A diffusible signal attracts olfactory sensory axons toward their target in the developing brain of the moth

The signals that olfactory receptor axons use to navigate to their target in the CNS are still not well understood. In the moth Manduca sexta, the primary olfactory pathway develops postembryonically, and the receptor axons navigate from an experimentally accessible sensory epithelium to the brain along a pathway long enough for detailed study of regions in which axon behavior changes. The current experiments ask whether diffusible factors contribute to receptor axon guidance. Explants were made from the antennal receptor epithelium and co-cultured in a collagen gel matrix with slices of various regions of the brain. Receptor axons were attracted toward the central regions of the brain, including the protocerebrum and antennal lobe. Receptor axons growing into a slice of the most proximal region of the antennal nerve, where axon sorting normally occurs, showed no directional preference. When the antennal lobe was included in the slice, the receptor axons entering the sorting region grew directly toward the antennal lobe. Taken together with the previous in vivo experiments, the current results suggest that an attractive diffusible factor can serve as one cue to direct misrouted olfactory receptor axons toward the medial regions of the brain, where local cues guide them to the antennal lobe. They also suggest that under normal circumstances, in which the receptor axons follow a pre-existing pupal nerve to the antennal lobe, the diffusible factor emanating from the lobe acts in parallel and at short range to maintain the fidelity of the path into the antennal lobe.     

Replication checkpoint kinase Cds1 regulates recombinational repair protein Rad60

Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks. How Cds1 accomplishes this task is unknown. We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast. Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization. A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest. Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair. These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1. We propose that control of Rad60 regulates recombination events at stalled forks.     

Regulation of mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint

The protein kinase Chk1 enforces the DNA damage checkpoint. This checkpoint delays mitosis until damaged DNA is repaired. Chk1 regulates the activity and localization of Cdc25, the tyrosine phosphatase that activates the cdk Cdc2. Here we report that Mik1, a tyrosine kinase that inhibits Cdc2, is positively regulated by the DNA damage checkpoint. Mik1 is required for checkpoint response in strains that lack Cdc25. Long-term DNA damage checkpoint arrest fails in Δmik1 cells. DNA damage increases Mik1 abundance in a Chk1-dependent manner. Ubiquitinated Mik1 accumulates in a proteasome mutant, which indicates that Mik1 normally has a short half-life. Thus, the DNA damage checkpoint might regulate Mik1 degradation. Mik1 protein and mRNA oscillate during the unperturbed cell cycle, with peak amounts detected around S phase. These data indicate that regulation of Mik1 abundance helps to couple mitotic onset to the completion of DNA replication and repair. Coordinated negative regulation of Cdc25 and positive regulation of Mik1 ensure the effective operation of the DNA damage checkpoint.     

Proportion estimation with confidence limits

A common task in microbiology involves determining the composition of a mixed population of individuals by drawing a sample from the population and using some procedure to identify the individuals in the sample. There may be a significant probability that the identification procedure misidentifies some members of the sample (for example, because the available data are insufficient unambiguously to identify an individual) which makes finding the proportions in the underlying population non-trivial. A further complication arises where individuals are present in the population that do not belong to any of the subpopulations recognised by use of the identification procedure. A simple algorithm is presented to address these problems and construct a maximum likelihood estimate of the proportions, together with confidence limits. The technique is illustrated using an example drawn from flow cytometry in which phytoplankton cells are identified from flow cytometry data by an RBF neural network, and the limitations of the approach are discussed.     

Interspecific combative interactions between wood-decaying basidiomycetes

Competition is the most common type of interaction occurring between wood-decaying higher fungi. Since competition for nutrients in organic resources is effectively brought about by competition for space, the common division into interference and exploitation competition is not very appropriate. Fungal competition can be divided into primary resource capture (obtaining uncolonized resources) and secondary resource capture (combat to obtain resources already colonized by other fungi). Combative mechanisms include antagonism at a distance, hyphal interference, mycoparasitism and gross mycelial contact. Interactions can result in deadlock or replacement, and a hierarchy of combative ability can be discerned amongst fungi that inhabit particular resources, but within this hierarchy there exists intransitivity, modification of outcome by other species and abiotic variables. Interactions can dramatically alter mycelial function, and have potential as biological control agents of fungal pathogens of trees and in service timber.     

Multiphyletic origins of methylotrophy in Alphaproteobacteria,exemplified by comparative genomics of Lake Washington isolates

We sequenced the genomes of 19 methylotrophic isolates from Lake Washington, which belong to nine genera within eight families of the Alphaproteobacteria, two of the families being the newly proposed families. Comparative genomic analysis with a focus on methylotrophy metabolism classifies these strains into heterotrophic and obligately or facultatively autotrophic methylotrophs. The most persistent metabolic modules enabling methylotrophy within this group are the N‐methylglutamate pathway, the two types of methanol dehydrogenase (MxaFI and XoxF), the tetrahydromethanopterin pathway for formaldehyde oxidation, the serine cycle and the ethylmalonyl‐CoA pathway. At the same time, a great potential for metabolic flexibility within this group is uncovered, with different combinations of these modules present. Phylogenetic analysis of key methylotrophy functions reveals that the serine cycle must have evolved independently in at least four lineages of Alphaproteobacteria and that all methylotrophy modules seem to be prone to lateral transfers as well as deletions.     

Proteolytic processing of the cilium adhesin MHJ_0194 (P123J) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules

Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non‐ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C‐terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells.     

Ethical considerations in fish research

Fishes are used in a wide range of scientific studies, from conservation research with potential benefits to the species used to biomedical research with potential human benefits. Fish research can take place in both laboratories and field environments and methods used represent a continuum from non-invasive observations, handling, through to experimental manipulation. While some countries have legislation or guidance regarding the use of fish in research, many do not and there exists a diversity of scientific opinions on the sentience of fish and how we determine welfare. Nevertheless, there is a growing pressure on the scientific community to take more responsibility for the animals they work with through maximising the benefits of their research to humans or animals while minimising welfare or survival costs to their study animals. In this review, we focus primarily on the refinement of common methods used in fish research based on emerging knowledge with the aim of improving the welfare of fish used in scientific studies. We consider the use of anaesthetics and analgesics and how we mark individuals for identification purposes. We highlight the main ethical concerns facing researchers in both laboratory and field environments and identify areas that need urgent future research. We hope that this review will help inform those who wish to refine their ethical practices and stimulate thought among fish researchers for further avenues of refinement. Improved ethics and welfare of fishes will inevitably lead to increased scientific rigour and is in the best interests of both fishes and scientists.     

An Arabidopsis quiescin-sulfhydryl oxidase regulates cation homeostasis at the root symplast-xylem interface

A genetic screen of Arabidopsis 'activation-tagging' mutant collection based on tolerance to norspermidine resulted in a dominant mutant (par1-1D) with increased expression of the QSO2 gene (At1g15020), encoding a member of the quiescin-sulfhydryl oxidase (QSO) family. The par1-1D mutant and transgenic plants overexpressing QSO2 cDNA grow better than wild-type Arabidopsis in media with toxic cations (polyamines, Li(+) and Na(+)) or reduced K(+) concentrations. This correlates with a decrease in the accumulation of toxic cations and an increase in the accumulation of K(+) in xylem sap and shoots. Conversely, three independent loss-of-function mutants of QSO2 exhibit phenotypes opposite to those of par1-1D. QSO2 is mostly expressed in roots and is upregulated by K(+) starvation. A QSO2Colon, two colonsGFP fusion ectopically expressed in leaf epidermis localized at the cell wall. The recombinant QSO2 protein, produced in yeast in secreted form, exhibits disulfhydryl oxidase activity. A plausible mechanism of QSO2 action consists on the activation of root systems loading K(+) into xylem, but different from the SKOR channel, which is not required for QSO2 action. These results uncover QSOs as novel regulators of ion homeostasis.