Discovery of psoralen as a quorum sensing inhibitor suppresses Pseudomonas aeruginosa virulence.

Pseudomonas aeruginosa is a common opportunistic pathogen with growing resistance and presents heightened treatment challenges. Quorum sensing (QS) is a cell-to-cell communication system that contributes to the production of a variety of virulence factors and is also related to biofilm formation of P. aeruginosa. Compared to traditional antibiotics which kill bacteria directly, the anti-virulence strategy by targeting QS is a promising strategy for combating pseudomonal infections. In this study, the QS inhibition potential of the compounds derived from the Traditional Chinese Medicines was evaluated by using in silico, in vitro, and in vivo analyses. The results showed that psoralen, a natural furocoumarin compound derived from Psoralea corylifolia L., was capable of simultaneously inhibiting the three main QS regulators, LasR, RhlR, and PqsR of P. aeruginosa. Psoralen had no bactericidal activity but could widely inhibit the production of extracellular proteases, pyocyanin, and biofilm, and the cell motilities of the model and clinical P. aeruginosa strains. RNA-sequencing and quantitative PCR analyses further demonstrated that a majority of QS-activated genes in P. aeruginosa were suppressed by psoralen. The supplementation of psoralen could protect Caenorhabditis elegans from P. aeruginosa challenge, especially for the hypervirulent strain PA14. Moreover, psoralen showed synergistic antibacterial effects with polymyxin B, levofloxacin, and kanamycin. In conclusions, this study identifies the anti-QS and antibiofilm effects of psoralen against P. aeruginosa strains and sheds light on the discovery of anti-pseudomonal drugs among Traditional Chinese Medicines. KEY POINTS: • Psoralen derived from Psoralea corylifolia L. inhibits the virulence-related phenotypes of P. aeruginosa. • Psoralen simultaneously targets the three core regulators of P. aeruginosa QS system and inhibits the expression of a large part of downstream genes. • Psoralen protects C. elegans from P. aeruginosa challenge and enhances the susceptibility of P. aeruginosa to antibiotics.

Multi-omics-based characterization of the influences of Mycobacterium tuberculosis virulence factors EsxB and PPE68 on host cells.

Mycobacterium tuberculosis, the ancient master of causing tuberculosis, is one of the most successful pathogens capable of persistently colonizing host lungs. The EsxB (CFP-10) of ESX-1 system and PPE68 of the PPE family contribute to the virulence of M. tuberculosis. However, the virulence potential and pathogenetic characteristics of these two proteins during M. tuberculosis infection remain unclear. In this study, two prokaryotic expression plasmids for EsxB or PPE68 of M. tuberculosis were constructed and the recombinant proteins His-EsxB or His-PPE68 were purified. The proteome and transcriptome of MH-S cells treated with His-EsxB or His-PPE68 were explored, followed by validating the expression of the identified differentially expressed genes (DEGs) using quantitative PCR. A total of 159/439 specific proteins or 633/1117 DEGs were obtained between control and His-EsxB or His-PPE68 treated groups in the MH-S proteomes and transcriptomes. Additionally, 37/60 signal pathways were predicted in the His-EsxB or His-PPE68 treated groups and "Cytokine-cytokine receptor interaction" was the most represented pathway. Furthermore, the expression of the DEGs (IL-1ß, IL-6, and TNF-α) was significantly upregulated, suggesting that these DEGs contributed to the host response during EsxB or PPE68 treatment. These findings provide detailed information on developing an effective intervention strategy to control M. tuberculosis infection.

Nonomuraea sediminis sp. nov., a novel actinobacterium with antimicrobial activity, isolated from sediment of Dianchi Lake.

A novel actinobacterium with antimicrobial activity, designated strain H16431T, was isolated from a sediment sample collected from Dianchi Lake, Yunnan Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain H16431T was most closely related to Nonomuraea rhizosphaerae CGMCC 4.7431T and Nonomuraea guangzhouensis CGMCC 4.7101T (98.1% similarity), but formed a monophyletic clade with Nonomuraea ceibae KCTC 39826T (98.0% similarity). Phylogenomic analysis based on whole-genome sequence showed that strain H16431T formed a separate clade within the genus Nonomuraea. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain H16431T and its closely related Nonomuraea species were 80.0-81.5%, 71.2-74.6%, and 23.2-25.0%, respectively, which were significantly lower than the widely accepted species-defined threshold. The DNA G + C content was 70.2% based on the whole-genome sequence. The menaquinones were identified as MK-9(H4), MK-9(H6), and MK-9(H2). The major fatty acids were iso-C16:0, 10 methyl-C17:0, and iso-C16:0 2OH. The phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, and phosphatidylinositol. These chemotaxonomic characteristics were corresponded to those of the genus Nonomuraea. On the basis of the taxonomic evidence, strain H16431T represents a novel species of the genus Nonomuraea, for which the name Nonomuraea sediminis sp. nov. is proposed. The type strain is H16431T (=JCM 34852T=CICC 25119T).

K-12 Science, Technology, Engineering, and Math characteristics and recommendations based on analyses of teaching cases in China.

Science, Technology, Engineering, and Math (STEM) education emphasizes solving problems in authentic contexts and developing 21st-century skills. It also helps to cultivate individuals possessing scientific curiosity and innovative abilities. These capacities align with China's core literacy training. Recent years have seen K-12 STEM cases flourish nationally. However, little attention has been paid to the shared characteristics of these practices, and suggestions for implementing STEM in primary and secondary schools are scarce. This paper presents commonalities in STEM practices within China from a curriculum perspective and offers recommendations for implementation based on these attributes. Specifically, this study first constructed analytical metrics via the Delphi method to assess STEM cases. Next, 51 typical STEM teaching cases in different regions of China were analyzed using these metrics. Based upon the statistical results, five characteristics of STEM cases were summarized: China's STEM education has an unbalanced geographical distribution; current practices benchmark the need for innovative talent training; most instructional content is drawn from real-world problems, but interdisciplinary integration deserves closer focus; the cases featured rich teaching activities and were conducted in a project-based learning fashion with insufficient emphasis on mathematical applications; and China seems to be holistically promoting STEM education, especially through new technologies and supplementary materials. Findings should allow instructors to better understand the intricacies of STEM implementation and to promote successful STEM cases. Recommendations are also provided to optimize the localization of STEM education in China in order to cultivate innovative and interdisciplinary talent.

Sphaerisporangium fuscum sp. nov., Isolated from Sediment of Anmucuo Lake in Tibet Autonomous Region of China.

A novel actinomycete strain, designated H8589T, was isolated from a lake sediment sample, and a polyphasic approach was employed to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene indicated that strain H8589T formed a monophyletic clade within the genus Sphaerisporangium and was most closely related to Sphaerisporangium siamense DSM 45784 T (97.9% similarity) and Sphaerisporangium rufum DSM 46862 T (97.7% similarity). The draft genome had a length of 10,134,050 bp with a G + C content of 71.2%. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain H8589T and its closely related Sphaerisporangium species were 80.6 ~ 83.2%, 73.9 ~ 78.4% and 24.5 ~ 29.0%, respectively, which were significantly lower than the widely accepted species-defined threshold. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. Whole-cell sugars were glucose, ribose and madurose. The menaquinones were MK-9(H4), MK-9(H2), MK-9(H6) and MK-9. The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, unidentified phospholipids and unidentified aminophospholipids. The major fatty acids were identified as iso-C16:0, 10-methyl-C17:0 and C17:0. The results of phenotypic properties, genotypic distinctiveness and chemotaxonomic features indicated that strain H8589T should represent a novel species within the genus Sphaerisporangium, Sphaerisporangium fuscum sp.nov. The type strain is H8589T (= JCM 34848 T = CICC 25115 T).

Acrocarpospora catenulata sp. nov., a novel actinobacterium isolated from lake sediment.

A novel actinobacterium, designated strain H8750T, was isolated from sediment sampled at Lugu Lake, southwest PR China and its polyphasic taxonomy was studied. Strain H8750T produced well-developed substrate mycelium, and formed club-shaped and hooked structures borne on the tip of the aerial mycelia. It contained meso-diaminopimelic, glucose, ribose and madurose in whole-cell hydrolysates. The predominant menaquinones were MK-9(H4), MK-9(H2) and MK-9(H6). The phospholipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, unidentified phospholipids and unidentified aminophospholipids. The major fatty acid (>10 %) were cis 9 C17 : 1, iso-C16 : 0 and C15 : 0. The DNA G+C content was 69.7 mol% based on the whole genome sequence. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences indicated that strain H8750T was closely related to Acrocarpospora macrocephala JCM 10982T (98.0 %), Acrocarpospora pleiomorpha JCM 10983T (97.9 %) and Acrocarpospora phusangensis DSM 45867 T (97.8 %) and formed a monophyletic clade within the genus Acrocarpospora in the phylogenetic trees. The average nucleotide identity and digital DNA-DNA hybridization values between strain H8750T and its closely related Acrocarpospora species were 79.8~87.2 % and 25.9~28.0 %, respectively, which showed that it belonged to a distinct species. Furthermore, the morphological and phenotypic characteristics allowed the isolate to be differentiated from its closely related species. Therefore, it is concluded that strain H8750T can be classified as representing a novel species of the genus Acrocarpospora, for which the name Acrocarpospora catenulata sp. nov. is proposed. The type strain is H8750T (=JCM 34849T=CICC 25116T). Moreover, based on the gene prediction results, strain H8750T may have the genetic potential to synthesize many new secondary metabolites, which further increases its bioactive value.

A novel antibiotic combination of linezolid and polymyxin B octapeptide PBOP against clinical Pseudomonas aeruginosa strains.

BACKGROUND: Antibiotic-resistant Gram-negative bacteria are becoming a major public health threat such as the important opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). The present study investigated enhancement of the linezolid spectrum, which is normally used to treat Gram-positive bacteria, at inhibiting P. aeruginosa growth. METHODS: The checkerboard test or time-kill assay were carried out to determine the antibacterial effects of linezolid in cooperation with polymyxin B octapeptide PBOP (LP) against P. aeruginosa based on in vitro model. The protective effect of LP against P. aeruginosa infection was assessed based on a Caenorhabditis elegans (C. elegans) model. RESULTS: The synergistic activity and antibacterial effects were significantly increased against P. aeruginosa by LP treatment, while linezolid and PBOP as monotherapies exhibited no remarkably bactericidal activity against the clinical strains. Additionally, LP treatment modified biofilm production, morphology, swimming motility of P. aeruginosa, and protected C. elegans from P. aeruginosa infection. CONCLUSIONS: This research demonstrates that LP combination has significant synergistic activity against P. aeruginosa, and PBOP is potential to be an activity enhancer. Notably, this strategy improved the antibacterial activity spectrum of linezolid and other anti-Gram-positive agents and represents an effective choice to surmount the antibiotic resistance of bacteria in the long term.

To Be Ethical and Responsible Digital Citizens or Not: A Linguistic Analysis of Cyberbullying on Social Media.

As a worldwide epidemic in the digital age, cyberbullying is a pertinent but understudied concern-especially from the perspective of language. Elucidating the linguistic features of cyberbullying is critical both to preventing it and to cultivating ethical and responsible digital citizens. In this study, a mixed-method approach integrating lexical feature analysis, sentiment polarity analysis, and semantic network analysis was adopted to develop a deeper understanding of cyberbullying language. Five cyberbullying cases on Chinese social media were analyzed to uncover explicit and implicit linguistic features. Results indicated that cyberbullying comments had significantly different linguistic profiles than non-bullying comments and that explicit and implicit bullying were distinct. The content of cases further suggested that cyberbullying language varied in the use of words, types of cyberbullying, and sentiment polarity. These findings offer useful insight for designing automatic cyberbullying detection tools for Chinese social networking platforms. Implications also offer guidance for regulating cyberbullying and fostering ethical and responsible digital citizens.

Extracellular products-mediated interspecific interaction between Pseudomonas aeruginosa and Escherichia coli.

The Gram-negative pathogen Pseudomonas aeruginosa adopts several elaborate strategies to colonize a wide range of natural or clinical niches and to overcome the neighboring bacterial competitors in polymicrobial communities. However, the relationship and interaction mechanism of P. aeruginosa with other bacterial pathogens remains largely unexplored. Here we explore the interaction dynamics of P. aeruginosa and Escherichia coli, which frequently coinfect the lungs of immunocompromised hosts, by using a series of on-plate proximity assays and RNA-sequencing. We show that the extracellular products of P. aeruginosa can inhibit the growth of neighboring E. coli and induce a large-scale of transcriptional reprogramming of E. coli, especially in terms of cellular respiration-related primary metabolisms and membrane components. In contrast, the presence of E. coli has no significant effect on the growth of P. aeruginosa in short-term culture, but causes a dysregulated expression of genes positively controlled by the quorum-sensing (QS) system of P. aeruginosa during subsequent pairwise culture. We further demonstrate that the divergent QS-regulation of P. aeruginosa may be related to the function of the transcriptional regulator PqsR, which can be enhanced by E. coli culture supernatant to increase the pyocyanin production by P. aeruginosa in the absence of the central las-QS system. Moreover, the extracellular products of E. coli promote the proliferation and lethality of P. aeruginosa in infecting the Caenorhabditis elegans model. The current study provides a general characterization of the extracellular products-mediated interactions between P. aeruginosa and E. coli, and may facilitate the understanding of polymicrobial infections.

Genetic and Functional Diversity of Pseudomonas aeruginosa in Patients With Chronic Obstructive Pulmonary Disease.

Pseudomonas aeruginosa is the most relevant pathogen to the severe exacerbations of patients with chronic obstructive pulmonary disease (COPD). However, the genetic and functional characteristics of P. aeruginosa isolates from COPD airways still remain less understood. In this study, the genetic, phylogenetic, phenotypic, and transcriptional features of P. aeruginosa isolates from COPD sputa were comprehensively explored by susceptibility testing, comparative-genomic analysis, phylogenetic analysis, phenotypic profiling, and comparative-transcriptomic analysis. We found that P. aeruginosa was prevalent in elder COPD patients and highly resisted to many commonly used antibiotics. P. aeruginosa COPD isolates harbored a substantial number of variant sites that might influence the primary metabolism and substance transport system. These isolates were discretely distributed in the phylogenetic tree and clustered with internationally collected P. aeruginosa in two major groups, and could be classified into three groups according to their differences in virulence-related phenotypes. Furthermore, the transcriptional patterns of COPD isolates could be classified into PAO1-like group with reduced protein secretion and motility and PAO1-distinct group with decreased substance transport but enhanced primary metabolism. In conclusion, this study demonstrates that P. aeruginosa isolates from COPD patients have abundant genetic and phenotypic diversity, and provides an important reference for further exploring the survival strategy of P. aeruginosa in COPD airways and the development of anti-pseudomonal therapy.

TEMPO-Mediated C-H Amination of Benzoxazoles with N-Heterocycles.

The direct amination of benzoxazoles at C2 using N-heterocycles as nitrogen sources has been developed for the first time. Several kinds of inexpensive oxidants and also electricity were effective for this transformation in the presence of 2,2,6,6-tetramethylpiperidine-N-oxyl. This metal-free and operationally simple reaction can afford a variety of important C,N'-linked bis-heteocycles in moderate to good yields under very mild reaction conditions. The in situ generated oxoammonium salt was proved to be important for this transformation.

Nutrient factor-dependent performance of bacterial quorum sensing system during population evolution.

Bacterial quorum sensing (QS) system regulates the production of most costly but sharable extracellular products (public goods) in a growth-phase-dependent manner, and the development of this energy-intensive process is susceptible to environmental changes. However, the role of nutrient factors in dominating the QS-mediated cooperative interaction and intracellular metabolism still remains less understood. Here we studied the performance of QS system by growing Pseudomonas aeruginosa under different nutrient and culture conditions. The results of comparative-transcriptomic analyses revealed that carbon source-limitation was the main factor suppressing the activation of QS system, and a substantial number of public-good-encoding genes were induced when phosphorus is limiting in short-term culture. By contrast, although the QS regulation of P. aeruginosa in all the cultures was generally decreased along with the enrichment of QS-deficient individuals during evolution, limitation of different nutrient factors had discrepant effects in directing the formation of population structure by coordinating the production of public goods and primary metabolism, especially the starch and sucrose metabolism. These findings demonstrate the pleiotropy of QS regulation in balancing the development of cooperative behavior and metabolism, and provide a reference for further understanding the role of QS system in causing persistent infections.

Catellatospora sichuanensis sp. nov., a novel actinobacterium isolated from soil.

A novel actinobacterium, designated strain H14505T, was isolated from a soil sample collected in Hong Yuan, Sichuan, southwest PR China. The temperature, pH and NaCl ranges for growth were determined to be 15-35 °C (optimum, 28 °C), 6.0-8.0 (optimum, pH 7.0) and 0-2 % (w/v; optimum without NaCl), respectively. The polar lipdis detected for strain H14505T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, glycolipid and four unidentified lipids. The predominant menaquinones of strain H14505T were MK-9(H4) and MK-9(H6), and the prevalent fatty acids (>10 %) were C18 : 1 ω9c, C17 : 1 ω8c, summed feature 5 (anteiso-C18 : 0/ C18 : 2 ω6,9c) and C16 : 0. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences indicated that strain H14505T showed high similarity to Catellatospora vulcania NEAU-JM1T (99.0 %) and Catellatospora paridis NEAU-CL2T (99.0 %), and formed a monophyletic clade within the the genus Catellatospora in the phylogenetic trees. However, the average nucleotide indentity and DNA-DNA hybridization values between strain H14505T and closely related Catellatospora species showed that it belonged to a distinct species. Furthermore, the results of morphological, physiological and biochemical tests allowed further phenotypic differentiation of strain H14505T from its closest relatives. Thus, it is proposed that strain H14505T represents a novel species of the genus Catellatospora, for which the name Catellatospora sichuanensis sp. nov. is proposed. The type strain of Catellatospora sichuanensis is H14505T (=JCM 32394T=CICC 11042T).

Population divergence of Pseudomonas aeruginosa can lead to the coexistence with Escherichia coli in animal suppurative lesions.

Purulent disease is the main factor that prevents the population increase of forest musk deer in artificial breeding, and especially the intracorporal suppurative lesions in late-stage with complex bacterial communities normally bring more difficulties for veterinary treatment. Although it is well-recognized that Pseudomonas aeruginosa and Escherichia coli are the two main bacterial pathogens which can be frequently co-isolated from the lung pus of forest musk deer, few studies have explored the interspecific relationship and coexistent mechanism of the two species. In this study, we identified a P. aeruginosa strain MYL-2, which harbored a loss-of-function mutation in the central regulator (LasR) of quorum-sensing (QS) system, from the lung pus of a dying forest musk deer with co-infecting E. coli strain MYL-58. Interestingly, P. aeruginosa MYL-2 could coexist with E. coli MYL-58 compared to the dominant role of lasR-intact P. aeruginosa strain MYL-1 in the competitive experiments. The results of in vitro coevolution assay further revealed that the QS-mediated competitive advantage of P. aeruginosa MYL-1 would be decreased along with the enrichment of lasR mutants in the communities, and P. aeruginosa could finally coexist with E. coli by forming a relatively stable equilibrium. Therefore, these findings provide an evolutionary explanation for the coexistence of P. aeruginosa and E. coli in the suppurative lesions of forest musk deer, and may also contribute to further understanding the pathology of animal purulent disease and the development of novel veterinary therapy.

Pseudomonas aeruginosa Quorum-Sensing and Type VI Secretion System Can Direct Interspecific Coexistence During Evolution.

It is reported that a wide range of bacterial infections are polymicrobial, and the members in a local microcommunity can influence the growth of neighbors through physical and chemical interactions. Pseudomonas aeruginosa is an important opportunistic pathogen that normally causes a variety of acute and chronic infections, and clinical evidences suggest that P. aeruginosa can be frequently coisolated with other pathogens from the patients with chronic infections. However, the interspecific interaction and the coexisting mechanism of P. aeruginosa with coinfecting bacterial species during evolution still remain largely unclear. In this study, the relationships of P. aeruginosa with other Gram-positive (Staphylococcus aureus) and Gram-negative (Klebsiella pneumoniae) are investigated by using a series of on-plate proximity assay, in vitro coevolution assay, and RNA-sequencing. We find that although the development of a quorum-sensing system contributes P. aeruginosa a significant growth advantage to compete with S. aureus and K. pneumoniae, the quorum-sensing regulation of P. aeruginosa will be decreased during evolution and thus provides a basis for the formation of interspecific coexistence. The results of comparative transcriptomic analyses suggest that the persistent survival of S. aureus in the microcommunity has no significant effect on the intracellular transcriptional pattern of P. aeruginosa, while a more detailed competition happens between P. aeruginosa and K. pneumoniae. Specifically, the population of P. aeruginosa with decreased quorum-sensing regulation can still restrict the proportion increase of K. pneumoniae by enhancing the type VI secretion system-elicited cell aggressivity during further coevolution. These findings provide a general explanation for the formation of a dynamic stable microcommunity consisting of more than two bacterial species, and may contribute to the development of population biology and clinical therapy.

Lipoxygenase-inhibiting phenolic glycosides and monoterpene glycosides from Paeonia lactiflora.

The EtOH extract of the roots of Paeonia lactiflora afforded a new phenolic glycoside paenoside A (1) and a new monoterpene glycoside paeonin D (2), and five known monoterpene glycosides. Their structures were elucidated on the basis of spectroscopic means and hydrolysis products. All compounds displayed inhibitory potential against enzyme lipoxygenase.