Functional activity and morphology of isolated rat cardiac mitochondria under calcium overload. Effect of naringin.

The function of mitochondria as a regulator of myocyte calcium homeostasis has been extensively discussed. The aim of the present work was further clarification of the details of modulation of the functional activity of rat cardiac mitochondria by exogenous Ca2+ ions either in the absence or in the presence of the plant flavonoid naringin. Low free Ca2+ concentrations (40-250 nM) effectively inhibited the respiratory activity of heart mitochondria, remaining unaffected the efficacy of oxygen consumption. In the presence of high exogenous Ca2+ ion concentrations (Ca2+ free was 550 µM), we observed a dramatic increase in mitochondrial heterogeneity in size and electron density, which was related to calcium-induced opening of the mitochondrial permeability transition pores (MPTP) and membrane depolarization (Ca2+free ions were from 150 to 750 µM). Naringin partially prevented Ca2+-induced cardiac mitochondrial morphological transformations (200 µM) and dose-dependently inhibited the respiratory activity of mitochondria (10-75 µM) in the absence or in the presence of calcium ions. Our data suggest that naringin (75 µM) promoted membrane potential dissipation, diminishing the potential-dependent accumulation of calcium ions by mitochondria and inhibiting calcium-induced MPTP formation. The modulating effect of the flavonoid on Ca2+-induced mitochondria alterations may be attributed to the weak-acidic nature of the flavonoid and its protonophoric/ionophoric properties. Our results show that the sensitivity of rat heart mitochondria to Ca2+ ions was much lower in the case of MPTP opening and much higher in the case of respiration inhibition as compared to liver mitochondria.

A new approach for sensitivity analysis of the StormWater Management Model applied in an airport.

Sensitivity analysis of urban flood model parameters is important for efficient and accurate flood simulation. In order to explore the problems of large sampling parameters and nonlinear correlation between input and output variables, this paper proposed a new correlation analysis approach. The type, strength, and the order of sensitive parameters to the four outputs are analyzed using the proposed approach. The results show that the R values of Manning-N are biggest, its distribution is linear in heat maps, and the Manning-N has a strong linear correlation with Average Depth, Hour of Maximum Flooding, and Time to Peak. For Average Depth, the second sensitive parameter is Conductivity. For Hour of Maximum Flooding, the second and third more sensitive parameters are Conductivity and N-perv; however, there are certain nonlinear correlations from heat maps. For Total Inflow, the R values of each parameter are between 0.021 and 0.534. Most sensitive parameters are none; however, the more sensitive parameters are Conductivity, N-perv, and initial deficit. For Time to Peak, the second and third more sensitive parameters are N-perv and N-Imperv; however, there are certain non-linear correlations from heat maps. The results can provide theoretical guidance for application and parameter calibration of SWMM in airport.

Cluster analysis and potential influencing factors of boars with different fertility.

The fertility of boars is intimately tied to the pig farm's economic benefits. This study aimed to rapidly categorize boars of different fertility and investigate the factors influencing the categorization using the production data in a large pig farm in northern China, including 11,163 semen collection records of Yorkshire boars (215), 11,163 breeding records and 8770 records of farrowing performance of Yorkshire sows (4505), as well as 4720 records of selection indices (sire line index and dam line index) for boars and sows (215 and 4505) between 2017 and 2020. The boar population was classified by two-step cluster analysis, followed by factor analysis to minimize the dimensionality of data variables and eliminate multicollinearity, and then using ordinal logistic regression model to investigate the risk variables impacting boar fertility categorization. Results showed that the two-step clustering divided the 215 boars into three subgroups: high-fertility (n = 61, 28.4%), medium-fertility (n = 127, 59.1%) and low-fertility (n = 27, 12.6%). The high-fertility boars were shown to be substantially greater than the medium-fertility or low-fertility boars (p < 0.05) in average total litter size, number of born alive, and number of healthy piglets of mated sows. Compared with low-fertility boars, the high-fertility boars were also significantly higher (p < 0.05) in the pregnancy rate and farrowing rate of mated sows. However, the three boar subgroups showed no difference (p > 0.05) in semen quality information (average sperm motility, average sperm density, and average sperm volume). Collinearity diagnosis indicated severe multicollinearity among the 20 data variables, which were reduced to 8 factor variables (factors 1-8) by factor analysis, and further collinearity diagnosis exhibited no multicollinearity among the 8 factor variables. Ordered logistic regression analysis revealed a significant and positive correlation (p < 0.05) of boar fertility with factor 2 (average total litter size, number of born alive, number of healthy piglets), factor 4 (average number of weak piglets and average weak piglet rate), factor 6 (sire line index of boars and dam line index of boars), factor 8 (pregnancy rate and farrowing rate), highlighting factor 2 as the most important factor influencing the classification of boar fertility. Our results indicate that the two-step cluster analysis can be used as a simple and effective method to screen boars with different fertility and that farm producers should pay attention to the recording of the reproductive performance of the mated sows due to its role as the risk factor for classification of boar fertility.

Mechanisms of Xiong-Pi-Fang in treating coronary heart disease associated with depression: A systematic pharmacology strategy and in vivo pharmacological validation.

BACKGROUND: Coronary heart disease (CHD) and depression are very common and often co-existing disorders. Xiong-Pi-Fang (XPF), a therapeutic classical traditional Chinese medicine (TCM) formula, has shown satisfactory efficacy in treating CHD associated with depression. However, its mechanism of action is still unknown. PURPOSE: To employ a systematic pharmacology approach for identifying the action mechanisms of XPF in treating CHD associated with depression. METHODS: We used a systematic pharmacology approach to identify the potential active mechanisms of XPF in treating CHD with depression. Potential active compounds in XPF and the diseases targets were screened using relevant databases to build corresponding pathways, following the experiments that were conducted to confirm whether the presumptive results of systemic pharmacology were correct. RESULTS: Network pharmacology predicted 42 key targets and 20 signaling pathways involved in XPF-mediated treatment, with IL-6/JAK2/STAT3/HIF-1α/VEGF-A pathway significantly affected. The common influences were hypothalamic-pituitary-adrenal axis (HPA axis) and glucocorticoid signaling, validated through chronic unexpected mild stress (CUMS) with isoprenaline (ISO) for inducing CHD within the depression model in rats. In addition, XPF intake reduced depressive-like behaviors and improved ECG ischemic changes. Furthermore, XPF exerted some anti-inflammatory effects by inhibiting the interleukin-6 (IL-6) induced phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), ultimately downregulating hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor-A (VEGF-A) activation. The dysfunctional HPA axis feedback loop was also regulated, which enhanced the glucocorticoid receptor (GR) expression. In contrast, it improved glucocorticoid resistance by reducing the mineralocorticoid receptor expression. CONCLUSIONS: Suppressing IL-6 release and maintaining the HPA feedback loop balance could be the primary mechanism of XPF against CHD with depression. The significance of the IL-6 and HPA axis identified indicates their potential as essential targets for CHD therapy with depression.

Identifying Candidate Genes for Short Gestation Length Trait in Chinese Qingping Pigs by Whole-Genome Resequencing and RNA Sequencing.

Gestation length is a complex polygenic trait that affects pig fetal development. The Qingping (QP) pig, a Chinese native black pig breed, is characterized by short gestation length. However, the genetic architecture of short gestation length is still not clear. The present study aimed to explore the genetic architecture of short gestation length in QP pigs. In this study, selective sweep analyses were performed to detect selective sweep signatures for short gestation length traits between 100 QP pigs and 219 pigs from 15 other breeds. In addition, differentially expressed genes for the short gestation length between QP pigs and Large White pigs were detected by RNA sequencing. Comparing candidate genes from these methods with known genes for preterm birth in the database, we obtained 111 candidate genes that were known preterm birth genes. Prioritizing other candidate genes, 839 novel prioritized candidate genes were found to have significant functional similarity to preterm birth genes. In particular, we highlighted EGFR, which was the most prioritized novel candidate relative to preterm birth genes. Experimental validations in placental and porcine trophectoderm cells suggest that EGFR is highly expressed in the QP pigs with short gestation length and could regulate the NF-κΒ pathway and downstream expression of PTGS2. These findings comprehensively identified candidate genes for short gestation length trait at the genomic and transcriptomic levels. These candidate genes provide an important new resource for further investigation and genetic improvement of gestation length.

Matrix stiffness exacerbates the proinflammatory responses of vascular smooth muscle cell through the DDR1-DNMT1 mechanotransduction axis.

Vascular smooth muscle cell (vSMC) is highly plastic as its phenotype can change in response to mechanical cues inherent to the extracellular matrix (ECM). VSMC may be activated from its quiescent contractile phenotype to a proinflammatory phenotype, whereby the cell secretes chemotactic and inflammatory cytokines, e.g. MCP1 and IL6, to functionally regulate monocyte and macrophage infiltration during the development of various vascular diseases including arteriosclerosis. Here, by culturing vSMCs on polyacrylamide (PA) substrates with variable elastic moduli, we discovered a role of discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that binds collagens, in mediating the mechanical regulation of vSMC gene expression, phenotype, and proinflammatory responses. We found that ECM stiffness induced DDR1 phosphorylation, oligomerization, and endocytosis to repress the expression of DNA methyltransferase 1 (DNMT1), very likely in a collagen-independent manner. The DDR1-to-DNMT1 signaling was sequentially mediated by the extracellular signal-regulated kinases (ERKs) and p53 pathways. ECM stiffness primed vSMC to a proinflammatory phenotype and this regulation was diminished by DDR1 inhibition. In agreement with the in vitro findings, increased DDR1 phosphorylation was observed in human arterial stiffening. DDR1 inhibition in mouse attenuated the acute injury or adenine diet-induced vascular stiffening and inflammation. Furthermore, mouse vasculature with SMC-specific deletion of Dnmt1 exhibited proinflammatory and stiffening phenotypes. Our study demonstrates a role of SMC DDR1 in perceiving the mechanical microenvironments and down-regulating expression of DNMT1 to result in vascular pathologies and has potential implications for optimization of engineering artificial vascular grafts and vascular networks.

Self-Organization of Tissue Growth by Interfacial Mechanical Interactions in Multilayered Systems.

Morphogenesis is a spatially and temporally regulated process involved in various physiological and pathological transformations. In addition to the associated biochemical factors, the physical regulation of morphogenesis has attracted increasing attention. However, the driving force of morphogenesis initiation remains elusive. Here, it is shown that during the growth of multilayered tissues, a morphogenetic process can be self-organized by the progression of compression gradient stemmed from the interfacial mechanical interactions between layers. In tissues with low fluidity, the compression gradient is progressively strengthened during growth and induces stratification by triggering symmetric-to-asymmetric cell division reorientation at the critical tissue size. In tissues with high fluidity, compression gradient is dynamic and induces cell rearrangement leading to 2D in-plane morphogenesis instead of 3D deformation. Morphogenesis can be tuned by manipulating tissue fluidity, cell adhesion forces, and mechanical properties to influence the progression of compression gradient during the development of cultured cell sheets and chicken embryos. Together, the dynamics of compression gradient arising from interfacial mechanical interaction provides a conserved mechanism underlying morphogenesis initiation and size control during tissue growth.

Exploration of a Nitromethane-Carbonylation Strategy during Route Design of an Atropisomeric KRASG12C Inhibitor.

Route design and proof of concept synthesis was conducted on a synthetically challenging atropisomeric KRASG12C inhibitor to support clinical API manufacture. Improvements to the synthesis of a chiral piperazine fragment gave reduced step count and streamlined protecting group strategy via the formation and methanol ring opening of an N-carboxy-anhydride (NCA). The complex atropisomeric nitroquinoline was accessed via an early stage salt-resolution followed by a formal two-part nitromethane-carbonylation, avoiding a high temperature Gould-Jacobs cyclization that previously led to atropisomer racemization. The substrate scope of the formal nitromethane-carbonylation strategy was further explored for a range of ortho-substituted bromo/iodo unprotected anilines.

Hypertonic pressure affects the pluripotency and self-renewal of mouse embryonic stem cells.

As an important mechanical cue in the extracellular microenvironment, osmotic stress directly affects the proliferation, migration, and differentiation of cells. In this paper, we focused on the influence of hypertonic pressure on the colony morphology, stemness, and self-renew of mouse embryonic stem cells (mESCs). Our results showed that culture media with hypertonic pressure are more conducive to the maintenance of 3D colony morphology and pluripotency of mESCs after withdrawing the glycogen synthase kinase 3ß (GSK3ß) inhibitor CHIR99021 and the mitogen-activated protein kinase (MEK) inhibitor PD0325901 (hereinafter referred to as 2i) for 48 h. Furthermore, we revealed the microscopic mechanisms of the this finding: hypertonic pressure resulted in the depolymerization of F-actin cytoskeleton and limits Yes-associated protein (hereinafter referred to as YAP) transmission into the nucleus which play a vital role in the regulation of cell proliferation, and resulting in cell-cycle arrest at last.

Wenxin Keli for the Treatment of Arrhythmia-Systems Pharmacology and In Vivo Pharmacological Assessment.

This study employed a systems pharmacology approach to identify the active compounds and action mechanisms of Wenxin Keli for arrhythmia treatment. Sixty-eight components identified in vivo and in vitro by UPLC/Q-TOF-MS were considered the potential active components of Wenxin Keli. Network pharmacology further revealed 33 key targets and 75 KEGG pathways as possible pathways and targets involved in WK-mediated treatment, with the CaMKII/CNCA1C/Ca2+ pathway being the most significantly affected. This finding was validated using an AC-induced rat arrhythmias model. Pretreatment with Wenxin Keli reduced the malignant arrhythmias and shortened RR, PR, and the QT interval. Wenxin Keli exerted some antiarrhythmic effects by inhibiting p-CaMKII and intracellular Ca2+ transients and overexpressing CNCA1C. Thus, suppressing SR Ca2+ release and maintaining intracellular Ca2+ balance may be the primary mechanism of Wenxin Keli against arrhythmia. In view of the significance of CaMKII and NCX identified in this experiment, we suggest that CaMKII and NCX are essential targets for treating arrhythmias.

AFM-based indentation method for measuring the relaxation property of living cells.

Probing the mechanical properties of cells is critical for understanding their deformation behaviors and biological functions. Although some methods have been proposed to characterize the elastic properties of cells, it is still difficult to measure their time-dependent properties. This paper investigates the use of atomic force microscope (AFM) to determine the reduced relaxation modulus of cells. In principle, AFM is hard to perform an indentation relaxation test that requires a constant indenter displacement during load relaxation, whereas the real AFM indenter displacement usually varies with time during relaxation due to the relatively small bending stiffness of its cantilever. We investigate this issue through a combined theoretical, computational, and experimental effort. A protocol relying on the choice of appropriate cantilever bending stiffness is proposed to perform an AFM-based indentation relaxation test of cells, which enables the measurement of reduced relaxation modulus with high accuracy. This protocol is first validated by performing nanoindentation relaxation tests on a soft material and by comparing the results with those from independent measurements. Then indentation tests of cartilage cells are conducted to demonstrate this method in determining time-dependent properties of living cells. Finally, the change in the viscoelasticity of MCF-7 cells under hyperthermia is investigated.

Piezo1 regulates migration and invasion of breast cancer cells via modulating cell mechanobiological properties.

Cell migration and invasion are two essential processes during cancer metastasis. Increasing evidence has shown that the Piezo1 channel is involved in mediating cell migration and invasion in some types of cancers. However, the role of Piezo1 in the breast cancer and its underlying mechanisms have not been clarified yet. Here, we show that Piezo1 is high-expressed in breast cancer cell (BCC) lines, despite its complex expression in clinical patient database. Piezo1 knockdown (Piezo1-KD) promotes unconfined BCC migration, but impedes confined cell migration. Piezo1 may mediate BCC migration through the balances of cell adhesion, cell stiffness, and contractility. Furthermore, Piezo1-KD inhibits BCC invasion by impairing the invadopodium formation and suppressing the expression of metalloproteinases (MMPs) as well. However, the proliferation and cell cycle of BCCs are not significantly affected by Piezo1. Our study highlights a crucial role of Piezo1 in regulating migration and invasion of BCCs, indicating Piezo1 channel might be a new prognostic and therapeutic target in BCCs.

Flavonoids modulate liposomal membrane structure, regulate mitochondrial membrane permeability and prevent erythrocyte oxidative damage.

In the present work, we investigated the interaction of flavonoids (quercetin, naringenin and catechin) with cellular and artificial membranes. The flavonoids considerably inhibited membrane lipid peroxidation in rat erythrocytes treated with tert-butyl hydroperoxide (700 µM), and the IC50 values for prevention of this process were equal to 9.7 ± 0.8 µM, 8.8 ± 0.7 µM, and 37.8 ± 4.4 µM in the case of quercetin, catechin and naringenin, respectively, and slightly decreased glutathione oxidation. In isolated rat liver mitochondria, quercetin, catechin and naringenin (10-50 µM) dose-dependently increased the sensitivity to Ca2+ ions - induced mitochondrial permeability transition. Using the probes TMA-DPH and DPH we showed that quercetin rather than catechin and naringenin strongly decreased the microfluidity of the 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomal membrane bilayer at different depths. On the contrary, using the probe Laurdan we observed that naringenin transfer the bilayer to a more ordered state, whereas quercetin dose-dependently decreased the order of lipid molecule packing and increased hydration in the region of polar head groups. The incorporation of the flavonoids, quercetin and naringenin and not catechin, into the liposomes induced an increase in the zeta potential of the membrane and enlarged the area of the bilayer as well as lowered the temperature and the enthalpy of the membrane phase transition. The effects of the flavonoids were connected with modification of membrane fluidity, packing, stability, electrokinetic properties, size and permeability, prevention of oxidative stress, which depended on the nature of the flavonoid molecule and the nature of the membrane.

A function of fascin1 in the colony formation of mouse embryonic stem cells.

Fascin1 is known to participate in the migration of cancer cells by binding to actin filaments. Recent studies evidenced that fascin1 also modulates processes such as the tumorigenesis and maintenance of pluripotency genes in cancer stem cells. However, the function of fascin1 in embryonic stem cells remains unclear. In this article, we report that fascin1 is highly expressed and widely distributed in mouse embryonic stem cells (mESCs), which are regulated by JAK-STAT3 and ß-catenin. We found that the overexpression of fascin1 impairs the formation of mESC colonies via the downregulation of intercellular adhesion molecules, and that mimicking the dephosphorylated mutation of fascin1 or inhibiting phosphorylation with Gö6983 significantly enhances colony formation. Hyperphosphorylated fascin1 can promote the maintenance of pluripotency in mESCs via nuclear localization and suppressing DNA methyltransferase expression. Our findings demonstrate a novel function of fascin1, as a vital regulator, in the colony formation and pluripotency of mESCs and provide insights into the molecular mechanisms underlying embryonic stem cell self-organization and development in vitro.

Involvement of p75NTR in the effects of Aß on L-type Ca2+ channel in cultured neuronal networks.

Ca2+ overload in neurons has been implicated in Alzheimer's Disease (AD). Upregulation of Ca2+ through L-type Ca2+ channels was known to be involved in the neurodegeneration induced by amyloid-ß (Aß) peptides in AD. However, little is known about the mechanism by which upregulation of L-type Ca2+ channel currents is linked to Aß-induced neuronal toxicity. In the present study, we found that the L-type Ca2+ current in transgenic AD mice (Tg2576) neurons is greater than in wild-type (WT) neurons, and this Ca2+ channel current change were rescued in Tg2576/p75NTR+/- (p75 neurotrophin receptor) neurons. We further examined the changes in the gating of L-type Ca2+ channels following Aß42 treatment, and the results showed that the L-type Ca2+ channel current was significantly increased by Aß42 treatment in WT hippocampal neurons. Blocking or decreasing the expression of p75NTR eliminated the influence of Aß42 on the L-type Ca2+ channel current in WT hippocampal neurons. We also evaluated how Aß42 affected the voltage-dependent activation and inactivation of L-type Ca2+ channels in cultured WT neurons. The results indicated that the half-maximal activation voltage (V1/2) was left shifted, and the half-inactivation voltage (V1/2) displayed a right shift in neuron treated by Aß42. Decreasing the expression of p75NTR eliminated the effect of Aß42 on voltage-dependent activation and inactivation of the L-type Ca2+ channel. These results indicate that Aß42 changes L-type Ca2+ channel currents by modulating the channel's activation and inactivation dynamics, while decreasing p75NTR expression can remove this effect.

Geometric Confinement Guides the Expression of Cancer Stem Cell Molecular Markers CD44 via Cell Traction Forces.

Cancer stem cells (CSCs) play a critical role in the cancer metastasis and account for tumor heterogeneity. Growing evidence indicates that the CSC phenotypes are related to the tumor microenvironment. In this study, we report that the gradient of mechanical stresses guides the spatial patterning of the expression of CD44 and Yes-associated protein (YAP) in the geometrically confined multicellular sheets. Our study shows that the cytoskeletal contraction regulates the expression of CD44 through the translocation of YAP into the nucleus. The results demonstrate that geometric confinement and mechanical stresses are the regulators in the spatial patterning of CSC. It may help to understand the relationship between the tumor microenvironment and oncogenesis.

Chemical activation of the Piezo1 channel drives mesenchymal stem cell migration via inducing ATP release and activation of P2 receptor purinergic signaling.

In this study, we examined the Ca2+ -permeable Piezo1 channel, a newly identified mechanosensing ion channel, in human dental pulp-derived mesenchymal stem cells (MSCs) and hypothesized that activation of the Piezo1 channel regulates MSC migration via inducing ATP release and activation of the P2 receptor purinergic signaling. The Piezo1 mRNA and protein were readily detected in hDP-MSCs from multiple donors and, consistently, brief exposure to Yoda1, the Piezo1 channel-specific activator, elevated intracellular Ca2+ concentration. Yoda1-induced Ca2+ response was inhibited by ruthenium red or GsMTx4, two Piezo1 channel inhibitors, and also by Piezo1-specific siRNA. Brief exposure to Yoda1 also induced ATP release. Persistent exposure to Yoda1 stimulated MSC migration, which was suppressed by Piezo1-specific siRNA, and also prevented by apyrase, an ATP scavenger, or PPADS, a P2 generic antagonist. Furthermore, stimulation of MSC migration induced by Yoda1 as well as ATP was suppressed by PF431396, a PYK2 kinase inhibitor, or U0126, an inhibitor of the mitogen-activated protein kinase MEK/ERK signaling pathway. Collectively, these results suggest that activation of the Piezo1 channel stimulates MSC migration via inducing ATP release and subsequent activation of the P2 receptor purinergic signaling and downstream PYK2 and MEK/ERK signaling pathways, thus revealing novel insights into the molecular and signaling mechanisms regulating MSC migration. Such findings provide useful information for evolving a full understanding of MSC migration and homing and developing strategies to improve MSC-based translational applications.

BK channels regulate calcium oscillations in ventricular myocytes on different substrate stiffness.

Substrate stiffness is essential for cell functions, but the mechanisms by which cell sense mechanical cues are still unclear. Here we show that the frequency and the amplitude of spontaneous Ca2+ oscillations were greater in chick cardiomyocytes cultured on the stiff substrates than that on the soft substrates. The spontaneous Ca2+ oscillations were increased on stiff substrates. However, an eliminated dependence of the Ca2+ oscillations on substrate stiffness was observed after applying blocker of the large-conductance Ca2+-activated K+ (BK) channels. In addition, the activity of BK channels in cardiomyocytes cultured on the stiff substrates was decreased. These results provide compelling evidences to show that BK channels are crucial in substrate stiffness-dependent regulation of the Ca2+ oscillation in cardiomyocytes.

Heat Stress-Induced Multiple Multipolar Divisions of Human Cancer Cells.

Multipolar divisions of heated cells has long been thought to stem from centrosome aberrations of cells directly caused by heat stress. In this paper, through long-term live-cell imaging, we provide direct cellular evidences to demonstrate that heat stress can promote multiple multipolar divisions of MGC-803 and MCF-7 cells. Our results show that, besides facilitating centrosome aberration, polyploidy induced by heat stress is another mechanism that causes multipolar cell divisions, in which polyploid cancer cells engendered by mitotic slippage, cytokinesis failure, and cell fusion. Furthermore, we also find that the fates of theses polyploid cells depend on their origins, in the sense that the polyploid cells generated by mitotic slippage experience bipolar divisions with a higher rate than multipolar divisions, while those polyploid cells induced by both cytokinesis failure and cell fusion have a higher frequency of multipolar divisions compared with bipolar divisions. This work indicates that heat stress-induced multiple multipolar divisions of cancer cells usually produce aneuploid daughter cells, and might lead to genetically unstable cancer cells and facilitate tumor heterogeneity.

Mechanism of Stiff Substrates up-Regulate Cultured Neuronal Network Activity.

Our previous work provided compelling evidence showing that substrate stiffness is crucial for regulating synaptic connectivity and excitatory synaptic transmission among neurons in the neuronal network. However, the underlying mechanisms remain elusive. In our study, polydimethylsiloxane (PDMS) substrates with different stiffness have been fabricated to investigate the mechanisms by which the substrate stiffness upregulates the formation and activity of the cultured neuronal network. Here we report that stiff substrate increased both the number of synapses and the efficacy of excitatory synaptic transmission. More colocalization of synaptotagmin and PSD-95 was observed in the neuronal network on stiff substrate, which indicated the synapse number has increased. We also found that the increased synapse number was mediated by Hevin and SPARC that are secreted from astrocyte. The increased efficacy of excitatory synaptic transmission induced by stiff substrate was explored in three aspects. First, stiff substrate enhanced the presynaptic activity through increasing the vesicular release probability (Pr) of neurotransmitters as well as the calcium influx. Second, stiff substrate reduced voltage-dependent Mg2+ blockade to N-methyl-d-aspartate receptor (NMDAR) channels, which led to higher postsynaptic activity. Third, our work suggested that the increased excitatory synaptic transmission in the neural network on stiff substrate involved the upregulated synaptic glutamate concentration. Taken together, these findings may provide a molecular mechanism underlying substrate stiffness regulation of excitatory synaptic transmission in the cultured neural network.