Substrates with patterned topography reveal metastasis of human cancer cells.

In this study, we aimed at studying the effects of engineered and patterned substrates on the migration characteristics of mammalian cancer cell lines. On the shallow topographical patterns, cells from different histological origins showed different migration speed and directionality. We also observed that cells from the same origin showed distinctive behaviour, suggesting these substrate topographies could distinguish cancer subtypes. To eliminate the influence of genetic background, we examined two isogenic subpopulations of ovarian cancer cell lines for their different metastatic activities. While these cell lines showed indistinguishable migration characteristics on a flat substrate, their motilities on the patterned substrates were highly different, suggesting that cancer cells' motilities on these substrates varied in a metastasis-dependent manner. While cells with different metastatic activities showed similar morphology and focal adhesion distribution on flat surface, vinculin aggregated into single cytoplasmic foci in metastatic cells cultured on the engineered substrates. This implies that the topographical patterns on the substrates induced vinculin redistribution in cancer cells with a higher invasive activity. The fabricated platforms with topographical patterns offer a novel in vitro technique for metastasis assessment. Moreover, such platforms could potentially provide the opportunity to sort cells in different metastatic states using advanced pattern designs and features.

Proteomic characterization of the interactions between fish serum proteins and waterborne bacteria reveals the suppression of anti-oxidative defense as a serum-mediated antimicrobial mechanism.

Fish blood is one of the crucial tissues of innate immune system, but the full repertoire of fish serum components involved in antibacterial defense is not fully identified. In this study, we demonstrated that turbot serum, but not the heat-inactivated control, significantly reduced the number of Edwardsiella tarda (E. tarda). By conjugating serum proteins with fluorescent dyes, we showed that E. tarda were coated with multiple fish proteins. In order to identify these proteins, we used E. tarda to capture turbot serum proteins and subjected the samples to shotgun proteomic analysis. A total of 76 fish proteins were identified in high confidence, including known antimicrobial proteins such as immunoglobins and complement components. 34 proteins with no previously known immunological functions were also identified. The expression of one of these proteins, IQ motif containing H (IQCH), was exclusively in fish brain and gonads and was induced during bacterial infection. This approach also allowed the study of the corresponding proteomic changes in E. tarda exposed to turbot serum, which is a general decrease of bacterial protein expression except for an upregulation of membrane components after serum treatment. Interestingly, while most other known stresses stimulate bacterial antioxidant enzymes, fish serum induced a rapid suppression of antioxidant proteins and led to an accumulation of reactive oxygen species. Heat treatment of fish serum eliminated this effect, suggesting that heat labile factors in the fish serum overrode bacterial antioxidant defenses. Taken together, this work offers a comprehensive view of the interactions between fish serum proteins and bacteria, and reveals previously unknown factors and mechanisms in fish innate immunity.

A Unidirectional Cell Switching Gate by Engineering Grating Length and Bending Angle.

On a microgrooved substrate, cells migrate along the pattern, and at random positions, reverse their directions. Here, we demonstrate that these reversals can be controlled by introducing discontinuities to the pattern. On "V-shaped grating patterns", mouse osteogenic progenitor MC3T3-E1 cells reversed predominately at the bends and the ends. The patterns were engineered in a way that the combined effects of angle- and length-dependence could be examined in addition to their individual effects. Results show that when the bend was placed closer to one end, migration behaviour of cells depends on their direction of approach. At an obtuse bend (135°), more cells reversed when approaching from the long segment than from the short segment. But at an acute bend (45°), this relationship was reversed. Based on this anisotropic behaviour, the designed patterns effectively allowed cells to move in one direction but blocked migrations in the opposing direction. This study demonstrates that by the strategic placement of bends and ends on grating patterns, we can engineer effective unidirectional switching gates that can control the movement of adherent cells. The knowledge developed in this study could be utilised in future cell sorting or filtering platforms without the need for chemotaxis or microfluidic control.

Comparative study of 17 ß-estradiol on endocrine disruption and biotransformation in fingerlings and juveniles of Japanese sea bass Lateolabrax japonicus.

Estrogenic contaminants in the aquatic environment are associated with endocrine disruption and feminization in fish. The effects of endocrine disrupting chemicals (EDCs) on fish have been well documented. However, very few studies have focused on 17 ß-estradiol (E2) and its effects on endocrine system and biotransformation in a single prolonged exposure. This study investigated changes in the levels of serotonin (5-hydroxytryptamine) and acetyl choline esterase (AchE) in brain, cortisol in plasma and Ethoxyresorufin-O-deethylase (EROD) activity in gill of two different size groups (fingerlings and juveniles) of Japanese sea bass (Lateolabrax japonicus) upon exposure to two sub-lethal concentrations (200 and 2000 ng L(-1)) of E2 for 30 d. The results indicate that cortisol level and EROD activity significantly increased in both groups, whereas serotonin level increased in juveniles and decreased in fingerlings due to E2 exposure. The correlation analysis revealed that E2 significantly affected the endocrine and biotransformation systems in both age groups.

Benzo[a]pyrene modulates the biotransformation, DNA damage and cortisol level of red sea bream challenged with lipopolysaccharide.

In animals, biotransformation and the immune system interact with each other, however, knowledge of the toxic mechanism of benzo[a]pyrene (BaP) on these two systems is not well known. The present study investigated the toxic effects of BaP on the biotransformation system, cortisol level and DNA integrity of red sea bream (Pagrus major). The results showed that cortisol level was induced under the challenge of lipopolysaccharide (LPS). Short-term exposure (96 h) of BaP at environmental concentration significantly increased the cortisol level, hepatic EROD activity and CYP1A1 mRNA expression. When P. major was exposed to BaP for 14 d followed by LPS challenge this increased the cortisol level, EROD activity and hepatic DNA damage except CYP1A1 mRNA expression. Combined with our previous data, which showed that BaP exposure can modulate the immunologic response in P. major challenged with LPS, a hypothetical adverse outcome pathway of BaP on fish was suggested.

Interactive effects of hypoxia and polybrominated diphenyl ethers (PBDEs) on microbial community assembly in surface marine sediments.

Hypoxia alters the oxidation-reduction balance and the biogeochemical processes in sediments, but little is known about its impacts on the microbial community that is responsible for such processes. In this study, we investigated the effects of hypoxia and the ubiquitously dispersed flame-retardant BDE47 on the bacterial communities in marine surface sediments during a 28-days microcosm experiment. Both hypoxia and BDE47 alone significantly altered the bacterial community and reduced the species and genetic diversity. UniFrac analysis revealed that BDE47 selected certain bacterial species and resulted in major community shifts, whereas hypoxia changed the relative abundances of taxa, suggesting slower but nonetheless significant community shifts. These two stressors targeted mostly different taxa, but they both favored Bacteroidetes and suppressed Gammaproteobacteria. Importantly, the impacts of BDE47 on bacterial communities were different under hypoxic and normoxic conditions, highlighting the need to consider risk assessments for BDE47 in a broader context of interaction with hypoxia.

Interactive effects of hypoxia and PBDE on larval settlement of a marine benthic polychaete.

Marine benthic polychaete Capitella sp. I is widely known to adapt to polluted habitats; however, its response to xenobiotics under hypoxic conditions has been rarely studied. This research aimed to test the hypothesis that interactive effects of hypoxia and congener BDE-47 of polybrominated diphenyl ethers (PBDE), which is ubiquitous in marine sediments, may alter the settlement of Capitella sp. I. Our results revealed that under hypoxic condition, settlement success and growth in body length of Capitella larvae were significantly reduced compared to those under normoxia of similar BDE-47 concentration. While no significant changes in morphology of settled larvae were noted in both exposure conditions, the presence of BDE-47 could enhance polychaete growth. The present findings demonstrated that the interactive effects of hypoxia and environmentally realistic concentrations of BDE-47 in sediments could affect polychaete settlement, which, in turn, reduce its recruitment and subsequent population size in the marine benthic ecosystem.

Benzo[a]pyrene modulation of acute immunologic responses in Red Sea bream pretreated with lipopolysaccharide.

The effects of polycyclic aromatic hydrocarbons (PAHs) have been reported to modulate the immune response in aquatic animals, but the collected information of their effects on fish immunity is so far ambiguous. This study demonstrated that Benzo[a]pyrene (BaP) exposure altered the expression pattern of an antimicrobial peptide hepcidin (PM-hepc) gene and the activities of some immune-associated parameters in the lipopolysaccharide (LPS)-challenged red sea bream (Pagrus major). It was observed that LPS could increase respiratory burst, lysozyme and antibacterial activity in P. major. However when the P. major was exposed to different concentrations of BaP (1, 4, or 8 µg L(-1) ) for 14 days and then challenged with LPS there was no significant change in the lysozyme and antibacterial activity. It was further observed that LPS could induce the PM-hepc mRNA expression at 3, 6, and 12-h post-LPS challenge. However, when P. major was exposed first to BaP for 14 days and then challenged with LPS, the expression of PM-hepc mRNA was delayed in the liver until 24 h and not significantly induced until 48 and 96 h. The mRNA expression pattern was completely different from that only with LPS challenge, showing that BaP exposure changed the PM-hepc mRNA expression pattern of fish with LPS challenge. This study demonstrated that BaP exposure can weaken or inhibit the induction of lysozyme and antibacterial activity in the LPS-challenged P. major; conversely BaP exposure could enhance the mRNA expression of PM-hepc gene, indicating that the effect of BaP has different modulatory mechanism on hepcidin genes and immune-associated parameters.

Medaka fish exhibits longevity gender gap, a natural drop in estrogen and telomere shortening during aging: a unique model for studying sex-dependent longevity.

INTRODUCTION: Females having a longer telomere and lifespan than males have been documented in many animals. Such linkage however has never been reported in fish. Progressive shortening of telomere length is an important aging mechanism. Mounting in vitro evidence has shown that telomere shortening beyond a critical length triggered replicative senescence or cell death. Estrogen has been postulated as a key factor contributing to maintenance of telomere and sex-dependent longevity in animals. This postulation remains unproven due to the lack of a suitable animal system for testing. Here, we introduce a teleost model, the Japanese medaka Oryzias latipes, which shows promise for research into the molecular mechanism(s) controlling sex difference in aging. RESULTS: Using the medaka, we demonstrate for the first time in teleost that (i) sex differences (female > male) in telomere length and longevity also exist in fish, and (ii) a natural, 'menopause'-like decline of plasma estrogen was evident in females during aging. Estrogen levels significantly correlated with telomerase activity as well as telomere length in female organs (not in males), suggesting estrogen could modulate telomere length via telomerase activation in a sex -specific manner. A hypothetical in vivo 'critical' terminal restriction fragment (TRF, representing telomere) length of approximately 4 kb was deduced in medaka liver for prediction of organismal mortality, which is highly comparable with that for human cells. An age conversion model was also established to enable age translation between medaka (in months) and human (in years). These novel tools are useful for future research on comparative biology of aging using medaka. CONCLUSION: The striking similarity in estrogen profile between aging female O. latipes and women enables studying the influence of "postmenopausal" decline of estrogen on telomere and longevity without the need of invasive ovariectomy. Medaka fish is advantageous for studying the direct effect of increased estrogen on telomere length and longevity without the breast cancer complications reported in rodents. The findings strongly support the notion that O. latipes is a unique non-mammalian model for validation of estrogenic influence on telomere and longevity in vertebrates. This laboratory model fish is of potential significance for deciphering the ostensibly conserved mechanism(s) of sex-associated longevity in vertebrates.

Modulation and Interaction of Immune-Associated Parameters with Antioxidant in the Immunocytes of Crab Scylla paramamosain Challenged with Lipopolysaccharides.

Invertebrates are dependent on cellular and humoral immune defences against microbial infection. Scylla paramamosain is an important commercial species, but the fundamental knowledge on its immune defense related to the antioxidant and immune-associated reactions is still lacking. The study was to differentiate the responses of immune-associated parameters of haemolymph components in S. paramamosain when challenged with bacterial lipopolysaccharides (LPSs). The immunostimulating effects of LPS in crab by triggering various immune parameters (phagocytosis, lysozyme, antibacterial activity, phenoloxidase, and the generation of superoxide and nitric oxide) were investigated. Results showed that the generation of free radicals, phenoloxidase, lysozyme and antibacterial activities was significantly increased through the exposure periods. Conversely, total hemocyte count and lysosomal membrane stability decreased significantly as the exposure period extended to 96 h. The relationship between the antioxidant enzymes and immune reactions due to LPS was highly significant. In addition, ROS production was positively correlated with antioxidant showing immediate response of antioxidant defense to the oxyradicals generated. Overall, the study indicated that nonspecific immune components in hemocytes of crab showed active response to the LPS stimulation, and their responses suggested that many immune-associated parameters could be modulated and interrelated with the influence of antioxidants in crustaceans.

Biochemical responses and DNA damage in red sea bream from coastal Fujian Province, China.

Relationships between measures of oxidative stress and adaptive antioxidant defenses were studied in red sea bream (Pagrus major, RSB) caged at eight locations along the Fujian coast, China. Oxidative damage in tissues was investigated by measuring concentrations of protein carbonyl (CP) and reactive oxygen species (ROS), lipid peroxidation (LPO), acetyl cholinesterase activity (AChE), concentration of thiol groups and activities of enzymes associated with antioxidant defense. Magnitudes of antioxidant defense parameters were significantly, positively correlated with oxidative stress. Significantly greater ROS, LPO and CP content was observed in most tissues of RSB caged at three locations relative to those at the other locations. RSB caged at one location, Meizhou, exhibited significantly more DNA damage than did RSB caged at other locations. AChE activities in liver, kidney and muscle were statistically different among locations. Multivariate analyses were used to categorize the environmental quality of different locations. The battery of measures of oxidative stress proved to be a useful integrator of oxidative stresses that could be compared among locations.

Effects of tributyltin and benzo[a]pyrene on the immune-associated activities of hemocytes and recovery responses in the gastropod abalone, Haliotis diversicolor.

Our previous study reports that short-term exposure to sublethal concentrations of benzo[a]pyrene (BaP) induces immunomodulation in the gastropod abalone, Haliotis diversicolor. In the present study, it was further observed that long-term chronic exposure to sublethal concentrations of BaP modulated the immunocompetence of abalones in terms of the change in activity of the antioxidant and immune associated parameters tested. In addition, the effect of tributyltin (TBT), another important genotoxicant in the aquatic environment, was investigated. Exposure of abalones to sublethal concentrations of TBT and BaP for 21 days resulted in significant decrease of total hemocyte count, phagocytosis, membrane stability and lysozyme activity. Conversely induction of extra and intra cellular superoxide generation, nitric oxide, nitric oxide synthase and myeloperoxidase activity was present when the abalones were exposed to TBT and BaP. Most of the immune associated parameters tested showed clear time dependent response to both toxicants. Within 14 days after the 21 day exposure to BaP, recovery was observed as evidenced by most of the parameters returning to their normal level. However, no recovery was observed within 14 days after the 21 day exposure to TBT as evidenced by continued elevation of intra cellular superoxide and nitrite production and decrease in THC, membrane stability and lysozyme activity. This suggested a prolonged TBT-induced impact on the immune reaction and possibly more damage than that caused by BaP. Overall the results suggest that chronic exposure to sublethal concentrations of TBT or BaP causes modulations in the immunocompetence of abalones with most of the immune associated parameters tested being stimulated, and this might be harmful to the host.

17ß estradiol induced ROS generation, DNA damage and enzymatic responses in the hepatic tissue of Japanese sea bass.

The importance of endocrine disrupting chemicals and their effects on fish has been documented in recent years. However, little is known about whether the estrogenic compound 17ß estradiol (E2) causes oxidative stress in the hepatic tissue of fish. Therefore, this work tested the hypothesis that E2 might cause oxidative stress in the Japanese sea bass Lateolabrax japonicus liver. To test this hypothesis, its effects on reactive oxygen species (ROS) production, DNA damage, antioxidants and biotransformation enzyme were investigated in two different size groups (fingerling and juvenile groups) following 30 days exposure. Results showed that there was a good relationship between the E2 exposure concentration, plasma E2 level and ROS generation. In addition ROS production correlated negatively with 7-ethoxyresorufin-O-deethylase activity and positively with DNA damage and lipid peroxidation (LPO). Antioxidant enzymes such as superoxide dismutase and catalase did not show any significant relation with ROS, LPO and DNA damage. In contrast, glutathione mediated enzymes showed a good relationship with the above parameters suggesting that the glutathione system in fish might be responsible for protection against the impact of E2 and also indicating a possible adaptive response during exposure periods. In addition, it was observed that fingerling was more susceptible to E2 exposure than juvenile fish. The present study provided strong evidence that the ROS level increased significantly in the liver of E2 exposed fish, and that ROS might serve as a biomarker to indicate estrogen contamination.

Antioxidant enzymes from the crab Scylla paramamosain: gene cloning and gene/protein expression profiles against LPS challenge.

Recent studies revealed that antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. Catalase is one of these key enzymes which has been characterized and highly conserved from invertebrates to vertebrates. In the present study, a full-length cDNA sequence of catalase was cloned from the hemocyte suppression subtractive hybridization library of the crab Scylla paramamosain. The Sp-catalase (Sp-CAT) cDNA sequence contained 2551bp with an open reading frame of 1551bp encoding 517 amino acid residues. The conserved catalytic active residues His-71, Asn-144 and Tyr-354 were predicted in the amino acid sequence of Sp-CAT. The deduced Sp-CAT protein had a calculated molecular mass of 59 kDa with an estimated isoelectric point of 6.4. Multiple alignment analysis revealed that the deduced amino acid sequence of Sp-CAT shared high identity (75.4%) with those of other species. The Sp-CAT mRNA transcripts were demonstrated in multiple tissues of normal S. paramamosain. After LPS challenge, the expression level of Sp-CAT gene was increased significantly in hemocyte at 3 and 6 h, and in hepatopancreas at 6 h, respectively, determined by quantitative real-time PCR. Furthermore, the activities of CAT and SOD were also measured in different tissues and serum after LPS challenge. The CAT activity was significantly increased at 3, 6, 24 and 48 h in hemocyte lysate, at 3 h in serum, and at 24 and 48 h in hepatopancreas after LPS challenge. In addition, the SOD activity was significantly induced at 3 and 6 h in hemocyte lysate, 3 and 12 h in serum, 12 and 48 h in hepatopancreas post LPS stimulation, indicating a tissue and time-dependent antioxidant response in the crab. Taken together, these data demonstrated that a strong antioxidant response occurred in the LPS-challenged crab, which might be involved in the protection of host against microbial infections.

First molecular cloning of a molluscan caspase from variously colored abalone (Haliotis diversicolor) and gene expression analysis with bacterial challenge.

Mammal caspases have been demonstrated to possess important functions in apoptosis and immune signaling, but there is less knowledge available on abalone caspases. In the present study, a molluscan caspase gene, abCaspase, was cloned for the first time from the variously colored abalone (Haliotis diversicolor) and its full-length cDNA sequence was 2427 bp, with a 1008 bp of open reading frame encoding a protein of 336 aa. The molecular mass of the deduced protein was approximately 36.97 kDa with an estimated pI of 5.28. The predicted amino acid sequence of abCaspase contained two domains of p20 and p10 which were conserved in the caspase family, including the cysteine active site pentapeptide "QSCRG" and the histidine active site signature "HTVYDCVVVIFLTHG". Homology analysis showed that abCaspase shared high similarity with apoptotic caspases and it was grouped together with vertebrate caspase-8s and caspase-10s using phylogenetic analysis, suggesting that abCaspase belonged to a typical apoptotic caspase and might possess the characteristic of human caspase-8 and -10. The mRNA transcripts of abCaspase were widely distributed in various tissues of H. diversicolor. Expression of the abCaspase gene was significantly induced in the tissues tested, especially in the hemocytes, gill and mantle with bacterial challenge. This study suggested that abCaspase may be an initiator caspase associated with the induction of apoptosis which is potentially involved in the immune defense of H. diversicolor.

Effect of 17beta-estradiol on the immunocompetence of Japanese sea bass (Lateolabrax japonicus).

Environmental contaminants can interfere with hormonal regulation in both vertebrates and invertebrates, and these contaminants may disrupt the endocrine system of human and other organisms. Evidence is growing that contaminants may be partly responsible for the observed increase of disease in marine organisms by adversely affecting their immunity. Fish are commonly used as sentinel organisms in vertebrate immunotoxicology; however, to date, studies have been undertaken only on a single size group of fish (juvenile/adult) and for acute exposure. In the present study, Lateolabrax japonicus fingerlings and juveniles were exposed to two sublethal concentrations (200 and 2,000 ng/L) of 17beta-estradiol (E2) for 30 d under laboratory conditions, and alterations in immune parameters comprising differential leukocyte count, respiratory burst, myeloperoxidase, immunoglobulin levels, serum lysozyme, and bactericidal activity were investigated to establish whether estrogen produced immunomodulation and to understand the effects of long-term exposure on these immune parameters in fish fingerlings and juveniles. The results revealed a significant elevation of respiratory burst activity, myeloperoxidase, immunoglobulin levels, and differential leukocyte counts of the fish exposed to estrogen compared to the control. The remaining parameters were significantly reduced in the experimental groups when compared to the control. The results indicated that sublethal E(2) exposure induced immunomodulation in both fingerling and juvenile L. japonicus, and the changes caused by estrogen might affect the function of immune system in fish.

Gene cloning of a sigma class glutathione S-transferase from abalone (Haliotis diversicolor) and expression analysis upon bacterial challenge.

Glutathione S-transferases (GSTs) are a multigene family of xenobiotic metabolizing phase II detoxification enzymes which take part in many pathological and physiological processes, and which can potentially be used as indicators and biomarkers for cancer diagnoses and organic or inorganic pollutant exposure. In this study, a full-length cDNA of a sigma class GST (abGSTsigma) (GenBank accession number EF546619) from variously colored abalone (Haliotis diversicolor) was identified. It was 1328bp containing an open reading frame of 624bp, encoding 208 amino acid residues with a predicted protein molecular weight of 23.67kDa and an estimated pI of 5.67. Sequence analysis showed that the predicted protein sequence of abGSTsigma cDNA contained the conserved domain of the GST_N_Sigma_like (PSSM: cd03039) and GST_C_Sigma_like (PSSM: cd03192). Alignment analysis demonstrated that the abGSTsigma of H. diversicolor was in a branch position with other known class sigma GSTs from different organisms. The abGSTsigma mRNA was distributed in multiple tissues tested and was highly demonstrated in the gill and mantle of normal abalones. In bacteria-challenged abalone, the abGSTsigma gene was significantly expressed in the hemocytes, gill, mantle and digestive gland and the total GSTs enzyme and SOD were also induced in the four tissues. The increased activities of SOD and GSTs can result in the elimination of reactive oxygen species (ROS) indicating antioxidant activities involved. The preliminary work revealed that the sigma class glutathione S-transferase gene abGSTsigma, a phase II detoxification enzyme, had a positive response to bacterial challenge, and that will lead to an insightful study on elucidating the interactions between immune responses and biotransformation exerted by abGSTsigma.

Immunomodulation in the marine gastropod Haliotis diversicolor exposed to benzo(a)pyrene.

It has been reported that environmental pollutants in the aquatic ecosystem could weaken immune competence of organisms. The purpose of the present study was to investigate the effects of benzo(a)pyrene [B(a)P] on immunomodulation in marine gastropods and to see if these effects are caused by or related to the generation of reactive oxygen species. In our present study, the marine gastropod Haliotis diversicolor was exposed to sublethal concentrations (0.01, 0.02, 0.04 and 0.08 mg L(-1)) of B(a)P for 7d under laboratory conditions and the alterations of hematological parameters like haemocyte count, haemocyte viability, protein content and immune components like phenoloxidase, phagocytosis and superoxide anion generation were measured. In addition, the changes in lysozyme activity, antibacterial activity due to the effect of B(a)P on abalone were analysed. B(a)P was found to decrease significantly the total number of circulating haemocytes. Intracellular superoxide anion generation and phenoloxidase significantly increased on exposure to B(a)P, whereas phagocytic activity was decreased significantly at higher concentration. Significant alterations were found in the uptake of neutral red and the observed alterations of hematological parameters and immune components tested indicated the generation of immunotoxicological effects on abalone due to B(a)P exposure. The results demonstrate a possible relationship between B(a)P and the immunological parameters of abalone studied.