Sativanine-a and sativanine-b,two new cyclopeptide alkaloids from the bark of Zizyphus sativa

From the bark of Zizyphus sativa, in addition to already described cyclopeptide alkaloids, two new compounds of this class, sativanine-A(1) and sativanine-B(2), were isolated. Both alkaloids contain 14-membered ring systems. 1 belongs to the integerrine type, while 2 is similar to nummularine-G, with an additional ring in the side chain.     

MgSlt2, a cellular integrity MAP kinase gene of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth

Among expressed sequence tag libraries of Mycosphaerella graminicola isolate IPO323, we identified a full-length cDNA clone with high homology to the mitogen-activated protein (MAP) kinase Slt2 in Saccharomyces cerevisiae. This MAP kinase consists of a 1242-bp open reading frame, and encodes a 414-amino-acid protein. We designated this homolog MgSlt2, generated MgSlt2 knockout strains in M. graminicola isolate IPO323, and found several altered phenotypes in vitro as well as in planta. In yeast glucose broth, MgSlt2 disruptants showed a defective polarized growth in the tip cells upon aging, causing substantial local enlargements culminating in large swollen cells containing two to four nuclei. The MgSlt2 disruptants showed a significantly increased sensitivity to several fungicides, including miconazole (2x), bifonazole (>4x), imazalil (5x), and cyproconazole (10x), and were hypersensitive to glucanase. Unlike the wild type, MgSlt2 disruptants did not produce aerial mycelia and did not melanize on potato dextrose agar. Although cytological analysis in planta showed normal penetration of wheat stomata by the germ tubes of the MgSlt2 disruptants, subsequently formed hyphal filaments frequently were unable to branch out and establish invasive growth resulting in highly reduced virulence, and prevented pycnidia formation. Therefore, we conclude that MgSlt2 is a new pathogenicity factor in M. graminicola.     

Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen

In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90 degrees C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear. To examine in more detail the structural and thermodynamic aspects of the binding mechanism, an extensive CD, ITC, and NMR study was initiated. In this study the interaction between the llama VHH -R2 fragment and its antigen, the dye Reactive Red-6 (RR6) has been explored. The data show clearly that most of the VHH-R2 population at 80 degrees C is in an unfolded conformation. In contrast, CD spectra representing the complex between VHH-R2 and the dye remained the same up to 80 degrees C. Interestingly, addition of the dye to the denatured VHH-R2 at 80 degrees C yielded the spectrum of the native complex. These results suggest an induced refolding of denatured VHH-R2 by its antigen under these extreme conditions. This induced refolding showed some similarities with the well established "induced fit" mechanism of antibody-antigen interactions at ambient temperature. However, the main difference with the "induced fit" mechanism is that at the start of the addition of the antigen most of the VHH molecules are in an unfolded conformation. The refolding capability under these extreme conditions and the stable complex formation make VHHs useful in a wide variety of applications.     

Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.     

Genomic relationships based on X chromosome markers and accuracy of genomic predictions with and without X chromosome markers

Background

Although the X chromosome is the second largest bovine chromosome, markers on the X chromosome are not used for genomic prediction in some countries and populations. In this study, we presented a method for computing genomic relationships using X chromosome markers, investigated the accuracy of imputation from a low density (7K) to the 54K SNP (single nucleotide polymorphism) panel, and compared the accuracy of genomic prediction with and without using X chromosome markers.

Methods

The impact of considering X chromosome markers on prediction accuracy was assessed using data from Nordic Holstein bulls and different sets of SNPs: (a) the 54K SNPs for reference and test animals, (b) SNPs imputed from the 7K to the 54K SNP panel for test animals, (c) SNPs imputed from the 7K to the 54K panel for half of the reference animals, and (d) the 7K SNP panel for all animals. Beagle and Findhap were used for imputation. GBLUP (genomic best linear unbiased prediction) models with or without X chromosome markers and with or without a residual polygenic effect were used to predict genomic breeding values for 15 traits.

Results

Averaged over the two imputation datasets, correlation coefficients between imputed and true genotypes for autosomal markers, pseudo-autosomal markers, and X-specific markers were 0.971, 0.831 and 0.935 when using Findhap, and 0.983, 0.856 and 0.937 when using Beagle. Estimated reliabilities of genomic predictions based on the imputed datasets using Findhap or Beagle were very close to those using the real 54K data. Genomic prediction using all markers gave slightly higher reliabilities than predictions without X chromosome markers. Based on our data which included only bulls, using a G matrix that accounted for sex-linked relationships did not improve prediction, compared with a G matrix that did not account for sex-linked relationships. A model that included a polygenic effect did not recover the loss of prediction accuracy from exclusion of X chromosome markers.

Conclusions

The results from this study suggest that markers on the X chromosome contribute to accuracy of genomic predictions and should be used for routine genomic evaluation.     

Alginate Microspheres Containing Temperature Sensitive Liposomes (TSL) for MR-Guided Embolization and Triggered Release of Doxorubicin

Objective

The objective of this study was to develop and characterize alginate microspheres suitable for embolization with on-demand triggered doxorubicin (DOX) release and whereby the microspheres as well as the drug releasing process can be visualized in vivo using MRI.

Methods and Findings

For this purpose, barium crosslinked alginate microspheres were loaded with temperature sensitive liposomes (TSL/TSL-Ba-ms), which release their payload upon mild hyperthermia. These TSL contained DOX and [Gd(HPDO3A)(H₂O)], a T₁ MRI contrast agent, for real time visualization of the release. Empty alginate microspheres crosslinked with holmium ions (T₂* MRI contrast agent, Ho-ms) were mixed with TSL-Ba-ms to allow microsphere visualization. TSL-Ba-ms and Ho-ms were prepared with a homemade spray device and sized by sieving. Encapsulation of TSL in barium crosslinked microspheres changed the triggered release properties only slightly: 95% of the loaded DOX was released from free TSL vs. 86% release for TSL-Ba-ms within 30 seconds in 50% FBS at 42°C. TSL-Ba-ms (76 ± 41 μm) and Ho-ms (64 ± 29 μm) had a comparable size, which most likely will result in a similar in vivo tissue distribution after an i.v. co-injection and therefore Ho-ms can be used as tracer for the TSL-Ba-ms. MR imaging of a TSL-Ba-ms and Ho-ms mixture (ratio 95:5) before and after hyperthermia allowed in vitro and in vivo visualization of microsphere deposition (T₂*-weighted images) as well as temperature-triggered release (T₁-weighted images). The [Gd(HPDO3A)(H₂O)] release and clusters of microspheres containing holmium ions were visualized in a VX₂ tumor model in a rabbit using MRI.

Conclusions

In conclusion, these TSL-Ba-ms and Ho-ms are promising systems for real-time, MR-guided embolization and triggered release of drugs in vivo.