Biochemical and structural characterization of the first-discovered metazoan DNA cytosine-N4 methyltransferase from the bdelloid rotifer Adineta vaga.

Much is known about the generation, removal, and roles of 5-methylcytosine (5mC) in eukaryote DNA, and there is a growing body of evidence regarding N6-methyladenine, but very little is known about N4-methylcytosine (4mC) in the DNA of eukaryotes. The gene for the first metazoan DNA methyltransferase generating 4mC (N4CMT) was reported and characterized recently by others, in tiny freshwater invertebrates called bdelloid rotifers. Bdelloid rotifers are ancient, apparently asexual animals, and lack canonical 5mC DNA methyltransferases. Here, we characterize the kinetic properties and structural features of the catalytic domain of the N4CMT protein from the bdelloid rotifer Adineta vaga. We find that N4CMT generates high-level methylation at preferred sites, (a/c)CG(t/c/a), and low-level methylation at disfavored sites, exemplified by ACGG. Like the mammalian de novo 5mC DNA methyltransferase 3A/3B (DNMT3A/3B), N4CMT methylates CpG dinucleotides on both DNA strands, generating hemimethylated intermediates and eventually fully methylated CpG sites, particularly in the context of favored symmetric sites. In addition, like DNMT3A/3B, N4CMT methylates non-CpG sites, mainly CpA/TpG, though at a lower rate. Both N4CMT and DNMT3A/3B even prefer similar CpG-flanking sequences. Structurally, the catalytic domain of N4CMT closely resembles the Caulobacter crescentus cell cycle-regulated DNA methyltransferase. The symmetric methylation of CpG, and similarity to a cell cycle-regulated DNA methyltransferase, together suggest that N4CMT might also carry out DNA synthesis-dependent methylation following DNA replication.

Molecular and epidemiological characterization of Staphylococcus aureus causing bovine mastitis in China.

BACKGROUND: Staphylococcus aureus is one of the most prevalent pathogens in bovine mastitis, which leads to substantial financial losses for the dairy industry. RESULTS: In this study, S. aureus (n = 72) was isolated from 18 dairy farms in 15 provinces across China in 2021. The identification of these isolates at the species level was achieved by employing 16S rRNA sequencing. An isothermal amplification method for auxiliary detection of S. aureus was established, which can be employed not only for laboratory detection but also for point-of-care testing (POCT). Molecular characteristics of S. aureus mastitis in Chinese dairy cows were determined through MLST and spa typing. Finally, methicillin-resistant Staphylococcus aureus (MRSA) and MRSA resistance genes were detected using MIC and PCR amplification techniques. 72 isolates were identified as 12 sequence types (STs) and 7 clonal complexes (CC). ST1/CC1 was the dominant prevalent accounting for 33.3 % of the total, and exhibiting a wide distribution range. In terms of spa types, t114 was the dominant type, accounting for 31.9 % of the total, followed by t529 as the second major type. Four S. aureus strains were classified as MRSA according to their levels of oxacillin resistance (MIC ≥4 µg/mL). Among these four MRSA strains, one of them was found to be mecA positive. However, the presence of drug-resistance genes mecA and mecC was not detected in the remaining three MRSA strains, indicating the possible existence of new resistance genes. CONCLUSIONS: Our study investigated the prevalence of S. aureus mastitis in dairy cows in China, while also examined the molecular characteristics and MRSA strains. This information will help with the clinical monitoring, prevention, and control of S. aureus mastitis in dairy cattle.

Kamebakaurin Suppresses Antigen-Induced Mast Cell Activation by Inhibition of FcεRI Signaling Pathway.

INTRODUCTION: Kamebakaurin is an active constituent of both Rabdosia japonica and Rabdosia excisa, which are utilized in Chinese traditional medicine for improving symptoms in patients with allergies. We investigated the molecular mechanisms of the anti-allergic effects of kamebakaurin using BMMCs. METHODS: The degranulation ratio, histamine release, and the interleukin (IL)-4, leukotriene B4 (LTB4), and cysteinyl leukotriene productions on antigen-triggered BMMC were investigated. Additionally, the effects of kamebakaurin on signal transduction proteins were examined by Western blot and binding to the Syk and Lyn kinase domain was calculated. The effects of kamebakaurin on antigen-induced hyperpermeability were investigated using mouse model. RESULTS: At 10 µm, kamebakaurin partially inhibited degranulation, histamine release, and IL-4 production. At 30 µm, kamebakaurin partially reduced LTB4 and cysteinyl leukotriene productions and suppressed degranulation, histamine release, and IL-4 production. Phosphorylation of both Syk Y519/520 and its downstream protein, Gab2, was reduced by kamebakaurin, and complete inhibition was observed with 30 µm kamebakaurin. In contrast, phosphorylation of Erk was only partially inhibited, even in the presence of 30 µm kamebakaurin. Syk Y519/520 is known to be auto-phosphorylated via intramolecular ATP present in its own ATP-binding site, and this auto-phosphorylation triggers degranulation, histamine release, and IL-4 production. Docking simulation study indicated kamebakaurin blocked ATP binding to the ATP-binding site in Syk. Therefore, inhibition of Syk auto-phosphorylation by kamebakaurin binding to the Syk ATP-binding site appeared to cause a reduction of histamine release and IL-4 production. Kamebakaurin inhibited antigen-induced vascular hyperpermeability in a dose-dependent fashion but did not reduce histamine-induced vascular hyperpermeability. CONCLUSION: Kamebakaurin ameliorates allergic symptoms via inhibition of Syk phosphorylation; thus, kamebakaurin could be a lead compound for the new anti-allergic drug.

Design and Synthesis of MarinePolybrominated Diphenyl Ether Derivatives as Potential Anti-inflammatory Agents.

Natural polybrominated diphenyl ethers are generally isolated from sponges and possess a broad range of biological activities. Through screening of our marine natural product library, we discovered that polybrominated diphenyl ethers 5 and 6 exhibit considerable anti-inflammatory activity. In order to expand our repertoire of derivatives for further biological activity studies, we designed and synthesized a series of 5-related polybrominated diphenyl ethers. Importantly, compound 5a showed comparable anti-inflammatory activity while much lower cytotoxicity on lipopolysaccharide (LPS)-induced RAW264.7 cells. Additionally, western blotting analysis showed that 5a reduced the expression of phosphorylated extracellular signal-regulated kinase (p-ERK). Besides, molecular docking experiments were conducted to predict and elucidate the potential mechanisms underlying the varying anti-inflammatory activities exhibited by compounds 5a, 5, and 6.

Anti-HBV Activities of Cembranoids from the South China Sea Soft Coral Sinularia pedunculata and Their Structure Activity Relationship.

Hepatitis B Virus (HBV) infection is a global public health challenge that seriously endangers human health. Soft coral, as a major source of terpenoids, contains many structurally novel and highly bioactive compounds. In previous studies, it has been demonstrated that cembranoid-type diterpenoids showed significant anti-inflammatory and anti-colorectal cancer activities. In this study, cembranoids isolated from Sinularia pedunculata was found with anti-HBV activity for the first time. Among them, compound 6 showed significant anti-HBV activity with an IC50 value of 5.57 µM without cytotoxicity. We analysed the preliminary structure-activity relationship (SAR). Furthermore, it is demonstrated that compound 6 can accelerate the formation of capsid, inhibit HBeAg, HBV core particle DNA, HBV total RNA and pregenomic RNA in a dose dependent manner. We also confirmed the anti-HBV activity in HepG2-NTCP infection system. Finally, we find the anti-HBV mechanism of these compounds by inhibiting the ENI/Xp enhancer/promoter.

Multi-Target Feeding-Behavior Recognition Method for Cows Based on Improved RefineMask.

Within the current process of large-scale dairy-cattle breeding, to address the problems of low recognition-accuracy and significant recognition-error associated with existing visual methods, we propose a method for recognizing the feeding behavior of dairy cows, one based on an improved RefineMask instance-segmentation model, and using high-quality detection and segmentation results to realize the recognition of the feeding behavior of dairy cows. Firstly, the input features are better extracted by incorporating the convolutional block attention module into the residual module of the feature extraction network. Secondly, an efficient channel attention module is incorporated into the neck design to achieve efficient integration of feature extraction while avoiding the surge of parameter volume computation. Subsequently, the GIoU loss function is used to increase the area of the prediction frame to optimize the convergence speed of the loss function, thus improving the regression accuracy. Finally, the logic of using mask information to recognize foraging behavior was designed, and the accurate recognition of foraging behavior was achieved according to the segmentation results of the model. We constructed, trained, and tested a cow dataset consisting of 1000 images from 50 different individual cows at peak feeding times. The method's effectiveness, robustness, and accuracy were verified by comparing it with example segmentation algorithms such as MSRCNN, Point_Rend, Cascade_Mask, and ConvNet_V2. The experimental results show that the accuracy of the improved RefineMask algorithm in recognizing the bounding box and accurately determining the segmentation mask is 98.3%, which is higher than that of the benchmark model by 0.7 percentage points; for this, the model parameter count size was 49.96 M, which meets the practical needs of local deployment. In addition, the technologies under study performed well in a variety of scenarios and adapted to various light environments; this research can provide technical support for the analysis of the relationship between cow feeding behavior and feed intake during peak feeding periods.

Recent advances on marine mollusk-derived natural products: chemistry, chemical ecology and therapeutical potential.

Covering: 2011-2021Marine mollusks, which are well known as rich sources of diverse and biologically active natural products, have attracted significant attention from researchers due to their chemical and pharmacological properties. The occurrence of some of these marine mollusk-derived natural products in their preys, predators, and associated microorganisms has also gained interest in chemical ecology research. Based on previous reviews, herein, we present a comprehensive summary of the recent advances of interesting secondary metabolites from marine mollusks, focusing on their structural features, possible chemo-ecological significance, and promising biological activities, covering the literature from 2011 to 2021.

Chemistry, Chemo-Ecology, and Biological Activities of Uncommon and Structurally Diverse Sesquiterpenoids from the Sanya Bay Nudibranch Hexabranchus sanguineus.

A detailed chemical investigation of the Sanya Bay nudibranch Hexabranchus sanguineus yielded thirteen new sesquiterpenoids, namely sanyagunins A-H, sanyalides A-C, and sanyalactams A and B, along with eleven known related ones. Sanyalactams A and B feature an unprecedented hexahydrospiro[indene-2,3'-pyrrolidine] core. The structures of new compounds were established by a combination of extensive spectroscopic data analysis, quantum mechanical-nuclear magnetic resonance methods, the modified Mosher's method, and X-ray diffraction analysis. Based on analysis of NOESY correlations and the modified Mosher's method, the stereochemistry of two known furodysinane-type sesquiterpenoids were revised. A plausible biogenetic relationship between these sesquiterpenoids wasproposed and discussed, and a chemo-ecological relationship of the title animal and its possible sponge preys has been analyzed. In bioassays, sanyagunin B showed moderate antibacterial activity, whereas 4α-formamidogorgon-11-ene exhibited potent cytotoxicity with IC50 values ranging from 0.87 to 1.95 µM.

Synthesis of 1-O-acyl- and 1-oxo-kamebanin analogues and their cytotoxic activity.

A series of 1-O-acyl- and 1-oxo-kamebanin analogues were prepared from kamebanin, isolated from Rabdosia excisa and their cytotoxicity was assayed on HL60 promyelocytic leukemia cells and HCT116 human colon cancer cells. The structure-activity relationship study showed that the presence of 1-O-acyl groups of a C3-C5 carbon chain increased the cytotoxic activity.

Assessing the prognostic impact of prostatic urethra involvement and developing a nomogram for T1 stage bladder cancer.

PURPOSE: To investigate prognostic values of prostatic urethra involvement (PUI) and construct a prognostic model that estimates the probability of cancer-specific survival for T1 bladder cancer patients. METHOD AND MATERIALS: We investigated the national Surveillance, Epidemiology, and End Results (SEER) database (2004-2015) to get patients diagnosed with T1 bladder cancer. An external validation cohort was obtained from the First Affiliated Hospital of Nanchang University. The Kaplan-Meier method with the log-rank test was applied to assess cancer-specific survival (CSS) and overall survival (OS). Moreover, the propensity score matching (PSM) and multivariable Cox proportional hazard model were performed. All patients were randomly divided into the development cohort and validation group at the ratio of 7:3. The performance of the model was internally validated by calibration curves and the concordance index (C-index). RESULTS: The PUI group had a lower survival rate of both CSS and overall survival OS before and after PSM when compared to non-involved patients (All P < 0.05). Multivariate analysis revealed a poor prognosis in the PUI group for cancer-specific mortality (CSM) and all-cause mortality (ACM) analyses before and after PSM (All P < 0.05). Seven variables, including age, surgery, radiotherapy, tumour size, PUI, and marital status, were incorporated in the final nomogram. The C-index in the development cohort was 0.715 (0.711-0.719), while it was 0.672 (0.667-0.677) in the validation group. Calibration plots for 3- and 5-year cancer-specific survival showed good concordance in the development and validation cohorts. CONCLUSIONS: PUI was an independent risk factor of ACM and CSM in T1 bladder cancer patients. In addition, a highly discriminative and precise nomogram that predicted the individualized probability of cancer-specific survival for patients with T1 bladder cancer was constructed.

New Glycerolipids from the Traditional Chinese Medicine of Syngnathus acus (Hai-Long).

Two new glycerolipids, syngaculipids A and B (1 and 2), one first naturally occurring metabolite (8), together with five known compounds (3-7) were isolated from the AcOEt fraction of Syngnathus acus L. (Hai-Long). Their structures were elucidated by comprehensive spectral analyses involving UV, IR, MS, 1D and 2D NMR spectra and ECD calculations. All the isolated compounds were evaluated for their cytotoxicity against A549 and HCT-116 cell lines. Compound 8 exhibited moderate cytotoxicity with IC50 values of 34.5 and 38.9 µM on the A549 and HCT-116 cell lines, respectively.

The Discovery and Anti-Colorectal Cancer Activities of Cembranolides from the South China Sea Soft Coral Sinularia pendunculata.

A new cembranolide, namely, sinupendunculide A (1), along with eight known related compounds (2-9), was isolated from the South China Sea Soft coral Sinularia pendunculata. The structure of sinupendunculide A (1) was established by extensive spectroscopic analysis and X-ray diffraction experiments. In a bioassay, anti-colorectal cancer (CRC) activity was performed, and the results showed that several compounds exhibited cytotoxicity against RKO cells, and a preliminary structure-activity relationship was analysed. Meanwhile, the most effective compound 7 was proven to increase reactive oxygen species levels, which promoted cell apoptosis and inhibited cell proliferation.

Design, synthesis and in vitro biological evaluation of marine phidianidine derivatives as potential anti-inflammatory agents.

Phidianidines A and B are novel marine indole alkaloids with various biological activities. Based on their potential anti-inflammatory properties, a series of phidianidine derivatives were designed, synthesized, and tested for their effects on IL-17A production in PMA/ionomycin-stimulated T-cell-lymphoma EL-4 cells. Compounds 9a and 22c exhibited excellent anti-inflammatory activity and low toxicity, with IC50 values of 7.7 µM and 5.3 µM for IL-17A production in PMA/ionomycin-stimulated EL-4 cells, respectively. Further mechanistic study showed that 9a could decrease the STAT3 phosphorylation at Y705 to inhibit IL-17A production in EL-4 cells, indicating its ability of preventing the differentiation of Th17 cells and their possible function. This research may give an insight for the discovery of marine indole alkaloid derived anti-inflammatory drug leads for the treatment of T cell-mediated diseases.

Weizhouochrones: Gorgonian-Derived Symmetric Dimers and Their Structure Elucidation Using Anisotropic NMR Combined with DP4+ Probability and CASE-3D.

Natural product dimers have intriguing structural features and often have remarkable pharmacological activities. We report here two uncommon marine gorgonian-derived symmetric dimers, weizhouochrones A (1) and B (2), with indenone-derived monomers, that were isolated from the coral Anthogorgia ochracea collected from the South China Sea. These dimers are difficult targets for structure elucidation that solely relies upon conventional NMR data such as NOEs and J-couplings. Here, to explore the application of emerging methods on the structure elucidation of challenging molecules, we explored a number of different anisotropic and computational NMR approaches. The measurements of anisotropic NMR parameters of weizhouochrone A, including residual dipolar couplings (RDCs) and residual chemical shift anisotropy (RCSA), allowed us to successfully determine the planar structure and its relative configuration. This result was corroborated by a computational NMR analysis based on DP4+ probability and computer-assisted 3D structure elucidation (CASE-3D).

Diversity-oriented synthesis of marine polybrominated diphenyl ethers as potential KCNQ potassium channel activators.

Natural polybrominated diphenyl ethers, often isolated from marine sponges, have been reported to possess various biological activities, such as antibacterial, antioxidant and antidiabetic effects. Via a high throughput screening of our marine natural product library, the polybrominated diphenyl ether 3 was found to display a KCNQ potassium channel activation effect. To obtain more compound 3 related natural products and their derivatives for further bioactivity study, a diversity-oriented synthesis was conducted, leading to the successful synthesis of five polybrominated diphenyl ether natural products (1-4, 6) and 30 new derivatives. Compound 3 was found to preferentially potentiate KCNQ1 potassium channel, whereas 17h relatively activated KCNQ2 potassium channel. The structure-activity relationship was analyzed assisted by molecular docking and 17h was further conducted for its agonistic mechanism study on KCNQ2 channel. This research work may give an insight for the discovery of marine polybrominated diphenyl ether derived new drug leads.

An efficient strategy based on two-stage chromatography and in vitro evaluation for rapid screening and isolation of acetylcholinesterase inhibitors from Scutellaria baicalensis Georgi.

The extraction of Scutellaria baicalensis Georgi was investigated using the response surface methodology-genetic algorithm mathematical regression model, and the extraction variables were optimized to maximize the flavonoid yield. Furthermore, a simple and efficient ultrafiltration-liquid chromatography-mass spectrometry and molecular docking methods were developed for the rapid screening and identification of acetylcholinesterase inhibitors present in Scutellaria baicalensis Georgi. Subsequently, four major chemical constituents, namely baicalein, norwogonin, wogonin, and oroxylin A, were identified as potent acetylcholinesterase inhibitors. This novel approach, involving the use of ultrafiltration-liquid chromatography-mass spectrometry and molecular docking methods combined with stepwise flow rate counter-current chromatography and semi-preparative high-performance liquid chromatography, could potentially provide a powerful tool for the screening and extraction of acetylcholinesterase inhibitors from complex matrices and be a useful platform for the production of bioactive and nutraceutical ingredients.

New Cladiellin-Type Diterpenoids from the South China Sea Soft Coral Cladiella krempfi: Structures and Molecular Docking Analysis in EGFRs.

Two new cladiellin-type diterpenoids (1 and 2) and four known related compounds 3-6, were isolated from the South China Sea soft coral Cladiella krempfi. Compound 2 is the third example of cladiellins of an unusual peroxy group in the C-6 position in C. krempfi. The structures and absolute configurations of the new compounds were established by extensive spectroscopic analysis, X-ray diffraction, and/or chemical correlation. In bioassay, all the compounds were evaluated for cytotoxicity and epidermal growth factor receptor (EGFR) inhibitory activity. A molecular docking experiment was conducted to study the structure-activity relationship of cladiellin-type diterpenoids on EGFR inhibitory activity.

Ximaoornatins A-C, Polyoxygenated Diterpenoids from the Hainan Soft Coral Sinularia ornata.

Three complex polyoxygenated diterpenoids possessing uncommon tetradecahydro-2,13:6,9-diepoxybenzo[10]annulene scaffold, namely ximaoornatins A-C (1-3), one new eunicellin-type diterpene, litophynin K (4), and a related known compound, litophynol B (5) were isolated from the South China Sea soft coral Sinularia ornata. The structures and absolute configurations of 1-4 were established by extensive spectroscopic analysis, X-ray diffraction analysis, and/or modified Mosher's method. A plausible biosynthetic relationship of 1 and its potential precursor 4 was proposed. In a bioassay, none of the isolated compounds showed obvious anti-inflammatory activity on LPS-induced TNF-α release in RAW264.7 macrophages and PTP1B inhibitory effects.

Sinuhirtone A, An Uncommon 17,19-Dinorxeniaphyllanoid, and Nine Related New Terpenoids from the Hainan Soft Coral Sinularia hirta.

Chemical investigation of the Hainan soft coral Sinularia hirta resulted in the isolation and identification of a library of sixteen structurally diverse terpenoids, including a dinorditerpenoid with an uncommon 17,19-dinorxeniaphyllane skeleton, namely sinuhirtone A (7), six new xeniaphyllane-type diterpenoids (1-6), one new norxeniaphyllanoid (8), two new norcaryophyllene-type sesquiterpenoids (9 and 10), together with six known related compounds (11-16). Compounds 1-3 are three new furanone-containing xeniaphyllane-type diterpenoids. The structures of the new compounds, including their absolute configurations, were determined by extensive spectroscopic analysis and a series of quantum chemical calculations, including quantum mechanical-nuclear magnetic resonance (QM-NMR), time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD), and optical rotatory dispersion (ORD) methods. A plausible biosynthetic connection between new compounds 1-9 was also proposed. New compounds 2-4, 7, and 8 were evaluated for in vitro cytotoxicity against four cancer cell lines.

Ocellatuperoxides A-F, Uncommon Anti-Tumoral γ-Pyrone Peroxides from a Photosynthetic Mollusk Placobranchus ocellatus.

Six new pairs of γ-pyrone polypropionate enantiomers with an unusual peroxyl bridge at the side chain, namely (±)-ocellatuperoxides A-F (1-6), were isolated and characterized from the South China Sea photosynthetic mollusk Placobranchus ocellatus. Extensive spectroscopic analysis, single crystal X-ray diffraction analysis, ECD- (electronic circular dichroism) comparison, and TDDFT (time-dependent density functional theory) ECD computation were used to determine the structures and absolute configurations of new compounds. In a cell viability assay, several compounds showed considerable anti-tumoral effects on human non-small cell lung cancer cells A549 with Gefitinib (7.4 µM) and Erlotinib (2.1 µM) as positive controls. Further RNA-sequencing analysis and gene expression evaluation indicated that the anti-tumoral activity of the most effective compound 3 was associated with the regulation of several important genes, such as FGFR1 and HDAC5.