Mitochondrial COI and morphological specificity of the mealy aphids (Hyalopterus ssp.) collected from different?hosts in Europe (Hemiptera,Aphididae)

Forty three European population samples of mealy aphids from various winter and summer host plants were attributed to respective species of Hyalopterus by means of their partial sequences of mitochondrial COI gene. Used Hyalopterus samples emerged as monophyletic relative to outgroup and formed three major clades representing three host specific mealy aphid species in the Neighbor joining, Maximum parsimony, Maximum likelihood and Bayesian inference trees. Hyalopterus pruni and Hyalopterus persikonus emerged as a sister species, whilst Hyalopterus amygdali was located basally. Samples representing different clades in the molecular trees were used for canonical discrimination analysis based on twenty two morphological characters. Length of the median dorsal head hair enabled a 97.3 % separation of Hyalopterus amygdali from the remaining two species. No single character enabled satisfactory discrimination between apterous viviparous females of Hyalopterus pruni and Hyalopterus persikonus. A modified key for the morphological identification of Hyalopterus species is suggested and their taxonomic status discussed.     

R-Type Calcium Channels Are Crucial for Semaphorin 3A–Induced DRG Axon Growth Cone Collapse

Semaphorin 3A (Sema3A) is a secreted protein involved in axon path-finding during nervous system development. Calcium signaling plays an important role during axonal growth in response to different guidance cues; however it remains unclear whether this is also the case for Sema3A. In this study we used intracellular calcium imaging to figure out whether Sema3A-induced growth cone collapse is a Ca²⁺ dependent process. Intracellular Ca²⁺ imaging results using Fura-2 AM showed Ca²⁺ increase in E15 mice dorsal root ganglia neurons upon Sema3A treatment. Consequently we analyzed Sema3A effect on growth cones after blocking or modifying intracellular and extracellular Ca²⁺ channels that are expressed in E15 mouse embryos. Our results demonstrate that Sema3A increased growth cone collapse rate is blocked by the non-selective R- and T- type Ca²⁺ channel blocker NiCl₂ and by the selective R-type Ca²⁺ channel blocker SNX482_. These Ca²⁺ channel blockers consistently decreased the Sema3A-induced intracellular Ca²⁺ concentration elevation. Overall, our results demonstrate that Sema3A-induced growth cone collapses are intimately related with increase in intracellular calcium concentration mediated by R-type calcium channels.     

Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane

DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double-stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.     

A novel lysis system in PM2, a lipid-containing marine double-stranded DNA bacteriophage

In this study we investigated the lysis system of the lipid-containing double-stranded DNA bacteriophage PM2 infecting Gram-negative marine Pseudoalteromonas species. We analysed wt and lysis-deficient phage-induced changes in the host physiology and ascribed functions to two PM2 gene products (gp) involved in lysis. We show that bacteriophage PM2 uses a novel system to disrupt the infected cell. The novelty is based on the following findings: (i) gp k is needed for the permeabilization of the cytoplasmic membrane and appears to play the role of a typical holin. However, its unique primary structure [53 aa, 1 transmembrane domain (TMD)] places it into a new class of holins. (ii) We have proposed that, unlike other bacteriophages studied, PM2 relies on lytic factors of the cellular origin for digestion of the peptidoglycan. (iii) gp l (51 aa, no TMDs) is needed for disruption of the outer membrane, which is highly rigidified by the divalent cations abundant in the marine environment. The gp l has no precedent in other phage lytic systems studied so far. However, the presence of open reading frame l-like genes in genomes of other bacterial viruses suggests that the same system might be used by other phages and is not unique to PM2.     

The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.     

Computational Optogenetics: Empirically-Derived Voltage- and Light-Sensitive Channelrhodopsin-2 Model

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q₁₀) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools.     

Influence of Plasmid Concentration on DNA Electrotransfer In Vitro Using High-Voltage and Low-Voltage Pulses

DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer experiments 50 μl of CHO cell suspension containing 100, 10 or 1 μg/ml of the plasmid were placed between plate electrodes and subjected to various combinations of HV and LV pulses. The results showed that at 100 μg/ml plasmid concentration LV pulse delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 μg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 μs) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 μg/ml and fivefold at 1 μg/ml. At 10 μg/ml concentration of plasmid, application of four LV pulses after HV pulse increased transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer can be investigated in vitro using HV and LV pulses.     

Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases

S-Adenosyl-L-methionine (AdoMet) is the major methyl donor for biological methylation reactions catalyzed by methyltransferases. We report the first chemical synthesis of AdoMet analogs with extended carbon chains replacing the methyl group and their evaluation as cofactors for all three classes of DNA methyltransferases. Extended groups containing a double or triple bond in the beta position to the sulfonium center were transferred onto DNA in a catalytic and sequence-specific manner, demonstrating a high utility of such synthetic cofactors for targeted functionalization of biopolymers.     

Spaced initial stimulus familiarization enhances novelty preference in Long-Evans rats

Berlyne [Berlyne, D.E., 1950. Novelty and curiosity as determinants of exploratory behaviour. Brit. J. Psychol. 41, 68–80] first illustrated that rats prefer to explore novel objects over ones with which they have had previous experience. Recently, variants on this novel object recognition (NOR) task have become widely popular and have been employed in numerous neuroscience and behavioral pharmacological studies investigating memory processes. Given this popularity, a thorough understanding of the various behavioral processes involved in novelty reaction and preference is essential. The current study compared the effects of spaced and massed initial stimulus exposures upon later object exploration and novel stimulus preference in Long-Evans rats. Results illustrated that a distributed initial stimulus familiarization procedure promoted greater novel object preference than did a massed procedure, and suggest that the novel object recognition task is sensitive to spacing effects in a similar fashion to more traditional learning paradigms. The mechanisms underlying such spacing effects are briefly discussed.