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Structure and assembly of calcium homeostasis modulator proteins
No full textThe biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer's disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore size, lipid accommodation and channel activity
Reconfigurable nanophotonic silicon probes for sub-millisecond deep-brain optical stimulation
No full textThe use of nanophotonics to rapidly and precisely reconfigure light beams for the optical stimulation of neurons in vivo has remained elusive. Here we report the design and fabrication of an implantable silicon-based probe that can switch and route multiple optical beams to stimulate identified sets of neurons across cortical layers and simultaneously record the produced spike patterns. Each switch in the device consists of a silicon nitride waveguide structure that can be rapidly (<20 mus) reconfigured by electrically tuning the phase of light. By using an eight-beam probe, we show in anaesthetized mice that small groups of single neurons can be independently stimulated to produce multineuron spike patterns at sub-millisecond precision. We also show that a probe integrating co-fabricated electrical recording sites can simultaneously optically stimulate and electrically measure deep-brain neural activity. The technology is scalable, and it allows for beam focusing and steering and for structured illumination via beam shaping. The high-bandwidth optical-stimulation capacity of the device might facilitate the probing of the spatiotemporal neural codes underlying behaviour
On the Importance of Variation: A High-Resolution Map of Copy Number Variants in Arabidopsis
No full textIn Brief Articl
Arabidopsis Retrotransposon Virus-Like Particles and Their Regulation by Epigenetically Activated Small RNA
No full textIn Arabidopsis, LTR retrotransposons are activated by mutations in the chromatin gene DECREASE in DNA METHYLATION 1 (DDM1), giving rise to 21- to 22-nt epigenetically activated siRNA (easiRNA) that depend on RNA DEPENDENT RNA POLYMERASE 6 (RDR6). We purified virus-like particles (VLPs) from ddm1 and ddm1rdr6 mutants in which genomic RNA is reverse transcribed into complementary DNA. High-throughput short-read and long-read sequencing of VLP DNA (VLP DNA-seq) revealed a comprehensive catalog of active LTR retrotransposons without the need for mapping transposition, as well as independent of genomic copy number. Linear replication intermediates of the functionally intact COPIA element EVADE revealed multiple central polypurine tracts (cPPTs), a feature shared with HIV in which cPPTs promote nuclear localization. For one member of the ATCOPIA52 subfamily (SISYPHUS), cPPT intermediates were not observed, but abundant circular DNA indicated transposon "suicide" by auto-integration within the VLP. easiRNA targeted EVADE genomic RNA, polysome association of GYPSY (ATHILA) subgenomic RNA, and transcription via histone H3 lysine-9 dimethylation. VLP DNA-seq provides a comprehensive landscape of LTR retrotransposons and their control at transcriptional, post-transcriptional, and reverse transcriptional levels
The molecular basis of mammary gland development and epithelial differentiation
No full textOur understanding of the molecular events underpinning the development of mammalian organ systems has been increasing rapidly in recent years. With the advent of new and improved next-generation sequencing methods, we are now able to dig deeper than ever before into the genomic and epigenomic events that play critical roles in determining the fates of stem and progenitor cells during the development of an embryo into an adult. In this review, we detail and discuss the genes and pathways that are involved in mammary gland development, from embryogenesis, through maturation into an adult gland, to the role of pregnancy signals in directing the terminal maturation of the mammary gland into a milk producing organ that can nurture the offspring. We also provide an overview of the latest research in the single-cell genomics of mammary gland development, which may help us to understand the lineage commitment of mammary stem cells (MaSCs) into luminal or basal epithelial cells that constitute the mammary gland. Finally, we summarize the use of 3D organoid cultures as a model system to study the molecular events during mammary gland development. Our increased investigation of the molecular requirements for normal mammary gland development will advance the discovery of targets to predict breast cancer risk and the development of new breast cancer therapies
Evolution of DNA Replication Origin Specification and Gene Silencing Mechanisms
Get PDFDNA replication in eukaryotic cells initiates from chromosomal locations, called replication origins, that bind the Origin Recognition Complex (ORC) prior to S phase. Origin establishment is guided by well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. At present, the mechanistic and evolutionary reasons for this difference are unclear. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed, among other things, that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove1. We show that this Orc4 α-helix mediates the sequence-specificity of origins in S. cerevisiae. Specifically, mutations were identified within this α-helix that alter the sequence-dependent activity of individual origins as well as change global genomic origin firing patterns. This was accomplished using a massively parallel origin selection assay analyzed using a custom mutual-information-based modeling approach and a separate analysis of whole-genome replication profiling and statistics. Interestingly, the sequence specificity of DNA replication initiation, as mediated by the Orc4 α-helix, has evolved in close conjunction with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference
Detection of incipient pancreatic cancer with novel tumor-specific antibodies in mouse models
No full textPancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy, as 90% of patients do not survive beyond five years from diagnosis. This dismal prognosis is largely due to the advanced stage of the disease at diagnosis, which precludes potentially curative surgical resection. Although early detection strategies hold significant promise for improving patient outcomes, there is still no accurate diagnostic tool to detect incipient PDAC. Here, we sought to develop antibodies for the early detection of PDAC by positron-emission tomography (PET) imaging. Accordingly, we establish a pipeline to generate novel tumor-specific monoclonal antibodies (mAbs) against cell-surface proteins of PDAC patient-derived organoids (PDOs). We identify a panel of 16 tumor organoid-binding antibodies (TOBi-bodies) that display high reactivity to human PDAC tissues but not to matched adjacent normal pancreas. We then employ biochemical, flow cytometric, mass spectrometric, and CRISPR/Cas9-mediated knockout methods to determine the cognate antigens of these TOBi-bodies. We identify two mAbs that bind to tumor-specific variants of the surface protein CEACAM6 and show minimal binding to normal tissues. PET imaging in mouse models using these TOBi-bodies enables the detection of incipient human organoid-derived PDAC tumors that are rather undetectable by palpation or high-resolution ultrasound imaging techniques. We propose that further development of these mAbs as PET radiotracers could facilitate the early detection and accurate staging of PDAC
Occupancy and fractal dimension analyses of the spatial distribution of cytotoxic (CD8+) T cells infiltrating the tumor microenvironment in triple negative breast cancer
No full textFavorable outcomes have been associated with high densities of tumor infiltrating lymphocytes (TILs) such as cytotoxic (CD8+) T cells. However, the clinical signifi- cance of the spatial distribution of TILs is less well understood. We have developed novel statistical techniques to characterize the spatial distribution of TILs at various length scales. These include a box counting method that we call “occupancy” and novel applications of fractal dimensions. We apply these techniques to the spatial distribution of CD8+ T cells in the tumor microenvironment of tissue resected from 35 triple negative breast cancer patients. We find that there is a distinct difference in the spatial distribution of CD8+ T cells between good clinical outcome (no recurrence within at least 5 years of diagnosis) and poor clinical outcome (recurrence within 3 years of diagnosis). The statistical significance of the difference between good and poor outcome in the occupancy, fractal dimension (FD), and FD difference of CD8+ T cells is comparable to that of the CD8+ T cell density. Even when we randomly exclude some of the cells so that the images have the same cell density, we still find that the fractal dimension at short length scales is correlated with cancer recurrence, implying that the actual spatial distribution of CD8+ cells, and not just the CD8+ cell density, is associated with clinical outcome. The occupancy and FD difference indicate that the CD8+ T cells are more spatially dispersed in good outcome and more aggregated in poor outcome. We discuss possible interpretations
Editorial for the Special Issue on Droplet Microfluidics
No full textEmulsions, which are collections of immiscible droplets, have elicited scientific and commercial interests for decades [...]
Immunogenicity and the CXCL12-coat control T cell accumulation in murine pancreatic cancer
No full textImproving the efficacy of immunotherapies in pancreatic cancer can increase patient survival rate. Microsatellite-stable pancreatic adenocarcinoma (PDA) exclude T cells and resist immunotherapy. This may occur by cancer cells coating themselves with CXCL12, a chemokine produced by cancer associated fibroblasts (CAFs). However, PDA with microsatellite instability (MSI-H) is infiltrated with T cells and responsive to PD-1 checkpoint inhibition. We found that human MSI-H pancreatic cancer cells display the CXCL12-coat, suggesting additional factors control pancreatic cancer immune accessibility. We hypothesized that expression of neoantigens by MSI-H PDA is an independent variable that overcomes T cell exclusion mediated by the CXCL12-coat. To test this, we established a mouse hepatic PDA metastases model in which PDA cells express doxycycline-induced ovalbumin (OVA), and adoptively transferred OVA-specific OT-I CD8+ T cells report intra-tumoral accumulation of T cells. Inducing OVA expression in established metastases increases intra-tumoral accumulation and activation of OT-I cells, despite the presence of the CXCL12-coat. We are currently investigating whether high- and low-affinity antigen-expressing cancers are subject to T cell mediated elimination in the presence and absence of the CXCL12-coat, respectively. The CXCL12-coat on PDA cells and the expression of neoantigens can independently control immune response. As the formation of the CXCL12-coat is facilitated by the Transglutaminase-2 (TGM2) and can be inhibited by TGM2 inhibitors (Z Wang et al., bioRxiv, 2019), these findings reveal potential combination therapies to overcome pancreatic cancer immunotherapy resistance