Bread matters: a national initiative to profile the genetic diversity of Australian wheat

The large and complex genome of wheat makes genetic and genomic analysis in this important species both expensive and resource intensive. The application of next-generation sequencing technologies is particularly resource intensive, with at least 17?Gbp of sequence data required to obtain minimal (1×) coverage of the genome. A similar volume of data would represent almost 40× coverage of the rice genome. Progress can be made through the establishment of consortia to produce shared genomic resources. Australian wheat genome researchers, working with Bioplatforms Australia, have collaborated in a national initiative to establish a genetic diversity dataset representing Australian wheat germplasm based on whole genome next-generation sequencing data. Here, we describe the establishment and validation of this resource which can provide a model for broader international initiatives for the analysis of large and complex genomes.     

Trivanillic polyphenols with anticancer cytostatic effects through the targeting of multiple kinases and intracellular Ca2+ release

Cancer cells exhibit de-regulation of multiple cellular signalling pathways and treatments of various types of cancers with polyphenols are promising. We recently reported the synthesis of a series of 33 novel divanillic and trivanillic polyphenols that displayed anticancer activity, at least in vitro, through inhibiting various kinases. This study revealed that minor chemical modifications of a trivanillate scaffold could convert cytotoxic compounds into cytostatic ones. Compound 13c, a tri-chloro derivative of trivanillic ester, displayed marked inhibitory activities against FGF-, VEGF-, EGF- and Src-related kinases, all of which are implicated not only in angiogenesis but also in the biological aggressiveness of various cancer types. The pan-anti-kinase activity of 13c occurs at less than one-tenth of its mean IC(50) in vitro growth inhibitory concentrations towards a panel of 12 cancer cell lines. Of the 26 kinases for which 13c inhibited their activity by >75%, eight (Yes, Fyn, FGF-R1, EGFR, Btk, Mink, Ret and Itk) are implicated in control of the actin cytoskeleton organization to varying degrees. Compound 13c accordingly impaired the typical organization of the actin cytoskeleton in human U373 glioblastoma cells. The pan-anti-kinase activity and actin cytoskeleton organization impairment provoked by 13c concomitantly occurs with calcium homeostasis impairment but without provoking MDR phenotype activation. All of these anticancer properties enabled 13c to confer therapeutic benefits in vivo in a mouse melanoma pseudometastatic lung model. These data argue in favour of further chemically modifying trivanillates to produce novel and potent anticancer drugs.     

CoMFA/CoMSIA 3D-QSAR of pyrimidine inhibitors of Pneumocystis carinii dihydrofolate reductase

Pneumocystis carinii is typically a non-pathogenic fungus found in the respiratory tract of healthy humans. However, it may cause P. carinii pneumonia (PCP) in people with immune deficiency, affecting mainly premature babies, cancer patients and transplant recipients, and people with acquired immunodeficiency syndrome (AIDS). In the latter group, PCP occurs in approximately 80% of patients, a major cause of death. Currently, there are many available therapies to treat PCP patients, including P. carinii dihydrofolate reductase (PcDHFR) inhibitors, such as trimetrexate (TMX), piritrexim (PTX), trimethoprim (TMP), and pyrimethamine (PMT). Nevertheless, the high percentage of adverse side effects and the limited therapeutic success of the current drug therapy justify the search for new drugs rationally planned against PCP. This work focuses on the study of pyrimidine inhibitors of PcDHFR, using both CoMFA and CoMSIA 3D-QSAR methods.     

Cannabinoid Receptor 1 Signaling in Embryo Neurodevelopment

In utero exposure to tetrahydrocannabinol, the psychoactive component of marijuana, is associated with an increased risk for neurodevelopmental defects in the offspring by interfering with the functioning of the endocannabinoid (eCB) system. At the present time, it is not clearly known whether the eCB system is present before neurogenesis. Using an array of biochemical techniques, we analyzed the levels of CB1 receptors, eCBs (AEA and 2‐AG), and the enzymes (NAPE‐PLD, DAGLα, DAGLβ, MAGL, and FAAH) involved in the metabolism of the eCBs in chick and mouse models during development. The findings demonstrate the presence of eCB system in early embryo before neurogenesis. The eCB system might play a critical role in early embryogenesis and there might be adverse developmental consequences of in utero exposure to marijuana and other drugs of abuse during this period.     

In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b⁺ F4/80⁺ MHC-II⁺ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS⁺ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.     

Multilevel Control of Organelle DNA Sequence Length in Plants

We have compared the length of noncoding organelle DNA spacers in a broad sample of plant species characterized by different life history traits to test hypotheses regarding the nature of the mechanisms driving changes in their size. We first demonstrate that the spacers do not evolve at random in size but have experienced directional evolutionary trends during plant diversification. We then study the relationships between spacer lengths and other molecular features and various species attributes by taking into account population genetic processes acting within cell lineages. Comparative techniques are used to test these relationships while controlling for species phylogenetic relatedness. The results indicate that spacer length depends on mode of organelle transmission, on population genetic structure, on nucleotide content, on rates of molecular evolution, and on life history traits, in conformity with predictions based on a model of intracellular competition among replicating organelle genomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Deletion of flagellin's hypervariable region abrogates antibody-mediated neutralization and systemic activation of TLR5-dependent immunity

TLRs trigger immunity by detecting microbe-associated molecular patterns (MAMPs). Flagellin is a unique MAMP because it harbors 1) an antigenic hypervariable region and 2) a conserved domain involved in TLR5-dependent systemic and mucosal proinflammatory and adjuvant activities. In this study, the contribution of the flagellin domains in TLR5 activation was investigated. We showed that TLR5 signaling can be neutralized in vivo by flagellin-specific Abs, which target the conserved domain. However, deletions of flagellin's hypervariable region abrogated the protein's intrinsic ability to trigger the production of neutralizing Abs. The fact that MAMP-specific Abs block TLR-mediated responses shows that this type of neutralization is a novel mechanism for down-regulating innate immunity. The stimulation of mucosal innate immunity and adjuvancy to foreign Ag was not altered by the hypervariable domain deletions. In contrast, this domain is essential to trigger systemic innate immunity, suggesting that there are distinct mechanisms for TLR5 activation in systemic and mucosal compartments. In summary, specific MAMP determinants control the production of neutralizing Abs and the compartmentalization of innate responses.