Characterization of Sesquiterpene Dimers from the Flowers of Inula japonica and the Structural Revisions of Related Compounds.

Sesquiterpene dimers are mainly found in the Asteraceae family. However, conflicting reports on the structures of these compounds can be found in the literature. Herein, we describe ten sesquiterpene dimers isolated from the flowers of Inula japonica, including configurational revisions of japonicone H (1-1), japonicone D (2-1), inulanolide A (4-1), japonicone X (5-1), and inulanolide F (5-2) to compounds 1, 2, 4, and 5, respectively. Five new related metabolites (3 and 6-9) are also described. Application of GIAO NMR/DP4+ analyses and ECD/OR calculations enabled us to revise the absolute configurations of an additional 13 sesquiterpene dimers isolated from plants of the genus Inula. Compounds 1, 2, 4, and 6 exhibited inhibition of nitric oxide production in lipopolysaccharide activated RAW264.7 macrophages with IC50 values of 4.07-10.00 µM.

TMEM147: A Promising Cancer Biomarker Associated with Immune Cell Infiltration and Prognosis in LIHC-Insights from a Comprehensive Pan-Cancer Genomic Analysis.

Previous studies have demonstrated the regulatory roles of Transmembrane protein 147 (TMEM147) in various diseases, including cancer. However, systematic pan-cancer analyses investigating the role of TMEM147 in diagnosis, prognosis, and immunological prediction are lacking. An analysis of data from The Cancer Genome Atlas (TCGA) revealed differential TMEM147 expression across various types of cancer as well as within immune and molecular cancer subtypes. Moreover, high TMEM147 expression was associated with poor disease-specific survival (DSS), overall survival (OS), and progression-free interval (PFI) across cancers, suggesting its potential as a prognostic biomarker. Our study further revealed a significant correlation between TMEM147 expression and T helper cell and Tcm cell infiltration in most cancer types. In the case of liver hepatocellular carcinoma (LIHC), the effect of TMEM147 on prognosis varied among different clinical subtypes. Additionally, functional enrichment analysis revealed an association between TMEM147 and metabolic pathways. Finally, experiments on the MIHA cell line and four LIHC cell lines confirmed the role of TMEM147 in promoting liver cancer cell proliferation, further confirming the clinical value of TMEM147 in liver cancer diagnosis. Our findings suggest that TMEM147 may serve as a diagnostic and prognostic biomarker across cancers while also playing a significant role in LIHC.

Discovery of natural AMPK activator from the fruits of Xanthium sibiricum Patr.: Xanthiumine A, protoberberine alkaloid with unique C28 skeleton.

Two protoberberine alkaloids with a unique C28 skeleton, named xanthiumines A (1) and B (2), respectively, were isolated from the fruits of Xanthium sibiricum Patr. Their structures including absolute configurations were unequivocally established by the comprehensive NMR and MS spectroscopic data analysis together with gauge-independent atomic orbital (GIAO) NMR calculations, and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are the first examples of natural protoberberine alkaloid with a phenolic acid group at C-13a. Their plausible biosynthetic pathway was proposed on the basis of the coexisting alkaloid monomer as the precursor. Furthermore, the effects and related molecular mechanism of compound 1 on hepatic lipid accumulation were also investigated in oleic acid (OA)-treated HepG2 cells.

Structure of the exopolyphosphatase (PPX) from Zymomonas mobilis reveals a two-magnesium-ions PPX.

Rapid adaptation of metabolic capabilities is crucial for bacterial survival in habitats with fluctuating nutrient availability. In such conditions, the bacterial stringent response is a central regulatory mechanism activated by nutrient starvation or other stressors. This response is primarily controlled by exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) enzymes. To gain further insight into these enzymes, the high-resolution crystal structure of PPX from Zymomonas mobilis (ZmPPX) was determined at 1.8 Å. The phosphatase activity of PPX was strictly dependent on the presence of divalent metal cations. Notably, the structure of ZmPPX revealed the presence of two magnesium ions in the active site center, which is atypical compared to other PPX structures where only one divalent ion is observed. ZmPPX exists as a dimer in solution and belongs to the "long" PPX group consisting of four domains. Remarkably, the dimer configuration exhibits a substantial and deep aqueduct with positive potential along its interface. This aqueduct appears to extend towards the active site region, suggesting that this positively charged aqueduct could potentially serve as a binding site for polyP.

Undescribed sesquiterpenoids with NO production inhibitory activity from oleo-gum resin of Commiphora myrrha.

Six undescribed cadinane sesquiterpenoids (1-6), two undescribed guaiane sesquiterpenoids (7-8), and an undescribed germacrane sesquiterpenoid (9) were isolated from the oleo-gum resin of Commiphora myrrha. Their structures were determined by the analysis of 1D/2D NMR and HRESIMS data, as well as quantum chemical ECD and NMR calculations. All the sesquiterpenoids were evaluated for their NO production inhibitory activity in LPS-stimulated RAW 264.7 mouse monocyte-macrophages. The results revealed that commiphone A (1) and commipholide D (7) exhibited significant inhibitory effect on NO generation with IC50 values of 18.6 ± 2.0 and 37.5 ± 1.5 µM, respectively. Furthermore, 1 and 7 dose-dependently inhibited the mRNA expression of inflammatory cytokines IL-1ß, IL-6 and TNF-α induced by LPS in the RAW264.7 cells, indicating that 1 and 7 possess potent anti-inflammatory activity in vitro.

Development of Cas13a-based therapy for cancer treatment.

Gene therapy has become a major focus of current biomedical research. CRISPR (Clustered Regularly Inter spaced Short Palindromic Repeats) systems have been extensively researched for disease treatment applications through genome editing specificity. Compared with Cas9 (CRISPR-associated proteins, Cas), a commonly used tool enzyme for genome editing, Cas13a exhibits RNA-dependent endonuclease activity, including collateral cleavage without obvious potential genetic risks. With its high specificity, Cas13a has significantly improved the sensitivity of viral diagnosis and shown potential to eliminate viruses. However, its efficacy in tumor therapy has not been determined. This review introduces the mechanism and research developments associated with the CRISPR-Cas13a system in tumor treatments and its potential to be used as a new tool for gene therapy. We hope more research would apply Cas13a-based therapy in cancer treatment in the future.

Anti-proliferative tirucallane triterpenoids from gum resin of Boswellia sacra.

Eight new tirucallane triterpenoids (1-2, 5-10) along with two known compounds (3-4) were isolated from the gum resin of Boswellia sacra. Their structures were elucidated by extensive physicochemical and spectroscopic analysis, as well as computational calculations, and single crystal X-ray diffraction. Spirosacraoic acid A (1) and B (2), possess an unusual 6/5/6/5 rearranged spirocyclic carbon skeleton. All the isolates were evaluated for their anti-proliferative activity against two tumor cell lines (HepG2 and HCT-116 cells). Compound 10 displayed remarkable inhibitory activity against HepG2 cells in a dose-dependent manner with the IC50 value of 28.01 µM. High content analysis (HCA) showed that 10 induces apoptosis in HepG2 cells. The western blotting results revealed that 10 could up-regulate the ratio of the expression of Bax/BCL-2, and promote the caspase 3 activation and PARP cleavage. Mechanically, molecular modeling studies demonstrated that 10 could dock into EGFR active site. Meanwhile 10 significantly decreased the protein expression of p-EGFR. Furthermore, inhibition of EGFR by addition of EGFR siRNA enhanced the growth inhibitory effects of 10 on HepG2 cells, indicating that the anti-tumor effect of 10 on HepG2 cells was mediated by inhibition of EGFR.

Meta-analysis of the efficacy of glycyrrhizin for postoperative liver preservation in patients with liver cancer.

Objective: This meta-analysis comprehensively summarizes the current clinical research on compound glycyrrhizin (CG) treatment for liver cancer and protecting liver function to guide clinical treatment. Methods: Eighteen English-language articles were retrieved from PubMed, SinoMed, Cochrane, Embase, Web of Science, and three Chinese databases: The Wan Fang database, China National Knowledge Infrastructure (CNKI), and the VIP database. Results: CG treatment improved the patient's alanine aminotransferase (ALT) level (in the metastatic liver cancer group: mean deviation (MD) = -13.78, 95% confidence interval (CI) = [-17.29, 10.27]; in the primary liver cancer group: MD = -32.15, 95% CI = [-35.48, 28.81]); aspartate aminotransferase (AST) level (in the primary liver cancer group: MD = -21.63, 95% CI = [-24.29, 18.96]; in the metastatic liver cancer group: MD = -15.64, 95% CI = [-19.08, -12.20]); serum total bilirubin (TBIL) level (MD = -1.61, 95% CI = [-2.71, -0.51]); and serum albumin (ALB) level (MD = 2.80, 95% CI = [1.85, 3.74]). CG treatment was efficient than the control (relative risk [RR] = 1.66, 95% CI = [1.35, 2.04]). Although adverse reactions, including fever, were higher than in the control group (RR = 1.13, 95% CI = [0.89, 1.43]), they were controllable. Conclusion: CG affects liver preservation in treating liver cancer, which can reduce ALT, AST, and TBIL levels in patients; increase the ALB level; and protect liver cells. The CG-treated group showed improvement compared with the control group; although adverse reactions occurred in the treated group, the duration was shortened.

Taurine protects dopaminergic neurons in paraquat-induced Parkinson's disease mouse model through PI3K/Akt signaling pathways.

Taurine (Tau) is one of the most abundant amino acids in the brain and regulates physiological functions in the central nervous system, including anti-inflammatory effects. There is growing evidence that microglia-mediated neuro-inflammatory responses are an integral part of Parkinson's disease (PD) onset and progression. Among the many factors regulating the inflammatory response, phosphatidylinositol-3 kinase (PI3K) is susceptible to activation by a variety of cytokines and physicochemical factors, and subsequently recruits signaling proteins containing the pleckstrin homology structural domain to further regulate protein kinase B (AKT) expression involved in the regulation of the intracellular immune response and inflammatory response. Therefore, we established a PD mouse model using paraquat (PQ) intraperitoneal injection staining to explore the mechanism of Tau action on PI3K/AKT signaling pathway. Our study showed that PD mice with Tau intervention recovered motor and non-motor functions to some extent, and the number of dopaminergic (DAc) neurons in the substantia nigra and the level of dopamine (DA) secretion in the striatum were also significantly increased compared with the PQ-dyed group, and the protein content of PI3K and PDK-1 and the phosphorylation level of AKT were reduced in parallel with the reduction in the expression of microglia and related inflammatory factors. In conclusion, our results suggest that Tau may regulate microglia-mediated inflammatory responses through inhibition of the PI3K/AKT pathway in the midbrain of PD mice, thereby reducing DAc neurons damage.

Mechanism Study of Thermally Induced Anti-Tumor Drug Loading to Engineered Human Heavy-Chain Ferritin Nanocages Aided by Computational Analysis.

Diverse drug loading approaches for human heavy-chain ferritin (HFn), a promising drug nanocarrier, have been established. However, anti-tumor drug loading ratio and protein carrier recovery yield are bottlenecks for future clinical application. Mechanisms behind drug loading have not been elaborated. In this work, a thermally induced drug loading approach was introduced to load anti-tumor drug doxorubicin hydrochloride (DOX) into HFn, and 2 functionalized HFns, HFn-PAS-RGDK, and HFn-PAS. Optimal conditions were obtained through orthogonal tests. All 3 HFn-based proteins achieved high protein recovery yield and drug loading ratio. Size exclusion chromatography (SEC) and transmission electron microscopy (TEM) results showed the majority of DOX loaded protein (protein/DOX) remained its nanocage conformation. Computational analysis, molecular docking followed by molecular dynamic (MD) simulation, revealed mechanisms of DOX loading and formation of by-product by investigating non-covalent interactions between DOX with HFn subunit and possible binding modes of DOX and HFn after drug loading. In in vitro tests, DOX in protein/DOX entered tumor cell nucleus and inhibited tumor cell growth.

Development of purification process for dual-function recombinant human heavy-chain ferritin by the investigation of genetic modification impact on conformation.

Ferritin is a promising drug delivery platform and has been functionalized through genetic modifications. This work has designed and expressed a dual-functional engineered human heavy-chain ferritin (HFn) with the inserted functional peptide PAS and RGDK to extend half-life and improve tumor targeted drug delivery. A facile and cost-effective two-step purification pathway for recombinant HFn was developed. The genetic modification was found to affect HFn conformation, and therefore varied the purification performance. Heat-acid precipitation followed by butyl fast flow hydrophobic interaction chromatography (HIC) has been developed to purify HFn and modified HFns. Nucleic acid removal reached above 99.8% for HFn and modified HFns. However, HFn purity reached above 95% and recovery yield (overall) above 90%, compared with modified HFns purity above 82% and recovery yield (overall) above 58%. It is interesting to find that the inserted functional peptides significantly changed the molecule conformation, where a putative turnover of the E-helix with the inserted functional peptides formed a "flop" conformation, in contrast with the "flip" conformation of HFn. It could be the cause of fragile stability of modified HFns, and therefore less tolerant to heat and acid condition, observed by the lower recovery yield in heat-acid precipitation.

Immunogenicity study of engineered ferritins with C- and N-terminus insertion of Epstein-Barr nuclear antigen 1 epitope.

Human ferritin heavy chain, an example of a protein nanoparticle, has recently been used as a vaccine delivery platform. Human ferritin has advantages of uniform architecture, robust thermal and chemical stabilities, and good biocompatibility and biodegradation. There is however a lack of understanding about the relationship between insertion sites in ferritin (N-terminus and C-terminus) and the corresponding humoral and cell-mediated immune responses. To bridge this gap, we utilized an Epstein-Barr Nuclear Antigen 1 (EBNA1) epitope as a model to produce engineered ferritin-based vaccines E1F1 (N-terminus insertion) and F1E1 (C-terminus insertion) for the prevention of Epstein-Barr virus (EBV) infections. X-ray crystallography confirmed the relative positions of the N-terminus insertion and C-terminus insertion. For N-terminus insertion, the epitopes were located on the exterior surface of ferritin, while for C-terminus insertion, the epitopes were inside the ferritin cage. Based on the results of antigen-specific antibody titers from in-vivo tests, we found that there was no obvious difference on humoral immune responses between N-terminus and C-terminus insertion. We also evaluated splenocyte proliferation and memory lymphocyte T cell differentiation. Both results suggested C-terminus insertion produced a stronger proliferative response and cell-mediated immune response than N-terminus insertion. C-terminus insertion of EBNA1 epitope was also processed more efficiently by dendritic cells (DCs) than N-terminus insertion. This provides new insight into the relationship between the insertion site and immunogenicity of ferritin nanoparticle vaccines.

Engineered Human Heavy-Chain Ferritin with Half-Life Extension and Tumor Targeting by PAS and RGDK Peptide Functionalization.

Ferritin, one of the most investigated protein nanocages, is considered as a promising drug carrier because of its advantageous stability and safety. However, its short half-life and undesirable tumor targeting ability has limited its usage in tumor treatment. In this work, two types of functional peptides, half-life extension peptide PAS, and tumor targeting peptide RGDK (Arg-Gly-Asp-Lys), are inserted to human heavy-chain ferritin (HFn) at C-terminal through flexible linkers with two distinct enzyme cleavable sites. Structural characterizations show both HFn and engineered HFns can assemble into nanoparticles but with different apparent hydrodynamic volumes and molecular weights. RGDK peptide enhanced the internalization efficiency of HFn and showed a significant increase of growth inhibition against 4T1 cell line in vitro. Pharmacokinetic study in vivo demonstrates PAS peptides extended ferritin half-life about 4.9 times in Sprague Dawley rats. RGDK peptides greatly enhanced drug accumulation in the tumor site rather than in other organs in biodistribution analysis. Drug loaded PAS-RGDK functionalized HFns curbed tumor growth with significantly greater efficacies in comparison with drug loaded HFn.

TWIST2 and the PPAR signaling pathway are important in the progression of nonalcoholic steatohepatitis.

BACKGROUND: To investigate the roles of the transcription factors twist family bHLH transcription factor 1 (TWIST1), twist family bHLH transcription factor 2 (TWIST2), and peroxisome proliferator activated receptor gamma (PPARγ) in the progression of nonalcoholic steatohepatitis. METHODS: The protein levels of TWIST1, TWIST2 and PPARγ were determined in the serum of nonalcoholic fatty liver disease (NAFLD) patients and healthy controls by enzyme-linked immunosorbent assay (ELISA). An in vivo model for fatty liver was established by feeding C57BL/6 J mice a high-fat diet (HFD). An in vitro model of steatosis was established by treating LO-2 cells with oleic acid (OA). RNA sequencing was performed on untreated and OA-treated LO-2 cells followed by TWIST1, TWIST2 and PPARγ gene mRNA levels analysis, Gene Ontology (GO) enrichment and pathway analysis. RESULTS: The TWIST2 serum protein levels decreased significantly in all fatty liver groups (P < 0.05), while TWIST1 varied. TWIST2 tended to be lower in mice fed an HFD and was significantly lower at 3 months. Similarly, in the in vitro model, the TWIST2 protein level was downregulated significantly at 48 and 72 h after OA treatment. RNA sequencing of LO-2 cells showed an approximately 2.3-fold decrease in TWIST2, with no obvious change in TWIST1 and PPARγ. The PPAR signaling pathway was enriched, with 4 genes upregulated in OA-treated cells (P = 0.0018). The interleukin (IL)-17 and tumor necrosis factor (TNF) signaling pathways were enriched in OA-treated cells. CONCLUSIONS: The results provide evidence that the TWIST2 and PPAR signaling pathways are important in NAFLD and shed light on a potential mechanism of steatosis.

A Population-Based Analysis of Mucoepidermoid Carcinoma of the Oral Cavity.

OBJECTIVES: To identify survival outcomes for patients with mucoepidermoid carcinoma (MEC) of the oral cavity and the effects of different prognostic factors on survival. STUDY DESIGN: Retrospective cohort study using a national database. METHODS: Retrospective, population-based cohort study of patients in the Surveillance, Epidemiology and End Results (SEER) database who were diagnosed with MEC of the oral cavity from 1973 to 2016. Overall survival (OS) and disease-specific survival (DSS) were calculated. RESULTS: A total of 1693 patients with MEC of the oral cavity were included. Of those, there are 696 males and 997 females, the average age at diagnosis being 52.4 years. The vast majority of cases (86.4%) presented with stage I and stage II disease. A total of 206 patients had received both surgical and radiation therapy (RT), while 1338 patients just had undergone only surgery and 149 with no treatment. On multivariate analysis, advanced age, stage, and histologic grade were associated with worse OS and DSS. Surgical therapy was an independent favorable predictor of OS and DSS. For radiotherapy, multivariate analysis showed that it was associated with worse DSS, whereas there was no significant difference in OS. CONCLUSION: MEC of the oral cavity is associated with a generally favorable prognosis. Advanced age, stage, and histologic grade were independent negative prognostic factors for survival, and surgery was the main treatment to improve survival. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E857-E863, 2021.

3D bioprinting of cell-laden electroconductive MXene nanocomposite bioinks.

MXenes, a new family of burgeoning two-dimensional (2D) transition metal carbides/nitrides, have been extensively explored in recent years owing to their outstanding properties such as a large specific surface area, high electrical conductivity, low toxicity, and biodegradability. Numerous efforts have been devoted to exploring MXenes for various biomedical applications such as cancer therapy, bioimaging, biosensing, and drug delivery. However, the potential application of MXene nanosheets in tissue engineering has been almost overlooked despite their excellent performance in other biomedical applications. The overarching goal of this paper is to demonstrate the potential of MXene cell-laden bioinks for tissue engineering and their ability to assemble functional scaffolds to regenerate damaged tissue via 3D bioprinting. We formulate a new electroconductive cell-laden bioink composed of Ti3C2 MXene nanosheets dispersed homogeneously within hyaluronic acid/alginate (HA/Alg) hydrogels and showed its performance for extrusion-based 3D bioprinting. The prepared hydrogel bioinks with MXenes display excellent rheological properties, which allows the fabrication of multilayered 3D structures with high resolution and shape retention. Moreover, the introduction of Ti3C2 MXene nanosheets within the HA/Alg hydrogel introduces electrical conductivity to the ink, addressing the poor electrical conductivity of the current bioinks that mismatch with the physico-chemical properties of tissue. In addition, the MXene nanocomposite ink with encapsulated Human Embryonic Kidney 293 (HEK-293) cells displayed high cell viability (>95%) in both bulk hydrogel and 3D bioprinted structures. These results suggest that MXene nanocomposite bioinks and their 3D bioprinting with high electrical conductivity, biocompatibility and degradability can synergize some new applications for tissue and neural engineering.

New withanolides from Physalis minima and their cytotoxicity against A375 human melanoma cells.

Seven previously undescribed withanolides, namely physaminilide A-G (1-7), and two artificial withanolides (8-9), along with 10 known analogues (10-19) were isolated from Physalis minima. The structures were established by spectroscopic analysis, including NMR and electronic circular dichroism (ECD) data. Cytotoxicity of all the isolates was evaluated against A375 human melanoma cells. Compounds 2, 5, 8, 10, 11 and 15 exhibited significant cytotoxic activities with IC50 values in the range of 1.2-9.4 µM.

Withapubesides A-D: natural inducible nitric oxide synthase (iNOS) inhibitors from Physalis pubescens.

Four new steroid glycosides, withapubesides A-D (1-4), were isolated from the stems of Physalis pubescens L. Their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2 were deduced by single-crystal X-ray diffraction and ECD data analysis, respectively. Compound 3 has shown significant inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages with an IC50 value of 12.8 µM and moderate cytostatic activity against human carcinoma cells (786-O, C4-2B, 22Rvl, A375 and A375S2) with IC50 values in the range of 3.05-9.47 µM. Molecular docking simulation demonstrated that 3 is bound in the inducible nitric oxide synthase (iNOS) active site heme pocket very well, which suggests that 3 might be a candidate for the development of iNOS inhibitors.

Comprehensive characterization and identification of antioxidants in Folium Artemisiae Argyi using high-resolution tandem mass spectrometry.

Antioxidants from natural sources, such as vegetables and fruits, are attracting more and more interest. In this work, we evaluated the antioxidant potential of Folium Artemisia Argyi, a traditional Chinese herb medicine and food supplement. The total phenolic content, total flavonoid content, and antioxidant ability of the crude extracts and fractions obtained from consecutively partition of n-hexane, ethyl acetate, and n-butanol were measured and compared. Ethyl acetate fraction shows the highest total phenolic and flavonoid contents and highest antioxidant capability with regard to DPPH, ABTS, superoxide anion free radical scavenging ability, and ferric-reducing antioxidant power. In addition, the potential antioxidant components were screened by DPPH-UHPLC-MS experiments and subsequently characterized by using high-resolution tandem mass spectrometry. This work finally identified 45 antioxidants, including organic acids, phenolic compounds, flavonoids, and methoxylated flavonoids. The results suggested that Folium Artemisiae Argyi is a potential inexpensive resource of natural antioxidants.

Tailored temozolomide therapy according to MGMT methylation status for elderly patients with acute myeloid leukemia.

Temozolomide sensitivity is determined by methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) promoter. This study assessed whether the temozolomide dose can be tailored by MGMT promoter status and whether protracted, low-dose temozolomide can "prime" blasts in patients with unmethylated MGMT (unMGMT). Elderly patients with high-risk AML were stratified by MGMT methylation. Patients with methylated MGMT (mMGMT) received temozolomide 200 mg/m(2) orally for 7 days every 4 weeks, while patients with unMGMT received temozolomide 100 mg/m(2) orally for 14 days followed by 200 mg/m(2) orally for 7 days every 6weeks. Of 36 patients (median age, 75 years), 31 (86%) had an unMGMT promoter. Overall response rate for the entire cohort was 36%. Patients with mMGMT and unMGMT had similar response rates (40% vs. 29%). Median duration of response and overall survival (OS) among responders were 29 and 35 weeks, respectively. Induction deaths (ID) occurred in 25% of patients, mostly caused by disease progression. Hematological toxicities were the most common adverse event. Toxicities were similar between patients on conventional versus protracted schedules. High HCT-CI scores were predictive of lower CR rate, higher ID, and shorter OS, while bone marrow blast count <50% at screening predicted for improved responses. Temozolomide, dosed according to MGMT methylation status, demonstrated modest clinical activity in elderly patients with AML, especially in those presenting with fewer comorbidities and low disease burden. The trial was registered on www.ClinicalTrials.gov as #NCT00611247.