New tools for studying osteoarthritis genetics in zebrafish.

OBJECTIVE: Increasing evidence points to a strong genetic component to osteoarthritis (OA) and that certain changes that occur in osteoarthritic cartilage recapitulate the developmental process of endochondral ossification. As zebrafish are a well validated model for genetic studies and developmental biology, our objective was to establish the spatiotemporal expression pattern of a number of OA susceptibility genes in the larval zebrafish providing a platform for functional studies into the role of these genes in OA. DESIGN: We identified the zebrafish homologues for Mcf2l, Gdf5, PthrP/Pthlh, Col9a2, and Col10a1 from the Ensembl genome browser. Labelled probes were generated for these genes and in situ hybridisations were performed on wild type zebrafish larvae. In addition, we generated transgenic reporter lines by modification of bacterial artificial chromosomes (BACs) containing full length promoters for col2a1 and col10a1. RESULTS: For the first time, we show the spatiotemporal expression pattern of Mcf2l. Furthermore, we show that all six putative OA genes are dynamically expressed during zebrafish larval development, and that all are expressed in the developing skeletal system. Furthermore, we demonstrate that the transgenic reporters we have generated for col2a1 and col10a1 can be used to visualise chondrocyte hypertrophy in vivo. CONCLUSION: In this study we describe the expression pattern of six OA susceptibility genes in zebrafish larvae and the generation of two new transgenic lines marking chondrocytes at different stages of maturation. Moreover, the tools used demonstrate the utility of the zebrafish model for functional studies on genes identified as playing a role in OA.

Autoradiographic evidence for a calcitonin receptor on testicular Leydig cells.

Previous studies have indicated that there is a relation between testicular function and adequate concentrations of zinc in testicular cells, and that calcitonin alters cellular zinc transfer in the testis. The present studies provide autoradiographic evidence that calcitonin binds in vivo to the cell membrane of testicular Leydig cells. The data thus confirm the presence of the testicular cell membrane calcitonin receptors that were previously demonstrated indirectly by Scatchard analysis of data collected from binding studies.

Ex vivo culture of adult CD34+ stem cells using functional highly porous polymer scaffolds to establish biomimicry of the bone marrow niche.

Haematopoiesis, the process of blood production, occurs from a tiny contingent of haematopoietic stem cells (HSC) in highly specialised three-dimensional niches located within the bone marrow. When haematopoiesis is replicated using in vitro two-dimensional culture, HSCs rapidly differentiate, limiting self-renewal. Emulsion-templated highly porous polyHIPE foam scaffolds were chosen to mimic the honeycomb architecture of human bone. The unmodified polyHIPE material supports haematopoietic stem and progenitor cell (HSPC) culture, with successful culture of erythroid progenitors and neutrophils within the scaffolds. Using erythroid culture methodology, the CD34+ population was maintained for 28 days with continual release of erythroid progenitors. These cells are shown to spontaneously repopulate the scaffolds, and the accumulated egress can be expanded and grown at large scale to reticulocytes. We next show that the polyHIPE scaffolds can be successfully functionalised using activated BM(PEG)2 (1,8-bismaleimido-diethyleneglycol) and then a Jagged-1 peptide attached in an attempt to facilitate notch signalling. Although Jagged-1 peptide had no detectable effect, the BM(PEG)2 alone significantly increased cell egress when compared to controls, without depleting the scaffold population. This work highlights polyHIPE as a novel functionalisable material for mimicking the bone marrow, and also that PEG can influence HSPC behaviour within scaffolds.

Fryns syndrome: a new variable multiple congenital anomaly (MCA) syndrome.

We report a brother and sister who died neonatally with a distinctive but variable multiple congenital anomaly (MCA) syndrome. Anomalies in both included similar facial changes, cleft palate, distal digital hypoplasia, lung hypoplasia, and urogenital abnormalities. They were discordant for cleft lip, diaphragmatic hernia, and Dandy-Walker anomaly. These sibs resemble three recently reported stillborn children and support the existence of a "new" autosomal recessive MCA syndrome with variable expressivity.

Familial amniotic bands.

Amniotic bands can cause a wide variety of deformities and mutilations. They are generally considered sporadic. Two families are presented with apparent familial amniotic band anomalies. Additional cases in the literature are reviewed. These aggregations may be coincidental, and recurrence risk is apparently low. However, amniotic band malformations may be an indication for caution in the use of amniocentesis in future pregnancies.

Relationships between acute sediment toxicity in laboratory tests and abundance and diversity of benthic infauna in marine sediments: a review.

Acute sediment toxicity tests have become important in regulatory, monitoring, and scientific programs, partly because it has been assumed that they are indicative of ecological damage to benthic infaunal resources. Data from tests of sediment toxicity and measures of benthic community structure were examined from > 1,400 saltwater samples to determine the relationships between acute toxicity and changes in the abundance and diversity of infauna resources. Data were compiled from studies conducted along portions of the Atlantic, Gulf of Mexico, and Pacific coasts of the United States. There was considerable variability among the data sets in the relationships between laboratory results and benthic measures. However, in 92% of the samples classified as toxic, at least one measure of benthic diversity or abundance was < 50% of the average reference value. In 67% of these samples, at least one measure of benthic infauna abundance or diversity was < 10% of average reference conditions. No amphipods were found in 39% of samples that were classified as toxic, whereas amphipods were absent from 28% of the nontoxic samples. In many survey areas, the abundance of crustaceans (notably the amphipods) decreased in the infauna as amphipod survival decreased in the laboratory tests. There appeared to be a break point in the data indicating that, generally, amphipod abundance in the field was lowest when survival in the laboratory tests dropped below 50% of controls. Based on the weight of evidence from all the data analyses, we conclude that ecologically relevant losses in the abundance and diversity of the benthic infauna frequently corresponded with reduced amphipod survival in the laboratory tests.

Evaluation of numerical sediment quality targets for the St. Louis River Area of Concern.

Numerical sediment quality targets (SQTs) for the protection of sediment-dwelling organisms have been established for the St. Louis River Area of Concern (AOC), 1 of 42 current AOCs in the Great Lakes basin. The two types of SQTs were established primarily from consensus-based sediment quality guidelines. Level I SQTs are intended to identify contaminant concentrations below which harmful effects on sediment-dwelling organisms are unlikely to be observed. Level II SQTs are intended to identify contaminant concentrations above which harmful effects on sediment-dwelling organisms are likely to be observed. The predictive ability of the numerical SQTs was evaluated using the matching sediment chemistry and toxicity data set for the St. Louis River AOC. This evaluation involved determination of the incidence of toxicity to amphipods ( Hyalella azteca) and midges (Chironomus tentans) within five ranges of Level II SQT quotients (i.e., mean probable effect concentration quotients [PEC-Qs]). The incidence of toxicity was determined based on the results of 10-day toxicity tests with amphipods (endpoints: survival and growth) and 10-day toxicity tests with midges (endpoints: survival and growth). For both toxicity tests, the incidence of toxicity increased as the mean PEC-Q ranges increased. The incidence of toxicity observed in these tests was also compared to that for other geographic areas in the Great Lakes region and in North America for 10- to 14-day amphipod (H. azteca) and 10- to 14-day midge (C. tentans or C. riparius) toxicity tests. In general, the predictive ability of the mean PEC-Qs was similar across geographic areas. The results of these predictive ability evaluations indicate that collectively the mean PEC-Qs provide a reliable basis for classifying sediments as toxic or not toxic in the St. Louis River AOC, in the larger geographic areas of the Great Lakes, and elsewhere in North America.

Predictions of sediment toxicity using consensus-based freshwater sediment quality guidelines.

The objectives of this study were to compare approaches for evaluating the combined effects of chemical mixtures on the toxicity in field-collected sediments and to evaluate the ability of consensus-based probable effect concentrations (PECs) to predict toxicity in a freshwater database on both a national and regional geographic basis. A database was developed from 92 published reports, which included a total of 1,657 samples with high-quality matching sediment toxicity and chemistry data from across North America. The database was comprised primarily of 10- to 14-day or 28- to 42-day toxicity tests with the amphipod Hyalella azteca (designated as the HA10 or HA28 tests) and 10- to 14-day toxicity tests with the midges Chironomus tentans or C. riparius (designated as the CS10 test). Mean PEC quotients were calculated to provide an overall measure of chemical contamination and to support an evaluation of the combined effects of multiple contaminants in sediments. There was an overall increase in the incidence of toxicity with an increase in the mean quotients in all three tests. A consistent increase in the toxicity in all three tests occurred at a mean quotient > 0.5, however, the overall incidence of toxicity was greater in the HA28 test compared to the short-term tests. The longer-term tests, in which survival and growth are measured, tend to be more sensitive than the shorter-term tests, with acute to chronic ratios on the order of six indicated for H. azteca. Different patterns were observed among the various procedures used to calculate mean quotients. For example, in the HA28 test, a relatively abrupt increase in toxicity was associated with elevated polychlorinated biphenyls (PCBs) alone or with elevated polycyclic aromatic hydrocarbons (PAHs) alone, compared to the pattern of a gradual increase in toxicity observed with quotients calculated using a combination of metals, PAHs, and PCBs. These analyses indicate that the different patterns in toxicity may be the result of unique chemical signals associated with individual contaminants in samples. Though mean quotients can be used to classify samples as toxic or nontoxic, individual quotients might be useful in helping identify substances that may be causing or substantially contributing to the observed toxicity. An increase in the incidence of toxicity was observed with increasing mean quotients within most of the regions, basins, and areas in North America for all three toxicity tests. The results of these analyses indicate that the consensus-based PECs can be used to reliably predict toxicity of sediments on both a regional and national basis.