Multiplexing Imaging of Closely Located Single-Nucleotide Mutations in Single Cells via Encoded in situ PCR.

Mutation accumulation in RNAs results in closely located single-nucleotide mutations (SNMs), which is highly associated with the drug resistance of pathogens. Imaging of SNMs in single cells has significance for understanding the heterogeneity of RNAs that are related to drug resistance, but the direct "see" closely located SNMs remains challenging. Herein, we designed an encoded ligation-mediated in situ polymerase chain reaction method (termed enPCR), which enabled the visualization of multiple closely located SNMs in bacterial RNAs. Unlike conventional ligation-based probes that can only discriminate a single SNM, this method can simultaneously image different SNMs at closely located sites with single-cell resolution using modular anchoring probes and encoded PCR primers. We tested the capacity of the method to detect closely located SNMs related to quinolone resistance in the gyrA gene of Salmonella enterica (S. enterica), and found that the simultaneous detection of the closely located SNMs can more precisely indicate the resistance of the S. enterica to quinolone compared to the detection of one SNM. The multiplexing imaging assay for SNMs can serve to reveal the relationship between complex cellular genotypes and phenotypes.

Precise, Sensitive Detection of Viable Foodborne Pathogenic Bacteria with a 6-Order Dynamic Range via Digital Rolling Circle Amplification.

The presence of viable pathogenic bacteria in food can lead to serious foodborne diseases, thus posing a risk to human health. Here, we develop a digital rolling circle amplification (dRCA) assay that enables the precise and sensitive quantification of viable foodborne pathogenic bacteria. Directly targeting pathogenic RNAs via a ligation-based padlock probe allows for precisely discriminating viable bacteria from dead one. The one-target-one-amplicon characteristic of dRCA enables high sensitivity and a broad quantitative detection range, conferring a detection limit of 10 CFU/mL and a dynamic range of 6 orders. dRCA can detect rare viable bacteria, even at a proportion as low as 0.1%, which is 50 times more sensitive than the live/dead staining method. The high sensitivity for detecting viable bacteria accommodates dRCA for assessing sterilization efficiency. Based on the assay, we found that, for pasteurization, slightly elevating the temperature to 68 °C can reduce the heating time to 10 min, which may minimize nutrient degradation caused by high-temperature exposure. The assay can serve as a precise tool for estimating the contamination by viable pathogenic bacteria and assessing sterilization, which facilitates food safety control.

Penthorum chinense Pursh leaf tea debittering mechanisms via green tea manufacturing process and its influence on NAFLD-alleviation activities.

The green-tea manufacturing process showed good effect of flavor improving, debittering and shaping in making Penthorum chinensePursh leaf (PL) tea (PLT), which serves as a polyphenol dietary supplement and beverage raw material. GC-MS results showed that its unpleasant grassy odor decreased by 42.8% due to dodecanal, geranylacetone, and (E)-2-nonenal reduction, coupled with 1-hexadecanol increasing. UPLC-ESI-TOF-MS identified 95 compounds and showed that the debittering effect of green-tea manufacturing process was attributed to decreasing of flavonols and lignans, especially quercetins, kaempferols and luteolins, and increasing of dihydrochalcones which act as sweeteners bitterness-masking agents, while astringency was weakened by reducing delphinidin-3,5-O-diglucoside chloride, kaempferol-7-O-ß-d-glucopyranoside, and tannins. The increase of pinocembrins and catechins in aqueous extracts of PLT, maintained its hepatoprotective, NAFLD-alleviation, and hepatofibrosis-prevention activities similar to PL in high fat-diet C57BL/6 mice, with flavonoids, tannins, tannic acids, and some newfound chemicals, including norbergenin, gomisin K2, pseudolaric acid B, tanshinol B, as functional ingredients.

Single-Cell Genotyping of Single-Nucleotide Mutations Using In Situ Allele-Specific Loop-Mediated Isothermal Amplification.

Single-nucleotide mutations (SNMs) in the bacterial genome may cause antibiotic resistance. The visualization of SNMs can indicate antibiotic resistance phenotypes at the single-cell level but remains challenging. Herein, we proposed an in situ allele-specific isothermal amplification proceeded inside cells, allowing us to image bacterial genes with single-nucleotide resolution. The primer for loop-mediated isothermal amplification (LAMP) was designed with artificial mismatch bases to serve as an allele-specific probe, endowing LAMP to specifically amplify genes with SNMs. Due to the high amplification efficiency of LAMP, the method termed AlleLAMP can generate high gain for imaging SNMs and precisely quantify mutated quinolone-resistant Salmonella in bacterial mixture. We utilized AlleLAMP to survey the selection of antibiotic resistance under the preservative stress and found that the mutant quinolone-resistant strain owned a survival advantage over the wild-type quinolone-sensitive strain under the stress of preservatives. AlleLAMP can serve as a single-cell tool for analyzing the relationship between bacterial genotype and phenotype.

Single Photon Emitters with Polarization and Orbital Angular Momentum Locking in Monolayer Semiconductors.

Excitons in monolayer transition metal dichalcogenide are endowed with intrinsic valley-orbit coupling between their center-of-mass motion and valley pseudospin. When trapped in a confinement potential, e.g., generated by strain field, we find that intralayer excitons are valley and orbital angular momentum (OAM) entangled. By tuning the trap profile and external magnetic field, one can engineer the exciton states at the ground state and realize a series of valley-OAM entangled states. We further show that the OAM of excitons can be transferred to emitted photons, and these novel exciton states can naturally serve as polarization-OAM locked single photon emitters, which under certain circumstance become polarization-OAM entangled, highly tunable by strain trap and magnetic field. Our proposal demonstrates a novel scheme to generate polarization-OAM locked/entangled photons at the nanoscale with a high degree of integrability and tunability, pointing to exciting opportunities for quantum information applications.

Evaluation of Enterprise Green Mine Construction Based on DPSIR Model.

As a new mode of mining development, green mine optimizes the development and utilization of mineral resources with a minimum of the environmental impact, and how to objectively evaluate the construction level of the green mine has become the key to promote green mine construction and it has also been an important path to achieve sustainable development of mineral resources. The evaluation system and methods of green mine construction, however, are not perfect at present as the existing green mine evaluation mostly adopts the index scoring accumulation method, with which the internal relations between the indicators are ignored, and the subjective influence it causes is too large. Based on the framework model of driving forces, pressure, state, impact and response, an indicator system is constructed in this paper to express the internal relationship between indicators more intuitively. Combined with subjective and objective combination weighting method to determine the index weight, TOPSIS and coupling coordination degree models are introduced to quantitatively evaluate the spatio-temporal evolution process of green mine construction and the coupling coordination between subsystems, analyze and obtain the main obstacle factors affecting the green mine construction of enterprises, and provide suggestions and countermeasures for the improvement of green mine construction of enterprises. The applicability of the model is verified by an actual case study of a mine in China. The model enriches the connotation of green mines, making the evaluation process and results fairer and more reliable, thus providing an effective way to promote the sustainable development of mines.

Primer-Engineered Transferase Enzyme for One-Pot and Amplified Detection of Cobalt Pollution and Peptide Remover Screening.

Extensive consumption of cobalt in the chemical field such as for battery materials, alloy, pigments, and dyes has aggravated the pollution of cobalt both in food and the environment, and assays for its on-site monitoring are urgently demanded. Herein, we utilized enzyme dependence on metal cofactors to develop terminal transferase (TdT) as a recognition element, achieving a one-pot sensitive and specific assay for detecting cobalt pollution. We engineered a 3'-OH terminus primer to improve the discrimination capacity of TdT for Co2+ from other bivalent cations. The TdT extension reaction amplified the recognition of Co2+ and yielded a limit of detection of 0.99 µM for Co2+ detection. Then, the TdT-based assay was designed to precisely detect cobalt in food and agricultural soil samples. By end-measurement of fluorescence using a microplate reader, the multiplexing assay enabled the rapid screening of the peptide remover for cobalt pollution. The TdT-based assay can be a promising tool for cobalt pollution monitoring and control.

Potential therapeutic effects and applications of Eucommiae Folium in secondary hypertension.

Eucommiae Folium (EF), a traditional Chinese medicine, has been used to treat secondary hypertension, including renal hypertension and salt-sensitive hypertension, as well as hypertension caused by thoracic aortic endothelial dysfunction, a high-fat diet, and oxidized low-density lipoprotein. The antihypertensive components of EF are divided into four categories: flavonoids, iridoids, lignans, and phenylpropanoids, such as chlorogenic acid, geniposide acid and pinoresinol diglucoside. EF regulates the occurrence and development of hypertension by regulating biological processes, such as inhibiting inflammation, regulating the nitric oxide synthase pathway, reducing oxidative stress levels, regulating endothelial vasoactive factors, and lowering blood pressure. However, its molecular antihypertensive mechanisms are still unclear and require further investigation. In this review, by consulting the relevant literature on the antihypertensive effects of EF and using network pharmacology, we summarized the active ingredients and pharmacological mechanisms of EF in the treatment of hypertension to clarify how EF is associated with secondary hypertension, the related components, and underlying mechanisms. The results of the network pharmacology analysis indicated that EF treats hypertension through a multi-component, multi-target and multi-pathway mechanism. In particular, we discussed the role of EF targets in the treatment of hypertension, including epithelial sodium channel, heat shock protein70, rho-associated protein kinase 1, catalase, and superoxide dismutase. The relevant signal transduction pathways, the ras homolog family member A (RhoA)/Rho-associated protein kinase (ROCK) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/eNOS/NO/Ca2+ pathways, are also discussed.

The explorations of dynamic interactions of paxillin at the focal adhesions.

Paxillin is one of the most important adapters in integrin-mediated adhesions that performs numerous crucial functions relying on its dynamic interactions. Its structural behavior serves different purposes, providing a base for several activities. The various domains of paxillin display different functions in the whole process of cell movements and have a significant role in cell adhesion, migration, signal transmission, and protein-protein interactions. On the other hand, some paxillin-associated proteins provide a unique spatiotemporal mechanism for regulating its dynamic characteristics in the tissue homeostasis and make it a more complex and decisive protein at the focal adhesions. This review briefly describes the structural adaptations and molecular mechanisms of recruitment of paxillin into adhesions, explains paxillin's binding dynamics and impact on adhesion stability and turnover, and reveals a variety of paxillin-associated regulatory mechanisms and how paxillin is embedded into the signaling networks.

Interaction Mechanisms and Application of Ozone Micro/Nanobubbles and Nanoparticles: A Review and Perspective.

Ozone micro/nanobubbles with catalytic processes are widely used in the treatment of refractory organic wastewater. Micro/nanobubble technology overcomes the limitations of ozone mass transfer and ozone utilization in the application of ozone oxidation, and effectively improves the oxidation efficiency of ozone. The presence of micro/nanobubbles keeps the catalyst particles in a dynamic discrete state, which effectively increases the contact frequency between the catalyst and refractory organic matter and greatly improves the mineralization efficiency of refractory organic matter. This paper expounds on the characteristics and advantages of micro/nanobubble technology and summarizes the synergistic mechanism of microbubble nanoparticles and the mechanism of catalyst ozone micro/nanobubble systems in the treatment of refractory organics. An interaction mechanism of nanoparticles and ozone microbubbles is suggested, and the proposed theories on ozone microbubble systems are discussed with suggestions for future studies on systems of nanoparticles and ozone microbubbles.

UPLC-MS analysis and network pharmacology-based investigation into the active ingredients and molecular mechanisms of anti-fatigue of male flowers with Eucommia ulmoides Oliv.

The male flowers of Eucommia ulmoides Oliv. (MFEU) was a natural product that could alleviate fatigue and accelerate fatigue alleviation. Nonetheless, the active ingredients and underlying pharmacological mechanisms remain unknown. This study aimed to decode the active ingredients and potential action mechanisms of MFEU in the therapy of anti-fatigue using an integrated UPLC-MS analysis, network pharmacology approach, and cell experiments. Characterizations of chemical constituents of MFEU extract were identified by UPLC-Q-TOF-MS. The corresponding drug targets were retrieved from the drug target database and used to construct the "composite-target-pathway" network. The Cytoscape was used to identify potential protein targets of these MFEU components, indicating that 24 anti-fatigue compounds in MFEU regulate 18 anti-fatigue-related targets in 10 signaling pathways. The 16 components of MFEU were verified at the cellular level. The results of cell experiments showed that MFEU extract (0.361 µg/ml), Caffeic acid, Deacetylasperulosidic acid, Naringenin, Acanthoside B, Geniposidic acid, Rutin, and Quercetin could promote testosterone secretion on Leydig cells at 50 µM. The MFEU extract and seven compounds in MFEU might play a role in anti-fatigue by participating in the regulation of testosterone secretion. Finally, the results of PCR analysis showed that MFEU promotes the secretion of testosterone, which is related to CYPIIa1 and 17ß-HSD, STAR in the signal pathway of testosterone synthesis. This study provides a basis for further exploring the anti-fatigue mechanism of MFEU, adopting the method of multi-compound and multi-target.

Dopamine-Decorated Ti3C2Tx MXene/Cellulose Nanofiber Aerogels Supported Form-Stable Phase Change Composites with Superior Solar-Thermal Conversion Efficiency and Extremely High Thermal Storage Density.

The exploitation of from-stable phase change materials (PCMs) with superior energy storage capacity and excellent solar-thermal conversion performance is crucial for the efficient exploitation of solar energy. Herein, 2D-layered polymerized dopamine-decorated Ti3C2Tx MXene nanosheets (P-MXene) with superior photothermal effects and excellent oxidation stability were synthesized from Ti3AlC2 particles by the selective etching and self-polymerization of dopamine. Then, novel biomass-derived PCM composites, eMPCMs, were fabricated by impregnating erythritol into P-MXene/cellulose nanofiber (CNF) hybrid aerogels. The porous and interconnected 3D aerogels adequately support erythritol and resist liquid leakage during thermal storage. Differential scanning calorimetry (DSC) results showed that the eMPCMs based on P-MXene/CNF aerogels exhibited an extremely high thermal storage density (325.4-330.6 J/g) and excellent PCM loading capacity (up to 1929%). The introduction of P-MXene nanosheets into eMPCMs significantly increased the solar-thermal conversion and storage efficiency, solar-thermal-electricity conversion capacity, and thermal conductivity of the synthesized PCM composites. Moreover, the P-MXene/CNF hybrid aerogel-based PCM composites possessed excellent long-term thermal reliability and thermostability. Hence, the synthesized eMPCMs reveal tremendous potential for efficient solar-thermal storage fields.

Label-Free Detection of Transgenic Crops Using an Isothermal Amplification Reporting CRISPR/Cas12 Assay.

Current tools for detecting transgenic crops, such as polymerase chain reaction (PCR), require professional equipment and complex operation. Herein, we introduce a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system to analyze transgenes by designing an isothermal amplification to serve as the amplified reporter, allowing an isothermal and label-free detection of transgenic crops. The use of Cas12a allowed direct and specific recognition of transgenes. To enhance the sensitivity of the assay, we used rolling circle amplification (RCA) to monitor the recognition of transgenes by designing the RCA primer as the cleavage substrate of Cas12a. The presence of transgenes can be detected by monitoring the G-quadruplex in RCA amplicon using a G-quadruplex binding dye, N-methyl mesoporphyrin IX (NMM). We termed the assay as isoCRISPR and showed that the assay allowed distinguishing transgenic corn cultivars ("Bt11" and "MON89034") from nontransgenic corn cultivars ("yellow", "shenyu", "xianyu", and "jingke"). The isoCRISPR assay will enrich the toolbox for transgenic crop identification and broaden the application of CRISPR/Cas in food authenticity and safety.

Network pharmacology, molecular docking technology integrated with pharmacodynamic study to reveal the potential targets of Schisandrol A in drug-induced liver injury by acetaminophen.

Schisandrae Chinensis Fructus (SCF) was a Traditional Chinese Medicine for protecting liver. However, underlying therapeutic mechanisms of these bioactive lignans from SCF similar hepatoprotective effects against drug-induced liver injury (DILI) by acetaminophen (APAP) are still unclear. This study aims to discover the potential regulation mechanisms of Schisandrol A in the treatment of DILI by APAP. The integrated UPLC-Q-TOF/MS, pharmacodynamic study, histopathological combination with network pharmacology and molecular docking technology were used to explore the potential mechanisms. The results showed that Schisandrol A reduced the level of AST, ALT, MDA, PNP, TNF-α and IL-1ß, increased the levels of the GSH against acute liver failure. Additionally, Schisandrol A could improve the morphological characteristics of DILI by APAP in mice with liver tissue. Molecular docking results had showed that Schisandrol A with high scores when docking with COX-2, ALOX5, CYP2E1, CYP2C9, CYP2C19, EGFR SRC, Nrf2, MAPK14 and MAPK8. The study demonstrated that Schisandrol A could play critical roles in DILI by APAP via regulating TNF signaling pathway, inhibiting oxidative stress, inflammation and inhibiting the activities of cytochrome P450 enzymes, which contributed to searching for leading compounds and the development of new drugs for DILI by APAP.

Direct Detection of Foodborne Pathogens via a Proximal DNA Probe-Based CRISPR-Cas12 Assay.

Foodborne pathogens can cause illnesses. Existing tools for detecting foodborne pathogens are typically time-consuming or require complex protocols. Here, we report an assay to directly analyze pathogenic genes based on CRISPR-Cas12. This new test, termed proximal DNA probe-based CRISPR-Cas12 (PPCas12), facilitates the detection of foodborne pathogens without amplification steps. The elimination of the nucleic acid amplification process dramatically reduced the processing time, complexity, and costs in the analysis of foodborne pathogens. The substitution of the frequently used dually labeled DNA reporter with a proximal DNA probe in the PPCas12 assay led to a 4-fold sensitivity enhancement. PPCas12 offered a limit of detection of 619 colony-forming units in the detection of Salmonella enterica (S. enterica) without the nucleic acid amplification process. The specific recognition of genes via PPCas12 allowed distinguishing S. enterica from other foodborne pathogens. The PPCas12 assay was applied in the screening of S. enterica contamination on fresh eggs with high precision. Hence, the new PPCas12 assay will be a valuable tool for on-site monitoring of foodborne pathogens.

Recyclable, Self-Healing, and Flame-Retardant Solid-Solid Phase Change Materials Based on Thermally Reversible Cross-Links for Sustainable Thermal Energy Storage.

Conventional polymeric phase change materials (PCMs) exhibit good shape stability, large energy storage density, and satisfactory chemical stability, but they cannot be recycled and self-healed due to their permanent cross-linking structure. Additionally, the high flammability of organic PCMs seriously restricts their applications for thermal energy storage (TES). Therefore, it is urgently required to explore PCM composites exhibiting superior recyclability, good self-healing capability, and excellent flame retardancy simultaneously. Herein, tri-maleimide end-capped cyclotriphosphazene flame retardant (TMCTP) was synthesized via the nucleophilic substitution between 1,3,5,2,4,6-triazatriphosphorine-2,2,4,4,6,6-hexachloride and N-(2-hydroxyethyl)maleimide. Then, novel dynamically cross-linked PCM composites (FPCMs) with superior recyclability, good self-healing capability, and excellent flame retardancy were fabricated by bonding PEG and TMCTP to polymeric skeleton via reversible furan/maleimide Diels-Alder (DA) reaction. TMCTP, which covalently and dynamically binding in the polymeric FPCMs, acted not only as an efficient flame retardant for reducing the flammability of PCM composites but also as dynamic cross-linking skeletons for thermally induced self-healing and recycling. Differential scanning calorimetry (DSC) analysis confirmed the reversible energy storage and release ability of FPCMs. Due to its reversible DA covalent bonds, the introduction of TMCTP endowed the FPCMs with considerably increased self-healing efficiency (up to 93.1%) and recyclability efficiency (94.6%). Moreover, with the introduction of TMCTP into FPCMs, the heat release rate (HRR) and total heat release (THR) significantly decreased, while the char residue and limiting oxygen index (LOI) value increased, confirming that the flame retardancy of FPCMs greatly improved. Hence, the synthesized FPCMs show enormous potential in TES applications.

A Novel Coumarin-based Fluorescent Probe with Aggregation Induced Emission for Detecting CN- and its Applications in Bioimaging.

Although cyanogen ion (CN-) plays important role in industry which also bring acute environmental pollution. More serious, trace CN- enters the human body can cause serious consequences and even death. Therefore, it is of great significance to detect trace CN- with high sensitivity. Herein, a novel aggregation-induced emission (AIE) probe C-BH was synthesized based on coumarin matrix. Probe C-BH showed high selectivity and sensitivity toward CN- by dual channel response due to the excited state intramolecular proton transfer (ESIPT). The low detection limit was calculated to be 0.05 µM. Moreover, probe C-BH was successfully used for imaging CN- in living cells and zebrafish due to its low toxicity and excellent optical properties.

Amplification Refractory Mutation System (ARMS)-PCR for Waxy Sorghum Authentication with Single-Nucleotide Resolution.

Waxy sorghum has greater economic value than wild sorghum in relation to their use in food processing and the brewing industry. Thus, the authentication of the waxy sorghum species is an important issue. Herein, a rapid and sensitive Authentication Amplification Refractory Mutation System-PCR (aARMS-PCR) method was employed to identify sorghum species via its ability to resolve single-nucleotide in genes. As a proof of concept, we chose a species of waxy sorghum containing the wxc mutation which is abundantly used in liquor brewing. The aARMS-PCR can distinguish non-wxc sorghum from wxc sorghum to guarantee identification of specific waxy sorghum species. It allowed to detect as low as 1% non-wxc sorghum in sorghum mixtures, which ar one of the most sensitive tools for food authentication. Due to its ability for resolving genes with single-nucleotide resolution and high sensitivity, aARMS-PCR may have wider applicability in monitoring food adulteration, offering a rapid food authenticity verification in the control of adulteration.