PhyloToAST: Bioinformatics Tools for Species-Level Analysis and Visualization of Complex Microbial Datasets

The 16S rRNA gene is widely used for taxonomic profiling of microbial ecosystems; and recent advances in sequencing chemistry have allowed extremely large numbers of sequences to be generated from minimal amounts of biological samples. Analysis speed and resolution of data to species-level taxa are two important factors in large-scale explorations of complex microbiomes using 16S sequencing. We present here new software, Phylogenetic Tools for Analysis of Species-level Taxa (PhyloToAST), that completely integrates with the QIIME pipeline to improve analysis speed, reduce primer bias (requiring two sequencing primers), enhance species-level analysis, and add new visualization tools. The code is free and open source, and can be accessed at http://phylotoast.org

Differentiation of the Lateral Compartment of the Cochlea Requires a Temporally Restricted FGF20 Signal

FGF20 signaling in mice is required specifically for the differentiation of cochlear outer hair cells, the cells most often damaged during age-related hearing loss

Multiple Wnts Redundantly Control Polarity Orientation in Caenorhabditis elegans Epithelial Stem Cells

During development, cell polarization is often coordinated to harmonize tissue patterning and morphogenesis. However, how extrinsic signals synchronize cell polarization is not understood. In Caenorhabditis elegans, most mitotic cells are polarized along the anterior-posterior axis and divide asymmetrically. Although this process is regulated by a Wnt-signaling pathway, Wnts functioning in cell polarity have been demonstrated in only a few cells. We analyzed how Wnts control cell polarity, using compound Wnt mutants, including animals with mutations in all five Wnt genes. We found that somatic gonadal precursor cells (SGPs) are properly polarized and oriented in quintuple Wnt mutants, suggesting Wnts are dispensable for the SGPs' polarity, which instead requires signals from the germ cells. Thus, signals from the germ cells organize the C. elegans somatic gonad. In contrast, in compound but not single Wnt mutants, most of the six seam cells, V1–V6 (which are epithelial stem cells), retain their polarization, but their polar orientation becomes random, indicating that it is redundantly regulated by multiple Wnt genes. In contrast, in animals in which the functions of three Wnt receptors (LIN-17, MOM-5, and CAM-1) are disrupted—the stem cells are not polarized and divide symmetrically—suggesting that the Wnt receptors are essential for generating polarity and that they function even in the absence of Wnts. All the seam cells except V5 were polarized properly by a single Wnt gene expressed at the cell's anterior or posterior. The ectopic expression of posteriorly expressed Wnts in an anterior region and vice versa rescued polarity defects in compound Wnt mutants, raising two possibilities: one, Wnts permissively control the orientation of polarity; or two, Wnt functions are instructive, but which orientation they specify is determined by the cells that express them. Our results provide a paradigm for understanding how cell polarity is coordinated by extrinsic signals

Disulphide Bridges of Phospholipase C of Chlamydomonas reinhardtii Modulates Lipid Interaction and Dimer Stability

BACKGROUND: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3- phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction. PRINCIPAL FINDINGS: We herein report that, the disruption of disulphide bond of a novel PI-specific PLC of C. reinhardtii (CrPLC) can modulate its interaction affinity with a set of phospholipids and also the stability of its dimer. CrPLC was found to form a mixture of higher oligomeric states with monomer and dimer as major species. Dimer adduct of CrPLC disappeared in the presence of DTT, which suggested the involvement of disulphide bond(s) in CrPLC oligomerization. Dimer-monomer equilibrium studies with the isolated fractions of CrPLC monomer and dimer supported the involvement of covalent forces in the dimerization of CrPLC. A disulphide bridge was found to be responsible for the dimerization and Cys7 seems to be involved in the formation of the disulphide bond. This crucial disulphide bond also modulated the lipid affinity of CrPLC. Oligomers of CrPLC were also captured in in vivo condition. CrPLC was mainly found to be localized in the plasma membrane of the cell. The cell surface localization of CrPLC may have significant implication in the downstream regulatory function of CrPLC. SIGNIFICANCE: This study helps in establishing the role of CrPLC (or similar proteins) in the quaternary structure of the molecule its affinities during lipid interactions

Stemness of the Organ of Corti Relates to the Epigenetic Status of Sox2 Enhancers

In the adult mammalian auditory epithelium, the organ of Corti, loss of sensory hair cells results in permanent hearing loss. The underlying cause for the lack of regenerative response is the depletion of otic progenitors in the cell pool of the sensory epithelium. Here, we show that an increase in the sequence-specific methylation of the otic Sox2 enhancers NOP1 and NOP2 is correlated with a reduced self-renewal potential in vivo and in vitro; additionally, the degree of methylation of NOP1 and NOP2 is correlated with the dedifferentiation potential of postmitotic supporting cells into otic stem cells. Thus, the stemness the organ of Corti is related to the epigenetic status of the otic Sox2 enhancers. These observations validate the continued exploration of treatment strategies for dedifferentiating or reprogramming of differentiated supporting cells into progenitors to regenerate the damaged organ of Corti

Neurod1 Suppresses Hair Cell Differentiation in Ear Ganglia and Regulates Hair Cell Subtype Development in the Cochlea

Background: At least five bHLH genes regulate cell fate determination and differentiation of sensory neurons, hair cells and supporting cells in the mammalian inner ear. Cross-regulation of Atoh1 and Neurog1 results in hair cell changes in Neurog1 null mice although the nature and mechanism of the cross-regulation has not yet been determined. Neurod1, regulated by both Neurog1 and Atoh1, could be the mediator of this cross-regulation. Methodology/Principal Findings: We used Tg(Pax2-Cre) to conditionally delete Neurod1 in the inner ear. Our data demonstrate for the first time that the absence of Neurod1 results in formation of hair cells within the inner ear sensory ganglia. Three cell types, neural crest derived Schwann cells and mesenchyme derived fibroblasts (neither expresses Neurod1) and inner ear derived neurons (which express Neurod1) constitute inner ear ganglia. The most parsimonious explanation is that Neurod1 suppresses the alternative fate of sensory neurons to develop as hair cells. In the absence of Neurod1, Atoh1 is expressed and differentiates cells within the ganglion into hair cells. We followed up on this effect in ganglia by demonstrating that Neurod1 also regulates differentiation of subtypes of hair cells in the organ of Corti. We show that in Neurod1 conditional null mice there is a premature expression of several genes in the apex of the developing cochlea and outer hair cells are transformed into inner hair cells. Conclusions/Significance: Our data suggest that the long noted cross-regulation of Atoh1 expression by Neurog1 migh

The Prosensory Function of Sox2 in the Chicken Inner Ear Relies on the Direct Regulation of Atoh1

The proneural gene Atoh1 is crucial for the development of inner ear hair cells and it requires the function of the transcription factor Sox2 through yet unknown mechanisms. In the present work, we used the chicken embryo and HEK293T cells to explore the regulation of Atoh1 by Sox2. The results show that hair cells derive from Sox2-positive otic progenitors and that Sox2 directly activates Atoh1 through a transcriptional activator function that requires the integrity of Sox2 DNA binding domain. Atoh1 activation depends on Sox transcription factor binding sites (SoxTFBS) present in the Atoh1 3′ enhancer where Sox2 directly binds, as shown by site directed mutagenesis and chromatin immunoprecipitation (ChIP). In the inner ear, Atoh1 enhancer activity is detected in the neurosensory domain and it depends on Sox2. Dominant negative competition (Sox2HMG-Engrailed) and mutation of the SoxTFBS abolish the reporter activity in vivo. Moreover, ChIP assay in isolated otic vesicles shows that Sox2 is bound to the Atoh1 enhancer in vivo. However, besides activating Atoh1, Sox2 also promotes the expression of Atoh1 negative regulators and the temporal profile of Atoh1 activation by Sox2 is transient suggesting that Sox2 triggers an incoherent feed-forward loop. These results provide a mechanism for the prosensory function of Sox2 in the inner ear. We suggest that sensory competence is established early in otic development through the activation of Atoh1 by Sox2, however, hair cell differentiation is prevented until later stages by the parallel activation of negative regulators of Atoh1 function