Wissen, wo man steht. Ergebnisse des Projektes "COMPASS".

Das Projekt "COMPASS" der Agrar- und Ernährungswissenschaftlichen Fakultät der Universität Kiel beschäftigt sich seit 2004 mit dem Monitoring von Leistungen und ökologischen Effekten auf 32 konventionellen und ökologischen Praxisbetrieben. Im vorliegenden Band sind die zentralen Ergebnisse aus den Bereichen Nährstoffmanagement, Pflanzenschutz, Rückstandsproblematik, Tiergesundheit und Biodiversität (EU-Projekt AVI-LAND) umfassend und gleichzeitig anschaulich dargestellt

Experimental Inoculation of Juvenile Rhesus Macaques with Primate Enteric Caliciviruses

Tissue culture-adapted Tulane virus (TV), a GI.1 rhesus enteric calicivirus (ReCV), and a mixture of GII.2 and GII.4 human norovirus (NoV)-containing stool sample were used to intrastomacheally inoculate juvenile rhesus macaques (Macaca mulatta) in order to evaluate infection caused by these viruses. METHODOLOGY & FINDINGS: Two of the three TV-inoculated macaques developed diarrhea, fever, virus-shedding in stools, inflammation of duodenum and 16-fold increase of TV-neutralizing (VN) serum antibodies but no vomiting or viremia. No VN-antibody responses could be detected against a GI.2 ReCV strain FT285, suggesting that TV and FT285 represent different ReCV serotypes. Both NoV-inoculated macaques remained asymptomatic but with demonstrable virus shedding in one animal. Examination of duodenum biopsies of the TV-inoculated macaques showed lymphocytic infiltration of the lamina propria and villous blunting. TV antigen-positive (TV+) cells were detected in the lamina propria. In most of the TV+ cells TV co-localized perinuclearly with calnexin--an endoplasmic reticulum protein. A few CD20+TV+ double-positive B cells were also identified in duodenum. To corroborate the authenticity of CD20+TV+ B cells, in vitro cultures of peripheral blood mononuclear cells (PBMCs) from healthy macaques were inoculated with TV. Multicolor flow cytometry confirmed the presence of TV antigen-containing B cells of predominantly CD20+HLA-DR+ phenotype. A 2-log increase of viral RNA by 6 days post inoculation (p<0.05) suggested active TV replication in cultured lymphocytes.Taken together, our results show that ReCVs represent an alternative cell culture and animal model to study enteric calicivirus replication, pathogenesis and immunity

Exposure to Inorganic Arsenic During Pregnancy Alters Structure and Function of the Murine Maternal Heart

Inorganic arsenic (iAs) is a drinking water contaminant in many countries throughout the world, including the U.S. The World Health Organization recommends a limit of 10µg/L iAs in drinking water yet estimates suggest that 140 million people worldwide consume water with iAs above this threshold. While iAs is a top public health contaminant of concern due to harmful health effects, iAs has been specifically established as a cardiotoxicant. The purpose of this study was to examine the effect(s) of iAs exposure on the maternal heart during pregnancy, with a focus on physiological and molecular changes using in vivo exposure paradigms. We sought to examine the impact of this environmental insult on the maternal heart as this is a relatively understudied area and can highlight potential hazards for pregnant mothers. Pregnant female C57BL/6J mice were exposed to either 1000, 100, or 0µg/L iAs (Control) after conception at embryonic day 2.5 (E2.5) with exposure ending at parturition. Dams were harvested at either E17.5 or postnatal day 12 (P12), and heart size and function were assessed using gravimetric measurement, histology, and transthoracic echocardiography. Markers of hypertrophy were examined at both time points using western blot and qRT-PCR, and single cell calcium and sarcomere shortening transients were examined postnatally. Offspring outcomes were also characterized. We found that prenatally, maternal hearts show a reduction in cardiac hypertrophy with downregulation of key markers of physiologic hypertrophy during pregnancy. Postnatally, we see that the hearts are enlarged compared to controls with some evidence of pathologic remodeling and calcium handling dysregulation. In offspring we find a reduction in postnatal growth with exposure to 100µg/L iAs and an increase in heart size in-utero at 1000µg/L iAs, as well as differential expression in proteins involved in one-carbon metabolism in the placenta. These findings underscore the detrimental impact of iAs on the cardiovascular system, as well as to reproductive and developmental health, and specifically highlight potential hazards posed by iAs exposure during pregnancy

Osteogenic induction from marmoset embryonic stem cells cultured in feeder-dependent and feeder-independent conditions

Summary Embryonic stem cells (ESCs) have become increasingly attractive for cell replacement therapies of osteodegenerative diseases; however, pre-clinical studies in large animal models to repair diseased or injured bone are lacking. As a first step into this direction, we describe here the feeder-free cultivation and directed osteogenic differentiation of marmoset ESCs. Introduction Owing to their potential to self-renew and their enormous differentiation capability, ESCs are an adequate cell source for cell replacement therapies. To implement stem cell technology clinically, standardized cultivation and differentiation protocols and appropriate animal models are needed. Here, we describe the feeder-free cultivation of Callithrix jacchus ESCs (cESCs) in a chemically defined medium and their subsequent osteogenic differentiation. Methods cESCs were maintained on mouse embryonic fibroblast feeder layers or in feeder-free conditions with activin A and basic fibroblast growth factor. Differentiation into mature osteoblasts was steered with ascorbic acid, β-glycerophosphate and 1α,25-(OH)2 vitamin D3 employing various induction strategies. Results In feeder-free conditions, cESCs maintained pluripotency as indicated by Oct-4 and Nanog expression, positive immunostaining for typical primate ESC markers and high telomerase activity. Cells also remained karyotypically normal after 40 passages without feeder cells. The hanging drop protocol as well as omitting the embryoid body step proved unsuccessful to initiate osteogenic differentiation. The highest degree of osteogenesis was achieved by formation of embryoid bodies employing the cell cluster technique as indicated by the amount of deposited calcium and bone marker gene expression. Early addition of retinoic acid further improved the yield of osteoblasts and led to an increase in calcium deposition. Conclusions The osteogenic differentiation potential of feeder-free cESCs was equal if not higher compared to cells grown on feeders. These findings open the field for near clinical transplantation studies in primate models to evaluate the effectiveness of ESC-derived osteoblasts

Do developmental temperatures affect redox level and lifespan in C. elegans through upregulation of peroxiredoxin?

Lifespan in poikilothermic organisms, such as Caenorhabditis elegans, can be substantially increased simply by decreasing growth temperature. To gain insights into the mechanistic underpinnings of this effect, we investigated the effects of temperature in development and adulthood on C. elegans lifespan. We found that worms exposed to 25 °C during development and shifted to 15 °C in adulthood exhibited an even longer lifespan than animals constantly kept at 15 °C. Analysis of the in vivo redox status demonstrated that at 25 °C, C. elegans larvae have a more reduced redox state and higher Prdx-2 expression levels than animals raised at 15 °C. Worms lacking prdx-2 fail to show the additional lifespan extension upon shift from 25 °C to 15 °C and reveal a lifespan similar to prdx-2 worms always kept at 15 °C. These results suggest that transiently altering the in vivo redox state during development can have highly beneficial long-term consequences for organisms. Keywords: Aging, Temperature, C. elegans, Oxidants, Peroxiredoxi

Altered PLP1 splicing causes hypomyelination of early myelinating structures

Objective: The objective of this study was to investigate the genetic etiology of the X-linked disorder "Hypomyelination of Early Myelinating Structures" (HEMS). Methods: We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients' fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results: All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. Interpretation: Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus-Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing