Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.     

Mechanism of chloride interaction with neurotransmitter:sodium symporters

Neurotransmitter:sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs. Whereas the non-homologous glutamate transporters mediate chloride conductance, in the eukaryotic NSS chloride is transported together with the neurotransmitter. In contrast, transport by the bacterial NSS family members LeuT, Tyt1 and TnaT is chloride independent. The crystal structure of LeuT reveals an occluded binding pocket containing leucine and two sodium ions, and is highly relevant for the neurotransmitter transporters. However, the precise role of chloride in neurotransmitter transport and the location of its binding site remain elusive. Here we show that introduction of a negatively charged amino acid at or near one of the two putative sodium-binding sites of the GABA (gamma-aminobutyric acid) transporter GAT-1 from rat brain (also called SLC6A1) renders both net flux and exchange of GABA largely chloride independent. In contrast to wild-type GAT-1, a marked stimulation of the rate of net flux, but not of exchange, was observed when the internal pH was lowered. Equivalent mutations introduced in the mouse GABA transporter GAT4 (SLC6A11) and the human dopamine transporter DAT (SLC6A3) also result in chloride-independent transport, whereas the reciprocal mutations in LeuT and Tyt1 render substrate binding and/or uptake by these bacterial NSS chloride dependent. Our data indicate that the negative charge, provided either by chloride or by the transporter itself, is required during binding and translocation of the neurotransmitter, probably to counterbalance the charge of the co-transported sodium ions.     

A genome-wide association study of metabolic traits in human urine

We present a genome-wide association study of metabolic traits in human urine, designed to investigate the detoxification capacity of the human body. Using NMR spectroscopy, we tested for associations between 59 metabolites in urine from 862 male participants in the population-based SHIP study. We replicated the results using 1,039 additional samples of the same study, including a 5-year follow-up, and 992 samples from the independent KORA study. We report five loci with joint P values of association from 3.2 × 10(-19) to 2.1 × 10(-182). Variants at three of these loci have previously been linked with important clinical outcomes: SLC7A9 is a risk locus for chronic kidney disease, NAT2 for coronary artery disease and genotype-dependent response to drug toxicity, and SLC6A20 for iminoglycinuria. Moreover, we identify rs37369 in AGXT2 as the genetic basis of hyper-β-aminoisobutyric aciduria.     

γδ T-APCs: a novel tool for immunotherapy?

The series of seminal articles in this book clearly illustrate the multi-functional nature of γδ T cells. Some of the functions correlate with the tissue tropism of distinct γδ T cell subsets whereas others appear to result from oligoclonal selection. Here, we discuss the antigen-presenting cell (APC) function of the major subset of circulating γδ T cells, Vγ9/Vδ2 T cells, present in human blood. During tissue culture, Vγ9/Vδ2 T cells uniformly respond to a class of non-peptide antigens, so-called prenyl pyrophosphates, derived from stressed host cells or from microbes. It is this feature that distinguishes human (and primate) Vγ9/Vδ2 T cells from αβ and γδ T cells of all other species and that forms the basis for detailed studies of human Vγ9/Vδ2 T cells. One of the consequences of Vγ9/Vδ2 T cell activation is the rapid acquisition of APC characteristics (γδ T-APCs) reminiscent of mature dendritic cells (DCs). In the following discussion, we will discriminate between the potential use of γδ T-APCs as a cellular vaccine in immunotherapy and their role in anti-microbial immunity. Exploiting the APC function in γδ T-APCs represents a true novelty in current immunotherapy research and may lead to effective, anti-tumor immunity in cancer patients.     

Common variants in P2RY11 are associated with narcolepsy

Growing evidence supports the hypothesis that narcolepsy with cataplexy is an autoimmune disease. We here report genome-wide association analyses for narcolepsy with replication and fine mapping across three ethnic groups (3,406 individuals of European ancestry, 2,414 Asians and 302 African Americans). We identify a SNP in the 3' untranslated region of P2RY11, the purinergic receptor subtype P2Y?? gene, which is associated with narcolepsy (rs2305795, combined P = 6.1 × 10?1?, odds ratio = 1.28, 95% CI 1.19-1.39, n = 5689). The disease-associated allele is correlated with reduced expression of P2RY11 in CD8(+) T lymphocytes (339% reduced, P = 0.003) and natural killer (NK) cells (P = 0.031), but not in other peripheral blood mononuclear cell types. The low expression variant is also associated with reduced P2RY11-mediated resistance to ATP-induced cell death in T lymphocytes (P = 0.0007) and natural killer cells (P = 0.001). These results identify P2RY11 as an important regulator of immune-cell survival, with possible implications in narcolepsy and other autoimmune diseases.     

Deciphering the evolution and metabolism of an anammox bacterium from a community genome

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycle's major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics--the reconstruction of genomic data directly from the environment--to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organism's special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.     

Control of four stereocentres in a triple cascade organocatalytic reaction

Efficient and elegant syntheses of complex organic molecules with multiple stereogenic centres continue to be important in both academic and industrial laboratories. In particular, catalytic asymmetric multi-component 'domino' reactions, used during total syntheses of natural products and synthetic building blocks, are highly desirable. These reactions avoid time-consuming and costly processes, including the purification of intermediates and steps involving the protection and deprotection of functional groups, and they are environmentally friendly and often proceed with excellent stereoselectivities. Therefore, the design of new catalytic and stereoselective cascade reactions is a continuing challenge at the forefront of synthetic chemistry. In addition, catalytic cascade reactions can be described as biomimetic, as they are reminiscent of tandem reactions that may occur during biosyntheses of complex natural products. Here we report the development of an asymmetric organocatalytic triple cascade reaction for the synthesis of tetra-substituted cyclohexene carbaldehydes. This three-component domino reaction proceeds by way of a catalysed Michael/Michael/aldol condensation sequence affording the products with good to moderate yields (25-58 per cent). During this sequence, four stereogenic centres are formed with high diastereoselectivity and complete enantioselectivity. In addition, variation of the starting materials can be used to obtain diverse polyfunctional cyclohexene derivatives, which can be used as building blocks in organic synthesis.     

Single-mode heat conduction by photons

The thermal conductance of a single channel is limited by its unique quantum value G(Q), as was shown theoretically in 1983. This result closely resembles the well-known quantization of electrical conductance in ballistic one-dimensional conductors. Interestingly, all particles-irrespective of whether they are bosons or fermions-have the same quantized thermal conductance when they are confined within dimensions that are small compared to their characteristic wavelength. The single-mode heat conductance is particularly relevant in nanostructures. Quantized heat transport through submicrometre dielectric wires by phonons has been observed, and it has been predicted to influence cooling of electrons in metals at very low temperatures due to electromagnetic radiation. Here we report experimental results showing that at low temperatures heat is transferred by photon radiation, when electron-phonon as well as normal electronic heat conduction is frozen out. We study heat exchange between two small pieces of normal metal, connected to each other only via superconducting leads, which are ideal insulators against conventional thermal conduction. Each superconducting lead is interrupted by a switch of electromagnetic (photon) radiation in the form of a DC-SQUID (a superconducting loop with two Josephson tunnel junctions). We find that the thermal conductance between the two metal islands mediated by photons indeed approaches the expected quantum limit of G(Q) at low temperatures. Our observation has practical implications-for example, for the performance and design of ultra-sensitive bolometers (detectors of far-infrared light) and electronic micro-refrigerators, whose operation is largely dependent on weak thermal coupling between the device and its environment.     

Viral immune modulators perturb the human molecular network by common and unique strategies

Viruses must enter host cells to replicate, assemble and propagate. Because of the restricted size of their genomes, viruses have had to evolve efficient ways of exploiting host cell processes to promote their own life cycles and also to escape host immune defence mechanisms. Many viral open reading frames (viORFs) with immune-modulating functions essential for productive viral growth have been identified across a range of viral classes. However, there has been no comprehensive study to identify the host factors with which these viORFs interact for a global perspective of viral perturbation strategies. Here we show that different viral perturbation patterns of the host molecular defence network can be deduced from a mass-spectrometry-based host-factor survey in a defined human cellular system by using 70 innate immune-modulating viORFs from 30 viral species. The 579 host proteins targeted by the viORFs mapped to an unexpectedly large number of signalling pathways and cellular processes, suggesting yet unknown mechanisms of antiviral immunity. We further experimentally verified the targets heterogeneous nuclear ribonucleoprotein?U, phosphatidylinositol-3-OH kinase, the WNK (with-no-lysine) kinase family and USP19 (ubiquitin-specific peptidase 19) as vulnerable nodes in the host cellular defence system. Evaluation of the impact of viral immune modulators on the host molecular network revealed perturbation strategies used by individual viruses and by viral classes. Our data are also valuable for the design of broad and specific antiviral therapies.