Transcriptome investigation on the multicellular behavior of Bacillus velezensis Bs916 anchoring surfactin.

AIMS: To provide valuable information for a comprehensive understanding of the multicellular behavior of Bacillus velezensis Bs916 regulated by surfactin and other natural signals by Transcriptome. METHODS AND RESULTS: Transcriptomics revealed a distinct effect on gene expression alterations caused by disruption of the surfactin gene cluster(Δsrf) and 100 µg/ml surfactin addition(Δsrf + SRF). A total of 1573 differential expression genes were identified among Bs916, Δsrf, and Δsrf + SRF and grouped into eight categories based on their expression profiles. RT-qPCR analysis of 30 candidate genes showed high consistency with those of transcriptome. Additionally, the expression of eight candidate genes regulated by surfactin in a dose-dependent manner was revealed by lacZ fusion. Based on the above evidence, we proposed that surfactin can act as an extracellular signal for monitoring biofilm formation in Bs916 by directly regulating the expression of AbrB, DegS-degU, and SinI-SinR, and indirectly regulating the phosphorylation of ComA and Spo0A. CONCLUSIONS: The biofilm of Δsrf was unable to restore significantly by surfactin addition, combined inclusion of surfactin (SRF), exopolysaccharide (EPS), and γ-poly-dl-glutamic acid (γ-PGA), results in significant restoration of Δsrf biofilm formation, thereby a preliminary model was presented about the molecular mechanism by which the signaling molecule surfactin regulates Bs916 multicellular behavior.

Focused ultrasound: a novel therapy for improving vaginal microecology in patients with high-risk HPV infection.

OBJECTIVES: To investigate changes in vaginal microecology in women with high-risk human papillomavirus (HR-HPV) infection after focused ultrasound (FU) treatment. MATERIALS AND METHODS: We collected vaginal secretions at the time of admission and 3 months after FU treatment from 169 women who received FU treatment for cervical HR-HPV infection between July 2020 and September 2022. Among them, there were 101 patients with cute vaginitis, we also collected their vaginal secretions after one week of drug treatment. These samples were evaluated for vaginal microecology and HPV-DNA examination. RESULTS: Of the 169 patients, 101 (59.7%) suffered from acute vaginitis at the time of admission. After one week of targeted antibiotics drug treatment, there were no pathogens or pus cells on the field of microscopic vision, but there was no significant difference(p > 0.05) in the diversity and density of vaginal flora, the proportion and function of Lactobacillus (H2O2 negative rate) between one week after treatment and at the time of admission. At the time of admission of the 169 patients, the normal flora rate was 40.3%, which increased to 93.5% three months after FU treatment. The differences in vaginal secretion parameters at the time of admission and 3 months after FU treatment were as follows: H2O2 negative rate (37.3% vs. 3.6%), leukocyte esterase positive rate (54.4% vs. 5.9%), sialidase positive rate (38.5% vs. 4.1%), bacterial vaginitis positive rate (55% vs. 4.7%), fungal vaginitis positive rate (44.4% vs. 5.9%), and trichomonal vaginitis positive rate (7.1% vs. 0). The difference was statistically significant (p < 0.01). The pH value and Nagent score at the time of admission were significantly higher than those three months after FU. Three months after FU, the positive rate of HPV was 5.8% in the group of patients with normal vaginal microecology at the time of admission and post-FU; it was 6.7% in the group of patients with abnormal vaginal microecology at the time of admission and normal vaginal microecology post-FU; and it was 100% in the group patients with abnormal vaginal microecology at the time of admission and post-FU. A significant difference was observed among the three groups (p < 0.01). CONCLUSION: FU is an effective treatment for patients with cervical HR-HPV infection. FU does not interfere with the vaginal microecology of HR-HPV positive patients with normal vaginal microecology.FU followed by antibiotic drug therapy for pathogens is beneficial to restore the function of Lactobacillus vaginalis in HR-HPV positive patients with acute vaginitis,so as to improve the vaginal microecology of HR-HPV positive patients with abnormal vaginal microecology.

Comparison of pregnancy outcomes in infertile patients with different types of adenomyosis treated with high-intensity focused ultrasound.

OBJECTIVE: This study assessed the improvement of symptoms and pregnancy outcomes in infertile patients with various types of adenomyosis who were treated with high-intensity focused ultrasound (HIFU). MATERIALS AND METHODS: Between October 2017 and January 2022, 129 infertile patients with adenomyosis who wished to conceive were treated with HIFU. Based on the relationship between the adenomyotic lesion, the endometrium, and the subserosa of the uterus on magnetic resonance imaging, the adenomyotic lesions were divided into internal, external, intramural, and full-thickness types. Menstruation pain score, menstruation blood volume score, anti-Müllerian hormone (AMH) levels, reproductive results, pregnancy and delivery complications, and other clinical variables were compared among these four groups. RESULTS: Patients with external adenomyosis had the greatest menstrual distress, whereas patients with internal adenomyosis had the greatest menstrual blood volume. Dysmenorrhea and heavy menstruation were significantly improved after HIFU treatment in all groups. AMH levels were not significantly different before and six months after HIFU. Of the 129 patients, 50 (38.7%) became pregnant after HIFU, and patients with internal adenomyosis had the highest pregnancy rate. Patients with adenomyotic lesions located in the posterior wall of the uterus had a higher pregnancy rate than those with lesions located in the fundus of the uterus. CONCLUSIONS: The classification of adenomyosis is closely related to distinctions in clinical symptoms and pregnancy outcomes. Infertile patients with different types of adenomyosis could be effectively treated with HIFU. HIFU can be considered as an option for infertile patients with adenomyosis who want to maintain their fertility.

Enhancing surfactin production in B. velezensis Bs916 combined cumulative mutagenesis and expression key enzymes.

Surfactin is a lipopeptide which has attracted massive attention due to its versatile bioactive properties, although it has less commercial application due to its low yield in wild strains. The B. velezensis Bs916 has enable commercial production of surfactin due to its outstanding capacity to synthesize lipopeptides and amenable to genetically engineering. In this study, 20 derivatives with high surfactin production were obtained firstly by transposon mutagenesis and knockout techniques, and the surfactin yield of the derivative H5 (△GltB) was increased approximately 7-folds, reaching to 1.48 g/L. The molecular mechanism of high yielding surfactin in △GltB was investigated by the transcriptomic and KEGG pathway analysis. The results indicated that △GltB enhanced its ability to synthesize surfactin mainly by promoting transcription of the srfA gene cluster and inhibiting degradation of some key precursors such as fatty acid. Secondly, we obtained a triple mutant derivative BsC3 by cumulative mutagenesis of the negative genes GltB, RapF, and SerA, and it could increase the surfactin titer by twofold, reaching to 2.98 g/L. Thirdly, we achieved overexpression of two key rate-limiting enzyme genes, YbdT, and srfAD, and the derivative BsC5 which further increased the surfactin titer by 1.3-fold, reaching to 3.79 g/L. Finally, the yield of surfactin by derivatives was significantly increased under the optimal medium, particularly the BsC5 increased the surfactin titer to 8.37 g/L. To the best of our knowledge, this is one of the highest yields that have been reported. Our work may pave way for large scale production of surfactin by B. velezensis Bs916. KEY POINTS: • Elucidation of the molecular mechanism of surfactin high-yielding transposon mutant. • Genetically engineering of B. velezensis Bs916 surfactin titer to 8.37 g/L for large scale preparation.

Genome Sequencing and Genetic Engineering Reveal the Contribution of Bacitracin Produced by Bacillus paralicheniformis CPL618 to Anti-Staphylococcus aureus Activity.

Staphylococcus aureus is one of the important pathogens causing human diseases, especially its treatment has great challenges due to its resistance to methicillin and vancomycin. The Bacillus strains are known to be major sources of second metabolites that can function as drugs. Therefore, it is of great value to excavate metabolites with good inhibitory activity against S. aureus from Bacillus strains. In this study, a strain Bacillus paralicheniformis CPL618 with good antagonistic activity against S. aureus was isolated and genome analysis showed that the size was 4,447,938 bp and contained four gene clusters fen, bac, dhb, and lch which are potentially responsible for four cyclic peptides fengycin, bacitracin, bacillibactin, and lichenysin biosynthesis, respectively. These gene clusters were knockout by homologous recombination. The bacteriostatic experiment results showed that the antibacterial activity of ∆bac decreased 72.3% while Δfen, Δdhb, and ΔlchA did not significantly changed as that of wild type. Interestingly, the maximum bacitracin yield was up to 92 U/mL in the LB medium, which was extremely unusual in wild type strains. To further improve the production of bacitracin, transcription regulators abrB and lrp were knocked out, the bacitracin produced by ΔabrB, Δlrp, and ΔabrB + lrp was 124 U/mL, 112 U/mL, and 160 U/ml, respectively. Although no new anti-S. aureus compounds was found by using genome mining in this study, the molecular mechanisms of high yield of bacitracin and anti-S. aureus in B. paralicheniformis CPL618 were clarified. Moreover, B. paralicheniformis CPL618 was further genetically engineered for industrial production of bacitracin.

Selective Inhibition of Lysine-Specific Demethylase 5A (KDM5A) Using a Rhodium(III) Complex for Triple-Negative Breast Cancer Therapy.

Lysine-specific demethylase 5A (KDM5A) has recently become a promising target for epigenetic therapy. In this study, we designed and synthesized metal complexes bearing ligands with reported demethylase and p27 modulating activities. The Rh(III) complex 1 was identified as a direct, selective and potent inhibitor of KDM5A that directly abrogate KDM5A demethylase activity via antagonizing the KDM5A-tri-/di-methylated histone 3 protein-protein interaction (PPI) in vitro and in cellulo. Complex 1 induced accumulation of H3K4me3 and H3K4me2 levels in cells, causing growth arrest at G1 phase in the triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and 4T1. Finally, 1 exhibited potent anti-tumor activity against TNBC xenografts in an in vivo mouse model, presumably via targeting of KDM5A and hence upregulating p27. Moreover, complex 1 was less toxic compared with two clinical drugs, cisplatin and doxorubicin. To our knowledge, complex 1 is the first metal-based KDM5A inhibitor reported in the literature. We anticipate that complex 1 may be used as a novel scaffold for the further development of more potent epigenetic agents against cancers, including TNBC.

A rhodium(III)-based inhibitor of autotaxin with antiproliferative activity.

BACKGROUND: Cancer of the skin is by far the most common of all cancers. Melanoma accounts for only about 1% of skin cancers but causes a large majority of skin cancer deaths. Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), regulates physiological and pathological functions of lysophosphatidic acid (LPA), and is thus an important therapeutic target. METHODS: We synthesized ten metal-based complexes and a novel cyclometalated rhodium(III) complex 1 was identified as an ATX enzymatic inhibitor using multiple methods, including ATX enzymatic assay, thermal shift assay, western immunoblotting and so on. RESULTS: Protein thermal shift assays showed that 1 increased the melting temperature (Tm) of ATX by 3.5°C. 1 also reduced ATX-LPA mediated downstream survival signal pathway proteins such as ERK and AKT, and inhibited the activation of the transcription factor nuclear factor κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3). 1 also exhibited strong anti-proliferative activity against A2058 melanoma cells (IC50=0.58µM). Structure-activity relationship indicated that both the rhodium(III) center and the auxiliary ligands of complex 1 are important for bioactivity. CONCLUSIONS: 1 represents a promising scaffold for the development of small-molecule ATX inhibitors for anti-tumor applications. To our knowledge, complex 1 is the first metal-based ATX inhibitor reported to date. GENERAL SIGNIFICANCE: Rhodium complexes will have the increased attention in therapeutic and bioanalytical applications.

In silico identification of natural product inhibitors of JAK2.

Emodic acid (1) and 6-chloroemodic acid (2) have been identified from a natural product database as useful scaffolds for the future development of novel JAK2 inhibitors using structure-based high-throughput virtual screening. Low-energy binding conformations of 1 and 2 in the JAK2 PTK domain were generated by virtual ligand docking and were found to overlap considerably with the binding pose of CMP6, a known JAK2 inhibitor. Compounds 1 and 2 displayed low micromolar efficacies against JAK2 enzyme activity and JAK2 autophosphorylation in human erythroleukemia cells, and inhibited STAT3 DNA-binding activity in a human hepatocarcinoma cell line.

Discovery of deoxyvasicinone derivatives as inhibitors of NEDD8-activating enzyme.

NEDD8-activating enzyme (NAE) controls the specific degradation of proteins regulated by cullin-RING ubiquitin E3 ligases, and has been considered as an attractive molecular target for the development of drugs against cancer. A pharmacophore model constructed from a training set of deoxyvasicinone derivatives was used to screen 376 compounds from an analogue database. From the initial screening, the valine-linked deoxyvasicinone derivative 9 and the N-isopropyl-linked deoxyvasicinone derivative 10 emerged as the top scoring candidates. Compounds 9 and 10 showed micromolar potencies in both cell-free and cell-based systems, with selectivity for NAE over the related enzymes SAE and UAE. Molecular modelling analysis suggested that 9 and 10 may inhibit NAE by blocking the ATP-binding domain. Thus, these deoxyvasicinone derivatives could be considered as promising lead molecules for the development of more potent inhibitors of NAE.

Discovery of a small-molecule inhibitor of STAT3 by ligand-based pharmacophore screening.

STAT3 modulates the transcription of a wide variety of regulatory genes involved in cell proliferation, differentiation, migration, apoptosis, and other critical cellular functions. Constitutive activation of STAT3 has been detected in a wide spectrum of human malignancies. A pharmacophore model constructed from a training set of STAT3 inhibitors binding to the SH2 domain was used to screen an in-house database of compounds, from which azepine 1 emerged as a top candidate. Compound 1 inhibited STAT3 DNA-binding activity in vitro and attenuated STAT3-directed transcription in cellulo with comparable potency to the well-known STAT3 inhibitor S3I-201. A fluorescence polarization assay revealed that compound 1 targeted the SH2 domain of STAT3. Furthermore, compound 1 inhibited STAT3 phosphorylation in cells without affecting the total expression of STAT3. This study also validates the use of pharmacophore modeling to identify inhibitors of protein-protein interactions.

A highly sensitive G-quadruplex-based luminescent switch-on probe for the detection of polymerase 3'-5' proofreading activity.

We report herein a luminescent switch-on label-free G-quadruplex-based assay for the rapid and sensitive detection of polymerase proofreading activity using a novel iridium(III) complex as a G-quadruplex-selective probe. The interaction of the iridium(III) complex with the G-quadruplex motif facilitates the highly sensitive switch-on detection of polymerase proofreading activity. Using T4 DNA polymerase (T4 pol) as a model enzyme, the assay achieved high sensitivity and selectivity for T4 pol over other tested enzymes.

Label-free luminescent oligonucleotide-based probes.

Breakthrough advances in chemistry and biology over the last two decades have vastly expanded the repertoire of nucleic acid structure and function with potential application in multiple areas of science and technology, including sensing and analytical applications. DNA oligonucleotides represent popular tools for the development of sensing platforms due to their low cost, rich structural polymorphism, and their ability to bind to cognate ligands with sensitivity and specificity rivaling those for protein enzymes and antibodies. In this review, we give an overview of the "label-free" approach that has been a particular focus of our group and others for the construction of luminescent DNA-based sensing platforms. The label-free strategy aims to overcome some of the drawbacks associated with the use of covalently-labeled oligonucleotides prevalent in electrochemical and optical platforms. Label-free DNA-based probes harness the selective interaction between luminescent dyes and functional oligonucleotides that exhibit a "structure-switching" response upon binding to analytes. Based on the numerous examples of label-free luminescent DNA-based probes reported recently, we envisage that this field would continue to thrive and mature in the years to come.

Antagonizing STAT3 dimerization with a rhodium(III) complex.

Kinetically inert metal complexes have arisen as promising alternatives to existing platinum and ruthenium chemotherapeutics. Reported herein, to our knowledge, is the first example of a substitutionally inert, Group 9 organometallic compound as a direct inhibitor of signal transducer and activator of transcription 3 (STAT3) dimerization. From a series of cyclometalated rhodium(III) and iridium(III) complexes, a rhodium(III) complex emerged as a potent inhibitor of STAT3 that targeted the SH2 domain and inhibited STAT3 phosphorylation and dimerization. Significantly, the complex exhibited potent anti-tumor activities in an in vivo mouse xenograft model of melanoma. This study demonstrates that rhodium complexes may be developed as effective STAT3 inhibitors with potent anti-tumor activity.

Oxyimperatorin attenuates LPS-induced microglial activation in vitro and in vivo via suppressing NF-κB p65 signaling.

BACKGROUND: Microglia-mediated neuroinflammation is an important pathological feature in many neurological diseases; thus, suppressing microglial activation is considered a possible therapeutic strategy for reducing neuronal damage. Oxyimperatorin (OIMP) is a member of furanocoumarin, isolated from the medicinal herb Glehnia littoralis. However, it is unknown whether OIMP can suppress the neuroinflammation. PURPOSE: To investigate the neuroprotective activity of oxyimperatorin (OIMP) in LPS-induced neuroinflammation in vitro and in vivo models. METHODS: In vitro inflammation-related assays were performed with OIMP in LPS-induced BV-2 microglia. In addition, intraperitoneal injection of LPS-induced microglial activation in the mouse brain was used to validate the anti-neuroinflammatory activity of OIMP. RESULTS: OIMP was found to suppress LPS-induced neuroinflammation in vitro and in vivo. OIMP significantly attenuated LPS-induced the production of free radicals, inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokines in BV-2 microglia without causing cytotoxicity. In addition, OIMP could reduce the M1 pro-inflammatory transition in LPS-stimulated BV-2 microglia. The mechanistic study revealed that OIMP inhibited LPS-induced NF-κB p65 phosphorylation and nuclear translocation. However, OIMP did not affect LPS-induced IκB phosphorylation and degradation. In addition, OIMP also was able to reduce LPS-induced microglial activation in mice brain. CONCLUSION: Our findings suggest that OIMP suppresses microglia activation and attenuates the production of pro-inflammatory mediators and cytokines via inhibition of NF-κB p65 signaling.

Quercetin exerts anti-tumor immune mechanism by regulating IL-6/JAK2/STAT3 signaling pathway to deplete Treg cells.

Breast cancer is still the leading cause of death among women worldwide. Due to the lack of effective drug targets, triple-negative breast cancer has a worse prognosis and higher mortality compared with other types of breast cancer, and chemotherapy is still the main treatment for triple-negative breast cancer at present. Quercetin (QUE) is a flavonoid compound found in a variety of fruits and vegetables. The mechanism of QUE has been extensively studied, such as prostate cancer, colon cancer, ovarian cancer, etc. However, the anti-tumor immune mechanism of QUE in triple-negative breast cancer remains unclear. Therefore, we assessed the anti-tumor immune effects of QUE on triple-negative breast cancer using both 4T1 cells and a xenograft mouse model of 4T1 cells. In vitro, we examined the inhibitory effects of QUE on 4T1 cells and its molecular mechanisms through MTT, Transwell, ELISA, and Western blotting. In vivo, by establishing a xenograft mouse model, we utilized flow cytometry, immunohistochemistry, ELISA, and Western blotting to evaluate the anti-tumor immune effects of QUE on triple-negative breast cancer. The results indicate that QUE inhibits the proliferation, migration, and invasion of 4T1 cells, concurrently significantly suppressing the IL-6/JAK2/STAT3 signaling pathway. Furthermore, it depletes Treg cell content in 4T1 xenograft mice, thereby improving the tumor immune microenvironment and promoting the cytotoxicity of relevant tumor immune cells. These findings suggest that QUE may serve as a potential adjuvant for immune therapy in triple-negative breast cancer.

Progress in the Preparation and Application of Inulin-Based Hydrogels.

Inulin, a natural polysaccharide, has emerged as a promising precursor for the preparation of hydrogels due to its biocompatibility, biodegradability, and structural versatility. This review provides a comprehensive overview of the recent progress in the preparation, characterization, and diverse applications of inulin-based hydrogels. Different synthesis strategies, including physical methods (thermal induction and non-thermal induction), chemical methods (free-radical polymerization and chemical crosslinking), and enzymatic approaches, are discussed in detail. The unique properties of inulin-based hydrogels, such as stimuli-responsiveness, antibacterial activity, and their potential as fat replacers, are highlighted. Special emphasis is given to their promising applications in drug delivery systems, especially for colon-targeted delivery, due to the selective degradation of inulin via colonic microflora. The ability to incorporate both hydrophilic and hydrophobic drugs further expands their therapeutic potential. In addition, the applications of inulin-based hydrogels in responsive materials, the food industry, wound dressings, and tissue engineering are discussed. While significant progress has been achieved, challenges and prospects in optimizing synthesis, improving mechanical properties, and exploring new functionalities are discussed. Overall, this review highlights the remarkable properties of inulin-based hydrogels as a promising class of biomaterials with immense potential in the biomedical, pharmaceutical, and materials science fields.

Apigenin promotes apoptosis of 4T1 cells through PI3K/AKT/Nrf2 pathway and improves tumor immune microenvironment in vivo.

The 2022 US Cancer Statistics show that breast cancer is one of the most common cancers in women. Epidemiology has shown that adding flavonoids to the diet inhibits cancers that arise in particular women, such as cervical cancer, ovarian cancer, and breast cancer. Although there have been research reports on apigenin (API) and breast cancer, its anti-tumor effect and potential mechanism on breast cancer have not yet been clarified. Therefore, in this study, we used 4T1 cells and a 4T1 xenograft tumor mouse model to investigate the antitumor effect of API on breast cancer and its underlying mechanism. In vitro, we used MTT, transwell, staining, and western blotting to investigate the inhibitory effect of apigenin on 4T1 and the underlying molecular mechanism. In vivo by establishing a xenograft tumor model, using immunohistochemistry, and flow cytometry to study the inhibitory effect of apigenin on solid breast tumors and its effect on the tumor immune microenvironment. The results showed that API can induce breast cancer cell apoptosis through the PI3K/AKT/Nrf2 pathway and can improve the tumor immune microenvironment in mice with breast tumors, thereby inhibiting the growth of breast cancer. Thus, API may be a promising agent for breast cancer treatment.

Recent Advances in Quaternary Ammonium Monomers for Dental Applications.

Resin-based dental materials have been one of the ideal choices among various materials in the treatment of dental caries. However, resin-based dental materials still have some drawbacks, such as the lack of inherent antibacterial activity. Extensive research has been conducted on the use of novel quaternary ammonium monomers (QAMs) to impart antibacterial activity to dental materials. This review provides a comprehensive overview of the recent advances in quaternary ammonium monomers (QAMs) for dental applications. The current progress and limitations of QAMs are discussed based on the evolution of their structures. The functional diversification and enhancement of QAMs are presented. QAMs have the potential to provide long-term antibacterial activity in dental resin composites, thereby prolonging their service life. However, there is a need to balance antibacterial performance with other material properties and the potential impact on the oral microbiome and general health. Finally, the necessity for further scientific progress in the development of novel quaternary ammonium monomers and the optimization of dental resin formulations is emphasized.

In silico screening of quadruplex-binding ligands.

Recent advances in computational processing power and molecular docking algorithms have facilitated the development of computer-aided methods for the rapid and efficient discovery of G-quadruplex-interacting molecules. In this article, we provide an introductory framework for the methodology of in silico screening for the identification of novel DNA G-quadruplex ligands from chemical libraries. We discuss aspects of model construction, database selection and molecular docking techniques, and highlight representative examples from this field. Finally, we offer a perspective on the potential application of in silico techniques for the discovery of RNA G-quadruplex-binding ligands in the future.

Strategies targeting endoplasmic reticulum stress to improve Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder with motor symptoms, which is caused by the progressive death of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Accumulating evidence shows that endoplasmic reticulum (ER) stress occurring in the SNpc DA neurons is an early event in the development of PD. ER stress triggers the activation of unfolded protein response (UPR) to reduce stress and restore ER function. However, excessive and continuous ER stress and UPR exacerbate the risk of DA neuron death through crosstalk with other PD events. Thus, ER stress is considered a promising therapeutic target for the treatment of PD. Various strategies targeting ER stress through the modulation of UPR signaling, the increase of ER's protein folding ability, and the enhancement of protein degradation are developed to alleviate neuronal death in PD models. In this review, we summarize the pathological role of ER stress in PD and update the strategies targeting ER stress to improve ER protein homeostasis and PD-related events.