High-throughput and genome-scale targeted mutagenesis using CRISPR in a nonmodel multicellular organism, Bombyx mori.

Large-scale genetic mutant libraries are powerful approaches to interrogating genotype-phenotype correlations and identifying genes responsible for certain environmental stimuli, both of which are the central goal of life science study. We produced the first large-scale CRISPR-Cas9-induced library in a nonmodel multicellular organism, Bombyx mori We developed a piggyBac-delivered binary genome editing strategy, which can simultaneously meet the requirements of mixed microinjection, efficient multipurpose genetic operation, and preservation of growth-defect lines. We constructed a single-guide RNA (sgRNA) plasmid library containing 92,917 sgRNAs targeting promoters and exons of 14,645 protein-coding genes, established 1726 transgenic sgRNA lines following microinjection of 66,650 embryos, and generated 300 mutant lines with diverse phenotypic changes. Phenomic characterization of mutant lines identified a large set of genes responsible for visual phenotypic or economically valuable trait changes. Next, we performed pooled context-specific positive screens for tolerance to environmental pollutant cadmium exposure, and identified KWMTBOMO12902 as a strong candidate gene for breeding applications in sericulture industry. Collectively, our results provide a novel and versatile approach for functional B. mori genomics, as well as a powerful resource for identifying the potential of key candidate genes for improving various economic traits. This study also shows the effectiveness, practicality, and convenience of large-scale mutant libraries in other nonmodel organisms.

Deciphering the tumor immune microenvironment from a multidimensional omics perspective: insight into next-generation CAR-T cell immunotherapy and beyond.

Tumor immune microenvironment (TIME) consists of intra-tumor immunological components and plays a significant role in tumor initiation, progression, metastasis, and response to therapy. Chimeric antigen receptor (CAR)-T cell immunotherapy has revolutionized the cancer treatment paradigm. Although CAR-T cell immunotherapy has emerged as a successful treatment for hematologic malignancies, it remains a conundrum for solid tumors. The heterogeneity of TIME is responsible for poor outcomes in CAR-T cell immunotherapy against solid tumors. The advancement of highly sophisticated technology enhances our exploration in TIME from a multi-omics perspective. In the era of machine learning, multi-omics studies could reveal the characteristics of TIME and its immune resistance mechanism. Therefore, the clinical efficacy of CAR-T cell immunotherapy in solid tumors could be further improved with strategies that target unfavorable conditions in TIME. Herein, this review seeks to investigate the factors influencing TIME formation and propose strategies for improving the effectiveness of CAR-T cell immunotherapy through a multi-omics perspective, with the ultimate goal of developing personalized therapeutic approaches.

High-Performance 2D Ambipolar MoTe2 Lateral Memristors by Mild Oxidation.

2D transition metal dichalcogenides (TMDCs) have been intensively explored in memristors for brain-inspired computing. Oxidation, which is usually unavoidable and harmful in 2D TMDCs, could also be used to enhance their memristive performances. However, it is still unclear how oxidation affects the resistive switching behaviors of 2D ambipolar TMDCs. In this work, a mild oxidation strategy is developed to greatly enhance the resistive switching ratio of ambipolar 2H-MoTe2 lateral memristors by more than 10 times. Such an enhancement results from the amplified doping due to O2 and H2O adsorption and the optimization of effective gate voltage distribution by mild oxidation. Moreover, the ambipolarity of 2H-MoTe2 also enables a change of resistive switching direction, which is uncommon in 2D memristors. Consequently, as an artificial synapse, the MoTe2 device exhibits a large dynamic range (≈200) and a good linearity (1.01) in long-term potentiation and depression, as well as a high-accuracy handwritten digit recognition (>96%). This work not only provides a feasible and effective way to enhance the memristive performance of 2D ambipolar materials, but also deepens the understanding of hidden mechanisms for RS behaviors in oxidized 2D materials.

Targeting HSP47 for cancer treatment.

Heat shock protein 47 (HSP47) serves as an endoplasmic reticulum residing collagen-specific chaperone and plays an important role in collagen biosynthesis and structural assembly. HSP47 is encoded by the SERPINH1 gene, which is located on chromosome 11q13.5, one of the most frequently amplified regions in human cancers. The expression of HSP47 is regulated by multiple cellular factors, including cytokines, transcription factors, microRNAs, and circular RNAs. HSP47 is frequently upregulated in a variety of cancers and plays an important role in tumor progression. HSP47 promotes tumor stemness, angiogenesis, growth, epithelial-mesenchymal transition, and metastatic capacity. HSP47 also regulates the efficacy of tumor therapies, such as chemotherapy, radiotherapy, and immunotherapy. Inhibition of HSP47 expression has antitumor effects, suggesting that targeting HSP47 is a feasible strategy for cancer treatment. In this review, we highlight the function and expression of regulatory mechanisms of HSP47 in cancer progression and point out the potential development of therapeutic strategies in targeting HSP47 in the future.

Highly Selective Acetylene-to-Ethylene Electroreduction Over Cd-Decorated Cu Catalyst with Efficiently Inhibited Carbon-Carbon Coupling.

Electrochemical acetylene reduction (EAR) employing Cu catalysts represents an environmentally friendly and cost-effective method for ethylene production and purification. However, Cu-based catalysts encounter product selectivity issues stemming from carbon-carbon coupling and other side reactions. We explored the use of secondary metals to modify Cu-based catalysts and identified Cd decoration as particular effective. Cd decoration demonstrated a high ethylene Faradaic efficiency (FE) of 98.38 % with well-inhibited carbon-carbon coupling reactions (0.06 % for butadiene FE at -0.5 V versus reversible hydrogen electrode) in a 5 vol % acetylene gas feed. Notably, ethylene selectivity of 99.99 % was achieved in the crude ethylene feed during prolonged stability tests. Theoretical calculations revealed that Cd metal accelerates the water dissociation on neighboring Cu surfaces allowing more H* to participate in the acetylene semi-hydrogenation, while increasing the energy barrier for carbon-carbon coupling, thereby contributing to a high ethylene semi-hydrogenation efficiency and significant inhibition of carbon-carbon coupling. This study provides a paradigm for a deeper understanding of secondary metals in regulating the product selectivity of EAR electrocatalysts.

Genome-wide CRISPR screening reveals genes essential for cell viability and resistance to abiotic and biotic stresses in Bombyx mori.

High-throughput genetic screens are powerful methods to interrogate gene function on a genome-wide scale and identify genes responsible to certain stresses. Here, we developed a piggyBac strategy to deliver pooled sgRNA libraries stably into cell lines. We used this strategy to conduct a screen based on genome-wide clustered regularly interspaced short palindromic repeat technology (CRISPR)-Cas9 in Bombyx mori cells. We first constructed a single guide RNA (sgRNA) library containing 94,000 sgRNAs, which targeted 16,571 protein-coding genes. We then generated knockout collections in BmE cells using the piggyBac transposon. We identified 1006 genes that are essential for cell viability under normal growth conditions. Of the identified genes, 82.4% (829 genes) were homologous to essential genes in seven animal species. We also identified 838 genes whose loss facilitated cell growth. Next, we performed context-specific positive screens for resistance to biotic or nonbiotic stresses using temperature and baculovirus separately, which identified several key genes and pathways from each screen. Collectively, our results provide a novel and versatile platform for functional annotations of B. mori genomes and deciphering key genes responsible for various conditions. This study also shows the effectiveness, practicality, and convenience of genome-wide CRISPR screens in nonmodel organisms.

Perforated Carbon Nanotube Film Assisted Growth of Uniform Monolayer MoS2.

Scaling up the chemical vapor deposition (CVD) of monolayer transition metal dichalcogenides (TMDCs) is in high demand for practical applications. However, for CVD-grown TMDCs on a large scale, there are many existing factors that result in their poor uniformity. In particular, gas flow, which usually leads to inhomogeneous distributions of precursor concentrations, has yet to be well controlled. In this work, the growth of uniform monolayer MoS2 on a large scale by the delicate control of gas flows of precursors, which is realized by vertically aligning a well-designed perforated carbon nanotube (p-CNT) film face-to-face with the substrate in a horizontal tube furnace, is achieved. The p-CNT film releases gaseous Mo precursor from the solid part and allows S vapor to pass through the hollow part, resulting in uniform distributions of both gas flow rate and precursor concentrations near the substrate. Simulation results further verify that the well-designed p-CNT film guarantees a steady gas flow and a uniform spatial distribution of precursors. Consequently, the as-grown monolayer MoS2 shows quite good uniformity in geometry, density, structure, and electrical properties. This work provides a universal pathway for the synthesis of large-scale uniform monolayer TMDCs, and will advance their applications in high-performance electronic devices.

Neutral-Coating Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Top-Down Identification of Hemoglobin Variants.

BACKGROUND: Identification of hemoglobin (Hb) variants is of significant value in the clinical diagnosis of hemoglobinopathy. However, conventional methods for identification of Hb variants in clinical laboratories can be inadequate due to the lack of structural characterization. We describe the use of neutral-coating capillary electrophoresis coupled with high-resolution mass spectrometry (CE-HR-MS) to achieve high-performance top-down identification of Hb variants. METHODS: An Orbitrap Q-Exactive Plus mass spectrometer was coupled with an ECE-001 capillary electrophoresis (CE) unit through an EMASS-II ion source. A PS1 neutral-coating capillary was used for CE. Samples of red blood cells were lysed in water and diluted in 10 mM ammonium formate buffer for analysis. Deconvolution of raw mass spectrometry data was carried out to merge multiple charge states and isotopic peaks of an analyte to obtain its monoisotopic mass. RESULTS: The neutral-coating CE could baseline separate individual Hb subunits dissociated from intact Hb forms, and the HR-MS could achieve both intact-protein analysis and top-down analysis of analytes. A number of patient samples that contain Hb subunit variants were analyzed, and the variants were successfully identified using the CE-HR-MS method. CONCLUSIONS: The CE-HR-MS method has been demonstrated as a useful tool for top-down identification of Hb variants. With the ability to characterize the primary structures of Hb subunits, the CE-HR-MS method has significant advantages to complement or partially replace the conventional methods for the identification of Hb variants.

Peripheral blood lymphocytes differentiation patterns in responses / outcomes to immune checkpoint blockade therapies in non-small cell lung cancer: a retrospective study.

OBJECTIVES: Programmed Cell Death-1/ Programmed Death-ligand 1 (PD-1 / PD-L1) inhibitor therapies targeting immunocytes induce persistent tumor remission in various cancers. However, the appropriate biomarkers for the therapeutic efficacy of PD-L1 and PD-1 blockade remain elusive. MATERIALS AND METHODS: For a comprehensive analysis of peri-treatment lymphocyte differentiation, in the current study, we enrolled 146 non-small cell lung cancer patients who received α-PD-1 therapies for exploring the peripheral blood lymphocyte differentiation pattern at baseline and post-treatment (dynamic changes) by flow cytometry. RESULTS: At baseline, CD4+ / CD8+ T cell ratio predicts good responses and outcomes, but activated T cell and cytotoxic T cell counts predict poor responses and outcomes. And for dynamic changes, after 6 weeks of immune checkpoint blockade (ICB) treatment, compared with baseline level, the elevation of total T and B cell counts indicate poor responses, and total T and TH cell counts indicate poor prognosis while activated T cell predicts good prognosis. And after 12 weeks, elevated total lymphocyte, cytotoxic T cell counts, and decreased total T cell counts and CD4+ / CD8+ T cell ratio predict good responses / outcomes. Our clinical predicting model shows good performance in predicting ICB treatment responses / outcomes. CONCLUSION: Patients with favorable clinical responses / outcomes have distinctive peripheral blood immunocyte differentiation characteristics, indicating the potential of utilizing the peripheral immunocyte differentiation patterns for predicting ICB responses / outcomes.

Selective Photocatalytic Oxidative Coupling of Methane via Regulating Methyl Intermediates over Metal/ZnO Nanoparticles.

Methane conversion to higher hydrocarbons requires harsh reaction conditions due to high energy barriers associated with C-H bond activation. Herein, we report a systematic investigation of photocatalytic oxidative coupling of methane (OCM) over transition-metal-loaded ZnO photocatalysts. A 1 wt % Au/ZnO delivered a remarkable C2 -C4 hydrocarbon production rate of 683 µmol g-1 h-1 (83 % C2 -C4 selectivity) under light irradiation with excellent photostability over two days. The metal type and its interaction with ZnO strongly influence the selectivity toward C-C coupling products. Photogenerated Zn+ -O- sites enable CH4 activation to methyl intermediates (*CH3 ) migrating onto adjacent metal nanoparticles. The nature of the *CH3 -metal interaction controls the OCM products. In the case of Au, strong d-σ orbital hybridization reduces metal-C-H bond angles and steric hindrance, thereby enabling efficient methyl coupling. Findings indicate the d-σ center may be a suitable descriptor for predicting product selectivity during OCM over metal/ZnO photocatalysts.

Plasmonic Cu Nanoparticles for the Low-temperature Photo-driven Water-gas Shift Reaction.

The activation of water molecules in thermal catalysis typically requires high temperatures, representing an obstacle to catalyst development for the low-temperature water-gas shift reaction (WGSR). Plasmonic photocatalysis allows activation of water at low temperatures through the generation of light-induced hot electrons. Herein, we report a layered double hydroxide-derived copper catalyst (LD-Cu) with outstanding performance for the low-temperature photo-driven WGSR. LD-Cu offered a lower activation energy for WGSR to H2 under UV/Vis irradiation (1.4 W cm-2 ) compared to under dark conditions. Detailed experimental studies revealed that highly dispersed Cu nanoparticles created an abundance of hot electrons during light absorption, which promoted *H2 O dissociation and *H combination via a carboxyl pathway, leading to the efficient production of H2 . Results demonstrate the benefits of exploiting plasmonic phenomena in the development of photo-driven low-temperature WGSR catalysts.

Liquid Precursor-Guided Phase Engineering of Single-Crystal VO2 Beams.

The study of VO2 flourishes due to its rich competing phases induced by slight stoichiometry variations. However, the vague mechanism of stoichiometry manipulation makes the precise phase engineering of VO2 still challenging. Here, stoichiometry manipulation of single-crystal VO2 beams in liquid-assisted growth is systematically studied. Contrary to previous experience, oxygen-rich VO2 phases are abnormally synthesized under a reduced oxygen concentration, revealing the important function of liquid V2 O5 precursor: It submerges VO2 crystals and stabilizes their stoichiometric phase (M1) by isolating them from the reactive atmosphere, while the uncovered crystals are oxidized by the growth atmosphere. By varying the thickness of liquid V2 O5 precursor and thus the exposure time of VO2 to the atmosphere, various VO2 phases (M1, T, and M2) can be selectively stabilized. Furthermore, this liquid precursor-guided growth can be used to spatially manages multiphase structures in single VO2 beams, enriching their deformation modes for actuation applications.

Long-Term Accuracy of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Interferon-γ Release Assay and Its Application in Household Investigation.

BACKGROUND: An immunodiagnostic assay that sensitively detects a cell-mediated immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed for epidemiological investigation and for clinical assessment of T- cell-mediated immune response to vaccines, particularly in the context of emerging variants that might escape antibody responses. METHODS: The performance of a whole blood interferon-gamma (IFN-γ) release assay (IGRA) for the detection of SARS-CoV-2 antigen-specific T cells was evaluated in coronavirus disease 2019 (COVID-19) convalescents tested serially up to 10 months post-infection and in healthy blood donors. SARS-CoV-2 IGRA was applied in contacts of households with index cases. Freshly collected blood in the lithium heparin tube was left unstimulated, stimulated with a SARS-CoV-2 peptide pool, and stimulated with mitogen. RESULTS: The overall sensitivity and specificity of IGRA were 84.5% (153/181; 95% confidence interval [CI]: 79.0-89.0) and 86.6% (123/142; 95% CI: 80.0-91.2), respectively. The sensitivity declined from 100% (16/16; 95% CI: 80.6-100) at 0.5-month post-infection to 79.5% (31/39; 95% CI: 64.4-89.2) at 10 months post-infection (P < .01). The IFN-γ response remained relatively robust at 10 months post-infection (3.8 vs 1.3 IU/mL, respectively). In 14 households, IGRA showed a positivity rate of 100% (12/12) and 65.2% (15/23), and IgG of 50.0% (6/12) and 43.5% (10/23) in index cases and contacts, respectively, exhibiting a difference of + 50% (95% CI: +25.4 to +74.6) and +21.7% (95% CI: +9.23 to +42.3), respectively. Either IGRA or IgG was positive in 100% (12/12) of index cases and 73.9% (17/23) of contacts. CONCLUSIONS: The SARS-CoV-2 IGRA is a useful clinical diagnostic tool for assessing cell-mediated immune response to SARS-CoV-2.

Targeting type I collagen for cancer treatment.

Collagen is the most abundant protein in animals. Interactions between tumor cells and collagen influence every step of tumor development. Type I collagen is the main fibrillar collagen in the extracellular matrix and is frequently upregulated during tumorigenesis. The binding of type I collagen to its receptors on tumor cells promotes tumor cell proliferation, epithelial-mesenchymal transition and metastasis. Type I collagen also regulates the efficacy of tumor therapies, such as chemotherapy, radiotherapy and immunotherapy. Furthermore, type I collagen fragments are diagnostic markers of metastatic tumors and have prognostic value. Inhibition of type I collagen synthesis has been reported to have antitumor effects in animal models. However, collagen has also been shown to possess antitumor activity. Therefore, the roles that type I collagen plays in tumor biology are complex and tumor type-dependent. In this review, we discuss the expression and regulation of synthesis of type I collagen, as well as the role upregulated type I collagen plays in various stages of cancer progression. We also discuss the role of collagen in tumor therapy. Finally, we highlight several recent approaches targeting type I collagen for cancer treatment.

Photothermal methane coupling into liquid fuels with hydrogen evolution over nanocatalysts based on layered double hydroxide (LDH).

The increasing energy and environmental problems have made clean energy-driven catalysis a hot research topic. Methane is an earth-abundant raw material but difficult to be converted by thermochemical processes. It is of great significance to seek novel strategies to convert methane into high-value chemicals. Herein, we synthesize a series of transition metal catalysts based on layered double hydroxide precursors which were used for photothermal methane nonoxidative coupling reactions. The strong photothermal and chemisorption effects of the derived transition metal nanostructures allow the efficient activation of methane molecules. Among them, alumina-supported metallic Ni and NiCo-alloy catalysts show excellent methane nonoxidative coupling activities, achieved hydrogen production rates of 4816.53µmol g-1h-1and 5130.9µmol g-1h-1, accompanied by liquid fuels production rates of 59.2 mg g-1h-1and 63 mg g-1h-1, respectively. The findings, therefore, provide a new strategy for methane nonoxidative coupling driven by light energy at mild conditions.

Cycloheximide promotes type I collagen maturation mainly via collagen prolyl 4-hydroxylase subunit α2.

Aberrant deposition of collagen is associated with cancer development and tissue fibrosis. Proline hydroxylation, catalyzed by collagen prolyl 4-hydroxylases (C-P4Hs), is necessary for collagen maturation and secretion. Here, we try to evaluate the mechanism of the regulation of CHX on collagen maturation. Using pepsin digestion, liquid chromatograph mass spectrometry and gene knockout, we find that treatment of mouse embryonic fibroblasts with cycloheximide (CHX) increases type I collagen proline hydroxylation partially via P4HA1 and mainly via P4HA2. Western blot analysis results show that CHX treatment reduces type I collagen but does not obviously impact the level of P4HA1/2 protein in the endoplasmic reticulum, which enhances the molar ratio of P4HA1/2 to type I collagen, and coimmunoprecipitation results confirm that more P4HA1/2 can bind to each type I collagen. Since C-P4Hs possess the capability to hydroxylate proline independent of ascorbate for a few cycles, this enhanced binding between P4HA1/2 and type I collagen can partially explain how CHX stimulates type I collagen maturation.

Triphase Photocatalytic CO2 Reduction over Silver-Decorated Titanium Oxide at a Gas-Water Boundary.

Photocatalytic CO2 reduction reaction (CO2 RR) is an attractive process to convert CO2 into valuable chemicals. But this reaction is often restricted by the poor mass transfer of CO2 in the liquid phase. Here, we have developed a triphase photocatalytic CO2 RR system by supporting Ag-decorated TiO2 nanoparticles at a gas-water boundary with hydrophobic-hydrophilic abrupt interfacial wettability. Such a triphase system allows the rapid delivery of gas-phase CO2 to the surface of photocatalysts while maintaining an efficient water supply and uncovered active sites. Ag-TiO2 supported at the gas-water boundary showed a CO2 reduction rate of 305.7 µmol g-1 h-1 , without hole scavengers, approximately 8 times higher than the nanoparticles dispersed in the liquid phase. Even using diluted CO2 (10 %) as the reactant, the CO2 RR activity was superior to most reported Ag-TiO2 based photocatalysts using pure CO2 . The findings provide a general strategy to promote the interfacial CO2 mass transfer to improve photoactivity and selectivity.

Large-Scale Testing of Asymptomatic Healthcare Personnel for Severe Acute Respiratory Syndrome Coronavirus 2.

Large-scale, 1-time testing of >12,000 asymptomatic healthcare personnel in California, USA, during April-June 2020 showed that prevalence of severe acute respiratory syndrome coronavirus 2 was low (<1%). Testing might identify asymptomatic and presymptomatic persons, including some with high viral burden, enabling prompt implementation of measures to limit nosocomial spread.

Integrated analysis of single-cell RNA-seq and bulk RNA-seq unravels tumour heterogeneity plus M2-like tumour-associated macrophage infiltration and aggressiveness in TNBC.

Triple-negative breast cancer (TNBC) is characterized by a more aggressive clinical course with extensive inter- and intra-tumour heterogeneity. Combination of single-cell and bulk tissue transcriptome profiling allows the characterization of tumour heterogeneity and identifies the association of the immune landscape with clinical outcomes. We identified inter- and intra-tumour heterogeneity at a single-cell resolution. Tumour cells shared a high correlation amongst stemness, angiogenesis, and EMT in TNBC. A subset of cells with concurrent high EMT, stemness and angiogenesis was identified at the single-cell level. Amongst tumour-infiltrating immune cells, M2-like tumour-associated macrophages (TAMs) made up the majority of macrophages and displayed immunosuppressive characteristics. CIBERSORT was applied to estimate the abundance of M2-like TAM in bulk tissue transcriptome file from The Cancer Genome Atlas (TCGA). M2-like TAMs were associated with unfavourable prognosis in TNBC patients. A TAM-related gene signature serves as a promising marker for predicting prognosis and response to immunotherapy. Two commonly used machine learning methods, random forest and SVM, were applied to find the genes that were mostly associated with M2-like TAM densities in the gene signature. A neural network-based deep learning framework based on the TAM-related gene signature exhibits high accuracy in predicting the immunotherapy response.

Efficient Combination of G-C3 N4 and CDs for Enhanced Photocatalytic Performance: A Review of Synthesis, Strategies, and Applications.

Recently, heterogeneous photocatalysts have achieved much interest on account of their great potential applications in resolving many tough energy and environmental troubles around the world through an ecologically sustainable way. Heterogeneous nanocomposites composed of graphitic carbon nitride (g-C3 N4 ) and carbon dots (CDs) possess broad spectrum absorption, appropriate electronic band structures, rapid carrier mobility, abundant reserves, excellent chemical stability, and facile synthesis methods, which make them promising composite photocatalysts for suitable applications such as photocatalytic solar fuels production and contaminant decomposition. With the rapid development in photocatalysis by hybridization of g-C3 N4 and CDs, a systematic summary and prospection of performance improvement are urgent and meaningful. This review first focuses on various kinds of effectively synthetic methods of composites. Following, the strategies available for enhanced performance, including morphology optimization, spectral absorption improvement, ternary or quaternary composition hybrid, lateral or vertical heterostructures construction, heteroatom doping, and so forth, are fully discussed. Then, the applications mainly in efficient photocatalytic hydrogen generation, photocatalytic carbon dioxide reduction, and organic pollutants degradation are systematically demonstrated. Finally, the remaining issues and prospect of further development are proposed as some kind of guidance for powerful combination of g-C3 N4 and CDs with high efficiency to photocatalysis.