Free energy estimation of short DNA duplex hybridizations

Background  

Estimation of DNA duplex hybridization free energy is widely used for predicting cross-hybridizations in DNA computing and microarray experiments. A number of software programs based on different methods and parametrizations are available for the theoretical estimation of duplex free energies. However, significant differences in free energy values are sometimes observed among estimations obtained with various methods, thus being difficult to decide what value is the accurate one.     

The origin of animals: an ancestral reconstruction of the unicellular-to-multicellular transition

How animals evolved from a single-celled ancestor, transitioning from a unicellular lifestyle to a coordinated multicellular entity, remains a fascinating question. Key events in this transition involved the emergence of processes related to cell adhesion, cell–cell communication and gene regulation. To understand how these capacities evolved, we need to reconstruct the features of both the last common multicellular ancestor of animals and the last unicellular ancestor of animals. In this review, we summarize recent advances in the characterization of these ancestors, inferred by comparative genomic analyses between the earliest branching animals and those radiating later, and between animals and their closest unicellular relatives. We also provide an updated hypothesis regarding the transition to animal multicellularity, which was likely gradual and involved the use of gene regulatory mechanisms in the emergence of early developmental and morphogenetic plans. Finally, we discuss some new avenues of research that will complement these studies in the coming years.     

The MrCYP52 cytochrome P450 monoxygenase gene of Metarhizium robertsii is important for utilizing insect epicuticular hydrocarbons

Fungal pathogens of plants and insects infect their hosts by direct penetration of the cuticle. Plant and insect cuticles are covered by a hydrocarbon-rich waxy outer layer that represents the first barrier against infection. However, the fungal genes that underlie insect waxy layer degradation have received little attention. Here we characterize the single cytochrome P450 monoxygenase family 52 (MrCYP52) gene of the insect pathogen Metarhizium robertsii, and demonstrate that it encodes an enzyme required for efficient utilization of host hydrocarbons. Expressing a green florescent protein gene under control of the MrCYP52 promoter confirmed that MrCYP52 is up regulated on insect cuticle as well as by artificial media containing decane (C10), extracted cuticle hydrocarbons, and to a lesser extent long chain alkanes. Disrupting MrCYP52 resulted in reduced growth on epicuticular hydrocarbons and delayed developmental processes on insect cuticle, including germination and production of appressoria (infection structures). Extraction of alkanes from cuticle prevented induction of MrCYP52 and reduced growth. Insect bioassays against caterpillars (Galleria mellonella) confirmed that disruption of MrCYP52 significantly reduces virulence. However, MrCYP52 was dispensable for normal germination and appressorial formation in vitro when the fungus was supplied with nitrogenous nutrients. We conclude therefore that MrCYP52 mediates degradation of epicuticular hydrocarbons and these are an important nutrient source, but not a source of chemical signals that trigger infection processes.     

Enhanced UV Resistance and Improved Killing of Malaria Mosquitoes by Photolyase Transgenic Entomopathogenic Fungi

The low survival of microbial pest control agents exposed to UV is the major environmental factor limiting their effectiveness. Using gene disruption we demonstrated that the insect pathogenic fungus Metarhizium robertsii uses photolyases to remove UV-induced cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) photoproducts [(6-4)PPs] from its DNA. However, this photorepair is insufficient to fix CPD lesions and prevent the loss of viability caused by seven hours of solar radiation. Expression of a highly efficient archaeal (Halobacterium salinarum) CPD photolyase increased photorepair >30-fold in both M. robertsii and Beauveria bassiana. Consequently, transgenic strains were much more resistant to sunlight and retained virulence against the malaria vector Anopheles gambiae. In the field this will translate into much more efficient pest control over a longer time period. Conversely, our data shows that deleting native photolyase genes will strictly contain M. robertsii to areas protected from sunlight, alleviating safety concerns that transgenic hypervirulent Metarhizium spp will spread from mosquito traps or houses. The precision and malleability of the native and transgenic photolyases allows design of multiple pathogens with different strategies based on the environments in which they will be used.     

Ischemic Postconditioning Fails to Protect against Neonatal Cerebral Stroke

The lack of efficient neuroprotective strategies for neonatal stroke could be ascribed to pathogenic ischemic processes differentiating adults and neonates. We explored this hypothesis using a rat model of neonatal ischemia induced by permanent occlusion of the left distal middle cerebral artery combined with 50 min of occlusion of both common carotid arteries (CCA). Postconditioning was performed by repetitive brief release and occlusion (30 s, 1 and/or 5 min) of CCA after 50 min of CCA occlusion. Alternative reperfusion was generated by controlled release of the bilateral CCA occlusion. Blood-flow velocities in the left internal carotid artery were measured using color-coded pulsed Doppler ultrasound imaging. Cortical perfusion was measured using laser Doppler. Cerebrovascular vasoreactivity was evaluated after inhalation with the hypercapnic gas or inhaled nitric oxide (NO). Whatever the type of serial mechanical interruptions of blood flow at reperfusion, postconditioning did not reduce infarct volume after 72 hours. A gradual perfusion was found during early re-flow both in the left internal carotid artery and in the cortical penumbra. The absence of acute hyperemia during early CCA re-flow, and the lack of NO-dependent vasoreactivity in P7 rat brain could in part explain the inefficiency of ischemic postconditioning after ischemia-reperfusion.     

Lack of host specialization in Aspergillus flavus

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22 degrees C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton balls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.     

The bumble bee microbiome increases survival of bees exposed to selenate toxicity

Bumble bees are important and widespread insect pollinators who face many environmental challenges. For example, bees are exposed to the metalloid selenate when foraging on pollen and nectar from plants growing in contaminated soils. As it has been shown that the microbiome of animals reduces metalloid toxicity, we assayed the ability of the bee microbiome to increase survivorship against selenate challenge. We exposed uninoculated or microbiota-inoculated Bombus impatiens workers to a field-realistic dose of 0.75 mg l⁻¹ selenate and found that microbiota-inoculated bees survive slightly but significantly longer than uninoculated bees. Using 16S rRNA gene sequencing, we found that selenate exposure altered gut microbial community composition and relative abundance of specific core bacteria. We also grew two core bumble bee microbes – Snodgrassella alvi and Lactobacillus bombicola – in selenate-spiked media and found that these bacteria grew in the tested concentrations of 0.001–10 mg l⁻¹ selenate. Furthermore, the genomes of these microbes harbour genes involved in selenate detoxification. The bumble bee microbiome slightly increases survivorship when the host is exposed to selenate, but the specific mechanisms and colony-level benefits under natural settings require further study.     

Low-resolution structural studies of human Stanniocalcin-1

Background  

Stanniocalcins (STCs) represent small glycoprotein hormones, found in all vertebrates, which have been functionally implicated in Calcium homeostasis. However, recent data from mammalian systems indicated that they may be also involved in embryogenesis, tumorigenesis and in the context of the latter especially in angiogenesis. Human STC1 is a 247 amino acids protein with a predicted molecular mass of 27 kDa, but preliminary data suggested its di- or multimerization. The latter in conjunction with alternative splicing and/or post-translational modification gives rise to forms described as STC₅₀ and "big STC", which molecular weights range from 56 to 135 kDa.