CLSSL-ResNet: Predicting malignancy of solitary pulmonary nodules from CT images by chimeric label with self-supervised learning.

BACKGROUND: Pulmonary granulomatous nodules (GN) with spiculation or lobulation have a similar morphological appearance to solid lung adenocarcinoma (SADC) under computed tomography (CT). However, these two kinds of solid pulmonary nodules (SPN) have different malignancies and are sometimes misdiagnosed. OBJECTIVE: This study aims to predict malignancies of SPNs by a deep learning model automatically. METHODS: A chimeric label with self-supervised learning (CLSSL) is proposed to pre-train a ResNet-based network (CLSSL-ResNet) for distinguishing isolated atypical GN from SADC in CT images. The malignancy, rotation, and morphology labels are integrated into a chimeric label and utilized to pre-train a ResNet50. The pre-trained ResNet50 is then transferred and fine-tuned to predict the malignancy of SPN. Two image datasets of 428 subjects (Dataset1, 307; Dataset2, 121) from different hospitals are collected. Dataset1 is divided into training, validation, and test data by a ratio of 7:1:2 to develop the model. Dataset2 is utilized as an external validation dataset. RESULTS: CLSSL-ResNet achieves an area under the ROC curve (AUC) of 0.944 and an accuracy (ACC) of 91.3%, which was much higher than that of the consensus of two experienced chest radiologists (77.3%). CLSSL-ResNet also outperforms other self-supervised learning models and many counterparts of other backbone networks. In Dataset2, AUC and ACC of CLSSL-ResNet are 0.923 and 89.3%, respectively. Additionally, the ablation experiment result indicates higher efficiency of the chimeric label. CONCLUSION: CLSSL with morphology labels can increase the ability of feature representation by deep networks. As a non-invasive method, CLSSL-ResNet can distinguish GN from SADC via CT images and may support clinical diagnoses after further validation.

A Novel Gene Alignment in Dorea sp. AM58-8 Produces 7-Dehydroxy-3ß Bile Acids from Primary Bile Acids.

Bile acids are essential metabolites and signaling molecules in mammals. Primary bile acids are synthesized from cholesterol in the liver. At the same time, the microbiota in the mammalian gut has many interactions with bile acid, including various biotransformation processes such as 7-dehydroxylation and 3-epimerization. 7-Dehydroxylation is mediated by a bile acid-inducible (bai) operon, while 7-dehydroxylation and 3-epimerization are independently observed in only a few strains. Herein, we describe a novel microbe, Dorea sp. AM58-8, that can accomplish a two-step transformation and turn primary bile acids into both 3α secondary bile acids like deoxycholic acid and lithocholic acid, and 3ß secondary bile acids like isodeoxycholic acid and isolithocholic acid. We subsequently characterized BaiA, BaiB, BaiE, and their substrate profiles biochemically. The potential bai gene clusters in the metagenomes were further mined. Their evolution, potential functions, and possible regulatory pathways were predicted using bioinformatics based on our understanding of the 7-dehydroxylation pathway in Dorea sp. AM58-8. This study of Dorea sp. AM58-8 also helps us distinguish the inactive bacteria that seem to have the 7-dehydroxylation pathway proteins and discover the 7-dehydroxylation pathway in other mammalian gut microbes.

De novo synthesis, structural assignment and biological evaluation of pseudopaline, a metallophore produced by Pseudomonas aeruginosa.

Pseudopaline is an opine carboxylate metallophore produced by Pseudomonas aeruginosa for harvesting divalent metals. However, the structure of pseudopaline is not fully elucidated. Herein, we report the first de novo total synthesis and isolation of pseudopaline, which allows unambiguous determination and confirmation of both the absolute and the relative configuration of the natural product. The synthesis highlights an efficient and stereocontrolled route using the asymmetric Tsuji-Trost reaction as the key step. The preliminary structure-activity relationship study indicated that one pseudopaline derivative shows comparable activity to pseudopaline. Moreover, a pseudopaline-fluorescein conjugate was prepared and evaluated, which confirmed that pseudopaline could be transported in the bacteria. Since the metal acquisition by P. aeruginosa is crucial for its ability to cause diseases, our extensive structural and functional studies of pseudopaline may pave the way for developing new therapeutic strategies such as the "Trojan horse" antibiotic conjugate against P. aeruginosa.

Development of an effective fluorescence probe for discovery of aminopeptidase inhibitors to suppress biofilm formation.

The human pathogen Pseudomonas aeruginosa can easily form biofilms. The extracellular matrix produced by the bacterial cells acts as a physical barrier to hinder the antibiotics treatment. It is necessary to destroy the biofilm in order to improve the efficacy of antibiotics. However, it has been a significant challenge to develop effective small molecules targeting the components of biofilm matrix. In this study, we report the development of a new effective fluorescence probe that could be used in the high throughput screening to identify novel small molecule inhibitors targeting the most abundant component in the biofilm formation: P. aeruginosa aminopeptidase (PaAP). Through screening of an in-house chemical library, a commercially available drug, balsalazide, has been identified as a novel PaAP inhibitor, which exhibited remarkable anti-biofilm effect. Our study indicated that the newly developed fluorescence probe is applicable in exploring new aminopeptidase inhibitors, and it also warrants further investigation of balsalazide as a new anti-biofilm agent to treat P. aeruginosa infection in combination with known antibiotics.

Extracellular aminopeptidase modulates biofilm development of Pseudomonas aeruginosa by affecting matrix exopolysaccharide and bacterial cell death.

Biofilm bacteria are embedded within a self-secreted extracellular matrix that contains a considerable amount of proteins including many extracellular enzymes. However, little is known about the roles of such enzymes in biofilm development. Here, we studied Pseudomonas aeruginosa aminopeptidase (PaAP, encoded by PA2939 that we named the gene as paaP in this study), a quorum-sensing-regulated enzyme and one of the most abundant extracellular proteins in the biofilm matrix of this opportunistic pathogen and environmental bacterium. We found that deletion of paaP in P. aeruginosa increased initial attachment and biofilm formation at early stages of biofilm development. After 24 h growth, loss of PaAP resulted in substantial cell death and biofilm disruption. Bacterial cell death was independent of biofilm matrix polysaccharide Psl, while biofilm disruption was due to the degradation of Psl matrix by dead-bacteria-released glycosyl hydrolase PslG, thereby leading to biofilm dispersion. PaAP functioned extracellularly and aminopeptidase catalytic activity was essential for its effect on biofilm development. Our data reveal an important role of extracellular aminopeptidase in biofilm development, suggesting PaAP as a therapeutic target for preventing P. aeruginosa infection and combating biofilm-related complications.

Dynamic Sessile-Droplet Habitats for Controllable Cultivation of Bacterial Biofilm.

Bacterial biofilms play essential roles in biogeochemical cycling, degradation of environmental pollutants, infection diseases, and maintenance of host health. The lack of quantitative methods for growing and characterizing biofilms remains a major challenge in understanding biofilm development. In this study, a dynamic sessile-droplet habitat is introduced, a simple method which cultivates biofilms on micropatterns with diameters of tens to hundreds of micrometers in a microfluidic channel. Nanoliter plugs are utilized, spaced by immiscible carrier oil to initiate and support the growth of an array of biofilms, anchored on and spatially confined to the micropatterns arranged on the bottom surface of the microchannel, while planktonic or dispersal cells are flushed away by shear force of aqueous plugs. The performance of the aforementioned method of cultivating biofilms is demonstrated by Pseudomonas aeruginosa PAO1 and its derived mutants, and quantitative antimicrobial susceptibility testing of PAO1 biofilms. This method could significantly eliminate corner effects, avoid microchannel clogging, and constrain the growth of biofilms for long-term observations. The controllable sessile droplet-based biofilm cultivation presented in this study should shed light on more quantitative and long-term studies of biofilms, and open new avenues for investigation of biofilm attachment, growth, expansion, and eradication.

A Survival Strategy for Pseudomonas aeruginosa That Uses Exopolysaccharides To Sequester and Store Iron To Stimulate Psl-Dependent Biofilm Formation.

Exopolysaccharide Psl is a critical biofilm matrix component in Pseudomonas aeruginosa, which forms a fiber-like matrix to enmesh bacterial communities. Iron is important for P. aeruginosa biofilm development, yet it is not clearly understood how iron contributes to biofilm development. Here, we showed that iron promoted biofilm formation via elevating Psl production in P. aeruginosa The high level of iron stimulated the synthesis of Psl by reducing rhamnolipid biosynthesis and inhibiting the expression of AmrZ, a repressor of psl genes. Iron-stimulated Psl biosynthesis and biofilm formation held true in mucoid P. aeruginosa strains. Subsequent experiments indicated that iron bound with Psl in vitro and in biofilms, which suggested that Psl fibers functioned as an iron storage channel in P. aeruginosa biofilms. Moreover, among three matrix exopolysaccharides of P. aeruginosa, Psl is the only exopolysaccharide that can bind with both ferrous and ferric ion, yet with higher affinity for ferrous iron. Our data suggest a survival strategy of P. aeruginosa that uses exopolysaccharide to sequester and store iron to stimulate Psl-dependent biofilm formation. IMPORTANCE: Pseudomonas aeruginosa is an environmental microorganism which is also an opportunistic pathogen that can cause severe infections in immunocompromised individuals. It is the predominant airway pathogen causing morbidity and mortality in individuals affected by the genetic disease cystic fibrosis (CF). Increased airway iron and biofilm formation have been proposed to be the potential factors involved in the persistence of P. aeruginosa in CF patients. Here, we showed that a high level of iron enhanced the production of the key biofilm matrix exopolysaccharide Psl to stimulate Psl-dependent biofilm formation. Our results not only make the link between biofilm formation and iron concentration in CF, but also could guide the administration or use of iron chelators to interfere with biofilm formation in P. aeruginosa in CF patients. Furthermore, our data also imply a survival strategy of P. aeruginosa under high-iron environmental conditions.

PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix.

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysaccharide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psl matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in improved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disassembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.

Characterization, antioxidant and hepatoprotective activities of polysaccharides from Ilex latifolia Thunb.

Crude polysaccharides from the leaves of Ilex latifolia Thunb (ILPS) was fractionated by DEAE cellulose-52 chromatography, affording four fractions of ILPS-1, ILPS-2, ILPS-3 and ILPS-4 in the recovery rates of 32.3, 20.6, 18.4 and 10.8%, respectively, based on the amount of crude ILPS used. The four fractions were mainly composed of arabinose and galactose in monosaccharide composition. Compared with ILPS-1 and ILPS-2, ILPS-3 and ILPS-4 had relative higher contents of sulfuric radical and uronic acid. The antioxidant activities in vitro of ILPS decreased in the order of crude ILPS>ILPS-4>ILPS-3>EPS-2>ILPS-1. Furthermore, the administration of crude ILPS significantly prevented the increase of serum alanine aminotransferase and aspartate aminotransferase levels, reduced the formation of malondialdehyde and enhanced the activities of superoxide dismutase and glutathione peroxidase in carbon tetrachloride-induced liver injury mice. The results suggested that ILPS should be a potent natural polymer with antioxidant and hepatoprotective activities.