Unraveling the influence of CRISPR/Cas13a reaction components on enhancing trans-cleavage activity for ultrasensitive on-chip RNA detection.

The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13's reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg2+ concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform.

Antimicrobial research of carbohydrate polymer- and protein-based hydrogels as reservoirs for the generation of reactive oxygen species: A review.

Reactive oxygen species (ROS) play an important role in biological milieu. Recently, the rapid growth in our understanding of ROS and their promise in antibacterial applications has generated tremendous interest in the combination of ROS generators with bulk hydrogels. Hydrogels represent promising supporters for ROS generators and can locally confine the nanoscale distribution of ROS generators whilst also promoting cellular integration via biomaterial-cell interactions. This review highlights recent efforts and progress in developing hydrogels derived from biological macromolecules with embedded ROS generators with a focus on antimicrobial applications. Initially, an overview of passive and active antibacterial hydrogels is provided to show the significance of proper hydrogel selection and design. These are followed by an in-depth discussion of the various approaches for ROS generation in hydrogels. The structural engineering and fabrication of ROS-laden hydrogels are given with a focus on their biomedical applications in therapeutics and diagnosis. Additionally, we discuss how a compromise needs to be sought between ROS generation and removal for maximizing the efficacy of therapeutic treatment. Finally, the current challenges and potential routes toward commercialization in this rapidly evolving field are discussed, focusing on the potential translation of laboratory research outcomes to real-world clinical outcomes.

Integrated network pharmacology and experimental validation to explore the potential pharmacological mechanism of Qihuang Granule and its main ingredients in regulating ferroptosis in AMD.

BACKGROUND: Qihuang Granule (QHG) is a traditional prescription  that has exhibited potential in safeguarding against age-related maculopathy (AMD). Salvia miltiorrhiza (SM) and Fructus lycii (FL) are the main components of QHG. Ferroptosis, a newly discovered, iron-dependent, regulated cell death pathway, have been implicated in the pathogenesis of AMD. This study delves into the intricate mechanism by which SM/FL and QHG confer protection against AMD by modulating the ferroptosis pathway, employing a combination of network pharmacology and experimental validation. METHODS: Bioactive compounds and potential targets of SM and FL were gathered from databases such as TCMSP, GeneCard, OMIM, and FerrDb, along with AMD-related genes and key genes responsible for ferroptosis regulation. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network were performed to discover the potential mechanism. The construction of an interaction network involving AMD, ferroptosis, SM/FL potential target genes was facilitated by the STRING database and realized using Cytoscape software. Subsequent validation was accomplished through molecular docking and in vitro cell experiments. RESULTS: Noteworthy active compounds including quercetin, tanshinone IIA, luteolin, cryptotanshinone, and hub targets such as HIF-1α, EGFR, IL6, and VEGFA were identified. KEGG enrichment unveiled the HIF-1 signalling pathway as profoundly enriched, and IL6 and VEGF were involved. The molecular docking revealed the significant active compounds with hub genes and quercetin showed good binding to HIF-1α, which is involved in inflammation and angiogenesis. Experimental results verified that both herbs and QHG could regulate key ferroptosis-related targets in the retinal pigment epithelium and inhibit the expression of HIF-1α, VEGFA, and IL-6, subsequently increase cell viability and decrease the ROS content induced by H2O2. CONCLUSION: This study demonstrates the molecular mechanism through which SM/FL and QHG protect against AMD and emerges as a plausible mechanism underlying this protection.

Ginsenosides Rc, as a novel SIRT6 activator, protects mice against high fat diet induced NAFLD.

Background: Hepatic lipid disorder impaired mitochondrial homeostasis and intracellular redox balance, triggering development of non-alcohol fatty liver disease (NAFLD), while effective therapeutic approach remains inadequate. Ginsenosides Rc has been reported to maintain glucose balance in adipose tissue, while its role in regulating lipid metabolism remain vacant. Thus, we investigated the function and mechanism of ginsenosides Rc in defending high fat diet (HFD)-induced NAFLD. Methods: Mice primary hepatocytes (MPHs) challenged with oleic acid & palmitic acid were used to test the effects of ginsenosides Rc on intracellular lipid metabolism. RNAseq and molecular docking study were performed to explore potential targets of ginsenosides Rc in defending lipid deposition. Wild type and liver specific sirtuin 6 (SIRT6, 50721) deficient mice on HFD for 12 weeks were subjected to different dose of ginsenosides Rc to determine the function and detailed mechanism in vivo. Results: We identified ginsenosides Rc as a novel SIRT6 activator via increasing its expression and deacetylase activity. Ginsenosides Rc defends OA&PA-induced lipid deposition in MPHs and protects mice against HFD-induced metabolic disorder in dosage dependent manner. Ginsenosides Rc (20mg/kg) injection improved glucose intolerance, insulin resistance, oxidative stress and inflammation response in HFD mice. Ginsenosides Rc treatment accelerates peroxisome proliferator activated receptor alpha (PPAR-α, 19013)-mediated fatty acid oxidation in vivo and in vitro. Hepatic specific SIRT6 deletion abolished ginsenoside Rc-derived protective effects against HFD-induced NAFLD. Conclusion: Ginsenosides Rc protects mice against HFD-induced hepatosteatosis by improving PPAR-α-mediated fatty acid oxidation and antioxidant capacity in a SIRT6 dependent manner, and providing a promising strategy for NAFLD.

Mining and Validation of Novel Hemp Seed-Derived DPP-IV-Inhibiting Peptides Using a Combination of Multi-omics and Molecular Docking.

Hemp seed-derived inhibitors of dipeptidyl peptidase IV (DPP-IV) demonstrate potential as novel therapeutics for diabetes; however, their proteome and genome remain uncharacterized. We used multi-omics technology to mine peptides capable of inhibiting DPP-IV. First, 1261 and 1184 proteins were identified in fresh and dry hemp seeds, respectively. Simulated protease cleavage of dry seed proteins yielded 185,446 peptides for virtual screening to select the potential DPP-IV-inhibiting peptides. Sixteen novel peptides were selected according to their DPP-IV-binding affinity determined via molecular docking. In vitro DPP-IV inhibition assays identified the peptides LPQNIPPL, YPYY, YPW, LPYPY, WWW, YPY, YPF, and WS with half-maximal inhibitory concentration (IC50) values lower than 0.5 mM, which were 0.08 ± 0.01, 0.18 ± 0.03, 0.18 ± 0.01, 0.20 ± 0.03, 0.22 ± 0.03, 0.29 ± 0.02, 0.42 ± 0.03, and 0.44 ± 0.09 mM, respectively. The dissociation constants (KD) of the 16 peptides ranged from 1.50 × 10-4 to 1.82 × 10-7 M. Furthermore, Caco2 and INS-1 cell assays showed that all 16 peptides could efficiently inhibit DPP-IV activity and increase insulin and glucagon-like peptide-1 concentrations. These results demonstrate a well-established and efficient method to isolate food-derived therapeutic DPP-IV-inhibiting peptides.

Base-free catalytic aerobic oxidation of mercaptans over MOF-derived Co/CN catalyst with controllable composition and structure.

The oxidation of mercaptans under mild and base-free conditions is of vital importance in terms of economy and environment for petroleum processing industry. Here, we developed a series of MOF-derived cobalt-based nitrogen-doped (N-doped) carbon (Co/CN-x) catalysts for the base-free catalytic oxidation of mercaptans. The optimal Co/CN-900 showed excellent catalytic activity for the oxidation of mercaptans under base-free conditions, yielding complete conversion of various mercaptans and > 99.0% selectivity of disulfides. The high performance can be contributed to the advantages of hierarchical pore structure for the diffusion and migration of substrates, self-carrying alkalinity for the formation of mercaptide anion, abundant active Co sites for catalytic oxidation of mercaptans as well as the synergistic effects between the Co nanoparticles (NPs) and N-doped carbon supports. Furthermore, a possible mechanism for base-free catalytic oxidation of mercaptans over Co/CN-x catalysts is proposed based on a set of control experiments and density functional theory (DFT) calculations.

Neutrophil-Based Drug Delivery Systems.

White blood cells (WBCs) are a major component of immunity in response to pathogen invasion. Neutrophils are the most abundant WBCs in humans, playing a central role in acute inflammation induced by pathogens. Adhesion to vasculature and tissue infiltration of neutrophils are key processes in acute inflammation. Many inflammatory/autoimmune disorders and cancer therapies have been found to be involved in activation and tissue infiltration of neutrophils. A promising strategy to develop novel targeted drug delivery systems is the targeting and exploitation of activated neutrophils. Herein, a new drug delivery platform based on neutrophils is reviewed. There are two types of drug delivery systems: neutrophils as carriers and neutrophil-membrane-derived nanovesicles. It is discussed how nanoparticles hijack neutrophils in vivo to deliver therapeutics across blood vessel barriers and how neutrophil-membrane-derived nanovesicles target inflamed vasculature. Finally, the potential applications of neutrophil-based drug delivery systems in treating inflammation and cancers are presented.

Quantitative structure-property relationship (QSPR) modeling of drug-loaded polymeric micelles via genetic function approximation.

Self-assembled nano-micelles of amphiphilic polymers represent a novel anticancer drug delivery system. However, their full clinical utilization remains challenging because the quantitative structure-property relationship (QSPR) between the polymer structure and the efficacy of micelles as a drug carrier is poorly understood. Here, we developed a series of QSPR models to account for the drug loading capacity of polymeric micelles using the genetic function approximation (GFA) algorithm. These models were further evaluated by internal and external validation and a Y-randomization test in terms of stability and generalization, yielding an optimization model that is applicable to an expanded materials regime. As confirmed by experimental data, the relationship between microstructure and drug loading capacity can be well-simulated, suggesting that our models are readily applicable to the quantitative evaluation of the drug-loading capacity of polymeric micelles. Our work may offer a pathway to the design of formulation experiments.