Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility for synthetic genome engineering, agricultural genomics, and biomedical applications. Although bacteria harboring CRISPR-Cas9 or ...CRISPR-Cas3 adaptive immune systems sometimes acquire mobile genetic elements encoding anti-CRISPR proteins that inhibit Cas9, Cas3, or the DNA-binding Cascade complex, no such inhibitors have been found for CRISPR-Cas12a. Here we use a comprehensive bioinformatic and experimental screening approach to identify three different inhibitors that block or diminish CRISPR-Cas12a-mediated genome editing in human cells. We also find a widespread connection between CRISPR self-targeting and inhibitor prevalence in prokaryotic genomes, suggesting a straightforward path to the discovery of many more anti-CRISPRs from the microbial world.
A new heteroleptic iridium complex demonstrated low cytotoxicity and near-infrared excitation (via two-photon absorption) for target-specific in vitro Golgi imaging in various cell lines (HeLa and ...A549 cells) with two-photon absorption cross section (~350 GM) in DMSO.
An AlCrFeCoNi high entropy alloy was prepared by vacuum arc melting. Only diffraction peak corresponding to a BCC crystal structure is observed for this AlCrFeCoNi high entropy alloy. The ...microstructure of this AlCrFeCoNi alloy is polygonal grains with intragranular dendritic segregation. Dendritic segregation area is found to be Al, Ni rich and Cr, Fe deplete, while interdendritic segregation area is Cr, Fe rich and Al, Ni deplete. The distribution of Co is essentially identical. The fine microstructure of dendritic segregation area and of interdendritic segregation area is found to be nanoscale spherical precipitates morphology and basket-weave morphology, respectively. Results of EDS attached on high resolution scanning electron microscope (SEM) revealed that these morphological characteristics are also resulted from elements segregation. This AlCrFeCoNi high entropy alloy exhibits excellent compressive properties. The yield stress, compressive strength and plastic strain of the alloy reaches 1250.96, 2004.23
MPa, and 32.7%, respectively. The fracture mechanism of this AlCrFeCoNi high entropy alloy is observed as cleavage fracture and slip separation.
Mn, Ti and V were added in an equal-molar ratio to the AlCrFeCoNiCu alloy and the effects of these added elements on microstructure and properties of AlCrFeCoNiCu high entropy alloy were studied. The ...results indicate that the AlCrFeCoNiCuMn alloy shows almost the same microstructure as in AlCrFeCoNiCu alloy except for a long-strip Cr-enriched phase. The additions of Ti change the AlCrFeCoNiCu alloy from a dendrite structure to eutectic-cell one. Hence two phases in the eutectic cells are detected as one Al, Ti, Co, Ni enriched phase and another Cr, Fe enriched phase. V added alloy is also a dendrite structure, but V additions change the morphology of dendritic area from modulated plates to ellipsoidal particles. The addition of V into the AlCrFeCoNiCu alloy shows the best strengthening effect and the lowest decrease in the ultimate strain in our experiments.
Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions on experimental animals, and one of the proposed disruption mechanisms is the competitive binding of ...PBDE metabolites to TH transport proteins. In this report, a nonradioactive, site-specific fluorescein–thyroxine (F–T4) conjugate was designed and synthesized as a fluorescence probe to study the binding interaction of hydroxylated PBDEs to thyroxine-binding globulin (TBG) and transthyretin (TTR), two major TH transport proteins in human plasma. Compared with free F–T4, the fluorescence intensity of TTR-bound conjugate was enhanced by as much as 2-fold, and the fluorescence polarization value of TBG-bound conjugate increased by more than 20-fold. These changes provide signal modulation mechanisms for F–T4 as a fluorescence probe. Based on fluorescence quantum yield and lifetime measurements, the fluorescence intensity enhancement was likely due to the elimination of intramolecular fluorescence quenching of fluorescein by T4 after F–T4 was bound to TTR. In circular dichroism and intrinsic tryptophan fluorescence measurements, F–T4 induced similar spectroscopic changes of the proteins as T4 did, suggesting that F–T4 bound to the proteins at the T4 binding site. By using F–T4 as the fluorescence probe in competitive binding assays, 11 OH–PBDEs with different levels of bromination and different hydroxylation positions were assessed for their binding affinity with TBG and TTR, respectively. The results indicate that the binding affinity generally increased with bromine number and OH position also played an important role. 3-OH–BDE-47 and 3′-OH–BDE-154 bound to TTR and TBG even stronger, respectively, than T4. With rising environmental level and high bioaccumulation capability, PBDEs have the potential to disrupt thyroid homeostasis by competitive binding with TH transport proteins.
The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or ...activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.
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•Cas9 can be circularly permuted (Cas9-CP) for optimized fusion protein construction•Cas9 circular permutation enables the engineering of protease-activated ProCas9s•ProCas9s can sense and respond to protease activity, such as during viral infection•Cas9-CPs provide a toolkit of safer and more efficient genome-modifying enzymes
Programmable Cas9 variants with improved functionality are generated by protein engineering—including versions that work as protease sensors responding to viral infection.
The adsorption of 1-naphthylamine, 1-naphthol and phenol on as-prepared and oxidized multiwalled carbon nanotubes (MWCNTs) has been investigated. The results illustrated that both as-prepared and ...oxidized MWCNTs showed high adsorption capacity for the three ionizable aromatic compounds (IACs) studied. Oxidation of MWCNTs increased the surface area and the pore volume, and introduced oxygen-containing functional groups to the surfaces of MWCNTs, which depressed the adsorption of IACs on MWCNTs. Both Langmuir and Freundlich models described the adsorption isotherms very well and the adsorption thermodynamic parameters (Δ
G°, Δ
H° and Δ
S°) were measured. The adsorption for 1-naphthylamine, 1-naphthol and phenol is general spontaneous and thermodynamically favorable. The adsorption of phenol is an exothermic process, whereas the adsorption of 1-naphthylamine and 1-naphthol is an endothermic process. Results of this work are of great significance for the environmental application of MWCNTs for the removal of IACs from large volume of aqueous solutions.
Core−shell Li3V2(PO4)3@C nanostructured composites were prepared via a sol−gel route followed by hydrothermal treatment. XRD patterns showed that Li3V2(PO4)3 has a monoclinic structure with space ...group P21/n. TEM images exhibited that Li3V2(PO4)3 particles are encapsulated with a carbon shell ∼10 nm in thickness. Compared with pure Li3V2(PO4)3, core−shell Li3V2(PO4)3@C composites presented enhanced electrochemical Li ion intercalation performances. Cyclic voltammetry and electrochemical impedance spectroscopy disclosed that carbon shells improved Li ion diffusion and electrical conductance significantly and also retarded formation of solid electrolyte interphase film of Li3V2(PO4)3 cathode materials.
CD4 T cells guide the development of CD8 T cells into memory by elaborating mitogenic and differentiation factors and by licensing professional antigen-presenting cells. CD4 T cells also act to stave ...off CD8 T cell dysfunction during repetitive antigen stimulation in persistent infection and cancer by mitigating generation of exhausted T cells (T
). CD4 T cell help is also required for establishing and maintaining tissue-resident memory T cells (T
), the nonrecirculating memory T cell subset parked in nonlymphoid tissues to provide frontline defense against reinvading pathogens. Interleukin (IL)-21 is the signature cytokine secreted by follicular helper CD4 T cells (T
) to drive B cell expansion and differentiation in germinal centers to mount high-affinity, isotype class-switched antibodies. In several infection models, IL-21 has been identified as the CD4 T help needed for formation and survival of T
and T
. In this review, we will explore the different memory subsets of CD8 T cells in persistent infections, the metabolic profiles associated with each, and evidence documenting the importance of CD4 T cell-derived IL-21 in regulating CD8 T
and T
development, homeostasis, and function.
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