A collagen network phase improves cell seeding of open-pore structure scaffolds under perfusion

Scaffolds with open-pore morphologies offer several advantages in cell-based tissue engineering, but their use is limited by a low cell seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open-pore architecture of polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds increases human bone marrow stromal cells (hBMSC) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL-TCP scaffolds, rapid prototyped with a honeycomb-like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen-free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSC directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSC under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5-fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell-based therapies, collagen-filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion-based bioreactor devices to streamline graft manufacture.

Molecular Structure of the mineral woodhouseite CaAl3(PO4,SO4)2(OH)6

The mineral woodhouseite CaAl3(PO4,SO4)2(OH)6 is a hydroxy phosphate-sulphate mineral belonging to the beudantite subgroup of alunites, and has been characterised by Raman spectroscopy, complimented with infrared spectroscopy. Bands at various wavenumbers were assigned to the different vibrational modes of woodhouseite, which were then associated to the molecular structure of the mineral. Bands were primarily assigned to phosphate and sulphate stretching and bending modes. Two symmetric stretching modes for both phosphate and sulphate supported the concept of non-equivalent phosphate and sulphate units in the mineral structure. Bands in the OH stretching region enabled hydrogen bond distances to be calculated.

Prediction of cis/trans isomerization in proteins using PSI-BLAST profiles and secondary structure information

Background The majority of peptide bonds in proteins are found to occur in the trans conformation. However, for proline residues, a considerable fraction of Prolyl peptide bonds adopt the cis form. Proline cis/trans isomerization is known to play a critical role in protein folding, splicing, cell signaling and transmembrane active transport. Accurate prediction of proline cis/trans isomerization in proteins would have many important applications towards the understanding of protein structure and function. Results In this paper, we propose a new approach to predict the proline cis/trans isomerization in proteins using support vector machine (SVM). The preliminary results indicated that using Radial Basis Function (RBF) kernels could lead to better prediction performance than that of polynomial and linear kernel functions. We used single sequence information of different local window sizes, amino acid compositions of different local sequences, multiple sequence alignment obtained from PSI-BLAST and the secondary structure information predicted by PSIPRED. We explored these different sequence encoding schemes in order to investigate their effects on the prediction performance. The training and testing of this approach was performed on a newly enlarged dataset of 2424 non-homologous proteins determined by X-Ray diffraction method using 5-fold cross-validation. Selecting the window size 11 provided the best performance for determining the proline cis/trans isomerization based on the single amino acid sequence. It was found that using multiple sequence alignments in the form of PSI-BLAST profiles could significantly improve the prediction performance, the prediction accuracy increased from 62.8% with single sequence to 69.8% and Matthews Correlation Coefficient (MCC) improved from 0.26 with single local sequence to 0.40. Furthermore, if coupled with the predicted secondary structure information by PSIPRED, our method yielded a prediction accuracy of 71.5% and MCC of 0.43, 9% and 0.17 higher than the accuracy achieved based on the singe sequence information, respectively. Conclusion A new method has been developed to predict the proline cis/trans isomerization in proteins based on support vector machine, which used the single amino acid sequence with different local window sizes, the amino acid compositions of local sequence flanking centered proline residues, the position-specific scoring matrices (PSSMs) extracted by PSI-BLAST and the predicted secondary structures generated by PSIPRED. The successful application of SVM approach in this study reinforced that SVM is a powerful tool in predicting proline cis/trans isomerization in proteins and biological sequence analysis.

Domestication to crop improvement: Genetic resources for sorghum and saccharum (Andropogoneae)

Background Both sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum) are members of the Andropogoneae tribe in the Poaceae and are each other's closest relatives amongst cultivated plants. Both are relatively recent domesticates and comparatively little of the genetic potential of these taxa and their wild relatives has been captured by breeding programmes to date. This review assesses the genetic gains made by plant breeders since domestication and the progress in the characterization of genetic resources and their utilization in crop improvement for these two related species. Genetic Resources The genome of sorghum has recently been sequenced providing a great boost to our knowledge of the evolution of grass genomes and the wealth of diversity within S. bicolor taxa. Molecular analysis of the Sorghum genus has identified close relatives of S. bicolor with novel traits, endosperm structure and composition that may be used to expand the cultivated gene pool. Mutant populations (including TILLING populations) provide a useful addition to genetic resources for this species. Sugarcane is a complex polyploid with a large and variable number of copies of each gene. The wild relatives of sugarcane represent a reservoir of genetic diversity for use in sugarcane improvement. Techniques for quantitative molecular analysis of gene or allele copy number in this genetically complex crop have been developed. SNP discovery and mapping in sugarcane has been advanced by the development of high-throughput techniques for ecoTILLING in sugarcane. Genetic linkage maps of the sugarcane genome are being improved for use in breeding selection. The improvement of both sorghum and sugarcane will be accelerated by the incorporation of more diverse germplasm into the domesticated gene pools using molecular tools and the improved knowledge of these genomes.

Massively parallel sequencing and analysis of expressed sequence tags in a successful invasive plant

Background Invasive species pose a significant threat to global economies, agriculture and biodiversity. Despite progress towards understanding the ecological factors associated with plant invasions, limited genomic resources have made it difficult to elucidate the evolutionary and genetic factors responsible for invasiveness. This study presents the first expressed sequence tag (EST) collection for Senecio madagascariensis, a globally invasive plant species. Methods We used pyrosequencing of one normalized and two subtractive libraries, derived from one native and one invasive population, to generate an EST collection. ESTs were assembled into contigs, annotated by BLAST comparison with the NCBI non-redundant protein database and assigned gene ontology (GO) terms from the Plant GO Slim ontologies. Key Results Assembly of the 221 746 sequence reads resulted in 12 442 contigs. Over 50 % (6183) of 12 442 contigs showed significant homology to proteins in the NCBI database, representing approx. 4800 independent transcripts. The molecular transducer GO term was significantly over-represented in the native (South African) subtractive library compared with the invasive (Australian) library. Based on NCBI BLAST hits and literature searches, 40 % of the molecular transducer genes identified in the South African subtractive library are likely to be involved in response to biotic stimuli, such as fungal, bacterial and viral pathogens. Conclusions This EST collection is the first representation of the S. madagascariensis transcriptome and provides an important resource for the discovery of candidate genes associated with plant invasiveness. The over-representation of molecular transducer genes associated with defence responses in the native subtractive library provides preliminary support for aspects of the enemy release and evolution of increased competitive ability hypotheses in this successful invasive. This study highlights the contribution of next-generation sequencing to better understanding the molecular mechanisms underlying ecological hypotheses that are important in successful plant invasions.