Evaluating different methods of microarray data normalization

Background  

With the development of DNA hybridization microarray technologies, nowadays it is possible to simultaneously assess the expression levels of thousands to tens of thousands of genes. Quantitative comparison of microarrays uncovers distinct patterns of gene expression, which define different cellular phenotypes or cellular responses to drugs. Due to technical biases, normalization of the intensity levels is a pre-requisite to performing further statistical analyses. Therefore, choosing a suitable approach for normalization can be critical, deserving judicious consideration.     

A comparison of nonlethal sampling methods for amphibian gut microbiome analyses

Noninvasive sampling methods for studying intestinal microbiomes are widely applied in studies of endangered species and in those conducting temporal monitoring during manipulative experiments. Although existing studies show that noninvasive sampling methods among different taxa vary in their accuracy, no studies have yet been published comparing nonlethal sampling methods in adult amphibians. In this study, we compare microbiomes from two noninvasive sample types (faeces and cloacal swabs) to that of the large intestine in adult cane toads, Rhinella marina. We use 16S rRNA gene sequencing to investigate how microbial communities change along the digestive tract and which nonlethal sampling method better represents large intestinal microbiota. We found that cane toads' intestinal microbiota was dominated by Bacteroidetes, Proteobacteria and Firmicutes and, interestingly, we also saw a high proportion of Fusobacteria, which has previously been associated with marine species and changes in frog immunity. The large and small intestine of cane toads had a similar microbial composition, but the large intestine showed higher diversity. Our results indicate that cloacal swabs were more similar to large intestine samples than were faecal samples, and small intestine samples were significantly different from both nonlethal sample types. Our study provides valuable information for future investigations of the cane toad gut microbiome and validates the use of cloacal swabs as a nonlethal method to study changes in the large intestine microbiome. These data provide insights for future studies requiring nonlethal sampling of amphibian gut microbiota.     

Regulation of AMPA Receptor Activity, Synaptic Targeting and Recycling: Role in Synaptic Plasticity

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors for the neurotransmitter glutamate are oligomeric structures responsible for most fast excitatory responses in the central nervous system. The activity of AMPA receptors can be directly regulated by protein phosphorylation, which may also affect the interaction with intracellular proteins and, consequently, their recycling and localization to defined postsynaptic sites. This review focuses on recent advances in understanding the dynamic regulation of AMPA receptors, on a short- and long-term basis, and its implications in synaptic plasticity.     

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.     

Causes of variation in mineral soil C content and turnover in differently managed beech dominated forests

Background and aims

Forest soils are important carbon stores and considered as net CO₂ sinks over decadal to centennial time scales. Intensive forest management is thought to reduce the carbon sequestration potential of forest soils. Here we study the effects of decades of forest management (as unmanaged forest, forest under selection cutting, forest under age class management) on the turnover of mineral associated soil organic matter (MOM) in German beech (Fagus sylvatica L.) dominated forests.

Methods

Radiocarbon contents were determined by accelerator mass spectrometry (AMS) in 79 Ah horizon MOM fractions of Cambisols (n?=?13), Luvisols (n?=?51) and Stagnosols (n?=?15). Mean residence times (MRTs) for soil organic carbon (SOC) were estimated with a 2-pool model using the litter input derived from a forest inventory.

Results

MOM fractions from Ah horizons contained 64?±?8.8 % of the bulk SOC. The radiocarbon content of MOM fractions in Ah horizons, expressed as Δ¹⁴C, ranged between ?2.8?‰ and 114?‰ for the three soil groups. Almost all samples contained a detectable proportion of ‘bomb’ carbon fixed from the atmosphere since 1963. Under the assumption that depending on the soil texture between 19 % and 24 % of the SOC from the labile pool is transferred to the stable SOC pool, the corresponding MRTs ranged between 72 and 723 years, with a median of 164 years.

Conclusions

Our results indicate that the MOM fraction of Ah horizons from beech forests contained a high proportion of young carbon, but we did not find a significant decadal effect of forest management on the radiocarbon signature and related turnover times. Instead, both variables were controlled by clay contents and associated SOC concentrations (p?<?0.01). This underlines the importance of pedogenic properties for SOC turnover in the MOM fraction.     

Rotavirus VP4 and VP7-Derived Synthetic Peptides as Potential Substrates of Protein Disulfide Isomerase Lead to Inhibition of Rotavirus Infection

Rotavirus infection of MA104 cells has been shown to be inhibited by cell membrane-impermeant thiol/disulfide exchange inhibitors and anti-PDI antibodies. To characterise the amino acid sequences of rotavirus structural proteins potentially mediating cell surface PDI?Csubstrate interactions, rotavirus-derived peptides from VP4 and VP7 (RRV) and VP7 (Wa), and their modified versions containing serine instead of cysteine were synthesized. Cysteine-containing VP7 peptides corresponding to residues 189?C210 or 243?C263 caused an infectivity inhibitory effect of about 64 and 85?%, respectively, when added to cells. Changing cysteine to serine significantly decreased the inhibitory effect. A cysteine-containing peptide corresponding to VP4 residues 200?C219 and its scrambled version reduced infectivity by 92 and 80?%, respectively. A cysteine to serine change in the original VP4 200?C219 peptide did not affect its inhibitory effect. Non-rotavirus related sequences containing cysteine residues efficiently inhibited rotavirus infectivity. Antibodies against VP7 residues 189?C210 or 243?C263 significantly inhibited rotavirus infectivity only after virus attachment to cells had occurred, whereas those against VP4 200?C219 peptide inhibited infectivity irrespective of whether virus or cell-attached virus was antibody-treated. A direct PDI?Cpeptide interaction was shown by ELISA for cysteine-containing VP7 and VP4 peptides. Virus?Ccell attachment was unaffected by the peptides inhibiting virus infectivity. The results showed that even though cysteine residues in the peptides tested are important in both virus infectivity inhibition and in vitro PDI?Cpeptide interaction, the accompanying amino acid sequence also plays some role. As a whole, our findings further support our hypothesis that cell surface PDI from MA104 cells might be contributing to rotavirus entry at a post-attachment step.     

Genetic evidence for the requirement of the endocytic pathway in the uptake of coenzyme Q6 in Saccharomyces cerevisiae

Coenzyme Q is an isoprenylated benzoquinone lipid that functions in respiratory electron transport and as a lipid antioxidant. Dietary supplementation with Q is increasingly used as a therapeutic for treatment of mitochondrial and neurodegenerative diseases, yet little is known regarding the mechanism of its uptake. As opposed to other yeast backgrounds, EG103 strains are unable to import exogenous Q₆ to the mitochondria. Furthermore, the distribution of exogenous Q₆ among endomembranes suggests an impairment of the membrane traffic at the level of the endocytic pathway. This fact was confirmed after the detection of defects in the incorporation of FM4-64 marker and CPY delivery to the vacuole. A similar effect was demonstrated in double mutant strains in Q₆ synthesis and several steps of endocytic process; those cells are unable to uptake exogenous Q₆ to the mitochondria and restore the growth on non-fermentable carbon sources. Additional data about the positive effect of peptone presence for exogenous Q₆ uptake support the hypothesis that Q₆ is transported to mitochondria through an endocytic-based system.     

Evaluation of an experimental product based on Bacillus thuringiensis sorovar. israelensis against Aedes aegypti larvae (Diptera:Culicidae)

The larvicidal activity of an experimental formulation of Bacillus thuringiensis israelensis (Bti) against Aedes aegypti larvae was evaluated under laboratory and simulated field conditions (SFC). Samples of technical powder (TP) were assayed to establish the LC₅₀ and the potency of the product. The larvicidal activity of the TP and the tablet (T) were evaluated under SFC to assess the efficacy and the residual activity, measured against Ae. aegypti larvae. Either a T or 250 mg of TP were added to 50 L of water in plastic containers. Containers were exposed to sunlight or kept in the shade. Results showed a LC₅₀ of 0.26 mg/L and a potency of 750 ITU/mg. In spite of differences in the toxicity amongst TP and T samples, all of them killed 98–100% of the larvae and the mortality remained high for six months, in the shade. The replacement of 20% or 60% of the water volume did not affect the activity of the product. Seasonal differences influenced the persistence of the product in containers exposed to sunlight. Both formulations showed an excellent performance, especially when kept in the shade. The Bti tablet evaluated in this study is potentially very useful in programs to control dengue vectors.