Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome

The serial analysis of gene expression (SAGE) method is used to study global gene expression in cells or tissues in various experimental conditions. However, its reproducibility has not yet been definitively assessed. In this study, we have evaluated the reproducibility of the SAGE method and identified the factors that affect it. The determination coefficient (R(2)) for the reproducibility of SAGE is 0.96. However, there are some factors that can affect the reproducibility of SAGE, such as the replication of concatemers and ditags, the number of sequenced tags and double PCR amplification of ditags. Thus, corrections for these factors must be made to ensure the reproducibility and accuracy of SAGE results. A bioinformatic analysis of SAGE data is also presented in order to eliminate these artifacts. Finally, the current study shows that increasing the number of sequenced tags improves the power of the method to detect transcripts and their regulation by experimental conditions

Host-linked soil viral ecology along a permafrost thaw gradient

Climate change threatens to release abundant carbon that is sequestered at high latitudes, but the constraints on microbial metabolisms that mediate the release of methane and carbon dioxide are poorly understood1,2,3,4,5,6,7. The role of viruses, which are known to affect microbial dynamics, metabolism and biogeochemistry in the oceans8,9,10, remains largely unexplored in soil. Here, we aimed to investigate how viruses influence microbial ecology and carbon metabolism in peatland soils along a permafrost thaw gradient in Sweden. We recovered 1,907 viral populations (genomes and large genome fragments) from 197 bulk soil and size-fractionated metagenomes, 58% of which were detected in metatranscriptomes and presumed to be active. In silico predictions linked 35% of the viruses to microbial host populations, highlighting likely viral predators of key carbon-cycling microorganisms, including methanogens and methanotrophs. Lineage-specific virus/host ratios varied, suggesting that viral infection dynamics may differentially impact microbial responses to a changing climate. Virus-encoded glycoside hydrolases, including an endomannanase with confirmed functional activity, indicated that viruses influence complex carbon degradation and that viral abundances were significant predictors of methane dynamics. These findings suggest that viruses may impact ecosystem function in climate-critical, terrestrial habitats and identify multiple potential viral contributions to soil carbon cycling

efam: an expanded, metaproteome-supported HMM profile database of viral protein families

Motivation: Viruses infect, reprogram and kill microbes, leading to profound ecosystem consequences, from elemental cycling in oceans and soils to microbiome-modulated diseases in plants and animals. Although metagenomic datasets are increasingly available, identifying viruses in them is challenging due to poor representation and annotation of viral sequences in databases. Results: Here, we establish efam, an expanded collection of Hidden Markov Model (HMM) profiles that represent viral protein families conservatively identified from the Global Ocean Virome 2.0 dataset. This resulted in 240 311 HMM profiles, each with at least 2 protein sequences, making efam >7-fold larger than the next largest, panecosystem viral HMM profile database. Adjusting the criteria for viral contig confidence from 'conservative' to 'eXtremely Conservative' resulted in 37 841 HMM profiles in our efam-XC database. To assess the value of this resource, we integrated efam-XC into VirSorter viral discovery software to discover viruses from less-studied, ecologically distinct oxygen minimum zone (OMZ) marine habitats. This expanded database led to an increase in viruses recovered from every tested OMZ virome by similar to 24% on average (up to similar to 42%) and especially improved the recovery of often-missed shorter contigs (<5 kb). Additionally, to help elucidate lesser-known viral protein functions, we annotated the profiles using multiple databases from the DRAM pipeline and virion-associated metaproteomic data, which doubled the number of annotations obtainable by standard, single-database annotation approaches. Together, these marine resources (efam and efam-XC) are provided as searchable, compressed HMM databases that will be updated bi-annually to help maximize viral sequence discovery and study from any ecosystem

Structural basis of nSH2 regulation and lipid binding in PI3Kα

We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP2, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP2 molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant

PDE-4 inhibition rescues aberrant synaptic plasticity in Drosophila and mouse models of fragile X syndrome.

Fragile X syndrome (FXS) is the leading cause of both intellectual disability and autism resulting from a single gene mutation. Previously, we characterized cognitive impairments and brain structural defects in a Drosophila model of FXS and demonstrated that these impairments were rescued by treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium. A well-documented biochemical defect observed in fly and mouse FXS models and FXS patients is low cAMP levels. cAMP levels can be regulated by mGluR signaling. Herein, we demonstrate PDE-4 inhibition as a therapeutic strategy to ameliorate memory impairments and brain structural defects in the Drosophila model of fragile X. Furthermore, we examine the effects of PDE-4 inhibition by pharmacologic treatment in the fragile X mouse model. We demonstrate that acute inhibition of PDE-4 by pharmacologic treatment in hippocampal slices rescues the enhanced mGluR-dependent LTD phenotype observed in FXS mice. Additionally, we find that chronic treatment of FXS model mice, in adulthood, also restores the level of mGluR-dependent LTD to that observed in wild-type animals. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of FXS is an important advance, in that this identifies and validates PDE-4 inhibition as potential therapeutic intervention for the treatment of individuals afflicted with FXS

Bax Inhibitor-1 down-regulation in the progression of chronic liver diseases

<p>Abstract</p> <p>Background</p> <p>Bax inhibitor-1 (BI-1) is an evolutionary conserved endoplasmic reticulum protein that, when overexpressed in mammalian cells, suppresses the apoptosis induced by Bax, a pro-apoptotic member of the Bcl-2 family. The aims of this study were: (1) to clarify the role of intrinsic anti- and pro-apoptotic mediators, evaluating Bax and BI-1 mRNA and protein expressions in liver tissues from patients with different degrees of liver damage; (2) to determine whether HCV and HBV infections modulate said expression.</p> <p>Methods</p> <p>We examined 62 patients: 39 with chronic hepatitis (CH) (31 HCV-related and 8 HBV-related); 7 with cirrhosis (6 HCV-related and 1 HBV-related); 13 with hepatocellular carcinoma (HCC) [7 in viral cirrhosis (6 HCV- and 1 HBV-related), 6 in non-viral cirrhosis]; and 3 controls. Bax and BI-1 mRNAs were quantified by real-time PCR, and BI-1 protein expression by Western blot.</p> <p>Results</p> <p>CH tissues expressed significantly higher BI-1 mRNA levels than cirrhotic tissues surrounding HCC (P < 0.0001) or HCC (P < 0.0001). Significantly higher Bax transcripts were observed in HCV-genotype-1-related than in HCV-genotype-3-related CH (P = 0.033). A positive correlation emerged between BI-1 and Bax transcripts in CH tissues, even when HCV-related CH and HCV-genotype-1-related CH were considered alone (P = 0.0007, P = 0.0005 and P = 0.0017, respectively).</p> <p>Conclusions</p> <p>BI-1 expression is down-regulated as liver damage progresses. The high BI-1 mRNAs levels observed in early liver disease may protect virus-infected cells against apoptosis, while their progressive downregulation may facilitate hepatocellular carcinogenesis. HCV genotype seems to have a relevant role in Bax transcript expression.</p

Positively Selected Codons in Immune-Exposed Loops of the Vaccine Candidate OMP-P1 of Haemophilus influenzae

The high levels of variation in surface epitopes can be considered as an evolutionary hallmark of immune selection. New computational tools enable analysis of this variation by identifying codons that exhibit high rates of amino acid changes relative to the synonymous substitution rate. In the outer membrane protein P1 of Haemophilus influenzae, a vaccine candidate for nontypeable strains, we identified four codons with this attribute in domains that did not correspond to known or assumed B- and T-cell epitopes of OMP-P1. These codons flank hypervariable domains and do not appear to be false positives as judged from parsimony and maximum likelihood analyses. Some closely spaced positively selected codons have been previously considered part of a transmembrane domain, which would render this region unsuited for inclusion in a vaccine. Secondary structure analysis, three-dimensional structural database searches, and homology modeling using FadL of E. coli as a structural homologue, however, revealed that all positively selected codons are located in or near extracellular looping domains. The spacing and level of diversity of these positively selected and exposed codons in OMP-P1 suggest that vaccine targets based on these and conserved flanking residues may provide broad coverage in H. influenzae

Scanning Angle Plasmon Waveguide Resonance Raman Spectroscopy for the Analysis of Thin Polystyrene Films

Scanning angle (SA) Raman spectroscopy was used to characterize thin polymer films at a sapphire/50 nm gold film/polystyrene/air interface. When the polymer thickness is greater than ∼260 nm, this interface behaves as a plasmon waveguide; Raman scatter is greatly enhanced with both p- and s-polarized excitation compared to an interface without the gold film. In this study, the reflected light intensities from the interface and Raman spectra were collected as a function of incident angle for three samples with different polystyrene thicknesses. The Raman peak areas were well modeled with the calculated mean-square electric field (MSEF) integrated over the polymer film at varying incident angles. A 412 nm polystyrene plasmon waveguide generated 3.34× the Raman signal at 40.52° (the plasmon waveguide resonance angle) compared to the signal measured at 70.4° (the surface plasmon resonance angle). None of the studied polystyrene plasmon waveguides produced detectable Raman scatter using a 180° backscatter collection geometry, demonstrating the sensitivity of the SA Raman technique. The data highlight the ability to measure polymer thickness, chemical content, and, when combined with calculations of MSEF as a function of distance from the interface, details of polymer structure and order. The SA Raman spectroscopy thickness measurements agreed with those obtained from optical interferometery with an average difference of 2.6%. This technique has the potential to impact the rapidly developing technologies utilizing metal/polymer films for energy storage and electronic devices