Systematic Approach toward Accurate and Efficient DNA Sequencing via Nanoconfinement.

Coarse-grained modeling tools are employed to simulate the mechanics of DNA loading within a nanoscale confinement and predict semiflexible polymer conformations within the confinement, providing design recommendations for DNA-sequencing devices. A workflow is developed to quantify competing requirements of efficiency and accuracy and extract metrics that guide design optimization. The mean first-passage time for DNA loading is calculated as a function of the nanochannel geometry and the applied electric field. We analyze the interplay between the free energy of confinement and the electric potential energy in achieving high-throughput, base-pair detection. The single-read probability is investigated as informative metrics for sequencing accuracy and for sensing-strategy design. High cost, low throughput, and low accuracy have so far limited the adoption of nanochannel analysis and other long-read technologies. Our work directly addresses these limitations with a systematic approach that is scalable to long molecules and complex geometries.

Correction: Dominant-Negative Effect of a Missense Variant in the TASK-2 (KCNK5) K+ Channel Associated with Balkan Endemic Nephropathy.

[This corrects the article DOI: 10.1371/journal.pone.0156456.].

Dominant-Negative Effect of a Missense Variant in the TASK-2 (KCNK5) K+ Channel Associated with Balkan Endemic Nephropathy.

TASK-2, a member of the Two-Pore Domain (K2P) subfamily of K+ channels, is encoded by the KCNK5 gene. The channel is expressed primarily in renal epithelial tissues and a potentially deleterious missense variant in KCNK5 has recently been shown to be prevalent amongst patients predisposed to the development of Balkan Endemic Nephropathy (BEN), a chronic tubulointerstitial renal disease of unknown etiology. In this study we show that this variant (T108P) results in a complete loss of channel function and is associated with a major reduction in TASK-2 channel subunits at the cell surface. Furthermore, these mutant subunits have a suppressive or 'dominant-negative' effect on channel function when coexpressed with wild-type subunits. This missense variant is located at the extracellular surface of the M2 transmembrane helix and by using a combination of structural modelling and further functional analysis we also show that this highly-conserved threonine residue is critical for the correct function of other K2P channels. These results therefore provide further structural and functional insights into the possible pathophysiological effects of this missense variant in TASK-2.

A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels.

Two-pore domain (K2P) K(+) channels are major regulators of excitability that endow cells with an outwardly rectifying background "leak" conductance. In some K2P channels, strong voltage-dependent activation has been observed, but the mechanism remains unresolved because they lack a canonical voltage-sensing domain. Here, we show voltage-dependent gating is common to most K2P channels and that this voltage sensitivity originates from the movement of three to four ions into the high electric field of an inactive selectivity filter. Overall, this ion-flux gating mechanism generates a one-way "check valve" within the filter because outward movement of K(+) induces filter opening, whereas inward movement promotes inactivation. Furthermore, many physiological stimuli switch off this flux gating mode to convert K2P channels into a leak conductance. These findings provide insight into the functional plasticity of a K(+)-selective filter and also refine our understanding of K2P channels and the mechanisms by which ion channels can sense voltage.

Influence of lipids on the hydrophobic barrier within the pore of the TWIK-1 K2P channel.

Several recent ion channel structures have revealed large side portals, or 'fenestrations' at the interface between their transmembrane helices that potentially expose the ion conduction pathway to the lipid core of the bilayer. In a recent study we demonstrated that functional activity of the TWIK-1 K2P channel is influenced by the presence of hydrophobic residues deep within the inner pore. These residues are located near the fenestrations in the TWIK-1 structure and promote dewetting of the pore by forming a hydrophobic barrier to ion conduction. During our previous MD simulations, lipid tails were observed to enter these fenestrations. In this addendum to that study, we investigate lipid contribution to the dewetting process. Our results demonstrate that lipid tails from both the upper and lower leaflets can occupy the fenestrations and partially penetrate into the pore. The lipid tails do not sterically occlude the pore, but there is an inverse correlation between the presence of water within the hydrophobic barrier and the number of lipids tails within the lining of the pore. However, dewetting still occurs in the absence of lipids tails, and pore hydration appears to be determined primarily by those side-chains lining the narrowest part of the pore cavity.

Exogenous α-synuclein decreases raft partitioning of Cav2.2 channels inducing dopamine release.

α-Synuclein is thought to regulate neurotransmitter release through multiple interactions with presynaptic proteins, cytoskeletal elements, ion channels, and synaptic vesicles membrane. α-Synuclein is abundant in the presynaptic compartment, and its release from neurons and glia has been described as responsible for spreading of α-synuclein-derived pathology. α-Synuclein-dependent dysregulation of neurotransmitter release might occur via its action on surface-exposed calcium channels. Here, we provide electrophysiological and biochemical evidence to show that α-synuclein, applied to rat neurons in culture or striatal slices, selectively activates Cav2.2 channels, and said activation correlates with increased neurotransmitter release. Furthermore, in vivo perfusion of α-synuclein into the striatum also leads to acute dopamine release. We further demonstrate that α-synuclein reduces the amount of plasma membrane cholesterol and alters the partitioning of Cav2.2 channels, which move from raft to cholesterol-poor areas of the plasma membrane. We provide evidence for a novel mechanism through which α-synuclein acts from the extracellular milieu to modulate neurotransmitter release and propose a unifying hypothesis for the mechanism of α-synuclein action on multiple targets: the reorganization of plasma membrane microdomains.

A hydrophobic barrier deep within the inner pore of the TWIK-1 K2P potassium channel.

Recent X-ray crystal structures of the two-pore domain (K2P) family of potassium channels have revealed a unique structural architecture at the point where the cytoplasmic bundle-crossing gate is found in most other tetrameric K(+) channels. However, despite the apparently open nature of the inner pore in the TWIK-1 (K2P1/KCNK1) crystal structure, the reasons underlying its low levels of functional activity remain unclear. In this study, we use a combination of molecular dynamics simulations and functional validation to demonstrate that TWIK-1 possesses a hydrophobic barrier deep within the inner pore, and that stochastic dewetting of this hydrophobic constriction acts as a major barrier to ion conduction. These results not only provide an important insight into the mechanisms which control TWIK-1 channel activity, but also have important implications for our understanding of how ion permeation may be controlled in similar ion channels and pores.

Physico-chemical characterization and biological evaluation of two fibroin materials.

Silk fibroin fibres from two different sources, Bombyx mori pure-breed silkworms and polyhybrid cross-bred silkworm cocoons, were treated with formic acid under planar stirring conditions to prepare non-woven nets. The treatment partially dissolved the fibres, which bound together and formed a non-woven micrometric net with fibres coated by a thin layer of low molecular weight fibroin matrix. The starting fibres, net materials and fibroin coating layer were characterized in terms of amino acid composition, molecular weight and calorimetric properties. In vitro cell culture tests with rat fibroblasts were performed to investigate cell proliferation, morphology and spreading. Moreover, host-rat fibroblasts were preseeded on the afore-mentioned nets and implanted in the thorax of rats for histological analysis. In spite of the chemical differences between the two starting fibroins, the response of the said materials in vitro and in vivo were very similar. These results suggest that the outcome is likely correlated with the modification of the processing technique; that during the formation of the net, a thin gel layer of similar amino acid composition was formed on the fibroin fibres.

Endogenous cholinergic tone modulates spontaneous network level neuronal activity in primary cortical cultures grown on multi-electrode arrays.

BACKGROUND: Cortical cultures grown long-term on multi-electrode arrays (MEAs) are frequently and extensively used as models of cortical networks in studies of neuronal firing activity, neuropharmacology, toxicology and mechanisms underlying synaptic plasticity. However, in contrast to the predominantly asynchronous neuronal firing activity exhibited by intact cortex, electrophysiological activity of mature cortical cultures is dominated by spontaneous epileptiform-like global burst events which hinders their effective use in network-level studies, particularly for neurally-controlled animat ('artificial animal') applications. Thus, the identification of culture features that can be exploited to produce neuronal activity more representative of that seen in vivo could increase the utility and relevance of studies that employ these preparations. Acetylcholine has a recognised neuromodulatory role affecting excitability, rhythmicity, plasticity and information flow in vivo although its endogenous production by cortical cultures and subsequent functional influence upon neuronal excitability remains unknown. RESULTS: Consequently, using MEA electrophysiological recording supported by immunohistochemical and RT-qPCR methods, we demonstrate for the first time, the presence of intrinsic cholinergic neurons and significant, endogenous cholinergic tone in cortical cultures with a characterisation of the muscarinic and nicotinic components that underlie modulation of spontaneous neuronal activity. We found that tonic muscarinic ACh receptor (mAChR) activation affects global excitability and burst event regularity in a culture age-dependent manner whilst, in contrast, tonic nicotinic ACh receptor (nAChR) activation can modulate burst duration and the proportion of spikes occurring within bursts in a spatio-temporal fashion. CONCLUSIONS: We suggest that the presence of significant endogenous cholinergic tone in cortical cultures and the comparability of its modulatory effects to those seen in intact brain tissues support emerging, exploitable commonalities between in vivo and in vitro preparations. We conclude that experimental manipulation of endogenous cholinergic tone could offer a novel opportunity to improve the use of cortical cultures for studies of network-level mechanisms in a manner that remains largely consistent with its functional role.

TRAIL up-regulation must be accompanied by a reciprocal PKCε down-regulation during differentiation of colonic epithelial cell: implications for colorectal cancer cell differentiation.

PKC isoenzymes play central roles in various cellular signalling pathways, participating in a variety of protein phosphorylation cascades that regulate/modulate cellular structure and gene expression. It has been firmly established that several isoforms of PKC have a role in the regulation of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) activity. Our interest in probing the role of the epsilon isoform of PKC in the colonic cell differentiation stems from the discovery that PKCε and TRAIL are involved in the differentiation of other cell types like hematopoietic stem cells. Although the role of PKCε and TRAIL in the gastrointestinal system is unclear, it has been observed that PKCε has oncogenic activity in colon epithelial cells (CEC), while TRAIL increases the death of intestinal epithelial cells during inflammation. Here we demonstrate a reciprocal expression of PKCε and TRAIL in human colon mucosa: CECs at the bottom of the colonic crypts show high levels of PKCε, being negative for TRAIL expression. On the contrary, luminal CECs are positive for TRAIL, while negative for PKCε. Indeed, TRAIL- and butyrate-induced differentiation of the human colorectal cancer cell line HT29 requires the decrease of PKCε expression, whose absence in turn increases cell sensitivity to TRAIL-induced apoptosis. Moreover, TRAIL preferentially promotes HT29 differentiation into goblet cells. Taken together, this data demonstrate that TRAIL and PKCε must be reciprocally regulated to ensure physiological CEC differentiation starting from the stem cell pool, and that the down-regulation of PKCε is however critical for the differentiation and apoptosis of cancer cells.

Inhibition of synaptic transmission and G protein modulation by synthetic CaV2.2 Ca²+ channel peptides.

Modulation of presynaptic voltage-dependent Ca2+ channels is a major means of controlling neurotransmitter release. The CaV2.2Ca2+ channel subunit contains several inhibitory interaction sites for Gßγ subunits, including the amino terminal (NT) and I-II loop. The NT and I-II loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV2 channel activity. Here, we investigate the effects of an amino terminal (CaV2.2[45-55]) 'NT peptide' and a I-II loop alpha interaction domain (CaV2.2[377-393]) 'AID peptide' on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV2.2 amino terminal and I-II loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation.

LXR as a novel antithrombotic target.

Liver X receptors (LXRs) are transcription factors involved in the regulation of cholesterol homeostasis. LXR ligands have athero-protective properties independent of their effects on cholesterol metabolism. Platelets are involved in the initiation of atherosclerosis and despite being anucleate express nuclear receptors. We hypothesized that the athero-protective effects of LXR ligands could be in part mediated through platelets and therefore explored the potential role of LXR in platelets. Our results show that LXR-ß is present in human platelets and the LXR ligands, GW3965 and T0901317, modulated nongenomically platelet aggregation stimulated by a range of agonists. GW3965 caused LXR to associate with signaling components proximal to the collagen receptor, GPVI, suggesting a potential mechanism of LXR action in platelets that leads to diminished platelet responses. Activation of platelets at sites of atherosclerotic lesions results in thrombosis preceding myocardial infarction and stroke. Using an in vivo model of thrombosis in mice, we show that GW3965 has antithrombotic effects, reducing the size and the stability of thrombi. The athero-protective effects of GW3965, together with its novel antiplatelet/thrombotic effects, indicate LXR as a potential target for prevention of athero-thrombotic disease.

Quantitative analysis of EAAT4 promoter activity in neurons and astrocytes of mouse somatic sensory cortex.

EAAT4-eGFP BAC reporter transgenic adult mice were used to detect EAAT4 gene expression in individual cells of cerebral cortex, and eGFP fluorescence was measured to compare EAAT4 promoter activity in different cells. Most eGFP+ cells were neurons; only rare GFAP+ profiles were eGFP+. About 10% of NeuN+ cells was eGFP+, and the percentage of NeuN/eGFP co-localization varied from 2 to 20% of NeuN+ cells throughout cortical layers: layers I and II-III showed the highest values of co-localization, layer IV the lowest. The intensity of eGFP fluorescence did not exhibit laminar variations. Finally, we observed that EAAT4 promoter activity in cortical neurons was 10% of that measured in cerebellar Purkinje cells, i.e., the cells displaying the highest intensity in the CNS. These results extend our knowledge on EAAT4 expression in the cerebral cortex of adult mice, and suggest that the role of EAAT4 in cortical glutamatergic transmission may be more important than previously thought.

A role for GABAA receptors in the modulation of Paramecium swimming behavior.

The presence in Paramecium of gamma-aminobutyric acid A-type receptors (GABA(A)) and the capability of the protozoon to synthesize and release the GABA neurotransmitter into the environment have already been demonstrated. This study investigates the involvement of the GABA(A) complex in the swimming control of the ciliated protozoon. The GABA(A) receptors were pharmacologically activated by the selective agonist muscimol and the effect on Paramecium primaurelia swimming behavior was analyzed. Paramecium normally swims forward, but the activation of GABA(A) receptors induced a peculiar response, characterized by alternate periods of whirling and forward swim. This effect was inhibited by the GABA(A) selective antagonists bicuculline and picrotoxin in a dose-dependent manner. Moreover, the application of benzodiazepines did not enhance the agonist action but only reduced it. Response to muscimol was also suppressed by nimodipine, a selective antagonist of dihydropyridine-sensitive calcium channels. This inhibition suggests that an inflow of calcium ions through L-type channels mediates the muscimol-induced swimming behavior.

Activation of gamma-aminobutyric acid GAT-1 transporters on glutamatergic terminals of mouse spinal cord mediates glutamate release through anion channels and by transporter reversal.

The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels.

Immunodetection of the expression of microsomal proteins encoded by the glucose 6-phosphate transporter gene.

Glucose 6-phosphate transport has been well characterized in liver microsomes. The transport is required for the functioning of the glucose-6-phosphatase enzyme that is situated in the lumen of the hepatic endoplasmic reticulum. The genetic deficiency of the glucose 6-phosphate transport activity causes a severe metabolic disease termed type 1b glycogen storage disease. The cDNA encoding a liver transporter for glucose 6-phosphate was cloned and was found to be mutated in patients suffering from glycogen storage disease 1b. While related mRNAs have been described in liver and other tissues, the encoded protein(s) has not been immunologically characterized yet. In the present study, we report (using antibodies against three different peptides of the predicted amino acid sequence) that a major protein encoded by the glucose 6-phosphate transporter gene is expressed in the endoplasmic reticulum membranes of rat and human liver. The protein has an apparent molecular mass of approx. 33 kDa using SDS/PAGE, but several lines of evidence indicate that its real molecular mass is 46 kDa, as expected. The glucose 6-phosphate transporter protein was also immunodetected in kidney microsomes, but not in microsomes derived from human fibrocytes, rat spleen and lung, and a variety of cell lines. Moreover, little or no expression of the glucose 6-phosphate transporter protein was found in liver microsomes obtained from three glycogen storage disease 1b patients, even bearing mutations that do not directly interfere with protein translation, which can be explained by a (proteasome-mediated) degradation of the mutated transporter.

DNA damage in tissues of rat treated with potassium canrenoate.

Potassium canrenoate (PC), a competitive aldosterone antagonist previously found to increase tumor incidence in rats and to produce genotoxic effects in in vitro systems, was examined in rats to acquire information on its genotoxic activity in vivo. Intragastric administration of 1/2 LD50 produced, as revealed by the Comet assay, a modest but statistically significant increase in the frequency of DNA lesions in liver but not in thyroid and bone marrow of male rats, and in thyroid and bone marrow but not in liver of female rats. In contrast with the frankly positive responses observed in primary cultures of rat hepatocytes (Martelli et al., Mutagenesis 14 (1999) 463-472) any evidence of DNA repair and micronuclei formation was absent in liver of rats treated with 1/2 LD50, and initiation of enzyme-altered liver preneoplastic lesions did not occur in the liver of rats given 100 mg/kg PC once a week for 6 successive weeks. A high and dose-dependent frequency of DNA lesions was found to occur in testes and ovaries of rats given single doses ranging from 1/8 to 1/2 LD50.