Probing the mechanism of drug/lipid membrane interactions using Biacore

Assay conditions were established to screen a panel of drugs for binding to liposome surfaces using a surface plasmon resonance (SPR) biosensor. Drugs were found to bind negligibly or reversibly or were retained on the liposome surface. Cationic amphiphilic drugs fell into the last class and correlated with drugs that induce phospholipidosis in vivo. To a first approximation, a single-site model yielded apparent binding affinities that adequately described a drug's dose-dependent binding to liposome surfaces. Affinities ranged at least 1000-fold within the drug panel. A liposome's drug-binding capacity and affinity depended on both the lipid headgroup and the drug's structure. Although a drug's charge state generally dominated whether or not it remained bound to the liposome, subtle structural differences between members of certain drug families led to them having widely differing binding affinities. A comparison between the dissociation of drugs from liposome surfaces by Biacore and the lipid retention measurements determined by a parallel artificial membrane permeability assay was drawn. The results from this study demonstrate the potential of using SPR-based assays to characterize drug/liposome-binding interactions.     

TGF-β Signaling Initiated in Dendritic Cells Instructs Suppressive Effects on Th17 Differentiation at the Site of Neuroinflammation

While the role of Transforming Growth Factor β (TGF-β) as an intrinsic pathway has been well established in driving de novo differentiation of Th17 cells, no study has directly assessed the capacity of TGF-β signaling initiated within dendritic cells (DCs) to regulate Th17 differentiation. The central finding of this study is the demonstration that Th17 cell fate during autoimmune inflammation is shaped by TGF-β extrinsic pathway via DCs. First, we provide evidence that TGF-β limits at the site of inflammation the differentiation of highly mature DCs as a means of restricting Th17 cell differentiation and controlling autoimmunity. Second, we demonstrate that TGF-β controls DC differentiation in the inflammatory site but not in the priming site. Third, we show that TGF-β controls DC numbers at a precursor level but not at a mature stage. While it is undisputable that TGF-β intrinsic pathway drives Th17 differentiation, our data provide the first evidence that TGF-β can restrict Th17 differentiation via DC suppression but such a control occurs in the site of inflammation, not at the site of priming. Such a demarcation of the role of TGF-β in DC lineage is unprecedented and holds serious implications vis-à-vis future DC-based therapeutic targets.     

Radiometrically determined dates and sedimentation rates for recent sediments in nine North African wetland lakes (the CASSARINA Project)

Sediment cores were collected from nine wetland lakes in Morocco, Tunisia and Egypt for the CASSARINA project investigating environmental change in Northern African wetlands. The cores were dated radiometrically by using natural (²¹⁰Pb) and artificial (¹³⁷Cs and ²⁴¹Am) radionuclides. At sites in Morocco and Tunisia with mean annual rainfall totals ranging from 500–1000 mm yr^–1, fallout records were generally satisfactory and it was possible to develop independent sediment chronologies based on the radiometric data alone. At the Egyptian sites, rainfall was less than 200 mm yr^–1 and fallout records were much less distinct. At these sites the radiometric data could only be used to give an indication of mean sedimentation rates during the past 30–40 years. By using a combination of fallout radionuclide, pollen, and macrofossil stratigraphic records it was however possible to determine a credible sediment chronology spanning the major part of the 20th century. Applying this chronology to records of spheroidal carbonaceous particles (SCP) from the same sediment cores, the onset of significant levels of atmospheric pollution in the Nile Delta is dated in all three cores to the mid 1950s. Results from a number of lakes (Sidi Bou Rhaba, Ichkeul and Korba) revealed high and accelerating siltation rates, threatening their continued existence beyond the next few decades. In contrast, sedimentation rates at all three Nile Delta sites appear to have declined in recent decades, most probably due to the impact of the Nile barrages.     

Daily variation in the concentration of neuropeptide Y in the rat atrium: effects of age and photoperiodic conditions

This study investigates the release characteristics of neuropeptide Y (NPY) from young (10 weeks) and old (22 months) rat atrium. Levels of NPY release from samples of atrium were studied by organ perifusion. Rats were exposed to light:dark (LD) cycles of 12:12 or 18:6 and sacrificed at different circadian stages: 0, 4, 7, 12, 18, and 20 h after dark onset (HADO) for LD 12:12 or 0, 2, 3.5, 6, 15, and 22 HADO for LD 18:6. The heart was collected, and the right atrium was removed, weighed, and perifused with Krebs-bicarbonate buffer for 100 min, including a period of 50 min for stabilization of secretion rate. NPY concentrations released by atrium did not differ between the two age groups. NPY exhibited daily variations in concentrations in LD 12:12, with a peak during the end of scotophase, at 12 HADO, in both the young and old rats. These variations were strongly modified in LD 18:6, where the pattern of the release exhibited two peaks occurring during the two thirds of dark (3.5 HADO) and light (22 HADO) periods. This strongly suggests that the NPY rhythm is dependent on the environmental light:dark cycle. In this paper we show that NPY concentrations in the rat atrium exhibit daily variations, which are maintained with ageing. Moreover, photoperiod greatly influences NPY levels in the atrium.     

Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors

We describe the use of four complementary biosensors (Biacore 3000, Octet QK, ProteOn XPR36, and KinExA 3000) in characterizing the kinetics of human nerve growth factor (NGF) binding to a humanized NGF-neutralizing monoclonal antibody (tanezumab, formerly known as RN624). Tanezumab is a clinical candidate as a therapy for chronic pain. Our measurements were consistent with the NGF/tanezumab binding affinity being tighter than 10 pM due to the formation of an extremely stable complex that had an estimated half-life exceeding 100 h, which was beyond the resolution of any of our methods. The system was particularly challenging to study because NGF is an obligate homodimer, and we describe various assay orientations and immobilization methods that were used to minimize avidity in our experiments while keeping NGF in as native a state as possible. We also explored the interactions of NGF with its natural receptors, TrkA and P75, and how tanezumab blocks them. The Biacore blocking assay that we designed was used to quantify the potency of tanezumab and is more precise and reproducible than the currently available cell-based functional assays.     

Selective Serotonin Reuptake Inhibitors and Violent Crime: A Cohort Study

BackgroundAlthough selective serotonin reuptake inhibitors (SSRIs) are widely prescribed, associations with violence are uncertain.ConclusionsThe association between SSRIs and violent crime convictions and violent crime arrests varied by age group. The increased risk we found in young people needs validation in other studies.     

Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast

Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role.     

Sphingosine 1-Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and Smad-independent Signaling

Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-β1. In contrast to TGF-β1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-β1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-β1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P₂ and S1P₃ receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P₃ receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-β1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P₃R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P₂R and S1P₃R stimulation using Smad-independent pathways.