Investigation of aspects of reactive gliosis in human astrocytoma cell lines:application for toxicity assessment

The process of astrogliosis, or reactive gliosis, is a typical response of astrocytes to a wide range of physical and chemical injuries. The up-regulation of the astrocyte specific glial fibrillary acidic protein (GFAP) is a hallmark of reactive gliosis and is widely used as a marker to identify the response. In order to develop a reliable, sensitive and high throughput astrocyte toxicity assay that is more relevant to the human response than existing animal cell based models, the U251-MG, U373-MG and CCF-STTG 1 human astrocytoma cell lines were investigated for their ability to exhibit reactive-like changes following exposure to ethanol, chloroquine diphosphate, trimethyltin chloride and acrylamide. Cytotoxicity analysis showed that the astrocytic cells were generally more resistant to the cytotoxic effects of the agents than the SH-SY5Y neuroblastoma cells. Retinoic acid induced differentiation of the SH-SY5Y line was also seen to confer some degree of resistance to toxicant exposure, particularly in the case of ethanol. Using a cell based ELISA for GFAP together with concurrent assays for metabolic activity and cell number, each of the three cell lines responded to toxicant exposure by an increase in GFAP immunoreactivity (GFAP-IR), or by increased metabolic activity. Ethanol, chloroquine diphosphate, trimethyltin chloride and bacterial lipopolysaccharide all induced either GFAP or MTT increases depending upon the cell line, dose and exposure time. Preliminary investigations of additional aspects of astrocytic injury indicated that IL-6, but not TNF-α. or nitric oxide, is released following exposure to each of the compounds, with the exception of acrylamide. It is clear that these human astrocytoma cell lines are capable of responding to toxicant exposure in a manner typical of reactive gliosis and are therefore a valuable cellular model in the assessment of in vitro neurotoxicity.

Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation

Oxidized phospholipids, such as the products of the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by nonenzymatic radical attack, are known to be formed in a number of inflammatory diseases. Interest in the bioactivity and signaling functions of these compounds has increased enormously, with many studies using cultured immortalized and primary cells, tissues, and animals to understand their roles in disease pathology. Initially, oxidized phospholipids were viewed largely as culprits, in line with observations that they have proinflammatory effects, enhancing inflammatory cytokine production, cell adhesion and migration, proliferation, apoptosis, and necrosis, especially in vascular endothelial cells, macrophages, and smooth muscle cells. However, evidence has emerged that these compounds also have protective effects in some situations and cell types; a notable example is their ability to interfere with signaling by certain Toll-like receptors (TLRs) induced by microbial products that normally leads to inflammation. They also have protective effects via the stimulation of small GTPases and induce up-regulation of antioxidant enzymes and cytoskeletal rearrangements that improve endothelial barrier function. Oxidized phospholipids interact with several cellular receptors, including scavenger receptors, platelet-activating factor receptors, peroxisome proliferator-activated receptors, and TLRs. The various and sometimes contradictory effects that have been observed for oxidized phospholipids depend on their concentration, their specific structure, and the cell type investigated. Nevertheless, the underlying molecular mechanisms by which oxidized phospholipids exert their effects in various pathologies are similar. Although our understanding of the actions and mechanisms of these mediators has advanced substantially, many questions do remain about their precise interactions with components of cell signaling pathways.

Visfatin regulates insulin secretion, insulin receptor signalling and mRNA expression of diabetes-related genes in mouse pancreatic beta-cells

The role of the adipocyte-derived factor visfatin in metabolism remains controversial, although some pancreatic ß-cell-specific effects have been reported. This study investigated the effects of visfatin upon insulin secretion, insulin receptor activation and mRNA expression of key diabetes-related genes in clonal mouse pancreatic ß-cells. ß-TC6 cells were cultured in RPMI 1640 and were subsequently treated with recombinant visfatin. One-hour static insulin secretion was measured by ELISA. Phospho-specific ELISA and western blotting were used to detect insulin receptor activation. Real-time SYBR Green PCR array technology was used to measure the expression of 84 diabetes-related genes in both treatment and control cells. Incubation with visfatin caused significant changes in the mRNA expression of several key diabetes-related genes, including marked up-regulation of insulin (9-fold increase), hepatocyte nuclear factor (HNF)1ß (32-fold increase), HNF4a (16-fold increase) and nuclear factor ?B (40-fold increase). Significant down-regulation was seen in angiotensin-converting enzyme (-3.73-fold) and UCP2 (-1.3-fold). Visfatin also caused a significant 46% increase in insulin secretion compared to control (P<0.003) at low glucose, and this increase was blocked by co-incubation with the specific nicotinamide phosphoribosyltransferase inhibitor FK866. Both visfatin and nicotinamide mononucleotide induced activation of both insulin receptor and extracellular signal-regulated kinase (ERK)1/2, with visfatin-induced insulin receptor/ERK1/2 activation being inhibited by FK866. We conclude that visfatin can significantly regulate insulin secretion, insulin receptor phosphorylation and intracellular signalling and the expression of a number of ß-cell function-associated genes in mouse ß-cells.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The Plasma Membrane Calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, up-regulation in the expression of the pro-apoptotic protein Fas Ligand, and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

Salmonella in companion animals

In an increasingly hygiene concerned society, a major barrier to pet ownership is the perceived role of companion animals in contributing to the risk of exposure to zoonotic bacterial pathogens, such as Salmonella. Manifestations of Salmonella can range from acute gastroenteritis to perfuse enteric fever, in both humans and dogs. Dogs are heavily associated with asymptomatic carriage of Salmonella as the microorganism can persist in the lower intestines of this host which can be then excreted into the environment. Studies in to the asymptomatic carriage of Salmonella in dogs are somewhat dated and there is limited UK data. The current UK carriage rate in dogs was investigated in a randomised dog population and it was revealed that the carriage rate in this population was very low with only one household dog positive for the carriage of Salmonella enterica arizonae (0.2%), out of 490 dogs sampled. Salmonella serotypes share phenotypic and genotypic similarities which are captured in epidemiological typing methods. Therefore, in parallel to the epidemiological investigations, a panel of clinical canine (VLA, UK) and human (Aston University, UK) Salmonella isolates were profiled based on their phenotypic and genotypic characteristics; using API 20E, Biolog Microbial ID System, antibiotic sensitivity testing and PFGE, respectively. Antibiotic sensitivity testing revealed a significant difference between the canine and human isolates with the canine group demonstrating a higher resistance to the panel of antibiotics tested. Further metabolic capabilities of the strains were tested using the Biolog Microbial ID System, which reveal no clear association between the two host groups. However, coupled with Principle Component Analysis two canine isolates were discriminated from the entire population on the basis of a high up-regulation of two carbohydrates. API 20E testing revealed no association between the two host groups. A PFGE harmonised protocol was used to genotypically profile the strains. A dendrogram depicting PFGE profiles of the panel of Salmonella isolates was performed where similarities were calculated by Dice coefficient and represented by UPGMA clustering. Clustering of the profiles from canine isolates and human isolates (HPA, UK) was diverse representing a natural heterogeneity of the genus, additionally, no clear clustering of the isolates was observed between host groups. Clustering was observed with isolates from the same serotype, independent of host origin. Host adaption is a common phenomenon in certain Salmonella serotypes, for example S. Typhi in humans and S. Dublin in cattle. It was of interest to investigate potential host adaptive or restricted strains for canine host by performing adhesion and invasion assays on Dog Intestinal Epithelial Cells (DIECs) (WALTHAM®, UK) and human CaCo-2 (HPA, UK) cell lines. Salmonella arizonae and Enteritidis from an asymptomatic dog and clinical isolate, respectively, demonstrated a significantly high proportion of invasion in DIEC in comparison to human CaCo-2 cells and other tested Salmonella serotypes. This may be suggestive of a potential host restrictive strain as their ability to invade the CaCo-2 cell line was significantly lower than the other serotypes. In conclusion to this thesis the investigations carried out suggest that asymptomatic carriage of Salmonella in UK dogs is low however the microorganism remains as a zoonotic and anthroponotic pathogen based on phenotypic and genotypic characterisation however there may be potential for particular serotype to become host restricted as observed in invasion assays

Amino acids in oral drug delivery:salts, ion-pairs and transcriptomics

Oral drug delivery is considered the most popular route of delivery because of the ease of administration, availability of a wide range of dosage forms and the large surface area for drug absorption via the intestinal membrane. However, besides the unfavourable biopharmaceutical properties of the therapeutic agents, efflux transporters such as Pglycoprotein (P-gp) and multiple resistance proteins (MRP) decrease the overall drug uptake by extruding the drug from the cells. Although, prodrugs have been investigated to improve drug partitioning by masking the polar groups covalently with pre-moieties promoting increased uptake, they present significant challenges including reduced solubility and increased toxicity. The current work investigates the use of amino acids as ion-pairs for three model drugs: indomethacin (weak acid), trimethoprim (weak base) and ciprofloxacin (zwitter ion) in an attempt to improve both solubility and uptake. Solubility was studied by salt formation while creating new routes for uptake across the membranes via amino acids transporter proteins or dipeptidyl transporters was the rationale to enhance absorption. New salts were prepared for the model drugs and the oppositely charged amino acids by freeze drying and they were characterised using FTIR, 1HNMR, DSC, SEM, pH solubility profile, solubility and dissolution. Permeability profiles were assessed using an in vitro cell based method; Caco-2 cells and the genetic changes occurring across the transporter genes and various pathways involved in the cellular activities were studied using DNA microarrays. Solubility data showed a significant increase in drug solubility upon preparing the new salts with the oppositely charged counter ions (ciprofloxacin glutamate salt exhibiting 2.9x103 fold enhancement when compared to the free drug). Moreover, permeability studies showed a 3 fold increase in trimethoprim and indomethacin permeabilities upon ion-pairing with amino acids and more than 10 fold when the zwitter ionic drug was paired with glutamic acid. Microarray data revealed that trimethoprim was absorbed actively via OCTN1 transporters while MRP7 is the main transporter gene that mediates its efflux. The absorption of trimethoprim from trimethoprim glutamic acid ion-paired formulations was affected by the ratio of glutamic acid in the formulation which was inversely proportional to the degree of expression of OCTN1. Interestingly, ciprofloxacin glutamic acid ion-pairs were found to decrease the up-regulation of ciprofloxacin efflux proteins (P-gp and MRP4) and over-express two solute carrier transporters; (PEPT2 and SLCO1A2) suggesting that a high aqueous binding constant (K11aq) enables the ion-paired formulations to be absorbed as one entity. In conclusion, formation of ion-pairs with amino acids can influence in a positive way solubility, transfer and gene expression effects of drugs.

Resveratrol inhibits the release of soluble fms-like tyrosine kinase (sFlt-1) from human placenta

Objective - Soluble vascular endothelial growth factor receptor–1 (also know as soluble fms-like tyrosine kinase [sFlt]-1) is a key causative factor of preeclampsia. Resveratrol, a plant phytoalexin, has antiinflammatory and cardioprotective properties. We sought to determine the effect of resveratrol on sFlt-1 release. Study Design - Human umbilical vein endothelial cells, transformed human trophoblast-8 (HTR/SVneo)-8/SVneo trophoblast cells, or placental explants were incubated with cytokines and/or resveratrol. Conditioned media were assayed for sFlt-1 by enzyme-linked immunosorbent assay and cell proteins used for Western blotting. Results - Resveratrol inhibited cytokine-induced release of sFlt-1 from normal placental explants and from preeclamptic placental explants. Preincubation of human umbilical vein endothelial cells or HTR-8/SVneo cells with resveratrol abrogated sFlt-1 release. Resveratrol prevented the up-regulation of early growth response protein-1 (Egr-1), a transcription factor necessary for induction of the vascular endothelial growth factor receptor–1 gene and caused up-regulation of heme oxygenase–1, a cytoprotective enzyme found to be dysfunctional in preeclampsia. Conclusion - In summary, resveratrol can inhibit sFlt-1 release and up-regulate heme oxygenase–1; thus, may offer therapeutic potential in preeclampsia.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Understanding the role of transforming growth factor beta signalling and epigenomics in osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis with a high socioeconomic burden, with an incompletely understood etiology. Evidence suggests a role for the transforming growth factor beta (TGF-ß) signalling pathway and epigenomics in OA. The aim of this thesis was to understand the involvement of the TGF-ß pathway in OA and to determine the DNA methylation patterns of OA-affected cartilage as compared to the OA-free cartilage. First, I found that a common SNP in the BMP2 gene, a ligand in the Bone morphogenetic protein (BMP) subunit of TGF-ß pathway, was associated with OA in the Newfoundland population. I also showed a genetic association between SMAD3 - a signal transducer in the TGF-ß subunit of the TGF-ß signalling pathway - and the total radiographic burden of OA. I further demonstrated that SMAD3 is over-expressed in OA cartilage, suggesting an over activation of the TGF-ß signalling in OA. Next, I examined the connection of these genes in the regulation of matrix metallopeptidase 13 (MMP13) - an enzyme known to destroy extracellular matrix in OA cartilage - in the context of the TGF-ß signalling. The analyses showed that TGF-ß, MMP13, and SMAD3 were overexpressed in OA cartilage, whereas the expression of BMP2 was significantly reduced. The expression of TGF-ß was positively correlated with that of SMAD3 and MMP13, suggesting that TGF-ß signalling is involved in up-regulation of MMP13. This regulation, however, appears not to be controlled by SMAD3 signals, possibly due to the involvement of collateral signalling, and to be suppressed by BMP regulation in healthy cartilage, whose levels were reduced in end-stage OA. In a genome-wide DNA methylation analysis, I reported CpG sites differentially methylated in OA and showed that the cartilage methylome has a potential to distinguish between OA-affected and non-OA cartilage. Functional clustering analysis of the genes harbouring differentially methylated loci revealed that they are enriched in the skeletal system morphogenesis pathway, which could be a potential candidate for further OA studies. Overall, the findings from the present thesis provide evidence that the TGF-ß signalling pathway is associated with the development of OA, and epigenomics might be involved as a potential mechanism in OA.

Diametrically opposed actions of the heme oxygenase-1 and endothelial nitric oxide synthase pathways in angiogenesis via p21WAF1

INTRODUCTION: Vascular endothelial growth factor (VEGF)-induced angiogenesis requires endothelial nitric oxide synthase (eNOS) activation, however, the mechanism is largely unknown. As nitric oxide(NO) inhibits endothelial proliferation to promote capillary formation (Am J Path,159:993-1008,2001) and p21WAF1 is an important cell cycle inhibitor, we hypothesised that eNOS-induced angiogenesis requires up regulation of p21WAF1. METHODS: Human and porcine endothelial cells were cultured on growth factor reduced Materigel for in vitro tube formation and in vivo angiogenesis was assessed by hind limb ligation ischemia model.Conversely, we propose that the cytoprotective enzyme, heme oxygenase-1(HO-1), may suppress p21WAF1 to limit angiogenesis. RESULTS: The expression of p21WAF1 was up regulated in porcine aorticenothelial cells stablely transfected with a constitutively activated form of eNOS (eNOSS1177D) as well as in HUVEC infected by adenovirus encoding eNOSS1177D. When these cells were plated on growth-factor reduced Matrigel (compaired to empty vector), they enhanced in vitro angiogenesis, which was inhibited following knockdown of p21WAF1. Furthermore, over expression of p21WAF1 led to increased tube formation while p21WAF1 knockdown abrogated vascular endothelial growth factor(VEGF) and fibroblast growth factor (FGF-2) mediated angiogenesis.Conversely, the cytoprotective enzyme, heme oxygenase-1 (HO-1) when over expressed decreased p21WAF1 expression and reduced VEGF, FGF-2 and eNOSS1177D-induced angiogenesis. CONCLUSIONS: These results demonstrate that eNOS-induced angiogenesis requires up regulation of p21WAF1/CIP1 wherease, induction of HO-1 will decrease the expression of p21WAF1/CIP1 to limit angiogenesisindicating that eNOS and HO-1 regulate angiogenesis via p21WAF1/CIP1 in adiametrically opposed manner and that p21WAF1/CIP1 appears to be a central regulator of angiogenesis that offers a new therapeutic target.

Pax3 expression enhances PDGF-B-induced brainstem gliomagenesis and characterizes a subset of brainstem glioma.

High-grade Brainstem Glioma (BSG), also known as Diffuse Intrinsic Pontine Glioma (DIPG), is an incurable pediatric brain cancer. Increasing evidence supports the existence of regional differences in gliomagenesis such that BSG is considered a distinct disease from glioma of the cerebral cortex (CG). In an effort to elucidate unique characteristics of BSG, we conducted expression analysis of mouse PDGF-B-driven BSG and CG initiated in Nestin progenitor cells and identified a short list of expression changes specific to the brainstem gliomagenesis process, including abnormal upregulation of paired box 3 (Pax3). In the neonatal mouse brain, Pax3 expression marks a subset of brainstem progenitor cells, while it is absent from the cerebral cortex, mirroring its regional expression in glioma. Ectopic expression of Pax3 in normal brainstem progenitors in vitro shows that Pax3 inhibits apoptosis. Pax3-induced inhibition of apoptosis is p53-dependent, however, and in the absence of p53, Pax3 promotes proliferation of brainstem progenitors. In vivo, Pax3 enhances PDGF-B-driven gliomagenesis by shortening tumor latency and increasing tumor penetrance and grade, in a region-specific manner, while loss of Pax3 function extends survival of PDGF-B-driven;p53-deficient BSG-bearing mice by 33%. Importantly, Pax3 is regionally expressed in human glioma as well, with high PAX3 mRNA characterizing 40% of human BSG, revealing a subset of tumors that significantly associates with PDGFRA alterations, amplifications of cell cycle regulatory genes, and is exclusive of ACVR1 mutations. Collectively, these data suggest that regional Pax3 expression not only marks a novel subset of BSG but also contributes to PDGF-B-induced brainstem gliomagenesis.

Discovery and Characterization of a Novel Fatty Acid Synthase Inhibitor with Antineoplastic Activity against Breast Cancer

During oncogenesis, cancer cells go through metabolic reprogramming to maintain their high growth rates and adapt to changes in the microenvironment and the lack of essential nutrients. Several types of cancer are dependent on de novo fatty acid synthesis to sustain their growth rates by providing precursors to construct membranes and produce vital signaling lipids. Fatty acid synthase (FASN) catalyze the terminal step of de novo fatty acid synthesis and it is highly expressed in many types of cancers where it’s up-regulation is correlated with cancer aggressiveness and low therapeutic outcome. Many FASN inhibitors were developed and showed potent anticancer activity however, only one inhibitor advanced to early stage clinical trials with some dose limiting toxicities. Using a modified fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, we identified HS-106, a thiophenopyrimiden FASN inhibitor that has anti-neoplastic activity against breast cancer in vitro and in vivo. HS-106 was able to inhibit both; purified human FASN activity and cellular fatty acid synthesis activity as evaluated by radioactive tracers incorporation into lipids experiments. In proliferation and apoptosis assays, HS-106 was able to block proliferation and induce apoptosis in several breast cancer cell lines. Several rescue experiment and global lipidome analysis were performed to probe the mechanism by which HS-106 induces apoptosis. HS-106 was found to induce several changes in lipids metabolism: (i) inhibit fatty acids synthesis. (ii) Inhibit fatty acids oxidation as indicated by the ability of inhibiting Malonyl CoA accumulation to block HS-106 induced apoptosis and the increase in the abundance of ceramides. (iii) Increase fatty acids uptake and neutral lipids formation as confirmed 14C Palmitate uptake assay and neutral lipids staining. (iv)Inhibit the formation of phospholipids by inhibiting de novo fatty acid synthesis and diverting exogenous fatty acids to neutral lipids. All of these events would lead to disruption in membranes structure and function. HS-106 was also tested in Lapatinib resistant cell lines and it was able to induce apoptosis and synergizes Lapatinib activity in these cell lines. This may be due the disruption of lipid rafts based on the observation that HS-106 reduces the expression of both HER2 and HER3. HS-106 was found to be well tolerated and bioavailable in mice with high elimination rate. HS-106 efficacy was tested in MMTV neu mouse model. Although did not significantly reduced tumor size (alone), HS-106 was able to double the median survival of the mice and showed potent antitumor activity when combined with Carboplatin. Similar results were obtained when same combinations and dosing schedule was used in C3Tag mouse model except for the inability of HS-106 affect mice survival.

From the above, HS-106 represent a novel FASN inhibitor that has anticancer activity both in vivo and in vitro. Being a chemically tractable molecule, the synthetic route to HS-106 is readily adaptable for the preparation of analogs that are similar in structure, suggesting that, the pharmacological properties of HS-106 can be improved.

Combined effects of solar UV radiation and CO2-induced seawater acidification on photosynthetic carbon fixation of phytoplankton assemblages in the South China Sea, 2010

We carried out short term pCO2/pH perturbation experiments in the coastal waters of the South China Sea to evaluate the combined effects of seawater acidification (low pH/high pCO2) and solar UV radiation (UVR, 280-400 nm) on photosynthetic carbon fixation of phytoplankton assemblages. Under photosynthetically active radiation (PAR) alone treatments, reduced pCO2 (190 ppmv) with increased pH resulted in a significant decrease in the photosynthetic carbon fixation rate (about 23%), while enriched pCO2 (700 ppmv) with lowered pH had no significant effect on the photosynthetic performance compared to the ambient level. The apparent photosynthetic efficiency decreased under the reduced pCO2 level, probably due to C-limitation as well as energy being diverged for up-regulation of carbon concentrating mechanisms (CCMs). In the presence of UVR, both UV-A and UV-B caused photosynthetic inhibition, though UV-A appeared to enhance the photosynthetic efficiency under lower PAR levels. UV-B caused less inhibition of photosynthesis under the reduced pCO2 level, probably because of its contribution to the inorganic carbon (Ci)-acquisition processes. Under the seawater acidification conditions (enriched pCO2), both UV-A and UV-B reduced the photosynthetic carbon fixation to higher extents compared to the ambient pCO2 conditions. We conclude that solar UV and seawater acidification could synergistically inhibit photosynthesis.

Seawater carbonate chemistry and Strongylocentrotus purpuratus body length and gene expression pattern changes during experiments, 2011

Extensive use of fossil fuels is leading to increasing CO2 concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO2. As a result, the oceans' surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO2 41 Pa e.g. 399 µatm) and CO2 acidified seawater with pH of 7.7 (pCO2 134 Pa e.g. 1318 µatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR. After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10 % reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (Two way ANOSIM: Global R = 1) while acidification effects were less pronounced (Global R = 0.518). Significant differences in gene expression patterns (ANOSIM R = 0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO2 treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO2 effect. We found an up regulation of metabolic genes (between 10 to 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23 and 36% in msp130, SM30B, SM50 at day 4). Ion regulation was mainly impacted by up regulation of Na+/K+-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%). We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts. This helps prevent misinterpretation of stressor effects on organism physiology.