Evolution of lasR mutants in polymorphic Pseudomonas aeruginosa populations facilitates chronic infection of the lung.

Chronic infection with the bacterial pathogen Pseudomonas aeruginosa often leads to coexistence of heterogeneous populations carrying diverse mutations. In particular, loss-of-function mutations affecting the quorum-sensing regulator LasR are often found in bacteria isolated from patients with lung chronic infection and cystic fibrosis. Here, we study the evolutionary dynamics of polymorphic P. aeruginosa populations using isolates longitudinally collected from patients with chronic obstructive pulmonary disease (COPD). We find that isolates deficient in production of different sharable extracellular products are sequentially selected in COPD airways, and lasR mutants appear to be selected first due to their quorum-sensing defects. Polymorphic populations including lasR mutants display survival advantages in animal models of infection and modulate immune responses. Our study sheds light on the multistage evolution of P. aeruginosa populations during their adaptation to host lungs.

Chronic pulmonary bacterial infection facilitates breast cancer lung metastasis by recruiting tumor-promoting MHCIIhi neutrophils.

Breast cancer can metastasize to various organs, including the lungs. The immune microenvironment of the organs to be metastasized plays a crucial role in the metastasis of breast cancer. Infection with pathogens such as viruses and bacteria can alter the immune status of the lung. However, the effect of chronic inflammation caused by bacteria on the formation of a premetastatic niche within the lung is unclear, and the contribution of specific immune mediators to tumor metastasis also remains largely undetermined. Here, we used a mouse model revealing that chronic pulmonary bacterial infection augmented breast cancer lung metastasis by recruiting a distinct subtype of tumor-infiltrating MHCIIhi neutrophils into the lung, which exhibit cancer-promoting properties. Functionally, MHCIIhi neutrophils enhanced the lung metastasis of breast cancer in a cell-intrinsic manner. Furthermore, we identified CCL2 from lung tissues as an important environmental signal to recruit and maintain MHCIIhi neutrophils. Our findings clearly link bacterial-immune crosstalk to breast cancer lung metastasis and define MHCIIhi neutrophils as the principal mediator between chronic infection and tumor metastasis.

Photocurable 3D-printed PMBG/TCP biphasic scaffold mimicking vasculature for bone regeneration.

Mesoporous bioglass (MBG) with excellent osteointegration, osteoinduction, and biodegradability is a promising material for bone regeneration. However, its clinical application is hindered by complex processing and a lack of personalization, low mechanical strength, and uncontrollable degradation rate. In this study, we developed a double-bond-functionalized photocurable mesoporous bioglass (PMBG) sol that enabled ultrafast photopolymerization within 5 s. By further integrating nanosized tricalcium phosphate (TCP) particles through three-dimensional (3D) printing technology, we fabricated personalized and highly porous PMBG/TCP biphasic scaffolds. The mechanical properties and degradation behavior of the scaffolds were regulated by varying the amount of TCP doping. In vitro and in vivo experiments verified that PMBG/TCP scaffolds slowly released SiO44- and Ca2+, forming a vascularized bone regeneration microenvironment within the fully interconnected pore channels of the scaffold. This microenvironment promoted angiogenesis and accelerated bone tissue regeneration. Overall, this work demonstrates the solution to the problem of complex processing and lack of personalization in bioglass scaffolds, and the developed PMBG/TCP biphasic scaffold is an ideal material for bone regeneration applications with broad clinical prospects.

Polydopamine-coated 3D-printed ß-tricalcium phosphate scaffolds to promote the adhesion and osteogenesis of BMSCs for bone-defect repair: mRNA transcriptomic sequencing analysis.

Cellular bioactivity and tissue regeneration can be affected by coatings on tissue-engineered scaffolds. Using mussel-inspired polydopamine (PDA) is a convenient and effective approach to surface modification. Therefore, 3D-printed ß-tricalcium phosphate (ß-TCP) scaffolds were coated with PDA in this study. The effects of the scaffolds on the adhesion and osteogenic differentiation of seeded bone marrow mesenchymal stem cells (BMSCs) in vitro and on new-bone formation in vivo were investigated. The potential mechanisms and related differential genes were assessed using mRNA sequencing. It was seen that PDA coating increased the surface roughness of the 3D-printed ß-TCP scaffolds. Furthermore, it prompted the adhesion and osteogenic differentiation of seeded BMSCs. mRNA sequencing analysis revealed that PDA coating might affect the osteogenic differentiation of BMSCs through the calcium signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, etc. Moreover, the expression of osteogenesis-related genes, such as R-spondin 1 and chemokine c-c-motif ligand 2, was increased. Finally, both the 3D-printed ß-TCP scaffolds and PDA-coated scaffolds could significantly accelerate the formation of new bone in critical-size calvarial defects in rats compared with the control group; and the new bone formation was obviously higher in the PDA-coated scaffolds than in ß-TCP scaffolds. In summary, 3D-printed ß-TCP scaffolds with a PDA coating can improve the physicochemical characteristics and cellular bioactivity of the scaffold surface for bone regeneration. Potential differential genes were identified, which can be used as a foundation for further research.

NEDD4L facilitates granulosa cell ferroptosis by promoting GPX4 ubiquitination and degradation.

Background: Polycystic ovary syndrome (PCOS) is an androgen disorder and ovarian dysfunction disease in women of reproductive age. The cell death of granulosa cells (GCs) plays an important role in the development of PCOS. However, the mechanism of GC death is still unclear. Methods: In the current study, NEDD4L was found to be elevated in PCOS GEO (Gene Expression Omnibus) databases and mouse models. The cell viability was analyzed by CCK-8 and FDA staining. The expression of ferroptosis markers was assessed by ELISA and immunofluorescence. The direct interaction of GPX4 and NEDD4L was verified by co-immunoprecipitation assay. Result: Functionally, results from CCK-8 and FDA staining demonstrated that NEDD4L inhibited the cell viability of KGN cells and NEDD4L increased the levels of iron, malonyldialdehyde, and reactive oxygen species and decreased glutathione levels. Moreover, the cell death of KGN induced by NEDD4L was blocked by ferroptosis inhibitor, suggesting that NEDD4L regulates KGN cell ferroptosis. Mechanistically, NEDD4L directly interacts with GPX4 and promotes GPX4 ubiquitination and degradation. Conclusion: Taken together, our study indicated that NEDD4L facilitates GC ferroptosis by promoting GPX4 ubiquitination and degradation and contributes to the development of PCOS.

Identification of a Multidrug Resistant Pseudomonas aeruginosa Isolate Harboring Infrequent Red Fluorescence Plasmid from COPD Patient.

Pseudomonas aeruginosa is a notorious Gram-negative opportunistic pathogen that normally causes acute and chronic infections in a wide range of hosts. In this study, a multi-resistant P. aeruginosa isolate L1a harboring an infrequent plasmid with red fluorescence was obtained from the bronchoalveolar lavage fluid of a patient with chronic obstructive pulmonary disease. The results of susceptibility testing and virulence-related phenotypic identification revealed that P. aeruginosa L1a was resistant to levofloxacin, cefepime, aztreonam, and imipenem and showed significantly stronger capacities for swimming and pyocyanin production than the reference strain PAO1. The genome of P. aeruginosa L1a was assembled into one circular chromosome (6,216,913 bp) and one circular plasmid (9111 bp). P. aeruginosa L1a was found to belong to the multilocus sequence type ST549, and serotype O5, and carried 8 drug resistance genes and 18 multidrug efflux pump-related genes in the chromosomal DNA. The plasmid pL1a harbored a tetracycline resistant gene tetA and a functional red fluorescence protein. This study reports a multidrug resistant P. aeruginosa clinical isolate harboring an infrequent red fluorescence plasmid for the first time.

Single-cell transcriptomics reveals distinct cell response between acute and chronic pulmonary infection of Pseudomonas aeruginosa.

Knowledge of the changes in the immune microenvironment during pulmonary bacterial acute and chronic infections is limited. The dissection of immune system may provide a basis for effective therapeutic strategies against bacterial infection. Here, we describe a single immune cell atlas of mouse lungs after acute and chronic Pseudomonas aeruginosa infection using single-cell transcriptomics, multiplex immunohistochemistry, and flow cytometry. Our single-cell transcriptomic analysis revealed large-scale comprehensive changes in immune cell composition and high variation in cell-cell interactions after acute and chronic P. aeruginosa infection. Bacterial infection reprograms the genetic architecture of immune cell populations. We identified specific immune cell types, including Cxcl2+ B cells and interstitial macrophages, in response to acute and chronic infection, such as their proportions, distribution, and functional status. Importantly, the patterns of immune cell response are drastically different between acute and chronic infection models. The distinct molecular signatures highlight the importance of the highly dynamic cell-cell interaction process in different pathological conditions, which has not been completely revealed previously. These findings provide a comprehensive and unbiased immune cellular landscape for respiratory pathogenesis in mice, enabling further understanding of immunologic mechanisms in infection and inflammatory diseases.

Repurposing Dimetridazole and Ribavirin to disarm Pseudomonas aeruginosa virulence by targeting the quorum sensing system.

Pseudomonas aeruginosa relies on its complex cellular regulatory network to produce a series of virulence factors and to cause various acute and chronic infections in a wide range of hosts. Compared with traditional antibiotics which frequently accompany with widespread antibiotic resistance, crippling the virulence system of bacteria is expected to be a promising anti-infective strategy. In this study, Dimetridazole and Ribavirin, which had poor antibacterial activities on P. aeruginosa reference isolate PAO1 in nutrient medium but significantly inhibited the growth of P. aeruginosa PAO1 in M9-adenosine, were selected from 40 marketed compounds with similar core structure (furan, benzofuran, or flavonoids) to the acyl-homoserine lactone signals of P. aeruginosa quorum sensing (QS) system. The production of QS-controlled proteases, pyocyanin, and biofilm formation of P. aeruginosa PAO1 and the clinical isolates were significantly decreased by the presence of Dimetridazole or Ribavirin. Correspondingly, the majority of QS-activated genes in P. aeruginosa, including the key regulatory genes lasR, rhlR, and pqsR and their downstream genes, were significantly inhibited by Ribavirin or Dimetridazole, as determined by RNA-sequencing and quantitative PCR. Furthermore, the susceptibilities of drug-resistant P. aeruginosa isolates to polymyxin B, meropenem, and kanamycin were remarkably promoted by the synergistic application of Dimetridazole or Ribavirin. Finally, the treatment of Ribavirin or Dimetridazole effectively protected Caenorhabditis elegans and mice from P. aeruginosa infection. In conclusion, this study reports the antivirulence potentials of Dimetridazole and Ribavirin on P. aeruginosa and provides structural basis and methodological reference for the development of anti-pseudomonal drugs.

Value of TCT combined with serum CA153 and CA50 in early diagnosis of cervical cancer and precancerous lesions.

Objectives: To determine the application value of thinprep cytologic test (TCT) combined with serum carbohydrate antigen 153 (CA153) and carbohydrate antigen 50 (CA50) detection in the early diagnosis and screening of cervical cancer and precancerous lesions. Methods: A total of 187 females with cervical lesions admitted to Shanghai 7th People's Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from January 2017 to December 2018 were selected and divided into two groups: the cervical cancer group and the cervical precancerous lesion group, with 16 cases in the cervical cancer group and 171 cases in the cervical precancerous lesion group (cervical precancerous lesions were divided into 63 cases of the CNI group, 59 cases of the CNII group and 49 cases of the CNIII group). During the same period, 106 healthy females were selected as the healthy group. The serum tumor markers CA153 and CA50 of all subjects were detected by chemiluminescence method; The diagnostic value of TCT combined with serum CA153 and CA50 in cervical cancer and precancerous lesions was analyzed with colposcopy pathological diagnosis results as gold standard; ROC curve was drawn to evaluate the diagnostic value of serum TCT, CA153 and CA50 in cervical cancer and precancerous lesions. Results: The levels of serum CA153 and CA50 in the cervical cancer group were significantly higher than those in the cervical precancerous lesion group and the healthy group (p< 0.05), and the levels of serum CA153 and CA50 in the cervical precancerous lesion group were significantly higher than those in the healthy group (p< 0.05). The sensitivity of TCT, serum CA153 and serum CA50 in the single detection of cervical cancer and precancerous lesions was 95.93%, 97.54% and 96.00%, the specificity was 59.41%, 60.23%, 60.12%, the accuracy was 74.74%, 75.77%, 75.43%, the positive predictive value was 62.03%, 63.64%, 63.10%, and the negative predictive value was 96.22%, 97.17% and 95.28%, respectively. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of TCT combined with serum CA153 and CA50 were 96.77%, 73.19%, 85.67%, 80.21% and 95.28%, respectively. ROC curve showed that the area under the curve (AUC) of TCT and serum CA153 and CA50 in the single detection of cervical cancer and precancerous lesions was 0.791, 0.864 and 0.787, respectively, the AUC of combined detection of TCT and serum CA153 and CA50 in patients with cervical cancer and precancerous lesions was 0.877, which was significantly higher than that of single detection (p< 0.05). Conclusions: TCT combined with serum CA153 and CA50 has been reported as a treatment regimen with high accuracy, which has a high diagnostic efficiency for early diagnosis of cervical cancer and precancerous lesions, and can significantly improve the sensitivity.

Antibacterial and anti-virulence effects of furazolidone on Trueperella pyogenes and Pseudomonas aeruginosa.

BACKGROUND: Trueperella pyogenes and Pseudomonas aeruginosa are two important bacterial pathogens closely relating to the occurrence and development of forest musk deer respiratory purulent disease. Although T. pyogenes is the causative agent of the disease, the subsequently invaded P. aeruginosa will predominate the infection by producing a substantial amount of quorum-sensing (QS)-controlled virulence factors, and co-infection of them usually creates serious difficulties for veterinary treatment. In order to find a potential compound that targets both T. pyogenes and P. aeruginosa, the antibacterial and anti-virulence capacities of 55 compounds, which have similar core structure to the signal molecules of P. aeruginosa QS system, were tested in this study by performing a series of in vitro screening experiments. RESULTS: We identified that furazolidone could significantly reduce the cell densities of T. pyogenes in mono-culture or in the co-culture with P. aeruginosa. Although the growth of P. aeruginosa could also be moderately inhibited by furazolidone, the results of phenotypic identification and transcriptomic analysis further revealed that sub-inhibitory furazolidone had remarkable inhibitory effect on the biofilm production, motility, and QS system of P. aeruginosa. Moreover, furazolidone could efficiently protect Caenorhabditis elegans models from P. aeruginosa infection under both fast-killing and slow-killing conditions. CONCLUSIONS: This study reports the antibacterial and anti-virulence abilities of furazolidone on T. pyogenes and P. aeruginosa, and provides a promising strategy and molecular basis for the development of novel anti-infectious drugs to dealing with forest musk deer purulent disease, or other diseases caused by T. pyogenes and P. aeruginosa co-infection.

Ectopic expression of an AGAMOUS homologue gene in Jatropha curcas causes early flowering and heterostylous phenotypes.

Jatropha curcasseeds are abundant in biodiesel, and low seed yields are linked to poor quality female flowers, which creates a bottleneck for Jatropha seed utilization. Therefore, identifying the genes associated with flowering is crucial for the genetic enrichment of seed yields. Here, we identified an AGAMOUS homologue gene (JcAG) from J. curcas. We found that reproductive organs had higher JcAG expression than vegetative organs, particularly the carpel. Rosette leaves were small and misshapen in 35S:JcAG transgenic lines in comparison with those in wild-type plants. JcAG overexpression caused an extremely early flowering, delayed perianth and stamen filament development, small flowers, and significantly shorter Arabidopsis plants with little fruit. In the JcAG-overexpressing line, the homeotic transformation of sepals into pistillate organs was observed, and floral meristem and organ identity genes were regulated. This study provides insights into the JcAG's function and benefits to our knowledge of the underlying the genetic mechanisms related to floral sex differentiation in Jatropha.