Marine Biological Monitoring in Broward County, Florida: Year 2 Annual Report

A study has been undertaken to monitor Broward County, Florida (southeast Florida) coral communities, reef fish assemblages and sedimentation rates in relation to possible effects from a proposed extensive beach renourishment (restoration) project. Coral communities and reef fish assemblages will be monitored at a total of 23 stations distributed offshore Broward County. This monitoring effort will characterize and quantify populations of scleractinian (stony) corals, octocorallian (gorgonian) corals, sponges, and reef fishes. In addition, sediment traps located at each station will be sampled and analyzed. This document reports the data collected during the second year of this project. Coral communities and fish assemblages were monitored at each of the 23 sites between September and October 2001. In addition, sedimentation analysis for the January, March, May, July and September 2001 collections were conducted. For September/October 2001, mean (± 1 S.D.) stony coral density for the 23 sites was 2.62 ± 1.85 colonies/m2. Mean stony coral coverage was 2.39 ± 3.96%. Mean gorgonian density was 7.91 ± 8.01 colonies/m2 and mean sponge density was 14.09 ± 6.93 colonies/m2. First Reef sites had greater mean stony coral coverage but lower gorgonian and sponge density than Second and Third Reef sites. First Reef coral cover was much lower than the Third Reef when the First reef site, FTL4, was removed from the analysis. FTL4 had much greater stony coral cover than the mean cover for the remaining First Reef sites (19.95% compared to 1.45%). Shannon-Weaver Diversity Indices performed on the overall transect data resulted in values of 1.45 ± 0.53 and 1.72 ± 0.44 for cover and number of species respectively. Overall evenness was 0.77 ± 0.14 for number of species and 0.64 ± 0.21 for cover. There was no significant difference determined between the January/February 2001 site visit data and the September/October 2001 site visit data for mean stony coral density and cover. Mean octocoral density also did not differ significantly between these site visits, but mean sponge density was significantly less in September/October 2001 than in January/February 2001. Stony coral density, stony coral coverage, gorgonian density and sponge density data collected from the 18 monitoring sites established in 1997 and visited yearly from 1997 to 1999 were analyzed. No significant difference in yearly mean stony coral density, mean stony coral cover and mean gorgonian density was determined. Mean sponge density did show significant differences with 1998 sponge density greater than 1997. Trends in fish density were similar to those trends identified within the coral community transects. The greatest density of fishes occurs on the Third Reef followed by the First and Second. A difference in richness was seen amongst the three Reefs with the First Reef having the lowest number of species. The differences noted in abundance, density, and richness between the data collected in January/February 2001 and in September/October 2001 confirm previous reports of temporal differences in the fish assemblage offshore Broward County (Spieler 1998). The First Reef had a statistically higher rate of sedimentation than both the Second and Third Reefs when data from January-September 2001 were pooled. Pooled site data showed that January 2001 and May 2001 samples had the greatest sedimentation rates. The grain size for sites on the Third Reef was significantly smaller than both the First and Second Reefs. When site data were pooled, January 2001 had a significantly larger mean grain size than the other four sampling intervals in 2001. Data collected and analyses completed during this monitoring project will be used to help evaluate effects from the proposed beach renourishment project

Automation of large scale transient protein expression in mammalian cells

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI⁻ cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative

Differential Regulation of Primitive Myelopoiesis in the Zebrafish by Spi-1/Pu.1 and C/ebp1

The zebrafish has become a powerful tool for analysis of vertebrate hematopoiesis. Zebrafish, unlike mammals, have a robust primitive myeloid pathway that generates both granulocytes and macrophages. It is not clear how this unique primitive myeloid pathway relates to mammalian definitive hematopoiesis. In this study, we show that the two myeloid subsets can be distinguished using RNA in situ hybridization. Using a morpholino-antisense gene knockdown approach, we have characterized the hematopoietic defects resulting from knockdown of the myeloid transcription factor gene pu.1 and the unique zebrafish gene c/ebp1. Severe reduction of pu.1 resulted in complete loss of primitive macrophage development, with effects on granulocyte development only with maximal knockdown. Reduction of c/ebp1 did not ablate initial macrophage or granulocyte development, but resulted in loss of expression of the secondary granule gene lys C. These data reveal strong functional conservation of pu.1 between zebrafish primitive myelopoiesis and mammalian definitive myelopoiesis. Further, these results are consistent with a conserved role between c/ebp1 and mammalian C/EBPE, whose ortholog in zebrafish has not been identified. These studies validate the examination of zebrafish primitive myeloid development as a model for human myelopoiesis, and form a framework for identification and analysis of myeloid mutants.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63190/1/zeb.2007.0505.pd

Adverse Impact of a History of Violence for Women with Breast, Cervical, Endometrial, or Overian Cancer

The experience of physical and sexual violence (victimization) is common among U.S. women and is associated with adverse health consequences. The study objectives were to estimate the prevalence of victimization in women with cancer and to examine associations with demographics, cancer screening, and cancer stage. METHODS: From 2004 to 2005, 101 women with breast, cervical, endometrial, or ovarian cancer were interviewed to collect demographics, cancer screening history, health care access/use, and violence history. Chisquare and Fisher exact tests were used test risk-factor associations. A multinomial logistic regression model was used for multivariable analysis. RESULTS: The prevalence of a history of violence was 48.5% (49/101 women), and within that group, 46.9% (23/49) had a positive childhood violence screen, 75.5% (37/49) had a positive adult screen, and 55% (27/49) reported sexual violence at any age. Women with a positive violence screen differed significantly from women with a negative screen in that they were younger (P .031), more often divorced (P.012), more likely to smoke (P.010), more often lacked commercial insurance (P.036), and had more advanced stage of disease (P.013), but they did not differ with regard to race, cancer type, education level, alcohol or drug use, or cancer screening compliance. Multivariable analysis revealed that only stage remained significant; women with a history of violence had a 2.6-fold increased chance of diagnosis in later stages (odds ratio 2.61, 95% confidence interval 1.03– 6.59). CONCLUSION: A history of violence in breast, ovarian, endometrial, and ovarian cancer patients was extremely common and correlated with advanced stage at diagnosis

Cluster analyses of the TCGA and a TMA dataset using the coexpression of HSP27 and CRYAB improves alignment with clinical-pathological parameters of breast cancer and suggests different epichaperome influences for each sHSP

Our cluster analysis of the Cancer Genome Atlas for co-expression of HSP27 and CRYAB in breast cancer patients identified three patient groups based on their expression level combination (high HSP27 + low CRYAB; low HSP27 + high CRYAB; similar HSP27 + CRYAB). Our analyses also suggest that there is a statistically significant inverse relationship between HSP27 and CRYAB and known clinicopathological markers in breast cancer. Screening an unbiased 248 breast cancer patient tissue microarray (TMA) for the protein expression of HSP27 and phosphorylated HSP27 (HSP27-82pS) with CRYAB also identified three patient groups based on HSP27 and CRYAB expression levels. TMA24 also had recorded clinical-pathological parameters, such as ER and PR receptor status, patient survival, and TP53 mutation status. High HSP27 protein levels were significant with ER and PR expression. HSP27-82pS associated with the best patient survival (Log Rank test). High CRYAB expression in combination with wild-type TP53 was significant for patient survival, but a different patient outcome was observed when mutant TP53 was combined with high CRYAB expression. Our data suggest that HSP27 and CRYAB have different epichaperome influences in breast cancer, but more importantly evidence the value of a cluster analysis that considers their coexpression. Our approach can deliver convergence for archival datasets as well as those from recent treatment and patient cohorts and can align HSP27 and CRYAB expression to important clinical-pathological features of breast cancer

A Novel Keratocan Mutation Causing Autosomal Recessive Cornea Plana

PURPOSE: Mutations in keratocan (KERA), a small leucine-rich proteoglycan, have recently been shown to be responsible for cases of autosomal recessive cornea plana (CNA2). A consanguineous pedigree in which cornea plana cosegregated with microphthalmia was investigated by linkage analysis and direct sequencing. METHODS: Linkage was sought to polymorphic microsatellite markers distributed around the CNA2 and microphthalmia loci (arCMIC, adCMIC, NNO1, and CHX10) using PCR and nondenaturing polyacrylamide gel electrophoresis before KERA was directly sequenced for mutations. RESULTS: Positive lod scores were obtained with markers encompassing the CNA2 locus, the maximum two-point lod scores of 2.18 at recombination fraction theta = 0 was obtained with markers D12S95 and D12S327. Mutation screening of KERA revealed a novel single-nucleotide substitution at codon 215, which results in the substitution of lysine for threonine at the start of a highly conserved leucine-rich repeat motif. Structural modeling predicts that the motifs are stacked into an arched beta-sheet array and that the effect of the mutation is to alter the length and position of one of these motifs. CONCLUSIONS: This report describes a novel mutation in KERA that alters a highly conserved motif and is predicted to affect the tertiary structure of the molecule. Normal corneal function is dependent on the regular spacing of collagen fibrils, and the predicted alteration of the tertiary structure of KERA is the probable mechanism of the cornea plana phenotype

Variant R94C in TNNT2‐encoded troponin T predisposes to pediatric restrictive dardiomyopathy and sudden death through impaired thin filament relaxation resulting in myocardial diastolic dysfunction

Background Pediatric-onset restrictive cardiomyopathy (RCM) is associated with high mortality, but underlying mechanisms of disease are under investigated. RCM-associated diastolic dysfunction secondary to variants i