Identifying biological landmarks using a novel cell measuring image analysis tool: Cell-o-Tape

Background

A large number of different plant lines are produced and maintained in a typical plant research laboratory, both as seed stocks and in active growth. These collections need careful and consistent management to track and maintain them properly, and this is a particularly pressing issue in laboratories undertaking research involving genetic manipulation due to regulatory requirements. Researchers and PIs need to access these data and collections, and therefore an easy-to-use plant-oriented laboratory information management system that implements, maintains and displays the information in a simple and visual format would be of great help in both the daily work in the lab and in ensuring regulatory compliance.

Results

Here, we introduce ??Phytotracker??, a laboratory management system designed specifically to organise and track plasmids, seeds and growing plants that can be used in mixed platform environments. Phytotracker is designed with simplicity of user operation and ease of installation and management as the major factor, whilst providing tracking tools that cover the full range of activities in molecular genetics labs. It utilises the cross-platform Filemaker relational database, which allows it to be run as a stand-alone or as a server-based networked solution available across all workstations in a lab that can be internet accessible if desired. It can also be readily modified or customised further. Phytotracker provides cataloguing and search functions for plasmids, seed batches, seed stocks and plants growing in pots or trays, and allows tracking of each plant from seed sowing, through harvest to the new seed batch and can print appropriate labels at each stage. The system enters seed information as it is transferred from the previous harvest data, and allows both selfing and hybridization (crossing) to be defined and tracked. Transgenic lines can be linked to their plasmid DNA source. This ease of use and flexibility helps users to reduce their time needed to organise their plants, seeds and plasmids and to maintain laboratory continuity involving multiple workers.

Conclusion

We have developed and used Phytotracker for over five years and have found it has been an intuitive, powerful and flexible research tool in organising our plasmid, seed and plant collections requiring minimal maintenance and training for users. It has been developed in an Arabidopsis molecular genetics environment, but can be readily adapted for almost any plant laboratory research.     

Effect of GnRH and its antagonist (Antarelix) on LH release from cultured bovine anterior pituitary cells

In the following investigations, the LH secretion of cells from pituitaries in heifers on days 16-18 of their oestrous cycle (n = 14) was analysed. Cells were dissociated with trypsin and collagenase and maintained in a static culture system. For the estimation of LH release, the cells were incubated with various concentrations of mammalian GnRH (Lutrelef) for 6 h. To determine the action of Antarelix (GnRH antagonist), the cells were preincubated for 1 h with concentrations of 10(-5) or 10(-4) M Antarelix followed by 10(-6) M GnRH coincubation for a further 6 h. At the end of each incubation, the medium was collected for LH analysis. Parallel, intracellular LH was qualitatively detected by immunocytochemistry. Changes in the intensity of LH staining within the cells in dependence of different GnRH concentrations were not observed, but a significant increase LH secretion in pituitary cells was measured at 10(-6) M GnRH. Antarelix had no effect on basal LH secretion at concentrations of 10(-4) and 10(-5) M. After coincubation of pituitary cells with Antarelix and GnRH, Antarelix blocked the GnRH-stimulated LH secretion with a maximal effect of 10(-4) M, but the staining of immunoreactive intracellular LH was detected at approximately the same level compared to the pituitary cells treated with exogenous GnRH alone. These data demonstrate that Antarelix is effective in influencing the GnRH-stimulated LH secretion of pituitary cells in vitro. After administration of Antarelix in vivo, the GnRH-stimulated LH secretion of cultured pituitary cells was not inhibited.     

Organic‐carbon and nitrogen stocks and organic‐carbon fractions in soil under mixed pine and oak forest stands of different ages in NE Germany

The objective of this work was to evaluate the C and N stocks and organic‐C fractions in soil under mixed forest stands of Scots pine (Pinus sylvestris L.) and Sessile oak (Quercus petraea [Matt.] Liebl.) of different ages in NE Germany. Treatments consisted of pure pine (age 102 y), and pine (age 90–102 y) mixed with 10‐, 35‐, 106‐, and 124‐y‐old oak trees. After sampling O layers, soils in the mineral layer were taken at two different depths (0–10 and 10–20 cm). Oak admixture did not affect total organic‐C (TOC) and N stocks considering the different layers separately. However, when the sum of TOC stocks in the organic and mineral layers was considered, TOC stocks decreased with increasing in oak age (r² = 0.58, p < 0.10). The microbial C (C_MB) was not directly correlated with increase of oak age, however, it was positively related with presence of oak species. There was an increase in the percentage of the C_MB‐to‐TOC ratio with increase of oak‐tree ages. On average, light‐fraction C (C_LF) comprised 68% of the soil TOC in upper layer corresponding to the highest C pool in the upper layer. C_LF and heavy‐fraction C (C_HF) were not directly affected by the admixture of oak trees in both layers. The C_HF accounted on average for 30% and 59% of the TOC at 0–10 and 10–20 cm depths, respectively. Despite low clay contents in the studied soils, the differences in the DCB‐extractable Fe and Al affected the concentrations of the C_HF and TOC in the 10–20 cm layers (p < 0.05). Admixture of oak in pine stands contributed to reduce topsoil C stocks, probably due to higher soil organic matter turnover promoted by higher quality of oak litter.     

DNA vaccines encoding the envelope protein of West Nile virus lineages 1 or 2 administered intramuscularly,via electroporation and with recombinant virus protein induce partial protection in large falcons (Falco spp.)

As West Nile virus (WNV) can cause lethal diseases in raptors, a vaccination prophylaxis of free-living and captive populations is desirable. In the absence of vaccines approved for birds, equine vaccines have been used in falcons, but full protection against WNV infection was not achieved. Therefore, two DNA vaccines encoding the ectodomain of the envelope protein of WNV lineages 1 and 2, respectively, were evaluated in 28 large falcons. Four different vaccination protocols were used, including electroporation and booster-injections of recombinant WNV domain III protein, before challenge with the live WNV lineage 1 strain NY99. Drug safety, plasmid shedding and antibody production were monitored during the vaccination period. Serological, virological, histological, immunohistochemical and molecular biological investigations were performed during the challenge trials. Antibody response following vaccination was low overall and lasted for a maximum of three weeks. Plasmid shedding was not detected at any time. Viremia, mortality and levels, but not duration, of oral virus shedding were reduced in all of the groups during the challenge trial compared to the non-vaccinated control group. Likewise, clinical scoring, levels of cloacal virus shedding and viral load in organs were significantly reduced in three vaccination groups. Histopathological findings associated with WNV infections (meningo-encephalitis, myocarditis, and arteritis) were present in all groups, but immunohistochemical detection of the viral antigen was reduced. In conclusion, the vaccines can be used safely in falcons to reduce mortality and clinical signs and to lower the risk of virus transmission due to decreased levels of virus shedding and viremia, but full protection was not achieved in all groups.     

Oligogenic control of resistance to soil‐borne viruses SBCMV and WSSMV in rye (Secale cereale L.)

Rye production in European growing areas is constrained by the soilborne cereal mosaic virus (SBCMV) and the wheat spindle streak mosaic virus (WSSMV). To date, no European rye cultivars are known to exhibit resistance against these viruses. In this study, we pursued a quantitative trait locus (QTL) mapping strategy to identify genomic regions for resistance to SBCMV and WSSMV in rye. Three populations, each comprising 100 lines segregating for resistance to SBCMV and/or WSSMV, were evaluated for disease response at two years in three locations in Germany where soils are naturally infested with SBCMV and WSSMV. In the combined analysis across environments, one QTL for SBCMV resistance on chromosome 5R explained 31.9% of the phenotypic variation in one of the populations. For WSSMV resistance, one QTL explaining up to 64.0% of the phenotypic variation was detected on chromosome 7R in each of the three populations. On the Triticeae homoeologous group 5, we found evidence for synteny of the major QTL for SBCMV resistance between the wheat and rye genomes.     

Pflanzenschutz mit Pflanzenextrakten (Botanicals): Hintergründe,zwei Fallstudien,Schlussfolgerungen und Standortbestimmung

Plant extracts (botanicals) are of special significance for plant protection, especially in organic farming. This is due to their origin, specific modes of action, toxicological and ecotoxicological properties as well as to ever changing legal, patent, economic and social/political issues. This paper surveys this complex background and presents data on two selected botanicals which pertain to the use of extract fractions from Glycyrrhiza glabra (sweet wood) for control of Bremia lactucae (downy mildew) in lettuce and azadirachtin for control of leafhoppers in Melissa officinalis L. Both compounds exhibited good effects in the greenhouse which were less pronounced or absent in the field. The effects of sweet wood were strongest when applied twice preventively at 5?% concentration. In the greenhouse, this resulted in a lower number of infected plants and smaller leaf areas attacked by the downy mildew pathogen at high disease pressure. Azadirachtin (plant protection chemical NeemAzal®-T/S in combination with the additive Trifolio S-forte; 1.5 l/ha + 3 l/ha in 600 l water per hektar, 3 applications, interval 7 days) was better in controlling leaf hoppers than natural pyrethrins in combination with rapeseed oil (Spruzit Schädlingsfrei; 6 l/ha in 1,000 l water per hektar, 1 application) in the greenhouse and exhibited very high insecticidal efficacy almost equivalent to that of thiacloprid (Calypso 480 SC; 0.12 l/ha in 400 l water per hektar, 1 application) at moderate disease pressure. Thiacloprid, however, is not applicable in organic farming. In order to introduce these promising results in practical plant protection further fundamental studies are needed. These can only be realized by a joint effort of private companies, research and plant protection institutions, as well as public funding bodies. This article is not meant as a presentation of “perfect” results, it is rather an example of the problems and pitfalls that need to be overcome during development of a botanical and its applications. They are the reason why the market for botanicals will remain small.     

Familial canine pituitary-dependent hyperadrenocorticism in wirehaired Dachshunds

Canine pituitary hyperadrenocorticism (Cushing's disease) caused by neoplasia of the corticotrope cells is one of the most common endocrine diseases especially in smaller dog breeds. Cushing's disease was diagnosed in eleven wire-haired Dachshunds and for further six wire-haired Dachshunds Cushing's disease was suspected on the basis of clinical signs. A joined pedigree could be ascertained for all these 17 dogs. Eleven of these dogs were so closely related to each other, that they were summarized in four nucleus families. Two fullsiblings were examined by means of clinical, laboratory diagnostic and morphological methods. The main lesions consisted of atrophic dermatosis with alopecia, increase of activity of liver enzymes in plasma and bilateral adrenocortical hyperplasia and therefore corresponded to the typical signs of a secondary hyperadrenocorticism. A rather unusual finding was the pituitary carcinoma in one of these dogs. Similarly to human patients affected by hyperadrenocorticism, real-time PCR analysis showed a 2.9-fold increase of expression of the canine MDR1 gene in the liver of one affected wirehaired Dachshund. This study documents the first familial occurrence of pituitary-dependent hyperadrenocorticism in wirehaired Dachshunds, the overexpression of the MDR1 gene in the dog and the third case of familial hyperadrenocorticism in dogs ever described.     

Untersuchungen zur systemischen Infektion von Fusarium culmorum an Winterweizen in Schleswig-Holstein

In north-west Germany Fusarium head blight of wheat (FHB) is frequently caused by Fusarium culmorum and F. graminearum. This damage has, however, mainly been caused by infections during flowering; the incidence of FHB has increased steadily for many years in crop rotations without maize. The authors postulate that F. culmorum can cause systemic infections in wheat. In 2003 they used real-time PCR to analyse more than 1?100 tissue samples from wheat fields in order to determine whether F. culmorum was present in the internodes and spikes of the wheat plants. More than 30% of all analysed samples contained F. culmorum-DNA, and many wheat stems were completely or nearly completely systemically colonized by F. culmorum. These findings are consistent with those of other researchers who have detected a systemic growth of F. culmorum during experiments under controlled conditions. It cannot be automatically assumed that the fungus spreads from the wheat head during flowering; the plant could have become infected through chlamydospores or mycelium on residual plant debris in the soil. Therefore this pathogen must be regarded as a crop rotation disease. F. culmorum is able to severely infect wheat through the roots and stem bases, especially when the soil is dry and warm. Thus, spraying fungicides during flowering is ineffective.     

Measurement of the elastic properties of the cancellous bone in the femoral head of the dog

The degenerative wear and pathologic damage of the joints are reasons for total endoprotheses in man as well as in dogs. The main problem is the aseptic loosening of the protheses. By usig the finite-element-method, the total endoprothesis is designed with new features, with the purpose of preventing loosening and being better adapted to load transmission. In order to simulate the femur of the dog for the numerical analysis, a material law is developed. By taking into account the anisotropy and the local density of the cancellous bone in the femoral head, the young's modules are experimentally determined. The measurements are performed by ultrasonic methods on femoral heads of euthanised dogs. The results show planar isotropic cancellous bone.     

Auftreten und Nachweis von Fusarium oxysporum und F. proliferatum an Steck- und Saatzwiebeln

The pathogen Fusarium oxysporum f. sp. cepae inducing the Fusarium basal rot mainly spreads in warmer cultivation regions due to its adaptibility to high temperature. Meanwhile the pathogen occurs in Germany as well, especially in years with relatively high average temperature during the growing season. Phytopathological investigations of 300 symptomless onion bulbs showed a contamination rate of approximately 10% with regard to Fusarium spp, with F.?oxysporum proving to be the predominant species. Onion sets planted in these fields were latently infected with F.?oxysporum at rates of 19?C98%. Unexpectedly, the contaminated sets did not indispensably lead to a high occurrence of plants exhibiting characteristic symptoms of Fusarium basal rot such as wet and dry rot. Presumably, the development of symptoms is particularly affected by given climatic conditions. The results of pathogenicity tests of isolated Fusarium spp. isolates under controlled conditions support this assumption. The inoculation of the substrate with selected Fusarium spp. isolates resulted in a reduction of emergence by up to 70% under controlled conditions, which are suboptimal with regard to the cultivation of onions. The emergence of plants was not affected by Fusarium spp. under optimal cultivation of onions. However, under optimal cultivation conditions a reduction of plant growth occurred in a subsequent growth stage. Beside F.?oxysporum, F.?proliferatum could be detected in onion bulbs as well as seeds. The proportion of contaminated seeds accounted to 62%. Both species F.?oxysporum and F.?proliferatum proved to be pathogenic in onion although their isolates varied much in their virulence.