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.     

Tg2576 cortical neurons that express human Ab are susceptible to extracellular Aβ-induced, K+ efflux dependent neurodegeneration

Background

One of the key pathological features of AD is the formation of insoluble amyloid plaques. The major constituent of these extracellular plaques is the beta-amyloid peptide (Aβ), although Aβ is also found to accumulate intraneuronally in AD. Due to the slowly progressive nature of the disease, it is likely that neurons are exposed to sublethal concentrations of both intracellular and extracellular Aβ for extended periods of time.

Results

In this study, we report that daily exposure to a sublethal concentration of Aβ_1-40 (1 µM) for six days induces substantial apoptosis of cortical neurons cultured from Tg2576 mice (which express substantial but sublethal levels of intracellular Aβ). Notably, untreated Tg2576 neurons of similar age did not display any signs of apoptosis, indicating that the level of intracellular Aβ present in these neurons was not the cause of toxicity. Furthermore, wildtype neurons did not become apoptotic under the same chronic Aβ_1-40 treatment. We found that this apoptosis was linked to Tg2576 neurons being unable to maintain K⁺ homeostasis following Aβ treatment. Furthermore, blocking K⁺ efflux protected Tg2576 neurons from Aβ-induced neurotoxicity. Interestingly, chronic exposure to 1 µM Aβ_1-40 caused the generation of axonal swellings in Tg2576 neurons that contained dense concentrations of hyperphosphorylated tau. These were not observed in wildtype neurons under the same treatment conditions.

Conclusions

Our data suggest that when neurons are chronically exposed to sublethal levels of both intra- and extra-cellular Aβ, this causes a K⁺-dependent neurodegeneration that has pathological characteristics similar to AD.     

Fluoxetine exerts age-dependent effects on behavior and amygdala neuroplasticity in the rat

The selective serotonin reuptake inhibitor (SSRI) Prozac® (fluoxetine) is the only registered antidepressant to treat depression in children and adolescents. Yet, while the safety of SSRIs has been well established in adults, serotonin exerts neurotrophic actions in the developing brain and thereby may have harmful effects in adolescents. Here we treated adolescent and adult rats chronically with fluoxetine (12 mg/kg) at postnatal day (PND) 25 to 46 and from PND 67 to 88, respectively, and tested the animals 7–14 days after the last injection when (nor)fluoxetine in blood plasma had been washed out, as determined by HPLC. Plasma (nor)fluoxetine levels were also measured 5 hrs after the last fluoxetine injection, and matched clinical levels. Adolescent rats displayed increased behavioral despair in the forced swim test, which was not seen in adult fluoxetine treated rats. In addition, beneficial effects of fluoxetine on wakefulness as measured by electroencephalography in adults was not seen in adolescent rats, and age-dependent effects on the acoustic startle response and prepulse inhibition were observed. On the other hand, adolescent rats showed resilience to the anorexic effects of fluoxetine. Exploratory behavior in the open field test was not affected by fluoxetine treatment, but anxiety levels in the elevated plus maze test were increased in both adolescent and adult fluoxetine treated rats. Finally, in the amygdala, but not the dorsal raphe nucleus and medial prefrontal cortex, the number of PSA-NCAM (marker for synaptic remodeling) immunoreactive neurons was increased in adolescent rats, and decreased in adult rats, as a consequence of chronic fluoxetine treatment. No fluoxetine-induced changes in 5-HT_1A receptor immunoreactivity were observed. In conclusion, we show that fluoxetine exerts both harmful and beneficial age-dependent effects on depressive behavior, body weight and wakefulness, which may relate, in part, to differential fluoxetine-induced neuroplasticity in the amygdala.     

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.     

Tetraspanin CD151 Mediates Papillomavirus Type 16 Endocytosis

Human papillomavirus type 16 (HPV16) is the primary etiologic agent for cervical cancer. The infectious entry of HPV16 into cells occurs via a so-far poorly characterized clathrin- and caveolin-independent endocytic pathway, which involves tetraspanin proteins and actin. In this study, we investigated the specific role of the tetraspanin CD151 in the early steps of HPV16 infection. We show that surface-bound HPV16 moves together with CD151 within the plane of the membrane before they cointernalize into endosomes. Depletion of endogenous CD151 did not affect binding of viral particles to cells but resulted in reduction of HPV16 endocytosis. HPV16 uptake is dependent on the C-terminal cytoplasmic region of CD151 but does not require its tyrosine-based sorting motif. Reexpression of the wild-type CD151 but not mutants affecting integrin functions restored virus internalization in CD151-depleted cells. Accordingly, short interfering RNA (siRNA) gene knockdown experiments confirmed that CD151-associated integrins (i.e., α3β1 and α6β1/4) are involved in HPV16 infection. Furthermore, palmitoylation-deficient CD151 did not support HPV16 cell entry. These data show that complex formation of CD151 with laminin-binding integrins and integration of the complex into tetraspanin-enriched microdomains are critical for HPV16 endocytosis.     

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.     

Diversity of Culturable Bacteria,from the Anaerobic Zone of the Meromictic Lake Pavin,Able to Perform Dissimilatory-Iron Reduction in Different in Vitro Conditions

A culture-dependent study was performed with the aim of assessing the carbon, electron and Fe(III) sources used for the dissimilatory Fe(III) reduction pathway and the diversity of culturable Fe(III)-reducers in the anoxic zone of the meromictic Lake Pavin. This metabolic pathway was investigated in enrichment cultures inoculated with water samples collected at 70 m depth in the anoxic zone of Lake Pavin. Combinations of different media, organic acids, and incubation gas phases were performed. The potential for Fe(III) reduction in the different growth conditions was assessed by measuring the accumulation of Fe(II) overtime. Bacterial community structure was determined in each growth conditions by Temporal Temperature gradient Gel Electrophoresis (TTGE) profiles of 16S rDNA genes and bands of interest in positive enrichments were sequenced. Comparisons of bacterial community structure between growth conditions revealed that the electron donor, the basal media as well as the Fe(III) source yielded to the selection of different bacterial populations, suggesting that Fe(III) reducers occupy different ecological niches in the anoxic zone of Lake Pavin. Facultative Fe(III) reducers, such as fermentative (e.g., Pseudomonas, Clostridium) and sulphate-reducing (e.g., Desulfovibrio sp.) bacteria, were retrieved in enrichments but well-known obligatory Fe(III) reducers (e.g., Geobacter) were not detected. A greater Fe(III) reduction was noted under H₂:CO₂ gas phase, suggesting that H₂ is used as an electron donor for Fe(III) reduction. Acetate was not used as a precursor for this terminal electron-accepting process, and a high Fe(III) reduction was observed with fumarate provided as the electron donor and carbon sources suggesting that this metabolite may be energetically more beneficial for Fe(III)-reducers.