Role and potential clinical utility of ARID1A in gastrointestinal malignancy.

ARID1A (AT-rich interactive domain 1A) is a newly discovered tumor suppressor gene, and its encoded product is an important component of the SWI/SNF chromatin remodeling complex. ARID1A plays an important role in cell proliferation, invasion and metastasis, apoptosis, cell cycle regulation, epithelial mesenchymal transition, and the regulation of other of biological behaviors. Recently, ARID1A mutations have been increasingly reported in esophageal adenocarcinoma, gastric cancer, colorectal cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, and other malignant tumors of the digestive system. This article reviews the relationship between ARID1A mutation and the molecular mechanisms of carcinogenesis, including microsatellite instability and the PI3K/ATK signaling pathway, and relates these mechanisms to the prognostic assessment of digestive malignancy. Further, this review describes the potential for molecular pathologic epidemiology (MPE) to provide new insights into environment-tumor-host interactions.

Current research progress in the role of reactive oxygen species in esophageal adenocarcinoma.

In the past few decades, the incidence of esophageal adenocarcinoma has increased by six-fold in western countries, as the proton pump inhibitor targeting the gastric acid reflux has failed to control the disease. It is currently suggested that deoxycholic acid reflux leads to esophageal adenocarcinoma. As an inflammation-related cancer, the formation and progression of esophageal adenocarcinoma are closely related to the concentration of reactive oxygen species (ROS). Meanwhile, the critical developmental stage of esophageal adenocarcinoma involves characteristic pathological changes in which the distal esophageal squamous epithelial cells are replaced by intestinal columnar epithelial cells, suggesting the involvement of cancer stem cells. Thus, esophageal adenocarcinoma is a good model to study the interplay between ROS and stem cells in cancer. Until now, some important questions related to ROS in esophageal adenocarcinoma remain unanswered. For example, the molecular mechanism by which deoxycholic acid induces malignant transformation in esophageal adenocarcinoma remains unclear. In addition, whether ROS are involved in the induction of cancer stem cell formation by chemotherapeutic drugs and deoxycholic acid stimulation in esophageal adenocarcinoma remains to be further explored. This review summarizes current research progress on ROS and stemness activity, regulation of ROS by stanniocalcin-1 (STC1)/uncoupling protein 2 (UCP2), and inspiration for ROS in esophageal adenocarcinoma to guide further research and provide insight into the clinical treatment of esophageal adenocarcinoma.

Deoxycholic Acid Upregulates the Reprogramming Factors KFL4 and OCT4 Through the IL-6/STAT3 Pathway in Esophageal Adenocarcinoma Cells.

Cancer stem cells, a special subgroup of cancer cells, have self-renewal capabilities and multidirectional potential, which may be reprogrammed from the dedifferentiation of cancer cells, contributing to the failure of clinical treatments. Esophageal adenocarcinoma grows in an inflammatory environment stimulated by deoxycholic acid, an important component of gastroesophageal reflux content, contributing to the transformation of esophageal squamous epithelium to the precancerous lesions of esophageal adenocarcinoma, that is, Barrett esophagus. In the present study, deoxycholic acid was used to investigate whether it could induce the expression of reprogramming factors Krüppel-like factor, OCT4, and Nanog; the transformation to cancer stem cells in esophageal adenocarcinoma; and the involvement of the interleukin-6/signal transduction and activation of transcription 3 inflammatory signaling pathway. OE33 cells were treated with deoxycholic acid (250 µM) for 0 hour, 3 hours, 6 hours, and 12 hours before evaluating the messenger RNA expression of Krüppel-like factor, OCT4, Nanog, interleukin-6, and Bcl-xL by reverse transcription-quantitative polymerase chain reaction. Interleukin-6 protein was detected by enzyme linked immunosorbent assay, while signal transduction and activation of transcription 3, phosphorylated signal transduction and activation of transcription 3, Krüppel-like factor, and OCT4 were detected by Western blot. Signal transduction and activation of transcription 3 small interfering RNA and human recombinant interleukin-6 were used to treat OE33 cells and to detect their effects on Krüppel-like factor, OCT4, Nanog, CD44, hypoxia-inducible factor 1-α, and Bcl-xL expression. Results showed that deoxycholic acid promotes the expression of reprogramming factors Krüppel-like factor and OCT4, which are regulated by the interleukin-6/signal transduction and activation of transcription 3 signaling pathway. Deoxycholic acid has a malignancy-inducing effect on the transformation of esophageal adenocarcinoma stem cells, improving the antiapoptotic ability of tumors, and increasing the malignancy of esophageal adenocarcinoma. Deactivating the regulatory signaling pathway of interleukin-6/signal transduction and activation of transcription 3 and neutralizing deoxycholic acid may be novel targets for improving the clinical efficacy of esophageal adenocarcinoma therapy.

Research progress of cancer stem cells and IL-6/STAT3 signaling pathway in esophageal adenocarcinoma.

Esophageal epithelial malignancy can be divided into esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). EAC is currently the seventh leading cause of death in US. Although ESCC occurs frequently in China and the population of EAC is mostly distributed in the western countries, with the change of lifestyle, diabetes, smoking, gastroesophageal exhalation disease, Barrett's esophagus (BE) and obesity have been confirmed as high-risk factors of EAC. The number of those high-risk groups in China is increasing, and the incidence of EAC in China is likely to increase in the future. At present, there is no clear epidemiological data of EAC in China. Attention should be paid to the pathogenesis, treatment plan and prognosis of EAC. In 2018, updated epidemiological data from the national institutes of health showed that the mortality rate of EAC patients diagnosed between 1975 and 2015 was as high as 91.6%. Currently, it is believed that the causes of poor prognosis, low survival rate, recurrence, metastasis, chemotherapy resistance and radiation resistance of malignant tumors may be closely related to the existence of cancer stem cells (CSCs). This paper reviews the source of BE, the CSC markers of EAC, the correlation among bile acid, IL-6/STAT3 signaling pathway and EAC, as well as the possible source of CSCs.

Pseudomonas aeruginosa displays a dormancy phenotype during long-term survival in water.

Pseudomonas aeruginosa is capable of long-term survival in water, which may serve as a reservoir for infection. Although viable cell counts of PAO1 incubated in water remain stable throughout 8 weeks, LIVE/DEAD staining indicated a high proportion of cells stained with propidium iodide (PI). The proportion of PI-stained cells increased by 4 weeks, then decreased again by 8 weeks, suggesting an adaptive response. This was also evident in an observed shift in cell morphology from a rod to a coccoid shape after 8 weeks. Fluorescence-activated cell sorting (FACS) was used to recover PI-stained cells, which were plated and shown to be viable, indicating that PI-stained cells were membrane-compromised but still cultivable. PAO1 mid-log cells in water were labeled with the dsDNA-binding dye PicoGreen to monitor viability as well as DNA integrity, which demonstrated that the population remains viable and transitions towards increased dsDNA staining. Metabolic activity was found to decrease significantly in water by 4 weeks. The PAO1 outer membrane became less permeable and more resistant to polymyxin B damage in water, and the profile of total membrane lipids changed over time. Among the ~1400 transcriptional lux fusions, gene expression in water revealed that the majority of genes were repressed, but subsets of genes were induced at particular time points. In summary, these results indicate that P. aeruginosa is dormant in water and this adaptation involves a complex pattern of gene regulation and changes to the cell to promote long-term survival and antibiotic tolerance. The approach of P. aeruginosa incubated in water may be useful to study antibiotic tolerance and the mechanisms of dormancy and survival in nutrient limiting conditions.

An intact microbiota is required for the gastrointestinal toxicity of the immunosuppressant mycophenolate mofetil.

BACKGROUND: Mycophenolate mofetil (MMF) is commonly prescribed after transplantation and has major advantages over other immunosuppressive drugs, but frequent gastrointestinal (GI) side-effects limit its use. The mechanism(s) underlying MMF-related GI toxicity have yet to be elucidated. METHODS: To investigate MMF-related GI toxicity, experimental mice were fed chow containing MMF (0.563%) and multiple indices of toxicity, including weight loss and colonic inflammation, were measured. Changes in intestinal microbial composition were detected using 16S rRNA Illumina sequencing, and downstream PICRUSt analysis was used to predict metagenomic pathways involved. Germ-free (GF) mice and mice treated with orally administered broad-spectrum antibiotics (ABX) were utilized to interrogate the importance of the microbiota in MMF-induced GI toxicity. RESULTS: Mice treated with MMF exhibited significant weight loss, related to loss of body fat and muscle, and marked colonic inflammation. MMF exposure was associated with changes in gut microbial composition, as demonstrated by a loss of overall diversity, expansion of Proteobacteria (specifically Escherichia/Shigella), and enrichment of genes involved in lipopolysaccharide (LPS) biosynthesis, which paralleled increased levels of LPS in the feces and serum. MMF-related GI toxicity was dependent on the intestinal microbiota, as MMF did not induce weight loss or colonic inflammation in GF mice. Furthermore, ABX prevented and reversed MMF-induced weight loss and colonic inflammation. CONCLUSIONS: An intact intestinal microbiota is required to initiate and sustain the GI toxicity of MMF. MMF treatment causes dynamic changes in the composition of the intestinal microbiota that may be a targetable driver of the GI side-effects of MMF.

Removal and metabolism of triclosan by three different microalgal species in aquatic environment.

Triclosan, an antimicrobial additive widely used in personal care products, has caused the contamination of various aquatic environment. Biodegradation was proved to play a vital role in the treatment of triclosan in wastewater. However, there is limited information about the metabolic pathway. In this study, three common freshwater microalgae including Chlorella pyrenoidosa (C. pyrenoidosa), Desmodesmus sp., and Scenedesmus obliquus (S. obliquus) were applied to remove and biodegrade triclosan in aqueous culture medium. High removal rate up to 99.7% was observed during the treatment of 400µgL-1 triclosan by the three microalgae for 1day. The removal of triclosan attributed to cellular uptake by C. pyrenoidosa, and biotransformation by Desmodesmus sp. and S. obliquus. Simultaneously, triclosan metabolites resulted from hydroxylation, reductive dechlorination, or ether bond cleavage and their conjugates produced through glucosylation and/or methylation were detected in the biodegradation samples. Metabolic pathway of triclosan by algae were firstly proposed in this work, shedding light on the environmental fate of triclosan.

Absence of the NOD2 protein renders epithelia more susceptible to barrier dysfunction due to mitochondrial dysfunction.

Irregular mitochondria structure and reduced ATP in some patients with IBD suggest that metabolic stress contributes to disease. Loss-of-function mutation in the nucleotide-binding oligomerization domain (NOD)-2 gene is a major susceptibility trait for IBD. Hence, we assessed if loss of NOD2 further impairs the epithelial barrier function instigated by disruption of mitochondrial ATP synthesis via the hydrogen ionophore dinitrophenol (DNP). NOD2 protein (virtually undetectable in epithelia under basal conditions) was increased in T84 (human colon cell line) cells treated with noninvasive Escherichia coli + DNP (16 h). Increased intracellular bacteria in wild-type (WT) and NOD2 knockdown (KD) cells and colonoids from NOD2-/- mice were mediated by reactive oxygen species (ROS) and the MAPK ERK1/2 pathways as determined by cotreatment with the antioxidant mitoTEMPO and the ERK inhibitor U0126: ROS was upstream of ERK1/2 activation. Despite increased E. coli in DNP-treated NOD2 KD compared with WT cells, there were no differences in the internalization of fluorescent inert beads or dead E. coli particles. This suggests that lack of killing in the NOD2 KD cells was responsible for the increased numbers of viable intracellular bacteria; a conclusion supported by evidence of reduced autophagy in NOD2 KD T84 epithelia. Thus, in a two-hit hypothesis, decreased barrier function due to dysfunctional mitochondrial is amplified by lack of NOD2 in transporting enterocytes: subsequently, greater numbers of bacteria entering the mucosa would be a significant inflammatory threat especially since individuals with NOD2 mutations have compromised macrophage and Paneth cell responses to bacteria.NEW & NOTEWORTHY Increased internalization of bacteria by epithelia with dysfunctional mitochondria (reduced ATP) is potentiated if the cells lack nucleotide-binding oligomerization domain 2 (NOD2), mutations in which are inflammatory bowel disease-susceptibility traits. Uptake of bacteria was dependent on reactive oxygen species and MAP-kinase activity, and the increased viable intracellular bacteria in NOD2-/- cells likely reflect a reduced ability to recognize and kill bacteria. Thus a significant barrier defect occurs with NOD2 deficiency in conjunction with metabolic stress that could contribute to inflammation.

Use of microbial fuel cells to monitor current production in Qi-deficient liver cells.

OBJECTIVE: To monitor current production in Qi-deficient liver cells, and to study how cellular proton leakage might affect electric current production. METHODS: Cells were placed in an microbial fuel cells (MFC) anode and the electric current was measured. Mitochondrial-affecting chemicals, 2,4-dinitrophenol (DNP) and resveratrol (RVT), were used to induce proton leakage in cells and their effect on current production observed. MCF-7 breast cancer cells exhibited higher proton leakage relative to normal liver cells. A mouse model for Qi-deficiency was prepared according to the Methodology of Animal Experiment in Chinese medicine. The Qi-tonics Buzhongyiqi Tang (BZYQT), which is used to treat the Qi-deficiency condition, was applied to Qi-deficient liver cells to examine how current production was altered. RESULTS: Adding either DNP or RVT to normal liver cells increased current production. MCF-7 cells that possessed high proton leakage were also found to produce higher currents than normal liver cells. Higher current production, lower cellular glucose content, and lower adenosine triphosphate (ATP) production rate were found in Qi-deficient liver cells, in which the use of DNP or RVT further increased current production. The use of BZYQT to treat Qi-deficient liver cells decreased current production, counteracted the action of DNP, and also improved cellular glucose content. CONCLUSION: High electric current production was found in liver cells with high cellular proton leakage. Positive current responses to both mitochondria-affecting chemicals, DNP and RVT, appeared to indicate proper mitochondrial function. The high proton leakage detected in Qi-deficient liver cells might have caused high energy losses, which served to explain the observed lower cellular glucose content and ATP production rate than in normal cells. These results might also explain the exhibited syndromes of low energy and fatigue in Qi-deficient patients. Proton leakage, induced by DNP or the Qi-deficient condition, was possibly caused by unusual uncoupling of oxidative phosphorylation and appeared to be inhibited by treatment with BZYOT such that decreased current production was observed after BZYQT treatment.

A Mechanism for the Temporal Potentiation of Genipin to the Cytotoxicity of Cisplatin in Colon Cancer Cells.

OBJECTIVES: To investigate the potentiation effect of Genipin to Cisplatin induced cell senescence in HCT-116 colon cancer cells in vitro. METHODS: Cell viability was estimated by Propidium iodide and Hoechst 3342, reactive oxygen species (ROS) with DHE, mitochondrial membrane potential (MMP) with JC-1 MMP assay Kit and electron current production with microbial fuel cells (MFC). RESULTS: Genipin inhibited the UCP2 mediated anti-oxidative proton leak significantly promoted the Cisplatin induced ROS and subsequent cell death, which was similar to that of UCP2-siRNA. Cells treated with Cisplatin alone or combined with Genipin, ROS negatively, while MMP positively correlated with cell viability. Cisplatin induced ROS was significantly decreased by detouring electrons to MFC, or increased by Genipin combined treatment. Compensatory effects of UCP2 up-regulation with time against Genipin treatment were suggested. Shorter the Genipin treatment before Cisplatin better promoted the Cisplatin induced ROS and subsequent cell death. CONCLUSION: The interaction of leaked electron with Cisplatin was important during ROS generation. Inhibition of UCP2-mediated proton leak with Genipin potentiated the cytotoxicity of Cisplatin. Owing to the compensatory effects against Genipin, shorter Genipin treatment before Cisplatin was recommended in order to achieve better potentiation effect.

Characterization and prediction of the mechanism of action of antibiotics through NMR metabolomics.

BACKGROUND: The emergence of antibiotic resistant pathogenic bacteria has reduced our ability to combat infectious diseases. At the same time the numbers of new antibiotics reaching the market have decreased. This situation has created an urgent need to discover novel antibiotic scaffolds. Recently, the application of pattern recognition techniques to identify molecular fingerprints in 'omics' studies, has emerged as an important tool in biomedical research and laboratory medicine to identify pathogens, to monitor therapeutic treatments or to develop drugs with improved metabolic stability, toxicological profile and efficacy. Here, we hypothesize that a combination of metabolic intracellular fingerprints and extracellular footprints would provide a more comprehensive picture about the mechanism of action of novel antibiotics in drug discovery programs. RESULTS: In an attempt to integrate the metabolomics approach as a classification tool in the drug discovery processes, we have used quantitative (1)H NMR spectroscopy to study the metabolic response of Escherichia coli cultures to different antibiotics. Within the frame of our study the effects of five different and well-known antibiotic classes on the bacterial metabolome were investigated both by intracellular fingerprint and extracellular footprint analysis. The metabolic fingerprints and footprints of bacterial cultures were affected in a distinct manner and provided complementary information regarding intracellular and extracellular targets such as protein synthesis, DNA and cell wall. While cell cultures affected by antibiotics that act on intracellular targets showed class-specific fingerprints, the metabolic footprints differed significantly only when antibiotics that target the cell wall were applied. In addition, using a training set of E. coli fingerprints extracted after treatment with different antibiotic classes, the mode of action of streptomycin, tetracycline and carbenicillin could be correctly predicted. CONCLUSION: The metabolic profiles of E. coli treated with antibiotics with intracellular and extracellular targets could be separated in fingerprint and footprint analysis, respectively and provided complementary information. Based on the specific fingerprints obtained for different classes of antibiotics, the mode of action of several antibiotics could be predicted. The same classification approach should be applicable to studies of other pathogenic bacteria.

UCP2- and non-UCP2-mediated electric current in eukaryotic cells exhibits different properties.

Using live eukaryotic cells, including cancer cells, MCF-7 and HCT-116, normal hepatocytes and red blood cells in anode and potassium ferricyanide in cathode of MFC could generate bio-based electric current. Electrons and protons generated from the metabolic reaction in both cytosol and mitochondria contributing to the leaking would mediate the generation of electric current. Both resveratrol (RVT) and 2,4-dinitrophenol (DNP) used to induce proton leak in mitochondria were found to promote electric current production in all cells except red blood cells without mitochondria. Proton leak might be important for electric current production by bringing the charge balance in cells to enhance the further electron leak. The induced electric current by RVT can be blocked by Genipin, an inhibitor of UCP2-mediated proton leak, while that induced by DNP cannot. RVT could reduce reactive oxygen species (ROS) level in cells better than that of DNP. In addition, RVT increased mitochondrial membrane potential (MMP), while DNP decreased it. Results highly suggested the existence of at least two types of electric current that showed different properties. They included UCP2-mediated and non-UCP2-mediated electric current. UCP2-mediated electric current exhibited higher reactive oxygen species (ROS) reduction effect per unit electric current production than that of non-UCP2-mediated electric current. Higher UCP2-mediated electric current observed in cancer cells might contribute to the mechanism of drug resistence. Correlation could not be established between electric current production with either ROS and MMP without distinguishing the types of electric current.

Using live algae at the anode of a microbial fuel cell to generate electricity.

Live green microalgae Chlorella pyrenoidosa was introduced in the anode of a microbial fuel cell (MFC) to act as an electron donor. By controlling the oxygen content, light intensity, and algal cell density at the anode, microalgae would generate electricity without requiring externally added substrates. Two models of algal microbial fuel cells (MFCs) were constructed with graphite/carbon electrodes and no mediator. Model 1 algal MFC has live microalgae grown at the anode and potassium ferricyanide at the cathode, while model 2 algal MFC had live microalgae in both the anode and cathode in different growth conditions. Results indicated that a higher current produced in model 1 algal MFC was obtained at low light intensity of 2500 lx and algal cell density of 5 × 10(6) cells/ml, in which high algal density would limit the electricity generation, probably by increasing oxygen level and mass transfer problem. The maximum power density per unit anode volume obtained in model 1 algal MFC was relatively high at 6030 mW/m(2), while the maximum power density at 30.15 mW/m(2) was comparable with that of previous reported bacteria-driven MFC with graphite/carbon electrodes. A much smaller power density at 2.5 mW/m(2) was observed in model 2 algal MFC. Increasing the algal cell permeability by 4-nitroaniline would increase the open circuit voltage, while the mitochondrial acting and proton leak promoting agents resveratrol and 2,4-dinitrophenol would increase the electric current production in algal MFC.

DNA is an antimicrobial component of neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) comprise an ejected lattice of chromatin enmeshed with granular and nuclear proteins that are capable of capturing and killing microbial invaders. Although widely employed to combat infection, the antimicrobial mechanism of NETs remains enigmatic. Efforts to elucidate the bactericidal component of NETs have focused on the role of NET-bound proteins including histones, calprotectin and cathepsin G protease; however, exogenous and microbial derived deoxyribonuclease (DNase) remains the most potent inhibitor of NET function. DNA possesses a rapid bactericidal activity due to its ability to sequester surface bound cations, disrupt membrane integrity and lyse bacterial cells. Here we demonstrate that direct contact and the phosphodiester backbone are required for the cation chelating, antimicrobial property of DNA. By treating NETs with excess cations or phosphatase enzyme, the antimicrobial activity of NETs is neutralized, but NET structure, including the localization and function of NET-bound proteins, is maintained. Using intravital microscopy, we visualized NET-like structures in the skin of a mouse during infection with Pseudomonas aeruginosa. Relative to other bacteria, P. aeruginosa is a weak inducer of NETosis and is more resistant to NETs. During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing. Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA. Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies.

Stress response of Chlorella pyrenoidosa to nitro-aromatic compounds.

Handling of two nitro-aromatic compounds, 4-nitroaniline (4NA) and 4-nitrophenol (4NP), simultaneously by Chlorella pyrenoidosa was investigated. Algae would secrete or degrade nitro-aromatic compounds depending on different environmental conditions, in which the mode of handling was determined by the relative formation and degradation rate of the compound. Repeated intermittent trigger with externally added 4NA would induce the continuous secretion of 4NA by algae. Simultaneous exposure of both 4NA and 4NP to algae at normal condition would induce the algae to secrete both compounds. An increase in 4NA exposure concentration would elevate both 4NA and 4NP secretion, and that would be inhibited by the stress conditions of starving or lack of oxygen. Increased 4NA degradation per production rate induced by starving or lack of oxygen might explain the subsequent decrease in 4NA secretion in the presence of 4NP in algae. For 4NP in the presence of 4NA, secretion at normal condition was completely stopped and turned to degradation mode in stress conditions. The decreased formation and increased degradation of 4NP during starving for replenishing energy would explain the net degradation of 4NP in starving condition. The condition of lack of oxygen would inhibit the 4NP formation from 4NA via oxidative deamination, while the degradation of 4NP might not be significantly affected because alternative pathway of degradation via nitro-reduction was available. It may lead to the degradation rate exceeding the formation and explain the net degradation of 4NP in the condition of lack of oxygen.

Removal and reductive dechlorination of triclosan by Chlorella pyrenoidosa.

Triclosan that is widely used as antimicrobial agent has been detected as contaminant in various aquatic environments. In this work, removal and biodegradation of triclosan in water by using a ubiquitous green alga, Chlorella pyrenoidosa was investigated. When C. pyrenoidosa was exposed to a series concentration of triclosan from 100 to 800ngmL(-1), more than 50% of triclosan was eliminated by algal uptake from the culture medium during the first 1h exposure and reached equilibrium after the 6h treatment. In the biodegradation experiments, a removal percentage of 77.2% was obtained after C. pyrenoidosa was cultivated with 800ngmL(-1) triclosan for 96h. A major metabolite from the reductive dechlorination of triclosan was identified by using liquid chromatography coupled with electrospray ionization-mass spectrometry. The ultrastructural morphology of algal cells grown in the presence of triclosan was observed by using transmission electron microscopy and the growth of algal cells was detected. It was found that the trilcosan treatment resulted in the disruption of the chloroplast and the release of organic material into aquatic environment, which indicated that triclosan may affect membrane metabolism.

Biodegradation and removal of 3,4-dichloroaniline by Chlorella pyrenoidosa based on liquid chromatography-electrospray ionization-mass spectrometry.

3,4-Dichloroaniline (3,4-DCA), widely used in the synthesis of dyes, textile and herbicides, is toxic to living organisms. The purpose of this study was to investigate the capability of green algae in degrading and removing 3,4-DCA in water. An environmentally ubiquitous green alga Chlorella pyrenoidosa was isolated from fresh aquatic environment. Then unicellular alga was incubated with 3,4-DCA at a concentration of 4.6 µg/ mL in water. The residual concentration of 3,4-DCA in the medium and the metabolites were analyzed. A removal percentage of 78.4 % was obtained over a 7-day period. Two major metabolites with less toxicity were identified as 3,4-dichloroformanilide and 3,4-dichloroacetanilide from the liquid chromatography-electrospray ionization-mass spectrometry analysis. The application of microalga C. pyrenoidosa may have potential for removing the environmental pollutant in aquatic environment.

A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus.

Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3-4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5-60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton-Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton-Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.

The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP-D-rhamnose biosynthesis pathway.

The rare 6-deoxysugar D-rhamnose is a component of bacterial cell surface glycans, including the D-rhamnose homopolymer produced by Pseudomonas aeruginosa, called A-band O polysaccharide. GDP-D-rhamnose synthesis from GDP-D-mannose is catalyzed by two enzymes. The first is a GDP-D-mannose-4,6-dehydratase (GMD). The second enzyme, RMD, reduces the GMD product (GDP-6-deoxy-D-lyxo-hexos-4-ulose) to GDP-d-rhamnose. Genes encoding GMD and RMD are present in P. aeruginosa, and genetic evidence indicates they act in A-band O-polysaccharide biosynthesis. Details of their enzyme functions have not, however, been previously elucidated. We aimed to characterize these enzymes biochemically, and to determine the structure of RMD to better understand what determines substrate specificity and catalytic activity in these enzymes. We used capillary electrophoresis and NMR analysis of reaction products to precisely define P. aeruginosa GMD and RMD functions. P. aeruginosa GMD is bifunctional, and can catalyze both GDP-d-mannose 4,6-dehydration and the subsequent reduction reaction to produce GDP-D-rhamnose. RMD catalyzes the stereospecific reduction of GDP-6-deoxy-D-lyxo-hexos-4-ulose, as predicted. Reconstitution of GDP-D-rhamnose biosynthesis in vitro revealed that the P. aeruginosa pathway may be regulated by feedback inhibition in the cell. We determined the structure of RMD from Aneurinibacillus thermoaerophilus at 1.8 A resolution. The structure of A. thermoaerophilus RMD is remarkably similar to that of P. aeruginosa GMD, which explains why P. aeruginosa GMD is also able to catalyze the RMD reaction. Comparison of the active sites and amino acid sequences suggests that a conserved amino acid side chain (Arg185 in P. aeruginosa GMD) may be crucial for orienting substrate and cofactor in GMD enzymes.