CTL clear virus-infected cells and tumorigenic cells by releasing potent cytotoxic enzymes stored in preformed lytic granules. The exocytosis process includes polarization of lytic granules toward the immunological synapse, tethering of lytic granules to the plasma membrane and finally fusion of lytic granules with the plasma membrane to release cytotoxic enzymes. Although much is known about the molecular machineries necessary for the earlier steps in lytic granule exocytosis, the molecular machinery governing the final step in the fusion process has not been identified. Here, we show using control and VAMP8 KO mice that VAMP8 is localized to the CTL lytic granules. While the immunological synapse and granule polarization appears normal in both VAMP8 KO and control CTL, CTL-mediated killing was reduced for the Vamp8 -/-CTL. Analysis of lytic enzyme secretion demonstrated that granzyme A and granzyme B secretion is significantly compromised in VAMP8 -/-CTL, while the levels of the lytic enzymes in the cells are unaffected. Our results clearly show that VAMP8 is one of the v-SNARE that regulate the lytic ability of CTL by influencing the ability of the lytic granules to fuse with the plasma membrane and release its contents.
Insertions of the human-specific subfamily of LINE-1 (L1) retrotransposon are highly polymorphic across individuals and can critically influence the human transcriptome. We hypothesized that L1 insertions could represent genetic variants determining important human phenotypic traits, and performed an integrated analysis of L1 elements and single nucleotide polymorphisms (SNPs) in several human populations. We found that a large fraction of L1s were in high linkage disequilibrium with their surrounding genomic regions and that they were well tagged by SNPs. However, L1 variants were only partially captured by SNPs on standard SNP arrays, so that their potential phenotypic impact would be frequently missed by SNP array-based genome-wide association studies. We next identified potential phenotypic effects of L1s by looking for signatures of natural selection linked to L1 insertions; significant extended haplotype homozygosity was detected around several L1 insertions. This finding suggests that some of these L1 insertions may have been the target of recent positive selection.human genetics | population genetics | evolution | L1-seq | extended haplotype homozygosity L INE-1 retrotransposons are mobile genetic elements that comprise almost 20% of the human genome (1). Most LINE-1 elements are either mutated or truncated and are retrotranposition incompetent. However, a human-specific subfamily of LINE-1 elements (L1Hs, referred to as "L1" below) is currently active in humans.Over the last years, the application of genome-wide approaches to identify mobile genetic elements has shed new light on retrotransposition in humans. Thousands of new polymorphic insertions have been identified, highlighting the differences in retrotransposon content across individual genomes (2, 3). There are an estimated 12,000 polymorphic L1 insertions with allele frequencies above 0.05 in humans (4) and L1 insertions represent a major source of structural variation between individuals (5, 6). Furthermore, polymorphic L1 elements can be highly active and retrotransposition thus continues to be an ongoing source of genetic variation in today's populations (7). The genome-wide search for genetic determinants of common human traits and diseases has been largely based on single-nucleotide polymorphisms (SNPs) and has sometimes failed to explain heritability of complex traits (8). In contrast, the association of phenotypic variability and disease susceptibility with structural variation remains relatively less explored (9). The recent realization of the extent and polymorphism of L1 insertions in humans thus make them a particularly interesting source of genetic variation.Mobile genetic elements were initially proposed to have no impact on phenotype and to be evolutionarily neutral (10, 11). The ongoing retrotransposition activity of L1, however, was shown to cause various genetic diseases by way of insertional mutagenesis (4). Such insertions are expected to be under strong purifying selection. In addition to these deleterious effects, the L1 seq...
Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.
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