CRISPR-Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a ...protospacer adjacent motif (PAM) and sequences complementary to the guide RNA. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing 'off-switch' tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.
The RNA-guided endonucleases of the CRISPR-Cas9 system, including the most widely used Cas9 from Streptococcus pyogenes (SpCas9), are becoming a robust genome editing tool in model organisms and hold ...immense promise for therapeutic applications. Many strategies have been employed to overcome the limitations caused by SpCas9's off-target effects and its stringent requirement for the protospacer adjacent motif (PAM) sequence. However, the structural mechanisms underlying these strategies remain undefined. Here, we present crystal structure of a SpCas9 variant, xCas9 3.7 that has broad PAM compatibility and high DNA targeting specificity, in complex with a single-guide RNA and its double-stranded DNA targets. Structural comparison revealed that salt bridge-stabilized R1335 is critical for the stringent selection of PAM sequence by SpCas9. Unrestricted rotamerization of this residue by the E1219V mutation in xCas9 3.7 lessens the stringency for PAM recognition and allows SpCas9 to recognize multiple PAM sequences as further supported by biochemical data. Compared to those in wild-type (WT) SpCas9, REC2 and REC3 domains in xCas9 3.7 undergo striking conformational changes, leading to reduced contact with DNA substrate. SpCas9 mutants engineered to display less interaction with DNA and have conformationally more flexible REC2 and REC3 domains display enhanced specificity for DNA substrates in both biochemical and cellular assays. Taken together, our findings reveal the structural mechanisms underlying the broadened PAM compatibility and high DNA fidelity of xCas9 3.7, which can assist rational engineering of more efficient SpCas9 variants and probably other Cas9 orthologs.
Accurate recognition of pest categories is crucial for effective pest control. Due to issues such as the large variation in pest appearance, low data quality, and complex real-world environments, ...pest recognition poses challenges in practical applications. At present, many models have made great efforts on the real scene dataset IP102, but the highest recognition accuracy is only 75%. To improve pest recognition in practice, this paper proposes a multi-image fusion recognition method. Considering that farmers have easy access to data, the method performs fusion recognition on multiple images of the same pest instead of the conventional single image. Specifically, the method first uses convolutional neural network (CNN) to extract feature maps from these images. Then, an effective feature localization module (EFLM) captures the feature maps outputted by all blocks of the last convolutional stage of the CNN, marks the regions with large activation values as pest locations, and then integrates and crops them to obtain the localized features. Next, the adaptive filtering fusion module (AFFM) learns gate masks and selection masks for these features to eliminate interference from useless information, and uses the attention mechanism to select beneficial features for fusion. Finally, the classifier categorizes the fused features and the soft voting (SV) module integrates these results to obtain the final pest category. The principle of the model is activation value localization, feature filtering and fusion, and voting integration. The experimental results indicate that the proposed method can train high-performance feature extractors and classifiers, achieving recognition accuracy of 73.9%, 99.8%, and 99.7% on IP102, D0, and ETP, respectively, surpassing most single models. The results also show that thanks to the positive role of each module, the accuracy of multi-image fusion recognition reaches the state-of-the-art level of 96.1%, 100%, and 100% on IP102, D0, and ETP using 5, 2, and 2 images, respectively, which meets the requirements of practical applications. Additionally, we have developed a web application that applies our research findings in practice to assist farmers in reliable pest identification and drive the advancement of smart agriculture.
Environment-friendly medium-density fiberboards (MDFs) prepared using sodium lignosulfonate/chitosan adhesives (L/C) show potential in environment-friendly wood-based panel application. However, the ...synthesis mechanism of this adhesive and the relationships between synthesis mechanism and bonding performance were not discussed in depth. Herein, the synthesis mechanism of L/C was explored in detail based on characterizations of L/C with different mass ratios of sodium lignosulfonate to chitosan by Fourier-transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction. For L/C with different mass ratios of sodium lignosulfonate to chitosan, the corresponding bonding performance was also determined based on characterizations of mechanical and dimensional performance of MDFs. Results showed a 3D network structure of L/C formed through the hydrogen linkages among hydroxyl groups in sodium lignosulfonate and hydroxyl and amino groups in chitosan, amide linkages resulted from reaction between carbonyl groups in sodium lignosulfonate and amino groups in chitosan, and sulfonamide linkages originated from reaction between sulfonic groups in sodium lignosulfonate and amino groups in chitosan. The mechanical performance of MDF was closely related to the 3D network and amino groups of L/C, while the dimensional performance of MDF was negatively affected by sodium lignosulfonate. The MDFs with 1:3 and 1:2 mass ratios of sodium lignosulfonate to chitosan showed superior mechanical properties and comparable dimensional performance with a commercial panel.
Layered lithium-rich oxide materials are regarded as one of the most promising cathode materials. However, inferior cycling stability and poor rate performance hinder their practical application ...prospect. In this study, Li
3
PO
4
-coated Li
1.2
Ni
0.2
Mn
0.6
O
2
cathode materials have been synthesized by sol–gel method together with a facile liquid-evaporation process. The results suggested that the Li
3
PO
4
coating layer, which could not only facilitate the lithium-ion diffusion rate and accelerate the diffusion kinetics but also act as a protective layer to protect it from corrosion by HF and other side reactions. Density functional theory (DFT) calculations confirmed the essence effect on lithium-ion diffusion coefficient and electronic conductivity. After modifying with an appropriate amount of Li
3
PO
4
, the Li-rich layered oxides showed enhanced electrochemical performance. Especially, the capacity retention of 5 wt% Li
3
PO
4
-coated Li
1.2
Ni
0.2
Mn
0.6
O
2
was significantly enhanced from 17.7% of the bare Li
1.2
Ni
0.2
Mn
0.6
O
2
to 73.8%. In terms of rate capabilities, 5 wt% Li
3
PO
4
-coated Li
1.2
Ni
0.2
Mn
0.6
O
2
retained capacities of 181.0, 165.9, 128.8, and 107.8 mAh g
−1
, while the bare Li
1.2
Ni
0.2
Mn
0.6
O
2
were only 137.4, 109.3, 75.6, and 45.9 mAh g
−1
, respectively, at rates of 0.5 C, 1 C, 2 C, and 5 C. Our research findings show that coating with an appropriate amount of lithium-ion conductor material is one of the effective measures to obtain improved performance of Li-rich and Mn-rich layered oxide materials.
The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading ...nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown. Here we report cryo-EM structures of the Csm-crRNA complex, with or without target ssRNA, at near-atomic resolution. Our cryo-EM maps allow us to build atomic models of the key macromolecular components, including Cas10, Csm2, Csm3, Csm4, crRNA and the invading ssRNA. Our structure resolves unambiguously the stoichiometry and tertiary structures of the Csm protein complex and the interactions between protein components and the crRNA/ssRNA. Interestingly, the new atomic structures of the Csm proteins presented here are similar to those of previously known Csm proteins in other species despite their low sequence similarity. Our combined structural and biochemical data suggest that ssRNA cleavage is preferentially carried out near its 5'-end, that the extent of interactions among the ssRNA, crRNA and the protein components regulates the DNase activity of the Csm complex, and that the 3' flanking sequence of ssRNA activates the Cas10 DNase activity allosterically.
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Patients with cancer survivors are at increased risk of cardiovascular disease(CVD). Cardio-oncology has developed as a new discipline with the advances in cancer treatment. There are ...many new challenges for the clinician and a new frontier for research and investigation. There is an urgent need for further study on the prevention of cardiovascular toxicity. Imperatorin (IMP) is a natural form of coumarin and extract from several plants with diver’s pharmacokinetic effects, including antioxidant and anti-inflammatory properties. This review focus on the molecular mechanisms and pharmacological effects of Imperatorin maybe provide potential cancer and cardiovascular protection that targets IMP. Further studies are required to elucidate the entire spectrum of cytotoxic activities of these compounds to validate and expand their preclinical and clinical applications and to clarify the potential role of IMP.
Driven by various environmental and economic factors, it is emerging to adopt an efficient and sustainable strategy to recycle carbon fibers (rCFs) from carbon fiber-reinforced polymer (CFRP) wastes ...and reuse them in high-value applications. This review summarized the latest progress of CFRP waste recycling methods (including mechanical, chemical, and thermal methods), discussed their advantages and disadvantages, influence parameters and possible environmental effects, and their potential effects on the mechanical and surface chemical properties of rCFs. In addition, the latest optimization schemes of leading recycling technologies were detailed. According to the literature, CFs are the key points in the structural support of semiconductor-based recyclable photocatalytic systems and the enhancement of performance, which means that rCFs have high reuse potential in sustainable photocatalysis. Therefore, this paper also emphasized the possibility and potential value of reusing recovered fibers for developing recyclable photocatalytic products, which may be a new way of reuse in environmental purification often ignored by researchers and decision-makers in the field of CFs.
Oil film vortex severely reduces the stability of hydrostatic bearings. A solid-liquid interface with drag and slip properties can weaken the oil film vortex of the bearing. Here, a combined ...picosecond laser ablation and chemical modification method is proposed to prepare surfaces with microbulge array structure on 6061 aluminum alloy substrates. Because of the low surface energy of the perfluorododecyltriethoxysilane modification and the bulge geometry of the microbulge array structure, the surface shows excellent superhydrophobicity. The optimum contact angle in air for water is 164°, and that for oil is 139°. Two surfaces with “lotus-leaf effect” and “rose-petal effect” were obtained by controlling the processing parameters. The drag reduction properties of superhydrophobic surfaces were systematically investigated with slip lengths of 22.26 and 36.25 µm for deionized water and VG5 lubricant, respectively. In addition, the superhydrophobic surface exhibits excellent mechanical durability and thermal stability. The proposed method provides a new idea for vortex suppression in hydrostatic bearings and improves the stability of bearings in high-speed operation.
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