Direct characterization of circulating DNA in blood plasma using μLAS technology

Circulating cell-free DNA (cfDNA) is a powerful cancer biomarker for establishing targeted therapies or monitoring patients' treatment. However, current cfDNA characterization is severely limited by its low concentration, requiring the extensive use of amplification techniques. Here we report that the μLAS technology allows us to quantitatively characterize the size distribution of purified cfDNA in a few minutes, even when its concentration is as low as 1 pg/μL. Moreover, we show that DNA profiles can be directly measured in blood plasma with a minimal conditioning process to speed up considerably speed up the cfDNA analytical chain

Bilateral hypertrophic olivary nucleus degeneration on magnetic resonance imaging in children with Leigh and Leigh-like syndrome

Objective: Bilateral hypertrophic olivary degeneration on brain MRI has been reported in a few metabolic, genetic and neurodegenerative disorders, including mitochondrial disorders. In this report, we sought to analyse whether bilateral symmetrical inferior olivary nucleus hypertrophy is specifically associated with mitochondrial disorders in children. Methods: This retrospective study included 125 children (mean age, 7.6  ±  5 years; male:female, 2.6:1) diagnosed with various metabolic and genetic disorders during 2005–2012. The routine MRI sequences (T1 weighted, T2 weighted and fluid-attenuated inversion–recovery sequences) were analysed for the presence of bilateral symmetrical olivary hypertrophy and central tegmental tract or dentate nuclei signal changes. The other imaging findings and the final diagnoses were noted. Results: The cohort included patients with Leigh and Leigh-like syndrome (n = 25), other mitochondrial diseases (n = 25), Wilson disease (n = 40), Type 1 glutaric aciduria (n = 14), maple syrup urine disease (n = 13), giant axonal neuropathy (n = 5) and L-2 hydroxy glutaric aciduria (n = 3). Bilateral inferior olivary nucleus hypertrophy was noted in 10 patients, all of whom belonged to the Leigh and Leigh-like syndrome group. Conclusion: Bilateral hypertrophic olivary degeneration on MRI is relatively often, but not routinely, seen in children with Leigh and Leigh-like syndrome. Early detection of this finding by radiologists and physicians may facilitate targeted metabolic testing in these children

10 simple rules to create a serious game, illustrated with examples from structural biology

Serious scientific games are games whose purpose is not only fun. In the field of science, the serious goals include crucial activities for scientists: outreach, teaching and research. The number of serious games is increasing rapidly, in particular citizen science games, games that allow people to produce and/or analyze scientific data. Interestingly, it is possible to build a set of rules providing a guideline to create or improve serious games. We present arguments gathered from our own experience ( Phylo , DocMolecules , HiRE-RNA contest and Pangu) as well as examples from the growing literature on scientific serious games

The effect of stimulation technique on sympathetic skin responses in healthy subjects

The aim of this study was to collect normative data for sympathetic skin responses (SSR) elicited by electrical stimulus of the ipsilateral and contralateral peripheral nerves, and by magnetic stimulus of cervical cord. SSRs were measured at the mid-palm of both hands following electrical stimulation of the left median nerve at the wrist and magnetic stimulation at the neck in 40 healthy adult volunteers (mean age 52.2 ± 12.2 years, 19 males). The onset latency, peak latency, amplitude and area were estimated in “P” type responses (i.e., waveforms with a larger positive, compared to negative, component). SSR onset and peak latency were prolonged when the electrical stimulus was applied at the contralateral side (i.e., the SSR recorded in the right palm P < 0.001). The onset latency was similar on both sides during cervical magnetic stimulation. However, peak latency was faster on the left side (P < 0.03). Comparison of electrical and magnetic stimulation revealed that both the onset and peak latency were shorter with magnetic stimulation (P < 0.001). The latency of a SSR varies depending on what type of stimulation is used and where the stimulus is applied. Electrically generated SSRs have a longer delay and the delay is prolonged at the contralateral side. These factors should be taken into account when interpreting SSR data

Endogenous cholinergic inputs and local circuit mechanisms govern the phasic mesolimbic dopamine response to nicotine

Nicotine exerts its reinforcing action by stimulating nicotinic acetylcholine receptors (nAChRs) and boosting dopamine (DA) output from the ventral tegmental area (VTA). Recent data have led to a debate about the principal pathway of nicotine action: direct stimulation of the DAergic cells through nAChR activation, or disinhibition mediated through desensitization of nAChRs on GABAergic interneurons. We use a computational model of the VTA circuitry and nAChR function to shed light on this issue. Our model illustrates that the α4β2-containing nAChRs either on DA or GABA cells can mediate the acute effects of nicotine. We account for in vitro as well as in vivo data, and predict the conditions necessary for either direct stimulation or disinhibition to be at the origin of DA activity increases. We propose key experiments to disentangle the contribution of both mechanisms. We show that the rate of endogenous acetylcholine input crucially determines the evoked DA response for both mechanisms. Together our results delineate the mechanisms by which the VTA mediates the acute rewarding properties of nicotine and suggest an acetylcholine dependence hypothesis for nicotine reinforcement.Peer reviewe

Motion and Flexibility in Human Cytochrome P450 Aromatase

The crystal structures of human placental aromatase in complex with the substrate androstenedione and exemestane have revealed an androgen-specific active site and the structural basis for higher order organization. However, X-ray structures do not provide accounts of movements due to short-range fluctuations, ligand binding and protein-protein association. In this work, we conduct normal mode analysis (NMA) revealing the intrinsic fluctuations of aromatase, deduce the internal modes in membrane-free and membrane-integrated monomers as well as the intermolecular modes in oligomers, and propose a quaternary organization for the endoplasmic reticulum (ER) membrane integration. Dynamics of the crystallographic oligomers from NMA is found to be in agreement with the isotropic thermal factors from the X-ray analysis. Calculations of the root mean square fluctuations of the C-alpha atoms from their equilibrium positions confirm that the rigid-core structure of aromatase is intrinsic regardless of the changes in steroid binding interactions, and that aromatase self-association does not deteriorate the rigidity of the catalytic cleft. Furthermore, NMA on membrane-integrated aromatase shows that the internal modes in all likelihood contribute to breathing of the active site access channel. The collective intermolecular hinge bending and twisting modes provide the flexibility in the quaternary association necessary for membrane integration of the aromatase oligomers. Taken together, fluctuations of the active site, the access channel, and the heme-proximal cavity, and a dynamic quaternary organization could all be essential components of the functional aromatase in its role as an ER membrane-embedded steroidogenic enzyme

Identification of a Negative Allosteric Site on Human α4β2 and α3β4 Neuronal Nicotinic Acetylcholine Receptors

Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs). These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human α4β2 and α3β4 nicotinic acetylcholine receptor (nAChR) extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the β2 subunit were independently mutated to the corresponding residues found on the β4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human α4β2 over α3β4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 µM. These results demonstrate the selectivity of KAB-18 for human α4β2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological diseases and disorders

Direction and magnitude of nicotine effects on the fMRI BOLD response are related to nicotine effects on behavioral performance

Considerable variability across individuals has been reported in both the behavioral and fMRI blood oxygen level-dependent (BOLD) response to nicotine. We aimed to investigate (1) whether there is a heterogeneous effect of nicotine on behavioral and BOLD responses across participants and (2) if heterogeneous BOLD responses are associated with behavioral performance measures. In this double-blind, placebo-controlled, cross-over study, 41 healthy participants (19 smokers)—drawn from a larger population-based sample—performed a visual oddball task after acute challenge with 1 mg nasal nicotine. fMRI data and reaction time were recorded during performance of the task. Across the entire group of subjects, we found increased activation in the anterior cingulate cortex, middle frontal gyrus, superior temporal gyrus, post-central gyrus, planum temporal and frontal pole in the nicotine condition compared with the placebo condition. However, follow-up analyses of this difference in activation between the placebo and nicotine conditions revealed that some participants showed an increase in activation while others showed a decrease in BOLD activation from the placebo to the nicotine condition. A reduction of BOLD activation from placebo to nicotine was associated with a decrease in reaction time and reaction time variability and vice versa, suggesting that it is the direction of BOLD response to nicotine which is related to task performance. We conclude that the BOLD response to nicotine is heterogeneous and that the direction of response to nicotine should be taken into account in future pharmaco-fMRI research on the central action of nicotine

Novel Strains of Mice Deficient for the Vesicular Acetylcholine Transporter: Insights on Transcriptional Regulation and Control of Locomotor Behavior

Defining the contribution of acetylcholine to specific behaviors has been challenging, mainly because of the difficulty in generating suitable animal models of cholinergic dysfunction. We have recently shown that, by targeting the vesicular acetylcholine transporter (VAChT) gene, it is possible to generate genetically modified mice with cholinergic deficiency. Here we describe novel VAChT mutant lines. VAChT gene is embedded within the first intron of the choline acetyltransferase (ChAT) gene, which provides a unique arrangement and regulation for these two genes. We generated a VAChT allele that is flanked by loxP sequences and carries the resistance cassette placed in a ChAT intronic region (FloxNeo allele). We show that mice with the FloxNeo allele exhibit differential VAChT expression in distinct neuronal populations. These mice show relatively intact VAChT expression in somatomotor cholinergic neurons, but pronounced decrease in other cholinergic neurons in the brain. VAChT mutant mice present preserved neuromuscular function, but altered brain cholinergic function and are hyperactive. Genetic removal of the resistance cassette rescues VAChT expression and the hyperactivity phenotype. These results suggest that release of ACh in the brain is normally required to “turn down” neuronal circuits controlling locomotion

Inherent Dynamics of the Acid-Sensing Ion Channel 1 Correlates with the Gating Mechanism

A combination of computational and experimental approaches reveals the dynamics of ASIC1 gating, involving a deformation of the channel that triggers “twist-to-open” motions of the channel pore