Replication and cytopathic effect of oncolytic vesicular stomatitis virus in hypoxic tumor cells in vitro and in vivo

Tumor hypoxia presents an obstacle to the effectiveness of most antitumor therapies, including treatment with oncolytic viruses. In particular, an oncolytic virus must be resistant to the inhibition of DNA, RNA, and protein synthesis that occurs during hypoxic stress. Here we show that vesicular stomatitis virus (VSV), an oncolytic RNA virus, is capable of replication under hypoxic conditions. In cells undergoing hypoxic stress, VSV infection produced larger amounts of mRNA than under normoxic conditions. However, translation of these mRNAs was reduced at earlier times postinfection in hypoxia-adapted cells than in normoxic cells. At later times postinfection, VSV overcame a hypoxia-associated increase in alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation and initial suppression of viral protein synthesis in hypoxic cells to produce large amounts of viral protein. VSV infection caused the dephosphorylation of the translation initiation factor eIF-4E and inhibited host translation similarly under both normoxic and hypoxic conditions. VSV produced progeny virus to similar levels in hypoxic and normoxic cells and showed the ability to expand from an initial infection of 1% of hypoxic cells to spread through an entire population. In all cases, virus infection induced classical cytopathic effects and apoptotic cell death. When VSV was used to treat tumors established in nude mice, we found VSV replication in hypoxic areas of these tumors. This occurred whether the virus was administered intratumorally or intravenously. These results show for the first time that VSV has an inherent capacity for infecting and killing hypoxic cancer cells. This ability could represent a critical advantage over existing therapies in treating established tumors.     

Preparation and characterization of lyophilized liposomes with incorporated quercetin

Liposomes composed of egg-phosphatidylcholine (EPC) incorporating quercetin (QR) were prepared by the thin-film hydration method (TFHM) and the monophase solution method (MSM). A rapid and slow freeze-drying process was applied for both laboratory and industrial scales. The purpose of this study was to compare the two methods of liposome preparation, and further determine whether the lyophilization process affects the liposome physicochemical characteristics (size, polydispersity index, and ?-potential) and incorporation of quercetin.     

A computational framework for the topological analysis and targeted disruption of signal transduction networks

In this article, optimization-based frameworks are introduced for elucidating the input-output structure of signaling networks and for pinpointing targeted disruptions leading to the silencing of undesirable outputs in therapeutic interventions. The frameworks are demonstrated on a large-scale reconstruction of a signaling network composed of nine signaling pathways implicated in prostate cancer. The Min-Input framework is used to exhaustively identify all input-output connections implied by the signaling network structure. Results reveal that there exist two distinct types of outputs in the signaling network that either can be elicited by many different input combinations or are highly specific requiring dedicated inputs. The Min-Interference framework is next used to precisely pinpoint key disruptions that negate undesirable outputs while leaving unaffected necessary ones. In addition to identifying disruptions of terminal steps, we also identify complex disruption combinations in upstream pathways that indirectly negate the targeted output by propagating their action through the signaling cascades. By comparing the obtained disruption targets with lists of drug molecules we find that many of these targets can be acted upon by existing drug compounds, whereas the remaining ones point at so-far unexplored targets. Overall the proposed computational frameworks can help elucidate input/output relationships of signaling networks and help to guide the systematic design of interference strategies.     

Effect of phosphorus dendrimers on DMPC lipid membranes

Large unilamellar liposomes and multilamellar vesicles consisting of DMPC interacted with cationic phosphorus-containing dendrimers CPDs G3 and G4. DSC and ζ-potential measurements have shown that liposomal-dendrimeric molecular recognition probably occurs due to the interaction between the complementary surface groups. Calorimetric studies indicate that the enthalpy of the transition of the lipids that interact with CPDs is dependent on the dendrimers generation. These results can be used in order to rationally design mixed modulatory liposomal locked-in dendrimeric, drug delivery nano systems.     

Gene flow at major transitional areas in sea bass (Dicentrarchus labrax) and the possible emergence of a hybrid swarm

The population genetic structure of sea bass (Dicentrarchus labrax) along a transect from the Atlantic Ocean (AO) to the Eastern Mediterranean (EM) Sea differs from that of most other marine taxa in this area. Three populations (AO, Western Mediterranean [WM], EM) are recognized today, which were originally two allopatric populations. How two ancestral genetic units have evolved into three distinct units has not been addressed yet. Therefore, to investigate mechanisms that lead to the emergence of the central WM population, its current status, and its connectivity with the two parental populations, we applied 20 nuclear loci that were either gene associated or gene independent. Results confirmed the existence of three distinct gene pools, with higher differentiation at two transitional areas, the Almeria‐Oran Front (AOF) and of the Siculo‐Tunisian Strait (STS), than within any population. Significant linkage disequilibrium and heterozygote excess indicated that the STS is probably another tension zone, as already described for the AOF. Neutrality tests fail to reveal marker loci that could be driven by selection within or among metapopulations, except for locus DLA0068. Collectively, results support that the central WM population arose by trapping two tensions zones at distinct geographic locations of limited connectivity. Population assignment further revealed that WM individuals were more introgressed than individuals from the other two metapopulations. This suggests that this population might result from hybrid swarming, and was or is still seeded by genes received through the filter of each tension zone.     

Effects of adaptation, chance, and history on the evolution of the toxic dinoflagellate Alexandrium minutum under selection of increased temperature and acidification

The roles of adaptation, chance, and history on evolution of the toxic dinoflagellate Alexandrium minutum Halim, under selective conditions simulating global change, have been addressed. Two toxic strains (AL1V and AL2V), previously acclimated for two years at pH 8.0 and 20°C, were transferred to selective conditions: pH 7.5 to simulate acidification and 25°C. Cultures under selective conditions were propagated until growth rate and toxin cell quota achieved an invariant mean value at 720 days (ca. 250 and ca. 180 generations for strains AL1V and AL2V, respectively). Historical contingencies strongly constrained the evolution of growth rate and toxin cell quota, but the forces involved in the evolution were not the same for both traits. Growth rate was 1.5-1.6 times higher than the one measured in ancestral conditions. Genetic adaptation explained two-thirds of total adaptation while one-third was a consequence of physiological adaptation. On the other hand, the evolution of toxin cell quota showed a pattern attributable to neutral mutations because the final variances were significantly higher than those measured at the start of the experiment. It has been hypothesized that harmful algal blooms will increase under the future scenario of global change. Although this study might be considered an oversimplification of the reality, it can be hypothesized that toxic blooms will increase but no predictions can be advanced about toxicity.     

Alcohol Affects the Brain's Resting-State Network in Social Drinkers

Acute alcohol intake is known to enhance inhibition through facilitation of GABA_A receptors, which are present in 40% of the synapses all over the brain. Evidence suggests that enhanced GABAergic transmission leads to increased large-scale brain connectivity. Our hypothesis is that acute alcohol intake would increase the functional connectivity of the human brain resting-state network (RSN). To test our hypothesis, electroencephalographic (EEG) measurements were recorded from healthy social drinkers at rest, during eyes-open and eyes-closed sessions, after administering to them an alcoholic beverage or placebo respectively. Salivary alcohol and cortisol served to measure the inebriation and stress levels. By calculating Magnitude Square Coherence (MSC) on standardized Low Resolution Electromagnetic Tomography (sLORETA) solutions, we formed cortical networks over several frequency bands, which were then analyzed in the context of functional connectivity and graph theory. MSC was increased (p<0.05, corrected with False Discovery Rate, FDR corrected) in alpha, beta (eyes-open) and theta bands (eyes-closed) following acute alcohol intake. Graph parameters were accordingly altered in these bands quantifying the effect of alcohol on the structure of brain networks; global efficiency and density were higher and path length was lower during alcohol (vs. placebo, p<0.05). Salivary alcohol concentration was positively correlated with the density of the network in beta band. The degree of specific nodes was elevated following alcohol (vs. placebo). Our findings support the hypothesis that short-term inebriation considerably increases large-scale connectivity in the RSN. The increased baseline functional connectivity can -at least partially- be attributed to the alcohol-induced disruption of the delicate balance between inhibitory and excitatory neurotransmission in favor of inhibitory influences. Thus, it is suggested that short-term inebriation is associated, as expected, to increased GABA transmission and functional connectivity, while long-term alcohol consumption may be linked to exactly the opposite effect.     

The Peripheral Binding of 14-3-3γ to Membranes Involves Isoform-Specific Histidine Residues

Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.     

Rapid adaptation of phytoplankters to geothermal waters is achieved by single mutations: were extreme environments 'Noah's Arks' for photosynthesizers during the Neoproterozoic 'snowball Earth'?

Geothermal waters often support remarkable communities of microalgae and cyanobacteria apparently living at the extreme limits of their tolerance. Little is known about the mechanisms allowing adaptation of mesophilic phytoplankters to such extreme conditions, but recent studies are challenging many preconceived notions about this. The aim of this study was to analyse mechanisms allowing adaptation of mesophilic microalgae and cyanobacteria to stressful geothermal waters. To distinguish between the pre-selective or post-selective origin of adaptation processes allowing the proliferation of mesophilic phytoplankters in geothermal waters, several Luria-Delbrück fluctuation analysis were performed with the microalga Dictyosphaerium chlorelloides and the cyanobacterium Microcystis aeruginosa, both isolated from nonextreme waters. Geothermal waters from seven places in Italy and five icebound places at Los Andes in Argentina were used as selective agents. Physiological adaptation was achieved in the least toxic waters. In contrast, rapid genetic adaptation was observed in waters ostensibly lethal for the experimental organisms. This adaptation was achieved as consequence of single mutations at one locus. It was hypothesized that a similar mechanism of rapid genetic adaptation could explain the survival of photosynthetic life during the Neoproterozoic 'snowball Earth,' where geothermal refuges such as those studied could have been 'Noah's Arks' for microalgae and cyanobacteria.