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We describe pLink 2, a search engine with higher speed and reliability for proteome-scale identification of cross-linked peptides. With a two-stage open search strategy facilitated by fragment indexing, pLink 2 is ~40 times faster than pLink 1 and 3~10 times faster than Kojak. Furthermore, using simulated datasets, synthetic datasets, 15 N metabolically labeled datasets, and entrapment databases, four analysis methods were designed to evaluate the credibility of ten state-of-the-art search engines. This systematic evaluation shows that pLink 2 outperforms these methods in precision and sensitivity, especially at proteome scales. Lastly, re-analysis of four published proteome-scale cross-linking datasets with pLink 2 required only a fraction of the time used by pLink 1, with up to 27% more cross-linked residue pairs identified. pLink 2 is therefore an efficient and reliable tool for cross-linking mass spectrometry analysis, and the systematic evaluation methods described here will be useful for future software development. The identification of cross-linked peptides at a proteome scale for interactome analyses represents a complex challenge. Here the authors report an efficient and reliable search engine pLink 2 for proteome-scale cross-linking mass spectrometry analyses, and demonstrate how to systematically evaluate the credibility of search engines.

Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.

Chemotherapeutic agents have been linked to immunogenic cell death (ICD) induction that is capable of augmenting anti-tumor immune surveillance. The cardiac glycoside oleandrin, which inhibits Na + /K + -ATPase pump (NKP), has been shown to suppress breast cancer growth via inducing apoptosis. In the present study, we showed that oleandrin treatment triggered breast cancer cell ICD by inducing calreticulin (CRT) exposure on cell surface and the release of high-mobility group protein B1 (HMGB1), heat shock protein 70/90 (HSP70/90), and adenosine triphosphate (ATP). The maturation and activation of dendritic cells (DCs) were increased by co-culturing with the oleandrin-treated cancer cells, which subsequently enhanced CD8 + T cell cytotoxicity. Murine breast cancer cell line EMT6 was engrafted into BALB/c mice, and tumor-bearing mice were administered with oleandrin intraperitoneally every day. Oleandrin inhibited tumor growth and increased tumor infiltrating lymphocytes including DCs and T cells. Furthermore, the differential mRNA expression incurred by oleandrin was investigated by mRNA sequencing and subsequently confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Mechanistically, oleandrin induced endoplasmic reticulum (ER) stress-associated, caspase-independent ICD mainly through PERK/elF2α/ATF4/CHOP pathway. Pharmacological and genetic inhibition of protein kinase R-like ER kinase (PERK) suppressed oleandrin-triggered ICD. Taken together, our findings showed that oleandrin triggered ER stress and induced ICD-mediated immune destruction of breast cancer cells. Oleandrin combined with immune checkpoint inhibitors might improve the efficacy of immunotherapy.

Background The prevalence of HIV among men who have sex with men (MSM) has become a significant public health challenge. The aim was to comprehensively estimate the national prevalence of HIV among MSM and its time trends through a large-scale systematic analysis. Methods Systematic search of Cochrane Library, PubMed, EMBASE, CNKI, VIP, and Wanfang Data databases without language restriction for studies on the prevalence of HIV among MSM published before Dec.31, 2018. Studies were eligible for inclusion if they were published in the peer-reviewed literature and used validated assessment methods to assess the prevalence of HIV among MSM. Estimates were pooled using random-effects analysis. Results Data were extracted from 355 cross-sectional studies (571,328 individuals) covered 59 cities from 30 provinces and municipalities of China. The overall national prevalence of HIV among MSM from 2001 to 2018 was estimated to be 5.7% (95% CI: 5.4–6.1%), with high between-study heterogeneity ( I 2  = 98.0%, P  <  0.001). Our study showed an increased tendency in the HIV prevalence as time progressed by meta-regression analysis ( I 2  = 95.9%, P  <  0.0001). HIV prevalence was the highest in those aged 50 years and older with HIV prevalence of 19.3% (95%CI: 13.1-27.4%, N  = 13). HIV was more prevalent in the illiterate population (16.8%), than in those who had received an education. Although the internet was a major venue for Chinese MSM seeking male sex partners (35.6, 95%CI: 32.3-39.9%, N  = 101), seeking MSM in bathhouses/saunas had the highest associated prevalence of HIV (13.4, 95%CI: 10.3-17.1%, N  = 22). The HIV prevalence among MSM varied by location: compared with other regions in China, HIV was highly prevalent among MSM in the southwest (10.7, 95%CI: 9.3-12.2%, N  = 91). Compared to participants who sometimes or always used condoms, participants who had never used a condom in the past 6 months had a higher risk of HIV infection, with odds ratios of 0.1 (95%CI: 0.08-0.14). Conclusions Our analysis provided reliable estimates of China’s HIV burden among MSM, which appears to present an increasing national public health challenge. Effective government responses are needed to address this challenge and include the implementation of HIV prevention.

Despite significant development recently, improving the power conversion efficiency of organic photovoltaics (OPVs) is still an ongoing challenge to overcome. One of the prerequisites to achieving this goal is to enable efficient charge separation and small voltage losses at the same time. In this work, a facile synthetic strategy is reported, where optoelectronic properties are delicately tuned by the introduction of electron-deficient-core-based fused structure into non-fullerene acceptors. Both devices exhibited a low voltage loss of 0.57 V and high short-circuit current density of 22.0 mA cm −2 , resulting in high power conversion efficiencies of over 13.4%. These unconventional electron-deficient-core-based non-fullerene acceptors with near-infrared absorption lead to low non-radiative recombination losses in the resulting organic photovoltaics, contributing to a certified high power conversion efficiency of 12.6%. Improving the power conversion efficiency is the main target of the organic solar cell research. Here Yuan et al. develop unconventional electron-deficient-core-based non-fullerene acceptors to achieve both low voltage loss and high current density, leading to a certified high efficiency of 12.6%.

The mechanosensitive Piezo channels function as key eukaryotic mechanotransducers. However, their structures and mechanogating mechanisms remain unknown. Here we determine the three-bladed, propeller-like electron cryo-microscopy structure of mouse Piezo1 and functionally reveal its mechanotransduction components. Despite the lack of sequence repetition, we identify nine repetitive units consisting of four transmembrane helices each—which we term transmembrane helical units (THUs)—which assemble into a highly curved blade-like structure. The last transmembrane helix encloses a hydrophobic pore, followed by three intracellular fenestration sites and side portals that contain pore-property-determining residues. The central region forms a 90?Å-long intracellular beam-like structure, which undergoes a lever-like motion to connect THUs to the pore via the interfaces of the C-terminal domain, the anchor-resembling domain and the outer helix. Deleting extracellular loops in the distal THUs or mutating single residues in the beam impairs the mechanical activation of Piezo1. Overall, Piezo1 possesses a unique 38-transmembrane-helix topology and designated mechanotransduction components, which enable a lever-like mechanogating mechanism. The electron cryo-microscopy structure of full-length mouse Piezo1 reveals unique topological features such as the repetitive transmembrane helical units that constitute the highly curved transmembrane region, and identifies regions and single residues that are crucial for the mechanical activation of the channel. Structure and mechanism of ion channel Piezo1 Mechanosensitive cation channels convert external mechanical stimuli into various biological actions, including touch, hearing, balance and cardiovascular regulation. The eukaryotic Piezo proteins are mechanotransduction channels, although their structure and gating mechanisms are not well elucidated. In related papers in this issue of Nature , two groups report cryo-electron microscopy structures of the full-length mouse Piezo1 and reveal three flexible propeller blades. Each blade is made up of at least 26 helices, forming a series of helical bundles, which adopt a curved transmembrane region. A kinked beam and anchor domain link these Piezo repeats to the pore, giving clues as to how the channel responds to membrane tension and mechanical force.

Liver cancer is one of the most common malignant tumors globally. Not only is it difficult to diagnose, but treatments are scarce and the prognosis is generally poor. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Aggressive cancer cells, such as those found in HCC, undergo extensive metabolic rewiring as tumorigenesis, the unique feature, ultimately causes adaptation to the neoplastic microenvironment. Intratumoral heterogeneity (ITH) is defined as the presence of distinct genetic features and different phenotypes in the same tumoral region. ITH, a property unique to malignant cancers, results in differences in many different features of tumors, including, but not limited to, tumor growth and resistance to chemotherapy, which in turn is partly responsible for metabolic reprogramming. Moreover, the different metabolic phenotypes might also activate the immune response to varying degrees and help tumor cells escape detection by the immune system. In this review, we summarize the reprogramming of glucose metabolism and tumoral heterogeneity and their associations that occur in HCC, to obtain a better understanding of the mechanisms of HCC oncogenesis.

Rett syndrome (RTT), a severe postnatal neurodevelopmental disorder, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). MeCP2 is a chromatin organizer regulating gene expression. RTT-causing mutations have been shown to affect this function. However, the mechanism by which MeCP2 organizes chromatin is unclear. In this study, we found that MeCP2 can induce compaction and liquid–liquid phase separation of nucleosomal arrays in vitro, and DNA methylation further enhances formation of chromatin condensates by MeCP2. Interestingly, RTT-causing mutations compromise MeCP2-mediated chromatin phase separation, while benign variants have little effect on this process. Moreover, MeCP2 competes with linker histone H1 to form mutually exclusive chromatin condensates in vitro and distinct heterochromatin foci in vivo. RTT-causing mutations reduce or even abolish the ability of MeCP2 to compete with histone H1 and to form chromatin condensates. Together, our results identify a novel mechanism by which phase separation underlies MeCP2-mediated heterochromatin formation and reveal the potential link between this process and the pathology of RTT.

Insulin/IGF-1 Signaling (IIS) is known to constrain longevity by inhibiting the transcription factor FOXO. How phosphorylation mediated by IIS kinases regulates lifespan beyond FOXO remains unclear. Here, we profile IIS-dependent phosphorylation changes in a large-scale quantitative phosphoproteomic analysis of wild-type and three IIS mutant Caenorhabditis elegans strains. We quantify more than 15,000 phosphosites and find that 476 of these are differentially phosphorylated in the long-lived daf-2/insulin receptor mutant. We develop a machine learning-based method to prioritize 25 potential lifespan-related phosphosites. We perform validations to show that AKT-1 pT492 inhibits DAF-16/FOXO and compensates the loss of daf-2 function, that EIF-2α pS49 potently inhibits protein synthesis and daf-2 longevity, and that reduced phosphorylation of multiple germline proteins apparently transmits reduced DAF-2 signaling to the soma. In addition, an analysis of kinases with enriched substrates detects that casein kinase 2 (CK2) subunits negatively regulate lifespan. Our study reveals detailed functional insights into longevity. How phosphorylation mediated by Insulin/IGF-1 Signaling kinases regulates lifespan remains unclear. Here the authors perform a large-scale quantitative phosphoproteomic analysis of wildtype and IIS mutant C. elegans strains to reveal detailed functional insights into longevity.

Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk. Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors. Ribosome biogenesis in eukaryotes is a complex process that involves more than 200 protein factors. Here the authors present a structural analysis of a collection of human pre-60S structures sampled through a nuclear export adaptor NMD3, representing structural snapshots of pre-60S particles immediately before and after passing through nuclear pore complex.