Transient Cluster Research Articles

Continuity of Short-Time Dynamics Crossing the Liquid-Liquid Phase Separation in Charge-Tuned Protein Solutions.

Liquid-liquid phase separation (LLPS) constitutes a crucial phenomenon in biological self-organization, not only intervening in the formation of membraneless organelles but also triggering pathological protein aggregation, which is a hallmark in neurodegenerative diseases. Employing incoherent quasi-elastic neutron spectroscopy (QENS), we examine the short-time self-diffusion of a model protein undergoing LLPS as a function of phase splitting and temperature to access information on the nanosecond hydrodynamic response to the cluster formation both within and outside the LLPS regime. We investigate the samples as they dissociate into microdroplets of a dense protein phase dispersed in a dilute phase as well as the separated dense and dilute phases obtained from centrifugation. By interpreting the QENS results in terms of the local concentrations in the two phases determined by UV-vis spectroscopy, we hypothesize that the short-time transient protein cluster size distribution is conserved at the transition point while the local volume fractions separate.

Diffusion and Viscosity in Mixed Protein Solutions.

The viscosity and diffusion properties of crowded protein systems were investigated with molecular dynamics simulations of SH3 mixtures with different crowders, and results were compared with experimental data. The simulations accurately reproduced experimental trends across a wide range of protein concentrations, including highly crowded environments up to 300 g/L. Notably, viscosity increased with crowding but varied little between different crowder types, while diffusion rates were significantly reduced depending on protein-protein interaction strength. Analysis using the Stokes-Einstein relation indicated that the reduction in diffusion exceeded what was expected from viscosity changes alone, with the additional slow-down attributable to transient cluster formation driven by weakly attractive interactions. Contact kinetics analysis further revealed that longer-lived interactions contributed more significantly to reduced diffusion rates than short-lived interactions. This study also highlights the accuracy of current computational methodologies for capturing the dynamics of proteins in highly concentrated solutions and provides insights into the molecular mechanisms affecting protein mobility in crowded environments.

Diffusion and Viscosity in Mixed Protein Solutions.

The viscosity and diffusion properties of crowded protein systems were investigated with molecular dynamics simulations of SH3 mixtures with different crowders, and results were compared with experimental data. The simulations accurately reproduced experimental trends across a wide range of protein concentrations, including highly crowded environments up to 300 g/L. Notably, viscosity increased with crowding but varied little between different crowder types, while diffusion rates were significantly reduced depending on protein-protein interaction strength. Analysis using the Stokes-Einstein relation indicated that the reduction in diffusion exceeded what was expected from viscosity changes alone, with the additional slow-down attributable to transient cluster formation driven by weakly attractive interactions. Contact kinetics analysis further revealed that longer-lived interactions contributed more significantly to reduced diffusion rates than short-lived interactions. This study also highlights the accuracy of current computational methodologies for capturing the dynamics of proteins in highly concentrated solutions and provides insights into the molecular mechanisms affecting protein mobility in crowded environments.

What does it take to stabilize a naphthalene anion?

We investigate attachment of slow electrons (0-10eV) to naphthalene (Np) clusters in a crossed beam experiment. Supersonic expansions under different conditions using different buffer gases generate the clusters: in He, Ne, and low pressure Ar, neat (Np)N clusters are formed, while we also observe mixed clusters of naphthalene with rare-gas atoms in co-expansion with Ar above 0.5bar and with Kr. Negatively charged (Np)n- and Rgm(Np)n- (Rg = Ar, Kr) clusters are analyzed by mass spectrometry, and electron energy dependent ion yields are measured. We show that the smallest stable naphthalene complex with an excess electron, the dimer (Np)2- anion, cannot be formed in a binary collision of a free electron with (Np)2 dimer, nor with (Np)3 trimer. Evaporation of a weakly bound Ar atom(s) from a mixed ArM(Np)2 cluster following electron attachment leads to the dimer (Np)2- anion. Larger (Np)n-, n > 3, transient cluster anions decay via evaporation of an Np unit on a timescale of tens of microseconds. The self-scavenging process opens around 6eV, where a naphthalene unit is electronically excited by the incoming electron, which is slowed down and trapped. However, the transient negative ion is efficiently stabilized only in the mixed clusters, from which Ar atom(s) can be evaporated.

Boundary cap neural crest stem cells promote angiogenesis after transplantation to avulsed dorsal roots in mice and induce migration of endothelial cells in 3D printed scaffolds

Dorsal root avulsion injuries lead to loss of sensation and to reorganization of blood vessels (BVs) in the injured area. The inability of injured sensory axons to re-enter the spinal cord results in permanent loss of sensation, and often also leads to the development of neuropathic pain. Approaches that restore connection between peripheral sensory axons and their CNS targets are thus urgently need. Previous research has shown that sensory axons from peripherally grafted human sensory neurons are able to enter the spinal cord by growing along BVs which penetrate the CNS from the spinal cord surface. In this study we analysed the distribution of BVs after avulsion injury and how their pattern is affected by implantation at the injury site of boundary cap neural crest stem cells (bNCSCs), a transient cluster of cells, which are located at the boundary between the spinal cord and peripheral nervous system and assist the growth of sensory axons from periphery into the spinal cord during development. The superficial dorsal spinal cord vasculature was examined using intravital microscopy and intravascular BV labelling. bNCSC transplantation increased vascular volume in a non-dose responsive manner, whereas dorsal root avulsion alone did not decrease the vascular volume. To determine whether bNCSC are endowed with angiogenic properties we prepared 3D printed scaffolds, containing bNCSCs together with rings prepared from mouse aorta. We show that bNCSC do induce migration and assembly of endothelial cells in this system. These findings suggest that bNCSC transplant can promote vascularization in vivo and contribute to BV formation in 3D printed scaffolds.

Bioinspired controllable CaCO3 synthesis from solid waste by an “all in one” amino acid-in strategy: Implication for CO2 mineralization

CO2 mineralization suffers from extensive consumption of acidic/alkaline reagents due to the limited kinetics, which necessitates the development of an atom/energy-efficient process. An “all in one” reagent amino acid-based Ca2+ leaching-CO2 mineralization process inspired by biomineralization was proposed. This study explored the interactions between amino acids and Ca leaching, CaCO3 pre-nucleation, nucleation, and growth by investigating the evolution of Ca concentration, solution turbidity and CaCO3 morphology in complex CFA. Promising Ca2+ leaching efficiencies were observed, being 51.1 %, 34.2 %, and 21.0 % for glycine (Gly), L-alanine (Ala), and L-arginine (Arg), respectively, negatively correlated with their pI but positively correlated with their bonding energy with Ca2+. These amino acids ultimately led to a CaCO3 production of Gly (114.8 g/kg) > Ala (78.2 g/kg) > Arg (54.5 g/kg) from CFA. Gly leachate remained transparent (1.23 NTU) in the first 3 min of CO2 bubbling even though the solution was supersaturated and then got turbid suddenly (6508.75 NTU in 10 min) as continued CO2 bubbling, indicating the inhibition of Gly on CaCO3 nucleation. Observations using Cryo-SEM confirm the presence of abundant CaCO3 nanoparticles in the early stages of mineralization, which later assemble into vaterite crystal particles with a sub-structure and coalesce into larger particles in Gly and Ala. Meanwhile, Arg inhibited calcite growth along the C-axis, leading to the formation of thin, blocky-like particles. Furthermore, 0.010 ∼ 0.027 mmol/g amino acids are occluded into the CaCO3, stabilizing the metastable vaterites and imparting thermal stability, which was believed to be associated with this multistep pathway involving a transient prenucleation cluster, nanoparticle precursor formation, and particle–particle coalescence.

An unsupervised real-time spike sorting system based on optimized OSort

Objective. The OSort algorithm, a pivotal unsupervised spike sorting method, has been implemented in dedicated hardware devices for real-time spike sorting. However, due to the inherent complexity of neural recording environments, OSort still grapples with numerous transient cluster occurrences during the practical sorting process. This leads to substantial memory usage, heavy computational load, and complex hardware architectures, especially in noisy recordings and multi-channel systems. Approach. This study introduces an optimized OSort algorithm (opt-OSort) which utilizes correlation coefficient (CC), instead of Euclidean distance as classification criterion. The CC method not only bolsters the robustness of spike classification amidst the diverse and ever-changing conditions of physiological and recording noise environments, but also can finish the entire sorting procedure within a fixed number of cluster slots, thus preventing a large number of transient clusters. Moreover, the opt-OSort incorporates two configurable validation loops to efficiently reject cluster outliers and track recording variations caused by electrode drifting in real-time. Main results. The opt-OSort significantly reduces transient cluster occurrences by two orders of magnitude and decreases memory usage by 2.5–80 times in the number of pre-allocated transient clusters compared with other hardware implementations of OSort. The opt-OSort maintains an accuracy comparable to offline OSort and other commonly-used algorithms, with a sorting time of 0.68 µs as measured by the hardware-implemented system in both simulated datasets and experimental data. The opt-OSort’s ability to handle variations in neural activity caused by electrode drifting is also demonstrated. Significance. These results present a rapid, precise, and robust spike sorting solution suitable for integration into low-power, portable, closed-loop neural control systems and brain–computer interfaces.

Self-Assembling Peptide-Appended Metallomacrocycle Pores for Selective Water Translocation.

Artificial water channels selectively transport water, excluding all ions. Unimolecular channels have been synthesized via complex synthetic steps. Ideally, simpler compounds requesting less synthetic steps should efficiently lead to selective channels by self-assembly. Herein, we report a self-assembled peptide-bound Ni2+ metallomacrocycle, 1, in which rim-peptide-bound units are connected to a central macrocycle obtained via condensation in the presence of Ni2+ ions. Compound 1 achieves a single-channel permeability up to 107-108 water/s/channel and insignificant ion transport, which is 1 order of magnitude lower than those for aquaporins. Molecular simulations probe that spongelike aggregates can form to generate transient cluster water pathways through the bilayer. Altogether, adaptive metallosupramolecular self-assembly is an efficient and simple way to construct selective channel superstructures.

Urban Mobility and Knowledge Extraction from Chaotic Time Series Data: A Comparative Analysis for Uncovering COVID-19 Effects

The COVID-19 pandemic exerted devastating effects on the global economy, public health, and urban life. The geographical consequences lie in spatial time series dimensions, but the chaotic characteristics hinder intuitive understanding. Aiming to discover the variance of urban mobility in the pandemic context, this article presents a hybrid clustering technique called whole time sequence mixture (WTSM). The combination of whole time and subsequence techniques ensures robustness to data volume, dimensionality, sampling, distortion tasks, and a prior constraint. For yellow taxi trips in New York City, the case study spanned the pre-, mid-, and postpandemic periods and compared the performance of the established methods (symbolic aggregate approximation and dynamic time warping) and WTSM techniques. Findings revealed COVID-19 trends and social restriction-induced mobility variations and determined that vaccine supply did not lead to immediate mobility restoration. Meanwhile, concepts of validation indexes and computational complexity corroborate the superiority of WTSM. The transient cluster arising from temporal dissimilarity is a unique finding of WTSM, which led to high cluster cohesion and separation. WTSM can obtain new knowledge and rationale for urgent government intervention and epidemic management, not sacrificing computational efficiency and clustering quality. With the improved storage capability and desire for multidimensional pattern extraction, the increased accessibility to the chaotic time series data sets could promote further studies and help administrative schemes.

Early childhood wheezing phenotypes and determinants in a South African birth cohort: longitudinal analysis of the Drakenstein Child Health Study

Developmental trajectories of childhood wheezing in low-income and middle-income countries (LMICs) have not been well described. We aimed to derive longitudinal wheeze phenotypes from birth to 5 years in a South African birth cohort and compare those with phenotypes derived from a UK cohort. We used data from the Drakenstein Child Health Study (DCHS), a longitudinal birth cohort study in a peri-urban area outside Cape Town, South Africa. Pregnant women (aged ≥18 years) were enrolled during their second trimester at two public health clinics. We followed up children from birth to 5 years to derive six multidimensional indicators of wheezing (including duration, temporal sequencing, persistence, and recurrence) and applied Partition Around Medoids clustering to derive wheeze phenotypes. We compared phenotypes with a UK cohort (the Avon Longitudinal Study of Parents and Children [ALSPAC]). We investigated associations of phenotypes with early-life exposures, including all-cause lower respiratory tract infection (LRTI) and virus-specific LRTI (respiratory syncytial virus, rhinovirus, adenovirus, influenza, and parainfluenza virus) up to age 5 years. We investigated the association of phenotypes with lung function at 6 weeks and 5 years. Between March 5, 2012, and March 31, 2015, we enrolled 1137 mothers and there were 1143 livebirths. Four wheeze phenotypes were identified among 950 children with complete data: never (480 children [50%]), early transient (215 children [23%]), late onset (104 children [11%]), and recurrent (151 children [16%]). Multivariate adjusted analysis indicated that LRTI and respiratory syncytial virus-LRTI, but not other respiratory viruses, were associated with increased risk of recurrent wheeze (odds ratio [OR] 2·79 [95% CI 2·05-3·81] for all LTRIs; OR 2·59 [1·30-5·15] for respiratory syncytial virus-LRTIs). Maternal smoking (1·88 [1·12-3·02]), higher socioeconomic status (2·46 [1·23-4·91]), intimate partner violence (2·01 [1·23-3·29]), and male sex (2·47 [1·50-4·04]) were also associated with recurrent wheeze. LRTI and respiratory syncytial virus-LRTI were also associated with early transient and late onset clusters. Wheezing illness architecture differed between DCHS and ALSPAC; children included in ALSPAC in the early transient cluster wheezed for a longer period before remission and late-onset wheezing started at an older age, and no persistent phenotype was identified in DCHS. At 5 years, airway resistance was higher in children with early or recurrent wheeze compared with children who had never wheezed. Airway resistance increased from 6 weeks to 5 years among children with recurrent wheeze. Effective strategies to reduce maternal smoking and psychosocial stressors and new preventive interventions for respiratory syncytial virus are urgently needed to optimise child health in LMICs. UK Medical Research Council; The Bill & Melinda Gates Foundation; National Institutes of Health Human Heredity and Health in Africa; South African Medical Research Council; Wellcome Trust.

Kölliker's organ-supporting cells and cochlear auditory development.

The Kölliker’s organ is a transient cellular cluster structure in the development of the mammalian cochlea. It gradually degenerates from embryonic columnar cells to cuboidal cells in the internal sulcus at postnatal day 12 (P12)–P14, with the cochlea maturing when the degeneration of supporting cells in the Kölliker’s organ is complete, which is distinct from humans because it disappears at birth already. The supporting cells in the Kölliker’s organ play a key role during this critical period of auditory development. Spontaneous release of ATP induces an increase in intracellular Ca2+ levels in inner hair cells in a paracrine form via intercellular gap junction protein hemichannels. The Ca2+ further induces the release of the neurotransmitter glutamate from the synaptic vesicles of the inner hair cells, which subsequently excite afferent nerve fibers. In this way, the supporting cells in the Kölliker’s organ transmit temporal and spatial information relevant to cochlear development to the hair cells, promoting fine-tuned connections at the synapses in the auditory pathway, thus facilitating cochlear maturation and auditory acquisition. The Kölliker’s organ plays a crucial role in such a scenario. In this article, we review the morphological changes, biological functions, degeneration, possible trans-differentiation of cochlear hair cells, and potential molecular mechanisms of supporting cells in the Kölliker’s organ during the auditory development in mammals, as well as future research perspectives.

Dynamic Stability of Copper Single-Atom Catalysts under Working Conditions.

The long-term stability of single-atom catalysts is a major factor affecting their large-scale commercial application. How to evaluate the dynamic stability of single-atom catalysts under working conditions is still lacking. Here, taking a single copper atom embedded in N-doped graphene as an example, the "constant-potential hybrid-solvation dynamic model" is used to evaluate the reversible transformation between copper single atoms and clusters under realistic reaction conditions. It is revealed that the adsorption of H is a vital driving force for the leaching of the Cu single atom from the catalyst surface. The more negative the electrode potential, the stronger the adsorption of H. As a result, the competitive hydrogen evolution reaction is inhibited, and Cu-N bonds are weakened, resulting in some Cu atoms being tethered on the catalyst surface and some being dissolved in the aqueous solution. The collision of the Cu atoms in the two states forms a transient Cu cluster structure as a true catalytic active site to promote CO2 reduction to ethanol. As the applied potential is released or switched to a positive value, hydroxyl radicals (OH•) play a dominant role in the oxidation process of the Cu cluster, and then Cu returns to the initial atomic dispersion state by redeposition, completing the reconstruction cycle of the copper catalyst. Our work provides a fundamental understanding of the dynamic stability of Cu single-atom catalysts under working conditions at the atomic level and calls for a reassessment of the stability of currently reported single-atom catalysts considering realistic reaction conditions.

Charge-State Dependence of Proton Uptake in Polyoxovanadate-alkoxide Clusters.

Here, we present an investigation of the thermochemistry of proton uptake in acetonitrile across three charge states of a polyoxovanadate-alkoxide (POV-alkoxide) cluster, [V6O7(OMe)12]n (n = 2–, 1–, and 0). The vanadium oxide assembly studied features bridging sites saturated by methoxide ligands, isolating protonation to terminal vanadyl moieties. Exposure of [V6O7(OMe)12]n to organic acids of appropriate strength results in the protonation of a terminal V=O bond, generating the transient hydroxide-substituted POV-alkoxide cluster [V6O6(OH)(OMe)12]n+1. Evidence for this intermediate proved elusive in our initial report, but here we present the isolation of a divalent anionic cluster that features hydrogen bonding to dimethylammonium at the terminal oxo site. Degradation of the protonated species results in the formation of equimolar quantities of one-electron-oxidized and oxygen-atom-efficient complexes, [V6O7(OMe)12]n+1 and [V6O6(OMe)12]n+1. While analogous reactivity was observed across the three charge states of the cluster, a dependence on the acid strength was observed, suggesting that the oxidation state of the vanadium oxide assembly influences the basicity of the cluster surface. Spectroscopic investigations reveal sigmoidal relationships between the acid strength and cluster conversion across the redox series, allowing for determination of the proton affinity of the surface of the cluster in all three charge states. The fully reduced cluster is found to be the most basic, with higher oxidation states of the assembly possessing substantially reduced proton affinities (∼7 pKa units per electron). These results further our understanding of the site-specific reactivity of terminal M=O bonds with protons in an organic solvent, revealing design criteria for engineering functional surfaces of metal oxide materials of relevance to energy storage and conversion.

Structural Basis of RICs Iron Donation for Iron-Sulfur Cluster Biogenesis.

Escherichia coli YtfE is a di-iron protein of the widespread Repair of Iron Centers proteins (RIC) family that has the capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. In this work we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron center to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters.

Hydroxy Channels-Adaptive Pathways for Selective Water Cluster Permeation.

Artificial water channels (AWCs) are known to selectively transport water, with ion exclusion. Similarly to natural porins, AWCs encapsulate water wires or clusters, offering continuous and iterative H-bonding that plays a vital role in their stabilization. Herein, we report octyl-ureido-polyol AWCs capable of self-assembly into hydrophilic hydroxy channels. Variants of ethanol, propanediol, and trimethanol are used as head groups to modulate the water transport permeabilities, with rejection of ions. The hydroxy channels achieve a single-channel permeability of 2.33 × 108 water molecules per second, which is within the same order of magnitude as the transport rates for aquaporins. Depending on their concentration in the membrane, adaptive channels are observed in the membrane. Over increased concentrations, a significant shift occurs, initiating unexpected higher water permeation. Molecular simulations probe that spongelike or cylindrical aggregates can form to generate transient cluster water pathways through the bilayer. Altogether, the adaptive self-assembly is a key feature influencing channel efficiency. The adaptive channels described here may be considered an important milestone contributing to the systematic discovery of artificial water channels for water desalination.

Insight into acrylate copolymer dispersion with multiple interactions using large-amplitude oscillation shear

The rheological behavior of hydroxyethyl acrylate sodium/acryloyldimethyl taurate copolymer aqueous dispersions with multiple interactions (hydrogen-bonding, van der Waals and electrostatic interactions) is investigated. An abnormal large-amplitude oscillation shear performance is observed, i.e., the linear viscoelastic region shortens with an increase in concentration, whereas the weak-strain overshoot moves to a high strain region. The synergistic effect of the three interactions facilitates the hydrophobic interaction and the formation of strong and enough clusters. Both the entire transient network structure relaxation and cluster relaxation (deformation, rupture, recovery, and friction) play vital roles in this unique response. Significant cluster relaxation is believed to have led to the abnormal weak-strain overshoot. The experimental results are similar to those derived from an anticipation based on friction dissipation of blobs inside clusters, which implies that the weak-strain overshoot exactly stems from the clusters relaxing and the associated energy dissipation. The mechanism proposed here offers an innovative analytical technique for complex biological fluids.

Clustering and Fibril Formation during GNNQQNY Aggregation: A Molecular Dynamics Study

The precise kinetic pathways of peptide clustering and fibril formation are not fully understood. Here we study the initial clustering kinetics and transient cluster morphologies during aggregation of the heptapeptide fragment GNNQQNY from the yeast prion protein Sup35. We use a mid-resolution coarse-grained molecular dynamics model of Bereau and Deserno to explore the aggregation pathways from the initial random distribution of free monomers to the formation of large clusters. By increasing the system size to 72 peptides we could follow directly the molecular events leading to the formation of stable fibril-like structures. To quantify those structures we developed a new cluster helicity parameter. We found that the formation of fibril-like structures is a cooperative processes that requires a critical number of monomers, , in a cluster. The terminal tyrosine residue is the structural determinant in the formation of helical fibril-like structures. This work supports and quantifies the two-step aggregation model where the initially formed amorphous clusters grow and, when they are large enough, rearrange into mature twisted structures. However, in addition to the nucleated fibrillation, growing aggregates undergo further internal reorganization, which leads to more compact structures of large aggregates.

Identification of different cell clusters in the endothelium of atherosclerotic vessels and determination of inter-cluster gradient of proliferative and inflammatory activity as new diagnostic markers

ABSTRACT To characterize atherogenesis functionally, we studied the functional heterogeneity of endotheliocytes in carotid vessels with atherosclerotic plaques and identified several distinct cell clusters. We measured the Ki-67 labeling index (Ki-67 LI), percentage of Bcl-2 cells (CP) and expression of CCL5, IL 6 and VCAM1 in each cell cluster. We also investigated how these indicators change when the plaque becomes unstable and how they affect the risk of adverse cerebrovascular events in patients. We evaluated the inter-cluster gradient of marker activity and its relation to patient prognosis. We identified five endothelial clusters: the under plaque cluster (UPC), peripheral cluster (PC), marginal cluster (MC), transient cluster (TC) and outside plaque cluster (OC). The UPC exhibited the greatest proliferative, proinflammatory and adhesive activity, but low anti-apoptotic activity. The PC exhibited the second greatest proliferative, adhesive and proinflammatory activity. Progression of atherosclerosis and transition of a stable atherosclerotic plaque to an unstable one was accompanied by increased expression of nearly all markers. The proliferative activity in the UPC, PC and OC, and the pro-inflammatory activity in UPC and anti-apoptotic activity in the PC, were correlated with prognosis. Also, two gradients of proliferative activity and a gradient of pro-inflammatory activity were associated with risk of adverse events.

Epicardium in Heart Development.

The epicardium, the outermost tissue layer that envelops all vertebrate hearts, plays a crucial role in cardiac development and regeneration and has been implicated in potential strategies for cardiac repair. The heterogenous cell population that composes the epicardium originates primarily from a transient embryonic cell cluster known as the proepicardial organ (PE). Characterized by its high cellular plasticity, the epicardium contributes to both heart development and regeneration in two critical ways: as a source of progenitor cells and as a critical signaling hub. Despite this knowledge, there are many unanswered questions in the field of epicardial biology, the resolution of which will advance the understanding of cardiac development and repair. We review current knowledge in cross-species epicardial involvement, specifically in relation to lineage specification and differentiation during cardiac development.

Heterogeneity of tumor cells in glioblastomas

To comprehensively assess the functional molecular biological status of different tumor cell populations in glioblastoma samples. The activity of Ki-67, Bcl-2, and BCL6 expression was determined in 20 tumor samples from patients with glioblastoma. After that, a spatial analysis of heterogeneity in the expression of these markers in different tumor cell populations was carried out using computer and software tools and calculating the percentage of cells (PC) expressing this marker (Ki-67 labeling index (LI)) and a modified histoscore in different cell clusters. Analysis of heterogeneity in the distribution of Ki-67, Bcl-2, and BCL6 expression could identify five cell clusters differing in the expression level of the above-mentioned markers. The most active cluster was the perivascular one (the highest mean Ki-67 LI (22.23±1.4%) and histoscore (118.59±3.36%); BCL6 PC and histoscore (17.4±1.4 and 79.32±4.86%, respectively). The least proliferative activity was observed in the perinecrotic cluster (Ki-67 LI (6.83±0.5%) and histoscore (62.46±2.25%)), while the neighboring transient necrotic cluster displayed sufficiently active proliferative processes (Ki-67 LI (18.39±0.56%) and histoscore (112.65±2.76%)). In addition, the transient vascular cluster was noted for a low proliferative activity (Ki-67 LI (8.37±0.35%) and histoscore (75.48±2.04%)). Finally, the intermediate cluster was characterized by the mean values of all parameters (Ki-67 LI (10.68±0.39%) and histoscore (95.73±2.37%). It should be noted that the differences in the expression activity for markers in these clusters were statistically significant. The perivascular cluster carries the greatest potential for tumor progression and recurrence, which agrees with the data available in the literature: the perivascular zone is the most important niche for glioma stem cells that make a considerable contribution to the malignant potential of glioblastoma. Tumor pathogenesis and morphogenesis are a complex interweaving of interrelated factors, the realization of which within the framework of a multi-level heterochronic pathological process leads to the segregation of tumor cells and to the appearance of separate cell populations described in this paper.