Daniel A. Brazeau

Localization and early time course of TGF-β1 mRNA expression in dystrophic muscle

Fibrosis is a common pathological feature observed in muscle from patients with Duchenne muscular dystrophy (DMD). In the dystrophic (mdx) mouse model of DMD, the diaphragm is more severely affected than other skeletal muscles. The level of transforming growth factor‐beta1 (TGF‐β1), an inflammatory cytokine, is significantly elevated in mdx diaphragm. However, little is known about the onset of TGF‐β1 messenger ribonucleic acid (mRNA) expression, or which cells express the mRNA. In this study, we characterized the location and time course of expression of TGF‐β1 mRNA in diaphragm from mdx mice. TGF‐β1 mRNA was significantly elevated in mdx diaphragm at 6 and 9 but not 12 weeks of age, and these changes corresponded with changes in type I collagen mRNA and hydroxyproline concentration. Mononucleated cells localized to areas of fiber necrosis highly expressed the TGF‐β1 transcript in mdx diaphragm. Neutralization of TGF‐β1 by decorin administration resulted in a 40% reduction in the level of diaphragm muscle type I collagen mRNA. These findings support a role for TGF‐β1 during the early stages of fibrogenesis in dystrophic diaphragm muscle. Therapeutic interventions aimed at neutralizing this cytokine may be beneficial in slowing the development of fibrosis in DMD. Muscle Nerve, 2004

Sexual reproduction and external brooding by the Caribbean gorgonianBriareum asbestinum

The reproductive biology of the gonochoricBriareum asbestinum (Pallas), a common Caribbean gorgonian, was studied in the San Blas Islands (Panama), La Paguera (Puerto Rico) and the Bahamas. Of 721 colonies examined at the height of the reproductive season, during the summers of 1985 to 1988, all were either male or female. No hermaphroditic colonies or polyps were observed. The sexratio of Panama populations was significantly biased in favor of males (2.1:1,n=454) over all four years of the study. A significant male bias was also found in populations sampled in Puerto Rico (2.3:1,n=143) and in the Bahamas (2.5:1,n=124). The consistency of this geographically widespread skewed sex-ratio is unique among octocorals and scleractinians. Ooyctes appear annually in September and reach maximum diameter (600 to 900µm) in June and July of the following year. The 3 to 5 mo developmental cycle of spermaries is shorter than that of females and begins in March. Spawning in males is synchronous and occurs following the full moons of June and July. Fertilization is internal. Embryos are released from polyps very early in development, often whilst still in the process of cleaving. Embryos remain attached to the outside of the colony for 3 to 5 d, many becoming entrapped in mucous sheets secreted by the colonies. Larvae are negatively buoyant and settle rapidly once displaced from the colony. Males become reproductive at smaller branch sizes than females. Reproductive effort (gonads polyp−1 and total gonad volume polyp−1) for both males and females increased with increasing branch size and in polyps away from the tip and base of the branch.

The Reproductive Cycle and Spawning in a Caribbean Gorgonian

The reproductive biology of Plexuara A, a common but undescribed Caribbean gorgonian, was studied in the San Blas Islands, Panama. Oocytes were present in the polyps throughout the year, though a seasonal developmental cycle is evident. Early stage oocytes appeared at the base of the polyps in November. In January total egg volume per polyp began to increase and reached a maximum in early May. Total egg volume per polyp then decreased through the summer as mature eggs were released. Spawning occurred over a period of 4-7 days following each full moon in May, June, and July. Egg release was synchronous, starting at approximately 18:30 and lasting 90 minutes. Larval development is initiated just prior to or at the time of release. Initiation of larval development at the time of release without brooding is unknown for any gorgonian or scleractinian. Of 265 colonies examined from 6 reefs near San Blas Point, all but 3 contained gonads and were female. No male or hermaphroditic colonies have been found in the ...

In situ Rates of Fertilization Among Broadcast Spawning Gorgonian Corals

Fertilization rates among marine benthic taxa have implicitly been assumed to be uniformly high in most analyses of life history evolution, but in situ fertilization rates during natural spawning events are rarely measured. Fertilization rates of the Caribbean gorgonians Plexaura kuna and Pseudoplexaura porosa were measured at a site in the San Blas Islands, Panama, by collecting eggs downstream of colonies during synchronous spawning events during the summer months in the years 1988-1994. Eggs collected by divers were incubated, and the proportion of eggs that developed was determined. Proportions of eggs developing suggest fertilization rates that vary from 0% to 100%. Monthly means ranged from 0% to 60.4%. Failure of gametes to develop can be attributed to sperm limitation, as eggs collected during spawning had higher fertilization rates if incubated with an excess of sperm. Plexaura kuna fertilization rates were highest during the July spawning events. Fertilization of Plexaura kuna eggs was usually lower during the first two nights of the 4-6 night spawning event. The proportion of eggs being fertilized when collected from a given place and time was highly variable, with one peak in the frequency distribution at or below 20% fertilization, and a second group of samples with greater fertilization rates. High variance in fertilization rates is evident at all levels of analysis: between replicate samples, times within nights, and among nights and months. This variance can be attributed to a combination of the effects of heterogeneity in the water column as gametes are diluted, spawning behavior of the gorgonians, and the current regime. Fertilization rates are often low and may represent a limiting step in recruitment during some years. Low fertilization rates may also be an important component of the life history evolution of these species.

Reproductive success in the Caribbean octocoral Briareum asbestinum

For most sessile marine invertebrates the number of offspring produced by an individual is often determined indirectly from counts of eggs present, with the assumption that all or most become viable offspring. Few field data on the actual number of offspring produced per individual (reproductive success) have been reported. We examined reproductive success in the octocoral, Briareum asbestinum (Pallas), by measuring embryo densities on 40 female colonies on two reefs (Pinnacles and House reefs) in the San Blas Islands, Panama from 1986 to 1988. The percentage of female colonies with mature eggs which subsequently released embryos was low, ranging from 46.6% in 1986 to 5.0% in 1988. Reproductive success was significantly different between reefs. Pinnacles reef had significantly more successful colonies (those releasing embryos) than House reef in all three years and significantly higher embryo densities in 1986 and 1987. There was a significant positive correlation between female reproductive success and the density and proximity of nearby males in both 1986 and 1987. In addition, fertile female branches placed 25 cm away from male branches produced significantly more embryos than female branches placed 50 cm away. In 1988 two groups of female branches were placed at distances of <50 cm and 5 m from four large male colonies at Pinnacles reef. Of the 20 female branches placed 5 m from the males none released embryos while 35% (7 of 20 branches) of the branches <50cm from males released embryos. These data indicate that simple counts of mature eggs present within female colonics prior to the reproductive season provide a poor estimate of reproductive success in B. ashestinum and that female reproductive success is positively influenced by the close proximity of males.

Environmental Symbiont Acquisition May Not Be the Solution to Warming Seas for Reef-Building Corals

Background Coral reefs worldwide are in decline. Much of the mortality can be attributed to coral bleaching (loss of the corals intracellular photosynthetic algal symbiont) associated with global warming. How corals will respond to increasing oceanic temperatures has been an area of extensive study and debate. Recovery after a bleaching event is dependent on regaining symbionts, but the source of repopulating symbionts is poorly understood. Possibilities include recovery from the proliferation of endogenous symbionts or recovery by uptake of exogenous stress-tolerant symbionts. Methodology/Principal Findings To test one of these possibilities, the ability of corals to acquire exogenous symbionts, bleached colonies of Porites divaricata were exposed to symbiont types not normally found within this coral and symbiont acquisition was monitored. After three weeks exposure to exogenous symbionts, these novel symbionts were detected in some of the recovering corals, providing the first experimental evidence that scleractinian corals are capable of temporarily acquiring symbionts from the water column after bleaching. However, the acquisition was transient, indicating that the new symbioses were unstable. Only those symbiont types present before bleaching were stable upon recovery, demonstrating that recovery was from the resident in situ symbiont populations. Conclusions/Significance These findings suggest that some corals do not have the ability to adjust to climate warming by acquiring and maintaining exogenous, more stress-tolerant symbionts. This has serious ramifications for the success of coral reefs and surrounding ecosystems and suggests that unless actions are taken to reverse it, climate change will lead to decreases in biodiversity and a loss of coral reefs.

Genetic structure in the coral, Montastraea cavernosa: assessing genetic differentiation among and within Mesophotic reefs.

Mesophotic coral reefs (30–150 m) have recently received increased attention as a potential source of larvae (e.g., the refugia hypothesis) to repopulate a select subset of the shallow water (<30 m) coral fauna. To test the refugia hypothesis we used highly polymorphic Amplified Fragment Length Polymorphism (AFLP) markers as a means to assess small-scale genetic heterogeneity between geographic locations and across depth clines in the Caribbean coral, Montastraea cavernosa. Zooxanthellae-free DNA extracts of coral samples (N = 105) were analyzed from four depths, shallow (3–10 m), medium (15–25 m), deep (30–50 m) and very deep (60–90 m) from Little Cayman Island (LCI), Lee Stocking Island (LSI), Bahamas and San Salvador (SS), Bahamas which range in distance from 170 to 1,600 km apart. Using AMOVA analysis there were significant differences in ΦST values in pair wise comparisons between LCI and LSI. Among depths at LCI, there was significant genetic differentiation between shallow and medium versus deep and very deep depths in contrast there were no significant differences in ΦST values among depths at LSI. The assignment program AFLPOP, however, correctly assigned 95.7% of the LCI and LSI samples to the depths from which they were collected, differentiating among populations as little as 10 to 20 m in depth from one another. Discriminant function analysis of the data showed significant differentiation among samples when categorized by collection site as well as collection depth. FST outlier analyses identified 2 loci under positive selection and 3 under balancing selection at LCI. At LSI 2 loci were identified, both showing balancing selection. This data shows that adult populations of M. cavernosa separated by depths of tens of meters exhibits significant genetic structure, indicative of low population connectivity among and within sites and are not supplying successful recruits to adjacent coral reefs less than 30 m in depth.

AMBIENCE: A Novel Approach and Efficient Algorithm for Identifying Informative Genetic and Environmental Associations With Complex Phenotypes

We developed a computationally efficient algorithm AMBIENCE, for identifying the informative variables involved in gene–gene (GGI) and gene–environment interactions (GEI) that are associated with disease phenotypes. The AMBIENCE algorithm uses a novel information theoretic metric called phenotype-associated information (PAI) to search for combinations of genetic variants and environmental variables associated with the disease phenotype. The PAI-based AMBIENCE algorithm effectively and efficiently detected GEI in simulated data sets of varying size and complexity, including the 10K simulated rheumatoid arthritis data set from Genetic Analysis Workshop 15. The method was also successfully used to detect GGI in a Crohns disease data set. The performance of the AMBIENCE algorithm was compared to the multifactor dimensionality reduction (MDR), generalized MDR (GMDR), and pedigree disequilibrium test (PDT) methods. Furthermore, we assessed the computational speed of AMBIENCE for detecting GGI and GEI for data sets varying in size from 100 to 105 variables. Our results demonstrate that the AMBIENCE information theoretic algorithm is useful for analyzing a diverse range of epidemiologic data sets containing evidence for GGI and GEI.

Inter- and intraspecific variation in gorgonian colony morphology: quantifying branching patterns in arborescent animals

Geomorphological methods for quantifying branching networks are used to describe inter- and intra-specific differences in branching patterns among two species of arborescent Caribbean gorgonian, Plexaura homomalla, P. flexuosa, and a third, undescribed plexaurid from the San Blas Islands, Panama. There were significant differences among species for first, second and third order branch lengths, and for tributary to source ratios for first and second order branches. Extrapolations from the branching parameters successfully predicted differences in the branch structure of naturally generated colony fragments. Within P. homomalla, significant differences with depth in the lengths and tributary to source ratios of first, second, and third order branches provide a measure of the greater bushiness of shallow water colonies. These measures can be used to quantify morphological differences in both ecological and systematic analyses. Data on the branching characters of these three gorgonian species demonstrate that gross colony form can be studied as a quantifiable component of phenotype and that gorgonians display both species level and ecophenotypic variation in colony form.

Multidrug resistance 1 polymorphisms and trough concentrations of atazanavir and lopinavir in patients with HIV

INTRODUCTION HIV-infected patients receiving protease inhibitors may benefit from therapeutic drug monitoring-assisted dose adjustment to achieve target plasma concentrations. However, efflux pumps such as permeability-glycoprotein, which is encoded by the multidrug resistance (MDR)1 gene, may decrease intracellular drug concentrations, thus reducing the amount of drug at the site of action. Plasma concentrations of protease inhibitors and CD4 cell count response have been associated with the T allele at the MDR1 C3435T locus. We examined MDR1 single nucleotide polymorphisms in a cohort of patients in whom therapeutic drug monitoring is ongoing through a research protocol. METHODS In a multicenter study, genotypic analyses at two MDR1 loci, C3435T and G2677T, were performed by a real-time polymerase chain reaction method using DNA from 103 patients categorized as substance users or nonusers on atazanavir or lopinavir as the primary antiretrovirals. Allelic frequencies were determined as a function of racial/ethnic background, substance use status and trough concentrations of atazanavir and lopinavir. RESULTS The C/T and G/T alleles at the MDR1 C3435T and G2677T loci were equally frequent in the Caucasian population, but the wild-type alleles were more prevalent in the African-American population (59% homozygous [CC] and 32% heterozygous [CT] for C3435T; 80% homozygous [GG] and 16% heterozygous [GT] for G2677T). The frequencies in the Hispanic population were 46% CC and 38% CT for C3435T, and 58% GG and 38% GT for G2677T. No significant differences were seen in allele frequencies for MDR1 polymorphisms in substance user versus nonuser groups. Trough plasma concentrations of atazanavir or lopinavir were not correlated with the variant T allele. CONCLUSIONS These data confirm the higher prevalence of wild-type alleles of the MDR1 gene in African-Americans and the linkage disequilibrium between C3435T and G2677T loci. The T allele at the MDR1 C3435T and G2677T loci was not associated with higher atazanavir or lopinavir trough concentrations.