Phylogenetic studies of the genus <Emphasis Type="Italic">Cebus</Emphasis>(Cebidae-Primates) using chromosome painting and G-banding

Background  

Chromosomal painting, using whole chromosome probes from humans and Saguinus oedipus, was used to establish karyotypic divergence among species of the genus Cebus, including C. olivaceus, C. albifrons, C. apella robustus and C. apella paraguayanus. Cytogenetic studies suggested that the species of this genus have conservative karyotypes, with diploid numbers ranging from 2n = 52 to 2n = 54.     

Selective maintenance of Drosophila tandemly arranged duplicated genes during evolution

Background  

The physical organization and chromosomal localization of genes within genomes is known to play an important role in their function. Most genes arise by duplication and move along the genome by random shuffling of DNA segments. Higher order structuring of the genome occurs in eukaryotes, where groups of physically linked genes are co-expressed. However, the contribution of gene duplication to gene order has not been analyzed in detail, as it is believed that co-expression due to recent duplicates would obscure other domains of co-expression.     

Development of nanostructured biomedical micro-drug testing device based on in situ cellular activity monitoring

Integration of micro and nanofabrication techniques with biotechnology has resulted in the development of in vitro analytical and diagnostic tools for biomedical applications. The focus of such technology has primarily been on therapeutic and sensing applications. The long-term integration of cells with inorganic materials provides the basis for novel sensing platforms. This paper describes the creation of, nanoporous, biocompatible, alumina membranes as a platform for incorporation into a cell based device targeted for in situ recording of cellular electrical activity variations due to the changes associated with the surrounding microenvironments more specifically due to the effect of therapeutic drugs. Studies described herein focus on the interaction of nanoporous alumina substrates embedded in silicon, patterned with cells of interest. The cells that have been used to develop the in vitro test platform are primary hippocampal neurons. Demonstrated here, is the fidelity of such a system in terms of determination of cell viability, proliferation, and functionality. The response of the cells to the "drug" molecules is electro-optically characterized in an in situ manner. The capability of such, micro fabricated nanoporous membranes as in vitro drug testing platforms, is first theoretically estimated using two dimensional finite element modeling of the diffusion of the molecules of interest through the nanoporous substrate using CFDRC. It is then experimentally established, using glucose and immunoglobulin G (IgG).     

Wg signaling via Zw3 and mad restricts self-renewal of sensory organ precursor cells in Drosophila

It is well known that the Dpp signal transducer Mad is activated by phosphorylation at its carboxy-terminus. The role of phosphorylation on other regions of Mad is not as well understood. Here we report that the phosphorylation of Mad in the linker region by the Wg antagonist Zw3 (homolog of vertebrate Gsk3-β) regulates the development of sensory organs in the anterior-dorsal quadrant of the wing. Proneural expression of Mad-RNA interference (RNAi) or a Mad transgene with its Zw3/Gsk3-β phosphorylation sites mutated (MGM) generated wings with ectopic sensilla and chemosensory bristle duplications. Studies with pMad-Gsk (an antibody specific to Zw3/Gsk3-β-phosphorylated Mad) in larval wing disks revealed that this phosphorylation event is Wg dependent (via an unconventional mechanism), is restricted to anterior-dorsal sensory organ precursors (SOP) expressing Senseless (Sens), and is always co-expressed with the mitotic marker phospho-histone3. Quantitative analysis in both Mad-RNAi and MGM larval wing disks revealed a significant increase in the number of Sens SOP. We conclude that the phosphorylation of Mad by Zw3 functions to prevent the self-renewal of Sens SOP, perhaps facilitating their differentiation via asymmetric division. The conservation of Zw3/Gsk3-β phosphorylation sites in vertebrate homologs of Mad (Smads) suggests that this pathway, the first transforming growth factor β-independent role for any Smad protein, may be widely utilized for regulating mitosis during development.     

Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation

Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or ‘antispacer’, PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.     

Phylogeny and species delineation in the marine diatom Pseudo‐nitzschia (Bacillariophyta) using cox1, LSU,and ITS2 rRNA genes: A perspective in character evolution

Analyses of the mitochondrial cox1, the nuclear‐encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo‐nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I–III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo‐nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence–structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.