Evaluation of automated cell disruptor methods for oomycetous and ascomycetous model organisms

Two automated cell disruptor-based methods for RNA extraction, disruption of thawed cells submerged in TRIzol Reagent (method QP), and direct disruption of frozen cells on dry ice (method CP), were optimized for a model oomycete, Phytophthora capsici, and a model filamentous ascomycete, Neurospora crassa. The results were compared with more conventional methods of manual grinding in a mortar and pestle under liquid nitrogen (method M&P) and those using lyophilized samples. A chip-based electrophoresis system showed that methods CP and M&P yielded high integrity RNA from both P. capsici and N. crassa. In contrast, method QP and lyophilized sample-based methods resulted in inconsistent RNA integrity between the two organisms, indicating they are not safe alternatives for method M&P. Microarray mRNA profiling for P. capsici revealed alterations in global mRNA profiles in those samples that the chip-based electrophoresis detected slight decreases in RNA integrity. Despite this, RNA integrity of these samples could still be high enough to pass conventional stringent quality control measures. This demonstrated the necessity of global mRNA profiling for the evaluation of RNA extraction protocols

Resistance of Mice to Infection with Friend Disease Virus After Subcutaneous Injection of Friend Virus and Friend Spleen-Cells

Swiss mice injected subcutaneously with suspensions of spleen cells or an extract of spleens from mice infected with Friend virus develop resistance to subsequent intravenous inoculation of Friend virus. A single injection of either Friend virus or Friend cells induces resistance. Immunized mice display resistance when challenged 6 months after immunization and survive for at least 20 weeks after infection. Neutralization tests indicate that serum, but not lymphoid cells of resistant animals, can neutralize Friend virus. In vitro neutralization tests indicate that residence of virus within the peritoneal cavity of immune mice for 1 h sharply reduces the infective titer of the virus

Apollo asteroids (1566) Icarus and 2007 MK6: Icarus family members?

Although it is more complicated to search for near-Earth object (NEO) families than main belt asteroid (MBA) families, since differential orbital evolution within a NEO family can cause current orbital elements to drastically differ from each other, we have found that Apollo asteroids (1566) Icarus and the newly discovered 2007 MK6 are almost certainly related. Specifically, their orbital evolutions show a similar profile, time shifted by only ~1000 yr, based on our time-lag theory. The dynamical relationship between Icarus and 2007 MK6 along with a possible dust band, the Taurid-Perseid meteor swarm, implies the first detection of an asteroidal NEO family, namely the "Icarus asteroid family".Comment: 11 pages, 1 figure, to appear on Astrophysical Journal Letters (journal info added

Long-oligomer microarray profiling in Neurospora crassa reveals the transcriptional program underlying biochemical and physiological events of conidial germination

To test the inferences of spotted microarray technology against a biochemically well-studied process, we performed transcriptional profiling of conidial germination in the filamentous fungus, Neurospora crassa. We first constructed a 70 base oligomer microarray that assays 3366 predicted genes. To estimate the relative gene expression levels and changes in gene expression during conidial germination, we analyzed a circuit design of competitive hybridizations throughout a time course using a Bayesian analysis of gene expression level. Remarkable consistency of mRNA profiles with previously published northern data was observed. Genes were hierarchically clustered into groups with respect to their expression profiles over the time course of conidial germination. A functional classification database was employed to characterize the global picture of gene expression. Consensus motif searches identified a putative regulatory component associated with genes involved in ribosomal biogenesis. Our transcriptional profiling data correlate well with biochemical and physiological processes associated with conidial germination and will facilitate functional predictions of novel genes in N.crassa and other filamentous ascomycete species. Furthermore, our dataset on conidial germination allowed comparisons to transcriptional mechanisms associated with germination processes of diverse propagules, such as teliospores of the phytopathogenic fungus Ustilago maydis and spores of the social amoeba Dictyostelium discoideum

Osteoblast-like cell responses to silicate ions released from 45S5-type bioactive glass and siloxane-doped vaterite

Silicate ions released from bioactive glasses and ceramics have been reported to stimulate osteogenic cell functions. Here, we evaluated osteoblast-like cell reactions to silicate ions released from two different types of materials, 45S5 bioactive glass (BG) and siloxane-doped vaterite (SiV), to investigate the influence of the ionic structure of silicate ions on osteoblast-like cell properties. BG and SiV powders were prepared by using melt-quenching and carbonation methods, respectively. Aminopropyltriethoxysilane was used as a siloxane source of SiV. MC3T3-E1 and SaOS-2 cells were cultured in media containing dissolved BG or SiV ions (10–50 ppm of Si). Cell proliferation (metabolic activity), differentiation (alkaline phosphatase activity) and mineralisation (Ca deposition) were examined. 29Si NMR spectra demonstrated that Q0,1 species and T0–3 species were released from BG and SiV, respectively. Proliferation and mineralisation of the two types of cells were influenced by silicate ions released from BG and SiV in a concentration-dependent manner. In particular, there were significant differences (P < 0.05) in the degree of proliferation and Ca deposition levels in SaOS-2 cells treated with dissolved BG and SiV ions. Furthermore, Ca deposition in SaOS-2 cells was influenced by both the presence of silicate ions and the duration of exposure of cells to them. The structure of silicate ions influenced the proliferation and mineralisation of SaOS-2 cells incubated for different time periods in culture media containing different Si concentrations. Understanding the effect of Si on bone cell behaviour will enable a design-led approach to further BG optimisation

Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes

AbstractTo explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function

Exploring the bZIP transcription factor regulatory network in Neurospora crassa

Transcription factors (TFs) are key nodes of regulatory networks in eukaryotic organisms, including filamentous fungi such as Neurospora crassa. The 178 predicted DNA-binding TFs in N. crassa are distributed primarily among six gene families, which represent an ancient expansion in filamentous ascomycete genomes; 98 TF genes show detectable expression levels during vegetative growth of N. crassa, including 35 that show a significant difference in expression level between hyphae at the periphery versus hyphae in the interior of a colony. Regulatory networks within a species genome include paralogous TFs and their respective target genes (TF regulon). To investigate TF network evolution in N. crassa, we focused on the basic leucine zipper (bZIP) TF family, which contains nine members. We performed baseline transcriptional profiling during vegetative growth of the wild-type and seven isogenic, viable bZIP deletion mutants. We further characterized the regulatory network of one member of the bZIP family, NCU03905. NCU03905 encodes an Ap1-like protein (NcAp-1), which is involved in resistance to multiple stress responses, including oxidative and heavy metal stress. Relocalization of NcAp-1 from the cytoplasm to the nucleus was associated with exposure to stress. A comparison of the NcAp-1 regulon with Ap1-like regulons in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Aspergillus fumigatus showed both conservation and divergence. These data indicate how N. crassa responds to stress and provide information on pathway evolution