Genomic and resistome analysis of Alcaligenes faecalis strain PGB1 by Nanopore MinION and Illumina Technologies.

BACKGROUND: Drug-resistant bacteria are important carriers of antibiotic-resistant genes (ARGs). This fact is crucial for the development of precise clinical drug treatment strategies. Long-read sequencing platforms such as the Oxford Nanopore sequencer can improve genome assembly efficiency particularly when they are combined with short-read sequencing data. RESULTS: Alcaligenes faecalis PGB1 was isolated and identified with resistance to penicillin and three other antibiotics. After being sequenced by Nanopore MinION and Illumina sequencer, its entire genome was hybrid-assembled. One chromosome and one plasmid was assembled and annotated with 4,433 genes (including 91 RNA genes). Function annotation and comparison between strains were performed. A phylogenetic analysis revealed that it was closest to A. faecalis ZD02. Resistome related sequences was explored, including ARGs, Insert sequence, phage. Two plasmid aminoglycoside genes were determined to be acquired ARGs. The main ARG category was antibiotic efflux resistance and ß-lactamase (EC 3.5.2.6) of PGB1 was assigned to Class A, Subclass A1b, and Cluster LSBL3. CONCLUSIONS: The present study identified the newly isolated bacterium A. faecalis PGB1 and systematically annotated its genome sequence and ARGs.

The evaluation of the shear bond strength between various Hybrid CAD/CAM restorative materials and repairing composite resins / 대한치과재료학회지

The purpose of this study was to evaluate the shear bond strengths between various hybrid computer-aided design (CAD)/computer-aided manufacturing (CAM) restorative materials and repairing resin. Two resin network-based hybrid (Lava Ultimate and Polyglass), one ceramic framework-based hybrid (Enamic), and one zirconia (Zenotec Zr bridge) CAD/CAM restorative materials were used in this study. The shear bond strength test and failure modes of four experimental groups designated LUS (Lava Ultimate), ENA (Enamic), PGB (Polyglass), and ZBR (zirconia control group) were characterized in this study. The hybrid CAD/CAM restorative materials showed stronger shear bond strengths in the sequence of PGB, LUS, and ENA (P < 0.05). The shear bond strengths of PGB and LUS groups showed significantly higher than those of ZBR (P < 0.05), while ENA did not show any significant difference from ZBR (P < 0.05). The PEG and LUS groups mostly exhibited cohesive failure, but the ENA and ZBR groups predominantly showed adhesive failure. Therefore, resin network-based hybrid CAD/CAM restorative materials such as Lava Ultimate and Polyglass should be more useful for intra-oral repairs.

Cord blood mesenchymal stem cells suppress DC-T Cell proliferation via prostaglandin B2.

Immune suppression is a very stable property of multipotent stromal cells also known as mesenchymal stem cells (MSCs). All cell lines tested showed robust immune suppression not affected by a long culture history. Several mechanisms were described to account for this capability. Since several of the described mechanisms were not causing the immune suppression, the expression pattern of cord-blood-derived MSCs by microarray experiments was determined. Dendritic cells cocultured with cord blood MSCs were compared with cord blood MSCs. Putative immune suppressive candidates were tested to explain this inhibition. We find that cord blood MSCs themselves are hardly immunogenic as tested with allogeneic T-cells. Dendritic cells cocultured with second-party T-cells evoked abundant proliferation that was inhibited by third-party cord blood MSCs. Optimal inhibition was seen with one cord blood MSC for every dendritic cell. Blocking human leukocyte antigen G only saw partial recovery of proliferation. Several cytokines, gangliosides, enzymes like arginase, NO synthase, and indole amine 2,3-dioxygenase as well as the induction of Treg were not involved in the inhibition. The inhibiting moiety was identified as prostaglandin B2 by lipid metabolite analysis of the culture supernatant and confirmed with purified prostaglandin B2.

Comparison of the Medication Effects between Milnacipran and Pregabalin in Fibromyalgia Syndrome Using a Functional MRI: a Follow-up Study

PURPOSE: In this study, the medication effects of Milnacipran and Pregabalin, as well known as fibromyalgia treatment medicine, in fibromyalgia syndrome patients were compared through the change of BOLD signal in pain related functional MRI. MATERIALS AND METHODS: Twenty fibromyalgia syndrome patients were enrolled in this study and they were separated into two groups according to the treatment medicine: 10 Milnacipran (MLN) treatment group and 7 Pregabalin (PGB) treatment group. For accurate diagnosis, all patients underwent several clinical tests. Pre-treated and post-treated fMRI image with block-designed pressure-pain stimulation for each group were obtained to conduct the statistical analysis of paired t-test and two sample t-test. All statistical significant level was less than 0.05. RESULTS: In clinical tests, the clinical scores of the two groups were not significantly different at pre-treatment stage. But, PGB treatment group had lower Widespread Pain Index (WPI) and Brief Fatigue Inventory (BFI) score than those of MLN treatment group at post-treatment stage. In functional image analysis, BOLD signal of PGB treatment group was higher BOLD signal at several regions including anterior cingulate and insula than MLN treatment group at post-treatment stage. Also, paired t-test values of the BOLD signal in MLN group decreased in several regions including insula and thalamus as known as 'pain network'. In contrast, size and number of regions in which the BOLD signal decreased in PGB treatment group were smaller than those of MLN treatment group. CONCLUSION: This study showed that MLN group and PGB group have different medication effects. It is not surprising that MLN and PGB have not the same therapeutic effects since these two drugs have different medicinal mechanisms such as antidepressants and anti-seizure medication, respectively, and different detailed target of fibromyalgia syndrome treatment. Therefore, it is difficult to say which medicine will work better in this study.

Participation of KATP Channels in the Antinociceptive Effect of Pregabalin in Rat Formalin Test

BACKGROUND: Pregabalin is an anticonvulsant and analgesic agent that interacts selectively with the voltage-sensitive-Ca(2+)-channel alpha-2-delta subunit. The aim of this study was to evaluate whether the analgesic action of intrathecal (IT) pregabalin is associated with KATP channels in the rat formalin test. METHODS: IT PE-10 catheters were implanted in male Sprague-Dawley rats (250-300 g) under inhalation anesthesia using enflurane. Nociceptive behavior was defined as the number of hind paw flinches during 60 min after formalin injection. Ten min before formalin injection, IT drug treatments were divided into 3 groups: normal saline (NS) 20 microl (CON group); pregabalin 0.3, 1, 3 and 10 microg in NS 10 microl (PGB group); glibenclamide 100 microg in DMSO 5 microl with pregabalin 0.3, 1, 3 and 10 microg in NS 5 microl (GBC group). All the drugs were flushed with NS 10 microl. Immunohistochemistry for the KATP channel was done with a different set of rats divided into naive, NS and PGB groups. RESULTS: IT pregabalin dose-dependently decreased the flinching number only in phase 2 of formalin test. The log dose response curve of the GBC group shifted to the right with respect to that of the PGB group. Immunohistochemistry for the KATP channel expression on the spinal cord dorsal horn showed no difference among the groups 1 hr after the formalin test. CONCLUSIONS: The antinociceptive effect of pregabalin in the rat formalin test was associated with the activation of the KATP channel. However, pregabalin did not induce KATP channel expression in the spinal cord dorsal horn.

The occurrence of 15-keto-prostaglandins in the red alga Gracilaria verrucosa.

New 15-keto-prostaglandins (1-4) were isolated from the MeOH extract of the red alga, Gracilaria verrucosa. Their structures were determined to be prostaglandin B congeners (1-3) and a prostaglandin E congener (4) based on the NMR and MS data. Prostaglandins with a C-15 keto function are rare from natural sources. The presence of these metabolites in the alga is notable because 15-keto-prostaglandins (15-keto-PGs) are considered to be the metabolic products of regular prostaglandins in mammals. The occurrence of different prostaglandins in this alga might be due to the existence of different oxidative enzymes, as previously mentioned for oxygenated fatty acids of the red alga Gracilariopsis lemaneiformis. The antiinflammatory activity of these prostaglandins was examined by evaluating their inhibitory effects on nitric oxide production in lipopolysaccharide (LPS)-activated RAW264.7 murine macrophage cells. These prostaglandins showed weak activity on nitric oxide production.

[Marine fungus Stilbella aciculosa as a potential producer of prostaglandins].

The amount and composition of fatty acids in the fungus Stilbella aciculosa associated with the marine macroorganism Apostichopus japonica (trepang) were determined by gas-liquid chromatography and gas chromatography-mass spectrometry. In the culture liquid of S. aciculosa, prostaglandins (PG) of groups E and F were revealed by UV spectroscopy. This finding was confirmed by the presence of direct precursors of PG, polyunsaturated eicosapentaenoic and docosahexaenoic acids, in the culture liquid. The biomass of this fungus contained PG of group B.

Glucuronidation of oxidized fatty acids and prostaglandins B1 and E2 by human hepatic and recombinant UDP-glucuronosyltransferases.

Arachidonic acid (AA) can be metabolized to various metabolites, which can act as mediators of cellular processes. The objective of this work was to identify whether AA, prostaglandin (PG) B1 and E2, and 15- and 20-hydroxyeicosatetraenoic acids (15- and 20-HETE) are metabolized via glucuronidation. Assays with human recombinant UDP-glucuronosyltransferase 1A (UGT1A) isoforms revealed that AA and 15-HETE were glucuronidated by UGT1A1, 1A3, 1A4, 1A9, and 1A10, whereas 20-HETE was glucuronidated by UGT1A1 and 1A4 and PGB1 was glucuronidated by UGT1A1, 1A9, and 1A10. All substrates were glucuronidated by recombinant UGT2B7, with AA and 20-HETE being the best substrates. Kinetic analysis of UGT1A1 and 1A9 with AA resulted in Km values of 37.9 and 45.8 microM, respectively. PGB1 was glucuronidated by UGT1A1 with a Km of 26.3 microM. The Km values for all substrates with UGT2B7 were significantly higher than with the UGT1A isoforms. Liquid chromatography-mass spectrometry of glucuronides biosynthesized from PGB1 and 15-HETE showed that hydroxyl groups were the major target of glucuronidation. This work demonstrates a novel metabolic pathway for HETEs and PGs and the role of UGT1A isoforms in this process. These results indicate that glucuronidation may play a significant role in modulation of the availability of these fatty acid derivatives for cellular processes.

Osteoblast-derived oxysterol is a migration-inducing factor for human breast cancer cells.

Bone metastasis is the major reason for death caused by breast cancer. We used human breast cancer (MCF-7) cells that are poorly metastatic but show highly inducible migration to determine bone-derived factors that induce migration of initially non-disseminating breast cancer cells. We have found that a lipid fraction from human osteoblast-like MG63 cell-conditioned medium (MG63CM) contains a migration-inducing factor for MCF-7 cells. In this fraction, we have identified oxysterol (OS) as a lipid mediator for tumor cell migration. In MCF-7 cells, insulin-like growth factor 1 elevates the expression of OS-binding protein-related protein 7. Binding of OS to OS-binding protein or OS-binding protein-related protein is known to trigger elevation of sphingomyelin, a sphingolipid that organizes lipid microdomains in the cell membrane. In MCF-7 cells, OS increases the intracellular concentration of sphingomyelin and other phospholipids and induces the translocation of the small GTPase p21Ras to GM1- and cholesterol-rich membrane areas. The induction of migration by MG63CM is prevented by incubation of MG63 cells with mevinolin, a statin-type cholesterol biosynthesis inhibitor that depletes the conditioned medium of OS. Osteoblast-derived OS may, thus, be a yet unrecognized lipid mediator for bone metastasis of breast cancer and a new target for anti-metastasis chemotherapy with statins.

Prostaglandin biosynthesis by midgut tissue isolated from the tobacco hornworm, Manduca sexta.

We describe prostaglandin (PG) biosynthesis by isolated midgut preparations from tobacco hornworms, Manduca sexta. Microsomal-enriched midgut preparations yielded four PGs, PGA/B(2), PGD(2), PGE(2) and PGF(2alpha), all of which were confirmed by analysis on gas chromatography--mass spectrometry (GC--MS). PGA and PGB are double bond isomers which do not resolve on TLC but do resolve by GC; for convenience, we use the single term PGA(2) for this product. PGA(2) was the major product under most conditions. The midgut preparations were sensitive to reaction conditions, including radioactive substrate, protein concentration (optimal at 1mg/reaction), reaction time (optimal at 0.5 min), temperature (optimal at 22 degrees C), buffer pH (highest at pH 6), and the presence of a co-factor cocktail composed of reduced glutathione, hydroquinine and hemoglobin. In vitro PG biosynthesis was inhibited by two cyclooxygenase inhibitors, indomethacin and naproxen. Subcellular localization of PG biosynthetic activity in midgut preparations, determined by ultracentrifugation, revealed the presence of PG biosynthetic activity in the cytosolic and microsomal fractions, although most activity was found in the cytosolic fractions. This is similar to other invertebrates, and different from mammalian preparations, in which the activity is exclusively associated with the microsomal fractions. Midgut preparations from M. sexta pupae, adult cockroach, Periplaneta americana, and corn ear worms, Helicoverpa zea, also produced the same four major PG products. We infer that insect midguts are competent to biosynthesize PGs, and speculate they exert important, albeit unrevealed, actions in midgut physiology.

Structural insights into human serum albumin-mediated prostaglandin catalysis.

Previous studies have shown that many arachidonic acid metabolites bind to human serum albumin (HSA) and that the metabolism of these molecules is altered as a result of binding. The present study attempted to gain insights into the mechanisms by which prostaglandins bound to subdomain 2A of HSA are metabolized by catalytic processes. The breakdown of the prostaglandin 15-keto-PGE(2) to 15-keto-PGA(2) and 15-keto-PGB(2) in the presence of wild-type HSA and a number of subdomain 2A mutants was examined using a previously validated spectroscopic method which monitors absorbance at 505 nm. The species examined using this method were wild-type HSA, K195M, K199M, F211V, W214L, R218M, R218P, R218H, R222M, H242V, R257M, and bovine serum albumin. Previous studies of HSA-mediated catalysis indicated that the breakdown of HSA-bound prostaglandins results from an alkaline microenvironment in the binding site. Our results show that the catalytic breakdown of HSA-bound 15-keto-PGE(2) to 15-keto-PGB(2) results from two specific processes which are modulated by specific amino acid residues. Specifically, some amino acid residues modulate the rate of step 1, the conversion of 15-keto-PGE(2) to 15-keto-PGA(2), while other residues modulate the rate of step 2, the conversion of 15-keto-PGA(2) to 15-keto-PGB(2). Some residues modulate the rate of steps 1 and 2. In total, while our results support the involvement of certain basic amino acid residues in the catabolism of HSA-bound 15-keto-PGE(2), our data suggest that metabolism of HSA-bound prostaglandins may be a more complex and specific process than previously thought.

A novel synthesis of racemic and enantiomeric forms of prostaglandin B1 methyl ester.

3-(Dimethoxyphosphorylmethyl)cyclopent-2-enone was converted into (+/-)-prostaglandin B1 methyl ester in two steps involving regioselective alkylation at C(2) with methyl 7-iodoheptanoate and subsequent Horner-Wittig reaction with dimer of 2-hydroxyheptanal (42% overall yield). The use of (R)- and (S)-2-(tert-butyldimethylsilyloxy)heptanal for the Horner olefination reaction gave, after deprotection of the hydroxy group, the enantiopure forms of the title compound in 28% overall yield.

Prostaglandin B(2) delivers a co-stimulatory signal leading to T cell activation.

Most of the data accumulated to date on the immunoregulatory effects of prostaglandins (PG) on T cell activation stem from the archetypal inhibitory effect of PGE(2). In this study we provide instead, the first evidence that exogenous PGB(2), a catabolic metabolite of PGE(2), synergizes with signals delivered by T cell receptor (TCR) engagement to induce interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) alpha-expression in Jurkat cells. Accordingly, PGB(2) enhances the proliferation of anti-CD3-activated peripheral blood lymphocytes (PBL). In terms of cellular signaling, we present evidence that PGB(2) activates tyrosine kinase activities and efficiently increases c-fos mRNA expression and nuclear factor-kappa B (NF-kappa B) translocation to the nucleus. Owing to these features, PGB(2) appears as a new lipid mediator capable of delivering an ancillary signal leading to T lymphocyte activation.

The wide binding properties of a wheat nonspecific lipid transfer protein. Solution structure of a complex with prostaglandin B2.

The 3D solution structure of wheat nonspecific lipid transfer protein (ns-LTP) complexed with prostaglandin B2, a lipid with both vinyl and hydroxylated groups, has been determined by 1H 2D NMR. The global fold of the protein is close to the previously published structures of wheat, maize, barley and rice ns-LTPs. The ligand is almost completely embedded in the hydrophobic core of the protein. Structure comparisons of free and bound wheat ns-LTP reveal that the binding of prostaglandin B2 hardly affects the global fold of the protein. The structural data on this unusual complex are discussed and compared with other known ns-LTP lipid-complexes.

The percutaneous penetration of prostaglandin E1 and its alkyl esters.

The percutaneous delivery of PGE1 and its alkyl esters in alcoholic saline solution through hairless mouse skin was compared. The quantification of alkyl esters was based on the same principle as that for PGE1, which was converted to PGB1 to enhance the sensitivity and minimize the interference. Results showed that it was PGE1 that appeared in the receiver compartment for all alkyl esters examined. The flux of all alkyl esters of PGE1 in the same concentration was higher than PGE1 itself at most of saline vehicle with various fractions of alcohol. The maximal flux for a fixed concentration of each alkyl ester appeared at different fractions of alcohol. When the fractions of alcohol was kept constant, the alkyl ester that showed the maximal flux at this concentration appeared to have a longer chain length with increasing the fraction of alcohol. But isopropyl ester deviated from this order. It was concluded that the alkyl ester derivatives promoted the penetration of PGE1 mainly as a result of enhancing the drug partitioning into the stratum corneum. The alcohol fraction that needed to achieve the maximal flux at the same concentration increased with the increase of alkyl chain length, which resulted in the decrease of solubility parameter. It is necessary to optimize the fraction of alcohol in the saline solution in order to achieve the maximal flux at a fixed concentration for these alkyl esters with different alkyl chain length.

LTB4 as marker of 5-LO inhibitory activity of two new N-omega-ethoxycarbonyl-4-quinolones.

The supposed 5-LO inhibitory activity of two N-omega-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from cell cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.3, containing 12.5 mM SDS as micelles generator. Therefore, following the decreasing of LTB4 it was possible to verify the 5-LO inhibitory activity of two quinolone derivatives. To asses the suitability of the use of LTB4 as marker of the activity of the new compounds, the analysis was repeated using quercetin, a well known 5-LO inhibitor.

On-line coupling of microdialysis sampling with liquid chromatography for the determination of peptide and non-peptide leukotrienes.

An automated on-line sampling method was developed using microdialysis as the simultaneous sampling and sample pre-treatment technique. The extraction fraction values of microdialysis probes sampling different eicosanoids were investigated. The impact of cyclodextrins in the perfusion liquid used for sampling hydrophobic eicosanoids in biological systems was also studied. The total time for one analysis was 7.6 min allowing seven measurements per hour for monitoring kinetic changes in biological systems.

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 µg/ml PGB2 inhibited virus yield by 60 percent, at the same dose PGA1 suppressed virus replication by more than 90 percent. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2

Absence of relation between the expression of cyclooxygenase isoforms and the synthesis of prostaglandin E2 in resident and thioglycollate-elicited macrophages in rats.

Macrophages exudating into inflammatory sites (thioglycollate-elicited macrophages, TGM) have a diminished ability to synthesize prostaglandins (PG) as compared with resident peritoneal macrophages (RM). Constitutive expression of cyclooxygenase-1 (COX-1) was lower in TGM than in RM but the releasability of arachidonic acid was not significantly different. Thus, the differences in expression of COX-1 were primarily responsible for the abilities of TGM and RM to synthesize PGE2 upon calcium ionophore (CaI) stimulation. COX-1 expression in RM and TGM was also correlated with their ability to synthesize PGE2 from exogenously added arachidonic acid. When exposed to lipopolysaccharide (LPS), the induction of COX-2 and the enhancement of PGE2 synthesis upon CaI were much lower in TGM as compared with RM the releasability of arachidonic acid upon CaI stimulation was relatively unchanged in RM but was reduced in TGM Thus, in TGM as compared with RM, a lower level of COX-1 expression and a lower level of COX-2 induction, and the reduction of arachidonate releasability by LPS exposure, are mainly responsible for lower PGE2 synthetic ability upon CaI stimulation. However, the different COX-2 induction by LPS in RM and TGM was not reflected in their increase in the ability to synthesize PGE2 from exogenously added arachidonic acid.

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells.

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 micrograms/ml PGB2 inhibited virus yield by 60%, at the same dose PGA1 suppressed virus replication by more than 90%. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2.