In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions.

How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.

Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer.

CD8+ cytotoxic T cells (CTLs) orchestrate antitumour immunity and exhibit inherent heterogeneity1,2, with precursor exhausted T (Tpex) cells but not terminally exhausted T (Tex) cells capable of responding to existing immunotherapies3-7. The gene regulatory network that underlies CTL differentiation and whether Tex cell responses can be functionally reinvigorated are incompletely understood. Here we systematically mapped causal gene regulatory networks using single-cell CRISPR screens in vivo and discovered checkpoints for CTL differentiation. First, the exit from quiescence of Tpex cells initiated successive differentiation into intermediate Tex cells. This process is differentially regulated by IKAROS and ETS1, the deficiencies of which dampened and increased mTORC1-associated metabolic activities, respectively. IKAROS-deficient cells accumulated as a metabolically quiescent Tpex cell population with limited differentiation potential following immune checkpoint blockade (ICB). Conversely, targeting ETS1 improved antitumour immunity and ICB efficacy by boosting differentiation of Tpex to intermediate Tex cells and metabolic rewiring. Mechanistically, TCF-1 and BATF are the targets for IKAROS and ETS1, respectively. Second, the RBPJ-IRF1 axis promoted differentiation of intermediate Tex to terminal Tex cells. Accordingly, targeting RBPJ enhanced functional and epigenetic reprogramming of Tex cells towards the proliferative state and improved therapeutic effects and ICB efficacy. Collectively, our study reveals that promoting the exit from quiescence of Tpex cells and enriching the proliferative Tex cell state act as key modalities for antitumour effects and provides a systemic framework to integrate cell fate regulomes and reprogrammable functional determinants for cancer immunity.

Metabolic control of TFH cells and humoral immunity by phosphatidylethanolamine.

T follicular helper (TFH) cells are crucial for B cell-mediated humoral immunity1. Although transcription factors such as BCL6 drive the differentiation of TFH cells2,3, it is unclear whether and how post-transcriptional and metabolic programs enforce TFH cell programming. Here we show that the cytidine diphosphate (CDP)-ethanolamine pathway co-ordinates the expression and localization of CXCR5 with the responses of TFH cells and humoral immunity. Using in vivo CRISPR-Cas9 screening and functional validation in mice, we identify ETNK1, PCYT2, and SELENOI-enzymes in the CDP-ethanolamine pathway for de novo synthesis of phosphatidylethanolamine (PE)-as selective post-transcriptional regulators of TFH cell differentiation that act by promoting the surface expression and functional effects of CXCR5. TFH cells exhibit unique lipid metabolic programs and PE is distributed to the outer layer of the plasma membrane, where it colocalizes with CXCR5. De novo synthesis of PE through the CDP-ethanolamine pathway co-ordinates these events to prevent the internalization and degradation of CXCR5. Genetic deletion of Pcyt2, but not of Pcyt1a (which mediates the CDP-choline pathway), in activated T cells impairs the differentiation of TFH cells, and this is associated with reduced humoral immune responses. Surface levels of PE and CXCR5 expression on B cells also depend on Pcyt2. Our results reveal that phospholipid metabolism orchestrates post-transcriptional mechanisms for TFH cell differentiation and humoral immunity, highlighting the metabolic control of context-dependent immune signalling and effector programs.

CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity.

Nutrients are emerging regulators of adaptive immunity1. Selective nutrients interplay with immunological signals to activate mechanistic target of rapamycin complex 1 (mTORC1), a key driver of cell metabolism2-4, but how these environmental signals are integrated for immune regulation remains unclear. Here we use genome-wide CRISPR screening combined with protein-protein interaction networks to identify regulatory modules that mediate immune receptor- and nutrient-dependent signalling to mTORC1 in mouse regulatory T (Treg) cells. SEC31A is identified to promote mTORC1 activation by interacting with the GATOR2 component SEC13 to protect it from SKP1-dependent proteasomal degradation. Accordingly, loss of SEC31A impairs T cell priming and Treg suppressive function in mice. In addition, the SWI/SNF complex restricts expression of the amino acid sensor CASTOR1, thereby enhancing mTORC1 activation. Moreover, we reveal that the CCDC101-associated SAGA complex is a potent inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains T cell quiescence in vivo. Specific deletion of Ccdc101 in mouse Treg cells results in uncontrolled inflammation but improved antitumour immunity. Collectively, our results establish epigenetic and post-translational mechanisms that underpin how nutrient transporters, sensors and transducers interplay with immune signals for three-tiered regulation of mTORC1 activity and identify their pivotal roles in licensing T cell immunity and immune tolerance.

Pipette-tip solid-phase extraction coupled with matrix-assisted laser desorption/ionization mass spectrometry enables rapid and high-throughput analysis of antidepressants in rat serum.

Therapeutic drug monitoring is essential for ensuring the efficacy and safety of medications. This study introduces a streamlined approach that combines pipette-tip solid-phase extraction (PT-SPE) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), facilitating rapid and high-throughput monitoring of drug concentrations. As a demonstration, this method was applied to the extraction and quantification of antidepressants in serum. Utilizing Zip-Tip C18, the method enabled the extraction of antidepressants from complex biological matrices in less than 2 min, with the subsequent MALDI-MS analysis yielding results in just 1 min. Optimal extraction recoveries were achieved using a sampling solution at pH 9.0 and a 10 µL ethanol desorption solution containing 0.1% phosphoric acid. For MALDI analysis, 2,5-dihydroxybenzoic acid was identified as the most effective matrix for producing the highest signal intensity. The quantification strategy exhibited robust linearities (R2 ≥ 0.997) and satisfactory limits of quantification, ranging from 0.05 to 0.5 µg/mL for a suite of antidepressants. The application for monitoring dynamic concentration changes of antidepressants in rat serum emphasized the method's efficacy. This strategy offers the advantages of high throughput, minimal sample usage, environmental sustainability, and simplicity, providing ideas and a reference basis for the subsequent development of methods for therapeutic drug monitoring.

Integration of Myrica rubra-based N-doped carbon dots with Fe3S4 as excellent peroxidase mimics for colorimetric assay and smartphone-based intelligent sensing of p-aminophenol in waters.

To realize the reutilization of waste Myrica rubra in the analytical field, we synthesized Myrica rubra-based N-doped carbon dots (MN-CDs) and further anchored them onto the surface of Fe3S4 to fabricate Fe3S4@MN-CD nanocomposites. The as-fabricated nanocomposites possessed higher peroxidase-mimetic activity than its two precursors, resulting from the synergistic effect between them, and could catalyze colorless 3,3',5,5'-tetramethylbenzidine (TMB) into deep blue oxTMB with a strong 652-nm absorption. Under optimized conditions (initial solution pH, 3.5; incubation temperature, 35 ℃; Fe3S4@MN-CD concentration, 50 µg mL-1, and 652-nm absorption), Fe3S4@MN-CDs were employed for colorimetric assay of p-aminophenol (p-AP) with wide linear range (LR, 2.9-100 µM), low detection limit (LOD, 0.87 µM), and satisfactory recoveries (86.3-105%) in environmental waters. Encouragingly, this colorimetric assay provided the relative accuracy of 97.0-99.4% as compared with  conventional HPLC-UV detection. A portable smartphone-based colorimetric application was developed by combining the Fe3S4@MN-CD-based visually chromogenic reaction with a "Thing Identify" APP software. Besides, we engineered an image-capturing device feasible for field use, in which the internal-compact sealing prevented external light source from entering photography chamber, thereby reducing light interference, and also the bottom light source enhanced the intensity of blue imaging. This colorimetric platform exhibited satisfactory LR (1-500 µM), low LOD (0.3 µM), and fortification recoveries (86.6-99.6%). In the chromogenic reaction catalyzed by Fe3S4@MN-CDs, ·O2- played a key role in concomitant with the participation of •OH and h+. Both the colorimetric assay and smartphone-based intelligent sensing show great promising in on-site monitoring of p-AP under field conditions.

mTOR signaling at the crossroads of environmental signals and T-cell fate decisions.

The evolutionarily conserved serine/threonine kinase mTOR (mechanistic target of rapamycin) forms the distinct protein complexes mTORC1 and mTORC2 and integrates signals from the environment to coordinate downstream signaling events and various cellular processes. T cells rely on mTOR activity for their development and to establish their homeostasis and functional fitness. Here, we review recent progress in our understanding of the upstream signaling and downstream targets of mTOR. We also provide an updated overview of the roles of mTOR in T-cell development, homeostasis, activation, and effector-cell fate decisions, as well as its important impacts on the suppressive activity of regulatory T cells. Moreover, we summarize the emerging roles of mTOR in T-cell exhaustion and transdifferentiation. A better understanding of the contribution of mTOR to T-cell fate decisions will ultimately aid in the therapeutic targeting of mTOR in human disease.

Classification and characterisation of extracellular vesicles-related tuberculosis subgroups and immune cell profiles.

Around the world, tuberculosis (TB) remains one of the most common causes of morbidity and mortality. The molecular mechanism of Mycobacterium tuberculosis (Mtb) infection is still unclear. Extracellular vesicles (EVs) play a key role in the onset and progression of many disease states and can serve as effective biomarkers or therapeutic targets for the identification and treatment of TB patients. We analysed the expression profile to better clarify the EVs characteristics of TB and explored potential diagnostic markers to distinguish TB from healthy control (HC). Twenty EVs-related differentially expressed genes (DEGs) were identified, and 17 EVs-related DEGs were up-regulated and three DEGs were down-regulated in TB samples, which were related to immune cells. Using machine learning, a nine EVs-related gene signature was identified and two EVs-related subclusters were defined. The single-cell RNA sequence (scRNA-seq) analysis further confirmed that these hub genes might play important roles in TB pathogenesis. The nine EVs-related hub genes had excellent diagnostic values and accurately estimated TB progression. TB's high-risk group had significantly enriched immune-related pathways, and there were substantial variations in immunity across different groups. Furthermore, five potential drugs were predicted for TB using CMap database. Based on the EVs-related gene signature, the TB risk model was established through a comprehensive analysis of different EV patterns, which can accurately predict TB. These genes could be used as novel biomarkers to distinguish TB from HC. These findings lay the foundation for further research and design of new therapeutic interventions aimed at treating this deadly infectious disease.

Ligand Torsion Engineering in Three Flexible Metal-Organic-Frameworks Exhibiting Distinct Breathing Behavior for Enhancing C2H2 Capture.

Acetylene is an important industrial gas for the production of vinyl chloride and 1,4-butynediol, but its storage remains a major challenge because it is highly explosive. Flexible metal-organic frameworks (FMOFs) are always at the forefront of porous materials due to the transformation of the structure under the external stimuli. In this work, divalent metal ions and multifunctional aromatic N,O-donor ligands were chosen, and three FMOFs [M(DTTA)2]·guest [M = Mn (1), Cd (2), and Cu (3)] (H2DTTA = 2,5-bis(1H-1,2,4-trazol-1-yl) terephthalic acid) have been successfully constructed. Single-crystal X-ray diffractions show that these compounds are isostructural and feature a three-dimensional framework. Topological analysis shows a (4, 6)-connected network with a Schläfli symbol of {44.610.8}{44.62}. All three compounds exhibit breathing behavior on N2 adsorption at 77 K, and due to the difference of ligand torsion angles, compounds 2 and 3 exhibit extraordinary adsorptions for C2H2 of 101 and 122 cm3 g-1 at 273 K under 1 bar, respectively. Compared with previous work, successfully obtaining compound 3 with an innovative structure can be attributed to the solvent-induced effect in the process of crystal synthesis, leading to the structure transformation promoting the significantly increased C2H2 adsorption performance. This study provides a platform for improvement of synthetic structures, which can effectively boost gas adsorption performance.

Highly efficient nanocomposites based on molecularly imprinted magnetic covalent organic frameworks for selective extraction of bisphenol A from liquid matrices.

Highly efficient nanocomposites, hydrophobic molecularly imprinted magnetic covalent organic frameworks (MI-MCOF), have been farbricated by a facile Schiff-base reaction. The MI-MCOF was based on terephthalaldehyde (TPA) and 1,3,5-tris(4-aminophenyl) benzene (TAPB) as functional monomer and crosslinker, anhydrous acetic acid as catalyst, bisphenol AF as dummy template, and NiFe2O4 as magnetic core. This organic framework significantly reduced the time consumption of conventional imprinted polymerization and avoided the use of traditional initiator and cross-linking agents. The synthesized MI-MCOF exhibited superior magnetic responsivity and affinity, as well as high selectivity and kinetics for bisphenol A (BPA) in water and urine samples. The equilibrium adsorption capacity (Qe) of BPA on the MI-MCOF was 50.65 mg g-1, which was 3-7-fold higher than of its three structural analogues. The imprinting factor of BPA reached up to 3.17, and the selective coefficients of three analogues were all > 2.0, evidencing the excellent selectivity of fabricated nanocomposites to BPA. Based on the MI-MCOF nanocomposites, the magnetic solid-phase extraction (MSPE), combined with HPLC and fluorescence detection (HPLC-FLD), offered superior analytical performance: wide linear range of 0.1-100 µg L-1, high correlation coefficient of 0.9996, low limit of detection of 0.020 µg L-1, good recoveries of 83.5-110%, and relative standard deviations (RSDs) of 0.5-5.7% in environmental water, beverage, and human urine samples. Consequently, the MI-MCOF-MSPE/HPLC-FLD method provides a good prospect in selective extraction of BPA from complex matrices while replacing traditional magnetic separation and adsorption materials.

Development and comparison of three 89Zr-labeled anti-CLDN18.2 antibodies to noninvasively evaluate CLDN18.2 expression in gastric cancer: a preclinical study.

BACKGROUND: Isoform 2 of claudin 18 (CLDN18.2) is overexpressed in gastric cancer and may be a promising imaging target. In this study, we constructed three anti-CLDN18.2 antibodies and compared them in preclinical experiments. METHODS: Screening from anti-CLDN18.2 nanobody library, we constructed three antibodies, anti-CLDN18.2 VHH (recombinant single-chain antibody fused with poly-histidine-tag), anti-CLDN18.2 VHH-ABD (recombinant single-chain antibody fused fused with albumin binding domain), and anti-CLDN18.2 VHH-Fc (recombinant single-chain antibody fused with IgG1-Fc) and radiolabeled with 89Zr. Affinity assay, in vitro stability, immunoactivity, blood pharmacokinetics, in vivo and ex vivo biodistribution study, specificity study, and immunohistochemical analysis were performed to assess these radiotracers. RESULTS: The EC50 were 12.21 nM, 2.48 nM, and 0.14 nM for anti-CLDN18.2 VHH, anti-CLDN18.2 VHH-ABD, and anti-CLDN18.2 VHH-Fc, respectively. 89Zr-anti-CLDN18.2 VHH demonstrated the lowest tumor uptake in PET imaging. Both 89Zr-anti-CLDN18.2 VHH-ABD and 89Zr-anti-CLDN18.2 VHH-Fc demonstrated high tumor accumulation, with highest ID%/g of 25.78 ± 5.60 at 24 h post-injection with 89Zr-anti-CLDN18.2 VHH-ABD and 49.43 ± 9.86 at 72 h post-injection with 89Zr-anti-CLDN18.2 VHH-Fc. The specificity of 89Zr-anti-CLDN18.2 VHH-Fc targeting CLDN18.2 was further confirmed by blocking study. The ex vivo biodistribution results were consistent with in vivo biodistribution data. For 89Zr-anti-CLDN18.2 VHH-ABD, tumor uptake was 21.46 ± 1.78 ID%/g at 12 h and 13.73 ± 2.22 ID%/g at 108 h. For 89Zr-anti-CLDN18.2 VHH-Fc, the tumor accumulation was 25.28 ± 3.83 ID%/g at 12 h and 40.13 ± 9.50 ID%/g at 108 h. Immunohistochemistry of the xenograft tissue revealed high and homogenous CLDN18.2 expression in CO-SNU620 tumor. CONCLUSION: Both anti-CLDN18.2 VHH-ABD and anti-CLDN18.2 VHH-Fc can be efficiently and stably radiolabeled with 89Zr for noninvasive imaging and quantification of CLDN18.2 expression in gastric cancer, of which 89Zr-anti-CLDN18.2 VHH-ABD seems to be the optimal choice balancing tumor uptake and liver background. They can provide essential information to select patients who are likely to benefit from CLDN18.2-targeted treatment.

Two Solvent-Induced In(III)-Based Metal-Organic Frameworks with Controllable Topology Performing High-Efficiency Separation of C2H2/CH4 and CO2/CH4.

For pure acetylene manufacturing and natural gas purification, the development of porous materials displaying highly selective C2H2/CH4 and CO2/CH4 separation is greatly important but remains a major challenge. In this work, a plausible strategy involving solvent-induced effects and using the flexibility of the ligand conformation to make two In(III) metal-organic frameworks (MOFs) is developed, showing topological diversity and different stability. The X-shaped tetracarboxylic ligand H4TPTA ([1,1':3',1″-terphenyl]-4,4',4″,6'-tetracarboxylic acid) was selected to construct two new heteroid In MOFs, namely, {[CH3NH3][In(TPTA)]·2(NMF)} (MOF 1) and {[In2(TPTA)(OH)2]·2(H2O)·(DMF)} (MOF 2). MOF 1 is a (4, 4)-connected net showing a pts topology with a large channel that is not conducive to fine gas separation. By contrast, with the reduction of SBU from uninucleated In to an {In-OH-In}n chain, MOF 2 has a (4, 6)-connected net with the fsc topology with an ∼5 Å suitable micropore to confine matching small gas. The permanent porosity of MOF 2 leads to the preferential adsorption of C2H2 over CO2 with superior C2H2/CH4 (332.3) and CO2/CH4 (31.2) separation selectivities. Meanwhile, the cycling dynamic breakthrough experiments showed that the high-purity C2H2 (>99.8%) capture capacities of MOF 2 were >1.92 mmol g-1 from a binary C2H2/CH4 mixture, and its separation factor reached 10.

Reuse of waste Myrica rubra for green synthesis of nitrogen-doped carbon dots as an "on-off-on" fluorescent probe for Fe3+ and ascorbic acid detection.

As one kind of high nutrition fruits, abandoned Myrica rubra causes great waste due to short storage period. For resource utilization, we herein fabricated the Myrica rubra-based N-doped carbon dots (MN-CDs) by a facile/green hydrothermal method. MN-CDs, fabricated from four regions of China, displayed significant differences in their corresponding fluorescence intensities (FIs). Interestingly, different batches of waxberry samples from the same region (Wenzhou, China) exhibited slight differences in their FIs, and also an excellent anti-photobleaching and anti-salt capacity. Based on Fe3+-triggered quenching effect and fluorescent recovery by redox reaction of AA and Fe3+, MN-CDs were employed to construct an "on-off-on" switch probe for sequential detection of Fe3+ and ascorbic acid (AA). Through Zeta potential, UV spectrum, Stern-Volmer equation, and valence-conduction band theory, the Fe3+-triggered quenching belonged to a static quenching process, which resulted from the synergistic contribution of inner filtering effect and photo-induced electron transfer mechanisms. The linear ranges for Fe3+ and AA detections were 1-1000 and 0.1-1000 mM. The limits of detection were 0.3 µM for Fe3+ in environmental waters, and 0.03 µM for AA in pharmaceutical tablets and fruit juice samples. Under 365-nm UV lamp, the color changes of test papers were easily observed from dark blue and bright blue in the presence of Fe3+ and AA, and thus the MN-CDs-based switch probe could be satisfactorily used for visually qualitative detection of Fe3+ and AA outdoor with our naked eyes. To sum up, MN-CDs not only realize resource reutilization of abandoned Myrica rubra, but also offer an convenient outdoor approach for qualitative detection of Fe3+ and AA in complex matrices.

POSS-based fluorescence sensor for rapid analysis of ß-carotene in health products.

In recent years, fluorescent organic-inorganic hybrid nanomaterials have received much interest as potential fluorescent sensor materials. In this study, fluorescent organic-inorganic hybrid nanomaterials (POSS@ANT) were created using polyhedral oligomeric silsesquioxane as the precursor and 9,10-bromoanthracene as the monomer. The morphology and composition of POSS@ANT, as well as its pore characteristics and fluorescence properties were studied. POSS@ANT displayed steady fluorescence emission at an excitation wavelength of 374 nm. Next, a ß-carotene fluorescence sensor was developed using the capacity of ß-carotene to quench the fluorescence of POSS@ANT. The quenching process is linked to acceptor electron transfer and energy transfer, and the sensor has a high selectivity for ß-carotene. This ß-carotene fluorescence analysis method that we established has a linear range of 0.2-4.3 mg/L and a detection limit of 0.081 mg/L. Finally, it was used to quantify ß-carotene in health products, the recovery rate was 91.1-109.9%, the relative standard deviation (RSD) was 2.2-4.3%, and the results were comparable with the results of high-performance liquid chromatography. The approach is reliable and can be used to determine ß-carotene in health products.

Sensitive colorimetric assay of hydrogen peroxide and glucose in humoral samples based on the enhanced peroxidase-mimetic activity of NH2-MIL-88-derived FeS2@CN nanocomposites compared to its precursors.

By employing NH2-MIL-88 as a template, we synthesized the intermediate Fe@CN under high-temperature calcination and further fabricated the FeS2@CN nanocomposites in the presence of sulfur powder. Under varying temperatures (300-600 °C) and Fe@CN-to-S ratios (1:3-6), FeS2@CN500-5 nanocomposites had the highest peroxidase-mimetic activity. Under optimized conditions (incubation temperature 40 °C; solution pH 4.0 and nanocomposite concentration 10 µg/mL; 652-nm absorption), the Michaelis-Menten constant (Km) of FeS2@CN was much lower than that of horseradish peroxidase (HRP), therefore demonstrating that it had a higher affinity for both chromogenic substrates than conventional HRP. The limits of detection for H2O2 and glucose were 0.15 and 0.30 µmol/L, respectively, and the recoveries for glucose were 91.8-103% with RSDs <5.2%. The novelty of this study lies in (1) the FeS2@CN was confirmed to possess stronger enzyme-mimetic activity than its precursors (NH2-MIL-88 and Fe@CN); (2) the enhanced activity resulted from the unsaturated sites of N and S doping and the plentiful defects on the porous carbon surface; and (3) free radical trapping experiments evidenced that •OH played a major role in the catalytic reaction, while h+ and •O2- simultaneously participated in the catalytic process. These convincing performance metrics lead us to postulate that the FeS2@CN-based colorimetric biosensor provides a promising approach for several real-world applications, such as point-of-care diagnosis and workplace health evaluations.

Anticoagulation may contribute to antimicrobial treatment of Lemierre syndrome: a case report.

BACKGROUND: Lemierre syndrome (LS) is characterized by multisystemic infection beginning in the oropharynx, local thrombophlebitis (typically, of the internal jugular vein) and peripheral embolism. No evidence-based guidelines exist for the management of this disease, and the use of anticoagulation therapy remains particularly controversial. CASE PRESENTATION: A 61-year-old man presenting with left neck swelling, odynophagia, and dyspnea underwent emergency surgery and received intravenous antibiotics. The primary infection was controlled on hospital day 5, but on day 6 sudden leukocytosis and hypoxemia were observed. CT angiography revealed an intraluminal filling defect in the pulmonary artery on day 8. LS was diagnosed and anticoagulation therapy was initiated. The WBC count, which had maintained its peak values in the previous 2 days, decreased instantly after initiation, and follow-up controls showed thrombus resolution. CONCLUSIONS: Our case supports the notion that anticoagulation therapy may be a valid supplement to antimicrobial therapy in LS, especially in the presence of a possibly young thrombus as suggested by clinical worsening.

Effervescence-assisted dual microextraction of PAHs in edible oils using lighter-than-water phosphonium-based ionic liquids and switchable hydrophilic/hydrophobic fatty acids.

Herein, we developed a novel effervescence-assisted dual microextraction method, abbreviated as EM-LPSH, using lighter-than-water phosphonium-based ionic liquids (LPILs) and switchable hydrophilic/hydrophobic fatty acids (SHFAs). The EM-LPSH method was utilized for quick enrichment/extraction of polycyclic aromatic hydrocarbons (PAHs) in edible oils. Owing to lower density than water, LPILs used as the first extractant were floated on the upper layer of the aqueous phase, leading to a convenient separation/collection compared with traditional heavier-than-water imidazolium-based ionic liquids. Interestingly, SHFAs play triple functions: a dispersive solvent in the microextraction process, an acidic source in effervescent reaction, and the second extractant in dual microextraction, due to switchability from hydrophilicity to hydrophobicity. Consequently, the integration of LPILs with SHFAs greatly enhanced the extraction efficiency for PAHs owing to the quick dual microextraction process. Some important variables were rigorously optimized using a one-factor-at-a-time approach. Under optimized conditions, the EM-LPSH/HPLC-FLD method provided a wide linear range (0.07~0.63-200 µg kg-1), satisfactory recovery (80.12-103.27%), and low limit of detection (0.02-0.19 µg kg-1), as well as high intra-day and inter-day precision (0.03-6.55) for six PAHs in edible oils. By using certified reference material in olive oil samples (GBW10162), the recoveries ranged from 97.40 to 98.39%, demonstrating high accuracy and precision. According to the detected levels of PAHs in six unheated and heated oils, their edible safety was evaluated in detail. In short, the newly developed method is simple, convenient, and highly efficient, thereby showing great prospects for application in conventional monitoring of trace-level PAHs in edible oils.

Magnetic amino-functionalized metal-organic frameworks as a novel solid support in ionic liquids-based effervescent tablets for efficient extraction of polycyclic aromatic hydrocarbons in milks.

Herein, a kind of novel multi-layer core-shell nanocomposites (NSPN) was prepared by employing SiO2 and polyvinylpyrrolidone (PVP) polymers as modifiers and amino-functionalized metal-organic frameworks (NH2-MIL101(Fe)) as coating. It was referred to as the NSPN and ILs-based effervescence-assisted dispersive solid-phase microextraction, hereafter abbreviated as NIE-DSM. In terms of extraction efficiency, SiO2 and PVP as modifiers and NH2-MIL(Fe) as coating onto the surface of NiFe2O4 cores played a synergistically enhancing effect on adsorption/extraction. Effervescent tablets were prepared by integrating the NSPN magnetic nanoparticles as adsorbents with imidazolium-based ionic liquids (ILs) as extractants as well as acidic and alkaline sources. Under vigorous dispersion of CO2 bubbles, the NIE-DSM method realized the goal of rapidly diffusing and separating the adsorbent/extractant (~3 min) without needing conventional vortexing or centrifugation step. Consequently, the NIE-DSM approach combined dispersion and adsorption/extractant in a synchronous way. Under optimized conditions, the NIE-DSM/HPLC-FLD method gave low limits of detection (0.008-0.034 µg kg-1) and satisfactory extraction recoveries (74.1-101.6%) for five polycyclic aromatic hydrocarbons (PAHs; fluorene, anthracene, pyrene, chrysene and benzo(a)pyrene) in milk samples. The intra-day and inter-day precision, expressed as relative standard deviations, was < 5.9% and 6.5%, respectively, demonstrating a high precision. Owing to no requirement for electrical power, this method shows great potential for outdoor monitoring of trace-level PAHs in food matrices.

Assessment of Clofazimine and TB47 Combination Activity against Mycobacterium abscessus Using a Bioluminescent Approach.

Mycobacterium abscessus is intrinsically resistant to most antimicrobial agents. The emerging infections caused by M. abscessus and the lack of effective treatment call for rapid attention. Here, we intended to construct a selectable marker-free autoluminescent M. abscessus strain (designated UAlMab) as a real-time reporter strain to facilitate the discovery of effective drugs and regimens for treating M. abscessus The UAlMab strain was constructed using the dif/Xer recombinase system. In vitro and in vivo activities of several drugs, including clofazimine and TB47, a recently reported cytochrome bc1 inhibitor, were assessed using UAlMab. Furthermore, the efficacy of multiple drug combinations, including the clofazimine and TB47 combination, were tested against 20 clinical M. abscessus isolates. The UAlMab strain enabled us to evaluate drug efficacy both in vitro and in live BALB/c mice in a real-time, noninvasive fashion. Importantly, although TB47 showed marginal activity either alone or in combination with clarithromycin, amikacin, or roxithromycin, the drug markedly potentiated the activity of clofazimine, both in vitro and in vivo This study demonstrates that the use of the UAlMab strain can significantly facilitate rapid evaluation of new drugs and regimens. The clofazimine and TB47 combination is effective against M. abscessus, and dual/triple electron transport chain (ETC) targeting can be an effective therapeutic approach for treating mycobacterial infections.

Akebia Saponin D Regulates the Metabolome and Intestinal Microbiota in High Fat Diet-Induced Hyperlipidemic Rats.

Hyperlipidemia is a major component of metabolic syndrome, and regarded as one of the main risk factors causing metabolic diseases. We have developed a therapeutic drug, akebia saponin D (ASD), and determined its anti-hyperlipidemia activity and the potential mechanism(s) of action by analyzing the metabolome and intestinal microbiota. Male Sprague-Dawley rats were fed a high fat diet to induce hyperlipidemia, and then given ASD orally for 8 weeks. Lipid levels in serum were determined biochemically. Metabolites in serum, urine and feces were analyzed by UPLC-Q/TOF-MS, and the structure of the intestinal microbiota was determined by 16S rRNA sequencing. The ASD treatment significantly decreased the levels of TC, TG and LDL-c and increased the serum level of HDL-c. Metabolomics analysis indicated that the ASD treatment mainly impacted seven differential metabolites in the serum, sixteen differential metabolites in the urine and four differential metabolites in feces compared to the model group. The ASD treatment significantly changed eight bacteria at the genus level compared to the model group. In conclusion, ASD treatment can significantly alleviate HFD-induced hyperlipidemia and the hypolipidemic effect of ASD treatment is certainly associated with a systematic change in the metabolism, as well as dynamic changes in the structure of the intestinal microbiota.