The ubiquitin E3 ligase APC/CCdc20 mediates mitotic degradation of OGT.

O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme that catalyzes all O-GlcNAcylation reactions intracellularly. Previous investigations have found that OGT levels oscillate during the cell division process. Specifically, OGT abundance is downregulated during mitosis, but the underlying mechanism is lacking. Here we demonstrate that OGT is ubiquitinated by the ubiquitin E3 ligase, anaphase promoting complex/cyclosome (APC/C)-cell division cycle 20 (Cdc20). We show that APC/CCdc20 interacts with OGT through a conserved destruction box (D-box): Arg-351/Leu-354, the abrogation of which stabilizes OGT. As APC/CCdc20-substrate binding is often preceded by a priming ubiquitination event, we also used mass spectrometry and mapped OGT Lys-352 to be a ubiquitination site, which is a prerequisite for OGT association with APC/C subunits. Interestingly, in The Cancer Genome Atlas, R351C is a uterine carcinoma mutant, suggesting that mutations of the D-box are linked with tumorigenesis. Paradoxically, we found that both R351C and the D-box mutants (R351A/L354A) inhibit uterine carcinoma in mouse xenograft models, probably due to impaired cell division and proliferation. In sum, we propose a model where OGT Lys-352 ubiquitination primes its binding with APC/C, and then APC/CCdc20 partners with OGT through the D-box for its mitotic destruction. Our work not only highlights the key mechanism that regulates OGT during the cell cycle, but also reveals the mutual coordination between glycosylation and the cell division machinery.

Immune characteristics of kidney transplant recipients with acute respiratory distress syndrome induced by COVID-19 at single-cell resolution.

BACKGROUND: COVID-19-induced acute respiratory distress syndrome (ARDS) can result in tissue damage and multiple organ dysfunction, especially in kidney transplant recipients (KTRs) receiving immunosuppressive drugs. Presently, single-cell research on COVID-19-induced ARDS is considerably advanced, yet knowledge about ARDS in KTRs is still constrained. METHODS: Single-cell RNA sequencing (scRNA-seq) analysis was performed to construct a comprehensive single-cell immune landscape of the peripheral blood mononuclear cells (PBMCs) of eight patients with COVID-19-induced ARDS, five KTRs with COVID-19-induced ARDS, and five healthy individuals. Subsequently, we conducted a comprehensive bioinformatics analysis, including cell clustering, enrichment analysis, trajectory analysis, gene regulatory network analysis, and cell-cell interaction analysis, to investigate the heterogeneity of the immune microenvironment in KTRs with ARDS. RESULT: Our study revealed that KTRs exhibit significant heterogeneity with COVID-19-induced ARDS compared with those of other individuals, with significant reductions in T cells, as well as an abnormal proliferation of B cells and monocytes. In the context of dual influences from immunosuppression and viral infection, KTRs exhibited more specific plasma cells, along with significant enrichment of dysfunctional GZMB and XAF1 double-positive effector T cells and IFI27-positive monocytes. Additionally, robust communication existed among T cells and monocytes in cytokine signaling. These effects impede the process of immune reconstitution in KTR patients. CONCLUSION: Our findings suggest that KTRs with COVID-19-induced ARDS show elevated antibody levels, impaired T cell differentiation, and dysregulation of innate immunity. In summary, this study provides a theoretical foundation for a comprehensive understanding of COVID-19-induced ARDS in KTRs.

Highly Sensitive and Selective Defect WS2 Chemical Sensor for Detecting HCHO Toxic Gases.

The gas sensitivity of the W defect in WS2 (VW/WS2) to five toxic gases-HCHO, CH4, CH3HO, CH3OH, and CH3CH3-has been examined in this article. These five gases were adsorbed on the VW/WS2 surface, and the band, density of state (DOS), charge density difference (CDD), work function (W), current-voltage (I-V) characteristic, and sensitivity of adsorption systems were determined. Interestingly, for HCHO-VW/WS2, the energy level contribution of HCHO is closer to the Fermi level, the charge transfer (B) is the largest (0.104 e), the increase in W is more obvious than other adsorption systems, the slope of the I-V characteristic changes more obviously, and the calculated sensitivity is the highest. To sum up, VW/WS2 is more sensitive to HCHO. In conclusion, VW/WS2 has a great deal of promise for producing HCHO chemical sensors due to its high sensitivity and selectivity for HCHO, which can aid in the precise and efficient detection of toxic gases.

O-GlcNAcylation stabilizes the autophagy-initiating kinase ULK1 by inhibiting chaperone-mediated autophagy upon HPV infection.

Human papillomaviruses (HPVs) cause a subset of head and neck squamous cell carcinomas (HNSCCs). Previously, we demonstrated that HPV16 oncogene E6 or E6/E7 transduction increases the abundance of O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT), but OGT substrates affected by this increase are unclear. Here, we focus on the effects of O-GlcNAcylation on HPV-positive HNSCCs. We found that upon HPV infection, Unc-51-like kinase 1 (ULK1), an autophagy-initiating kinase, is hyper-O-GlcNAcylated, stabilized, and linked with autophagy elevation. Through mass spectrometry, we identified that ULK1 is O-GlcNAcylated at Ser409, which is distinct from the previously reported Thr635/Thr754 sites. It has been demonstrated that PKCα mediates phosphorylation of ULK1 at Ser423, which attenuates its stability by shunting ULK1 to the chaperone-mediated autophagy (CMA) pathway. Using biochemical assays, we demonstrate that ULK1 Ser409Ser410 O-GlcNAcylation antagonizes its phosphorylation at Ser423. Moreover, mutations of Ser409A and its neighboring site Ser410A (2A) render ULK1 less stable by promoting interaction with the CMA chaperone HSC70 (heat shock cognate 70 kDa protein). Furthermore, ULK1-2A mutants attenuate the association of ULK1 with STX17, which is vital for the fusion between autophagosomes and lysosomes. Analysis of The Cancer Genome Atlas (TCGA) database reveals that ULK1 is upregulated in HPV-positive HNSCCs, and its level positively correlates with HNSCC patient survival. Overall, our work demonstrates that O-GlcNAcylation of ULK1 is altered in response to environmental changes. O-GlcNAcylation of ULK1 at Ser409 and perhaps Ser410 stabilizes ULK1, which might underlie the molecular mechanism of HPV-positive HNSCC patient survival.

Chemoproteomic profiling of O-GlcNAcylated proteins and identification of O-GlcNAc transferases in rice.

O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous post-translation modification occurring in both animals and plants. Thousands of proteins along with their O-GlcNAcylation sites have been identified in various animal systems, yet the O-GlcNAcylated proteomes in plants remain poorly understood. Here, we report a large-scale profiling of protein O-GlcNAcylation in a site-specific manner in rice. We first established the metabolic glycan labelling (MGL) strategy with N-azidoacetylgalactosamine (GalNAz) in rice seedlings, which enabled incorporation of azides as a bioorthogonal handle into O-GlcNAc. By conjugation of the azide-incorporated O-GlcNAc with alkyne-biotin containing a cleavable linker via click chemistry, O-GlcNAcylated proteins were selectively enriched for mass spectrometry (MS) analysis. A total of 1591 unambiguous O-GlcNAcylation sites distributed on 709 O-GlcNAcylated proteins were identified. Additionally, 102 O-GlcNAcylated proteins were identified with their O-GlcNAcylation sites located within serine/threonine-enriched peptides, causing ambiguous site assignment. The identified O-GlcNAcylated proteins are involved in multiple biological processes, such as transcription, translation and plant hormone signalling. Furthermore, we discovered two O-GlcNAc transferases (OsOGTs) in rice. By expressing OsOGTs in Escherichia coli and Nicotiana benthamiana leaves, we confirmed their OGT enzymatic activities and used them to validate the identified rice O-GlcNAcylated proteins. Our dataset provides a valuable resource for studying O-GlcNAc biology in rice, and the MGL method should facilitate the identification of O-GlcNAcylated proteins in various plants.

Elastohydrodynamic propulsion of a filament magnetically driven at both ends.

The elastohydrodynamic interaction between an elastic filament and its surrounding fluid was exploited to develop the first microswimmers. These flexible microswimmers are typically actuated magnetically at one end and their propulsion behavior is relatively well understood. In this work, we move beyond the traditional single-end actuation setup and explore the propulsion characteristics of an elastic filament driven by magnetic torques at both ends. We report the emergence of new modes of propulsion behaviors in different physical regimes, depending on the balance of elastic and viscous forces as well as the arrangement of the magnetic moments at the filament ends. In particular, under the same magnetic actuation, a filament driven at both ends can propel either forward or backward depending on its relative stiffness. Moreover, this new backward propulsion mode can generate a magnitude of propulsion that is unattainable by the traditional single-end actuation setup. We characterize these new propulsion behaviors and provide some physical insights into how they emerge from the complex interplay between viscous and elastic forces and magnetic actuation in various configurations. Taken together, these findings could guide the development of soft microrobots with enhanced propulsion performance and maneuverability for future biomedical applications.

Chemoproteomic and Transcriptomic Analysis Reveals that O-GlcNAc Regulates Mouse Embryonic Stem Cell Fate through the Pluripotency Network.

Self-renewal and differentiation of embryonic stem cells (ESCs) are influenced by protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification, but the underlying mechanism remains incompletely understood. Herein, we report the identification of 979 O-GlcNAcylated proteins and 1340 modification sites in mouse ESCs (mESCs) by using a chemoproteomics method. In addition to OCT4 and SOX2, the third core pluripotency transcription factor (PTF) NANOG was found to be modified and functionally regulated by O-GlcNAc. Upon differentiation along the neuronal lineage, the O-GlcNAc stoichiometry at 123 sites of 83 proteins-several of which were PTFs-was found to decline. Transcriptomic profiling reveals 2456 differentially expressed genes responsive to OGT inhibition during differentiation, of which 901 are target genes of core PTFs. By acting on the core PTF network, suppression of O-GlcNAcylation upregulates neuron-related genes, thus contributing to mESC fate determination.

General Label-Free Fluorescent Aptamer Binding Assay Using Cationic Conjugated Polymers.

With more and more new aptamers being reported, a general, cost-effective yet reliable aptamer binding assay is still needed. Herein, we studied cationic conjugated polymer (CCP)-based binding assays taking advantage of the conformational change of aptamer after binding with a target, which is reflected by the fluorescence change of the CCP. Poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) was used as a model CCP in this study, and the optimal buffer was close to physiological conditions with 100 mM NaCl and 10 mM MgCl2. We characterized four aptamers for K+, adenosine, cortisol, and caffeine. For cortisol and caffeine, the drop in the 580 nm peak intensity was used for quantification, whereas for K+ and adenosine, the fluorescence ratio at 580 over 530 nm was used. The longer stem of the stem-loop structured aptamer facilitated binding of the target and enlarged the detection signal. High specificity was achieved in differentiating targets with analogues. Compared with the SYBR Green I dye-based staining method, our method achieved equal or even higher sensitivity. Therefore, this assay is practicable as a general aptamer binding assay. The simple, label-free, quick response, and cost-effective features will make it a useful method to evaluate aptamer binding. At the same time, this system can also serve as label-free biosensors for target detection.

Necrostatin-1 attenuates Caspase-1-dependent pyroptosis induced by the RIPK1/ZBP1 pathway in ventilator-induced lung injury.

BACKGROUND: Ventilator-induced lung injury (VILI) is a complex pathophysiological process leading to acute respiratory distress syndrome (ARDS) and poor outcomes in affected patients. As a form of programmed cell death, pyroptosis is proposed to play an important role in the development of ARDS. Here we investigated whether treating mice with the specific RIPK1 inhibitor Necrostatin-1 (Nec-1) before mechanical ventilation could inhibit pyroptosis and alleviate lung injury in a mouse model. METHODOLOGYS: Anesthetized C57BL/6J mice received a transtracheal injection of Nec-1 (5 mg/kg) or vehicle (DMSO) 30 min before the experiment which was ventilated for up to 4 h. Lung damage was assessed macroscopically and histologically with oedema measured as the wet/dry ratio of lung tissues. The release of inflammatory mediators into bronchoalveolar lavage fluid (BALF) was assessed by ELISA measurements of TNF-α,interleukin-1ß (IL-1ß), and IL-6. The expression of RIPK1, ZBP1, caspase-1, and activated (cleaved) caspase-1 were analyzed using western blot and immunohistochemistry, and the levels of gasdermin-D (GSDMD) and IL-1ß were analyzed by immunofluorescence staining. RESULTS: High tidal ventilation produced time-dependent inflammation and lung injury in mice which could be significantly reduced by pretreatment with Nec-1. Notably, Nec-1 reduced the expression of key pyroptosis mediator proteins in lung tissues exposed to mechanical ventilation, including caspase-1, cleaved caspase-1, and GSDMD together with inhibiting the release of inflammatory cytokines. CONCLUSION: Nec-1 pretreatment alleviates pulmonary inflammatory responses and protects the lung from mechanical ventilation damage. The beneficial effects were mediated at least in part by inhibiting caspase-1-dependent pyroptosis through the RIPK1/ZBP1 pathway.

Functional characterization of cAMP signaling of variant porcine MC1R alleles in PK15 cells.

The melanocortin 1 receptor (MC1R), encoded by the classical extension (E) coat color locus, is expressed on the surface of melanocytes and plays a critical role in switching melanin synthesis from pheomelanin (red/yellow) to eumelanin (black/brown). Different MC1R alleles associated with various coat color patterns in pigs have been identified over the past decades. However, functional analysis of variant porcine MC1R alleles has not yet been performed. Therefore, in this study, we examined the subcellular localization and cyclic adenosine monophosphate (cAMP) signaling capability of MC1R variants in porcine kidney epithelial cells (PK15) overexpressing different MC1R alleles. Transcriptional slippage may partially restore the reading frame of the EP allele, possibly accounting for the observed spot phenotype. The A243T substitution in the e allele severely disrupted the membrane localization of the MC1R receptor, resulting in a severely impaired cAMP signaling capability. Both the V95M and L102P substitutions in the ED1 allele may contribute to the constitutively active function of MC1R, thus accounting for the dominant black phenotype. The D124N substitution in the ED2 allele severely attenuated the cAMP signaling capability of MC1R; however, whether this mutation contributes to the distinct phenotype of Hampshire pigs requires further investigation. Thus, our results provide new insights into the functional characteristics of MC1R variants and their roles in porcine coat color formation.

Genetic ancestry differences in pediatric asthma readmission are mediated by socioenvironmental factors.

BACKGROUND: Social and financial hardships, combined with disease managment and environmental factors explain approximately 80% of the observed disparity in asthma-related readmissions between Black and White children. OBJECTIVE: We sought to determine whether asthma-related readmissions differed by degree of African ancestry and the extent to which such an association would also be explained by socioenvironmental risk factors. METHODS: This study used data from a prospective cohort study of 695 Black and White children aged 1 to 16 years with an asthma-related admission. The primary outcome was a similar readmission within 12 months. Each subject's African ancestry was determined by single nucleotide polymorphisms on a continuous scale ranging from 0 to 1 (0 = no African ancestry; 1 = 100% African ancestry). We also assessed 37 social, environmental, and clinical variables that we clustered into 6 domains (for example, hardship, disease management). Survival and mediation analyses were conducted. RESULTS: A total of 134 children (19.3%) were readmitted within 12 months. Higher African ancestry was associated with asthma readmission (odds ratio 1.11, 95% confidence interval 1.05-1.18 for every 10% increase in African ancestry) with adjustment for age and gender. The association between African ancestry and readmission was mediated by hardship (sß = 3.42, P < .001) and disease management (sß = 0.046, P = .001), accounting for >50% of African ancestry's effect on readmission. African ancestry was no longer significantly associated with readmission (sß = 0.035, P = .388) after accounting for these mediators. CONCLUSIONS: African ancestry was strongly associated with readmission, and the association was mediated by family hardship and disease management. These results are consistent with the notion that asthma-related racial disparities are driven by factors like structural racism and social adversity.

School resources, self-control and problem behaviors in Chinese adolescents: a longitudinal study in the post-pandemic era.

The outbreak of COVID-19 has brought many challenges to youth development. During this specific period, adolescents have suffered from numerous behavioral problems, which will lead to more maladaptive consequences. It is necessary to explore several protective factors to prevent or reduce the occurrence of problem behaviors in adolescence. The current study combined school resources and self-control to evaluate the multiple protective effects on adolescents' problematic behaviors in a two-wave longitudinal study. A sample of 789 Chinese adolescents (Mage = 14.00 years, SD = 2.05, 418 boys) were recruited via the random cluster sampling method to participate in the survey. The results confirmed the assumptions about the multiple protective effects of school resources and self-control on adolescents' problem behaviors. Specifically, school resources could negatively predict IGD and victimization, and self-control mediated these associations. Moreover, one problematic behavior could also mediate the associations between self-control and another problematic behavior. This is the first study to focus on the multiple protective effects of positive factors on adolescents' problem behaviors during the post-pandemic period, which has made several contributions to the literature and practice.

S-glycosylation-based cysteine profiling reveals regulation of glycolysis by itaconate.

Itaconate has been recently recognized as an anti-inflammatory metabolite involved in the pathogen-macrophage interface. Due to its weak electrophilicity, itaconate could modify cysteines of the protein KEAP1 and glutathione, which contribute to its anti-inflammatory effect. However, the substrates of itaconate modification in macrophages have not been systematically profiled, which largely impedes the understanding of its roles in immune responses. Here, we developed a specific thiol-reactive probe, 1-OH-Az, for quantitative chemoproteomic profiling of cysteine modifications by itaconate, and provided a global portrait of its proteome reactivity. We found that itaconate covalently modifies key glycolytic enzymes and impairs glycolytic flux mainly through inhibition of fructose-bisphosphate aldolase A (ALDOA). Moreover, itaconate attenuates the inflammatory response in stimulated macrophages by impairing the glycolysis. Our study provides a valuable resource of protein targets of itaconate in macrophages and establishes a negative-feedback link between glycolysis and itaconate, elucidating new functional insights for this anti-inflammatory metabolite.

Propulsion of an elastic filament in a shear-thinning fluid.

Some micro-organisms and artificial micro-swimmers propel at low Reynolds numbers (Re) via the interaction of their flexible appendages with the surrounding fluid. While their locomotion has been extensively studied with a Newtonian fluid assumption, in realistic biological environments these micro-swimmers invariably encounter rheologically complex fluids. In particular, many biological fluids such as blood and different types of mucus have shear-thinning viscosities. The influence of this ubiquitous non-Newtonian rheology on the performance of flexible swimmers remains largely unknown. Here, we present a first study to examine how shear-thinning rheology alters the fluid-structure interaction and hence the propulsion performance of elastic swimmers at low Re. Via a simple elastic swimmer actuated magnetically, we demonstrate that shear-thinning rheology can either enhance or hinder elastohydrodynamic propulsion, depending on the intricate interplay between elastic and viscous forces as well as the magnetic actuation. We also use a reduced-order model to elucidate the mechanisms underlying the enhanced and hindered propulsion observed in different physical regimes. These results and improved understanding could guide the design of flexible micro-swimmers in non-Newtonian fluids.

Protein S-Glyco-Modification through an Elimination-Addition Mechanism.

Per-O-acetylated unnatural monosaccharides containing a bioorthogonal group have been widely used for metabolic glycan labeling (MGL) in live cells for two decades, but it is only recently that we discovered the existence of an artificial "S-glycosylation" between protein cysteines and per-O-acetylated sugars. While efforts are being made to avoid this nonspecific reaction in MGL, the reaction mechanism remains unknown. Here, we present a detailed mechanistic investigation, which unveils the "S-glycosylation" being an atypical glycosylation termed S-glyco-modification. In alkaline protein microenvironments, per-O-acetylated monosaccharides undergo base-promoted ß-elimination to form thiol-reactive α,ß-unsaturated aldehydes, which then react with cysteine residues via Michael addition. This S-glyco-modification produces 3-thiolated sugars in hemiacetal form, rather than typical glycosides. The elimination-addition mechanism guides us to develop 1,6-di-O-propionyl-N-azidoacetylgalactosamine (1,6-Pr2GalNAz) as an improved unnatural monosaccharide for MGL.

Blue-Phosphorescent Pt(II) Complexes of Tetradentate Pyridyl-Carbolinyl Ligands: Synthesis, Structure, Photophysics, and Electroluminescence.

Blue phosphorescent tetradentate pyridyl-carbolinyl Pt(II) complexes, Pt(ppzOclpy-Me), Pt(ppzOclpy-iPr), and Pt(ppzOclpy-mesi), were purposefully synthesized and investigated with their photophysical and luminescent properties. The complexes, incorporating with carbolinyl moieties, have twisted planar structure. X-ray crystallography revealed that the intraligand N···H-C hydrogen bond reversely turned the twisty pyridyl moiety back into the chelating plane. Computational analyses confirmed that the metal-to-ligand charge-transfer transition character appears in the singlet manifolds. However, the ligand-centered transitions rule in their triplet states, which accounts for the phosphorescent emission. The Pt(II) complexes emit blue light with peak wavelengths (λmax) of 461-481 nm and moderate photoluminescent quantum yields (Φ = 34-46% in dichloromethane and Φ = 44-52% in films). The electroluminescent devices were fabricated by solution processes, giving blue emissions peaking at around 470 nm.

Quantitative time-resolved chemoproteomics reveals that stable O-GlcNAc regulates box C/D snoRNP biogenesis.

O-linked GlcNAcylation (O-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of O-GlcNAcylated proteins, we developed a quantitative time-resolved O-linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an O-GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 O-GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of O-GlcNAc or degradation of protein backbones. The stability of those hyperstable O-GlcNAcylated proteins was more sensitive to O-GlcNAcylation inhibition compared with the more dynamic populations. Among the hyperstable population were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs): fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). We showed that O-GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking O-GlcNAcylation on FBL altered the 2'-O-methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo.

[The Effects and Mechanisms of Dihydroartemisinin on Influenza A Virus H1N1 Induces TNF-α and IL-6 Expression in Bronchial Epithelial Cells].

OBJECTIVE: To investigate the effects of dihydroartemis (DHA) on influenza A virus (IAV) A/PR/8/34 (H1N1) induces the pro-inflammatory factor and protein of extracellular signal regulated kinase (ERK) signaling pathway expression in bronchial epithelial cells. METHODS: The BEAS-2B cells were treated with different concentrations of DHA (i.e.,0， 12.5, 25，50 and 100 µmol/L) for 24 h and the effect of DHA on the viability of BEAS-2B cells were measure by CCK8 method. The BEAS-2B cells were absorbed with IAV for 1 h, and then were treated with different concentrations of DHA (i.e., 12.5, 25 and 50 µmol/L) for 24 h, meanwhile, the normal control group and IAV group were established. The mRNA and protein expression levels of tumor necrosis factor-α (TNF-α) and interleukin (IL-6) were measured by real time quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), the expression levels of phospho-ERK (p-ERK) proteins were tested by Western blot (WB). Then, an ERK agonist (20 ng/mL) was used to treat BEAS-2B cells (the groups were divided into normal control group, DHA group, DHA+IAV group, ERK agonist group and DHA+IAV+ERK agonist group) for 24 h, and to observe the effect of DHA on inhibiting IAV induce the TNF-α, IL-6 and p-ERK expression in the BEAS-2B cells. RESULTS: The BEAS-2B cells viability was not significantly different from that of the normal control group after treatment with DHA (i.e., 12.5, 25, and 50 µmol/L). The expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein in IAV group were significantly up-regulated compared with that in the normal control group ( P<0.05), meanwhile, compared with the IAV group, the expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein showed dose-dependent decrease in IAV+DHA group ( P<0.05). However, ERK agonists attenuated the DHA inhibit IAV induced the proinflammatory factors TNF-α, IL-6 secretion and the p-ERK protein expression of ERK signaling pathway in BEAS-2B cells. CONCLUSION: These data suggest that DHA can inhibit IAV induces the TNF-α and IL-6 expression in BEAS-2B cells through ERK signaling pathway.

Visible Light-Initiated Bioorthogonal Photoclick Cycloaddition.

Here we report a visible light-triggered, catalyst free bioorthogonal reaction that proceeds via a distinct pathway from reported bioorthogonal reactions. The prototype of this bioorthogonal reaction was the photocycloaddition of 9,10-phenanthrenequinone with electron-rich alkenes to form fluorogenic [4+2] cycloadducts. The bioorthogonal photoclick cycloaddition was readily initiated using a conventional visible light source such as a hand-held LED lamp. The reaction proceeded rapidly under biocompatible conditions, without observable competition from side reactions such as nucleophilic additions by water or common nucleophilic species. The bioorthogonal functionality in this reaction did not cross react with various alkynes and electron-deficient alkenes such as monomethyl fumarate. We demonstrated orthogonal labeling of two proteins using this reaction together with a strain promoting azide-alkyne click reaction or the UV-triggered reaction of tetrazole with monomethyl fumarate. The application of this reaction in the temporal and spatial labeling of live cells was also demonstrated.

Quantitative Profiling of Protein Carbonylations in Ferroptosis by an Aniline-Derived Probe.

Ferroptosis is a regulated form of necrotic cell death implicated in carcinogenesis and neurodegeneration that is driven by phospholipid peroxidation. Lipid-derived electrophiles (LDEs) generated during this process can covalently modify proteins ("carbonylation") and affect their functions. Here we report the development of a quantitative chemoproteomic method to profile carbonylations in ferroptosis by an aniline-derived probe. Using the method, we established a global portrait of protein carbonylations in ferroptosis with >400 endogenously modified proteins and for the first time, identified >20 residue sites with endogenous LDE modifications in ferroptotic cells. Specifically, we discovered and validated a novel cysteine site of modification on voltage-dependent anion-selective channel protein 2 (VDAC2) that might play an important role in sensitizing LDE signals and mediating ferroptosis. Our results will contribute to the understanding of ferroptotic signaling and pathogenesis and provide potential biomarkers for ferroptosis detection.