Phylogenetics of asterids based on 3 coding and 3 non-coding chloroplast DNA markers and the utility of non-coding DNA at higher taxonomic levels

Asterids comprise 1/4-1/3 of all flowering plants and are classified in 10 orders and >100 families. The phylogeny of asterids is here explored with jackknife parsimony analysis of chloroplast DNA from 132 genera representing 103 families and all higher groups of asterids. Six different markers were used, three of the markers represent protein coding genes, rbcL, ndhF, and matK, and three other represent non-coding DNA; a region including trnL exons and the intron and intergenic spacers between trnT (UGU) to trnF (GAA); another region including trnV exons and intron, trnM and intergenic spacers between trnV (UAC) and atpE, and the rps16 intron. The three non-coding markers proved almost equally useful as the three coding genes in phylogenetic reconstruction at the high level of orders and families in asterids, and in relation to the number of aligned positions the non-coding markers were even more effective. Basal interrelationships among Cornales, Ericales, lamiids (new name replacing euasterids I), and campanulids (new name replacing euasterids II) are resolved with strong support. Family interrelationships are fully or almost fully resolved with medium to strong support in Cornales, Garryales, Gentianales, Solanales, Aquifoliales, Apiales, and Dipsacales. Within the three large orders Ericales, Lamiales, and Asterales, family interrelationships remain partly unclear. The analysis has contributed to reclassification of several families, e.g., Tetrameristaceae, Ebenaceae, Styracaceae, Montiniaceae, Orobanchaceae, and Scrophulariaceae (by inclusion of Pellicieraceae, Lissocarpaceae, Halesiaceae, Kaliphoraceae, Cyclocheilaceae, and Myoporaceae+Buddlejaceae, respectively), and to the placement of families that were unplaced in the APG-system, e.g., Sladeniaceae, Pentaphylacaceae, Plocospermataceae, Cardiopteridaceae, and Adoxaceae (in Ericales, Ericales, Lamiales, Aquifoliales, and Dipsacales, respectively), and Paracryphiaceae among campanulids. Several families of euasterids remain unclassified to order.     

Gravity and cyclic GMP levels in melanocytic cells

Guanosine 3',5'-cyclic monophosphate (cyclic GMP) is a major second messenger molecule, that is believed to play a role in various physiological and pathophysiological processes. Here we report that hypergravity induces differential effects on cyclic GMP turnover in melanocytic cells. Nonmetastatic melanoma cells responded to long-time exposure (24 h) of hypergravity (up to 5 x g) with decrease in intracellular cyclic GMP accumulation in the presence of an universal inhibitor of phosphodiesterases (IBMX), whereas the extracellular cyclic GMP increase. In contrast, there were no changes in cyclic GMP turnover in metastatic melanocytes. The expression of the guanylyl cyclases appeared to be not affected. These results suggest that cyclic GMP signaling may be involved in adaptation of human melanocytes to altered gravity conditions.     

Identification of Two Novel Mutations in PKHD1 Gene from Two Families with Polycystic Kidney Disease by Whole Exome Sequencing

Background Polycystic kidney disease (PKD) is an autosomal recessive disorder resulting from mutations in the PKHD1 gene on chromosome 6 (6p12), a large gene spanning 470 kb of genomic DNA.ObjectiveThe aim of the present study was to report newly identified mutations in the PKHD1 gene in two Iranian families with PKD.Materials and Methods Genetic alterations of a 3-month-old boy and a 27-year-old girl with PKD were evaluated using whole-exome sequencing. The PCR direct sequencing was performed to analyse the co-segregation of the variants with the disease in the family. Finally, the molecular function of the identified novel mutations was evaluated by in silico study. ResultsIn the 3 month-old boy, a novel homozygous frameshift mutation was detected in the PKHD1 gene, which can cause PKD. Moreover, we identified three novel heterozygous missense mutations in ATIC, VPS13B, and TP53RK genes. In the 27-year-old woman, with two recurrent abortions history and two infant mortalities at early weeks due to metabolic and/or renal disease, we detected a novel missense mutation on PKHD1 gene and a novel mutation in ETFDH gene.Conclusion In general, we have identified two novel mutations in the PKHD1 gene. These molecular findings can help accurately correlate genotype and phenotype in families with such disease in order to reduce patient births through preoperative genetic diagnosis or better management of disorders.     

Hydrophilic carotenoids: surface properties and aqueous aggregation of a rigid, long-chain, highly unsaturated dianionic bolaamphiphile with a carotenoid spacer

The water dispersibility of astaxanthin was greatly enhanced by converting it to a disodium disuccinate salt. This carotenoid salt behaved as a bolaamphiphile in water; dynamic light scattering (DLS) revealed the formation of stable aggregates with an average hydrodynamic radius close to 1 microm. Larger aggregates were observed in solutions of increased osmolarity. Absorption spectra demonstrated that the aggregates could withstand the addition of 20% acetonitrile before disintegrating to monomers. The physicochemical properties of this astaxanthin derivative in solution were comprehensively studied by measuring surface tension, critical aggregate concentration, surface concentration, molecule area, free energy of adsorption and micellation, adsorption-aggregate energy relationship, and equilibrium constants, and then compared with similar compounds reported previously in the literature.     

Murine segmental duplications are hot spots for chromosome and gene evolution

Mouse and rat genomic sequences permit us to obtain a global view of evolutionary rearrangements that have occurred between the two species and to define hallmarks that might underlie these events. We present a comparative study of the sequence assemblies of mouse and rat genomes and report an enrichment of rodent-specific segmental duplications in regions where synteny is not preserved. We show that segmental duplications present higher rates of molecular evolution and that genes in rearranged regions have evolved faster than those located elsewhere. Previous studies have shown that synteny breakpoints between the mouse and the human genomes are enriched in human segmental duplications, suggesting a causative connection between such structures and evolutionary rearrangements. Our work provides further evidence to support the role of segmental duplications in chromosomal rearrangements in the evolution of the architecture of mammalian chromosomes and in the speciation processes that separate the mouse and the rat.     

A Novel Syntaxin 6‐Interacting Protein,SHIP164, Regulates Syntaxin 6‐Dependent Sorting from Early Endosomes

Membrane fusion is dependent on the function of SNAREs and their α‐helical SNARE motifs that form SNARE complexes. The Habc domains at the N‐termini of some SNAREs can interact with their associated SNARE motif, Sec1/Munc18 (SM) proteins, tethering proteins or adaptor proteins, suggesting that they play an important regulatory function. We screened for proteins that interact with the Habc domain of Syntaxin 6, and isolated an uncharacterized 164‐kDa protein that we named SHIP164. SHIP164 is part of a large (∼700 kDa) complex, and interacts with components of the Golgi‐associated retrograde protein (GARP) tethering complex. Depletion of GARP subunits or overexpression of Syntaxin 6 results in a redistribution of soluble SHIP164 to endosomal structures. Co‐overexpression of Syntaxin 6 and SHIP164 produced excessive tubulation of endosomes, and perturbed the transport of cation‐independent mannose‐6‐phosphate receptor (CI‐MPR) and transferrin receptor. Thus, we propose that SHIP164 functions in trafficking through the early/recycling endosomal system.     

Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.     

Construction of naïve camelids VHH repertoire in phage display-based library

Camelids have unique antibodies, namely HCAbs (VHH) or commercially named Nanobodies^® (Nb) that are composed only of a heavy-chain homodimer. As libraries based on immunized camelids are time-consuming, costly and likely redundant for certain antigens, we describe the construction of a naïve camelid VHHs library from blood serum of non-immunized camelids with affinity in the subnanomolar range and suitable for standard immune applications. This approach is rapid and recovers VHH repertoire with the advantages of being more diverse, non-specific and devoid of subpopulations of specific antibodies, which allows the identification of binders for any potential antigen (or pathogen). RNAs from a number of camelids from Saudi Arabia were isolated and cDNAs of the diverse vhh gene were amplified; the resulting amplicons were cloned in the phage display pSEX81 vector. The size of the library was found to be within the required range (10⁷) suitable for subsequent applications in disease diagnosis and treatment. Two hundred clones were randomly selected and the inserted gene library was either estimated for redundancy or sequenced and aligned to the reference camelid vhh gene (acc. No. ADE99145). Results indicated complete non-specificity of this small library in which no single event of redundancy was detected. These results indicate the efficacy of following this approach in order to yield a large and diverse enough gene library to secure the presence of the required version encoding the required antibodies for any target antigen. This work is a first step towards the construction of phage display-based biosensors useful in disease (e.g., TB or tuberculosis) diagnosis and treatment.