CoolMPS for robust sequencing of single-nuclear RNAs captured by droplet-based method

Massively-parallel single-cell and single-nucleus RNA sequencing (scRNA-seq, snRNA-seq) requires extensive sequencing to achieve proper per-cell coverage, making sequencing resources and availability of sequencers critical factors for conducting deep transcriptional profiling. CoolMPS is a novel sequencing-by-synthesis approach that relies on nucleotide labeling by re-usable antibodies, but whether it is applicable to snRNA-seq has not been tested. Here, we use a low-cost and off-the-shelf protocol to chemically convert libraries generated with the widely-used Chromium 10X technology to be sequenceable with CoolMPS technology. To assess the quality and performance of converted libraries sequenced with CoolMPS, we generated a snRNA-seq dataset from the hippocampus of young and old mice. Native libraries were sequenced on an Illumina Novaseq and libraries that were converted to be compatible with CoolMPS were sequenced on a DNBSEQ-400RS. CoolMPS-derived data faithfully replicated key characteristics of the native library dataset, including correct estimation of ambient RNA-contamination, detection of captured cells, cell clustering results, spatial marker gene expression, inter- and intra-replicate differences and gene expression changes during aging. In conclusion, our results show that CoolMPS provides a viable alternative to standard sequencing of RNA from droplet-based libraries

Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity

Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins

Fission Yeast Sec3 and Exo70 Are Transported on Actin Cables and Localize the Exocyst Complex to Cell Poles

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Cu and Zn isotope fractionation during extreme chemical weathering

Copper and Zn are trace metal micronutrients whose stable isotope systematics are receiving increasing attention as possible paleoenvironmental tracers. However, to realise this potential, their behaviour during chemical weathering must be better constrained. We present coupled Cu and Zn isotope data for a well-characterised Indian laterite weathering profile, which includes a full suite of samples from unaltered greywacke bedrock to indurated lateritic duricrust. This sample set provides an exceptional opportunity to interrogate Cu and Zn isotope compositions during an extreme example of chemical weathering. Despite their occurrence in different host phases within the parent greywacke, Cu and Zn isotopes behave coherently during weathering. We observe preferential loss of heavy isotopes at increasing degrees of alteration, with 0.6‰ total variability in δ66Zn and 0.9‰ in δ65Cu. Given the absence of evidence for CuS or ZnS phases in the parent lithology, we attribute the liberation of heavy isotopes to organic complexation in the aqueous phase and/or incorporation of light isotopes in secondary aluminous Fe-oxides. Strong enrichment of both metals is also associated with a peak in Mn at a previously identified paleo-water table horizon. This dataset confirms that weathering under oxygenated conditions releases isotopically heavy Cu, regardless of the host phase. Meanwhile, Zn isotopes are only fractionated to any significant extent at the most extreme degrees of chemical weathering reached during lateritization. We conclude that the isotopic composition of the weathering-derived input of Zn to rivers should be largely insensitive to climate change on geological timescales

Firn on ice sheets

Most of the Greenland and Antarctic ice sheets are covered with firn — the transitional material between snow and glacial ice. Firn is vital for understanding ice-sheet mass balance and hydrology, and palaeoclimate. In this Review, we synthesize knowledge of firn, including its formation, observation, modelling and relevance to ice sheets. The refreezing of meltwater in the pore space of firn currently prevents 50% of meltwater in Greenland from running off into the ocean and protects Antarctic ice shelves from catastrophic collapse. Continued atmospheric warming could inhibit future protection against mass loss. For example, warming in Greenland has already contributed to a 5% reduction in firn pore space since 1980. All projections of future firn change suggest that surface meltwater will have an increasing impact on firn, with melt occurring tens to hundreds of kilometres further inland in Greenland, and more extensively on Antarctic ice shelves. Although progress in observation and modelling techniques has led to a well-established understanding of firn, the large uncertainties associated with meltwater percolation processes (refreezing, ice-layer formation and storage) must be reduced further. A tighter integration of modelling components (firn, atmosphere and ice-sheet models) will also be needed to better simulate ice-sheet responses to anthropogenic warming and to quantify future sea-level rise

Firn on ice sheets

Most of the Greenland and Antarctic ice sheets are covered with firn — the transitional material between snow and glacial ice. Firn is vital for understanding ice-sheet mass balance and hydrology, and palaeoclimate. In this Review, we synthesize knowledge of firn, including its formation, observation, modelling and relevance to ice sheets. The refreezing of meltwater in the pore space of firn currently prevents 50% of meltwater in Greenland from running off into the ocean and protects Antarctic ice shelves from catastrophic collapse. Continued atmospheric warming could inhibit future protection against mass loss. For example, warming in Greenland has already contributed to a 5% reduction in firn pore space since 1980. All projections of future firn change suggest that surface meltwater will have an increasing impact on firn, with melt occurring tens to hundreds of kilometres further inland in Greenland, and more extensively on Antarctic ice shelves. Although progress in observation and modelling techniques has led to a well-established understanding of firn, the large uncertainties associated with meltwater percolation processes (refreezing, ice-layer formation and storage) must be reduced further. A tighter integration of modelling components (firn, atmosphere and ice-sheet models) will also be needed to better simulate ice-sheet responses to anthropogenic warming and to quantify future sea-level rise