Yi Zhang

<h2>Summary</h2><h3>Background</h3> Since December, 2019, Wuhan, China, has experienced an outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Epidemiological and clinical characteristics of patients with COVID-19 have been reported but risk factors for mortality and a detailed clinical course of illness, including viral shedding, have not been well described. <h3>Methods</h3> In this retrospective, multicentre cohort study, we included all adult inpatients (≥18 years old) with laboratory-confirmed COVID-19 from Jinyintan Hospital and Wuhan Pulmonary Hospital (Wuhan, China) who had been discharged or had died by Jan 31, 2020. Demographic, clinical, treatment, and laboratory data, including serial samples for viral RNA detection, were extracted from electronic medical records and compared between survivors and non-survivors. We used univariable and multivariable logistic regression methods to explore the risk factors associated with in-hospital death. <h3>Findings</h3> 191 patients (135 from Jinyintan Hospital and 56 from Wuhan Pulmonary Hospital) were included in this study, of whom 137 were discharged and 54 died in hospital. 91 (48%) patients had a comorbidity, with hypertension being the most common (58 [30%] patients), followed by diabetes (36 [19%] patients) and coronary heart disease (15 [8%] patients). Multivariable regression showed increasing odds of in-hospital death associated with older age (odds ratio 1·10, 95% CI 1·03–1·17, per year increase; p=0·0043), higher Sequential Organ Failure Assessment (SOFA) score (5·65, 2·61–12·23; p<0·0001), and d-dimer greater than 1 μg/mL (18·42, 2·64–128·55; p=0·0033) on admission. Median duration of viral shedding was 20·0 days (IQR 17·0–24·0) in survivors, but SARS-CoV-2 was detectable until death in non-survivors. The longest observed duration of viral shedding in survivors was 37 days. <h3>Interpretation</h3> The potential risk factors of older age, high SOFA score, and d-dimer greater than 1 μg/mL could help clinicians to identify patients with poor prognosis at an early stage. Prolonged viral shedding provides the rationale for a strategy of isolation of infected patients and optimal antiviral interventions in the future. <h3>Funding</h3> Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences; National Science Grant for Distinguished Young Scholars; National Key Research and Development Program of China; The Beijing Science and Technology Project; and Major Projects of National Science and Technology on New Drug Creation and Development.

<h2>Abstract</h2> The rapid development of high-throughput sequencing techniques has led biology into the big-data era. Data analyses using various bioinformatics tools rely on programming and command-line environments, which are challenging and time-consuming for most wet-lab biologists. Here, we present TBtools (a Toolkit for Biologists integrating various biological data-handling tools), a stand-alone software with a user-friendly interface. The toolkit incorporates over 130 functions, which are designed to meet the increasing demand for big-data analyses, ranging from bulk sequence processing to interactive data visualization. A wide variety of graphs can be prepared in TBtools using a new plotting engine ("JIGplot") developed to maximize their interactive ability; this engine allows quick point-and-click modification of almost every graphic feature. TBtools is platform-independent software that can be run under all operating systems with Java Runtime Environment 1.6 or newer. It is freely available to non-commercial users at https://github.com/CJ-Chen/TBtools/releases.

No therapeutics have yet been proven effective for the treatment of severe illness caused by SARS-CoV-2.We conducted a randomized, controlled, open-label trial involving hospitalized adult patients with confirmed SARS-CoV-2 infection, which causes the respiratory illness Covid-19, and an oxygen saturation (Sao2) of 94% or less while they were breathing ambient air or a ratio of the partial pressure of oxygen (Pao2) to the fraction of inspired oxygen (Fio2) of less than 300 mm Hg. Patients were randomly assigned in a 1:1 ratio to receive either lopinavir-ritonavir (400 mg and 100 mg, respectively) twice a day for 14 days, in addition to standard care, or standard care alone. The primary end point was the time to clinical improvement, defined as the time from randomization to either an improvement of two points on a seven-category ordinal scale or discharge from the hospital, whichever came first.A total of 199 patients with laboratory-confirmed SARS-CoV-2 infection underwent randomization; 99 were assigned to the lopinavir-ritonavir group, and 100 to the standard-care group. Treatment with lopinavir-ritonavir was not associated with a difference from standard care in the time to clinical improvement (hazard ratio for clinical improvement, 1.31; 95% confidence interval [CI], 0.95 to 1.80). Mortality at 28 days was similar in the lopinavir-ritonavir group and the standard-care group (19.2% vs. 25.0%; difference, -5.8 percentage points; 95% CI, -17.3 to 5.7). The percentages of patients with detectable viral RNA at various time points were similar. In a modified intention-to-treat analysis, lopinavir-ritonavir led to a median time to clinical improvement that was shorter by 1 day than that observed with standard care (hazard ratio, 1.39; 95% CI, 1.00 to 1.91). Gastrointestinal adverse events were more common in the lopinavir-ritonavir group, but serious adverse events were more common in the standard-care group. Lopinavir-ritonavir treatment was stopped early in 13 patients (13.8%) because of adverse events.In hospitalized adult patients with severe Covid-19, no benefit was observed with lopinavir-ritonavir treatment beyond standard care. Future trials in patients with severe illness may help to confirm or exclude the possibility of a treatment benefit. (Funded by Major Projects of National Science and Technology on New Drug Creation and Development and others; Chinese Clinical Trial Register number, ChiCTR2000029308.).

We examined the hypothesis that a failure of the immune system to eradicate tumors is due to the immunosuppressive environment created by the growing tumor, which is influenced by the site of tumor growth. We demonstrated that T cell responses to a bystander Ag in mice were suppressed by a growing CT26 tumor. T cells purified from the growing tumor expressed mRNA for IL-10, TGF-beta, and Foxp3. Intracellular cytokine staining revealed a high frequency of IL-10-secreting macrophages, dendritic cells, and CD4+ and CD8+ T cells infiltrating the tumor. In contrast, T cell IFN-gamma production was weak and CD8+ CTL responses were undetectable in mice with CT26 lung metastases and weak and transient following s.c. injection of CT26 cells, but were enhanced in the presence of anti-IL-10 and anti-TGF-beta. Consistent with this, removal of CD8+ T cells abrogated CTL responses and promoted progression of the s.c. tumor. However, in the lung model, depletion of CD8+ T cells significantly reduced the tumor burden. Furthermore, depletion of CD4+ or CD25+ T cells in vivo reduced tumor burden in s.c. and lung models, and this was associated with significantly enhanced IFN-gamma production by CD8+ T cells. These findings suggest that tumor growth facilitates the induction or recruitment of CD4+ regulatory T cells that secrete IL-10 and TGF-beta and suppress effector CD8+ T cell responses. However, CD8+ T regulatory cells expressing IL-10 and TGF-beta are also recruited or activated by the immunosuppressive environment of the lung, where they may suppress the induction of antitumor immunity.

No specific antiviral drug has been proven effective for treatment of patients with severe coronavirus disease 2019 (COVID-19). Remdesivir (GS-5734), a nucleoside analogue prodrug, has inhibitory effects on pathogenic animal and human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro, and inhibits Middle East respiratory syndrome coronavirus, SARS-CoV-1, and SARS-CoV-2 replication in animal models.We did a randomised, double-blind, placebo-controlled, multicentre trial at ten hospitals in Hubei, China. Eligible patients were adults (aged ≥18 years) admitted to hospital with laboratory-confirmed SARS-CoV-2 infection, with an interval from symptom onset to enrolment of 12 days or less, oxygen saturation of 94% or less on room air or a ratio of arterial oxygen partial pressure to fractional inspired oxygen of 300 mm Hg or less, and radiologically confirmed pneumonia. Patients were randomly assigned in a 2:1 ratio to intravenous remdesivir (200 mg on day 1 followed by 100 mg on days 2-10 in single daily infusions) or the same volume of placebo infusions for 10 days. Patients were permitted concomitant use of lopinavir-ritonavir, interferons, and corticosteroids. The primary endpoint was time to clinical improvement up to day 28, defined as the time (in days) from randomisation to the point of a decline of two levels on a six-point ordinal scale of clinical status (from 1=discharged to 6=death) or discharged alive from hospital, whichever came first. Primary analysis was done in the intention-to-treat (ITT) population and safety analysis was done in all patients who started their assigned treatment. This trial is registered with ClinicalTrials.gov, NCT04257656.Between Feb 6, 2020, and March 12, 2020, 237 patients were enrolled and randomly assigned to a treatment group (158 to remdesivir and 79 to placebo); one patient in the placebo group who withdrew after randomisation was not included in the ITT population. Remdesivir use was not associated with a difference in time to clinical improvement (hazard ratio 1·23 [95% CI 0·87-1·75]). Although not statistically significant, patients receiving remdesivir had a numerically faster time to clinical improvement than those receiving placebo among patients with symptom duration of 10 days or less (hazard ratio 1·52 [0·95-2·43]). Adverse events were reported in 102 (66%) of 155 remdesivir recipients versus 50 (64%) of 78 placebo recipients. Remdesivir was stopped early because of adverse events in 18 (12%) patients versus four (5%) patients who stopped placebo early.In this study of adult patients admitted to hospital for severe COVID-19, remdesivir was not associated with statistically significant clinical benefits. However, the numerical reduction in time to clinical improvement in those treated earlier requires confirmation in larger studies.Chinese Academy of Medical Sciences Emergency Project of COVID-19, National Key Research and Development Program of China, the Beijing Science and Technology Project.

<h2>Summary</h2> Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

IDH1 and IDH2 mutations occur frequently in gliomas and acute myeloid leukemia, leading to simultaneous loss and gain of activities in the production of α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG), respectively. Here we demonstrate that 2-HG is a competitive inhibitor of multiple α-KG-dependent dioxygenases, including histone demethylases and the TET family of 5-methlycytosine (5mC) hydroxylases. 2-HG occupies the same space as α-KG does in the active site of histone demethylases. Ectopic expression of tumor-derived IDH1 and IDH2 mutants inhibits histone demethylation and 5mC hydroxylation. In glioma, IDH1 mutations are associated with increased histone methylation and decreased 5-hydroxylmethylcytosine (5hmC). Hence, tumor-derived IDH1 and IDH2 mutations reduce α-KG and accumulate an α-KG antagonist, 2-HG, leading to genome-wide histone and DNA methylation alterations.

Accessing and analyzing the exponentially expanding genomic sequence and functional data pose a challenge for biomedical researchers. Here we describe an interactive system, Galaxy, that combines the power of existing genome annotation databases with a simple Web portal to enable users to search remote resources, combine data from independent queries, and visualize the results. The heart of Galaxy is a flexible history system that stores the queries from each user; performs operations such as intersections, unions, and subtractions; and links to other computational tools. Galaxy can be accessed at http://g2.bx.psu.edu.

Genome-wide measures of gene expression can identify patterns of gene activity that subclassify tumours and might provide a better means than is currently available for individual risk assessment in patients with lymph-node-negative breast cancer.We analysed, with Affymetrix Human U133a GeneChips, the expression of 22000 transcripts from total RNA of frozen tumour samples from 286 lymph-node-negative patients who had not received adjuvant systemic treatment.In a training set of 115 tumours, we identified a 76-gene signature consisting of 60 genes for patients positive for oestrogen receptors (ER) and 16 genes for ER-negative patients. This signature showed 93% sensitivity and 48% specificity in a subsequent independent testing set of 171 lymph-node-negative patients. The gene profile was highly informative in identifying patients who developed distant metastases within 5 years (hazard ratio 5.67 [95% CI 2.59-12.4]), even when corrected for traditional prognostic factors in multivariate analysis (5.55 [2.46-12.5]). The 76-gene profile also represented a strong prognostic factor for the development of metastasis in the subgroups of 84 premenopausal patients (9.60 [2.28-40.5]), 87 postmenopausal patients (4.04 [1.57-10.4]), and 79 patients with tumours of 10-20 mm (14.1 [3.34-59.2]), a group of patients for whom prediction of prognosis is especially difficult.The identified signature provides a powerful tool for identification of patients at high risk of distant recurrence. The ability to identify patients who have a favourable prognosis could, after independent confirmation, allow clinicians to avoid adjuvant systemic therapy or to choose less aggressive therapeutic options.

Thermally activated delayed fluorescence: harvesting dark triplet excitons to generate bright emissive singlet excitons.

The broadband and tunable high-performance microwave absorption properties of an ultralight and highly compressible graphene foam (GF) are investigated. Simply via physical compression, the microwave absorption performance can be tuned. The qualified bandwidth coverage of 93.8% (60.5 GHz/64.5 GHz) is achieved for the GF under 90% compressive strain (1.0 mm thickness). This mainly because of the 3D conductive network. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Predictive modeling with electronic health record (EHR) data is anticipated to drive personalized medicine and improve healthcare quality. Constructing predictive statistical models typically requires extraction of curated predictor variables from normalized EHR data, a labor-intensive process that discards the vast majority of information in each patient's record. We propose a representation of patients' entire raw EHR records based on the Fast Healthcare Interoperability Resources (FHIR) format. We demonstrate that deep learning methods using this representation are capable of accurately predicting multiple medical events from multiple centers without site-specific data harmonization. We validated our approach using de-identified EHR data from two US academic medical centers with 216,221 adult patients hospitalized for at least 24 h. In the sequential format we propose, this volume of EHR data unrolled into a total of 46,864,534,945 data points, including clinical notes. Deep learning models achieved high accuracy for tasks such as predicting: in-hospital mortality (area under the receiver operator curve [AUROC] across sites 0.93-0.94), 30-day unplanned readmission (AUROC 0.75-0.76), prolonged length of stay (AUROC 0.85-0.86), and all of a patient's final discharge diagnoses (frequency-weighted AUROC 0.90). These models outperformed traditional, clinically-used predictive models in all cases. We believe that this approach can be used to create accurate and scalable predictions for a variety of clinical scenarios. In a case study of a particular prediction, we demonstrate that neural networks can be used to identify relevant information from the patient's chart.

Mechanofluorochromic materials, which are dependent on changes in physical molecular packing modes, have attracted considerable interest over the past ten years. In this review, recent progress in the area of pure organic mechanofluorochromism is summarized, and majority of the reported organic mechanofluorochromic systems are discussed, along with their derived structure-property relationships. The existence of a structural relationship between aggregation-induced emission compounds and mechanofluorochromism is recognized based on our recent results, which considered aggregation-induced emission compounds as a well of mechanofluorochromic materials. The established structure-property relationship will guide researchers in identifying and synthesizing more mechanofluorochromic materials.

Since its debut in 2004, graphene has attracted enormous interest because of its unique properties. Chemical vapor deposition (CVD) has emerged as an important method for the preparation and production of graphene for various applications since the method was first reported in 2008/2009. In this Account, we review graphene CVD on various metal substrates with an emphasis on Ni and Cu. In addition, we discuss important and representative applications of graphene formed by CVD, including as flexible transparent conductors for organic photovoltaic cells and in field effect transistors.Growth on polycrystalline Ni films leads to both monolayer and few-layer graphene with multiple layers because of the grain boundaries on Ni films. We can greatly increase the percentage of monolayer graphene by using single-crystalline Ni(111) substrates, which have smooth surface and no grain boundaries. Due to the extremely low solubility of carbon in Cu, Cu has emerged as an even better catalyst for the growth of monolayer graphene with a high percentage of single layers. The growth of graphene on Cu is a surface reaction. As a result, only one layer of graphene can form on a Cu surface, in contrast with Ni, where more than one layer can form through carbon segregation and precipitation. We also describe a method for transferring graphene sheets from the metal using polymethyl methacrylate (PMMA).CVD graphene has electronic properties that are potentially valuable in a number of applications. For example, few-layer graphene grown on Ni can function as flexible transparent conductive electrodes for organic photovoltaic cells. In addition, because we can synthesize large-grain graphene on Cu foil, such large-grain graphene has electronic properties suitable for use in field effect transistors.

The gapless surface states of topological insulators could enable quantitatively different types of electronic device. A study of the topological insulating Bi2Se3 thin films finds that a gap in these states opens up in films below a certain thickness. This in turn suggests that in thicker films, gapless states exist on both upper and lower surfaces. A topological insulator1,2,3,4,5,6,7,8,9 is a new state of quantum matter that is characterized by a finite energy gap in the bulk and gapless modes flowing along the boundaries that are robust against disorder scattering. The topological protection of the surface state could be useful for both low-power electronics10 and error-tolerant quantum computing11,12. For a thin slab of three-dimensional topological insulator, the boundary modes from the opposite surfaces may be coupled by quantum tunnelling, so that a small, thickness-dependent gap is opened up13,14,15. Here we report such results from angle-resolved photoemission spectroscopy on Bi2Se3 films of various thicknesses grown by molecular beam epitaxy. The energy gap opening is clearly seen when the thickness is below six quintuple layers. The gapped surface states also exhibit sizeable Rashba-type spin–orbit splitting because of the substrate-induced potential difference between the two surfaces. The tunable gap and the spin–orbit coupling make these topological thin films ideal for electronic and spintronic device applications.

Abstract Mesenchymal stem cells (MSCs), in addition to their multilineage differentiation, have a direct immunosuppressive effect on T-cell proliferation in vitro. However, it is unclear whether they also modulate the immune system by acting on the very first step. In this investigation, we addressed the effects of human MSCs on the differentiation, maturation, and function of dendritic cells (DCs) derived from CD14+ monocytes in vitro. Upon induction with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4), MSC coculture could strongly inhibit the initial differentiation of monocytes to DCs, but this effect is reversible. In particular, such suppression could be recapitulated with no intercellular contact at a higher MSC/monocyte ratio (1:10). Furthermore, mature DCs treated with MSCs were significantly reduced in the expression of CD83, suggesting their skew to immature status. Meanwhile, decreased expression of presentation molecules (HLA-DR and CD1a) and costimulatory molecules (CD80 and CD86) and down-regulated IL-12 secretion were also observed. In consistence, the allostimulatory ability of MSC-treated mature DCs on allogeneic T cells was impaired. In conclusion, our data suggested for the first time that human MSCs could suppress monocyte differentiation into DCs, the most potent antigen-presenting cells (APCs), thus indicating the versatile regulation of MSCs on the ultimate specific immune response.

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity1. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant2 SARS-CoV-2 as well as CD8+ T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy. mRNA-1273, an mRNA vaccine that encodes a stabilized prefusion-state severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, elicits robust immune responses and protects mice against replication of SARS-CoV-2 in the upper and lower airways.

We report the implementation of continuous, highly flexible, and transparent graphene films obtained by chemical vapor deposition (CVD) as transparent conductive electrodes (TCE) in organic photovoltaic cells. Graphene films were synthesized by CVD, transferred to transparent substrates, and evaluated in organic solar cell heterojunctions (TCE/poly-3,4-ethylenedioxythiophene:poly styrenesulfonate (PEDOT:PSS)/copper phthalocyanine/fullerene/bathocuproine/aluminum). Key to our success is the continuous nature of the CVD graphene films, which led to minimal surface roughness (∼0.9 nm) and offered sheet resistance down to 230 Ω/sq (at 72% transparency), much lower than stacked graphene flakes at similar transparency. In addition, solar cells with CVD graphene and indium tin oxide (ITO) electrodes were fabricated side-by-side on flexible polyethylene terephthalate (PET) substrates and were confirmed to offer comparable performance, with power conversion efficiencies (η) of 1.18 and 1.27%, respectively. Furthermore, CVD graphene solar cells demonstrated outstanding capability to operate under bending conditions up to 138°, whereas the ITO-based devices displayed cracks and irreversible failure under bending of 60°. Our work indicates the great potential of CVD graphene films for flexible photovoltaic applications.

The continuous development and extensive use of computed tomography (CT) in medical practice has raised a public concern over the associated radiation dose to the patient. Reducing the radiation dose may lead to increased noise and artifacts, which can adversely affect the radiologists' judgment and confidence. Hence, advanced image reconstruction from low-dose CT data is needed to improve the diagnostic performance, which is a challenging problem due to its ill-posed nature. Over the past years, various low-dose CT methods have produced impressive results. However, most of the algorithms developed for this application, including the recently popularized deep learning techniques, aim for minimizing the mean-squared error (MSE) between a denoised CT image and the ground truth under generic penalties. Although the peak signal-to-noise ratio is improved, MSE- or weighted-MSE-based methods can compromise the visibility of important structural details after aggressive denoising. This paper introduces a new CT image denoising method based on the generative adversarial network (GAN) with Wasserstein distance and perceptual similarity. The Wasserstein distance is a key concept of the optimal transport theory and promises to improve the performance of GAN. The perceptual loss suppresses noise by comparing the perceptual features of a denoised output against those of the ground truth in an established feature space, while the GAN focuses more on migrating the data noise distribution from strong to weak statistically. Therefore, our proposed method transfers our knowledge of visual perception to the image denoising task and is capable of not only reducing the image noise level but also trying to keep the critical information at the same time. Promising results have been obtained in our experiments with clinical CT images.

Silicene, a two-dimensional (2D) honeycomb structure similar to graphene, has been successfully fabricated on an Ir(111) substrate. It is characterized as a (√7×√7) superstructure with respect to the substrate lattice, as revealed by low energy electron diffraction and scanning tunneling microscopy. Such a superstructure coincides with the (√3×√3) superlattice of silicene. First-principles calculations confirm that this is a (√3×√3)silicene/(√7×√7)Ir(111) configuration and that it has a buckled conformation. Importantly, the calculated electron localization function shows that the silicon adlayer on the Ir(111) substrate has 2D continuity. This work provides a method to fabricate high-quality silicene and an explanation for the formation of the buckled silicene sheet.

Vaccines to prevent coronavirus disease 2019 (Covid-19) are urgently needed. The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines on viral replication in both upper and lower airways is important to evaluate in nonhuman primates.Nonhuman primates received 10 or 100 μg of mRNA-1273, a vaccine encoding the prefusion-stabilized spike protein of SARS-CoV-2, or no vaccine. Antibody and T-cell responses were assessed before upper- and lower-airway challenge with SARS-CoV-2. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens were assessed by polymerase chain reaction, and histopathological analysis and viral quantification were performed on lung-tissue specimens.The mRNA-1273 vaccine candidate induced antibody levels exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory dilution (ID50) geometric mean titers of 501 in the 10-μg dose group and 3481 in the 100-μg dose group. Vaccination induced type 1 helper T-cell (Th1)-biased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-μg dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group.Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathologic changes in the lung. (Funded by the National Institutes of Health and others.).

Parkinson's disease is a common neurodegenerative disorder in which familial-linked genes have provided novel insights into the pathogenesis of this disorder. Mutations in Parkin, a ring-finger-containing protein of unknown function, are implicated in the pathogenesis of autosomal recessive familial Parkinson's disease. Here, we show that Parkin binds to the E2 ubiquitin-conjugating human enzyme 8 (UbcH8) through its C-terminal ring-finger. Parkin has ubiquitin-protein ligase activity in the presence of UbcH8. Parkin also ubiquitinates itself and promotes its own degradation. We also identify and show that the synaptic vesicle-associated protein, CDCrel-1, interacts with Parkin through its ring-finger domains. Furthermore, Parkin ubiquitinates and promotes the degradation of CDCrel-1. Familial-linked mutations disrupt the ubiquitin-protein ligase function of Parkin and impair Parkin and CDCrel-1 degradation. These results suggest that Parkin functions as an E3 ubiquitin-protein ligase through its ring domains and that it may control protein levels via ubiquitination. The loss of Parkin's ubiquitin-protein ligase function in familial-linked mutations suggests that this may be the cause of familial autosomal recessive Parkinson's disease.

Abstract All‐solid‐state lithium batteries (ASSLBs) have the potential to revolutionize battery systems for electric vehicles due to their benefits in safety, energy density, packaging, and operable temperature range. As the key component in ASSLBs, inorganic lithium‐ion‐based solid‐state electrolytes (SSEs) have attracted great interest, and advances in SSEs are vital to deliver the promise of ASSLBs. Herein, a survey of emerging SSEs is presented, and ion‐transport mechanisms are briefly discussed. Techniques for increasing the ionic conductivity of SSEs, including substitution and mechanical strain treatment, are highlighted. Recent advances in various classes of SSEs enabled by different preparation methods are described. Then, the issues of chemical stabilities, electrochemical compatibility, and the interfaces between electrodes and SSEs are focused on. A variety of research addressing these issues is outlined accordingly. Given their importance for next‐generation battery systems and transportation style, a perspective on the current challenges and opportunities is provided, and suggestions for future research directions for SSEs and ASSLBs are suggested.

Abstract Purpose: Recently, a 76-gene prognostic signature able to predict distant metastases in lymph node–negative (N−) breast cancer patients was reported. The aims of this study conducted by TRANSBIG were to independently validate these results and to compare the outcome with clinical risk assessment. Experimental Design: Gene expression profiling of frozen samples from 198 N− systemically untreated patients was done at the Bordet Institute, blinded to clinical data and independent of Veridex. Genomic risk was defined by Veridex, blinded to clinical data. Survival analyses, done by an independent statistician, were done with the genomic risk and adjusted for the clinical risk, defined by Adjuvant! Online. Results: The actual 5- and 10-year time to distant metastasis were 98% (88-100%) and 94% (83-98%), respectively, for the good profile group and 76% (68-82%) and 73% (65-79%), respectively, for the poor profile group. The actual 5- and 10-year overall survival were 98% (88-100%) and 87% (73-94%), respectively, for the good profile group and 84% (77-89%) and 72% (63-78%), respectively, for the poor profile group. We observed a strong time dependence of this signature, leading to an adjusted hazard ratio of 13.58 (1.85-99.63) and 8.20 (1.10-60.90) at 5 years and 5.11 (1.57-16.67) and 2.55 (1.07-6.10) at 10 years for time to distant metastasis and overall survival, respectively. Conclusion: This independent validation confirmed the performance of the 76-gene signature and adds to the growing evidence that gene expression signatures are of clinical relevance, especially for identifying patients at high risk of early distant metastases.

Protein function is regulated by diverse posttranslational modifications. The mitochondrial sirtuin SIRT5 removes malonyl and succinyl moieties from target lysines. The spectrum of protein substrates subject to these modifications is unknown. We report systematic profiling of the mammalian succinylome, identifying 2,565 succinylation sites on 779 proteins. Most of these do not overlap with acetylation sites, suggesting differential regulation of succinylation and acetylation. Our analysis reveals potential impacts of lysine succinylation on enzymes involved in mitochondrial metabolism; e.g., amino acid degradation, the tricarboxylic acid cycle (TCA) cycle, and fatty acid metabolism. Lysine succinylation is also present on cytosolic and nuclear proteins; indeed, we show that a substantial fraction of SIRT5 is extramitochondrial. SIRT5 represses biochemical activity of, and cellular respiration through, two protein complexes identified in our analysis, pyruvate dehydrogenase complex and succinate dehydrogenase. Our data reveal widespread roles for lysine succinylation in regulating metabolism and potentially other cellular functions.

Recycling of spent lithium-ion batteries (LIBs) has attracted significant attention in recent years due to the increasing demand for corresponding critical metals/materials and growing pressure on the environmental impact of solid waste disposal. A range of investigations have been carried out for recycling spent LIBs to obtain either battery materials or individual compounds. For the effective recovery of materials to be enhanced, physical pretreatment is usually applied to obtain different streams of waste materials ensuring efficient separation for further processing. Subsequently, a metallurgical process is used to extract metals or separate impurities from a specific waste stream so that the recycled materials or compounds can be further prepared by incorporating principles of materials engineering. In this review, the current status of spent LIB recycling is summarized in light of the whole recycling process, especially focusing on the hydrometallurgy. In addition to understanding different hydrometallurgical technologies including acidic leaching, alkaline leaching, chemical precipitation, and solvent extraction, the existing challenges for process optimization during the recycling are critically analyzed. Moreover, the energy consumption of different processes is evaluated and discussed. It is expected that this research could provide a guideline for improving spent LIB recycling, and this topic can be further stimulated for industrial realization.

Breast cancer arising in young women is correlated with inferior survival and higher incidence of negative clinicopathologic features. The biology driving this aggressive disease has yet to be defined.Clinically annotated, microarray data from 784 early-stage breast cancers were identified, and prospectively defined, age-specific cohorts (young: </= 45 years, n = 200; older: >/= 65 years, n = 211) were compared by prognosis, clinicopathologic variables, mRNA expression values, single-gene analysis, and gene set enrichment analysis (GSEA). Univariate and multivariate analyses were performed.Using clinicopathologic variables, young women illustrated lower estrogen receptor (ER) positivity (immunohistochemistry [IHC], P = .027), larger tumors (P = .012), higher human epidermal growth factor receptor 2 (HER-2) overexpression (IHC, P = .075), lymph node positivity (P = .008), higher grade tumors (P < .0001), and trends toward inferior disease-free survival (DFS; hazard ratio = 1.32; P = .094). Using genomic expression analysis, tumors arising in young women had significantly lower ERalpha mRNA (P < .0001), ERbeta (P = .02), and progesterone receptor (PR) expression (P < .0001), but higher HER-2 (P < .0001) and epidermal growth factor receptor (EGFR) expression (P < .0001). Exploratory analysis (GSEA) revealed 367 biologically relevant gene sets significantly distinguishing breast tumors arising in young women. Combining clinicopathologic and genomic variables among tumors arising in young women demonstrated that younger age and lower ERbeta and higher EGFR mRNA expression were significant predictors of inferior DFS.This large-scale genomic analysis illustrates that breast cancer arising in young women is a unique biologic entity driven by unifying oncogenic signaling pathways, is characterized by less hormone sensitivity and higher HER-2/EGFR expression, and warrants further study to offer this poor-prognosis group of women better preventative and therapeutic options.

Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

We explored whether the five previously reported molecular subtypes in breast cancer show a preference for organ-specific relapse and searched for molecular pathways involved. The "intrinsic" gene list describing the subtypes was used to classify 344 primary breast tumors of lymph node-negative patients. Fisher exact tests were used to determine the association between a tumor subtype and a particular site of distant relapse in these patients who only received local treatment. Modulated genes and pathways were identified in the various groups using Significance Analysis of Microarrays and Global Testing. Bone relapse patients were most abundant in the luminal subtypes but were found less than expected in the basal subtype. The reverse was true for lung and brain relapse patients with the remark that absence of lung relapse was luminal A specific. Finally, a pleura relapse, although rare, was found almost exclusively in both luminal subtypes. Many differentially expressed genes were identified, of which several were in common in a subtype and the site to which the subtype preferentially relapsed. WNT signaling was up-regulated in the basal subtype and in brain-specific relapse, and down-modulated in the luminal B subtype and in bone-specific relapse. Focal adhesion was found up-regulated in the luminal A subtype but down-regulated in lung relapse. The five major molecular subtypes in breast cancer are evidently different with regard to their ability to metastasize to distant organ(s), and share biological features and pathways with their preferred distant metastatic site.

Efficient solar water evaporation was achieved by antifouling hybrid hydrogels with capillarity facilitated water transport and heat concentration in a polymeric network.

We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed that Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.

Oxygen-vacant nanocrystalline MnO(2) has been prepared by the simple process of annealing pristine oxide in Ar or O(2) . Both experimental and computational studies indicate that the catalytic activity of MnO(2) towards oxygen reduction is enhanced by introducing a modest concentration of oxygen vacancies.

Protein post-translational modifications (PTMs) at the lysine residue, such as lysine methylation, acetylation, and ubiquitination, are diverse, abundant, and dynamic. They play a key role in the regulation of diverse cellular physiology. Here we report discovery of a new type of lysine PTM, lysine malonylation (Kmal). Kmal was initially detected by mass spectrometry and protein sequence-database searching. The modification was comprehensively validated by Western blot, tandem MS, and high-performance liquid chromatography of synthetic peptides, isotopic labeling, and identification of multiple Kmal substrate proteins. Kmal is a dynamic and evolutionarily conserved PTM observed in mammalian cells and bacterial cells. In addition, we demonstrate that Sirt5, a member of the class III lysine deacetylases, can catalyze lysine demalonylation and lysine desuccinylation reactions both in vitro and in vivo. This result suggests the possibility of nondeacetylation activity of other class III lysine deacetylases, especially those without obvious acetylation protein substrates. Our results therefore reveal a new type of PTM pathway and identify the first enzyme that can regulate lysine malonylation and lysine succinylation status.

Abstract: Exosomes are nano-sized small extracellular vesicles secreted by cells, carrying nucleic acids, proteins, lipids and other bioactive substances to play a role in the body’s physiological and pathological processes. Compared to synthetic carriers such as liposomes and nanoparticles, the endogeneity and heterogeneity of exosomes give them extensive and unique advantages in the field of disease diagnosis and treatment. However, the storage stability, low yield, low purity, and weak targeting of exosomes limit its clinical application. For this reason, further exploration is needed to optimize the above problems and facilitate future functional studies of exosomes. In this paper, the origin, classification, preparation and characterization, storage stability and applications of exosome delivery system are summarized and discussed by searching a large number of literatures. Keywords: exosomes, extraction and purification, storage stability, biomarkers, targeted drug delivery, surface modification

In order to reduce the potential radiation risk, low-dose CT has attracted an increasing attention. However, simply lowering the radiation dose will significantly degrade the image quality. In this paper, we propose a new noise reduction method for low-dose CT via deep learning without accessing original projection data. A deep convolutional neural network is here used to map low-dose CT images towards its corresponding normal-dose counterparts in a patch-by-patch fashion. Qualitative results demonstrate a great potential of the proposed method on artifact reduction and structure preservation. In terms of the quantitative metrics, the proposed method has showed a substantial improvement on PSNR, RMSE and SSIM than the competing state-of-art methods. Furthermore, the speed of our method is one order of magnitude faster than the iterative reconstruction and patch-based image denoising methods.

The recent novel coronavirus disease (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is seeing a rapid increase in infected patients worldwide. The host immune response to SARS-CoV-2 appears to play a critical role in disease pathogenesis and clinical manifestations. SARS-CoV-2 not only activates antiviral immune responses, but can also cause uncontrolled inflammatory responses characterized by marked pro-inflammatory cytokine release in patients with severe COVID-19, leading to lymphopenia, lymphocyte dysfunction, and granulocyte and monocyte abnormalities. These SARS-CoV-2-induced immune abnormalities may lead to infections by microorganisms, septic shock, and severe multiple organ dysfunction. Therefore, mechanisms underlying immune abnormalities in patients with COVID-19 must be elucidated to guide clinical management of the disease. Moreover, rational management of the immune responses to SARS-CoV-2, which includes enhancing anti-viral immunity while inhibiting systemic inflammation, may be key to successful treatment. In this review, we discuss the immunopathology of COVID-19, its potential mechanisms, and clinical implications to aid the development of new therapeutic strategies against COVID-19.

Collaborative Filtering(CF)-based recommendation algorithms, such as Latent Factor Models (LFM), work well in terms of prediction accuracy. However, the latent features make it difficulty to explain the recommendation results to the users. Fortunately, with the continuous growth of online user reviews, the information available for training a recommender system is no longer limited to just numerical star ratings or user/item features. By extracting explicit user opinions about various aspects of a product from the reviews, it is possible to learn more details about what aspects a user cares, which further sheds light on the possibility to make explainable recommendations.

Mechanochromic luminescent metal complexes have recently attracted considerable interest as smart materials for use in the fields of luminescence switches, mechanosensors, mechanohistory indicators, security papers, optoelectronic devices, and data storage. In this review, recent advancements in metal complex mechanochromic luminescence are summarized. The majority of the reported mechanochromic luminescent metal complex systems are discussed, including Zn(II), Au(I), Pt(II), Ag(I), Cu(I), Al(III), and Ir(III) complexes. Aggregation-induced emission (AIE) complexes are described based on the existence of a structural relationship between AIE compounds and mechanochromism. It is expected that AIE complexes may become important alternative sources of mechanochromic luminescent complexes.

Abstract Although persistent room‐temperature phosphorescence (RTP) emission has been observed for a few pure crystalline organic molecules, there is no consistent mechanism and no universal design strategy for organic persistent RTP (pRTP) materials. A new mechanism for pRTP is presented, based on combining the advantages of different excited‐state configurations in coupled intermolecular units, which may be applicable to a wide range of organic molecules. By following this mechanism, we have developed a successful design strategy to obtain bright pRTP by utilizing a heavy halogen atom to further increase the intersystem crossing rate of the coupled units. RTP with a remarkably long lifetime of 0.28 s and a very high quantum efficiency of 5 % was thus obtained under ambient conditions. This strategy represents an important step in the understanding of organic pRTP emission.

Leptospirosis is a widely spread disease of global concern. Infection causes flu-like episodes with frequent severe renal and hepatic damage, such as haemorrhage and jaundice. In more severe cases, massive pulmonary haemorrhages, including fatal sudden haemoptysis, can occur1. Here we report the complete genomic sequence of a representative virulent serovar type strain (Lai)2 of Leptospira interrogans serogroup Icterohaemorrhagiae consisting of a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with a total of 4,768 predicted genes. In terms of the genetic determinants of physiological characteristics, the facultatively parasitic L. interrogans differs extensively from two other strictly parasitic pathogenic spirochaetes, Treponema pallidum3 and Borrelia burgdorferi4, although similarities exist in the genes that govern their unique morphological features. A comprehensive analysis of the L. interrogans genes for chemotaxis/motility and lipopolysaccharide synthesis provides a basis for in-depth studies of virulence and pathogenesis. The discovery of a series of genes possibly related to adhesion, invasion and the haematological changes that characterize leptospirosis has provided clues about how an environmental organism might evolve into an important human pathogen.

Abstract Background No clinically proven effective antiviral strategy exists for the epidemic Coronavirus Disease 2019 (COVID-19). Methods We conducted a prospective, randomized, controlled, open-label multicenter trial involving adult patients with COVID-19. Patients were randomly assigned in a 1:1 ratio to receive conventional therapy plus Umifenovir (Arbidol) (200mg*3/day) or Favipiravir (1600mg*2/first day followed by 600mg*2/day) for 10 days. The primary outcome was clinical recovery rate of Day 7. Latency to relief for pyrexia and cough, the rate of auxiliary oxygen therapy (AOT) or noninvasive mechanical ventilation (NMV) were the secondary outcomes. Safety data were collected for 17 days. Results 240 enrolled COVID-19 patients underwent randomization; 120 patients were assigned to receive Favipiravir (116 assessed), and 120 to receive Arbidol (120 assessed). Clinical recovery rate of Day 7 does not significantly differ between Favipiravir group (71/116) and Arbidol group (62/120) (P=0.1396, difference of recovery rate: 0.0954; 95% CI: -0.0305 to 0.2213). Favipiravir led to shorter latencies to relief for both pyrexia (difference: 1.70 days, P&lt;0.0001) and cough (difference: 1.75 days, P&lt;0.0001). No difference was observed of AOT or NMV rate (both P&gt;0.05). The most frequently observed Favipiravir-associated adverse event was raised serum uric acid (16/116, OR: 5.52, P=0.0014). Conclusions Among patients with COVID-19, Favipiravir, compared to Arbidol, did not significantly improve the clinically recovery rate at Day 7. Favipiravir significantly improved the latency to relief for pyrexia and cough. Adverse effects caused Favipiravir are mild and manageable. This trial is registered with Chictr.org.cn (ChiCTR2000030254).

Ligand binding to cell surface receptors initiates a cascade of signaling events regulated by dynamic phosphorylation events on a multitude of pathway proteins. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues, may play a role in determining cellular response, but experimental data required for analysis of these features have not previously been available. To understand the dynamic operation of signaling cascades, we have developed a method enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins in a time-resolved manner, downstream of epidermal growth factor receptor (EGFR) activation. Tryptic peptides from four different EGFR stimulation time points were labeled with four isoforms of the iTRAQ reagent to enable downstream quantification. After mixing of the labeled samples, tyrosine-phosphorylated peptides were immunoprecipitated with an anti-phosphotyrosine antibody and further enriched by IMAC before LC/MS/MS analysis. Database searching and manual confirmation of peptide phosphorylation site assignments led to the identification of 78 tyrosine phosphorylation sites on 58 proteins from a single analysis. Replicate analyses of a separate biological sample provided both validation of this first data set and identification of 26 additional tyrosine phosphorylation sites and 18 additional proteins. iTRAQ fragment ion ratios provided time course phosphorylation profiles for each site. The data set of quantitative temporal phosphorylation profiles was further characterized by self-organizing maps, which resulted in identification of several cohorts of tyrosine residues exhibiting self-similar temporal phosphorylation profiles, operationally defining dynamic modules in the EGFR signaling network consistent with particular cellular processes. The presence of novel proteins and associated tyrosine phosphorylation sites within these modules indicates additional components of this network and potentially localizes the topological action of these proteins. Additional analysis and modeling of the data generated in this study are likely to yield more sophisticated models of receptor tyrosine kinase-initiated signal transduction, trafficking, and regulation.

Norihiro Kato and colleagues perform a meta-analysis of genome-wide association studies to identify common variants associated with blood pressure variation in east Asians. They identify five new genome-wide significant signals and replicate seven loci previously discovered in populations of European ancestry. We conducted a meta-analysis of genome-wide association studies of systolic (SBP) and diastolic (DBP) blood pressure in 19,608 subjects of east Asian ancestry from the AGEN-BP consortium followed up with de novo genotyping (n = 10,518) and further replication (n = 20,247) in east Asian samples. We identified genome-wide significant (P < 5 × 10−8) associations with SBP or DBP, which included variants at four new loci (ST7L-CAPZA1, FIGN-GRB14, ENPEP and NPR3) and a newly discovered variant near TBX3. Among the five newly discovered variants, we obtained significant replication in the independent samples for all of these loci except NPR3. We also confirmed seven loci previously identified in populations of European descent. Moreover, at 12q24.13 near ALDH2, we observed strong association signals (P = 7.9 × 10−31 and P = 1.3 × 10−35 for SBP and DBP, respectively) with ethnic specificity. These findings provide new insights into blood pressure regulation and potential targets for intervention.

We have recently identified and clonedFoxp3, the gene defective in mice with thescurfy mutation. The immune dysregulation documented in these mice and in humans with mutations in the orthologous gene indicates that the foxp3 gene product, scurfin, is involved in the regulation of T cell activation and differentiation. The autoimmune state observed in these patients with the immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome, or X-linked autoimmunity-allergic dysregulation syndrome also points to a critical role for scurfin in the regulation of T cell homeostasis.FOXP3 encodes a novel member of the forkhead family of transcription factors. Here we demonstrate that this structural domain is required for nuclear localization and DNA binding. Scurfin, transiently expressed in heterologous cells, represses transcription of a reporter containing a multimeric forkhead binding site. Upon overexpression in CD4 T cells, scurfin attenuates activation-induced cytokine production and proliferation. We have identified FKH binding sequences adjacent to critical NFAT regulatory sites in the promoters of several cytokine genes whose expression is sensitive to changes in SFN abundance. Our findings indicate that the ability of scurfin to bind DNA, and presumably repress transcription, plays a paramount role in determining the amplitude of the response of CD4 T cells to activation. We have recently identified and clonedFoxp3, the gene defective in mice with thescurfy mutation. The immune dysregulation documented in these mice and in humans with mutations in the orthologous gene indicates that the foxp3 gene product, scurfin, is involved in the regulation of T cell activation and differentiation. The autoimmune state observed in these patients with the immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome, or X-linked autoimmunity-allergic dysregulation syndrome also points to a critical role for scurfin in the regulation of T cell homeostasis.FOXP3 encodes a novel member of the forkhead family of transcription factors. Here we demonstrate that this structural domain is required for nuclear localization and DNA binding. Scurfin, transiently expressed in heterologous cells, represses transcription of a reporter containing a multimeric forkhead binding site. Upon overexpression in CD4 T cells, scurfin attenuates activation-induced cytokine production and proliferation. We have identified FKH binding sequences adjacent to critical NFAT regulatory sites in the promoters of several cytokine genes whose expression is sensitive to changes in SFN abundance. Our findings indicate that the ability of scurfin to bind DNA, and presumably repress transcription, plays a paramount role in determining the amplitude of the response of CD4 T cells to activation. scurfy scurfin immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome X-linked autoimmunity-allergic dysregulation syndrome forkhead glutathione S-transferase V1 immunoglobulin heavy chain variable-region (VH) promoter fetal calf serum tetracycline-inducible green fluorescence protein anti electromobility shift assay transthyretin site nuclear factor of activated T cells granulocyte macrophage-colony stimulating factor interleukin tumor necrosis factor α phorbol myristate acetate signal transducer and activator of transcription base pair(s) scurfy(sf),1 a naturally occurring, X-linked, recessive mutation, has been described previously (1Lyon M. Peters J. Glenister P. Ball S. Wright E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2433-2437Crossref PubMed Scopus (151) Google Scholar, 2Godfrey V.L. Wilkinson J.E. Russell L.B. Am. J. Pathol. 1991; 138: 1379-1387PubMed Google Scholar, 3Godfrey, V. L., Wilkinson, J. E., Rinchik, E. M., and Russell, L. B. (1991) Proc. Natl. Acad. Sci. U. S. A.1991 88, 5528–5532Google Scholar, 4Godfrey V.L. Rouse B.T. Wilkinson J.E. Am. J. Pathol. 1994; 145: 281-286PubMed Google Scholar, 5Blair P.J. Carpenter D.A. Godfrey V.L. Russell L.B. Wilkinson J.E. Rinchik E.M. Mamm. Genome. 1994; 5: 652-654Crossref PubMed Scopus (25) Google Scholar, 6Blair P.J. Bultman S.J. Haas J.C. Rouse B.T. Wilkinson J.E. Godfrey V.L. J. Immunol. 1994; 153: 3764-3774PubMed Google Scholar, 7Derry J.M. Wiedemann P. Blair P. Wang Y. Kerns J.A. Lemahieu V. Godfrey V.L. Wilkinson J.E. Francke U. Genomics. 1995; 29: 471Crossref PubMed Scopus (39) Google Scholar, 8Kanangat S. Blair P. Reddy R. Deheshia J. Godfrey V. Rouse B.T. Wilkinson J.E. Eur. J. Immunol. 1996; 26: 161-165Crossref PubMed Scopus (106) Google Scholar, 9Clark L.B. Appleby M.W. Brunkow M.E. Wilkinson J.E. Ziegler S.F. Ramsdell F. J. Immunol. 1999; 162: 2546-2554PubMed Google Scholar). The disease observed in hemizygous mutant males features massive lymphoproliferation and subsequent infiltration of several organs, and results in death by ∼3 weeks of age (1Lyon M. Peters J. Glenister P. Ball S. Wright E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2433-2437Crossref PubMed Scopus (151) Google Scholar, 4Godfrey V.L. Rouse B.T. Wilkinson J.E. Am. J. Pathol. 1994; 145: 281-286PubMed Google Scholar). Both depletion and adoptive transfer experiments indicate that CD4 T cells are primarily responsible for the pathology observed in sfmice (2Godfrey V.L. Wilkinson J.E. Russell L.B. Am. J. Pathol. 1991; 138: 1379-1387PubMed Google Scholar, 6Blair P.J. Bultman S.J. Haas J.C. Rouse B.T. Wilkinson J.E. Godfrey V.L. J. Immunol. 1994; 153: 3764-3774PubMed Google Scholar). sf CD4 T cells are chronically activated, expressing up-regulated levels of several activation markers and secreting increased levels of several cytokines directly ex vivo (8Kanangat S. Blair P. Reddy R. Deheshia J. Godfrey V. Rouse B.T. Wilkinson J.E. Eur. J. Immunol. 1996; 26: 161-165Crossref PubMed Scopus (106) Google Scholar, 9Clark L.B. Appleby M.W. Brunkow M.E. Wilkinson J.E. Ziegler S.F. Ramsdell F. J. Immunol. 1999; 162: 2546-2554PubMed Google Scholar). sf-derived CD4 T cell effector function is also refractory to inhibition with several pharmacological reagents, particularly the immunosuppresants cyclosporin A and rapamycin (9Clark L.B. Appleby M.W. Brunkow M.E. Wilkinson J.E. Ziegler S.F. Ramsdell F. J. Immunol. 1999; 162: 2546-2554PubMed Google Scholar). The phenotype of sf mutant mice is strikingly similar to that observed in mice deficient in expression of CTLA-4, indicating that scurfin may also be an important regulator of the T cell activation program (10Waterhouse P. Penninger J.M. Timms E. Wakeham A. Shahinian A. Lee K.P. Thompson C.B. Griesser H. Mak T.W. Science. 1995; 270: 985-988Crossref PubMed Scopus (2376) Google Scholar, 11Tivol E.A. Borriello F. Schweitzer A.N. Lynch W.P. Bluestone J.A. Sharpe A.H. Immunity. 1995; 3: 541-547Abstract Full Text PDF PubMed Scopus (2393) Google Scholar, 12Chambers C.A. Sullivan T.J. Allison J.P. Immunity. 1997; 7: 885-895Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 13Walunas T.L. Lenschow D.J. Bakker C.Y. Linsley P.S. Freeman G.J. Green J.M. Thompson C.B. Bluestone J.A. Immunity. 1994; 1: 405-413Abstract Full Text PDF PubMed Scopus (1791) Google Scholar, 14Thompson C.B. Allison J.P. Immunity. 1997; 7: 445-450Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). Recently we have positionally cloned the sf gene (15Jeffery E.W. Hjerrild K.A. Paeper B. Clark L.B. Yasayko S.A. Wilkinson J.E. Galas D. Ziegler S.F. Ramsdell F. Brunkow M.E. Nat. Genet. 2001; 27: 68-73PubMed Google Scholar). The wild type sf gene (Foxp3) encodes a novel 48-kDa protein, scurfin (SFN). The protein is expressed at low levels, primarily in CD4 T cells (15Jeffery E.W. Hjerrild K.A. Paeper B. Clark L.B. Yasayko S.A. Wilkinson J.E. Galas D. Ziegler S.F. Ramsdell F. Brunkow M.E. Nat. Genet. 2001; 27: 68-73PubMed Google Scholar). Interestingly, SFN expression levels do not appear to be modulated following activation. The sfmutation is a 2-bp insertion that results in a premature stop codon. It is not known if the predicted truncated protein is stably expressed in mutants. However, overexpression of a transgene encoding the mutant form of the SFN gene in wild type mice yielded no phenotype (15Jeffery E.W. Hjerrild K.A. Paeper B. Clark L.B. Yasayko S.A. Wilkinson J.E. Galas D. Ziegler S.F. Ramsdell F. Brunkow M.E. Nat. Genet. 2001; 27: 68-73PubMed Google Scholar). This finding suggests that the sf phenotype most likely results from a loss of SFN function. The observations that females heterozygous for the sf mutation are phenotypically normal and display random X-inactivation also support this interpretation. Concurrent with the cloning of the sf gene, several reports of mutations in the human ortholog of the Foxp3 gene have been made (16Chatila T.A. Blaeser F. Ho N. Lederman H.M. Voulgaropoulos C. Helms C. Bowcock A.M. J. Clin. Invest. 2000; 106: R75-R81Crossref PubMed Scopus (737) Google Scholar, 17Wildin R.S. Ramsdell F. Peake J. Faravelli F. Casanova J.L. Buist N. Levy-Lahad E. Mazzella M. Goulet O. Perroni L. Dagna Bricarelli F. Byrne G. McEuen M. Proll S. Appleby M. Brunkow M.E. Wildin R.S. Nat. Genet. 2001; 27: 18-20Crossref PubMed Scopus (1481) Google Scholar, 18Bennett C.L. Christie J. Ramsdell F. Brunkow M.E. Ferguson P.J. Whitesell L. Kelly T.E. Saulsbury F.T. Chance P.F. Ochs H.D. Nat. Genet. 2001; 27: 20-21Crossref PubMed Scopus (2639) Google Scholar). Affected males present symptoms similar to those observed in sf mice and include a predilection to autoimmune diseases and allergy. The majority of the mutations in these patients also result in premature stop codons. Although the phenotype ofsf mice and of patients with the immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), or X-linked autoimmunity-allergic dysregulation syndrome (XLAAD) indicate a critical role for the foxp3 gene product in the negative regulation of T cell activation, it is not immediately evident how the protein exacts this effect. The most prominent structural feature of SFN is a forkhead/winged helix domain at the C-terminal end of the protein. Forkhead/winged helix domain-containing proteins are members of a rapidly growing family of DNA binding factors. Since the identification of the original forkhead (FKH) protein in Drosophila in the early 1990s, over 80 FKH family proteins, classified into 17 different subfamilies, have been described in a variety of species, including nematodes, yeast, and mammals (Ref. 19Kaestner K.H. Knochel W. Martinez D.E. Genes Dev. 2000; 14: 142-146PubMed Google Scholar and www.biology.pomona.edu/fox.html). Although both transcriptional activators and repressors have been identified in this family, FKH domain-containing proteins typically function in the regulation of lineage commitment and developmental differentiation (reviewed in Ref. 20Kaufmann E. Knochel W. Mech. Dev. 1996; 57: 3-20Crossref PubMed Scopus (573) Google Scholar). SFN, with an FKH domain of only 84 amino acids, is an atypical winged helix family member. QRF-1, a previously described partial protein sequence (21Li C. Tucker P.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11583-11587Crossref PubMed Scopus (56) Google Scholar), contains a similarly truncated FKH domain. Li and Tucker (21Li C. Tucker P.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11583-11587Crossref PubMed Scopus (56) Google Scholar) have demonstrated that the FKH domain of QRF-1 is necessary and sufficient for binding to oligonucleotides containing DNA sequences described to bind FKH domain-containing proteins. The sequence identity between the FKH domain of QRF-1 and scurfin suggested that the latter would also function as a DNA binding protein. Another distinct feature of SFN is the location of the FKH domain at its C terminus. Generally, the FKH domain is located near the amino terminus of proteins in this family. This unique structural feature may be significant in the protein's function and help distinguish SFN from other FKH family proteins active in CD4 T cells. With the ultimate goal of identifying how SFN acts to regulate T cell function, we have carried out fundamental analyses of this protein. Here, we report that scurfin is a DNA binding protein that can repress transcription. The FKH domain is required for this activity. Complimentary to the observation of hyper-responsive T cells in mice carrying the inactivating sf mutation, and the description of T cell-mediated autoimmunity in humans with the IPEX or XLAAD syndrome, overexpression of scurfin attenuates the T cell activation response, as evidenced by reduced cytokine production in response to activating stimuli. The ability to bind DNA, i.e. inclusion of the FKH domain, is required for this effect. Although the exact mechanisms by which SFN regulates T cell activation remain to be elucidated, the findings presented here suggest that the ability of scurfin to act as a negative regulator of cytokine production by CD4 T cells may involve direct repression of NFAT-mediated transcription. cDNA encoding full-length human scurfin (amino acids 1–431) or a fragment lacking the FKH domain (amino acids 1–327) was inserted into the expression vector pIRES2-EGFP (CLONTECH, Palo Alto, CA), the GST fusion vector pGEX-4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ), and the tetracycline-responsive vector pREV-TRE (CLONTECH). A multimeric FKH binding site construct was created by annealing complementary oligonucleotides containing three tandem repeats of the FKH binding site V1P (sequence given below). Restriction sites were included at the ends of the oligonucleotides to facilitate subcloning of the multimer into the SV40 promoter-driven luciferase reporter, pGL-3 Promoter (Promega, Madison, WI) to create 3xFKH-luc. Sequencing confirmed all constructs to be correct and free of mutation. HEK 293T cells and COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS, penicillin, and streptomycin. The Jurkat subline D4 was maintained in RPMI 1640 supplemented with 10% FCS, penicillin, and streptomycin. Tetracycline-inducible (Teti) Jurkat clones were generated following neomycin selection of D4 cells transfected with pREV-Tet-On (CLONTECH). Co-expression of a Tet-responsive GFP construct allowed selection of a clone yielding minimal promoter activity in the absence of treatment with the tetracycline analog, doxycycline (CLONTECH). This clone, hereafter referred to as TO, was used to create Tetiexpressers of empty vector (TO.TRE), full-length scurfin (TO.SFN), or scurfin lacking the FKH domain (TO.ΔFKH). The full-length (SFN), deletion mutant (ΔFKH), or empty pIRES2-EGFP construct was transiently expressed in HEK 293T cells using a calcium phosphate transfection kit (5 Prime → 3 Prime, Inc., Boulder, CO). After 24 h of culture, ∼5000 cells/well were plated on poly-lysine-coated microscope slides and allowed to adhere overnight at 37 °C. 48 h post-transfection, cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% saponin. Cells were stained with pre-immune sera (Pre) or polyclonal antisera raised against full-length human scurfin (αSFN), followed by goat α-rabbit IgG Alexa-568, the actin stain phalloidin-Alexa 488, and the DNA intercalating dye Toto-3 (all from Molecular Probes, Eugene, OR). After several phosphate-buffered saline washes, mount medium (50% glycerol in phosphate-buffered saline) was added to each well. A coverslip was then applied and sealed. The Laser Scanning Confocal Imaging System, MRC 1024 (Bio-Rad, Hercules, CA), was used to visualize subcellular localization of full-length and ΔFKH scurfin. Cytoplasmic and nuclear protein was isolated from HEK 293T transient transfectants as previously described (22Penix L. Weaver W.M. Pang Y. Young H.A. Wilson C.B. J. Exp. Med. 1993; 178: 1483-1496Crossref PubMed Scopus (178) Google Scholar). Protein concentration was determined by Bradford assay. Equal quantities of cytoplasmic and nuclear protein were resolved by polyacrylamide gel electrophoresis and transferred to nitrocellulose. The subcellular localization of scurfin was assessed by immunoblotting with αSFN, followed by detection with a chemiluminescent reagent. Single-stranded oligonucleotides containing the consensus FKH binding sites (21 and references therein) TTR-S (5′-TCG AGT TGA CTA AGT CAA TAA TCA GAA TCA G) and V1P (5′-TCA AAA ATA TTG AAG TGT TAT CAC ATA CAC), an irrelevant sequence (5′-ATG AAT ATG CAA ATC AGG TG), or putative FKH/NFAT composite binding sites in the human GM-CSF enhancer (5′-TCT GCC CTG CCA CAA CCC CAT CGG AGC CCC TGA GTC AGC ATG G), the human IL-2 promoter (5′-ATC AGA AGA GGA AAA ATG AAG GTA ATG TTT TTT CAG ACT GGT AA), or the NFAT multimeric reporter construct (5′-AAG AGG AAA ATT TGT TTC ATA CAG AAG GCG) were annealed with their complementary strand and used as cold competitors or radiolabeled probe in gel shift assays with Jurkat-derived nuclear protein. A dimer of the TTR-S oligonucleotide (5′-CTG AAT TCT GAT TAT TGA CTT AGT CAA CAT TCT GAT TAT TGA CTT AGT CAA C) and a different irrelevant competitor (5′-GAT TTA AAA GTG TGT CCC AGC AGC CCT GGT CCA GCC CTC T) were annealed with their complementary strand, filled in with Klenow, and used in EMSA assays performed with recombinant proteins. GST fusion proteins were affinity purified from isopropyl-1-thio-β-d-galactopyranoside-induced bacterial lysates in the presence of protease inhibitors using pre-swelled glutathione-agarose beads (Sigma Chemical Co., St. Louis, MO). Nuclear extracts were prepared from D4 cells as previously described (22Penix L. Weaver W.M. Pang Y. Young H.A. Wilson C.B. J. Exp. Med. 1993; 178: 1483-1496Crossref PubMed Scopus (178) Google Scholar). Nuclear protein (4 μg/lane) or a comparable amount of purified GST fusion protein was preincubated at room temperature with binding buffer supplemented with 500 ng of poly(dI-dC) (Roche Molecular Biochemicals, Indianapolis, IN) and a 50- to 100-fold molar excess of cold oligonucleotide competitor for 10 min (23Schubert L.A. King G. Cron R.Q. Lewis D.B. Aruffo A. Hollenbaugh D. J. Biol. Chem. 1995; 270: 29624-29627Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Probe (2 × 104 cpm, end-labeled with [γ-32P]ATP) was then added to a final volume of 25 μl, and the incubation continued for an additional 15 min at room temperature. In some reactions, rabbit pre-immune sera or antisera raised against full-length SFN was incubated with nuclear protein for 30 min on ice prior to addition of the probe. All reactions were analyzed by polyacrylamide gel electrophoresis using 4% gels in 0.027 m Tris borate, 0.6 mm EDTA buffer. The activity of the 3xFKH-luc or the pGL-3 Promoter luciferase reporter construct was assessed in COS-7 cells. Calcium phosphate transfection was used to transiently express the β-galactosidase control vector, pSV-β-gal (Promega), the luciferase reporter pGL-3 Promoter or 3xFKH-luc, and the SFN, ΔFKH, or empty pIRES2-EGFP expression construct in COS-7 cells. 48 h post-transfection, cells were harvested and lysates were prepared in 1× Reporter lysis buffer according to the manufacturer's specifications (Promega). Luciferase activity was measured in cellular lysates using the Luciferase assay system (Promega) and the EG & G Berthold Lumat 9507 luminometer (PerkinElmer Life Sciences, Boston, MA) according to the manufacturers' specifications. The β-galactosidase activity in duplicates of each lysate was determined using the β-galactosidase enzyme assay system (Promega) and used to normalize measured luciferase activity. The Jurkat subline D4 was transiently transfected with the expression construct encoding full-length human SFN or the parent vector as described previously (23Schubert L.A. King G. Cron R.Q. Lewis D.B. Aruffo A. Hollenbaugh D. J. Biol. Chem. 1995; 270: 29624-29627Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). After 24 h of culture in the absence or presence of 1 or 10 μg/ml CD3 antibody (OKT3), culture supernatants were collected. IL-2 secretion was evaluated by bioassay using the IL-2-dependent murine cell line HT-2 (9Clark L.B. Appleby M.W. Brunkow M.E. Wilkinson J.E. Ziegler S.F. Ramsdell F. J. Immunol. 1999; 162: 2546-2554PubMed Google Scholar). The activity of a luciferase reporter containing a multimer of a regulatory NFAT site (−280) from the murine IL-2 gene (NFAT-luc) (24Petrak D. Memon S.A. Birrer M.J. Ashwell J.D. Zacharchuk C.M. J. Immunol. 1994; 153: 2046-2051PubMed Google Scholar) or the SV40 promoter-driven luciferase reporter, pGL-3 Promoter, was assessed in the TO.TRE, TO.SFN, and TO.ΔFKH cell lines. Cells were cultured in 1 μg/ml doxycycline for 24 h prior to transfection. Equal concentrations (107 cells/0.4 ml) of each clone were electroporated with 10 μg of the control vector pSV-β-gal and either NFAT-luc or pGL-3 Promoter as previously described (23Schubert L.A. King G. Cron R.Q. Lewis D.B. Aruffo A. Hollenbaugh D. J. Biol. Chem. 1995; 270: 29624-29627Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Cells were rested for 2 h at 37 °C. Cell viability was determined by trypan blue exclusion, and 106 cells per well were cultured in the absence or presence of ionomycin (1.5 μm) and PMA (25 ng/ml) for 6 h. Cells were harvested, and lysates were prepared in 1× reporter lysis buffer. Luciferase activity was measured and normalized based on the β-galactosidase activity detected in duplicate lysates. Distinct sequences present in the FKH domain have been described to mediate nuclear localization and DNA binding by proteins containing this structural feature (25Hellqvist M. Mahlapuu M. Blixt A. Enerback S. Carlsson P. J. Biol. Chem. 1998; 273: 23335-23343Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To analyze scurfin localization, we first assessed the ability of transiently expressed scurfin to localize to the nucleus. A construct directing expression of scurfin (SFN), a fragment lacking the FKH domain (ΔFKH), or the parent vector pIRES2-EGFP (293) was transiently transfected into HEK 293T cells as described under “Experimental Procedures.” Scurfin localization was assessed at both the single cell level by confocal microscopy (Fig.1, A–D) and at the level of the transfected population by fractionation of total cellular protein into nuclear and cytoplasmic components and subsequent Western blotting (Fig. 1E). Scurfin (in red) predominantly localizes to the nucleus of HEK 293T cells (Fig. 1C). As predicted, removal of the FKH domain results in the exclusion of the majority of scurfin from the nucleus (Fig. 1D). The residual red staining observed in the nucleus of ΔFKH-transfected cells could reflect staining of scurfin endogenously expressed in HEK 293T cells or, more likely, cross-reactivity of the antisera with other FKH domain-containing proteins expressed in these cells. A similar, low level of reactivity with the SFN antisera is observed in the nucleus of cells transfected with vector alone (Fig. 1B). Finally, staining with the scurfin antisera is specific, because no detectable signal is observed when pre-immune sera is used for staining (Fig.1A). To demonstrate that the results observed by confocal microscopy were representative of the entire transfected population, cytoplasmic and nuclear protein were concomitantly isolated from transfectants. Equal quantities of cytoplasmic (c) and nuclear (n) protein were immunoblotted with scurfin antisera (Fig. 1E). As observed with confocal microscopy, the majority of scurfin (∼48 kDa) localizes to the nuclear protein fraction, and removal of the FKH domain (∼34-kDa band) results in its retention in the cytoplasm. These findings indicate that scurfin can localize to the nucleus and that the FKH domain is required for this to occur. Confocal microscopy and Western blotting of nuclear protein isolated from Jurkat cells both indicate that endogenously expressed SFN also localizes to the nucleus (data not shown). Based on the sequence identity between the FKH domain of the previously described partial protein QRF-1 and scurfin, we predicted that scurfin would form a complex with the same DNA sequences (21Li C. Tucker P.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11583-11587Crossref PubMed Scopus (56) Google Scholar). We tested the ability of both recombinant GST fusions with scurfin and endogenously expressed scurfin to bind to oligonucleotides containing these sequences. As shown in Fig.2A, a fusion of GST with full-length scurfin binds to a probe containing a dimer of the FKH consensus binding site from the TTR-S gene (Fig. 2A,lane 3). Addition of a 50-fold molar excess of cold competitor of the same sequence eliminates formation of the lowest band (Fig. 2A, lane 4), but inclusion of the same amount of an irrelevant DNA sequence does not (Fig. 2A,lane 5). Addition of polyclonal antisera raised against SFN in the gel shift reaction results in retardation of the lowest band (Fig. 2A, lane 6). This indicates that SFN is a primary component of the lowest DNA-protein complex and that the two higher bands may represent nonspecific binding activity. As expected, a fusion of GST with a deletion mutant of scurfin, which lacks the FKH domain, fails to form a complex with the probe (Fig. 2A,lane 2). Finally a fusion between GST and c-Jun, a protein known to bind a distinct DNA sequence, fails to form a complex with the TTR-S probe (Fig. 2A, lane 1). These results suggest that scurfin is a DNA binding protein that specifically complexes with a consensus FKH binding site and that the FKH domain is required for this activity. The FKH domain-dependent ability of recombinant scurfin to bind to DNA was not surprising. However, this finding did not prove that scurfin binds DNA under more physiologically relevant circumstances. Because CD4 T cells express scurfin and are most affected by the inactivating sf mutation, we next studied the DNA binding ability of endogenously expressed scurfin. Nuclear protein was extracted from Jurkat cells activated with αCD3 for 2 h and used in gel shift assays. As observed with recombinant full-length protein, endogenously expressed scurfin forms a complex with a probe that contains a FKH protein consensus DNA binding site from the V1P promoter (Fig. 2B, lanes 1 and5). A 50-fold molar excess of cold oligonucleotide competitor of the same DNA sequence as the probe (Fig. 2B,lane 2) or the FKH binding site from the TTR-S promoter (Fig. 2B, lane 3) competes away the protein/probe complex, indicating that a specific complex is formed. Furthermore, addition of an irrelevant DNA sequence does not disrupt complex formation (Fig. 2B, lane 4). The same findings were attained when the FKH binding site from the TTR-S promoter was used as the probe (data not shown). Inclusion of antisera raised against scurfin (Fig. 2B, lane 6), but not pre-immune sera (Fig. 2B, lane 7), results in retardation of the migration of the complex, demonstrating that scurfin is a major component of the protein-DNA complex. These findings confirm that scurfin is present in the nucleus of CD4 T cells and is capable of binding DNA. The same DNA binding activity is observed in nuclear protein isolated from unstimulated Jurkat cells (data not shown). Next we investigated the functional significance of the ability of scurfin to bind DNA. The FKH family of DNA binding factors contains transcriptional activators as well as repressors of transcription. The documented transactivation domains of other FKH family proteins are located C-terminal of the FKH domain (26Mahlapuu M. Pelto-Huikko M. Aitola M. Enerback S. Carlsson P. Dev. Biol. 1998; 202: 183-195Crossref PubMed Scopus (72) Google Scholar, 27Tang E.D. Nunez G. Barr F.G. Guan K., L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 28Schuddekopf K. Schorpp M. Boehm T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9661-9664Crossref PubMed Scopus (78) Google Scholar). As previously mentioned, the scurfin FKH domain is unique in that it ends only 11 amino acids from the C terminus of the protein. We hypothesized that SFN might lack a transactivation domain and possibly inhibit transcription. To address this question, we examined the effect of SFN on the expression of the SV40 minimal promoter reporter flanked by multiple FKH binding sites. COS-7 cells were transiently transfected with an expression construct directing the production of scurfin (SFN), a mutant lacking the FKH domain (ΔFKH), or the parent vector pIRES2-EGFP (Control). The control vector pSV-β-gal and either 3xFKH-luc (Fig. 3,speckled bars) or the parent luciferase reporter pGL-3 Promoter (Fig. 3, solid bars) were co-transfected with each expression construct. Lysates were harvested and luciferase and β-galactosidase activity measured 48 h later. Comparable levels of luciferase activity are detected in cells transfected with the pGL-3 Promoter, irrespective of the expression construct co-transfected (Fig.3, solid bars). Activity of the 3xFKH-lucconstruct in COS-7 cells transfected with the parent vector (Fig. 3,Control, speckled bar) and SFN lacking the DNA binding domain (Fig. 3, ΔFKH, speckled bar) is comparable. However, overexpression of full-length scurfin consistently results in more than a 50% reduction of transcription of the reporter gene (Fig. 3, SFN, speckled bar), suggesting that SFN can repress transcription. The observation that SFN overexpression does not affect promoter activity in the absence of FKH binding sites (Fig. 3, SFN, solid bar) and that removal of the DNA binding domain eliminates the reduction in expression of the 3xFKH-luc reporter gene (Fig. 3, ΔFKH,speckled bar) both indicate that this repression is specific and most likely mediated by the FKH domain. Although Fig. 3 shows an apparent reduction in expression of the 3xFKH-luc in ΔFKH-transfected cells, relative to that observed in control transfected cells, this trend has not been

It is widely accepted that the familial Parkinson's disease (PD)-linked gene product, parkin, functions as a ubiquitin ligase involved in protein turnover via the ubiquitin-proteasome system. Substrates ubiquitinated by parkin are hence thought to be destined for proteasomal degradation. Because we demonstrated previously that parkin interacts with and ubiquitinates synphilin-1, we initially expected synphilin-1 degradation to be enhanced in the presence of parkin. Contrary to our expectation, we found that synphilin-1 is normally ubiquitinated by parkin in a nonclassical, proteasomal-independent manner that involves lysine 63 (K63)-linked polyubiquitin chain formation. Parkin-mediated degradation of synphilin-1 occurs appreciably only at an unusually high parkin to synphilin-1 expression ratio or when primed for lysine 48 (K48)-linked ubiquitination. In addition we found that parkin-mediated ubiquitination of proteins within Lewy-body-like inclusions formed by the coexpression of synphilin-1, alpha-synuclein, and parkin occurs predominantly via K63 linkages and that the formation of these inclusions is enhanced by K63-linked ubiquitination. Our results suggest that parkin is a dual-function ubiquitin ligase and that K63-linked ubiquitination of synphilin-1 by parkin may be involved in the formation of Lewy body inclusions associated with PD.

<h2>Summary</h2> The induction of pluripotency or <i>trans</i>-differentiation of one cell type to another can be accomplished with cell-lineage-specific transcription factors. Here, we report that repression of a single RNA binding polypyrimidine-tract-binding (PTB) protein, which occurs during normal brain development via the action of miR-124, is sufficient to induce <i>trans</i>-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby derepressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in nonneuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage.

The impact of phase-locked loop (PLL) parameters on the dynamic and steady-state behavior of a voltage-source converter (VSC) in an HVDC transmission system is determined as a function of the system strength [parameterized by the short-circuit ratio (SCR)]. This is achieved by using a linearized small-signal model of the converter system and its controls. The model is validated via electromagnetic transients simulation of the fully detailed large signal model. An interesting result from this study is that the maximum power transfer capability of the VSC-HVDC converter is affected by the PLL gains, and that the theoretical limit (obtained from static voltage stability analysis) is approachable as the PLL gains become very small. This paper shows that gains of the PLL, particularly at low SCRs, greatly affect the operation of the VSC-HVDC converter and that operation at low SCRs approaching 1.3 is very difficult.

GRP78/BiP is a major endoplasmic reticulum (ER) chaperone protein critical for protein quality control of the ER, as well as controlling the activation of the ER-transmembrane signaling molecules. Through creation of mouse models targeting the Grp78 allele, the function of GRP78 in development and disease has been investigated. These led to the discovery that GRP78 function is obligatory for early embryonic development. However, in adult animals, GRP78 is preferably required for cancer cell survival under pathologic conditions such as tumor progression and drug resistance. The discovery of surface localization of GRP78 in cancer cells reveals potential novel function, interaction with cell-surface receptors, and possible therapeutic implications. Mouse models also reveal that GRP78 controls maturation and secretion of neuronal factors for proper neural migration and offers neuroprotection.

Due to the rapid expanding of plug-in hybrid electric vehicles (PHEVs), hybrid electric vehicles (HEVs) and electric vehicles (EVs), the projectfed demand for lithium-ion batteries (LIBs) is huge and might result in supply risks for natural lithium-containing reserves. After the service life, spent LIBs continuously accumulate in the market, and they are excellent secondary resources for lithium recovery. To alleviate resource shortage and to decrease potential environmental pollution caused by improper solid waste disposal, recycling of spent LIBs is motivated world widely in recent years. Previous studies have usually focused on the recovery of cobalt and nickel, which create high economic benefit. Recovery of lithium, however, has not been highlighted. In this article, state-of-the-art on spent LIBs recycling is discussed with emphasis on lithium recovery. In addition to understanding underlying mechanisms and physiochemistry features of various recycling methods, the possibility for industrial realization of each method is also evaluated. The complex processing steps limit the industrial implementation of hydrometallurgy-dominant methods, which usually reclaim lithium in the last step, resulting in a poor recovery efficiency of lithium. The pyrometallurgy-dominant approach is readily to scale up but lithium is lost in the slag phase. Therefore, the mild recycling (cleaner production) methods are recommended for future study since they take advantages of traditional pyrometallurgy and hydrometallurgy, and could decrease treatment temperature as well as acid/alkaline usage.

The rapid growth of lithium ion batteries (LIBs) for portable electronic devices and electric vehicles has resulted in an increased number of spent LIBs. Spent LIBs contain not only dangerous heavy metals but also toxic chemicals that pose a serious threat to ecosystems and human health. Therefore, a great deal of attention has been paid to the development of an efficient process to recycle spent LIBs for both economic aspects and environmental protection. In this paper, we review the state-of-the-art processes for metal recycling from spent LIBs, introduce the structure of a LIB, and summarize all available technologies that are used in different recovery processes. It is notable that metal extraction and pretreatment play important roles in the whole recovery process, based on one or more of the principles of pyrometallurgy, hydrometallurgy, biometallurgy, and so forth. By further comparing different recycling methods, existing challenges are identified and suggestions for improving the recycling effectiveness can be proposed.

Article Free Access Share on Entropy-based subspace clustering for mining numerical data Authors: Chun-Hung Cheng Department of Computer Science and Engineering, The Chinese University of Hong Kong Department of Computer Science and Engineering, The Chinese University of Hong KongView Profile , Ada Waichee Fu Department of Computer Science and Engineering, The Chinese University of Hong Kong Department of Computer Science and Engineering, The Chinese University of Hong KongView Profile , Yi Zhang Department of Computer Science and Engineering, The Chinese University of Hong Kong Department of Computer Science and Engineering, The Chinese University of Hong KongView Profile Authors Info & Claims KDD '99: Proceedings of the fifth ACM SIGKDD international conference on Knowledge discovery and data miningAugust 1999 Pages 84–93https://doi.org/10.1145/312129.312199Published:01 August 1999Publication History 321citation3,260DownloadsMetricsTotal Citations321Total Downloads3,260Last 12 Months226Last 6 weeks36 Get Citation AlertsNew Citation Alert added!This alert has been successfully added and will be sent to:You will be notified whenever a record that you have chosen has been cited.To manage your alert preferences, click on the button below.Manage my AlertsNew Citation Alert!Please log in to your account Save to BinderSave to BinderCreate a New BinderNameCancelCreateExport CitationPublisher SiteeReaderPDF

TET dioxygenases successively oxidize 5-methylcytosine (5mC) in mammalian genomes to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired to regenerate unmodified cytosines by thymine-DNA glycosylase (TDG) and base excision repair (BER) pathway, but it is unclear to what extent and at which part of the genome this active demethylation process takes place. Here, we have generated genome-wide distribution maps of 5hmC/5fC/5caC using modification-specific antibodies in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs). In wild-type mouse ESCs, 5fC/5caC accumulates to detectable levels at major satellite repeats but not at nonrepetitive loci. In contrast, Tdg depletion in mouse ESCs causes marked accumulation of 5fC and 5caC at a large number of proximal and distal gene regulatory elements. Thus, these results reveal the genome-wide view of iterative 5mC oxidation dynamics and indicate that TET/TDG-dependent active DNA demethylation process occurs extensively in the mammalian genome.

Using a panel of Chinese banks over the 1997–2004 period, we assess the effect of bank ownership on performance. Specifically, we conduct a joint analysis of the static, selection, and dynamic effects of (domestic) private, foreign and state ownership. We find that the “Big Four” state-owned commercial banks are less profitable, are less efficient, and have worse asset quality than other types of banks except the “policy” banks (static effect). Further, the banks undergoing a foreign acquisition or public listing record better pre-event performance (selection effect); however, we find little performance change in either the short or the long term.

Polymeric materials containing quaternary ammonium and/or phosphonium salts have been extensively studied and applied to a variety of antimicrobial-relevant areas. With various architectures, polymeric quaternary ammonium/phosphonium salts were prepared using different approaches, exhibiting different antimicrobial activities and potential applications. This review focuses on the state of the art of antimicrobial polymers with quaternary ammonium/phosphonium salts. In particular, it discusses the structure and synthesis method, mechanisms of antimicrobial action, and the comparison of antimicrobial performance between these two kinds of polymers.

The development of two-dimensional (2D) materials has opened up possibilities for their application in electronics, optoelectronics and photovoltaics, because they can provide devices with smaller size, higher speed and additional functionalities compared with conventional silicon-based devices1. The ability to grow large, high-quality single crystals for 2D components-that is, conductors, semiconductors and insulators-is essential for the industrial application of 2D devices2-4. Atom-layered hexagonal boron nitride (hBN), with its excellent stability, flat surface and large bandgap, has been reported to be the best 2D insulator5-12. However, the size of 2D hBN single crystals is typically limited to less than one millimetre13-18, mainly because of difficulties in the growth of such crystals; these include excessive nucleation, which precludes growth from a single nucleus to large single crystals, and the threefold symmetry of the hBN lattice, which leads to antiparallel domains and twin boundaries on most substrates19. Here we report the epitaxial growth of a 100-square-centimetre single-crystal hBN monolayer on a low-symmetry Cu (110) vicinal surface, obtained by annealing an industrial copper foil. Structural characterizations and theoretical calculations indicate that epitaxial growth was achieved by the coupling of Cu <211> step edges with hBN zigzag edges, which breaks the equivalence of antiparallel hBN domains, enabling unidirectional domain alignment better than 99 per cent. The growth kinetics, unidirectional alignment and seamless stitching of the hBN domains are unambiguously demonstrated using centimetre- to atomic-scale characterization techniques. Our findings are expected to facilitate the wide application of 2D devices and lead to the epitaxial growth of broad non-centrosymmetric 2D materials, such as various transition-metal dichalcogenides20-23, to produce large single crystals.

There has been an urgent need to eliminate toxic lead from the prevailing halide perovskite solar cells (PSCs), but the current lead-free PSCs are still plagued with the critical issues of low efficiency and poor stability. This is primarily due to their inadequate photovoltaic properties and chemical stability. Herein we demonstrate the use of the lead-free, all-inorganic cesium tin-germanium triiodide (CsSn0.5Ge0.5I3) solid-solution perovskite as the light absorber in PSCs, delivering promising efficiency of up to 7.11%. More importantly, these PSCs show very high stability, with less than 10% decay in efficiency after 500 h of continuous operation in N2 atmosphere under one-sun illumination. The key to this striking performance of these PSCs is the formation of a full-coverage, stable native-oxide layer, which fully encapsulates and passivates the perovskite surfaces. The native-oxide passivation approach reported here represents an alternate avenue for boosting the efficiency and stability of lead-free PSCs.

A multiple exp-function method for exact multiple wave solutions of nonlinear partial differential equations is proposed. The method is oriented towards the ease of use and capability of computer algebra systems and provides a direct and systematic solution procedure that generalizes Hirota's perturbation scheme. With the help of Maple, applying the approach to the (3+1)-dimensional potential-Yu–Toda–Sasa–Fukuyama equation yields exact explicit one-wave, two-wave and three-wave solutions, which include one-soliton, two-soliton and three-soliton type solutions. Two cases with specific values of the involved parameters are plotted for each of the two-wave and three-wave solutions.

A novel white-light-emitting organic molecule, which consists of carbazolyl- and phenothiazinyl-substituted benzophenone (OPC) and exhibits aggregation-induced emission-delayed fluorescence (AIE-DF) and mechanofluorochromic properties was synthesized. The CIE color coordinates of OPC were directly measured with a non-doped powder, which presented white-emission coordinates (0.33, 0.33) at 244 K to 252 K and (0.35, 0.35) at 298 K. The asymmetric donor-acceptor-donor' (D-A-D') type of OPC exhibits an accurate inherited relationship from dicarbazolyl-substituted benzophenone (O2C, D-A-D) and diphenothiazinyl-substituted benzophenone (O2P, D'-A-D'). By purposefully selecting the two parent molecules, that is, O2C (blue) and O2P (yellow), the white-light emission of OPC can be achieved in a single molecule. This finding provides a feasible molecular strategy to design new AIE-DF white-light-emitting organic molecules.

Gas bubbles of nanometer size were produced on atomically flat solid surfaces and imaged by atomic force microscopy (AFM) in tapping mode in water. In AFM images, nanobubbles appeared like bright spheres. Some of the bubbles remained stable for hours during the experiments. The bubbles were disturbed under high load during AFM imaging. A related mechanism is discussed.

Abstract The rapid development of high-throughput sequencing (HTS) techniques has led biology into the big-data era. Data analyses using various bioinformatics tools rely on programming and command-line environments, which are challenging and time-consuming for most wet-lab biologists. Here, we present TBtools (a T oolkit for B iologists integrating various biological data handling tools ), a stand-alone software with a user-friendly interface. The toolkit incorporates over 100 functions, which are designed to meet the increasing demand for big-data analyses, ranging from bulk sequence processing to interactive data visualization. A wide variety of graphs can be prepared in TBtools, with a new plotting engine (“JIGplot”) developed to maximum their interactive ability, which allows quick point-and-click modification to almost every graphic feature. TBtools is a platform-independent software that can be run under all operating systems with Java Runtime Environment 1.6 or newer. It is freely available to non-commercial users at https://github.com/CJ-Chen/TBtools/releases .

The polarization of the ferroelectric BiFeO(3) sub-jected to different electrical boundary conditions by heterointerfaces is imaged with atomic resolution using a spherical aberration-corrected transmission electron microscope. Unusual triangular-shaped nanodomains are seen, and their role in providing polarization closure is understood through phase-field simulations. Heterointerfaces are key to the performance of ferroelectric devices, and this first observation of spontaneous vortex nanodomain arrays at ferroelectric heterointerfaces reveals properties unlike the surrounding film including mixed Ising-Néel domain walls, which will affect switching behavior, and a drastic increase of in-plane polarization. The importance of magnetization closure has long been appreciated in multidomain ferromagnetic systems; imaging this analogous effect with atomic resolution at ferroelectric heterointerfaces provides the ability to see device-relevant interface issues. Extension of this technique to visualize domain dynamics is envisioned.

Homozygous familial hypercholesterolemia is characterized by premature cardiovascular disease caused by markedly elevated levels of low-density lipoprotein (LDL) cholesterol. This disorder is associated with genetic variants that result in virtually absent (null–null) or impaired (non-null) LDL-receptor activity. Loss-of-function variants in the gene encoding angiopoietin-like 3 (ANGPTL3) are associated with hypolipidemia and protection against atherosclerotic cardiovascular disease. Evinacumab, a monoclonal antibody against ANGPTL3, has shown potential benefit in patients with homozygous familial hypercholesterolemia.

Recent transcriptome analysis indicates that > 90% of human genes undergo alternative splicing, underscoring the contribution of differential RNA processing to diverse proteomes in higher eukaryotic cells. The polypyrimidine tract-binding protein PTB is a well-characterized splicing repressor, but PTB knockdown causes both exon inclusion and skipping. Genome-wide mapping of PTB-RNA interactions and construction of a functional RNA map now reveal that dominant PTB binding near a competing constitutive splice site generally induces exon inclusion, whereas prevalent binding close to an alternative site often causes exon skipping. This positional effect was further demonstrated by disrupting or creating a PTB-binding site on minigene constructs and testing their responses to PTB knockdown or overexpression. These findings suggest a mechanism for PTB to modulate splice site competition to produce opposite functional consequences, which may be generally applicable to RNA-binding splicing factors to positively or negatively regulate alternative splicing in mammalian cells.

Purpose The 5-year survival rate of patients with Dukes' B colon cancer is approximately 75%. Identification of the patients at high risk of recurrence in this group would allow better staging and more informed use of adjuvant chemotherapy. In this study, we used DNA chip technology to systematically identify new prognostic markers for tumor relapse in Dukes' B patients. Patients and Methods Using Affymetrix U133a GeneChip containing approximately 22,000 transcripts (Affymetrix, Santa Clara, CA), RNA samples from 74 patients with Dukes' B colon cancer were analyzed. Thirty-one patients developed tumor relapse in less than 3 years, whereas 43 patients remained disease-free for more than 3 years after surgery. Two supervised class prediction approaches were used to identify gene markers that can best discriminate between patients who would experience relapse and patients who would remain disease-free. A multivariate Cox model was built to predict recurrence. Results Gene expression profiling identified a 23-gene signature that predicts recurrence in Dukes'B patients. This signature was validated in 36 independent patients. The overall performance accuracy was 78%. Thirteen of 18 relapse patients and 15 of 18 disease-free patients were predicted correctly, giving an odds ratio of 13 (95% CI, 2.6 to 65; P = .003). The log-rank test indicated a significant difference in disease-free time between the predicted relapse and disease-free patients (P = .0001). Conclusion The clinical value of these markers is that the patients at a high predicted risk of relapse (13-fold risk) could be upstaged to receive adjuvant therapy, similar to Dukes' C patients. Our data highlight the feasibility of a prognostic assay that could focus more intensive treatment for localized colon cancer.

Abstract Dielectric energy‐storage capacitors have received increasing attention in recent years due to the advantages of high voltage, high power density, and fast charge/discharge rates. Here, a new environment‐friendly 0.76NaNbO 3 –0.24(Bi 0.5 Na 0.5 )TiO 3 relaxor antiferroelectric (AFE) bulk ceramic is studied, where local orthorhombic Pnma symmetry (R phase) and nanodomains are observed based on high‐resolution transmission electron microscopy, selected area electron diffraction, and in/ex situ synchrotron X‐ray diffraction. The orthorhombic AFE R phase and relaxor characteristics synergistically contribute to the record‐high energy‐storage density W rec of ≈12.2 J cm −3 and acceptable energy efficiency η ≈ 69% at 68 kV mm −1 , showing great advantages over currently reported bulk dielectric ceramics. In comparison with normal AFEs, the existence of large random fields in the relaxor AFE matrix and intrinsically high breakdown strength of NaNbO 3 ‐based compositions are thought to be responsible for the observed energy‐storage performances. Together with the good thermal stability of W rec (&gt;7.4 J cm −3 ) and η (&gt;73%) values at 45 kV mm −1 up to temperature of 200 °C, it is demonstrated that NaNbO 3 ‐based relaxor AFE ceramics will be potential lead‐free dielectric materials for next‐generation pulsed power capacitor applications.

Signal transducers and activators of transcription (STATs) comprise a family of cytoplasmic signaling proteins that participates in normal cellular responses to cytokines and growth factors. Frequently, however, constitutive activation of certain STAT family members, particularly Stat3, has accompanied a wide variety of human malignancies. To identify small molecule inhibitors of Stat3, we investigated the ability of the Stat3 SH2 domain-binding peptide, PY*LKTK (where Y* represents phosphotyrosine), to disrupt Stat3 activity <i>in vitro</i>. The presence of PY*LKTK, but not PYLKTK or PFLKTK, in nuclear extracts results in significant reduction in the levels of DNA binding activities of Stat3, to a lesser extent of Stat1, and with no effect on that of Stat5. Analyses of alanine scanning mutagenesis and deletion derivatives of PY*LKTK reveal that the Leu residue at the Y+1 position and a substituent at the Y−1 position (but not necessarily Pro) are essential for the disruption of active Stat3, thereby mapping the minimum active sequence to the tripeptide, <i>X</i>Y*L. Studies involving bead-coupled PY*LKTK peptide demonstrate that this phosphopeptide directly complexes with Stat3 monomers <i>in vitro</i>, suggesting that PY*LKTK disrupts Stat3:Stat3 dimers. As evidence for the functional importance of peptide-directed inhibition of Stat3, PY*LKTK-mts (mts,membrane translocating sequence) selectively inhibits constitutive and ligand-induced Stat3 activation<i>in vivo</i>. Furthermore, PY*LKTK-mts suppresses transformation by the Src oncoprotein, which has been shown previously to require constitutive Stat3 activation. Altogether, we have identified a minimal peptide that inhibits Stat3 signaling and provides the conceptual basis for use of this peptide as a lead for novel peptidomimetic drug design.

The development of metal-free catalyst for oxygen reduction reaction (ORR) is one of the most challenging tasks in fuel cells. Heteroatom doped graphenes have been recognized as the promising candidate. In this work, we have developed a one-pot hydrothermal approach towards three-dimensional nitrogen and sulfur co-doped graphene frameworks (N/S-GFs) employing graphene oxide and ammonium thiocyanate as the precursors. N/S-GFs manifest excellent catalytic behavior with mainly four electron transfer pathway in ORR in alkaline condition.

Two-dimensional (2D) materials such as graphene and transition-metal dichalcogenides reveal the electronic phases that emerge when a bulk crystal is reduced to a monolayer1-4. Transition-metal oxide perovskites host a variety of correlated electronic phases5-12, so similar behaviour in monolayer materials based on transition-metal oxide perovskites would open the door to a rich spectrum of exotic 2D correlated phases that have not yet been explored. Here we report the fabrication of freestanding perovskite films with high crystalline quality almost down to a single unit cell. Using a recently developed method based on water-soluble Sr3Al2O6 as the sacrificial buffer layer13,14 we synthesize freestanding SrTiO3 and BiFeO3 ultrathin films by reactive molecular beam epitaxy and transfer them to diverse substrates, in particular crystalline silicon wafers and holey carbon films. We find that freestanding BiFeO3 films exhibit unexpected and giant tetragonality and polarization when approaching the 2D limit. Our results demonstrate the absence of a critical thickness for stabilizing the crystalline order in the freestanding ultrathin oxide films. The ability to synthesize and transfer crystalline freestanding perovskite films without any thickness limitation onto any desired substrate creates opportunities for research into 2D correlated phases and interfacial phenomena that have not previously been technically possible.

Macroscopic lossy foam has been expected to be the most promising candidate for lightweight high-performance microwave absorption (MA). However, inferior MA behaviors of conventional foams reported previously are disappointing. The emerging graphene foam (GF) has broken this paradoxical state of affairs. Here, series of GFs with various chemical compositions and physical structures have been prepared via a facile and controllable method and their MA performance is investigated in 2–18 GHz. The in-depth analyses of the GF’s composition, structure and MA property demonstrate that the MA performance of the GF is strongly correlated with the C/O ratio, conjugated carbon domain size and graphene framework’s microstructure. A maximum absorption value of −34.0 dB as well as 14.3 GHz qualified bandwidth with reflection loss below −10 dB is achieved for the GF with an ultralow bulk density of 1.6 mg/cm3, of which the average absorption intensity and the specific MA efficiency are much higher than those of the best available MA materials in previous literature. The composition & structure–performance relationship of MA foams is revealed. The balance between small interfacial impedance gap and high loss characteristic has wide implications in improving the MA performance of the GF and other porous materials.

Parkinson's Disease (PD) is the second most common chronic neurodegenerative disease characterized by the progressive loss of dopamine neurons, leading to rigidity, slowness of movement, rest tremor, gait disturbances, and imbalance. Although there is effective symptomatic treatment for PD, there is no proven preventative or regenerative therapy. The etiology of this disorder remains unknown. Recent genetic studies have identified mutations in alpha-synuclein as a rare cause of autosomal dominant familial PD and mutations in parkin as a cause of autosomal recessive familial PD. The more common sporadic form of PD is thought to be due to oxidative stress and derangements in mitochondrial complex I activity. Understanding the mechanism by which familial linked mutations and oxidative stress cause PD has tremendous potential for unraveling the mechanisms of dopamine cell death in PD. In this article, we review recent advances in the understanding of the role of genetics and oxidative stress in the pathogenesis of PD.

This paper addresses the problem of extending an adaptive information filtering system to make decisions about the novelty and redundancy of relevant documents. It argues that relevance and redundance should each be modelled explicitly and separately. A set of five redundancy measures are proposed and evaluated in experiments with and without redundancy thresholds. The experimental results demonstrate that the cosine similarity metric and a redundancy measure based on a mixture of language models are both effective for identifying redundant documents.

Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by in vitro fertilization (IVF) but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells and its removal by ectopically expressed H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency.

The identification of stem-cell-like cancer cells through conventional methods that depend on stem cell markers is often unreliable. We developed a mechanical method for selecting tumorigenic cells by culturing single cancer cells in fibrin matrices of ~100 Pa in stiffness. When cultured within these gels, primary human cancer cells or single cancer cells from mouse or human cancer cell lines grew within a few days into individual round colonies that resembled embryonic stem cell colonies. Subcutaneous or intravenous injection of 10 or 100 fibrin-cultured cells in syngeneic or severe combined immunodeficiency mice led to the formation of solid tumours at the site of injection or at the distant lung organ much more efficiently than control cancer cells selected using conventional surface marker methods or cultured on conventional rigid dishes or on soft gels. Remarkably, as few as ten such cells were able to survive and form tumours in the lungs of wild-type non-syngeneic mice. Conventional methods for the selection of tumorigenic cells from cancer cell lines rely on stem-cell markers. It is now shown that soft fibrin gels promote the growth of colonies of tumorigenic cells from single cancer cells from mouse or human cancer cell lines, and that as few as ten fibrin-cultured cells can lead to the formation of tumours in mice more efficiently than marker-selected cells.

MicroRNAs are well known to mediate translational repression and mRNA degradation in the cytoplasm. Various microRNAs have also been detected in membrane-compartmentalized organelles, but the functional significance has remained elusive. Here, we report that miR-1, a microRNA specifically induced during myogenesis, efficiently enters the mitochondria where it unexpectedly stimulates, rather than represses, the translation of specific mitochondrial genome-encoded transcripts. We show that this positive effect requires specific miR:mRNA base-pairing and Ago2, but not its functional partner GW182, which is excluded from the mitochondria. We provide evidence for the direct action of Ago2 in mitochondrial translation by crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq), functional rescue with mitochondria-targeted Ago2, and selective inhibition of the microRNA machinery in the cytoplasm. These findings unveil a positive function of microRNA in mitochondrial translation and suggest a highly coordinated myogenic program via miR-1-mediated translational stimulation in the mitochondria and repression in the cytoplasm.

Violet electroluminescence is rare in both inorganic and organic light-emitting diodes (LEDs). Low-cost and room-temperature solution-processed lead halide perovskites with high-efficiency and color-tunable photoluminescence are promising for LEDs. Here, we report room-temperature color-pure violet LEDs based on a two-dimensional lead halide perovskite material, namely, 2-phenylethylammonium (C6H5CH2CH2NH3(+), PEA) lead bromide [(PEA)2PbBr4]. The natural quantum confinement of two-dimensional layered perovskite (PEA)2PbBr4 allows for photoluminescence of shorter wavelength (410 nm) than its three-dimensional counterpart. By converting as-deposited polycrystalline thin films to micrometer-sized (PEA)2PbBr4 nanoplates using solvent vapor annealing, we successfully integrated this layered perovskite material into LEDs and achieved efficient room-temperature violet electroluminescence at 410 nm with a narrow bandwidth. This conversion to nanoplates significantly enhanced the crystallinity and photophysical properties of the (PEA)2PbBr4 samples and the external quantum efficiency of the violet LED. The solvent vapor annealing method reported herein can be generally applied to other perovskite materials to increase their grain size and, ultimately, improve the performance of optoelectronic devices based on perovskite materials.

Abstract Non–small-cell lung cancers (NSCLC) compose 80% of all lung carcinomas with squamous cell carcinomas (SCC) and adenocarcinoma representing the majority of these tumors. Although patients with early-stage NSCLC typically have a better outcome, 35% to 50% will relapse within 5 years after surgical treatment. We have profiled primary squamous cell lung carcinomas from 129 patients using Affymetrix U133A gene chips. Unsupervised analysis revealed two clusters of SCC that had no correlation with tumor stage but had significantly different overall patient survival (P = 0.036). The high-risk cluster was most significantly associated with down-regulation of epidermal development genes. Cox proportional hazard models identified an optimal set of 50 prognostic mRNA transcripts using a 5-fold cross-validation procedure. Quantitative reverse transcription-PCR and immunohistochemistry using tissue microarrays were used to validate individual gene candidates. This signature was tested in an independent set of 36 SCC samples and achieved 84% specificity and 41% sensitivity with an overall predictive accuracy of 68%. Kaplan-Meier analysis showed clear stratification of high-risk and low-risk patients [log-rank P = 0.04; hazard ratio (HR), 2.66; 95% confidence interval (95% CI), 1.01-7.05]. Finally, we combined the SCC classifier with our previously identified adenocarcinoma prognostic signature and showed that the combined classifier had a predictive accuracy of 71% in 72 NSCLC samples also showing significant differences in overall survival (log-rank P = 0.0002; HR, 3.54; 95% CI, 1.74-7.19). This prognostic signature could be used to identify patients with early-stage high-risk NSCLC who might benefit from adjuvant therapy following surgery. (Cancer Res 2006; 66(15): 7466-72)

Development of immature T-cell precursors (thymocytes) to either the CD4 helper or CD8 killer T-cell lineages correlates precisely with their T-cell receptor specificity for major histocompatibility complex class II or class I molecules, respectively, indicating that the process is carefully regulated. Although intensively studied owing to its importance in determining the composition of the mature T-cell compartment and as a general model of binary lineage decisions, the underlying molecular pathways remain obscure. We have previously reported a spontaneous mouse mutant (HD (helper deficient) mice) in which lineage commitment is specifically perturbed without affecting positive selection. Here we show that a point mutation in the zinc finger transcription factor Th-POK (T-helper-inducing POZ/Krüppel-like factor) is responsible for redirection of class-II-restricted thymocytes to the CD8 lineage in HD mice. Furthermore, we demonstrate that constitutive expression of this factor during thymic development leads to redirection of class-I-restricted thymocytes to the CD4 lineage, indicating that Th-POK is a master regulator of lineage commitment.

Topological defects, such as domain walls and vortices, have long fascinated physicists. A novel twist is added in quantum systems such as the B-phase of superfluid helium He3, where vortices are associated with low-energy excitations in the cores. Similarly, cosmic strings may be tied to propagating fermion modes. Can analogous phenomena occur in crystalline solids that host a plethora of topological defects? Here, we show that indeed dislocation lines are associated with one-dimensional fermionic excitations in a ‘topological insulator’, a novel phase of matter believed to be realized in the material Bi0.9Sb0.1. In contrast to fermionic excitations in a regular quantum wire, these modes are topologically protected and not scattered by disorder. As dislocations are ubiquitous in real materials, these excitations could dominate spin and charge transport in topological insulators. Our results provide a novel route to creating a potentially ideal quantum wire in a bulk solid. Topological insulators are band insulators in which spin–orbit coupling takes the role of the applied magnetic field in the integer quantum Hall effect. Theory now predicts that dislocations in such systems can give rise to one-dimensional topologically protected states, resembling helical modes at the edge of a two-dimensional quantum spin Hall insulator.

Multiple genetic loci associated with obesity or body mass index (BMI) have been identified through genome-wide association studies conducted predominantly in populations of European ancestry. We performed a meta-analysis of associations between BMI and approximately 2.4 million SNPs in 27,715 east Asians, which was followed by in silico and de novo replication studies in 37,691 and 17,642 additional east Asians, respectively. We identified ten BMI-associated loci at genome-wide significance (P < 5.0 × 10(-8)), including seven previously identified loci (FTO, SEC16B, MC4R, GIPR-QPCTL, ADCY3-DNAJC27, BDNF and MAP2K5) and three novel loci in or near the CDKAL1, PCSK1 and GP2 genes. Three additional loci nearly reached the genome-wide significance threshold, including two previously identified loci in the GNPDA2 and TFAP2B genes and a newly identified signal near PAX6, all of which were associated with BMI with P < 5.0 × 10(-7). Findings from this study may shed light on new pathways involved in obesity and demonstrate the value of conducting genetic studies in non-European populations.

We investigated the cellular basis for secretion of inflammatory cytokines in mice following intravenous administration of adenoviral vectors (Ad). Serum inflammatory cytokines including interleukin-6 (IL-6), IL-12, and tumor necrosis factor-alpha (TNF-alpha) were detected as early as 6 h following intravenous injection of Ad-expressing Escherichia coli beta-galactosidase (Ad-lacZ). Ad-lacZ readily accumulated in the splenic marginal zone 1 h after intravenous infusion, where both dendritic cells (DCs) and macrophages were transduced and activated within 6 h. Flow cytometric analyses showed that the expression of Ia and CD86 antigens was markedly enhanced on splenic DCs indicating their activation in vivo by Ad-lacZ. Upon ex vivo culture, these early-activated splenic DCs spontaneously produced high levels of IL-6 and IL-12. By contrast, activated splenic macrophages spontaneously secreted only IL-6. Elimination of tissue macrophages and splenic DCs in vivo considerably reduced the early release of IL-12, IL-6, and TNF-alpha and significantly blocked the specific cellular immune response to Ad and the transgene product in vivo. Our findings indicate that preferential activation of DCs and macrophages may account for Ad-triggered acute inflammatory response in vivo in mice. Moreover, DCs and macrophages may play different roles in this process in terms of their abilities to produce distinct patterns of inflammatory cytokines.

Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-beta-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd(2+)) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd(2+)-sensitive phenotype. Further analyses showed that Cd(2+) stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd(2+) tolerance.

Blood-based analytes may be indicators of pathological processes in Alzheimer disease (AD).To identify plasma proteins associated with AD pathology using a combined proteomic and neuroimaging approach.Discovery-phase proteomics to identify plasma proteins associated with correlates of AD pathology. Confirmation and validation using immunodetection in a replication set and an animal model.A multicenter European study (AddNeuroMed) and the Baltimore Longitudinal Study of Aging.Patients with AD, subjects with mild cognitive impairment, and healthy controls with standardized clinical assessments and structural neuroimaging.Association of plasma proteins with brain atrophy, disease severity, and rate of clinical progression. Extension studies in humans and transgenic mice tested the association between plasma proteins and brain amyloid.Clusterin/apolipoprotein J was associated with atrophy of the entorhinal cortex, baseline disease severity, and rapid clinical progression in AD. Increased plasma concentration of clusterin was predictive of greater fibrillar amyloid-beta burden in the medial temporal lobe. Subjects with AD had increased clusterin messenger RNA in blood, but there was no effect of single-nucleotide polymorphisms in the gene encoding clusterin with gene or protein expression. APP/PS1 transgenic mice showed increased plasma clusterin, age-dependent increase in brain clusterin, as well as amyloid and clusterin colocalization in plaques.These results demonstrate an important role of clusterin in the pathogenesis of AD and suggest that alterations in amyloid chaperone proteins may be a biologically relevant peripheral signature of AD.

A hydrometallurgical process for the recovery of cobalt oxalate from spent lithium-ion batteries (LIBs) has been developed using alkali leaching followed by reductive acid leaching, solvent extraction and chemical deposition of cobalt oxalate. The active cathode powder material was first leached with 5 wt.% NaOH solution for the selective removal of aluminum; and the residues were further leached with 4 M H2SO4 + 10% v/v H2O2 solution. The leaching efficiency of cobalt was 95% and lithium was 96% under optimum conditions of liquid/solid ratio 10:1, leaching time 120 min and a temperature 85 °C. The impurity ions of Fe(III), Cu(II), Mn(II) in the leach liquor were precipitated by adjusting the pH value. Cobalt(II) was then extracted selectively from the purified aqueous phase with saponified P507 (2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester) and chemically deposited as oxalate from the strip liquor with a yield of ~ 93% and purity > 99.9%. This process is simple, environmentally friendly and adequate for the recovery of valuable metals from spent LIBs.

Single-phase, insulating Bi1−xLaxFeO3 (BLFOx, x=0.05, 0.10, 0.15, 0.20, 0.30, and 0.40) ceramics were prepared. An obvious phase transition from rhombohedral to orthorhombic phase was observed near x=0.30. It is found that the phase transition destructs the spin cycloid of BiFeO3 (BFO), and therefore, releases the locked magnetization and enhances magnetoelectric interaction. As a result, improved multiferroic properties of the BLFO0.30 ceramics with remnant polarization and magnetization (2Pr and 2Mr) of 22.4μC∕cm2 and 0.041emu∕g, respectively, were established.

We previously identified in a single-center study a 76-gene prognostic signature for lymph node-negative (LNN) breast cancer patients. The aim of this study was to validate this gene signature in an independent more diverse population of LNN patients from multiple institutions.Using custom-designed DNA chips we analyzed the expression of the 76 genes in RNA of frozen tumor samples from 180 LNN patients who did not receive adjuvant systemic treatment.In this independent validation, the 76-gene signature was highly informative in identifying patients with distant metastasis within 5 years (hazard ratio, [HR], 7.41; 95% CI, 2.63 to 20.9), even when corrected for traditional prognostic factors in multivariate analysis (HR, 11.36; 95% CI, 2.67 to 48.4). The actuarial 5- and 10-year distant metastasis-free survival were 96% (95% CI, 89% to 99%) and 94% (95% CI, 83% to 98%), respectively, for the good profile group and 74% (95% CI, 64% to 81%) and 65% (53% to 74%), respectively for the poor profile group. The sensitivity for 5-yr distant metastasis-free survival was 90%, and the specificity was 50%. The positive and negative predictive values were 38% (95% CI, 29% to 47%) and 94% (95% CI, 86% to 97%), respectively. The 76-gene signature was confirmed as a strong prognostic factor in subgroups of estrogen receptor-positive patients, pre- and postmenopausal patients, and patients with tumor sizes 20 mm or smaller. The subgroup of patients with estrogen receptor-negative tumors was considered too small to perform a separate analysis.Our data provide a strong methodologic and clinical multicenter validation of the predefined prognostic 76-gene signature in LNN breast cancer patients.

The association between large tumor size and metastatic risk in a majority of clinical cancers has led to questions as to whether these observations are causally related or whether one is simply a marker for the other. This is partly due to an uncertainty about how metastasis-promoting gene expression changes can arise in primary tumors. We investigated this question through the analysis of a previously defined "lung metastasis gene-expression signature" (LMS) that mediates experimental breast cancer metastasis selectively to the lung and is expressed by primary human breast cancer with a high risk for developing lung metastasis. Experimentally, we demonstrate that the LMS promotes primary tumor growth that enriches for LMS(+) cells, and it allows for intravasation after reaching a critical tumor size. Clinically, this corresponds to LMS(+) tumors being larger at diagnosis compared with LMS(-) tumors and to a marked rise in the incidence of metastasis after LMS(+) tumors reach 2 cm. Patients with LMS-expressing primary tumors selectively fail in the lung compared with the bone or other visceral sites and have a worse overall survival. The mechanistic linkage between metastasis gene expression, accelerated tumor growth, and likelihood of metastatic recurrence provided by the LMS may help to explain observations of prognostic gene signatures in primary cancer and how tumor growth can both lead to metastasis and be a marker for cells destined to metastasize.

We report a comparative study and Raman characterization of the formation of graphene on single crystal Ni (111) and polycrystalline Ni substrates using chemical vapor deposition (CVD). Preferential formation of monolayer/bilayer graphene on the single crystal surface is attributed to its atomically smooth surface and the absence of grain boundaries. In contrast, CVD graphene formed on polycrystalline Ni leads to a higher percentage of multilayer graphene (≥3 layers), which is attributed to the presence of grain boundaries in Ni that can serve as nucleation sites for multilayer growth. Micro-Raman surface mapping reveals that the area percentages of monolayer/bilayer graphene are 91.4% for the Ni (111) substrate and 72.8% for the polycrystalline Ni substrate under comparable CVD conditions. The use of single crystal substrates for graphene growth may open ways for uniform high-quality graphene over large areas.

Functionalized carbon nanotubes display unique properties that enable a variety of medicinal applications, including the diagnosis and treatment of cancer, infectious diseases and central nervous system disorders, and applications in tissue engineering. These potential applications are particularly encouraged by their ability to penetrate biological membranes and relatively low toxicity.

Inverted Generational Distance (IGD) has been widely considered as a reliable performance indicator to concurrently quantify the convergence and diversity of multi- and many-objective evolutionary algorithms. In this paper, an IGD indicator-based evolutionary algorithm for solving many-objective optimization problems (MaOPs) has been proposed. Specifically, the IGD indicator is employed in each generation to select the solutions with favorable convergence and diversity. In addition, a computationally efficient dominance comparison method is designed to assign the rank values of solutions along with three newly proposed proximity distance assignments. Based on these two designs, the solutions are selected from a global view by linear assignment mechanism to concern the convergence and diversity simultaneously. In order to facilitate the accuracy of the sampled reference points for the calculation of IGD indicator, we also propose an efficient decomposition-based nadir point estimation method for constructing the Utopian Pareto front which is regarded as the best approximate Pareto front for real-world MaOPs at the early stage of the evolution. To evaluate the performance, a series of experiments is performed on the proposed algorithm against a group of selected state-of-the-art many-objective optimization algorithms over optimization problems with $8$-, $15$-, and $20$-objective. Experimental results measured by the chosen performance metrics indicate that the proposed algorithm is very competitive in addressing MaOPs.

Biomarkers to improve the risk-benefit of extended adjuvant endocrine therapy for late recurrence in patients with oestrogen-receptor-positive breast cancer would be clinically valuable. We compared the prognostic ability of the breast-cancer index (BCI) assay, 21-gene recurrence score (Oncotype DX), and an immunohistochemical prognostic model (IHC4) for both early and late recurrence in patients with oestrogen-receptor-positive, node-negative (N0) disease who took part in the Arimidex, Tamoxifen, Alone or in Combination (ATAC) clinical trial.In this prospective comparison study, we obtained archival tumour blocks from the TransATAC tissue bank from all postmenopausal patients with oestrogen-receptor-positive breast cancer from whom the 21-gene recurrence score and IHC4 values had already been derived. We did BCI analysis in matched samples with sufficient residual RNA using two BCI models-cubic (BCI-C) and linear (BCI-L)-using previously validated cutoffs. We assessed prognostic ability of BCI for distant recurrence over 10 years (the primary endpoint) and compared it with that of the 21-gene recurrence score and IHC4. We also tested the ability of the assays to predict early (0-5 years) and late (5-10 years) distant recurrence. To assess the ability of the biomarkers to predict recurrence beyond standard clinicopathological variables, we calculated the change in the likelihood-ratio χ(2) (LR-Δχ(2)) from Cox proportional hazards models.Suitable tissue was available from 665 patients with oestrogen-receptor-positive, N0 breast cancer for BCI analysis. The primary analysis showed significant differences in risk of distant recurrence over 10 years in the categorical BCI-C risk groups (p<0·0001) with 6·8% (95% CI 4·4-10·0) of patients in the low-risk group, 17·3% (12·0-24·7) in the intermediate group, and 22·2% (15·3-31·5) in the high-risk group having distant recurrence. The secondary analysis showed that BCI-L was a much stronger predictor for overall (0-10 year) distant recurrence compared with BCI-C (interquartile HR 2·30 [95% CI 1·62-3·27]; LR-Δχ(2)=22·69; p<0·0001). When compared with BCI-L, the 21-gene recurrence score was less predictive (HR 1·48 [95% CI 1·22-1·78]; LR-Δχ(2)=13·68; p=0·0002) and IHC4 was similar (HR 1·69 [95% CI 1·51-2·56]; LR-Δχ(2)=22·83; p<0·0001). All further analyses were done with the BCI-L model. In a multivariable analysis, all assays had significant prognostic ability for early distant recurrence (BCI-L HR 2·77 [95% CI 1·63-4·70], LR-Δχ(2)=15·42, p<0·0001; 21-gene recurrence score HR 1·80 [1·42-2·29], LR-Δχ(2)=18·48, p<0·0001; IHC4 HR 2·90 [2·01-4·18], LR-Δχ(2)=29·14, p<0·0001); however, only BCI-L was significant for late distant recurrence (BCI-L HR 1·95 [95% CI 1·22-3·14], LR-Δχ(2)=7·97, p=0·0048; 21-gene recurrence score HR 1·13 [0·82-1·56], LR-Δχ(2)=0·48, p=0·47; IHC4 HR 1·30 [0·88-1·94], LR-Δχ(2)=1·59, p=0·20).BCI-L was the only significant prognostic test for risk of both early and late distant recurrence and identified two risk populations for each timeframe. It could help to identify patients at high risk for late distant recurrence who might benefit from extended endocrine or other therapy.Avon Foundation, National Institutes of Health, Breast Cancer Foundation, US Department of Defense Breast Cancer Research Program, Susan G Komen for the Cure, Breakthrough Breast Cancer through the Mary-Jean Mitchell Green Foundation, AstraZeneca, Cancer Research UK, and the National Institute for Health Research Biomedical Research Centre at the Royal Marsden (London, UK).