Comparative transcriptome profiling of amyloid precursor protein family members in the adult cortex

Background

SOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem cells. SOX2 appears to re-activate in several human cancers including glioblastoma multiforme (GBM), however, the detailed response program of SOX2 in GBM has not yet been defined.

Results

We show that knockdown of the SOX2 gene in LN229 GBM cells reduces cell proliferation and colony formation. We then comprehensively characterize the SOX2 response program by an integrated analysis using several advanced genomic technologies including ChIP-seq, microarray profiling, and microRNA sequencing. Using ChIP-seq technology, we identified 4883 SOX2 binding regions in the GBM cancer genome. SOX2 binding regions contain the consensus sequence wwTGnwTw that occurred 3931 instances in 2312 SOX2 binding regions. Microarray analysis identified 489 genes whose expression altered in response to SOX2 knockdown. Interesting findings include that SOX2 regulates the expression of SOX family proteins SOX1 and SOX18, and that SOX2 down regulates BEX1 (brain expressed X-linked 1) and BEX2 (brain expressed X-linked 2), two genes with tumor suppressor activity in GBM. Using next generation sequencing, we identified 105 precursor microRNAs (corresponding to 95 mature miRNAs) regulated by SOX2, including down regulation of miR-143, -145, -253-5p and miR-452. We also show that miR-145 and SOX2 form a double negative feedback loop in GBM cells, potentially creating a bistable system in GBM cells.

Conclusions

We present an integrated dataset of ChIP-seq, expression microarrays and microRNA sequencing representing the SOX2 response program in LN229 GBM cells. The insights gained from our integrated analysis further our understanding of the potential actions of SOX2 in carcinogenesis and serves as a useful resource for the research community.     

The antiviral efficacy of HIV-specific CD8⁺ T-cells to a conserved epitope is heavily dependent on the infecting HIV-1 isolate

A major challenge to developing a successful HIV vaccine is the vast diversity of viral sequences, yet it is generally assumed that an epitope conserved between different strains will be recognised by responding T-cells. We examined whether an invariant HLA-B8 restricted Nef_90–97 epitope FL8 shared between five high titre viruses and eight recombinant vaccinia viruses expressing Nef from different viral isolates (clades A–H) could activate antiviral activity in FL8-specific cytotoxic T-lymphocytes (CTL). Surprisingly, despite epitope conservation, we found that CTL antiviral efficacy is dependent on the infecting viral isolate. Only 23% of Nef proteins, expressed by HIV-1 isolates or as recombinant vaccinia-Nef, were optimally recognised by CTL. Recognition of the HIV-1 isolates by CTL was independent of clade-grouping but correlated with virus-specific polymorphisms in the epitope flanking region, which altered immunoproteasomal cleavage resulting in enhanced or impaired epitope generation. The finding that the majority of virus isolates failed to present this conserved epitope highlights the importance of viral variance in CTL epitope flanking regions on the efficiency of antigen processing, which has been considerably underestimated previously. This has important implications for future vaccine design strategies since efficient presentation of conserved viral epitopes is necessary to promote enhanced anti-viral immune responses.     

Using an adherent cell culture of the mouse subependymal zone to study the behavior of adult neural stem cells on a single-cell level

A comprehensive understanding of the cell biology of adult neural stem cells (aNSCs) requires direct observation of aNSC division and lineage progression in the absence of niche-dependent signals. Here we describe a culture preparation of the adult mouse subependymal zone (SEZ), which allows for continuous single-cell tracking of aNSC behavior. The protocol involves the isolation (approximately 3 h) and culture of cells from the adult SEZ at low density in the absence of mitogenic growth factors in chemically defined medium and subsequent live imaging using time-lapse video microscopy (5-7 d); these steps are followed by postimaging immunocytochemistry to identify progeny (approximately 7 h). This protocol enables the observation of the progression from slow-dividing aNSCs of radial/astroglial identity up to the neuroblast stage, involving asymmetric and symmetric cell divisions of distinct fast-dividing precursors. This culture provides an experimental system for studying instructive or permissive effects of signal molecules on aNSC modes of cell division and lineage progression.     

A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae

We have discovered a ubiquitin (Ub)-specific cysteine protease encoded within the N-terminal approximately 500 residues of the UL36 gene product, the largest (3164 aa) tegument protein of herpes simplex virus 1 (HSV-1). Enzymatic activity of this fragment, UL36USP, is detectable only after cleavage of UL36USP from full-length UL36 and occurs late during viral replication. UL36USP bears no homology to known deubiquitinating enzymes (DUBs) or Ub binding proteins. Sequence alignment of the large tegument proteins across the family Herpesviridae indicates conservation of key catalytic residues amongst these viruses. Recombinant UL36USP exhibits hydrolytic activity toward Ub-AMC and ubiquitinated branched peptides in vitro. In addition, recombinant UL36USP can cleave polyUb chains and appears to be specific for Lys48 linkages. Mutation of the active site cysteine residue (Cys65) to alanine abolishes this enzymatic activity. The lack of homology between UL36USP and eukaryotic DUBs makes this new family of herpesvirus ubiquitin-specific proteases attractive targets for selective inhibition.     

Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast

The MDM31 and MDM32 genes are required for normal distribution and morphology of mitochondria in the yeast Saccharomyces cerevisiae. They encode two related proteins located in distinct protein complexes in the mitochondrial inner membrane. Cells lacking Mdm31 and Mdm32 harbor giant spherical mitochondria with highly aberrant internal structure. Mitochondrial DNA (mtDNA) is instable in the mutants, mtDNA nucleoids are disorganized, and their association with Mmm1-containing complexes in the outer membrane is abolished. Mutant mitochondria are largely immotile, resulting in a mitochondrial inheritance defect. Deletion of either one of the MDM31 and MDM32 genes is synthetically lethal with deletion of either one of the MMM1, MMM2, MDM10, and MDM12 genes, which encode outer membrane proteins involved in mitochondrial morphogenesis and mtDNA inheritance. We propose that Mdm31 and Mdm32 cooperate with Mmm1, Mmm2, Mdm10, and Mdm12 in maintenance of mitochondrial morphology and mtDNA.     

Why are rare traits unilaterally expressed?: Trait frequency and unilateral expression for cranial nonmetric traits in humans

Based on an analysis of nonmetric trait databases from several large skeletal series in Northern Europe and South America, representing 27 bilateral traits, we report a predictable relationship between the frequency of nonmetric traits and the probability that they are expressed bilaterally. In a wider sampling of traits and populations, this study thus confirms the findings of an earlier study by Ossenberg ([1981] Am. J. Phys. Anthropol. 54:471-479), which reported the same relationship for two mandibular traits. This trend was previously explained by extending the multifactorial threshold model for discontinuous traits to incorporate either separate thresholds for unilateral or bilateral expression, or by a fuzzy threshold in which the probability of bilateral expression increases away from the median threshold value. We show that the trend is produced under the standard multifactorial threshold model for discontinuous traits simply if the within-individual or developmental instability variance remains relatively constant across the range of liability. Under this assumption, the number of individuals in which one side but not the other is pushed over the threshold for trait formation will be a larger proportion of the number of individuals expressing the trait when the trait frequency is low. As trait frequency increases, the significance of within-individual variance as a determinant of trait formation decreases relative to the genetic and among-individual environmental variance. These results have implications for interpreting nonmetric trait data as well as for understanding the prevalence of unilateral vs. bilateral expression of a wide variety of discontinuous traits, including dysmorphologies in humans.     

Using stable isotopes to investigate migratory connectivity of the globally threatened aquatic warbler Acrocephalus paludicola

Understanding the links between breeding and wintering areas of migratory species has important ecological and conservation implications. Recently, stable isotope technology has been used to further our understanding. Stable isotope ratios vary geographically with a range of biogeochemical factors and isotope profiles in organisms reflect those in their food and environment. For inert tissues like feathers, isotope profiles reflect the environment in which they were formed. Following large-scale habitat destruction, the globally threatened aquatic warbler Acrocephalus paludicola has a fragmented breeding population across central Europe, largely in Belarus, Poland and Ukraine. The species sub-Saharan African wintering grounds have not yet been discovered, and this significantly hampers conservation efforts. Aquatic warblers grow their flight feathers on their wintering grounds, and we analysed stable isotope ratios (¹⁵N, ¹³C, D) in rectrices of adults from six main breeding sites (subpopulations) across Europe to determine whether different breeding subpopulations formed a single mixed population on the wintering grounds. ¹⁵N varies considerably with dietary trophic level and environmental factors, and D with the D in rainfall; neither varied between aquatic warbler subpopulations. Uniform feather ¹⁵N signatures suggest no major variation in dietary trophic level during feather formation. High variance and inter-annual differences in mean D values hinder interpretation of these data. Significant differences in mean ¹³C ratios existed between subpopulations. We discuss possible interpretations of this result, and consider differences in moulting latitude of different subpopulations to be the most parsimonious. ¹³C in plants and animals decreases with latitude, along a steep gradient in sub-Saharan Africa. Birds from the most north-westerly breeding subpopulation (Karsibor, Poland) had significantly lower variance in ¹³C and ¹⁵N than birds from all other sites, suggesting either that birds from Karsibor are less geographically dispersed during moult, or moult in an area with less isotopic heterogeneity. Mean ¹³C signatures from winter-grown feathers of different subpopulations were positively correlated with the latitude and longitude of breeding sites, suggesting a strong relationship between European breeding and African winter moulting latitudes. The use of stable isotopes provides novel insights into migratory connectivity and migration patterns in this little-known threatened species.     

Activation of the lectin pathway by natural IgM in a model of ischemia/reperfusion injury

Reperfusion of ischemic tissues elicits an acute inflammatory response involving serum complement, which is activated by circulating natural IgM specific to self-Ags exposed by ischemia. Recent reports demonstrating a role for the lectin pathway raise a question regarding the initial events in complement activation. To dissect the individual roles of natural IgM and lectin in activation of complement, mice bearing genetic deficiency in early complement, IgM, or mannan-binding lectin were characterized in a mesenteric model of ischemia reperfusion injury. The results reveal that IgM binds initially to ischemic Ag providing a binding site for mannan-binding lectin which subsequently leads to activation of complement and injury.