Mediated Signal Transduction Pathways Research Articles

Comparative analyses of dynamic transcriptome profiles highlight key response genes and dominant isoforms for muscle development and growth in chicken

BackgroundModern breeding strategies have resulted in significant differences in muscle mass between indigenous chicken and specialized broiler. However, the molecular regulatory mechanisms that underlie these differences remain elusive. The aim of this study was to identify key genes and regulatory mechanisms underlying differences in breast muscle development between indigenous chicken and specialized broiler.ResultsTwo time-series RNA-sequencing profiles of breast muscles were generated from commercial Arbor Acres (AA) broiler (fast-growing) and Chinese indigenous Lushi blue-shelled-egg (LS) chicken (slow-growing) at embryonic days 10, 14, and 18, and post-hatching day 1 and weeks 1, 3, and 5. Principal component analysis of the transcriptome profiles showed that the top four principal components accounted for more than 80% of the total variance in each breed. The developmental axes between the AA and LS chicken overlapped at the embryonic stages but gradually separated at the adult stages. Integrative investigation of differentially-expressed transcripts contained in the top four principal components identified 44 genes that formed a molecular network associated with differences in breast muscle mass between the two breeds. In addition, alternative splicing analysis revealed that genes with multiple isoforms always had one dominant transcript that exhibited a significantly higher expression level than the others. Among the 44 genes, the TNFRSF6B gene, a mediator of signal transduction pathways and cell proliferation, harbored two alternative splicing isoforms, TNFRSF6B-X1 and TNFRSF6B-X2. TNFRSF6B-X1 was the dominant isoform in both breeds before the age of one week. A switching event of the dominant isoform occurred at one week of age, resulting in TNFRSF6B-X2 being the dominant isoform in AA broiler, whereas TNFRSF6B-X1 remained the dominant isoform in LS chicken. Gain-of-function assays demonstrated that both isoforms promoted the proliferation of chicken primary myoblasts, but only TNFRSF6B-X2 augmented the differentiation and intracellular protein content of chicken primary myoblasts.ConclusionsFor the first time, we identified several key genes and dominant isoforms that may be responsible for differences in muscle mass between slow-growing indigenous chicken and fast-growing commercial broiler. These findings provide new insights into the regulatory mechanisms underlying breast muscle development in chicken.

Redox regulation, thioredoxins and glutaredoxins in retrograde signalling and in gene transcription.

Integration of reactive oxygen species (ROS)-mediated signal transduction pathways via redox sensors and the thiol-dependent signalling network is of increasing interest in cell biology for their implications in plant growth and productivity. Redox regulation is an important point of control in the protein structure, interactions, cellular location and function, thioredoxins (TRXs) and glutaredoxins (GRXs) being key players in the maintenance of cellular redox homeostasis. The crosstalk between second messengers, ROS, thiol redox signalling and redox homeostasis-related genes, controls almost every aspect of plant development and stress response. We review the emerging roles of TRXs and GRXs in redox-regulated processes interacting with other cell signalling systems such as organellar retrograde communication and gene expression, especially in plants during their development and under stressful environments. This approach will throw light on the specific role of these proteins as redox signalling components, and their importance in different developmental processes during abiotic stress.

Enteroaggregative Escherichia coli induced activation of epidermal growth factor receptor contributes to IL-8 secretion by cultured human intestinal epithelial cells

Enteroaggregative Escherichia coli (EAEC) has been identified as a new enteropathogen that causes acute and chronic diarrhea in children and travelers. One defining aspect of EAEC-pathogenesis is the induction of an inflammatory response in intestinal epithelium. In this study, we have found that EAEC-induced EGFR activation in human small intestinal and colonic epithelial was attenuated in the presence of a specific inhibitor of EGFR (Tyrphostin AG1478). Further, the aggregative stacked-brick type of adherence of this organism to both the cell lines and this pathogen-induced cytoskeletal rearrangement of these cells was also reduced in the presence of Tyrphostin AG1478. Moreover, EAEC-induced activation of downstream effectors (ERK-1/2, PI3K and Akt) of EGFR mediated cell signaling pathways were found to be suppressed in the presence of EGFR inhibitor. A decrease in IL-8 response in EAEC infected both the cell types were also noted in the presence of specific inhibitors of these downstream effectors, transcription factors and Tyrphostin AG1478. We propose that EAEC-induced activation of EGFR is quintessential for stacked-brick adherence of EAEC to human intestinal epithelial cells, their cytoskeletal rearrangements and stimulation of ERK-1/2 and PI3K/Akt mediated signal transduction pathways, resulting in the activation of NF-κB, AP-1, STAT-3 and finally IL-8 secretion by these cells.

Sulforaphane suppresses paraquat-induced oxidative damage in bovine in vitro-matured oocytes through Nrf2 transduction pathway

Sulforaphane (SFN), a bioactive phytocompound extracted from cruciferous plants, has received increasing attention due to its vital cytoprotective role in eliminating oxidative free radical through activation of nuclear factor erythroid 2-related factor (Nrf2)-mediated signal transduction pathway. This study aims at a better insight into the protective benefit of SFN in attenuating paraquat (PQ)-caused impairment in bovine in vitro-matured oocytes and the possible mechanisms involved therein. Results showed that addition of 1 μM SFN during oocyte maturation obtained higher proportions of matured oocytes and in vitro-fertilized embryos. SFN application attenuated the toxicological effects of PQ on bovine oocytes, as manifested by enhanced extending capability of cumulus cell and increased extrusion proportion of first polar body. Following incubation with SFN, oocytes exposed to PQ exhibited reduced intracellular ROS and lipid accumulation levels, and elevated T-SOD and GSH contents. SFN also effectively inhibited PQ-mediated increase in BAX and CASPASE-3 protein expressions. Besides, SFN promoted the transcription of NRF2 and its downstream antioxidative-related genes GCLC, GCLM, HO-1, NQO-1, and TXN1 in a PQ-exposed environment, indicating that SFN prevents PQ-caused cytotoxicity through activation of Nrf2 signal transduction pathway. The mechanisms underlying the role of SFN against PQ-induced injury included the inhibition of TXNIP protein and restoration of the global O-GlcNAc level. Collectively, these findings provide novel evidence for the protective role of SFN in alleviating PQ-caused injury, and suggest that SFN application may be an efficacious intervention strategy against PQ cytotoxicity.

From protein-protein interactions to immune modulation: Therapeutic prospects of targeting Neuropilin-1 in high-grade glioma.

In the past several years there has been a marked increase in our understanding of the pathophysiological hallmarks of glioblastoma development and progression, with specific respect to the contribution of the glioma tumor microenvironment to the rapid progression and treatment resistance of high-grade gliomas. Despite these strides, standard of care therapy still only targets rapidly dividing tumor cells in the glioma, and does little to curb the pro-tumorigenic functions of non-cancerous cells entrenched in the glioma microenvironment. This tumor promoting environment as well as the heterogeneity of high-grade gliomas contribute to the poor prognosis of this malignancy. The interaction of non-malignant cells in the microenvironment with the tumor cells accentuate phenotypes such as rapid proliferation or immunosuppression, so therapeutically modulating one target expressed on one cell type may be insufficient to restrain these rapidly developing neoplasias. With this in mind, identifying a target expressed on multiple cell types and understanding how it governs tumor-promoting functions in each cell type may have great utility in better managing this disease. Herein, we review the physiology and pathological effects of Neuropilin-1, a transmembrane co-receptor which mediates signal transduction pathways when associated with multiple other receptors. We discuss its effects on the properties of endothelial cells and on immune cell types within gliomas including glioma-associated macrophages, microglia, cytotoxic T cells and T regulatory cells. We also consider its effects when elaborated on the surface of tumor cells with respect to proliferation, stemness and treatment resistance, and review attempts to target Neuroplin-1 in the clinical setting.

Role of a Disease-associated ST3Gal-4 in Non-small Cell Lung Cancer.

Sialylation promotes tumorigenesis by affecting various cancer-related events, including apoptosis inhibition, cell growth, invasion, migration, metastasis, chemo-resistance, and immunomodulation in favor of tumor progression. An altered expression of sialyltransferase enzymes is responsible for synthesizing various tumor-associated sialylated structures. In the present study, our findings have revealed a significant up-regulation of ST3Gal-4 transcript in the two major subtypes of NSCLC cell lines [squamous cell carcinoma cell line (NCI-H520) and adenocarcinoma cell line (A549)]. Thus, the role of the ST3Gal-4 gene was assessed on cancer-associated signal transduction pathways in these cells in view of proliferation, invasion, and migration. ST3Gal-4 was silenced by transfection of both the cell lines with esi-ST3Gal-4-RNA, which RT-PCR and western immunoblotting confirmed. Silencing of ST3Gal-4 resulted in a decreased expression of MAL-I interacting membrane-HSP60, identified earlier as an α2,3-sialylated glycoprotein, thus pointing towards the possible role of ST3Gal-4 in its sialylation. The proliferation, invasion, and migration of both types of NSCLC cells were reduced significantly in the ST3Gal-4 silenced cells. Our findings were substantiated by the down-regulation of β-catenin and E-cadherin, a reduced expression of activated AKT1, ERK1/2, and NF-ƙB in these cells. We propose that ST3Gal-4 may be the disease-associated sialyltransferase involved in α2,3 sialylation of the membrane proteins, including HSP60 of the NSCLC cells. This may lead to the conformational alteration of these proteins, required for the activation of E-cadherin/β-catenin, AKT, and ERK/NF-ƙB mediated signal transduction pathways in these cells, resulting in their proliferation, invasion, and migration.

Multiple Components of Protein Homeostasis Pathway Can Be Targeted to Produce Drug Synergies with VCP Inhibitors in Ovarian Cancer.

Simple SummaryThe increased dependency of cancer cells on protein homeostasis creates a vulnerability that can be exploited using protein homeostasis inhibiting drugs. Recently, valosin-containing protein has emerged as a key component of the pathway and a novel therapeutic target for cancer treatment. The aim of our research is to explore the potential synergies between VCP inhibitors and other agents that disrupt protein homeostasis and to provide mechanistic understanding of drug synergies targeting multiple components of protein homeostasis. Our results indicate that VCP inhibitors can be combined with other endoplasmic reticulum stress-inducing agents to produce synergistic cytotoxicity in ovarian cancer cells.Protein quality control mechanisms play an important role in cancer progression by providing adaptive responses and morphologic stability against genome-wide copy number alterations, aneuploidy, and conformation-altering somatic mutations. This dependency on protein quality control mechanisms creates a vulnerability that may be exploited for therapeutic benefits by targeting components of the protein quality control mechanism. Recently, valosin-containing protein (VCP), also known at p97 AAA-ATPase, has emerged as a druggable target in cancer cells to affect their dependency on protein quality control. Here, we show that VCP inhibitors induce cytotoxicity in several ovarian cancer cell lines and these compounds act synergistically with mifepristone, a drug previously shown to induce an atypical unfolded protein response. Although mifepristone at a clinically achievable dose induces a weak unfolded protein response, it enhances the cytotoxic effects of VCP inhibitor CB-5083. Mechanistically, mifepristone blocks the cytoprotective effect of ATF6 in response to endoplasmic reticulum (ER) stress while activating the cytotoxic effects of ATF4 and CHOP through the HRI (EIF2AK1)-mediated signal transduction pathway. In contrast, CB-5083 activates ATF4 and CHOP through the PERK (EIF2AK3)-mediated signaling pathway. This combination activates ATF4 and CHOP while blocking the adaptive response provided by ATF6, resulting in increased cytotoxic effects and synergistic drug interaction.

Transcriptome changes in ERGIC3-knockdown hepatocellular carcinoma cells: ERGIC3 is a novel immune function related gene.

ObjectiveThe expression of ERGIC3 is increased in a variety of tumors and promotes the growth and metastasis of liver cancer, but the molecular mechanism needs to be further studied.In this study, we aimed to analyze the molecular mechanism of ERGIC3 regulating the proliferation of human hepatocellular carcinoma (HCC) SMMC-7721 cells using transcriptomics.MethodsERGIC3 was knocked down in SMMC-7721 cells by RNAi technique, and the expression of ERGIC3 was detected by Q-RT-PCR and Western Blot. RNA sequencing was performed in the Illumina HiSeq platform in the control group and the ERGIC3i group and bioinformatics methods were selected to analyze the data.ResultsThe expression of ERGIC3 was reduced to 10% in SMMC-7721 cells by RNAi technique, and 176 genes were up-regulated and 34 genes were down-regulated in ERGIC3i group compared with the control group. Analysis of the pathways and biological processes that enrich the function of differentially expressed genes showed thatthese differentially expressed genes were mainly involved in vesicular transport, growth factors, PI3K-Akt, NOD-like, Jak-STAT, NF-kappa B and other protein kinase-coupled receptors mediated signal transduction pathways, tumor immune response, collagen-integrin receptor-actin axis, and miRNA pathways. More importantly, most of the significantly altered pathways were related to immunity. ERGIC3 may be a key immune-related gene.ConclusionBased on the transcriptomic analysis, the mechanism of ERGIC3 promoting the growth of HCC is link with the transport of growth factor receptor, cytokine receptor and collagen. Then it is involved in signal transduction pathways mediated by protein kinase-coupled receptors, PI3K-Akt, NOD-like, Jak-STAT and NF-kappa B. In particular, the mechanism is also involved in the ERGIC3-dependent immune pathways. ERGIC3 is a potential target for prevention and treatment of HCC.

Casein kinases are required for the stability of the glucose-sensing receptor Rgt2 in yeast

In yeast, glucose induction of HXT (glucose transporter gene) expression is achieved via the Rgt2 and Snf3 glucose sensing receptor (GSR)-mediated signal transduction pathway. The membrane-associated casein kinases Yck1 and Yck2 (Ycks) are involved in this pathway, but their exact role remains unclear. Previous work suggests that the Ycks are activated by the glucose-bound GSRs and transmit the glucose signal from the plasma membrane to the nucleus. However, here we provide evidence that the YCks are constitutively active and required for the stability of the Rgt2 receptor. Cell surface levels of Rgt2 are significantly decreased in a yck1Δyck2ts mutant, but this is not due to endocytosis-mediated vacuolar degradation of the receptor. Similar observations are made in an akr1Δ mutant, where the Ycks are no longer associated with the membrane, and in a sod1Δ mutant in which the kinases are unstable. Of note, in an akr1Δ mutant, both the Ycks and Rgt2 are mislocalized to the cytoplasm, where Rgt2 is stable and functions as an effective receptor for glucose signaling. We also demonstrate that Rgt2 is phosphorylated on the putative Yck consensus phosphorylation sites in its C-terminal domain (CTD) in a Yck-dependent manner and that this glucose-induced modification is critical for its stability and function. Thus, these results indicate a role for the Ycks in stabilizing Rgt2 and suggest that Rgt2 may use glucose binding as a molecular switch not to activate the Ycks but to promote Yck-dependent interaction and phosphorylation of the CTD that increases its stability.

Galanin enhanced insulin-mediated intracellular signaling by regulating the stability of membrane-localized insulin/IR.

Previous studies have shown that insulin has the important regulatory effect on the intestinal tract. However, until now, the biological properties of insulin on intestinal cell has not been revealed. Therefore, in the current research, we first studied the cell characteristics and signaling profiles of insulin in the intestinal cell model, and found that insulin can be internalized into the cytoplasm in a time-dependent manner. After internalization, insulin transported into different type of endosomes. More importantly, we explored the effect of galanin on insulin-mediated signaling pathways (galanin is a polypeptide composed of 29 amino acid residues, galanin is widely distributed in the central and peripheral nervous system and has a variety of biological activities), and found that galanin can increase insulin sensitivity by regulating insulin receptor (IR)-mediated signal transduction pathways. We further study the potential molecular mechanism by which galanin enhances insulin sensitivity, and found that galanin could increase the time of insulin acting on the cell membrane. Further experiments showed that galanin could stabilize the membrane-localized insulin/IR, which may be an important new potential mechanism by which galanin improves the biological activity of insulin. This study laid the foundation for exploring the relationship between galanin and insulin sensitivity.

The Stomatal Openings Occurred from Blue Light Photoreceptors Mediated Signal Transduction Pathway May be Enhanced by a Blue Light Stimulated Photosynthesis

The Stomatal Openings Occurred from Blue Light Photoreceptors Mediated Signal Transduction Pathway May be Enhanced by a Blue Light Stimulated Photosynthesis

The kinase CIPK14 functions as a negative regulator of plant immune responses to Pseudomonas syringae in Arabidopsis

As a critical second messenger in plants, Ca2+ is involved in numerous biological processes including biotic and abiotic stress responses. The CBL-interacting protein kinases, known as CIPKs, are essential components in Ca2+-mediated signal transduction pathways. Here, we found that CIPK14 plays a role in the process of regulating immune response in Arabidopsis. The CIPK14 loss-of-function mutants exhibited enhanced resistance to the P. syringae, whereas CIPK14 overexpression plants were more susceptible to bacterial pathogen. Enhanced resistance in cipk14 mutants were accompanied by increased accumulation of SA and elevated expression of defense marker genes (PR1, EDS1, EDS5, ICS1). Overexpression of CIPK14 suppressed Pst DC3000, Pst DC3000 hrcC and flg22 induced generation of ROS and callose deposition. As compared with wild type plants, the expression levels of MPK3/6-dependent PTI marker genes (FRK1, CYP81F2, WAK2, FOX) were up-regulated in cipk14 mutants but down-regulated in CIPK14 overexpression plants after flg22 and elf18 treatment. Additionally, both loss-of-function and gain-of-function of CIPK14 significantly altered the phosphorylation status of MPK3/6 under flg22 treatment, suggesting that CIPK14 is a general modulator of plant immunity at both transcriptional and post-transcriptional level. Taken together, our results uncover that CIPK14 acts as a negative regulator in plant immune response.

The Effects of Apelin and Elabela Ligands on Apelin Receptor Distinct Signaling Profiles.

Apelin and Elabela are endogenous peptide ligands for Apelin receptor (APJ), a widely expressed G protein-coupled receptor. They constitute a spatiotemporal dual ligand system to control APJ signal transduction and function. We investigated the effects of Apelin-13, pGlu1-apelin-13, Apelin-17, Apelin-36, Elabela-21 and Elabela-32 peptides on APJ signal transduction. Whether different ligands are biased to different APJ mediated signal transduction pathways was studied. We observed the different changes of G protein dependent and β-arrestin dependent signaling pathways after APJ was activated by six peptide ligands. We demonstrated that stimulation with APJ ligands resulted in dose-dependent increases in both G protein dependent [cyclic AMP (cAMP), Ca2+ mobilization, and the early phase extracellular related kinase (ERK) activation] and β-arrestin dependent [GRKs, β-arrestin 1, β-arrestin 2, and β2 subunit of the clathrin adaptor AP2] signaling pathways. However, the ligands exhibited distinct signaling profiles. Elabela-32 showed a >1000-fold bias to the β-statin-dependent signaling pathway. These data provide that Apelin-17 was biased toward β-arrestin dependent signaling. Eabela-21 and pGlu1-Apelin-13 exhibited very distinct activities on the G protein dependent pathway. The activity profiles of these ligands could be valuable for the development of drugs with high selectivity for specific APJ downstream signaling pathways.

Tensins - emerging insights into their domain functions, biological roles and disease relevance.

Tensins are a family of focal adhesion proteins consisting of four members in mammals (TNS1, TNS2, TNS3 and TNS4). Their multiple domains and activities contribute to the molecular linkage between the extracellular matrix and cytoskeletal networks, as well as mediating signal transduction pathways, leading to a variety of physiological processes, including cell proliferation, attachment, migration and mechanical sensing in a cell. Tensins are required for maintaining normal tissue structures and functions, especially in the kidney and heart, as well as in muscle regeneration, in animals. This Review discusses our current understanding of the domain functions and biological roles of tensins in cells and mice, as well as highlighting their relevance to human diseases.

Biased Opioid Ligands.

Achieving effective pain management is one of the major challenges associated with modern day medicine. Opioids, such as morphine, have been the reference treatment for moderate to severe acute pain not excluding chronic pain modalities. Opioids act through the opioid receptors, the family of G-protein coupled receptors (GPCRs) that mediate pain relief through both the central and peripheral nervous systems. Four types of opioid receptors have been described, including the μ-opioid receptor (MOR), κ-opioid receptor (KOR), δ-opioid receptor (DOR), and the nociceptin opioid peptide receptor (NOP receptor). Despite the proven success of opioids in treating pain, there are still some inherent limitations. All clinically approved MOR analgesics are associated with adverse effects, which include tolerance, dependence, addiction, constipation, and respiratory depression. On the other hand, KOR selective analgesics have found limited clinical utility because they cause sedation, anxiety, dysphoria, and hallucinations. DOR agonists have also been investigated but they have a tendency to cause convulsions. Ligands targeting NOP receptor have been reported in the preclinical literature to be useful as spinal analgesics and as entities against substance abuse disorders while mixed MOR/NOP receptor agonists are useful as analgesics. Ultimately, the goal of opioid-related drug development has always been to design and synthesize derivatives that are equally or more potent than morphine but most importantly are devoid of the dangerous residual side effects and abuse potential. One proposed strategy is to take advantage of biased agonism, in which distinct downstream pathways can be activated by different molecules working through the exact same receptor. It has been proposed that ligands not recruiting β-arrestin 2 or showing a preference for activating a specific G-protein mediated signal transduction pathway will function as safer analgesic across all opioid subtypes. This review will focus on the design and the pharmacological outcomes of biased ligands at the opioid receptors, aiming at achieving functional selectivity.

Heat Shock Protein 90 Involvement in the Development of Idiopathic Epiretinal Membranes

PurposeThis work was aimed to further characterize cells of idiopathic epiretinal membranes (iERMs). We wanted to determine the contribution of 90-kDa heat shock protein (HSP90) to sustain the transforming growth factor-β (TGF-β)-mediated signal transduction pathway in iERM.MethodsImmunofluorescence and confocal microscopy were carried out on deplasticized sections from 36 epiretinal membranes processed for electron microscopy and on frozen sections from five additional samples with antibodies against α-smooth muscle actin (αSMA), vimentin, glial fibrillary acidic protein (GFAP), SMAD2, HSP90α, type-II TGF-β1 receptor (TβRII), type-I collagen, and type-IV collagen. In addition, Müller MIO-M1 cells were transfected with HSP90 and challenged with TGF-β1.ResultsDouble and triple labeling experiments showed that a variable number of TβRII+ cells were present in 94.1% of tested iERMs and they were mostly GFAP−/αSMA+/vimentin+/HSP90α+. In almost half of the cases these cells contained type-I collagen, suggesting their involvement in matrix deposition. HSP90 overexpressing MIO-M1 cells challenged with TGF-β1 showed increased levels of TβRII, SMAD2, SMAD3, and phosphor-SMAD2. Nuclear SMAD2 staining could be observed in HSP90α+ cells on frozen sections of iERMs.ConclusionsCells in iERMs that express TβRII are also HSP90α+ and show the antigenic profile of myofibroblast-like cells as they are GFAP−/αSMA+/vimentin+. HSP90α-overexpressing MIO-M1 cells challenged with TGF-β1 showed an increased activation of the SMAD pathway implying that HSP90α might play a role in sustaining the TGF-β1-induced fibrotic response of iERM cells.

Salt Stress Signals on Demand: Cellular Events in the Right Context.

Plant stress is a real dilemma; it puzzles plant biologists and is a global problem that negatively affects people’s daily lives. Of particular interest is salinity, because it represents one of the major water-related stress types. We aimed to determine the signals that guide the cellular-related events where various adaptation mechanisms cross-talk to cope with salinity-related water stress in plants. In an attempt to unravel these mechanisms and introduce cellular events in the right context, we expansively discussed how salt-related signals are sensed, with particular emphasis on aquaporins, nonselective cation channels (NSCCs), and glycosyl inositol phosphorylceramide (GIPC). We also elaborated on the critical role Ca2+, H+, and ROS in mediating signal transduction pathways associated with the response and tolerance to salt stress. In addition, the fragmentary results from the literature were compiled to develop a harmonized, informational, and contemplative model that is intended to improve our perception of these adaptative mechanisms and set a common platform for plant biologists to identify intriguing research questions in this area.

Reactive oxygen species in male reproduction: A boon or a bane?

Reactive oxygen species (ROS) are free radicals derived from oxygen during normal cellular metabolism. ROS play a crucial role in the physiological processes and signalling pathways associated with male fertility. At physiological concentrations, ROS act as molecular mediators of signal transduction pathways involved in the regulation of the hypothalamic-pituitary-gonadal axis, spermatogenesis and steroidogenesis. Theyalso trigger the morphological changes required for sperm maturation, such as DNA compaction and flagellar modification. Furthermore, ROS modulate crucial processes involved in the attainment of sperm fertilising ability such as capacitation, hyperactivation, acrosome reaction and sperm-oocyte fusion. Conversely, oxidative stress prevails when the concentration of ROS overwhelms the body's antioxidant defence. Various endogenous and exogenous factors enhance the synthesis of ROS resulting in the disruption of structural and functional integrity of spermatozoa through the induction of apoptotic pathway and oxidation of molecules, such as lipids, proteins and DNA. Therefore, maintenance of a balanced redox state is critical for normal male reproductive functions. This article discusses the dual role of ROS in male reproduction, highlighting the physiological role as well as theirpathological implications on male fertility.

SPARC: Atomistic Modeling of Autonomic Stimulation for a Multi‐Scale Neurocardiovascular Simulator.

Heart rhythm and vascular tone are modulated by stimulation of the autonomic nervous system, and dysregulation of sympathetic or parasympathetic stimuli is associated with multiple cardiovascular disorders such as arrhythmias and hypertension. At the molecular level, such neuromodulation is established by stimulating myocyte G protein‐coupled receptor (GPCR) mediated signal transduction pathways with neurotransmitters like norepinephrine or acetylcholine. In this study, funded by NIH Common Fund program “Stimulating Peripheral Activity to Relieve Conditions” (SPARC), we used atomistic modeling to identify structural, energetic, and kinetic determinants of several molecular processes crucial for cardiovascular neuromodulation such as neurotransmitter interactions with GPCR / G protein complexes at the neuroeffector junction, as well as G protein interactions with adenylyl cyclase (AC), a key enzyme for downstream subcellular signaling. To determine these parameters, we developed atomistic models of human β1‐ and β2‐adrenergic receptors (β1AR and β2AR) as well as muscarinic acetylcholine receptor M2 (M2R) in different conformational states with and without bound Gs or Gi protein using available structures and Rosetta structural modeling. These models were validated via molecular docking of βAR agonists norepinephrine and isoproterenol and MR agonists acetylcholine and carbachol to compare with experimental crystal structures. Model structural stabilities were assessed via microsecond‐long all‐atom molecular dynamics (MD) simulations with and without bound ligand. Enhanced sampling atomistic MD simulations were used to estimate state‐dependent receptor ‐ ligand binding affinities and association / dissociation rates, which were compared with available experimental values. Also, coarse‐grained and Brownian dynamics implicit‐solvent techniques were utilized to assess G protein interactions with AC and GPCRs. This information will be used to inform functional kinetic models of autonomic control of myocyte subcellular signaling, a crucial component of our predictive multi‐scale neurocardiovascular simulator.Support or Funding InformationNIH SPARC 1OT2OD026580 (C.E.C., I.V.), UC Davis Pharmacology T32GM099608 (J.R.D.D.), AHA career development award 19CDA34770101 (I.V.), NIH NHLBI 5R01HL128537 (C.E.C., V.Y.Y.) and 5U01HL126273 (C.E.C., V.Y.Y.). Computer time allocations: Anton 2 PSCA18077P (I.V., C.E.C., K.R.D.), XSEDE MCB170095 (I.V., K.R.D.), NCSA Blue Waters Broadening Participation Allocation (C.E.C., I.V., K.R.D.).

LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells.

LDL receptor-related proteins 6 (LRP6) is a type I transmembrane receptor (C-terminus in cytosol), which appears to be essential in numerous biological processes, since it is an essential co-receptor of Wnt ligands for canonical β-catenin dependent signal transduction. It was shown that tissue plasminogen activator (tPA), physically interacting with LRP6, induces protein phosphorylation, which may have large implication in the regulation of neural processes. In this investigation we analyzed whether LRP6 is associated with lipid rafts following tPA triggering in neuroblastoma cells and the role of raft integrity in LRP6 cell signaling. Sucrose gradient separation revealed that phosphorylated LRP6 was mainly, but not exclusively present in lipid rafts; this enrichment became more evident after triggering with tPA. In these microdomains LRP6 is strictly associated with ganglioside GM1, a paradigmatic component of these plasma membrane compartments, as revealed by coimmunoprecipitation experiments. As expected, tPA triggering induced LRP6 phosphorylation, which was independent of LRP1, as revealed by knockdown experiments by siRNA, but strictly dependent on raft integrity. Moreover, tPA induced β-catenin phosphorylation was also significantly prevented by previous pretreatment with methyl-β-cyclodextrin. Our results demonstrate that LRP6 mediated signal transduction pathway triggered by tPA acts through lipid rafts in neuroblastoma cells. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents. Indeed, these data, pointing to the key role of lipid rafts in tPA triggered signaling involving β-catenin, may have pharmacological implications, suggesting that cyclodextrins, and potentially other drugs, such as statins, may represent an useful tool.