Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate.

Type III CRISPR systems are innate immune systems found in bacteria and archaea, which produce cyclic oligoadenylate (cOA) second messengers in response to viral infections. In these systems, Csm6 proteins serve as ancillary nucleases that degrade single-stranded RNA (ssRNA) upon activation by cOA. In addition, Csm6 proteins also possess cOA-degrading activity as an intrinsic off-switch to avoid degradation of host RNA and DNA that would eventually lead to cell dormancy or cell death. Here, we present the crystal structures of Thermus thermophilus (Tt) Csm6 alone, and in complex with cyclic tetra-adenylate (cA4) in both pre- and post-cleavage states. These structures establish the molecular basis of the long-range allosteric activation of TtCsm6 ribonuclease by cA4. cA4 binding induces significant conformational changes, including closure of the CARF domain, dimerization of the HTH domain, and reorganization of the R-X4-6-H motif within the HEPN domain. The cleavage of cA4 by the CARF domain restores each domain to a conformation similar to its apo state. Furthermore, we have identified hyperactive TtCsm6 variants that exhibit sustained cA4-activated RNase activity, showing great promise for their applications in genome editing and diagnostics.

Fat analogue emulsions stabilized by peanut protein microgel particles: microscale and nanoscale structure and stabilization process analysis.

BACKGROUND: Biopolymer-based microgels are being regarded increasingly as promising building blocks in food applications. This study aimed to clarify the evolution process of the network for fat analogue emulsions stabilized by peanut protein isolate (PPI) microgel particles. It also investigated the interfacial structure and characteristics of emulsions (50% oil phase, w/w) stabilized by microgels under different pH conditions. RESULTS: There was an increasing interfacial adsorption capacity for PPI microgels over time (from 85.26% to the maximum of 89.78% at 24 h of storage) due to the aggregation of microgels around droplets and the development of cross-linking microgel chains between adjacent interfaces. The increased ß-sheet content (from 35.51% to 41.12%) of adsorbed microgels indicated unfolding and the enhanced aggregation of microgels, which led to stronger droplet interaction. The network evolution observed with different microscopes clarified the transition to a self-supporting emulsion. The uneven adsorption of large microgel aggregates at the oil-water interface promoted larger and deformed droplets, so more fat-like medium internal phase emulsion stabilized by PPI microgel could be obtained by adjusting the microgel pH to 4.5. The interfacial membranes observed by scanning electron microscopy were thicker and coarser at pH 3.0 and 4.5 than those at pH 7.0 and 9.0. The adsorption of PPI microgel aggregates enhanced the structural strength and improved emulsion stability. CONCLUSION: This work could form a basis for further studies relating physical properties to the design of plant protein-based fat analogues. © 2024 Society of Chemical Industry.

Edible polysaccharide-based oleogels and novel emulsion gels as fat analogues: A review.

Polysaccharide-based oleogels and emulsion gels have become novel strategies to replace solid fats due to safe and plentiful raw material, healthier fatty acid composition, controllable viscoelasticity, and more varied nutrition/flavor embedding. Recently, various oleogelation techniques and novel emulsion gels have been reported further to enrich the potential of polysaccharides in oil structuring, in which a crucial step is to promote the formation of polysaccharide networks determining gel properties through different media. Meanwhile, polysaccharide-based oleogels and emulsion gels have good oil holding, nutrient/flavor embedding, and 3D food printability, and their applications as fat substitutes have been explored in foods. This paper comprehensively reviews the types, preparation methods, and mechanisms of various polysaccharide-based oleogels and emulsion gels; meanwhile, the food applications and new trends of polysaccharide-based gels are discussed. Moreover, some viewpoints about potential developments and application challenges of polysaccharide-based gels are mentioned. In the future, polysaccharide-based gels may be flexible materials for customized nutritional foods and molecular gastronomy. However, it is still a challenge to select the appropriate oleogels or emulsion gels to meet the requirements of the products. Once this issue is addressed, oleogels and emulsion gels are anticipated to be used widely.

Regulate structure and properties of κ-carrageenan/konjac glucomannan composite hydrogel by filling effects of Quillaja saponin-stabilized solid lipid nanostructure.

κ-Carrageenan/konjac glucomannan (κ-CA/KGM) composite hydrogels often fail to meet industrial requirements due to their low gel strength and poor mechanical properties, while solid lipid nanoparticles are potential materials to address this challenge due to their good biocompatibility. In the study, we propose using Quillaja saponin-stabilized solid lipid nanoparticle (QSLN) as nanofillers to enhance properties of κ-carrageenan/konjac glucan (κ-CA/KGM) composite hydrogels, and with emphasis on the effect of QSLN filling concentration on the structure and properties of composite hydrogels and the possible mechanisms were investigated. The best performance of QSLN-filled composite hydrogels was achieved at the QSLN concentration of 2.4 %. QSLN was uniformly distributed in the hydrogel matrix and formed electrostatic interactions and hydrogen bonding interactions with the matrix at an appropriate filling level, which enhanced the textural and rheological properties of the hydrogel greatly. In addition, the results of low-field NMR experiments showed that the filling of QSLN reduced the water mobility by enhancing the entanglement of polymer chains in the hydrogel matrix, which improved the freeze-thaw stability and regulated the swelling and deswelling behavior of the composite hydrogel. However, with the increasing of QSLN filling concentration, the above improvements were weakened by the depletion of van der Waals interactions due to the large amount of QSLN aggregation and the weakening of electrostatic interaction. In turn, the hydrogel was found to modulate the crystalline behavior of QSLN by X-ray diffraction and differential scanning calorimeter monitoring. Overall, the optimal synergistic effect between structure and properties could be achieved when the QSLN filling concentration was 2.4 %. These results provide a basis for the development of products that require excellent gel properties and structure.

A Nonlinear-Model-Based High-Bandwidth Current Sensor Design for Switching Current Measurement of Wide Bandgap Devices.

With the growing adoption of wide bandgap devices in power electronic applications, current sensor design for switching current measurement has become more important. The demands for high accuracy, high bandwidth, low cost, compact size, and galvanic isolation pose significant design challenges. The conventional modeling approach for bandwidth analysis of current transformer sensors assumes that the magnetizing inductance remains constant, which does not always hold true in high-frequency operations. This can result in inaccurate bandwidth estimation and affect the overall performance of the current sensor. To address this limitation, this paper provides a comprehensive analysis of nonlinear modeling and bandwidth, considering the varying magnetizing inductance in a wide frequency range. A precise and straightforward arctangent-based fitting algorithm was proposed to accurately emulate the nonlinear feature, and the fitting results were compared with the magnetic core's datasheet to confirm its accuracy. This approach contributes to more accurate bandwidth prediction in field applications. In addition, the droop phenomenon of the current transformer and saturation effects are analyzed in detail. For high-voltage applications, different insulation methods are compared and an optimized insulation process is proposed. Finally, the design process is experimentally validated. The bandwidth of the proposed current transformer is around 100 MHz and the cost is around $20, making it a low-cost and high-bandwidth solution for switching current measurements in power electronic applications.

Thermal-reversible lacquer wax-based oleofoams in dual stabilization with high ambient stability.

In this study, the effect of the content of the lacquer wax and whipping time on the overrun was explored. It was found that an appropriate amount of wax content and whipping time could promote crystal dual stabilization through the Pickering mechanism and the close packing in the bulk phase. Otherwise, it would result in low overrun caused by high viscous and crystal bridging. The addition of polyglycerol polyricinoleate (PGPR) could effectively enhance the overrun by apace absorbing. At the same time, adding PGPR also improved the contact angle, which was beneficial to the adsorption at the A-O interface. The 8 wt% oleogel was partially substituted by high-melting fat palm stearin (POs) and oleofoams were prepared based on blended fat. POs increased the melting point, structural strength, and ß'-form crystal of oleofoams, thus improving the storage and temperature stability. The oleofoam has a maximum overrun of 189% and could maintain the shape of the decorating over 15 d at the ambient temperature, showing great potential in low-fat food applications and other delivery systems.

Comparative study of natural wax-based W/O emulsion gels: Microstructure and macroscopic properties.

In this paper, six kinds of natural wax, including sunflower wax (SFX), rice bran wax (RBX), carnauba Brazilian wax (CBX), beeswax (BWX), candelilla wax (CDX), and sugarcane wax (SGX) were used to prepare water-in-oil (W/O) emulsion gels. Microstructures and rheological properties of all emulsion gels were investigated by microscopy, confocal laser scanning microscope (CLSM), scanning electron microscopy (SEM), and rheometer, respectively. By comparing polarized light images of wax-based emulsion gels and corresponding wax-based oleogels, it could be found that dispersed water droplets greatly affected the crystal distribution and hindered crystal growth. Polarized light microscopy and CLSM images proved that natural wax could perform a dual-stabilization mechanism by interfacial crystallization and crystal networks. SEM images illustrated all waxes except SGX were platelets and formed networks by stacking on top of each other, while flocs-like SGX was easier to adsorb on the interface and formed a "crystalline shell". The surface area and pore formed by different wax varied wildly, which accounted for their differences in the gelation ability, oil binding capacity, and strength of the crystal network. The rheological study showed that all wax had solid-like properties and wax-based oleogels with denser crystal networks correspond to emulsion gels with higher modules. The dense crystal network and interfacial crystallization could improve the stability of W/O emulsion gels proved by recovery rates and critical strain. All the above proved that natural wax-based emulsion gels can be used as stable, low-fat, and thermal-sensitive fat mimics.

Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081.

The cyclic oligoadenylates (cOAs) act as second messengers of the type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an 'off-switch' regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the founding member of CRISPR-associated ring nuclease 1 (Crn1) Sso2081 from Saccharolobus solfataricus, alone, bound to phosphate ions or cA4 in both pre-cleavage and cleavage intermediate states. These structures together with biochemical characterizations establish the molecular basis of cA4 recognition and catalysis by Sso2081. The conformational changes in the C-terminal helical insert upon the binding of phosphate ions or cA4 reveal a gate-locking mechanism for ligand binding. The critical residues and motifs identified in this study provide a new insight to distinguish between cOA-degrading and -nondegrading CARF domain-containing proteins.

A Hypoxia-Activated Prodrug Conjugated with a BODIPY-Based Photothermal Agent for Imaging-Guided Chemo-Photothermal Combination Therapy.

Hypoxia-activated prodrugs (HAPs) have drawn increasing attention for improving the antitumor effects while minimizing side effects. However, the heterogeneous distribution of the hypoxic region in tumors severely impedes the curative effect of HAPs. Additionally, most HAPs are not amenable to optical imaging, and it is difficult to precisely trace them in tissues. Herein, we carefully designed and synthesized a multifunctional therapeutic BAC prodrug by connecting the chemotherapeutic drug camptothecin (CPT) and the fluorescent photothermal agent boron dipyrromethene (BODIPY) via hypoxia-responsive azobenzene linkers. To enhance the solubility and tumor accumulation, the prepared BAC was further encapsulated into a human serum albumin (HSA)-based drug delivery system to form HSA@BAC nanoparticles. Since the CPT was caged by a BODIPY-based molecule at the active site, the BAC exhibited excellent biosafety. Importantly, the activated CPT could be quickly released from BAC and could perform chemotherapy in hypoxic cancer cells, which was ascribed to the cleavage of the azobenzene linker by overexpressed azoreductase. After irradiation with a 730 nm laser, HSA@BAC can efficiently generate hyperthermia to achieve irreversible cancer cell death by oxygen-independent photothermal therapy. Under fluorescence imaging-guided local irradiation, both in vitro and in vivo studies demonstrated that HSA@BAC exhibited superior antitumor effects with minimal side effects.

Ionic-liquid-based effervescence-enhanced magnetic solid-phase extraction for organophosphorus pesticide detection in water samples.

Herein, an ionic-liquid-based effervescence-enhanced magnetic solid-phase extraction (ILE-MSPE) approach for the extraction/concentration of organophosphorus pesticides in waters is reported with high stability and portability for rapid sample pretreatment in the field. The ionic-liquid-based magnetic effervescent tablet, composed of magnetic nanoparticles (Fe3O4), sodium carbonate (Na2CO3) as an alkaline source, and an ionic liquid ([C6MIM][PF6]), played triple functions: extractant, dispersant, and retrieving agent. Based on the one-factor-at-a-time method, the important variables for the ILE-MSPE approach were optimized as follows: as an extractant, 70 µL of [C6MIM][PF6]; molar ratio of alkaline to acidic sources (Na2CO3 : H2C4H4O6) as 1 : 1; and mass of magnetic nanoparticles (MNPs) of 30 mg. By integrating HPLC-DAD detection, the ILE-MSPE approach offered the limits of detection of 0.14-0.22 µg L-1 and fortified recoveries of 81.4-97.6% for three representative species (methamidophos, phoxim, and parathion) in water samples. The relative standard deviations were lower than 4.9% for both the intra-day and inter-day precision. Overall, the newly developed method is environmentally benign, time-saving, and feasible for outdoor application.

Discovery of a novel Aurora B inhibitor GSK650394 with potent anticancer and anti-aspergillus fumigatus dual efficacies in vitro.

Invasive fungal infections including Candidiasis and Aspergillosis are associated with considerable morbidity and mortality in immunocompromised individuals, such as cancer patients. Aurora B is a key mitotic kinase required for the cell division of eukaryotes from fungus to man. Here, we identified a novel Aurora B inhibitor GSK650394 that can inhibit the recombinant Aurora B from human and Aspergillus fumigatus, with IC50 values of 5.68 and 1.29 µM, respectively. In HeLa and HepG2 cells, GSK650394 diminishes the endogenous Aurora B activity and causes cell cycle arrest in G2/M phase. Further cell-based assays demonstrate that GSK650394 efficiently suppresses the proliferation of both cancer cells and Aspergillus fumigatus. Finally, the molecular docking calculation and site-directed mutagenesis analyses reveal the molecular mechanism of Aurora B inhibition by GSK650394. Our work is expected to provide new insight into the combinational therapy of cancer and Aspergillus fumigatus infection.

Synergetic effects of water-soluble polysaccharides for intensifying performances of oleogels fabricated by oil-absorbing cryogels.

Oleogels were prepared by the cryogel-templated method from porous cryogels, which were co-structured using hydroxypropyl-methylcellulose and structural enhancers (such as flaxseed gum, κ-carrageenan, carboxymethyl-cellulose, arabic gum, and guar gum). The hardness, network density, pore size, pore volume, and SEM micrographs of cryogels showed that κ-carrageenan and flaxseed gum could keep the integrity of aqueous foams during freeze-drying and endow cryogels with the high hardness and content to hold oils (>98%). Oil absorption curves indicated that flaxseed gum and guar gum-enhanced cryogels provided the fastest oil absorption rate due to bigger pores. The absorption model was fitted well with all experimental data. Physical and mechanical properties of cryogels were positively related to the rheological property and oil bonding capacity of oleogels. κ-carrageenan and flaxseed gum were more suitable as structural enhancers to improve hydroxypropyl-methylcellulose-based cryogels for preparing oleogels to replace plastic fats in foods.

Soft κ-carrageenan microgels stabilized pickering emulsion gels: Compact interfacial layer construction and particle-dominated emulsion gelation.

HYPOTHESIS: The well-known gelling ability of κ-carrageenan can make aqueous solutions into soft materials, which are crisp and can be mechanically treated into the nano-sized microgel particle (MP) as the building block for constructing the Pickering emulsion gel (PEG). MPs are expected to adhere and further create the network structure in PEGs due to their viscoelastic texture. Herein, properties of PEGs should be possibly altered by using MPs with different pH and ionic strength. EXPERIMENTS: MPs were prepared by shearing and gelling κ-carrageenan solutions simultaneously. Effects of pH and ionic strength on MPs were formulated, and physical properties of PEGs prepared from corresponding MPs were investigated. The interaction between κ-carrageenan molecules was analyzed by FTIR, and the formation process of the interfacial layer was traced by the interfacial rheological technique. FINDINGS: The moderate K+ could increase the flocculation and hardness of MPs to improve the viscoelasticity of PEGs. Prepared MPs were more favorable for forming PEGs when pH was 8 and 9. The oil fraction impacted physical properties of PEGs slightly. MPs constantly moved to the interface from the continuous phase, forming the compact adsorption layer due to the extrusion of MPs.

Effects of polar compounds in fried palm oil on liver lipid metabolism in C57 mice.

Polar components (PCs) are produced during the frying of oil, affecting the quality of edible oil and posing a hazard to human health. In this study, C57 mice were fed a high-fat (HF) diet containing purified PCs for nine weeks. Their effects on lipid metabolism and liver function in animals were analyzed. Our results indicated that the contents of total PCs and saturated fatty acid increased from 6.07 ± 0.6% and 58.27 ± 0.35% to 19.17 ± 1.8% and 69.91 ± 0.51%, respectively (P < 0.01). PC intake resulted an 18.56% higher liver index in mice than that in the HF group. The PC group had the highest malondialdehyde (MDA) content (1.94 ± 0.11 nmol/mg protein) and the liver nonalcoholic fatty liver disease (NAFLD) activity score (NAS) was 4, which already showed NAFLD characteristics. In addition, the expression levels of lipid metabolism-related genes, including sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthetase (FAS), peroxisome proliferator-activated receptor-alpha, and peroxisome acyl-CoA oxidase 1, indicated that PC increased hepatic lipid accumulation by upregulating the transcriptional level of fat synthesis genes and further leads to liver damage by affecting mitochondrial function. Our results provided important information about the effects of PCs produced in the frying process of PO on animal health, which is critical for assessing the biosafety of fried products. PRACTICAL APPLICATION: The research will help promote the industrial upgrading of fried foods and help consumers build healthy lifestyles.

Lipid metabolism disorder induced by up-regulation of miR-125b and miR-144 following ß-diketone antibiotic exposure to F0-zebrafish (Danio rerio).

ß-Diketone antibiotics (DKAs) are widely used in human and veterinary medicine to prevent and treat a large variety of infectious diseases. Long-term DKA exposure to zebrafish can result in lipid metabolism disorders and liver function abnormalities. Based on our previous miRNA-seq analyses, miR-144 and miR-125b were identified as target genes regulating lipid metabolism. DKA-exposure at 12.5 and 25 mg/L significantly increased the expressions of miR-144 and miR-125b. The expression levels for the two miRNAs exhibited an inverse relationship with their lipid-metabolism-related target genes (ppardb, bcl2a, pparaa and pparda). Over-expression and inhibition of miR-144 and miR-125b were observed by micro-injection of agomir-144, agomir-125b, antagomir-144 and antagomir-125b. The over-expression of miR-144 and miR-125b enhanced lipid accumulation and further induced lipid-metabolism-disorder syndrome in F1-zebrafish. The expression of ppardb and bcl2a in whole-mount in situ hybridization was in general agreement with results from qRT-PCR and was concentration-dependent. Oil red O and H&E staining, as well as related physiological and biochemical indexes, showed that chronic DKA exposure resulted in lipid-metabolism-disorder in F0-adults, and in F1-larvae fat accumulation, increased lipid content, abnormal liver function and obesity. The abnormal levels of triglyceride (TG) and total cholesterol (TCH) in DKA-exposed zebrafish increased the risk of hyperlipidemia, atherosclerosis and coronary heart disease. These observations improve our understanding of mechanisms leading to liver disease from exposure to environmental pollution, thereby having relevant practical significance in health prevention, early intervention, and gene therapy for drug-induced diseases.

A novel naphthalene carboxylic acid-based ionic liquid mixed disperser combined with ultrasonic-enhanced in-situ metathesis reaction for preconcentration of triclosan and methyltriclosan in milk and eggs.

A microextraction method was developed based on utilization of a novel ionic liquid (IL) [C4MIM][NCA] as disperser and conventional ILs as extractor (IL-IL-DLLME). This method was integrated with an in-situ metathesis reaction to achieve high extraction efficiency by eliminating the loss of analytes in the discarded disperser after microextraction. Ultrasonic energy was compared to traditional mechanical shaking to accelerate the in-situ metathesis reaction. A 3-min ultrasonic treatment provided similar extraction efficiency as a 120-min mechanical shaking. Due to their strong acidity and lower solubility than traditional hydrophilic ILs, utilization of [C4MIM][NCA] in the IL-IL-DLLME procedure increased extraction recoveries (ERs) for triclosan (TCS) and methyltriclosan (MTCS) by 10-12% and also avoided an extra pH adjustment step. A series of operational parameters were optimized using single-factor screening and central composite design as follows: 65 µL extraction solvent, 150 µL [C4MIM][BF4] and [C4MIM][NCA] (132/18, v/v, µL) as dispersive solvent, 0.16 g NH4PF6 and 3.3 min ultrasonic time. Under optimized conditions with a fortification of 100 µg kg-1, ERs were 92.6-93.4% for TCS and 92.7-94.2% for MTCS in bovine milk and chicken egg samples. LODs for TCS and MTCS were 0.16-0.24 µg kg-1 and the enrichment factors were 21.8-23.1. Inter- and intra-day precisions had relative standard deviations of 3.3-5.4% for the optimized method. Overall, this newly developed IL-IL-DLLME method was effective for detecting trace levels of TCS and MTCS in real-world, animal-based foods. Prominent advantages of the new method include high precision and accuracy, high extraction efficiency, simple analytical operations, and no use of organic solvents making the procedure environmentally benign.