An archaeal virus-encoded anti-CRISPR protein inhibits type III-B immunity by inhibiting Cas RNP complex turnover.

CRISPR-Cas systems are widespread in prokaryotes and provide adaptive immune against viral infection. Viruses encode a type of proteins called anti-CRISPR to evade the immunity. Here, we identify an archaeal virus-encoded anti-CRISPR protein, AcrIIIB2, that inhibits Type III-B immunity. We find that AcrIIIB2 inhibits Type III-B CRISPR-Cas immunity in vivo regardless of viral early or middle-/late-expressed genes to be targeted. We also demonstrate that AcrIIIB2 interacts with Cmr4α subunit, forming a complex with target RNA and Cmr-α ribonucleoprotein complex (RNP). Furtherly, we discover that AcrIIIB2 inhibits the RNase activity, ssDNase activity and cOA synthesis activity of Cmr-α RNP in vitro under a higher target RNA-to-Cmr-α RNP ratio and has no effect on Cmr-α activities at the target RNA-to-Cmr-α RNP ratio of 1. Our results suggest that once the target RNA is cleaved by Cmr-α RNP, AcrIIIB2 probably inhibits the disassociation of cleaved target RNA, therefore blocking the access of other target RNA substrates. Together, our findings highlight the multiple functions of a novel anti-CRISPR protein on inhibition of the most complicated CRISPR-Cas system targeting the genes involved in the whole life cycle of viruses.

Aqueous microdroplets promote C-C bond formation and sequences in the reverse tricarboxylic acid cycle.

The reverse tricarboxylic acid cycle (rTCA) is a central anabolic network that uses carbon dioxide (CO2) and may have provided complex carbon substrates for life before the advent of RNA or enzymes. However, non-enzymatic promotion of the rTCA cycle, in particular carbon fixation, remains challenging, even with primordial metal catalysis. Here, we report that the fixation of CO2 by reductive carboxylation of succinate and α-ketoglutarate was achieved in aqueous microdroplets under ambient conditions without the use of catalysts. Under identical conditions, the aqueous microdroplets also facilitated the sequences in the rTCA cycle, including reduction, hydration, dehydration and retro-aldol cleavage and linked with the glyoxylate cycle. These reactions of the rTCA cycle were compatible with the aqueous microdroplets, as demonstrated with two-reaction and four-reaction sequences. A higher selectivity giving higher product yields was also observed. Our results suggest that the microdroplets provide an energetically favourable microenvironment and facilitate a non-enzymatic version of the rTCA cycle in prebiotic carbon anabolism.

Integration of Cobalt Phthalocyanine, Acetylene Black and Cu2 O Nanocubes for Efficient Electroreduction of CO2 to C2 H4.

Suppressing side reactions and simultaneously enriching key intermediates during CO2 reduction reaction (CO2 RR) has been a challenge. Here, we propose a tandem catalyst (Cu2 O NCs-C-Copc) consisting of acetylene black, cobalt phthalocyanine (Copc) and cuprous oxide nanocubes (Cu2 O NCs) for efficient CO2 -to-ethylene conversion. Density-functional theory (DFT) calculation combined with experimental verification demonstrated that Copc can provide abundant CO to nearby copper sites while acetylene black successfully reduces the formation energies of key intermediates, leading to enhanced C2 H4 selectivity. X-ray photoelectron spectroscopy (XPS) and potentiostatic tests indicated that the catalytic stability of Cu2 O NCs-C-Copc was significantly enhanced compared with Cu2 O NCs. Finally, the industrial application prospect of the catalyst was evaluated using gas diffusion electrolyzers. The F E C 2 H 4 ${{\rm { F}}{{\rm { E}}}_{{{\rm { C}}}_{{\rm { 2}}}{{\rm { H}}}_{{\rm { 4}}}}}$ of Cu2 O NCs-C-Copc can reach to 58.4 % at -1.1 V vs. RHE in 0.1 M KHCO3 and 70.3 % at -0.76 V vs. RHE in 1.0 M KOH. This study sheds new light on the design and development of highly efficient CO2 RR tandem catalytic systems.

Cu2O Nanoparticles Wrapped by N-Doped Carbon Nanotubes for Efficient Electroreduction of CO2 to C2 Products.

Electroreduction of carbon dioxide (CO2) to C2 products (ethylene and ethanol) using efficient catalysts is a feasible approach to alleviate the climate crisis. Cuprous oxide nanoparticles (Cu2O NPs) are a promising catalyst for C2 production but suffer from inherent selectivity and durability. To address this challenge, a Cu2O NPs-nitrogen-doped carbon nanotube (Cu2O NPs-NCNT) composite was prepared with carbon nanotubes (CNTs), Cu2O NPs, and phthalocyanine (Pc). The results indicate that Cu2O NPs-NCNT has excellent Faradic efficiency of C2 products (77.61%) at -1.1 V vs RHE, which is 103.43% higher than that of Cu2O NPs. In the potentiostatic electrolysis combined with Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) measurements, Cu2O NPs-NCNT exhibited structural and catalytic current stability over 10 h. Finally, density functional theory calculations combined with XPS demonstrated that the NCNT in Cu2O NPs-NCNT can selectively absorb CO2 through specific N-CO2 interactions. Our work provides a unique strategy to promote the selectivity of Cu2O NPs for C2 production by introducing N-doped linear carbon materials to fabricate composite.

Electrochemiluminescence Distance and Reactivity of Coreactants Determine the Sensitivity of Bead-Based Immunoassays.

Herein we report the study of electrochemiluminescence (ECL) generation by tris(2,2'-bipyridyl)ruthenium (Ru(bpy)3 2+ ) and five tertiary amine coreactants. The ECL distance and lifetime of coreactant radical cations were measured by ECL self-interference spectroscopy. And the reactivity of coreactants was quantitatively evaluated in terms of integrated ECL intensity. By statistical analysis of ECL images of single Ru(bpy)3 2+ -labeled microbeads, we propose that ECL distance and reactivity of coreactant codetermine the emission intensity and thus the sensitivity of immunoassay. 2,2-bis(hydroxymethyl)-2,2',2''-nitrilotriethanol (BIS-TRIS) can well balance ECL distance-reactivity trade-off and enhance the sensitivity by 236 % compared with tri-n-propylamine (TPrA) in the bead-based immunoassay of carcinoembryonic antigen. The study brings an insightful understanding of ECL generation in bead-based immunoassay and a way of maximizing the analytical sensitivity from the aspect of coreactant.

Development and Optimization of a Prognostic Model Associated with Stemness Genes in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide, which is associated with a variety of risk factors. Cancer stem cells are self-renewal cells, which can promote the occurrence and metastasis of tumors and enhance the drug resistance of tumor treatment. This study aimed to develop a stemness score model to assess the prognosis of hepatocellular carcinoma (HCC) patients for the optimization of treatment. The single-cell sequencing data GSE149614 was downloaded from the GEO database. Then, we compared the gene expression of hepatic stem cells and other hepatocytes in tumor samples to screen differentially expressed genes related to stemness. R package "clusterProfiler" was used to explore the potential function of stemness-related genes. We then constructed a prognostic model using LASSO regression analysis based on the TCGA and GSE14520 cohorts. The associations of stemness score with clinical features, drug sensitivity, gene mutation, and tumor immune microenvironment were further explored. R package "rms" was used to construct the nomogram model. A total of 18 stemness-related genes were enrolled to construct the prognosis model. Kaplan-Meier analysis proved the good performance of the stemness score model at predicting overall survival (OS) of HCC patients. The stemness score was closely associated with clinical features, drug sensitivity, and tumor immune microenvironment of HCC. The infiltration level of CD8+ T cells was lower, and tumor-associated macrophages were higher in patients with high-stemness score, indicating an immunosuppressive microenvironment. Our study established an 18 stemness-related gene model that reliably predicts OS in HCC. The findings may help clarify the biological characteristics and progression of HCC and help the future diagnosis and therapy of HCC.

DNA Motifs and an Accessory CRISPR Factor Determine Cas1 Binding and Integration Activity in Sulfolobus islandicus.

CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the leader-proximal end remains largely unknown. Here, in the Sulfolobus islandicus subtype I-A system, we show that mutations destroying the proximal region reduce CRISPR adaptation in vivo. We identify that a stem-loop structure is present on the leader-proximal end, and we demonstrate that Cas1 preferentially binds the stem-loop structure in vitro. Moreover, we demonstrate that the integrase activity of Cas1 is modulated by interacting with a CRISPR-associated factor Csa3a. When translocated to the CRISPR array, the Csa3a-Cas1 complex is separated by Csa3a binding to the leader-distal motif and Cas1 binding to the leader-proximal end. Mutation at the leader-distal motif reduces CRISPR adaptation efficiency, further confirming the in vivo function of leader-distal motif. Together, our results suggest a general model for binding of Cas1 protein to a leader motif and modulation of integrase activity by an accessory factor.

Early Age Shrinkage and Mechanical Effect of Ultra-High-Performance Concrete Composite Deck: A Case Study with In Situ Test and Numerical Simulation.

For the Honghe Bridge project located in Yunnan Province, Southwest China, a steel/ultrahigh-performance concrete (UHPC) composite deck is used in the suspension bridge with a 700 m main span, and the steel stud connectors are used in the 50 mm-thick UHPC layer. To investigate the shrinkage behavior of UHPC and the relevant influence, the in situ time-dependent strain is measured continuously, and within the 20-day curing time, the material behavior is summarized based on test results. This paper proposes a prediction model for UHPC shrinkage which is refined from the widely used B3 model for normal concrete material, and the parameter values are modified and optimized by experimental comparison. Combining the numerical model and the finite element analysis model of the composite deck, the detailed mechanical state in structural parts is studied. For the practical construction, the simulation results indicate that the small thickness of UHPC above the stud and weak bond strength can influence the eventual structural performance greatly. In the discussion of stress distribution at different locations of the deck, the potential crack on the edge and the corner of the UHPC-steel interface and the mechanical damage on the stud connector around are also indicated.

Effect of the Flame Retardants and Glass Fiber on the Polyamide 66/Polyphenylene Oxide Composites.

In this work, polyamide 66/polyphenylene oxide (PA66/PPO) composites, including the flame retardants 98 wt% aluminum diethylphosphinate + 2 wt% polydimethylsiloxane (P@Si), Al(OH)3-coated red phosphorus (RP*), and glass fiber (GF), were systematically studied, respectively. The limiting oxygen index (LOI), UL-94 vertical burning level, and thermal and mechanical properties of the PA66/PPO composites were characterized. The results showed that the P@Si and RP flame retardants both improved the LOI value and UL-94 vertical burning level of the PA66/PPO composites, and PA66/PPO composites passed to the UL-94 V-0 level when the contents of P@Si and RP* flame retardants were 16 wt% and 8 wt%. On the other hand, the mechanical properties of the PA66/PPO composites were reduced from a ductile to a brittle fracture mode. The addition of GF effectively made up for these defects and improved the mechanical properties of the PA66/PPO composites containing the P@Si and RP*, but it did not change the fracture mode. P@Si and RP* flame retardants improved the thermal decomposition of PA66/PPO/GF composites and reduced the maximum mass loss rates, showing that the PA66/PPO/GF composites containing the P@Si and RP* flame retardants could be used in higher-temperature fields.

Mass spectrometric observation on free radicals during electrooxidation of dopamine.

It has been challenging to directly observe the o-semiquinone radicals and transient intermediates produced during the oxidation of dopamine (DA). To achieve this goal, we developed an electrochemistry-neutral reionization-mass spectrometry (EC-NR-MS) technique for online studying the electrooxidation process of DA. The EC-NR device mainly composed by a self-designed EC flow cell, a sonic spray ionization source, a heating tube, an ion deflector and an electrospray ionization source. By precisely controlling the oxidation potential at 0.55 V, a series of reaction products include o-quinone (DAQ), Leukodopaminochrome (LDAC), Dopaminochrome (DAC), 5,6-dihydroxyindole (DHI) and DA dimer clearly appeared in the MS spectrum. Based on the ion deflector of EC-NR setup, the neutral o-semiquinone radical DA● and neutral Leukodopaminochrome radical LDAC● were successfully extracted from these ionic products and allowed to be detected by MS. Such finding was further confirmed by spin trapping experiment. The formation of o-semiquinone radical anion (DA●-) has been identified as its metal complex with Zn2+. These results provide conclusive evidence for the DA oxidation mechanism, which consists of proton coupled electron transfer and sequential proton-electron transfer steps.

Correction: Mass spectrometric detection of fleeting neutral intermediates generated in electrochemical reactions.

[This corrects the article DOI: 10.1039/D1SC01385H.].

Mass spectrometric detection of fleeting neutral intermediates generated in electrochemical reactions.

Towards the goal of on-line monitoring of transient neutral intermediates during electrochemical reactions, an electrochemistry-neutral reionization-mass spectrometry (EC-NR-MS) technique was developed in this work. The EC-NR setup consisted of a customized EC flow cell, a sonic spray ionization source, a heating tube, an ion deflector and an electrospray ionization source, which were respectively used for the precise control of the electrochemical reaction, solution nebulization, droplet desolvation, ion deflection and neutral intermediate ionization. Based on the EC-NR-MS approach, some long-sought neutral radicals including TPrA˙, DBAE˙ and TEOA˙, which belong to important reductive intermediates in electrochemiluminescence (ECL) reactions, were successfully identified which helps to clarify the previously unproven ECL reaction mechanism. These findings were also supported by spin-trapping experiments and the tandem MS technique. Accordingly, the EC-NR-MS method provides a direct solution for studying complicated electrochemical reactions, especially for detecting short-lived neutral radicals as well as ionic intermediates.

CRISPR-Associated Factor Csa3b Regulates CRISPR Adaptation and Cmr-Mediated RNA Interference in Sulfolobus islandicus.

Acquisition of spacers confers the CRISPR-Cas system with the memory to defend against invading mobile genetic elements. We previously reported that the CRISPR-associated factor Csa3a triggers CRISPR adaptation in Sulfolobus islandicus. However, a feedback regulation of CRISPR adaptation remains unclear. Here we show that another CRISPR-associated factor, Csa3b, binds a cyclic oligoadenylate (cOA) analog (5'-CAAAA-3') and mutation at its CARF domain, which reduces the binding affinity. Csa3b also binds the promoter of adaptation cas genes, and the cOA analog enhances their binding probably by allosteric regulation. Deletion of the csa3b gene triggers spacer acquisition from both plasmid and viral DNAs, indicating that Csa3b acted as a repressor for CRISPR adaptation. Moreover, we also find that Csa3b activates the expression of subtype cmr-α and cmr-ß genes according to transcriptome data and demonstrate that Csa3b binds the promoters of cmr genes. The deletion of the csa3b gene reduces Cmr-mediated RNA interference activity, indicating that Csa3b acts as a transcriptional activator for Cmr-mediated RNA interference. In summary, our findings reveal a novel pathway for the regulation of CRISPR adaptation and CRISPR-Cmr RNA interference in S. islandicus. Our results also suggest a feedback repression of CRIPSR adaptation by the Csa3b factor and the cOA signal produced by the Cmr complex at the CRISPR interference stage.

A CRISPR-associated factor Csa3a regulates DNA damage repair in Crenarchaeon Sulfolobus islandicus.

CRISPR-Cas system provides acquired immunity against invasive genetic elements in prokaryotes. In both bacteria and archaea, transcriptional factors play important roles in regulation of CRISPR adaptation and interference. In the model Crenarchaeon Sulfolobus islandicus, a CRISPR-associated factor Csa3a triggers CRISPR adaptation and activates CRISPR RNA transcription for the immunity. However, regulation of DNA repair systems for repairing the genomic DNA damages caused by the CRISPR self-immunity is less understood. Here, according to the transcriptome and reporter gene data, we found that deletion of the csa3a gene down-regulated the DNA damage response (DDR) genes, including the ups and ced genes. Furthermore, in vitro analyses demonstrated that Csa3a specifically bound the DDR gene promoters. Microscopic analysis showed that deletion of csa3a significantly inhibited DNA damage-induced cell aggregation. Moreover, the flow cytometry study and survival rate analysis revealed that the csa3a deletion strain was more sensitive to the DNA-damaging reagent. Importantly, CRISPR self-targeting and DNA transfer experiments revealed that Csa3a was involved in regulating Ups- and Ced-mediated repair of CRISPR-damaged host genomic DNA. These results explain the interplay between Csa3a functions in activating CRISPR adaptation and DNA repair systems, and expands our understanding of the lost link between CRISPR self-immunity and genome stability.

Thermally stable monolithic Doppler asymmetric spatial heterodyne interferometer: optical design and laboratory performance.

We report on a thermally stable monolithic Doppler asymmetric spatial heterodyne (DASH) interferometer with field-widening prisms for thermospheric wind measurements by observing the Doppler shift of the airglow emission. Analytical deduction and numerical simulation are applied to determine the central optical path difference, the thermal compensation condition and the field-widening design. A monolithic interferometer with optimized configuration was built and tested in the laboratory. Laboratory tests show that the best visibility of 0.94 was realized with the 9 ° field-of-view illumination, while the thermal responses of the spatial frequency and the optical phase offset are 0.0154 cm-1/°C and 0.469 rad/°C, respectively.

Ocular Inflammatory Response to Intravitreal Injection of Adeno-Associated Virus Vector: Relative Contribution of Genome and Capsid.

Both subretinal dosing and intravitreal (IVT) dosing of adeno-associated virus (AAV) in higher species induce mild and transient inflammatory responses that increase with dose. Foreign protein and foreign DNA are known inducers of inflammation, which is also true in the immune-privileged ocular environment. We explored which component(s) of AAV vectors, viral capsid, or viral DNA drive inflammatory responses. Recombinant AAV with three tyrosine to phenylalanine substitutions in the capsid of AAV serotype 2 (rAAV2tYF), and with a generic ubiquitous promoter (cytomegalovirus [CMV]) controlling the expression of humanized green fluorescent protein (hGFP), was processed to enrich for AAV capsids containing genome (full capsids), capsids without genome (empty capsids), and residual material. Nonhuman primate eyes were injected by IVT in both eyes. During in-life, ocular inflammation and development of neutralizing antibodies (NAb) were measured. Following termination, lymph node immunophenotyping was performed, vitreous was processed for cytokine and RNAseq analyses, and ocular sections were assessed for transgene expression (by in situ hybridization) and histopathology. IVT dosing of AAV vectors transiently raised cellular inflammation in the aqueous and induced a more sustained inflammation in the vitreous. Lowering the total capsid dose by removing empty AAV capsids reduced inflammation and improved viral transduction. IVT dosing of AAV induced systemic NAb to AAV irrespective of the vector preparation. Similarly, lymph node immunophenotyping revealed identical profiles irrespective of viral preparation used for dosing. Immune cells in the vitreous were identified based on RNAseq analysis. Three months postdose, cytokine levels were low, indicative of minimal levels of inflammation in agreement with histopathological assessment of the retina.

Preparation and application of a highly sensitive conjugated polymer-copper (â ¡) composite fluorescent sensor for detecting hydrazine in aqueous solution.

At first, a conjugated polymer (poly(2,5-bis(4-(bis(pyridin-2-ylmethyl)amino) butoxyl)-1,4-Phenylethynylene-1,4-poly(phenylene ethynylene), PPE-DPA) was synthesized based on our earlier report. In this work, a highly selective and sensitive composite fluorescent sensor (PPE-DPA-Cu2+), which was PPE-DPA complexed with copper ions, has been developed and used to measure trace amounts of hydrazine in environmental water samples. The experiment conditions were investigated. Under optimized conditions, the sensor shows satisfactory detection performance, which can highly sensitively detect hydrazine with a linear range of 0-70 nmol L-1 and detection limit as low as 5.4 × 10-10 mol L-1 (S/N = 3). Meanwhile, the sensor exhibits high selectivity and strong anti-interference ability for various cations, anions, common reducing agents and organic amines. This sensor has been applied to detect hydrazine in the real water samples, and the recovery of hydrazine was 92.3%-112.0% after added standard solution of hydrazine. Satisfactory results indicate that the sensor is expected to be utilized for quantitative detection of hydrazine in environmental samples.

Developed a novel sensor based on fluorescent graft conjugated polymer for the determination of aristolochic acid in traditional Chinese medicine.

A novel fluorescent graft conjugated polymer (poly (2, 5-bis (Polyethylene glycol oxybutyrate)-1, 4-phenylethynylene-alt-1, 4-phenyleneethynylene, PPE-OB-PEG) has been designed and synthesized for the determination of aristolochic acid (AA). The detection conditions and detection characters of PPE-OB-PEG were systematically explored in this work. The fluorescence intensity of PPE-OB-PEG changes with the different concentration of AA. PPE-OB-PEG has a good linear range towards AA from 1.00 × 10-7 to 8.00 × 10-5 mol L-1 and the limit of detection (LOD) is 3.00 × 10-8 mol L-1 (S/N = 3). PPE-OB-PEG have been applied to detect AA in traditional Chinese medicine samples and the results are satisfactory. The experimental results show that PPE-OB-PEG can be used as a fluorescence probe for rapid and sensitive detection of AA.

Analysis and correction of distortions in a spatial heterodyne spectrometer system.

In this paper a method for correcting the radial distortion of interferograms generated by a spatial heterodyne spectrometer system is presented. Instead of utilizing calibration patterns, the distortion model parameters are estimated based on the distorted fringe features generated by projecting the straight interference stripes onto the detector. Comparisons between polynomial models and division models indicate that division models can deliver competitive performance on the reconstructed image with fewer parameters. Simulated interferograms based on ray-tracing are used to demonstrate the correction of errors in the spatial, phase, and spectral domain caused by optical distortion.

A Conjugated Polymer Fluorescent Sensor for Continuous Identification of Copper(II) and Pyrophosphate in Blood Serum and Synovial Fluid.

A novel "on-off-on" super-sensitive conjugated polymer fluorescence sensor (PPE-DPA) was developed and it was applied to realize the continuous recognition of Cu2+ and pyrophosphate (PPi). The fluorescence intensity decreased linearly with the change of Cu2+ from 0.05 to 5.0 µmol L-1 and the limit of detection was 24 nmol L-1. The fluorescence intensity was linearly enhanced with the increase of PPi from 0.5 to 12.0 µmol L-1 and the limit of detection was 230 nmol L-1. In addition, this method was applied to detect PPi in the blood serum and synovial fluid of patients with arthritis and satisfactory results were obtained. Thus, the PPE-DPA is not only an effective tool for detecting Cu2+ and PPi in samples, but also presents a potential way to diagnose arthritis.