Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2).

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca(2+) oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP2) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca(2+) oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP2 hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP2, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca(2+) oscillation inducing activity and in vitro interaction with PIP2 without affecting its Ca(2+) sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca(2+) frequency and the binding ability of different PLCζ mutants to PIP2. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP2, leading to complete abolishment of its Ca(2+) oscillation inducing activity.

Human PLCζ exhibits superior fertilization potency over mouse PLCζ in triggering the Ca(2+) oscillations required for mammalian oocyte activation.

A sperm-specific phospholipase C-zeta (PLCζ) is believed to play an essential role in oocyte activation during mammalian fertilization. Sperm PLCζ has been shown to trigger a prolonged series of repetitive Ca(2+) transients or oscillations in oocytes that precede activation. This remarkable intracellular Ca(2+) signalling phenomenon is a distinctive characteristic observed during in vitro fertilization by sperm. Previous studies have notably observed an apparent differential ability of PLCζ from disparate mammalian species to trigger Ca(2+) oscillations in mouse oocytes. However, the molecular basis and confirmation of the apparent PLCζ species difference in activity remains to be provided. In the present study, we provide direct evidence for the superior effectiveness of human PLCζ relative to mouse PLCζ in generating Ca(2+) oscillations in mouse oocytes. In addition, we have designed and constructed a series of human/mouse PLCζ chimeras to enable study of the potential role of discrete PLCζ domains in conferring the enhanced Ca(2+) signalling potency of human PLCζ. Functional analysis of these human/mouse PLCζ domain chimeras suggests a novel role of the EF-hand domain in the species-specific differences in PLCζ activity. Our empirical observations are compatible with a basic mathematical model for the Ca(2+) dependence of generating cytoplasmic Ca(2+) oscillations in mammalian oocytes by sperm PLCζ.

Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization.

Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca(2+) oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca(2+) in eggs. To investigate how discrete protein domains contribute to Ca(2+) release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca(2+) oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca(2+) sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca(2+) oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca(2+) releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca(2+) sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca(2+) oscillations in eggs, we have developed a mathematical model that incorporates Ca(2+)-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca(2+) oscillatory activity associated with specific PLC variants.

A network-oriented perspective on cardiac calcium signaling.

The normal contractile, electrical, and energetic function of the heart depends on the synchronization of biological oscillators and signal integrators that make up cellular signaling networks. In this review we interpret experimental data from molecular, cellular, and transgenic models of cardiac signaling behavior in the context of established concepts in cell network architecture and organization. Focusing on the cellular Ca(2+) handling machinery, we describe how the plasticity and adaptability of normal Ca(2+) signaling is dependent on dynamic network configurations that operate across a wide range of functional states. We consider how (mal)adaptive changes in signaling pathways restrict the dynamic range of the network such that it cannot respond appropriately to physiologic stimuli or perturbation. Based on these concepts, a model is proposed in which pathologic abnormalities in cardiac rhythm and contractility (e.g., arrhythmias and heart failure) arise as a consequence of progressive desynchronization and reduction in the dynamic range of the Ca(2+) signaling network. We discuss how a systems-level understanding of the network organization, cellular noise, and chaotic behavior may inform the design of new therapeutic modalities that prevent or reverse the disease-linked unraveling of the Ca(2+) signaling network.

Deterministic nonlinear features of cutaneous perfusion are lost in diabetic subjects with neuropathy.

The aim of this study was to analyze and compare the deterministic nonlinear structure of cutaneous laser Doppler flowmetry signals obtained from the forearm and foot of normal subjects and diabetic patients without neuropathy (D), with peripheral neuropathy (DPN) and with combined autonomic and peripheral neuropathy (DAN). Flow oscillations were evaluated under baseline conditions, after local warming of the skin to 44 °C and after warming plus iontophoresis of phenylephrine. The presence of nonlinearity was investigated by three complementary approaches: (i) attractor reconstruction, (ii) calculation of largest Lyapunov exponents (LLEs), and (iii) correlation dimension analysis. Conclusions were validated against surrogate stochastic time series generated by randomizing the Fourier phase of the raw data. In the control and D groups, the combination of phenylephrine and warming unmasked flowmotion with a prominent component at 0.1 Hz. Attractor reconstruction revealed toroidal structure and estimated LLEs were positive. LLEs decreased to zero and dimension estimates increased for surrogate data, consistent with loss of determinism. In diabetic subjects with neuropathy estimates of LLE were not significantly different from zero and dimensions were unaffected by phase randomization. Evidence for nonlinear structure was also obtained under baseline conditions in normal and D subjects, but was lost on warming alone. We conclude that deterministic control mechanisms contribute to cutaneous flowmotion, particularly when pseudo-quasiperiodic behavior is enhanced by phenylephrine. Nonlinear analysis of laser Doppler signals may provide previously unrecognized insights into the effects of diabetic neuropathy on perfusion because it can identify loss of complexity independently of the amplitude of the signals recorded.

Aquaglyceroporin-3's Expression and Cellular Localization Is Differentially Modulated by Hypoxia in Prostate Cancer Cell Lines.

Aquaporins are required by cells to enable fast adaptation to volume and osmotic changes, as well as microenvironmental metabolic stimuli. Aquaglyceroporins play a crucial role in supplying cancer cells with glycerol for metabolic needs. Here, we show that AQP3 is differentially expressed in cells of a prostate cancer panel. AQP3 is located at the cell membrane and cytoplasm of LNCaP cell while being exclusively expressed in the cytoplasm of Du145 and PC3 cells. LNCaP cells show enhanced hypoxia growth; Du145 and PC3 cells display stress factors, indicating a crucial role for AQP3 at the plasma membrane in adaptation to hypoxia. Hypoxia, both acute and chronic affected AQP3's cellular localization. These outcomes were validated using a machine learning classification approach of the three cell lines and of the six normoxic or hypoxic conditions. Classifiers trained on morphological features derived from cytoskeletal and nuclear labeling alongside corresponding texture features could uniquely identify each individual cell line and the corresponding hypoxia exposure. Cytoskeletal features were 70-90% accurate, while nuclear features allowed for 55-70% accuracy. Cellular texture features (73.9% accuracy) were a stronger predictor of the hypoxic load than the AQP3 distribution (60.3%).

Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function.

AIMS: The cardiac ryanodine receptor (RyR2), which mediates intracellular Ca2+ release to trigger cardiomyocyte contraction, participates in development of acquired and inherited arrhythmogenic cardiac disease. This study was undertaken to characterize the network of inter- and intra-subunit interactions regulating the activity of the RyR2 homotetramer. METHODS AND RESULTS: We use mutational investigations combined with biochemical assays to identify the peptide sequence bridging the ß8 with ß9 strand as the primary determinant mediating RyR2 N-terminus self-association. The negatively charged side chains of two aspartate residues (D179 and D180) within the ß8-ß9 loop are crucial for the N-terminal inter-subunit interaction. We also show that the RyR2 N-terminus domain interacts with the C-terminal channel pore region in a Ca2+-independent manner. The ß8-ß9 loop is required for efficient RyR2 subunit oligomerization but it is dispensable for N-terminus interaction with C-terminus. Deletion of the ß8-ß9 sequence produces unstable tetrameric channels with subdued intracellular Ca2+ mobilization implicating a role for this domain in channel opening. The arrhythmia-linked R176Q mutation within the ß8-ß9 loop decreases N-terminus tetramerization but does not affect RyR2 subunit tetramerization or the N-terminus interaction with C-terminus. RyR2R176Q is a characteristic hypersensitive channel displaying enhanced intracellular Ca2+ mobilization suggesting an additional role for the ß8-ß9 domain in channel closing. CONCLUSION: These results suggest that efficient N-terminus inter-subunit communication mediated by the ß8-ß9 loop may constitute a primary regulatory mechanism for both RyR2 channel activation and suppression.

Stroma-derived extracellular vesicle mRNA signatures inform histological nature of prostate cancer.

Histological assessment of prostate cancer is the key diagnostic test and can predict disease outcome. This is however an invasive procedure that carries associated risks, hence non-invasive assays to support the diagnostic pathway are much needed. A key feature of disease progression, and subsequent poor prognosis, is the presence of an altered stroma. Here we explored the utility of prostate stromal cell-derived vesicles as indicators of an altered tumour environment. We compared vesicles from six donor-matched pairs of adjacent-normal versus disease-associated primary stromal cultures. We identified 19 differentially expressed transcripts that discriminate disease from normal stromal extracellular vesicles (EVs). EVs isolated from patient serum were investigated for these putative disease-discriminating mRNA. A set of transcripts including Caveolin-1 (CAV1), TMP2, THBS1, and CTGF were found to be successful in discriminating clinically insignificant (Gleason = 6) disease from clinically significant (Gleason > 8) prostate cancer. Furthermore, correlation between transcript expression and progression-free survival suggests that levels of these mRNA may predict disease outcome. Informed by a machine learning approach, combining measures of the five most informative EV-associated mRNAs with PSA was shown to significantly improve assay sensitivity and specificity. An in-silico model was produced, showcasing the superiority of this multi-modal liquid biopsy compared to needle biopsy for predicting disease progression. This proof of concept highlights the utility of serum EV analytics as a companion diagnostic test with prognostic utility, which may obviate the need for biopsy.

Influence of ageing on human body blood flow and heat transfer: A detailed computational modelling study.

Ageing plays a fundamental role in arterial blood transport and heat transfer within a human body. The aim of this work is to provide a comprehensive methodology, based on biomechanical considerations, for modelling arterial flow and energy exchange mechanisms in the body accounting for age-induced changes. The study outlines a framework for age-related modifications within several interlinked subsystems, which include arterial stiffening, heart contractility variations, tissue volume and property changes, and thermoregulatory system deterioration. Some of the proposed age-dependent governing equations are directly extrapolated from experimental data sets. The computational framework is demonstrated through numerical experiments, which show the impact of such age-related changes on arterial blood pressure, local temperature distribution, and global body thermal response. The proposed numerical experiments show that the age-related changes in arterial convection do not significantly affect the tissue temperature distribution. Results also highlight age-related effects on the sweating mechanism, which lead to a significant reduction in heat dissipation and a subsequent rise in skin and core temperatures.

A multiscale active structural model of the arterial wall accounting for smooth muscle dynamics.

Arterial wall dynamics arise from the synergy of passive mechano-elastic properties of the vascular tissue and the active contractile behaviour of smooth muscle cells (SMCs) that form the media layer of vessels. We have developed a computational framework that incorporates both these components to account for vascular responses to mechanical and pharmacological stimuli. To validate the proposed framework and demonstrate its potential for testing hypotheses on the pathogenesis of vascular disease, we have employed a number of pharmacological probes that modulate the arterial wall contractile machinery by selectively inhibiting a range of intracellular signalling pathways. Experimental probes used on ring segments from the rabbit central ear artery are: phenylephrine, a selective α1-adrenergic receptor agonist that induces vasoconstriction; cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; and ryanodine, a diterpenoid that modulates Ca2+ release from the sarcoplasmic reticulum. These interventions were able to delineate the role of membrane versus intracellular signalling, previously identified as main factors in smooth muscle contraction and the generation of vessel tone. Each SMC was modelled by a system of nonlinear differential equations that account for intracellular ionic signalling, and in particular Ca2+ dynamics. Cytosolic Ca2+ concentrations formed the catalytic input to a cross-bridge kinetics model. Contractile output from these cellular components forms the input to the finite-element model of the arterial rings under isometric conditions that reproduces the experimental conditions. The model does not account for the role of the endothelium, as the nitric oxide production was suppressed by the action of L-NAME, and also due to the absence of shear stress on the arterial ring, as the experimental set-up did not involve flow. Simulations generated by the integrated model closely matched experimental observations qualitatively, as well as quantitatively within a range of physiological parametric values. The model also illustrated how increased intercellular coupling led to smooth muscle coordination and the genesis of vascular tone.

Modelling accidental hypothermia effects on a human body under different pathophysiological conditions.

Accidental exposure to cold water environment is one of the most challenging situations in which hypothermia occurs. In the present work, we aim to characterise the energy balance of a human body subjected to such extreme environmental conditions. This study is carried out using a recently developed computational model and by setting boundary conditions needed to simulate the effect of cold surrounding environment. A major finding is the capacity of the body core regions to maintain their temperature high for a substantial amount of time, even under the most extreme environmental conditions. We also considered two disease states that highlight the spectrum of possible pathologies implicated in thermal regulation of the human body. These states are (i) cardiomyopathy, which affects the operating capacity of the heart, and (ii) malnutrition, which directly impairs the body's ability to regulate heat exchange with the environment. We have found that cardiomyopathy has little influence on the thermal balance of the human body, whereas malnutrition has a profound negative effect on the thermal balance and leads to dramatic reduction in core temperature.

An advanced computational bioheat transfer model for a human body with an embedded systemic circulation.

In the present work, an elaborate one-dimensional thermofluid model for a human body is presented. By contrast to the existing pure conduction-/perfusion-based models, the proposed methodology couples the arterial fluid dynamics of a human body with a multi-segmental bioheat model of surrounding solid tissues. In the present configuration, arterial flow is included through a network of elastic vessels. More than a dozen solid segments are employed to represent the heat conduction in the surrounding tissues, and each segment is constituted by a multilayered circular cylinder. Such multi-layers allow flexible delineation of the geometry and incorporation of properties of different tissue types. The coupling of solid tissue and fluid models requires subdivision of the arterial circulation into large and small arteries. The heat exchange between tissues and arterial wall occurs by convection in large vessels and by perfusion in small arteries. The core region, including the heart, provides the inlet conditions for the fluid equations. In the proposed model, shivering, sweating, and perfusion changes constitute the basis of the thermoregulatory system. The equations governing flow and heat transfer in the circulatory system are solved using a locally conservative Galerkin approach, and the heat conduction in the surrounding tissues is solved using a standard implicit backward Euler method. To investigate the effectiveness of the proposed model, temperature field evolutions are monitored at different points of the arterial tree and in the surrounding tissue layers. To study the differences due to flow-induced convection effects on thermal balance, the results of the current model are compared against those of the widely used modelling methodologies. The results show that the convection significantly influences the temperature distribution of the solid tissues in the vicinity of the arteries. Thus, the inner convection has a more predominant role in the human body heat balance than previously thought. To demonstrate its capabilities, the proposed new model is used to study different scenarios, including thermoregulation inactivity and variation in surrounding atmospheric conditions.

Synergy Between Intercellular Communication and Intracellular Ca(2+) Handling in Arrhythmogenesis.

Calcium is the primary signalling component of excitation-contraction coupling, the process linking electrical excitability of cardiac muscle cells to coordinated contraction of the heart. Understanding [Formula: see text] handling processes at the cellular level and the role of intercellular communication in the emergence of multicellular synchronization are key aspects in the study of arrhythmias. To probe these mechanisms, we have simulated cellular interactions on large scale arrays that mimic cardiac tissue, and where individual cells are represented by a mathematical model of intracellular [Formula: see text] dynamics. Theoretical predictions successfully reproduced experimental findings and provide novel insights on the action of two pharmacological agents (ionomycin and verapamil) that modulate [Formula: see text] signalling pathways via distinct mechanisms. Computational results have demonstrated how transitions between local synchronisation events and large scale wave formation are affected by these agents. Entrainment phenomena are shown to be linked to both intracellular [Formula: see text] and coupling-specific dynamics in a synergistic manner. The intrinsic variability of the cellular matrix is also shown to affect emergent patterns of rhythmicity, providing insights into the origins of arrhythmogenic [Formula: see text] perturbations in cardiac tissue in situ.

Arterial distensibility: acute changes following dynamic exercise in normal subjects.

The time course of acute changes in large artery distensibility immediately and for 60 min following maximum treadmill exercise in normal subjects was characterized by simultaneously measuring upper and lower limb pulse wave velocity (PWV). A new oscillometric technique was used, which has proven to be sensitive to changes in distensibility induced by acute changes in vascular tone independently of blood pressure. The observed changes in PWV are attributable to changes in vascular tone corresponding to recovery from a systemic net constrictor response and a local net dilator response to exercise with persisting postexercise vasodilatation. They are inadequately explained by associated changes in blood pressure and cannot be attributed to changes in heart rate or viscosity. Modeled as a system of n coupled linear differential equations, the minimum (and adequate) order required to reproduce these patterns was n = 1 for the upper and n = 2 for the exercising lower limb. The economy of the solution suggests entrainment among the multiple interactive mechanisms governing vasomotor control.