Embryogenic suspensions of adult cork oak: the first step towards mass propagation.

Abstract Protocols have been established to clone adult cork oak trees by somatic embryogenesis using semisolid medium. However, for economically viable mass propagation, embryogenic cultures in liquid medium need to be developed. In this study, suspension cultures were initiated from embryo clusters obtained by secondary embryogenesis on a gelled medium lacking plant growth regulators. After 6 days of culture, these embryo clusters generated high cell density suspensions that also contained small organized structures (embryos and embryogenic clumps). As the culture duration increased, tissue necrosis and fewer embryogenic structures were observed and the establishment of suspension cultures failed. An alternative method was found adequate for initiation of embryogenic suspensions: embryo clusters from gelled medium were briefly shaken in liquid medium and detached cells and embryogenic masses of 41?800 lm were used as inoculum. Maintenance of embryogenic suspensions was achieved using a low-density inoculum (43 mg l-1) by subculturing four embryogenic clumps of 0.8?1.2 mm per 70 ml of medium. Proliferation ability was maintained for almost 1 year through ten consecutive subcultures. The initiation and maintenance protocols first developed for a single genotype were effective when tested on 11 cork oak genotypes.

Negative density dependence and environmental heterogeneity effects on tree ferns across succession in a tropical montane forest.

Although tree ferns are an important component of temperate and tropical forests, very little is known about their ecology. Their peculiar biology (e.g., dispersal by spores and two-phase life cycle) makes it difficult to extrapolate current knowledge on the ecology of other tree species to tree ferns. In this paper, we studied the effects of negative density dependence (NDD) and environmental heterogeneity on populations of two abundant tree fern species, Cyathea caracasana and Alsophila engelii, and how these effects change across a successional gradient. Species patterns harbor information on processes such as competition that can be easily revealed using point pattern analysis techniques. However, its detection may be difficult due to the confounded effects of habitat heterogeneity. Here, we mapped three forest plots along a successional gradient in the montane forests of Southern Ecuador. We employed homogeneous and inhomogeneous K and pair correlation functions to quantify the change in the spatial pattern of different size classes and a case-control design to study associations between juvenile and adult tree ferns. Using spatial estimates of the biomass of four functional tree types (short- and long-lived pioneer, shade- and partial shade-tolerant) as covariates, we fitted heterogeneous Poisson models to the point pattern of juvenile and adult tree ferns and explored the existence of habitat dependencies on these patterns. Our study revealed NDD effects for C. caracasana and strong environmental filtering underlying the pattern of A. engelii. We found that adult and juvenile populations of both species responded differently to habitat heterogeneity and in most cases this heterogeneity was associated with the spatial distribution of biomass of the four functional tree types. These findings show the effectiveness of factoring out environmental heterogeneity to avoid confounding factors when studying NDD and demonstrate the usefulness of covariate maps derived from mapped communities.

Evolution of foraging behavior in Drosophila by density-dependent selection

One of the rare examples of a single major gene underlying a naturally occurring behavioral polymorphism is the foraging locus of Drosophila melanogaster. Larvae with the rover allele, forR, have significantly longer foraging path lengths on a yeast paste than do those homozygous for the sitter allele, fors. These variants do not differ in general activity in the absence of food. The evolutionary significance of this polymorphism is not as yet understood. Here we examine the effect of high and low animal rearing densities on the larval foraging path-length phenotype and show that density-dependent natural selection produces changes in this trait. In three unrelated base populations the long path (rover) phenotype was selected for under high-density rearing conditions, whereas the short path (sitter) phenotype was selected for under low-density conditions. Genetic crosses suggested that these changes resulted from alterations in the frequency of the fors allele in the low-density-selected lines. Further experiments showed that density-dependent selection during the larval stage rather than the adult stage of development was sufficient to explain these results. Density-dependent mechanisms may be sufficient to maintain variation in rover and sitter behavior in laboratory populations.

Acetylcholine receptor ɛ-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice

In mammalian muscle a postnatal switch in functional properties of neuromuscular transmission occurs when miniature end plate currents become shorter and the conductance and Ca2+ permeability of end plate channels increases. These changes are due to replacement during early neonatal development of the γ-subunit of the fetal acetylcholine receptor (AChR) by the ɛ-subunit. The long-term functional consequences of this switch for neuromuscular transmission and motor behavior of the animal remained elusive. We report that deletion of the ɛ-subunit gene caused in homozygous mutant mice the persistence of γ-subunit gene expression in juvenile and adult animals. Neuromuscular transmission in these animals is based on fetal type AChRs present in the end plate at reduced density. Impaired neuromuscular transmission, progressive muscle weakness, and atrophy caused premature death 2 to 3 months after birth. The results demonstrate that postnatal incorporation into the end plate of ɛ-subunit containing AChRs is essential for normal development of skeletal muscle.

Recruitment and replacement of hippocampal neurons in young and adult chickadees: an addition to the theory of hippocampal learning.

We used [3H]thymidine to document the birth of neurons and their recruitment into the hippocampal complex (HC) of juvenile (4.5 months old) and adult blackcapped chickadees (Parus atricapillus) living in their natural surroundings. Birds received a single dose of [3H]thymidine in August and were recaptured and killed 6 weeks later, in early October. All brains were stained with Cresyl violet, a Nissl stain. The boundaries of the HC were defined by reference to the ventricular wall, the brain surface, or differences in neuronal packing density. The HC of juveniles was as large as or larger than that of adults and packing density of HC neurons was 31% higher in juveniles than in adults. Almost all of the 3H-labeled HC neurons were found in a 350-m-wide layer of tissue adjacent to the lateral ventricle. Within this layer the fraction of 3H-labeled neurons was 50% higher in juveniles than in adults. We conclude that the HC of juvenile chickadees recruits more neurons and has more neurons than that of adults. We speculate that juveniles encounter greater environmental novelty than adults and that the greater number of HC neurons found in juveniles allows them to learn more than adults. At a more general level, we suggest that (i) long-term learning alters HC neurons irreversibly; (ii) sustained hippocampal learning requires the periodic replacement of HC neurons; (iii) memories coded by hippocampal neurons are transferred elsewhere before the neurons are replaced.

Transient prenatal vitamin D deficiency is associated with hyperlocomotion in adult rats

Rat experiments have shown that prenatal Vitamin D deficiency leads to altered neonatal brain morphology, cell density and neurotrophin expression. In the current study we examined the hypothesis that Vitamin D deficiency during early development alters adult behaviour even when there is an intervening period in which the animal receives normal Vitamin D in later development. Rats were conceived and born to Vitamin D deficient dams (Birth); conceived, born and weaned from Vitamin D deficient dams (Weaning); or deficient in Vitamin D from conception to 10 weeks of age (Life). Litters were standardized to three males and three females per litter. All rat offspring were rendered normocalcaemic with calcium supplemented water (2 mM) after weaning. Control animals were born to mothers fed a normal diet but subject to similar litter size and calcium supplementation. At 10 weeks all animals were tested on the holeboard test, elevated plus maze test, social interaction observation, acoustic startle response test, prepulse inhibition of the acoustic startle response and a forced swim test. Early Vitamin D deficiency (Birth group) enhanced locomotion in the holeboard test and increased activity in the elevated plus maze. Thus, transient prenatal Vitamin D deficiency induces hyperlocomotion in adulthood, without severe motor abnormalities. (C) 2004 Elsevier B.V. All rights reserved.

Temporal dynamics of rabbit haemorrhagic disease virus infection in a low-density population of wild rabbits (Oryctolagus cuniculus) in New Zealand

A longitudinal capture-mark-recapture study was conducted to determine the temporal dynamics of rabbit haemorrhagic disease (RHD) in a European rabbit (Oryctolagus cuniculus) population of low to moderate density on sand-hill country in the lower North Island of New Zealand. A combination of sampling ( trapping and radio-tracking) and diagnostic (cELISA, PCR and isotype ELISA) methods was employed to obtain data weekly from May 1998 until June 2001. Although rabbit haemorrhagic disease virus ( RHDV) infection was detected in the study population in all 3 years, disease epidemics were evident only in the late summer or autumn months in 1999 and 2001. Overall, 20% of 385 samples obtained from adult animals older than 11 weeks were seropositive. An RHD outbreak in 1999 contributed to an estimated population decline of 26%. A second RHD epidemic in February 2001 was associated with a population decline of 52% over the subsequent month. Following the outbreaks, the seroprevalence in adult survivors was between 40% and 50%. During 2000, no deaths from RHDV were confirmed and mortalities were predominantly attributed to predation. Influx of seronegative immigrants was greatest in the 1999 and 2001 breeding seasons, and preceded the RHD epidemics in those years. Our data suggest that RHD epidemics require the population immunity level to fall below a threshold where propagation of infection can be maintained through the population.

The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture

The adult human intervertebral disc (IVD) is normally avascular. Changes to the extracellular matrix in degenerative disc disease may promote vascularisation and subsequently alter cell nutrition and disc homeostasis. This study examines the influence of cell density and the presence of glucose and serum on the proliferation and survival of IVD cells in 3D culture. Bovine nucleus pulposus (NP) cells were seeded at a range of cell densities (1.25 × 10(5)-10(6) cells/mL) and cultured in alginate beads under standard culture conditions (with 3.15 g/L glucose and 10 % serum), or without glucose and/or 20% serum. Cell proliferation, apoptosis and cell senescence were examined after 8 days in culture. Under standard culture conditions, NP cell proliferation and cluster formation was inversely related to cell seeding density, whilst the number of apoptotic cells and enucleated "ghost" cells was positively correlated to cell seeding density. Increasing serum levels from 10% to 20% was associated with increased cluster size and also an increased prevalence of apoptotic cells within clusters. Omitting glucose produced even larger clusters and also more apoptotic and senescent cells. These studies demonstrate that NP cell growth and survival are influenced both by cell density and the availability of serum or nutrients, such as glucose. The observation of clustered, senescent, apoptotic or "ghost" cells in vitro suggests that environmental factors may influence the formation of these phenotypes that have been previously reported in vivo. Hence this study has implications for both our understanding of degenerative disc disease and also cell-based therapy using cells cultured in vitro.

The influence of neighbourhood formality status and socio-economic position on self-rated health among adult men and women: a multilevel, cross sectional, population study from Aleppo, Syria

Background There is substantial evidence from high income countries that neighbourhoods have an influence on health independent of individual characteristics. However, neighbourhood characteristics are rarely taken into account in the analysis of urban health studies from developing countries. Informal urban neighbourhoods are home to about half of the population in Aleppo, the second largest city in Syria (population>2.5 million). This study aimed to examine the influence of neighbourhood socioeconomic status (SES) and formality status on self-rated health (SRH) of adult men and women residing in formal and informal urban neighbourhoods in Aleppo. Methods The study used data from 2038 survey respondents to the Aleppo Household Survey, 2004 (age 18–65 years, 54.8% women, response rate 86%). Respondents were nested in 45 neighbourhoods. Five individual-level SES measures, namely education, employment, car ownership, item ownership and household density, were aggregated to the level of neighbourhood. Multilevel regression models were used to investigate associations. Results We did not find evidence of important SRH variation between neighbourhoods. Neighbourhood average of household item ownership was associated with a greater likelihood of reporting excellent SRH in women; odds ratio (OR) for an increase of one item on average was 2.3 (95% CI 1.3-4.4 (versus poor SRH)) and 1.7 (95% CI 1.1-2.5 (versus normal SRH)), adjusted for individual characteristics and neighbourhood formality. After controlling for individual and neighbourhood SES measures, women living in informal neighbourhoods were less likely to report poor SRH than women living in formal neighbourhoods (OR= 0.4; 95% CI (0.2- 0.8) (versus poor SRH) and OR=0.5; 95%; CI (0.3-0.9) (versus normal SRH). Conclusions Findings support evidence from high income countries that certain characteristic of neighbourhoods affect men and women in different ways. Further research from similar urban settings in developing countries is needed to understand the mechanisms by which informal neighbourhoods influence women’s health.

Suppression of the Insulin Receptors in Adult Schistosoma japonicum Impacts on Parasite Growth and Development: Further Evidence of Vaccine Potential

To further investigate the importance of insulin signaling in the growth, development, sexual maturation and egg production of adult schistosomes, we have focused attention on the insulin receptors (SjIRs) of Schistosoma japonicum, which we have previously cloned and partially characterised. We now show, by Biolayer Interferometry, that human insulin can bind the L1 subdomain (insulin binding domain) of recombinant (r)SjIR1 and rSjIR2 (designated SjLD1 and SjLD2) produced using the Drosophila S2 protein expression system. We have then used RNA interference (RNAi) to knock down the expression of the SjIRs in adult S. japonicum in vitro and show that, in addition to their reduced transcription, the transcript levels of other important downstream genes within the insulin pathway, associated with glucose metabolism and schistosome fecundity, were also impacted substantially. Further, a significant decrease in glucose uptake was observed in the SjIR-knockdown worms compared with luciferase controls. In vaccine/challenge experiments, we found that rSjLD1 and rSjLD2 depressed female growth, intestinal granuloma density and faecal egg production in S. japonicum in mice presented with a low dose challenge infection. These data re-emphasize the potential of the SjIRs as veterinary transmission blocking vaccine candidates against zoonotic schistosomiasis japonica in China and the Philippines.

Satellite tracking of adult loggerheads (Caretta caretta L.) around waters of Cape Verde Archipielago (Western Africa)

[EN] During the 1999 nesting season (from June to October), four satellite transmitters were attached to two males and two females of Caretta caretta from Boavista Island (Cape Verde Archipelago, western Africa), where density of nesting females is the highest in the whole archipelago. Transmitters did not work as well as expected, resulting in a low number of locations, possibly due to the neritic behavior of adult loggerheads at reproductive habitat, with the antennae exposed to continuous rubbing. Therefore, we establish a conservative hypothesis about movements of C.caretta in Cape Verde.

Propofol causes neuronal degeneration in neonatal mice and long-term neurocognitive consequences in adult mice

Purpose: To investigate the effect of propofol on brain development in neonatal mice and long-term neurocognitive impact in adult mice. Method: The offspring of female C57Bl/6 and male CD-1 mice were administered propofol at concentrations of 2.5 and 5.0 mg/kg (treatment group) or normal saline (control) on postnatal day 7. Thereafter, histological and immunohistochemical examinations were performed on the mice brain. Apoptotic assay, neuronal nuclei antigen immunohistochemistry (to assess neuron density), and behavioral and neurocognitive tests were conducted on the adult mice. Results: Propofol induced cellular degeneration and apoptosis in the brains of neonatal mice. It also modulated physiological parameters (pH, PO2, glucose and lactate), among which decreased blood glucose might be associated with cellular degeneration in the brain. Propofol also caused long-term neuronal deficits in adults, which showed impaired neurocognitive functions. Upon reaching adulthood, propofol-treated mice showed slow learning response and poor memory compared to controls. Conclusion: Propofol causes neurodegeneration in neonatal mice and has long-term neurocognitive consequences in adults, indicating that the use of propofol anesthetics in neonates requires careful consideration.

Cell Pattern in Adult Human Corneal Endothelium

A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.

Exploratory Study Of The Effect Of Lifestyle Counselling On Bone Mineral Density And Body Composition In Users Of The Contraceptive Depot-medroxyprogesterone Acetate.

To compare variations in bone mineral density (BMD) and body composition (BC) in depot-medroxyprogesterone acetate (DMPA) users and nonusers after providing counselling on healthy lifestyle habits. An exploratory study in which women aged 18 to 40 years participated: 29 new DMPA users and 25 new non-hormonal contraceptive users. All participants were advised on healthy lifestyle habits: sun exposure, walking and calcium intake. BMD and BC were assessed at baseline and 12 months later. Statistical analysis included the Mann-Whitney test or Student's t-test followed by multiple linear regression analysis. Compared to the controls, DMPA users had lower BMD at vertebrae L1 and L4 after 12 months of use. They also had a mean increase of 2 kg in total fat mass and an increase of 2.2% in body fat compared to the non-hormonal contraceptive users. BMD loss at L1 was less pronounced in DMPA users with a calcium intake ≥ 1 g/day compared to DMPA users with a lower calcium intake. DMPA use was apparently associated with lower BMD and an increase in fat mass at 12 months of use. Calcium intake ≥ 1 g/day attenuates BMD loss in DMPA users. Counselling on healthy lifestyle habits failed to achieve its aims.

Exposure To Bisphenol-a During Pregnancy Partially Mimics The Effects Of A High-fat Diet Altering Glucose Homeostasis And Gene Expression In Adult Male Mice.

Bisphenol-A (BPA) is one of the most widespread EDCs used as a base compound in the manufacture of polycarbonate plastics. The aim of our research has been to study how the exposure to BPA during pregnancy affects weight, glucose homeostasis, pancreatic β-cell function and gene expression in the major peripheral organs that control energy flux: white adipose tissue (WAT), the liver and skeletal muscle, in male offspring 17 and 28 weeks old. Pregnant mice were treated with a subcutaneous injection of 10 µg/kg/day of BPA or a vehicle from day 9 to 16 of pregnancy. One month old offspring were divided into four different groups: vehicle treated mice that ate a normal chow diet (Control group); BPA treated mice that also ate a normal chow diet (BPA); vehicle treated animals that had a high fat diet (HFD) and BPA treated animals that were fed HFD (HFD-BPA). The BPA group started to gain weight at 18 weeks old and caught up to the HFD group before week 28. The BPA group as well as the HFD and HFD-BPA ones presented fasting hyperglycemia, glucose intolerance and high levels of non-esterified fatty acids (NEFA) in plasma compared with the Control one. Glucose stimulated insulin release was disrupted, particularly in the HFD-BPA group. In WAT, the mRNA expression of the genes involved in fatty acid metabolism, Srebpc1, Pparα and Cpt1β was decreased by BPA to the same extent as with the HFD treatment. BPA treatment upregulated Pparγ and Prkaa1 genes in the liver; yet it diminished the expression of Cd36. Hepatic triglyceride levels were increased in all groups compared to control. In conclusion, male offspring from BPA-treated mothers presented symptoms of diabesity. This term refers to a form of diabetes which typically develops in later life and is associated with obesity.