Multiple Signaling Roles of CD3ε and Its Application in CAR-T Cell Therapy.

A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.

FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.

Flow cytometry analysis of protein expression using antibody-derived tags followed by CITE-Seq.

Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) is a single-cell phenotyping method that uses antibody-derived tags (ADTs) to quantitatively detect cell surface protein expression and generate transcriptomic data at the single-cell level. Despite the increased popularity of this technique to study cellular heterogeneity and dynamics, detailed methods on how to choose ADT markers and ensuring reagent performance in biological relevant systems prior to sequencing is not available. Here we describe a novel and easy-to-use multiplex flow proxy assay in which multiple protein markers can be measured simultaneously using a combination of ADT reagents and dye-oligo conjugates by flow cytometry. Using dye-oligo conjugates with sequences complementary to the ADT reagents, we can achieve specific binding and evaluate protein marker expression in a multiplex way. This quality control assay is useful for guiding ADT marker choice and confirming protein expression prior to sequencing. Importantly, the labeled cells can be directly isolated based on the specific fluorescence from dye-oligo conjugates using a flow cytometry cell sorter and processed for downstream single-cell multiomics. Using this streamlined workflow, we sorted natural killer cells and T cells efficiently using only ADT and dye-oligo reagents, avoiding the possibility of decreased marker resolution from co-staining cells with ADT and fluorescent antibodies. This novel workflow provides a viable option for improving ADT marker choice and cell sorting efficiency, allowing subsequent CITE-Seq.

Diketopiperazine Alkaloids and Bisabolene Sesquiterpenoids from Aspergillus versicolor AS-212, an Endozoic Fungus Associated with Deep-Sea Coral of Magellan Seamounts.

Two new quinazolinone diketopiperazine alkaloids, including versicomide E (2) and cottoquinazoline H (4), together with ten known compounds (1, 3, and 5-12) were isolated and identified from Aspergillus versicolor AS-212, an endozoic fungus associated with the deep-sea coral Hemicorallium cf. imperiale, which was collected from the Magellan Seamounts. Their chemical structures were determined by an extensive interpretation of the spectroscopic and X-ray crystallographic data as well as specific rotation calculation, ECD calculation, and comparison of their ECD spectra. The absolute configurations of (-)-isoversicomide A (1) and cottoquinazoline A (3) were not assigned in the literature reports and were solved in the present work by single-crystal X-ray diffraction analysis. In the antibacterial assays, compound 3 exhibited antibacterial activity against aquatic pathogenic bacteria Aeromonas hydrophilia with an MIC value of 18.6 µM, while compounds 4 and 8 exhibited inhibitory effects against Vibrio harveyi and V. parahaemolyticus with MIC values ranging from 9.0 to 18.1 µM.

Sulfonated covalent organic framework modified separators suppress the shuttle effect in lithium-sulfur batteries.

Lithium-sulfur batteries (LSBs) have gained intense research enthusiasm due to their high energy density. Nevertheless, the 'shuttle effect' of soluble polysulfide (a discharge product) reduces their cycling stability and capacity, thus restricting their practical application. To tackle this challenging issue, we herein report a sulfonated covalent organic framework modified separator (SCOF-Celgard) that alleviates the shuttling of polysulfide anions and accelerates the migration of Li+ions. Specifically, the negatively charged sulfonate can inhibit the same charged polysulfide anion through electrostatic repulsion, thereby improving the cycle stability of the battery and preventing the Li-anode from being corroded. Meanwhile, the sulfonate groups may facilitate the positively charged lithium ions to pass through the separator. Consequently, the battery assembled with the SCOF-Celgard separator exhibits an 81.1% capacity retention after 120 cycles at 0.5 C, which is far superior to that (55.7%) of the battery with a Celgard separator. It has a low capacity degradation of 0.067% per cycle after 600 cycles at 1 C, and a high discharge capacity (576 mAh g-1) even at 2 C. Our work proves that the modification of a separator with a SCOF is a viable and effective route for enhancing the electrochemical performance of a LSB.

Highly oxygenated polyketides produced by Trichoderma koningiopsis QA-3, an endophytic fungus obtained from the fresh roots of the medicinal plant Artemisia argyi.

Eight new highly oxygenated fungal polyketides, namely, 15-hydroxy-1,4,5,6-tetra-epi-koninginin G (1), 14-hydroxykoninginin E (2), koninginin U (3), 4'-hydroxykoninginin U (4), koninginin V (5), 14-ketokoninginin B (6), 14-hydroxykoninginin B (7), and 7-O-methylkoninginin B (8), together with six known related analogues (9-14), were isolated from Trichoderma koningiopsis QA-3, a fungus obtained from the inner root tissue of the well known medicinal plant Artemisia argyi. All these compounds are bicyclic polyketides, with compound 1 contains unusual hemiketal moiety at C-5 and compounds 2-14 having ketone group at C-1 and double bond at C-5(6). The structures and absolute configurations of the new compounds were established by spectroscopic analysis, X-ray crystal diffraction, modified Mosher's method, and ECD calculation. The absolute configurations of the known compounds 9, 10, and 12 were determined by X-ray crystal diffractions for the first time. The antimicrobial activities against human pathogen, marine-derived aquatic bacteria, and plant-pathogenic fungi of compounds 1-14 were evaluated, and compound 1 showed remarkable activity against aquatic pathogen Vibrio alginolyticus with MIC value 1 µg/mL, which is as active as that of the positive control.

Antibacterial Alkaloids and Polyketide Derivatives from the Deep Sea-Derived Fungus Penicillium cyclopium SD-413.

Nine secondary metabolites (1-9), including two new polyketide derivatives 9-dehydroxysargassopenilline A (4) and 1,2-didehydropeaurantiogriseol E (5), along with seven known related secondary metabolites (1-3 and 6-9), were isolated and identified from the deep sea-derived fungus Penicillium cyclopium SD-413. Their structures were elucidated on the basis of 1D/2D NMR spectroscopic and mass spectrometric analysis and the absolute configurations were determined by the combination of NOESY correlations and time-dependent density functional (TDDFT) ECD calculations. Compounds 1-9 inhibited some pathogenic bacteria including Escherichia coli, E. ictaluri, Edwardsiella tarda, Micrococcus luteus, Vibrio anguillarum, and V. harveyi, with MIC (minimum inhibitory concentration) values ranging from 4 to 32 µg/mL.

Ionic CD3-Lck interaction regulates the initiation of T-cell receptor signaling.

Antigen-triggered T-cell receptor (TCR) phosphorylation is the first signaling event in T cells to elicit adaptive immunity against invading pathogens and tumor cells. Despite its physiological importance, the underlying mechanism of TCR phosphorylation remains elusive. Here, we report a key mechanism regulating the initiation of TCR phosphorylation. The major TCR kinase Lck shows high selectivity on the four CD3 signaling proteins of TCR. CD3ε is the only CD3 chain that can efficiently interact with Lck, mainly through the ionic interactions between CD3ε basic residue-rich sequence (BRS) and acidic residues in the Unique domain of Lck. We applied a TCR reconstitution system to explicitly study the initiation of TCR phosphorylation. The ionic CD3ε-Lck interaction controls the phosphorylation level of the whole TCR upon antigen stimulation. CD3ε BRS is sequestered in the membrane, and antigen stimulation can unlock this motif. Dynamic opening of CD3ε BRS and its subsequent recruitment of Lck thus can serve as an important switch of the initiation of TCR phosphorylation.

Polyketides and Terpenoids with Potent Antibacterial Activities from the Artemisia argyi-Derived Fungus Trichoderma koningiopsis QA-3.

The AcOEt extract of Artemisia argyi-derived fungus Trichoderma koningiopsis QA-3 showed potent inhibitory activity against pathogenic bacteria. Fractionation of the extract resulted in the isolation of three new polyketides (1-3) and two new terpenoids (4 and 5), together with three known metabolites (6-8). Their chemical structures were analyzed by NMR spectra, ECD, HR-ESI-MS or HR-EI-MS, optical rotation, and X-ray crystallographic data, as well as by comparison with literature reports. In the antibacterial assays, 3-hydroxyharziandione (4) showed potent activity against human pathogen Escherichia coli with an MIC value of 0.5 µg/mL, while 6-(3-hydroxypent-1-en-1-yl)-2H-pyran-2-one exhibited strong activity against marine-derived aquatic pathogen Micrococcus luteus with an MIC value of 1.0 µg/mL.

Trichocadinins B-G: Antimicrobial Cadinane Sesquiterpenes from Trichoderma virens QA-8, an Endophytic Fungus Obtained from the Medicinal Plant Artemisia argyi.

Trichocadinins B-G (1-6), six new cadinane-type sesquiterpene derivatives, each with C-14 carboxyl functionality, were isolated from the culture extract of Trichoderma virens QA-8, an endophytic fungus obtained from the fresh inner tissue of the medicinal plant Artemisia argyi. Their structures were elucidated by interpretation of the NMR spectroscopic and mass spectrometric data. The structures and absolute configurations of compounds 1 and 3 were confirmed by X-ray crystallographic analysis. Compounds 1-3 showed antibacterial and antifungal activity.

Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids.

Ionic protein-lipid interactions are critical for the structure and function of membrane receptors, ion channels, integrins and many other proteins. However, the regulatory mechanism of these interactions is largely unknown. Here we show that Ca(2+) can bind directly to anionic phospholipids and thus modulate membrane protein function. The activation of T-cell antigen receptor-CD3 complex (TCR), a key membrane receptor for adaptive immunity, is regulated by ionic interactions between positively charged CD3ε/ζ cytoplasmic domains (CD3(CD)) and negatively charged phospholipids in the plasma membrane. Crucial tyrosines are buried in the membrane and are largely protected from phosphorylation in resting T cells. It is not clear how CD3(CD) dissociates from the membrane in antigen-stimulated T cells. The antigen engagement of even a single TCR triggers a Ca(2+) influx and TCR-proximal Ca(2+) concentration is higher than the average cytosolic Ca(2+) concentration. Our biochemical, live-cell fluorescence resonance energy transfer and NMR experiments showed that an increase in Ca(2+) concentration induced the dissociation of CD3(CD) from the membrane and the solvent exposure of tyrosine residues. As a consequence, CD3 tyrosine phosphorylation was significantly enhanced by Ca(2+) influx. Moreover, when compared with wild-type cells, Ca(2+) channel-deficient T cells had substantially lower levels of CD3 phosphorylation after stimulation. The effect of Ca(2+) on facilitating CD3 phosphorylation is primarily due to the charge of this ion, as demonstrated by the fact that replacing Ca(2+) with the non-physiological ion Sr(2+) resulted in the same feedback effect. Finally, (31)P NMR spectroscopy showed that Ca(2+) bound to the phosphate group in anionic phospholipids at physiological concentrations, thus neutralizing the negative charge of phospholipids. Rather than initiating CD3 phosphorylation, this regulatory pathway of Ca(2+) has a positive feedback effect on amplifying and sustaining CD3 phosphorylation and should enhance T-cell sensitivity to foreign antigens. Our study thus provides a new regulatory mechanism of Ca(2+) to T-cell activation involving direct lipid manipulation.

Ionic protein-lipid interaction at the plasma membrane: what can the charge do?

Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms.

Mycobacterium tuberculosis copper-regulated protein SocB is an intrinsically disordered protein that folds upon interaction with a synthetic phospholipid bilayer.

Multiple genes in Mycobacterium tuberculosis (Mtb) are regulated by copper including socAB (small orf induced by copper A and B), which is induced by copper and repressed by RicR (regulated in copper repressor). socA and socB encode hypothetical proteins of 61 and 54 amino acids, respectively. Here, we use biophysical and computational methods to evaluate the SocB structure. We find that SocB lacks evidence for secondary structure, with no thermal cooperative unfolding event, according to circular dichroism measurements. 2D NMR spectra similarly exhibit hallmarks of a disordered structural state, which is also supported by analyzing SocB diffusion. Altogether, these findings suggest that by itself SocB is intrinsically disordered. Interestingly, SocB interacts with a synthetic phospholipid bilayer and becomes helical, which suggests that it may be membrane-associated.

Co-staining with Fluorescent Antibodies and Antibody-Derived Tags for Cell Sorting Prior to CITE-Seq.

The paired detection of the transcriptome and proteome at single-cell resolution provides exquisite insight to immune mechanisms in health and disease. Here, we describe a detailed protocol wherein we combine cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), a technique utilizing antibody-derived tags (ADTs) to profile mRNA and proteins simultaneously via sequencing, with fluorescence-activated cell sorting to enrich cell populations. Our protocol provides step-by-step guidance on co-staining cells with both fluorescent antibodies and ADTs simultaneously, instructions on cell sorting and an overview of the single-cell capture workflow using the BD Rhapsody™ system. This method is useful for in-depth single-cell characterization on sorted rare cell populations.

Comparison of dexmedetomidine and a dexmedetomidine-esketamine combination for reducing dental anxiety in preschool children undergoing dental treatment under general anesthesia: A randomized controlled trial.

BACKGROUND: Dental anxiety is a widespread complication occurring in pediatric patients during dental visits and may lead to undesirable complications. Esketamine may be effective in anxiety. OBJECTIVE: The objective of this study was to investigate the effect of premedication with a dexmedetomidine-esketamine combination compared with dexmedetomidine alone on dental anxiety in preschool children undergoing dental treatment under general anesthesia. METHODS: This is a prospective, double-blinded, randomized controlled trial. A total of 84 patients were scheduled for elective outpatient dental caries treatment under general anesthesia. Patients were randomly premedicated with intranasal dexmedetomidine (group D) or intranasal dexmedetomidine-esketamine (group DS). The primary outcome was the level of dental anxiety assessed by the Modified Child Dental Anxiety Scale (MCDAS) at 2 h after surgery. Secondary outcomes included level of dental anxiety at 1 day and 7 days after surgery, the incidence of dental anxiety at 2 h, 1 day, and 7 days after surgery, sedation onset time, overall success of sedation, acceptance of mask induction, postoperative pain intensity, incidence of emergence agitation in PACU, adverse reactions, HR, and SpO2 before premedication (baseline) and at 10, 20, and 30 min after the end of study drug delivery. RESULTS: The dental anxiety in group DS was lower than that in group D at 2 h, 1 day, and 7 days postoperatively (P = 0.04, 0.004, and 0.006, respectively). The incidences of dental anxiety in group DS were lower than those in group D at 2 h (53 % vs 76 %, P = 0.03), 1 day (47 % vs 71 %, P = 0.04), and 7 days (44 % vs 71 %, P = 0.02) after surgery. Group DS had a higher success rate of sedation (P = 0.03) but showed a lower MAS score (P = 0.005) and smoother hemodynamics (P < 0.01) after drug administration than group D. Group DS showed a significantly lower incidence rate of emergence agitation (P = 0.03) and postoperative pain intensity (P = 0.006) than that in group D during the anesthesia recovery time. The occurrence of adverse reactions was similar in both groups (P > 0.05). LIMITATIONS: We did not analyze and correct for the learning effect caused by repeated applications of the MCDAS and MCDAS scores on the 1 day after surgery were obtained by telephone follow-up. CONCLUSIONS: Compared to premedication with dexmedetomidine alone, premedication with intranasal dexmedetomidine combined with esketamine could significantly improve dental anxiety in preschool children undergoing dental treatment under general anesthesia.

Curvularin derivatives from the marine mangrove derived fungus Penicillium sumatrense MA-325.

Sumalarins D-G (1-4), four previously undescribed curvularin derivatives, along with two known related metabolites, curvularin (5) and dehydrocurvularin (6), were isolated and identified from the mangrove-derived fungus Penicillium sumatrense MA-325. Among them, sumalarin D (1) represents a unique example of curvularin derivative featuring a 5-methylfuran-2-yl-methyl group. Their structures were elucidated based on analysis of NMR and MS data as well as comparison of ECD spectra and quantum chemical calculations of NMR, and compound 1 was confirmed by X-ray crystallographic analysis. Compounds 1, 2, 5, and 6 are active against aquatic pathogenic bacteria Vibrio alginolyticus and V. harveyi with MIC values ranging from 4 to 64 µg/mL, while compound 6 is cytotoxic against tumor cell lines 5673, HCT 116, 786-O, and Hela with IC50 values of 3.5, 10.6, 10.9, and 14.9 µM, respectively.

A review on synthesis and antibacterial potential of bio-selenium nanoparticles in the food industry.

Effective control of foodborne pathogen contamination is a significant challenge to the food industry, but the development of new antibacterial nanotechnologies offers new opportunities. Notably, selenium nanoparticles have been extensively studied and successfully applied in various food fields. Selenium nanoparticles act as food antibacterial agents with a number of benefits, including selenium as an essential trace element in food, prevention of drug resistance induction in foodborne pathogens, and improvement of shelf life and food storage conditions. Compared to physical and chemical methods, biogenic selenium nanoparticles (Bio-SeNPs) are safer and more multifunctional due to the bioactive molecules in Bio-SeNPs. This review includes a summarization of (1) biosynthesized of Bio-SeNPs from different sources (plant extracts, fungi and bacteria) and their antibacterial activity against various foodborne bacteria; (2) the antibacterial mechanisms of Bio-SeNPs, including penetration of cell wall, damage to cell membrane and contents leakage, inhibition of biofilm formation, and induction of oxidative stress; (3) the potential antibacterial applications of Bio-SeNPs as food packaging materials, food additives and fertilizers/feeds for crops and animals in the food industry; and (4) the cytotoxicity and animal toxicity of Bio-SeNPs. The related knowledge contributes to enhancing our understanding of Bio-SeNP applications and makes a valuable contribution to ensuring food safety.

Mott-Schottky MXene@WS2 Heterostructure: Structural and Thermodynamic Insights and Application in Ultra Stable Lithium-Sulfur Batteries.

Due to the "shuttle effect" and low conversion kinetics of polysulfides, the cycle stability of lithium sulfur (Li-S) battery is unsatisfactory, which hinders its practical application. The Mott-Schottky heterostructures for Li-S batteries not only provide more catalytic/adsorption active sites, but also facilitate electrons transport by a built-in electric field, which are both beneficial for polysulfides conversion and long-term cycle stability. Here, MXene@WS2 heterostructure was constructed by in-situ hydrothermal growth for separator modification. In-depth ultraviolet photoelectron spectroscopy and ultraviolet visible diffuse reflectance spectroscopy analysis reveals that there is an energy band difference between MXene and WS2 , confirming the heterostructure nature of MXene@WS2 . DFT calculations indicate that the Mott-Schottky MXene@WS2 heterostructure can effectively promote electron transfer, improve the multi-step cathodic reaction kinetics, and further enhance polysulfides conversion. The built-in electric field of the heterostructure plays an important role in reducing the energy barrier of polysulfides conversion. Thermodynamic studies reveal the best stability of MXene@WS2 during polysulfides adsorption. As a result, the Li-S battery with MXene@WS2 modified separator exhibits high specific capacity (1613.7 mAh g-1 at 0.1 C) and excellent cycling stability (2000 cycles with 0.0286 % decay per cycle at 2 C). Even at a high sulfur loading of 6.3 mg cm-2 , the specific capacity could be retained by 60.0 % after 240 cycles at 0.3 C. This work provides deep structural and thermodynamic insights into MXene@WS2 heterostructure and its promising prospect of application in high performance Li-S batteries.

MOF-Derived Hollow Carbon Supported Nickel-Cobalt Alloy Catalysts Driving Fast Polysulfide Conversion for Lithium-Sulfur Batteries.

Transition-metal compounds can be used as electrocatalysts to expedite polysulfide conversion effectively in lithium-sulfur batteries. However, insufficient conductivity and tedious preparation process still limit their practical applications. In this work, NiCo alloy nanoparticles embedded in hollow carbon spheres (NiCo@HCS) are fabricated via a facile, template-free strategy from the NiCo-metal-organic framework (MOF) precursor and used as electrocatalysts for separator modification. NiCo@HCS can not only improve the adsorption capacity of polysulfides by d-band deviation to the Fermi level but also reduce the energy barrier in the process of catalytic polysulfide conversion. Due to favorable three-dimensional (3-D) morphology, improved adsorption, and promoted kinetics of NiCo@HCS, the battery containing the NiCo@HCS-modified separator gives a starting capacity of 1377 mAh g-1 at 0.2C, which is retained by 72% over 500 charge/discharge operations at 1.0C current density. Moreover, the battery's start capacity reaches 1180 mAh g-1 (5.9 mAh cm-2) with a high sulfur content of 5.0 mg cm-1 at 0.2C.

Oxepine-containing pyrazinopyrimidine alkaloids and quinolinone derivatives produced by Aspergillus versicolor AS-212, a deep-sea-derived endozoic fungus.

Four new oxepine-containing pyrazinopyrimidine alkaloids, versicoxepines A - D (1-4), two quinolinone alkaloid analogs including 3-hydroxy-6-methoxy-4-phenylquinolin-2(1H)-one (5) and 3-methoxy-6-hydroxy-4-phenylquinolin-2(1H)-one (6) which were new naturally occurring compounds, together with two known compounds (7 and 8) were isolated from Aspergillus versicolor AS-212, an endozoic fungus isolated from the deep-sea coral Hemicorallium cf. imperiale, which was collected from the Magellan Seamounts in the Western Pacific Ocean. Their structures were determined by extensive analysis of the spectroscopic and X-ray crystallographic data as well as by chiral HPLC analysis, ECD calculation, and DP4+ probability prediction. Structurally, versicoxepines B and C (2 and 3) represent the first example of a new oxepine-containing pyrazinopyrimidine alkaloid whose cyclic dipeptide moiety is composed of the same type of amino acid (Val or Ile). Compound 5 displayed antibacterial activity against aquatic pathogens, Vibrio harveyi and V. alginolyticus, with MICs of 8 µg/mL.