N6-Cycloalkyl-2-substituted adenosine derivatives as selective, high affinity adenosine A1 receptor agonists

A series of new selective, high affinity A(1)-AdoR agonists is reported. Compound 23 that incorporated a carboxylic acid functionality in the 4-position of the pyrazole ring displayed K(iL) value of 1 nM for the A(1)-AdoR and >5000-fold selectivity over the A(3) and A(2A)-AdoRs. In addition, compound 19 that incorporated a carboxamide functionality in the 4-position of the pyrazole ring displayed subnanomolar affinity for the A(1)-AdoR (K(iL)=0.6 nM) and >600-fold selectivity over the A(3) and A(2A)-AdoRs.     

The maize floury1 gene encodes a novel endoplasmic reticulum protein involved in zein protein body formation

The maize (Zea mays) floury1 (fl1) mutant was first reported almost 100 years ago, but its molecular identity has remained unknown. We report the cloning of Fl1, which encodes a novel zein protein body membrane protein with three predicted transmembrane domains and a C-terminal plant-specific domain of unknown function (DUF593). In wild-type endosperm, the FL1 protein accumulates at a high level during the period of zein synthesis and protein body development and declines to a low level at kernel maturity. Immunogold labeling showed that FL1 resides in the endoplasmic reticulum surrounding the protein body. Zein protein bodies in fl1 mutants are of normal size, shape, and abundance. However, mutant protein bodies ectopically accumulate 22-kD alpha-zeins in the gamma-zein-rich periphery and center of the core, rather than their normal discrete location in a ring at outer edge of the core. The 19-kD alpha-zein is uniformly distributed throughout the core in wild-type protein bodies, and this distribution is unaffected in fl1 mutants. Pairwise yeast two-hybrid experiments showed that FL1 DUF593 interacts with the 22-kD alpha-zein. Results of these studies suggest that FL1 participates in protein body formation by facilitating the localization of 22-kD alpha-zein and that this is essential for the formation of vitreous endosperm.     

Primers with 5' flaps improve real-time PCR

Primers that contain portions noncomplementary to the target region are usually used to add to the PCR product a utility sequence such as a restriction site or a universal probe binding site. We have demonstrated that primers with short 5'AT-rich overhangs increase real-time PCR fluorescent signal. The improvement is particularly significant for difficult to amplify templates, such as highly variable viral sequences or bisulfite-treated DNA.     

Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacteria-containing vacuoles

Strains of the Burkholderia cepacia complex (Bcc) are opportunistic bacteria that can cause life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Previous work has shown that Bcc isolates can persist in membrane-bound vacuoles within amoeba and macrophages without bacterial replication, but the detailed mechanism of bacterial persistence is unknown. In this study, we have investigated the survival of the Burkholderia cenocepacia strain J2315 within RAW264.7 murine macrophages. Strain J2315 is a prototypic isolate of the widespread and transmissible ET12 clone. Unlike heat-inactivated bacteria, which reach lysosomes shortly after internalization, vacuoles containing live B. cenocepacia J2315 accumulate the late endosome/lysosome marker LAMP-1 and start fusing with lysosomal compartments only after 6 h post internalization. Using fluorescent fluid-phase probes, we also demonstrated that B. cenocepacia-containing vacuoles continued to interact with newly formed endosomes, and maintained a luminal pH of 6.4 +/- 0.12. In contrast, vacuoles containing heat-inactivated bacteria had an average pH of 4.8 +/- 0.03 and rapidly merged with lysosomes. Additional experiments using concanamycin A, a specific inhibitor of the vacuolar H+-ATPase, revealed that vacuoles containing live bacteria did not exclude the H+-ATPase. This mode of bacterial survival did not require type III secretion, as no differences were found between wild type and a type III secretion mutant strain. Collectively, our results suggest that intracellular B. cenocepacia cause a delay in the maturation of the phagosome, which may contribute to facilitate bacterial escape from the microbicidal activities of the host cell.     

Micromechanical Modeling Study of Mechanical Inhibition of Enzymatic Degradation of Collagen Tissues

This study investigates how the collagen fiber structure influences the enzymatic degradation of collagen tissues. We developed a micromechanical model of a fibrous collagen tissue undergoing enzymatic degradation based on two central hypotheses. The collagen fibers are crimped in the undeformed configuration. Enzymatic degradation is an energy activated process and the activation energy is increased by the axial strain energy density of the fiber. We determined the intrinsic degradation rate and characteristic energy for mechanical inhibition from fibril-level degradation experiments and applied the parameters to predict the effect of the crimped fiber structure and fiber properties on the degradation of bovine cornea and pericardium tissues under controlled tension. We then applied the model to examine the effect of the tissue stress state on the rate of tissue degradation and the anisotropic fiber structures that developed from enzymatic degradation.     

Protocadherins control the modular assembly of neuronal columns in the zebrafish optic tectum

Cell–cell recognition guides the assembly of the vertebrate brain during development. δ-Protocadherins comprise a family of neural adhesion molecules that are differentially expressed and have been implicated in a range of neurodevelopmental disorders. Here we show that the expression of δ-protocadherins partitions the zebrafish optic tectum into radial columns of neurons. Using in vivo two-photon imaging of bacterial artificial chromosome transgenic zebrafish, we show that pcdh19 is expressed in discrete columns of neurons, and that these columnar modules are derived from proliferative pcdh19⁺ neuroepithelial precursors. Elimination of pcdh19 results in both a disruption of columnar organization and defects in visually guided behaviors. These results reveal a fundamental mechanism for organizing the developing nervous system: subdivision of the early neuroepithelium into precursors with distinct molecular identities guides the autonomous development of parallel neuronal units, organizing neural circuit formation and behavior.     

Optimized microRNA purification from TRIzol-treated plasma

Background

MicroRNAs (miRNAs) represent new and potentially informative diagnostic targets for disease detection and prognosis. However, little work exists documenting the effect of TRIzol, a common viral inactivation and nucleic acid extraction reagent, on miRNA purification. Here, we developed an optimized protocol for miRNA extraction from plasma samples by evaluating five different RNA extraction kits, TRIzol phase separation, purification additives, and initial plasma sample volume. This method was then used for downstream profiling of plasma miRNAs found in archived samples from one nonhuman primate (NHP) experimentally challenged with Ebola virus by the aerosol route.

Results

Comparison of real-time RT-PCR results for spiked-in and endogenous miRNA sequences determined extraction efficiencies from five different RNA purification kits. These experiments showed that 50 μL plasma processed using the QIAGEN miRNeasy Mini Kit with 5 μg of glycogen as a co-precipitant yielded the highest recovery of endogenous miRNAs. Using this optimized protocol, miRNAs from archived plasma samples of one rhesus macaque challenged with aerosolized Ebola virus was profiled using a targeted real-time PCR array. A total of 519 of the 752 unique miRNAs assayed were present in the plasma samples at day 0 and day 7 (time of death) post-exposure. Statistical analyses revealed 25 sequences significantly up- or down-regulated between day 0 and day 7 post infection, validating the utility of the extraction method for plasma miRNA profiling.

Conclusions

This study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized an extraction protocol for miRNAs from TRIzol-inactivated plasma samples that can be used for highly pathogenic viruses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1299-5) contains supplementary material, which is available to authorized users.