Alignment, calibration, and validation of an adaptive optics scanning laser ophthalmoscope for high-resolution human foveal imaging.

In prior art, advances in adaptive optics scanning laser ophthalmoscope (AOSLO) technology have enabled cones in the human fovea to be resolved in healthy eyes with normal vision and low to moderate refractive errors, providing new insight into human foveal anatomy, visual perception, and retinal degenerative diseases. These high-resolution ophthalmoscopes require careful alignment of each optical subsystem to ensure diffraction-limited imaging performance, which is necessary for resolving the smallest foveal cones. This paper presents a systematic and rigorous methodology for building, aligning, calibrating, and testing an AOSLO designed for imaging the cone mosaic of the central fovea in humans with cellular resolution. This methodology uses a two-stage alignment procedure and thorough system testing to achieve diffraction-limited performance. Results from retinal imaging of healthy human subjects under 30 years of age with refractive errors of less than 3.5 diopters using either 680 nm or 840 nm light show that the system can resolve cones at the very center of the fovea, the region where the cones are smallest and most densely packed.

AutophagyNet: high-resolution data source for the analysis of autophagy and its regulation.

Macroautophagy/autophagy is a highly-conserved catabolic procss eliminating dysfunctional cellular components and invading pathogens. Autophagy malfunction contributes to disorders such as cancer, neurodegenerative and inflammatory diseases. Understanding autophagy regulation in health and disease has been the focus of the last decades. We previously provided an integrated database for autophagy research, the Autophagy Regulatory Network (ARN). For the last eight years, this resource has been used by thousands of users. Here, we present a new and upgraded resource, AutophagyNet. It builds on the previous database but contains major improvements to address user feedback and novel needs due to the advancement in omics data availability. AutophagyNet contains updated interaction curation and integration of over 280,000 experimentally verified interactions between core autophagy proteins and their protein, transcriptional and post-transcriptional regulators as well as their potential upstream pathway connections. AutophagyNet provides annotations for each core protein about their role: 1) in different types of autophagy (mitophagy, xenophagy, etc.); 2) in distinct stages of autophagy (initiation, expansion, termination, etc.); 3) with subcellular and tissue-specific localization. These annotations can be used to filter the dataset, providing customizable download options tailored to the user's needs. The resource is available in various file formats (e.g. CSV, BioPAX and PSI-MI), and data can be analyzed and visualized directly in Cytoscape. The multi-layered regulation of autophagy can be analyzed by combining AutophagyNet with tissue- or cell type-specific (multi-)omics datasets (e.g. transcriptomic or proteomic data). The resource is publicly accessible at http://autophagynet.org.Abbreviations: ARN: Autophagy Regulatory Network; ATG: autophagy related; BCR: B cell receptor pathway; BECN1: beclin 1; GABARAP: GABA type A receptor-associated protein; IIP: innate immune pathway; LIR: LC3-interacting region; lncRNA: long non-coding RNA; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; miRNA: microRNA; NHR: nuclear hormone receptor; PTM: post-translational modification; RTK: receptor tyrosine kinase; TCR: T cell receptor; TLR: toll like receptor.

Ultra-fine resolution of pre-saccadic attention in the fovea.

Microsaccades, the tiny gaze relocations that occurr during fixation, have been linked to covert attention deployed degrees away from the center of gaze. However, the link between attention and microsaccades is deeper in that it also unfolds at the foveal scale. Here, we have examined the spatial grain of pre-microsaccadic attention across the 1° foveola. Through the use of high-precision eye-tracking and gaze-contingent display system that achieves arcminute precision in gaze localization, we have shown that the spotlight of attention at this scale can reach a strikingly high resolution, in the order of 0.17°. Further, when a microsaccade occurs, vision is modulated in a peculiar way across the foveola; whereas fine spatial vision is enhanced at the microsaccade goal location, it drops at the very center of gaze, where acuity is normally highest. These results reveal the finesse of the visuomotor system and of the interplay between eye movements and attention.

FAK family proteins regulate in vivo breast cancer metastasis via distinct mechanisms.

Breast cancer is the most commonly diagnosed malignancy and the major leading cause of tumor-related deaths in women. It is estimated that the majority of breast tumor-related deaths are a consequence of metastasis, to which no cure exists at present. The FAK family proteins Proline-rich tyrosine kinase (PYK2) and focal adhesion kinase (FAK) are highly expressed in breast cancer, but the exact cellular and signaling mechanisms by which they regulate in vivo tumor cell invasiveness and consequent metastatic dissemination are mostly unknown. Using a PYK2 and FAK knockdown xenograft model we show here, for the first time, that ablation of either PYK2 or FAK decreases primary tumor size and significantly reduces Tumor MicroEnvironment of Metastasis (TMEM) doorway activation, leading to decreased intravasation and reduced spontaneous lung metastasis. Intravital imaging analysis further demonstrates that PYK2, but not FAK, regulates a motility phenotype switch between focal adhesion-mediated fast motility and invadopodia-dependent, ECM-degradation associated slow motility within the primary tumor. Furthermore, we validate our in vivo and intravital imaging results with integrated transcriptomic and proteomic data analysis from xenograft knockdown tumors and reveal new and distinct pathways by which these two homologous kinases regulate breast tumor cell invasiveness and consequent metastatic dissemination. Our findings identify PYK2 and FAK as novel mediators of mammary tumor progression and metastasis and as candidate therapeutic targets for breast cancer metastasis.

An eye for detail: Eye movements and attention at the foveal scale.

Human vision relies on a tiny region of the retina, the 1-deg foveola, to achieve high spatial resolution. Foveal vision is of paramount importance in daily activities, yet its study is challenging, as eye movements incessantly displace stimuli across this region. Here I will review work that, building on recent advances in eye-tracking and gaze-contingent display, examines how attention and eye movements operate at the foveal level. This research highlights how exploration of fine spatial detail unfolds following visuomotor strategies reminiscent of those occurring at larger scales. It shows that, together with highly precise control of attention, this motor activity is linked to non-homogenous processing within the foveola and selectively modulates sensitivity both in space and time. Overall, the picture emerges of a highly dynamic foveal perception in which fine spatial vision, rather than simply being the result of placing a stimulus at the center of gaze, is the result of a finely tuned and orchestrated synergy of motor, cognitive, and attentional processes.

Eye drift during fixation predicts visual acuity.

Visual acuity is commonly assumed to be determined by the eye optics and spatial sampling in the retina. Unlike a camera, however, the eyes are never stationary during the acquisition of visual information; a jittery motion known as ocular drift incessantly displaces stimuli over many photoreceptors. Previous studies have shown that acuity is impaired in the absence of retinal image motion caused by eye drift. However, the relation between individual drift characteristics and acuity remains unknown. Here, we show that a) healthy emmetropes exhibit a large variability in their amount of drift and that b) these differences profoundly affect the structure of spatiotemporal signals to the retina. We further show that c) the spectral distribution of the resulting luminance modulations strongly correlates with individual visual acuity and that d) natural intertrial fluctuations in the amount of drift modulate acuity. As a consequence, in healthy emmetropes, acuity can be predicted from the motor behavior elicited by a simple fixation task, without directly measuring it. These results shed new light on how oculomotor behavior contributes to fine spatial vision.

Everything You Always Wanted to Know About Organoid-Based Models (and Never Dared to Ask).

Homeostatic functions of a living tissue, such as the gastrointestinal tract, rely on highly sophisticated and finely tuned cell-to-cell interactions. These crosstalks evolve and continuously are refined as the tissue develops and give rise to specialized cells performing general and tissue-specific functions. To study these systems, stem cell-based in vitro models, often called organoids, and non-stem cell-based primary cell aggregates (called spheroids) appeared just over a decade ago. These models still are evolving and gaining complexity, making them the state-of-the-art models for studying cellular crosstalk in the gastrointestinal tract, and to investigate digestive pathologies, such as inflammatory bowel disease, colorectal cancer, and liver diseases. However, the use of organoid- or spheroid-based models to recapitulate in vitro the highly complex structure of in vivo tissue remains challenging, and mainly restricted to expert developmental cell biologists. Here, we condense the founding knowledge and key literature information that scientists adopting the organoid technology for the first time need to consider when using these models for novel biological questions. We also include information that current organoid/spheroid users could use to add to increase the complexity to their existing models. We highlight the current and prospective evolution of these models through bridging stem cell biology with biomaterial and scaffold engineering research areas. Linking these complementary fields will increase the in vitro mimicry of in vivo tissue, and potentially lead to more successful translational biomedical applications. Deepening our understanding of the nature and dynamic fine-tuning of intercellular crosstalks will enable identifying novel signaling targets for new or repurposed therapeutics used in many multifactorial diseases.

Mapping the epithelial-immune cell interactome upon infection in the gut and the upper airways.

Increasing evidence points towards the key role of the epithelium in the systemic and over-activated immune response to viral infection, including SARS-CoV-2 infection. Yet, how viral infection alters epithelial-immune cell interactions regulating inflammatory responses, is not well known. Available experimental approaches are insufficient to properly analyse this complex system, and computational predictions and targeted data integration are needed as an alternative approach. In this work, we propose an integrated computational biology framework that models how infection alters intracellular signalling of epithelial cells and how this change impacts the systemic immune response through modified interactions between epithelial cells and local immune cell populations. As a proof-of-concept, we focused on the role of intestinal and upper-airway epithelial infection. To characterise the modified epithelial-immune interactome, we integrated intra- and intercellular networks with single-cell RNA-seq data from SARS-CoV-2 infected human ileal and colonic organoids as well as from infected airway ciliated epithelial cells. This integrated methodology has proven useful to point out specific epithelial-immune interactions driving inflammation during disease response, and propose relevant molecular targets to guide focused experimental analysis.

All eyes on attention.

Eye movements are neither necessary nor sufficient to account for the neural effects associated with covert attention.

Plating human iPSC lines on micropatterned substrates reveals role for ITGB1 nsSNV in endoderm formation.

Quantitative analysis of human induced pluripotent stem cell (iPSC) lines from healthy donors is a powerful tool for uncovering the relationship between genetic variants and cellular behavior. We previously identified rare, deleterious non-synonymous single nucleotide variants (nsSNVs) in cell adhesion genes that are associated with outlier iPSC phenotypes in the pluripotent state. Here, we generated micropatterned colonies of iPSCs to test whether nsSNVs influence patterning of radially ordered germ layers. Using a custom-built image analysis pipeline, we quantified the differentiation phenotypes of 13 iPSC lines that harbor nsSNVs in genes related to cell adhesion or germ layer development. All iPSC lines differentiated into the three germ layers; however, there was donor-specific variation in germ layer patterning. We identified one line that presented an outlier phenotype of expanded endodermal differentiation, which was associated with a nsSNV in ITGB1. Our study establishes a platform for investigating the impact of nsSNVs on differentiation.

Transient perceptual enhancements resulting from selective shifts of exogenous attention in the central fovea.

Exogenous attention, a powerful adaptive tool that quickly and involuntarily orients processing resources to salient stimuli, has traditionally been studied in the lower-resolution parafoveal and peripheral visual field.1-4 It is not known whether and how it operates across the 1° central fovea where visual resolution peaks.5,6 Here we investigated the dynamics of exogenous attention in the foveola. To circumvent the challenges posed by fixational eye movements at this scale, we used high-precision eye-tracking and gaze-contingent display control for retinal stabilization.7 High-acuity stimuli were briefly presented foveally at varying delays following an exogenous cue. Attended and unattended locations were just a few arcminutes away from the preferred locus of fixation. Our results show that for short temporal delays, observers' ability to discriminate fine detail is enhanced at the cued location. This enhancement is highly localized and does not extend to the nearby locations only 16' away. On a longer timescale, instead, we report an inverse effect: paradoxically, acuity is sharper at the unattended locations, resembling the phenomenon of inhibition of return at much larger eccentricities.8-10 Although exogenous attention represents a mechanism for low-cost monitoring of the environment in the extrafoveal space, these findings show that, in the foveola, it transiently modulates vision of detail with a high degree of resolution. Together with inhibition of return, it may aid visual exploration of complex foveal stimuli.11.

SARS-CoV-2 Causes a Different Cytokine Response Compared to Other Cytokine Storm-Causing Respiratory Viruses in Severely Ill Patients.

Hyper-induction of pro-inflammatory cytokines, also known as a cytokine storm or cytokine release syndrome (CRS), is one of the key aspects of the currently ongoing SARS-CoV-2 pandemic. This process occurs when a large number of innate and adaptive immune cells activate and start producing pro-inflammatory cytokines, establishing an exacerbated feedback loop of inflammation. It is one of the factors contributing to the mortality observed with coronavirus 2019 (COVID-19) for a subgroup of patients. CRS is not unique to the SARS-CoV-2 infection; it was prevalent in most of the major human coronavirus and influenza A subtype outbreaks of the past two decades (H5N1, SARS-CoV, MERS-CoV, and H7N9). With a comprehensive literature search, we collected changing the cytokine levels from patients upon infection with the viral pathogens mentioned above. We analyzed published patient data to highlight the conserved and unique cytokine responses caused by these viruses. Our curation indicates that the cytokine response induced by SARS-CoV-2 is different compared to other CRS-causing respiratory viruses, as SARS-CoV-2 does not always induce specific cytokines like other coronaviruses or influenza do, such as IL-2, IL-10, IL-4, or IL-5. Comparing the collated cytokine responses caused by the analyzed viruses highlights a SARS-CoV-2-specific dysregulation of the type-I interferon (IFN) response and its downstream cytokine signatures. The map of responses gathered in this study could help specialists identify interventions that alleviate CRS in different diseases and evaluate whether they could be used in the COVID-19 cases.

ViralLink: An integrated workflow to investigate the effect of SARS-CoV-2 on intracellular signalling and regulatory pathways.

The SARS-CoV-2 pandemic of 2020 has mobilised scientists around the globe to research all aspects of the coronavirus virus and its infection. For fruitful and rapid investigation of viral pathomechanisms, a collaborative and interdisciplinary approach is required. Therefore, we have developed ViralLink: a systems biology workflow which reconstructs and analyses networks representing the effect of viruses on intracellular signalling. These networks trace the flow of signal from intracellular viral proteins through their human binding proteins and downstream signalling pathways, ending with transcription factors regulating genes differentially expressed upon viral exposure. In this way, the workflow provides a mechanistic insight from previously identified knowledge of virally infected cells. By default, the workflow is set up to analyse the intracellular effects of SARS-CoV-2, requiring only transcriptomics counts data as input from the user: thus, encouraging and enabling rapid multidisciplinary research. However, the wide-ranging applicability and modularity of the workflow facilitates customisation of viral context, a priori interactions and analysis methods. Through a case study of SARS-CoV-2 infected bronchial/tracheal epithelial cells, we evidence the functionality of the workflow and its ability to identify key pathways and proteins in the cellular response to infection. The application of ViralLink to different viral infections in a context specific manner using different available transcriptomics datasets will uncover key mechanisms in viral pathogenesis.

Organoid-based Models to Study the Role of Host-microbiota Interactions in IBD.

The gut microbiota appears to play a central role in health, and alterations in the gut microbiota are observed in both forms of inflammatory bowel disease [IBD], namely Crohn's disease and ulcerative colitis. Yet, the mechanisms behind host-microbiota interactions in IBD, especially at the intestinal epithelial cell level, are not yet fully understood. Dissecting the role of host-microbiota interactions in disease onset and progression is pivotal, and requires representative models mimicking the gastrointestinal ecosystem, including the intestinal epithelium, the gut microbiota, and immune cells. New advancements in organoid microfluidics technology are facilitating the study of IBD-related microbial-epithelial cross-talk, and the discovery of novel microbial therapies. Here, we review different organoid-based ex vivo models that are currently available, and benchmark their suitability and limitations for specific research questions. Organoid applications, such as patient-derived organoid biobanks for microbial screening and 'omics technologies, are discussed, highlighting their potential to gain better mechanistic insights into disease mechanisms and eventually allow personalised medicine.

Accuracy and precision of small saccades.

Despite recent advances on the mechanisms and purposes of fine oculomotor behavior, a rigorous assessment of the precision and accuracy of the smallest saccades is still lacking. Yet knowledge of how effectively these movements shift gaze is necessary for understanding their functions and is helpful in further elucidating their motor underpinnings. Using a combination of high-resolution eye-tracking and gaze-contingent control, here we examined the accuracy and precision of saccades aimed toward targets ranging from [Formula: see text] to [Formula: see text] eccentricity. We show that even small saccades of just 14-[Formula: see text] are very effective in centering the stimulus on the retina. Furthermore, we show that for a target at any given eccentricity, the probability of eliciting a saccade depends on its efficacy in reducing the foveal offset. The pattern of results reported here is consistent with current knowledge on the motor mechanisms of microsaccade production.

Modulations of foveal vision associated with microsaccade preparation.

It is known that attention shifts prior to a saccade to start processing the saccade target before it lands in the foveola, the high-resolution region of the retina. Yet, once the target is foveated, microsaccades, tiny saccades maintaining the fixated object within the fovea, continue to occur. What is the link between these eye movements and attention? There is growing evidence that these eye movements are associated with covert shifts of attention in the visual periphery, when the attended stimuli are presented far from the center of gaze. Yet, microsaccades are primarily used to explore complex foveal stimuli and to optimize fine spatial vision in the foveola, suggesting that the influences of microsaccades on attention may predominantly impact vision at this scale. To address this question we tracked gaze position with high precision and briefly presented high-acuity stimuli at predefined foveal locations right before microsaccade execution. Our results show that visual discrimination changes prior to microsaccade onset. An enhancement occurs at the microsaccade target location. This modulation is highly selective and it is coupled with a drastic impairment at the opposite foveal location, just a few arcminutes away. This effect is strongest when stimuli are presented closer to the eye movement onset time. These findings reveal that the link between attention and microsaccades is deeper than previously thought, exerting its strongest effects within the foveola. As a result, during fixation, foveal vision is constantly being reshaped both in space and in time with the occurrence of microsaccades.

Task-driven visual exploration at the foveal scale.

Humans use saccades to inspect objects of interest with the foveola, the small region of the retina with highest acuity. This process of visual exploration is normally studied over large scenes. However, in everyday tasks, the stimulus within the foveola is complex, and the need for visual exploration may extend to this smaller scale. We have previously shown that fixational eye movements, in particular microsaccades, play an important role in fine spatial vision. Here, we investigate whether task-driven visual exploration occurs during the fixation pauses in between large saccades. Observers judged the expression of faces covering approximately 1°, as if viewed from a distance of many meters. We use a custom system for accurately localizing the line of sight and continually track gaze position at high resolution. Our findings reveal that active spatial exploration, a process driven by the goals of the task, takes place at the foveal scale. The scanning strategies used at this scale resemble those used when examining larger scenes, with idiosyncrasies maintained across spatial scales. These findings suggest that the visual system possesses not only a coarser priority map of the extrafoveal space to guide saccades, but also a finer-grained priority map that is used to guide microsaccades once the region of interest is foveated.

Monocular microsaccades: Do they really occur?

Small saccades, known as microsaccades, occur frequently during fixation. Several recent studies have argued that a considerable fraction of these movements are present in the traces from one eye only. This claim contrasts with the findings of older reports, which concluded that microsaccades, like larger saccades, are virtually always binocular events. Here we examined the characteristics of small saccades by means of two of the most established high-resolution eye-tracking techniques available. A binocular Dual Purkinje Image eye-tracker was used to record eye movements while observers fixated, with their head immobilized, on markers displayed on a monitor. A specially designed eye-coil system was used to measure eye movements during normal head-free viewing, while subjects fixated on markers at various distances. Monocular microsaccades were virtually absent in both datasets. In the head-fixed data, not a single monocular microsaccade was observed. In the head-free data, only one event appeared to be monocular out of more than a thousand saccades. Monocular microsaccades do not seem to occur during normal head-free or head-immobilized fixation.

Microsaccades during reading.

Recent research has shown that microsaccades contribute to high acuity vision. However, little is known about whether microsaccades also play a role in daily activities, such as reading, that do not involve stimuli at the limit of spatial resolution. While the functions of larger saccades in reading have been extensively examined, microsaccades are commonly regarded as oculomotor noise in this context. We used high-resolution eyetracking and precise gaze localization to investigate fine oculomotor behavior during reading. Our findings show that microsaccade characteristics differ from those measured during sustained fixation: microsaccades are larger in size and primarily leftwards during reading, i.e. they move the line of sight backward on the text. Analysis of how microsaccades shift gaze relative to the text suggests that these movements serve two important functions: (1) a corrective function, by moving the gaze regressively within longer words when the preceding saccade lands too far toward the end of these words, and (2) an exploratory function, by shifting the gaze on adjacent words to gain additional information before the execution of the next saccade. Thus, microsaccades may benefit reading by enhancing the visibility of nearby words. This study highlights the importance of examining fine oculomotor behavior in reading, and calls for further research to investigate the possible roles of microsaccades in reading difficulties.

Selective attention within the foveola.

Efficient control of attentional resources and high-acuity vision are both fundamental for survival. Shifts in visual attention are known to covertly enhance processing at locations away from the center of gaze, where visual resolution is low. It is unknown, however, whether selective spatial attention operates where the observer is already looking-that is, within the high-acuity foveola, the small yet disproportionally important rod-free region of the retina. Using new methods for precisely controlling retinal stimulation, here we show that covert attention flexibly improves and speeds up both detection and discrimination at loci only a fraction of a degree apart within the foveola. These findings reveal a surprisingly precise control of attention and its involvement in fine spatial vision. They show that the commonly studied covert shifts of attention away from the fovea are the expression of a global mechanism that exerts its action across the entire visual field.