Use of RAD sequencing for delimiting species

RAD-tag sequencing is a promising method for conducting genome-wide evolutionary studies. However, to date, only a handful of studies empirically tested its applicability above the species level. In this communication, we use RAD tags to contribute to the delimitation of species within a diverse genus of deep-sea octocorals, Chrysogorgia, for which few classical genetic markers have proved informative. Previous studies have hypothesized that single mitochondrial haplotypes can be used to delimit Chrysogorgia species. On the basis of two lanes of Illumina sequencing, we inferred phylogenetic relationships among 12 putative species that were delimited using mitochondrial data, comparing two RAD analysis pipelines (Stacks and PyRAD). The number of homologous RAD loci decreased dramatically with increasing divergence, as >70% of loci are lost when comparing specimens separated by two mutations on the 700-nt long mitochondrial phylogeny. Species delimitation hypotheses based on the mitochondrial mtMutS gene are largely supported, as six out of nine putative species represented by more than one colony were recovered as discrete, well-supported clades. Significant genetic structure (correlating with geography) was detected within one putative species, suggesting that individuals characterized by the same mtMutS haplotype may belong to distinct species. Conversely, three mtMutS haplotypes formed one well-supported clade within which no population structure was detected, also suggesting that intraspecific variation exists at mtMutS in Chrysogorgia. Despite an impressive decrease in the number of homologous loci across clades, RAD data helped us to fine-tune our interpretations of classical mitochondrial markers used in octocoral species delimitation, and discover previously undetected diversity.     

Characterization of photo-cross-linked oligo[poly(ethylene glycol) fumarate] hydrogels for cartilage tissue engineering

Photo-cross-linkable oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels have been developed for use in tissue engineering applications. We demonstrated that compressive modulus of these hydrogels increased with increasing polymer concentration, and hydrogels with different mechanical properties were formed by altering the ratio of cross-linker/polymer in precursor solution. Conversely, swelling of hydrogels decreased with increasing polymer concentration and cross-linker/polymer ratio. These hydrogels are degradable and degradation rates vary with the change in cross-linking level. Chondrocyte attachment was quantified as a method for evaluating adhesion of cells to the hydrogels. These data revealed that cross-linking density affects cell behavior on the hydrogel surfaces. Cell attachment was greater on the samples with increased cross-linking density. Chondrocytes on these samples exhibited spread morphology with distinct actin stress fibers, whereas they maintained their rounded morphology on the samples with lower cross-linking density. Moreover, chondrocytes were photoencapsulated within various hydrogel networks. Our results revealed that cells encapsulated within 2-mm thick OPF hydrogel disks remained viable throughout the 3-week culture period, with no difference in viability across the thickness of hydrogels. Photoencapsulated chondrocytes expressed the mRNA of type II collagen and produced cartilaginous matrix within the hydrogel constructs after three weeks. These findings suggest that photo-cross-linkable OPF hydrogels may be useful for cartilage tissue engineering and cell delivery applications.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

3D Culture Histology Cryosectioned Well Insert Technology Preserves the Structural Relationship between Cells and Biomaterials for Time‐Lapse Analysis of 3D Cultures

When performing histology of softer biomaterials, aspiration disrupts the cellular and molecular location information. This study aims to develop a cryosectionable well insert able to preserve the biomaterial and cell's original 3D conformation from the well to histology analysis. The well insert is composed of a paraffin‐coated gelatine pill. Within the coated capsule, the human epithelial cell line (NS‐SV‐AC) is cultured in Matrigel, GrowDex, Myogel, Myogel + GrowDex, or cell culture media for 14 days. At 0 and 14 days, the samples are frozen in liquid nitrogen and cryotome is used to create sections. The slides are stained by Sirius Red and immunohistochemistry using antibodies human collagens I–V and human Ki‐67. Sirius Red shows pink shades of biomaterials and the best cellular vertical distribution throughout the sagittal section of the well is achieved with Matrigel, GrowDex, and Myogel + GrowDex; in Myogel and media, the cells sink. For collagen protein expression, only Matrigel induces a notable difference while in the other materials, collagen staining is weak or difficult to distinguish from endogenous collagens. Ki‐67 expression is maintained over time. The 3D‐cryo well insert provides a new time‐lapse histology perspective of analysis for liquid or gel cultures that maintains cells and macromolecules in their unaltered in‐well configuration.     

Biological activities of Schottenol and Spinasterol,two natural phytosterols present in argan oil and in cactus pear seed oil,on murine miroglial BV2 cells

The objective of this study was to evaluate the biological activities of the major phytosterols present in argan oil (AO) and in cactus seed oil (CSO) in BV2 microglial cells. Accordingly, we first determined the sterol composition of AO and CSO, showing the presence of Schottenol and Spinasterol as major sterols in AO. While in CSO, in addition to these two sterols, we found mainly another sterol, the Sitosterol. The chemical synthesis of Schottenol and Spinasterol was performed. Our results showed that these two phytosterols, as well as sterol extracts from AO or CSO, are not toxic to microglial BV2 cells. However, treatments by these phytosterols impact the mitochondrial membrane potential. Furthermore, both Schottenol and Spinasterol can modulate the gene expression of two nuclear receptors, liver X receptor (LXR)-α and LXRβ, their target genes ABCA1 and ABCG1. Nonetheless, only Schottenol exhibited a differential activation vis-à-vis the nuclear receptor LXRβ. Thus Schottenol and Spinasterol can be considered as new LXR agonists, which may play protective roles by the modulation of cholesterol metabolism.     

Molecular modelling,synthesis and acetylcholinesterase inhibition of ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8]naphthyridine-3-carboxylate

In silico analysis of ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8]naphthyridine-3-carboxylate (2) predicts that this molecule should be successfully docked in the PAS, and easily accommodated in the CAS of AChE. The synthesis and the AChE/BuChE inhibition studies are reported, confirming that compound 2 is a potent and selective AChE inhibitor, and consequently, a new lead compound for further development into new dual CAS/PAS cholinergic agents for the treatment of Alzheimer’s disease.