GENETIC ADAPTATION AND ACCLIMATION OF PHYTOPLANKTON ALONG A STRESS GRADIENT IN THE EXTREME WATERS OF THE AGRIO RIVER–CAVIAHUE LAKE (ARGENTINA)1

We tested if different adaptation strategies were linked to a stress gradient in phytoplankton cells. For this purpose, we studied the adaptation and acclimation of Dictyosphaerium chlorelloides (Naumann) Komárek et Perman (Chlorophyta) and Microcystis aeruginosa (Kütz.) Kütz. (Cyanobacteria) to different water samples (from extremely acid, metal‐rich water to moderate stressful conditions) of the Agrio River–Caviahue Lake system (Neuquén, Argentina). Both experimental strains were isolated from pristine, slightly alkaline waters. To distinguish between physiological acclimation and genetic adaptation (an adaptive evolution event), a modified Luria‐Delbrück fluctuation analysis was carried out with both species by using as selective agent sample waters from different points along the stress gradient. M. aeruginosa did not acclimate to any of the waters tested from different points along the stress gradient nor did D. chlorelloides to the two most acidic and metal‐rich waters. However, D. chlorelloides proliferated by rapid genetic adaptation, as the consequence of a single mutation (5.4 × 10^?7 resistant mutants per cell per division) at one locus, in less extreme water and also by acclimation in the least extreme water. It is hypothesized that the stress gradient resulted in different strategies of adaptation in phytoplankton cells from nonextreme waters. Thus, very extreme conditions were lethal for both organisms, but as stressful conditions decreased, adaptation of D. chlorelloides cells was possible by the selection of resistant mutants, and in less extreme conditions, by acclimation.     

A Splicing Mutation in the Novel Mitochondrial Protein DNAJC11 Causes Motor Neuron Pathology Associated with Cristae Disorganization,and Lymphoid Abnormalities in Mice

Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.     

Recent changes in the distribution of carboxylesterase genes and associated chromosomal rearrangements in Greek populations of the tobacco aphid Myzus persicae nicotianae

We present data on the frequency of amplified E4 and FE4 carboxylesterase genes in Myzus persicae s.l. clones collected during the years 2002–2007 and 2012 in Greece. Most clones were of the tobacco aphid, Myzus persicae nicotianae. Samples from 2012 were genotyped with microsatellite DNA markers and a number of them were karyotyped. Aphid clones with amplified FE4 genes predominated in all years, whereas E4 was present in only 3.5% of all samples and always occurred in clones with FE4. Most of the clones examined showed high carboxylesterase activity levels (R2 resistant category). The results showed marked changes in the frequencies of the two carboxylesterase genes in the tobacco aphid populations compared to published data that were collected in Greece in the mid 1990s, when E4 was recorded on its own in 20% of all samples and in 32% of samples from tobacco. A parallel change in karyotype was also observed because the A1,3 translocation, which had a worldwide association with amplified E4 genes in the 1990s, was not detected in the clones analyzed in 2012. Possible causes for these changes are discussed, although selection as a result of pest management practices appears to be the major one. Novel chromosomal rearrangements were also found in M. persicae nicotianae clones. These rearrangements could be a result of clastogenic effects of nicotine, which could persist because of the holocentric nature of aphid chromosomes. The results are discussed in relation to rapid evolution events that have taken place in the tobacco aphid in Greece during the last two decades. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 455–470.     

The limit of the genetic adaptation to copper in freshwater phytoplankton

Copper is one of the most frequently used algaecides to control blooms of toxic cyanobacteria in water supply reservoirs. Among the negative impacts derived from the use of this substance is the increasing resistance of cyanobacteria to copper toxicity, as well as changes in the community structure of native phytoplankton. Here, we used the ratchet protocol to investigate the differential evolution and maximum adaptation capacity of selected freshwater phytoplankton species to the exposure of increasing doses of copper. Initially, a dose of 2.5 μM CuSO₄·5H₂O was able to completely inhibit growth in three strains of the toxic cyanobacterium Microcystis aeruginosa, whereas growth of the chlorophyceans Dictyosphaerium chlorelloides and Desmodesmus intermedius (represented by two different strains) was completely abolished at 12 μM. A significant increase in resistance was achieved in all derived populations during the ratchet experiment. All the chlorophyceans were able to adapt to up to 270 μM of copper sulfate, but 10 μM was the highest concentration that M. aeruginosa strains were able to cope with, although one of the replicates adapted to 30 μM. The recurrent use and increasing doses of copper in water reservoirs could lead to the selection of copper-resistant mutants of both chlorophyceans and cyanobacteria. However, under high concentrations of copper, the composition of phytoplankton community could undergo a drastic change with cyanobacteria being replaced by copper-resistant chlorophyceans. This result stems from a distinct evolutionary potential of these species to adapt to this substance.     

Label-Free Imaging of Lipid Depositions in C. elegans Using Third-Harmonic Generation Microscopy

Elucidation of the molecular mechanisms regulating lipid storage and metabolism is essential for mitigating excess adiposity and obesity, which has been associated with increased prevalence of severe pathological conditions such as cardiovascular disorders and type II diabetes, worldwide. However, imaging fatty acid distribution and dynamics in vivo, at the cellular or organismal level is challenging. We developed a label-free method for visualizing lipid depositions in vivo, based on third harmonic generation (THG) microscopy. THG imaging requires a single pulsed-laser light source, alleviating the technical challenges of implementing coherent anti-Stokes Raman scattering spectroscopy (CARS) to detect fat stores in living cells. We demonstrate that THG can be used to efficiently and reliably visualize lipid droplets in Caenorhabditis elegans. Thus, THG microscopy offers a versatile alternative to fluorescence and dye-based approaches for lipid biology research.     

Controlled Ultrasound-Induced Blood-Brain Barrier Disruption Using Passive Acoustic Emissions Monitoring

The ability of ultrasonically-induced oscillations of circulating microbubbles to permeabilize vascular barriers such as the blood-brain barrier (BBB) holds great promise for noninvasive targeted drug delivery. A major issue has been a lack of control over the procedure to ensure both safe and effective treatment. Here, we evaluated the use of passively-recorded acoustic emissions as a means to achieve this control. An acoustic emissions monitoring system was constructed and integrated into a clinical transcranial MRI-guided focused ultrasound system. Recordings were analyzed using a spectroscopic method that isolates the acoustic emissions caused by the microbubbles during sonication. This analysis characterized and quantified harmonic oscillations that occur when the BBB is disrupted, and broadband emissions that occur when tissue damage occurs. After validating the system''s performance in pilot studies that explored a wide range of exposure levels, the measurements were used to control the ultrasound exposure level during transcranial sonications at 104 volumes over 22 weekly sessions in four macaques. We found that increasing the exposure level until a large harmonic emissions signal was observed was an effective means to ensure BBB disruption without broadband emissions. We had a success rate of 96% in inducing BBB disruption as measured by in contrast-enhanced MRI, and we detected broadband emissions in less than 0.2% of the applied bursts. The magnitude of the harmonic emissions signals was significantly (P<0.001) larger for sonications where BBB disruption was detected, and it correlated with BBB permeabilization as indicated by the magnitude of the MRI signal enhancement after MRI contrast administration (R² = 0.78). Overall, the results indicate that harmonic emissions can be a used to control focused ultrasound-induced BBB disruption. These results are promising for clinical translation of this technology.     

Numerical analysis of electrophoretic protein patterns of Group EF-4 bacteria, predominantly from dog-bite wounds of humans

Thirty-seven strains of Group EF-4 bacteria (from various countries) were characterized by one-dimensional SDS-PAGE of cellular proteins. They comprised 21 from dog-bite wounds of humans, three from cat-bite wounds of humans and five from human limb wounds which may have been inflicted by dogs or cats; there was also one each from a pet monkey, a tiger lung (fatal), a dog tonsil, a mouse, a cat liver, a wallaby mandible, a human vagina and one from a human limb wound which was apparently not inflicted by an animal. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible and were used as the basis for three numerical analyses. In the first, in which the principal protein bands (in the 34.8 to 41.3 kD range) were excluded, the 37 Group EF-4 strains formed, at the 62% S level, two major clusters corresponding to strains producing a dihydrolase for arginine and those not doing so. In the second analysis, which included all the protein bands and which was performed only on the 22 arginine-positive strains, two phenons formed (one of which could be further divided into two sub-phenons) at the 56% S level. The third analysis, also based on all the protein bands, divided the 15 arginine-negative strains into three clusters at the 56% S level. We conclude that high resolution PAGE combined with computerised analysis of protein patterns correlates exactly with the separation of Group EF-4 into two biovars (also with the distinction of the biovars on the basis of G + C content). Reference strains of each of the PAGE types identified are available from the NCTC for inclusion in future studies.