Interaction of Methylene Blue with Severe Acute Respiratory Syndrome Coronavirus 2 Envelope Revealed by Molecular Modeling.

Methylene blue has multiple antiviral properties against Severe Acute Respiratory Syndrome-related Coronavirus 2 (SARS-CoV-2). The ability of methylene blue to inhibit different stages of the virus life cycle, both in light-independent and photodynamic processes, is used in clinical practice. At the same time, the molecular aspects of the interactions of methylene blue with molecular components of coronaviruses are not fully understood. Here, we use Brownian dynamics to identify methylene blue binding sites on the SARS-CoV-2 envelope. The local lipid and protein composition of the coronavirus envelope plays a crucial role in the binding of this cationic dye. Viral structures targeted by methylene blue include the S and E proteins and negatively charged lipids. We compare the obtained results with known experimental data on the antiviral effects of methylene blue to elucidate the molecular basis of its activity against coronaviruses.

Photodynamic inactivation of bacteria: Why it is not enough to excite a photosensitizer.

BACKGROUND: The development of multidrug resistance (MDR) in infectious agents is one of the most serious global problems facing humanity. Antimicrobial photodynamic therapy (APDT) shows encouraging results in the fight against MDR pathogens, including those in biofilms. METHODS: Photosensitizers (PS), monocationic methylene blue, polycationic and polyanionic derivatives of phthalocyanines, electroneutral and polycationic derivatives of bacteriochlorin were used to study photodynamic inactivation of Gram-positive and Gram-negative planktonic bacteria and biofilms under LED irradiation. Zeta potential measurements, confocal fluorescence imaging, and coarse-grained modeling were used to evaluate the interactions of PS with bacteria. PS aggregation and photobleaching were studied using absorption and fluorescence spectroscopy. RESULTS: The main approaches to ensure high efficiency of bacteria photosensitization are analyzed. CONCLUSIONS: PS must maintain a delicate balance between binding to exocellular and external structures of bacterial cells and penetration through the cell wall so as not to get stuck on the way to photooxidation-sensitive structures of the bacterial cell.

Insights into the Formation of Intermolecular Complexes of Fluorescent Probe 10-N-Nonyl Acridine Orange with Cardiolipin and Phosphatidylglycerol in Bacterial Plasma Membrane by Molecular Modeling.

In this article, we used molecular dynamics (MD), one of the most common methods for simulations of membranes, to study the interaction of fluorescent membranotropic biological probe 10-N-nonyl acridine orange (NAO) with the bilayer, mimicking a plasma membrane of Gram-negative bacteria. Fluorescent probes serve as an effective tool to study the localization of different components in biological membranes. Revealing the molecular details of their interaction with membrane phospholipids is important both for the interpretation of experimental results and future design of lipid-specific stains. By means of coarse-grained (CG) MD, we studied the interactions of NAO with a model membrane, imitating the plasma membrane of Gram-negative bacteria. In our simulations, we detected different NAO forms: monomers, dimers, and stacks. NAO dimers had the central cardiolipin (CL) molecule in a sandwich-like structure. The stacks were formed by NAO molecules interlayered with anionic lipids, predominantly CL. Use of the CG approach allowed to confirm the ability of NAO to bind to both major negatively charged phospholipids, phosphatidylglycerol (PG) and CL, and to shed light on the exact structure of previously proposed NAO-lipid complexes. Thus, CG modeling can be useful for the development of new effective and highly specific molecular probes.

Mathematical Simulation of Electron Transport in the Primary Photosynthetic Processes.

Summarized results of investigation of regulation of electron transport and associated processes in the photosynthetic membrane using methods of mathematical and computer modeling carried out at the Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, are presented in this review. Detailed kinetic models of processes in the thylakoid membrane were developed using the apparatus of differential equations. Fitting of the model curves to the data of spectral measurements allowed us to estimate the values of parameters that were not determined directly in experiments. The probabilistic method of agent-based Monte Carlo modeling provides ample opportunities for studying dynamics of heterogeneous systems based on the rules for the behavior of individual elements of the system. Algorithms for simplified representation of Big Data make it possible to monitor changes in the photosynthetic apparatus in the course of culture growth in a photobioreactor and for the purpose of environmental monitoring. Brownian and molecular models describe movement and interaction of individual electron carrier proteins and make it possible to study electrostatic, hydrophobic, and other interactions leading to regulation of conformational changes in the reaction complexes. Direct multiparticle models explicitly simulate Brownian diffusion of the mobile protein carriers and their electrostatic interactions with multienzyme complexes both in solution and in heterogeneous interior of a biomembrane. The combined use of methods of kinetic and Brownian multiparticle and molecular modeling makes it possible to study the mechanisms of regulation of an integral system of electron transport processes in plants and algae at molecular and subcellular levels.

Molecular, Brownian, kinetic and stochastic models of the processes in photosynthetic membrane of green plants and microalgae.

The paper presents the results of recent work at the Department of Biophysics of the Biological Faculty, Lomonosov Moscow State University on the kinetic and multiparticle modeling of processes in the photosynthetic membrane. The detailed kinetic models and the rule-based kinetic Monte Carlo models allow to reproduce the fluorescence induction curves and redox transformations of the photoactive pigment P700 in the time range from 100 ns to dozens of seconds and make it possible to reveal the role of individual carriers in their formation for different types of photosynthetic organisms under different illumination regimes, in the presence of inhibitors, under stress conditions. The fitting of the model curves to the experimental data quantifies the reaction rate constants that cannot be directly measured experimentally, including the non-radiative thermal relaxation reactions. We use the direct multiparticle models to explicitly describe the interactions of mobile photosynthetic carrier proteins with multienzyme complexes both in solution and in the biomembrane interior. An analysis of these models reveals the role of diffusion and electrostatic factors in the regulation of electron transport, the influence of ionic strength and pH of the cellular environment on the rate of electron transport reactions between carrier proteins. To describe the conformational intramolecular processes of formation of the final complex, in which the actual electron transfer occurs, we use the methods of molecular dynamics. The results obtained using kinetic and molecular models supplement our knowledge of the mechanisms of organization of the photosynthetic electron transport processes at the cellular and molecular levels.

Electrostatic Map of the SARS-CoV-2 Virion Specifies Binding Sites of the Antiviral Cationic Photosensitizer.

Electrostatics is an important part of virus life. Understanding the detailed distribution of charges over the surface of a virus is important to predict its interactions with host cells, antibodies, drugs, and different materials. Using a coarse-grained model of the entire viral envelope developed by D. Korkin and S.-J. Marrink's scientific groups, we created an electrostatic map of the external surface of SARS-CoV-2 and found a highly heterogeneous distribution of the electrostatic potential field of the viral envelope. Numerous negative patches originate mainly from negatively charged lipid domains in the viral membrane and negatively charged areas on the "stalks" of the spike (S) proteins. Membrane (M) and envelope (E) proteins with the total positive charge tend to colocalize with the negatively charged lipids. In the E protein pentamer exposed to the outer surface, negatively charged glutamate residues and surrounding lipids form a negative electrostatic potential ring around the channel entrance. We simulated the interaction of the antiviral octacationic photosensitizer octakis(cholinyl)zinc phthalocyanine with the surface structures of the entire model virion using the Brownian dynamics computational method implemented in ProKSim software (version r661). All mentioned negatively charged envelope components attracted the photosensitizer molecules and are thus potential targets for reactive oxygen generated in photosensitized reactions.

Mitochondrial complex II in intestinal epithelial cells regulates T cell-mediated immunopathology.

Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.

What Binds Cationic Photosensitizers Better: Brownian Dynamics Reveals Key Interaction Sites on Spike Proteins of SARS-CoV, MERS-CoV, and SARS-CoV-2.

We compared the electrostatic properties of the spike proteins (S-proteins) of three coronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, and their interactions with photosensitizers (PSs), octacationic octakis(cholinyl)zinc phthalocyanine (Zn-PcChol8+) and monocationic methylene blue (MB). We found a major common PS binding site at the connection of the S-protein stalk and head. The molecules of Zn-PcChol8+ and MB also form electrostatic encounter complexes with large area of negative electrostatic potential at the head of the S-protein of SARS-CoV-2, between fusion protein and heptad repeat 1 domain. The top of the SARS-CoV spike head demonstrates a notable area of electrostatic contacts with Zn-PcChol8+ and MB that corresponds to the N-terminal domain. The S-protein protomers of SARS-CoV-2 in "open" and "closed" conformations demonstrate different ability to attract PS molecules. In contrast with Zn-PcChol8+, MB possesses the ability to penetrate inside the pocket formed as a result of SARS-CoV-2 receptor binding domain transition into the "open" state. The existence of binding site for cationic PSs common to the S-proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV creates prospects for the wide use of this type of PSs to combat the spread of coronaviruses.

Multiple in vivo Effects of Cadmium on Photosynthetic Electron Transport in Pea Plants.

The inhibitory effects of cadmium (CdSO4 ) on the primary photosynthetic processes were studied in vivo in Pisum sativum by using Multi-function Plant Efficiency Analyser (M-PEA-2). Photosynthetic parameters related to photosystem (PS) II, PS I and intersystem electron carriers were calculated from the light-induced kinetics of prompt chlorophyll a fluorescence (OJIP transient), delayed chlorophyll a fluorescence (DF), and 820 nm modulated reflection (MR). Low-dose exposure to cadmium (20 µm CdSO4 for 48 h) reduced probability of electron transfer from plastoquinones to the terminal electron acceptors of PSI (δRo ) accompanied by a decrease in the rate of P700 + and PC reduction (Vred ) and the magnitude of the I2 step on the DF kinetics. Electron transport through PSI remained unaltered. The obtained results allowed us to propose existence of the potential site of inhibition of photosynthetic electron flow by cadmium between PSII and PSI. We propose to use parameters δRo , Vred , and I2 /I1 as sensitive indicators of an early contamination by heavy metals.

α-tubulin tail modifications regulate microtubule stability through selective effector recruitment, not changes in intrinsic polymer dynamics.

Microtubules are non-covalent polymers of αß-tubulin dimers. Posttranslational processing of the intrinsically disordered C-terminal α-tubulin tail produces detyrosinated and Δ2-tubulin. Although these are widely employed as proxies for stable cellular microtubules, their effect (and of the α-tail) on microtubule dynamics remains uncharacterized. Using recombinant, engineered human tubulins, we now find that neither detyrosinated nor Δ2-tubulin affect microtubule dynamics, while the α-tubulin tail is an inhibitor of microtubule growth. Consistent with the latter, molecular dynamics simulations show the α-tubulin tail transiently occluding the longitudinal microtubule polymerization interface. The marked differential in vivo stabilities of the modified microtubule subpopulations, therefore, must result exclusively from selective effector recruitment. We find that tyrosination quantitatively tunes CLIP-170 density at the growing plus end and that CLIP170 and EB1 synergize to selectively upregulate the dynamicity of tyrosinated microtubules. Modification-dependent recruitment of regulators thereby results in microtubule subpopulations with distinct dynamics, a tenet of the tubulin code hypothesis.

The Photosensitizer Octakis(cholinyl)zinc Phthalocyanine with Ability to Bind to a Model Spike Protein Leads to a Loss of SARS-CoV-2 Infectivity In Vitro When Exposed to Far-Red LED.

Photodynamic inactivation of pathogenic microorganisms can be successfully used to eradicate pathogens in localized lesions, infected liquid media, and on various surfaces. This technique utilizes the photosensitizer (PS), light, and molecular oxygen to produce reactive oxygen species that kill pathogens. Here, we used the PS, water soluble octakis(cholinyl)zinc phthalocyanine (Zn-PcChol8+), to inactivate an initial 4.75-5.00 IgTCID50/mL titer of SARS-CoV-2, thereby preventing viral infection when tested in Vero E6 cell cultures. Zn-PcChol8+ in a minimally studied concentration, 1 µM and LED 3.75 J/cm2, completely destroyed the infectivity of SARS-CoV-2. To detect possible PS binding sites on the envelope of SARS-CoV-2, we analyzed electrostatic potential and simulated binding of Zn-PcChol8+ to the spike protein of this coronavirus by means of Brownian dynamics software, ProKSim (Protein Kinetics Simulator). Most of the Zn-PcChol8+ molecules formed clusters at the upper half of the stalk within a vast area of negative electrostatic potential. Positioning of the PS on the surface of the spike protein at a distance of no more than 10 nm from the viral membrane may be favorable for the oxidative damage. The high sensitivity of SARS-CoV-2 to photodynamic inactivation by Zn-PcChol8+ is discussed with respect to the application of this PS to control the spread of COVID-19.

High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions.

Metabolic flux technology with the Seahorse bioanalyzer has emerged as a standard technique in cellular metabolism studies, allowing for simultaneous kinetic measurements of respiration and glycolysis. Methods to extend the utility and versatility of the metabolic flux assay would undoubtedly have immediate and wide-reaching impacts. Herein, we describe a platform that couples the metabolic flux assay with high-content fluorescence imaging to simultaneously provide means for normalization of respiration data with cell number; analyze cell cycle distribution; and quantify mitochondrial content, fragmentation state, membrane potential, and mitochondrial reactive oxygen species. Integration of fluorescent dyes directly into the metabolic flux assay generates a more complete data set of mitochondrial features in a single assay. Moreover, application of this integrated strategy revealed insights into mitochondrial function following PGC1a and PRC1 inhibition in pancreatic cancer and demonstrated how the Rho-GTPases impact mitochondrial dynamics in breast cancer.

SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism.

SLC25A32 is a member of the solute carrier 25 family of mitochondrial transporters. SLC25A32 transports tetrahydrofolate (THF) as well as FAD into mitochondria and regulates mitochondrial one-carbon metabolism and redox balance. While it is known that cancer cells require one-carbon and FAD-dependent mitochondrial metabolism to sustain cell proliferation, the role of SLC25A32 in cancer cell growth remains unexplored. Our results indicate that the SLC25A32 gene is highly amplified in different tumors and that amplification correlates with increased mRNA expression and reduced patients´ survival. siRNA-mediated knock-down and CRISPR-mediated knock-out of SLC25A32 in cancer cells of different origins, resulted in the identification of cell lines sensitive and resistant to SLC25A32 inhibition. Mechanistically, tracing of deuterated serine revealed that SLC25A32 knock-down does not affect the mitochondrial/cytosolic folate flux as measured by Liquid Chromatography coupled Mass Spectrometry (LC-MS). Instead, SLC25A32 inhibition results in a respiratory chain dysfunction at the FAD-dependent complex II enzyme, induction of Reactive Oxygen Species (ROS) and depletion of reduced glutathione (GSH), which impairs cancer cell proliferation. Moreover, buthionine sulfoximine (BSO) treatment further sensitizes cells to ROS-mediated inhibition of cell proliferation upon SLC25A32 knock-down. Treatment of cells with the FAD precursor riboflavin and with GSH rescues cancer cell proliferation upon SLC25A32 down-regulation. Our results indicate that the reduction of mitochondrial FAD concentrations by targeting SLC25A32 has potential clinical applications as a single agent or in combination with approved cancer drugs that lead to increased oxidative stress and reduced tumor growth.

Tissue of origin dictates GOT1 dependence and confers synthetic lethality to radiotherapy.

BACKGROUND: Metabolic programs in cancer cells are influenced by genotype and the tissue of origin. We have previously shown that central carbon metabolism is rewired in pancreatic ductal adenocarcinoma (PDA) to support proliferation through a glutamate oxaloacetate transaminase 1 (GOT1)-dependent pathway. METHODS: We utilized a doxycycline-inducible shRNA-mediated strategy to knockdown GOT1 in PDA and colorectal cancer (CRC) cell lines and tumor models of similar genotype. These cells were analyzed for the ability to form colonies and tumors to test if tissue type impacted GOT1 dependence. Additionally, the ability of GOT1 to impact the response to chemo- and radiotherapy was assessed. Mechanistically, the associated specimens were examined using a combination of steady-state and stable isotope tracing metabolomics strategies and computational modeling. Statistics were calculated using GraphPad Prism 7. One-way ANOVA was performed for experiments comparing multiple groups with one changing variable. Student's t test (unpaired, two-tailed) was performed when comparing two groups to each other. Metabolomics data comparing three PDA and three CRC cell lines were analyzed by performing Student's t test (unpaired, two-tailed) between all PDA metabolites and CRC metabolites. RESULTS: While PDA exhibits profound growth inhibition upon GOT1 knockdown, we found CRC to be insensitive. In PDA, but not CRC, GOT1 inhibition disrupted glycolysis, nucleotide metabolism, and redox homeostasis. These insights were leveraged in PDA, where we demonstrate that radiotherapy potently enhanced the effect of GOT1 inhibition on tumor growth. CONCLUSIONS: Taken together, these results illustrate the role of tissue type in dictating metabolic dependencies and provide new insights for targeting metabolism to treat PDA.

Visualization of the Anisotropy of the Velocity Dispersion and Characteristics of the Multi-Velocity Continuum in the Regions of Multi-Stream Flows of Gas-Dust Media with Polydisperse Dust.

Collisionless media devoid of intrinsic stresses, for example, a dispersed phase in a multiphase medium, have a much wider variety of space-time structures and features formed in them than collisional media, for example, a carrier, gas, or liquid phase. This is a consequence of the fact that evolution in such media occurs in phase space, i.e., in a space of greater dimensions than the usual coordinate space. As a consequence, the process of the formation of features in collisionless media (clustering or vice versa, a loss of continuity) can occur primarily in the velocity space, which, in contrast to the features in the coordinate space (folds, caustics, or voids), is poorly observed directly. To identify such features, it is necessary to use visualization methods that allow us to consider, in detail, the evolution of the medium in the velocity space. This article is devoted to the development of techniques that allow visualizing the degree of anisotropy of the velocity fields of collisionless interpenetrating media. Simultaneously tracking the behavior of different fractions in such media is important, as their behavior can be significantly different. We propose three different techniques for visualizing the anisotropy of velocity fields using the example of two- and three-continuum dispersed media models. We proposed the construction of spatial distributions of eccentricity fields (scalar fields), or fields of principal directions of the velocity dispersion tensor (tensor fields). In the first case, we used some simple eccentricity functions for dispersion tensors for two fractions simultaneously, which we call surrogate entropy. In the second case, to visualize the anisotropy of the velocity fields of three fractions simultaneously, we used an ordered array (3-vector) of eccentricities for the color representation through decomposition in three basic colors. In the case of a multi-stream flow, we used cluster analysis methods to identify sections of a multi-stream flow (beams) and used glyphs to visualize the entire set of beams (vector-tensor fields).

Chromium effects on photosynthetic electron transport in pea (Pisum sativum L.).

MAIN CONCLUSION: Components of the photosynthetic electron transport chain in pea (Pisum sativum L.) leaves under in vivo conditions showed the following sensitivity to the inhibitory action of chromium(VI): intersystem electron transport > photosystem I > photosystem II. Inhibitory effects of chromium (VI) (K2Cr2O7, Cr) on the light reactions of photosynthesis were studied in vivo in Pisum sativum L. by using Multi-function Plant Efficiency Analyser (M-PEA-2). Photosynthetic parameters related to photosystem (PS) II, PSI and intersystem electron carriers were calculated from the light-induced kinetics of prompt chlorophyll a fluorescence (OJIP transient), delayed chlorophyll a fluorescence (DF), and 820 nm modulated reflection (MR). We showed that the I2 step of DF induction is sensitive to inhibition of the Q0 site of the cytochrome b6f complex. Such parameters as δRo of the JIP test related to the functional state of photosynthetic reactions beyond the PQ pool, Vred of the MR induction assigned to the overall rate of P700+ and plastocyanin reduction, and I2 step of the DF induction were significantly altered in the presence of low-dose Cr(VI). Moderate doses of Cr affected mainly PSI-related parameters including Vox and ΔMR parameters of the MR induction, whereas high-dose treatment influenced JIP test parameters φPo(= FV/FM) and ψEo related to PSII. The obtained results showed that the earliest Cr(VI) effect on the photosynthetic electron transport chain manifests itself by inhibition of the intersystem electron transport, rather, at the level of the cytochrome b6f complex. Inhibitory effects of Cr on PSI were more pronounced than those on PSII. Sensitivity of the used kinetic parameters toward the functional state of photosynthetic reactions makes this approach suitable for early diagnostics of toxic action of pollutants on plants.

Explicit measurement of the endotoxin adsorption efficiency detects non-Langmuir behavior at low concentrations.

Lipopolysaccharides (LPS) are the Gram-negative bacteria cell wall components capable to induce the system inflammatory response even at picomolar concentrations. LPS detection at these concentrations is necessary to develop new sorbents for the efficient purification of the biological fluids. LAL-test widely used for LPS concentration estimation is based on the LPS biological activity measurement and thus may depend on the LPS concentration in a non-linear way. Here we propose a new explicit method for the LPS concentration measurement based on fluorescently labeled LPS and direct photon counting and develop the new protocol for LPS adsorption efficiency measurement. Following the suggested protocol in the experiments on novel sorbents, we demonstrate that LPS adsorption at small biologically relevant concentrations is non-Langmuir.

Mechanical properties of tubulin intra- and inter-dimer interfaces and their implications for microtubule dynamic instability.

Thirteen tubulin protofilaments, made of αß-tubulin heterodimers, interact laterally to produce cytoskeletal microtubules. Microtubules exhibit the striking property of dynamic instability, manifested in their intermittent growth and shrinkage at both ends. This behavior is key to many cellular processes, such as cell division, migration, maintenance of cell shape, etc. Although assembly and disassembly of microtubules is known to be linked to hydrolysis of a guanosine triphosphate molecule in the pocket of ß-tubulin, detailed mechanistic understanding of corresponding conformational changes is still lacking. Here we take advantage of the recent generation of in-microtubule structures of tubulin to examine the properties of protofilaments, which serve as important microtubule assembly and disassembly intermediates. We find that initially straight tubulin protofilaments, relax to similar non-radially curved and slightly twisted conformations. Our analysis further suggests that guanosine triphosphate hydrolysis primarily affects the flexibility and conformation of the inter-dimer interface, without a strong impact on the shape or flexibility of αß-heterodimer. Inter-dimer interfaces are significantly more flexible compared to intra-dimer interfaces. We argue that such a difference in flexibility could be key for distinct stability of the plus and minus microtubule ends. The higher flexibility of the inter-dimer interface may have implications for development of pulling force by curving tubulin protofilaments during microtubule disassembly, a process of major importance for chromosome motions in mitosis.

A Pan-ALDH1A Inhibitor Induces Necroptosis in Ovarian Cancer Stem-like Cells.

Ovarian cancer is typified by the development of chemotherapy resistance. Chemotherapy resistance is associated with high aldehyde dehydrogenase (ALDH) enzymatic activity, increased cancer "stemness," and expression of the stem cell marker CD133. As such, ALDH activity has been proposed as a therapeutic target. Although it remains controversial which of the 19 ALDH family members drive chemotherapy resistance, ALDH1A family members have been primarily linked with chemotherapy resistant and stemness. We identified two ALDH1A family selective inhibitors (ALDH1Ai). ALDH1Ai preferentially kills CD133+ ovarian cancer stem-like cells (CSCs). ALDH1Ai induce necroptotic CSC death, mediated, in part, by the induction of mitochondrial uncoupling proteins and reduction in oxidative phosphorylation. ALDH1Ai is highly synergistic with chemotherapy, reducing tumor initiation capacity and increasing tumor eradication in vivo. These studies link ALDH1A with necroptosis and confirm the family as a critical therapeutic target to overcome chemotherapy resistance and improve patient outcomes.