Force-enhanced sensitive and specific detection of DNA-intercalative agents directly from microorganisms at single-molecule level.

Microorganisms can produce a vast array of bioactive secondary metabolites, including DNA-intercalating agents like actinomycin D, doxorubicin, which hold great potential for cancer chemotherapy. However, discovering novel DNA-intercalating compounds remains challenging due to the limited sensitivity and specificity of conventional activity assays, which require large-scale fermentation and purification. Here, we introduced the single-molecule stretching assay (SMSA) directly to microbial cultures or extracts for discovering DNA-intercalating agents, even in trace amounts of microbial cultures (5 µl). We showed that the unique changes of dsDNA in contour length and overstretching transition enable the specific detection of intercalators from complex samples without the need for extensive purification. Applying force to dsDNA also enhanced the sensitivity by increasing both the binding affinity Ka and the quantity of ligands intercalation, thus allowing the detection of weak intercalators, which are often overlooked using traditional methods. We demonstrated the effectiveness of SMSA, identified two DNA intercalator-producing strains: Streptomyces tanashiensis and Talaromyces funiculosus, and isolated three DNA intercalators: medermycin, kalafungin and ligustrone B. Interestingly, both medermycin and kalafungin, classified as weak DNA intercalators (Ka ∼103 M-1), exhibited potent anti-cancer activity against HCT-116 cancer cells, with IC50 values of 52 ± 6 and 70 ± 7 nM, respectively.

The total xanthones extracted from Gentianella acuta alleviates HFpEF by activating the IRE1α/Xbp1s pathway.

Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome characterized by pulmonary and systemic congestion resulting from left ventricular diastolic dysfunction and increased filling pressure. Currently, however, there is no evidence on effective pharmacotherapy for HFpEF. In this study, we aimed to investigate the therapeutic effect of total xanthones extracted from Gentianella acuta (TXG) on HFpEF by establishing an high-fat diet (HFD) + L-NAME-induced mouse model. Echocardiography was employed to assess the impact of TXG on the cardiac function in HFpEF mice. Haematoxylin and eosin staining, wheat germ agglutinin staining, and Masson's trichrome staining were utilized to observe the histopathological changes following TXG treatment. The results demonstrated that TXG alleviated HFpEF by reducing the expressions of genes associated with myocardial hypertrophy, fibrosis and apoptosis. Furthermore, TXG improved cardiomyocyte apoptosis by inhibiting the expression of apoptosis-related proteins. Mechanistic investigations revealed that TXG could activate the inositol-requiring enzyme 1α (IRE1α)/X-box-binding protein 1 (Xbp1s) signalling pathway, but the knockdown of IRE1α using the IRE1α inhibitor STF083010 or siRNA-IRE1α impaired the ability of TXG to ameliorate cardiac remodelling in HFpEF models. In conclusion, TXG alleviates myocardial hypertrophy, fibrosis and apoptosis through the activation of the IRE1α/Xbp1s signalling pathway, suggesting its potential beneficial effects on HFpEF patients.

Molecular Networking-Guided Discovery of a New Antitumor Pyranonaphthoquinone from Streptomyces tanashiensis DSM 731: Insights from Single-Molecule Stretching Assays.

Guided by molecular networking based on single-molecule stretching assay, an unprecedented pyranonaphthoquinone, methyl kalafunginate (1) and five known compounds 2-6 were isolated from Streptomyces tanashiensis DSM 731. Compound 1 was characterized through a combination of spectroscopic techniques, including 1D and 2D NMR analysis, ECD calculation, and X-ray crystallography. Interestingly, we discovered that compound 1 was spontaneously converted from kalafungin (4) in methanol solution. All isolated compounds were assessed for their cytotoxic potential against a panel of five human cancer cell lines: A549, HepG2, BxPC-3, SW620, and C4-2B. Compounds 1, 2, 4, and 5 exhibited remarkable cytotoxicity with IC50 values below 2.382 µM, suggesting their potential as promising anticancer agents. These findings highlight the significance of using a combined approach of single-molecule stretching assays and molecular networking for efficiently discovering novel natural products with potential therapeutic applications.

The matrix protein of respiratory syncytial virus suppresses interferon signaling via RACK1 association.

IMPORTANCE: Respiratory syncytial virus (RSV) matrix (M) protein is indispensable for virion assembly and release. It is localized to the nucleus during early infection to perturb host transcription. However, the function of RSV M protein in other cellular activities remains poorly understood. In this study, several interferon response-associated host factors, including RACK1, were identified by proteomic analysis as RSV M interactors. Knockdown of RACK1 attenuates RSV-restricted IFN signaling leading to enhanced host defense against RSV infection, unraveling a role of M protein in antagonizing IFN response via association with RACK1. Our study uncovers a previously unrecognized mechanism of immune evasion by RSV M protein and identifies RACK1 as a novel host factor recruited by RSV, highlighting RACK1 as a potential new target for RSV therapeutics development.

The prevalence of disability in older adults with multimorbidity: a meta-analysis.

BACKGROUND: Disability is typically correlated with lower quality of life and decreased capacity for self-care. It has been demonstrated that multimorbidity is closely linked to a variety of unfavorable events, such as disability. Researchers are still figuring out how and to what extent co-morbidities impact disability, though. In order to fill up this gap, this study examines the prevalence and contributing variables of disability in older patients who have multimorbidity. METHODS: We conducted a systematic search of Pubmed, Cochrane Library, Web of Science, Embase, and CINAL databases for articles from their inception until September 2023. We selected co-morbid older adults aged > 60 years and used the ADL scale or any scale that assesses disability as an assessment tool. We excluded literature that did not meet the criteria, and literature that could not be included in the data we needed. We extracted data from the included literature and calculated synthetic prevalence rates, ORs, and 95% confidence intervals. RESULTS: A total of 32 papers (71,135 older adults) were included in the study. The prevalence of disability among older patients with multimorbidity was around 34.9% (95% CI = 25.8-43.9%). Subgroup analysis showed higher rates of disability among comorbidities who were older, female, unmarried, and long-term users of health services. And the incidence of disability increased each year. Meanwhile, the regions of the United States, China, and Spain showed higher rates of disability. CONCLUSIONS: Disability rates in older patients with multimorbidity are higher, thus it's critical to focus on risk factors while fully accounting for regional variances.

KLF6 aggravates myocardial fibrosis by promoting mitochondrial division.

The dysregulation of mitochondrial dynamics in cardiac fibroblasts (CFs) is closely linked to myocardial fibrosis, which can induce cardiac dysfunction and even lead to heart failure. As an essential multifunctional zinc-finger transcriptional factor of cardiovascular remodeling, the role of KLF6 mediating the link between mitochondrial fission and myocardial fibrosis remains unclear. Next, we want to explore whether the effect of KLF6 on mitochondrial fission might influence cardiac fibroblasts, we established a model of Transforming growth factor ß1 (TGF-ß1) and Isoprenaline (ISO)-induced myocardial fibrosis. Here, we found that KLF6 up-regulation in CFs is correlated with myocardial fibrosis. While knockdown of KLF6 suppresses mitochondrial fission and the Keap1/Nrf2 pathway molecules, which alleviates myocardial fibrosis induced by TGF-ß1. Our findings not only clarified the regulation mechanism of mitochondrial fission by KLF6 but also provided a potential therapeutic target for cardiovascular disease.

High-yield production of FK228 and new derivatives in a Burkholderia chassis.

FK228 (romidepsin) is the only natural histone deacetylases (HDACs) inhibitor approved by FDA to treat cutaneous and peripheral T-cell lymphoma. However, the limited supply and severe cardiotoxicity of FK228 underscore the importance to develop an effective synthetic biology platform for the manufacturing and fine-tuning of this drug lead. In this work, we constructed a Burkholderia chassis for the high-yield production of FK228-family (unnatural) natural products. By virtue of the optimized Burkholderia-specific recombineering system, the biosynthetic gene cluster (BGC) encoding the FK228-like skeleton thailandepsins (tdp) in Burkholderia thailandensis E264 was replaced with an attB integration site to afford the basal chassis KOGC1. The tdp BGC directly captured from E264 was hybridized with the FK228-encoding BGC (dep) using the versatile Red/ET technology. The hybrid BGC (tdp-dep) was integrated into the attB site of KOGC1, resulting in the heterologous expression of FK228. Remarkably, the titer reached 581 mg/L, which is 30-fold higher than that of native producer Chromobacterium violaceum No. 968. This success encouraged us to further engineer the NRPS modules 4 or 6 of hybrid tdp-dep BGC by domain units swapping strategy, and eight new FK228 derivatives (1-8) varying in the composition of amino acids were generated. Especially, the titers of 2 and 3 in KOGC1 were up to 985 mg/L and 453 mg/L, respectively. 2 and 3 displayed stronger cytotoxic activity than FK228. All in all, this work established a robust platform to produce FK228 and its new derivatives in sufficient quantities for anticancer drug development.

Genome-driven discovery of new serrawettin W2 analogues from Serratia fonticola DSM 4576.

By expressing a multimodular NRPS gene sefA from Serratia fonticola DSM 4576 in E. coli, four new serrawettin W2 analogues, namely sefopeptides A-D (1-4), were isolated and structurally characterized and their biosynthesis was proposed. A bioactivity assay showed that sefopeptide C (3) exhibits moderate cytotoxic activity against acute promyelocytic leukemia NB4 cells.

Diterpenoids from Streptomyces: Structures, Biosyntheses and Bioactivities.

Bacteria, especially Streptomyces spp., have emerged as a rich source for natural diterpenoids with diverse structures and broad bioactivities. Here, we review diterpenoids biosynthesized by Streptomyces, with an emphasis on their structures, biosyntheses, and bioactivities. Although diterpenoids from Streptomyces are relatively rare compared to those from plants and fungi, their novel skeletons, biosyntheses and bioactivities present opportunities for discovering new drugs, enzyme mechanisms, and applications in biocatalysis and metabolic pathway engineering.

Isolation, complete genome sequencing and in silico genome mining of Burkholderia for secondary metabolites.

Recent years, Burkholderia species have emerged as a new source of natural products (NPs) with increasing attractions. Genome mining suggests the Burkholderia genomes include many natural product biosynthetic gene clusters (BGCs) which are new targets for drug discovery. In order to collect more Burkholderia, here, a strain S-53 was isolated from the soil samples on a mountain area in Changde, P.R. China and verified by comparative genetic analysis to belong to Burkholderia. The complete genome of Burkholderia strain S-53 is 8.2 Mbps in size with an average G + C content of 66.35%. Its taxonomy was both characterized by 16S rRNA- and whole genome-based phylogenetic trees. Bioinformatic prediction in silico revealed it has a total of 15 NP BGCs, some of which may encode unknown products. It is expectable that availability of these BGCs will speed up the identification of new secondary metabolites from Burkholderia and help us understand how sophisticated BGC regulation works.

IDO1 can impair NK cells function against non-small cell lung cancer by downregulation of NKG2D Ligand via ADAM10.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the rate-limiting step in tryptophan catabolism along the kynurenine (Kyn) pathway and exerts immunosuppressive properties mainly via activation of transcription factor aryl hydrocarbon receptor (AhR) pathway. IDO1 induces NK cells dysfunction via downregulation of the activating receptor NKG2D on NK cells, but whether and how it affects the expression of NKG2D Ligand (NKG2DL) on tumor cells remains unclear. Since a disintegrin and metalloprotease 10 (ADAM10) plays a potential role in the shedding of NKG2DL and the releasing of soluble NKG2DL (sNKG2DL), we investigated how IDO1 modulates the expression of NKG2DL via ADAM10 in non-small cell lung cancer (NSCLC). We found that IDO1 expression was negatively correlated with NKG2DL expression while positively correlated with ADAM10 expression with human lung cancer brain metastasis tissue, NSCLC cells and LLC tumor-bearing mice. IDO1 could regulate ADAM10 expression via IDO1-Kyn-AhR signaling pathway and subsequently regulate NKG2DL expression. IDO1 deficiency led to retarded tumor growth and improved NK cells function in NSCLC mice. IDO1 inhibitors improved NK cells function in vitro and in vivo. The combo of IDO1 inhibitor and NK cells exhibited more therapeutic efficacy than either of the single IDO1 inhibitor or NK cells treatment.

ITGB1 Suppresses Autophagy Through Inhibiting The mTORC2/AKT Signaling Pathway In H9C2 Cells.

Cardiomyocyte autophagy is closely related to myocardial infarction and hypertrophy. To study the molecular mechanism of autophagy is helpful for the prevention and treatment of these diseases. As a cell surface receptor, the function of ITGB1 gene in cardiomyocyte autophagy is not clear. The purpose of this research was to investigate the function and molecular mechanism of ITGB1 on autophagy. The autophagy-related marker proteins and signaling molecules were detected using western blot with knockdown and overexpression of ITGB1 in H9C2 cells. The results suggested that ITGB1 could inhibit autophagy and the mTORC2/Akt pathway molecules. To further investigate whether the effect of ITGB1 on autophagy might affect myocardial hypertrophy, we constructed AngII induced H9C2 cells and TAC induced rats models. The results showed that ITGB1 inhibited myocardial hypertrophy in both H9C2 cells and heart tissues of disease model. These data highlight the regulation mechanism on autophagy by ITGB1 and the potential usefulness of the gene as a potential target for preventing heart disease.

Comparative genomics with evolutionary lineage in Streptomyces bacteria reveals high biosynthetic potentials.

Genome mining in silico approaches allow scientists to proficiently evaluate the genomic potency of secondary bioactive chemical producers and find new bioactive compounds in different bacteria. Streptomyces is one of the most ubiquitous bacterial genera in the environments, and well-known as prolific producers of diverse and valuable natural products (NPs) with significant biological activities. Mining and prioritizing of NP biosynthetic gene clusters (BGCs) would be the most important stage in the identification of novel compounds. Comparative genomics and genetic similarity network analysis of 62 Streptomyces public reference genomes demonstrated that individuals of these species exhibit a huge number of distinct NP BGCs, the most of which are cryptic and unconnected to any reported NPs with high phylogenetic variation among individuals. It was assumed that substantial heterogeneity across the varieties of species of Streptomyces drives outstanding biosynthetic and metabolic potential, making them plausible candidates for the identification of novel molecules.

Impact of MtrA on phosphate metabolism genes and the response to altered phosphate conditions in Streptomyces.

Phosphate metabolism is known to be regulated by the PhoPR regulatory system in Streptomyces and some other bacteria. In this study, we report that MtrA also regulates phosphate metabolism in Streptomyces. Our data showed that, in Streptomyces coelicolor, MtrA regulates not only phosphate metabolism genes such as phoA but also phoP under different phosphate conditions, including growth on rich complex media without added inorganic phosphate and on defined media with low or high concentrations of inorganic phosphate. Cross-regulation was also observed among mtrA, phoP and glnR under these conditions. We demonstrated both in vitro and in vivo binding of MtrA to the promoter regions of genes associated with phosphate metabolism and to the intergenic region between phoR and phoU, indicating that these phosphate metabolism genes are targets of MtrA. We further showed that MtrA in S. lividans and S. venezuelae has detectable regulatory effects on expression of phosphate metabolism genes. Additionally, the MtrA homologue from Corynebacterium glutamicum bound predicted MtrA sites of multiple phosphate metabolism genes, implying its potential for regulating phosphate metabolism in this species. Overall, our findings support MtrA as a major regulator for phosphate metabolism in Streptomyces and also potentially in other actinobacteria.

Genome-Guided Discovery of Highly Oxygenated Aromatic Polyketides, Saccharothrixins D-M, from the Rare Marine Actinomycete Saccharothrix sp. D09.

Angucyclines and angucyclinones are aromatic polyketides with intriguing structures and therapeutic value. Genome mining of the rare marine actinomycete Saccharothrix sp. D09 led to the identification of a type II polyketide synthase biosynthetic gene cluster, sxn, which encodes several distinct subclasses of oxidoreductases, implying that this strain has the potential to produce novel polycyclic aromatic polyketides with unusual redox modifications. The "one strain-many compounds" (OSMAC) strategy and comparative metabolite analysis facilitated the discovery of 20 angucycline derivatives from the D09 strain, including six new highly oxygenated saccharothrixins D-I (1-6), four new glycosylated saccharothrixins J-M (7-10), and 10 known analogues (11-20). Their structures were elucidated based on detailed HRESIMS, NMR spectroscopic, and X-ray crystallographic analysis. With the help of gene disruption and heterologous expression, we proposed their plausible biosynthetic pathways. In addition, compounds 3, 4, and 8 showed antibacterial activity against Helicobacter pylori with MIC values ranging from 16 to 32 µg/mL. Compound 3 also revealed anti-inflammatory activity by inhibiting the production of NO with an IC50 value of 28 µM.

Effect of galectin-3 in the pathogenesis of arteriovenous fistula stenosis formation.

OBJECTIVE: This study sought to investigate the effect of local expression of galectin-3 in the development of stenotic arteriovenous fistula (AVF). METHODS: We collected stenotic venous tissues, adjacent nonstenotic venous tissues, and blood samples from end-stage renal disease (ESRD) patients with AVF stenosis, while normal venous tissues and blood samples were collected from ESRD patients before AVF creation as controls. Also blood samples were collected from ESRD patients with nonstenosis functional AVF. Galectin-3, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-9 (MMP-9), and α-SMA expression in the venous tissues were examined by immunohistochemistry, and the ERK1/2 pathway activity in the intima was accessed by western blot. Serum galectin-3 level was measured by ELISA. Thereafter, human pulmonary arterial smooth muscle cells (HPASMCs) were cultured in vitro, and the interaction between Galectin-3 and ERK1/2 pathway in HPASMCs was estimated by western blot. RESULTS: ESRD patients with stenotic AVF had a significant higher serum galectin-3 level than normal controls, and patients with non-stenotic functional AVF. The expression levels of galectin-3, phosphorylated ERK1/2, PCNA, MMP-9, and α-SMA in the stenotic venous tissues were higher than that in the normal venous tissues or the adjacent nonstenotic AVF venous tissues. Correlation analysis showed that the expression of galectin-3 of the neointima was positively correlated with PCNA and α-SMA in the stenotic AVF venous tissues. In HPASMCs, galectin-3 can increase the activity of phosphorylated ERK1/2 and promote the expression of α-SMA. CONCLUSION: In the stenotic AVF of ESRD patients, expression of the galectin-3 was significantly increased, showing a positive relation with neointima development.

Strategies for Natural Products Discovery from Uncultured Microorganisms.

Microorganisms are highly regarded as a prominent source of natural products that have significant importance in many fields such as medicine, farming, environmental safety, and material production. Due to this, only tiny amounts of microorganisms can be cultivated under standard laboratory conditions, and the bulk of microorganisms in the ecosystems are still unidentified, which restricts our knowledge of uncultured microbial metabolism. However, they could hypothetically provide a large collection of innovative natural products. Culture-independent metagenomics study has the ability to address core questions in the potential of NP production by cloning and analysis of microbial DNA derived directly from environmental samples. Latest advancements in next generation sequencing and genetic engineering tools for genome assembly have broadened the scope of metagenomics to offer perspectives into the life of uncultured microorganisms. In this review, we cover the methods of metagenomic library construction, and heterologous expression for the exploration and development of the environmental metabolome and focus on the function-based metagenomics, sequencing-based metagenomics, and single-cell metagenomics of uncultured microorganisms.

Genome Mining of Pseudomonas Species: Diversity and Evolution of Metabolic and Biosynthetic Potential.

Microbial genome sequencing has uncovered a myriad of natural products (NPs) that have yet to be explored. Bacteria in the genus Pseudomonas serve as pathogens, plant growth promoters, and therapeutically, industrially, and environmentally important microorganisms. Though most species of Pseudomonas have a large number of NP biosynthetic gene clusters (BGCs) in their genomes, it is difficult to link many of these BGCs with products under current laboratory conditions. In order to gain new insights into the diversity, distribution, and evolution of these BGCs in Pseudomonas for the discovery of unexplored NPs, we applied several bioinformatic programming approaches to characterize BGCs from Pseudomonas reference genome sequences available in public databases along with phylogenetic and genomic comparison. Our research revealed that most BGCs in the genomes of Pseudomonas species have a high diversity for NPs at the species and subspecies levels and built the correlation of species with BGC taxonomic ranges. These data will pave the way for the algorithmic detection of species- and subspecies-specific pathways for NP development.

Gentianella acuta prevents acute myocardial infarction induced by isoproterenol in rats via inhibition of galectin-3/TLR4/MyD88/NF-кB inflammatory signalling.

Gentianella acuta (G. acuta), as a folk medicine, was used to treat heart disease by the Ewenki people in Inner Mongolia. However, the effect of G. acuta on acute myocardial infarction (AMI) is not clear. To explore the mechanisms of G. acuta on isoproterenol (ISO)-induced AMI, rats were administered G. acuta for 28 days, then injected intraperitoneally with ISO (85 mg/kg) on days 29 and 30. An electrocardiogram helped to evaluate the myocardial injury. Serum lactate dehydrogenase (LDH), creatinine kinase (CK) and aspartate aminotransferase (AST) levels were evaluated, and haematoxylin eosin, Masson's trichrome staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining were used to detect myocardial histological changes. Radioimmunoassay was used to measure serum tumour necrosis factor alpha (TNFα) and interleukin (IL)-6. An enzyme-linked immunosorbent assay kit was used to analyse serum galectin-3 (Gal-3) levels. Immunohistochemistry, Western blotting and reverse transcription polymerase chain reaction were used to examine relevant molecular events. The results revealed that pre-treatment with G. acuta decreased the elevation in the ST segment; reduced serum LDH, CK and AST levels; alleviated cardiac structure disorder; and reduced inflammatory infiltration, abnormal collagen deposition and cardiomyocyte apoptosis that were induced by ISO. Furthermore, pre-treatment with G. acuta inhibited serum Gal-3 levels and Gal-3 expression in heart tissue, and also impeded TLR4/MyD88/NF-кB signalling activation, which ultimately prevented the expression of inflammatory cytokines. The study indicated that pre-treatment with G. acuta protects against ISO-induced AMI, and the protective role may be related to inhibiting Gal-3/TLR4/MyD88/NF-кB inflammatory signalling.

Genomics-Driven Activation of Silent Biosynthetic Gene Clusters in Burkholderia gladioli by Screening Recombineering System.

The Burkholderia genus possesses ecological and metabolic diversities. A large number of silent biosynthetic gene clusters (BGCs) in the Burkholderia genome remain uncharacterized and represent a promising resource for new natural product discovery. However, exploitation of the metabolomic potential of Burkholderia is limited by the absence of efficient genetic manipulation tools. Here, we screened a bacteriophage recombinase system Redγ-BAS, which was functional for genome modification in the plant pathogen Burkholderia gladioli ATCC 10248. By using this recombineering tool, the constitutive promoters were precisely inserted in the genome, leading to activation of two silent nonribosomal peptide synthetase gene clusters (bgdd and hgdd) and production of corresponding new classes of lipopeptides, burriogladiodins A-H (1-8) and haereogladiodins A-B (9-10). Structure elucidation revealed an unnatural amino acid Z- dehydrobutyrine (Dhb) in 1-8 and an E-Dhb in 9-10. Notably, compounds 2-4 and 9 feature an unusual threonine tag that is longer than the predicted collinearity assembly lines. The structural diversity of burriogladiodins was derived from the relaxed substrate specificity of the fifth adenylation domain as well as chain termination conducted by water or threonine. The recombinase-mediating genome editing system is not only applicable in B. gladioli, but also possesses great potential for mining meaningful silent gene clusters from other Burkholderia species.