Discovery of novel ocotillol derivatives modulating glucocorticoid receptor/NF-κB signaling for the treatment of sepsis.

Glucocorticoids (GCs) have been used in the treatment of sepsis because of their potent anti-inflammatory effects. However, their clinical efficacy against sepsis remains controversial because of glucocorticoid receptor (GR) downregulation and side effects. Herein, we designed and synthesized 30 ocotillol derivatives and evaluated their anti-inflammatory activities. Ocotillol 24(R/S) differential isomers were stereoselective in their pharmacological action. Specifically, 24(S) derivatives had better anti-inflammatory activity than their corresponding 24(R) derivatives. Compound 20 most effectively inhibited NO release (85.97% reduction), and it exerted dose-dependent inhibitory effects on interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels. Mechanistic studies revealed that compound 20 reduces the degradation of GR mRNA and GR protein. Meanwhile, compound 20 inhibited the activation of nuclear factor-κB (NF-κB) signaling, thereby inhibiting the nuclear translocation of p65 and attenuating the inflammatory response. In vivo studies revealed that compound 20 attenuated hepatic, pulmonary, and renal pathology damage in mice with sepsis and suppressed the production of inflammatory mediators. These results indicated that compound 20 is a promising lead compound for designing and developing anti-sepsis drugs.

[Analysis of Photochemical Characteristics and Sensitivity of Atmospheric Ozone in Nanjing in Summer].

Tropospheric ozone (O3) is mainly produced through a series of photochemical reactions of nitrogen oxides (NOx) and volatile organic compounds (VOCs). The reaction process presents complex non-linear relationships. In this work, datasets of atmospheric ozone and volatile organic compounds (VOCs) observed during the summer of 2018 in Nanjing were used. Combining with the framework for 0-D atmospheric model-master chemical mechanism (F0AM-MCM), the characteristics of photochemical reactions for ozone (O3) formation in Nanjing during the O3 episode days and non-episode days were investigated. The results showed that φ(O3) and φ(TVOCs) in the O3 episode days were 47.8×10-9 and 49.0×10-9, respectively, exceeding those in the non-episode days by factors of 1.8 and 1.6. Furthermore, F0AM, the empirical kinetic modeling approach (EKMA), and relative incremental reactivity (RIR) were utilized for the calculation of ozone chemical sensitivity. It was found that O3 formation in Nanjing was attributed to both VOCs and NOx limitation. In addition, the modeled ·OH and HO2 concentrations in the O3 episode days were 1.3 and 1.8 times higher than those in the non-episode days. The higher formation and loss rates of ·OH and HO2 were also found during O3 episode days. These findings reflected that the enhancements of atmospheric oxidation capacity resulted in increased production rates of O3, providing an explanation for the enhancements of O3 concentrations in Nanjing during the O3 episode days. The findings also improved the understanding of the O3 photochemical characteristics over Nanjing in the summer during the O3 episode days.

Neuronal miR-9 promotes HSV-1 epigenetic silencing and latency by repressing Oct-1 and Onecut family genes.

Herpes simplex virus 1 (HSV-1) latent infection entails repression of viral lytic genes in neurons. By functional screening using luciferase-expressing HSV-1, we identify ten neuron-specific microRNAs potentially repressing HSV-1 neuronal replication. Transfection of miR-9, the most active candidate from the screen, decreases HSV-1 replication and gene expression in Neuro-2a cells. Ectopic expression of miR-9 from lentivirus or recombinant HSV-1 suppresses HSV-1 replication in male primary mouse neurons in culture and mouse trigeminal ganglia in vivo, and reactivation from latency in the primary neurons. Target prediction and validation identify transcription factors Oct-1, a known co-activator of HSV transcription, and all three Onecut family members as miR-9 targets. Knockdown of ONECUT2 decreases HSV-1 yields in Neuro-2a cells. Overexpression of each ONECUT protein increases HSV-1 replication in Neuro-2a cells, human induced pluripotent stem cell-derived neurons, and primary mouse neurons, and accelerates reactivation from latency in the mouse neurons. Mutagenesis, ChIP-seq, RNA-seq, ChIP-qPCR and ATAC-seq results suggest that ONECUT2 can nonspecifically bind to viral genes via its CUT domain, globally stimulate viral gene transcription, reduce viral heterochromatin and enhance the accessibility of viral chromatin. Thus, neuronal miR-9 promotes viral epigenetic silencing and latency by targeting multiple host transcription factors important for lytic gene activation.

Structural characterization and ferroptosis-related immunomodulatory of a novel exopolysaccharide isolated from marine fungus Aspergillus medius.

Marine fungal exopolysaccharides play a crucial role in immunoregulation. In this investigation, a novel polysaccharide was extracted from the culture medium of the marine fungus Aspergillus medius SCAU-236. Compositional analysis revealed a structure composed of glucose units with (1,4)-α-D-Glcp, (1,3,4)-ß-D-Glcp, and (1,4,6)-α-D-Glcp, along with side chains of 1-α-D-Glcp linked to carbon 6 of (1,4,6)-α-D-Glcp and carbon 3 of (1,3,4)-ß-D-Glcp. Functional evaluations on RAW264.7 macrophage cells demonstrated Aspergillus medius polysaccharide (ASMP)'s effects on cell proliferation, nitric oxide levels, and the secretion of TNF-α, IL-6, and IL-1ß cytokines. Additionally, metabolomics indicated ASMP's potential to modulate macrophage immune function by impacting key regulatory molecules, including COX-2, iNOS, Nrf2, SLC7A11, GPX4, and ACSL4. The Nrf2/SLC7A11/GPX4 axis and ACSL4 were suggested to be involved in ASMP-induced ferroptosis, leading to increased reactive oxygen species (ROS) levels and lipid peroxidation. These findings propose a unique mechanism by which ASMP exerts immunomodulatory effects through ferroptosis induction, contributing to the understanding of marine-derived compounds in immunomodulation research.

Current-Controlled Electrochemical Approach Toward Mono- and Trifluorinated Isoindolin-1-one Derivatives.

An electrochemical intramolecular 5-exo-dig aza-cyclization of 2-alkynylbenzamides and subsequent nucleophilic fluorination have been developed to afford the highly selective synthesis of mono- and trifluorinated isoindolin-1-one derivatives. This work demonstrates the unique capability of synthetic electrochemistry in controlling reaction selectivity through the applied electrolytic parameters. In addition, the obtained monofluorinated 3-methyleneisoindolin-1-one (19) displays interesting photophysical properties that are not observed in its nonfluorinated analog.

A New Exopolysaccharide of Marine Coral-Associated Aspergillus pseudoglaucus SCAU265: Structural Characterization and Immunomodulatory Activity.

Recent studies have found that many marine microbial polysaccharides exhibit distinct immune activity. However, there is a relative scarcity of research on the immunomodulatory activity of marine fungal exopolysaccharides. A novel water-soluble fungal exopolysaccharide ASP-1 was isolated from the fermentation broths of marine coral-associated fungus Aspergillus pseudoglaucus SCAU265, and purified by Diethylaminoethyl-Sepharose-52 (DEAE-52) Fast Flow and Sephadex G-75. Structural analysis revealed that ASP-1 had an average molecular weight of 36.07 kDa and was mainly composed of (1→4)-linked α-D-glucopyranosyl residues, along with highly branched heteropolysaccharide regions containing 1,4,6-glucopyranosyl, 1,3,4-glucopyranosyl, 1,4,6-galactopyranosyl, T(terminal)-glucopyranosyl, T-mannopyranosyl, and T-galactopyranosyl residues. ASP-1 demonstrated significant effects on the proliferation, nitric oxide levels, and the secretion of cytokines TNF-α and IL-6 in macrophage RAW264.7 cells. Metabolomic analysis provided insights into the potential mechanisms of the immune regulation of ASP-1, suggesting its involvement in regulating immune function by modulating amino acid anabolism, particularly arginine synthesis and metabolism. These findings provide fundamental scientific data for further research on its accurate molecular mechanism of immunomodulatory activity.

Tetramerization of upstream stimulating factor USF2 requires the elongated bent leucine zipper of the bHLH-LZ domain.

Upstream stimulating factors (USFs), including USF1 and USF2, are key components of the transcription machinery that recruit coactivators and histone-modifying enzymes. Using the classic basic helix-loop-helix leucine zipper (bHLH-LZ) domain, USFs bind the E-box DNA and form tetramers that promote DNA looping for transcription initiation. The structural basis by which USFs tetramerize and bind DNA, however, remains unknown. Here, we report the crystal structure of the complete bHLH-LZ domain of USF2 in complex with E-box DNA. We observed that the leucine zipper (LZ) of USF2 is longer than that of other bHLH-LZ family transcription factors and that the C-terminus of USF2 forms an additional α-helix following the LZ region (denoted as LZ-Ext). We also found the elongated LZ-Ext facilitates compact tetramer formation. In addition to the classic interactions between the basic region and DNA, we show a highly conserved basic residue in the loop region, Lys271, participates in DNA interaction. Together, these findings suggest that USF2 forms a tetramer structure with a bent elongated LZ-Ext region, providing a molecular basis for its role as a key component of the transcription machinery.

Synthesis of 2-Indolyl C-Glycoside Neopetrosins A and C and Congeners via Ni-Catalyzed Photoreductive Cross-Coupling.

The synthesis of neopetrosins A and C, two 2-indolyl C-α-d-mannopyranosides, and their congeners has been realized via a direct Ni/photoredox-catalyzed reductive coupling of 3-methoxycarbonyl-2-iodo-1H-indoles with pyranosyl bromides.

Elucidating the mechanisms underlying the anti-hyperlipidemic effects of Laportea bulbifera using integrated serum metabolomics and network pharmacology.

Hyperlipidemia is a chronic metabolic disorder characterized by alterations in lipid metabolism as well as other pathways. Laportea bulbifera, an indigenous medicinal plant of Chinese herbal medicine, exhibits therapeutic effects on hyperlipidemia, but the mechanisms remain unclear. This study investigated the potential mechanisms underlying the anti-hyperlipidemic effects of L. bulbifera using an integrated strategy based on metabolomics and network pharmacology methods that were established to investigate the potential mechanism of anti-hyperlipidemia effect of L. bulbifera. First, the therapeutic effects of L. bulbifera on body weight reduction and biochemical indices were assessed. Next, 18 significant metabolites distinguishing the control and model groups were identified based on serum metabolomics and multivariate analyses. Then, a compound-target network was constructed by linking L. bulbifera and hyperlipidemia using network pharmacology. Three metabolic pathways involved in treating hyperlipidemia were identified. Finally, five crucial targets were selected by constructing a bionetwork starting from the compounds and ending in the metabolites. This study established an integrated strategy based on metabolomics coupled with network pharmacology and revealed the mechanism underlying the protective effects of L. bulbifera against hyperlipidemia for the first time.

Diet Restriction Impact on High-Fat-Diet-Induced Obesity by Regulating Mitochondrial Cardiolipin Biosynthesis and Remodeling.

Diet restriction (DR) ameliorates obesity by regulating mitochondrial function. Cardiolipin (CL), a mitochondrial phospholipid, is closely associated with mitochondrial function. This study aimed to evaluate the anti-obesity effects of graded levels of DR based on mitochondrial CL levels in the liver. Obese mice were treated with 0%, 20%, 40%, and 60% reductions in the normal diet compared to normal animals (0 DR, 20 DR, 40 DR, and 60 DR groups, respectively). Biochemical and histopathological analyses were performed to evaluate the ameliorative effects of DR on obese mice. The altered profile of mitochondrial CL in the liver was explored using a targeted metabolomics strategy by ultra-high-pressure liquid chromatography MS/MS coupled with quadrupole time-of-flight mass spectrometry. Finally, gene expression associated with CL biosynthesis and remodeling was quantified. Tissue histopathology and biochemical index evaluations revealed significant improvements in the liver after DR, except for the 60 DR group. The variation in mitochondrial CL distribution and DR levels showed an inverted U-shape, and the CL content in the 40 DR group was the most upregulated. This result is consistent with the results of the target metabolomic analysis, which showed that 40 DR presented more variation. Furthermore, DR led to increased gene expression associated with CL biosynthesis and remodeling. This study provides new insights into the mitochondrial mechanisms underlying DR intervention in obesity.

Inhibiting autophagy before it starts.

Autophagy, an important cellular stress response mechanism, is often exploited by a variety of cancer cells to sustain rapid growth under stresses such as nutrient deprivation and hypoxia. Autophagy also plays a key role in tumor resistance to chemotherapy, radiotherapy or targeted therapy. Inhibition of autophagy is therefore a promising tumor treatment strategy. However, there is still a lack of effective autophagy inhibitors suitable for clinical use. Most drug development has focused on enzymes like the VPS34 and ULK1 kinases, or the cysteine protease ATG4B, which plays different roles in autophagy. We discovered a drug molecule Eltrombopag that inhibits the expression of autophagic lysosomal genes at the stage of transcriptional level, where the synthesis of these proteins has not really begun, by directly inhibiting the TFEB (transcription factor EB). This drug can improve the therapeutic effect of Temozolomide on glioblastoma treatment, further confirming the value of inhibiting autophagy in the treatment of cancer.Abbreviation: VPS34: vacuolar protein sorting 34; ULK1: unc-51 like autophagy activating kinase 1; TFEB: transcription factor EB; MITF: microphthalmia-associated transcription factor; TFE3: transcription factor E3; EO: Eltrombopag; ITC: isothermal titration calorimetry; bHLH-LZ: basic helix-loop-helix leucine zipper; LAMP1: lysosomal-associated membrane protein 1; CTSF: cathepsin F; HEXA: hexosaminidase subunit alpha.

Isoalantolactone suppresses gallbladder cancer progression via inhibiting the ERK signalling pathway.

CONTEXT: Gallbladder cancer (GBC) is the most common malignant tumour of the biliary tract. Isoalantolactone (IAL), an active sesquiterpene lactone compound isolated from the roots of Inula helenium L. (Asteraceae), has antitumour effects. OBJECTIVE: This study investigates the effects of IAL on GBC. MATERIALS AND METHODS: In vitro, NOZ and GBC-SD cells were treated with IAL (0, 10, 20 and 40 µM) for 24 h. The DMSO-treated cells were selected as a control. Cell proliferation, migration, invasion and apoptosis were measured by the CCK-8 assay, transwell assay, flow cytometry and western blot. In vivo, subcutaneous tumour xenografts were constructed by injecting nude mice (BALB/C) with 5 × 106 NOZ cells. Mice were divided into the control group (equal amount of DMSO), the IAL group (10 mg/kg/day) and the IAL + Ro 67-7476 group (IAL, 10 mg/kg/day; Ro 67-7476, 4 mg/kg/day). The study duration was 30 days. RESULTS: Compared with the DMSO group, cell proliferation of NOZ (IC50 15.98 µM) and GBC-SD (IC50 20.22 µM) was inhibited by about 70% in the IAL 40 µM group. Migration and invasion were suppressed by about 80%. Cell apoptosis rate was increased about three-fold. The phosphorylation level of ERK was decreased to 30-35%. Tumour volume and weight (about 80% reduction) were suppressed by IAL in vivo. Moreover, the effects of IAL were abolished by Ro 67-7476 in vitro and in vivo. DISCUSSION AND CONCLUSIONS: Our findings indicate that IAL could inhibit GBC progression in vitro and in vivo by inhibiting the ERK signalling pathway.

A small-molecule drug inhibits autophagy gene expression through the central regulator TFEB.

Autophagy supports the fast growth of established tumors and promotes tumor resistance to multiple treatments. Inhibition of autophagy is a promising strategy for tumor therapy. However, effective autophagy inhibitors suitable for clinical use are currently lacking. There is a high demand for identifying novel autophagy drug targets and potent inhibitors with drug-like properties. The transcription factor EB (TFEB) is the central transcriptional regulator of autophagy, which promotes lysosomal biogenesis and functions and systematically up-regulates autophagy. Despite extensive evidence that TFEB is a promising target for autophagy inhibition, no small molecular TFEB inhibitors were reported. Here, we show that an United States Food and Drug Administration (FDA)-approved drug Eltrombopag (EO) binds to the basic helix-loop-helix-leucine zipper domain of TFEB, specifically the bottom surface of helix-loop-helix to clash with DNA recognition, and disrupts TFEB-DNA interaction in vitro and in cellular context. EO selectively inhibits TFEB's transcriptional activity at the genomic scale according to RNA sequencing analyses, blocks autophagy in a dose-dependent manner, and increases the sensitivity of glioblastoma to temozolomide in vivo. Together, this work reveals that TFEB is targetable and presents the first direct TFEB inhibitor EO, a drug compound with great potential to benefit a wide range of cancer therapies by inhibiting autophagy.

A unique hyperdynamic dimer interface permits small molecule perturbation of the melanoma oncoprotein MITF for melanoma therapy.

Microphthalmia transcription factor (MITF) regulates melanocyte development and is the "lineage-specific survival" oncogene of melanoma. MITF is essential for melanoma initiation, progression, and relapse and has been considered an important therapeutic target; however, direct inhibition of MITF through small molecules is considered impossible, due to the absence of a ligand-binding pocket for drug design. Here, our structural analyses show that the structure of MITF is hyperdynamic because of its out-of-register leucine zipper with a 3-residue insertion. The dynamic MITF is highly vulnerable to dimer-disrupting mutations, as we observed that MITF loss-of-function mutations in human Waardenburg syndrome type 2 A are frequently located on the dimer interface and disrupt the dimer forming ability accordingly. These observations suggest a unique opportunity to inhibit MITF with small molecules capable of disrupting the MITF dimer. From a high throughput screening against 654,650 compounds, we discovered compound TT-012, which specifically binds to dynamic MITF and destroys the latter's dimer formation and DNA-binding ability. Using chromatin immunoprecipitation assay and RNA sequencing, we showed that TT-012 inhibits the transcriptional activity of MITF in B16F10 melanoma cells. In addition, TT-012 inhibits the growth of high-MITF melanoma cells, and inhibits the tumor growth and metastasis with tolerable toxicity to liver and immune cells in animal models. Together, this study demonstrates a unique hyperdynamic dimer interface in melanoma oncoprotein MITF, and reveals a novel approach to therapeutically suppress MITF activity.

Structural characterization and immunomodulatory activity of an exopolysaccharide from marine-derived Aspergillus versicolor SCAU141.

Until now, relatively little is known about marine-derived fungal polysaccharides and their activities. Exopolysaccharide AVP141-A was isolated from the broth of marine-derived fungus Aspergillus versicolor SCAU141 and purified by Diethylaminoethyl-Sepharose Fast Flow and Sephadex G-100. The structural characteristics of AVP141-A was studied by chemical analysis together with high-performance gel permeation chromatography, ion chromatography, Fourier-transform infrared spectroscopy, gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The results showed that AVP141-A with the molecular weight of 5.10 kDa was mainly composed of →4)-α-D-Glcp-(1→, branched by α-D-Glcp-(1→ and →6)-α-D-Glcp-(1→ at C-6 positions of the glucan backbone. In particular, sulfate ester (approximately 3.62 %) was found in AVP141-A, which was frequently considered to occur in marine-derived microbial polysaccharides rather than other microbial polysaccharides. Furthermore, AVP141-A significantly enhanced the activity of the inflammatory factors NO, COX-2 and TNF-α in RAW264.7 macrophages by activating the MAPK/p38 and NF-κB/p65 pathways. In addition, metabolomic analysis revealed that most of the pathways with significant changes in RAW264.7 macrophages treated with AVP141-A were amino acid-related pathways, and arginine was the characteristic metabolite. In conclusion, this study identified AVP141-A as a marine fungus-derived sulfated exopolysaccharide with potential for development as an immune activator.

Anti-colon cancer effects of Spirulina polysaccharide and its mechanism based on 3D models.

Spirulina polysaccharides (PSP) possess significant biological properties. However, it is still a lack of investigation on the anti-colorectal cancer effect and mechanism. In this study, PSP showed significant effects on LoVo cell spheroids with an IC50 value of 0.1943 mg/mL. The analysis of transcriptomics and metabolomics indicated the impact of PSP on LoVo spheroid cells through involvement in the two pathways of "glycine, serine, and threonine metabolism" and "ABC transporters". And, the q-PCR data further verified the pointed mechanism of PSP on colon cancer (CC) by regulating the expression levels of relevant genes in the synthesis pathways of serine and glycine in tumor cells. Furthermore, the anti-colon cancer effects of PSP were verified via other human colon cancer cell lines HCT116 and HT29 spheroids (IC50 = 0.0646 mg/mL and 0.2213 mg/mL, respectively), and three patient-derived organoids (PDOs) with IC50 values ranging from 3.807 to 7.788 mg/mL. In addition, this study found that a mild concentration of PSP cannot enhance the anti-tumor effect of 5-Fu. And a significant inhibition was found of PSP in 5-Fu resistance organoids. These results illustrated that PSP could be a treatment or supplement for 5-Fu resistant colorectal cancer (CRC).

Exploration of the hypoglycemic mechanism of Fuzhuan brick tea based on integrating global metabolomics and network pharmacology analysis.

Introduction: Fuzhuan brick tea (FBT) is a worldwide popular beverage which has the appreciable potential in regulating glycometabolism. However, the reports on the hypoglycemic mechanism of FBT remain limited. Methods: In this study, the hypoglycemic effect of FBT was evaluated in a pharmacological experiment based on Kunming mice. Global metabolomics and network pharmacology were combined to discover the potential target metabolites and genes. In addition, the real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed for verification. Results: Seven potential target metabolites and six potential target genes were screened using the integrated approach. After RT-qPCR analysis, it was found that the mRNA expression of VEGFA, KDR, MAPK14, and PPARA showed significant differences between normal and diabetes mellitus mice, with a retracement after FBT treatment. Conclusion: These results indicated that the hypoglycemic effect of FBT was associated with its anti-inflammatory activities and regulation of lipid metabolism disorders. The exploration of the hypoglycemic mechanism of FBT would be meaningful for its further application and development.