MODULATION OF OSTEOGENIC DIFFERENTIATION IN HUMAN SKELETAL CELLS IN VITRO BY 5-AZACYTIDINE

Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system.     

Visual displays and taxonomic affinities in the parrot genus Trichoglossus

Variation in visual signalling behaviour in ten different species and subspecies was used re-assess taxonomic affinities within the Loriine parrot genus Trichoglossus. Within the genus, visual signals are derived from a range of non-signalling source movements from which a range of both unritualized and ritualized derivative signals have evolved. This evolution of signal movement can be viewed as occurring in a series of stages, beginning with a number of unritualized primary signals and followed by a succession of increasingly ritualized secondary, tertiary and even quaternary derivatives. By treating each primary signal and its subsequent derivatives as a separate, multi-state, taxonomic character, coefficients of similarity were calculated between each of the different taxa. In general, the results of this analysis confirmed taxonomic affinities derived previously from morphological comparisons. In some cases, however, behavioural resemblances suggested plausible alternatives to existing taxonomic theories. The validity of using culturally inherited behaviour patterns for systematic purposes is discussed.     

Adaptive microclimatic structural and expressional dehydrin 1 evolution in wild barley, Hordeum spontaneum, at 'Evolution Canyon', Mount Carmel, Israel

'Evolution Canyon' (ECI) at Lower Nahal Oren, Mount Carmel, Israel, is an optimal natural microscale model for unravelling evolution in action highlighting the twin evolutionary processes of adaptation and speciation. A major model organism in ECI is wild barley, Hordeum spontaneum , the progenitor of cultivated barley, which displays dramatic interslope adaptive and speciational divergence on the 'African' dry slope (AS) and the 'European' humid slope (ES), separated on average by 200 m. Here we examined interslope single nucleotide polymorphism (SNP) sequences and the expression diversity of the drought resistant dehydrin 1 gene ( Dhn1 ) between the opposite slopes. We analysed 47 plants (genotypes), 4–10 individuals in each of seven stations (populations) in an area of 7000 m², for Dhn1 sequence diversity located in the 5' upstream flanking region of the gene. We found significant levels of Dhn1 genic diversity represented by 29 haplotypes, derived from 45 SNPs in a total of 708 bp sites. Most of the haplotypes, 25 out of 29 (= 86.2%), were represented by one genotype; hence, unique to one population. Only a single haplotype was common to both slopes. Genetic divergence of sequence and haplotype diversity was generally and significantly different among the populations and slopes. Nucleotide diversity was higher on the AS, whereas haplotype diversity was higher on the ES. Interslope divergence was significantly higher than intraslope divergence. The applied Tajima D rejected neutrality of the SNP diversity. The Dhn1 expression under dehydration indicated interslope divergent expression between AS and ES genotypes, reinforcing Dhn1 associated with drought resistance of wild barley at 'Evolution Canyon'. These results are inexplicable by mutation, gene flow, or chance effects, and support adaptive natural microclimatic selection as the major evolutionary divergent driving force.     

The long-term costs of traumatic stress: intertwined physical and psychological consequences

The gradual emergence of symptoms following exposure to traumatic events has presented a major conceptual challenge to psychiatry. The mechanism that causes the progressive escalation of symptoms with the passage of time leading to delayed onset post-traumatic stress disorder (PTSD) involves the process of sensitization and kindling. The development of traumatic memories at the time of stress exposure represents a major vulnerability through repeated environmental triggering of the increasing dysregulation of an individual’s neurobiology. An increasing body of evidence demonstrates how the increased allostatic load associated with PTSD is associated with a significant body of physical morbidity in the form of chronic musculoskeletal pain, hypertension, hyperlipidaemia, obesity and cardiovascular disease. This increasing body of literature suggests that the effects of traumatic stress need to be considered as a major environmental challenge that places individual’s physical and psychological health equally at risk. This broader perspective has important implications for developing treatments that address the underlying dysregulation of cortical arousal and neurohormonal abnormalities following exposure to traumatic stress.     

Re‐Evaluation of the EUK516 Probe for the Domain Eukarya Results in a Suitable Probe for the Detection of Kinetoplastids,an Important Group of Parasitic and Free‐Living Flagellates

ABSTRACT. Two frequently used universal eukaryote probes, EUK1209 and EUK516, are not consistent with one branch of the eukaryotic phylogenetic tree, the Kinetoplastida, which has undergone rapid evolution of their small subunit rRNA gene. Kinetoplastids include medically important parasitic organisms (e.g. Trypanosoma, Leishmania) and free‐living flagellates that occur in all aquatic environments and in soils (e.g. Bodo, Neobodo, Rhynchomonas). A modified probe presented here as KIN516, now based on the kinetoplastid sequence, provides a strong signal with Neobodo designis, Leishmania donovani, and Trypanosoma cruzi using the catalyzed reporter deposition protocol. EUK516 and KIN516 function as competitor probes, thereby greatly increasing discriminatory power when used in combination. The probe pair was tested in field samples collected in a freshwater pond in Norfolk, the mesohaline Elizabeth River, Norfolk, Virginia, and a tropical lagoon in Belize. The combined probes bound to 58–84% of organisms identified as eukaryotic based on having large DAPI‐stained nuclei. The contribution of kinetoplastids to total eukaryotes (positive signal of EUK516+KIN516) was much higher in marine samples (ca. 17%) than in either the freshwater or brackish water sites (<0.2%).     

Effects of coalbed natural gas development on fish assemblages in tributary streams of the Powder and Tongue rivers

1. Extraction of coalbed natural gas (CBNG) often results in disposal of large quantities of CBNG product water, which may affect aquatic ecosystems. We evaluated the effects of CBNG development on fish assemblages in tributary streams of the Powder and Tongue rivers. We used treatment and control, impact versus reference sites comparisons, surveys of CBNG product‐water streams and in situ fish survival approaches to determine if CBNG development affected fish assemblages. 2. Several of our results suggested that CBNG development did not affect fish assemblages. Species richness and index of biotic integrity (IBI) scores were similar in streams with and streams without CBNG development, and overall biotic integrity was not related to the number or density of CBNG wells. Fish occurred in one stream that was composed largely or entirely of CBNG product water. Sentinel fish survived in cages at treatment sites where no or few fish were captured, suggesting that factors such as lack of stream connectivity rather than water quality limited fish abundance at these sites. Fish species richness did not differ significantly from 1994 to 2006 in comparisons of CBNG‐developed and undeveloped streams. Biotic integrity declined from 1994 to 2006; however, declines occurred at both impact and reference sites, possibly because of long‐term drought. 3. Some evidence suggested that CBNG development negatively affected fish assemblages, or may do so over time. Specific conductivity was on average higher in treatment streams and was negatively related to biotic integrity. Four IBI species richness metrics were negatively correlated with the number or density of CBNG wells in the catchment above sampling sites. Bicarbonate, one of the primary ions in product water, was significantly higher in developed streams and may have limited abundance of longnose dace (Rhinichthys cataractae). Total dissolved solids, alkalinity, magnesium and sulphate were significantly higher in developed streams. 4. Biological monitoring conducted before the development of CBNG, and continuing through the life of development and reclamation, together with data on the quantity, quality and fate of CBNG product water will allow robust assessment of potential effects of future CBNG development worldwide.     

Survival of Colonizing Wolves in the Northern Rocky Mountains of the United States, 1982–2004

Abstract: After roughly a 60-year absence, wolves (Canis lupus) immigrated (1979) and were reintroduced (1995-1996) into the northern Rocky Mountains (NRM), USA, where wolves are protected under the Endangered Species Act. The wolf recovery goal is to restore an equitably distributed metapopulation of ≥30 breeding pairs and 300 wolves in Montana, Idaho, and Wyoming, while minimizing damage to livestock; ultimately, the objective is to establish state-managed conservation programs for wolf populations in NRM. Previously, wolves were eradicated from the NRM because of excessive human killing. We used Andersen–Gill hazard models to assess biological, habitat, and anthropogenic factors contributing to current wolf mortality risk and whether federal protection was adequate to provide acceptably low hazards. We radiocollared 711 wolves in Idaho, Montana, and Wyoming (e.g., NRM region of the United States) from 1982 to 2004 and recorded 363 mortalities. Overall, annual survival rate of wolves in the recovery areas was 0.750 (95% CI = 0.728-0.772), which is generally considered adequate for wolf population sustainability and thereby allowed the NRM wolf population to increase. Contrary to our prediction, wolf mortality risk was higher in the northwest Montana (NWMT) recovery area, likely due to less abundant public land being secure wolf habitat compared to other recovery areas. In contrast, lower hazards in the Greater Yellowstone Area (GYA) and central Idaho (CID) likely were due to larger core areas that offered stronger wolf protection. We also found that wolves collared for damage management purposes (targeted sample) had substantially lower survival than those collared for monitoring purposes (representative sample) because most mortality was due to human factors (e.g., illegal take, control). This difference in survival underscores the importance of human-caused mortality in this recovering NRM population. Other factors contributing to increased mortality risk were pup and yearling age class, or dispersing status, which was related to younger age cohorts. When we included habitat variables in our analysis, we found that wolves having abundant agricultural and private land as well as livestock in their territory had higher mortality risk. Wolf survival was higher in areas with increased wolf density, implying that secure core habitat, particularly in GYA and CID, is important for wolf protection. We failed to detect changes in wolf hazards according to either gender or season. Maintaining wolves in NWMT will require greater attention to human harvest, conflict resolution, and illegal mortality than in either CID or GYA; however, if human access increases in the future in either of the latter 2 areas hazards to wolves also may increase. Indeed, because overall suitable habitat is more fragmented and the NRM has higher human access than many places where wolves roam freely and are subject to harvest (e.g., Canada and AK), monitoring of wolf vital rates, along with concomitant conservation and management strategies directed at wolves, their habitat, and humans, will be important for ensuring long-term viability of wolves in the region.