Whole transcriptome profiling of Late-Onset Alzheimer's Disease patients provides insights into the molecular changes involved in the disease.

Alzheimer's Disease (AD) is the most common cause of dementia affecting the elderly population worldwide. We have performed a comprehensive transcriptome profiling of Late-Onset AD (LOAD) patients using second generation sequencing technologies, identifying 2,064 genes, 47 lncRNAs and 4 miRNAs whose expression is specifically deregulated in the hippocampal region of LOAD patients. Moreover, analyzing the hippocampal, temporal and frontal regions from the same LOAD patients, we identify specific sets of deregulated miRNAs for each region, and we confirm that the miR-132/212 cluster is deregulated in each of these regions in LOAD patients, consistent with these miRNAs playing a role in AD pathogenesis. Notably, a luciferase assay indicates that miR-184 is able to target the 3'UTR NR4A2 - which is known to be involved in cognitive functions and long-term memory and whose expression levels are inversely correlated with those of miR-184 in the hippocampus. Finally, RNA editing analysis  reveals a general RNA editing decrease in LOAD hippocampus, with 14 recoding sites significantly and differentially edited in 11 genes. Our data underline specific transcriptional changes in LOAD brain and provide an important source of information for understanding the molecular changes characterizing LOAD progression.

Seasonal rather than spatial variability drives planktonic and benthic bacterial diversity in a microtidal lagoon and the adjacent open sea.

Coastal lagoons are highly productive ecosystems, which are experiencing a variety of human disturbances at increasing frequency. Bacteria are key ecological players within lagoons, yet little is known about the magnitude, patterns and drivers of diversity in these transitional environments. We carried out a seasonal study in the Venice Lagoon (Italy) and the adjacent sea, to simultaneously explore diversity patterns in different domains (pelagic, benthic) and their spatio-temporal variability, and test the role of environmental gradients in structuring assemblages. Community composition differed between lagoon and open sea, and between domains. The dominant phyla varied temporally, with varying trends for the two domains, suggesting different environmental constraints on the assemblages. The percentage of freshwater taxa within the lagoon increased during higher river run-off, pointing at the lagoon as a dynamic mosaic of microbial taxa that generate the metacommunity across the whole hydrological continuum. Seasonality was more important than spatial variability in shaping assemblages. Network analyses indicated more interactions between several genera and environmental variables in the open sea than the lagoon. Our study provides evidences for a temporally dynamic nature of bacterial assemblages in lagoons and suggests that an interplay of seasonally influenced environmental drivers shape assemblages in these vulnerable ecosystems.

Geographic Population Structure in Epstein-Barr Virus Revealed by Comparative Genomics.

Epstein-Barr virus (EBV) latently infects the majority of the human population and is implicated as a causal or contributory factor in numerous diseases. We sequenced 27 complete EBV genomes from a cohort of Multiple Sclerosis (MS) patients and healthy controls from Italy, although no variants showed a statistically significant association with MS. Taking advantage of the availability of ∼130 EBV genomes with known geographical origins, we reveal a striking geographic distribution of EBV sub-populations with distinct allele frequency distributions. We discuss mechanisms that potentially explain these observations, and their implications for understanding the association of EBV with human disease.

The sea urchin mitochondrial transcription factor A binds and bends DNA efficiently despite its unusually short C-terminal tail.

Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM.

Transcriptional Analysis of Acinetobacter sp. neg1 Capable of Degrading Ochratoxin A.

Ochratoxin A (OTA) is a nephrotoxic and potentially carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium, contaminating grapes, wine and a variety of food products. We recently isolated from OTA contaminated soil vineyard a novel free-living strain of Acinetobacter sp. neg1, ITEM 17016, able to degrade OTA into the non-toxic catabolic product ochratoxin α. Biochemical studies suggested that the degradation reaction proceeds via peptide bond hydrolysis with phenylalanine (Phe) release. In order to identify genes responsible for OTA degradation we performed a differential gene expression analysis of ITEM 17016 grown in the presence or absence of the toxin. Among the differentially expressed genes, six peptidases up-regulated at 6 h were identified. The degrading activity of the carboxypeptidase PJ_1540 was confirmed in vitro in a heterologous system. The enrichment analysis for Gene Ontology terms confirmed that OTA degradation proceeds through peptidase activities and revealed the over-representation of pathways related to Phe catabolism. These results indicate that Phe may represent an energy source for this Acinetobacter sp. neg1 strain and that OTA degrading reaction triggers the modulation of further catabolic activities.

Expression and purification of large active GST fusion enzymes.

GST fusion proteins expressed in bacteria often tend to form aggregates and are inefficiently purified by standard procedures, which employ a mixture of detergents that compromise the binding efficiency to the affinity resin and the biological activity of the recombinant proteins. Moreover, the binding to the resin is negatively affected by the molecular weight of the fusion protein. Here we report a simple and efficient method to purify active large GST-tagged proteins, which uses high ionic strength buffer to solubilize the protein aggregates in a bacterial lysate. Affinity-chromatography purification is achieved by adopting two columns connected in series, which facilitate the binding of large GST fused molecules. This approach was applied to purify the 180-kDa GST-tagged mitochondrial RNA polymerase. We also report conditions for simple and efficient GST tag removal from the eluted protein. Finally we demonstrate that the recombinant enzyme is capable to catalyze RNA synthesis.

Characterization of the sea urchin mitochondrial transcription factor A reveals unusual features.

Sea urchin mtDNA is transcribed via a different mechanism compared to vertebrates. To gain information on the apparatus of sea urchin mitochondrial transcription we have characterized the DNA binding properties of the mitochondrial transcription factor A (TFAM). The protein contains two HMG box domains but, differently from vertebrates, displays a very short C-terminal tail. Phylogenetic analysis showed that the distribution of tail length is mixed in the different lineages, indicating that it is a trait that undergoes rapid changes during evolution. Homology modeling suggests that the protein adopts the same configuration of the human counterpart and possibly a similar mode of binding to DNA. DNase I footprinting showed that TFAM specifically contacts mtDNA at a fixed distance from three AT-rich consensus sequences that were supposed to act as transcriptional initiation sites. Bound sequences are homologous and contain an inverted repeat motif, which resembles that involved in the intercalation of human TFAM in LSP DNA. The here reported data indicate that sea urchin TFAM specifically binds mtDNA. The protein could intercalate residues at the DNA inverted motif and, despite its short tail, might have a role in mitochondrial transcription.

A modified method for the purification of active large enzymes using the glutathione S-transferase expression system.

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase.