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.

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.

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.

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.

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.

Genomics-Driven Discovery of Benzoxazole Alkaloids from the Marine-Derived Micromonospora sp. SCSIO 07395.

Benzoxazole alkaloids exhibit a diverse array of structures and interesting biological activities. Herein we report the identification of a benzoxazole alkaloid-encoding biosynthetic gene cluster (mich BGC) in the marine-derived actinomycete Micromonospora sp. SCSIO 07395 and the heterologous expression of this BGC in Streptomyces albus. This approach led to the discovery of five new benzoxazole alkaloids microechmycin A-E (1-5), and a previously synthesized compound 6. Their structures were elucidated by HRESIMS and 1D and 2D NMR data. Microechmycin A (1) showed moderate antibacterial activity against Micrococcus luteus SCSIO ML01 with the minimal inhibitory concentration (MIC) value of 8 µg mL-1.

A Widespread Glycosidase Confers Lobophorin Resistance and Host-Dependent Structural Diversity.

Identifying new environmental resistance determinants is significant to combat rising antibiotic resistance. Herein we report the unexpected correlation of a lobophorin (LOB) resistance-related glycosidase KijX with the host-dependent chemical diversity of LOBs, by a process of glycosylation, deglycosylation and reglycosylation. KijX homologues are widespread among bacteria, archaea and fungi, and encode the same glycohydrolytic activity on LOBs. The crystal structure of AcvX (a KijX homologue) shows a similar fold to that of the glycoside hydrolase family 113 and a special negatively charged groove to accommodate and deglycosylate LOBs. Antagonistic assays indicate kijX as a defense weapon of actinomycetes to combat LOB producers in environment, reflecting an elegant coevolution relationship. Our study provides insight into the KijX-related glycosidases as preexisting resistance determinants and represents an example of resistance genes accidentally integrated into natural product assembly.

A Mechanistic Understanding of the Distinct Regio- and Chemoselectivity of Multifunctional P450s by Structural Comparison of IkaD and CftA Complexed with Common Substrates.

Regio- and chemoselective C-H activation at multi-positions of a single molecule is fascinating but chemically challenging. The homologous cytochrome P450 enzymes IkaD and CftA catalyze multiple C-H oxidations on the same polycyclic tetramate macrolactam (PoTeM) ikarugamycin, with distinct regio- and chemoselectivity. Herein we provide mechanistic understanding of their functional differences by solving crystal structures of IkaD and CftA in complex with ikarugamycin and unnatural substrates. Distinct conformations of the F/G region in IkaD and CftA are found to differentiate the orientation of PoTeM substrates, by causing different binding patterns with polar moieties to determine site selection, oxidation order, and chemoselectivity. Fine-tuning the polar subpocket altered the regioselectivity of IkaD, indicating that substrate re-orientation by mutating residues distal to the oxidation site could serve as an important method in future engineering of P450 enzymes.

Discovery of Efrotomycin Congeners and Heterologous Expression-Based Insights into the Self-Resistance Mechanism.

Four new efrotomycins, A1-A4 (1-4), were isolated from the salt mine-derived Amycolatopsis cihanbeyliensis DSM 45679 and structurally determined. Efrotomycins A3 (3) and A4 (4) feature a tetrahydrofuran ring configured distinctly from known elfamycins. Heterologous expression of the efrotomycin gene cluster (efr BGC) in Streptomyces lividans SBT18 led to efrotomycin B1 (5), the yield of which was improved several fold upon introduction of the transporter gene efrT, a putative self-resistance determinant outside of the efr BGC.

Development and validation of a risk nomogram for postoperative acute kidney injury in older patients undergoing liver resection: a pilot study.

BACKGROUND: Postoperative acute kidney injury (AKI) is associated with poor clinical outcomes. Early identification of high-risk patients of developing postoperative AKI can optimize perioperative renal management and facilitate patient survival. The present study aims to develop and validate a nomogram to predict postoperative AKI after liver resection in older patients. METHODS: A retrospective observational study was conducted involving data from 843 older patients scheduled for liver resection at a single tertiary high caseload general hospital between 2012 and 2019. The data were randomly divided into training (70%, n = 599) and validation (30%, n = 244) datasets. The training cohort was used to construct a predictive nomogram for postoperative AKI with the logistic regression model which was confirmed by a validation cohort. The model was evaluated by receiver operating characteristic (ROC) curve, calibration plot, and decision curve analysis in the validation cohort. A summary risk score was also constructed for identifying postoperative AKI patients. RESULTS: Postoperative AKI occurred in 155 (18.4%) patients and was highly associated with in-hospital mortality (5.2% vs. 0.7%, P <  0.001). The six predictors selected and assembled into the nomogram included age, preexisting chronic kidney disease (CKD), non-steroidal anti-inflammatory drugs (NSAIDs) usage, intraoperative hepatic inflow occlusion, blood loss, and transfusion. The predictive nomogram performed well in terms of discrimination with area under ROC curve (AUC) in training (0.73, 95% confidence interval (CI): 0.68-0.78) and validation (0.71, 95% CI: 0.63-0.80) datasets. The nomogram was well-calibrated with the Hosmer-Lemeshow chi-square value of 9.68 (P = 0.47). Decision curve analysis demonstrated a significant clinical benefit. The summary risk score calculated as the sum of points from the six variables (one point for each variable) performed as well as the nomogram in identifying the risk of AKI (AUC 0.71, 95% CI: 0.66-0.76). CONCLUSION: This nomogram and summary risk score accurately predicted postoperative AKI using six clinically accessible variables, with potential application in facilitating the optimized perioperative renal management in older patients undergoing liver resection. TRIAL REGISTRATION: NCT04922866 , retrospectively registered on clinicaltrials.gov on June 11, 2021.