Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells

The uptake of calcium phosphate nanoparticles (diameter 120 nm) with different charge by HeLa cells was studied by flow cytometry. The amount of uptaken nanoparticles increased with increasing concentration of nanoparticles in the cell culture medium. Several inhibitors of endocytosis and macropinocytosis were applied to elucidate the uptake mechanism of nanoparticles into HeLa cells: wortmannin, LY294002, nocodazole, chlorpromazine and nystatin. Wortmannin and LY294002 strongly reduced the uptake of anionic nanoparticles, which indicates macropinocytosis as uptake mechanism. For cationic nanoparticles, the uptake was reduced to a lesser extent, indicating a different uptake mechanism. The localization of nanoparticles inside the cells was investigated by conjugating them with the pH-sensitive dye SNARF-1. The nanoparticles were localized in lysosomes after 3 h of incubation.     

PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes

Perspectives for the use of carbon nanotubes in biomedical applications depend largely on their ability to degrade in the body into products that can be easily cleared out. Carboxylated single-walled carbon nanotubes (c-SWCNTs) were shown to be degraded by oxidants generated by peroxidases in the presence of hydrogen peroxide. In the present study we demonstrated that conjugation of poly(ethylene glycol) (PEG) to c-SWCNTs does not interfere with their degradation by peroxidase/H₂O₂ system or by hypochlorite. Comparison of different heme-containing proteins for their ability to degrade PEG-SWCNTs has led us to conclude that the myeloperoxidase (MPO) product hypochlorous acid (HOCl) is the major oxidant that may be responsible for biodegradation of PEG-SWCNTs in vivo. MPO is secreted mainly by neutrophils upon activation. We hypothesize that SWCNTs may enhance neutrophil activation and therefore stimulate their own biodegradation due to MPO-generated HOCl. PEG-SWCNTs at concentrations similar to those commonly used in in vivo studies were found to activate isolated human neutrophils to produce HOCl. Both PEG-SWCNTs and c-SWCNTs enhanced HOCl generation from isolated neutrophils upon serum-opsonized zymosan stimulation. Both types of nanotubes were also found to activate neutrophils in whole blood samples. Intraperitoneal injection of a low dose of PEG-SWCNTs into mice induced an increase in percentage of circulating neutrophils and activation of neutrophils and macrophages in the peritoneal cavity, suggesting the evolution of an inflammatory response. Activated neutrophils can produce high local concentrations of HOCl, thereby creating the conditions favorable for degradation of the nanotubes.     

Preconditioning-mimetics bradykinin and DADLE activate PI3-kinase through divergent pathways

We previously reported that pharmacological preconditioning of rabbit hearts with acetylcholine involves activation of phosphatidylinositol 3-kinase (PI3-K) through transactivation of the epidermal growth factor receptor (EGFR). Transactivation is thought to be initiated by cleavage of membrane-bound pro-heparin-binding EGF-like growth factor (HB-EGF) by a membrane metalloproteinase thus releasing HB-EGF which binds to the EGFR. This pathway leads to redox signaling with the generation of reactive oxygen species (ROS) by mitochondria. We tested whether preconditioning's physiological triggers, bradykinin and opioid, also signal through the EGFR. Both bradykinin and the synthetic delta-opioid agonist DADLE increased ROS production in isolated cardiomyocytes by approximately 50%. DADLE's effect was abrogated by either metalloproteinase inhibitor III (MPI) or the diphtheria toxin mutant CRM-197 which blocks heparin-binding EGF shedding indicating that DADLE signals through EGFR transactivation. MPI also blocked DADLE's infarct-sparing effect in whole hearts. Additionally, blocking Src kinase (a component of the EGFR's signaling complex) with PP2 or PI3-K with wortmannin blocked DADLE's effect on cardiomyocyte ROS production and PP2 blocked DADLE's salvage of ischemic myocardium. Finally, DADLE increased phosphorylation of Akt and extracellular signal-regulated protein kinases (ERK) 1/2 in left ventricular myocardium, and this increase was blocked by the EGFR antagonist AG1478. On the other hand, neither MPI nor CRM-197 prevented bradykinin from increasing ROS production, and MPI did not affect bradykinin's infarct-sparing effect in intact hearts. Conversely, both PP2 and wortmannin blocked bradykinin's effect on ROS generation and also aborted bradykinin's cardioprotective effect in intact hearts. While bradykinin also increased phosphorylation of Akt and ERK in myocardium, that increase was not affected by AG1478. Hence bradykinin, unlike acetylcholine or opioid, does not transactivate EGFR, although all 3 agonists do signal through Src and PI3-K.     

Evaluating the multivisceral involvement on adult-onset Still’s disease to retrieve imaging-based differences in patients with and without macrophage activation syndrome: results from a single-centre observational study

Clinical Rheumatology - In this study, we aimed at describing the multivisceral involvement on adult-onset Still’s disease (AOSD) to retrieve imaging-based differences in patients with and...     

Evaluation of diffusion kurtosis and diffusivity from baseline staging MRI as predictive biomarkers for response to neoadjuvant chemoradiation in locally advanced rectal cancer

Abdominal Radiology - To evaluate the role of diffusion kurtosis and diffusivity as potential imaging biomarkers to predict response to neoadjuvant chemoradiation therapy (CRT) from baseline...     

BRET-based effector membrane translocation assay monitors GPCR-promoted and endocytosis-mediated Gq activation at early endosomes

G protein–coupled receptors (GPCRs) are gatekeepers of cellular homeostasis and the targets of a large proportion of drugs. In addition to their signaling activity at the plasma membrane, it has been proposed that their actions may result from translocation and activation of G proteins at endomembranes—namely endosomes. This could have a significant impact on our understanding of how signals from GPCR-targeting drugs are propagated within the cell. However, little is known about the mechanisms that drive G protein movement and activation in subcellular compartments. Using bioluminescence resonance energy transfer (BRET)–based effector membrane translocation assays, we dissected the mechanisms underlying endosomal G_q trafficking and activity following activation of G_q-coupled receptors, including the angiotensin II type 1, bradykinin B₂, oxytocin, thromboxane A₂ alpha isoform, and muscarinic acetylcholine M₃ receptors. Our data reveal that GPCR-promoted activation of G_q at the plasma membrane induces its translocation to endosomes independently of β-arrestin engagement and receptor endocytosis. In contrast, G_q activity at endosomes was found to rely on both receptor endocytosis-dependent and -independent mechanisms. In addition to shedding light on the molecular processes controlling subcellular G_q signaling, our study provides a set of tools that will be generally applicable to the study of G protein translocation and activation at endosomes and other subcellular organelles, as well as the contribution of signal propagation to drug action.

G protein–coupled receptors (GPCRs) act as signaling hubs that direct molecular events to maintain cellular homeostasis. Historically, signal transduction has been thought to take place exclusively at the plasma membrane—where receptors activate heterotrimeric G proteins and β-arrestins (βARRs) desensitize them (1). This canonical view of receptor signaling has been challenged in recent years by observations that GPCRs can activate G proteins at endomembranes such as early endosomes, the Golgi apparatus, mitochondria, and the nucleus and that βARRs modulate signal amplitude and duration (2). These findings are important in the context of pharmacology and drug discovery because they will redefine how we understand cell signaling and may impact drug development in the future. Our understanding of the mechanisms controlling endomembrane signaling has been limited by the lack of tools required to quantitatively measure not only the presence of G protein but also their signaling at a subcellular level.Studies aimed at better understanding G protein trafficking have largely focused on G_s, which is known to leave the plasma membrane upon activation, become cytoplasmic, and sample multiple endomembrane compartments (3, 4). Mechanistic insight into this phenomenon has revealed that G_s becomes reactivated at endosomes following translocation (5). However, less is known about the activation status of other G protein families found in subcellular organelles. In particular, G_q has been shown to transit from the plasma membrane to endosomes upon receptor activation and as a consequence of activating mutations (6–8). Yet, the contribution of βARR and receptor endocytosis to G protein trafficking and activation at endosomes has not been documented. In the absence of tools that directly measure G protein activation, current evidence for endosomal G_q activity has been limited to amplified downstream signaling events.Nanobodies have been used as crystallization chaperones and as live cell imaging tools to visualize the distribution of active-state proteins (9). These tools have been particularly effective for G_s-coupled receptors in the absence of tools directly measuring the activity of the G protein in organelles (i.e., known effector regulators of G_s signaling). In particular, nanobodies or engineered G proteins that bind the active conformation of the receptor and nanobodies that stabilize the nucleotide-free state of G_s were used to suggest that receptor-G_s complexes were active at endomembranes (5, 10–12). In order to unearth more direct evidence of activation and a better understanding of the mechanisms for the other G protein families, tools that directly monitor the molecular events that immediately follow G protein activation would offer much-needed insight.Enhanced bystander bioluminescence resonance energy transfer (ebBRET) was developed as a way to provide more sensitive and robust measurements of protein trafficking and activity in living cells. This technique relies on the natural association of the luciferase and green fluorescent protein (GFP) that both come from Renilla reniformis (Rluc and rGFP, respectively). These two proteins do not interact spontaneously unless they are concentrated in the same compartment and thus can be used to monitor protein movement when the energy acceptor is anchored to a specific subcellular compartment (13). To measure the activity of G proteins at endosomes, we engineered an ebBRET-based effector membrane translocation assay (EMTA) for subcellular organelles. In addition, we developed organelle-targeted inhibitors to block G protein activity in a compartment-specific manner. Using these tools, we demonstrate the direct activation of G_q/11 by angiotensin II type 1 (AT₁R), bradykinin B₂ (B₂R), oxytocin (OXTR), thromboxane A₂ alpha isoform (TPαR), and muscarinic acetylcholine M₃ (M₃R) receptors in endosomes. Using cells devoid of βARRs (14), our results show that in contrast to the GPCRs, for which trafficking to endosomes is βARR-dependent, the trafficking of G_q to this compartment was not. These findings indicate that G_q translocation can occur through nonconventional receptor endocytosis–independent pathways. Interestingly, activation of G_q in the endosomal compartment was two-tiered, displaying a component that was βARR-independent and another that was further promoted by βARR, supporting the notion that the formation of a receptor–G_q complex in endosomes can promote G protein activation. Moreover, we show that G_q signaling at the plasma membrane not only exhibits a faster onset than the signaling that takes places in endosomes, but it is also functionally different. We anticipate that our methodological advances and mechanistic understanding of endosomal G_q signaling will be of general interest to the study of other GPCRs and to the study of G protein signaling in other compartments.     

Focal amyotrophy in multiple sclerosis

Introduction: The assumption that multiple sclerosis (MS) is purely a white matter disease has been challenged in recent years by observations of axonal damage and neuronal loss in gray matter of the cortex, subcortex, and spinal cord. Methods: We report the case of a 71‐year‐old man with primary progressive MS and longstanding right arm weakness who presented with intermittent right arm pain. Results: Neurological examination showed atrophy, weakness, and hyporeflexia, and electromyography (EMG) showed acute and chronic partial denervation in multiple segments of the right arm. Magnetic resonance imaging (MRI) demonstrated asymmetric volume loss and increased T2 signal in the right anterior spinal cord from C3 to C7, with no evidence of nerve root compression. Conclusions: Lower motor neuron involvement of his right arm was caused by MS with involvement of either the anterior horn cells or the intraspinal motor nerve roots. Muscle Nerve 51 : 137–140, 2015