Development of a highly efficient prime editor system in mice and rabbits.

The recently developed prime-editing (PE) technique is more precise than previously available techniques and permits base-to-base conversion, replacement, and insertions and deletions in the genome. However, previous reports show that the efficiency of prime editing is insufficient to produce genome-edited animals. In fact, prime-guide RNA (pegRNA) designs have posed a challenge in achieving favorable editing efficiency. Here, we designed prime binding sites (PBS) with a melting temperature (Tm) of 42 °C, leading to optimal performance in cells, and we found that the optimal Tm was affected by the culture temperature. In addition, the ePE3max system was developed by updating the PE architecture to PEmax and expressing engineered pegRNA (epegRNA) based on the original PE3 system. The updated ePE3max system can efficiently induce gene editing in mouse and rabbit embryos. Furthermore, we successfully generated a Hoxd13 (c. 671 G > T) mutation in mice and a Tyr (c. 572 del) mutation in rabbits by ePE3max. Overall, the editing efficiency of modified ePE3max systems is superior to that of the original PE3 system in producing genome-edited animals, which can serve as an effective and versatile genome-editing tool for precise genome modification in animal models.

A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9.

BACKGROUND: Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size. RESULTS: We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes. CONCLUSIONS: We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.

Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope.

BACKGROUND: Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency. RESULTS: Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797-C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice. CONCLUSIONS: These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.

Mixed solvent fabrication of tobermorite and the fixation of heavy metals in water and soil.

Here, tobermorite was prepared by a solvothermal technology using calcite and quartz with a mixed solvent of ethanol and water. Factors including reaction temperature, time and KOH content were studied to optimize the preparation procedure. To study the relationship between ethanol content-material structural characteristics-adsorption capacity, a series of materials were prepared in different mixed solvent proportions of ethanol and water, and their structural characteristics and adsorption capacity were compared. We found that the adsorption capacity of different samples for Pb2+ and Cd2+ was positively correlated with negatively correlated with the surface area and negatively correlated with the crystallinity of materials. Then, the material prepared by 30% ethanol solution (30-T) with the best adsorption performance was used for further research; the results were fitted by kinetic and thermodynamic models, and adsorbed materials were analyzed by various characterizations, suggesting that the adsorption process was ascribed to comprehensive pathways including ion exchange, chemical precipitation, and surface-complexation. Then, the 30-T was further used to remediate heavy metals contaminated soil, and the remediation effect was examined by the DTPA-extractable method and the European Community Bureau of Reference (BCR) sequential extraction method. The DTPA-extractable results showed that tobermorite observably reduced the bioavailability of Pb and Cd, and the BCR results suggested that the acid-soluble and reducible fractions of Pb and Cd were transformed to the oxidizable and residual fractions after remediation. In summary, tobermorite has great potential in the remediation of heavy metal polluted-aquatic environment/system and soil.

On-Cell Catalytic Detection of Epithelial-to-Mesenchymal Transition by a Clusterzyme Bioprobe.

We construct a peptide-conjugated metal cluster as an enzyme-like catalytic bioprobe to enhance quantitative analysis of a membrane protein biomarker and detect epithelial-to-mesenchymal transition of tumor cells. This bioprobe with atomically precise formula, termed clusterzyme, possesses selective recognition and intrinsic enzyme-like activity. These favorable features facilitate sensitive quantitative analysis of the membrane protein in situ through on-cell catalytic signal amplification. This clusterzyme-based analytical method exhibits excellent compatibility with a traditional enzyme-linked immunosorbent assay and improved detection sensitivity with accuracy and robustness. Further, the expression level of the membrane protein reflects the ability of migration and invasion of model tumor cells, revealing epithelial-to-mesenchymal transition process. This work offers a facile and sensitive approach to monitor tumor cell type evolution at the molecular level, demonstrating a potential application of early cancer diagnosis and therapy assessment.

Assessing environmental fate of hexavalent chromium as influenced by fractionation of ferrihydrite with dissolved organic matter.

The dynamic interactions among iron (Fe) oxides, dissolved organic matter (DOM) and toxic trace metals play crucial roles in risk assessment and environmental remediation. Although the inhibitory effects of DOM on the iron oxides transformation process have been studied previously, there is still a lack of mechanistic and quantitative understanding on the kinetics of Cr(VI) and ferrihydrite transformation in the present of DOM. In this study, we investigated the fractionation process of DOM on ferrihydrite and its influence on the fate of Cr(VI) and transformation of ferrihydrite. The result of three-dimension excitation emission matrix (3D-EEM), Q-Exactive LC-MS/MS, X-ray diffraction (XRD) and high-resolution transmission electron microscopy (HR-TEM) indicated that fulvic acid-like compounds of DOM were the mainly fractionated compounds on the surface of ferrihydrite, which further inhibited the transformation of ferrihydrite. Besides, bracewellite (CrO(OH)) generated as an accompanied mineral during the transformation of ferrihydrite in the present of Cr(VI). Based on the DFT theoretical calculation, we concluded that Cr(VI) mainly in the form of HCr O4- was more inclined to be adsorbed on iron-oxide tetrahedron by inner-sphere monodentate mononuclear configurations. The findings on the dynamic coupling among Fe oxide transformation and Cr(VI) sequestration under the effect of DOM provided the basis for accurately predicting the fate of trace elements and iron mineral.

The Phage Holin HolGH15 Exhibits Potential As an Antibacterial Agent to Control Listeria monocytogenes.

Listeria monocytogenes is an important foodborne pathogen that is a serious threat to public health security, and new strategies to control this bacterium in food are needed. HolGH15, derived from Staphylococcus aureus phage GH15, has shown antibacterial activity against several bacterial species. In this work, the antilisterial behavior and effectiveness of HolGH15 are further studied. To elucidate its antimicrobial modes against L. monocytogenes, cell integrity and membrane permeabilization assays were performed. When treated with HolGH15, the release of 260-nm-absorbing materials of L. monocytogenes was rapidly increased. HolGH15 triggered a significant increase in fluorescence intensity by flow cytometry. In membrane permeabilization assays, the cytoplasmic ß-galactosidase of L. monocytogenes treated with HolGH15 was released via an increase in the permeability of the membrane. HolGH15 caused changes in the structural properties of L. monocytogenes cells resulting in shrinkage, which evoked the release and removal of cellular contents and finally lead to cell death. Electron microscopy observations indicated that HolGH15 exhibited excellent bactericidal potency by permeabilizing the cell membrane, damaging membrane integrity, and inducing cellular content shrinkage or loss. Moreover, HolGH15 (at the final concentration of 240 µg/mL) reduced L. monocytogenes (at the initial concentration of 106 colony-forming unit/mL) to an undetectable level at 4°C. Collectively, HolGH15 has potential as a novel antimicrobial agent against L. monocytogenes in the manufacture and store of food by spraying or soaking, especially at refrigerated temperature.

Is GSH Chelated Pt Molecule Inactive in Anti-Cancer Treatment? A Case Study of Pt6 GS4.

Platinum (Pt) drugs are widely used in anti-cancer treatment although many reports advocated that tumor cells could inactivate Pt drugs via glutathione-Pt (GSH-Pt) adducts formation. To date, GSH chelated Pt molecules have not been assessed in cancer treatment because GSH-Pt adducts are not capable of killing cancer cells, which is widely accepted and well followed. In this report, endogenous biothiol is utilized to precisely synthesize a GSH chelated Pt molecule (Pt6 GS4 ). This Pt6 GS4 molecule can be well taken up by aggressive triple negative breast cancer (TNBC) cells. Subsequently, its metabolites could enter nuclei to interact with DNA, finally the DNA-Pt complex triggers TNBC cell apoptosis via the p53 pathway. Impressively, high efficacy for anti-cancer treatment is achieved by Pt6 GS4 both in vitro and in vivo when compared with traditional first-line carboplatin in the same dosage. Compared with carboplatin, Pt6 GS4 keeps tumor bearing mice alive for a longer time and is non-toxic for the liver and kidneys. This work opens a route to explore polynuclear Pt compound with accurate architecture for enhancing therapeutic effects and reducing systemic toxicity.

Pressure induced excellent thermoelectric behavior in skutterudites CoSb3 and IrSb3.

Utilizing the first-principle calculations combined with Boltzmann transport equation (BTE) and semiclassical analysis, we have systematically investigated the electronic structure, lattice thermal conductivity κL, Seebeck coefficient S, and the dimensionless figure of merit zT as a function of hydrostatic pressure P in crystalline skutterudites CoSb3 and IrSb3. Interestingly, as the pressure increases, the band gap and κL show an approximate parabolic trend, which results in extraordinarily high S and excellent thermoelectric properties, and zT even exceeds 1.4(1.09) in IrSb3(CoSb3) at 54(58) GPa. This anomalous behavior arises from the electron distribution and intrinsic scattering processes. Further analyses indicate that (i) nonbonding electron pairs of Sb atoms are gradually transferred to the region between Co(Ir) and Sb atoms as the pressure increases, which leads to the formation of a partial metallic bond and thus the band gap first expands and then shrinks; (ii) the change of the strength of the anharmonic phonon scattering process results in the variation of κL. As a result, these behaviors cause excellent thermoelectric properties. Our results provide insight into the thermal transport properties of skutterudites, meanwhile, forecast potential high pressure applications for thermoelectric materials.

Water-Regulated Lead Halide Perovskites Precursor Solution: Perovskite Structure Making and Breaking.

Identifying the impact of water on iodoplumbate complexes in various solutions is essential for linking the coordination environment of the perovskite precursor to its final perovskite solar cell (PSC) properties. In this study, we propose a digital twin approach based on X-ray absorption fine structure and molecular dynamic simulation to investigate the structure evolution of iodoplumbate complexes in precursor solutions as a function of storage time under a constant humidity environment. A full picture about what water does in the perovskite formation process is brought out, and the "making and breaking" role of water molecules is uncovered to link the structure of iodoplumbate complexes to its final properties. This study sheds light on a full picture about what water does in the perovskite formation process and the role of water, which will lead to developing water-involved strategies for consistent PSC fabrication under ambient conditions.

Fast extraction of three-dimensional nanofiber orientation from WAXD patterns using machine learning.

Structural disclosure of biological materials can help our understanding of design disciplines in nature and inspire research for artificial materials. Synchrotron microfocus X-ray diffraction is one of the main techniques for characterizing hierarchically structured biological materials, especially the 3D orientation distribution of their interpenetrating nanofiber networks. However, extraction of 3D fiber orientation from X-ray patterns is still carried out by iterative parametric fitting, with disadvantages of time consumption and demand for expertise and initial parameter estimates. When faced with high-throughput experiments, existing analysis methods cannot meet the real time analysis challenges. In this work, using the assumption that the X-ray illuminated volume is dominated by two groups of nanofibers in a gradient biological composite, a machine-learning based method is proposed for fast and automatic fiber orientation metrics prediction from synchrotron X-ray micro-focused diffraction data. The simulated data were corrupted in the training procedure to guarantee the prediction ability of the trained machine-learning algorithm in real-world experimental data predictions. Label transformation was used to resolve the jump discontinuity problem when predicting angle parameters. The proposed method shows promise for application in the automatic data-processing pipeline for fast analysis of the vast data generated from multiscale diffraction-based tomography characterization of textured biomaterials.

The Precise Detection of HER-2 Expression in Breast Cancer Cell via Au25 Probes.

Triple-negative breast cancer (TNBC) accounts for nearly one-quarter of all breast cancer cases, but effective targeted therapies for this disease remain elusive because TNBC cells lack the expression of the most common three receptors seen in other subtypes of breast cancers. The medium-term diagnosis of breast cancers is essential for development and prognosis. According to reports, patients with TNBC may be converted to a positive epidermal growth factor receptor 2(HER-2) after chemotherapy, and trastuzumab treatment will have a better prognosis. Therefore, it is important to accurately quantify the expression of HER-2 in breast cancer cells. Herein, we design a red fluorescent Au25 probe synthesized with BSA-biotin as the ligand, which is accurately quantified by HER-2 primary antibody-biotin using the avidin system. The quantitative detection of the expression of HER-2 in breast cancers is helpful for the companion diagnostic of breast cancer treatment and provides follow-up treatment.

Activatable fluorogenic probe for accurate imaging of ulcerative colitis hypoxia in vivo.

A simple but efficient fluorogenic probe is reported for accurate imaging of ulcerative colitis via hypoxia detection. The hypoxia produced by ulcerative colitis can lead to the upregulation of nitroreductase (NTR). NB-NO2 provides a unique response to NTR, enabling accurate imaging of Dextran sulphate sodium (DSS)-induced ulcerative colitis in vivo.

Acrylamide fragment inhibitors that induce unprecedented conformational distortions in enterovirus 71 3C and SARS-CoV-2 main protease.

RNA viruses are critically dependent upon virally encoded proteases to cleave the viral polyproteins into functional proteins. Many of these proteases exhibit a similar fold and contain an essential catalytic cysteine, offering the opportunity to inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold and a similar active site. The hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. We demonstrate that targeting the active site cysteine of Mpro can have profound allosteric effects, distorting secondary structures to disrupt the active dimeric unit.

A comparison of Remdesivir versus gold cluster in COVID-19 animal model: A better therapeutic outcome of gold cluster.

While gold compound have been approved for Rheumatoid arthritis treatment as it well suppresses inflammatory cytokines of patients, no such treatment is currently available for COVID-19 treatment in vivo . We firstly disclose gold cluster yields better therapeutic outcome than Remdesivir in COVID-19 hamster treatments as it is armed with direct inhibition viral replication and intrinsic suppression inflammatory cytokines expression. Crystal data reveals that Au (I), released from gold cluster (GA), covalently binds thiolate of Cys145 of SARS-CoV-2 Mpro. GA directly decreases SARS-CoV-2 viral replication and intrinsically down-regulates NFκB pathway therefore significantly inhibiting expression of inflammatory cytokines in cells. The inflammatory cytokines in GA-treated COVID-19 transgenic mice are found to be significantly lower than that of control mice. When COVID-19 golden hamsters are treated by GA, the lung inflammatory cytokines levels are significantly lower than that of Remdesivir. The pathological results show that GA treatment significantly reduce lung inflammatory injuries when compared to that of Remdesivir-treated COVID-19 hamsters.

Metal Cluster-Based Electrochemical Biosensing System for Detecting Epithelial-to-Mesenchymal Transition.

N-cadherin serves as an important oncobiomarker of epithelial-to-mesenchymal transition (EMT) progression, which identifies invasion and metastasis of malignant tumor cells. Although many efforts have been devoted to quantitative detection of N-cadherin, efforts to analyzing the protein of interest at intact cellular levels are scarce. Herein, a metal cluster-based electrochemical biosensing system is developed to determine the expressing levels of N-cadherin during the EMT process of tumor cells. To be specific, a peptide with a unique sequence and function is designed as a reductant and an anchor to synthesize metal clusters in a precise manner. Consequently, peptide-modified metal clusters possess N-cadherin-targeting, photoluminescence, and electrocatalytic properties. Especially, the redox-active metal clusters function as both an electron-transfer mediator and an electronic conductor for enhanced electrochemical sensing. These favorable features enable them as a rapid, sensitive, and reliable whole-cell biosensor, which integrates the fluorescence and electrochemical signals. This cytosensor can accurately quantify the expression levels of N-cadherin on at least 5000 tumor cells. Further, the current signals of model cancer cells gradually increase with EMT progression, indicating tumor cell-type evolution. Our study represents the advanced bioprobe and analytical methods for accurate quantitation of a biomarker to identify tumor progression.

Differential regulation and the underlying mechanisms of clay minerals to Escherichia coli under the stress of polymyxin B: Comparing halloysite with kaolinite.

The reuse of polymyxin B (PMB) has attracted extensive attention. Although the resistance mechanism to PMB is clear, there are few reports on the regulation mechanisms and effects of clay minerals on bacteria induced by PMB. The focus of this study is to investigate the multidrug resistance, cell morphology and physiological modification of Escherichia coli (E. coli) exposed to PMB in the presence and absence of clay minerals. To be specific, E. coli was cultured serially for 15 days in the increasing concentration of PMB, with or without halloysite or kaolinite. The potential influence mechanisms of halloysite and kaolinite on E. coli was analyzed by proteomics, antibiotic resistance testing, confocal laser scanning microscopy, scanning electron microscopy and Fourier transform infrared. The results showed that kaolinite could obviously promote the growth of bacteria. Moreover, compared with halloysite, kaolinite could stimulate the overexpression of PMB resistance-related proteins ArnA, ArnB and EptA in E. coli exposed to PMB, and promote the synthesis of peptidoglycan and activate glycolysis pathway to produce energy. In contrast, halloysite was able to regulate the production of low molecular weight thiols by E. coli to prevent bacteria from producing excessive reactive oxygen species, activate the oxidative phosphorylation pathway to supply energy for bacterial life activities, and reduce multidrug resistance of E. coli in a variety of ways. These findings are essential for exploring the impacts of clay minerals on the emergence and spread of multi-drug resistant strains in the environment.

Enhancing peroxymonosulfate activation of Fe-Al layered double hydroxide by dissolved organic matter: Performance and mechanism.

In this study, peroxymonosulfate (PMS) activation of FeAl layered double hydroxide (FeAl-LDH) was enhanced by compounding dissolved organic matter (DOM). The characterization and catalytic performance of FeAl-LDH and DOM-LDH were investigated. The results revealed that the physicochemical properties of DOM-LDH were superior to FeAl-LDH: (i) The higher proportion of Fe(II) was found in DOM-LDH, mainly existed in the form of trans-coordinated octahedral Fe(II); (ii) DOM-LDH showed a flower-like morphology with larger specific surface area, pore width and pore volume; (iii) More functional groups and surface oxygen vacancies were found in DOM-LDH. Moreover, DOM promoted the process of PMS activation by accelerating Fe(III) reduction with humic acid-like compounds. The results of electron paramagnetic resonance (EPR) and quenching experiments indicated that more reactive oxygen species (ROS) were generated in DOM-LDH/PMS system, •OH was considered as the dominant ROS for Bisphenol A (BPA) degradation. As a result, the degradation efficiency for BPA (20 mg L-1) in FeAl-LDH/PMS system was increased from 60% to 93% within 60 min after the introduction of DOM. This work is expected to facilitate the design and application of Fe(II)/PMS system for environmental protection.

An artificial metalloenzyme for catalytic cancer-specific DNA cleavage and operando imaging.

Metalloenzymes are promising anticancer candidates to overcome chemoresistance by involving unique mechanisms. To date, it is still a great challenge to obtain synthetic metalloenzymes with persistent catalytic performance for cancer-specific DNA cleavage and operando imaging. Here, an artificial metalloenzyme, copper cluster firmly anchored in bovine serum albumin conjugated with tumor-targeting peptide, is exquisitely constructed. It is capable of persistently transforming hydrogen peroxide in tumor microenvironment to hydroxyl radical and oxygen in a catalytic manner. The stable catalysis recycling stems from the electron transfer between copper cluster and substrate with well-matched energy levels. Notably, their high biocompatibility, tumor-specific recognition, and persistent catalytic performance ensure the substantial anticancer efficacy by triggering DNA damage. Meanwhile, by coupling with enzyme-like reactions, the operando therapy effect is expediently traced by chemiluminescence signal with high sensitivity and sustainability. It provides new insights into synthesizing biocompatible metalloenzymes on demand to visually monitor and efficiently combat specific cancers.

Peptide-Templated Gold Clusters as Enzyme-Like Catalyst Boost Intracellular Oxidative Pressure and Induce Tumor-Specific Cell Apoptosis.

Anticancer metallodrugs that aim to physiological characters unique to tumor microenvironment are expected to combat drug tolerance and side-effects. Recently, owing to the fact that reactive oxygen species' is closely related to the development of tumors, people are committed to developing metallodrugs with the capacity of improving the level of reactive oxygen species level toinduce oxidative stress in cancer cells. Herein, we demonstrated that peptide templated gold clusters with atomic precision preferably catalyze the transformation of hydrogen peroxide into superoxide anion in oxidative pressure-type tumor cells. Firstly, we successfully constructed gold clusters by rationally designing peptide sequences which targets integrin ανß3 overexpressed on glioblastoma cells. The superoxide anion, radical derived from hydrogen peroxide and catalyzed by gold clusters, was confirmed in vitro under pseudo-physiological conditions. Then, kinetic parameters were evaluated to verify the catalytic properties of gold clusters. Furthermore, these peptide decorated clusters can serve as special enzyme-like catalyst to convert endogenous hydrogen peroxide into superoxide anion, elevated intracellular reactive oxygen species levels, lower mitochondrial membrane potential, damage biomacromolecules, and trigger tumor cell apoptosis consequently.