FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre– and post–power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.     

Natural products as modulators of the nuclear receptors and metabolic sensors LXR,FXR and RXR

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets.This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.     

Combining ontogenetic and evolutionary scales of morphological disparity: a study of early Jurassic ammonites

Two major research themes in Evolutionary Developmental Biology and in Paleobiology, respectively, have each become central for the analysis and interpretation of morphological changes in evolution: the study of ontogeny/phylogeny connections, mainly within the widespread and controversial framework of heterochrony; and the study of morphological disparity, the morphological signal of biodiversity, describing secular changes in morphospace occupation during the history of any given clade. Although enriching in their respective fields, these two themes have remained rather isolated to date, despite the potential value of integrating them as some recent studies begin to suggest. Here, we explore the recent notion of developmental morphospace-morphospace carrying ontogenetic information-as a potential tool for bridging the gap between disparity dynamics and developmental dynamics. We elaborate this approach with a case study of Early Jurassic ammonite family Hildoceratidae (Mollusca, Cephalopoda). Morphometric analyses of the shell shape of 20 species spanning the morphological spectrum of the family are used to quantify and contrast juvenile and adult disparity levels. Adult disparity is significantly greater than juvenile disparity at the family level; yet, some subclades also display different patterns. In addition, comparisons of ontogenetic trajectories underline the prevalence of heterochrony-based evolutionary modifications within subfamilies (via ontogenetic scaling); they also point to the probable existence of pervasive developmental constraints structuring inhomogeneous morphospace occupation.     

Inhibition of toll-like receptor 7- and 9-mediated alpha/beta interferon production in human plasmacytoid dendritic cells by respiratory syncytial virus and measles virus

Human plasmacytoid dendritic cells (PDC) are key sentinels alerting both innate and adaptive immune responses through production of huge amounts of alpha/beta interferon (IFN). IFN induction in PDC is triggered by outside-in signal transduction pathways through Toll-like receptor 7 (TLR7) and TLR9 as well as by recognition of cytosolic virus-specific patterns. TLR7 and TLR9 ligands include single-stranded RNA and CpG-rich DNA, respectively, as well as synthetic derivatives thereof which are being evaluated as therapeutic immune modulators promoting Th1 immune responses. Here, we identify the first viruses able to block IFN production by PDC. Both TLR-dependent and -independent IFN responses are abolished in human PDC infected with clinical isolates of respiratory syncytial virus (RSV), RSV strain A2, and measles virus Schwarz, in contrast to RSV strain Long, which we previously identified as a potent IFN inducer in human PDC (Hornung et al., J. Immunol. 173:5935-5943, 2004). Notably, IFN synthesis of PDC activated by the TLR7 and TLR9 agonists resiquimod (R848) and CpG oligodeoxynucleotide 2216 is switched off by subsequent infection by RSV A2 and measles virus. The capacity of RSV and measles virus of human PDC to shut down IFN production should contribute to the characteristic features of these viruses, such as Th2-biased immune pathology, immune suppression, and superinfection.     

A new, versatile field immunosensor for environmental pollutants: development and proof of principle with TNT, diuron, and atrazine

This paper presents a new, versatile, portable miniaturized flow-injection immunosensor which is designed for field analysis. The temperature-controlled field prototype can run for 6h without external power supply. The bio-recognition element is an analyte-specific antibody immobilized on a gold surface of pyramidal structures inside an exchangeable single-use chip, which hosts also the enzyme-tracer and the sample reservoirs. The competition between the enzyme-tracer and the analyte for the antigen-binding sites of the antibodies yields in the final step a chemiluminescence signal that is inversely proportional to the concentration of analyte in the given range of detection. A proof of principle is shown for nitroaromatics and pesticides. The detection limits (DL; IC20) reached with the field prototype in the laboratory was below 0.1 microg l(-1) for 2,4,6-trinitrotoluene (TNT), and about 0.2 microg l(-1) for diuron and atrazine, respectively. Important aspects in this development were the design of the competition between analyte and enzyme-tracer, the unspecific signal due to unspecific binding and/or luminescence background signal, and the flow pattern inside the chip.     

Plasmacytoid dendritic cells control TLR7 sensitivity of naive B cells via type I IFN

Detailed information of human B cell activation via TLR may lead to a better understanding of B cell involvement in autoimmunity and malignancy. In this study we identified a fundamental difference in the regulation of TLR7- and TLR9-mediated B cell stimulation: whereas the induction of polyclonal naive B cell proliferation by the TLR7 ligands resiquimod (R848) and loxoribine required the presence of plasmacytoid dendritic cells (PDCs), activation via the TLR9 ligand CpG was independent of PDCs. We found that PDC-derived type I IFN enhanced TLR7 sensitivity of B cells by selectively up-regulating TLR7 expression. In contrast the expression levels of TLR9 and of other TLRs studied remained unchanged. In the presence of type I IFN, TLR7 ligation triggered polyclonal B cell expansion and B cell differentiation toward Ig-producing plasma cells; notably, this occurred independently of T cell help and B cell Ag. Human B cells did not respond to ligands of other TLRs including TLR2, TLR4 and TLR6 with and without type I IFN. In conclusion, our results reveal a distinct regulation of TLR7 and TLR9 function in human B cells and highlight TLR7 and TLR9 as unique targets for therapeutic intervention in B cell-mediated immunity and disease.