Leptospirosis serology in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia

The common brushtail possum (Trichosurus vulpecula) is indeed a common marsupial in major cities of Australia. This species is known to be susceptible to leptospirosis and often lives in close contact with humans, raising concerns about the potential for transmission of this disease in urban areas. A total of 192 brushtail possum blood samples were collected from 136 individuals in suburban areas of metropolitan Sydney from November 2002 to November 2004. Sera were screened against a reference panel of 21 Leptospira spp. using the microscopic agglutination test. Leptospiral antibodies were detected in 9.6% (13/136) of tested brushtail possums and represented two serovars; antibodies to Leptospira interrogans serovar Hardjo were most frequently identified (11/136). A representative of the exotic sero-group Ballum, most likely serovar Arborea, was found in two of 136 brushtail possums. Exposure to leptospirosis seemed to be associated with age, as older animals had a higher incidence, but there was no distinction in relation to gender. Antibody prevalence varied between the different sampling sites and seropositive animals were clustered and restricted to a few sites. These data support the possible role of brushtail possums as a maintenance host for Leptospira spp. in urban environments and also identified them as a previously unknown and potential source of serovar Arborea.     

Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae

Dicarboxylic acids that are produced from renewable resources are becoming attractive building blocks for the polymers industry. In this respect, fumaric acid is very interesting. Its low aqueous solubility facilitates product recovery. To avoid excessive waste salt production during downstream processing, a low pH for fumaric acid fermentation will be beneficial. Studying the influence of pH, working volume and shaking frequency on cell cultivation helped us to identify the best conditions to obtain appropriate pellet morphologies of a wild type strain of Rhizopus oryzae. Using these pellets, the effects of pH and CO(2) addition were studied to determine the best conditions to produce fumaric acid in batch fermentations under nitrogen-limited conditions with glucose as carbon source. Decreasing either the fermentation pH below 5 or increasing the CO(2) content of the inlet air above 10% was unfavourable for the cell-specific productivity, fumaric acid yield, and fumaric acid titer. However, switching off the pH control late in the batch phase did not affect these performance parameters and allowed achieving pH of 3.6. A concentration of 20 gL(-1) of fumaric acid was obtained at pH 3.6 while the average cell mass specific productivity and fumaric acid yield were the same as at pH 5.0. Consequently, relatively modest amounts of inorganic base were required for pH control, while recovery of the acid should be relatively easy at pH 3.6.     

A New β-Estradiol-lnducible Vector Set that Facilitates Easy Construction and Efficient Expression of Transgenes Reveals CBL3-Dependent Cytoplasm to Tonoplast Translocation of CIPK5

Transient and stable expression of transgenes is central to many investigations in plant biology research. Chemical regulation of expression can circumvent problems of plant lethality caused by constitutive overexpression or allow inducible knock （out/down） approaches. Several chemically inducible or repressible systems have been described and successfully applied. However, cloning and application-specific modification of most available inducible expression systems have been limited and remained complicated due to restricted cloning options. Here we describe a new set of 57 vectors that enable transgene expression in transiently or stably transformed cells. All vectors harbor a synthetically optimized XVE expression cassette, allowing I~-estradiol mediated protein expression. Plasmids are equipped with the reporter genes GUS, GFP, mCherry, or with HA and Strepll epitope tags and harbor an optimized multiple cloning site for flexible and simple clon- ing strategies. Moreover, the vector design allows simple substitution of the driving promoter to achieve tissue-specificity or to modulate expression ranges of inducible transgene expression. We report details of the kinetics and dose-dependence of expression induction in Arabidopsis leaf mesophyll protoplasts, transiently transformed Nicotiana benthamiana leaves, and stably transformed Arabidopsis plants. Using these vectors, we investigated the influence of CBL （Calcineurin B-like） protein expression on the subcellular localization of CIPKs （Calcineurin B-like interacting protein kinases）. These analyses uncovered that induced co-expression of CBL3 is fully sufficient for dynamic translocation of CIPK5 from the cytoplasm to the tonoplast. Thus, the vector system presented here facilitates a broad range of research applications.     

The PI3K inhibitor pictilisib and the multikinase inhibitors pazopanib and sorafenib have an impact on Rac1 level and migration of medulloblastoma in vitro

Metastatic disease is the leading cause of death in children suffering from medulloblastoma and a major treatment challenge. The evidence of leptomeningeal dissemination defines the most aggressive tumours and is associated with increased mortality; thus, inhibition of migration as a factor involved in the process of metastatic disease is fundamental for the treatment and prevention of metastatic dissemination. Targeting the small Rho GTPases Rac1 has been shown to effectively impair medulloblastoma cell migration in vitro. Yet clinically applicable selective Rac1 inhibitors are still lacking. In view of the pertinent oncogenic role of the PI3K signalling cascade and tyrosine kinase‐mediated signalling pathways in medulloblastoma, we explored clinically available targeted therapeutics to this effect. Here, we show that Rac1 is expressed in both the cytoplasm and nucleus in the medulloblastoma cell lines Daoy and MEB‐Med‐8A representative of two high risk medulloblastoma entities. We demonstrate that activated Rac1 is subject to substantial downmodulation following administration of the clinically available inhibitor of the PI3K pathway Pictilisib (GDC‐0941) and the multityrosine kinase inhibitors Pazopanib and Sorafenib. The application of those drugs was associated with reduced mobility of the medulloblastoma cells and alterations of the actin skeleton. Of note, PI3K inhibition reveals the strongest anti‐migratory effect in Daoy cells. Thus, our in vitro observations provide new insights into different strategies of blocking Rac1 and inhibiting migration in medulloblastoma employing clinically available agents paving the way for confirmatory studies in in vivo models.     

Phenotyping of Left and Right Ventricular Function in Mouse Models of Compensated Hypertrophy and Heart Failure with Cardiac MRI

Background

Left ventricular (LV) and right ventricular (RV) function have an important impact on symptom occurrence, disease progression and exercise tolerance in pressure overload-induced heart failure, but particularly RV functional changes are not well described in the relevant aortic banding mouse model. Therefore, we quantified time-dependent alterations in the ventricular morphology and function in two models of hypertrophy and heart failure and we studied the relationship between RV and LV function during the transition from hypertrophy to heart failure.

Methods

MRI was used to quantify RV and LV function and morphology in healthy (n = 4) and sham operated (n = 3) C57BL/6 mice, and animals with a mild (n = 5) and a severe aortic constriction (n = 10).

Results

Mice subjected to a mild constriction showed increased LV mass (P<0.01) and depressed LV ejection fraction (EF) (P<0.05) as compared to controls, but had similar RVEF (P>0.05). Animals with a severe constriction progressively developed LV hypertrophy (P<0.001), depressed LVEF (P<0.001), followed by a declining RVEF (P<0.001) and the development of pulmonary remodeling, as compared to controls during a 10-week follow-up. Myocardial strain, as a measure for local cardiac function, decreased in mice with a severe constriction compared to controls (P<0.05).

Conclusions

Relevant changes in mouse RV and LV function following an aortic constriction could be quantified using MRI. The well-controlled models described here open opportunities to assess the added value of new MRI techniques for the diagnosis of heart failure and to study the impact of new therapeutic strategies on disease progression and symptom occurrence.     

Time‐Continuous Phosphorus Flows in the Indian Agri‐Food Sector: Long‐Term Drivers and Management Options

Phosphorus (P) is a major agricultural nutrient and, in its mineable form, a potentially scarce resource. Countries with limited physical access to P should hence develop an effective national P governance. This requires analyses of trends and variations in P flows and stocks over time. Here, we present a long‐term P flow analysis for the Indian agri‐food sector from 1988 to 2011. Major P flows are imports of mineral P, fertilizer application, and uptake of animal fodder. The mineral P import dependency ratio is constant at around 93%. On average, 20% of P inputs to soils are lost through erosion. Key drivers of changes in P flows include population growth, dietary change, and agricultural intensification. To reduce its P fertilizer import dependence, India could, for example, substitute up to 19% of the presently applied mineral P if manure used as a household fuel were recycled, and up to 21% if P was fully recovered from wastewater and household waste. Comparing selected indicators for P use in agriculture with China and the European Union (EU) reveals that there are structural similarities, such as increasing fertilizer application rates and P accumulation in soils, with the first but large differences compared to the latter. The analyses highlight that in contrast to static indicators, the time‐continuous tracking of P flows provides substantial advantages, such as the identification of long‐term trends, drivers, and intervention options for sustainable P management, given that it allows for the interpretation of present indicators in the context of past trends and legacies.     

Switching on the lights for gene therapy

Strategies for non-invasive and quantitative imaging of gene expression in vivo have been developed over the past decade. Non-invasive assessment of the dynamics of gene regulation is of interest for the detection of endogenous disease-specific biological alterations (e.g., signal transduction) and for monitoring the induction and regulation of therapeutic genes (e.g., gene therapy). To demonstrate that non-invasive imaging of regulated expression of any type of gene after in vivo transduction by versatile vectors is feasible, we generated regulatable herpes simplex virus type 1 (HSV-1) amplicon vectors carrying hormone (mifepristone) or antibiotic (tetracycline) regulated promoters driving the proportional co-expression of two marker genes. Regulated gene expression was monitored by fluorescence microscopy in culture and by positron emission tomography (PET) or bioluminescence (BLI) in vivo. The induction levels evaluated in glioma models varied depending on the dose of inductor. With fluorescence microscopy and BLI being the tools for assessing gene expression in culture and animal models, and with PET being the technology for possible application in humans, the generated vectors may serve to non-invasively monitor the dynamics of any gene of interest which is proportionally co-expressed with the respective imaging marker gene in research applications aiming towards translation into clinical application.     

Cascading effects of climate extremes on vertebrate fauna through changes to low‐latitude tree flowering and fruiting phenology

Forest vertebrate fauna provide critical services, such as pollination and seed dispersal, which underpin functional and resilient ecosystems. In turn, many of these fauna are dependent on the flowering phenology of the plant species of such ecosystems. The impact of changes in climate, including climate extremes, on the interaction between these fauna and flora has not been identified or elucidated, yet influences on flowering phenology are already evident. These changes are well documented in the mid to high latitudes. However, there is emerging evidence that the flowering phenology, nectar/pollen production, and fruit production of long‐lived trees in tropical and subtropical forests are also being impacted by changes in the frequency and severity of climate extremes. Here, we examine the implications of these changes for vertebrate fauna dependent on these resources. We review the literature to establish evidence for links between climate extremes and flowering phenology, elucidating the nature of relationships between different vertebrate taxa and flowering regimes. We combine this information with climate change projections to postulate about the likely impacts on nectar, pollen and fruit resource availability and the consequences for dependent vertebrate fauna. The most recent climate projections show that the frequency and intensity of climate extremes will increase during the 21st century. These changes are likely to significantly alter mass flowering and fruiting events in the tropics and subtropics, which are frequently cued by climate extremes, such as intensive rainfall events or rapid temperature shifts. We find that in these systems the abundance and duration of resource availability for vertebrate fauna is likely to fluctuate, and the time intervals between episodes of high resource availability to increase. The combined impact of these changes has the potential to result in cascading effects on ecosystems through changes in pollinator and seed dispersal ecology, and demands a focused research effort.     

The IL-2 diphtheria toxin fusion protein denileukin diftitox modulates the onset of diabetes in female nonobese diabetic animals in a time-dependent manner and breaks tolerance in male nonobese diabetic animals

Denileukin diftitox, also known as DAB(389)IL-2 or Ontak, is a fusion protein toxin consisting of the full-length sequence of the IL-2 protein and as toxophore the truncated diphtheria toxin. As a consequence, it delivers the toxic agent to CD25-bearing cells, whereby CD25 represents the high-affinity α-subunit of the IL-2 receptor. Initially it was developed for the treatment of patients with cutaneous T cell lymphoma. Meanwhile, denileukin diftitox is also used as an adjuvant in other tumor therapies and neoplastic disorders. In this study, to our knowledge we report for the first time that denileukin diftitox has also dramatic effects regarding the pathology of type 1 diabetes using the NOD mouse model. Repeated injections of denileukin diftitox into female NOD mice at 12 wk of age led to a clear acceleration of disease onset, whereas injection at 7 wk of age did not. Using male NOD mice, which are much less susceptible to diabetes, we demonstrate that the injection of denileukin diftitox leads to a dramatic development of type 1 diabetes within days after injection, thereby obviously breaking pre-existing tolerance mechanisms. This is accompanied by an increased IFN-γ production of autoreactive splenic cells and a decreased presence of regulatory CD4(+)CD25(+)Foxp3(+) T cells. In contrast, transfer of CD4(+)CD25(+)Foxp3(+) T cells could correct the defect after denileukin diftitox treatment. Furthermore, whereas IFN-γ production was increased in the pancreata of treated animals, insulin expression was strongly reduced. These finding should be considered when denileukin diftitox is used for the treatment of patients suffering from tumors and/or autoimmune disorders.