TNFAIP3 interacting protein 2 relieves lipopolysaccharide (LPS)-induced inflammatory injury in endometritis by inhibiting NF-kappaB activation.

BACKGROUND: Endometritis seriously affects the health of women, and it is important to identify new targets for its treatment. OBJECTIVE: This study aimed to explore the role of TNFAIP3 interacting protein 2 (TNIP2) in endometritis through human endometrial epithelial cells (hEECs) stimulated by lipopolysaccharide (LPS). METHODS: hEECs were induced with LPS to build a cellular model of endometritis. Cell growth and apoptosis were detected by cell counting kit-8 and flow cytometry. The TNIP2 mRNA and protein levels were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. The caspase3 activity was calculated using a Caspase3 activity kit. Interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were determined by enzyme-linked-immunosorbent-assay. The reactive oxygen species (ROS), lactate dehydrogenase (LDH), catalase (CAT), and superoxide dismutase (SOD) levels were determined using the corresponding kits. Nuclear factor-kappaB (NF-κB) pathway was determined by western blot assay. RESULTS: TNIP2 was downregulated in the LPS-induced endometritis cell model. Cell viability was reduced, apoptosis was enhanced, and IL-6, IL-1ß, and TNF-α levels increased in LPS-induced hEECs. Additionally, LDH activity and ROS concentration were upregulated, whereas CAT and SOD activities were downregulated in LPS-induced hEECs. These results were reversed by TNIP2 overexpression. Moreover, the results hinted that NF-κB was involved in the effects of TNIP2 on the LPS-induced endometritis cell model. CONCLUSION: TNIP2 alleviated endometritis by inhibiting the NF-κB pathway, suggesting a potential therapeutic target for endometritis.

Flagellar brake protein YcgR interacts with motor proteins MotA and FliG to regulate the flagellar rotation speed and direction.

In E. coli and related species, flagellar brake protein YcgR responds to the elevated intracellular c-di-GMP, decreases the flagellar rotation speed, causes a CCW rotation bias, and regulates bacterial swimming. Boehm et al. suggested that c-di-GMP-activated YcgR directly interacted with the motor protein MotA to curb flagellar motor output. Paul et al. proposed that YcgR disrupted the organization of the FliG C-terminal domain to bias the flagellar rotation. The target proteins are controversial, and the role of motor proteins remains unclear in flagellar rotation speed and direction regulation by YcgR. Here we assayed the motor proteins' affinity via a modified FRET biosensor and accessed the role of those key residue via bead assays. We found that YcgR could interact with both MotA and FliG, and the affinities could be enhanced upon c-di-GMP binding. Furthermore, residue D54 of YcgR-N was needed for FliG binding. The mutation of the FliG binding residue D54 or the MotA binding ones, F117 and E232, restored flagellar rotation speed in wild-type cells and cells lacking chemotaxis response regulator CheY that switched the flagellar rotation direction and decreased the CCW ratio in wild-type cells. We propose that c-di-GMP-activated YcgR regulated the flagellar rotation speed and direction via its interaction with motor proteins MotA and FliG. Our work suggest the role of YcgR-motor proteins interaction in bacterial swimming regulation.

Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.

Genetic Foundations of Direct Ammonia Oxidation (Dirammox) to N2 and MocR-Like Transcriptional Regulator DnfR in Alcaligenes faecalis Strain JQ135.

Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (direct ammonia oxidation, NH3→NH2OH→N2) has been identified in Alcaligenes ammonioxydans. However, whether Dirammox is present in other microbes, as well as its genetic regulation, remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N2 via NH2OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N2O and trace amounts of N2, with nitrite as the sole nitrogen source. Deletion of the nirK and nosZ genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 5'-TGGTCTGT-3' in the promoter region. A bioinformatic survey showed that homologs of dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR. IMPORTANCE Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previously known ammonia oxidation pathways (i.e., nitrification, anaerobic ammonia oxidation [Anammox], and complete ammonia oxidation [Comammox]) are mediated by autotrophic microbes. However, the genetic foundations of ammonia oxidation by heterotrophic microorganisms have not been investigated in depth. Recently, a previously unknown pathway, termed direct ammonia oxidation to N2 (Dirammox), has been identified in the heterotrophic bacterium Alcaligenes ammonioxydans HO-1. This paper shows that, in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by dnf genes, which are independent of the denitrification pathway. A bioinformatic survey suggests that homologs of dnf genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N2.

Structural Modification of Nanomicelles through Phosphatidylcholine: The Enhanced Drug-Loading Capacity and Anticancer Activity of Celecoxib-Casein Nanoparticles for the Intravenous Delivery of Celecoxib.

This study aims to stabilize loaded celecoxib (CX) by modifying the structure of casein nanoparticles through phosphatidylcholine. The results show that Egg yolk phosphatidylcholine PC98T (PC) significantly increased the stability of CX-PC-casein nanoparticles (NPs) (192.6 nm) from 5 min (CX-ß-casein-NPs) to 2.5 h at 37 °C. In addition, the resuspended freeze-dried NPs (202.4 nm) remained stable for 2.5 h. Scanning electron microscopy indicated that PC may block the micropore structures in nanoparticles by ultrasonic treatment and hence improve the physicochemical stability of CX-PC-casein-NPs. The stability of the NPs was positively correlated with their inhibiting ability for human malignant melanoma A375 cells. The structural modification of CX-PC-casein-NPs resulted in an increased intracellular uptake of CX by 2.4 times than that of the unmodified ones. The pharmacokinetic study showed that the Area Under Curve (AUC) of the CX-PC-casein-NPs was 2.9-fold higher in rats than that of the original casein nanoparticles. When CX-PC-casein-NPs were intravenously administrated to mice implanted with A375 tumors (CX dose = 16 mg/kg bodyweight), the tumor inhibition rate reached 56.2%, which was comparable to that of paclitaxel (57.3%) at a dose of 4 mg/kg bodyweight. Our results confirm that the structural modification of CX-PC-casein-NPs can effectively prolong the remaining time of specific drugs, and may provide a potential strategy for cancer treatment.

Juxtaposed membranes underpin cellular adhesion and display unilateral cell division of multicellular magnetotactic prokaryotes.

Multicellular magnetotactic prokaryotes (MMPs) exhibit peculiar coordination of swimming along geomagnetic field lines. Approximately 40-80 cells assemble, with a helical geometry or axisymmetry, into spherical or ellipsoidal MMPs respectively. To contribute to a comprehensive understanding of bacterial multicellularity here we took multiple microscopic approaches to study the diversity, assembly, reproduction and motility of ellipsoidal MMPs. Using correlative fluorescence in situ hybridization and scanning electron microscopy analysis, we found an unexpected diversity in populations of ellipsoidal MMPs in the Mediterranean Sea. The high-pressure freezing/freeze substitution fixation technique allowed us to show, for the first time, that cells adhere via juxtaposed membranes and are held together by a rimming lattice. Fluorescence confocal microscopy and ultrathin section images revealed not only the one-layer hollow three-dimensional architecture, but also periphery-core unilateral constriction of constituent cells and unidirectional binary fission of the ellipsoidal MMPs. This finding suggests the evolution toward MMPs multicellularity via the mechanism of incomplete separation of offspring. Remarkably, thousands of flagellar at the periphery surface of cells underpin the coordinated swimming of MMPs in response to mechanical, chemical, magnetic and optical stimuli, including a magnetotactic photokinesis behaviour. Together these results unveil the unique structure and function property of ellipsoidal MMPs.

Identification of novel species of marine magnetotactic bacteria affiliated with Nitrospirae phylum.

Magnetotactic bacteria (MTB) are a group of Gram-negative bacteria characterized by synthesizing magnetosomes and swimming along geomagnetic field lines. Phylogenetically, they belong to different taxonomic lineages including Proteobacteria, Nitrospirae, Omnitrophica, Latescibacteria and Planctomycetes phyla on the phylogenetic tree. To date, six Nitrospirae MTB phylotypes have been identified from freshwater or low-salinity environments and described in the literature. Here, we report the identification of two Nitrospirae MTB phylotypes collected, for the first time, from the marine environment. Both have a spherical morphology with a cell size of ~ 5 µM and similar motility but are different colours (black-brown and ivory-white) under the optic microscope. They synthesized bullet-shaped iron-oxide magnetosomes that were arranged in multiple bundles of chains. Moreover, the cytoplasm of the black-brown Nitrospirae MTB contained sulphur inclusions that conferred on cells a rough, granular appearance. Phylogenetic analysis based on their 16S rRNA gene sequences revealed that they are two novel species and cluster with the previously reported MTB affiliated with the phylum Nitrospirae, thus extending the distribution of Nitrospirae MTB from freshwater to the marine environment.

Efficient heterologous expression of an alkaline lipase and its application in hydrolytic production of free astaxanthin.

BACKGROUND: Astaxanthin, a naturally occurring carotenoid pigment molecule, displays strong antioxidant, anti-cancer, and immunity-enhancing properties, and is often utilized in food, biomedical, cosmetic, and other industries. Free astaxanthin has better solubility than astaxanthin esters (Ast-E), and is a useful auxiliary ingredient in health foods and medicines. Our goal was to establish an improved enzymatic method for preparation of free astaxanthin from natural sources (e.g., the microalga Haematococcus pluvialis), to expand the potential applications of free astaxanthin. RESULTS: The alkaline lipase gene proalip and its propeptide were cloned and successfully fusion-expressed in Pichia pastoris X-33. The recombinant lipase was termed Lipase-YH. Through optimization of culture conditions (medium formulation, pH, added methanol concentration), cell growth (OD600) and secreted enzyme activity respectively reached to 280 and 2050 U/mL in a 50-L autofermentor. Activity of Lipase-YH enzyme powder was about 40,000 U/g. Hydrolysis of Ast-E (extracted from H. pluvialis) by Lipase-YH occurred in aqueous phase, and reaction conditions were optimized based on emulsification method and enzyme/substrate ratio. The highest enzymatic reaction rate was observed for substrate concentration 200 µg/mL, with maximal free astaxanthin yield (80%) at 1 h, and maximal Ast-E hydrolysis rate 96%, as confirmed by TLC, HPLC, and mass spectroscopy. CONCLUSION: A novel, efficient enzymatic process was developed for production of free astaxanthin through hydrolysis of Ast-E. Lipase activity was enhanced, and production cost was greatly reduced. The unique structure of free astaxanthin allows linkage to various functional compounds, which will facilitate development of novel pharmaceutical and food products in future studies.

Glycosylate and move! The glycosyltransferase Maf is involved in bacterial flagella formation.

The flagella of various Gram-negative bacteria are decorated with diverse glycan structures, amongst them nonulosonic acids related to the sialic acid family. Although nonulosonic sugar biosynthesis pathways have been dissected in various pathogens, the enzymes transferring the sugars onto flagellin are still poorly characterized. The deletion of genes coding for motility associated factors (Mafs) found in many pathogenic strains systematically gives rise to nonflagellated bacteria lacking specific nonulosonic sugars on the flagellins, therefore, relating Maf function to flagellin glycosylation and bacterial motility. We investigated the role of Maf from our model organism, Magnetospirillum magneticum AMB-1, in the glycosylation and formation of the flagellum. Deletion of the gene amb0685 coding for Maf produced a nonflagellated bacterium where the flagellin was still produced but no longer glycosylated. Our X-ray structure analysis revealed that the central domain of Maf exhibits similarity to sialyltransferases from Campylobacter jejuni. Glycan analysis suggested that the nonulosonic carbohydrate structure transferred is pseudaminic acid or a very close derivative. This work describes the importance of glycosylation in the formation of the bacterial flagellum and provides the first structural model for a member of a new bacterial glycosyltransferase family involved in nonulosonic acids transfer onto flagellins.