Proteomic analysis of extracellular matrices used in stem cell culture

Numerous matrices for the growth of human embryonic stem cells (hESC) in vitro have been described. However, their exact composition is typically unknown. Information on the components of these matrices will aid in the development of a fully defined growth surface for hESCs. These matrices typically consist of mixture of proteins present in a wide range of abundance making their characterization challenging. In this study, we performed the proteomic analysis of five previously uncharacterized matrices: CellStart, Human Basement Membrane Extract (Human BME), StemXVivo, Bridge Human Extracellular Matrix (BridgeECM), and mouse embryonic fibroblast conditioned matrix (MEF-CMTX). Based on a proteomics protocol optimized using lysates from HeLa cells, we undertook the analysis of the five complex extracellular matrix (ECM) samples using a combination of strong anion and cation exchange chromatography and SDS-PAGE. For each of these matrices, we identify numerous proteins, indicating their complex nature. We also compared these results with a similar proteomics analysis of the growth matrix, Matrigel?. From these analyses, we observed that fibronectin is a primary component of nearly all hESC supportive matrices. This observation led to the investigation of the suitability of fibronectin as a defined ECM for the growth of hESCs. We found that fibronectin promotes the maintenance of pluripotent H9 and CA1 hESCs in an undifferentiated state using mTeSR1 medium. This finding validates the utility of characterizing matrices used for hESC growth in revealing ECM components required for culturing hESCs in a universally applicable defined system.     

Marine biodiversity characteristics

Oceans contain the largest living volume of the “blue” planet, inhabited by approximately 235–250,000 described species, all groups included. They only represent some 13% of the known species on the Earth, but the marine biomasses are really huge. Marine phytoplankton alone represents half the production of organic matter on Earth while marine bacteria represent more than 10%. Life first appeared in the oceans more than 3.8 billion years ago and several determining events took place that changed the course of life, ranging from the development of the cell nucleus to sexual reproduction going through multi-cellular organisms and the capture of organelles. Of the 31 animal phyla currently listed, 12 are exclusively marine phyla and have never left the ocean. An interesting question is to try to understand why there are so few marine species versus land species? This pattern of distribution seems pretty recent in the course of Evolution. From an exclusively marine world, since the beginning until 440 million years ago, land number of species much increased 110 million years ago. Specific diversity and ancestral roles, in addition to organizational models and original behaviors, have made marine organisms excellent reservoirs for identifying and extracting molecules (> 15,000 today) with pharmacological potential. They also make particularly relevant models for both fundamental and applied research. Some marine models have been the source of essential discoveries in life sciences. From this diversity, the ocean provides humankind with renewable resources, which are highly threatened today and need more adequate management to preserve ocean habitats, stocks and biodiversity.     

Worldwide genotyping in the planktonic foraminifer Globoconella inflata: implications for life history and paleoceanography

The planktonic foraminiferal morpho-species Globoconella inflata is widely used as a stratigraphic and paleoceanographic index. While G. inflata was until now regarded as a single species, we show that it rather constitutes a complex of two pseudo-cryptic species. Our study is based on SSU and ITS rDNA sequence analyses and genotyping of 497 individuals collected at 49 oceanic stations covering the worldwide range of the morpho-species. Phylogenetic analyses unveil the presence of two divergent genotypes. Type I inhabits transitional and subtropical waters of both hemispheres, while Type II is restricted to the Antarctic subpolar waters. The two genetic species exhibit a strictly allopatric distribution on each side of the Antarctic Subpolar Front. On the other hand, sediment data show that G. inflata was restricted to transitional and subtropical environments since the early Pliocene, and expanded its geographic range to southern subpolar waters ～700 kyrs ago, during marine isotopic stage 17. This datum may correspond to a peripatric speciation event that led to the partition of an ancestral genotype into two distinct evolutionary units. Biometric measurements performed on individual G. inflata from plankton tows north and south of the Antarctic Subpolar Front indicate that Types I and II display slight but significant differences in shell morphology. These morphological differences may allow recognition of the G. inflata pseudo-cryptic species back into the fossil record, which in turn may contribute to monitor past movements of the Antarctic Subpolar Front during the middle and late Pleistocene.     

Selection for earlier flowering crop associated with climatic variations in the Sahel

Climate changes will have an impact on food production and will require costly adaptive responses. Adapting to a changing environment will be particularly challenging in sub-Saharan Africa where climate change is expected to have a major impact. However, one important phenomenon that is often overlooked and is poorly documented is the ability of agro-systems to rapidly adapt to environmental variations. Such an adaptation could proceed by the adoption of new varieties or by the adaptation of varieties to a changing environment. In this study, we analyzed these two processes in one of the driest agro-ecosystems in Africa, the Sahel. We performed a detailed study in Niger where pearl millet is the main crop and covers 65% of the cultivated area. To assess how the agro-system is responding to recent recurrent drought, we analyzed samples of pearl millet landraces collected in the same villages in 1976 and 2003 throughout the entire cultivated area of Niger. We studied phenological and morphological differences in the 1976 and 2003 collections by comparing them over three cropping seasons in a common garden experiment. We found no major changes in the main cultivated varieties or in their genetic diversity. However, we observed a significant shift in adaptive traits. Compared to the 1976 samples, samples collected in 2003 displayed a shorter lifecycle, and a reduction in plant and spike size. We also found that an early flowering allele at the PHYC locus increased in frequency between 1976 and 2003. The increase exceeded the effect of drift and sampling, suggesting a direct effect of selection for earliness on this gene. We conclude that recurrent drought can lead to selection for earlier flowering in a major Sahelian crop. Surprisingly, these results suggest that diffusion of crop varieties is not the main driver of short term adaptation to climatic variation.     

A novel SERRS sandwich-hybridization assay to detect specific DNA target

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.     

γ-Hemolysin oligomeric structure and effect of its formation on supported lipid bilayers: An AFM Investigation

γ-Hemolysins are bicomponent β-barrel pore forming toxins produced by Staphylococcus aureus as water-soluble monomers, which assemble into oligomeric pores on the surface of lipid bilayers. Here, after investigating the oligomeric structure of γ-hemolysins on supported lipid bilayers (SLBs) by atomic force microscopy (AFM), we studied the effect produced by this toxin on the structure of SLBs. We found that oligomeric structures with different number of monomers can assemble on the lipid bilayer being the octameric form the stablest one. Moreover, in this membrane model we found that γ-hemolysins can form clusters of oligomers inducing a curvature in the lipid bilayer, which could probably enhance the aggressiveness of these toxins at high concentrations.     

Functional Modeling Identifies Paralogous Solanesyl-diphosphate Synthases That Assemble the Side Chain of Plastoquinone-9 in Plastids

It is a little known fact that plastoquinone-9, a vital redox cofactor of photosynthesis, doubles as a precursor for the biosynthesis of a vitamin E analog called plastochromanol-8, the physiological significance of which has remained elusive. Gene network reconstruction, GFP fusion experiments, and targeted metabolite profiling of insertion mutants indicated that Arabidopsis possesses two paralogous solanesyl-diphosphate synthases, AtSPS1 (At1g78510) and AtSPS2 (At1g17050), that assemble the side chain of plastoquinone-9 in plastids. Similar paralogous pairs were detected throughout terrestrial plant lineages but were not distinguished in the literature and genomic databases from mitochondrial homologs involved in the biosynthesis of ubiquinone. The leaves of the atsps2 knock-out were devoid of plastochromanol-8 and displayed severe losses of both non-photoactive and photoactive plastoquinone-9, resulting in near complete photoinhibition at high light intensity. Such a photoinhibition was paralleled by significant damage to photosystem II but not to photosystem I. In contrast, in the atsps1 knock-out, a small loss of plastoquinone-9, restricted to the non-photoactive pool, was sufficient to eliminate half of the plastochromanol-8 content of the leaves. Taken together, these results demonstrate that plastochromanol-8 originates from a subfraction of the non-photoactive pool of plastoquinone-9. In contrast to other plastochromanol-8 biosynthetic mutants, neither the single atsps knock-outs nor the atsps1 atsps2 double knock-out displayed any defects in tocopherols accumulation or germination.     

The Green Microalga Chlamydomonas reinhardtii Has a Single ω-3 Fatty Acid Desaturase That Localizes to the Chloroplast and Impacts Both Plastidic and Extraplastidic Membrane Lipids

The ω-3 polyunsaturated fatty acids account for more than 50% of total fatty acids in the green microalga Chlamydomonas reinhardtii, where they are present in both plastidic and extraplastidic membranes. In an effort to elucidate the lipid desaturation pathways in this model alga, a mutant with more than 65% reduction in total ω-3 fatty acids was isolated by screening an insertional mutant library using gas chromatography-based analysis of total fatty acids of cell pellets. Molecular genetics analyses revealed the insertion of a TOC1 transposon 113 bp upstream of the ATG start codon of a putative ω-3 desaturase (CrFAD7; locus Cre01.g038600). Nuclear genetic complementation of crfad7 using genomic DNA containing CrFAD7 restored the wild-type fatty acid profile. Under standard growth conditions, the mutant is indistinguishable from the wild type except for the fatty acid difference, but when exposed to short-term heat stress, its photosynthesis activity is more thermotolerant than the wild type. A comparative lipidomic analysis of the crfad7 mutant and the wild type revealed reductions in all ω-3 fatty acid-containing plastidic and extraplastidic glycerolipid molecular species. CrFAD7 was localized to the plastid by immunofluorescence in situ hybridization. Transformation of the crfad7 plastidial genome with a codon-optimized CrFAD7 restored the ω-3 fatty acid content of both plastidic and extraplastidic lipids. These results show that CrFAD7 is the only ω-3 fatty acid desaturase expressed in C. reinhardtii, and we discuss possible mechanisms of how a plastid-located desaturase may impact the ω-3 fatty acid content of extraplastidic lipids.Research on lipid metabolism in microalgae has flourished in recent years due to their potential as a rich source of ω-3 fatty acids (Guschina and Harwood, 2006; Khozin-Goldberg et al., 2011) and as a feedstock for biodiesel (Hu et al., 2008b; Rosenberg et al., 2008; Beer et al., 2009; Radakovits et al., 2010; Wijffels and Barbosa, 2010; Merchant et al., 2012; Work et al., 2012). Oils produced by microalgae resemble that of plants (Hu et al., 2008b), with the exception that they contain higher proportions of polyunsaturated fatty acid (PUFA) species (Harwood and Guschina, 2009). Desaturation of acyl groups in glycerolipids is catalyzed by fatty acid desaturases (FADs), which insert a C=C bond at a specifically defined position of an acyl chain (Shanklin and Cahoon, 1998). The degree of unsaturation of fatty acid components largely determines the chemical property and thus the utility of the oils produced. FADs have been one of the major tools for the genetic engineering of oil composition in land crops (Shanklin and Cahoon, 1998; Napier et al., 1999). In view of biodiesel applications, low PUFA content is advantageous in algal oil because of oxidation issues (Frankel, 1991).With the suites of sophisticated molecular genetic and genomic tools developed in the green microalga Chlamydomonas reinhardtii and the existence of substantial literature related to its cell biology, physiology, and biochemistry, this organism has emerged as a major model for research on algal oil (Radakovits et al., 2010; Merchant et al., 2012; Liu and Benning, 2013). Although the understanding of lipid metabolism in C. reinhardtii largely relies on sequence homologies to other models (Riekhof et al., 2005) and is still rather limited compared with the model plant Arabidopsis (Arabidopsis thaliana; Li-Beisson et al., 2010), functional studies based on mutants have started to provide important insights into the biosynthesis and turnover of membrane and storage lipids in this model alga (Riekhof et al., 2005; Work et al., 2010; Fan et al., 2011; Goodson et al., 2011; Boyle et al., 2012; Li et al., 2012a, 2012b; Yoon et al., 2012).In C. reinhardtii, C16 and C18 PUFAs (ω-3 + ω-6) make up to 60 mol% of total membrane fatty acids, of which more than 80% are ω-3 species (Giroud and Eichenberger, 1988; Siaut et al., 2011). Biochemical evidence for lipid-linked desaturation of fatty acyl chains has been established in C. reinhardtii over 20 years (Giroud and Eichenberger, 1989), but only two C. reinhardtii mutants affected in fatty acid desaturation have been described to date. These are crfad6 (hf-9), an insertional mutant for the plastidial ω-6 desaturase FAD6 (Sato et al., 1995), and microRNA-based silenced lines for the Δ4 desaturase CrΔ4FAD (Zäuner et al., 2012). The putative microsomal Δ12 desaturase FAD2 (Chi et al., 2008) and front-end ω-13 desaturase (Kajikawa et al., 2006) have been characterized by heterologous expression in the methylotrophic yeast Pichia pastoris, but no mutant is available. Moreover, although ω-3 PUFA is the most abundant fatty acid class in C. reinhardtii, the ω-3 desaturase remains uncharacterized, and no mutant with specific reduction in ω-3 content has been isolated so far.In Arabidopsis and C. reinhardtii, ω-3 PUFAs are present in both plastidic and extraplastidic lipids such as monogalactosyldiacylglycerol (MGDG) and phosphatidylethanolamine (PtdEtn), respectively (Mendiola-Morgenthaler et al., 1985; Giroud et al., 1988). While in plants there are distinct genes for plastidial and extraplastidial ω-3 FADs (Wallis and Browse, 2002), only one putative ω-3 desaturase seems encoded in the C. reinhardtii genome (version 5.0; Merchant et al., 2007). This raises several intriguing possibilities, including the existence of a mechanism to export ω-3 acyls from their site of biogenesis to other membranes or a dual localization of the ω-3 desaturase homolog (plastid and endoplasmic reticulum [ER]). In this study, we report the identification and characterization of a C. reinhardtii mutant defective in the promoter region of the putative ω-3 FAD encoded by the Cre01.g038600 locus. We show that while this enzyme is localized to plastids, impairment in its expression leads to a reduction of ω-3 fatty acids acylated to both plastidial and ER lipids. Additionally, using plastidial transformation of the mutant, it is demonstrated that the location of this desaturase in the plastid alone is sufficient to ensure normal ω-3 fatty acid content in extraplastidic lipids. Possible acyl desaturation and trafficking mechanisms implied by these findings are discussed.