How Old are Colonizing Hypothenemus hampei (Ferrari) Females When They Leave the Native Coffee Fruit?

The age of colonizing Hypothenemus hampei (Ferrari) females when they leave the native coffee fruit was determined under laboratory conditions. This biological parameter might be useful for planning experiments with this species because certain physiological statuses are expressed from or until a determined age, which in turn may determine the response of individuals to different treatments. An experimental device was used to simulate the conditions inside a coffee berry and to permit the observation of the abandonment behavior of the beetles. Virgin and mated females with or without melanized cuticles were used in the experiments. On average, colonizing coffee berry borers were 15-days-old at the moment of host abandonment. Females at this age were mated, had full dark cuticles, and were able to display flight and lay viable eggs. Interestingly, H. hampei females that mated before they acquire a fully dark cuticle abandoned the host 1.7 times faster than females that mated after they reach this physiological status. Further studies into food conditions and their impacts on the pre- abandonment of H. hampei females are encouraged.     

96-well microtiter plates for biofouling simulation in biomedical settings

Microtiter plates with 96 wells are routinely used in biofilm research mainly because they enable high-throughput assays. These platforms are used in a variety of conditions ranging from static to dynamic operation using different shaking frequencies and orbital diameters. The main goals of this work were to assess the influence of nutrient concentration and flow conditions on biofilm formation by Escherichia coli in microtiter plates and to define the operational conditions to be used in order to simulate relevant biomedical scenarios. Assays were performed in static mode and in incubators with distinct orbital diameters using different concentrations of glucose, peptone and yeast extract. Computational fluid dynamics (CFD) was used to simulate the flow inside the wells for shaking frequencies ranging from 50 to 200?rpm and orbital diameters from 25 to 100?mm. Higher glucose concentrations enhanced adhesion of E. coli in the first 24?h, but variation in peptone and yeast extract concentration had no significant impact on biofilm formation. Numerical simulations indicate that 96-well microtiter plates can be used to simulate a variety of biomedical scenarios if the operating conditions are carefully set.     

Virus‐induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation

We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so‐called virus‐induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5′ end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3′ end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3′ end, and an overall increase in CG methylation in the 5′ end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced.     

How much can history constrain adaptive evolution? A real‐time evolutionary approach of inversion polymorphisms in Drosophila subobscura

Chromosomal inversions are present in a wide range of animals and plants, having an important role in adaptation and speciation. Although empirical evidence of their adaptive value is abundant, the role of different processes underlying evolution of chromosomal polymorphisms is not fully understood. History and selection are likely to shape inversion polymorphism variation to an extent yet largely unknown. Here, we perform a real‐time evolution study addressing the role of historical constraints and selection in the evolution of these polymorphisms. We founded laboratory populations of Drosophila subobscura derived from three locations along the European cline and followed the evolutionary dynamics of inversion polymorphisms throughout the first 40 generations. At the beginning, populations were highly differentiated and remained so throughout generations. We report evidence of positive selection for some inversions, variable between foundations. Signs of negative selection were more frequent, in particular for most cold‐climate standard inversions across the three foundations. We found that previously observed convergence at the phenotypic level in these populations was not associated with convergence in inversion frequencies. In conclusion, our study shows that selection has shaped the evolutionary dynamics of inversion frequencies, but doing so within the constraints imposed by previous history. Both history and selection are therefore fundamental to predict the evolutionary potential of different populations to respond to global environmental changes.     

Wohlfahrtiimonas larvae sp. nov., isolated from the larval gut of Hermetia illucens (Diptera: Stratiomyidae)

A novel, Gram-negative, facultative anaerobic, motile and short rod-shaped bacterium, strain KBL006^T was isolated from the larval gut of Hermetia illucens, Black soldier fly. The 16S rRNA gene sequence of strain KBL006^T showed 96.4 % similarity to that of Wohlfahrtiimonas chitiniclastica S5^T. Strain KBL006^T grew optimally at 30 °C, at pH 8.0 and in the presence of 1–2 % (w/v) NaCl. Oxidase activity and catalase activity were positive. The major fatty acids were C_18:1 ω7c, C_14:0, and C_16:0. The major respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol, and two phospholipids. The G+C content of the genomic DNA was 45.2 mol%. Based on these polyphasic data, strain KBL006^T is considered to represent a novel species in the genus Wohlfahrtiimonas, for which the name Wohlfahrtiimonas larvae sp. nov. is proposed. The type strain is KBL006^T (= KACC 16839^T = JCM 18424^T).     

LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl⁻-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser⁷³⁷ in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl⁻ secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser⁷³⁷ mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl⁻ channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl⁻ channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.     

Physiology of Lichtheimia ramosa obtained by solid-state bioprocess using fruit wastes as substrate

Due to the amount of nutrients available in the agroindustrial wastes, these can be converted into high added-value products by the action of microorganisms in solid-state bioprocesses. The aim of this work was to evaluate the growth physiology and lipase production of the fungus Lichtheimia ramosa using the following Brazilian savannah fruit wastes as substrates: bocaiuva (Acrocomia aculeata), pequi (Caryocar brasiliense), guavira (Campomanesia pubescens), araticum (Annona crassiflora) and seriguela (Spondias purpurea). These residues were triturated, homogenized, adjusted to pH 5.0 and 60 % moisture, sterilized and packaged in plastic tray-type bioreactors before inoculation with 10 % (w/v) of L. ramosa pre-culture medium. The cultivations were conducted in a bacteriological incubator at 30 °C for 40 days. Samples were taken every 5 days and fungi and bacteria contents, proximate composition and lipase activity were evaluated. The maximum fungal counting was observed between 25 and 35 days. L. ramosa reached the stationary phase next to 40 days in all substrates. Mesophilic and psicrophilic aerobic bacteria were not detected. Protein enrichment was obtained for all media, being superior in seriguela residues (391.66 %), followed by pequi (160.04 %), araticum (143.31 %), guavira (102.42 %), and bocaiuva (67.88 %). Lipase production was observed in all cultivated media, except in pequi residues that showed decreasing lipase activity. The higher production was observed in guavira (1.12 U/g) followed by araticum (0.58 U/g), seriguela (0.41 U/g) and bocaiuva (0.21 U/g) waste substrates. It was concluded that the studied fruit wastes have been successfully utilized as substrates for protein enrichment and lipase production with L. ramosa.     

Non-typeable pneumococci circulating in Portugal are of cps type NCC2 and have genomic features typical of encapsulated isolates

Background

Pneumococcus is a major human pathogen and the polysaccharide capsule is considered its main virulence factor. Nevertheless, strains lacking a capsule, named non-typeable pneumococcus (NT), are maintained in nature and frequently colonise the human nasopharynx. Interest in these strains, not targeted by any of the currently available pneumococcal vaccines, has been rising as they seem to play an important role in the evolution of the species. Currently, there is a paucity of data regarding this group of pneumococci. Also, questions have been raised on whether they are true pneumococci. We aimed to obtain insights in the genetic content of NT and the mechanisms leading to non-typeability and to genetic diversity.

Results

A collection of 52 NT isolates representative of the lineages circulating in Portugal between 1997 and 2007, as determined by pulsed-field gel electrophoresis and multilocus sequence typing, was analysed. The capsular region was sequenced and comparative genomic hybridisation (CGH) using a microarray covering the genome of 10 pneumococcal strains was carried out. The presence of mobile elements was investigated as source of intraclonal variation. NT circulating in Portugal were found to have similar capsular regions, of cps type NCC2, i.e., having aliB-like ORF1 and aliB-like ORF2 genes. The core genome of NT was essentially similar to that of encapsulated strains. Also, competence genes and most virulence genes were present. The few virulence genes absent in all NT were the capsular genes, type-I and type-II pili, choline-binding protein A (cbpA/pspC), and pneumococcal surface protein A (pspA). Intraclonal variation could not be entirely explained by the presence of prophages and other mobile elements.

Conclusions

NT circulating in Portugal are a homogeneous group belonging to cps type NCC2. Our observations support the theory that they are bona-fide pneumococcal isolates that do not express the capsule but are otherwise essentially similar to encapsulated pneumococci. Thus we propose that NT should be routinely identified and reported in surveillance studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-863) contains supplementary material, which is available to authorized users.     

Glial cell type-specific responses to menadione-induced oxidative stress

Glial cell types in the central nervous system are continuously exposed to reactive oxygen species (ROS) due to their high oxygen metabolism and demonstrate differential susceptibility to certain pathological conditions believed to involve oxidative stress. The purpose of the current studies was to test the hypothesis that mtDNA damage could contribute to the differential susceptibility of glial cell types to apoptosis induced by oxidative stress. Primary cultures of rat astrocytes, oligodendrocytes, and microglia were utilized, and menadione was used to produce the oxidative stress. Apoptosis was detected and quantitated in menadione-treated oligodendrocytes and microglia (but not astrocytes) using either positive annexin-V staining or positive staining for 3'-OH groups in DNA. The apoptotic pathway that was activated involved the release of cytochrome c from the intermitochondrial space and activation of caspase 9. Caspase 8 was not activated after exposure to menadione in any of the cells. Using equimolar concentrations of menadione, more initial damage was observed in mtDNA from oligodendrocytes and microglia. Additionally, using concentrations of menadione that resulted in comparable initial mtDNA damage, more efficient repair was observed in astrocytes compared to either oligodendrocytes or microglia. The differential susceptibility of glial cell types to oxidative damage and apoptosis did not appear related to cellular antioxidant capacity, because under the current culture conditions astrocytes had lower total glutathione content and superoxide dismutase activity than oligodendrocytes and microglia. These results show that the differential susceptibility of glial cell types to menadione-induced oxidative stress and apoptosis appears to correlate with increased oxidative mtDNA damage and support the hypothesis that mtDNA damage could participate in the initiation of apoptosis through the enhanced release of cytochrome c and the activation of caspase 9.     

The isolation of structural genes from libraries of eucaryotic DNA

We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of β-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked β-globin genes.