Search Results (27)

Pancreatic beta cells regulate insulin secretion in response to glucose by generating ATP, which modulates ATP-sensitive potassium channels (K_ATP) channel activity and Ca²⁺ dynamics. We present a model of ATP production in pancreatic beta cells, focusing on ATP dynamics within the bulk cytosol, submembrane region, and microdomains near K_ATP channels. ATP is generated through glycolysis, mitochondrial oxidative phosphorylation (OxPhos), and glycolytic pyruvate kinase-mediated phosphoenolpyruvate (PEP) production, supported by PEP cycling between mitochondria and the cytosol. The model examines ATP production in relation to Ca²⁺ oscillations, elucidating their interdependent dynamics. Our findings demonstrate that both mitochondrial OxPhos and PEP-mediated ATP production contribute substantially to cellular ATP levels. Specifically, glycolysis and mitochondrial OxPhos are crucial for the initial (first-phase) increase in bulk and subplasmalemmal ATP, effectively “filling up” the ATP pool in beta cells. In the second phase, coordinated cycling between OxPhos and PEP pathways enables cost-effective fine-tuning of ATP levels, with localized effects in the K_ATP channel microdomains. This model addresses and clarifies the recent debate regarding the mechanisms by which sufficient ATP concentrations are achieved to close K_ATP channels in glucose-stimulated beta cells, offering novel insights into the regulation of energy production and K_ATP channel activity. Full article

Regulation of blood glucose levels depends on the property of beta cells to couple glucose sensing with insulin secretion. This is accomplished by the concentration-dependent flux of glucose through glycolysis and oxidative phosphorylation, generating ATP. The resulting rise in cytosolic ATP/ADP inhibits K_ATP channels, inducing membrane depolarization and Ca²⁺ influx, which prompts insulin secretion. Evidence suggests that this coupling of glucose sensing with insulin secretion may be compartmentalized in the submembrane regions of the beta cell. We investigated the subcellular responses of key components involved in this coupling and found mitochondria in the submembrane zone, some tethered to the cytoskeleton near capillaries. Using Fluorescent Lifetime Imaging Microscopy (FLIM), we observed that submembrane mitochondria were the fastest to respond to glucose. In the most glucose-responsive beta cells, glucose triggers rapid, localized submembrane increases in ATP and Ca²⁺ as synchronized ~4-min oscillations, consistent with pulsatile insulin release after meals. These findings are consistent with the hypothesis that glucose sensing is coupled with insulin secretion in the submembrane zone of beta cells. This zonal adaptation would enhance both the speed and energy efficiency of beta cell responses to glucose, as only a subset of the most accessible mitochondria would be required to trigger insulin secretion. Full article

The spontaneous firing of the sinoatrial (SA) node, the physiological pacemaker of the heart, is generated within sinoatrial nodal cells (SANCs) and is regulated by a “coupled-clock” pacemaker system, which integrates a “membrane clock”, the ensemble of ion channel currents, and an intracellular “Ca²⁺ clock”, sarcoplasmic reticulum-generated local submembrane Ca²⁺ releases via ryanodine receptors. The interactions within a “coupled-clock” system are modulated by phosphorylation of surface membrane and sarcoplasmic reticulum proteins. Though the essential role of a high basal cAMP level and PKA-dependent phosphorylation for basal spontaneous SANC firing is well recognized, the role of basal CaMKII-dependent phosphorylation remains uncertain. This is a critical issue with respect to how cardiac pacemaker cells fire spontaneous action potentials. This review aspires to explain and unite apparently contradictory results of pharmacological studies in the literature that have demonstrated a fundamental role of basal CaMKII activation for basal cardiac pacemaker function, as well as studies in mice with genetic CaMKII inhibition which have been interpreted to indicate that basal spontaneous SANC firing is independent of CaMKII activation. The assessment of supporting and opposing data regarding CaMKII effects on phosphorylation of Ca²⁺-cycling proteins and spontaneous firing of SANC in the basal state leads to the necessary conclusion that CaMKII activity and CaMKII-dependent phosphorylation do regulate basal cardiac pacemaker function. Full article

Sialodacryoadenitis virus (SDAV) has been identified as the etiological agent responsible for the respiratory system and salivary gland infections in rats. The existing literature on SDAV infections is insufficient to address the topic adequately, particularly in relation to the central nervous system. In order to ascertain how SDAV gains access to neuronal cells and subsequently exits, our attention was focused on the small molecule valosin-containing protein (VCP), which is an ATPase. VCP is acknowledged for its function in the ubiquitin-mediated proteasomal degradation of proteins, including those of viral origin. To ascertain the potential influence of VCP on SDAV replication and egress, high-content screening was employed to determine the viral titer and protein content. Western blot analysis was employed to ascertain the relative expression of VCP. Real-time imaging of SDAV-infected cells and confocal imaging for qualitative morphological analysis were conducted. The Eeyarestatin I (EerI) inhibitor was employed to disrupt VCP involvement in the endoplasmic reticulum-associated protein degradation pathway (ERAD) in both pre- and post-incubation systems, with concentrations of 5 μM/mL and 25 μM/mL, respectively. We demonstrated for the first time that SDAV productively replicates in cultured primary neurons. VCP expression is markedly elevated during SDAV infection. The application of 5 μM/mL EerI in the post-treatment system yielded a statistically significant inhibition of the SDAV yield. It is likely that this modulates the efficacy of virion assembly by arresting viral proteins in the submembrane area. Full article

As one of the most important food crops, the potato is widely planted in the oasis agricultural region of Northwest China. To ascertain the impact of regulated deficit irrigation (RDI) on various facets including dry matter accumulation, tuber yield, quality and water use efficiency (WUE) of potato plants, a two-growth season field experiment under mulched drip irrigation was conducted in the desert oasis region of Northwest China. Water deficits, applied at the seedling, tuber formation, tuber expansion and starch accumulation stages, encompassed two distinctive levels: mild (55–65% of field capacity, F_C) and moderate (45–55% F_C) deficit, with full irrigation (65–75% F_C) throughout the growing season as the control (CK). The results showed that water deficit significantly reduced (p < 0.05) above-ground dry matter, water consumption and tuber yield compared to CK, and the reduction increased with the increasing water deficit. A mild water deficit at the tuber formation stage, without significantly reducing (p > 0.05) yield, could significantly increase WUE and irrigation water use efficiency (IWUE), with two-year average increases of 25.55% and 32.33%, respectively, compared to CK. Water deficit at the tuber formation stage increased starch content, whereas water deficit at tuber expansion stage significantly reduced starch, protein and reducing sugar content. Additionally, a comprehensive evaluation showed that a mild water deficit at the tuber formation stage is the optimal RDI strategy for potato production, providing a good balance between yield, quality and WUE. The results of this study can provide theoretical support for efficient and sustainable potato production in the desert oasis regions of Northwest China. Full article

C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs’ activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg¹⁰⁷ site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones. Full article

Tethered bilayer lipid membranes (tBLMs) are increasingly used in biosensor applications where electrochemical impedance spectroscopy (EIS) is the method of choice for amplifying and recording the activity of membrane-damaging agents such as pore-forming toxins or disrupting peptides. While the activity of these biological agents may depend on the pH of the analytes, there is increasing evidence that the sensitivity of tethered bilayer sensors depends on the pH of the solutions. In our study, we addressed the question of what are the fundamental reasons for the variability of the EIS signal of the tBLMs with pH. We designed an experiment to compare the EIS response of tBLMs with natural membrane defects and two different membrane disruptors: vaginolysin and melittin. Our experimental design ensured that the same amount of protein and peptide was present in the tBLMs, while the pH was varied by replacing the buffers with different pH values. Using a recently developed EIS data analysis algorithm from our research group, we were able to demonstrate that, in contrast to previous literature which relates the variability of tBLM, EIS response to the variation in defect density, the main reason for the observed variability in EIS response is the change in the sub-membrane properties of tBLMs with pH. Using surface-enhanced infrared absorption spectroscopy (SEIRAS), we have shown that pH changes from neutral to slightly acidic leads to an expulsion of water, presumably bound to ions, from the sub-membrane reservoir, resulting in a marked decrease in the carrier concentration and specific conductance of the sub-membrane reservoir. Such a decrease is recorded by the EIS as a decrease in the conductance of the tBLM complex and affects the sensitivity of a biosensor. Our data provide important evidence of pH-sensitive effects that should be considered in both the development and operation of biosensors. Full article

Dendritic cells (DC) are critical cellular mediators of host immunity, notably by expressing a broad panel of pattern recognition receptors. One of those receptors, the C-type lectin receptor DC-SIGN, was previously reported as a regulator of endo/lysosomal targeting through functional connections with the autophagy pathway. Here, we confirmed that DC-SIGN internalization intersects with LC3⁺ autophagy structures in primary human monocyte-derived dendritic cells (MoDC). DC-SIGN engagement promoted autophagy flux which coincided with the recruitment of ATG-related factors. As such, the autophagy initiation factor ATG9 was found to be associated with DC-SIGN very early upon receptor engagement and required for an optimal DC-SIGN-mediated autophagy flux. The autophagy flux activation upon DC-SIGN engagement was recapitulated using engineered DC-SIGN-expressing epithelial cells in which ATG9 association with the receptor was also confirmed. Finally, Stimulated emission depletion (STED) microscopy performed in primary human MoDC revealed DC-SIGN-dependent submembrane nanoclusters formed with ATG9, which was required to degrade incoming viruses and further limit DC-mediated transmission of HIV-1 infection to CD4⁺ T lymphocytes. Our study unveils a physical association between the Pattern Recognition Receptor DC-SIGN and essential components of the autophagy pathway contributing to early endocytic events and the host’s antiviral immune response. Full article

Chromate and dichromate solutions used for the activation and passivation of cadmium and zinc galvanic coatings of metal products are widely used due to their ability to form corrosion-protective films. Therefore, in this article, we examined the kinetic features of the cathodic deposition of Cd and Zn during membrane electrolysis. As a result of comprehensive experimental and theoretical studies, the features of Cd and Zn cathodic depositions were analyzed under different hydrodynamic conditions in a submembrane zone of an anolyte. Experimental physicochemical methods such as the experimental analysis of solutions, analytical modeling, and a statistical analysis were used during the research. A regression dependence for evaluating a reaction rate constant was assessed based on the least-square approximation of the proposed model. As a result, the peculiarities of the cathodic formations for Cd and Zn during the membrane electrolysis process were analyzed. The effect of mechanical mixing with different values of the Reynolds number on the deposition of Cd and Zn on a cathode was evaluated. A change in Cd²⁺ and Zn²⁺ ion concentrations was also considered during the research. Overall, the obtained results increased the Cd deposition rate by 2.2 times using an active hydrodynamic environment with the anolyte. Full article

An extended membrane system with a dynamic nested membrane structure, which is integrated with the evolution-communication mechanism of a cell-like P system with evolutional symport/antiport rules and active membranes (ECP), and the evolutionary mechanisms of particle swarm optimization (PSO) and improved PSO inspired by starling flock behavior (SPSO), named DSPSO-ECP, is designed and developed to try to break application restrictions of P systems in this paper. The purpose of DSPSO-ECP is to enhance the performance of extended membrane system in solving optimization problems. In the proposed DSPSO-ECP, the updated model of velocity and position of standard PSO, as basic evolution rules, are adopted to evolve objects in elementary membranes. The modified updated model of the velocity of improved SPSO is used as local evolution rules to evolve objects in sub-membranes. A group of sub-membranes for elementary membranes are specially designed to avoid prematurity through membrane creation and dissolution rules with promoter/inhibitor. The exchange and sharing of information between different membranes are achieved by communication rules for objects based on evolutional symport rules of ECP. At last, computational results, which are made on numerical benchmark functions and classic test images, are discussed and analyzed to validate the efficiency of the proposed DSPSO-ECP. Full article

Aiming to investigate the disadvantage of the optimization algorithm of membrane computing (a P system) in which it is difficult to take advantage of parallelism in MATLAB, leading to a slow optimization speed, a digital-specific hardware solution (field-programmable gate array, FPGA) is proposed to design and implement the single-cell-membrane algorithm (SCA). Because the SCA achieves extensive global searches by the symmetric processing of the solution set, with independent and symmetrically distributed submembrane structures, the FPGA-hardware-based design of the SCA system includes a control module, an HSP module, an initial value module, a fitness module, a random number module, and multiple submembrane modules with symmetrical structures. This research utilizes the inherent parallel characteristics of the FPGA to achieve parallel computations of multiple submembrane modules with a symmetric structure inside the SCA, and it achieves a high degree of parallelism of rules inside the modules by using a non-blocking allocation. This study uses the benchmark Sphere function to verify the performance of the FPGA-designed SCA system. The experimental results show that, when the FPGA platform and the MATLAB platform obtain a similar calculation accuracy, the average time-consuming of the FPGA is 0.00041 s, and the average time-consuming of MATLAB is 0.0122 s, and the calculation speed is improved by nearly 40 times. This study uses the FPGA design to implement the SCA, and it verifies the advantages of the membrane-computing maximum-parallelism theory and distributed structures in computing speed. The realization platform of membrane computing is expanded, which provides a theoretical basis for further development of the distributed computing model of population cells. Full article

SARS-CoV-2 infection inhibits interferon expression, while hyper-activating innate-immune signalling and expression of pro-inflammatory cytokines. SARS-CoV-2 proteins: Spike, M and nsp6, nsp12 and nsp13 inhibit IFR3-mediated Type-1-interferon defence, but hyper-activate intracellular signalling, which leads to dysfunctional expression of pro-inflammatory cytokines, particularly IL-1β IL-6, IL-8, and TNFα. Ezrin, a sub-membrane adaptor-protein, organises multi-protein-complexes such as ezrin+NHERF1+NHE+CFTR, which control the density and location of ACE2 receptor expression on the luminal surface of airway-epithelial-cells, as well as determining susceptibility to SARS-CoV-2 infection. This protein complex is vital for lung-surfactant production for efficient gas-exchange. Ezrin also forms multi-protein-complexes that regulate signalling kinases; Ras, PKC, PI3K, and PKA. m-RAGE is a pattern-recognition-receptor of the innate immune system that is triggered by AGEs, which are chemically modified proteins common in the elderly and obese. m-RAGE forms multi-protein complexes with ezrin and TIRAP, a toll-like-receptor adaptor-protein. The main cause of COVID-19 is not viral infection but pro-inflammatory p38MAPK signalling mediated by TLRs and RAGE. In contrast, it appears that activated ezrin+PKA signalling results in the activation of transcription-factor CREB, which suppresses NFκB mediated pro-inflammatory cytokine expression. In addition, competition between ezrin and TIRAP to form multi-protein-complexes on membrane PIP2-lipid-rafts is a macromolecular-switch that changes the priority from innate immune activation programs to adaptive immune activation programs. Human Vasoactive Intestinal Peptide (VIP), and Human Ezrin Peptides (HEP-1 and RepG3) probably inhibit COVID-19 by activating the ezrin+PKA and ras>Raf>MEK>ERK>RSK>CREB>IL-10 signalling, which favours activation of adaptive immunity programs and inhibition of the dysfunctional innate-inflammation, the cause of COVID-19. HEP-1, RepG3, and VIP in individual human volunteers and in small clinical studies have been shown to be effective COVID-19 therapies, and seem to have a closely related mechanism of action. Full article

Surface-enhanced Raman spectroscopy (SERS) is a promising tool that can be used in the detection of molecular changes triggered by disease development. Cardiovascular diseases (CVDs) are caused by multiple pathologies originating at the cellular level. The identification of these deteriorations can provide a better understanding of CVD mechanisms, and the monitoring of the identified molecular changes can be employed in the development of novel biosensor tools for early diagnostics. We applied plasmonic SERS nanosensors to assess changes in the properties of erythrocytes under normotensive and hypertensive conditions in the animal model. We found that spontaneous hypertension in rats leads (i) to a decrease in the erythrocyte plasma membrane fluidity and (ii) to a decrease in the mobility of the heme of the membrane-bound hemoglobin. We identified SERS parameters that can be used to detect pathological changes in the plasma membrane and submembrane region of erythrocytes. Full article

GP.Mur is a clinically important red blood cell (RBC) phenotype in Southeast Asia. The molecular entity of GP.Mur is glycophorin B-A-B hybrid protein that promotes band 3 expression and band 3–AQP1 interaction, and alters the organization of band 3 complexes with Rh/RhAG complexes. GP.Mur+ RBCs are more resistant to osmotic stress. To explore whether GP.Mur+ RBCs could be structurally more resilient, we compared deformability and osmotic fragility of fresh RBCs from 145 adults without major illness (47% GP.Mur). We also evaluated potential impacts of cellular and lipid factors on RBC deformability and osmotic resistivity. Contrary to our anticipation, these two physical properties were independent from each other based on multivariate regression analyses. GP.Mur+ RBCs were less deformable than non-GP.Mur RBCs. We also unexpectedly found 25% microcytosis in GP.Mur+ female subjects (10/40). Both microcytosis and membrane cholesterol reduced deformability, but the latter was only observed in non-GP.Mur and not GP.Mur+ normocytes. The osmotic fragility of erythrocytes was not affected by microcytosis; instead, larger mean corpuscular volume (MCV) increased the chances of hypotonic burst. From comparison with GP.Mur+ RBCs, higher band 3 expression strengthened the structure of RBC membrane and submembranous cytoskeletal networks and thereby reduced cell deformability; stronger band 3–AQP1 interaction additionally supported osmotic resistance. Thus, red cell deformability and osmotic resistivity involve distinct structural–functional roles of band 3. Full article

Changes in plasma membrane curvature and intracellular ionic strength are two key features of cell volume perturbations. In this hypothesis we present a model of the responsible molecular apparatus which is assembled of two molecular motors [non-muscle myosin II (NMMII) and protrusive actin polymerization], a spring [a complex between the plasma membrane (PM) and the submembrane actin-based cytoskeleton (smACSK) which behaves like a viscoelastic solid] and the associated signaling proteins. We hypothesize that this apparatus senses changes in both the plasma membrane curvature and the ionic strength and in turn activates signaling pathways responsible for regulatory volume increase (RVI) and regulatory volume decrease (RVD). During cell volume changes hydrostatic pressure (HP) changes drive alterations in the cell membrane curvature. HP difference has opposite directions in swelling versus shrinkage, thus allowing distinction between them. By analogy with actomyosin contractility that appears to sense stiffness of the extracellular matrix we propose that NMMII and actin polymerization can actively probe the transmembrane gradient in HP. Furthermore, NMMII and protein-protein interactions in the actin cortex are sensitive to ionic strength. Emerging data on direct binding to and regulating activities of transmembrane mechanosensors by NMMII and actin cortex provide routes for signal transduction from transmembrane mechanosensors to cell volume regulatory mechanisms. Full article