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.

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.

Comparative pharmacognosy and secondary metabolite analysis of Balanophorae herbs from different sources.

The Balanophorae are not only traditional Chinese herbal medicines but also functional foods with diverse sources. This study aimed to distinguish pharmacognostic characteristics and secondary metabolites among different species of Balanophorae. Eight species of Balanophorae herbs were harvested, including 21 batches with 209 samples. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used to analyze secondary metabolites of Balanophorae from 21 sources. Targeted metabolomic analysis was performed to compare differences among the groups. Rhopalocnemis phalloide and B. indica can be identified by their pharmacognostic characteristics. Then, 41 secondary metabolites were identified or characterized in the mixed extracts of the 209 samples, mainly phenolic acids, flavonoids, and their derivatives. The distribution of these secondary metabolites revealed apparent differences among different species. In addition, targeted metabolomic analysis suggested that the secondary metabolite profiles of seven species of Balanophorae showed noticeable differences, and differences were also observed among different growing regions. Finally, five important metabolic markers were screened to successfully distinguish B. laxiflora, B. harlandii, and B. polyandra, including three phenolic acids and two flavonoids. This is the first study to systematically compare both the morphology and secondary metabolites among different sources of Balanophorae, which could provide effective information for identifying diverse species.

A study on the impact of open source metaverse immersive teaching method on emergency skills training for medical undergraduate students.

BACKGROUND: In recent years, the traditional simulation-based medical teaching approach has faced challenges in meeting the requirements of practical emergency medicine education. This study utilized open-source tools and software to develop immersive panoramic videos using virtual reality technology for emergency medical teaching. It aims to investigate the efficacy of this novel teaching methodology. This transformation shifted the focus from physical simulation to virtual simulation in medical education, establishing a metaverse for emergency medical teaching. METHODS: In accordance with the curriculum guidelines, the instructors produced panoramic videos demonstrating procedures such as spinal injury management, humeral fracture with abdominal wall intestinal tube prolapse, head and chest composite injuries, cardiopulmonary resuscitation, and tracheal intubation. Using Unity software, a virtual training application for bronchoscopy was developed and integrated into the PICO4 VR all-in-one device to create a metaverse teaching environment. Fourth-year medical undergraduate students were allocated into either an experimental group (n = 26) or a control group (n = 30) based on student IDs. The experimental group received instruction through the metaverse immersive teaching method, while the control group followed the traditional simulation-based medical teaching approach. Both groups participated in theoretical and practical lessons as usual. Subsequently, all students underwent a four-station Objective Structured Clinical Examination (OSCE) to assess the effectiveness of the teaching methods based on their performance. Additionally, students in the experimental group provided subjective evaluations to assess their acceptance of the new teaching approach. RESULTS: Before the training commenced, there were no significant statistical differences in the first aid test scores between the experimental and control groups. Following the training, the experimental group outperformed the control group in the four-station OSCE examination, with all P-values being less than 0.05. The satisfaction rate among the experimental group regarding the new teaching method reached 88.46%, reflecting levels of satisfaction and extreme satisfaction. CONCLUSION: The open-source metaverse immersive teaching method has demonstrated a positive impact on enhancing the emergency skills of medical undergraduate students, with a high level of acceptance among students. In comparison to traditional simulated medical teaching methods, this approach requires less time and space, incurring lower costs, and is deemed worthy of wider adoption.

Neuronal miR-138 Represses HSV-2 Lytic Infection by Regulating Viral and Host Genes with Mechanistic Differences from HSV-1.

Herpes simplex virus 2 (HSV-2) establishes latent infection in dorsal root ganglion (DRG) neurons after productive (lytic) infection in peripheral tissues. A neuron-specific microRNA, miR-138, favors HSV-1 latency by repressing viral ICP0 and host Oct-1 and Foxc1 genes, yet the role of miR-138 in HSV-2 infection was unknown. The ICP0 mRNAs of HSV-1, HSV-2, and chimpanzee herpesvirus each have one to two canonical miR-138 binding sites. The sites are 100% conserved in 308 HSV-1 and 300 HSV-2 published sequences of clinical isolates. In cotransfection assays, miR-138 repressed HSV-2 ICP0 expression through the seed region and surrounding interactions that are different from HSV-1. An HSV-2 mutant with disrupted miR-138 binding sites on ICP0 showed increased ICP0 expression in Neuro-2a cells. Photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation confirmed miR-138 binding to HSV-2 ICP0 and identified UL19 and UL20 as additional targets whose expression was repressed by miR-138 during cotransfection. In Neuro-2a cells, transfected miR-138 and its antagomir decreased and increased HSV-2 replication, respectively, and a knockout experiment showed that miR-138's host targets OCT-1 and FOXC1 were important for HSV-2 replication. In primary mouse DRG neurons, both ICP0 and FOXC1 positively regulated HSV-2 replication, but both overexpressed and endogenous miR-138 suppressed HSV-2 replication primarily by repressing ICP0 expression. Thus, miR-138 can suppress HSV-2 neuronal replication through multiple viral and host pathways. These results reveal functional similarities and mechanistic differences in how miR-138 regulates HSV-1 and HSV-2 infection and indicate an evolutionary advantage of using miR-138 to repress lytic infection in neurons. IMPORTANCE HSV-1 and HSV-2 are closely related viruses with major differences. Both viruses establish latency in neurons from which they reactivate to cause disease. A key aspect of HSV latency is repression of productive infection in neurons. Based on previous work with HSV-1, we investigated the role of a neuron-specific microRNA, miR-138, in HSV-2 infection and established it as a repressor of HSV-2 productive infection in neuronal cells. This repression is mediated mainly by targeting viral ICP0 and host Foxc1 mRNAs, but other pathways also contribute. Despite functional conservation of the role of miR-138 between HSV-1 and HSV-2, many molecular mechanisms differ, including how miR-138 represses ICP0 expression and miR-138 targeting of HSV-2 but not HSV-1 UL19 and UL20. To our knowledge, this study provides the first example of host microRNA regulation of HSV-2 infection.

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.

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.

Ferroptosis Related Immunomodulatory Effect of a Novel Extracellular Polysaccharides from Marine Fungus Aureobasidium melanogenum.

Marine fungi represent an important and sustainable resource, from which the search for novel biological substances for application in the pharmacy or food industry offers great potential. In our research, novel polysaccharide (AUM-1) was obtained from marine Aureobasidium melanogenum SCAU-266 were obtained and the molecular weight of AUM-1 was determined to be 8000 Da with 97.30% of glucose, 1.9% of mannose, and 0.08% galactose, owing to a potential backbone of α-D-Glcp-(1→2)-α-D-Manp-(1→4)-α-D-Glcp-(1→6)-(SO3-)-4-α-D-Glcp-(1→6)-1-ß-D-Glcp-1→2)-α-D-Glcp-(1→6)-ß-D-Glcp-1→6)-α-D-Glcp-1→4)-α-D-Glcp-6→1)-[α-D-Glcp-4]26→1)-α-D-Glcp and two side chains that consisted of α-D-Glcp-1 and α-D-Glcp-(1→6)-α-D-Glcp residues. The immunomodulatory effect of AUM-1 was identified. Then, the potential molecular mechanism by which AUM-1 may be connected to ferroptosis was indicated by metabonomics, and the expression of COX2, SLC7A11, GPX4, ACSL4, FTH1, and ROS were further verified. Thus, we first speculated that AUM-1 has a potential effect on the ferroptosis-related immunomodulatory property in RAW 264.7 cells by adjusting the expression of GPX4, regulated glutathione (oxidative), directly causing lipid peroxidation owing to the higher ROS level through the glutamate metabolism and TCA cycle. Thus, the ferroptosis related immunomodulatory effect of AUM-1 was obtained.

Sex Differences of Cardiolipin in Tissue Distribution Based on Targeted Lipidomic Analysis by UHPLC-QTOF-MS/MS.

Cardiolipins (CLs) are involved in ATP production, mitochondria biogenesis, apoptosis and mitophagy. Their tissue distribution can provide insight into the function of mitochondria and related diseases. However, the reports on tissue distribution of CLs remain limited. In this research, CLs were identified from heart, liver, kidney, spleen, lung, skeletal muscle, and brain using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS). Then, the distribution and sex difference of CLs in seven tissues were compared by a targeted lipidomic approach. A total of 88 CLs were identified, of which 58, 51, 57, 58, 50, 61 and 52 CLs were found in heart, liver, kidney, spleen, lung, skeletal muscle, and brain, respectively. Compared with the distribution of CLs in heart, liver, kidney, and skeletal muscle, the CLs in spleen, lung, and brain showed significant differences. Moreover, the results indicated that there were sex differences of CLs in liver and kidney. A total of 16 CLs in liver tissue and 21 CLs in kidney tissue, with significant sex differences, were screened. Our findings in the targeted lipidomic analysis demonstrated that tissue distribution of CLs was essential in the dynamic states and sex differences of CLs, which might provide evidence for the mitochondrial-related mechanism under physiological and pathological conditions.

Characterization of a carboxyl methyltransferase in Fusarium graminearum provides insights into the biosynthesis of fusarin A.

Fusarium graminearum is a major fungal pathogen that causes a series of devastating crop diseases by producing a variety of mycotoxins. Fusarins are a class of polyketide-nonribosomal peptide hybrids. In Fusarium mycotoxins, a variable 2-pyrrolidone ring conjugates with a polyene chain substituted with a methyl ester moiety. The enzymatic route through which fusarin A, a major member of the fusarin family with a characteristic tetrohydrofuran-coupled pyrrolidone ring, is formed in F. graminearum has not been established. By targeting the final step in the biosynthesis of fusarin A, we report here an S-adenosyl methionine-dependent carboxyl methyltransferase responsible for the formation of the methyl ester moiety by in vivo gene inactivation, isolation and characterization of a key fusarin intermediate, and in vitro biochemical characterization. Related findings provide insights into the poorly understood biosynthetic pathway of fusarin A. Additionally, bioactivity assays demonstrate that the methyl ester is necessary for fusarin cytotoxicity.

Correction: Characterization of a carboxyl methyltransferase in Fusarium graminearum provides insights into the biosynthesis of fusarin A.

Correction for 'Characterization of a carboxyl methyltransferase in Fusarium graminearum provides insights into the biosynthesis of fusarin A' by Qian Yang et al., Org. Biomol. Chem., 2021, DOI: 10.1039/d1ob01010g.

Follow-up study on serum cholesterol profiles and potential sequelae in recovered COVID-19 patients.

BACKGROUND: COVID-19 patients develop hypolipidemia. However, it is unknown whether lipid levels have improved and there are potential sequlae in recovered patients. OBJECTIVE: In this follow-up study, we evaluated serum lipidemia and various physiopathological laboratory values in recovered patients. METHODS: A 3-6 month follow-up study was performed between June 15 and September 3, 2020, to examine serum levels of laboratory values in 107 discharged COVID-19 patients (mild = 59; severe/critical = 48; diagnoses on admission). Sixty-one patients had a revisit chest CT scan. A Wilcoxon signed-rank test was used to analyze changes in laboratory values at admission and follow-up. RESULTS: LDL-c and HDL-c levels were significantly higher at follow-up than at admission in severe/critical cases (p <  0.05). LDL-c levels were significantly higher at follow-up than at admission in mild cases (p <  0.05). Coagulation and liver functional values were significantly improved at follow-up than at admission for patients (p <  0.05). Increases in HDL-c significantly correlated with increases in numbers of white blood cells (p <  0.001) during patients' recovery. With exclusion of the subjects taking traditional Chinese medicines or cholesterol-lowering drugs, LDL-c and HDL-c levels were significantly increased at follow-up than at admission in severe/critical cases (p <  0.05). Residue lesions were observed in CT images in 72% (44 of 61) of follow-up patients. CONCLUSIONS: Improvements of LDL-c, HDL-c, liver functions, and incomplete resolution of lung lesions were observed at 3-6 month follow-up for recovered patients, indicating that a long-term recovery process could be required and the development of sequelae such as pulmonary fibrosis could be expected in some patients.

Fecal occult blood and urinary cytology tests for rapid screening of inflammatory infection in the gastrointestinal and urological systems in patients with Coronavirus disease 2019.

BACKGROUND: Gastrointestinal infections (GI) and urological infections (UI) have not been fully addressed in COVID-19 patients. We aimed to evaluate the values of routine fecal occult blood (FOB) test and urinary cytology test (UCT) for screening of GI and UI in COVID-19 patients. METHODS: In this retrospective study, COVID-19 patients without associated comorbidities were divided into FOB- or UCT-positive or FOB- or UCT-negative groups. Their clinical characteristics and laboratory findings were then compared. RESULTS: A total of 13.6% of patients (47 of 345) tested positive for FOB, and 57.4% (27 of 47) of these patients lacked gastrointestinal symptoms. A total of 30.1% of patients (104 of 345) exhibited gastrointestinal symptoms, and 38.0% (131 of 345) were positive for either FOB or gastrointestinal symptoms. FOB-positive patients possessed significantly higher levels of C-reactive protein and fewer lymphocytes than FOB-negative patients. A total of 36.9% of patients (80 of 217) exhibited positive UCT, and 97.5% (78 of 80) of these patients possessed normal levels of serum markers for renal injuries. Significant differences in age and sex ratios were observed between the UCT-positive and UCT-negative groups, and 72.4% (42 of 58) of female patients over 60 years old were UCT-positive. CONCLUSIONS: Fecal occult blood test in combination with gastrointestinal symptoms could serve as a simple and useful screening approach for GI diagnoses for COVID-19. Age and sex are risk factors for UI in COVID-19 patients. UCT could be a sensitive tool for assessing early UI at a stage in which serum markers for renal injuries appear normal.

Multi-Omics Analysis Reveals Anti-Staphylococcus aureus Activity of Actinomycin D Originating from Streptomyces parvulus.

Staphylococcus aureus (S. aureus) is a common pathogen that causes various serious diseases, including chronic infections. Discovering new antibacterial agents is an important aspect of the pharmaceutical field because of the lack of effective antibacterial drugs. In our research, we found that one anti-S. aureus substance is actinomycin D, originating from Streptomyces parvulus (S. parvulus); then, we further focused on the anti-S. aureus ability and the omics profile of S. aureus in response to actinomycin D. The results revealed that actinomycin D had a significant inhibitory activity on S. aureus with a minimum inhibitory concentration (MIC) of 2 µg/mL and a minimum bactericidal concentration (MBC) of 64 µg/mL. Bacterial reactive oxygen species (ROS) increased 3.5-fold upon treatment with actinomycin D, as was measured with the oxidation-sensitive fluorescent probe DCFH-DA, and H2O2 increased 3.5 times with treatment by actinomycin D. Proteomics and metabolomics, respectively, identified differentially expressed proteins in control and treatment groups, and the co-mapped correlation network of proteomics and metabolomics annotated five major pathways that were potentially related to disrupting the energy metabolism and oxidative stress of S. aureus. All findings contributed to providing new insight into the mechanisms of the anti-S. aureus effects of actinomycin D originating from S. parvulus.

[Analysis of medication characteristics of traditional Chinese medicine in treating COVID-19 based on data mining].

OBJECTIVE: To analysis the medication characteristics of the prescriptions issued via open channel by the National and Provincial Health Committee and the State Administration of Traditional Chinese Medicine in treating coronavirus disease 2019 (COVID-19). METHODS: We collected the data of traditional Chinese medicine related to treatment plans published by the National and Provincial Health Committee and the State Administration of Traditional Chinese Medicine from the start of COVID-19 outbreak to February 19, 2020. The frequency analysis, cluster analysis and association analysis were performed. RESULTS: The study collected 4 national and 34 regional prevention and treatment plans, 578 items, 84 traditional Chinese formulations, 60 Chinese patent medicines, and 230 Chinese herbs. The high frequently used herbs were Liquorice, Scutellariabaicalensis, Semen armeniacaeamarae, and Gypsum. The commonly used traditional formulations included Maxing Shigan decoction, Yin Qiao powder, and Xuanbai Chengqi decoction. The Chinese patent drugs included Angong Niuhuang pill, Xuebijing injection, and Lianhua Qingwen capsule. The most common paired medications were Ephedra and Semen armeniacaeamarae, Fructusforsythiae and Liquorice. Two core combinations and one novel formula were discovered in the study. CONCLUSIONS: Yin Qiao powder and Huopo Xialing decoction are the basic formulations for Weifen syndrome of COVID-19. In addition, Maxing Shigan decoction, Liang Ge powder, Qingwen Baidu decoction and Da Yuan decoction are the basic formulations for Qifen syndrome of COVID-19. The main medication characteristics are clearing heat, entilating lung, removing toxicity and removing turbidity. It shows that removing toxicity and eliminating evil are the prescription thought in treating epidemic disease of traditional Chinese medicine.

Hypervalent iodine catalysis enabled iterative multi-fluorination: not just a simple alternative for the electrochemical approach.

Unlike the well-established fluoroalkylation, the direct incorporation of multiple fluorine atoms into small molecules via iterative fluorinations has been much less investigated. Herein, we report a hypervalent iodine catalytically selective multi-fluorination in which the fluorination degree is controlled by the delicate balance between the HF/amine ratios. The pros and cons of hypervalent iodine catalysis and the previously established electrochemical approach in the iterative multi-fluorinations are also provided.