Regulation of c-Src activity by the expression of wild-type v-Src and its kinase-dead double Y416F-K295N mutant

Active, wild-type v-Src and its kinase-dead double Y416F-K295N mutant were expressed in hamster fibroblasts. Expression of the active v-Src induced activation of endogenous c-Src and increased general protein-tyrosine phosphorylation in the infected cells. Expression of the kinase-dead mutant induced hypophosphorylation of Tyr416 of the endogenous c-Src. The inactivation of c-Src was reversible, as confirmed by in vitro kinase activity of c-Src immunoprecipitated from the kinase-dead v-Src-expressing cells. Both activation and inactivation of c-Src may be explained by direct interaction of the v-Src and c-Src that may either facilitate transphosphorylation of the regulatory Tyr416 in the activation loop, or prevent it by formation of transient dead-end complexes of the Y416F-K295N mutant with c-Src. The interaction was also indicated by co-localization of v- and c-Src proteins in immunofluorescent images of the infected cells. These results suggest that dimerization of Src plays an important role in the regulation of Src tyrosine kinase activity.     

Signature of an early genetic bottleneck in a population of Moroccan sardines (Sardina pilchardus)

Fishery assessment models meant to determine sustainability of commercial marine fish failed to predict recent stock collapses due to overexploitation. One flaw of assessment models is that they strongly rely on catch and age-composition statistics, but largely ignore the genetic background of the studied populations. We examined population genetic structure of sardine (Sardina pilchardus) in the centraleastern and northeastern Atlantic Ocean and Mediterranean Sea to aid fishery management of this heavily fished small pelagic species. We found that sardine has a striking mitochondrial control region, and sequenced a fragment of 387 bp of its 5'-end in 261 individuals collected off the coasts of Morocco (Dakhla, Tantan, Safi, Larache, and Nador), Portugal (Quarteira), Spain (Pasajes, Barcelona), and Greece (Kavala). High levels of haplotypic diversity rendered a rather unresolved NJ phylogeny. The recovered tree had no phylogeographic structuring except for the clustering of 13 individuals of Safi. In contrast, individuals grouped together according to the presence or absence of a 13-bp insertion in the sequence. Phi(ST) pairwise comparisons and molecular variance analyses supported genetic differentiation between the population of Pasajes (Bay of Biscay), and those of the Mediterranean Sea and Moroccan coast, with a contact zone around the Strait of Gibraltar. This result confirms the existence of two subspecies, S. pilchardus pilchardus and S. pilchardus sardina that were previously identified based on meristics and morphometry. Mismatch distribution analysis showed that sardine populations are expanding since the Pleistocene. Surprisingly, the population of Safi showed strong and statistically significant levels of genetic differentiation that could be related with isolation and genetic drift. Comparative analysis of the Safi population versus the rest including mismatch distributions, and a Bayesian skyline plot suggest that the Safi population likely underwent an early genetic bottleneck. The genetic singularity of the Safi population could have been responsible for the historical collapse of this sardine stock in the 1970s.     

Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge

Previously uncultured nitrite-oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira-like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira-like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 microg ml(-1). The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA-targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira-like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as 'Candidatus Nitrospira defluvii'.     

Replicability of dominant bacterial populations after long-term surfactant-enrichment in lab-scale activated sludge

Bacterial communities were examined in replicate lab-scale activated sludge reactors after a period of several months of enrichment with non-ionic nonylphenol ethoxylate (NPE) surfactants. Four sequential batch reactors were fed with synthetic sewage, two of which received additionally NPE. Small subunit rDNA-derived denaturing gel gradient electrophoresis (DGGE) profiles and 16S rDNA clone libraries were dominated by clones of Gammaproteobacteria class. Sequences of the other codominant rDNA phylotypes observed only in DGGE from NPE-amended reactors were, respectively, associated with the Group III of the Acidobacteria phylum. Intriguingly, 16S rRNA content from abundant Gammaproteobacteria cells was unexpectedly low. In addition to Acidobacteria, rRNA-derived DGGE profiles were dominated by members of the order Burkholderiales (of the Betaproteobacteria) and of the genus Sphingomonas (a member of the Alphaproteobacteria). Specific oligonucleotide probes for the selected ribotypes were designed and applied for quantitative real time polymerase chain reaction and fluorescence in situ hybridization, confirming their dominance in treated reactors. The parallel abundance of unique phylotypes in replicate reactors implies a distinctive selection of dominant organisms, which are better adapted to specialized niches in the highly selective environment.     

Enlarged processing window of plasticized wheat gluten using salicylic acid

The temperature window for the extrusion of glycerol-plasticized wheat gluten was increased by the use of salicylic acid, a known scorch retarder and radical scavenger. It was possible to extrude 30 wt % glycerol-wheat gluten films with a die-head temperature as high as 135 degrees C, rather than 95 degrees C, by incorporating only 1 wt % salicylic acid. Small effects of shear-induced heating during extrusion at the higher temperatures suggested that the acid acted as a lubricant and viscosity reducer. The latter was suggested to originate primarily from the salicylic-acid-induced reduction in the degree of protein aggregation/cross-linking, as indicated by size-exclusion high-performance liquid chromatography and chemiluminescence. Electron paramagnetic resonance spectroscopy on extruded films indicated that the beneficial effect of salicylic acid was due to its radical scavenging effect. Tensile tests on extrudates revealed that the materials produced at the substantially higher processing temperature were still ductile. The complex shear modulus increased more slowly with increasing salicylic acid content above 110-120 degrees C, indicating that the aggregation/cross-linking rate was slower with salicylic acid, that is, that it did have a scorch-retarding effect, besides yielding a lower final degree/complexity of aggregation.     

Run-Off Replication of Host-Adaptability Genes Is Associated with Gene Transfer Agents in the Genome of Mouse-Infecting Bartonella grahamii

The genus Bartonella comprises facultative intracellular bacteria adapted to mammals, including previously recognized and emerging human pathogens. We report the 2,341,328 bp genome sequence of Bartonella grahamii, one of the most prevalent Bartonella species in wild rodents. Comparative genomics revealed that rodent-associated Bartonella species have higher copy numbers of genes for putative host-adaptability factors than the related human-specific pathogens. Many of these gene clusters are located in a highly dynamic region of 461 kb. Using hybridization to a microarray designed for the B. grahamii genome, we observed a massive, putatively phage-derived run-off replication of this region. We also identified a novel gene transfer agent, which packages the bacterial genome, with an over-representation of the amplified DNA, in 14 kb pieces. This is the first observation associating the products of run-off replication with a gene transfer agent. Because of the high concentration of gene clusters for host-adaptation proteins in the amplified region, and since the genes encoding the gene transfer agent and the phage origin are well conserved in Bartonella, we hypothesize that these systems are driven by selection. We propose that the coupling of run-off replication with gene transfer agents promotes diversification and rapid spread of host-adaptability factors, facilitating host shifts in Bartonella.     

A dynamical model for the basal ganglia-thalamo-cortical oscillatory activity and its implications in Parkinson’s disease

We propose to investigate brain electrophysiological alterations associated with Parkinson’s disease through a novel adaptive dynamical model of the network of the basal ganglia, the cortex and the thalamus. The model uniquely unifies the influence of dopamine in the regulation of the activity of all basal ganglia nuclei, the self-organised neuronal interdependent activity of basal ganglia-thalamo-cortical circuits and the generation of subcortical background oscillations. Variations in the amount of dopamine produced in the neurons of the substantia nigra pars compacta are key both in the onset of Parkinson’s disease and in the basal ganglia action selection. We model these dopamine-induced relationships, and Parkinsonian states are interpreted as spontaneous emergent behaviours associated with different rhythms of oscillatory activity patterns of the basal ganglia-thalamo-cortical network. These results are significant because: (1) the neural populations are built upon single-neuron models that have been robustly designed to have eletrophysiologically-realistic responses, and (2) our model distinctively links changes in the oscillatory activity in subcortical structures, dopamine levels in the basal ganglia and pathological synchronisation neuronal patterns compatible with Parkinsonian states, this still remains an open problem and is crucial to better understand the progression of the disease.Electronic supplementary materialThe online version of this article (10.1007/s11571-020-09653-y) contains supplementary material, which is available to authorized users.     

Plasma chemistry in booted eagle (Hieraaetus pennatus) during breeding season

Most studies that have examined raptor plasma chemistry have been conducted on birds living in captivity. In this study, we describe typical plasma chemistry values indicators of body condition in free-living Booted Eagles, Hieraaetus pennatus, from Do?ana National Park (Spain). Values are compared with those of other raptors. Mean concentrations of creatinine, uric acid and urea were lower in adults than in nestlings, while glucose, DAT and AAT were lower in nestlings than in adults. Interactions of age/sex affected plasma mean levels of creatine kinase, glucose, AAT, uric acid and urea. Adult females showed significantly lower levels of creatine kinase, uric acid and urea than adult males and nestlings. Adult males had significantly higher levels of AAT than the other groups. The lowest levels of glucose and the highest levels of uric acid were found in nestling females. We think the differences in blood parameters can be explained by differences in size of species, of individuals (because of both body condition and sexual dimorphism) and diet.     

Glycoprotein production in moss bioreactors

Complex multimeric recombinant proteins such as therapeutic antibodies require a eukaryotic expression system. Transgenic plants may serve as promising alternatives to the currently favored mammalian cell lines or hybridomas. In contrast to prokaryotic systems, posttranslational modifications of plant and human proteins resemble each other largely, among those, protein N-glycosylation of the complex type. However, a few plant-specific sugar residues may cause immune reactions in humans, representing an obstacle for the broad use of plant-based systems as biopharmaceutical production hosts. The moss Physcomitrella patens represents a flexible tissue-culture system for the contained production and secretion of recombinant biopharmaceuticals in photobioreactors. The recent synthesis of therapeutic proteins as a scFv antibody fragment or the large and heavily modified complement regulator factor H demonstrate the versatility of this expression system. A uniquely efficient gene targeting mechanism can be employed to precisely engineer the glycosylation machinery for recombinant products. In this way, P. patens lines with non-immunogenic optimized glycan structures were created. Therapeutic antibodies produced in these strains exhibited antibody-dependent cellular cytotoxicity superior to the same molecules synthesized in mammalian cell lines.