Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems.

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.

An anti-CRISPR that pulls apart a CRISPR-Cas complex.

In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications1. Anti-CRISPRs-proteins that block the activity of CRISPR-Cas systems-are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms2-4. Here, we characterize the anti-CRISPR AcrIF25, and we show that it inhibits the type I-F CRISPR-Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications.

Anti-CRISPR AcrIF9 functions by inducing the CRISPR-Cas complex to bind DNA non-specifically.

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complementarity to the CRISPR RNA. Induction of non-specific DNA-binding is a shared property of diverse AcrIF9 homologues. Substitution of a conserved positively charged surface on AcrIF9 abrogated non-specific dsDNA-binding of the Csy:AcrIF9 complex, but specific dsDNA binding was maintained. AcrIF9 mutants with impaired non-specific dsDNA binding activity in vitro displayed a reduced ability to inhibit CRISPR-Cas activity in vivo. We conclude that misdirecting the CRISPR-Cas complex to bind non-specific DNA is a key component of the inhibitory mechanism of AcrIF9. This inhibitory mechanism is distinct from a previously characterized anti-CRISPR, AcrIF1, that sterically blocks DNA-binding, even though AcrIF1and AcrIF9 bind to the same site on the Csy complex.

Highly Effective OER Electrocatalysts Generated from a Two-Dimensional Metal-Organic Framework Including a Sulfur-Containing Linker without Doping.

Metal-organic frameworks (MOFs) with different topologies formed by the self-assembly of sulfur-containing inorganic ligands, cobalt ions, and large ligands can be used to prepare electrocatalysts for water splitting in order to fully explore the advantages of MOFs in terms of structural tailoring and quantitative assembly. It is possible to avoid using an extradoped sulfur source to reduce waste as well as to disperse Co and sulfur elements evenly and controllably throughout the final material to maximize the overall synergistic effect. In this work, different kinds of bimetallic MOF materials containing sulfur can be synthesized very conveniently by using an economical and practical diffusion method. These materials are directly used as OER electrocatalysts, and the bimetallic MOFs have the best electrocatalytic performance when the ratio of Co to Fe is 6:4. The overpotential at a current density of 10 mA cm-2 was 260 mV, with a Tafel slope of 56 mV dec-1 and good stability. It was assembled with 20% commercial Pt/C material into a two-electrode system for all-water decomposition, and the decomposition voltage at 10 mA cm-2 was 1.81 V. From the electronic configuration microscopic point of view, the introduction of iron ions changed the original synergistic effect for Co-S-Co, which more easily led to the formation of high-valence Co3+ and finally produced highly active electrocatalytic sites. From a macroscopic point of view, the material produced in situ during the electrochemical reaction process not only retains the original 2D layered structure but also utilizes bubbles to produce a loose structure with defective sites. These structural features are advantageous because they provide not only an abundance of active sites and permeable channels but also the necessary interfaces and electron-transport channels for the formation of electrostatic charge-separation layers, making it easier to intercalate and delaminate the hydroxide ions. Furthermore, the changed hydroxyl ions and nitrogen and sulfur atoms on the channel surface may operate as interaction sites, increasing the surface characteristics, facilitating electron transfer, and reducing electron-transfer resistance. To summarize, the rational design of sulfur-containing layered MOF materials directly as water-splitting catalysts is a crucial next step in developing cost-effective, environmentally friendly, and low-energy-consumption electrocatalysts based on the findings of this study.

Binding affinity and conformation of a conjugated AS1411 aptamer at a cationic lipid bilayer interface.

Aptamers have been widely used in the detection, diagnosis, and treatment of cancer. Owing to their special binding affinity toward cancer-related biomarkers, aptamers can be used for targeted drug delivery or bio-sensing/bio-imaging in various scenarios. The interfacial properties of aptamers play important roles in controlling the surface charge, recognition efficiency, and binding affinity of drug-delivering lipid-based carriers. In this research, the interfacial behaviors, such as surface orientation, molecular conformation, and adsorption kinetics of conjugated AS1411 molecules at different cationic lipid bilayer interfaces were investigated by sum frequency generation vibrational spectroscopy (SFG-VS) in situ and in real-time. It is shown that the conjugated AS1411 molecules at the DMTAP bilayer interface show a higher binding affinity but with slower binding kinetics compared to the DMDAP bilayer interface. The analysis results also reveal that the thymine residues of cholesteryl conjugated AS1411 molecules show higher conformational ordering compared to the thymine residues of the alkyl chain conjugated AS1411 molecules. These understandings provide unique molecular insight into the aptamer-lipid membrane interactions, which may help researchers to improve the efficiency and safety of aptamer-related drug delivery systems.

Ni17 W3 -W Interconnected Hybrid Prepared by Atmosphere- and Thermal-Induced Phase Separation for Efficient Electrocatalysis of Alkaline Hydrogen Evolution.

The development of efficient and stable noble-metal-free electrocatalysts for hydrogen evolution reaction (HER) in alkaline media is still a challenge. Herein, a hybrid material formed by the interconnection of Ni17 W3 intermetallic compound with metallic W is demonstrated for HER. The Ni17 W3 -W hybrid is prepared by the atmosphere- and thermal-induced phase-separation strategy from a single-phase precursor (NiWO4 ), which gives Ni17 W3 -W hybrid abundant and tight interfaces. The theoretical calculation manifests that Ni17 W3 shows more optimized energetics for adsorbed H atom, while W has lower energy barrier for water dissociation, and the synergistic effect between them is believed to facilitate the HER kinetics. Moreover, Ni17 W3 presents a proper adsorption strength for both adsorbed OH and H, and thus Ni17 W3 may also act as a high HER catalyst by itself. As a result, the Ni17 W3 -W hybrid demonstrates high activity and durability for HER in liquid alkaline electrolyte; the electrolyzer assembled by Ni17 W3 -W hybrid and Ni-Fe-layered double hydroxide (LDH) as, respectively, the cathode and anode electrocatalysts presents superior performance to Pt/C-IrO2 benchmark. In addition, the Ni17 W3 -W hybrid also works well in the water electrolyzer based on solid hydroxide exchange membrane. The present work provides a promising pathway to the design of high-performance electrocatalysts.

The electric double layer structure modulates poly-dT25 conformation and adsorption kinetics at the cationic lipid bilayer interface.

The conformation and adsorption kinetics of oligonucleotides at lipid membrane interfaces are crucial to their biological functions, but are yet not clearly understood. Poly-dT oligonucleotide molecules have been widely used as primers for reverse translation of RNA molecules, as well as a surface recognition agent for mRNA purification and extraction. In this research, the adsorption processes of poly-dT25 on lipid membranes in different ionic solutions were investigated by sum frequency generation vibrational spectroscopy (SFG-VS) together with a single molecule tracking technique in situ and in real time. These systematic studies provide us with molecular insight into the chemical and physical nature of oligonucleotide-membrane interactions, and show us how the electric double layer (EDL) structure changes the conformation and adsorption kinetics of oligonucleotides. The SFG-VS results indicate that an increase of ionic concentration not only decreases the adsorption density of oligonucleotides but also changes the conformation of oligonucleotides from an elongated conformation to a coiled conformation, causing stronger thermodynamic interactions with membranes, as demonstrated by single molecule tracking techniques. It is also shown that the ionic solution can tune the balance between the surface diffusion rate and solution diffusion rate of oligonucleotides significantly. These results demonstrated that the spectra and kinetics collected by in situ label-free SFG-VS detection and the single molecular tracking technique can provide new molecular insights into the mechanisms of oligonucleotide-membrane interactions. These new understandings may help researchers to control the assembly of oligonucleotide-liposome complexes and to improve the efficiency of transportation and delivery of oligonucleotide molecules.

Correction: Assembly and relaxation behaviours of phosphatidylethanolamine monolayers investigated by polarization and frequency resolved SFG-VS.

Correction for 'Assembly and relaxation behaviours of phosphatidylethanolamine monolayers investigated by polarization and frequency resolved SFG-VS' by Feng Wei et al., Phys. Chem. Chem. Phys., 2015, 17, 25114-25122.

Assembly and relaxation behaviours of phosphatidylethanolamine monolayers investigated by polarization and frequency resolved SFG-VS.

The assembly conformation and kinetics of phosphatidylethanolamine (PE) lipids are the key to their membrane curvatures and activities, such as exocytosis, endocytosis and Golgi membrane fusion. In the current study, a polarization and frequency resolved (bandwidth ≈ 1 cm(-1)) picosecond sum frequency generation (SFG) system was developed to characterize phosphatidylethanolamine monolayers. In addition to obtaining π-A isotherms and Brewster angle microscopy (BAM) images, the conformational changes and assembly behaviors of phosphatidylethanolamine molecules are investigated by analyzing the SFG spectra collected at various surface pressures (SPs). The compression kinetics and relaxation kinetics of phosphatidylethanolamine monolayers are also reported. The conformational changes of PE molecules during the monolayer compression are separated into several stages: reorientation of the head group PO2(-) in the beginning of the liquid-expanded (LE) phase, conformational changes of head group alkyl chains in the LE phase, and conformational changes of tail group alkyl chains in the LE-liquid condensed (LE-LC) phase. Such an understanding may help researchers to effectively control the lipid molecular conformation and membrane curvatures during the exocytosis/endocytosis processes.

Enzymatic fluorescent supramolecular hydrogel with aggregation-induced emission characteristics for sensing alkaline phosphatase.

Alkaline phosphatase is an important biomarker for medical diagnosis. An enzymatic fluorescence supramolecular hydrogel with AIE properties was developed and used for sensing alkaline phosphatase in vitro and in living cells. In the presence of ALP, K(TPE)EFYp was partially converted to the hydrogelator K(TPE)EFY and self-assembled into nanofibers to form Hydrogel. With the sol-gel transition and the AIE effect, the fluorescence emission was turned on. The linear concentration range of ALP activity in vitro quantified by this method was determined as 0-3 U/L with aLODat 0.02 U/L. In addition, cell imaging and serum experiment showed that K(TPE)EFYp could also be used to detect ALP activity in living cells and biological samples.

Cyclodextrin-conjugated low-molecular-weight polyethyleneimine as a macromolecular contrast agent for tumor-targeted magnetic resonance imaging.

Macromolecular contrast agents (CAs) usually possess excellent contrast ability and tumor-targeting ability in comparison with small-molecule CAs, especially for early tumor detection. Herein, cyclodextrin-conjugated low-molecular-weight polyethyleneimine was synthesized as a macromolecular backbone. Afterward, a linear polymer with adamantane terminal and Gd chelates was synthesized, followed by conjugating with the backbone via host-guest interaction. Finally, folic acid was conjugated onto the as-prepared CAs through bioorthogonal chemistry, which endowed the CAs with the capability to accumulate into the tumor region. Compared to Magnevist (r1 = 4.25 mM-1 s-1) used in clinic, the PC/Ad-PEG2000-PLL(DTPA-Gd)-FA exhibited higher longitudinal relaxivity (r1 = 11.62 mM-1 s-1) with excellent biocompatibility. Furthermore, in vivo experiments demonstrated that PC/Ad-PEG2000-PLL(DTPA-Gd)-FA could effectively accumulate in the tumor region and produce a brighter image than that of Magnevist. The H&E staining and metabolic data further illustrated that this CA possessed excellent biocompatibility in vivo. Finally, these results above suggest that this macromolecular CA could be a potential candidate as a MRI CA for tumor-targeted diagnosis.

Efficient recovery of gold from solution with a thiocyanate-modified Zr-MOF: adsorption properties and DFT calculations.

The design and development of new large-capacity and selective materials for extracting rare precious metals via electronic waste is practically essential. In this paper, a new efficient UiO-66-NCS has been obtained as a consequence of the modification of the classical Zr-MOF (UiO-66-NH2), and its ability to recover gold has been investigated. These batch results adequately illustrated that UiO-66-NCS exhibited good adsorption capacity (675.5 mg g-1) and exceptional selectivity. In addition, UiO-66-NCS achieved faster adsorption equilibrium times of about 120 min. Adsorption kinetics and isotherms demonstrated that the pseudo-second-order adsorption scheme and a Langmuir-type procedure were shown by the adsorption of Au(III) on UiO-66-NCS. Characterized by pH effect experiments, TEM, XRD, and XPS, the adsorption of UiO-66-NCS with Au(III) relies on coordination, which further results in reduction, and the generated Au(0) is uniformly dispersed in the MOF. The adsorbent has considerable advantages for cyclic regeneration. Finally, DFT fitting results showed that the adsorption binding energy of UiO-66-NCS with [AuCl4]- was -8.63 kcal mol-1 for the adsorption process. UiO-66-NCS is likely to be an ideal substance for gold recovery.

Ni/TiO2 heterostructures derived from phase separation for enhanced electrocatalysis of hydrogen evolution and biomass oxidative upgrading in anion exchange membrane electrolyzers.

The construction of heterostructures is an effective strategy to enhance electrocatalysis for hydrogen evolution reactions (HERs) and biomass oxidative upgrading. In this work, a Ni/TiO2 heterostructure prepared by a phase-separation strategy was adopted as a bifunctional electrocatalyst for HERs and biomass oxidation in alkaline media. Due to the optimized hydrogen adsorption energetics as well as the interfacial water structure and hydrogen bond connectivity in the electrical double layer, Ni/TiO2 exhibited high activity for HERs with an overpotential of 28 mV at 10 mA cm-2 and good durability at 1000 mA cm-2 for over 100 h in an anion exchange membrane (AEM) electrolyzer. In addition, Ni/TiO2 showed high catalytic performance for the oxidation of biomass-based platform compound 5-hydroxymethylfurfural (HMF) to high-value added compound 2,5-furandicarboxylic acid (FDCA). Continuous production of FDCA with a yield >95% was achieved in the AEM electrolyzer for over 50 h. The superior HMF oxidation performance on the Ni/TiO2 heterostructure compared to Ni resulted from stronger HMF adsorption, lower Ni3+-O formation potential, longer Ni3+-O bond and smaller Ni crystal size.

Recent Progress on Natural α-glucosidase Inhibitors Derived from the Plants and Microorganisms.

α-Glucosidase inhibitors (AGIs) showcase versatile biochemical activities with respect to antidiabetic, anticancerous, antiobese and antiviral effects. They have drawn a great deal of attention from the scientific community. While α-glucosidase inhibitors are mostly discovered from plants and microorganisms, the recent advance in natural αglucosidase inhibitors over the past five years has been reviewed in this article, and 139 distinct α-glucosidase inhibitors from the plants and microorganisms were classified into ten groups based on their chemical structures, including flavonoids (34), xanthones (6), alkaloids (8), benzopyrones / benzofuranones (8), terpenes (23), saponins (8), phenols / alcohols (25), esters (18), chalcone (5) and other compounds (4). In this review, we mainly focused on the novel chemical structures and the various biological activities of theses natural AGIs. Some of the selected natural compounds exhibit powerful α-glucosidase inhibitory activity and anti-tumor activity, may hold promise to become the candidate drugs for treating type II diabetes and cancer in future.

Magnesium Ion Responses of Zwitterionic Phosphatidylethanolamine Head and Tail Groups Elucidated by Frequency-Resolved SFG-VS.

In the present study, the effects of magnesium ions on the conformational changes of the deuterated 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (D54-DMPE) monolayer were elucidated by frequency-resolved sum frequency generation vibrational spectroscopy (SFG-VS) and surface pressure-area isotherm measurements. It is found that the tilt angles of the methyl in tail groups decrease, while the tilt angles of the phosphate and methylene in head groups increase during the compression of the DMPE monolayers at both the air/water interface and the air/MgCl2 solution interfaces. It is also shown that the tilt angle of the methyl in the tail groups slightly decreases, while the tilt angles of the phosphate and methylene in the head groups significantly increase as the MgCl2 concentration increases from 0 to 1.0 M. These results indicate that both the tail groups and the head groups of the DMPE molecules become closer to the surface normal, as the MgCl2 concentration increases in the subphase.

In situ preparation of a Co4S3-based electrocatalyst by taking advantage of the controllable components of metal-organic frameworks.

In order to give full play to the advantages in structure tailoring and quantitative assembly, metal-organic frameworks (MOFs) with different topological structures formed by the self-assembly of inorganic ligands containing sulfur, cobalt ions and large-size ligands were used to prepare electrocatalyst materials for hydrolysis with controllable composition and performance. According to the synthesis proposition, we can not only avoid using additional doped sulfur sources to reduce waste but also make it very convenient for Co and sulfur elements to be uniformly and controllably distributed in the whole material, and enhance the overall synergistic effects. Based on the above considerations, two-dimensional layered and three-dimensional MOFs, Co-MOF-1, and Co-MOF-2, with the same chemical compositions were utilized as the templates, and a series of Co/S-based materials with variable compositions and properties were obtained only by controlling the pyrolysis temperature. For each MOF series, it can be observed that with the increase in the pyrolysis temperature, the derivatives gradually change from Co4S3 to Co9S8 composites, which could be proven by PXRD studies. The electrocatalytic properties of two series of derivatives were also investigated, and the results indicate that the materials containing Co4S3 can indeed show better water-splitting performance than Co9S8 ones. Furthermore, the macroscopic stacking form of the MOF template also plays an important role in determining the electrocatalytic performance of the derived materials. Through an overall comparison, it is found that the electrocatalytic performance of the Co-MOF-1 series is better than that of the Co-MOF-2 series at various temperatures, which should be only caused by the natural packing modes of the pristine MOF template. For Co-MOF-1 derivatives, the retention of the two-dimensional layered structure is favorable to form an electrostatic charge separation layer and electron transport channel, which is beneficial to the intercalation and delamination of hydroxide ions, thus improving the storage capacity of materials, promoting electron transfer, and producing less electron transfer resistance. Therefore, based on the research results, the reasonable design of layered MOF materials containing the specific sulfur-containing linker as water-splitting catalysts is an applicable route for the preparation of economical, environmentally friendly, and low energy consumption electrocatalysts, which should receive increasing attention in the future.

In-situ Electrochemical Transformed Cu Oxide from Cu Sulfide for Efficient Upgrading of Biomass Derived 5-Hydroxymethylfurfural in Anion Exchange Membrane Electrolyzer.

The electrochemical transformation of biomass to high value-added products is attractive. Herein, Cu sulfide-mediated in-situ synthesis of Cu oxide was achieved for efficient electro-oxidation of biomass derived 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA). The copper foam-supported Cu sulfide (Cu-S/CF) was in-situ converted to Cu oxide during the electro-oxidation process. The in-situ formed Cu oxide presented high HMF conversion, FDCA yield, and faradaic efficiency in 1 m KOH with HMF concentration up to 100 mm. The oxidation of HMF on Cu oxide started with the formation of high-valence Cu species with the assistance of OH- , which then oxidized HMF spontaneously. An anion exchange membrane (AEM) electrolyzer with Cu-S/CF as the anode was assembled to continuously produce FDCA with H2 generation at the cathode. The AEM electrolyzer ran stably for 60 h with FDCA content higher than 85 % at a cell voltage between 1.50 and 1.60 V.

A sensitive platform for DNA detection based on organic electrochemical transistor and nucleic acid self-assembly signal amplification.

Highly sensitive detection of DNA is of great importance for the detection of genetic damage and errors for the diagnosis of many diseases. Traditional highly sensitive organic electrochemical transistor (OECT)-based methods mainly rely on good conductivity materials, which may be limited by complex synthesis and modification steps. In this work, DNA biosensor based on OECT and hybridization chain reaction (HCR) signal amplification was demonstrated for the first time. Au nanoparticles were electrochemically deposited on the Au gate electrode to increase the surface area. Then, the HCR products, long negatively charged double-stranded DNA, were connected to the target by hybridization, which can increase the effective gate voltage offset of OECT. This sensor exhibited high sensitivity and even 0.1 pM target DNA could be directly detected with a significant voltage shift. In addition, it could discriminate target DNA from the mismatched DNA with good selectivity. This proposed method based on HCR in DNA detection exhibited an efficient amplification performance on OECT, which provided new opportunities for highly sensitive and selective detection of DNA.

Amorphous Ni-Fe-Mo Suboxides Coupled with Ni Network as Porous Nanoplate Array on Nickel Foam: A Highly Efficient and Durable Bifunctional Electrode for Overall Water Splitting.

It is a great challenge to fabricate electrode with simultaneous high activity for the hydrogen evolution reaction (HER) and the oxygen evolution reaction (OER). Herein, a high-performance bifunctional electrode formed by vertically depositing a porous nanoplate array on the surface of nickel foam is provided, where the nanoplate is made up by the interconnection of trinary Ni-Fe-Mo suboxides and Ni nanoparticles. The amorphous Ni-Fe-Mo suboxide and its in situ transformed amorphous Ni-Fe-Mo (oxy)hydroxide acts as the main active species for HER and OER, respectively. The conductive network built by Ni nanoparticles provides rapid electron transfer to active sites. Moreover, the hydrophilic and aerophobic electrode surface together with the hierarchical pore structure facilitate mass transfer. The corresponding water electrolyzer demonstrates low cell voltage (1.50 V @ 10 mA cm-2 and 1.63 V @ 100 mA cm-2) with high durability at 500 mA cm-2 for at least 100 h in 1 m KOH.

AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex.

Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.