Neuronal Exosomal miRNA-dependent Translational Regulation of Astroglial Glutamate Transporter GLT1

Perisynaptic astrocytes express important glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.     

Loss-of-function mutations in RAB18 cause Warburg micro syndrome

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.     

Histidine 416 of the periplasmic binding protein NikA is essential for nickel uptake in Escherichia coli

Escherichia coli require nickel for the synthesis of [NiFe] hydrogenases under anaerobic growth conditions. Nickel import depends on the specific ABC-transporter NikABCDE encoded by the nik operon, which deletion causes the complete abolition of hydrogenase activity. We have previously postulated that the periplasmic binding protein NikA binds a natural metallophore containing three carboxylate functions that coordinate a Ni(II) ion, the fourth ligand being His416, the only direct metal-protein contact, completing a square-planar coordination for the metal. The crystal structure of the H416I mutant showed no electron density corresponding to a metal-chelator complex. In vivo experiments indicate that the mutation causes a significant decrease in nickel uptake and hydrogenase activity. These results confirm the essential role of His416 in nickel transport by NikA.     

Variations in spatial and temporal distribution of Archaea in the North Sea in relation to environmental variables

The spatial and temporal distribution of pelagic Archaea was studied in the southern North Sea by rRNA hybridization, sequencing and quantification of 16S rRNA gene and membrane lipid analyses and related to physical, chemical and biological parameters to determine the factors influencing archaeal biogeography. A clear temporal variability was observed, with marine Crenarchaeota (Group I.1a) being relatively more abundant in winter and Euryarchaeota dominating the archaeal assemblage in spring and summer. Spatial differences in the lateral distribution of Crenarchaeota were also evident. In fact, their abundance was positively correlated with the copy number of the gene encoding the alpha subunit of crenarchaeotal ammonia monooxygenase (amoA) and with concentrations of ammonia, nitrate, nitrite and phosphorus. This suggests that most Crenarchaeota in the North Sea are nitrifiers and that their distribution is determined by nutrient concentrations. However, Crenarchaeota were not abundant when larger phytoplankton (>3 microm) dominated the algal population. It is hypothesized that together with nutrient concentration, phytoplankton biomass and community structure can predict crenarchaeotal abundance in the southern North Sea. Euryarchaeotal abundance was positively correlated with chlorophyll a concentrations, but not with phytoplankton community structure. Whether this is related to the potential of Euryarchaeota to perform aerobic anoxygenic phototrophy remains to be shown, but the conspicuous seasonal distribution pattern of Crenarchaeota and Euryarchaeota suggests that they occupy a different ecological niche.     

Mobilisation of nutrients and transport via the xylem sap in a shrub (Ligustrum ovalifolium) during spring growth: N and C compounds and interactions

In open-field soilless culture there can be great deal of leaching, particularly in rainy springs. Ligneous plants have the capacity to store large quantities of nutrients in perennial organs. Knowledge of the plant's internal nutrient mobilisation during spring to supply growing organs could lead to reduction of fertiliser application. To quantify the fraction of storage mobilisation available for growth of new organs during spring, Ligustrum ovalifolium shrubs were grown for 2 years with or without fertilisation in the second spring. Nitrogen (N) absorption and N and carbon (C) mobilisation from storage were followed during spring growth via the sap quality. A mathematical combination of the sap composition with flow velocity provided the transported quantities of N and C. Nitrogen and C mobilisation towards new shoots took place during all the spring growth from bud break onwards. In unfertilised plants, C was mobilised primarily as sugars (stachyose, mannose and sucrose) and starch. In fertilised plants, the same sugars were transported in the xylem sap, but at lower concentrations. Stachyose concentration was lower in fertilised than in unfertilised plants and decreased during spring growth. Nitrogen was transported in the xylem sap mainly as amino acids in both fertilisation treatments. Glutamine was the predominant form at bud break and during shoot elongation. In fertilised plants, arginine became predominant after shoot elongation, and was related to low C availability. The interactions of N with C are discussed; specifically, insufficient availability of N limits the use of C, more of which is directed to aerial organs by sap flow.     

Applications of a microplate reader in yeast physiology research

Microplate readers have been useful assistants of researchers for several decades. This work is focused on the applications of a simple absorbance microplate reader in yeast physiology research, and its advantages and limitations in comparison with alternative methods are discussed. The two main procedures involved are measuring growth curves and monitoring the pH changes of medium using two different pH indicators. We suggest mathematical formulas for converting absorbance data into pH values. With a microplate reader as many as 96 samples can be simultaneously analyzed, while medium consumption is minimized to 100 microL per sample. The results can be observed in 24-48 h (for growth curves) or in 1-3 h (for pH changes) with minimal hands-on time required.     

Association of Shifting Populations in the Root Zone Microbiome of Millet with Enhanced Crop Productivity in the Sahel Region (Africa)

This study characterized specific changes in the millet root zone microbiome stimulated by long-term woody-shrub intercropping at different sites in Senegal. At the two study sites, intercropping with woody shrubs and shrub residue resulted in a significant increase in millet [Pennisetum glaucum (L.) R. Br.] yield (P < 0.05) and associated patterns of increased diversity in both bacterial and fungal communities in the root zone of the crop. Across four experiments, operational taxonomic units (OTUs) belonging to Chitinophaga were consistently significantly (P < 0.001) enriched in the intercropped samples, and “Candidatus Koribacter” was consistently significantly enriched in samples where millet was grown alone. Those OTUs belonging to Chitinophaga were enriched more than 30-fold in residue-amended samples and formed a distinct subgroup from all OTUs detected in the genus. Additionally, OTUs belonging to 8 fungal genera (Aspergillus, Coniella, Epicoccum, Fusarium, Gibberella, Lasiodiplodia, Penicillium, and Phoma) were significantly (P < 0.005) enriched in all experiments at all sites in intercropped samples. The OTUs of four genera (Epicoccum, Fusarium, Gibberella, and Haematonectria) were consistently enriched at sites where millet was grown alone. Those enriched OTUs in intercropped samples showed consistently large-magnitude differences, ranging from 30- to 1,000-fold increases in abundance. Consistently enriched OTUs in intercropped samples in the genera Aspergillus, Fusarium, and Penicillium also formed phylogenetically distinct subgroups. These results suggest that the intercropping system used here can influence the recruitment of potentially beneficial microorganisms to the root zone of millet and aid subsistence farmers in producing higher-yielding crops.     

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis.