Deep Learning-Enabled Raman Spectroscopic Identification of Pathogen-Derived Extracellular Vesicles and the Biogenesis Process.

Pathogenic bacterial infections, exacerbated by increasing antimicrobial resistance, pose a major threat to human health worldwide. Extracellular vesicles (EVs), secreted by bacteria and acting as their "long-distance weapons", play an important role in the occurrence and development of infectious diseases. However, no efficient methods to rapidly detect and identify EVs of different bacterial origins are available. Here, label-free Raman spectroscopy in combination with a new deep learning model of the attentional neural network (aNN) was developed to identify pathogen-derived EVs at Gram±, species, strain, and even down to physiological levels. By training the aNN model with a large Raman data set from six typical pathogen-derived EVs, we achieved the identification of EVs with high accuracies at all levels: exceeding 96% at the Gram and species levels, 93% at the antibiotic-resistant and sensitive strain levels, and still above 87% at the physiological level. aNN enabled Raman spectroscopy to interrogate the bacterial origin of EVs to a much higher level than previous methods. Moreover, spectral markers underpinning EV discrimination were uncovered from subtly different EV spectra via an interpretation algorithm of the integrated gradient. A further comparative analysis of the rich Raman biochemical signatures of EVs and parental pathogens clearly revealed the biogenesis process of EVs, including the selective encapsulation of biocomponents and distinct membrane compositions from the original bacteria. This developed platform provides an accurate and versatile means to identify pathogen-derived EVs, spectral markers, and the biogenesis process. It will promote rapid diagnosis and allow the timely treatment of bacterial infections.

MiR-26a functions as a tumor suppressor in ambient particulate matter-bound metal-triggered lung cancer cell metastasis by targeting LIN28B-IL6-STAT3 axis.

Exposure to ambient particulate matter (PM) has been linked to the increasing incidence and mortality of lung cancer, but the principal toxic components and molecular mechanism remain to be further elucidated. In this study, human lung adenocarcinoma A549 cells were treated with serial concentrations of water-extracted PM10 (WE-PM10) collected from Beijing, China. Our results showed that exposure to 25 and 50 µg/ml of WE-PM10 for 48 h significantly suppressed miR-26a to upregulate lin-28 homolog B (LIN28B), and in turn activated interleukin 6 (IL6) and signal transducer and activator of transcription 3 (STAT3) in A549 cells, subsequently contributing to enhanced epithelial-mesenchymal transition and accelerated migration and invasion. In vivo pulmonary colonization assay further indicated that WE-PM10 enhanced the metastatic ability of A549 cells. In addition, luciferase reporter assay demonstrated that 3' untranslated region of LIN28B was a direct target of miR-26a. Last but not the least, the key toxic contribution of metals in WE-PM10 was confirmed by the finding that removal of metals through chelation significantly rescued WE-PM10-mediated inflammatory, carcinogenic and metastatic responses. Taken together, miR-26a could act as the tumor suppressor in PM10-related lung cancer, and PM10-bound metals promoted lung cancer cell metastasis through downregulation of miR-26a that directly mediated LIN28B expression.

The GH18 family of chitinases: their domain architectures, functions and evolutions.

The glycoside hydrolase 18 (GH18) family of chitinases is a multigene family that plays various roles, such as ecdysis, embryonic development, allergic inflammation and so on. Efforts are still needed to reveal their functional diversification in an evolutionary and systematic manner. We collected 85 GH18 genes from eukaryotic representatives. The domain architectures of GH18 proteins were analyzed and several conserved patterns were identified. It was observed that some (11 proteins) GH18 members in Ecdysozoa or fungi possess repeats of catalytic domains and/or chitin-binding domains (ChtBs). The domain repeats are likely to meet requirements for higher efficiency of chitin degradation in chitin-containing species. On the contrary, all vertebrate GH18 proteins contain no more than one catalytic domain or ChtB. The results from homologous analysis, domain architectures, exon arrangements and synteny loci supported two evolutionary paths for the GH18 family. One path experienced gene expansion and contraction several times during evolution, covering most of GH18 members except CHID1 (stabilin-1 interacting partner) and its homologs. Proteins in this path underwent frequent domain gain and loss, as well as domain recombination, that could achieve versatility in function. The other path is comparatively conserved. The CHID1 gene evolved without gene duplication except in Danio rerio. Domain architectures of CHID1 orthologs are all identical. The diverse phylogeny of the GH18 family in arthropod is also presented.

Inhibitory kinetics of citric acid on beta-N-acetyl-d-glucosaminidase from prawn (Litopenaeus vannamei).

NAGase (EC.3.2.1.52) from crustaceans has the important roles in immunity, molting and digestion of chitinous foods. In this paper, the effects of citric acid on the activity of NAGase from Litopenaeus vannamei for the hydrolysis of pNP-NAG have been studied. The results showed that appropriate concentrations of citric acid could lead to reversible inhibition on NAGase and IC(50) was estimated to be 5.00 +/- 0.35 mM. Using the plots of Lineweaver-Burk, the inhibition of NAGase by citric acid belongs to competitive type, the inhibitory equilibrium constant for citric acid binding with free NAGase, K(I), is 3.26 +/- 0.25 mM. The inhibitory kinetics of citric acid on NAGase in the appropriate concentrations of citric acid has been studied using the kinetic method of substrate reaction. The time course of NAGase for the hydrolysis of pNP-NAG in the presence of different concentrations of citric acid showed that at each citric acid concentration, the rate decreased with increasing time until a straight line was approached. The results show that the inhibition of NAGase by citric acid is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction on citric acid with NAGase.

Cloning and tissue expressions of seven chitinase family genes in Litopenaeus vannamei.

GH18 chitinase is a multi-gene family. The family plays important physiological roles in Crustacea, e.g. ecdysis and defense against pathogen. However, data about GH18 family are rather limited in Crustacea. In the study, different cloning strategies were adopted to clone chitinase genes of Litopenaeus vannamei, which is the most widely cultured shrimp. Seven chitinase family members were identified. Analysis of domain architectures showed the repeated CBM18 modules and catalytic domain of enzymatically inactive chitolectin in Crustacea for the first time. Comparing to the three known groups of crustacean chitinase, four of the seven members are located on new evolutionary clades thus enriched the chitinase family of Crustacea. Tissue expression profiles were investigated in eight tissues. Expression of CHT5 and CHID1 were both detected in the hemocyte by which the innate immunity activity was carried out. The domain architectures, evolutionary relationships and tissue expression patterns all provide reasonable explanation for the existence of multiple genes in crustacean chitinase family.

Inhibitory kinetics of beta-N-acetyl-D-glucosaminidase from prawn (Litopenaeus vannamei) by zinc ion.

Prawn (Litopenaeus vannamei) beta-N-acetyl-D-glucosaminidase (NAGase, EC 3.2.1.52) is involved in the digestion and molting processes. Zinc is one of the most important metals often found in the pollutant. In this article, the effects of Zn(2+) on prawn NAGase activity for the hydrolysis of pNP-NAG have been investigated. The results showed that Zn(2+) could reversibly and noncompetitively inhibit the enzyme activity at appropriate concentrations and its IC(50) value was estimated to be 6.00 +/- 0.25 mM. The inhibition model was set up, and the inhibition kinetics of the enzyme by Zn(2+) has been studied using the kinetic method of the substrate reaction. The inhibition constant was determined to be 11.96 mM and the microscopic rate constants were also determined for inactivation and reactivation. The rate constant of the inactivation (k(+0)) is much larger than that of the reactivation (k(-0)). Therefore, when the Zn(2+) concentration is sufficiently large, the enzyme is completely inactivated. On increasing the concentration of Zn(2+), the fluorescence emission peak and the UV absorbance peak are not position shifted, but the intensity decreased, indicating that the conformation of Zn(2+)-bound inactive NAGase is stable and different from that of native NAGase. We presumed that Zn(2+) made changes in the activity and conformation of prawn NAGase by binding with the histidine or cysteine residues of the enzyme.

Modification and modificatory kinetics of the active center of prawn beta-N-acetyl-D-glucosaminidase.

Beta-N-acetyl-D-glucosaminidase (NAGase, EC3.2.1.52) plays important role in molting, digestion of chitinous foods, and defense systems against parasites in prawn (Litopenaeus vannamei). However, study on functional groups and catalytic mechanism of NAGase are yet limited. The modification of the active center of NAGase from prawn has been first studied. The results demonstrate that the disulfide bonds and the carbamidine groups of arginine residues are not essential to the enzyme's activity. The modification of indole group of tryptophan of the enzyme by N-bromosuccinimide (NBS) can lead to the complete inactivation, accompanying the absorption decreasing at 276 nm, indicating that tryptophan is essential residue to the enzyme. The modificatory kinetics of NAGase in the appropriate concentrations of NBS solution has been studied and the numbers of essential tryptophan residues have been determined using the kinetic method of the substrate reaction. The result shows that only one tryptophan residue is essential for enzyme activity. And the modifications of histidine, lysine residue, and the carboxyl groups also inactivate the enzyme completely or incompletely. The results showed that the carboxyl groups of acidic amino acid, imidazole groups of histidine residue, amino groups of lysine residue, and indole group of tryptophan were essential for the activity of enzyme.

Inactivation kinetics of ß-N-acetyl-D-glucosaminidase from green crab (Scylla serrata) by guanidinium chloride.

ß-N-acetyl-D-glucosaminidase (NAGase) is a major member in chitinolytic enzymes system, which plays an important role in the hatching and molting processes of marine organism. The effects of guanidinium chloride (GuHCl) on the activity of NAGase from green crab (Scylla serrata) were investigated in this study. In results, GuHCl causes reversible inactivation of the enzyme at below 0.8 M concentrations, and the IC50 is estimated to be 0.15 M. The relationship between the enzyme activity and conformation was charaterized by monitoring the change of protein fluorescence spectra. With increasing GuHCl concentration, the fluorescence intensity of the enzyme distinctly decreases , and the maximal emission peaks appear red-shifted (from 338 nm to 343 nm). The enzyme inactivation precedes conformational changes, indicating that the enzyme active site is more flexible than the whole enzyme molecule. The result of the kinetics of inactivation shows that the value of k(+0) is larger than that of k(+0)'. It suggests that the substrate could protect the enzyme to a certain extent during guanidine denaturation. Our results provide important new insights in marine organism culture, especially in crustacean growth.

Evaluation of cellular response to perfluorooctane sulfonate in human umbilical vein endothelial cells.

Perfluorooctane sulfonate (PFOS), a stable fluorosurfactant and global pollutant, can be bioaccumulated. Recently, the possible adverse effects of PFOS on human health have raised concern. In this study, we examined the effects of PFOS on the expression of three important receptors related to endocrine-disrupting chemicals, six inflammatory-related genes, the production of reactive oxygen species (ROS) and the ability of cell adhesion in human umbilical vein endothelial cells (HUVECs). The results demonstrated that the expression of PPARγ and ERα were up-regulated after exposure to PFOS. After a high dose exposure (100 mg/L) for 48 h, the expression of IL-1ß, IL-6, COX-2, NOS3, ICAM-1 and P-Selectin were all notably up-regulated, whereas all of them did not show any significant changes after a low dose exposure (50 mg/L) for 24 h. Moreover, the expression of IL-1ß, COX-2 and NOS3 were significantly up-regulated, when cells were exposed to 100 mg/L PFOS for 24 h. Meanwhile, the amount of ROS induced by the exposure to high-dose PFOS was significantly increased with increasing incubation times. Furthermore, the adhesion of THP-1 cells onto HUVECs was significantly increased after exposure to 100 mg/L PFOS for 48 h, as observed for the expression of ICAM-1 and P-Selectin. In conclusion, our data suggest that PFOS exposure may play an important role in the vascular inflammatory disorders and endothelial dysfunctions.

Change of proinflammatory cytokines follows certain patterns after induction of endometriosis in a mouse model.

OBJECTIVE: To examine the change in proinflammatory cytokines in the pathologic processes of endometriosis in mice. DESIGN: A dynamic study on a murine model of endometriosis. SETTING: Medical school. ANIMAL(S): Female BALB/c mice. INTERVENTION(S): Endometriosis was induced by injecting endometrial fragments of syngenic mice into the peritoneal cavity of model mice; in control group, phosphate-buffered saline instead of fragments was injected. The peritoneal fluid and the endometriotic lesions were harvested 1 to 21 days after the induction. MAIN OUTCOME MEASURE(S): The endometriotic lesions were weighed, the gene and protein levels of some proinflammatory cytokines, including interleukin 1beta, tumor necrosis factor alpha, vascular endothelial growth factor, and monocyte chemoattractant protein 1, were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULT(S): The levels of these cytokines reached the first peak on the first day and no endometriotic lesions were found. The lesions began to appear on the second day, presenting red color during the initial 6 days, and then they turned dark-red, brown, or bluish. The adhesion took place on the 9th day, and all the lesions evolved into white or transparent cysts on the 15th day. Corresponding to these changes, the second and the third peaks were identified during the 3rd-6th day and the 12th-15th day, respectively. CONCLUSION(S): The change pattern of cytokines over time might bear some relationship with the development and progression of the endometriosis.

15-Epi-lipoxin A(4) inhibits the progression of endometriosis in a murine model.

OBJECTIVE: To examine the pro-resolution actions of 15-epi-lipoxin A(4) (LXA(4)) on endometriotic lesions, on the concentrations and activities of matrix metalloproteinases (MMP-2 and MMP-9) in murine endometriosis. DESIGN: Prospective, vehicle-controlled experimental study. SETTING: Animal research facility. ANIMAL(S): BALB/c mice. INTERVENTION(S): Endometriosis (EM) was induced in 30 mice. Fifteen of them were administered LXA(4) for 24 days (LXA(4) group), whereas the other 15 served as a control group (EM group). Another 15 sham-operated mice (sham-operated group) were treated with vehicles. MAIN OUTCOME MEASURE(S): The weight of the endometriotic lesions was measured. The concentrations, mRNA, and activities of MMP-2 and MMP-9 were determined by enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and gelatin zymography, respectively. RESULT(S): Compared with EM group, the weight of the endometriotic lesions was decreased, the concentrations of MMP-2 and MMP-9 dropped, the mRNA levels of MMP-2 and MMP-9 in the peritoneal fluid cells and the endometriotic lesions were reduced, and the activities of MMP-2 and MMP-9 were inhibited in the LXA(4) group. CONCLUSION(S): LXA(4) may inhibit the progression of endometriosis possibly by lowering the concentrations and the activities of MMP-2 and MMP-9.

Inhibitory kinetics of betaine on beta-N-acetyl-D-glucosaminidase from prawn (Litopenaeus vannamei).

The effects of betaine on prawn beta-N-acetyl-D-glucosaminidase (NAGase) activity for the hydrolysis of p-nitrophenyl-N-acetyl- beta-D-glucosaminide (pNP-NAG) have been studied. The results showed that appropriate concentrations of betaine could lead to reversible inhibition against NAGase, and the IC(50) value was estimated to be 15.00 +/- 0.30 mM. The inhibitory kinetics assay showed that betaine was a mixed type inhibitor with a K(I) value of 9.17 +/- 0.85 mM and a K(IS) value of 45.58 +/- 2.52 mM. The inhibitory model was set, and the microscopic rate constants were determined using the kinetic method of the substrate reaction. The time course of the hydrolysis of pNP-NAG catalyzed by NAGase in the presence of different betaine concentrations showed that at each betaine concentration, the rate decreased with an increase in time until a straight line was approached, indicating that the inhibition of NAGase by betaine is a slow, reversible reaction with fractional residual activity. The fact that k(+0) is much larger than k(+0)(') indicated that the free enzyme molecule is more fragile than the enzyme-substrate complex against betaine. It is suggested that the presence of the substrate offers marked protection of NAGase against inhibition by betaine.

Inhibitory effects of methyl trans-cinnamate on mushroom tyrosinase and its antimicrobial activities.

The control of food browning and growth of disease-causing microorganisms is critical to maintaining the quality and safety of food. Tyrosinase is the key enzyme in food browning. The inhibitory effect of methyl trans-cinnamate on the activity of tyrosinase has been investigated. Methyl trans-cinnamate can strongly inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. When the concentration of methyl trans-cinnamate reached 2.5 mM, the lag time was lengthened from 32 to 160 s and the steady-state activity was lost about 65%. The IC(50) value was 1.25 mM. For the diphenolase activity, the inhibition of methyl trans-cinnamate displayed a reversible and noncompetitive mechanism. The IC(50) value was 1.62 mM, and the inhibition constant (K(I)) was determined to be 1.60 mM. Moreover, the antibacterial activity against Escherichia coli, Bacillus subtilis and Staphyloccocus aureus and antifungal activity against Candida albicans were tested. The results showed that methyl trans-cinnamate possessed an antimicrobial ability.