Precise genome editing of the Kozak sequence enables bidirectional and quantitative modulation of protein translation to anticipated levels without affecting transcription.

None of the existing approaches for regulating gene expression can bidirectionally and quantitatively fine-tune gene expression to desired levels. Here, on the basis of precise manipulations of the Kozak sequence, which has a remarkable influence on translation initiation, we proposed and validated a novel strategy to directly modify the upstream nucleotides of the translation initiation codon of a given gene to flexibly alter the gene translation level by using base editors and prime editors. When the three nucleotides upstream of the translation initiation codon (named KZ3, part of the Kozak sequence), which exhibits the most significant base preference of the Kozak sequence, were selected as the editing region to alter the translation levels of proteins, we confirmed that each of the 64 KZ3 variants had a different translation efficiency, but all had similar transcription levels. Using the ranked KZ3 variants with different translation efficiencies as predictors, base editor- and prime editor-mediated mutations of KZ3 in the local genome could bidirectionally and quantitatively fine-tune gene translation to the anticipated levels without affecting transcription in vitro and in vivo. Notably, this strategy can be extended to the whole Kozak sequence and applied to all protein-coding genes in all eukaryotes.

AGBE: a dual deaminase-mediated base editor by fusing CGBE with ABE for creating a saturated mutant population with multiple editing patterns.

Establishing saturated mutagenesis in a specific gene through gene editing is an efficient approach for identifying the relationships between mutations and the corresponding phenotypes. CRISPR/Cas9-based sgRNA library screening often creates indel mutations with multiple nucleotides. Single base editors and dual deaminase-mediated base editors can achieve only one and two types of base substitutions, respectively. A new glycosylase base editor (CGBE) system, in which the uracil glycosylase inhibitor (UGI) is replaced with uracil-DNA glycosylase (UNG), was recently reported to efficiently induce multiple base conversions, including C-to-G, C-to-T and C-to-A. In this study, we fused a CGBE with ABE to develop a new type of dual deaminase-mediated base editing system, the AGBE system, that can simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels with a single sgRNA in mammalian cells. AGBEs can be used to establish saturated mutant populations for verification of the functions and consequences of multiple gene mutation patterns, including single-nucleotide variants (SNVs) and indels, through high-throughput screening.

Engineered Pigs Carrying a Gain-of-Function NLRP3 Homozygous Mutation Can Survive to Adulthood and Accurately Recapitulate Human Systemic Spontaneous Inflammatory Responses.

The NLRP3 inflammasome is associated with a variety of human diseases, including cryopyrin-associated periodic syndrome (CAPS). CAPS is a dominantly inherited disease with NLRP3 missense mutations. Currently, most studies on the NLRP3-inflammasome have been performed with mice, but the activation patterns and the signaling pathways of the mouse NLRP3 inflammasome are not always identical with those in humans. The NLRP3 inflammasome activation in pigs is similar to that in humans. Therefore, pigs with precise NLRP3-point mutations may model human CAPS more accurately. In this study, an NLRP3 gain-of-function pig model carrying a homozygous R259W mutation was generated by combining CRISPR/Cpf1-mediated somatic cell genome editing with nuclear transfer. The newborn NLRP3 R259W homozygous piglets showed early mortality, poor growth, and spontaneous systemic inflammation symptoms, including skin lesion, joint inflammation, severe contracture, and inflammation-mediated multiorgan failure. Severe myocardial fibrosis was also observed. The tissues of inflamed skins and several organs showed significantly increased expressions of NLRP3, Caspase-1, and inflammation-associated cytokines and factors (i.e., IL-1ß, TNF-α, IL-6, and IL-17). Notably, approximately half of the homozygous piglets grew up to adulthood and even gave birth to offspring. Although the F1 heterozygous piglets showed improved survival rate and normal weight gain, 39.1% (nine out of 23) of the piglets died early and exhibited spontaneous systemic inflammation symptoms. In addition, similar to homozygotes, adult heterozygotes showed increased delayed hypersensitivity response. Thus, the NLRP3 R259W pigs are similar to human CAPS and can serve as an ideal animal model to bridge the gap between rodents and humans.

CRISPR/Cas9-Mediated Gene Correction in Newborn Rabbits with Hereditary Tyrosinemia Type I.

Patients with hereditary tyrosinemia type I (HT1) present acute and irreversible liver and kidney damage during infancy. CRISPR-Cas9-mediated gene correction during infancy may provide a promising approach to treat patients with HT1. However, all previous studies were performed on adult HT1 rodent models, which cannot authentically recapitulate some symptoms of human patients. The efficacy and safety should be verified in large animals to translate precise gene therapy to clinical practice. Here, we delivered CRISPR-Cas9 and donor templates via adeno-associated virus to newborn HT1 rabbits. The lethal phenotypes could be rescued, and notably, these HT1 rabbits reached adulthood normally without 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione administration and even gave birth to offspring. Adeno-associated virus (AAV)-treated HT1 rabbits displayed normal liver and kidney structures and functions. Homology-directed repair-mediated precise gene corrections and non-homologous end joining-mediated out-of-frame to in-frame corrections in the livers were observed with efficiencies of 0.90%-3.71% and 2.39%-6.35%, respectively, which appeared to be sufficient to recover liver function and decrease liver and kidney damage. This study provides useful large-animal preclinical data for rescuing hepatocyte-related monogenetic metabolic disorders with precise gene therapy.

The Role of Graphene Oxide in the Exothermic Mechanism of Al/CuO Nanocomposites.

Metastable intermixed composites (MICs) have received increasing attention in the field of energy materials in recent years due to their high energy and good combustion performance. The exploration of ways of improving their potential release of heat is still underway. In this study, Al-CuO/graphene oxide (GO) nanocomposites were prepared using a combination of the self-assembly and in-suit synthesis methods. The formulation and experimental conditions were also optimized to maximize the exothermic heat. The DSC analysis shows that the addition of the GO made a significant contribution to the exothermic effect of the nanothermite. Compared with the Al-CuO nanothermite, the exothermic heat of the Al-CuO/GO nanocomposites increase by 306.9-1166.3 J/g and the peak temperatures dropped by 7.9-26.4 °C with different GO content. The reaction mechanism of the nanocomposite was investigated using a DSC and thermal reaction kinetics analysis. It was found that, compared with typical thermite reactions, the addition of the GO changed the reaction pathway of the nanothermite. The reaction products included CuAlO2. Moreover, the combustion properties of nanocomposite were investigated. This work reveals the unique mechanism of GO in thermite reactions, which may promote the application of carbon materials in nanothermite.

Exploring the Interfacial Reaction of Nano Al/CuO Energetic Films through Thermal Analysis and Ab Initio Molecular Dynamics Simulation.

The effect of the interface layer on energy release in nanoenergetic composite films is important and challenging for the utilization of energy. Nano Al/CuO composite films with different modulation periods were prepared by magnetron sputtering and tested by differential scanning calorimetry. With the increase in the modulation period of the nano Al/CuO energetic composite films, the interface layer contained in the energetic composite film decreased meaningfully, increasing the total heat release meaningfully. Ab initio molecular dynamics (AIMD) simulation were carried out to study the preparation process changes and related properties of the nano Al/CuO energetic composite films under different configurations at 400 K. The results showed that the diffusion of oxygen atoms first occurred at the upper and lower interfaces of CuO and Al, forming AlOx and CuxAlyOz. The two-modulation-period structure changed more obviously than the one-modulation-period structure, and the reaction was faster. The propagation rate and reaction duration of the front end of the diffusion reaction fronts at the upper and lower interfaces were different. The Helmholtz free energy loss of the nano Al/CuO composite films with a two-modulation-period configuration was large, and the number of interfacial layers had a great influence on the Helmholtz free energy, which was consistent with the results of the thermal analysis. Current molecular dynamics studies may provide new insights into the nature and characteristics of fast thermite reactions in atomic detail.

Transitory restless arms syndrome in a patient with antipsychotics and antidepressants: a case report.

BACKGROUND: Restless arms syndrome (RAS) is characterized by uncomfortable aching or burning sensations in the arms. RAS is regarded as an upper limb variant of restless legs syndrome (RLS). The lack of specific diagnostic criteria makes it difficult to recognize the RAS. Therefore, RAS is usually neglected in clinical practice. Moreover, when a patient was diagnosed with RAS, the adjustment of medications was the first choice for doctors, which may make the patient's condition unstable. CASE PRESENTATION: A 33-year-old woman was diagnosed with schizophrenia and major depressive disorder. Starting with 0.6 g/d amisulpride, 0.1 g/d quetiapine, 75 mg/d venlafaxine sustained-release tablets, the patient reported symptoms of RAS (itching arms) on the fourth day since the latest hospitalization. After ruling out other factors, her RAS was suspected to be induced by antidepressants or antipsychotics. Without medication adjustment, RAS spontaneously remitted. CONCLUSIONS: This case suggests that psychiatrists should pay attention to RAS when using antipsychotics and/or antidepressants. Moreover, RAS may be transitory. When a patient manifests RAS, observation may be one choice instead of an immediate medication adjustment.

ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems.

BACKGROUND: Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site. RESULTS: In this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system. CONCLUSION: The newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

Probing the reaction mechanism of Al/CuO nanocomposites doped with ammonium perchlorate.

The oxide shell of Al nanoparticles (Al NPs) prevents further reaction of Al/CuO nanothermites which reduces Al utilization efficiency and the performance of the nanothermites. However, the performance of Al/CuO nanothermites can be improved by adding ammonium perchlorate (AP). In this work, in order to confirm and explain the enhancement mechanism of AP on Al/CuO nanothermites, Al/CuO/NC and Al/CuO/NC/AP composites were prepared using the electrospray method. The composites were characterized by differential scanning calorimetry/thermogravimetric, x-ray diffraction, scanning electron microscope and transmission electron microscopy. Meanwhile, the ignition temperature and the time-resolved analysis of the rapid pyrolysis chemistry of the composites were tested using T-jump and time-of-flight mass spectrometry, respectively. The results show that Al NPs of Al/CuO/NC/AP composite are hollow compared to Al/CuO/NC composite after reaction. Al NPs and CuO NPs reduce the decomposition temperature and facilitate the rapid decomposition of the AP, and the decomposition products of the AP can destroy the oxidation layer of Al NPs. This result facilitates the further conduct of the thermite reaction. A mutually reinforcing relationship exists between the Al/CuO/NC composites and AP.

From nanoparticles to on-chip 3D nanothermite: electrospray deposition of reactive Al/CuO@NC onto semiconductor bridge and its application for rapid ignition.

Nanothermites composed of nano-fuels and oxidants are attractive energetic materials, which have potential applications in microscale energy-demanding systems. Herein, nano-Al/CuO with nitrocellulose (NC) binder have been bottom-up assembled on semiconductor bridge (SCB) chip by electrospray, from nanoparticles to three-dimensional (3D) deposited structure. The morphological and compositional characterization confirms the constituents in Al/CuO@NC are homogeneously mixed at nano scale and the 3D structure at micro scale is tunable. The as-deposited Al/CuO@NC exhibits excellent energy output and superior chemical reactivity. Specifically, the heat release of Al/CuO@NC (1179.5 J g-1) is higher than that of random mixed Al/CuO (730.9 J g-1). Benefiting from outstanding exothermic properties, the material integrated with SCB initiator chip (Al/CuO@NC-SCB) for potential ignition application was investigated. The Al/CuO@NC-SCB micro energetic initiator can be functioned rapidly (with delay time of 2.8 µs) and exhibits superb ignition performances with violent explosion process, high combustion temperature (4636 °C) and successful ignition of B/KNO3 propellant, in comparison to SCB initiator. The strategy provides promising route to introduce nano reactive particles into various functional energy-demanding systems for potential energetic applications.

A Deep Learning Approach on Building Detection from Unmanned Aerial Vehicle-Based Images in Riverbank Monitoring.

Buildings along riverbanks are likely to be affected by rising water levels, therefore the acquisition of accurate building information has great importance not only for riverbank environmental protection but also for dealing with emergency cases like flooding. UAV-based photographs are flexible and cloud-free compared to satellite images and can provide very high-resolution images up to centimeter level, while there exist great challenges in quickly and accurately detecting and extracting building from UAV images because there are usually too many details and distortions on UAV images. In this paper, a deep learning (DL)-based approach is proposed for more accurately extracting building information, in which the network architecture, SegNet, is used in the semantic segmentation after the network training on a completely labeled UAV image dataset covering multi-dimension urban settlement appearances along a riverbank area in Chongqing. The experiment results show that an excellent performance has been obtained in the detection of buildings from untrained locations with an average overall accuracy more than 90%. To verify the generality and advantage of the proposed method, the procedure is further evaluated by training and testing with another two open standard datasets which have a variety of building patterns and styles, and the final overall accuracies of building extraction are more than 93% and 95%, respectively.

Examining relations between locus of control, loneliness, subjective well-being, and preference for online social interaction.

The unprecedented popularity of online communication has raised interests and concerns among the public as well as in scholarly circles. Online communications have pushed people farther away from one another. This study is a further examination of the effects of online communications on well-being, in particular: Locus of control, Loneliness, Subjective well-being, and Preference for online social interaction. Chinese undergraduate students (N = 260; 84 men, 176 women; M age = 20.1 yr., SD = 1.2) were questioned about demographic information and use of social media as well as four previously validated questionnaires related to well-being. Most participants used QQ, a popular social networking program, as the major channel for online social interactions. Locus of control was positively related to Loneliness and Preference for online social interaction, but negatively related to Subjective well-being; Loneliness (positively) and Subjective well-being (negatively) were related to Preference for online social interaction; and Loneliness and Subjective well-being had a full mediating effect between the relationships of Locus of control and Preference for online social interaction. The findings of the study showed that more lonely, unhappy, and externally controlled students were more likely to be engaged in online social interaction. Improving students' locus of control, loneliness, and happiness may help reduce problematic Internet use.

Structure of sorting nexin 11 (SNX11) reveals a novel extended phox homology (PX) domain critical for inhibition of SNX10-induced vacuolation.

Sorting nexins are phox homology (PX) domain-containing proteins involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols and is recruited to vesicles rich in these lipids. The structure of the PX domain is highly conserved, containing a three-stranded ß-sheet, followed by three α-helices. Here, we report the crystal structures of truncated human SNX11 (sorting nexin 11). The structures reveal that SNX11 contains a novel PX domain, hereby named the extended PX (PXe) domain, with two additional α-helices at the C terminus. We demonstrate that these α-helices are indispensible for the in vitro functions of SNX11. We propose that this PXe domain is present in SNX10 and is responsible for the vacuolation activity of SNX10. Thus, this novel PXe domain constitutes a structurally and functionally important PX domain subfamily.

Structure of human SNX10 reveals insights into its role in human autosomal recessive osteopetrosis.

Sorting nexin 10 (SNX10), the unique member of the SNX family having vacuolation activity in cells, was shown to be involved in the development of autosomal recessive osteopetrosis (ARO) in recent genetic studies. However, the molecular mechanism of the disease-related mutations affecting the biological function of SNX10 is unclear. Here, we report the crystal structure of human SNX10 to 2.6 Å resolution. The structure reveals that SNX10 contains the extended phox-homology domain we previously proposed. Our study provides the structural details of those disease-related mutations. Combined with the vacuolation study of those mutations, we found that Tyr32 and Arg51 are important for the protein stability and both play a critical role in vacuolation activity, while Arg16Leu may affect the function of SNX10 in osteoclast through protein-protein interactions.

Role of career decision-making self-efficacy and risk of career options on career decision-making of Chinese graduates.

This study investigated the influence of Chinese graduates' career decision-making self-efficacy (CDMSE) and the moderating effect of career options on career decisions. Graduates (92 men, 119 women, 5 unreported gender; M age = 223 yr., SD = 1.2) from four different types of universities in Zhejiang Province participated in the study. CDMSE was measured with the CDMSE Scale for University Students, and participants rated their choices on 3 career options with different levels of risk. The results showed that participants were more likely to choose a high-risk option, and that career options moderated the relation between graduates' CDMSE and career decision. Graduate career counseling programs should encourage students to develop more reasonable career goals that match their skills.

Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos.

Cas9 protein without sgRNAs can induce genomic damage at the cellular level in vitro. However, whether the detrimental effects occur in embryos after Cas9 treatment remains unknown. Here, using pig embryos as subjects, we observed that Cas9 protein transcribed from injected Cas9 mRNA can persist until at least the blastocyst stage. Cas9 protein alone can induce genome damage in preimplantation embryos, represented by the increased number of phosphorylated histone H2AX foci on the chromatin fiber, which led to apoptosis and decreased cell number of blastocysts. In addition, single-blastocyst RNA sequencing confirmed that Cas9 protein without sgRNAs can cause changes in the blastocyst transcriptome, depressing embryo development signal pathways, such as cell cycle, metabolism, and cellular communication-related signal pathways, while activating apoptosis and necroptosis signal pathways, which together resulted in impaired preimplantation embryonic development. These results indicated that attention should be given to the detrimental effects caused by the Cas9 protein when using CRISPR-Cas9 for germline genome editing, especially for the targeted correction of human pathological mutations using germline gene therapy.

Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation.

BACKGROUND: CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals. RESULTS: Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53-sgRNA, LKB1-sgRNA, and mutant human KRAS gene into the adult pancreases. CONCLUSIONS: Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.

An investigation into the fabrication and combustion performance of porous silicon nanoenergetic array chips.

An investigation into the ignitions and combustions of porous silicon (PS) nanoenergetic material array chips (nECs) at different ignition voltages was performed. The PS nECs were fabricated by integrating PS nanoenergetic material (nEMs) matrices and Cr-microbridges (microigniters) on the surface of silicon substrates. The combustion of PS nECs was in ambient air. Its ignition and combustion were investigated by a testing system and an optical high-speed camera. Experimental results revealed that the combustion delay time of PS nEMs increased from 8.0 × 10(-5) s to 1.1 × 10(-4) s with the decrement of ignition voltages from 140 to 80 V. The scope of ignition energy ranged from 0.153 to 0.287 mJ by calculations. The reaction type was deflagration, from the analysis of the high-speed video of PS nECs. The comprehensive experimental results indicated that the combustion of PS nECs was ignited by the synergic effect of the heat and the plasma. The ignition experiments suggested that Cr-microbridges were reliable igniters to trigger the self-sustained combustion of PS nECs. The strong plume of flame emitted from the surface of PS nECs indicated that the PS nECs may be applied as micro/nano igniter chips and microthruster chips.

Fear of Missing Out and Irrational Procrastination in the Mobile Social Media Environment: A Moderated Mediation Analysis.

This study explored the relationship between Fear of Missing Out (FoMO) and irrational procrastination in a mobile social media environment and its underlying mechanism: the mediating role of cognitive failure. The study was conducted with 817 college students using the FoMO Scale, Irrational Procrastination Scale, Cognitive Failures Questionnaire, and Self-Control Scale. The results showed that (a) FoMO positively predicted irrational procrastination in the mobile social media environment; (b) cognitive failure had a complete mediating effect on the relationship between FoMO and irrational procrastination; and (c) self-control had a moderating effect on the relationship between FoMO and cognitive failure.