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Planaria tissue regeneration

Planaria (Dugesia japonica) were kept in water (Poland Spring; Nestle Waters North America, Stamford, CT, USA) at 18°C. All animals were starved for at least 7 days before the experiments. Tissue regeneration was assessed as described []. In brief, planaria were subjected to head resection postocularly (surgical injury). The posterior portions of the planaria were then placed in spring water containing 0.01% EtOH, or MCTR at indicated concentrations. The extent of tissue regeneration during a 6-day period was determined using captured images of regenerating blastemas at regular intervals (24 h). These images were analyzed using ImageJ software (NIH, Bethesda, MD, USA) to give a tissue regeneration index (TRI) [].

Results

Having matched the physical properties of synthetic, stereochemically defined materials with those of endogenously produced MCTRs from both human and murine systems, we next required assessment of their potent biological actions. Given that MCTRs display tissue regenerative actions, we assessed the ability of each synthetic MCTR to promote tissue regeneration. Planaria were surgically injured by rapidly removing the head portion of the animals and tissue regeneration was followed over time []. Planaria undergo both physiological and restorative regeneration via evolutionarily conserved pathways, making this an ideal system to characterize novel chemical signals involved in tissue regeneration []. When planaria were kept with vehicle alone, maximum tissue regeneration (TRI max) occurred within ~6 days with time to 50% regeneration (T50) occurring by ~2.7 days. Incubation of planaria with MCTR1 led to acceleration in tissue regeneration with a reduction in T50 from ~2.7 days to ~2 days when planaria were incubated with 100nM of MCTR1 and to ~1.9 days when incubated with 1nM (). MCTR2 also accelerated tissue regeneration with planaria, where incubation of surgically injured planaria with MCTR2 reduced T50 to ~1.8 days () and at 1nM reduced T50 to ~1.9 days. The new MCTR3 also demonstrated potent tissue regenerative actions that were dose dependent (). Assessment of their relative potencies at stimulating tissue regeneration demonstrated that MCTR3 and MCTR2 were the most potent of the three, followed by MCTR1. Hence, MCTR3 ≈ MCTR2>MCTR1 ().

E. coli peritonitis

Peritonitis experiments were conducted as in []. Briefly, FVB mice (6-8-weeks old), purchased from Charles River Laboratories (Wilmington, MA, USA) were fed ad libitum Laboratory RodentDiet 20–5058 (LabDiet; Purina Mills, St. Louis, MO, USA) and housed at a maximum of four animals per cage and maintained in 12:12 hour light-dark cycles. Mouse experimental procedures were approved by the Standing Committee on Animals of Harvard Medical School (Protocol 02570) and complied with institutional and U.S. National Institutes of Health (NIH) guidelines. No surgical procedures were conducted and no analgesics were administered. Mice were euthanized by overdose of isoflurane via inhalation in saturated atmosphere, followed by cervical dislocation. If recovery from procedure is compromised as assessed by increase in self grooming, aggressive behavior or rapid movements, self-mutilation, absent activity, lack of eating or drinking, abdominal distention or redness and heat over abdominal area, humane endpoint in Protocol 02570 requires that mice must be euthanized immediately by isoflurane overdose, followed by cervical dislocation. During this procedure, animals did not experience more than slight or momentary pain or distress and no animals died prior to experimental endpoint. E. coli (serotypeO6:K2:H1) was cultured in Luria-Bertani broth and harvested at midlog phase (OD600 nm, ~0.5 absorbance units). In determined experiments mice were administered MCTR1, MCTR2, MCTR3 (50ng/mouse) or vehicle via intraperitoneal (i.p) injection and after 5 minutes inoculated with E. coli (105 CFU/mouse). Exudates were collected at the indicated intervals and leukocyte populations as well as bacterial phagocytosis and macrophage efferocytosis determined using flow cytometry and fluorescent-labeled antibodies []. In select experiments, mice were inoculated with E. coli (105 CFU/mouse), after 12h administered MCTR1, MCTR2, MCTR3 (100ng/mouse) or vehicle i.p. and exudates collected after 24h with leukocyte populations and bacterial phagocytosis determined as above.

Results

Given the increased incidence of bacterial infections [, ] and that MCTR1 and MCTR2 each displays potent actions promoting resolution of infections, we could now test whether each of the stereochemically pure MCTR1, 2 and 3 also carried these actions in murine infections. E. coli (1x105 CFU/mouse) inoculation gave a self-resolving inflammatory response with maximal neutrophil infiltration at ~12 h that subsequently declined, which gave a calculated resolution interval (Ri) of ~12h (). MCTR1 (50ng/mouse) injection immediately prior to E. coli inoculation gave a significant reduction in neutrophil infiltration at the 12 h interval, and shortened the resolution interval from ~12h to ~4h (). Administration of MCTR2 also gave significant reductions in exudate leukocyte counts (~50%), a shortening in the Ri to ~2h and an increase in phagocytosis of E. coli (~50%) by exudate leukocytes. These results with synthetic MCTRs are in accordance with published findings with biologically produced MCTRs [] and confirm the potent actions of these novel mediators in infections. Of note, MCTR3 given immediately prior to E. coli inoculation reduced exudate neutrophil levels by ~54%, shortening the Ri from 12 h to 3 h (). In addition to reducing neutrophil infiltration, each of three MCTR significantly increased leukocyte phagocytosis of E. coli, with MCTR2 and MCTR3 displaying the highest potencies (). MCTR1 was the most potent in stimulating macrophage efferocytosis (), a key defining pro-resolving action.

E. coli peritonitis

Peritonitis experiments were conducted as in []. Briefly, FVB mice (6-8-weeks old), purchased from Charles River Laboratories (Wilmington, MA, USA) were fed ad libitum Laboratory RodentDiet 20–5058 (LabDiet; Purina Mills, St. Louis, MO, USA) and housed at a maximum of four animals per cage and maintained in 12:12 hour light-dark cycles. Mouse experimental procedures were approved by the Standing Committee on Animals of Harvard Medical School (Protocol 02570) and complied with institutional and U.S. National Institutes of Health (NIH) guidelines. No surgical procedures were conducted and no analgesics were administered. Mice were euthanized by overdose of isoflurane via inhalation in saturated atmosphere, followed by cervical dislocation. If recovery from procedure is compromised as assessed by increase in self grooming, aggressive behavior or rapid movements, self-mutilation, absent activity, lack of eating or drinking, abdominal distention or redness and heat over abdominal area, humane endpoint in Protocol 02570 requires that mice must be euthanized immediately by isoflurane overdose, followed by cervical dislocation. During this procedure, animals did not experience more than slight or momentary pain or distress and no animals died prior to experimental endpoint. E. coli (serotypeO6:K2:H1) was cultured in Luria-Bertani broth and harvested at midlog phase (OD600 nm, ~0.5 absorbance units). In determined experiments mice were administered MCTR1, MCTR2, MCTR3 (50ng/mouse) or vehicle via intraperitoneal (i.p) injection and after 5 minutes inoculated with E. coli (105 CFU/mouse). Exudates were collected at the indicated intervals and leukocyte populations as well as bacterial phagocytosis and macrophage efferocytosis determined using flow cytometry and fluorescent-labeled antibodies []. In select experiments, mice were inoculated with E. coli (105 CFU/mouse), after 12h administered MCTR1, MCTR2, MCTR3 (100ng/mouse) or vehicle i.p. and exudates collected after 24h with leukocyte populations and bacterial phagocytosis determined as above.

Results

We also tested whether MCTR carry pro-resolving actions when administered at the peak of inflammation during E. coli infections. Administration of MCTR1 at the peak of inflammation (12h interval) gave reductions in exudate neutrophil counts at 24h and shortened Ri to ~10 h (). In mice given MCTR2 we found reductions in exudate neutrophil counts (~50%) giving an Ri of ~7 h (). At equal doses, MCTR3 also promoted the resolution of E. coli infections, reducing exudate neutrophil counts by ~50% and shortening the Ri to ~9 h (). Together these results demonstrate the potent biological actions of MCTR1 and MCTR2, confirming their original structural assignments (). Moreover, we describe here the unexpected actions of MCTR3 in accelerating the resolution of E. coli infections and establish the rank order potencies in promoting the resolution of infectious-inflammation.

Phagocytosis

Human macrophages and neutrophils were prepared from peripheral blood leukocytes [] and in accordance with the approved Partners Human Research Committee Protocol (1999P001297). Briefly, macrophages were plated in 96-well plates (5x104 cells per well) for 24h, neutrophils were plated in 96-well plates (1x105 cells per well) for 30 min and phagocytosis or efferocytosis was assessed as in []. For efferocytosis, apoptotic PMNs were obtained by culturing neutrophils overnight in PBS-/- (5x106 cells/ml). Apoptotic human PMNs were labeled with bisBenzimide trihydrochloride (Sigma-Aldrich). For efferocytosis, human macrophages were incubated with MCTR1, MCTR2, MCTR3 (1pM-10nM), or vehicle then labeled apoptotic PMNs (2.5x105 cells) and incubated for 60 min (37°C pH 7.45). Fluorescence was measured using a SpectraMax M3 plate reader (Molecular Devices, Sunnyvale, CA, USA), and results were analyzed using SoftMax Pro (Molecular Devices). In select experiments, macrophages were incubated for 15 minutes with vehicle, MCTR1, MCTR2, MCTR3 (1pM-10nM) or a mixture of MCTR1+MCTR2, MCTR2+MCTR3, or MCTR1+MCTR3 (0.1nM each). BacLight Green Bacterial Stain (Life Technologies, Eugene, OR, USA) labeled E. coli (2.5x 106 CFU/well) were added, cells incubated for 60 min (37°C pH 7.45) and fluorescence assessed using the fluorescence plate reader. In separate experiments, human neutrophils were incubated with MCTR1, MCTR2, MCTR3 (1pM-10nM), or vehicle then with BacLight Green Bacterial Stain-labeled E. coli (2.5x 106 CFU/well; 60 min; 37°C pH 7.45) and fluorescence assessed as above.

Abstract

The cell wall is one major determinant of plant cell morphology, and is an attractive bioresource. Here, we report a novel strategy to modify plant cell wall property by small molecules. Lasalocid sodium (LS) was isolated by chemical screening to identify molecules that affect the cell morphology of tobacco BY-2 cells. LS treatment led to an increase in cell wall thickness, whilst the quantity and sugar composition of the cell wall remained unchanged in BY-2 cells. The chemical also disordered the cellular arrangement of hypocotyls of Arabidopsis plants, resulting in a decrease in hypocotyl length. LS treatment enhanced enzymatic saccharification efficiency in both BY-2 cells and Arabidopsis plants. Microarray analysis on Arabidopsis showed that exposure to LS upregulated type III peroxidase genes, of which some are involved in lignin biogenesis, and jasmonic acid response genes, and phloroglucinol staining supported the activation of lignification by the LS treatment. As jasmonic acid-mediated lignification is a typical reaction to cell wall damage, it is possible that LS induces cell wall loosening, which can trigger cell wall damage response. Thus, LS is a unique chemical for modification of cell wall and morphology through changes in cell wall architecture.

LS changed cell wall thickness without obvious alteration to wall quantity and sugar composition

To investigate the effect of LS on cell wall structure, we observed BY-2 cell walls by transmission electron microscopy after LS treatment (). Cell wall thickness increased in a dose-dependent manner, consistent with observations obtained from the confocal microscope. Thickness when treated with 1.0 μM LS was approximately twice that of non-treated cells (). Solid-state NMR analysis showed that LS treatment hardly changed the cellulose crystallinity of the cell wall (). This result indicated that change in wall thickness was not related to cellulose crystallinity.

LS changed cell wall thickness without obvious alteration to wall quantity and sugar composition

Next, we checked the quantity of the cell wall and its cellulose, hemicellulose and pectin content with and without LS treatment. No significant differences were found in the amount of cell wall fraction or in the contents of each cell wall component between these samples ( and ). Analysis of the sugar composition of the matrix polysaccharide fraction also showed that the amounts of sugars in the LS treated samples were comparable to the non-treated samples (). Therefore, increase in cell wall thickness by LS does not result from changes in the amount or the chemical composition of the cell wall.

LS increased saccharification efficiency

To further analyze the effect of LS on properties of the cell wall, we investigated enzymatic saccharification efficiency of the cell wall material. Preparations from BY-2 cells with and without chemical treatment were subjected to enzymatic saccharification analysis. Results showed that LS-treated samples released approximately 1.2 times as much glucose as non-treated samples (). This increase was found from early stages of incubation with the enzymes. Taken together with observations that LS affected neither cellulose crystallinity nor compositional properties of the cell wall ( and and ), the increase in enzymatic saccharification efficiency by the treatment is caused by changes in structural properties of cell wall components other than cellulose crystallinity.

Effects of LS treatment on Arabidopsis seedlings

To investigate effects of LS on plant growth, Arabidopsis thaliana seeds were germinated and grown continuously on MS agar medium containing 0, 0.1, 0.5, 1.0 or 5.0 μM of LS under dark conditions. Germination was severely inhibited by 5.0 μM, and elongation of hypocotyls was affected on medium containing 0.5 and 1.0 μM (upper panels in ). Abnormal-shaped cells and reduced cell length were observed in hypocotyls grown in the presence of 0.5 and 1.0 μM LS, (lower panels in ), suggesting that inhibition of hypocotyl elongation is mainly attributed to abnormal cell elongation and reduction of cell length. Moreover, we found that LS induced opening of cotyledons even in the dark, when cotyledons generally remain closed (). We further checked enzymatic saccharification efficiency of Arabidopsis samples treated with or without LS. As with the BY-2 cells, chemical-treated samples showed higher amounts of released glucose compared with non-treated samples (approximately 1.3 times) from early stages of incubation with the enzymes (). These results indicated that LS treatment can change cell wall properties not only in culture cells but also in plants.

LS induced cell wall damage response in Arabidopsis plants

To examine the effects of LS on gene expression, we performed microarray analysis using 3-day-old dark-grown Arabidopsis seedlings treated with or without the chemical for 24 hours. The results showed that 1,430 and 1,526 transcripts were significantly increased and decreased, respectively, by LS treatment (threshold value of fold change is 1.5 and p < 0.05). Gene ontology analysis showed that up-regulated genes were enriched in extracellular, membrane-related and cell wall-related components in “Cellular Component” categories and that genes annotated with enzymatic activities and transporter activity were enriched in the “Molecular Component” categories (). The most remarkable finding from these enrichment analyses is that, of the transcripts that were increased, 23 encode class III secretable peroxidase family proteins, a part of which are presumed to function in lignification,,, and 27 encode jasmonic acid (JA)-related proteins including JA biosynthetic enzymes and JA-mediated signaling proteins ( and ). Besides, some transcripts for lignin biosynthesis-related genes were also increased by LS treatment (). Previous works have suggested that reactive oxygen species (ROS) production and JA signaling are important cross-talk modules to regulate cell wall damage-induced lignin biosynthesis,. Thus, it was possible that LS triggers cell wall damage response in plants.

LS induced cell wall damage response in Arabidopsis plants

To check if high levels of peroxidase gene expression induced by LS are associated with increased peroxidase activity, mm2d Arabidopsis suspension cells were incubated with different concentrations of 0, 0.1, 0.5, 1.0 and 5.0 μM LS for 24 h, and then subjected to diaminobenzidine (DAB) staining analysis. The results showed that DAB staining signal increased as concentration of LS increased, indicating that the chemical activates peroxidase activity through up-regulation of peroxidase gene expression and releases ROS (). Next, we checked whether LS treatment induced lignin accumulation by staining with phloroglucinol-HCl, which dyes lignin-containing tissues a red-violet color. We observed that phloroglucinol staining was more apparent in vascular tissues of plants grown on medium containing 1 μM LS compared with control plants (). These facts show that LS increases peroxidase activity by up-regulation of class III peroxidase genes in plant cells, and that the increased peroxidase activity would increase lignification activity.

Abstract

[18F]Fluciclovine (trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid; anti-[18F]FACBC), a positron emission tomography tracer used for the diagnosis of recurrent prostate cancer, is transported via amino acid transporters (AATs) with high affinity (Km: 97–230 μM). However, the mechanism underlying urinary excretion is unknown. In this study, we investigated the involvement of AATs and drug transporters in renal [18F]fluciclovine reuptake. [14C]Fluciclovine (trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid) was used because of its long half-life. The involvement of AATs in [14C]fluciclovine transport was measured by apical-to-basal transport using an LLC-PK1 monolayer as model for renal proximal tubules. The contribution of drug transporters herein was assessed using vesicles/cells expressing the drug transporters P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated protein 4 (MRP4), organic anion transporter 1 (OAT1), organic anion transporter 3 (OAT3) , organic cation transporter 2 (OCT2), organic anion transporting polypeptide 1B1 (OATP1B1), and organic anion transporting polypeptide 1B3 (OATP1B3). The apical-to-basal transport of [14C]fluciclovine was attenuated by l-threonine, the substrate for system alanine-serine-cysteine (ASC) AATs. [14C]Fluciclovine uptake by drug transporter-expressing vesicles/cells was not significantly different from that of control vesicles/cells. Fluciclovine inhibited P-gp, MRP4, OAT1, OCT2, and OATP1B1 (IC50 > 2.95 mM). Therefore, system ASC AATs may be partly involved in the renal reuptake of [18F]fluciclovine. Further, given that [18F]fluciclovine is recognized as an inhibitor with millimolar affinity for the tested drug transporters, slow urinary excretion of [18F]fluciclovine may be mediated by system ASC AATs, but not by drug transporters.

3. Discussion

We conducted a series of studies to assess the involvement of AATs and drug transporters in the distribution of [14C]fluciclovine in vitro, thereby specifically focusing on renal reuptake. The apical-to-basal transport of [14C]fluciclovine in the LLC-PK1 monolayer model was inhibited by Thr, a substrate representative for system ASC AATs [,]. Furthermore, control vesicles/cells and ABC/SLC drug transporter-expressing vesicles/cells showed no significant differences in drug transporter-mediated [14C]fluciclovine uptake. Fluciclovine at millimolar concentrations inhibited the uptake of specific substrates by some of the drug transporters tested. Our study suggests that the slow urinary excretion of [18F]fluciclovine is partly mediated by system ASC AATs, which serve as high-affinity transporters for [18F]fluciclovine uptake/efflux, but that the contribution of drug transporters to [18F]fluciclovine distribution is negligible compared to that of AATs.

Abstract

The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). However, unlike the other P[II] RVs, which mainly infect humans, P[19] RVs commonly infect animals (pigs), making P[19] unique for the study of RV diversity and host ranges. Through in vitro binding assays and saturation transfer difference (STD) nuclear magnetic resonance (NMR), we found that P[19] could bind mucin cores 2, 4, and 6, as well as type 1 histo-blood group antigens (HBGAs). The common sequences of these glycans serve as minimal binding units, while additional residues, such as the A, B, H, and Lewis epitopes of the type 1 HBGAs, can further define the binding outcomes and therefore likely the host ranges for P[19] RVs. This complex binding property of P[19] is shared with the other three P[II] RVs (P[4], P[6], and P[8]) in that all of them recognized the type 1 HBGA precursor, although P[4] and P[8], but not P[6], also bind to mucin cores. Moreover, while essential for P[4] and P[8] binding, the addition of the Lewis epitope blocked P[6] and P[19] binding to type 1 HBGAs. Chemical-shift NMR of P[19] VP8* identified a ligand binding interface that has shifted away from the known RV P-genotype binding sites but is conserved among all P[II] RVs and two P[I] RVs (P[10] and P[12]), suggesting an evolutionary connection among these human and animal RVs. Taken together, these data are important for hypotheses on potential mechanisms for RV diversity, host ranges, and cross-species transmission.

Expression and purification of VP8* proteins in Escherichia coli.

The VP8* sequences (amino acids 46 to 231) from a human RV P[19] (GenBank accession number ) was expressed in E. coli BL21 and purified by glutathione S-transferase (GST) tag affinity purification, as described previously (). Recombinant VP8* proteins of other P[II] RVs (P[4], P[6], and P[8]) and a P[10] RV were also purified using the same expression system. 15N- or 15N,13C-labeled P[19] VP8* proteins were also made for the NMR studies using the same E. coli culture procedures, with the addition of 15N-minimal growth medium (22 mM KH2PO4, 50 mM Na2HPO4, 8.5 mM NaCl, 0.1 mM CaCl2, 2 mM MgSO4, 1× BME vitamins (catalog no. B6891; Sigma), 18 mM 15NH4Cl, and 22 mM glucose) or 15N,13C-minimal growth medium supplied with 22 mM [13C]glucose (). The GST tag of the labeled VP8* proteins was removed for the NMR study by cutting with thrombin (Sigma-Aldrich Co., St. Louis, MO), followed by fast protein liquid chromatography (FPLC) gel filtration to collect the VP8* protein fractions and concentration by ultracentrifugation. The purified VP8* proteins were stored at −80°C until needed.

STD NMR experiments.

All saturation transfer difference (STD) NMR spectra were acquired in Shigemi Tubes (Shigemi, USA) with a Bruker 600-MHz Avance III spectrometer at 10°C using a conventional HCN probe and the TopSpin 3.1 pulse sequence STDDIFFGG19.3. The NMR sample for mucin core 2 in complex with P[19] VP8* was prepared with 200 μM protein and 6.0 mM ligand in a final volume of 300 μl of Tris NMR buffer. The LNFPI STD sample was prepared with 130 μM P[19] VP8* and 6.6 mM LNFPI in a final volume of 300 μl of phosphate NMR buffer. This results in total protein-ligand ratios of 1:35 and 1:50 for the mucin core 2 and LNFPI samples, respectively. The protein resonances were saturated at 0 ppm with a cascade of 40 Gaussian-shaped pulses with duration of 50 ms, resulting in a total saturation time of 2 s. The off-resonance saturation was applied to 30 ppm, and 1,024 experiments were acquired for each with a 5-s recycle delay. A spin-lock filter with strength of 10 kHz and duration of 100 to 200 ms was applied to suppress the broad protein resonance signals, and Watergate 3-9-19 was used to suppress the residual water signal. The STD spectra were obtained by subtracting the on-resonance spectrum from the off-resonance reference spectrum. STD amplification factors were determined by the equation (Io − Isat)/Io, where Io and Isat are the intensities of the signals in the reference and saturated spectra, respectively, and the normalized STD amplification percentage was determined by dividing by the largest STD amplification factor for each ligand. The protons with the largest STD amplification percentage along with data from the 2D STD TOCSY spectrum, which were used to resolve overlap from the 1D spectra, were used to assign the binding epitopes.

P[19] VP8* recognizes mucin cores and the type 1 HBGA precursor.

The ligand binding specificity of the P[19] VP8* was further characterized by STD NMR. Data for a type 1 HBGA pentasaccharide, LNFPI (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), demonstrated that the extended type 1 chain of an internal Gal (Fucα1-2Galβ1-3GlcNAcβ1-3Gal) is also involved in the interaction with P[19] VP8*, in addition to the GlcNAc and the terminal (H) fucose ( and ). Similarly, STD NMR of P[19] VP8* with mucin core 2 showed a strong interaction with the disaccharide GlcNAcβ1-6GalNAc, in which the GlcNAc residue was highly involved ( and ).

Abstract

The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). However, unlike the other P[II] RVs, which mainly infect humans, P[19] RVs commonly infect animals (pigs), making P[19] unique for the study of RV diversity and host ranges. Through in vitro binding assays and saturation transfer difference (STD) nuclear magnetic resonance (NMR), we found that P[19] could bind mucin cores 2, 4, and 6, as well as type 1 histo-blood group antigens (HBGAs). The common sequences of these glycans serve as minimal binding units, while additional residues, such as the A, B, H, and Lewis epitopes of the type 1 HBGAs, can further define the binding outcomes and therefore likely the host ranges for P[19] RVs. This complex binding property of P[19] is shared with the other three P[II] RVs (P[4], P[6], and P[8]) in that all of them recognized the type 1 HBGA precursor, although P[4] and P[8], but not P[6], also bind to mucin cores. Moreover, while essential for P[4] and P[8] binding, the addition of the Lewis epitope blocked P[6] and P[19] binding to type 1 HBGAs. Chemical-shift NMR of P[19] VP8* identified a ligand binding interface that has shifted away from the known RV P-genotype binding sites but is conserved among all P[II] RVs and two P[I] RVs (P[10] and P[12]), suggesting an evolutionary connection among these human and animal RVs. Taken together, these data are important for hypotheses on potential mechanisms for RV diversity, host ranges, and cross-species transmission.

Expression and purification of VP8* proteins in Escherichia coli.

The VP8* sequences (amino acids 46 to 231) from a human RV P[19] (GenBank accession number ) was expressed in E. coli BL21 and purified by glutathione S-transferase (GST) tag affinity purification, as described previously (). Recombinant VP8* proteins of other P[II] RVs (P[4], P[6], and P[8]) and a P[10] RV were also purified using the same expression system. 15N- or 15N,13C-labeled P[19] VP8* proteins were also made for the NMR studies using the same E. coli culture procedures, with the addition of 15N-minimal growth medium (22 mM KH2PO4, 50 mM Na2HPO4, 8.5 mM NaCl, 0.1 mM CaCl2, 2 mM MgSO4, 1× BME vitamins (catalog no. B6891; Sigma), 18 mM 15NH4Cl, and 22 mM glucose) or 15N,13C-minimal growth medium supplied with 22 mM [13C]glucose (). The GST tag of the labeled VP8* proteins was removed for the NMR study by cutting with thrombin (Sigma-Aldrich Co., St. Louis, MO), followed by fast protein liquid chromatography (FPLC) gel filtration to collect the VP8* protein fractions and concentration by ultracentrifugation. The purified VP8* proteins were stored at −80°C until needed.

STD NMR experiments.

All saturation transfer difference (STD) NMR spectra were acquired in Shigemi Tubes (Shigemi, USA) with a Bruker 600-MHz Avance III spectrometer at 10°C using a conventional HCN probe and the TopSpin 3.1 pulse sequence STDDIFFGG19.3. The NMR sample for mucin core 2 in complex with P[19] VP8* was prepared with 200 μM protein and 6.0 mM ligand in a final volume of 300 μl of Tris NMR buffer. The LNFPI STD sample was prepared with 130 μM P[19] VP8* and 6.6 mM LNFPI in a final volume of 300 μl of phosphate NMR buffer. This results in total protein-ligand ratios of 1:35 and 1:50 for the mucin core 2 and LNFPI samples, respectively. The protein resonances were saturated at 0 ppm with a cascade of 40 Gaussian-shaped pulses with duration of 50 ms, resulting in a total saturation time of 2 s. The off-resonance saturation was applied to 30 ppm, and 1,024 experiments were acquired for each with a 5-s recycle delay. A spin-lock filter with strength of 10 kHz and duration of 100 to 200 ms was applied to suppress the broad protein resonance signals, and Watergate 3-9-19 was used to suppress the residual water signal. The STD spectra were obtained by subtracting the on-resonance spectrum from the off-resonance reference spectrum. STD amplification factors were determined by the equation (Io − Isat)/Io, where Io and Isat are the intensities of the signals in the reference and saturated spectra, respectively, and the normalized STD amplification percentage was determined by dividing by the largest STD amplification factor for each ligand. The protons with the largest STD amplification percentage along with data from the 2D STD TOCSY spectrum, which were used to resolve overlap from the 1D spectra, were used to assign the binding epitopes.

P[19] VP8* recognizes mucin cores and the type 1 HBGA precursor.

The ligand binding specificity of the P[19] VP8* was further characterized by STD NMR. Data for a type 1 HBGA pentasaccharide, LNFPI (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), demonstrated that the extended type 1 chain of an internal Gal (Fucα1-2Galβ1-3GlcNAcβ1-3Gal) is also involved in the interaction with P[19] VP8*, in addition to the GlcNAc and the terminal (H) fucose ( and ). Similarly, STD NMR of P[19] VP8* with mucin core 2 showed a strong interaction with the disaccharide GlcNAcβ1-6GalNAc, in which the GlcNAc residue was highly involved ( and ).

Abstract

Activation of an aberrant glycosylation pathway in cancer cells can lead to expression of the onco-foetal sialyl-Tn (sTn) antigen. STn is a truncated O-glycan containing a sialic acid α-2,6 linked to GalNAc α-O-Ser/Thr and is associated with an adverse outcome and poor prognosis in cancer patients. The biosynthesis of the sTn antigen has been linked to the expression of the sialytransferase ST6GalNAc1, and also to mutations in and loss of heterozygosity of the COSMC gene. sTn neo- or over-expression occurs in many types of epithelial cancer including gastric, colon, breast, lung, oesophageal, prostate and endometrial cancer. sTn is believed to be carried by a variety of glycoproteins and may influence protein function and be involved in tumour development. This review discusses how the role of sTn in cancer development and tumour cell invasiveness might be organ specific and occur through different mechanisms depending on each cancer type or subtype. As the sTn-antigen is expressed early in carcinogenesis targeting sTn in cancer may enable the targeting of tumours from the earliest stage.

5. The Role of sTn in Cancer Progression

Targeting sTn carrier proteins is a potential cancer therapeutic option. It remains open whether sTn might be carried on cancer stem cell marker proteins but as these proteins can be modified with O-glycans this seems a likely possibility []. The expression of sTn is generally reported to be heterogeneous within tumours [,,,,,] and could be regulated in the tumour via the expression of carrier proteins. As sTn can be carried by different glycoproteins in different compartments, its role in cancer progression may be different in different tumour types, and the effect of sTn expression on tumour cell invasiveness might be organ specific. Together these findings suggest that the biological function of sTn in promoting cancer development may occur through different mechanisms depending on each cancer type or subtype. Future studies using animal models expressing sTn in multiple cancer types and cell backgrounds will help further elucidate the role in sTn in cancer cell invasion and metastasis in different cancers.

Methods

To clarify the exposure of ERGO to brain and the possible antidepressant‐like effect after oral ingestion, ERGO or GOME (golden oyster mushroom extract) which contains 1.2% (w/w) ERGO was mixed with feed and provided to mice for 2 weeks, and then ERGO concentration and antidepressant‐like effect were evaluated by LC‐MS/MS and FST (forced swimming test) or TST (tail suspension test), respectively.

Results

Diet containing ERGO or GOME greatly increased the ERGO concentrations in plasma and brain, and significantly decreased the immobility time in both FST and TST. The required amount of GOME (~37 mg/day) to show the antidepressant‐like effect corresponds to at most 8 g/day in humans. In mice receiving GOME‐containing diet, doublecortin‐positive cells showed a significant increase from the basal level, suggesting promotion of neuronal differentiation.

Animals

C57BL/6J mice were purchased from Sankyo Labo Service Co. (Tokyo, Japan). About 5–8‐week‐old male mice were housed under pathogen‐free conditions at controlled temperature (21–25°C) with a 12 h light/dark cycle. The lights remained on from 8:00 to 20:00, and food and water were available ad libitum. The animals were cared for in strict compliance with the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal procedures used in this work were approved by the Kanazawa University Animal Care Committee.

Antidepressant‐like effect of ERGO

Next, we examined the antidepressant‐like effect of ERGO. GOME or ERGO was fed to mice in the diet for 2 weeks, and FST, which is a well‐established tool for assessing antidepressant‐like activity in rodents, was performed (Fig. B). The immobility time of mice given diet containing 10% GOME was significantly lower than that of mice given normal diet [F(5,70) = 2.9, P < 0.05] (Fig. A). To examine whether this activity of GOME is due to ERGO present in GOME, mice were fed diet containing authentic ERGO, the amount of which (120 mg ERGO/100 g diet) was equivalent to that in diet containing 10% GOME. The immobility time in mice given diet containing 10% GOME was similar to that given diet containing the equivalent amount of authentic ERGO (Fig. A). In addition, ERGO concentration in the brain in mice given diet containing 10% GOME was also similar to that given diet containing the equivalent amount of authentic ERGO (Fig. ). These data suggested that the antidepressant‐like effect of GOME is predominantly due to ERGO contained in the extract.

Abstract

Gamma tocopherol (gT) exhibits beneficial cardiovascular effects partly due to its anti-inflammatory activity. Important sources of gT are vegetable oils. However, little is known to what extent gT can be transferred into marine animal species such as Atlantic salmon by feeding. Therefore, in this study we have investigated the transfer of dietary gT into salmon. To this end, fish were fed a diet supplemented with 170 ppm gT for 16 weeks whereby alpha tocopherol levels were adjusted to 190 ppm in this and the control diet. Feeding gT-rich diets resulted in a three-fold increase in gT concentrations in the liver and fillet compared to non-gT-supplemented controls. Tissue alpha tocopherol levels were not decreased indicating no antagonistic interaction between gamma- and alpha tocopherol in salmon. The concentration of total omega 3 fatty acids slightly increased in response to dietary gT. Furthermore, dietary gT significantly decreased malondialdehyde in the fillet, determined as a biomarker of lipid peroxidation. In the liver of gT fed salmon we observed an overall down-regulation of genes involved in lipid homeostasis. Additionally, gT improved the antioxidant capacity by up-regulating Gpx4a gene expression in the pyloric caeca. We suggest that Atlantic salmon may provide a marine functional source capable of enriching gT for human consumption.

2.2. Tissue Levels of Tocopherols, Malondialdehyde, Antioxidant Enzymes and Fatty Acid Composition

In order to evaluate the transfer of Vitamin E from the diet into the tissues of Atlantic salmon we determined the gamma and alpha tocopherol concentrations in fish fillet and liver. As summarized in , feeding the gT enriched diet resulted in a three-fold increase of gT concentrations in both fillet and liver (p < 0.0001). However, alpha tocopherol levels in fillet and liver were similar between the groups indicating no antagonistic interaction between gamma and alpha tocopherol.

2.2. Tissue Levels of Tocopherols, Malondialdehyde, Antioxidant Enzymes and Fatty Acid Composition

Most methods for analyzing lipid peroxidation in animal tissues lack specificity and sensitivity []. However, MDA is an important biomarker of lipid peroxidation and may also partly reflect the sensory quality of fish []. Using a validated HPLC method [] to measure MDA, we found that MDA concentrations were significantly decreased by 25% in the fillet of gT-fed salmon as compared to the controls (3.3 ± 0.2 mmol·MDA/kg fillet in the control vs. 2.5 ± 0.2 mmol·MDA/kg fillet in gT-fed salmon, p < 0.02).

3. Discussion

Our data indicate that feeding Atlantic salmon with gT-rich diets resulted in a significant increase in gT concentrations in fish fillet, thereby improving its nutritional value. Thus, Atlantic salmon could be considered as a potential marine functional source of gT for human nutrition. However, future human trials (e.g., bioavailability studies, intervention studies) are required to test the hypothesis whether the consumption of gT-rich Atlantic salmon results in increased plasma levels of gT that may lead to potential anti-inflammatory and anti-atherogenic effects.

3. Discussion

The enrichment of fat soluble pro-vitamins and vitamins into the food matrix is a common practice in aquaculture. However, the transfer of micronutrients including Vitamin E from the diet into the animal tissue is always accompanied by a loss in assimilation. In order to quantify the apparent Vitamin E absorption, digestibility experiments need to be conducted which was beyond the scope of our study. Furthermore, Vitamin E transfer may be experimentally determined by its whole body retention. In order to do so, Vitamin E levels need to be quantified in all tissues at the beginning and the end of a feeding trial. However, within this study Vitamin E was only determined in fillet and liver at the end of the study. Thus, it was not possible to pinpoint the exact Vitamin E retention. However, analyzed alpha and gamma tocopherol levels in liver and fillet suggest that the tissue retention of alpha tocopherol was higher as compared to gamma tocopherol which is in line with literature data []. In terms of alpha tocopherol, it has been suggested that around 40% of dietary alpha tocopherol is retained in the fillet of Atlantic salmon []. Since in the fillet of our salmon following dietary supplementation, the gamma tocopherol concentration was 2.8 fold lower as compared to alpha tocopherol a transfer efficiency of around 15% may be assumed.

Abstract

YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.

6.1. Antioxidant Activity

Given the fact that chemical compounds belonging to the sesquiterpene group are well known for their antioxidant properties [,], perhaps antioxidant activity may be expected from nerolidol. Furthermore, EOs containing nerolidol derived from medicinal plants were found to exhibit antioxidant activity, suggesting its plausible utilization as an antioxidant agent. Indeed, it has been demonstrated that nerolidol exhibits potent antioxidant properties in counterbalancing the effect of ROS by protecting the cells against oxidative damage to lipids, proteins and DNA [,,]. According to a study conducted by Vinholes et al., the antioxidant activity of cis-nerolidol was evaluated using 1,1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging assay. The study revealed that cis-nerolidol exhibited DPPH scavenging activity []. In another study, cis-nerolidol was found to possess higher scavenging activity towards hydroxyl radicals with IC50 measured at 1.48 mM []. Due to the ability in scavenging several types of free radicals, cis-nerolidol was evidenced to protect Caco-2 cells against oxidative stress induced by tert-butyl hydroperoxide (tert-BuOOH), suggesting that nerolidol is a good antioxidant that exerts protection against oxidative damage []. Another study conducted by Vinholes et al. reported that cis-nerolidol mediated its strong antioxidant activity in protecting the hepatocytes through the inhibition of lipid peroxidation induced by tert-BuOOH, thereby 1mM of cis-nerolidol resulted in 36.50% ± 4.47% of malonaldehyde (MDA) reduction [].