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Electrolytic aluminum operation accidents have caused great losses to countries and people. Therefore, taking two typical accidents of explosion and leakage in electrolytic aluminum production as the research object, the form of accident risk characterization was explored and a risk characterization visualization system was designed. Based on the methods of risk assessment and characterization, the likelihood and consequence of accidents were studied. The influencing factors of likelihood and a type of Euclidean distance formula were used to characterize the likelihood. The consequence portion was characterized by two components, with one being the failure degree determined by the accident and the other being the exposure degree of people and equipment. The intensity of event was used to characterize the failure degree and the spatial search model was used to characterize the exposure degree. Finally, the visual system of electrolytic aluminum operation accidents was established based on the risk characterization model.

Survivin protein determination in multiple neuroblastoma lines

Purpose: mTORC1 inhibitors are promising agents for neuroblastoma therapy; however, they have shown limited clinical activity as monotherapy, thus rational drug combinations need to be explored to improve efficacy. Importantly, neuroblastoma maintains both an active p53 and an aberrant mTOR signaling.Experimental Design: Using an orthotopic xenograft model and modulating p53 levels, we investigated the antitumor effects of the mTORC1 inhibitor temsirolimus in neuroblastoma expressing normal, decreased, or mutant p53, both as single agent and in combination with first- and second-generation MDM2 inhibitors to reactivate p53.Results: Nongenotoxic p53 activation suppresses mTOR activity. Moreover, p53 reactivation via RG7388, a second-generation MDM2 inhibitor, strongly enhances the in vivo antitumor activity of temsirolimus. Single-agent temsirolimus does not elicit apoptosis, and tumors rapidly regrow after treatment suspension. In contrast, our combination therapy triggers a potent apoptotic response in wild-type p53 xenografts and efficiently blocks tumor regrowth after treatment completion. We also found that this combination uniquely led to p53-dependent suppression of survivin whose ectopic expression is sufficient to rescue the apoptosis induced by our combination.Conclusions: Our study supports a novel highly effective strategy that combines RG7388 and temsirolimus in wild-type p53 neuroblastoma, which warrants testing in early-phase clinical trials. Clin Cancer Res; 23(21); 6629–39. ©2017 AACR.

Effect of combined RG7388 and temsirolimus on human PBMCs viability and apoptosis

Supplementary Material and Figures "clean"

Temsirolimus blocks in vivo tumor growth by suppressing mTOR activity.

The combination therapy temsirolimus and RG7388 does not affect the expression of other apoptotic/anti-apoptotic proteins

Effect of combined RG7388 and temsirolimus on viability of human PBMCs subpopulations

Effect of combined RG7388 and temsirolimus on colony-forming unit generation of CD34+ Hematopoietic Stem-Progenitor Cells

Effect of combined treatment on cell viability

p53 knockdown in neuroblastoma cells

Bioluminescence data of xenograft tumors upon treatment suspension

Integrating hydrogen evolution reaction (HER) with hydrazine oxidation reaction (HzOR) can construct novel electrolytic system and thus accomplish the energy-efficient H2 production, which necessitates exploring excellent bifunctional electrocatalysts for HER...

The electrolytic aluminum operation accidents have caused great losses to the country and people. Therefore, taking two typical accidents of explosion and leakage in electrolytic aluminum production as the research object, the form of accident risk characterization was explored and a risk characterization visualization system was designed. Based on the methods of risk assessment and characterization, the likelihood and the consequence of the accident were studied. The influencing factors of likelihood and a kind of Euclidean distance formula were used to characterize the likelihood. The consequence portion was characterized by two components, with one being the failure degree determined by the accident and the other being the exposure degree of people and equipment. The intensity of event was used to characterize the failure degree and the spatial search model was used to characterize the exposure degree. Finally, the visual system of electrolytic aluminum operation accidents was established that based on the risk characterization model.

β-Thalassemia pathology is due not only to loss of β-globin (HBB), but also to erythrotoxic accumulation and aggregation of the β-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.

Workplace accidents constitute a major problem worldwide and pose a threat to human life, the social economy, and the ecological environment. This study proposes a method of risk characterization to assess the risk of accidents. First, the concepts of likelihood, event intensity, and event exposure are proposed based on the energy transfer theory. Second, the factors influencing these concepts are identified through a statistical analysis of cases of accidents and used to develop the proposed method of risk evaluation. Third, a combination of Dempster–Shafer (DS) evidence theory, the analytic hierarchy process, and the Euclidean distance method is used to develop the AHP-DS and ED-DS evidence theory models. Finally, experimental scenarios involving a storage facility for tailings and a section of converter steelmaking are used to verify the effectiveness of the proposed method.

Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the β-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16–19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.

Additional file 2: Figure S2. Functional enrichment of 39 unique aging-related genes using the Metascape database. (A) GO enrichment analysis; (B) KEGG enrichment analysis.

International audience; The recognition of substituted phosphates underpins many processes including DNA binding, enantioselective catalysis, and recently template-directed rotaxane synthesis. Beyond ATP and a few commercial substrates, however, little is known about how substituents effect organophosphate recognition. Here, we examined alcohol substituents and their impact on recognition by cyanostar macrocycles. The organophosphates were disubstituted by alcohols of various chain lengths, dipropanol, dihexanol, and didecanol phosphate, each accessed using modular solid-phases syntheses. Based on the known size-selective binding of phosphates by π-stacked dimers of cyanostars, threaded [3]pseudorotaxanes were anticipated. While seen with butyl substituents, pseudorotaxane formation was disrupted by competitive OH···O– hydrogen bonding between both terminal hydroxyls and the anionic phosphate unit. Crystallography also showed formation of a backfolded propanol conformation resulting in an 8-membered ring and a perched cyanostar assembly. Motivated by established entropic penalties accompanying ring formation, we reinstated [3]pseudorotaxanes by extending the size of the substituent to hexanol and decanol. Chain entropy overcomes the enthalpically favored OH···O– contacts to favor random-coil conformations required for seamless, high-fidelity threading of dihexanol and didecanol phosphates inside cyanostars. These studies highlight how chain length and functional groups on phosphate’s substituents can be powerful design tools to regulate binding and control assembly formation during phosphate recognition.

Additional file 1: Figure S1. The optimal cut-off value of the ARGPs risk-score obtained by the time-dependent ROC curve analysis.

Based on the actual industrial site and roadway engineering layout of the mine, this work constructs a high-precision 3D virtual simulation roaming scene of metal mine filling production, and stereoscopically reproduces the paste filling process so that students can establish a global understanding of the filling production environment, technological process, key steps and main equipment. This work breaks the limitations of the teaching method which mainly focuses on indoor experimental teaching and supplemented by industrial experimental demonstration, creates a new way to adapt to the characteristics of mining personnel training in the new era, creates a virtual reality, all-weather, online and offline integrated learning mode, platform and environment, and explores new methods, new directions and new ways for traditional industry teaching reformation.

The dynamic visualization system for risk characterization and accident evolution of molten metal operation takes steel smelting and electrolytic aluminum as scenes, taking the four typical accidents of explosion, spitting, tilting and leakage as the objects, 3Dmax+Substance Painter2 are used for modeling and mapping, and Unity3d+C# are used for development. The system consists of steel smelting and electrolytic aluminum subsystems, including the functional modules such as hazard & risk characterization, accident rehearsal, accident reconstruction, safety cognition, etc. It is expected to provide operators, managers and other relevant personnel with effective tools for accident evolution analysis, accident deduction analysis, and safety cognition, to reduce accidents.

Purpose An increasing number of long non-coding RNAs (lncRNAs) are thought to be associated with gastric cancer (GC). A lncRNA subclass that promotes enhancer function is called enhancer RNA (eRNA). We aimed to identify an eRNA that can predict GC prognosis and response to immune checkpoint inhibitors (ICIs).Methods Kaplan–Meier survival analysis was utilized to screen eRNA which can predict the prognosis of GC (P 0.4, P Results WAKMAR2 and its target gene TNFAIP3 entered the subsequent analysis. Patients with high-WAKMAR2 expression had a favorable prognosis compared to patients with low-WAKMAR2 expression (P = 0.048). Immune checkpoint-related genes (PD-L1, CTLA4, PDCD1, LAG3) in the WAKMAR2 high-expression group were also highly expressed, except for B7-H3. In addition, infiltration levels of B cells naive, T cells CD8, T cells CD4 memory activated, as well as Macrophages M1 in high-WAKMAR2 group were greater than in low-WAKMAR2 group. Last, the expression of WAKMAR2 in GC was significantly correlated with TMB and MSI.Conclusion WAKMAR2, a new eRNA, is a promising biomarker that can be used to predict the overall survival (OS) of GC patients, and WAKMAR2 expression can be utilized to identify ICB responders in GC, providing new insights for immunotherapy strategies.

β-thalassemia pathology is not only due to loss of β-globin (HBB), but also erythrotoxic accumulation and aggregation of the β-globin binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin mRNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in mice, establishing proof-of-concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.

With the continuous development and improvement of power grid technology, intelligence has become the development goal of power grid technology. By designing an intelligent substation inspection system, simplifies the operational tasks of inspectors, and makes inspection of power equipment more accurate, efficient, and traceable. In the implementation of the system, the mobile terminal is responsible for the collection, input, and upload of inspection data, and the server completes the data reception and statistics and displays it visually. And the application of the case proves that the intelligent patrol system is convenient and quick to operate, the social and economic benefits produced are significant, and it has strong practicability.

In order to evaluate risk and prevent accidents, a visualization system of accidents and risk in molten metal operations is proposed in this paper. Based on risk identification and analysis, the system characterizes the evolution process of hazards, and realizes dynamic visualization and risk evaluation of accidents through fluid simulation and three-dimensional (3D) visualization technology. And on the basis of many times of simulation and attempts, risk decisions that are technically feasible, economically reasonable and socially acceptable can be obtained. The system consists of four functional subsystems: production scene roaming subsystem, hazards evolution characterization subsystem, accident dynamic visualization subsystem, risk evaluation and decision-making subsystem. The system is applied to the production safety management of a steel plant. The results show that it can realize accident preview, playback and risk evaluation, and make appropriate risk decisions based on different acceptable levels of risk. It provides new ideas and methods for production safety management.

Induction of red blood cell (RBC) fetal hemoglobin (HbF; α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by reducing the concentration of sickle hemoglobin (HbS; α2β(S)2) to inhibit its polymerization. Hydroxyurea (HU), the only US Food and Drug Administration (FDA)-approved drug for SCD, acts in part by inducing HbF; however, it is not fully effective, reflecting the need for new therapies. Whole-exome sequence analysis of rare genetic variants in SCD patients identified FOXO3 as a candidate regulator of RBC HbF. We validated these genomic findings through loss- and gain-of-function studies in normal human CD34(+) hematopoietic stem and progenitor cells induced to undergo erythroid differentiation. FOXO3 gene silencing reduced γ-globin RNA levels and HbF levels in erythroblasts, whereas overexpression of FOXO3 produced the opposite effect. Moreover, treatment of primary CD34(+) cell–derived erythroid cultures with metformin, an FDA-approved drug known to enhance FOXO3 activity in nonerythroid cells, caused dose-related FOXO3-dependent increases in the percentage of HbF protein and the fraction of HbF-immunostaining cells (F cells). Combined HU and metformin treatment induced HbF additively and reversed the arrest in erythroid maturation caused by HU treatment alone. HbF induction by metformin in erythroid precursors was dependent on FOXO3 expression and did not alter expression of BCL11A, MYB, or KLF1. Collectively, our data implicate FOXO3 as a positive regulator of γ-globin expression and identify metformin as a potential therapeutic agent for SCD.

Introduction: Several gene editing strategies have been developed to cure sickle cell disease (SCD), including the use of CRISPR/Cas9 to edit beta-globin (HBB), gamma-globin (HBG), or B-cell lymphoma/leukemia 11A (BCL11A) in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Although high gene-editing rates can be achieved and off-target effects reduced, new challenges in applying the gene-editing strategies, including unintended gene modifications, need to be addressed in order to cure SCD with high efficacy and safety. To date, due to limitations in sequencing methods, studies on CRISPR/Cas9 genome editing for treating SCD only identified small insertions/deletions (INDELs); the extent and consequences of unintended large gene modifications are generally unknown. Here we provide accurate quantification and profiling of unintended gene modifications due to Cas9 induced double-stranded breaks (DSBs) in SCD HSPCs, including large deletions, insertions, and complex chromosomal arrangements, and the comparison of different approaches. Methods: R-66S gRNA targets the sickle mutation on the HBB. R-02 gRNA generates a DSB 16 bp away from the sickle mutation site. SD-02 gRNA introduces a 13-bp Hereditary Persistence of Fetal Hemoglobin (HPFH) deletion as a major INDEL in the HBG1/HBG2 promoter to reactivate fetal hemoglobin (HbF). BCL11A gRNA targets the GATA1 site at the BCL11A erythroid enhancer to induce HbF. R-66S, R-02, SD-02, and BCL11A gRNAs were respectively complexed with SpyCas9 and delivered as ribonucleoprotein (RNP) to SCD HSPCs. To accurately quantify CRISPR/Cas9 induced large modifications in gene-edited SCD HSPCs, we used PacBio Single Molecule, Real-Time (SMRT) Sequencing with Unique Molecular Identifiers (UMI). The 5-6 kb region around the Cas9 cut-site was dual-UMI tagged using two PCR cycles. The second and third PCR was performed with minimal cycle numbers to enrich the UMI-tagged template molecules. The SMRTbell library composed of edited and unedited SCD HSPCs samples was sequenced on a PacBio Sequel II 8M flowcell using the circular consensus sequencing (CCS) mode. The PacBio subreads were converted to HiFi reads and subjected to UMI consensus read generation and variant calling. Results: SMRT-seq with UMI revealed high rates and broad spectra of unintended large deletions (> 200 bp) induced by Cas9 cutting at HBB, HBG1, and BCL11A genes in RNP treated samples, with respectively R-66S RNP, 31.7%; R-02 RNP, 17.4%; SD-02 RNP, 13.3%; BCL11A RNP, 40%. The large deletions have a very broad distribution of sizes and locations. In addition, we found large insertions (> 50 bp) and local complex chromosomal rearrangements at the Cas9 cut-sites. Therefore, the current assessment of gene-editing rates using short-read Next Generation Sequencing (NGS) misses a substantial proportion of Cas9-cutting induced large gene modifications, resulting in an inaccurate measure of both allele and genotype frequencies. Discussions: We found that unintended on-target large deletions occur at high rates at HBB, HBG1, and BCL11A in gene-edited SCD HSPCs. These results raise significant safety concerns regarding gene-editing of HSPCs to treat SCD. Our results demonstrate the importance of detecting and quantifying all possible CRISPR/Cas9 gene-editing outcomes to ensure the efficient and safe translation of gene-editing-based strategies to cure SCD and other human diseases. Additional work is required to determine the functional consequences of the unintended gene modifications and the persistence of the unintended large gene modifications at the on-target cut-sites. Disclosures Sheehan: Forma Therapeutics: Research Funding; Beam Therapeutics: Research Funding; Novartis: Research Funding.

Introduction: Sickle cell disease (SCD) is a red blood cell disorder caused by a single nucleotide mutation in the β-globin gene (HBB). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available cure, but is available to only a minority of patients and can be associated with high morbidity and mortality. CRISPR/Cas9 mediated genome editing may provide a permanent cure for SCD patients by correcting the sickle mutation in HBB in hematopoietic stem and progenitor cells (HSPCs). Previously, we achieved ~39% sickle mutation correction in SCD HSPCs by delivering S. pyogenes (Spy) Cas9/R-66S gRNA as ribonucleoprotein (RNP) and single-stranded oligodeoxynucleotides (ssODN) corrective donor template. S. aureus (Sau) Cas9 has potentially advantageous properties to improve therapeutic gene editing efficiency and safety, including smaller size allowing for efficient in vivo delivery and longer Protospacer Adjacent Motif (PAM) sequence for higher specificity. However, although in general, the cutting efficiency of SauCas9 is lower than SpyCas9, the differences in gene correction and other gene-editing outcomes between SpyCas9 and SauCas9 have not been well studied. Methods: With our R-66S gRNA sequence targeting the sickle mutation, the PAM sequence of SauCas9 (NGGRRT) is mutually permissive with that of SpyCas9 (NGG), allowing the same sequence to be targeted by both Cas9 nucleases. We delivered R-66S gRNA with SpyCas9 and SauCas9 respectively as RNP, along with corrective ssODN donor template into SCD HSPCs. We analyzed sickle mutation correction rate and small insertions and deletions (INDELs) profile by Next Generation Sequencing (NGS). Results/discussions: We found that although the INDEL rate of SpyCas9 is higher than SauCas9 at the same molar concentration of RNP, SauCas9 gave 43% sickle mutation correction, slightly higher than SpyCas9 (39%), demonstrating efficient homology-directed repair (HDR) mediated gene correction by SauCas9. To further investigate the potential for clinical translation, we will perform in-depth efficiency and safety characterization comparing SauCas9 and SpyCas9 mediated sickle mutation correction therapy in SCD HSPCs. Conclusion: In this work, we showed that, compared with the highly-optimized and widely-used SpyCas9, SauCas9 leads to a higher sickle mutation correction in SCD HSPCs, demonstrating the therapeutic potential of SauCas9 for treating SCD. We will further investigate the efficiency and safety of gene-edited therapy mediated by these two Cas9 orthologs, including in-depth characterization of off-target effects, chromosomal rearrangement and aberrations, and large genomic modifications. We will differentiate gene-corrected SCD HSPCs to study erythropoiesis and red cell phenotype, including normal hemoglobin production and reduced sickling under hypoxic conditions. Lastly, we will evaluate the engraftment efficiency of gene-edited cells in Nonirradiated NOD,B6.SCID Il2rγ -/- Kit (W41/W41) (NBSGW) mice that support the engraftment of human hematopoietic stem cells. Disclosures Sheehan: Forma Therapeutics: Research Funding; Beam Therapeutics: Research Funding; Novartis: Research Funding.