Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike.

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct "S-R/x3" to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

[Establishment of a fast discriminant model with electronic nose for Polygonati Rhizoma mildew based on odor variation].

The odor fingerprint of Pollygonati Rhizoma samples with different mildewing degrees was analyzed and the relationship between the odor variation and the mildewing degree was explored. A fast discriminant model was established according to the response intensity of electronic nose. The α-FOX3000 electronic nose was applied to analyze the odor fingerprint of Pollygonati Rhizoma samples with different mildewing degrees and the radar map was used to analyze the main contributors among the volatile organic compounds. The feature data were processed and analyzed by partial least squares discriminant analysis(PLS-DA), K-nearest neighbor(KNN), sequential minimal optimization(SMO), random forest(RF) and naive Bayes(NB), respectively. According to the radar map of the electronic nose, the response values of three sensors, namely T70/2, T30/1, and P10/2, increased with the mildewing, indicating that the Pollygonati Rhizoma produced alkanes and aromatic compounds after the mildewing. According to PLS-DA model, Pollygonati Rhizoma samples of three mildewing degrees could be well distinguished in three areas. Afterwards, the variable importance analysis of the sensors was carried out and then five sensors that contributed a lot to the classification were screened out: T70/2, T30/1, PA/2, P10/1 and P40/1. The classification accuracy of all the four models(KNN, SMO, RF, and NB) was above 90%, and KNN was most accurate(accuracy: 97.2%). Different volatile organic compounds were produced after the mildewing of Pollygonati Rhizoma, and they could be detected by electronic nose, which laid a foundation for the establishment of a rapid discrimination model for mildewed Pollygonati Rhizoma. This paper shed lights on further research on change pattern and quick detection of volatile organic compounds in moldy Chinese herbal medicines.

[Identification of Curcuma herbs using XGBoost algorithm in electronic nose odor fingerprint].

This article aims to identify four commonly applied herbs from Curcuma genus of Zingiberaceae family,namely Curcumae Radix( Yujin),Curcumae Rhizoma( Ezhu),Curcumae Longae Rhizoma( Jianghuang) and Wenyujin Rhizoma Concisum( Pianjianghuang). The odor fingerprints of those four herbal medicines were collected by electronic nose,respectively. Meanwhile,XGBoost algorithm was introduced to data analysis and discriminant model establishment,with four indexes for performance evaluation,including accuracy,precision,recall,and F-measure. The discriminant model was established by XGBoost with positive rate of returning to 166 samples in the training set and 69 samples in the test set were 99. 39% and 95. 65%,respectively. The top four of the contribution to the discriminant model were LY2/g CT,P40/1,LY2/Gh and LY2/LG,the least contributing sensor was T70/2. Compared with support vector machine,random forest and artificial neural network,XGBoost algorithms shows better identification capacity with higher recognition efficiency. The accuracy,precision,recall and F-measure of the XGBoost discriminant model forecast set were 95. 65%,95. 25%,93. 07%,93. 75%,respectively. The superiority of XGBoost in the identification of Curcuma herbs was verified. Obviously,this new method could not only be suitable for digitization and objectification of traditional Chinese medicine( TCM) odor indicators,but also achieve the identification of different TCM based on their odor fingerprint in electronic nose system. The introduction of XGBoost algorithm and more excellent algorithms provide more ideas for the application of electronic nose in data mining for TCM studies.

[Effects of nitrogen and sulfur combined application on nutritional components and active components of Isatis indigotica at seedling stage].

Using split plot design, a pot experiment with sand culture was conducted to investigate the effects ofnitrogen and sulfur combined application on nutritional components and active component of Isatis indigotica at seedling stage under different N (5，15，25 mmol·L⁻¹)and S(0.00，1.25，2.50，5.00，7.50 mmol·L⁻¹) levels. The results showed thatthe two elements had obvious effects and the leaf and root dry weights of I. indigotica seedlings increased greatly at N2 level. Under the same nitrogen concentration, the leaf and root dry weights increased firstly and decreased with the rising of sulfur concentrations in which S2 was conducive to the growth and biomass accumulation. Soluble sugar, soluble protein, soluble amino acids contents were the highest in N1, N2 and N3 treatments, respectively. The influence of sulfur concentrations on nutritional components was same as biomass, but the peak of different nutritional components was diversity in different nitrogen levels. The effects on secondary metabolites (total flavones, indigo, indriubin, epigotrin contents) were not obvious significantly, in which these indexes by N1S3，N1S2，N3S0，N3S1were the highest, respectively. In conclusion, the combination of nitrogen and sulfur of N2S2(N 15 mmol·L⁻¹ and S 2.5 mmol·L⁻¹) was beneficial to the growth and secondary metabolites accumulation of I. indigotica. These results could provide a theoretical basis for rational fertilization and cultivation of I. indigotica seedling.

Cryo-EM structure and functional analysis of the chromatin remodeler RSF.

The RSF complex belongs to the ISWI chromatin-remodeling family and is composed of two subunits: RSF1 (remodeling and spacing factor 1) and SNF2h (sucrose nonfermenting protein 2 homolog). The RSF complex participates in nucleosome spacing and assembly, and subsequently promotes nucleosome maturation. Although SNF2h has been extensively studied in the last few years, the structural and functional properties of the remodeler RSF1 still remain vague. Here, a cryo-EM structure of the RSF-nucleosome complex is reported. The 3D model shows a two-lobe architecture of RSF, and the structure of the RSF-nucleosome (flanked with linker DNA) complex shows that the RSF complex moves the DNA away from the histone octamer surface at the DNA-entry point. Additionally, a nucleosome-sliding assay and a restriction-enzyme accessibility assay show that the RSF1 subunit may cause changes in the chromatin-remodeling properties of SNF2h. As a `nucleosome ruler', the results of an RSF-dinucleosome binding affinity test led to the proposal that the critical distance that RSF `measures' between two nucleosomes is about 24 base pairs.

Structural basis of nucleosome deacetylation and DNA linker tightening by Rpd3S histone deacetylase complex.

In Saccharomyces cerevisiae, cryptic transcription at the coding region is prevented by the activity of Sin3 histone deacetylase (HDAC) complex Rpd3S, which is carried by the transcribing RNA polymerase II (RNAPII) to deacetylate and stabilize chromatin. Despite its fundamental importance, the mechanisms by which Rpd3S deacetylates nucleosomes and regulates chromatin dynamics remain elusive. Here, we determined several cryo-EM structures of Rpd3S in complex with nucleosome core particles (NCPs), including the H3/H4 deacetylation states, the alternative deacetylation state, the linker tightening state, and a state in which Rpd3S co-exists with the Hho1 linker histone on NCP. These structures suggest that Rpd3S utilizes a conserved Sin3 basic surface to navigate through the nucleosomal DNA, guided by its interactions with H3K36 methylation and the extra-nucleosomal DNA linkers, to target acetylated H3K9 and sample other histone tails. Furthermore, our structures illustrate that Rpd3S reconfigures the DNA linkers and acts in concert with Hho1 to engage the NCP, potentially unraveling how Rpd3S and Hho1 work in tandem for gene silencing.

The tumor immune microenvironment of nasopharyngeal carcinoma after gemcitabine plus cisplatin treatment.

Gemcitabine plus cisplatin (GP) chemotherapy is the standard of care for nasopharyngeal carcinoma (NPC). However, the mechanisms underpinning its clinical activity are unclear. Here, using single-cell RNA sequencing and T cell and B cell receptor sequencing of matched, treatment-naive and post-GP chemotherapy NPC samples (n = 15 pairs), we show that GP chemotherapy activated an innate-like B cell (ILB)-dominant antitumor immune response. DNA fragments induced by chemotherapy activated the STING type-I-interferon-dependent pathway to increase major histocompatibility complex class I expression in cancer cells, and simultaneously induced ILB via Toll-like receptor 9 signaling. ILB further expanded follicular helper and helper type 1 T cells via the ICOSL-ICOS axis and subsequently enhanced cytotoxic T cells in tertiary lymphoid organ-like structures after chemotherapy that were deficient for germinal centers. ILB frequency was positively associated with overall and disease-free survival in a phase 3 trial of patients with NPC receiving GP chemotherapy ( NCT01872962 , n = 139). It also served as a predictor for favorable outcomes in patients with NPC treated with GP and immunotherapy combined treatment (n = 380). Collectively, our study provides a high-resolution map of the tumor immune microenvironment after GP chemotherapy and uncovers a role for B cell-centered antitumor immunity. We also identify and validate ILB as a potential biomarker for GP-based treatment in NPC, which could improve patient management.

Long Noncoding RNA TINCR-Mediated Regulation of Acetyl-CoA Metabolism Promotes Nasopharyngeal Carcinoma Progression and Chemoresistance.

Frontier evidence suggests that dysregulation of long noncoding RNAs (lncRNA) is ubiquitous in all human tumors, indicating that lncRNAs might have essential roles in tumorigenesis. Therefore, an in-depth study of the roles of lncRNA in nasopharyngeal carcinoma (NPC) carcinogenesis might be helpful to provide novel therapeutic targets. Here we report that lncRNA TINCR was significantly upregulated in NPC and was associated positively with poor survival. Silencing TINCR inhibited NPC progression and cisplatin resistance. Mechanistically, TINCR bound ACLY and protected it from ubiquitin degradation to maintain total cellular acetyl-CoA levels. Accumulation of cellular acetyl-CoA promoted de novo lipid biosynthesis and histone H3K27 acetylation, which ultimately regulated the peptidyl arginine deiminase 1 (PADI1)-MAPK-MMP2/9 pathway. In addition, insulin-like growth factor 2 mRNA-binding protein 3 interacted with TINCR and slowed its decay, which partially accounted for TINCR upregulation in NPC. These findings demonstrate that TINCR acts as a crucial driver of NPC progression and chemoresistance and highlights the newly identified TINCR-ACLY-PADI1-MAPK-MMP2/9 axis as a potential therapeutic target in NPC. SIGNIFICANCE: TINCR-mediated regulation of a PADI1-MAPK-MMP2/9 signaling pathway plays a critical role in NPC progression and chemoresistance, marking TINCR as a viable therapeutic target in this disease.

Comprehensive characterization of the alternative splicing landscape in head and neck squamous cell carcinoma reveals novel events associated with tumorigenesis and the immune microenvironment.

Alternative splicing (AS) has emerged as a key event in tumor development and microenvironment formation. However, comprehensive analysis of AS and its clinical significance in head and neck squamous cell carcinoma (HNSC) is urgently required. Methods: Genome-wide profiling of AS events using RNA-Seq data from The Cancer Genome Atlas (TCGA) program was performed in a cohort of 464 patients with HNSC. Cancer-associated AS events (CASEs) were identified between paired HNSC and adjacent normal tissues and evaluated in functional enrichment analysis. Splicing networks and prognostic models were constructed using bioinformatics tools. Unsupervised clustering of the CASEs identified was conducted and associations with clinical, molecular and immune features were analyzed. Results: We detected a total of 32,309 AS events and identified 473 CASEs in HNSC; among these, 91 were validated in an independent cohort (n = 15). Functional protein domains were frequently altered, especially by CASEs affecting cancer drivers, such as PCSK5. CASE parent genes were significantly enriched in pathways related to HNSC and the tumor immune microenvironment, such as the viral carcinogenesis (FDR < 0.001), Human Papillomavirus infection (FDR < 0.001), chemokine (FDR < 0.001) and T cell receptor (FDR < 0.001) signaling pathways. CASEs enriched in immune-related pathways were closely associated with immune cell infiltration and cytolytic activity. AS regulatory networks suggested a significant association between splicing factor (SF) expression and CASEs and might be regulated by SF methylation. Eighteen CASEs were identified as independent prognostic factors for overall and disease-free survival. Unsupervised clustering analysis revealed distinct correlations between AS-based clusters and prognosis, molecular characteristics and immune features. Immunogenic features and immune subgroups cooperatively depict the immune features of AS-based clusters. Conclusion: This comprehensive genome-wide analysis of the AS landscape in HNSC revealed novel AS events related to carcinogenesis and immune microenvironment, with implications for prognosis and therapeutic responses.