GAMBIT (Genomic Approximation Method for Bacterial Identification and Tracking): A methodology to rapidly leverage whole genome sequencing of bacterial isolates for clinical identification.

Whole genome sequencing (WGS) of clinical bacterial isolates has the potential to transform the fields of diagnostics and public health. To realize this potential, bioinformatic software that reports identification results needs to be developed that meets the quality standards of a diagnostic test. We developed GAMBIT (Genomic Approximation Method for Bacterial Identification and Tracking) using k-mer based strategies for identification of bacteria based on WGS reads. GAMBIT incorporates this algorithm with a highly curated searchable database of 48,224 genomes. Herein, we describe validation of the scoring methodology, parameter robustness, establishment of confidence thresholds and the curation of the reference database. We assessed GAMBIT by way of validation studies when it was deployed as a laboratory-developed test in two public health laboratories. This method greatly reduces or eliminates false identifications which are often detrimental in a clinical setting.

Diverse ETS transcription factors mediate FGF signaling in the Ciona anterior neural plate.

The ascidian Ciona intestinalis is a marine invertebrate belonging to the sister group of the vertebrates, the tunicates. Its compact genome and simple, experimentally tractable embryos make Ciona well-suited for the study of cell-fate specification in chordates. Tunicate larvae possess a characteristic chordate body plan, and many developmental pathways are conserved between tunicates and vertebrates. Previous studies have shown that FGF signals are essential for neural induction and patterning at sequential steps of Ciona embryogenesis. Here we show that two different ETS family transcription factors, Ets1/2 and Elk1/3/4, have partially redundant activities in the anterior neural plate of gastrulating embryos. Whereas Ets1/2 promotes pigment cell formation in lateral lineages, both Ets1/2 and Elk1/3/4 are involved in the activation of Myt1L in medial lineages and the restriction of Six3/6 expression to the anterior-most regions of the neural tube. We also provide evidence that photoreceptor cells arise from posterior regions of the presumptive sensory vesicle, and do not depend on FGF signaling. Cells previously identified as photoreceptor progenitors instead form ependymal cells and neurons of the larval brain. Our results extend recent findings on FGF-dependent patterning of anterior-posterior compartments in the Ciona central nervous system.

Islet is a key determinant of ascidian palp morphogenesis.

The anterior-most ectoderm of ascidian larvae contains the adhesive papillae, or palps, which play an important role in triggering the metamorphosis of swimming tadpoles. In Ciona intestinalis, the palps consist of three conical protrusions within a field of thickened epithelium that form late in embryogenesis, as tailbuds mature into larvae. The palp protrusions express the LIM-homeodomain transcription factor Islet. Protrusion occurs through differential cell elongation, probably mediated by Islet, as we find that ectopic expression of Islet is sufficient to promote cell lengthening. FGF signaling is required for both Islet expression and palp morphogenesis. Importantly, we show that Islet expression can rescue the palp-deficient phenotype that results from inhibition of FGF signaling. We conclude that Islet is a key regulatory factor governing morphogenesis of the palps. It is conceivable that Islet is also essential for the cellular morphogenesis of placode-derived sensory neurons in vertebrates.

Identification of a rudimentary neural crest in a non-vertebrate chordate.

Neural crest arises at the neural plate border, expresses a core set of regulatory genes and produces a diverse array of cell types, including ectomesenchyme derivatives that elaborate the vertebrate head. The evolution of neural crest has been proposed to be a key event leading to the appearance of new cell types that fostered the transition from filter feeding to active predation in ancestral vertebrates. However, the origin of neural crest remains controversial, as homologous cell types have not been unambiguously identified in non-vertebrate chordates. Here we show that the tunicate Ciona intestinalis possesses a cephalic melanocyte lineage (a9.49) similar to neural crest that can be reprogrammed into migrating 'ectomesenchyme' by the targeted misexpression of Twist (also known as twist-like 2). Our results suggest that the neural crest melanocyte regulatory network pre-dated the divergence of tunicates and vertebrates. We propose that the co-option of mesenchyme determinants, such as Twist, into the neural plate ectoderm was crucial to the emergence of the vertebrate 'new head'.

FGF signaling establishes the anterior border of the Ciona neural tube.

The Ciona tadpole is constructed from simple, well-defined cell lineages governed by provisional gene networks that have been defined via extensive gene disruption assays. Here, we examine the patterning of the anterior neural plate, which produces placodal derivatives such as the adhesive palps and stomodeum, as well as the sensory vesicle (simple brain) of the Ciona tadpole. Evidence is presented that the doublesex-related gene DMRT is expressed throughout the anterior neural plate of neurulating embryos. It leads to the activation of FoxC and ZicL in the palp placode and anterior neural tube, respectively. This differential expression depends on FGF signaling, which inhibits FoxC expression in the anterior neural tube. Inhibition of FGF signaling leads to expanded expression of FoxC, the loss of ZicL, and truncation of the anterior neural tube.

Neural tube patterning by Ephrin, FGF and Notch signaling relays.

The motor ganglion (MG) controls the rhythmic swimming behavior of the Ciona intestinalis tadpole. Despite its cellular simplicity (five pairs of neurons), the MG exhibits conservation of transcription factor expression with the spinal cord of vertebrates. Evidence is presented that the developing MG is patterned by sequential Ephrin/FGF/MAPK and Delta/Notch signaling events. FGF/MAPK attenuation by a localized EphrinAb signal specifies posterior neuronal subtypes, which in turn relay a Delta2/Notch signal that specifies anterior fates. This short-range relay is distinct from the patterning of the vertebrate spinal cord, which is a result of opposing BMP and Shh morphogen gradients. Nonetheless, both mechanisms lead to localized expression of related homeodomain codes for the specification of distinct neuronal subtypes. This MG regulatory network provides a foundation for elucidating the genetic and cellular basis of a model chordate central pattern generator.

Suppression of in vivo beta-amyloid peptide toxicity by overexpression of the HSP-16.2 small chaperone protein.

Expression of the human beta-amyloid peptide (Abeta) in a transgenic Caenorhabditis elegans Alzheimer disease model leads to the induction of HSP-16 proteins, a family of small heat shock-inducible proteins homologous to vertebrate alphaB crystallin. These proteins also co-localize and co-immunoprecipitate with Abeta in this model (Fonte, V., Kapulkin, V., Taft, A., Fluet, A., Friedman, D., and Link, C. D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 9439-9444). To investigate the molecular basis and biological function of this interaction between HSP-16 and Abeta, we generated transgenic C. elegans animals with high level, constitutive expression of HSP-16.2. We find that constitutive expression of wild type, but not mutant, HSP-16.2 partially suppresses Abeta toxicity. Wild type Abeta-(1-42), but not Abeta single chain dimer, was observed to become sequestered in HSP-16.2-containing inclusions, indicating a conformation-dependent interaction between HSP-16.2 and Abeta in vivo. Constitutive expression of HSP-16.2 could reduce amyloid fibril formation, but it did not reduce the overall accumulation of Abeta peptide or alter the pattern of the predominant oligomeric species. Studies with recombinant HSP-16.2 demonstrated that HSP-16.2 can bind directly to Abeta in vitro, with a preferential affinity for oligomeric Abeta species. This interaction between Abeta and HSP-16.2 also influences the formation of Abeta oligomers in in vitro assays. These studies are consistent with a model in which small chaperone proteins reduce Abeta toxicity by interacting directly with the Abeta peptide and altering its oligomerization pathways, thereby reducing the formation of a minor toxic species.

An unconventional human Ccr4-Caf1 deadenylase complex in nuclear cajal bodies.

mRNA deadenylation is a key process in the regulation of translation and mRNA turnover. In Saccharomyces cerevisiae, deadenylation is primarily carried out by the Ccr4p and Caf1p/Pop2p subunits of the Ccr4-Not complex, which is conserved in eukaryotes including humans. Here we have identified an unconventional human Ccr4-Caf1 complex containing hCcr4d and hCaf1z, distant human homologs of yeast Ccr4p and Caf1p/Pop2p, respectively. The hCcr4d-hCaf1z complex differs from conventional Ccr4-Not deadenylase complexes, because (i) hCaf1z and hCcr4d concentrate in nuclear Cajal bodies and shuttle between the nucleus and cytoplasm and (ii) the hCaf1z subunit, in addition to rapid deadenylation, subjects substrate RNAs to slow exonucleolytic degradation from the 3' end in vitro. Exogenously expressed hCaf1z shows both of those activities on reporter mRNAs in human HeLa cells and stimulates general mRNA decay when restricted to the cytoplasm by deletion of its nuclear localization signal. These observations suggest that the hCcr4d-hCaf1z complex may function either in the nucleus or in the cytoplasm after its nuclear export, to degrade polyadenylated RNAs, such as mRNAs, pre-mRNAs, or those RNAs that are polyadenylated prior to their degradation in the nucleus.

Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1.

In human cells, a critical pathway in gene regulation subjects mRNAs with AU-rich elements (AREs) to rapid decay by a poorly understood process. AREs have been shown to directly activate deadenylation, decapping, or 3'-to-5' exonucleolytic decay. We demonstrate that enzymes involved in all three of these mRNA decay processes, as well as 5'-to-3' exonucleolytic decay, associate with the protein tristetraprolin (TTP) and its homolog BRF-1, which bind AREs and activate mRNA decay. TTP and BRF-1 each contain two activation domains that can activate mRNA decay after fusion to a heterologous RNA-binding protein, and inhibit ARE-mediated mRNA decay when overexpressed. Both activation domains employ trans-acting factors to trigger mRNA decay, and the N-terminal activation domain functions as a binding platform for mRNA decay enzymes. Our data suggest that the TTP protein family functions as a molecular link between ARE-containing mRNAs and the mRNA decay machinery by recruitment of mRNA decay enzymes, and help explain how deadenylation, decapping, and exonucleolytic decay can all be independently activated on ARE-containing mRNAs. This describes a potentially regulated step in activation of mRNA decay.

Culture-independent molecular analysis of microbial constituents of the healthy human outer ear.

Molecular-phylogenetic sequence analyses have provided a new perspective on microbial communities by allowing the detection and identification of constituent microorganisms in the absence of cultivation. In this study we used broad-specificity amplification of ribosomal DNA (rDNA) genes to survey organisms present in the human outer ear canal. Samples were obtained from 24 individuals, including members of three extended families, in order to survey the resident microbiota and to examine microbial population structures in individuals related by familial or household associations. To examine the stability of the microbial populations, one individual was sampled four times and another twice over a 14-month period. We found that a distinct set of microbial types was present in the majority of the subjects sampled. The two most prevalent rDNA sequence types that were identified in multiple individuals corresponded closely to those of Alloiococcus otitis and Corynebacterium otitidis, commonly thought to be associated exclusively with infections of the middle ear. Our results suggest, therefore, that the outer ear canal may serve as a reservoir for normally commensal microbes that can contribute to pathogenesis upon introduction into the middle ear. Alternatively, culture analyses of diseases of the middle ear may have been confounded by these contaminating commensal organisms.

mRNA surveillance: the perfect persist.

In eukaryotes, an elaborate set of mechanisms has evolved to ensure that the multistep process of gene expression is accurately executed and adapted to cellular needs. The mRNA surveillance pathway works in this context by assessing the quality of mRNAs to ensure that they are suitable for translation. mRNA surveillance facilitates the detection and destruction of mRNAs that contain premature termination codons by a process called nonsense-mediated decay. Moreover, recent studies have shown that a distinct mRNA surveillance process, called nonstop decay, is responsible for depleting mRNAs that lack in-frame termination codons. mRNA surveillance thereby prevents the synthesis of truncated and otherwise aberrant proteins, which can have dominant-negative and other deleterious effects.