Interleukin‐6 (IL‐6) is critically involved in liver regeneration after partial hepatectomy (PHX). Previous reports suggest that IL‐6 trans‐signaling through the soluble IL‐6/IL‐6R complex is ...involved in this process. However, the long‐term contribution of IL‐6 trans‐signaling for liver regeneration after PHX is unknown. PHX‐induced generation of the soluble IL‐6R by ADAM (a disintegrin and metallo) proteases enables IL‐6 trans‐signaling, in which IL‐6 forms an agonistic complex with the soluble IL‐6 receptor (sIL‐6R) to activate all cells expressing the signal‐transducing receptor chain glycoprotein 130 (gp130). In contrast, without activation of ADAM proteases, IL‐6 in complex with membrane‐bound IL‐6R and gp130 activates classic signaling. Here, we describe the generation of IL‐6 trans‐signaling mice, which exhibit boosted IL‐6 trans‐signaling and abrogated classic signaling by genetic conversion of all membrane‐bound IL‐6R into sIL‐6R proteins phenocopying hyperactivation of ADAM‐mediated shedding of IL‐6R as single substrate. Importantly, although IL‐6R deficient mice were strongly affected by PHX, survival and regeneration of IL‐6 trans‐signaling mice was indistinguishable from control mice, demonstrating that IL‐6 trans‐signaling fully compensates for disabled classic signaling in liver regeneration after PHX. Moreover, we monitored the long‐term consequences of global IL‐6 signaling inhibition versus IL‐6 trans‐signaling selective blockade after PHX by IL‐6 monoclonal antibodies and soluble glycoprotein 130 as fragment crystallizable fusion, respectively. Both global IL‐6 blockade and selective inhibition of IL‐6 trans‐signaling results in a strong decrease of overall survival after PHX, accompanied by decreased signal transducer and activator of transcription 3 phosphorylation and proliferation of hepatocytes. Mechanistically, IL‐6 trans‐signaling induces hepatocyte growth factor production by hepatic stellate cells. Conclusion: IL‐6 trans‐signaling, but not classic signaling, controls liver regeneration following PHX.
Silencing of the fragile X mental retardation 1 (FMR1) gene and consequently lack of synthesis of FMR protein (FMRP) are associated with fragile X syndrome, which is one of the most prevalent ...inherited intellectual disabilities, with additional roles in increased viral infection, liver disease, and reduced cancer risk. FMRP plays critical roles in chromatin dynamics, RNA binding, mRNA transport, and mRNA translation. However, the underlying molecular mechanisms, including the (sub)cellular FMRP protein networks, remain elusive. Here, we employed affinity pull‐down and quantitative LC‐MS/MS analyses with FMRP. We identified known and novel candidate FMRP‐binding proteins as well as protein complexes. FMRP interacted with 180 proteins, 28 of which interacted with its N terminus. Interaction with the C terminus of FMRP was observed for 102 proteins, and 48 proteins interacted with both termini. This FMRP interactome comprises known FMRP‐binding proteins, including the ribosomal proteins FXR1P, NUFIP2, Caprin‐1, and numerous novel FMRP candidate interacting proteins that localize to different subcellular compartments, including CARF, LARP1, LEO1, NOG2, G3BP1, NONO, NPM1, SKIP, SND1, SQSTM1, and TRIM28. Our data considerably expand the protein and RNA interaction networks of FMRP, which thereby suggest that, in addition to its known functions, FMRP participates in transcription, RNA metabolism, ribonucleoprotein stress granule formation, translation, DNA damage response, chromatin dynamics, cell cycle regulation, ribosome biogenesis, miRNA biogenesis, and mitochondrial organization. Thus, FMRP seems associated with multiple cellular processes both under normal and cell stress conditions in neuronal as well as non‐neuronal cell types, as exemplified by its role in the formation of stress granules.
FMRP appears to play a critical role in diverse intracellular processes in both neuronal and non‐neuronal cells. Here, we propose its involvement in transcription (A), DNA damage response (B), cell cycle (C), actin dynamics and transport (D), mitochondrial organization (E), immune response (F), RNA metabolism (G), ribosome biogenesis (H), RNA granules (I), translation (J), and miRNA biogenesis (K). ER, endoplasmic reticulum; Mt, mitochondrion; N, nucleus; No, nucleolus; PM, plasma membrane; SG, stress granules.
Human pluripotent stem cells (PSCs) open new windows for basic research and regenerative medicine due to their remarkable properties, i.e. their ability to self-renew indefinitely and being ...pluripotent. There are different, conflicting data related to the role of basic fibroblast growth factor (bFGF) in intracellular signal transduction and the regulation of pluripotency of PSCs. Here, we investigated the effect of bFGF and its downstream pathways in pluripotent vs. differentiated human induced (hi) PSCs.
bFGF downstream signaling pathways were investigated in long-term culture of hiPSCs from pluripotent to differentiated state (withdrawing bFGF) using immunoblotting, immunocytochemistry and qPCR. Subcellular distribution of signaling components were investigated by simple fractionation and immunoblotting upon bFGF stimulation. Finally, RAS activity and RAS isoforms were studied using RAS assays both after short- and long-term culture in response to bFGF stimulation.
Our results revealed that hiPSCs were differentiated into the ectoderm lineage upon withdrawing bFGF as an essential pluripotency mediator. Pluripotency markers OCT4, SOX2 and NANOG were downregulated, following a drastic decrease in MAPK pathway activity levels. Notably, a remarkable increase in phosphorylation levels of p38 and JAK/STAT3 was observed in differentiated hiPSCs, while the PI3K/AKT and JNK pathways remained active during differentiation. Our data further indicate that among the RAS paralogs, NRAS predominantly activates the MAPK pathway in hiPSCs.
Collectively, the MAPK pathway appears to be the prime signaling pathway downstream of bFGF for maintaining pluripotency in hiPSCs and among the MAPK pathways, the activity of NRAS-RAF-MEK-ERK is decreased during differentiation, whereas p38 is activated and JNK remains constant.
Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu ...substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1
and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1-associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1
cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1
-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.
•NP coating with APTES increases biocompatibility and stability of nanoparticles.•Cellular uptake of particles is cell type dependent.•Different pattern of NP internalization are observed after and ...before coating.
Among nanoparticles, magnetic nanoparticles are the most appealing candidate for diagnosis and cancer therapy. The researchers are tempting to improve the particles properties, including the size, shape, coating, and magnetic behavior or heating characteristics. Core shell type of magnetic nanoparticle is an important property that modulates their internalization via normal and cancer cells. In this study, magnetite nanoparticles (MNP) covered by N-(2-aminoethyl)-3-aminopropyltriethoxysilane (aminosilane – APTES) were synthesized by co-precipitation of aqueous solution of ferric chloride and ferrous sulfate iron salts with ammonium hydroxide as a base and functionalized by APTES to increase the viability and affinity of the particles to the cancer cells. The structural and morphological properties of these particles were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transformed infrared spectroscopy (FTIR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT assay) was carried out, to check the viability of the cells treatment with MNP and APTES-MNP. To study the cellular uptake in vitro, two prostate cell lines were investigated: PC3 as a cancerous cell line and BPH1 as a benign epithelial cell line (normal cells). Both cell lines were incubated for 24 h with different concentrations of MNP and APTES-MNP (100 and 500 μg/ml and one untreated sample as control). TEM and flow cytometry (FC) analyses were subsequently carried out to monitor the cellular uptake of MNP and APTES-MNP. FC data revealed an increase in cell granularity following the treatment with high concentration of the particles. Data showed that PC3 cancer cells take up more APTES-MNP with respect to control cells than BPH1 benign cells and in contrast BPH1 cell uptake MNP correlated to control cells more efficient than PC3 cells. The results from FC and TEM analyses demonstrate increasing of affinity of particles to cancer cell line (PC3). In this project we investigated the effect of surface functionalization of NP to affinity of the MNP and APTES-MNP on PC3 cells as a malignancy prostate cell and BPH1 benign cells as a normal cells. This approach may help to optimize the efficiency of hyperthermia for prostate cancer through internalization of particles to the cells or attaching to the membrane.
Abstract
Aims
Atrial fibrillation (AF) is a commonly occurring arrhythmia after cardiac surgery (postoperative AF, poAF) and is associated with poorer outcomes. Considering that reduced atrial ...contractile function is a predictor of poAF and that Ca2+ plays an important role in both excitation–contraction coupling and atrial arrhythmogenesis, this study aims to test whether alterations of intracellular Ca2+ handling contribute to impaired atrial contractility and to the arrhythmogenic substrate predisposing patients to poAF.
Methods and results
Right atrial appendages were obtained from patients in sinus rhythm undergoing open-heart surgery. Cardiomyocytes were investigated by simultaneous measurement of Ca2+i and action potentials (APs, patch-clamp). Patients were followed-up for 6 days to identify those with and without poAF. Speckle-tracking analysis of preoperative echocardiography revealed reduced left atrial contraction strain in poAF patients. At the time of surgery, cellular Ca2+ transients (CaTs) and the sarcoplasmic reticulum (SR) Ca2+ content were smaller in the poAF group. CaT decay was slower in poAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting preserved sodium-calcium exchanger function. In agreement, western blots revealed reduced SERCA2a expression in poAF patients but unaltered phospholamban expression/phosphorylation. Computational modelling indicated that reduced SERCA activity promotes occurrence of CaT and AP alternans. Indeed, alternans of CaT and AP occurred more often and at lower stimulation frequencies in atrial myocytes from poAF patients. Resting membrane potential and AP duration were comparable between both groups at various pacing frequencies (0.25–8 Hz).
Conclusions
Biochemical, functional, and modelling data implicate reduced SERCA-mediated Ca2+ reuptake into the SR as a major contributor to impaired preoperative atrial contractile function and to the pre-existing arrhythmogenic substrate in patients developing poAF.
Graphical Abstract
Abstract
Aims
Atrial fibrillation (AF) is associated with tachycardia-induced cellular electrophysiology alterations which promote AF chronification and treatment resistance. Development of novel ...antiarrhythmic therapies is hampered by the absence of scalable experimental human models that reflect AF-associated electrical remodelling. Therefore, we aimed to assess if AF-associated remodelling of cellular electrophysiology can be simulated in human atrial-like cardiomyocytes derived from induced pluripotent stem cells in the presence of retinoic acid (iPSC-aCM), and atrial-engineered human myocardium (aEHM) under short term (24 h) and chronic (7 days) tachypacing (TP).
Methods and results
First, 24-h electrical pacing at 3 Hz was used to investigate whether AF-associated remodelling in iPSC-aCM and aEHM would ensue. Compared to controls (24 h, 1 Hz pacing) TP-stimulated iPSC-aCM presented classical hallmarks of AF-associated remodelling: (i) decreased L-type Ca2+ current (ICa,L) and (ii) impaired activation of acetylcholine-activated inward-rectifier K+ current (IK,ACh). This resulted in action potential shortening and an absent response to the M-receptor agonist carbachol in both iPSC-aCM and aEHM subjected to TP. Accordingly, mRNA expression of the channel-subunit Kir3.4 was reduced. Selective IK,ACh blockade with tertiapin reduced basal inward-rectifier K+ current only in iPSC-aCM subjected to TP, thereby unmasking an agonist-independent constitutively active IK,ACh. To allow for long-term TP, we developed iPSC-aCM and aEHM expressing the light-gated ion-channel f-Chrimson. The same hallmarks of AF-associated remodelling were observed after optical-TP. In addition, continuous TP (7 days) led to (i) increased amplitude of inward-rectifier K+ current (IK1), (ii) hyperpolarization of the resting membrane potential, (iii) increased action potential-amplitude and upstroke velocity as well as (iv) reversibly impaired contractile function in aEHM.
Conclusions
Classical hallmarks of AF-associated remodelling were mimicked through TP of iPSC-aCM and aEHM. The use of the ultrafast f-Chrimson depolarizing ion channel allowed us to model the time-dependence of AF-associated remodelling in vitro for the first time. The observation of electrical remodelling with associated reversible contractile dysfunction offers a novel platform for human-centric discovery of antiarrhythmic therapies.
Graphical Abstract
Graphical Abstract
Among the signaling molecules indirectly linked to many different cell surface receptors, RAS proteins essentially respond to a diverse range of extracellular cues. They control activities of ...multiple signaling pathways and consequently a wide array of cellular processes, including survival, growth, adhesion, migration, and differentiation. Any dysregulation of these pathway leads, thus, to cancer, developmental disorders, metabolic, and cardiovascular diseases. The biochemistry of RAS family proteins has become multifaceted since the discovery of the first members, more than 40 years ago. Substantial knowledge has been attained about molecular mechanisms underlying post-translational modification, membrane localization, regulation, and signal transduction through diverse effector molecules. However, the increasing complexity of the underlying signaling mechanisms is considerable, in part due to multiple effector pathways, crosstalks between them and eventually feedback mechanisms. Here, we take a broad view of regulatory and signaling networks of all RAS family proteins that extends beyond RAS paralogs. As described in this review, a lot is known but a lot has to be discovered yet.
Graphical abstract: The RAS paralogs, KRAS4B, NRAS, and HRAS, are the best investigated members of the RAS family, not only because of their oncogenic capacity. This protein family, however, contains 22 additional isoforms and paralogs, most of which are distantly related, with typically 20-30% amino acid identity, although they share a conserved GTP-binding domain the color spectrum goes from white (for identical) through yellow and orange (for partially conserved) to red (for highly variable amino acids). RAS family proteins control a wide array of signaling pathways and cellular processes distinct from those controlled by RAS paralogs. This review focuses on common features and differences of RAS family proteins regarding their structure, function, regulation, signaling, and involvement in diseases.
Organophosphorus (OPs) compounds are widely used in many pesticides, insecticides, and chemical nerve agents. These compounds are hazardous for humans and the environment. There are many reports on ...detoxification of these compounds, among them enzymatic cleavage of these compounds with organophosphorus hydrolase (OPH) has been taken into more consideration. Several studies have been performed to improve OPH secretion in Escherichia coli by different signal peptides, but have not been successful. In this study, to achieve the extracellular secretion of OPH in E. coli, the complete opd gene along with its native signal peptide was codon optimized and expressed in E. coli BL21(DE3)pLysS. The culture medium showed OPH activity after 2, 4, and 6 H of induction time. The extracellular secretion of OPH was also confirmed by SDS‐PAGE and Western blot analysis. The effects of different factors in growth medium were also investigated regarding expression and extracellular secretion of OPH. It appears that the secretion of OPH into the extracellular medium is highly affected by culture conditions. Therefore, our results revealed that the recombinant OPH was successfully secreted into the extracellular medium. This secretion system can be considered as a high efficiency biocatalyst for detoxification of OPs compounds.
Chemotherapy is one of the main strategies for reducing the rate of cancer progression or, in some cases, curing the tumour. Since a great number of chemotherapeutic agents are cytotoxic compounds, ...i. e. similarly affect normal and neoplastic cells, application of antitumour drugs is preferred in cancer management and therapy. In this study, the cytotoxicity of diversin was evaluated in 5637 cells, a transitional cell carcinoma (TCC) subline (bladder carcinoma), and normal human fibroblast cells using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Chromatin condensation and DNA damage induced by diversin were also determined by means of 4’,6-diamidino-2- phenylindole (DAPI) staining and the comet assay, respectively. In addition, the mechanism of action of diversin was studied in more detail by the caspase 3 colourimetric assay and flow cytometry-based cell-cycle analyses (PI staining). Our results revealed that diversin has considerable cytotoxic effects in 5637 cells, but not on HFF3 (human foreskin fibroblast) and HDF1 (human dermal fibroblast) cells. Further studies showed that diversin exerts its cytotoxicity via induction of chromatin condensation, DNA damage, and activation of caspase 3 in 5637 cells. In addition, flow cytometric analyses revealed that 5637 cells are mostly arrested at the G2 phase of the cell cycle in the presence of diversin.
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