Functional state of the myometrium of rats under chronic in vivo effect of nanostructured ZnO and ТіО2 materials

The specificities of the structure and blood supply of the uterus facilitate a considerable accumulation of nanosized xenobiotics, including nanoparticles of metal oxides, in its tissues. Numerous in vitro and in vivo experiments demonstrated that nanoparticles of metal oxides (ZnO and TiO2) have significant cytotoxic activity, caused by oxidative stress induction. However, there is no information about the impact of these nanomaterials on the functional state of the myometrium under chronic exposure on the organism. Tenzometric methods and mechanokinetic analysis were used in our work to investigate the contractile activity of the myometrium of non-pregnant rats. The contractile activity was either spontaneous or induced by oxytocin (the uterotonic hormone) and acetylcholine (the agonist of muscarinic choline receptors) under chronic peroral intake of the ZnO and TiO2 aqueous nanocolloids into the organism. It was found that after burdening of rats with ZnO and ТіО2 aqueous nanocolloids there were no changes in the pacemaker-dependent mechanisms forming the frequency of spontaneous contractions in the myometrium, but there was a considerably induced increase in the AU index of contractions. It was shown that during the oxytocin-induced excitation of the myometrium under both chronic and short-term burdening of the rats with ZnO and TiO2 aqueous nanocolloids, the mechanisms that regulate the intracellular concentration of Ca2+ ions are the target for the nanomaterials. When the rats were burdened with ZnO aqueous nanocolloids for 6 months, during cholinergic excitation there was hyperstimulation of both M3-receptor-dependent mechanisms of Са2+ ions intake via the potential-governed Са2+-channels of L-type into the smooth muscles of the myometrium, and M2-receptor-dependent mechanisms, controlling the intracellular concentration of these cations. Thus, the regularities and mechanisms of the change in the functioning of uterine smooth muscles under chronic intake of the ZnO and TiO2 aqueous nanocolloids were determined in this study.

Tumor-Derived miR-378a-3p-Containing Extracellular Vesicles Promote Osteolysis by Activating a Dyrk1a/Nfatc1/Angptl2 Axis for Bone Metastasis

Background The majority of the deaths of prostate cancer (PCa) are caused by progression to bone metastatic PCa. The importance of extracellular vesicles (EVs) in the formation of the pre-metastatic niche has been demonstrated in recent years. However, whether and how tumor-derived EVs interact with bone marrow macrophages (BMMs) to release EV-delivered microRNAs to promote osteolysis and to activate pre-metastatic niche formation for PCa bone metastasis remain unclear. Methods Bioinformatics and qRT-PCR analyses were used to screen microRNAs and to identify the elevated expression of miR-378a-3p in both serum-derived EVs from PCa patients and in culture medium-derived EVs from PCa cell lines. Functional assays in vitro and in vivo were performed to investigate the functions of miR-378a-3p during PCa progression. IF staining and Dual-luciferase reporter, co-IP, western blot, RIP and ChIP assays were conducted to reveal the underlying mechanism. Results We found that EV-mediated release of miR-378a-3p from tumor cells was upregulated in bone-metastatic PCa which keeps a low intracellular concentration of miR-378a-3p, to promote proliferation and the MAOA-mediated epithelial-to-mesenchymal transition (EMT) in PCa cells. In addition, we demonstrated that the enrichment of miR-378a-3p in tumor derived EVs was induced by overexpression of hnRNPA2B1 as a transfer chaperone. After miR-378a-3p-enriched EVs were taken in by BMMs, elevated intracellular concentration of miR-378a-3p promoted osteolytic progression by targeting the Dyrk1a/Nfatc1 pathway. Mechanistically, inhibition of Dyrk1a by miR-378a-3p improved the nuclear translocation of Nfatc1 to promote expression of the downstream target gene Angptl2. As a feedback, increased secretion of Angptl2 into the tumor environment promoted PCa progression. Conclusions Our findings indicate that tumor-derived miR-378a-3p-containing EVs play a significant role in promoting prostate cancer bone metastasis by activating a Dyrk1a/Nfatc1/Angptl2 axis in BMMs to induce osteolytic progression, which implicates that miR-378a-3p may be a potential predictor of metastatic PCa. Moreover, reducing the release of miR-378a-3p-containing EVs or inhibiting the recruitment of miR-378a-3p into tumor-derived EVs might be a potential therapeutic strategy for PCa metastasis.

OpaR Controls the Metabolism of c-di-GMP in Vibrio parahaemolyticus

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis worldwide, has a strong ability to form biofilms on surfaces. Quorum sensing (QS) is a process widely used by bacteria to communicate with each other and control gene expression via the secretion and detection of autoinducers. OpaR is the master QS regulator of V. parahaemolyticus operating under high cell density (HCD). OpaR regulation of V. parahaemolyticus biofilm formation has been reported, but the regulatory mechanisms are still not fully understood. bis-(3′-5′)-cyclic di-GMP (c-di-GMP) is an omnipresent intracellular second messenger that regulates diverse behaviors of bacteria including activation of biofilm formation. In this work, we showed that OpaR repressed biofilm formation and decreased the intracellular concentration of c-di-GMP in V. parahaemolyticus RIMD2210633. The OpaR box-like sequences were detected within the regulatory DNA regions of scrA, scrG, VP0117, VPA0198, VPA1176, VP0699, and VP2979, encoding a group of GGDEF and/or EAL-type proteins. The results of qPCR, LacZ fusion, EMSA, and DNase I footprinting assays demonstrated that OpaR bound to the upstream DNA regions of scrA, VP0117, VPA0198, VPA1176, and VP0699 to repress their transcription, whereas it positively and directly regulated the transcription of scrG and VP2979. Thus, transcriptional regulation of these genes by OpaR led directly to changes in the intracellular concentration of c-di-GMP. The direct association between QS and c-di-GMP metabolism in V. parahaemolyticus RIMD2210633 would be conducive to precise control of gene transcription and bacterial behaviors such as biofilm formation.

The inhibition of ABCB1/MDR1 or ABCG2/BCRP enables doxorubicin to eliminate liver cancer stem cells

Two ATP-binding cassette transporters, ABCB1/MDR1 and ABCG2/BCRP, are considered the most critical determinants for chemoresistance in hepatocellular carcinoma. However, their roles in the chemoresistance in liver cancer stem cells remain elusive. Here we explored the role of inhibition of MDR1 or ABCG2 in sensitizing liver cancer stem cells to doxorubicin, the most frequently used chemotherapeutic agent in treating liver cancer. We show that the inhibition of MDR1 or ABCG2 in Huh7 and PLC/PRF/5 cells using either pharmacological inhibitors or RNAi resulted in the elevated level of intracellular concentration of doxorubicin and the accompanied increased apoptosis as determined by confocal microscopy, high-performance liquid chromatography, flow cytometry, and annexin V assay. Notably, the inhibition of MDR1 or ABCG2 led to the reversal of the chemoresistance, as evident from the enhanced death of the chemoresistant liver cancer stem cells in tumorsphere-forming assays. Thus, the elevation of effective intracellular concentration of doxorubicin via the inhibition of MDR1 or ABCG2 represents a promising future strategy that transforms doxorubicin from a traditional chemotherapy agent into a robust killer of liver cancer stem cells for patients undergoing transarterial chemoembolization.

Cholinergic regulation of cardiac pacemaker activity by L-type Cav1.3 channels

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondation Recherche Médicale Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including "funny" f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the role of L-type Cav1.3 channels in cholinergic regulation of heart rate. Methods We recorded the frequency of activation and position of pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice at baseline or during ACh perfusion. We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. We performed in vivo telemetric recordings of heart rate (HR) in WT and GIRK4KO/Cav1.3KO animals. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all sinus nodes tested, regardless of genotype, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3µM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. In vivo data indicate that HR reduction induced by combined injection of Hexamethonium ( a Nicotinic acetylcholine receptor blocker) with Carbamoylcholine (CCH, M2 receptor agonist) or with 2-Chloro-N6-Cyclopentyladenosine (CCPA, A1 receptor agonist) is higher in WT than in GIRK4KO/Cav1.3KO animals (68% vs 48% CCH, and 79% vs 62% CCPA, respectively). Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), cholinergic regulation of sinus node pacemaking is reliant on Cav1.3 and KACh channels.

ROLE OF MACROLIDES (CLARITHROMYCIN) ON BIOFILMS IN CHRONIC RHINOSINUSITIS

Background: Rhinosinusitis, a group of disorders characterized by inammation of the nose and paranasal sinuses can be classied as acute, sub-acute or chronic and recurrent based on the duration of inammation. Biolm with formation of a physical barrier prevent antibiotics from penetrating it. Macrolides achieve high intracellular concentration and have a spectrum of activity against Gram positive cocci. This study aims to describe effect of macrolide antibiotics (clarithromycin) on eradication of suspected biolms in chronic rhinosinusitis.

Calcium homeostasis

Calcium and phosphorus are the most abundant minerals in the body. An adult body contains over 1 kg of calcium and it has various functions throughout the body. The average plasma concentration of calcium is about 2.5 mmol/l. The intracellular concentration of calcium is approximately 100 nmol/ml. The control of both intracellular and extracellular calcium concentrations is vital.

Modelling of combination therapy using implantable anticancer drug delivery with thermal ablation in solid tumor

Local implantable drug delivery system (IDDS) can be used as an effective adjunctive therapy for solid tumor following thermal ablation for destroying the residual cancer cells and preventing the tumor recurrence. In this paper, we develop comprehensive mathematical pharmacokinetic/pharmacodynamic (PK/PD) models for combination therapy using implantable drug delivery system following thermal ablation inside solid tumors with the help of molecular communication paradigm. In this model, doxorubicin (DOX)-loaded implant (act as a transmitter) is assumed to be inserted inside solid tumor (acts as a channel) after thermal ablation. Using this model, we can predict the extracellular and intracellular concentration of both free and bound drugs. Also, Impact of the anticancer drug on both cancer and normal cells is evaluated using a pharmacodynamic (PD) model that depends on both the spatiotemporal intracellular concentration as well as characteristics of anticancer drug and cells. Accuracy and validity of the proposed drug transport model is verified with published experimental data in the literature. The results show that this combination therapy results in high therapeutic efficacy with negligible toxicity effect on the normal tissue. The proposed model can help in optimize development of this combination treatment for solid tumors, particularly, the design parameters of the implant.

Cholinergic regulation of cardiac pacemaker activity by l-type cav1.3 channels

Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including “funny” f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the importance of L-type Cav1.3 channels in the cholinergic regulation of heart rate. Methods We recorded the frequency and the position of the pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice in control or during ACh perfusion. We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all the sinus node tissues tested, regardless of the genotypes, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3μM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), the cholinergic regulation of sinus node pacemaking is predominantly ensured by Cav1.3 and KACh channels. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): “Fondation pour la recherche medicale” FRM