Inbreeding Ratio and Genetic Relationships among Strains of the Western Clawed Frog, Xenopus tropicalis.

The Western clawed frog, Xenopus tropicalis, is a highly promising model amphibian, especially in developmental and physiological research, and as a tool for understanding disease. It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s. The major strains thus far known include the Nigerian and Ivory Coast strains. However, due to its short history as an experimental animal, the genetic relationship among the various strains has not yet been clarified, and establishment of inbred strains has not yet been achieved. Since 2003 the Institute for Amphibian Biology (IAB), Hiroshima University has maintained stocks of multiple X. tropicalis strains and conducted consecutive breeding as part of the National BioResource Project. In the present study we investigated the inbreeding ratio and genetic relationship of four inbred strains at IAB, as well as stocks from other institutions, using highly polymorphic microsatellite markers and mitochondrial haplotypes. Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains. The Ivory Coast strains clearly differed from the Nigerian strains genetically, and three subgroups were identified within both the Nigerian and Ivory Coast strains. It is noteworthy that the Ivory Coast strains have an evolutionary divergent genetic background. Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

Heat capacity anomaly in a self-aggregating system: Triblock copolymer 17R4 in water.

The reverse Pluronic, triblock copolymer 17R4 is formed from poly(propylene oxide) (PPO) and poly(ethylene oxide) (PEO): PPO14 - PEO24 - PPO14, where the number of monomers in each block is denoted by the subscripts. In water, 17R4 has a micellization line marking the transition from a unimer network to self-aggregated spherical micelles which is quite near a cloud point curve above which the system separates into copolymer-rich and copolymer-poor liquid phases. The phase separation has an Ising-like, lower consolute critical point with a well-determined critical temperature and composition. We have measured the heat capacity as a function of temperature using an adiabatic calorimeter for three compositions: (1) the critical composition where the anomaly at the critical point is analyzed, (2) a composition much less than the critical composition with a much smaller spike when the cloud point curve is crossed, and (3) a composition near where the micellization line intersects the cloud point curve that only shows micellization. For the critical composition, the heat capacity anomaly very near the critical point is observed for the first time in a Pluronic/water system and is described well as a second-order phase transition resulting from the copolymer-water interaction. For all compositions, the onset of micellization is clear, but the formation of micelles occurs over a broad range of temperatures and never becomes complete because micelles form differently in each phase above the cloud point curve. The integrated heat capacity gives an enthalpy that is smaller than the standard state enthalpy of micellization given by a van't Hoff plot, a typical result for Pluronic systems.

Deficient induction response in a Xenopus nucleocytoplasmic hybrid.

Incompatibilities between the nucleus and the cytoplasm of sufficiently distant species result in developmental arrest of hybrid and nucleocytoplasmic hybrid (cybrid) embryos. Several hypotheses have been proposed to explain their lethality, including problems in embryonic genome activation (EGA) and/or nucleo-mitochondrial interactions. However, conclusive identification of the causes underlying developmental defects of cybrid embryos is still lacking. We show here that while over 80% of both Xenopus laevis and Xenopus (Silurana) tropicalis same-species androgenetic haploids develop to the swimming tadpole stage, the androgenetic cybrids formed by the combination of X. laevis egg cytoplasm and X. tropicalis sperm nucleus invariably fail to gastrulate properly and never reach the swimming tadpole stage. In spite of this arrest, these cybrids show quantitatively normal EGA and energy levels at the stage where their initial gastrulation defects are manifested. The nucleocytoplasmic incompatibility between these two species instead results from a combination of factors, including a reduced emission of induction signal from the vegetal half, a decreased sensitivity of animal cells to induction signals, and differences in a key embryonic protein (Xbra) concentration between the two species, together leading to inefficient induction and defective convergence-extension during gastrulation. Indeed, increased exposure to induction signals and/or Xbra signalling partially rescues the induction response in animal explants and whole cybrid embryos. Altogether, our study demonstrates that the egg cytoplasm of one species may not support the development promoted by the nucleus of another species, even if this nucleus does not interfere with the cytoplasmic/maternal functions of the egg, while the egg cytoplasm is also capable of activating the genome of that nucleus. Instead, our results provide evidence that inefficient signalling and differences in the concentrations of key proteins between species lead to developmental defects in cybrids. Finally, they show that the incompatibilities of cybrids can be corrected by appropriate treatments.

Large variations occur in bone density measurements of children when using different software.

BACKGROUND: Paediatric dual X-ray absorptiometry (DXA) studies present a number of technical problems. One of these is that the edge detection algorithms designed for the adult skeleton may fail for paediatric studies. Hologic provide alternative algorithms for low bone density studies. AIM: To assess low-density software for the analysis of paediatric DXA studies and to compare with the adult protocol. METHODS: Our centre has scanned 450 normal children as part of a normal range study. A subgroup of 103 children was selected using a random number generator. The group was distributed evenly between males and females and across the age range 5-17 years. Each individual underwent both a lumbar spine and a whole-body scan on a Hologic QDR-4500W DXA scanner. Both scans were analysed using the standard adult protocol and then re-analysed using the Hologic experimental paediatric protocol for whole body and the Hologic low-density protocol for lumbar spine. RESULTS: Both lumbar spine protocols showed an increase in bone mineral density with age; however, the low-density protocol always produced a lower bone mineral density result than the adult protocol. Bland-Altman analysis showed limits of agreement of 0.031-0.093 g x cm(-2) (male, 0.032-0.089 g x cm(-2); female, 0.031-0.096 g x cm(-2)). This represents a mean difference of 9%. Five results showed differences greater than the upper limit of agreement. All these cases were children under 11 years of age who had large areas of spine not identified as bone by the adult protocol. These children were all below the 30th percentile for the body mass index. The whole-body protocols showed similar increases in bone mineral density with age; however, the experimental paediatric protocol always produced a lower bone mineral density result than the adult protocol. Paired results showed limits of agreement of 0.0668-0.130 g x cm(-2) (male, 0.063-0.124 g x cm(-2); female, 0.073-0.134 g x cm(-2)). This represents a mean difference of 11%. Five results showed differences greater than the upper limit of agreement. CONCLUSIONS: For anteroposterior (AP) lumbar spine scans, the use of the paediatric algorithm in children under 11 years of age would prevent the largest failures in analysis. For whole-body scanning, the adult algorithm showed no major failures in children of 11 years or older. It is hoped that forthcoming improvements in whole-body density analysis will improve the results for those under 11 years of age. Normal range data should be generated for any new algorithm to allow proper interpretation of clinical studies.

Cystatin C improves the detection of mild renal dysfunction in older patients.

BACKGROUND: Conventional estimates of glomerular dysfunction, including serum creatinine and creatinine clearance, are inadequate in older people. In this study we have compared the diagnostic accuracy of a novel test of kidney disease, cystatin C, against these markers in older patients with a range of renal function. METHODS: Fifty-three patients (mean age 79.6 years, range 69-92 years) with a variety of medical diagnoses were recruited via outpatient clinics. Exclusion criteria included active rheumatoid disease, known current malignancy, renal replacement therapy/renal transplantation and cognitive impairment. (51)Cr-EDTA was used as the reference method against which the other markers of glomerular filtration rate were compared using regression analyses. RESULTS: The best fit with glomerular filtration rate was given by Cockcroft and Gault calculated clearance (R(2) = 0.83), followed by serum cystatin C (R(2) = 0.79), serum creatinine (R(2) = 0.76) and creatinine clearance (R(2) = 0.73). The accuracy for glomerular filtration rate prediction was poor for all markers. Serum cystatin C detected nearly all patients with mild renal impairment whereas serum creatinine only detected half of these cases. Regression modelling predicted that the upper limit of normal for serum cystatin C would be exceeded as glomerular filtration rate fell below 64 mL/min/1.73 m(2), compared with 44 mL/min/1.73 m(2) for serum creatinine. CONCLUSION: Serum cystatin C is a simple and sensitive screening test for kidney dysfunction in older people.

Estimation of glomerular filtration rate in older patients with chronic renal insufficiency: is the modification of diet in renal disease formula an improvement?

OBJECTIVES: To evaluate a new formula for glomerular filtration rate (GFR), derived from the Modification of Diet in Renal Disease (MDRD) study in older people. DESIGN: An observational study of the performance of the MDRD formula compared with other formulae and creatinine clearance (ClCr) as measures of the GFR. SETTING: Volunteers were recruited via outpatient clinics. PARTICIPANTS: Fifty-two patients (27 men, 25 women: mean age 80, range 69-92) with a variety of medical diagnoses. Mean GFR was 53.3 mL/min/1.73 m2 (range 15.9-100.2). Exclusion criteria included renal replacement therapy/renal transplantation and cognitive impairment. MEASUREMENTS: 51Chromium ethylenediaminetetraacetic acid (51Cr EDTA) was used as the reference method against which the formulaic estimates of GFR were compared using bias plot and regression analyses. RESULTS: The MDRD and Cockcroft and Gault formulae (both coefficient of determination (R2) = 0.84) gave the best fit with GFR, followed by the Jelliffe formula (R2 = 0.81), ClCr (R2 = 0.73) and the Baracskay formula (R2 = 0.56). ClCr (-1.2%) demonstrated minimal bias compared with the MDRD (8.0%) and Cockcroft and Gault (-10.4%) formulae. However, imprecision compared with 51Cr EDTA was lowest for the Cockcroft and Gault formula, with 50% of estimates lying between -9.5 and -0.5 mL/min/1.73 m2 of measured 51Cr EDTA clearance. This compares with -6.7 and 10.1 mL/min/1.73 m2 for ClCr and 0.0 and 12.7 mL/min/1.73 m2 for the MDRD formula. CONCLUSION: Calculated estimates of GFR are an improvement over ClCr estimation. On balance, the MDRD formula does not improve the estimate of GFR compared with the Cockcroft and Gault formula in older Caucasian patients with chronic renal insufficiency.