Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3).

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

GhEXL3 participates in brassinosteroids regulation of fiber elongation in Gossypium hirsutum.

Cotton fiber (Gossypium hirsutum) serves as an ideal model for investigating the molecular mechanisms of plant cell elongation at the single-cell level. Brassinosteroids (BRs) play a crucial role in regulating plant growth and development. However, the mechanism by which BR influences cotton fiber elongation remains incompletely understood. In this study, we identified EXORDIUM-like (GhEXL3) through transcriptome analysis of fibers from BR-deficient cotton mutant pagoda 1 (pag1) and BRI1-EMS-SUPPRESSOR 1 (GhBES1.4, encoding a central transcription factor of BR signaling) overexpression cotton lines. Knockout of GhEXL3 using CRISPR/Cas9 was found to impede cotton fiber elongation, while its overexpression promoted fiber elongation, suggesting a positive regulatory function for GhEXL3 in fiber elongation. Furthermore, in vitro ovule culture experiments revealed that the overexpression of GhEXL3 partially counteracted the inhibitory effects of brassinazole (BRZ) on cotton fiber elongation, providing additional evidence of GhEXL3 involvement in BR signaling pathways. Moreover, our findings demonstrate that GhBES1.4 directly binds to the E-box (CACGTG) motif in the GhEXL3 promoter region and enhances its transcription. RNA-seq analysis revealed that overexpression of GhEXL3 upregulated the expression of EXPs, XTHs, and other genes associated with fiber cell elongation. Overall, our study contributes to understanding the mechanism by which BR regulates the elongation of cotton fibers through the direct modulation of GhEXL3 expression by GhBES1.4.

N6 -Methyladenosine mRNA modification regulates transcripts stability associated with cotton fiber elongation.

N6 -Methyladenosine (m6 A) is the most abundant methylation modification in eukaryotic mRNA. The discovery of the dynamic and reversible regulatory mechanism of m6 A has greatly promoted the development of m6 A-led epitranscriptomics. However, the characterization of m6 A in cotton fiber is still unknown. Here, we reveal the potential link between m6 A modification and cotton fiber elongation by parallel m6 A-immunoprecipitation-sequencing (m6 A-seq) and RNA-seq analysis of fibers from the short fiber mutants Ligonliness-2 (Li2 ) and wild-type (WT). This study demonstrated a higher level of m6 A in the Li2 mutant, with the enrichment of m6 A modifications in the stop codon, 3'-untranslated region and coding sequence regions than in WT cotton. In the correlation analysis between genes containing differential m6 A modifications and differentially expressed genes, we identified several genes that could potentially regulate fiber elongation, including cytoskeleton, microtubule binding, cell wall and transcription factors (TFs). We further confirmed that the methylation of m6 A affected the mRNA stability of these fiber elongation-related genes including the TF GhMYB44, which showed the highest expression level in the RNA-seq data and m6 A methylation in the m6 A-seq data. Next, the overexpression of GhMYB44 reduces fiber elongation, whereas the silencing of GhMYB44 produces longer fibers. In summary, these results uncover that m6 A methylation regulated the expression of genes related to fiber development by affecting mRNA's stability, ultimately affecting cotton fiber elongation.

Discovery and Biosynthesis of Cihanmycins Reveal Cytochrome P450-Catalyzed Intramolecular C-O Phenol Coupling Reactions.

Cinnamoyl-containing nonribosomal peptides (CCNPs) constitute a unique family of natural products. The enzyme mechanism for the biaryl phenol coupling reaction of the bicyclic CCNPs remains unclear. Herein, we report the discovery of two new arabinofuranosylated bicyclic CCNPs cihanmycins (CHMs) A (1) and B (2) from Amycolatopsis cihanbeyliensis DSM 45679 and the identification of the CHM biosynthetic gene cluster (cih BGC) by heterologous expression in Streptomyces lividans SBT18 to afford CHMs C (3) and D (4). The structure of 1 was confirmed by X-ray diffraction analysis. Three cytochrome P450 enzyme (CYP)-encoding genes cih26, cih32, and cih33 were individually inactivated in the heterologous host to produce CHMs E (5), F (6), and G (7), respectively. The structures of 5 and 6 indicated that Cih26 was responsible for the hydroxylation and epoxidation of the cinnamoyl moiety, and Cih32 should catalyze the ß-hydroxylation of three amino acid residues. Cih33 and its homologues DmlH and EpcH were biochemically verified to convert CHM G (7) with a monocyclic structure to a bicyclic skeleton of CHM C (3) through an intramolecular C-O phenol coupling reaction. The substrate 7-bound crystal structure of DmlH not only established the structure of 7, which was difficult for NMR analysis for displaying anomalous splitting signals, but also provided the binding mode of macrocyclic peptides recognized by these intramolecular C-O coupling CYPs. In addition, computational studies revealed a water-mediated diradical mechanism for the C-O phenol coupling reaction. These findings have shed important mechanistic insights into the CYP-catalyzed phenol coupling reactions.

Achieving Regioselectivity for Remote C-H Activation by Substructure Conformations: an Approach of Paralogous Cytochrome P450 Enzymes.

For advanced synthetic intermediates or natural products with multiple unactivated and energetically similar C(sp3)-H bonds, controlling regioselectivity for the C-H activation is particularly challenging. The use of cytochrome P450 enzymes (CYPs) is a promising solution to the 'regioelectivity' challenge in remote C-H activation. Notably, CYPs and organic catalysts share a fundamental principle: they strive to control the distance and geometry between the metal reaction center and the target C-H site. Most structural analyses of the regioselectivity of CYPs are limited to the active pocket, particularly when explaining why regioselectivity could be altered by enzyme engineering through mutagenesis. However, the substructures responsible for forming the active pocket in CYPs are well known to display complex dynamic changes and substrate-induced plasticity. In this context, we highlight a comparative study of the recently reported paralogous CYPs, IkaD and CftA, which achieve different regioselectivity towards the same substrate ikarugamycin by distinct substructure conformations. We propose that substructural conformation-controlled regioselectivity might also be present in CYPs of other natural product biosynthesis pathways, which should be considered when engineering CYPs for regioselective modifications.

Non-enzymatic synthesis of C-methylated fluostatins: discovery and reaction mechanism.

Two C-methylated fluostatins (FSTs) B3 (1) and B4 (2) were synthesized from flavin-mediated nonenzymatic epoxide ring-opening reactions of FST C. The structures of 1 and 2 were elucidated by HRESIMS, NMR, and ECD spectroscopic analyses. A subsequent 13C labeling study demonstrated that the C-methyl groups of 1 and 2 were derived from DMSO and enabled the mechanistic proposal of a nonenzymatic C-methylation.

Target-Specific De Novo Peptide Binder Design with DiffPepBuilder.

Despite the exciting progress in target-specific de novo protein binder design, peptide binder design remains challenging due to the flexibility of peptide structures and the scarcity of protein-peptide complex structure data. In this study, we curated a large synthetic data set, referred to as PepPC-F, from the abundant protein-protein interface data and developed DiffPepBuilder, a de novo target-specific peptide binder generation method that utilizes an SE(3)-equivariant diffusion model trained on PepPC-F to codesign peptide sequences and structures. DiffPepBuilder also introduces disulfide bonds to stabilize the generated peptide structures. We tested DiffPepBuilder on 30 experimentally verified strong peptide binders with available protein-peptide complex structures. DiffPepBuilder was able to effectively recall the native structures and sequences of the peptide ligands and to generate novel peptide binders with improved binding free energy. We subsequently conducted de novo generation case studies on three targets. In both the regeneration test and case studies, DiffPepBuilder outperformed AfDesign and RFdiffusion coupled with ProteinMPNN, in terms of sequence and structure recall, interface quality, and structural diversity. Molecular dynamics simulations confirmed that the introduction of disulfide bonds enhanced the structural rigidity and binding performance of the generated peptides. As a general peptide binder de novo design tool, DiffPepBuilder can be used to design peptide binders for given protein targets with three-dimensional and binding site information.

Discovery of Kebanmycins with Antibacterial and Cytotoxic Activities from the Mangrove-Derived Streptomyces sp. SCSIO 40068.

Mangrove derived actinomycetes are a rich reservoir of bioactive natural products and play important roles in pharmaceutical chemistry. In a screen of actinomycetes from mangrove rhizosphere sedimental environments, the isolated strain Streptomyces sp. SCSIO 40068 displayed strong antibacterial activity. Further fractionation of the extract yielded four new compounds kebanmycins A-D (1-4) and two known analogues FD-594 (5) and the aglycon (6). The structures of 1-6 were determined based on extensive spectroscopic data and single-crystal X-ray diffraction analysis. 1-3 featured a fused pyranonaphthaxanthene as an integral part of a 6/6/6/6/6/6 polycyclic motif, and showed bioactivity against a series of Gram-positive bacteria and cytotoxicity to several human tumor cells. In addition, the kebanmycins biosynthetic gene cluster (keb) was identified in Streptomyces sp. SCSIO 40068, and KebMT2 was biochemically characterized as a tailoring sugar-O-methyltransferase, leading to a proposed biosynthetic route to 1-6. This study paves the way to further investigate 1 as a potential lead compound.

Expanding the Chemical Diversity of Grisechelins via Heterologous Expression.

Thiazole scaffold-based small molecules exhibit a range of biological activities and play important roles in drug discovery. Based on bioinformatics analysis, a putative biosynthetic gene cluster (BGC) for thiazole-containing compounds was identified from Streptomyces sp. SCSIO 40020. Heterologous expression of this BGC led to the production of eight new thiazole-containing compounds, grisechelins E, F, and I-N (1, 2, 5-10), and two quinoline derivatives, grisechelins G and H (3 and 4). The structures of 1-10, including their absolute configurations, were elucidated by HRESIMS, NMR spectroscopic data, ECD calculations, and single-crystal X-ray diffraction analysis. Grisechelin F (2) is a unique derivative, distinguished by the presence of a salicylic acid moiety. The biosynthetic pathway for 2 was proposed based on bioinformatics analysis and in vivo gene knockout experiments. Grisechelin E (1) displayed moderate antimycobacterial activity against Mycobacterium tuberculosis H37Ra (MIC of 8 µg mL-1).

Public Knowledge and Awareness of Anesthesiology and Anesthesiologists in China: A Cross-Sectional National Survey of 1 Million Participants.

BACKGROUND: In the past 20 years, anesthesiology has become one of the most advanced specialties and has undergone rapid development. However, public awareness regarding anesthesiology and anesthesiologists is limited, especially in developing countries. It is important for anesthesiologists to make the public aware of their role during surgery. Therefore, a nationwide survey was set up to investigate public awareness of anesthesiology and anesthesiologists in China. METHOD: A cross-sectional nationwide survey was performed from June 2018 to June 2019 in 34 provinces, municipalities, and autonomous regions across China and an overseas region. The questionnaires of the survey were divided into 2 main parts: general items and research items. General items included the demographic characteristics of the participants; research items consisted of 10 questions about the public's awareness of anesthesiologists and anesthesiology. Data quality control was undertaken by the investigation committee throughout the survey process. RESULTS: The nationwide survey enrolled 1,001,279 participants (male, 40.7%). We found that most of the participants regarded anesthesiologists as doctors. However, public knowledge of anesthesiologists' work and duties during surgery was quite low, with correct response rate ranging from 16.5% to 52.9%, and anesthesiologist responsibilities were often mistakenly attributed to surgeons or nurses. It is disappointing that more than half of participants still thought that, once the patient fell asleep after receiving anesthetics, the anesthesiologist could leave the operating room. Finally, the correct response rate was positively correlated with the economic levels of the regions. CONCLUSIONS: Public awareness regarding anesthesiology and anesthesiologists in China remains inadequate. Due to the biases and characteristics of the participants, the actual situation of the general Chinese public is likely even worse than reflected here. Therefore, extensive measures should be undertaken to improve public knowledge of anesthesiology and anesthesiologists.

A ratiometric fluorescence and colorimetry dual-signal sensing strategy based on o-phenylenediamine and AuNCs for determination of Cu2+ and glyphosate.

A ratiometric fluorescence sensing strategy has been developed for the determination of Cu2+ and glyphosate with high sensitivity and specificity based on OPD (o-phenylenediamine) and glutathione-stabilized gold nanoclusters (GSH-AuNCs). Water-soluble 1.75-nm size GSH-AuNCs with strong red fluorescence and maximum emission wavelength at 682 nm were synthesized using GSH as the template. OPD was oxidized by Cu2+, which produced the bright yellow fluorescence oxidation product 2,3-diaminophenazine (DAP) with a maximum fluorescence emission peak at 570 nm. When glyphosate existed in the system, the chelation between glyphosate and Cu2+ hindered the formation of DAP and reduced the fluorescence intensity of the system at the wavelength of 570 nm. Meanwhile, the fluorescence intensity at the wavelength of 682 nm remained basically stable. It exhibited a good linear relationship towards Cu2+ and glyphosate in water in the range 1.0-10 µM and 0.050-3.0 µg/mL with a detection limit of 0.547 µM and 0.0028 µg/mL, respectively. The method was also used for the semi-quantitative determination of Cu2+ and glyphosate in water by fluorescence color changes visually detected by the naked eyes in the range 1.0-10 µM and 0.30-3.0 µg/mL, respectively. The sensing strategy showed higher sensitivity, more obvious color changes, and better disturbance performance, satisfying with the detection demands of Cu2+ and glyphosate in environmental water samples. The study provides a reliable detection strategy in the environment safety fields.

VCD Analysis of Axial Chirality in Synthetic Stereoisomeric Biaryl-Type bis-Isochroman Heterodimers with Isolated Blocks of Central and Axial Chirality.

Optically active heterodimeric 5,5'-linked bis-isochromans, containing a stereogenic ortho-trisubstituted biaryl axis and up to four chirality centers, were synthesized stereoselectively by using a Suzuki-Miyaura biaryl coupling reaction of optically active isochroman and 1-arylpropan-2-ol derivatives, providing the first access to synthetic biaryl-type isochroman dimers. Enantiomeric pairs and stereoisomers up to seven derivatives were prepared with four different substitution patterns, which enabled us to test how OR, ECD, and VCD measurements and DFT calculations can be used to determine parallel central and axial chirality elements in three isolated blocks of chirality. In contrast to natural penicisteckins A-D and related biaryls, the ECD spectra and OR data of (aS) and (aR) atropodiastereomers did not reflect the opposite axial chirality, but they were characteristic of the central chirality. The atropodiastereomers showed consistently near-mirror-image VCD curves, allowing the determination of axial chirality with the aid of DFT calculation or by comparison of characteristic VCD transitions.

All-Atom Protein Sequence Design Based on Geometric Deep Learning.

Designing sequences for specific protein backbones is a key step in creating new functional proteins. Here, we introduce GeoSeqBuilder, a deep learning framework that integrates protein sequence generation with side chain conformation prediction to produce the complete all-atom structures for designed sequences. GeoSeqBuilder uses spatial geometric features from protein backbones and explicitly includes three-body interactions of neighboring residues. GeoSeqBuilder achieves native residue type recovery rate of 51.6 %, comparable to ProteinMPNN and other leading methods, while accurately predicting side chain conformations. We first used GeoSeqBuilder to design sequences for thioredoxin and a hallucinated three-helical bundle protein. All the 15 tested sequences expressed as soluble monomeric proteins with high thermal stability, and the 2 high-resolution crystal structures solved closely match the designed models. The generated protein sequences exhibit low similarity (minimum 23 %) to the original sequences, with significantly altered hydrophobic cores. We further redesigned the hydrophobic core of glutathione peroxidase 4, and 3 of the 5 designs showed improved enzyme activity. Although further testing is needed, the high experimental success rate in our testing demonstrates that GeoSeqBuilder is a powerful tool for designing novel sequences for predefined protein structures with atomic details. GeoSeqBuilder is available at https://github.com/PKUliujl/GeoSeqBuilder.

An Enzymatic Carbon-Carbon Bond Cleavage and Aldol Reaction Cascade Converts an Angular Scaffold into the Linear Tetracyclic Core of Ochraceopones.

Meroterpenoids of the ochraceopones family featuring a linear tetracyclic scaffold exhibit exceptional antiviral and anti-inflammatory activities. The biosynthetic pathway and chemical logic to generate this linear tetracycle, however, remain unknown. In this study, we identified and characterized all biosynthetic enzymes to afford ochraceopones and elucidated the complete biosynthetic pathway. We demonstrated that the linear tetracyclic scaffold of ochraceopones was derived from an angular tetracyclic precursor. A multifunctional cytochrome P450 OchH was validated to catalyze the free-radical-initiated carbon-carbon bond cleavage of the angular tetracycle. Then, a new carbon-carbon bond was verified to be constructed using a new aldolase OchL, which catalyzes an intramolecular aldol reaction to form the linear tetracycle. This carbon-carbon bond fragmentation and aldol reaction cascade features an unprecedented strategy for converting a common angular tetracycle to a distinctive linear tetracyclic scaffold in meroterpenoid biosynthesis.

Deciphering Deoxynybomycin Biosynthesis Reveals Fe(II)/α-Ketoglutarate-Dependent Dioxygenase-Catalyzed Oxazoline Ring Formation and Decomposition.

The antibacterial agents deoxynybomycin (DNM) and nybomycin (NM) have a unique tetracyclic structure featuring an angularly fused 4-oxazoline ring. Here, we report the identification of key enzymes responsible for forming the 4-oxazoline ring in Embleya hyalina NBRC 13850 by comparative bioinformatics analysis of the biosynthetic gene clusters encoding structurally similar natural products DNM, deoxynyboquinone (DNQ), and diazaquinomycins (DAQs). The N-methyltransferase DnmS plays a crucial role in catalyzing the N-dimethylation of a tricyclic precursor prenybomycin to generate NM D; subsequently, the Fe(II)/α-ketoglutarate-dependent dioxygenase (Fe/αKGD) DnmT catalyzes the formation of a 4-oxazoline ring from NM D to produce DNM; finally, a second Fe/αKGD DnmU catalyzes the C-12 hydroxylation of DNM to yield NM. Strikingly, DnmT is shown to display unexpected functions to also catalyze the decomposition of the 4-oxazoline ring and the N-demethylation, thereby converting DNM back to prenybomycin, to putatively serve as a manner to control the intracellular yield of DNM. Structure modeling, site-directed mutagenesis, and quantum mechanics calculations provide mechanistic insights into the DnmT-catalyzed reactions. This work expands our understanding of the functional diversity of Fe/αKGDs in natural product biosynthesis.

Biosynthetic Diversification of Fidaxomicin Aglycones by Heterologous Expression and Promoter Refactoring.

Fidaxomicin (Dificid) is a commercial macrolide antibiotic for treating Clostridium difficile infection. Total synthesis of fidaxomicin and its aglycone had been achieved through different synthetic schemes. In this study, an alternative biological route to afford the unique 18-membered macrolactone aglycone of fidaxomicin was developed. The promoter refactored fidaxomicin biosynthetic gene cluster from Dactylosporangium aurantiacum was expressed in the commonly used host Streptomyces albus J1074, thereby delivering five structurally diverse fidaxomicin aglycones with the corresponding titers ranging from 4.9 to 15.0 mg L-1. In general, these results validated a biological strategy to construct and diversify fidaxomicin aglycones on the basis of promoter refactoring and heterologous expression.

Aromatic Polyketides from the Mangrove-Derived Streptomyces sp. SCSIO 40069.

A chemical investigation of Streptomyces sp. SCSIO 40069 resulted in the isolation of a series of aromatic polyketides with rare skeletons, including five new compounds RM18c-RM18g (1-5) and three known ones (6-8). Their structures and absolute configurations were determined by diverse methods, including HRMS and NMR spectra, chemical reaction, Snatzke's method, quantum mechanical-nuclear magnetic resonance (QM-NMR), and X-ray crystallographic analysis. Compounds 1, 2, 4b, and 8 displayed moderate or weak antibacterial activities.

Genome Mining and Biosynthetic Reconstitution of Fungal Depsidone Mollicellins Reveal a Dual Functional Cytochrome P450 for Ether Formation.

Depsidones are significant in structural diversity and broad in biological activities; however, their biosynthetic pathways have not been well understood and have attracted considerable attention. Herein, we heterologously reconstituted a depsidone encoding gene cluster from Ovatospora sp. SCSIO SY280D in Aspergillus nidulans A1145, leading to production of mollicellins, a representative family of depsidones, and discovering a bifunctional P450 monooxygenase that catalyzes both ether formation and hydroxylation in the biosynthesis of the mollicellins. The functions of a decarboxylase and an aromatic prenyltransferase are also characterized to understand the tailoring modification steps. This work provides important insights into the biosynthesis of mollicellins.

Totopotensamide Congeners from a Halogenase-Inactivated Mutant.

The installation of halogen atoms into aromatic and less activated polyketide substrates by halogenases is a powerful strategy to tune the bioactivity, bioavailability, and reactivity of compounds. In the biosynthetic pathway of totopotensamide A (1), the halogenase TotH was confirmed in vivo to catalyze the C-4 chlorination to form the nonproteinogenic amino acid ClMeDPG. Herein, we report the isolation, structure elucidation, and bioactivity evaluation of six new deschloro totopotensamide (TPM) congeners TPMs H2-H7 (5-10) from the totH-inactivated strain and the proposed absolute configuration of the polyketide chain in TPMs using 4 as a model compound by a combination of the JBCA and bioinformatic analysis. Compounds 5, 6, 8, and 9 displayed cytotoxicity against the A549, PANC-1, Calu3, and BXPC3 cell lines with IC50 values ranging from 2.3 to 9.7 µM.

Individualised positive end-expiratory pressure titrated intra-operatively by electrical impedance tomography optimises pulmonary mechanics and reduces postoperative atelectasis: A randomised controlled trial.

BACKGROUND: A protective intra-operative lung ventilation strategy has been widely recommended for laparoscopic surgery. However, there is no consensus regarding the optimal level of positive end-expiratory pressure (PEEP) and its effects during pneumoperitoneum. Electrical impedance tomography (EIT) has recently been introduced as a bedside tool to monitor lung ventilation in real-time. OBJECTIVE: We hypothesised that individually titrated EIT-PEEP adjusted to the surgical intervention would improve respiratory mechanics during and after surgery. DESIGN: Randomised controlled trial. SETTING: First Medical Centre of Chinese PLA General Hospital, Beijing. PATIENTS: Seventy-five patients undergoing robotic-assisted laparoscopic hepatobiliary and pancreatic surgery under general anaesthesia. INTERVENTIONS: Patients were randomly assigned 2 : 1 to individualised EIT-titrated PEEP (PEEPEIT; n = 50) or traditional PEEP 5 cmH2O (PEEP5 cmH2O; n = 25). The PEEPEIT group received individually titrated EIT-PEEP during pneumoperitoneum. The PEEP5 cmH2O group received PEEP of 5 cmH2O during pneumoperitoneum. MAIN OUTCOME MEASURES: The primary outcome was respiratory system compliance during laparoscopic surgery. Secondary outcomes were individualised PEEP levels, oxygenation, respiratory and haemodynamic status, and occurrence of postoperative pulmonary complications (PPCs) within 7 days. RESULTS: Compared with PEEP5 cmH2O, patients who received PEEPEIT had higher respiratory system compliance (mean values during surgery of 44.3 ±â€Š11.3 vs. 31.9 ±â€Š6.6, ml cmH2O-1; P < 0.001), lower driving pressure (11.5 ±â€Š2.1 vs. 14.0 ±â€Š2.4 cmH2O; P < 0.001), better oxygenation (mean PaO2/FiO2 427.5 ±â€Š28.6 vs. 366.8 ±â€Š36.4; P = 0.003), and less postoperative atelectasis (19.4 ±â€Š1.6 vs. 46.3 ±â€Š14.8 g of lung tissue mass; P = 0.003). Haemodynamic values did not differ significantly between the groups. No adverse effects were observed during surgery. CONCLUSION: Individualised PEEP by EIT may improve intra-operative pulmonary mechanics and oxygenation without impairing haemodynamic stability, and decrease postoperative atelectasis. TRIAL REGISTRATION: Chinese Clinical Trial Registry (www.chictr.org.cn) identifier: ChiCTR2100045166.