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1.  Influence of Environmental Factors on the Paralytic Shellfish Toxin Content and Profile of Alexandrium catenella (Dinophyceae) Isolated from the Mediterranean Sea 
Marine Drugs  2013;11(5):1583-1601.
Laboratory experiments were designed to study the toxin content and profile of the Alexandrium catenella strain ACT03 (isolated from Thau Lagoon, French Mediterranean) in response to abiotic environmental factors under nutrient-replete conditions. This dinoflagellate can produce various paralytic shellfish toxins with concentrations ranging from 2.9 to 50.3 fmol/cell. The toxin profile was characterized by carbamate toxins (GTX3, GTX4 and GTX5) and N-sulfocarbamoyl toxins (C1, C2, C3 and C4). C2 dominated at 12–18 °C, but only for salinities ranging from 10 to 25 psu, whereas GTX5 became dominant at temperatures ranging from 21 to 30 °C at almost all salinities. There was no significant variation in the cellular toxin amount from 18 °C to 27 °C for salinities ranging between 30 and 40 psu. At salinities of 10 to 25 psu, the toxin concentrations always remained below 20 fmol/cell. Toxin content was stable for irradiance ranging from 10 to 70 μmol photons/m2/s then slightly increased. Overall, the toxin profile was more stable than the toxin content (fmol/cell), except for temperature and/or salinity values different from those recorded during Alexandrium blooms in Thau Lagoon.
PMCID: PMC3707163  PMID: 23676417
Alexandrium catenella; PSP toxins; temperature; salinity; light
2.  Climate Change and Eutrophication Induced Shifts in Northern Summer Plankton Communities 
PLoS ONE  2013;8(6):e66475.
Marine ecosystems are undergoing substantial changes due to human-induced pressures. Analysis of long-term data series is a valuable tool for understanding naturally and anthropogenically induced changes in plankton communities. In the present study, seasonal monitoring data were collected in three sub-basins of the northern Baltic Sea between 1979 and 2011 and statistically analysed for trends and interactions between surface water hydrography, inorganic nutrient concentrations and phyto- and zooplankton community composition. The most conspicuous hydrographic change was a significant increase in late summer surface water temperatures over the study period. In addition, salinity decreased and dissolved inorganic nutrient concentrations increased in some basins. Based on redundancy analysis (RDA), warming was the key environmental factor explaining the observed changes in plankton communities: the general increase in total phytoplankton biomass, Cyanophyceae, Prymnesiophyceae and Chrysophyceae, and decrease in Cryptophyceae throughout the study area, as well as increase in rotifers and decrease in total zooplankton, cladoceran and copepod abundances in some basins. We conclude that the plankton communities in the Baltic Sea have shifted towards a food web structure with smaller sized organisms, leading to decreased energy available for grazing zooplankton and planktivorous fish. The shift is most probably due to complex interactions between warming, eutrophication and increased top-down pressure due to overexploitation of resources, and the resulting trophic cascades.
PMCID: PMC3680480  PMID: 23776676
3.  Evidence for isolated evolution of deep-sea ciliate communities through geological separation and environmental selection 
BMC Microbiology  2013;13:150.
Deep hypersaline anoxic basins (DHABs) are isolated habitats at the bottom of the eastern Mediterranean Sea, which originate from the ancient dissolution of Messinian evaporites. The different basins have recruited their original biota from the same source, but their geological evolution eventually constituted sharp environmental barriers, restricting genetic exchange between the individual basins. Therefore, DHABs are unique model systems to assess the effect of geological events and environmental conditions on the evolution and diversification of protistan plankton. Here, we examine evidence for isolated evolution of unicellular eukaryote protistan plankton communities driven by geological separation and environmental selection. We specifically focused on ciliated protists as a major component of protistan DHAB plankton by pyrosequencing the hypervariable V4 fragment of the small subunit ribosomal RNA. Geospatial distributions and responses of marine ciliates to differential hydrochemistries suggest strong physical and chemical barriers to dispersal that influence the evolution of this plankton group.
Ciliate communities in the brines of four investigated DHABs are distinctively different from ciliate communities in the interfaces (haloclines) immediately above the brines. While the interface ciliate communities from different sites are relatively similar to each other, the brine ciliate communities are significantly different between sites. We found no distance-decay relationship, and canonical correspondence analyses identified oxygen and sodium as most important hydrochemical parameters explaining the partitioning of diversity between interface and brine ciliate communities. However, none of the analyzed hydrochemical parameters explained the significant differences between brine ciliate communities in different basins.
Our data indicate a frequent genetic exchange in the deep-sea water above the brines. The “isolated island character” of the different brines, that resulted from geological events and contemporary environmental conditions, create selective pressures driving evolutionary processes, and with time, lead to speciation and shape protistan community composition. We conclude that community assembly in DHABs is a mixture of isolated evolution (as evidenced by small changes in V4 primary structure in some taxa) and species sorting (as indicated by the regional absence/presence of individual taxon groups on high levels in taxonomic hierarchy).
PMCID: PMC3707832  PMID: 23834625
Ciliates; Hypersaline; Deep-sea anoxic basins; DHABs; Brine; Species sorting; Environmental filtering; Niche separation
4.  Sponge-Microbe Associations Survive High Nutrients and Temperatures 
PLoS ONE  2012;7(12):e52220.
Coral reefs are under considerable pressure from global stressors such as elevated sea surface temperature and ocean acidification, as well as local factors including eutrophication and poor water quality. Marine sponges are diverse, abundant and ecologically important components of coral reefs in both coastal and offshore environments. Due to their exceptionally high filtration rates, sponges also form a crucial coupling point between benthic and pelagic habitats. Sponges harbor extensive microbial communities, with many microbial phylotypes found exclusively in sponges and thought to contribute to the health and survival of their hosts. Manipulative experiments were undertaken to ascertain the impact of elevated nutrients and seawater temperature on health and microbial community dynamics in the Great Barrier Reef sponge Rhopaloeides odorabile. R. odorabile exposed to elevated nutrient levels including 10 µmol/L total nitrogen at 31°C appeared visually similar to those maintained under ambient seawater conditions after 7 days. The symbiotic microbial community, analyzed by 16S rRNA gene pyrotag sequencing, was highly conserved for the duration of the experiment at both phylum and operational taxonomic unit (OTU) (97% sequence similarity) levels with 19 bacterial phyla and 1743 OTUs identified across all samples. Additionally, elevated nutrients and temperatures did not alter the archaeal associations in R. odorabile, with sequencing of 16S rRNA gene libraries revealing similar Thaumarchaeota diversity and denaturing gradient gel electrophoresis (DGGE) revealing consistent amoA gene patterns, across all experimental treatments. A conserved eukaryotic community was also identified across all nutrient and temperature treatments by DGGE. The highly stable microbial associations indicate that R. odorabile symbionts are capable of withstanding short-term exposure to elevated nutrient concentrations and sub-lethal temperatures.
PMCID: PMC3527390  PMID: 23284943
5.  UVB Induces a Genome-Wide Acting Negative Regulatory Mechanism That Operates at the Level of Transcription Initiation in Human Cells 
PLoS Genetics  2014;10(7):e1004483.
Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair (TCR) has been described to stop transcription and quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. Moreover, numerous efforts investigated the fate of blocked RNA polymerase II (Pol II) during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed and degraded or bypass the lesions to allow TCR. We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We found that the used UVB dose did not induce Pol II degradation however surprisingly at about 93% of the promoters of all expressed genes Pol II occupancy was seriously reduced 2–4 hours following UVB irradiation. The presence of Pol II at these cleared promoters was restored 5–6 hours after irradiation, indicating that the negative regulation is very dynamic. We also identified a small set of genes (including several p53 regulated genes), where the UVB-induced Pol II clearing did not operate. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH, but not TBP, follows the behavior of Pol II, suggesting that at these genes upon UVB treatment TFIIH is sequestered for DNA repair by the TCR machinery. In agreement, in cells where the TCR factor, the Cockayne Syndrome B protein, was depleted UVB did not induce Pol II and TFIIH clearance at promoters. Thus, our study reveals a UVB induced negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed gene promoters, and a small subset of genes, where Pol II escapes this negative regulation.
Author Summary
Our genome is continuously exposed to genotoxic attacks that generate aberrant DNA structures. These can block the transcribing DNA-dependent RNA polymerase II (Pol II) enzyme and can lead to deleterious cellular processes. Cells have developed several mechanisms to stop Pol II, repair the roadblocks and to restore normal polymerase traffic. Numerous efforts investigated the fate of blocked Pol II during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed or bypass the lesions to allow repair. We carried out a genome-wide analysis of Pol II behavior upon a DNA damaging stress, UVB, which is relevant from the public health standpoint. Thus, we could follow UVB-induced Pol II behavior changes on every human gene over time. We uncovered a novel UV induced negative regulatory mechanism, which inhibits the recruitment of Pol II to the promoters of about 93% of all transcribed genes, and a small subset of gene (including regulators of repair, cell growth and survival) that escapes this negative regulation, probably because their gene products are required during/after UVB irradiation. Thus, we uncover how a cell induces a global negative regulation at the level of transcription initiation in response to a genotoxic stress.
PMCID: PMC4109906  PMID: 25058334
6.  Ultraviolet B-induced expression of amphiregulin and growth differentiation factor 15 in human lens epithelial cells 
Molecular Vision  2011;17:159-169.
Epidemiological and experimental studies have revealed that exposure to ultraviolet B (UVB) light can induce cataractogenesis. The objective of this study was to determine gene expression changes in human lens epithelial cells in response to UVB exposure and identify factors that can be involved in UVB-induced cataractogenesis.
SV40 T-antigen-transformed human lens epithelial cells (SRA01/04) were irradiated at various UVB-energy levels (10–80 mJ/cm2) and checked for viability. An irradiation condition of 30 mJ/cm2 was adopted for transcriptome analysis. Total RNAs isolated from UVB-exposed and unexposed cells at 12 h and 24 h after UVB exposure were examined for global gene expression changes using Affymetrix Human Gene 1.0 ST array. mRNA levels of specific genes were examined by RT–PCR and real-time PCR, and protein levels in the conditioned media were assayed by ELISA. To examine mRNA expression in human lens, primary cultured human lens epithelial (HLE) cells were prepared from surgically removed lens epithelium, and used for UVB-irradiation and expression analysis. Effects of certain gene products on SRA01/04 cell metabolism were examined using commercially available recombinant proteins.
Expression of most the genes analyzed was essentially unchanged (between 0.5 and 2.0 fold) in UVB-irradiated cells compared to non-irradiated cells at both 12 and 24 h after UVB exposure. Sixty one and 44 genes were upregulated more than twofold by UVB exposure at 12 h and 24 h, respectively. Emphasis was placed on genes encoding extracellular proteins, especially growth factors and cytokines. A total of 18 secreted protein genes were upregulated more than twofold at either or both time points. Amphiregulin (AREG) and growth differentiation factor 15 (GDF15) were chosen because of their higher upregulation and novelty, and their upregulation was confirmed in SRA01/04 cells using RT–PCR and real-time PCR analysis. AREG and GDF15 protein levels in conditioned media significantly increased at all UVB-energy points at 24 h, while they were scarcely detectable at 12 h. AREG and GDF15 mRNA levels were also significantly upregulated in UVB-irradiated primary cultured HLE cells compared with the corresponding control culture. AREG significantly stimulated 3H-thymidine and 3H-leucine uptake in SRA01/04 cells as did a positive control epidermal growth factor (EGF). Recombinant GDF15 did not stimulate 3H-thymidine incorporation at any concentration tested, but significantly stimulated 3H-leucine uptake. RT–PCR analysis demonstrated that primary cultured HLE and SRA01/04 cells expressed not only epidermal growth factor receptor (EGFR) mRNA but also transforming growth factor β receptors (TGFBR1 and TGFBR2) mRNAs.
These results indicate that AREG and GDF15 produced in response to UVB exposure can affect the growth and protein synthesis of lens epithelial cells, suggesting that they have autocrine and paracrine roles related to pathological changes of lens tissue during long-term UVB exposure.
PMCID: PMC3021579  PMID: 21245963
7.  Synergistic Interactions within Disturbed Habitats between Temperature, Relative Humidity and UVB Radiation on Egg Survival in a Diadromous Fish 
PLoS ONE  2011;6(9):e24318.
Anthropogenic impacts, including urbanization, deforestation, farming, and livestock grazing have altered riparian margins worldwide. One effect of changes to riparian vegetation is that the ground-level light, temperature, and humidity environment has also been altered. Galaxias maculatus, one of the most widely distributed fishes of the southern hemisphere, lays eggs almost exclusively beneath riparian vegetation in tidally influenced reaches of rivers. We hypothesized that the survival of these eggs is greatly affected by the micro-environment afforded by vegetation, particularly relating to temperature, humidity and UVB radiation. We experimentally reduced riparian vegetation height and altered shading characteristics, tracked egg survival, and used small ground-level temperature, humidity and UVB sensors to relate survival to ground-level effects around egg masses. The ground-level physical environment was markedly different from the surrounding ambient conditions. Tall dense riparian vegetation modified ambient conditions to produce a buffered temperature regime with constant high relative humidity, generally above 90%, and negligible UVB radiation at ground-level. Where vegetation height was reduced, frequent high temperatures, low humidity, and high UVB irradiances reduced egg survival by up to 95%. Temperature effects on egg survival were probably indirect, through reduced humidity, because developing eggs are known to survive in a wide range of temperatures. In this study, it was remarkable how such small variations in relatively small sites could have such a large effect on egg survival. It appears that modifications to riparian vegetation and the associated changes in the physical conditions of egg laying sites are major mechanisms affecting egg survival. The impacts associated with vegetational changes through human-induced disturbances are complex yet potentially devastating. These effects are particularly important because they affect a very small portion of habitat that is required to complete the life history of a species, despite the wide distribution of adults and juveniles across aquatic and marine environments.
PMCID: PMC3169581  PMID: 21931680
8.  Expression and Regulation of Cornified Envelope Proteins in Human Corneal Epithelium 
Stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery, termed the cornified envelope. The purpose of this study was to evaluate the presence and distribution of cornified envelope precursors in human corneal epithelium, their expression in human corneal epithelial cell cultures, and the effect of ultraviolet radiation (UVB) and transglutaminase (TG) inhibition on their expression.
Tissue distribution of small proline-rich proteins (SPRRs) and filaggrin and involucrin was studied in human cornea sections by immunofluorescence staining. Primary human corneal epithelial cells (HCECs) from limbal explants were used in cell culture experiments. A single dose of UVB at 20 mJ/cm2 was used to stimulate these cells, in the presence or absence of mono-dansyl cadaverine (MDC), a TG inhibitor. SPRR2 and involucrin protein levels were studied by immunofluorescence staining and Western blot analysis. Gene expression of 12 proteins was investigated by semiquantitative reverse transcription–polymerase chain reaction.
In human cornea tissue, SPRR1, SPRR2, filaggrin, and involucrin protein expression were detected in the central and peripheral corneal and limbal epithelium. In HCECs, SPRR2 and involucrin proteins were detected in the cytosolic fraction, and involucrin levels increased after UVB. Both SPRR2 and involucrin levels accumulated in the presence of MDC. Nine genes including involucrin, SPRR (types 1A, 1B, 2A, 2B, and 3), late envelope protein (LEP) 1 and 16, and filaggrin were expressed by HCECs. SPRR 4, loricrin, and LEP 6 transcripts were not detected. UVB downregulated SPRR (2A, 2B) and LEP 1 transcripts.
Various envelope precursors are expressed in human corneal epithelium and in HCECs, acute UVB stress differentially alters their expression in HCECs. The expression of envelope precursors and their rapid modulation by UVB supports the role of these proteins in the regulation of ocular surface stress. TG function may be relevant in the regulation of soluble precursors in UVB-stimulated corneal epithelium.
PMCID: PMC2906387  PMID: 16639001
9.  Dose-Response on the Chemopreventive Effects of Sarcophine-Diol on UVB-Induced Skin Tumor Development in SKH-1 Hairless Mice 
Marine Drugs  2012;10(9):2111-2125.
Sarcophine-diol (SD) is a lactone ring-opened analogue of sarcophine. It has shown chemopreventive effects on chemically-induced skin tumor development in female CD-1 mice, as well as in a UVB-induced skin tumor development model in hairless SKH-1 mice at a dose of 30 μg SD applied topically and 180 mJ/cm2 UVB. The objective of this study was to determine the dose-response on the chemopreventive effects of SD on SKH-1 hairless mice when exposed to a UVB radiation dose of 30 mJ/cm2. This UVB dose better represents chronic human skin exposure to sunlight leading to skin cancer than previous studies applying much higher UVB doses. Carcinogenesis was initiated and promoted by UVB radiation. Female hairless SKH-1 mice were divided into five groups. The control group was topically treated with 200 μL of acetone (vehicle), and the SD treatment groups were topically treated with SD (30 μg, 45 μg, and 60 μg dissolved in 200 μL of acetone) 1 h before UVB radiation (30 mJ/cm2). The last group of animals received 60 μg SD/200 μL acetone without UVB exposure. These treatments were continued for 27 weeks. Tumor multiplicity and tumor volumes were recorded on a weekly basis for 27 weeks. Weight gain and any signs of toxicity were also closely monitored. Histological characteristics and the proliferating cell nuclear antigen (PCNA) were evaluated in the mice skin collected at the end of the experiment. The dose-response study proved a modest increase in chemopreventive effects with the increase in SD dose. SD reduced the number of cells positively stained with PCNA proliferation marker in mice skin. The study also showed that SD application without UVB exposure has no effect on the structure of skin. The results from this study suggest that broader range doses of SD are necessary to improve the chemopreventive effects.
PMCID: PMC3475277  PMID: 23118725
sarcophine-diol; skin cancer; chemopreventive agent; SKH-1 mice; UVB radiation
10.  Involvement of Platelet-Activating Factor in Ultraviolet B-Induced Hyperalgesia 
Ultraviolet B (UVB) radiation causes cutaneous inflammation. One important clinical consequence of UVB-induced inflammation is increased pain or hyperalgesia, which is likely mediated by enhanced sensitivity of cutaneous sensory neurons. Previous studies have demonstrated that UVB radiation generates the lipid mediator, platelet-activating factor (PAF), as well as oxidized phospholipids that act as PAF-mimetics. These substances exert effects through the PAF receptor (PAF-R). This study was designed to assess whether PAF-R is involved in UVB-induced hyperalgesia. Intradermal injection of carbamoyl PAF (CPAF; 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine) resulted in an enhanced response to mechanical stimuli in wild-type mice but not in PAF-R knockout (KO) mice. There was no significant change in paw withdrawal to noxious thermal stimuli in either genotype after intradermal injection of CPAF. Exposure of the hind paw to 1,500 J m−2 UVB radiation caused an increased sensitivity to both mechanical and thermal stimulation in wild-type mice but not in PAF-R KO mice. The thermal hyperalgesia caused by UVB irradiation was inhibited in mice that lacked PAF-R in bone marrow-derived cells. These data demonstrate that the PAF-R is important for UVB-induced hyperalgesia. Further investigation of the role of PAF-R signaling in UVB-induced hyperalgesia could provide better understanding of the pathological processes initiated by UVB-induced skin damage.
PMCID: PMC2717090  PMID: 18580961
11.  Narrow-band UVB radiation promotes dendrite formation by activating Rac1 in B16 melanoma cells 
Molecular and Clinical Oncology  2013;1(5):858-862.
Melanocytes are found scattered throughout the basal layer of the epidermis. Following hormone or ultraviolet (UV) light stimulation, the melanin pigments contained in melanocytes are transferred through the dendrites to the surrounding keratinocytes to protect against UV light damage or carcinogenesis. This has been considered as a morphological indicator of melanocytes and melanoma cells. Small GTPases of the Rho family have been implicated in the regulation of actin reorganization underlying dendrite formation in melanocytes and melanoma cells. It has been proven that ultraviolet light plays a pivotal role in melanocyte dendrite formation; however, the molecular mechanism underlying this process has not been fully elucidated. The effect of small GTPases, such as Rac1 and RhoA, on the morphology of B16 melanoma cells treated with narrow-band UVB radiation was investigated. The morphological changes were observed under a phase contrast microscope and the F-actin microfilament of the cytoskeleton was observed under a laser scanning confocal microscope. The pull-down assay was performed to detect the activity of the small GTPases Rac1 and RhoA. The morphological changes were evident, with globular cell bodies and increased numbers of tree branch-like dendrites. The cytoskeletal F-actin appeared disassembled following narrow-band UVB irradiation of B16 melanoma cells. Treatment of B16 melanoma cells with narrow-band UVB radiation resulted in the activation of Rac1 in a time-dependent manner. In conclusion, the present study may provide a novel method through which narrow-band UVB radiation may be used to promote dendrite formation by activating the Rac1 signaling pathway, resulting in F-actin rearrangement in B16 melanoma cells.
PMCID: PMC3916207  PMID: 24649261
narrow-band ultraviolet B radiation; Rac1; F-actin rearrangement; dendricity; B16 melanoma cells
12.  Cell membrane is a major locus for ultraviolet B-induced alterations in accessory cells. 
Journal of Clinical Investigation  1990;85(5):1529-1536.
In vitro ultraviolet B (UVB) irradiation of human blood monocytes inhibits their accessory cell function for antigen- and mitogen-induced T cell responses. These studies were designed to characterize the nature of the UVB-induced defect in human monocyte accessory cell function. Irradiated monocytes were deficient in their ability to serve as accessory cells for OKT3-induced T cell activation. In vitro exposure of monocytes to 100 J/m2 UVB completely inhibited the T cell proliferative response (51502 cpm, non-UVB-irradiated; 302 cpm, UVB-irradiated). Analysis of the accessory signals altered by UVB indicated that irradiated monocytes were incapable of binding to OKT3 molecules attached to the CD3 antigen on T cells. Provision of an alternative mechanism for binding of OKT3 molecules by attaching anti-mouse IgG to the bottom of microtiter wells completely restored accessory cell function. Further characterization of the defect demonstrated that UVB radiation did not deplete p72 Fc receptors from the surface of irradiated monocytes. However, UVB exposure did produce a dose-dependent decrease in monocyte membrane expression of ICAM-1. It is proposed that UVB radiation leads to changes within the cell membrane that inhibit the ability of monocytes to express selected molecules necessary for binding of T cells.
PMCID: PMC296601  PMID: 1970582
13.  IMOS National Reference Stations: A Continental-Wide Physical, Chemical and Biological Coastal Observing System 
PLoS ONE  2014;9(12):e113652.
Sustained observations allow for the tracking of change in oceanography and ecosystems, however, these are rare, particularly for the Southern Hemisphere. To address this in part, the Australian Integrated Marine Observing System (IMOS) implemented a network of nine National Reference Stations (NRS). The network builds on one long-term location, where monthly water sampling has been sustained since the 1940s and two others that commenced in the 1950s. In-situ continuously moored sensors and an enhanced monthly water sampling regime now collect more than 50 data streams. Building on sampling for temperature, salinity and nutrients, the network now observes dissolved oxygen, carbon, turbidity, currents, chlorophyll a and both phytoplankton and zooplankton. Additional parameters for studies of ocean acidification and bio-optics are collected at a sub-set of sites and all data is made freely and publically available. Our preliminary results demonstrate increased utility to observe extreme events, such as marine heat waves and coastal flooding; rare events, such as plankton blooms; and have, for the first time, allowed for consistent continental scale sampling and analysis of coastal zooplankton and phytoplankton communities. Independent water sampling allows for cross validation of the deployed sensors for quality control of data that now continuously tracks daily, seasonal and annual variation. The NRS will provide multi-decadal time series, against which more spatially replicated short-term studies can be referenced, models and remote sensing products validated, and improvements made to our understanding of how large-scale, long-term change and variability in the global ocean are affecting Australia's coastal seas and ecosystems. The NRS network provides an example of how a continental scaled observing systems can be developed to collect observations that integrate across physics, chemistry and biology.
PMCID: PMC4269483  PMID: 25517905
14.  Phylogenetic Differences in Attached and Free-Living Bacterial Communities in a Temperate Coastal Lagoon during Summer, Revealed via High-Throughput 16S rRNA Gene Sequencing 
Most of what is known about coastal free-living and attached bacterial diversity is based on open coasts, with high particulate and nutrient riverine supply, terrestrial runoffs, and anthropogenic activities. The Magdalen Islands in the Gulf of St. Lawrence (Canada) are dominated by shallow lagoons with small, relatively pristine catchments and no freshwater input apart from rain. Such conditions provided an opportunity to investigate coastal free-living and attached marine bacterial diversity in the absence of confounding effects of steep freshwater gradients. We found significant differences between the two communities and marked temporal patterns in both. Taxonomic richness and diversity were greater in the attached than in the free-living community, increasing over summer, especially within the least abundant bacterial phyla. The highest number of reads fell within the SAR 11 clade (Pelagibacter, Alphaproteobacteria), which dominated free-living communities. The attached communities had deeper phylum-level diversity than the free-living fraction. Distance-based redundancy analysis indicated that the particulate organic matter (POM) concentration was the main variable separating early and late summer samples with salinity and temperature changes also significantly correlated to bacterial community structure. Our approach using high-throughput sequencing detected differences in free-living versus attached bacteria in the absence of riverine input, in keeping with the concept that marine attached communities are distinct from cooccurring free-living taxa. This diversity likely reflects the diverse microhabitats of available particles, implying that the total bacterial diversity in coastal systems is linked to particle supply and variability, with implications for understanding microbial biodiversity in marine systems.
PMCID: PMC3993158  PMID: 24463966
15.  Extensive genetic diversity and rapid population differentiation during blooms of Alexandrium fundyense (Dinophyceae) in an isolated salt pond on Cape Cod, MA, USA 
Ecology and Evolution  2012;2(10):2588-2599.
In Massachusetts, paralytic shellfish poisoning (PSP) is annually recurrent along the coastline, including within several small embayments on Cape Cod. One such system, the Nauset Marsh System (NMS), supports extensive marshes and a thriving shellfishing industry. Over the last decade, PSP in the NMS has grown significantly worse; however, the origins and dynamics of the toxic Alexandrium fundyense (Balech) populations that bloom within the NMS are not well known. This study examined a collection of 412 strains isolated from the NMS and the Gulf of Maine (GOM) in 2006–2007 to investigate the genetic characteristics of localized blooms and assess connectivity with coastal populations. Comparisons of genetic differentiation showed that A. fundyense blooms in the NMS exhibited extensive clonal diversity and were genetically distinct from populations in the GOM. In both project years, genetic differentiation was observed among temporal samples collected from the NMS, sometimes occurring on the order of approximately 7 days. The underlying reasons for temporal differentiation are unknown, but may be due, in part, to life-cycle characteristics unique to the populations in shallow embayments, or possibly driven by selection from parasitism and zooplankton grazing; these results highlight the need to investigate the role of selective forces in the genetic dynamics of bloom populations. The small geographic scale and limited connectivity of NMS salt ponds provide a novel system for investigating regulators of blooms, as well as the influence of selective forces on population structure, all of which are otherwise difficult or impossible to study in the adjacent open-coastal waters or within larger estuaries.
PMCID: PMC3492784  PMID: 23145343
Alexandrium; Amoebophrya; dinoflagellate; Gulf of Maine; microsatellites; Nauset Marsh; paralytic shellfish poisoning.
16.  Role of AMPK in UVB-induced DNA damage repair and growth control 
Oncogene  2012;32(21):2682-2689.
Skin cancer is the most common cancer in the U.S., while DNA-damaging UVB radiation from the sun remains the major environmental risk factor. Reducing skin cancer incidence is becoming an urgent issue. The energy-sensing enzyme 5’-AMP-activated protein kinase (AMPK) plays a key role in the regulation of cellular lipid and protein metabolism in response to stimuli such as exercise and changes in fuel availability. However, the role AMPK in the response of skin cells to UVB damage and in skin cancer prevention remains unknown. Here we show that AMPK activation is reduced in human and mouse squamous cell carcinoma as compared with normal skin, and by UVB irradiation, suggesting that AMPK is a tumor suppressor. At the molecular level, AMPK deletion reduced the expression of the DNA repair protein xeroderma pigmentosum C (XPC) and UVB-induced DNA repair. AMPK activation by its activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin (N’,N’-dimethylbiguanide), the most widely used anti-diabetic drug, increased the expression of XPC expression and UVB-induced DNA repair in mouse skin, normal human epidermal keratinocytes, and AMPK wild-type cells but not in AMPK deficient cells, indicating an AMPK-dependent mechanism. Topical treatment with AICAR and metformin not only delayed onset of UVB-induced skin tumorigenesis but also reduced tumor multiplicity. Furthermore, AMPK deletion increased ERK activation and cell proliferation, while AICAR and metformin inhibited ERK activation and cell proliferation in keratinocytes, mouse skin, AMPK wild-type and AMPK deficient cells, suggesting an AMPK-independent mechanism. Finally, in UVB-damaged tumor-bearing mice, both topical and systemic metformin prevented the formation of new tumors and suppressed growth of established tumors. Our findings not only suggest that AMPK is a tumor suppressor in the skin by promoting DNA repair and controlling cell proliferation, but also demonstrate previously unknown mechanisms by which the AMPK activators prevent UVB-induced skin tumorigenesis.
PMCID: PMC3465498  PMID: 22751115
AMPK; Tumorigenesis; UVB; DNA repair; XPC; proliferation; ERK
17.  Gardenia jasminoides Extract Attenuates the UVB-Induced Expressions of Cytokines in Keratinocytes and Indirectly Inhibits Matrix Metalloproteinase-1 Expression in Human Dermal Fibroblasts 
Ultraviolet radiation (UV) is a major cause of photoaging, which also involves inflammatory cytokines and matrix metalloproteinases (MMP). The present study was undertaken to examine the UVB-protecting effects of yellow-colored plant extracts in cell-based assays. HaCaT keratinocytes were exposed to UVB in the absence or presence of plant extracts, and resulting changes in cell viability and inflammatory cytokine expression were measured. Of the plant extracts tested, Gardenia jasminoides extract showed the lowest cytotoxicity and dose-dependently enhanced the viabilities of UVB-exposed cells. Gardenia jasminoides extract also attenuated the mRNA expressions of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in HaCaT cells stimulated by UVB. Conditioned medium from UVB-exposed HaCaT cells was observed to stimulate MMP-1 protein expression in human dermal fibroblasts, and this effect was much smaller for the conditioned medium of HaCaT cells exposed to UVB in the presence of Gardenia jasminoides extract. Gardenia jasminoides extract also exhibited antioxidative and antiapoptotic effects in HaCaT cells exposed to UVB. These results indicated that UVB-induced injury and inflammatory responses of skin cells can be attenuated by yellow-colored plant extracts, such as Gardenia jasminoides extract.
PMCID: PMC3966326  PMID: 24711853
18.  Do high temperatures enhance the negative effects of ultraviolet-B radiation in embryonic and larval amphibians? 
Biology Open  2012;1(9):897-903.
For the embryos and tadpoles of amphibian species, exposure to ultraviolet-B radiation (UVBR) can be lethal, or cause a variety of sublethal effects. Low temperatures enhance the detrimental effects of UVBR and this is most likely because the enzyme-mediated processes involved in the repair of UVBR-induced damage function less effectively at low temperatures. Whether these repair processes are also impaired, and thus the negative effects of UVBR similarly enhanced, at high temperatures is not known, but is an ecologically relevant question to ask given that organisms that inhabit environments where the temperature fluctuates widely on a daily timescale are likely to experience high doses of UVBR when temperatures are high. Here we examined the thermal-dependence of UVBR effects in the context of an ecologically-relevant fluctuating UVBR and temperature regime to test the hypothesis that exposure to peak UVBR levels while the temperature is high (35°C) is more detrimental to embryonic and larval Limnodynastes peronii than exposure to peak UVBR levels while the temperature is moderate (25°C). Embryos exposed to peak UVBR levels at 35°C hatched 10 h later than those exposed to peak UVBR levels at 25°C and, as tadpoles, were smaller and consequently swam more slowly but, in an environment with predators, exhibited no difference in survival time. There was also no effect of experimental treatment on the hatching success of embryos, nor on the post-hatch survival of tadpoles. These findings, therefore, are not sufficiently strong to support our hypothesis that high temperatures enhance the negative effects of UVBR in embryonic and larval amphibians.
PMCID: PMC3507240  PMID: 23213484
Amphibian declines; Ultraviolet radiation; Temperature; Locomotor performance; Predation
19.  MicroRNAs in Skin Response to UV Radiation 
Current drug targets  2013;14(10):1128-1134.
Solar ultraviolet (UV) radiation, an ubiquitous environmental carcinogen, is classified depending on the wave-length, into three regions; short-wave UVC (200–280 nm), mid-wave UVB (280–320 nm), and long-wave UVA (320–400 nm). The human skin, constantly exposed to UV radiation, particularly the UVB and UVA components, is vulnerable to its various deleterious effects such as erythema, photoaging, immunosuppression and cancer. To counteract these and for the maintenance of genomic integrity, cells have developed several protective mechanisms including DNA repair, cell-cycle arrest and apoptosis. The network of damage sensors, signal transducers, mediators, and various effector proteins is regulated through changes in gene expression. MicroRNAs (miRNAs), a group of small non-coding RNAs, act as post-transcriptional regulators through binding to complementary sequences in the 3′-untranslated region of their target genes, resulting in either translational repression or target degradation. Recent studies show that miRNAs add an additional layer of complexity to the intricately controlled cellular responses to UV radiation. This review summarizes our current knowledge of the role of miRNAs in the regulation of the human skin response upon exposure to UV radiation.
PMCID: PMC3985496  PMID: 23834148
MicroRNAs; skin; UV
20.  UVB irradiation does not directly induce detectable changes of DNA methylation in human keratinocytes 
F1000Research  2013;2:45.
Unprotected exposure to UVB radiation from the sun and the resulting DNA damage are thought to be responsible for physiological changes in the skin and for a variety of skin cancers, including basal cell and squamous cell carcinoma and malignant melanoma. Although the mutagenic effects of UVB have been well documented and studied mechanistically, there is only limited information as to whether UV light may also be responsible for inducing epigenetic changes in the genome of exposed cells. DNA methylation is a stable epigenetic modification involved in gene control. To study the effects of UVB radiation on DNA methylation, we repeatedly exposed normal human keratinocytes to a UVB light source. After a recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA) method in combination with high-resolution microarrays. Bioinformatics analysis revealed only a limited number of possible differences between UVB-exposed and control cells. However, these minor apparent changes could not be independently confirmed by bisulfite sequencing-based approaches. This study reveals that UVB irradiation of keratinocytes has no recognizable global effect on DNA methylation patterns and suggests that changes in DNA methylation, as observed in skin cancers, are not immediate consequences of human exposure to solar UVB irradiation.
PMCID: PMC3901454  PMID: 24555035
21.  Interleukin-12-Deficiency Exacerbates Inflammatory Responses in UV-Irradiated Skin and Skin Tumors 
Interleukin (IL)-12-deficiency has been shown to promote photocarcinogenesis in mice. As UVB-induced inflammation is an important tumor-promoting event in the development of skin tumors, we determined the susceptibility of IL-12-deficiency on UVB-induced inflammatory responses in mice. For this purpose IL-12-knockout (IL-12 KO) and their wild-type counterparts were subjected to photocarcinogenesis protocol; skin and tumor samples were collected at the termination of the experiment, and analyzed for biomarkers of inflammation and their mediators. We found that the levels of infiltrating leukocytes, myeloperoxidase, proliferating cell-nuclear antigen (PCNA), COX-2, PGE2, proinflammatory cytokines IL-1β, TNF-α, and IL-6 were higher in UVB-exposed skin of IL-12 KO mice than wild-types. In a short-term experiment, pretreatment of IL-12 KO mice with rIL-12 (50 ng/mouse) before each exposure of UVB increased the repair rate of UVB-induced cyclobutane pyrimidine dimers while inhibited UVB-induced increase of myeloperoxidase, COX-2, PGE2, PCNA, TNF-α and IL-1β compared to non-rIL-12-treated IL-12 KO mouse skin. Similarly, the tumors of IL-12 KO mice expressed higher levels of inflammatory responses compared to the tumors from wild-types. Together, our data suggest that IL-12 KO mice are more susceptible to both UVB-induced inflammation and photocarcinogenesis because of the deficiency in the repair of UVB-induced DNA damage.
PMCID: PMC2574589  PMID: 18509359
22.  High temporal and spatial diversity in marine RNA viruses implies that they have an important role in mortality and structuring plankton communities 
Viruses in the order Picornavirales infect eukaryotes, and are widely distributed in coastal waters. Amplicon deep-sequencing of the RNA dependent RNA polymerase (RdRp) revealed diverse and highly uneven communities of picorna-like viruses in the coastal waters of British Columbia (BC), Canada. Almost 300 000 pyrosequence reads revealed 145 operational taxonomic units (OTUs) based on 95% sequence similarity at the amino-acid level. Each sample had between 24 and 71 OTUs and there was little overlap among samples. Phylogenetic analysis revealed that some clades of OTUs were only found at one site; whereas, other clades included OTUs from all sites. Since most of these OTUs are likely from viruses that infect eukaryotic phytoplankton, and viral isolates infecting phytoplankton are strain-specific; each OTU probably arose from the lysis of a specific phytoplankton taxon. Moreover, the patchiness in OTU distribution, and the high turnover of viruses in the mixed layer, implies continuous infection and lysis by RNA viruses of a diverse array of eukaryotic phytoplankton taxa. Hence, these viruses are likely important elements structuring the phytoplankton community, and play a significant role in nutrient cycling and energy transfer.
PMCID: PMC4266044  PMID: 25566218
RNA viruses; phytoplankton mortality; viral ecology; pyrosequencing; viral diversity; Picornavirales
23.  Distinct Pigmentary and Melanocortin 1 Receptor–Dependent Components of Cutaneous Defense against Ultraviolet Radiation 
PLoS Genetics  2007;3(1):e9.
Genetic variation at the melanocortin 1 receptor (MC1R) is an important risk factor for developing ultraviolet (UV) radiation–induced skin cancer, the most common form of cancer in humans. The underlying mechanisms by which the MC1R defends against UV-induced skin cancer are not known. We used neonatal mouse skin (which, like human skin, contains a mixture of melanocytes and keratinocytes) to study how pigment cells and Mc1r genotype affect the genome-level response to UV radiation. Animals without viable melanocytes (KitW-v/KitW-v) or animals lacking a functional Mc1r (Mc1re/Mc1re) were exposed to sunburn-level doses of UVB radiation, and the patterns of large-scale gene expression in the basal epidermis were compared to each other and to nonmutant animals. Our analysis revealed discrete Kit- and Mc1r-dependent UVB transcriptional responses in the basal epidermis. The Kit-dependent UVB response was characterized largely by an enrichment of oxidative and endoplasmic reticulum stress genes, highlighting a distinctive role for pigmented melanocytes in mediating antioxidant defenses against genotoxic stresses within the basal epidermal environment. By contrast, the Mc1r-dependent UVB response contained an abundance of genes associated with regulating the cell cycle and oncogenesis. To test the clinical relevance of these observations, we analyzed publicly available data sets for primary melanoma and melanoma metastases and found that the set of genes specific for the Mc1r-dependent UVB response was able to differentiate between different clinical subtypes. Our analysis also revealed that the classes of genes induced by UVB differ from those repressed by UVB with regard to their biological functions, their overall number, and their size. The findings described here offer new insights into the transcriptional nature of the UV response in the skin and provide a molecular framework for the underlying mechanisms by which melanocytes and the Mc1r independently mediate and afford protection against UV radiation.
Author Summary
Skin cancer is the most common type of cancer in humans and annually accounts for approximately 60,000 deaths worldwide. The most important factors causally linked to skin cancer susceptibility are inadequate protection against ultraviolet (UV) B radiation, fair skin color, and variation of the melanocortin 1 receptor (MC1R) gene. We used cDNA microarrays to measure the genome-wide transcriptional responses to UVB irradiation in the epidermis of neonatal mice (which approximates the human basal epidermis in its cellular composition and general physiology). To investigate how pigment cells (melanocytes) and MC1R afford protection against UVB radiation, we compared results from normal mice to those from mutant mice that lacked either melanocytes (KitW-v/KitW-v) or a functional Mc1r (Mc1re/Mc1re). We identified melanocyte- and Mc1r-dependent UVB gene expression profiles in the basal epidermis. Surprisingly, the melanocyte- and Mc1r-dependent UVB responses highlighted distinct functions, with the former largely mediating antioxidant defenses and the latter regulating the cell cycle and susceptibility to oncogenesis. We also demonstrated that a subset of Mc1r-dependent UVB-responsive genes could discriminate among human melanoma subtypes, thereby suggesting a mechanism by which MC1R gene variants may predispose toward skin cancer.
PMCID: PMC1774588  PMID: 17222061
24.  Mitigating Harmful Cyanobacterial Blooms in a Human- and Climatically-Impacted World 
Life  2014;4(4):988-1012.
Bloom-forming harmful cyanobacteria (CyanoHABs) are harmful from environmental, ecological and human health perspectives by outcompeting beneficial phytoplankton, creating low oxygen conditions (hypoxia, anoxia), and by producing cyanotoxins. Cyanobacterial genera exhibit optimal growth rates and bloom potentials at relatively high water temperatures; hence, global warming plays a key role in their expansion and persistence. CyanoHABs are regulated by synergistic effects of nutrient (nitrogen:N and phosphorus:P) supplies, light, temperature, vertical stratification, water residence times, and biotic interactions. In most instances, nutrient control strategies should focus on reducing both N and P inputs. Strategies based on physical, chemical (nutrient) and biological manipulations can be effective in reducing CyanoHABs; however, these strategies are largely confined to relatively small systems, and some are prone to ecological and environmental drawbacks, including enhancing release of cyanotoxins, disruption of planktonic and benthic communities and fisheries habitat. All strategies should consider and be adaptive to climatic variability and change in order to be effective for long-term control of CyanoHABs. Rising temperatures and greater hydrologic variability will increase growth rates and alter critical nutrient thresholds for CyanoHAB development; thus, nutrient reductions for bloom control may need to be more aggressively pursued in response to climatic changes globally.
PMCID: PMC4284478  PMID: 25517134
harmful cyanobacteria; nitrogen; phosphorus; water quality management; mitigation; climate change
25.  Ultraviolet B Preconditioning Enhances the Hair Growth-Promoting Effects of Adipose-Derived Stem Cells Via Generation of Reactive Oxygen Species 
Stem Cells and Development  2012;22(1):158-168.
Hypoxia induces the survival and regenerative potential of adipose-derived stem cells (ASCs), but there are tremendous needs to find alternative methods for ASC preconditioning. Therefore, this work investigated: (1) the ability of low-dose ultraviolet B (UVB) radiation to stimulate the survival, migration, and tube-forming activity of ASCs in vitro; (2) the ability of UVB preconditioning to enhance the hair growth-promoting capacity of ASCs in vivo; and (3) the mechanism of action for ASC stimulation by UVB. Although high-dose UVB decreased the proliferation of ASCs, low-dose (10 or 20 mJ/cm2) treatment increased their survival, migration, and tube-forming activity. In addition, low-dose UVB upregulated the expression of ASC-derived growth factors, and a culture medium conditioned by UVB-irradiated ASCs increased the proliferation of dermal papilla and outer root sheet cells. Notably, injection of UVB-preconditioned ASCs into C3H/HeN mice significantly induced the telogen-to-anagen transition and increased new hair weight in vivo. UVB treatment significantly increased the generation of reactive oxygen species (ROS) in cultured ASCs, and inhibition of ROS generation by diphenyleneiodonium chloride (DPI) significantly attenuated UVB-induced ASC stimulation. Furthermore, NADPH oxidase 4 (Nox4) expression was induced in ASCs by UVB irradiation, and Nox4 silencing by small interfering RNA, like DPI, significantly reduced UVB-induced ROS generation. These results suggest that the primary involvement of ROS generation in UVB-mediated ASC stimulation occurs via the Nox4 enzyme. This is the first indication that a low dose of UVB radiation and/or the control of ROS generation could potentially be incorporated into a novel ASC preconditioning method for hair regeneration.
PMCID: PMC3528093  PMID: 22784094

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