Minimizing off-target signals in RNA fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) techniques are becoming extremely sensitive, to the point where individual RNA or DNA molecules can be detected with small probes. At this level of sensitivity, the elimination of ‘off-target’ hybridization is of crucial importance, but typical probes used for RNA and DNA FISH contain sequences repeated elsewhere in the genome. We find that very short (e.g. 20 nt) perfect repeated sequences within much longer probes (e.g. 350–1500 nt) can produce significant off-target signals. The extent of noise is surprising given the long length of the probes and the short length of non-specific regions. When we removed the small regions of repeated sequence from either short or long probes, we find that the signal-to-noise ratio is increased by orders of magnitude, putting us in a regime where fluorescent signals can be considered to be a quantitative measure of target transcript numbers. As the majority of genes in complex organisms contain repeated k-mers, we provide genome-wide annotations of k-mer-uniqueness at http://cbio.mskcc.org/∼aarvey/repeatmap

Mediterranean seagrass vulnerable to regional climate warming

The Mediterranean Sea, one of the regions warming fastest under climate change, harbours lush seagrass (Posidonia oceanica) meadows that form the basis for a key ecosystem in the region. Recent field results have shown that increased maximum annual seawater temperature in the Mediterranean has already led to increased seagrass mortality. Here we project the trajectory of P. oceanica meadows under the warming expected in the western Mediterranean through the twenty-first century to conclude that warming will lead to the functional extinction of P. oceanica meadows by the middle of this century (year 2049±10) even under a relatively mild greenhouse-gas emissions scenario. Efforts to alleviate local stresses adding to the loss of P. oceanica meadows will have a limited effect in conserving the meadows under climate change. Efforts to mitigate climate change are urgently needed to preserve this key ecosystem. © 2012 Macmillan Publishers Limited. All rights reserved.Peer Reviewe

Stuttering associated with a pathogenic variant in the chaperone protein cyclophilin 40

Stuttering is a common speech disorder that interrupts speech fluency and tends to cluster in families. Typically, stuttering is characterized by speech sounds, words or syllables which may be repeated or prolonged and speech that may be further interrupted by hesitations or 'blocks'. Rare variants in a small number of genes encoding lysosomal pathway proteins have been linked to stuttering. We studied a large four-generation family in which persistent stuttering was inherited in an autosomal dominant manner with disruption of the cortico-basal-ganglia-thalamo-cortical network found on imaging. Exome sequencing of three affected family members revealed the PPID c.808C>T (p.Pro270Ser) variant that segregated with stuttering in the family. We generated a Ppid p.Pro270Ser knock-in mouse model and performed ex vivo imaging to assess for brain changes. Diffusion-weighted MRI in the mouse revealed significant microstructural changes in the left corticospinal tract, as previously implicated in stuttering. Quantitative susceptibility mapping also detected changes in cortico-striatal-thalamo-cortical loop tissue composition, consistent with findings in affected family members. This is the first report to implicate a chaperone protein in the pathogenesis of stuttering. The humanized Ppid murine model recapitulates network findings observed in affected family members