A High Throughput Genotyping Approach Reveals Distinctive Autosomal Genetic Signatures for European and Near Eastern Wild Boar

The lack of a Near Eastern genetic signature in modern European porcine breeds indicates that, although domestic pigs from the Fertile Crescent entered Europe during the Neolithic, they were completely replaced by their European counterparts in a short window of time. Whilst the absence of such genetic signature has been convincingly demonstrated at the mitochondrial level, variation at the autosomal genomes of European and Near Eastern Sus scrofa has not been compared yet. Herewith, we have explored the genetic relationships among 43 wild boar from Europe (N = 21), Near East (N = 19) and Korea (N = 3), and 40 Iberian (N = 16), Canarian (N = 4) and Mangalitza (N = 20) pigs by using a high throughput SNP genotyping platform. After data filtering, 37,167 autosomal SNPs were used to perform population genetics analyses. A multidimensional scaling plot based on genome-wide identity-by-state pairwise distances inferred with PLINK showed that Near Eastern and European wild boar populations are genetically differentiated. Maximum likelihood trees built with TreeMix supported this conclusion i.e. an early population split between Near Eastern and European Sus scrofa was observed. Moreover, analysis of the data with Structure evidenced that the sampled Iberian, Canarian and Mangalitza pigs did not carry any autosomal signature compatible with a Near Eastern ancestry, a finding that agrees well with previous mitochondrial studies.     

Lettuce‐produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination

The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV‐related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium‐mediated transient expression technology. The wild‐type dimer (E1E2) and a variant without an N‐glycosylation site in the E2 polypeptide (E1E2?N6) were expressed, and appropriate N‐glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell‐expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti‐HCV IgM for both antigens; however, the E1E2?N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N‐glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti‐HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low‐cost oral vaccine development.     

MALDI-TOF-MS analysis of sialylated glycans and glycopeptides using 4-chloro-α-cyanocinnamic acid matrix

For MALDI analysis of glycans and glycopeptides, the choice of matrix is crucial in minimizing desialylation by mass spectrometric in-source and metastable decay. Here, we evaluated the potential of 4-chloro-α-cyanocinnamic acid (Cl-CCA) for MALDI-TOF-MS analysis of labile sialylated tryptic N-glycopeptides and released N- and O-glycans. Similar to DHB, but in contrast to CHCA, the Cl-CCA matrix allowed the analysis of sialylated N-glycans and glycopeptides in negative ion mode MALDI-TOF-MS. Dried droplet preparations of Cl-CCA provided microcrystals with a homogeneous spatial distribution and high shot-to-shot repeatability similar to CHCA, which simplified the automatic measurement and improved the resolution and mass accuracy. Interestingly, reflectron-positive ion mode analysis of 1-phenyl-3-methyl-5-pyrazolone (PMP)-labeled O-glycans with Cl-CCA revealed more complete profiles than with DHB and CHCA. In conclusion, we clearly demonstrate the high potential of this rationally designed matrix for glycomics and glycoproteomics.     

Phenotypical changes during in vitro cultivation of monocytes derived immature dendritic cells

Dendritic cells (DC) form a link between the first line of host defence and cellular immunity. In the present study we investigated the effect of cultivation time in generation of immature dendritic cells (iDCs) in vitro from human peripheral blood (PB) monocytes and the influence of iDCs on the oxygen free radicals (OFR) release by polymorphonuclear granulocytes (PMN) stimulated with opsonized zymozan (OZ) and concanavalin A (ConA). Our data suggest that the differentiation in vitro of PB monocytes in iDCs is influenced by the health status of the cell donor, and more by the concentration of cytokines GM-CSF and IL-4 in the system than by the time of cultivation. The in vitro experiments performed demonstrated the interactions between iDC and PMN granulocytes, evaluated by enhanced release of OFR.     

Pancreatic beta cell allotransplant in double transgenic mouse model of type 1 diabetes mellitus

The purpose of this study was to evaluate the therapeutic response to intra-hepatic and intra-portal allotransplant with pancreatic beta cells in double transgenic mice (dTg) with autoimmune diabetes. The results showed an improvement in metabolic and somatic parameters and an increase in survival rate. Histopathology analysis revealed the presence of transplanted islets and the absence of the inflammatory infiltrate 5 days after the procedure and an increase in insulinemia. In the absence of immunosuppressive drugs, rejection of the transplanted islet seems to appear after 10 weeks, being marked by an increase in blood glucose level. A re-transplantation was performed in one mouse of each group and the glycemia levels recorded after the second transplant were less successful.     

Slo1 tail domains,but not the Ca2+ bowl,are required for the beta 1 subunit to increase the apparent Ca2+ sensitivity of BK channels

Functional large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels can be assembled from four alpha subunits (Slo1) alone, or together with four auxiliary beta1 subunits to greatly increase the apparent Ca(2+) sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the alpha subunit, which includes the RCK2 (regulator of K(+) conductance) domain and Ca(2+) bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca(2+) bowl and high affinity Ca(2+) sensitivity. In the second, the Ca(2+) bowl was disrupted by mutations that greatly reduce the apparent Ca(2+) sensitivity. We found that the beta1 subunit increased the apparent Ca(2+) sensitivity of Slo1 channels, independently of whether the alpha subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca(2+) bowl was mutated. In contrast, beta1 subunits no longer increased Ca(2+) sensitivity when Slo1 tails were replaced by Slo3 tails. The beta1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 beta-estradiol activated these channels in the presence of beta1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca(2+) sensitivity induced by the beta1 subunit does not require either the Ca(2+) bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The beta1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the beta1 subunit-induced increase in apparent Ca(2+) sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the beta1 subunit may be different.     

Impact of diet and stress on the development of preeclampsia-like symptoms in p57kip2 mice

The cyclin-dependent kinase inhibitor p57(kip2) regulates the cell cycle of trophoblastic cells. It has been established by a Japanese group that the heterozygous p57(kip2) knockout (p57(-/+)) mice are a good model of preeclampsia as they develop hypertension, proteinuria, and placental pathology. However, apart from the placental pathology, we could not observe these symptoms in our laboratory. Hence, we investigated the impact of diet and stress on this model. To do so, we compared the effects of the Japanese diet to that of the North American diet used by our animal facility. Furthermore, the impact of stress was determined by placing the mice in a restraining device before and at the end of gestation. Although the Japanese diet did not have any impact on blood pressure or proteinuria, the mice did develop endothelial dysfunction, left ventricular hypertrophy, as well as increased placental pathology. Also, all mice had smaller litters when fed the Japanese diet. However, stress response of these mice was not increased during gestation; in fact, a decrease was observed in the p57(-/+) mice, suggesting that this was probably not a player in the development of the pathology. Taken together, these results suggest that other environmental factors may have been implicated in the development of preeclampsia-like symptoms in this animal model. Moreover, we demonstrated that placental pathology and genetic factors are not sufficient to trigger preeclampsia-like symptoms in this model and that the diet might play an important part in the development of this multifactorial disease.     

Geometric enantiomerism in cyclic compounds: Chiral dibrominated 1,3‐dioxanes

The first geometric enantiomers in the cyclic compounds series are reported. The investigated compounds are 2,2‐disubstituted‐5‐methyl‐1,3‐dioxane derivatives in which the substituents at position 2 bear chiral centers with identical substituents but with opposite configurations. The structure of the unlike isomers was determined from the solid state molecular structure of the compounds obtained by single crystal X‐ray diffractometry and the enantiomers of these diastereoisomers were observed by chiral column HPLC base‐line separation. The investigated compounds were obtained by a diastereoselective bromination reaction of the corresponding 2,2‐dialkyl and 2,2‐dibenzyl‐5‐methyl‐1,3‐dioxanes. Chirality, 2011. © 2010 Wiley‐Liss, Inc.