Syk is downstream of intercellular adhesion molecule-1 and mediates human rhinovirus activation of p38 MAPK in airway epithelial cells

The airway epithelium is the primary target of inhaled pathogens such as human rhinovirus (HRV). Airway epithelial cells express ICAM-1, the major receptor for HRV. HRV binding to ICAM-1 mediates not only viral entry and replication but also a signaling cascade that leads to enhanced inflammatory mediator production. The specific signaling molecules and pathways activated by HRV-ICAM-1 interactions are not well characterized, although studies in human airway epithelia implicate a role for the p38 MAPK in HRV-induced cytokine production. In the current study, we report that Syk, an important immunoregulatory protein tyrosine kinase, is highly expressed by primary and cultured human airway epithelial cells and is activated in response to infection with HRV16. Biochemical studies revealed that ICAM-1 engagement by HRV and cross-linking Abs enhanced the coassociation of Syk with ICAM-1 and ezrin, a cytoskeletal linker protein. In polarized airway epithelial cells, Syk is diffusely distributed in the cytosol under basal conditions but, following engagement of ICAM-1 by cross-linking Abs, is recruited to the plasma membrane. The enhanced Syk-ICAM-1 association following HRV exposure is accompanied by Syk phosphorylation. ICAM-1 engagement by HRV and cross-linking Abs also induced phosphorylation of p38 in a Syk-dependent manner, and conversely, knockdown of Syk by short interfering (si)RNA substantially diminished p38 activation and IL-8 gene expression. Taken together, these observations identify Syk as an important mediator of the airway epithelial cell inflammatory response by modulating p38 phosphorylation and IL-8 gene expression following ICAM-1 engagement by HRV.     

Multiple traits of agronomic importance in transgenic indica rice plants: analysis of transgene integration patterns,expression levels and stability

We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) -endotoxin genes cry1Ac and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R₀ and R₁ plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.     

Accurate detection of subclonal single nucleotide variants in whole genome amplified and pooled cancer samples using HaloPlex target enrichment

Background

Target enrichment and resequencing is a widely used approach for identification of cancer genes and genetic variants associated with diseases. Although cost effective compared to whole genome sequencing, analysis of many samples constitutes a significant cost, which could be reduced by pooling samples before capture. Another limitation to the number of cancer samples that can be analyzed is often the amount of available tumor DNA. We evaluated the performance of whole genome amplified DNA and the power to detect subclonal somatic single nucleotide variants in non-indexed pools of cancer samples using the HaloPlex technology for target enrichment and next generation sequencing.

Results

We captured a set of 1528 putative somatic single nucleotide variants and germline SNPs, which were identified by whole genome sequencing, with the HaloPlex technology and sequenced to a depth of 792–1752. We found that the allele fractions of the analyzed variants are well preserved during whole genome amplification and that capture specificity or variant calling is not affected. We detected a large majority of the known single nucleotide variants present uniquely in one sample with allele fractions as low as 0.1 in non-indexed pools of up to ten samples. We also identified and experimentally validated six novel variants in the samples included in the pools.

Conclusion

Our work demonstrates that whole genome amplified DNA can be used for target enrichment equally well as genomic DNA and that accurate variant detection is possible in non-indexed pools of cancer samples. These findings show that analysis of a large number of samples is feasible at low cost, even when only small amounts of DNA is available, and thereby significantly increases the chances of indentifying recurrent mutations in cancer samples.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-856) contains supplementary material, which is available to authorized users.     

Thermodynamic parameters for salt‐induced reversible protein precipitation from automated microscale experiments

Reversible precipitation can be used as an efficient purification tool for proteins. In addition, identifying conditions under which precipitation or aggregation occurs is of key importance in the bioprocessing and pharmaceutical industry, as this can aid in better formulations and hinder aggregation in chromatography. We have evaluated the precipitation of proteins as determined by light scattering in microplates as a tool for the high‐throughput determination of thermodynamic parameters for protein precipitation, with the potential for screening of formulation additives and relevant bioprocess conditions such as pH. This provides a useful complementary technique to existing microplate‐based protein thermostability measurements. Using hen egg‐white lysozyme and alcohol dehydrogenase as model proteins we have determined the extent of reversible precipitation as a function of ammonium sulfate and sodium chloride concentrations, and also demonstrated global fitting of the data to generate a model where the fraction precipitated can be predicted for any given condition. The global fit provided thermodynamic parameters, including the free energy for protein precipitation, and also allowed an approximate determination of the average size of the structural nucleus that contributes to the free energy of precipitation for each protein. The rapid collection of thermodynamic parameters for protein precipitation, in parallel with protein thermostability measurements, will provide a powerful platform for protein formulation, and also lead to datasets useful for testing theoretical predictions of reversible precipitation based on the molecular modeling of specific protein structure interactions. Biotechnol. Bioeng. 2011;108: 322–332. © 2010 Wiley Periodicals, Inc.     

A micropropagation protocol for <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl. from root explants

The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl., an important medicinal legume using root explant as starting material. Root explants taken from 30-day-old aseptic seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators: 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Organogenic nodular calli obtained on MS + TDZ (1.0 μM) were transferred to shoot regeneration medium supplemented with different cytokinins (BA, Kn or TDZ) either alone or in combination with auxin:indole-3-acetic acid or α-naphthalene acetic acid. Maximum shoot regeneration frequency (90%) was obtained on MS + BA (2.5 μM) + NAA (0.6 μM) wherein a maximum of 42.76 ± 1.47 shoot buds per explant were induced with a maximum conversion rate of 35.63 ± 0.75 shoots per explant and average shoot length of 5.43 ± 0.20 cm. Elongated microshoots were successfully rooted under ex vitro conditions by pulse treatment in 200 μM of indole-3-butyric acid for half an hour. Microshoots were rooted, acclimatized and hardened off simultaneously in sterilized soilrite inside the growth room and then established in pots containing sterilized soil and manure (1:1) and grown under greenhouse condition with 90% survival rate. The histological sections at different developmental stages of shoot buds revealed the organization of nodular meristematic zone leading to the orientation and differentiation of shoot buds in large number and thereafter conversion into healthy shoots.     

Siansivirga zeaxanthinifaciens gen. nov., sp. nov., a novel zeaxanthin-producing member of the family Flavobacteriaceae isolated from coastal seawater of Taiwan

A strictly aerobic, Gram-negative, rod-shaped bacterium (strain CC-SAMT-1(T)) showing gliding motility was isolated from coastal seawater of China Sea, Taiwan. Strain CC-SAMT-1(T) synthesizes all-trans-zeaxanthin (6.5 ± 0.5 mg g(-1) dry biomass) as a predominant xanthophyll carotenoid. As determined by 16S rRNA gene analysis, strain CC-SAMT-1(T) shared very high sequence similarity to the members of the genera Mariniflexile (96.1-95.3%) and Gaetbulibacter (96.0-95.9%); however, it formed a distinct phyletic lineage distantly associated with Mariniflexile species. Polar lipid profile constitutes phosphatidylethanolamine, four unidentified aminolipids, four unidentified lipids, and an unidentified glycolipid. Strain CC-SAMT-1(T) contains excessive unidentified aminolipid lipid (AL2-4) and glycolipid contents, and therefore clearly distinct from Mariniflexile species. Major fatty acids (> 5% of total fatty acids) were iso-C(15:0) (14.8%), iso-C(17:0) 3-OH (11.8%), iso-C(15:1) G (10.6%), anteiso-C(15:0) (9.7%), C(16:0) (8.1%), iso-C(16:0) 3-OH (7.9%), iso-C(15:0) 3-OH (7.5%), and summed feature 3 (containing C(16:1) ω6c and/or C(16:1) ω7c) (7.5%). Menaquinone-6 (MK-6) was major respiratory quinone. DNA G+C content was 33.7 mol%. Based on polyphasic taxonomy, strain CC-SAMT-1(T) represents a novel genus and species in the family Flavobacteriaceae for which the name Siansivirga zeaxanthinifaciens gen. nov., sp. nov. is proposed. The type strain is CC-SAMT-1(T) (= BCRC 80315(T) = JCM 17682(T)).     

Biotechnology: Genetic improvement of sorghum (Sorghum bicolor (L.) Moench)

Summary This report reviews the contributions to the improvement of sorghum (Sorghum bicolor (L.) Moench) through traditional approaches with emphasis on the application of biotechnological methods. Strategies include breeding for higher yield, improved grain quality, and biotic and abiotic stress tolerance. Hybrid development and polyploidy breeding are also discussed. Plant breeders, working in concert with biotechnologists, have developed new powerful tools for plant genetic manipulation and genotype evaluation that will significantly improve the efficiency of plant breeding. Improving sorghum through biotechnology is the latest in a long series of technologies that have been applied to this crop. Five basic tools of technology have been developed for sorghum improvement: (1) in vitro protocols for efficient plant regeneration; (2) molecular markers; (3) gene identification and cloning; (4) genetic engineering and gene transfer technology to integrate desirable traits into the sorghum genome; and (5) genomics and germplasm databases. Reports on studies involving the problems, progress, and prospects for utilizing the biotechnological methods for sorghum improvement are discussed.