Engineering temporal accumulation of a low recalcitrance polysaccharide leads to increased C6 sugar content in plant cell walls

Reduced cell wall recalcitrance and increased C6 monosaccharide content are desirable traits for future biofuel crops, as long as these biomass modifications do not significantly alter normal growth and development. Mixed‐linkage glucan (MLG), a cell wall polysaccharide only present in grasses and related species among flowering plants, is comprised of glucose monomers linked by both β‐1,3 and β‐1,4 bonds. Previous data have shown that constitutive production of MLG in barley (Hordeum vulgare) severely compromises growth and development. Here, we used spatio‐temporal strategies to engineer Arabidopsis thaliana plants to accumulate significant amounts of MLG in the cell wall by expressing the rice CslF6 MLG synthase using secondary cell wall and senescence‐associated promoters. Results using secondary wall promoters were suboptimal. When the rice MLG synthase was expressed under the control of a senescence‐associated promoter, we obtained up to four times more glucose in the matrix cell wall fraction and up to a 42% increase in saccharification compared to control lines. Importantly, these plants grew and developed normally. The induction of MLG deposition at senescence correlated with an increase of gluconic acid in cell wall extracts of transgenic plants in contrast to the other approaches presented in this study. MLG produced in Arabidopsis has an altered structure compared to the grass glucan, which likely affects its solubility, while its molecular size is unaffected. The induction of cell wall polysaccharide biosynthesis in senescing tissues offers a novel engineering alternative to enhance cell wall properties of lignocellulosic biofuel crops.     

A quantum dot-aptamer beacon using a DNA intercalating dye as the FRET reporter: application to label-free thrombin detection

A new quantum dot (QD)-aptamer (apt) beacon that acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3(B), is demonstrated with label-free thrombin detection. The beacon, denoted as QD-apt:B, is constructed by (1) coupling of a single-stranded thrombin aptamer to Qdot 565 via EDC/Sulfo-NHS chemistry and (2) staining the duplex regions of the aptamer on QD with excess BOBO-3 before thrombin binding. When mixing a thrombin sample with QD-apt:B, BOBO-3 is competed away from the beacon due to target-induced aptamer folding, which then causes a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission and achieves thrombin quantitation. In this work, the effects of Mg(2+), coupling time, and aptamer type on the beacon's performances are investigated and discussed thoroughly with various methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and two-color differential gel electrophoresis. Using the best aptamer beacon (HTQ37), we attain highly specific and wide-range detection (from nM to μM) of thrombin in buffer, and the beacon can sense nM-range thrombin in 15% diluted serum. Compared to the reported QD aptamer assays, our method is advantageous from the aspect of using a simple sensory unit design without losing the detection sensitivity. Therefore, we consider the QD-apt:B beacon a potential alternative to immuno-reagents and an effective tool to study nucleic acid folding on QD as well.     

Enhanced immunostimulatory and antitumor activity of different derivatives of κ-carrageenan oligosaccharides from <Emphasis Type="Italic">Kappaphycus striatum</Emphasis>

Chemical modification of carbohydrates can lead to differences in their biological activities. We previously showed that κ-carrageenan oligosaccharides from Kappaphycus striatum have antitumor and immunomodulation effects on S180-bearing mice. In this study, we tested the hypothesis that different chemical modifications of carrageenan oligosaccharides enhance their activities. The mice inoculated with S180 cell suspension were treated p.o. with carrageenan oligosaccharides and their sulfated, acetylated, and phosphorylated derivatives (50, 100, and 200 μg g⁻¹) for 14 days. Transplantable tumor inhibition rate and macrophage phagocytosis, quantitative hemolysis of sheep red blood cells, lymphocyte proliferation, the activity of natural killer cells, production of interleukin-2, and tumor necrosis factor-α were also analyzed. As expected, treatment with different κ-carrageenan oligosaccharides derivatives resulted in an increase in tumor inhibition rate and macrophage phagocytosis and cellular immunity, especially on spleen lymphocyte proliferation. The sulfated derivative at the dose 200 μg g⁻¹ per day showed the highest antitumor activity with the 54.12% tumor weight inhibition and elicited an increase in nature killer cells activity up to 76.1% on S180-bearing mice, which were both significantly higher than the unmodified oligosaccharides. It suggested that chemical modification (especially sulfation) of carrageenan oligosaccharides can enhance their antitumor effect and boost their antitumor immunity.     

Next generation genome-wide association tool: design and coverage of a high-throughput European-optimized SNP array

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.     

Identification of Mycobacterium tuberculosis-specific Th1, Th17 and Th22 cells using the expression of CD40L in tuberculous pleurisy

Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4(+) T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4(+)CD40L(+) but not CD4(+)CD40L(-) T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4(+)CD40L(+) T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA(-)CD45RO(+)CCR7(-)CD62L(-)ICOS(-)). To determine the specificity of CD4(+)CD40L(+) T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4(+)CD40L(+) and CD4(+)CD40L(-) T cells by flow cytometry. We further demonstrated that sorted CD4(+)CD40L(+), but not CD4(+)CD40L(-) fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4(+) T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB.     

Remodeling of the vascular tunica media is essential for development of collateral vessels in the canine heart

Previous studies have shown that neointima formation and adventitial remodeling play an important role in the enlargement of collateral vessels (CVs) during coronary arteriogenesis in the dog heart. In this study, we investigated the importance of remodeling of the tunica media in the same model. Basal membrane (BM), contractile and cytoskeletal components of smooth muscle cells (SMCs) were studied in growth of coronary CVs induced by chronic occlusion of the left circumflex (LCX) coronary artery by routine histology, electron microscopy (EM), and immunoconfocal microscopy using antibodies against α-smooth actin (α-SM actin), calponin, desmin, and laminin. In addition, matrix metalloproteinase-2 (MMP-2) and tissue inhibitor-1 of matrix metalloproteinase (TIMP-1) were investigated. The data showed that (1) in normal small arteries (NVs) laminin formed a network in which SMCs were encaged;α-SM actin, calponin and desmin were evenly expressed in SMCs; (2) in early (2 weeks) growing CVs the laminin network was disrupted, desmin was significantly reduced in SMCs, but α-SM actin and calponin still highly expressed; (3) in actively (6 weeks) growing CVs laminin was still weak in the tunica media (TM), but without network-like structure. Desmin was further reduced in SMCs of TM, whereas α-SM actin and calponin showed little changes, although they were significantly decreased in intimal SMCs; (4) in mature CVs, the network-like structure was re-formed, and α-SM actin, calponin, and desmin were all similar to that in normal vessels; (5) histology for BM confirmed laminin staining; (6) EM revealed that in NVs the SMCs contained abundant contractile filaments and were surrounded by a layer of BM whereas in growing CVs, BM structure was not observed, but the SMCs in the media still contained many myofilaments; (7) MMP-2 was highly expressed in the media of early growing vessels, but decreased in TM of actively growing vessels where TIMP-1 expression was high. In conclusion, our data revealed features of TM of growing CVs. Disruption and degradation of BM facilitate SMC proliferation, and together with reduction of desmin and fragmentation of the internal elastic lamina enable the vascular wall to expand and enlarge when blood pressure and shear stress increase. MMP2 may be an important player in regulating SMC phenotype, proliferation, migration and maintaining integrity of the vascular wall through governing proteolysis during arteriogenesis. (Mol Cell Biochem 264: 201–210, 2004)     

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmoniers algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.     

Encapsulating live cells with water-soluble chitosan in physiological conditions

A new class of microcapsules was prepared under physiological conditions by polyelectrolyte complexation between two oppositely-charged, water-soluble polymers. The microcapsules consisted of an inner core of half N-acetylated chitosan and an outer shell of methacrylic acid (MAA) (20.4%)-hydroxyethyl methacrylate (HEMA) (27.4%)-methyl methacrylate (MMA) (52.2%) (MAA-HEMA-MMA) terpolymer. Both 400 and 150 kDa half N-acetylated chitosans maintained good water solubility and supplied enough protonated amino groups to coacervate with terpolymer at pH 7.0-7.4, in contrast to other chitosan-based microcapsules which must be prepared at pH <6.5. The viscosity of half N-acetylated chitosan solutions between 80 and 3000 cPas allowed the formation of microcapsules with spherical shape. Molar mass, pH and concentration of half N-acetylated chitosan, and reaction time, influenced the morphology, thickness and porosity of the microcapsules. Microcapsules formed with high concentration of half N-acetylated chitosan exhibited improved mechanical stability, whereas microcapsules formed with low concentration of half N-acetylated chitosan exhibited good permeability. This 3D microenvironment has been configured to cultivate sensitive anchorage-dependent cells such as hepatocytes to maintain high level of functions.     

Yellow pigments used in rapid identification of aflatoxin-producing Aspergillus strains are anthraquinones associated with the aflatoxin biosynthetic pathway

Studies on biological control of aflatoxin production in crops by pre-infection with non-toxigenic Aspergillus flavus strains have created a need for improved methods to screen isolates for aflatoxigenicity. We have evaluated two empirical aflatoxigenicity tests: (i) yellow pigment production, and (ii) the appearance of a plum-red color in colonies exposed to ammonium hydroxide vapor. Yellow pigments from aflatoxigenic A. flavus were shown to function as pH indicator dyes. Seven pigments representing most of the pigmentation in extracts have been isolated using color changes when chromatography spots were exposed to ammonium hydroxide vapor to guide fractionation. Their structures have been shown to be norsolorinic acid, averantin, averufin, versicolorin C, versicolorin A, versicolorin A hemiacetal and nidurufin, all of which are known anthraquinone pigments on, or associated with, the aflatoxin biosynthetic pathway in Aspergillus spp. Thus, the basis of both empirical tests for aflatoxigenicity is detecting production of excess aflatoxin biosynthetic intermediates.