Immune recognition of self nucleic acids driven by endogenous antimicrobial peptides: role in autoimmunity

Innate immune recognition of extracellular host-derived self-DNA and self-RNA is prevented by endosomal seclusion of the Toll-like receptors (TLRs) in the dendritic cells (DCs). However, in psoriasis plasmacytoid dendritic cells have been found to be able to sense self-DNA molecules in complex with the endogenous cationic antimicrobial peptide LL37, which are internalized into the endosomal compartments and thus can access TLR9. We investigated whether this endogenous peptide can also interact with extracellular self-RNA and lead to DC activation. We found that LL37 binds self-RNA as well as self-DNA going into an electrostatic interaction; forms micro-aggregates of nano-scale particles protected from enzymatic degradation and transport it into the endosomal compartments of both plasmacytoid and myeloid dendritic cells. In the plasmacytoid DCs, the self-RNA-LL37 complexes activate TLR7 and like the self-DNA-LL37 complexes, trigger the production of IFN-α in the absence of induction of maturation or production of IL-6 and TNF-α. In contrast to the self-DNA-LL37 complexes, the self-RNA-LL37 complexes are also internalized into the endosomal compartments of myeloid dendritic cells and trigger activation through TLR8, leading to the production of TNF-α and IL-6, and the maturation of the myeloid DCs. Furthermore, we found that these self nucleic acid-LL37 complexes can be found in vivo in the skin lesions of the cutaneous autoimmune disease psoriasis, where they are associated with mature mDCs in situ. On the other hand, in the systemic autoimmune disease systemic lupus erythematosus, self-DNA-LL37 complexes were found to be a constituent of the circulating immune complexes isolated from patient sera. This interaction between the endogenous peptide with the self nucleic acid molecules present in the immune complexes was found to be electrostatic and it confers resistance to enzymatic degradation of the nucleic acid molecules in the immune complexes. Moreover, autoantibodies to these endogenous peptides were found to trigger neutrophil activation and release of neutrophil extracellular traps composed of DNA, which are potential sources of the self nucleic acid-LL37 complexes present in SLE immune complexes. Our results demonstrate that the cationic antimicrobial peptide LL37 drives the innate immune recognition of self nucleic acid molecules through toll-like receptors in human dendritic cells, thus elucidating a pathway for innate sensing of host cell death. This pathway of autoreactivity was found to be pathologically relevant in human autoimmune diseases psoriasis and SLE, and thus this study provides new insights into the mechanisms autoimmune diseases.

Multi-scale micro-structure generation strategy for up-scaling transport in clays

Clays and claystones are used as backfill and barrier materials in the design of waste repositories, because they act as hydraulic barriers and retain contaminants. Transport through such barriers occurs mainly by molecular diffusion. There is thus an interest to relate the diffusion properties of clays to their structural properties. In previous work, we have developed a concept for up-scaling pore-scale molecular diffusion coefficients using a grid-based model for the sample pore structure. Here we present an operational algorithm which can generate such model pore structures of polymineral materials. The obtained pore maps match the rock’s mineralogical components and its macroscopic properties such as porosity, grain and pore size distributions. Representative ensembles of grains in 2D or 3D are created by a lattice Monte Carlo (MC) method, which minimizes the interfacial energy of grains starting from an initial grain distribution. Pores are generated at grain boundaries and/or within grains. The method is general and allows to generate anisotropic structures with grains of approximately predetermined shapes, or with mixtures of different grain types. A specific focus of this study was on the simulation of clay-like materials. The generated clay pore maps were then used to derive upscaled effective diffusion coefficients for non-sorbing tracers using a homogenization technique. The large number of generated maps allowed to check the relations between micro-structural features of clays and their effective transport parameters, as is required to explain and extrapolate experimental diffusion results. As examples, we present a set of 2D and 3D simulations and investigated the effects of nanopores within particles (interlayer pores) and micropores between particles. Archie’s simple power law is followed in systems with only micropores. When nanopores are present, additional parameters are required; the data reveal that effective diffusion coefficients could be described by a sum of two power functions, related to the micro- and nanoporosity. We further used the model to investigate the relationships between particle orientation and effective transport properties of the sample.

Reconstruction of a flash flood with large wood transport and its influence on hazard patterns in an ungauged mountain basin

The reconstruction of past flash floods in ungauged basins leads to a high level of uncertainty, which increases if other processes are involved such as the transport of large wood material. An important flash flood occurred in 1997 in Venero Claro (Central Spain), causing significant economic losses. The wood material clogged bridge sections, raising the water level upstream. The aim of this study was to reconstruct this event, analysing the influence of woody debris transport on the flood hazard pattern. Because the reach in question was affected by backwater effects due to bridge clogging, using only high water mark or palaeostage indicators may overestimate discharges, and so other methods are required to estimate peak flows. Therefore, the peak discharge was estimated (123 ± 18 m3 s–1) using indirect methods, but one-dimensional hydraulic simulation was also used to validate these indirect estimates through an iterative process (127 ± 33 m3 s–1) and reconstruct the bridge obstruction to obtain the blockage ratio during the 1997 event (~48%) and the bridge clogging curves. Rainfall–Runoff modelling with stochastic simulation of different rainfall field configurations also helped to confirm that a peak discharge greater than 150 m3 s–1 is very unlikely to occur and that the estimated discharge range is consistent with the estimated rainfall amount (233 ± 27 mm). It was observed that the backwater effect due to the obstruction (water level ~7 m) made the 1997 flood (~35-year return period) equivalent to the 50-year flood. This allowed the equivalent return period to be defined as the recurrence interval of an event of specified magnitude, which, where large woody debris is present, is equivalent in water depth and extent of flooded area to a more extreme event of greater magnitude. These results highlight the need to include obstruction phenomena in flood hazard analysis.

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells.

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5'-to-3' degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells.

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5'-to-3' degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.

A conserved motif at the 3$\sp\prime$ end of genomic RNA of mouse hepatitis virus required for host protein binding and viral RNA replication

The initial step in coronavirus-mouse hepatitis virus (MHV) replication is the synthesis of negative strand RNA from a positive strand genomic RNA template. Our approach to studying MHV RNA replication is to identify the cis-acting signals for RNA synthesis and the protein(s) which recognizes these signals at the 3$\sp\prime$ end of genomic RNA of MHV. To determine whether host cellular and/or virus-specific proteins interact with the 3$\sp\prime$ end of the coronavirus genome, an RNase T$\sb1$ protection/gel mobility shift electrophoresis assay was used to examine cytoplasmic extracts from either mock- or MHV-JHM-infected 17Cl-1 murine cells for the ability to form complexes with defined regions of the genomic RNA. A conserved 11 nucleotide sequence UGAAUGAAGUU at nucleotide positions 36 to 26 from the 3$\sp\prime$ end of genomic RNA was identified to be responsible for the specific binding of host proteins, by using a series of RNA probes with deletions and mutations in this region. The RNA probe containing the 11 nucleotide sequence bound approximately four host cellular proteins with a highly labeled 120 kDa and three minor species with sizes of 103, 81 and 55 kDa, assayed by UV-induced covalent cross-linking. Mutation of the 11 nucleotide motif strongly inhibited cellular protein binding, and decreased the amount of the 103 and 81 kDa proteins in the complex to undetectable levels and strongly reduced the binding of the 120 kDa protein. Less extensive mutations within this 11 nucleotide motif resulted in variable decreases in RNA-protein complex formation depending on each probe tested. The RNA-protein complexes observed with cytoplasmic extracts from MHV-JHM-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were indistinguishable to those observed with extracts from uninfected cells.^ To investigate the possible role of this 3$\sp\prime$ protein binding element in viral RNA replication in vivo, defective interfering RNA molecules with complete or partial mutations of the 11 nucleotide conserved sequence were transcribed in vitro, transfected to host 17Cl-1 cells in the presence of helper virus MHV-JHM and analyzed by agarose gel electrophoresis, competitive RT-PCR and direct sequencing of the RT-PCR products. Both negative strand synthesis and positive strand replication of DI RNA were affected by mutation that disrupts RNA-protein complex formation, even though the 11 mutated nucleotides were converted to wild type sequence, presumably by recombination with helper virus. Kinetic analysis indicated that recombination between DI RNA and helper virus occurred 5.5 to 7.5 hours post infection when replication of positive strand DI RNA was barely observed. Replication of positive strand DI RNAs carrying partial mutations within the 11 nucleotide motif was dependent upon recombination events after transfection. Replication was strongly inhibited when reversion to wild type sequence did not occur, and after recombination, reached similar levels as wild type DI RNA. A DI RNA with mutation upstream of the protein binding motif replicated as efficiently as wild type without undergoing recombination. Thus the conserved 11 nucleotide host protein binding motif appears to play an important role in viral RNA replication. ^

Lissencephaly-1 promotes the recruitment of dynein and dynactin to transported mRNAs

Microtubule-based transport mediates the sorting and dispersal of many cellular components and pathogens. However, the mechanisms by which motor complexes are recruited to and regulated on different cargos remain poorly understood. Here we describe a large-scale biochemical screen for novel factors associated with RNA localization signals mediating minus end-directed mRNA transport during Drosophila development. We identified the protein Lissencephaly-1 (Lis1) and found that minus-end travel distances of localizing transcripts are dramatically reduced in lis1 mutant embryos. Surprisingly, given its well-documented role in regulating dynein mechanochemistry, we uncovered an important requirement for Lis1 in promoting the recruitment of dynein and its accessory complex dynactin to RNA localization complexes. Furthermore, we provide evidence that Lis1 levels regulate the overall association of dynein with dynactin. Our data therefore reveal a critical role for Lis1 within the mRNA localization machinery and suggest a model in which Lis1 facilitates motor complex association with cargos by promoting the interaction of dynein with dynactin.

Mitochondrial outer membrane proteome of T.brucei reveals novel factors required to maintain mitochondrial morphology.

The mitochondrial outer membrane (MOM) separates the mitochondria from the cytoplasm, serving both as a barrier and as a gateway. Protein complexes residing in the MOM orchestrate protein and tRNA import, metabolite exchange and lipid metabolism. African trypanosomes are among the earliest diverging eukaryotes that have bona fide mitochondria capable of oxidative phosphorylation. The MOM of T. brucei is essentially unchartered territory. It lacks a canonical TOM-complex and proteins are imported across the MOM using ATOM, which is related to both Tom40 and to the bacterial Omp85-protein family. The beta barrel membrane proteins ATOM, VDAC and Sam50 are the only MOM proteins that have been characterized in T. brucei so far. Using biochemical fractionation and correlated protein abundance-profiling we were able to identify a cluster of 82 candidate proteins that can be localized to the trypanosomal MOM with high confidence Two-thirds of these polypeptides have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the MOM of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and therefore directly or indirectly are involved in the regulation of mitochondrial morphology in T. brucei.

Improved sampling for gradient-domain metropolis light transport

We present a generalized framework for gradient-domain Metropolis rendering, and introduce three techniques to reduce sampling artifacts and variance. The first one is a heuristic weighting strategy that combines several sampling techniques to avoid outliers. The second one is an improved mapping to generate offset paths required for computing gradients. Here we leverage the properties of manifold walks in path space to cancel out singularities. Finally, the third technique introduces generalized screen space gradient kernels. This approach aligns the gradient kernels with image structures such as texture edges and geometric discontinuities to obtain sparser gradients than with the conventional gradient kernel. We implement our framework on top of an existing Metropolis sampler, and we demonstrate significant improvements in visual and numerical quality of our results compared to previous work.

InvB is required for type III-dependent secretion of SopA in Salmonella enterica serovar Typhimurium.

The Salmonella effector protein SopA is translocated into host cells via the SPI-1 type III secretion system (TTSS) and contributes to enteric disease. We found that the chaperone InvB binds to SopA and slightly stabilizes it in the bacterial cytosol and that it is required for its transport via the SPI-1 TTSS.

VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens.

VirB6 from Agrobacterium tumefaciens is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants. Due to its predicted topology as a polytopic inner membrane protein, it was proposed to form the transport pore for cell-to-cell transfer of genetic material and proteinaceous virulence factors. Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively. Overexpression of virB6 in trans restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore VirB3 levels. Thus, VirB6 has a stabilizing effect on VirB5 accumulation, thereby regulating T pilus assembly. In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodimer formation was abolished. This effect could not be restored by expression of VirB6 in trans. Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a virB6 deletion strain. VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required.

Identification of Trypanosoma brucei components involved in trypanolysis by normal human serum

Normal human serum (NHS) confers human resistance to infection by the parasite Trypanosoma brucei owing to the trypanolytic activity of apolipoprotein L1 (APOL1), present in two serum complexes termed Trypanolytic Factors (TLF-1 and -2). In order to identify parasite components involved in the intracellular trafficking and activity of TLFs, an inducible RNA interference (RNAi) genomic DNA library constructed in bloodstream form T. brucei was subjected to RNAi induction and selection for resistant parasites under NHS conditions favouring either TLF-1 or TLF-2 uptake. While TLF-1 conditions readily selected the haptoglobin-haemoglobin (HP-HB) surface receptor TbHpHbR as expected, given its known ability to bind TLF-1, under TLF-2 conditions no specific receptor for TLF-2 was identified. Instead, the screen allowed the identification of five distinct factors expected to be involved in the assembly of the vacuolar proton pump V-ATPase and consecutive endosomal acidification. These data confirm that lowering the pH during endocytosis is required for APOL1 toxic activity.

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.

Solvation-Driven Charge Transfer and Localization in Metal Complexes

In any physicochemical process in liquids, the dynamical response of the solvent to the solutes out of equilibrium plays a crucial role in the rates and products: the solvent molecules react to the changes in volume and electron density of the solutes to minimize the free energy of the solution, thus modulating the activation barriers and stabilizing (or destabilizing) intermediate states. In charge transfer (CT) processes in polar solvents, the response of the solvent always assists the formation of charge separation states by stabilizing the energy of the localized charges. A deep understanding of the solvation mechanisms and time scales is therefore essential for a correct description of any photochemical process in dense phase and for designing molecular devices based on photosensitizers with CT excited states. In the last two decades, with the advent of ultrafast time-resolved spectroscopies, microscopic models describing the relevant case of polar solvation (where both the solvent and the solute molecules have a permanent electric dipole and the mutual interaction is mainly dipole−dipole) have dramatically progressed. Regardless of the details of each model, they all assume that the effect of the electrostatic fields of the solvent molecules on the internal electronic dynamics of the solute are perturbative and that the solvent−solute coupling is mainly an electrostatic interaction between the constant permanent dipoles of the solute and the solvent molecules. This well-established picture has proven to quantitatively rationalize spectroscopic effects of environmental and electric dynamics (time-resolved Stokes shifts, inhomogeneous broadening, etc.). However, recent computational and experimental studies, including ours, have shown that further improvement is required. Indeed, in the last years we investigated several molecular complexes exhibiting photoexcited CT states, and we found that the current description of the formation and stabilization of CT states in an important group of molecules such as transition metal complexes is inaccurate. In particular, we proved that the solvent molecules are not just spectators of intramolecular electron density redistribution but significantly modulate it. Our results solicit further development of quantum mechanics computational methods to treat the solute and (at least) the closest solvent molecules including the nonperturbative treatment of the effects of local electrostatics and direct solvent−solute interactions to describe the dynamical changes of the solute excited states during the solvent response.

SEVERE HEAD TRAUMA, EMERGENCY TRANSPORT, AND PATIENT OUTCOME

Trauma and severe head injuries are important issues because they are prevalent, because they occur predominantly in the young, and because variations in clinical management may matter. Trauma is the leading cause of death for those under age 40. The focus of this head injury study is to determine if variations in time from the scene of accident to a trauma center hospital makes a difference in patient outcomes.^ A trauma registry is maintained in the Houston-Galveston area and includes all patients admitted to any one of three trauma center hospitals with mild or severe head injuries. A study cohort, derived from the Registry, includes 254 severe head injury cases, for 1980, with a Glasgow Coma Score of 8 or less.^ Multiple influences relate to patient outcomes from severe head injury. Two primary variables and four confounding variables are identified, including time to emergency room, time to intubation, patient age, severity of injury, type of injury and mode of transport to the emergency room. Regression analysis, analysis of variance, and chi-square analysis were the principal statistical methods utilized.^ Analysis indicates that within an urban setting, with a four-hour time span, variations in time to emergency room do not provide any strong influence or predictive value to patient outcome. However, data are suggestive that at longer time periods there is a negative influence on outcomes. Age is influential only when the older group (55-64) is included. Mode of transport (helicopter or ambulance) did not indicate any significant difference in outcome.^ In a multivariate regression model, outcomes are influenced primarily by severity of injury and age which explain 36% (R('2)) of variance. Inclusion of time to emergency room, time to intubation, transport mode and type injury add only 4% (R('2)) additional contribution to explaining variation in patient outcome.^ The research concludes that since the group most at risk to head trauma is the young adult male involved in automobile/motorcycle accidents, more may be gained by modifying driving habits and other preventive measures. Continuous clinical and evaluative research are required to provide updated clinical wisdom in patient management and trauma treatment protocols. A National Institute of Trauma may be required to develop a national public policy and evaluate the many medical, behavioral and social changes required to cope with the country's number 3 killer and the primary killer of young adults.^