Cellular Ensembles Research Articles

Diagnostic value of the gingival cytogram in school-age children suffering from chronic gastritis and duodenitis

In recent years, there has been an increase in the incidence of morbidity associated with digestive organ pathology in the pediatric population. The oral cavity is the digestive tract opening, sharing a common ectodermal origin with it. The changes in the cytogram of the oral mucous membranes can signal about exacerbation of pathological processes in the gastrointestinal tract. The aim of this work: to identify the features of gingival cellular composition in school-age patients with chronic gastritis and duodenitis in order to improve diagnostic methods at the disease stages. Examinations of the gingival cytogram in children aged 12-17 years with chronic gastritis and duodenitis (27 individuals with the disease in acute stage, 30 – in remission) were carried out. The control group included 28 children who did not have any somatic pathology by the results of the examination. For cytological examination, imprint smears from the gingival vestibular surface of the upper and lower jaws were made. The smears were fixed and stained by the May-Grunwald Pappenheim method, and then examined using a microscope with an im­mersion system. The percentage of epithelial cells and connective tissue cells was calculated per 100 cells. The number of pathologically altered epithelial cells was determined: with vacuolated cytoplasm, with nucleus deformation. In children with chronic gastritis and duodenitis disorders in the gingival cellular composition with a predominance of dystrophic components were found, that was manifested by a decrease in the proportion of epithelial cells at terminal stages of differentiation, an increase in the proportion of cells with cytopathological phenomena, polymorphonuclear leukocytes and lymphocytes. Changes in the cytogram were especially expressed in the phase of disease exacerbation. Knowing the phases of the wound process, based on the timing and sequence of the cellular ensembles seen in the cytogram, it is possible to control the disease periods, and timely apply measures to prevent exacerbations. This diag­nostic method is especially indicated for children who have relative and absolute contraindications for fibroesogastroduodenoscopy.

Spatial Patterning Analysis of Cellular Ensembles (SPACE) discovers complex spatial organization at the cell and tissue levels.

Spatial patterns of cells and other biological elements drive both physiologic and pathologic processes within tissues. While many imaging and transcriptomic methods document tissue organization, discerning these patterns is challenging, especially when they involve multiple elements in complex arrangements. To address this challenge, we present Spatial Patterning Analysis of Cellular Ensembles (SPACE), an R package for analysis of high-plex spatial data. SPACE is compatible with any data collection modality that records values (i.e., categorical cell/structure types or quantitative expression levels) at fixed spatial coordinates (i.e., 2d pixels or 3d voxels). SPACE detects not only broad patterns of co-occurrence but also context-dependent associations, quantitative gradients and orientations, and other organizational complexities. Via a robust information theoretic framework, SPACE explores all possible ensembles of tissue elements - single elements, pairs, triplets, and so on - and ranks the most strongly patterned ensembles. For single images, rankings reflect patterns that differ from random assortment. For sets of images, rankings reflect patterns that differ across sample groups (e.g., genotypes, treatments, timepoints, etc.). Further tools then thoroughly characterize the nature of each pattern for intuitive interpretation. We validate SPACE and demonstrate its advantages using murine lymph node images for which ground truth has been defined. We then use SPACE to detect new patterns across varied datasets, including tumors and tuberculosis granulomas.

Confinement induces internal flows in adherent cell aggregates.

During mesenchymal migration, F-actin protrusion at the leading edge and actomyosin contraction determine the retrograde flow of F-actin within the lamella. The coupling of this flow to integrin-based adhesions determines the force transmitted to the extracellular matrix and the net motion of the cell. In tissues, motion may also arise from convection, driven by gradients in tissue-scale surface tensions and pressures. However, how migration coordinates with convection to determine the net motion of cellular ensembles is unclear. To explore this, we study the spreading of cell aggregates on adhesive micropatterns on compliant substrates. During spreading, a cell monolayer expands from the aggregate towards the adhesive boundary. However, cells are unable to stabilize the protrusion beyond the adhesive boundary, resulting in retraction of the protrusion and detachment of cells from the matrix. Subsequently, the cells move upwards and rearwards, yielding a bulk convective flow towards the centre of the aggregate. The process is cyclic, yielding a steady-state balance between outward (protrusive) migration along the surface, and 'retrograde' (contractile) flows above the surface. Modelling the cell aggregates as confined active droplets, we demonstrate that the interplay between surface tension-driven flows within the aggregate, radially outward monolayer flow and conservation of mass leads to an internal circulation.

Hippocampal Engrams and Contextual Memory.

Memories are not formed in a vacuum and often include rich details about the time and place in which events occur. Contextual stimuli promote the retrieval of events that have previously occurred in the encoding context and limit the retrieval of context-inappropriate information. Contexts that are associated with traumatic or harmful events both directly elicit fear and serve as reminders of aversive events associated with trauma. It has long been appreciated that the hippocampus is involved in contextual learning and memory and is central to contextual fear conditioning. However, little is known about the underlying neuronal mechanisms underlying the encoding and retrieval of contextual fear memories. Recent advancements in neuronal labeling methods, including activity-dependent tagging of cellular ensembles encoding memory ("engrams"), provide unique insight into the neural substrates of memory in the hippocampus. Moreover, these methods allow for the selective manipulation of memory ensembles. Attenuating or erasing fear memories may have considerable therapeutic value for patients with post-traumatic stress disorder or other trauma- or stressor-related conditions. In this chapter, we review the role of the hippocampus in contextual fear conditioning in rodents and explore recent work implicating hippocampal ensembles in the encoding and retrieval of aversive memories.

Engrams: From Behavior to Brain-Wide Networks.

Animals utilize a repertoire of behavioral responses during everyday experiences. During a potentially dangerous encounter, defensive actions such as "fight, flight, or freeze" are selected for survival. The successful use of behavior is determined by a series of real-time computations combining an animal's internal (i.e., body) and external (i.e., environment) state. Brain-wide neural pathways are engaged throughout this process to detect stimuli, integrate information, and command behavioral output. The hippocampus, in particular, plays a role in the encoding and storing of the episodic information surrounding these encounters as putative "engram" or experience-modified cellular ensembles. Recalling a negative experience then reactivates a dedicated engram ensemble and elicits a behavioral response. How hippocampus-based engrams modulate brain-wide states and an animal's internal/external milieu to influence behavior is an exciting area of investigation for contemporary neuroscience. In this chapter, we provide an overview of recent technological advancements that allow researchers to tag, manipulate, and visualize putative engram ensembles, with an overarching goal of casually connecting their brain-wide underpinnings to behavior. We then discuss how hippocampal fear engrams alter behavior in a manner that is contingent on an environment's physical features as well as how they influence brain-wide patterns of cellular activity. Overall, we propose here that studies on memory engrams offer an exciting avenue for contemporary neuroscience to casually link the activity of cells to cognition and behavior while also offering testable theoretical and experimental frameworks for how the brain organizes experience.

Use of artificial neural networks to identify and analyze polymerized actin‐based cytoskeletal structures in 3D confocal images

We propose an artificial neural network (ANN) as a kernel function of the recognizer of legitimate CABs from candidate CABs, that does not need human interventions. The performance of the recognizer shows noticeable recognition accuracy and addresses shortcomings of previous methods, including the need for human visual validation to recognize CABs from candidate CABs. Further, it helps to find and reduce errors resulting from human visual validation, which in turn would provide biologists/biophysicists a more comprehensive. understanding of a CAB.BackgroundLiving cells need to undergo subtle shape adaptations in response to the topography of their substrates. These shape changes are mainly determined by reorganization of their internal cytoskeleton, with a major contribution from filamentous (F) actin. Bundles of F‐actin play a major role in determining cell shape and their interaction with substrates, either as “stress fibers,” or as our newly discovered “Concave Actin Bundles” (CABs), which mainly occur while endothelial cells wrap micro‐fibers in culture.MethodsTo better understand the morphology and functions of these CABs, it is necessary to recognize and analyze as many of them as possible in complex cellular ensembles, which is a demanding and time‐consuming task. In this study, we present a novel algorithm to automatically recognize CABs without further human intervention. We developed and employed a multilayer perceptron artificial neural network (“the recognizer”), which was trained to identify CABs.ResultsThe recognizer demonstrated high overall recognition rate and reliability in both randomized training, and in subsequent testing experiments.ConclusionIt would be an effective replacement for validation by visual detection which is both tedious and inherently prone to errors.

THE ROLE OF INTERCELLULAR INTERACTIONS IN THE FORMATION OF KELOID SCARS

Pathological scarring at the present stage does not have a clear pathogenetic justification, it is characterized by unpredictability and constant growth of keloid scars, the absence of regression for many years and reasonable pathogenetic treatment. Despite the fact that there is the possibility of extirpation of excess tissue, it is almost impossible to remove keloid scars without recurrence. The paper presents phenotyping data in the structure of cellular ensembles of scars with the disclosure of signaling mechanisms of fibroblast differentiation into specialized cells that secrete collagen and the main matrix of the intercellular substance with pronounced hyalinization. It has been shown that the number of macrophages in the tissue of developing keloid scars is reduced in comparison with areas of normal reparative skin regeneration and in the structure of hypertrophic scars. Given the responsibility of macrophages for the secretion of angiogenesis factors and, accordingly, further oxygenation of the tissue healing zone, the authors point to a decrease in the number of macrophages as one of the main factors inducing keloid scarring and connective tissue metaplasia.

Bridging the In Vitro to In Vivo gap: Using the Chick Embryo Model to Accelerate Nanoparticle Validation and Qualification for In Vivo studies.

We postulate that nanoparticles (NPs) for use in therapeutic applications have largely not realized their clinical potential due to an overall inability to use in vitro results to predict NP performance in vivo. The avian embryo and associated chorioallantoic membrane (CAM) has emerged as an in vivo preclinical model that bridges the gap between in vitro and in vivo, enabling rapid screening of NP behavior under physiologically relevant conditions and providing a rapid, accessible, economical, and more ethical means of qualifying nanoparticles for in vivo use. The CAM is highly vascularized and mimics the diverging/converging vasculature of the liver, spleen, and lungs that serve as nanoparticle traps. Intravital imaging of fluorescently labeled NPs injected into the CAM vasculature enables immediate assessment and quantification of nano-bio interactions at the individual NP scale in any tissue of interest that is perfused with a microvasculature. In this review, we highlight how utilization of the avian embryo and its CAM as a preclinical model can be used to understand NP stability in blood and tissues, extravasation, biocompatibility, and NP distribution over time, thereby serving to identify a subset of NPs with the requisite stability and performance to introduce into rodent models and enabling the development of structure-property relationships and NP optimization without the sacrifice of large populations of mice or other rodents. We then review how the chicken embryo and CAM model systems have been used to accelerate the development of NP delivery and imaging agents by allowing direct visualization of targeted (active) and nontargeted (passive) NP binding, internalization, and cargo delivery to individual cells (of relevance for the treatment of leukemia and metastatic cancer) and cellular ensembles (e.g., cancer xenografts of interest for treatment or imaging of cancer tumors). We conclude by showcasing emerging techniques for the utilization of the CAM in future nano-bio studies.

Reactivating hippocampal-mediated memories during reconsolidation to disrupt fear

Memories are stored in the brain as cellular ensembles activated during learning and reactivated during retrieval. Using the Tet-tag system in mice, we label dorsal dentate gyrus neurons activated by positive, neutral or negative experiences with channelrhodopsin-2. Following fear-conditioning, these cells are artificially reactivated during fear memory recall. Optical stimulation of a competing positive memory is sufficient to update the memory during reconsolidation, thereby reducing conditioned fear acutely and enduringly. Moreover, mice demonstrate operant responding for reactivation of a positive memory, confirming its rewarding properties. These results show that interference from a rewarding experience can counteract negative affective states. While memory-updating, induced by memory reactivation, involves a relatively small set of neurons, we also find that activating a large population of randomly labeled dorsal dentate gyrus neurons is effective in promoting reconsolidation. Importantly, memory-updating is specific to the fear memory. These findings implicate the dorsal dentate gyrus as a potential therapeutic node for modulating memories to suppress fear.

Social reactivation of fear engrams enhances memory recall

For group-living animals, the social environment provides salient experience that can weaken or strengthen aspects of cognition such as memory recall. Although the cellular substrates of individually acquired fear memories in the dentate gyrus (DG) and basolateral amygdala (BLA) have been well-studied and recent work has revealed circuit mechanisms underlying the encoding of social experience, the processes by which social experience interacts with an individual’s memories to alter recall remain unknown. Here we show that stressful social experiences enhance the recall of previously acquired fear memories in male but not female mice, and that social buffering of conspecifics’ distress blocks this enhancement. Activity-dependent tagging of cells in the DG during fear learning revealed that these ensembles were endogenously reactivated during the social experiences in males, even after extinction. These reactivated cells were shown to be functional components of engrams, as optogenetic stimulation of the cells active during the social experience in previously fear-conditioned and not naïve animals was sufficient to drive fear-related behaviors. Taken together, our findings suggest that social experiences can reactivate preexisting engrams to thereby strengthen discrete memories.

What Neuroscientists Think, and Don't Think, About Consciousness.

The approach the majority of neuroscientists take to the question of how consciousness is generated, it is probably fair to say, is to ignore it. Although there are active research programs looking at correlates of consciousness, and explorations of informational properties of what might be relevant neural ensembles, the tacitly implied mechanism of consciousness in these approaches is that it somehow just happens. This reliance on a “magical emergence” of consciousness does not address the “objectively unreasonable” proposition that elements that have no attributes or properties that can be said to relate to consciousness somehow aggregate to produce it. Neuroscience has furnished evidence that neurons are fundamental to consciousness; at the fine and gross scale, aspects of our conscious experience depend on specific patterns of neural activity – in some way, the connectivity of neurons computes the features of our experience. So how do we get from knowing that some specific configurations of cells produce consciousness to understanding why this would be the case? Behind the voltages and currents electrophysiologists measure is a staggeringly complex system of electromagnetic fields – these are the fundamental physics of neurons and glia in the brain. The brain is entirely made of electromagnetism (EM) phenomena from the level of the atoms up. The EM field literally manifests the computations, or signaling, or information processing/activities performed by connected cellular ensembles that generate a 1st-person perspective. An investigation into the EM field at the cellular scale provides the possibility of identifying the outward signs of a mechanism in fundamental terms (physics), as opposed to merely describing the correlates of our mental abstractions of it.

In Vitro Assays for Measuring Intercellular Coupling Among Peripheral Circadian Oscillators.

Circadian clocks can be found in nearly all eukaryotic organisms, as well as certain bacterial strains, including commensal microbiota. Exploring intercellular coupling among cell-autonomous circadian oscillators is crucial for understanding how cellular ensembles generate and sustain coherent circadian rhythms on the tissue level, and thus, rhythmic organ functions. Here we describe a protocol for studying intercellular coupling among peripheral circadian oscillators using three-dimensional spheroid cultures in order to measure coupling strength within peripheral clock networks. We use cell spheroids to simulate in vivo tissue integrity, as well as to increase complexity of cell-cell interactions and the abundance of potential coupling factors. Circadian rhythms are monitored using live-cell imaging of spheroids equipped with circadian reporters over several days.

Label-Free, Real-Time Measurement of Metabolism of Adherent and Suspended Single Cells by In-Cell Fourier Transform Infrared Microspectroscopy.

We used infrared (IR) microscopy to monitor in real-time the metabolic turnover of individual mammalian cells in morphologically different states. By relying on the intrinsic absorption of mid-IR light by molecular components, we could discriminate the metabolism of adherent cells as compared to suspended cells. We identified major biochemical differences between the two cellular states, whereby only adherent cells appeared to rely heavily on glycolytic turnover and lactic fermentation. We also report spectroscopic variations that appear as spectral oscillations in the IR domain, observed only when using synchrotron infrared radiation. We propose that this effect could be used as a reporter of the cellular conditions. Our results are instrumental in establishing IR microscopy as a label-free method for real-time metabolic studies of individual cells in different morphological states, and in more complex cellular ensembles.

Neurophysiological mechanisms and features of autonomic support of productive cognitive activity of intuitive type in young adults

Is intuition a conscious or subconscious process, a “sixth sense” or a product of learning? This article contains an answer to this question which is based on the disclosure of neurophysiological mechanisms of one of the least understandable types of human cognitive activity. For the first time with the use of cerebral cortex electrical activity mapping, a comprehensive study of the organization of cortical neural networks and the functional state of the autonomic nervous system of female biology students engaged in intuitive thinking has been conducted. The productivity of intuitive thinking is associated with increasing the spectral power of beta2-, delta-, theta-components of the electroencephalogram. The synchronization of the activity of most frequency bands is manifested in the frontal and motor areas of the cortex, which have close connections with the basal ganglia, which are responsible for the formation of skills. In the beta1-band there are probable intrahemispheric long coherences between the anterior and posterior parts of the right hemisphere, at the frequency of beta1,2- and theta-bands of the electroencephalogram they are combined into large cellular ensembles that cover the central-parietal-temporal loci of the cortex. Synchronization of biopotentials in the delta range covers large areas of the cerebral cortex. It has been established that in intuitive thinking the female students with a low standard of efficiency had a high level of central rhythm regulation, and the female students with a high standard of efficiency had a high level of autonomous regulation. Higher performance under intuitive thinking negatively correlated with low frequency findings and positively correlated with high frequency and the value of the square root of the mean squares of the intervals between heartbeats (rMSSD) in the structure of cardiorhythm. Correlation analysis found that productive mental activity is conditioned by the specific integration of cortico-visceral processes: productive intuitive thinking is associated with the activation of autonomic regulation of heart rate variability and coherence in the evolutionarily older delta and delta-theta systems of the brain. Thus, intuition is a scientific set of skills and knowledge, and the topographic signs of synchronization of electrical processes of the cerebral cortex can serve as objective criteria for successful intuitive thinking, which allow one to predict both individual abilities and the state that contributes to their realization.

Anesthetics fragment hippocampal network activity, alter spine dynamics, and affect memory consolidation.

General anesthesia is characterized by reversible loss of consciousness accompanied by transient amnesia. Yet, long-term memory impairment is an undesirable side effect. How different types of general anesthetics (GAs) affect the hippocampus, a brain region central to memory formation and consolidation, is poorly understood. Using extracellular recordings, chronic 2-photon imaging, and behavioral analysis, we monitor the effects of isoflurane (Iso), medetomidine/midazolam/fentanyl (MMF), and ketamine/xylazine (Keta/Xyl) on network activity and structural spine dynamics in the hippocampal CA1 area of adult mice. GAs robustly reduced spiking activity, decorrelated cellular ensembles, albeit with distinct activity signatures, and altered spine dynamics. CA1 network activity under all 3 anesthetics was different to natural sleep. Iso anesthesia most closely resembled unperturbed activity during wakefulness and sleep, and network alterations recovered more readily than with Keta/Xyl and MMF. Correspondingly, memory consolidation was impaired after exposure to Keta/Xyl and MMF, but not Iso. Thus, different anesthetics distinctly alter hippocampal network dynamics, synaptic connectivity, and memory consolidation, with implications for GA strategy appraisal in animal research and clinical settings.

A Molecular Calcium Integrator Reveals a Striatal Cell Type Driving Aversion

The ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here, we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCRE's modular design, we expressed an optogenetic channel selectively in this cell type and showed that direct recruitment of this otherwise genetically inaccessible population elicits behavioral aversion. The specificity and minute resolution of FLiCRE enables molecularly informed characterization, manipulation, and reprogramming of activated cellular ensembles.

Exploring the interdependence between self-organization and functional morphology in cellular systems.

All living matter is subject to continuous adaptation and functional optimization via natural selection. Consequentially, structures with close morphological resemblance repeatedly appear across the phylogenetic tree. How these designs emerge at the cellular level is not fully understood. Here, we explore core concepts of functional morphology and discuss its cause and consequences, with a specific focus on emerging properties of self-organizing systems as the potential driving force. We conclude with open questions and limitations that are present when studying shape-function interdependence in single cells and cellular ensembles.

Dynamic and Programmable Cellular-Scale Granules Enable Tissue-like Materials

Summary Living tissues are an integrated, multiscale architecture consisting of dense cellular ensembles and extracellular matrices (ECMs). The cells and ECMs cooperate to enable specialized mechanical properties and dynamic responsiveness. However, the mechanical properties of living tissues are difficult to replicate. A particular challenge is identification of a cell-like synthetic component, which is tightly integrated with its matrix and also responsive to external stimuli. Here, we demonstrate that cellular-scale hydrated starch granules, an underexplored component in materials science, can turn conventional hydrogels into tissue-like materials when composites are formed. By using several synchrotron-based X-ray techniques, we reveal the mechanically induced organization and training dynamics of the starch granules in the hydrogel matrix. These dynamic behaviors enable multiple tissue-like properties such as programmability, anisotropy, strain-stiffening, mechanochemistry, and self-healability.

On a Simple General Principle of Brain Organization.

A possible framework to characterize nervous system dynamics and its organization in conscious and unconscious states is introduced, derived from a high level perspective on the coordinated activity of brain cell ensembles. Some questions are best addressable in a global framework and here we build on past observations about the structure of configurations of brain networks in conscious and unconscious states and about neurophysiological results. Aiming to bind some results together into some sort of coherence with a central theme, the scenario that emerges underscores the crucial importance of the creation and dissipation of energy gradients in brain cellular ensembles resulting in maximization of the configurations in the functional connectivity among those networks that favor conscious awareness and healthy conditions. These considerations are then applied to indicate approaches that can be used to improve neuropathological syndromes.

Angularly resolved, finely sampled elastic scattering measurements of single cells: requirements for robust organelle size extractions.

.Angularly resolved elastic light scattering is an established technique for probing the average size of organelles in biological tissue and cellular ensembles. Focusing of the incident light to illuminate no more than one cell at a time restricts the minimum forward-scattering angle that can be detected. Series of simulated single-cell angular-scattering patterns have been generated to explore how size estimates vary as a function of . At a setting of , the size estimates hop unstably between multiple minima in the solution space as simulated noise (mimicking experimentally observed levels) is varied. As is reduced from 20 deg to 10 deg, the instability vanishes, and the variance of estimates near the correct answer also decreases. The simulations thus suggest that robust Mie theory fits to single-cell scattering at 785 nm excitation require measurements down to at least 15 deg. Notably, no such instability was observed at for narrow bead distributions. Accurate sizing of traditional calibration beads is, therefore, insufficient proof that an angular-scattering system is capable of robust analysis of single cells. Experimental support for the simulation results is also presented using measurements on cells fixed with formaldehyde.