Tests.

Biomedical personnel can become contaminated with nonhazardous reagents used in the laboratory. We describe molecular studies performed on nasal secretions collected longitudinally from asymptomatic laboratory coworkers to determine if they were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulating in the community or with SARS-CoV-2 DNA from a plasmid vector. Participants enrolled in a prospective study of incident SARS-CoV-2 infection had nasal swabs collected aseptically by study staff at enrollment, followed by weekly self-collection of anterior nasal swabs. SARS-CoV-2 diagnosis was performed by a real-time PCR test targeting the nucleocapsid gene. PCR tests targeting SARS-CoV-2 nonstructural protein 10 (nsp10), nsp14, and envelope and three regions of the plasmid vector were performed to differentiate amplification of SARS-CoV-2 RNA from the plasmid vector's DNA. Nasal swabs from four asymptomatic coworkers with positive real-time PCR results for the SARS-CoV-2 nucleocapsid targets were negative when tested for SARS-CoV-2 nsp10, nsp14, and envelope protein. However, nucleic acids extracted from these nasal swabs amplified DNA regions of the plasmid vector used by the coworkers, including the ampicillin and neomycin/kanamycin resistance genes, the promoter-nucleocapsid junction, and unique codon-optimized regions. Nasal swabs from these individuals tested positive repeatedly, including during isolation. Longitudinal detection of plasmid DNA with SARS-CoV-2 nucleocapsid in nasal swabs suggests persistence in nasal tissues or colonizing bacteria. Nonviral plasmid vectors, while regarded as safe laboratory reagents, can interfere with molecular diagnostic tests. These reagents should be handled using proper personal protective equipment to prevent contamination of samples or laboratory personnel. IMPORTANCE Asymptomatic laboratory workers who tested positive for SARS-CoV-2 for days to months were found to harbor a laboratory plasmid vector containing SARS-CoV-2 DNA, which they had worked with in the past, in their nasal secretions. While prior studies have documented contamination of research personnel with PCR amplicons, our observation is novel, as these individuals shed the laboratory plasmid over days to months, including during isolation in their homes. This suggests that the plasmid was in their nasal tissues or that bacteria containing the plasmid had colonized their noses. While plasmids are generally safe, our detection of plasmid DNA in the nasal secretions of laboratory workers for weeks after they had stopped working with the plasmid shows the potential for these reagents to interfere with clinical tests and emphasizes that occupational exposures in the preceding months should be considered when interpreting diagnostic clinical tests.