BackgroundThe objective of this study was to evaluate potential long-term (110 days) and age-specific effects of feeding genetically modified Bt maize on peripheral immune response in pigs and to determine the digestive fate of the cry1Ab gene and truncated Bt toxin.Methodology/Principal FindingsForty day old pigs (n = 40) were fed one of the following treatments: 1) isogenic maize-based diet for 110 days (isogenic); 2) Bt maize-based diet (MON810) for 110 days (Bt); 3) Isogenic maize-based diet for 30 days followed by Bt maize-based diet for 80 days (isogenic/Bt); and 4) Bt maize-based diet (MON810) for 30 days followed by isogenic maize-based diet for 80 days (Bt/isogenic). Blood samples were collected during the study for haematological analysis, measurement of cytokine and Cry1Ab-specific antibody production, immune cell phenotyping and cry1Ab gene and truncated Bt toxin detection. Pigs were sacrificed on day 110 and digesta and organ samples were taken for detection of the cry1Ab gene and the truncated Bt toxin. On day 100, lymphocyte counts were higher (P<0.05) in pigs fed Bt/isogenic than pigs fed Bt or isogenic. Erythrocyte counts on day 100 were lower in pigs fed Bt or isogenic/Bt than pigs fed Bt/isogenic (P<0.05). Neither the truncated Bt toxin nor the cry1Ab gene were detected in the organs or blood of pigs fed Bt maize. The cry1Ab gene was detected in stomach digesta and at low frequency in the ileum but not in the distal gastrointestinal tract (GIT), while the Bt toxin fragments were detected at all sites in the GIT.Conclusions/SignificancePerturbations in peripheral immune response were thought not to be age-specific and were not indicative of Th 2 type allergenic or Th 1 type inflammatory responses. There was no evidence of cry1Ab gene or Bt toxin translocation to organs or blood following long-term feeding.
BackgroundWe aimed to determine the effect of feeding transgenic maize to sows during gestation and lactation on maternal and offspring immunity and to assess the fate of transgenic material.Methodology/Principal FindingsOn the day of insemination, sows were assigned to one of two treatments (n = 12/treatment); 1) non-Bt control maize diet or 2) Bt-MON810 maize diet, which were fed for ∼143 days throughout gestation and lactation. Immune function was assessed by leukocyte phenotyping, haematology and Cry1Ab-specific antibody presence in blood on days 0, 28 and 110 of gestation and at the end of lactation. Peripheral-blood mononuclear cell cytokine production was investigated on days 28 and 110 of gestation. Haematological analysis was performed on offspring at birth (n = 12/treatment). Presence of the cry1Ab transgene was assessed in sows' blood and faeces on day 110 of gestation and in blood and tissues of offspring at birth. Cry1Ab protein presence was assessed in sows' blood during gestation and lactation and in tissues of offspring at birth. Blood monocyte count and percentage were higher (P<0.05), while granulocyte percentage was lower (P<0.05) in Bt maize-fed sows on day 110 of gestation. Leukocyte count and granulocyte count and percentage were lower (P<0.05), while lymphocyte percentage was higher (P<0.05) in offspring of Bt maize-fed sows. Bt maize-fed sows had a lower percentage of monocytes on day 28 of lactation and of CD4+CD8+ lymphocytes on day 110 of gestation, day 28 of lactation and overall (P<0.05). Cytokine production was similar between treatments. Transgenic material or Cry1Ab-specific antibodies were not detected in sows or offspring.Conclusions/SignificanceTreatment differences observed following feeding of Bt maize to sows did not indicate inflammation or allergy and are unlikely to be of major importance. These results provide additional data for Bt maize safety assessment.
In order to characterize unauthorized genetically modified petunia, an integrated strategy has been applied here on several suspected petunia samples from the European market. More precisely, DNA fragments of interest were produced by DNA walking anchored on key targets, earlier detected by real-time PCR screening analysis, to be subsequently sequenced using the MinION platform from Oxford Nanopore Technologies. This way, the presence of genetically modified petunia was demonstrated via the characterization of their transgene flanking regions as well as unnatural associations of elements from their transgenic cassette.
The target of this article is to summarize the chemical and pharmaceutical (formulation) approaches that were found to be suitable for increasing the bioavailability of a model weak base (SAR1) with a very narrow good solubility range at physiologically relevant pH. 1 As part of the preformulation and formulation development to support toxicological and first in man studies of a free base (SAR1), several formulation approaches, including particle size reduction, emulsions, permeability enhancers, amorphous dispersions, salt formation, and co-crystal formation, were screened by in vitro dissolution methods and in vivo pharmacokinetic (PK) studies to evaluate and rank formulation performance. From the PK studies, it was observed that a suspension formulation containing a SAR1 fumaric acid (1:1) co-crystal provided the best oral exposure. Sensitivity of the co-crystal to physical disintegration into base and fumaric acid was solved by including Cremophor ELP as a solubility enhancer, surfactant, and co-crystal protector. This formulation was well-tolerated in rat. A flow-through dissolution method was more discriminating than the paddle type dissolution equipment for evaluation of co-crystal containing solid formulations.
The whole genome of Mangalica animals has been screened on the Illumina porcine chip giving the possibility (1) to replace the previously applied ten microsatellite markers by nine SNP loci to classify the Blond, Swallow-Belly and Red Mangalica individuals into three different breed groups (P>0.95); (2) to propose 54 SNP loci for parentage testing in Mangalica pigs where the exclusion probability is 0.999115 if one parent is known and the probability of identity is 1.54×10 -23 .
BackgroundWith the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.ResultsOf the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands).ConclusionsFrom the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible.
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