Agrobacterium-mediated transformation of the CrDAT gene and selection of transgenic periwinkle lines with a high vincristine accumulation

Catharanthus roseus contains vincristine and vinblastine, which are outstanding drugs for cancer. In the biosynthetic pathways of terpenoid indole alkaloids (TIAs) in C. roseus, deacetylvindoline 4-O-acetyltransferase (DAT) is a key enzyme that catalyses the last reaction of vindoline biosynthesis to form vinblastine and vincristine. In this study, the CrDAT transgene was transferred into the periwinkle by Agrobacterium-mediated transformation and generated transgenic periwinkle lines with an increase in vincristine accumulation. The C. roseus DAT gene was introduced into C. roseus plants and it was confirmed that CrDAT was successfully transferred into the genome of periwinkle plants and efficiently translated to synthesise recombinant DAT protein. Four transgenic periwinkle lines in T1 generation, T1-1, T1-3, T1-6, and T1-7, expressed recombinant DAT protein with the total protein content in the range of 2.86 μg.mg^?1 to 5.12 μg.mg^?1. Moreover, the vincristine contents of four transgenic lines increased by 1.63?2.48-fold compared to non-transgenic plants, ranging from 6.91 µg.g^?1 (fresh weight) to 10.53 µg.g^?1 (fresh weight). The T1-1 line had the highest vincristine content. Hence, the overexpression of the recombinant DAT protein can improve the vincristine accumulation of transgenic C. roseus plants.

Abbreviation: CrDAT - Catharanthus roseus Deacetylvindoline-4-O-Acetyl Transferase; D4H - Deacetoxyvindoline 4-hydroxylase; ELISA - Enzyme-Linked Immunosorbent Assay Monoterpene indole alkaloid; T0, T1 - Generations of transgenic plants; TIAs - Terpenoid indole alkaloids; WT- The wild-type tobacco plants (non transgenic plant); 35S - Cauliflower mosaic virus 35S promoter     

Variability in the performance of juvenile Chinook salmon is explained primarily by when and where they resided in estuarine habitats

Estuarine habitats provide rearing opportunities for the juvenile life stage of anadromous fishes. Because survival is positively correlated with juvenile performance, these estuarine habitats play an important role in population abundance and productivity. To provide information for the recovery of several depressed stocks of Chinook salmon in the Columbia River Basin, we sought to identify the factors that explain variability in performance. Using otolith‐derived estimates of juvenile somatic growth rate as an index of recent performance, we observed a negative nonlinear relationship between growth rate and day of year, and a decreasing and increasing trend of growth rate over the 8 years of this study and distance from the river mouth respectively. Using a generalised linear modelling approach, we found that variability in juvenile somatic growth rate was best explained by where and when individuals were collected, their body size, contaminant loads, stock of origin, and whether a fish was hatchery produced or unmarked. Lastly, we argue that a considerable improvement to the growth rate of juveniles in estuarine habitats is physiologically possible. The results of this 8‐year study provide a baseline of the performance of juvenile Chinook salmon to evaluate habitat restoration programs and to compare against future anthropogenic conditions.     

Comparing the behavioural thermoregulation response to heat stress by Atlantic salmon parr (Salmo salar) in two rivers

Climate change is expected to increase the frequency and magnitude of extreme thermal events in rivers. The Little Southwest Miramichi River (LSWM) and the Ouelle River (OR) are two Atlantic salmon (Salmo salar) rivers located in eastern Canada, where in recent years, water temperatures have exceeded known thermal limits (~23°C). Once temperature surpasses this threshold, juvenile salmon exploit thermal heterogeneity to behaviourally thermoregulate, forming aggregations in coolwater refuges. This study aimed to determine whether the behavioural thermoregulation response is universal across rivers, arising from common thermal cues. We detailed the temperature and discharge patterns of two geographically distinct rivers from 2010 to 2012 and compared these with aggregation onset temperature. PIT telemetry and snorkelling were used to confirm the presence of aggregations. Mean daily maximum temperature in 2010 was significantly greater in the OR versus the LSWM (p = 0.005), but not in other years (p = 0.090–0.353). Aggregations occurred on 14 and 9 occasions in the OR and LSWM respectively. Temperature at onset of aggregation was significantly greater in the OR (T_onset = 28.3°C) than in the LSWM (T_onset = 27.3°C; p = 0.049). Logistic regression models varied by river and were able to predict the probability of aggregation based on the preceding number of hours >23°C (R² = 0.61 & 0.65; P₅₀ = 27.4°C & 28.9°C; in the OR and LSWM respectively). These results imply the preceding local thermal regime may influence behaviour and indicate a degree of phenotypic plasticity, illustrating a need for localised management strategies.     

Ecosystem services as the products of land system dynamics: lessons from a longitudinal study of coupled human–environment systems

Context

We address understanding of whole-system and landscape-based approaches to the ecosystem services framework by considering the supply of provisioning services and the dynamics of agricultural land use in Scotland between 1940 and 2016.

Objectives

To characterise and understand the dynamics of change in provisioning services from agriculture in Scotland over the period 1940–2016. To identify ways in which funds of capitals and flows of inputs and output ecosystem goods are linked to land management practices and policies at a national scale.

Methods

Data describing agricultural land use, production, financial and energy inputs and outputs, and drivers of change in land use in Scotland are analysed with an accounting framework that links funds of natural, human, physical and financial capital, with flows of goods and services. Flow–fund ratios are used as benchmarks of system performance and dynamics.

Results

Scotland’s agriculture has modernised since 1940 and become more efficient in conversion of resources, with a consequent increase in delivery of provisioning goods and services. Although the energy ratio, and flow of goods per unit hectare and per unit labour have increased, the inputs necessary to maintain those flows of ecosystem goods are also increasing, even as their relative economic costs decrease. Increases in use of fertiliser suggests that production from the soil, as a natural capital fund, is not being conserved without a large, and increasing, input. Analysis of the complexity of the coupled agricultural land system also suggests that land management rather than biodiversity is a necessary subject for evaluation of provisioning services from agriculture. Understanding of ecosystem services based on accounts that integrate inputs, outputs and flows from funds of natural, human, social, financial and physical capitals, provides a process-based foundation for improved understanding of ecosystem services and human–environmental relationships.

Conclusions

Adopting an accounting approach for understanding the role of agricultural land use for supply of provisioning services, and particularly examining a long time-series of accounts, enables understanding of land changes and underlying drivers, as well as the contribution of cultural and other aspects of human systems coupled with environment systems. Accounting for ecosystem services using costs as well as benefits, and use of metrics beyond financial benefit, supports debate and evaluation of trade-offs between services and has direct relevance for decision- and policy-making.

     

No effect of turkey herpesvirus infections on the outcome of avian pneumovirus infections in turkeys.

To determine whether turkey herpesvirus (HVT) impairs the aspecific and specific defense against an avian pneumovirus (APV) infection, specific-pathogen-free turkeys were inoculated at 7 days of age with HVT and 1, 5, or 7 wk later with APV. Clinical signs, APV replication, and development of antibodies against APV were evaluated. No differences were found between the birds that received both HVT and APV and those that received only APV.     

Culture of bovine bone marrow progenitor cells in vitro.

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400 g). The use of 3% bovine leucocyte-conditioned medium, produced by stimulation of blood lymphocytes with 4 microg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte-conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose-based medium requires different culture conditions, which are different from the culture conditions for human cells.     

A Tremor Syndrome With a Central Axonopathy in Scottish Terriers

A central axonopathy in 2 male and 1 female Scottish Terrier puppies from 3 different but related litters is reported. Clinical signs consisting of severe whole-body tremors and ataxia were first detected at the age of 10 to 12 weeks. They worsened with activity and excitement and diminished during rest or sleep. Two dogs also had paraparesis. In 1 dog the neurological deficits progressed over several months. Neuropathological examination revealed widespread axonal changes, vacuolation, and gliosis in the white matter of the central nervous system.