TGFβ alters growth and differentiation related gene expression in proliferating osteoblasts in vitro,preventing development of the mature bone phenotype

This study examines the mechanism by which TGF-β1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-β1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-β1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-β1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and α1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-β1 response throughout the course of osteoblast differentiation. The initiation of a TGF-β1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-β1. These in vitro results indicate that TGF-β1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml. © 1994 Wiley-Liss, Inc.     

Validation of ecological status concepts in UK rivers using historic diatom samples

We assessed the feasibility of using herbarium specimens to validate reference conditions in the UK by comparing diatom community composition of river sites with both recent and historic diatom samples. The question of substrate specificity was addressed by comparing epilithon (stone-derived) and epiphyton (plant-derived) samples from a number of rivers. No significant differences were found between the Trophic Diatom Index (TDI), species richness, species diversity, and percentage of motile valves between paired diatom samples (epilithic and epiphytic) from contemporary samples. Significant differences were recorded between a number of indices derived from analysis of the historic diatom samples on plant material sampled pre-1930 compared with diatoms from stones collected post-1990 from the same river location. The TDI, mean species richness, and species diversity and percentage of motile valves and nutrient tolerant valves were all significantly greater in the contemporary samples (p ≤ 0.05). The percentage of nutrient sensitive valves was significantly lower in the contemporary samples (p ≤ 0.05).The relative abundance of Achnanthidium minutissimum and Cocconeis placentula var. lineata was significantly greater on the herbarium material compared to matched contemporary samples. Calculated values for the TDI (43 ± 3) expected at reference conditions were similar to the observed TDI values derived from herbarium material (44 ± 12) showing no significant deviation in ecological status.     

Comparison of two Mn porphyrin-based mimics of superoxide dismutase in pulmonary radioprotection

Development of radiation therapy (RT)-induced lung injury is associated with chronic production of reactive oxygen and nitrogen species (ROS/RNS). MnTE-2-PyP5+ is a catalytic Mn porphyrin mimic of SOD, already shown to protect lungs from RT-induced injury by scavenging ROS/RNS. The purpose of this study was to compare MnTE-2-PyP5+ with a newly introduced analogue MnTnHex-2-PyP5+, which is expected to be a more effective radioprotector due to its lipophilic properties. This study shows that Fischer rats which were irradiated to their right hemithorax (28 Gy) have less pulmonary injury as measured using breathing frequencies when treated with daily subcutaneous injections of MnTE-2-PyP5+ (3 and 6 mg/kg) or MnTnHex-2-PyP5+ (0.3, 0.6, or 1.0 mg/kg) for 2 weeks after RT. However, at 16 weeks post-RT, only MnTE-2-PyP5+ at a dose of 6 mg/kg is able to ameliorate oxidative damage, block activation of HIF-1alpha and TGF-beta, and impair upregulation of CA-IX and VEGF. MnTnHex-2-PyP5+ at a dose of 0.3 mg/kg is effective only in reducing RT-induced TGF-beta and CA-IX expression. Significant loss of body weight was observed in animals receiving MnTnHex-2-PyP5+ (0.3 and 0.6 mg/kg). MnTnHex-2-PyP5+ has the ability to dissolve lipid membranes, causing local irritation/necrosis at injection sites if given at doses of 1 mg/kg or higher. In conclusion, both compounds show an ability to ameliorate lung damage as measured using breathing frequencies and histopathologic evaluation. However, MnTE-2-PyP5+ at 6 mg/kg proved to be more effective in reducing expression of key molecular factors known to play an important role in radiation-induced lung injury.     

Evaluation of the Growth Assessment Protocol (GAP) for antenatal detection of small for gestational age: The DESiGN cluster randomised trial

BackgroundAntenatal detection and management of small for gestational age (SGA) is a strategy to reduce stillbirth. Large observational studies provide conflicting results on the effect of the Growth Assessment Protocol (GAP) in relation to detection of SGA and reduction of stillbirth; to the best of our knowledge, there are no reported randomised control trials. Our aim was to determine if GAP improves antenatal detection of SGA compared to standard care.Methods and findingsThis was a pragmatic, superiority, 2-arm, parallel group, open, cluster randomised control trial. Maternity units in England were eligible to participate in the study, except if they had already implemented GAP. All women who gave birth in participating clusters (maternity units) during the year prior to randomisation and during the trial (November 2016 to February 2019) were included. Multiple pregnancies, fetal abnormalities or births before 24⁺¹ weeks were excluded. Clusters were randomised to immediate implementation of GAP, an antenatal care package aimed at improving detection of SGA as a means to reduce the rate of stillbirth, or to standard care. Randomisation by random permutation was stratified by time of study inclusion and cluster size. Data were obtained from hospital electronic records for 12 months prerandomisation, the washout period (interval between randomisation and data collection of outcomes), and the outcome period (last 6 months of the study). The primary outcome was ultrasound detection of SGA (estimated fetal weight <10th centile using customised centiles (intervention) or Hadlock centiles (standard care)) confirmed at birth (birthweight <10th centile by both customised and population centiles). Secondary outcomes were maternal and neonatal outcomes, including induction of labour, gestational age at delivery, mode of birth, neonatal morbidity, and stillbirth/perinatal mortality. A 2-stage cluster–summary statistical approach calculated the absolute difference (intervention minus standard care arm) adjusted using the prerandomisation estimate, maternal age, ethnicity, parity, and randomisation strata. Intervention arm clusters that made no attempt to implement GAP were excluded in modified intention to treat (mITT) analysis; full ITT was also reported. Process evaluation assessed implementation fidelity, reach, dose, acceptability, and feasibility. Seven clusters were randomised to GAP and 6 to standard care. Following exclusions, there were 11,096 births exposed to the intervention (5 clusters) and 13,810 exposed to standard care (6 clusters) during the outcome period (mITT analysis). Age, height, and weight were broadly similar between arms, but there were fewer women: of white ethnicity (56.2% versus 62.7%), and in the least deprived quintile of the Index of Multiple Deprivation (7.5% versus 16.5%) in the intervention arm during the outcome period. Antenatal detection of SGA was 25.9% in the intervention and 27.7% in the standard care arm (adjusted difference 2.2%, 95% confidence interval (CI) −6.4% to 10.7%; p = 0.62). Findings were consistent in full ITT analysis. Fidelity and dose of GAP implementation were variable, while a high proportion (88.7%) of women were reached. Use of routinely collected data is both a strength (cost-efficient) and a limitation (occurrence of missing data); the modest number of clusters limits our ability to study small effect sizes.ConclusionsIn this study, we observed no effect of GAP on antenatal detection of SGA compared to standard care. Given variable implementation observed, future studies should incorporate standardised implementation outcomes such as those reported here to determine generalisability of our findings.Trial registrationThis trial is registered with the ISRCTN registry, ISRCTN67698474.

Matias C Vieira and colleagues evaluate the Growth Assessment Protocol (GAP) for antenatal detection of small for gestational age in the DESiGN cluster randomised trial.     

A keystone predator controls bacterial diversity in the pitcher-plant (Sarracenia purpurea) microecosystem

The community of organisms inhabiting the water-filled leaves of the carnivorous pitcher-plant Sarracenia purpurea includes arthropods, protozoa and bacteria, and serves as a model system for studies of food web dynamics. Despite the wealth of data collected by ecologists and zoologists on this food web, very little is known about the bacterial assemblage in this microecosystem. We used terminal restriction fragment length polymorphism (T-RFLP) analysis to quantify bacterial diversity within the pitchers as a function of pitcher size, pH of the pitcher fluid and the presence of the keystone predator in this food web, larvae of the pitcher-plant mosquito Wyeomyia smithii. Results were analysed at two spatial scales: within a single bog and across three isolated bogs. Pitchers were sterile before they opened and composition of the bacterial assemblage was more variable between different bogs than within bogs. Measures of bacterial richness and diversity were greater in the presence of W. smithii and increased with increasing pitcher size. Our results suggest that fundamental ecological concepts derived from macroscopic food webs can also be used to predict the bacterial assemblages in pitcher plants.