Two Cytochrome P450 Enzymes Form the Tricyclic Nested Skeleton of Meroterpenoids by Sequential Oxidative Reactions.

Meroterpenoid clavilactones feature a unique benzo-fused ten-membered carbocyclic ring unit with an α,ß-epoxy-γ-lactone moiety, forming an intriguing 10/5/3 tricyclic nested skeleton. These compounds are good inhibitors of the tyrosine kinase, attracting a lot of chemical synthesis studies. However, the natural enzymes involved in the formation of the 10/5/3 tricyclic nested skeleton remain unexplored. Here, we identified a gene cluster responsible for the biosynthesis of clavilactone A in the basidiomycetous fungus Clitocybe clavipes. We showed that a key cytochrome P450 monooxygenase ClaR catalyzes the diradical coupling reaction between the intramolecular hydroquinone and allyl moieties to form the benzo-fused ten-membered carbocyclic ring unit, followed by the P450 ClaT that exquisitely and stereoselectively assembles the α,ß-epoxy-γ-lactone moiety in clavilactone biosynthesis. ClaR unprecedentedly acts as a macrocyclase to catalyze the oxidative cyclization of the isopentenyl to the nonterpenoid moieties to form the benzo-fused macrocycle, and a multifunctional P450 ClaT catalyzes a ten-electron oxidation to accomplish the biosynthesis of the 10/5/3 tricyclic nested skeleton in clavilactones. Our findings establish the foundation for the efficient production of clavilactones using synthetic biology approaches and provide the mechanistic insights into the macrocycle formation in the biosynthesis of fungal meroterpenoids.

Neuronal CFL1 upregulation in head and neck squamous cell carcinoma enhances tumor-nerve crosstalk and promotes tumor growth.

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type, marked by a pronounced nerve density within the tumor microenvironment and a high rate of perineural invasion (PNI). Growing evidence suggests that the nervous system plays a vital role in HNSCC progression. Yet, the mechanisms governing cancer-nerve interactions remain largely elusive. Our research revealed that cofilin-1 (CFL1) is significantly overexpressed in HNSCC and correlates with both PNI and unfavorable prognosis. Utilizing multiplex fluorescent immunohistochemistry, we have localized CFL1 chiefly to the nerves adjacent to tumor sites. Significantly, it is the elevated expression of CFL1 in neuronal structures, rather than in the tumor cells, that aligns with diminished patient survival rates. We observed that HNSCC cells induced the expression of neuronal CFL1 and that the conditional knockout of neuronal CFL1 impedes tumor-nerve interactions. Both Gene Ontology functional enrichment analyses and Gene Set Enrichment Analysis demonstrate that CFL1 expression in HNSCC is associated with specific biological processes, including "RIBOSOME," "PROTEASOME," and "cadherin binding." In summary, HNSCC promotes the expression of CFL1 in nerves, which is essential for cancer-nerve interactions. The neuronal CFL1 is associated with PNI and may be a potential molecular prognostic marker of poor survival in HNSCC.

Anti-HIV Potential of Beesioside I Derivatives as Maturation Inhibitors: Synthesis, 3D-QSAR, Molecular Docking and Molecular Dynamics Simulations.

HIV-1 maturation is the final step in the retroviral lifecycle that is regulated by the proteolytic cleavage of the Gag precursor protein. As a first-in-class HIV-1 maturation inhibitor (MI), bevirimat blocks virion maturation by disrupting capsid-spacer peptide 1 (CA-SP1) cleavage, which acts as the target of MIs. Previous alterations of beesioside I (1) produced (20S,24S)-15ꞵ,16ꞵ-diacetoxy-18,24; 20,24-diepoxy-9,19-cyclolanostane-3ꞵ,25-diol 3-O-3',3'-dimethylsuccinate (3, DSC), showing similar anti-HIV potency compared to bevirimat. To ascertain the binding modes of this derivative, further modification of compound 1 was conducted. Three-dimensional quantitative structure−activity relationship (3D-QSAR) analysis combined with docking simulations and molecular dynamics (MD) were conducted. Five new derivatives were synthesized, among which compound 3b showed significant activity against HIV-1NL4-3 with an EC50 value of 0.28 µM. The developed 3D-QSAR model resulted in great predictive ability with training set (r2 = 0.99, q2 = 0.55). Molecular docking studies were complementary to the 3D-QSAR analysis, showing that DSC was differently bound to CA-SP1 with higher affinity than that of bevirimat. MD studies revealed that the complex of the ligand and the protein was stable, with root mean square deviation (RMSD) values <2.5 Å. The above results provided valuable insights into the potential of DSC as a prototype to develop new antiviral agents.

Four Meroterpenoids with Novel Aminoglycoside Moiety from the Basidiomycete Clitocybe clavipes with Cytotoxic Activity.

Four new meroterpenoids, Clavilactone M-P, possessing novel aminoglycoside moiety (1-4) and a 10-membered carbocycle fused with an α,ß-epoxy-γ-lactone, were isolated from Clitocybe clavipes, a basidiomycete. Their structures with absolute configurations were determined by extensive analysis of their spectroscopic data, and the ECD method. All the isolated compounds (1-4) were evaluated for their antitumor activity against three human cancer cell lines using the MTT assay. Compound 1 and 2 exhibited a significant suppression of cell viability in the Hela (IC50 = 22.8 and 19.7 µM) cell line.

Gram-Scale Total Synthesis of TAB with Cardioprotective Activity and the Structure-Activity Relationship of Its Analogs.

Traditional Chinese medicine has been proven to be of great significance in cardioprotective effects. Clinopodium chinense (Lamiaceae) has unique advantages in the treatment and prevention of cardiovascular diseases. Tournefolic acid B (TAB) was proven to be a potent component against myocardial ischemia reperfusion injury (MIRI) from Clinopodium chinense (Lamiaceae). This article will attempt to establish a gram-scale synthesis method of TAB and discuss the structure-activity relationship of its analogs. The total synthesis of TAB was completed in 10 steps with an overall yield of 13%. In addition, analogs were synthesized, and their cardioprotective activity was evaluated on the hypoxia/reoxygenation of H9c2 cells. Amidation of the acid position is helpful to the activity, while methylation of phenolic hydroxyl groups greatly decreased the cardioprotective activity. The easily prepared azxepin analogs also showed cardioprotective activity. Most of the clogP values calculated by Molinspiration ranged from 2.5 to 5, which is in accordance with Lipinski's rule of 5. These findings represent a novel kind of cardioprotective agent that is worthy of further study.

Concise total synthesis of (+)-Zeylenone with antitumor activity and the structure-activity relationship of its derivatives.

Natural products--polyoxygenated cyclohexenes exhibited potent anti-tumor activity, such as zeylenone, which is a natural product isolated from Uvaria grandiflora Roxb. This article will attempt to establish a gram-scale synthesis method of (+)-zeylenone and explain the structure-activity relationship of this kind of compound. Total synthesis of (+)-zeylenone was completed in 13 steps with quinic acid as the starting material in 9.8% overall yield. The highlight of the route was the control of the three carbon's chirality by single step dihydroxylation. In addition, different kinds of derivatives were designed and synthesized. Cell Counting Kit-8 (CCK8) assay was used for evaluating antitumor activity against three human cancer cell lines. The structure--activity relationship suggested that compounds with both absolute configurations exhibited tumor-suppressive effects. Moreover, hydroxyls at the C-1/C-2 position were crucial to the activity, and the esterification of large groups at C-1 hydroxyl eliminated the activity. Hydroxyl at the C-3 position was also important as proper ester substituent could increase the potency.

Blocking Effect of Demethylzeylasteral on the Interaction between Human ACE2 Protein and SARS-CoV-2 RBD Protein Discovered Using SPR Technology.

The novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019, and there is no sign that the epidemic is abating. Targeting the interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor is a promising therapeutic strategy. In this study, surface plasmon resonance (SPR) was used as the primary method to screen a library of 960 compounds. A compound 02B05 (demethylzeylasteral, CAS number: 107316-88-1) that had high affinities for S-RBD and ACE2 was discovered, and binding affinities (KD, µM) of 02B05-ACE2 and 02B05-S-RBD were 1.736 and 1.039 µM, respectively. The results of a competition experiment showed that 02B05 could effectively block the binding of S-RBD to ACE2 protein. Furthermore, pseudovirus infection assay revealed that 02B05 could inhibit entry of SARS-CoV-2 pseudovirus into 293T cells to a certain extent at nontoxic concentration. The compoundobtained in this study serve as references for the design of drugs which have potential in the treatment of COVID-19 and can thus accelerate the process of developing effective drugs to treat SARS-CoV-2 infections.

Prediction of Marine Pycnocline Based on Kernel Support Vector Machine and Convex Optimization Technology.

With the explosive growth of ocean data, it is of great significance to use ocean observation data to analyze ocean pycnocline data in military field. However, due to natural factors, most of the time the ocean hydrological data is not complete. In this case, predicting the ocean hydrological data by partial data has become a hot spot in marine science. In this paper, based on the traditional statistical analysis literature, we propose a machine-learning ocean hydrological data processing process under big data. At the same time, based on the traditional pycnocline gradient determination method, the open Argo data set is analyzed, and the local characteristics of pycnocline are verified from several aspects combined with the current research about pycnocline. Most importantly, in this paper, the combination of kernel function and support vector machine(SVM) is extended to nonlinear learning by using the idea of machine learning and convex optimization technology. Based on this, the known pycnocline training set is trained, and an accurate model is obtained to predict the pycnocline in unknown domains. In the specific steps, this paper combines the classification problem with the regression problem, and determines the proportion of training set and test formula set by polynomial regression. Subsequently, the feature scaling of the input data accelerated the gradient convergence, and a grid search algorithm with variable step size was proposed to determine the super parameter c and gamma of the SVM model. The prediction results not only used the confusion matrix to analyze the accuracy of GridSearch-SVM with variable step size, but also compared the traditional SVM and the similar algorithm. At the end of the experiment, two features which have the greatest influence on the Marine density thermocline are found out by the feature ranking algorithm based on learning.

Quantitative Evaluation of Twelve Major Components of Sulfur-Fumigated Astragali Radix with Different Durations by UPLC-MS.

In this study, an improved UPLC-MS (Ultra-high performance liquid chromatography-tandem mass spectrometry) method for simultaneously quantifying twelve major components belonging to two chemical types was developed and validated, and was applied to quantitatively compare the quality of sulfur-fumigated Astragali Radix of different durations and of the fresh reference sample. The results showed that the contents of triterpenes astragaloside III and astragaloside IV decreased moderately, while the flavonoids calycosin, formononetin, and 7,2'-dihydroxy-3',4'-dimethoxyisoflavane decreased significantly. The corresponding flavonoid glycosides increased accordingly, which indicated the occurrence of chemical transformation of flavonoids and glycosides in the process of sulfur-fumigation. These transformations were further confirmed by the synthesis of flavonoid glycosides under simulated sulfur-fumigation circumstances. Furthermore, the sulfur-fumigated duration varied in proportion with the contents of compounds 7, 11, and 12. These results suggest that the established method was precise, accurate and sensitive enough for the global quality evaluation of sulfur-fumigated Astragali Radix. Further, sulfur-fumigation not only changes the proportions of bioactive components, but also causes chemical transformation in Astragali Radix.

Pyrrole Alkaloids from the Edible Mushroom Phlebopus portentosus with Their Bioactive Activities.

Seven pyrrole alkaloids, three of which are novel (phlebopines A⁻C (1⁻3)), were isolated from the fruiting bodies of the edible mushroom Phlebopus portentosus. Their structures were determined on the basis of spectroscopic data. All the isolated compounds were tested for their neuroprotective properties and acetylcholine esterase (AChE) inhibition activities. Compound 7 displayed remarkable neuroprotective effects against hydrogen peroxide (H2O2)-induced neuronal-cell damage in human neuroblastoma SH-SY5Y cells.

Semisynthesis and Biological Evaluation of Oleanolic Acid 3-O-ß-d-Glucuronopyranoside Derivatives for Protecting H9c2 Cardiomyoblasts against H2O2-Induced Injury.

A series of novel oleanolic acid 3-O-ß-d-glucuronopyranoside derivatives have been designed and synthesized. Biological evaluation has indicated that some of the synthesized compounds exhibit moderate to good activity against H2O2-induced injury in rat myocardial cells (H9c2). Particularly, derivative 28-N-isobutyl ursolic amide 3-O-ß-d-galactopyranoside (8a) exhibited a greater protective effect than the positive control oleanolic acid 3-O-ß-d-glucuronopyranoside, indicating that it possesses a great potential for further development as a cardiovascular disease modulator by structural modification.

New Cassane Diterpenoids from Caesalpinia sappan and their Antiplasmodial Activity.

One new cassane diterpene possessing an unusual N bridge between C-19 and C-20 named caesalsappanin R (1), as well as another new diterpene caesalsappanin S (2), were isolated from the seeds of Caesalpinia sappanwith methanol extract. Their structures were determined by spectroscopic analysis and examined alongside existing data from prior studies. Their biological activities were profiled by their antiplasmodial activity.

[Studies on chemical constituents of Clinopodium chinense].

Twenty-eight compounds were isolated and purified from Clinopodium chinense by Sephedax LH-20, ODS, MCI and preparative HPLC. Their structures were identified as apigenin (1), apigenin-7-O-ß-D-glucopyranoside (2), apigenin-7-O-ß-D-glucuronopyranoside (3), thellungianol (4), apigenin-7-O-ß-D-rutinoside (5), luteolin (6), luteolin-4'-O-ß-D-glucopyranoside (7), apigenin-7-O-ß-D-pyranglycuronate butyl ester (8), luteolin-7-O-ß-D-rutinoside (9), luteolin-7-O-ß-D-noehesperidoside (10), acacetin (11), acacetin-7-O-ß-D-glucuronopyranoside (12), buddleoside (13), naringenin (14), pruning (15), nairutin (16), isosakuranetin (17), isosakuranin (18), didymin (19), hesperidin (20), kaempferol (21), quercetin (22), kaempferol-3-O-α-L-rahmnoside (23), p-hydroxycinnamic acid (24), caffeic acid (25), cis-3-[2-[1-(3,4-dihydroxy-phenyl)-1 -hydroxymethyl]-1,3-ben-zodioxol-5-yl]-(E)-2-propenoic acid (26), mesaconic acid (27), gentisic acid 5-O-ß-D-(6'-salicylyl)-glucopyranoside (28). Among them, compounds 7, 9-10, 12, 23, 26-28 were isolated from the Clinopodium for the first time. The protective effects of compounds 1-6, 8-17 and 19 against H2O2-induced H9c2 cardiomyocyte injury were tested, compounds 15 exhibited significantly protective effects. Compared with the cell viability of (62.12±6.18)% in the model, pruning exhibited viabilities of (84.25±7.36)% at 25.0 mg•L⁻¹, respectively, using quercetin as a positive control [cell viability of (84.55±8.26)%, 20 mg•L⁻¹].

Antimalarial and Antiproliferative Cassane Diterpenes of Caesalpinia sappan.

Bioassay-guided fractionation of a methanol extract of the seeds of Caesalpinia sappan led to the isolation of 12 new cassane-type diterpenes, caesalsappanins A-L (1-12). Their structures were elucidated on the basis of NMR and HRESIMS analysis, and the absolute configuration of compound 1 was determined by single-crystal X-ray crystallography. All isolated compounds were tested against a chloroquine-resistant Plasmodium falciparum strain for antiplasmodial activities and against a small panel of human cancer cell lines for antiproliferative activities. Compounds 7 and 8 displayed antimalarial activity against the chloroquine-resistant K1 strain of P. falciparum with IC50 values of 0.78 and 0.52 µM and selectivity indices of 17.6 and 16.4, respectively. Compound 10 showed antiproliferative activity against the KB cancer cell line with an IC50 value of 7.4 µM.

[Cassae-type diterpenes from seeds of Caesalpinia minax].

Fifteen cassaen-type diterpenes were isolated from the 95% ethanolic extract of the seeds of C. minax through various chromatographic techniques. Their structures were identified on the basis of spectroscopic data as pulcherralpin (1), caesalpinin ML (2), chamaetexane C (3), chamaetexane D (4), 6ß, 18-diacetoxycassan-13, 15-diene (5), neocaesalpin K (6), neocaesalpin MP (7), neocaesalpin M (8), neocaesalpin Q (9), neocaesalpin P (10), neocaesalpin R (11), caesaldekarin D (12), caesaldekarin A (13), caesaldekarin b (14), 3ß,6α-diacetoxyvouacapane (15). Among them, compounds 14, 9-11 were isolated from this plant for the first time.

Two new degradative cassane-type diterpenes isolated from Caesalpinia minax.

Cassane-type diterpenes are main bioactive constituents of Caesalpinia minax HANCE. As a part of our ongoing chemical investigation of C. minax, two new degradative cassane-type diterpenes, named caesalpins I (1) and J (2), were isolated from the EtOAc extract of the seeds of C. minax. The structures were elucidated by means of spectroscopic analysis.

Advances in colorimetric biosensors of exosomes: novel approaches based on natural enzymes and nanozymes.

Exosomes are 30-150 nm vesicles derived from diverse cell types, serving as one of the most important biomarkers for early diagnosis and prognosis of diseases. However, the conventional detection method for exosomes faces significant challenges, such as unsatisfactory sensitivity, complicated operation, and the requirement of complicated devices. In recent years, colorimetric exosome biosensors with a visual readout underwent rapid development due to the advances in natural enzyme-based assays and the integration of various types of nanozymes. These synthetic nanomaterials show unique physiochemical properties and catalytic abilities, enabling the construction of exosome colorimetric biosensors with novel principles. This review will illustrate the reaction mechanisms and properties of natural enzymes and nanozymes, followed by a detailed introduction of the recent advances in both types of enzyme-based colorimetric biosensors. A comparison between natural enzymes and nanozymes is made to provide insights into the research that improves the sensitivity and convenience of assays. Finally, the advantages, challenges, and future directions of enzymes as well as exosome colorimetric biosensors are highlighted, aiming at improving the overall performance from different approaches.

Phenolic constituents with antibacterial activity from Eleutherine bulbosa.

Eleutherine bulbosa (Mill.) Urb. is a medicinal and edible plant with various benefits for humans and animals. In this work, four new phenolic constituents (1-4), along with six known phenolic compounds (5-10) were obtained from the red bulbs of E. bulbosa. Their structures with absolute configurations were characterized by extensive spectroscopic analysis, combined with HR-ESI-MS and quantum mechanical electronic circular dichroism (ECD). Compounds 1 and 2 are novel homologous and heterodimers, respectively, featuring an unusual spiro ring system. All isolated phenolic constituents were tested for their antibacterial effects. The results revealed four phenolic compounds 1-3 and 7 showed moderate antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Escherichia coli with minimum inhibitory concentration (MIC) values ranging from 15.6 to 250.0 µg/mL.

Changes of serum cortisol during pregnancy and labor initiation: an onsite cross-sectional study.

Background: Increased maternal cortisol secretion has been observed during pregnancy and labor. However, due to the limitations in diagnostic methods, the dynamic change of cortisol during the short period between threatened labor and labor is unknown. In this study, we aim to evaluate the changes in serum cortisol during late pregnancy and full-term labor initiation, verifying if cortisol could serve as a biomarker for the diagnosis of labor initiation from threatened labor. Methods: This cross-sectional onsite study involved 564 participants of 6 different gestational stages (C: Control; T1: Trimester 1; T3: Trimester 3; E: expectant; TL: threatened labor; L: labor), all patients in the E, TL, and L groups were at full term. The serum cortisol concentration was quantified with a point-of-care test (POCT), and the gestation, age, parity, and BMI of participants were documented. Morning serum cortisol was collected between 8:00 and 10:00 a.m., except for the TL and L group women who were tested upon arrival or during latent labor. With cortisol levels or all five variables, L was distinguished from TL using machine learning algorithms. Results: Significant elevation of cortisol concentration was observed between T1 and T3, or TL and L group (P< 0.001). Women belonging to the E and TL group showed similar gestation week and cortisol levels. Diagnosis of labor initiation using cortisol levels (cutoff = 21.46 µg/dL) yielded sensitivity, specificity, and AUC of 86.50%, 88.60%, and 0.934. With additional variables, a higher specificity (89.29%) was achieved. The diagnostic accuracy of all methods ranged from 85.93% to 87.90%. Conclusion: Serum cortisol could serve as a potential biomarker for diagnosis of L form TL. The rapid onsite detection of serum cortisol with POCT could facilitate medical decision-making for admission and special treatments, either as an additional parameter or when other technical platforms are not available.