Kevin B. Strychar

Enhanced gene transfection efficiency by polyamidoamine (PAMAM) dendrimers modified with ornithine residues

Aim of the study was to prepare and to evaluate gene transfection efficiency and cytotoxicity of the ornithine-conjugated PAMAMG4 dendrimers. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. A comparative gene transfection study between PAMAMG4 dendrimers and the surface modified dendrimers was conducted in HEK 293T, GM7373 and NCI H157G cell lines. Effect of excess of ornithine (100muM) on transfection efficiency of the ornithine-conjugated PAMAMG4 dendrimers was investigated in separate experiment. Cytotoxicity of the dendriplexes was tested in HEK 293T cells by MTT assay. (1)H NMR and MALDI-TOF spectral analysis showed that about 60 molecules of ornithine (PAMAMG4-ORN60) were conjugated to a PAMAMG4 dendrimer. Preliminary studies indicated that dendriplexes at charge ratio (N/P 10) show higher transfection efficiency and presence of serum does not affect the transfection efficiency of the dendriplexes. Transfection efficiency of PAMAMG4-ORN60 dendriplexes was slightly higher in cancer cells (NCI H157G) as compared to HEK 293T cells. Transfection efficiency of the PAMAMG4-ORN60 dendrimers decreased in presence of excess of ornithine while there was no effect on the parent PAMAMG4 dendrimers. Cytotoxicity assay has shown that PAMAMG4-ORN60 dendriplexes at N/P 10 were safe at concentrations <or=50 microg/mL. It may be concluded that the ornithine-conjugated dendrimers possess the potential to be novel gene carrier.

Responses to high seawater temperatures in zooxanthellate octocorals.

Increases in Sea Surface Temperatures (SSTs) as a result of global warming have caused reef-building scleractinian corals to bleach worldwide, a result of the loss of obligate endosymbiotic zooxanthellae. Since the 1980’s, bleaching severity and frequency has increased, in some cases causing mass mortality of corals. Earlier experiments have demonstrated that zooxanthellae in scleractinian corals from three families from the Great Barrier Reef, Australia (Faviidae, Poritidae, and Acroporidae) are more sensitive to heat stress than their hosts, exhibiting differential symptoms of programmed cell death – apoptosis and necrosis. Most zooxanthellar phylotypes are dying during expulsion upon release from the host. The host corals appear to be adapted or exapted to the heat increases. We attempt to determine whether this adaptation/exaptation occurs in octocorals by examining the heat-sensitivities of zooxanthellae and their host octocoral alcyonacean soft corals – Sarcophyton ehrenbergi (Alcyoniidae), Sinularia lochmodes (Alcyoniidae), and Xenia elongata (Xeniidae), species from two different families. The soft coral holobionts were subjected to experimental seawater temperatures of 28, 30, 32, 34, and 36°C for 48 hrs. Host and zooxanthellar cells were examined for viability, apoptosis, and necrosis (in hospite and expelled) using transmission electron microscopy (TEM), fluorescent microscopy (FM), and flow cytometry (FC). As experimental temperatures increased, zooxanthellae generally exhibited apoptotic and necrotic symptoms at lower temperatures than host cells and were expelled. Responses varied species-specifically. Soft coral hosts were adapted/exapted to higher seawater temperatures than their zooxanthellae. As with the scleractinians, the zooxanthellae appear to be the limiting factor for survival of the holobiont in the groups tested, in this region. These limits have now been shown to operate in six species within five families and two orders of the Cnidaria in the western Pacific. We hypothesize that this relationship may have taxonomic implications for other obligate zooxanthellate cnidarians subject to bleaching.

Loss of Symbiodinium from bleached Australian scleractinian corals (Acropora hyacinthus, Favites complanata and Porites solida)

Coral bleaching results from the dissociation of Symbiodinium and is primarily related to sea surface temperatures above mean yearly maximums. The numbers of live, dead, and mitotic Symbiodinium cells lost from three scleractinian corals from three different families (Acropora hyacinthus, Favites complanata, and Porites solida), which have not been studied previously in central Queensland (Australia), were compared at 28, 30, 32, and 34 ◦ C. Specific expulsion rates, growth rates, and mitotic indices were compared for each host at each temperature. Porites solida was the most robust coral, A. hyacinthus bleached more readily at low temperatures and F. complanata showed levels of intermediate bleaching tolerance to elevated temperatures. However, the timing of Symbiodinium cell loss was similar between all corals tested. Mitotic indices and specific growth rates were found to be positively associated with increasing temperature; thus, symbiont reproduction increased despite elevated losses of Symbiodinium from the host. Because all corals in the present study were symbiotic with Symbiodinium from clade C, different levels of stress tolerance to temperature suggests that bleaching resistance is an attribute associated with the coral host and, to a lesser degree, the symbiont.

Effects of climate change/global warming on coral reefs: adaptation/exaptation in corals, evolution in zooxanthellae, and biogeographic shifts.

Increased sea-surface temperatures (SSTs) associated with climate change/global warming have caused bleaching in scleractinian corals (the loss of obligate symbiotic zooxanthellae) on a global basis, resulting in mass mortality of corals and decimation of reefs. This symbiotic relationship makes these corals an excellent bioindicator of climate change. It has been hypothesized that bleaching is a mechanism by which corals can adapt to changing environmental conditions via the “shuffling” of symbiont clades and acquisition of better-adapted symbionts. Experimental research has confirmed that zooxanthellae are sensitive to increases in seawater temperatures, exhibiting apoptosis (a form of programmed cell death) at temperatures of ≥30οC while in situ. The coral hosts, however, tolerate experimental temperatures up to 34οC, not showing signs of apoptosis and necrosis until 36οC. Thus, zooxanthellae currently appear to be poorly adapted to temperature increases, while the corals are resistant to higher temper...

Apoptotic and necrotic stages of Symbiodinium (Dinophyceae) cell death activity: bleaching of soft and scleractinian corals

Abstract Mechanisms which regulate abundances of Symbiodinium populations during bleaching are not yet fully understood. Currently proposed mechanisms include cell exocytosis, host-cell detachment, apoptosis and necrosis. Here we have analysed zooxanthellae of soft and scleractinian corals under thermal stress using light, fluorescence and transmission electron microscopy (TEM) and confirmed that different stages of apoptotic and necrotic cell death of Symbiodinium occur. The diagnostic characteristic of apoptosis within Symbiodinium cells included nuclear and organelle condensation, membrane blebbing, the formation of apoptotic bodies and mitochondrial swelling. Necrotic cells swell, cytoplasmic organelles fuse and burst, followed by Symbiodinium cell membrane disintegration and release of cytosolic contents. In addition, TEM showed that apoptotic and necrotic processes are initiated in situ. Symbiont cells in the process of dying in situ therefore reduce the likelihood of recolonization upon expulsion from the host.

Corals and Their Potential Applications to Integrative Medicine

Over the last few years, we have pursued the use and exploitation of invertebrate immune systems, most notably their humoral products, to determine what effects their complex molecules might exert on humans, specifically their potential for therapeutic applications. This endeavor, called “bioprospecting,” is an emerging necessity for biomedical research. In order to treat the currently “untreatable,” or to discover more efficient treatment modalities, all options and potential sources must be exhausted so that we can provide the best care to patients, that is, proceed from forest and ocean ecosystems through the laboratory to the bedside. Here, we review current research findings that have yielded therapeutic benefits, particularly as derived from soft and hard corals. Several applications have already been demonstrated, including anti-inflammatory properties, anticancer properties, bone repair, and neurological benefits.

Ulcerated yellow spot syndrome: implications of aquaculture-related pathogens associated with soft coral Sarcophyton ehrenbergi tissue lesions

We introduce a new marine syndrome called ulcerated yellow spot, affecting the soft coral Sarcophyton ehrenbergi. To identify bacteria associated with tissue lesions, tissue and mucus samples were taken during a 2009 Indo-Pacific research expedition near the Wakatobi Island chain, Indonesia. Polymerase chain reaction targeting the 16S rDNA gene indicated associations with the known fish-disease-causing bacterium Photobacterium damselae, as well as multiple Vibrio species. Results indicate a shift toward decreasing diversity of bacteria in lesioned samples. Photobacterium damselae ssp. piscicida, formerly known as Pasteurella piscicida, is known as the causative agent of fish pasteurellosis and in this study, was isolated solely in lesioned tissues. Globally, fish pasteurellosis is one of the most damaging fish diseases in marine aquaculture. Vibrio alginolyticus, a putative pathogen associated with yellow band disease in scleractinian coral, was also isolated from lesioned tissues. Lesions appear to be inflicting damage on symbiotic zooxanthellae (Symbiodinium sp.), measurable by decreases in mitotic index, cell density and photosynthetic efficiency. Mitotic index of zooxanthellae within infected tissue samples was decreased by ~80%, while zooxanthellae densities were decreased by ~40% in lesioned tissue samples compared with healthy coral. These results provide evidence for the presence of known aquaculture pathogens in lesioned soft coral and may be a concern with respect to cross-species epizootics in the tropics.

Coral Bleaching Susceptibility Is Decreased following Short-Term (1–3 Year) Prior Temperature Exposure and Evolutionary History

Coral exposed to short periods of temperature stress (≥1.0°C above mean monthly maximum) and/or increased frequencies of high temperatures may bolster resilience to global warming associated with climate change. We compared Montastraea cavernosa (Linnaeus, 1767; Cnidaria, Scleractinia, Faviidae) from the Florida Keys National Marine Sanctuary (FKNMS) and the Flower Garden Banks National Marine Sanctuary (FGBNMS). Thermal stress has been reported frequently within the FKNMS; however, corals in the FGBNMS experience nominal exposures to similar stressors. Corals were exposed to three temperatures (27°C, 31°C, and 35°C) for 72 h. Colonies from the FKNMS lost significantly fewer viable and necrotic zooxanthellae under conditions of acute stress (35°C) than the FGBNMS colonies. This indicates that the FKNMS corals are less temperature-sensitive than those in the FGBNMS. The observed differences point to greater prior temperature exposure and adaptation in the former versus the latter site when correlated to previous years of thermal exposure.

Mechanism of gene transfection by polyamidoamine (PAMAM) dendrimers modified with ornithine residues

The aim of this study was to prepare and investigate the mechanism of uptake of the dendriplexes prepared with ornithine-conjugated polyamidoamine (PAMAM) G4 dendrimers. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. A comparative transfection study in NCI H157G cells and polyamine transport-deficient cell line NCI H157R was performed to confirm the role of the polyamine transporter system (PAT) in the dendriplex uptake. Transfection efficiency significantly increased with increase in generation number and extent of ornithine conjugation. Transfection efficiency of the PAMAMG4-ORN60 dendrimers significantly decreased in presence of excess of ornithine (P < 0.05) and paraquat (P < 0.01) but not of PAMAMG4 dendrimers. Transfection efficiency of PAMAMG4-ORN60 was significantly low in NCI H157R (31.66 ± 3.95%, RFU: 17.87 ± 1.34) as compared to NCI H157G cell line (63.07 ± 6.8%, relative fluorescence units (RFU): 23.28 ± 0.66). Results indicate the role of PAT in addition to charge-mediated endocytosis in the internalization of ornithine-conjugated PAMAMG4 dendrimers. Cytotoxicity analysis (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay) in human embryonic kidney cell line (HEK) 293T cells showed that the dendriplexes were non-toxic at N/P 10.

Genetic circumscription of deep-water coral species in Canada using 18S rRNA

Many deep-water coral species have very broad global distributions and are eurybathic from depths of meters to kilometers. Such ecological breadth may be confounded by the presence of cryptic species. We are currently comparing the genetic distances between Paragorgia sp. and Primnoa sp. across their distribution and depth range in Canada using 18S ribosomal DNA (rDNA) sequences. Initial results show a confusing picture amongst the geographically distant Paragorgia taxa. Specimens of P. arborea from the Canadian Atlantic are very divergent from the specimen from the Canadian Pacific. The placement of Pennatula and Anthomastus relative to these taxa is also unexpected. We expect this topology to alter with the addition of more taxa and further testing.