NKG2D discriminates diverse ligands through selectively mechano-regulated ligand conformational changes.

Stimulatory immune receptor NKG2D binds diverse ligands to elicit differential anti-tumor and anti-virus immune responses. Two conflicting degeneracy recognition models based on static crystal structures and in-solution binding affinities have been considered for almost two decades. Whether and how NKG2D recognizes and discriminates diverse ligands still remain unclear. Using live-cell-based single-molecule biomechanical assay, we characterized the in situ binding kinetics of NKG2D interacting with different ligands in the absence or presence of mechanical force. We found that mechanical force application selectively prolonged NKG2D interaction lifetimes with the ligands MICA and MICB, but not with ULBPs, and that force-strengthened binding is much more pronounced for MICA than for other ligands. We also integrated steered molecular dynamics simulations and mutagenesis to reveal force-induced rotational conformational changes of MICA, involving formation of additional hydrogen bonds on its binding interface with NKG2D, impeding MICA dissociation under force. We further provided a kinetic triggering model to reveal that force-dependent affinity determines NKG2D ligand discrimination and its downstream NK cell activation. Together, our results demonstrate that NKG2D has a discrimination power to recognize different ligands, which depends on selective mechanical force-induced ligand conformational changes.

A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2.

An outbreak of coronavirus disease 2019 (COVID-19)1-3, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, has spread globally. Countermeasures are needed to treat and prevent further dissemination of the virus. Here we report the isolation of two specific human monoclonal antibodies (termed CA1 and CB6) from a patient convalescing from COVID-19. CA1 and CB6 demonstrated potent SARS-CoV-2-specific neutralization activity in vitro. In addition, CB6 inhibited infection with SARS-CoV-2 in rhesus monkeys in both prophylactic and treatment settings. We also performed structural studies, which revealed that CB6 recognizes an epitope that overlaps with angiotensin-converting enzyme 2 (ACE2)-binding sites in the SARS-CoV-2 receptor-binding domain, and thereby interferes with virus-receptor interactions by both steric hindrance and direct competition for interface residues. Our results suggest that CB6 deserves further study as a candidate for translation to the clinic.

Defining a de novo non-RBM antibody as RBD-8 and its synergistic rescue of immune-evaded antibodies to neutralize Omicron SARS-CoV-2.

Currently, monoclonal antibodies (MAbs) targeting the SARS-CoV-2 receptor binding domain (RBD) of spike (S) protein are classified into seven classes based on their binding epitopes. However, most of these antibodies are seriously impaired by SARS-CoV-2 Omicron and its subvariants, especially the recent BQ.1.1, XBB and its derivatives. Identification of broadly neutralizing MAbs against currently circulating variants is imperative. In this study, we identified a "breathing" cryptic epitope in the S protein, named as RBD-8. Two human MAbs, BIOLS56 and IMCAS74, were isolated recognizing this epitope with broad neutralization abilities against tested sarbecoviruses, including SARS-CoV, pangolin-origin coronaviruses, and all the SARS-CoV-2 variants tested (Omicron BA.4/BA.5, BQ.1.1, and XBB subvariants). Searching through the literature, some more RBD-8 MAbs were defined. More importantly, BIOLS56 rescues the immune-evaded antibody, RBD-5 MAb IMCAS-L4.65, by making a bispecific MAb, to neutralize BQ.1 and BQ.1.1, thereby producing an MAb to cover all the currently circulating Omicron subvariants. Structural analysis reveals that the neutralization effect of RBD-8 antibodies depends on the extent of epitope exposure, which is affected by the angle of antibody binding and the number of up-RBDs induced by angiotensin-converting enzyme 2 binding. This cryptic epitope which recognizes non- receptor binding motif (non-RBM) provides guidance for the development of universal therapeutic antibodies and vaccines against COVID-19.

SNX27 suppresses SARS-CoV-2 infection by inhibiting viral lysosome/late endosome entry.

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27-retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.

LCDR regulates the integrity of lysosomal membrane by hnRNP K-stabilized LAPTM5 transcript and promotes cell survival.

Lysosome plays important roles in cellular homeostasis, and its dysregulation contributes to tumor growth and survival. However, the understanding of regulation and the underlying mechanism of lysosome in cancer survival is incomplete. Here, we reveal a role for a histone acetylation-regulated long noncoding RNA termed lysosome cell death regulator (LCDR) in lung cancer cell survival, in which its knockdown promotes apoptosis. Mechanistically, LCDR binds to heterogenous nuclear ribonucleoprotein K (hnRNP K) to regulate the stability of the lysosomal-associated protein transmembrane 5 (LAPTM5) transcript that maintains the integrity of the lysosomal membrane. Knockdown of LCDR, hnRNP K, or LAPTM5 promotes lysosomal membrane permeabilization and lysosomal cell death, thus consequently resulting in apoptosis. LAPTM5 overexpression or cathepsin B inhibitor partially restores the effects of this axis on lysosomal cell death in vitro and in vivo. Similarly, targeting LCDR significantly decreased tumor growth of patient-derived xenografts of lung adenocarcinoma (LUAD) and had significant cell death using nanoparticles (NPs)-mediated systematic short interfering RNA delivery. Moreover, LCDR/hnRNP K/LAPTM5 are up-regulated in LUAD tissues, and coexpression of this axis shows the increased diagnostic value for LUAD. Collectively, we identified a long noncoding RNA that regulates lysosome function at the posttranscriptional level. These findings shed light on LCDR/hnRNP K/LAPTM5 as potential therapeutic targets, and targeting lysosome is a promising strategy in cancer treatment.

Brassisterol A, a new ergosterol from co-cultivation of fungi attenuates neuroinflammation via targeting NLRP3/caspase-1/GSDMD pathway.

Three new ergosterol derivatives brassisterol A-C (1-3) and two new epimeric bicycle-lactones brassictones A and B (4 and 5), were isolated from the co-cultivation of Alternaria brassicicola and Penicillium granulatum. The absolute configurations of these isolates were confirmed by extensive NMR spectra, TD-DFT ECD calculation, and the single crystal XRD data analysis. Amongst the metabolites, compound 1 exhibited potential anti-Parkinson's disease activity in both MPTP-induced zebrafish and MPP+-induced SH-SY5Y cells. Molecular mechanism studies in vitro showed that 1 attenuated the increase of α-synuclein, NLRP3, ASC, caspase-1, IL-1ß, IL-18, and GSDMD expression in the MPP+ induced PD model. Molecular docking in silico simulations exhibited that 1 was well accommodated to one of the binding pockets of NLRP3 8ETR in an appropriate conformation via forming typical hydrogen bonds as well as possessing a high negative binding affinity (-8.97 kcal/mol). Thus, our work suggested that 1 protected dopaminergic cell from neuroinflammation via targeting NLRP3/caspase-1/GSDMD signaling pathway.

Granulathiazole A protects 6-OHDA-induced Parkinson's disease from ferroptosis via activating Nrf2/HO-1 pathway.

Two pairs of enantiomers (1a-2b), namely (±)-alterpyrone F and (±)-alterpyrone G, along with a rare benzothiazole meroterpenoid granulathiazole A (3, GA), and two undescribed compounds called respectively granulahydeoate (4) and granulaone (5), were obtained from the co-cultivation of Alternaria brassicicola and Penicillium sp. HUBU0120. Exhaustive analyses of NMR, single crystal XRD, Mo2(OAc)4-induced circular dichroism data, and a modified Mosher's method distinguished the absolute configurations of isolates. Bioactive evaluations exhibited that GA possessed promising anti-PD activity in both in vitro and in vivo PD models viz. 6-OHDA-induced SH-SY5Y cells and 6-OHDA-induced zebrafish, respectively. Moreover, our research demonstrated that ferroptosis activated by 6-OHDA was mitigated in PD models after treated with GA. Extensive molecular mechanism studies in PD-modelled cells manifested that GA attenuated the decreased expressions of SLC7A11, GPX4, and FSP-1, and the increased level of ACSL4 via activating Nrf2/HO-1 pathway as well as ameliorated the accumulation of α-synuclein.

Mechanisms of adsorption and functionalization of biochar for pesticides: A review.

Agricultural production relies heavily on pesticides. However, factors like inefficient application, pesticide resistance, and environmental conditions reduce their effective utilization in agriculture. Subsequently, pesticides transfer into the soil, adversely affecting its physicochemical properties, microbial populations, and enzyme activities. Different pesticides interacting can lead to combined toxicity, posing risks to non-target organisms, biodiversity, and organism-environment interactions. Pesticide exposure may cause both acute and chronic effects on human health. Biochar, with its high specific surface area and porosity, offers numerous adsorption sites. Its stability, eco-friendliness, and superior adsorption capabilities render it an excellent choice. As a versatile material, biochar finds use in agriculture, environmental management, industry, energy, and medicine. Added to soil, biochar helps absorb or degrade pesticides in contaminated areas, enhancing soil microbial activity. Current research primarily focuses on biochar produced via direct pyrolysis for pesticide adsorption. Studies on functionalized biochar for this purpose are relatively scarce. This review examines biochar's pesticide absorption properties, its characteristics, formation mechanisms, environmental impact, and delves into adsorption mechanisms, functionalization methods, and their prospects and limitations.

Hydroquinone-induced skin irritant reaction could be achieved by activating mast cells via mas-related G protein-coupled receptor X2.

Hydroquinone (HQ) is one of the most effective drugs to treat hyperpigmentary disorders, but often causes skin irritation in clinic. Mast cell plays an important role in contact dermatitis and triggering pseudo-allergic reactions via MRGPRX2. Whether HQ-induced skin irritant reaction through activating mast cells via MRGPRX2 remains unknown. To investigate the role of mast cells in HQ-induced skin irritant reaction and verify whether MRGPRX2 participated in the HQ effect on mast cells which contributed to the pathogenesis of skin irritant reaction, a mouse model of HQ-induced skin irritation was established to observe the local and systemic inflammation associated with mast cell receptor MrgprB2. Human mast cell LAD2 was used to verify the effect of HQ on mast cells via MRGPRX2 by knocking down with siRNA. As a result, mast cells were involved in the development of HQ-induced irritant reaction, and local inflammation is closely related to mast cell receptor MrgprB2. HQ could activate mast cells via MRGPRX2, causing changes in calcium concentration, degranulation and release of inflammatory cytokines which lead to skin irritant reaction. In conclusion, HQ-induced skin irritant reaction could be skin pseudo-allergic reactions achieved by activating mast cells via MRGPRX2.

Chaetocochin J exhibits anti-hepatocellular carcinoma effect independent of hypoxia.

The most studied epipolythiodioxopiperazine (ETP) alkaloids, such as chetomin, gliotoxin and chaetocin, were reported to exert their antitumor effects through targeting HIF-1α. Chaetocochin J (CJ) is another ETP alkaloid, of which the effect and mechanism on cancer are not fully elucidated. Considering the high incidence and mortality of hepatocellular carcinoma (HCC) in China, in the present study, using HCC cell lines and tumor-bearing mice as models, we explored the anti-HCC effect and mechanism of CJ. Particularly, we investigated whether HIF-1α is related to the function of CJ. The results showed that, both under normoxic and CoCl2 induced-hypoxic conditions, CJ in low concentrations (<1 µM) inhibits the proliferation, induces G2/M phase arrest, leading to the disorder of metabolism, migration, invasion, and caspase-dependent apoptosis in HepG2 and Hep3B cells. CJ also showed anti-tumor effect on a nude xenograft mice model without significant toxicity. Moreover, we demonstrated that the key to CJ's function is mainly associate with its inhibition of PI3K/Akt/mTOR/p70S6K/4EBP1 pathway independent of hypoxia, and it also could suppress the expression of HIF-1α as well as disrupt the binding of HIF-1α/p300 and subsequently inhibits the expression of its target genes under hypoxic condition. These results demonstrated that CJ possessed a hypoxia-independent anti-HCC effects in vitro and in vivo, which was mainly attributable to its inhibition on the upstream pathways of HIF-1α.

High-precision optical frequency transfer over a 96 km urban fiber link.

We demonstrated the optical frequency transfer over a 96 km urban business network in Shanghai. The key factors affecting the optical frequency transmission system, such as fiber link quality, feedback compensation strength, and out-of-loop fiber temperature variation, are studied for the urban fiber link characteristics. The effective suppression technique of complex urban fiber link noise with different feedback compensation parameters is studied. With active phase noise suppression, the optical frequency stability can reach 1.9×10-16 at 1 s and 2.2×10-18 at 10,000 s over a 96 km urban fiber link. This work potentially plays an important role in optical clock frequency comparison, and it makes a good foundation for future research on long-distance optical frequency transfer over an urban business network.

Molecular basis of Coxsackievirus A10 entry using the two-in-one attachment and uncoating receptor KRM1.

KREMEN1 (KRM1) has been identified as a functional receptor for Coxsackievirus A10 (CV-A10), a causative agent of hand-foot-and-mouth disease (HFMD), which poses a great threat to infants globally. However, the underlying mechanisms for the viral entry process are not well understood. Here we determined the atomic structures of different forms of CV-A10 viral particles and its complex with KRM1 in both neutral and acidic conditions. These structures reveal that KRM1 selectively binds to the mature viral particle above the canyon of the viral protein 1 (VP1) subunit and contacts across two adjacent asymmetry units. The key residues for receptor binding are conserved among most KRM1-dependent enteroviruses, suggesting a uniform mechanism for receptor binding. Moreover, the binding of KRM1 induces the release of pocket factor, a process accelerated under acidic conditions. Further biochemical studies confirmed that receptor binding at acidic pH enabled CV-A10 virion uncoating in vitro. Taken together, these findings provide high-resolution snapshots of CV-A10 entry and identify KRM1 as a two-in-one receptor for enterovirus infection.

Tumor cell-intrinsic PD-1 receptor is a tumor suppressor and mediates resistance to PD-1 blockade therapy.

The programmed cell death 1 (PD-1) receptor on the surface of immune cells is an immune checkpoint molecule that mediates the immune escape of tumor cells. Consequently, antibodies targeting PD-1 have shown efficacy in enhancing the antitumor activity of T cells in some types of cancers. However, the potential effects of PD-1 on tumor cells remain largely unknown. Here, we show that PD-1 is expressed across a broad range of tumor cells. The silencing of PD-1 or its ligand, PD-1 ligand 1 (PD-L1), promotes cell proliferation and colony formation in vitro and tumor growth in vivo. Conversely, overexpression of PD-1 or PD-L1 inhibits tumor cell proliferation and colony formation. Moreover, blocking antibodies targeting PD-1 or PD-L1 promote tumor growth in cell cultures and xenografts. Mechanistically, the coordination of PD-1 and PD-L1 activates its major downstream signaling pathways including the AKT and ERK1/2 pathways, thus enhancing tumor cell growth. This study demonstrates that PD-1/PD-L1 is a potential tumor suppressor and potentially regulates the response to anti-PD-1/PD-L1 treatments, thus representing a potential biomarker for the optimal cancer immunotherapeutic treatment.

Residual Behavior and Dietary Risk Assessment of Chlorfenapyr and Its Metabolites in Radish.

Chlorfenapyr, as a highly effective and low-toxicity insect growth regulation inhibitor, has been used to control cross-cruciferous vegetable pests. However, the pesticide residue caused by its application threatens human health. In this paper, the residue digestion and final residue of chlorfenapyr in radish were studied in a field experiment. The results of the dynamic digestion test showed that the half-life of chlorfenapyr in radish leaves ranged from 6.0 to 6.4 days, and the digestion rate was fast. The median residual values of chlorfenapyr in radish and radish leaves at 14 days after treatment were 0.12 and 3.92 mg/kg, respectively. The results of the dietary intake risk assessment showed that the national estimated daily intake (NEDI) of chlorfenapyr in various populations in China were 0.373 and 5.66 µg/(kg bw·d), respectively. The risk entropy (RQ) was 0.012 and 0.147, respectively, indicating that the chronic dietary intake risk of chlorfenapyr in radish was low. The results of this study provided data support and a theoretical basis for guiding the scientific use of chlorfenapyr in radish production and evaluating the dietary risk of chlorfenapyr in vegetables.

Construction of TME and Identification of crosstalk between malignant cells and macrophages by SPP1 in hepatocellular carcinoma.

Liver cancer accounts for 6% of all malignancies causing death worldwide, and hepatocellular carcinoma (HCC) is the most common histological type. HCC is a heterogeneous cancer, but how the tumour microenvironment (TME) of HCC contributes to the progression of HCC remains unclear. In this study, we investigated the immune microenvironment by multiomics analysis. The tumour immune infiltration characteristics of HCC were determined at the genomic, epigenetic, bulk transcriptome and single-cell levels by data from The Cancer Genome Atlas portal and the Gene Expression Omnibus (GEO). An epigenetic immune-related scoring system (EIRS) was developed to stratify patients with poor prognosis. SPP1, one gene in the EIRS system, was identified as an immune-related predictor of poor survival in HCC patients. Through receptor-ligand pair analysis in single-cell RNA-seq, SPP1 was indicated to mediate the crosstalk between HCC cells and macrophages via SPP1-CD44 and SPP1-PTGER4 association. In vitro experiments further validate SPP1 can trigger the polarization of macrophages to M2-phenotype tumour-associated macrophages (TAMs).

Stable optical and radio frequency joint transfer based on a passive phase compensation.

We propose a novel scheme that uses only a single passive phase compensation device to achieve stable optical and radio frequency joint transfer. The phase noises of optical and radio frequency can be simultaneously compensated by passively embedding their phase information on the two optical carrier sidebands generated by an electro-optical modulator without using the phase discrimination and active servo controller. As a result, this scheme has many advantages, such as high spectral purity, short settling time and infinite compensation accuracy. We experimentally demonstrate the joint transfer of optical and 1 GHz RF over 120 km fiber spools. The optical frequency stability achieves 6.9 × 10-17 at 1 s and 7.03 × 10-19 at 10000 s, while the 1 GHz RF is 6.47 × 10-13 at 1 s and 3.96 × 10-16 at 10000 s.

Cu(I)-Catalyzed C-H Alkenylation of Tertiary C(sp3)-H Bonds of 3-Aryl Benzofuran-2(3H)-ones to Give Z- and E-Styrene Containing Quaternary Carbon Centers with 99/1 Regioselectivity.

The synthesis of isomerically pure olefins containing all-carbon quaternary centers is a challenging issue. Herein, we developed an efficient protocol for the synthesis of (Z)-olefins (27 examples, yield up to 97%, Z/E up to 99/1) and (E)-olefins (16 examples, yield up to 94%, E/Z up to 99/1) containing all-carbon quaternary centers. This protocol is adopted for the copper-catalyzed regioselective C-H alkenylation of the tertiary C(sp3)-H bond of 3-aryl benzofuran-2(3H)-ones with alkyne and alkenes. A diverse range of functional groups in the substrates is well-tolerated, such as F, Cl, Br, Me, OMe, ester, CF3, etc. A gram scale experiment was performed in good yield, and the radical mechanisms are also proposed based on the control experiments.

Integrated Bioinformatics Analysis Identifies microRNA-200a-5p as a New Plasma Marker in Patients with Venous Thromboembolism.

BACKGROUND: Venous thromboembolism (VTE) is a major global health problem with high incidence and mortality. Vein endothelial cell (VEC) dysfunction is the primary cause of VTE. MicroRNAs (miRNAs) assist in the regulation of VEC functional pathways. Our objective was to identify potential miRNA target genes associated with VTE. MATERIALS AND METHODS: To explore the association between mRNAs and miRNAs in VTE, we performed an mRNA or miRNA microarray analysis and experiments in vitro. In addition, five online bioinformatics tools identified the target genes of differentially expressed miRNAs, and a miRNA-gene network was constructed. As a result, hub miRNA and mRNA were confirmed. Finally, wound healing assay and transwell migration assay were performed to elucidate the effect of hub miRNA in VEC. Luciferase reporter assay and real-time quantitative polymerase chain reaction (RT-qPCR) were performed to decide the role of miRNA in the expression of hub mRNA. RESULTS: Screening identified eight overlapping dysregulated genes in patients with VTE, three of which demonstrated a significantly decreased expression of miR-200a-5p. Low expression miR-200a-5p in VTE patients is confirmed by a receiver operating characteristic analysis (AUC = 0.800, P = 0.023) and binary logistic regression (OR = 0.359, 95% confidence interval: 0.605-0.995). RT-qPCR results showed that the miR-200a-5p level was decreased in hypoxia VEC (P = 0.038). MiR-200a-5p significantly promoted the migration ability of VEC. The result of Dual-luciferase reporter assay showed that cytochrome coxidase Ⅶc (COX7C) directly inhibit the miR-200a-5p expression by binding 5'UTR of miR-200a-5p (P = 0.011). CONCLUSIONS: We anticipate that the miR-200a-5p-COX7C pair might be involved in the progression of VTE. Moreover, miR-200a-5p might be a therapeutic target for VTE.

Novel Prenylated Indole Alkaloids with Neuroprotection on SH-SY5Y Cells against Oxidative Stress Targeting Keap1-Nrf2.

Oxidative stress has been implicated in the etiology of Parkinson's disease (PD). Molecules non-covalently binding to the Keap1-Nrf2 complex could be a promising therapeutic approach for PD. Herein, two novel prenylated indole alkaloids asperpenazine (1), and asperpendoline (2) with a scarce skeleton of pyrimido[1,6-a]indole were discovered from the co-cultivated fungi of Aspergillus ochraceus MCCC 3A00521 and Penicillium sp. HUBU 0120. Compound 2 exhibited potential neuroprotective activity on SH-SY5Y cells against oxidative stress. Molecular mechanism research demonstrated that 2 inhibited Keap1 expression, resulting in the translocation of Nrf2 from the cytoplasm to the nucleus, activating the downstream genes expression of HO-1 and NQO1, leading to the reduction in reactive oxygen species (ROS) and the augment of glutathione. Molecular docking and dynamic simulation analyses manifested that 2 interacted with Keap1 (PDB ID: 1X2R) via forming typical hydrogen and hydrophobic bonds with residues and presented less fluctuation of RMSD and RMSF during a natural physiological condition.

Stereoselective bioactivity, toxicity and degradation of novel fungicide sedaxane with four enantiomers under rice-wheat rotation mode.

Sedaxane was a novel chiral fungicide that contains four enantiomers. Unfortunately, the stereoselective bioactivity, toxicity and degradation of sedaxane have not been clarified. In this study, we identified the absolute configuration of the four sedaxane enantiomers at first time. The stereoselective bioactivity toward three wheat and rice pathogens, stereoselective acute toxicity to aquatic organisms (Selenastrum capricornutum and Daphnia magna), and stereoselective degradation of sedaxane were studied. The 1 S,2S-(+)-sedaxane possessed 5.4-7.3 times greater bioactivity than 1 R,2R-(-)-sedaxane to Rhizoctonia solani and Rhizoctonia cerealis. Contrarily, the 1 R,2S-(+)-sedaxane had 4.2 times greater activity than 1 S,2S-(+)-sedaxane against Fusarium graminearum. The 1 R,2R-(-)-sedaxane had 2.8 times greater toxicity than 1 S,2S-(+)-sedaxane to S. capricornutum. The chiral determination method used ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The recovery of sedaxane stereoisomers ranged from 83.1 % to 98.2 %, with RSDs (Relative standard deviations) of 1.2 %- 8.4 %. The trans-sedaxane existed stereoselective degradation phenomenon in the rice-wheat rotation mode, and 1 S,2S-(+)-sedaxane was preferentially degraded. Our results would provide scientific importance and practical guidance to the safety evaluation of chiral pesticides.