MybA,a transcription factor involved in conidiation and conidial viability of the human pathogen Aspergillus fumigatus

Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA‐sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane‐associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.     

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage

Background

Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown.

Principal Findings

We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

Significance

Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.     

Pleiotropic phenotypes of a <Emphasis Type="Italic">Yersinia enterocolitica flhD</Emphasis> mutant include reduced lethality in a chicken embryo model

Background  

The Yersinia enterocolitica flagellar master regulator FlhD/FlhC affects the expression levels of non-flagellar genes, including 21 genes that are involved in central metabolism. The sigma factor of the flagellar system, FliA, has a negative effect on the expression levels of seven plasmid-encoded virulence genes in addition to its positive effect on the expression levels of eight of the flagellar operons. This study investigates the phenotypes of flhD and fliA mutants that result from the complex gene regulation.     

Beta-lactam type molecular scaffolds for antiproliferative activity: synthesis and cytotoxic effects in breast cancer cells

A series of novel beta-lactam containing compounds are described as antiproliferative agents and potential selective modulators of the oestrogen receptor. The purpose of the study is to evaluate the antiproliferative effects of these compounds on human MCF-7 and MDA MB-231 breast cancer cells. The compounds are designed to contain three aryl ring substituents arranged on the heterocyclic azetidin-2-one (beta-lactam), thus providing conformationally restrained analogues of the triarylethylene arrangement exemplified in the tamoxifen type structure. The compounds demonstrated potency in antiproliferative assays against MCF-7 human breast cancer cell line at low micromolar to nanomolar concentrations with low cytotoxicity and moderate binding affinity to the oestrogen receptor. The effect of a number of aryl and amine functional group substitutions on the antiproliferative activity of the beta-lactam products was explored and a brief computational structure-activity relationship investigation with molecular simulation was investigated.     

Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe

Cre/lox site-specific recombination systems provide important tools for genetic manipulation. Here we present an efficient method for gene tagging and gene replacement using Cre recombinase-mediated cassette exchange (RMCE). The cassette consists of the S. pombe ura4(+) selectable marker flanked by a wild-type loxP site at one end and by a modified heterospecific lox site (loxM3) at the other. The cassette is stable because the flanking lox sites cannot recombine with each other. Following integration of the cassette at the chosen chromosomal locus, exchange is achieved by introducing a Cre-expression plasmid containing an equivalent cassette containing the required tag or gene sequence. Recombinants are selected by uracil prototrophy using the reagent 5-fluoroorotic acid (5-FOA). The cassette exchange system provides for repetitive integrations at the same locus, allowing different protein tags or gene sequences to be integrated quickly and efficiently. We have established a range of reagents and verified utility by C-terminally tagging the S. pombe rad4 and swi1 genes with yEGFP and the yEGFP derivatives yECFP and yECitrine and by transferring the coding sequence for both genes.     

p21 activated kinase 5 activates Raf-1 and targets it to mitochondria

Raf-1 is an important effector of Ras mediated signaling and is a critical regulator of the ERK/MAPK pathway. Raf-1 activation is controlled in part by phosphorylation on multiple residues, including an obligate phosphorylation site at serine 338. Previously PAK1 and casein kinase II have been implicated as serine 338 kinases. To identify novel kinases that phosphorylate this site, we tested the ability of group II PAKs (PAKs 4-6) to control serine 338 phosphorylation. We observed that all group II PAKs were efficient serine 338 kinases, although only PAK1 and PAK5 significantly stimulated Raf-1 kinase activity. We also showed that PAK5 forms a tight complex with Raf-1 in the cell, but not A-Raf or B-Raf. Importantly, we also demonstrated that the association of Raf-1 with PAK5 targets a subpopulation of Raf-1 to mitochondria. These data indicate that PAK5 is a potent regulator of Raf-1 activity and may control Raf-1 dependent signaling at mitochondria.     

Confused about NMDA and addiction? Targeted knockouts provide answers and new questions

NMDA-dependent plasticity in VTA dopamine neurons has been hypothesized to be an important first step in the development of long-term changes in the brain reward circuitry that underlie addiction. Two papers from Zweifel et al. and Engblom et al. in this issue of Neuron raise new questions concerning the role of NMDA receptors within VTA dopamine neurons in mediating the behavioral effects of drugs of abuse.     

Diversification of photoperiodic response patterns in a collection of early-flowering mutants of Arabidopsis

Many plant species exhibit seasonal variation of flowering time in response to daylength. Arabidopsis (Arabidopsis thaliana) flowers earlier under long days (LDs) than under short days (SDs). This quantitative response to photoperiod is characterized by two parameters, the critical photoperiod (Pc), below which there is a delay in flowering, and the ceiling photoperiod (Pce), below which there is no further delay. Thus Pc and Pce define the thresholds beyond which maximum LD and SD responses are observed, respectively. We studied the quantitative response to photoperiod in 49 mutants selected for early flowering in SDs. Nine of these mutants exhibited normal Pce and Pc, showing that their precocious phenotype was not linked to abnormal measurement of daylength. However, we observed broad diversification in the patterns of quantitative responses in the other mutants. To identify factors involved in abnormal measurement of daylength, we analyzed the association of these various patterns with morphogenetic and rhythmic defects. A high proportion of mutants with altered Pce exhibited abnormal hypocotyl elongation in the dark and altered circadian periods of leaf movements. This suggested that the circadian clock and negative regulators of photomorphogenesis may contribute to the specification of SD responses. In contrast, altered Pc correlated with abnormal hypocotyl elongation in the light and reduced photosynthetic light-input requirements for bolting. This indicated that LD responses may be specified by positive elements of light signal transduction pathways and by regulators of resource allocation. Furthermore, the frequency of circadian defects in mutants with normal photoperiodic responses suggested that the circadian clock may regulate the number of leaves independently of its effect on daylength perception.     

Farm Households and Land Use in a Core Conservation Zone of the Maya Biosphere Reserve,Guatemala

This paper employs cross-tabular analysis, and multivariate and logistic regression to explore demographic, political-economic, socioeconomic, and ecological patterns of farm households and land use outcomes in an emergent agricultural frontier: the Sierra de Lacandón National Park (SLNP)-a core conservation zone of the Maya Biosphere Reserve (MBR), Petén, Guatemala. Data were obtained from a 1998 probability sample of 241 farm households, the first large detailed household land use survey in Guatemala’s Selva Maya-the largest lowland tropical forest in Central America. Virtually all settler households were poor maize farmers who colonized the SLNP in search of land for subsistence. While they faced similar ecological and economic conditions, land use strategies and patterns of forest clearing varied with demographic, household, and farm characteristics. Findings support and refute elements from previous frontier land use theory and offer policy implications for conservation and development initiatives in the Maya Forest specifically, and in tropical agricultural frontiers in general.