Safari with an Electron Gun: Visualization of Protein and Membrane Interactions in Mitochondria in Natural Environment.

This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari. The paper highlights two areas of research that can only be accomplished using these methods. The study visualized location of the Aß42 amyloid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in Alzheimer's disease. The results showed that the Aß42 aggregates do not interact with mitochondria, although some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not directly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-resolution tomography expands and complements existing ideas about the structural and functional organization of mitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with each other and with membranes in vivo.

PMF-seq: a highly scalable screening strategy for linking genetics to mitochondrial bioenergetics.

Our current understanding of mitochondrial organelle physiology has benefited from two broad approaches: classically, cuvette-based measurements with suspensions of isolated mitochondria, in which bioenergetic parameters are monitored acutely in response to respiratory chain substrates and inhibitors1-4, and more recently, highly scalable genetic screens for fitness phenotypes associated with coarse-grained properties of the mitochondrial state5-10. Here we introduce permeabilized-cell mitochondrial function sequencing (PMF-seq) to combine strengths of these two approaches to connect genes to detailed bioenergetic phenotypes. In PMF-seq, the plasma membranes within a pool of CRISPR mutagenized cells are gently permeabilized under conditions that preserve mitochondrial physiology, where detailed bioenergetics can be probed in the same way as with isolated organelles. Cells with desired bioenergetic parameters are selected optically using flow cytometry and subjected to next-generation sequencing. Using PMF-seq, we recover genes differentially required for mitochondrial respiratory chain branching and reversibility. We demonstrate that human D-lactate dehydrogenase specifically conveys electrons from D-lactate into cytochrome c to support mitochondrial membrane polarization. Finally, we screen for genetic modifiers of tBID, a pro-apoptotic protein that acts directly and acutely on mitochondria. We find the loss of the complex V assembly factor ATPAF2 acts as a genetic sensitizer of tBID's acute action. We anticipate that PMF-seq will be valuable for defining genes critical to the physiology of mitochondria and other organelles.

Photoinduced Transition from Quasi-Two-Dimensional Ruddlesden-Popper to Three-Dimensional Halide Perovskites for the Optical Writing of Multicolor and Light-Erasable Images.

Optical data storage, information encryption, and security labeling technologies require materials that exhibit local, pronounced, and diverse modifications of their structure-dependent optical properties under external excitation. Herein, we propose and develop a novel platform relying on lead halide Ruddlesden-Popper phases that undergo a light-induced transition toward bulk perovskite and employ this phenomenon for the direct optical writing of multicolor patterns. This transition causes the weakening of quantum confinement and hence a reduction in the band gap. To extend the color gamut of photoluminescence, we use mixed-halide compositions that exhibit photoinduced halide segregation. The emission of the films can be tuned across the range of 450-600 nm. Laser irradiation provides high-resolution direct writing, whereas continuous-wave ultraviolet exposure is suitable for recording on larger scales. The luminescent images created on such films can be erased during the visualization process. This makes the proposed writing/erasing platform suitable for the manufacturing of optical data storage devices and light-erasable security labels.

Association of 2-oxoacid dehydrogenase complexes with respirasomes in mitochondria.

In the present study, cryo-electron tomography was used to investigate the localization of 2-oxoacid dehydrogenase complexes (OADCs) in cardiac mitochondria and mitochondrial inner membrane samples. Two classes of ordered OADC inner cores with different symmetries were distinguished and their quaternary structures modeled. One class corresponds to pyruvate dehydrogenase complexes and the other to dehydrogenase complexes of α-ketoglutarate and branched-chain α-ketoacids. OADCs were shown to be localized in close proximity to membrane-embedded respirasomes, as observed both in densely packed lamellar cristae of cardiac mitochondria and in ruptured mitochondrial samples where the dense packing is absent. This suggests the specificity of the OADC-respirasome interaction, which allows localized NADH/NAD+ exchange between OADCs and complex I of the respiratory chain. The importance of this local coupling is based on OADCs being the link between respiration, glycolysis and amino acid metabolism. The coupling of these basic metabolic processes can vary in different tissues and conditions and may be involved in the development of various pathologies. The present study shows that this important and previously missing parameter of mitochondrial complex coupling can be successfully assessed using cryo-electron tomography.

Directed proton transfer from Fo to F1 extends the multifaceted proton functions in ATP synthase.

The role of protons in ATP synthase is typically considered to be energy storage in the form of an electrochemical potential, as well as an operating element proving rotation. However, this review emphasizes that protons also act as activators of conformational changes in F1 and as direct participants in phosphorylation reaction. The protons transferred through Fo do not immediately leave to the bulk aqueous phase, but instead provide for the formation of a pH gradient between acidifying Fo and alkalizing F1. It facilitates a directed inter-subunit proton transfer to F1, where they are used in the ATP synthesis reaction. This ensures that the enzyme activity is not limited by a lack of protons in the alkaline mitochondrial matrix or chloroplast stroma. Up to one hundred protons bind to the carboxyl groups of the F1 subunit, altering the electrical interactions between the amino acids of the enzyme. This removes the inhibition of ATP synthase caused by the electrostatic attraction of charged amino acids of the stator and rotor and also makes the enzyme more prone to conformational changes. Protonation occurs during ATP synthesis initiation and during phosphorylation, while deprotonation blocks the rotation inhibiting both synthesis and hydrolysis. Thus, protons participate in the functioning of all main components of ATP synthase molecular machine making it effectively a proton-driven electric machine. The review highlights the key role of protons as a coupling factor in ATP synthase with multifaceted functions, including charge and energy transport, torque generation, facilitation of conformational changes, and participation in the ATP synthesis reaction.

Quantifying dynamic pro-inflammatory gene expression and heterogeneity in single macrophage cells.

Macrophages must respond appropriately to pathogens and other pro-inflammatory stimuli in order to perform their roles in fighting infection. One way in which inflammatory stimuli can vary is in their dynamics-that is, the amplitude and duration of stimulus experienced by the cell. In this study, we performed long-term live cell imaging in a microfluidic device to investigate how the pro-inflammatory genes IRF1, CXCL10, and CXCL9 respond to dynamic interferon-gamma (IFNγ) stimulation. We found that IRF1 responds to low concentration or short duration IFNγ stimulation, whereas CXCL10 and CXCL9 require longer or higherconcentration stimulation to be expressed. We also investigated the heterogeneity in the expression of each gene and found that CXCL10 and CXCL9 have substantial cell-to-cell variability. In particular, the expression of CXCL10 appears to be largely stochastic with a subpopulation of nonresponding cells across all the stimulation conditions tested. We developed both deterministic and stochastic models for the expression of each gene. Our modeling analysis revealed that the heterogeneity in CXCL10 can be attributed to a slow chromatin-opening step that is on a similar timescale to that of adaptation of the upstream signal. In this way, CXCL10 expression in individual cells can remain stochastic in response to each pulse of repeated stimulation, which we also validated by experiments. Together, we conclude that pro-inflammatory genes in the same signaling pathway can respond to dynamic IFNγ stimulus with very different response features and that upstream signal adaptation can contribute to shaping heterogeneous gene expression.

Enhanced cellular longevity arising from environmental fluctuations.

Cellular longevity is regulated by both genetic and environmental factors. However, the interactions of these factors in the context of aging remain largely unclear. Here, we formulate a mathematical model for dynamic glucose modulation of a core gene circuit in yeast aging, which not only guided the design of pro-longevity interventions, but also revealed the theoretical principles underlying these interventions. We introduce the dynamical systems theory to capture two general means for promoting longevity - the creation of a stable fixed point in the "healthy" state of the cell and the dynamic stabilization of the system around this healthy state through environmental oscillations. Guided by the model, we investigate how both of these can be experimentally realized by dynamically modulating environmental glucose levels. The results establish a paradigm for theoretically analyzing the trajectories and perturbations of aging that can be generalized to aging processes in diverse cell types and organisms.

Clinical Outcomes in Patients With COVID-19 and Hematologic Disease.

BACKGROUND: Patients with hematologic diseases are at higher risk of the SARS-CoV-2 infection and more severe clinical outcomes of the coronavirus disease. CHRONOS19 is an observational prospective cohort study with the aim to determine the short and longer-term clinical outcomes, risk factors for disease severity and mortality, and rates of postinfectious immunity in patients with malignant and nonmalignant hematologic diseases and COVID-19. PATIENTS AND METHODS: Overall, 666 patients were enrolled in the study, of which 626 were included in the final data analysis. The primary endpoint was 30-days all-cause mortality. Secondary endpoints included COVID-19 complications, rates of ICU admission and mechanical ventilation, outcomes of a hematologic disease in SARS-CoV-2 infected patients, overall survival, and risk factors for disease severity and mortality. Data from 15 centers were collected at 30, 90, and 180 days after COVID-19 was diagnosed and were managed using a web-based e-data capture platform. All evaluations were performed in the pre-omicron period of COVID-19 pandemic. RESULTS: Thirty-days all-cause mortality was 18.9%. The predominant cause of death (in 80% of cases) were COVID-19 complications. At 180 days, the majority (70%) of additional deaths were due to hematologic disease progression. At a median follow-up of 5.7 [0.03-19.04] months, 6-months overall survival was 72% [95% CI: 0.69-0.76]. One-third of patients had severe SARS-CoV-2 disease. The rate of ICU admission was 22% with 77% of these patients requiring mechanical ventilation, with poor survival rate. A univariate analysis revealed that older age (≥ 60 years), male sex, malignant hematologic disease, myelotoxic agranulocytosis, transfusion dependence, refractory disease or relapse, diabetes among comorbidities, any complications, especially ARDS alone or in combination with CRS, admission to an ICU, and mechanical ventilation were associated with higher risks of mortality. Treatment of the hematologic disease was changed, postponed, or canceled in 63% of patients. At a longer follow-up (90 and 180 days), the status of the hematologic disease changed in 7.5% of patients. CONCLUSION: Patients with hematologic disease and COVID-19 have high mortality rates, predominantly due to COVID-19 complications. At a longer-term follow-up, no significant impact of COVID-19 on the course of a hematologic disease was revealed.

Engineering longevity-design of a synthetic gene oscillator to slow cellular aging.

Synthetic biology enables the design of gene networks to confer specific biological functions, yet it remains a challenge to rationally engineer a biological trait as complex as longevity. A naturally occurring toggle switch underlies fate decisions toward either nucleolar or mitochondrial decline during the aging of yeast cells. We rewired this endogenous toggle to engineer an autonomous genetic clock that generates sustained oscillations between the nucleolar and mitochondrial aging processes in individual cells. These oscillations increased cellular life span through the delay of the commitment to aging that resulted from either the loss of chromatin silencing or the depletion of heme. Our results establish a connection between gene network architecture and cellular longevity that could lead to rationally designed gene circuits that slow aging.

Statistical Theory of Asymmetric Damage Segregation in Clonal Cell Populations.

Asymmetric damage segregation (ADS) is ubiquitous among unicellular organisms: After a mother cell divides, its two daughter cells receive sometimes slightly, sometimes strongly different fractions of damaged proteins accumulated in the mother cell. Previous studies demonstrated that ADS provides a selective advantage over symmetrically dividing cells by rejuvenating and perpetuating the population as a whole. In this work we focus on the statistical properties of damage in individual lineages and the overall damage distributions in growing populations for a variety of ADS models with different rules governing damage accumulation, segregation, and the lifetime dependence on damage. We show that for a large class of deterministic ADS rules the trajectories of damage along the lineages are chaotic, and the distributions of damage in cells born at a given time asymptotically becomes fractal. By exploiting the analogy of linear ADS models with the Iterated Function Systems known in chaos theory, we derive the Frobenius-Perron equation for the stationary damage density distribution and analytically compute the damage distribution moments and fractal dimensions. We also investigate nonlinear and stochastic variants of ADS models and show the robustness of the salient features of the damage distributions.

Statistical theory of asymmetric damage segregation in clonal cell populations.

Asymmetric damage segregation (ADS) is ubiquitous among unicellular organisms: After a mother cell divides, its two daughter cells receive sometimes slightly, sometimes strongly different fractions of damaged proteins accumulated in the mother cell. Previous studies demonstrated that ADS provides a selective advantage over symmetrically dividing cells by rejuvenating and perpetuating the population as a whole. In this work we focus on the statistical properties of damage in individual lineages and the overall damage distributions in growing populations for a variety of ADS models with different rules governing damage accumulation, segregation, and the lifetime dependence on damage. We show that for a large class of deterministic ADS rules the trajectories of damage along the lineages are chaotic, and the distributions of damage in cells born at a given time asymptotically becomes fractal. By exploiting the analogy of linear ADS models with the Iterated Function Systems known in chaos theory, we derive the Frobenius-Perron equation for the stationary damage density distribution and analytically compute the damage distribution moments and fractal dimensions. We also investigate nonlinear and stochastic variants of ADS models and show the robustness of the salient features of the damage distributions.

Early Fetal Corpus Callosum: Demonstrating Normal Growth and Detecting Pathologies in Early Pregnancy.

BACKGROUND AND PURPOSE: A malformed corpus callosum carries a risk for abnormal neurodevelopment. The advent of high-frequency transducers offers the opportunity to assess corpus callosum development in early pregnancy. The aim of the study was to construct a reference chart of the fetal corpus callosum length on ultrasound between 13 and 19 weeks of gestation and to prospectively examine growth patterns in pathologic cases. MATERIALS AND METHODS: We performed a prospective cross-sectional study between 2020 and 2022 in well-dated, low-risk, singleton pregnancies between 13 and 19 weeks of gestation. A standardized image was obtained in the midsagittal plane. Imaging criteria were used as a confirmation of the early corpus callosum. Measurements were taken by 4 trained sonographers. Intra- and interobserver variability was assessed. Corpus callosum length in centiles were calculated for each gestational week. RESULTS: One hundred eighty-seven fetuses were included in the study. All cases met inclusion criteria. At 13 weeks of gestation, the margins of the early corpus callosum were sufficiently clear to be measured in 80% (20/25) of fetuses. A cubic polynomial regression model best described the correlation between corpus length and gestational age. The correlation coefficient (r 2) was 0.929 (P < .001). Intra- and interobserver variability had high interclass correlation coefficients (>0.99). Presented is the earliest published case of agenesis of corpus callosum and a case of dysgenetic corpus callosum in Rubinstein-Taybi syndrome. CONCLUSIONS: Provided is a nomogram of the early fetal corpus callosum. Applying imaging criteria helped to identify a case of complete agenesis of the corpus callosum as early as 14 weeks.

Ag4B7O12X (X = Cl, Br, I) Heptaborate Family: Comprehensive Crystal Chemistry, Thermal Stability Trends, Topology, and Vibrational Anharmonicity.

Using glass crystallization and solid-state techniques, we were able to complete the family of salt-inclusion silver halide borates, Ag4B7O12X, by the X = Cl and I members. The new compounds are characterized by differential scanning calorimetry, single-crystal and high-temperature powder X-ray diffraction, optical spectroscopy, and density functional theory calculations. In all structures, the silver atoms exhibit strong anharmonicity of thermal vibrations, which could be modeled using Gram-Charlier expansion, and its asymmetry was characterized by the skewness vector. The topology of the silver halide and borate sublattices has been analyzed separately for the first time. Along the I → Br → Cl series, we observe a decrease of the melting point and configuration entropy and an increase of thermal expansion and its anisotropy and thermal vibration anharmonicity, which indicates decreasing stability.

Contribution of the Collective Excitations to the Coupled Proton and Energy Transport along Mitochondrial Cristae Membrane in Oxidative Phosphorylation System.

The results of many experimental and theoretical works indicate that after transport of protons across the mitochondrial inner membrane (MIM) in the oxidative phosphorylation (OXPHOS) system, they are retained on the membrane-water interface in nonequilibrium state with free energy excess due to low proton surface-to-bulk release. This well-established phenomenon suggests that proton trapping on the membrane interface ensures vectorial lateral transport of protons from proton pumps to ATP synthases (proton acceptors). Despite the key role of the proton transport in bioenergetics, the molecular mechanism of proton transfer in the OXPHOS system is not yet completely established. Here, we developed a dynamics model of long-range transport of energized protons along the MIM accompanied by collective excitation of localized waves propagating on the membrane surface. Our model is based on the new data on the macromolecular organization of the OXPHOS system showing the well-ordered structure of respirasomes and ATP synthases on the cristae membrane folds. We developed a two-component dynamics model of the proton transport considering two coupled subsystems: the ordered hydrogen bond (HB) chain of water molecules and lipid headgroups of MIM. We analytically obtained a two-component soliton solution in this model, which describes the motion of the proton kink, corresponding to successive proton hops in the HB chain, and coherent motion of a compression soliton in the chain of lipid headgroups. The local deformation in a soliton range facilitates proton jumps due to water molecules approaching each other in the HB chain. We suggested that the proton-conducting structures formed along the cristae membrane surface promote direct lateral proton transfer in the OXPHOS system. Collective excitations at the water-membrane interface in a form of two-component soliton ensure the coupled non-dissipative transport of charge carriers and elastic energy of MIM deformation to ATP synthases that may be utilized in ATP synthesis providing maximal efficiency in mitochondrial bioenergetics.

Autoquenching of spherical photon density waves during propagation in a turbid medium.

The effect of the formation of deep minima in frequency characteristics of photon density waves (PDWs) during their propagation in scattering media with different optical characteristics has been studied by statistical Monte Carlo modeling. The simulation was performed for the Henyey-Greenstein scattering phase function with the anisotropy factor value varying in the range of 0-0.93. The dependence of the position and magnitude of the minimum in the frequency response of PDWs on the combination of the parameters of the scattering medium and the distance to the radiation source is demonstrated.

Age-dependent aggregation of ribosomal RNA-binding proteins links deterioration in chromatin stability with challenges to proteostasis.

Chromatin instability and protein homeostasis (proteostasis) stress are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of Saccharomyces cerevisiae, a challenge to proteostasis occurs specifically in the fraction of cells with decreased stability within the ribosomal DNA (rDNA). A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)-binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability induced by inhibition of a conserved lysine deacetylase Sir2. Further, loss of rDNA stability induces age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates contribute to age-induced proteostasis decline and limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis stress and highlight the importance of cell-to-cell variability in aging processes.

Optimal transcriptional regulation of dynamic bacterial responses to sudden drug exposures.

Cellular responses to the presence of toxic compounds in their environment require prompt expression of the correct levels of the appropriate enzymes, which are typically regulated by transcription factors that control gene expression for the duration of the response. The characteristics of each response dictate the choice of regulatory parameters such as the affinity of the transcription factor to its binding sites and the strength of the promoters it regulates. Although much is known about the dynamics of cellular responses, we still lack a framework to understand how different regulatory strategies evolved in natural systems relate to the selective pressures acting in each particular case. Here, we analyze a dynamical model of a typical antibiotic response in bacteria, where a transcriptionally repressed enzyme is induced by a sudden exposure to the drug that it processes. We identify strategies of gene regulation that optimize this response for different types of selective pressures, which we define as a set of costs associated with the drug, enzyme, and repressor concentrations during the response. We find that regulation happens in a limited region of the regulatory parameter space. While responses to more costly (toxic) drugs favor the usage of strongly self-regulated repressors, responses where expression of enzyme is more costly favor the usage of constitutively expressed repressors. Only a very narrow range of selective pressures favor weakly self-regulated repressors. We use this framework to determine which costs and benefits are most critical for the evolution of a variety of natural cellular responses that satisfy the approximations in our model and to analyze how regulation is optimized in new environments with different demands.

Mechanism of Energy Storage and Transformation in the Mitochondria at the Water-Membrane Interface.

In this review, we discuss the mechanisms of generation of membrane-bound protons using different energy sources in model and natural systems. Analysis of these mechanisms revealed that all three types of reactions include the same principal stage, which is dissociation of electrically neutral Brønsted acids at the interface during transition from the hydrophobic phase to water with a low dielectric constant. Special attention is paid to the fact that in one of the analyzed model systems, membrane-bound protons provide energy for the reaction of ATP synthesis. Similar mechanism for the generation of membrane-bound protons has been found in natural membranes involved in oxidative phosphorylation, in particular, on the membranes of mitoplasts and mitochondria. The energy of oxidative reactions required for ATP synthesis, is stored at the intermediate stage not only in the form of transmembrane electrochemical potential of protons, but also and perhaps mostly, as protons attached to the inner mitochondrial membrane. The process of energy storage in mitochondria is linked to the transfer of protons that simultaneously perform two functions. Protons on the membrane surface carry free energy and, at the same time, act as substrates facilitating the movement of F1F0-ATP-synthase biological machine.

Structural and functional roles of non-bilayer lipid phases of chloroplast thylakoid membranes and mitochondrial inner membranes.

The 'standard' fluid-mosaic membrane model can provide a framework for the operation of the photosynthetic and respiratory electron transport systems, the generation of the proton motive force (pmf) and its utilization for ATP synthesis according to the chemiosmotic theory. However, this model, with the bilayer organization of all lipid molecules, assigns no function to non-bilayer lipids - while in recent years it became clear that the two fundamental energy transducing membranes of the biosphere, chloroplast thylakoid membranes (TMs) and inner mitochondrial membranes (IMMs), contain large amounts of non-bilayer (non-lamellar) lipid phases. In this review, we summarize our understanding on the role of non-lamellar phases in TMs and IMMs: (i) We propose that for these membrane vesicles the dynamic exchange model (DEM) provides a more suitable framework than the 'standard' model; DEM complements the 'standard' model by assuming the co-existence of bilayer and non-bilayer phases and their interactions, which contribute to the structural dynamics of the membrane systems and safe-guard the membranes' high protein:lipid ratios. (ii) Non-bilayer phases play pivotal roles in membrane fusion and intermembrane lipid exchanges - essential processes in the self-assembly of these highly folded intricate membranes. (iii) The photoprotective, lipocalin-like lumenal enzyme, violaxanthin de-epoxidase, in its active state requires the presence of non-bilayer lipid phase. (iv) Cardiotoxins, water-soluble polypeptides, induce non-bilayer phases in mitochondria. (v) ATP synthesis, in mammalian heart IMMs, is positively correlated with the amount of non-bilayer packed lipids with restricted mobility. (vi) The hypothesized sub-compartments, due to non-lamellar phases, are proposed to enhance the utilization of pmf and might contribute to the recently documented functional independence of individual cristae within the same mitochondrion. Further research is needed to identify and characterize the structural entities associated with the observed non-bilayer phases; and albeit fundamental questions remain to be elucidated, non-lamellar lipid phases should be considered on a par with the bilayer phase, with which they co-exist in functional TMs and IMMs.