Scar conducting channel characterization to predict arrhythmogenicity during ventricular tachycardia ablation.

AIMS: Heterogeneous tissue channels (HTCs) detected by late gadolinium enhancement cardiac magnetic resonance (LGE-CMR) are related to ventricular arrhythmias, but there are few published data about their arrhythmogenic characteristics. METHODS AND RESULTS: We enrolled 34 consecutive patients with ischaemic and non-ischaemic cardiomyopathy who were referred for ventricular tachycardia (VT) ablation. LGE-CMR was performed prior to ablation, and the HTCs were analyzed. Arrhythmogenic HTCs linked to induced VT were identified during the VT ablation procedure. The characteristics of arrhythmogenic HTCs were compared with those of non-arrhythmogenic HTCs. Three patients were excluded due to low-quality LGE-CMR images. A total of 87 HTCs were identified on LGE-CMR in 31 patients (age:63.8 ± 12.3 years; 96.8% male; left ventricular ejection fraction: 36.1 ± 10.7%). Of the 87 HTCs, only 31 were considered arrhythmogenic because of their relation to a VT isthmus. The HTCs related to a VT isthmus were longer [64.6 ± 49.4 vs. 32.9 ± 26.6 mm; OR: 1.02; 95% CI: (1.01-1.04); P < 0.001] and had greater mass [2.5 ± 2.2 vs. 1.2 ± 1.2 grams; OR: 1.62; 95% CI: (1.18-2.21); P < 0.001], a higher degree of protectedness [26.19 ± 19.2 vs. 10.74 ± 8.4; OR 1.09; 95% CI: (1.04-1.14); P < 0.001], higher transmurality [number of wall layers with CCs: 3.8 ± 2.4 vs. 2.4 ± 2.0; OR: 1.31; 95% CI: (1.07-1.60); P = 0.008] and more ramifications [3.8 ± 2.0 vs. 2.7 ± 1.1; OR: 1.59; 95% CI: (1.15-2.19); P = 0.002] than non-arrhythmogenic HTCs. Multivariate logistic regression analysis revealed that protectedness was the strongest predictor of arrhythmogenicity. CONCLUSION: The protectedness of an HTC identified by LGE-CMR is strongly related to its arrhythmogenicity during VT ablation.

Characterization of the Features of Water Inside the SecY Translocon.

Despite extended experimental and computational studies, the mechanism regulating membrane protein folding and stability in cell membranes is not fully understood. In this review, I will provide a personal and partial account of the scientific efforts undertaken by Dr. Stephen White to shed light on this topic. After briefly describing the role of water and the hydrophobic effect on cellular processes, I will discuss the physical chemistry of water confined inside the SecY translocon pore. I conclude with a review of recent literature that attempts to answer fundamental questions on the pathway and energetics of translocon-guided membrane protein insertion.

The structure of the TOM core complex in the mitochondrial outer membrane.

In the past three decades, significant advances have been made in providing the biochemical background of TOM (translocase of the outer mitochondrial membrane)-mediated protein translocation into mitochondria. In the light of recent cryoelectron microscopy-derived structures of TOM isolated from Neurospora crassa and Saccharomyces cerevisiae, the interpretation of biochemical and biophysical studies of TOM-mediated protein transport into mitochondria now rests on a solid basis. In this review, we compare the subnanometer structure of N. crassa TOM core complex with that of yeast. Both structures reveal remarkably well-conserved symmetrical dimers of 10 membrane protein subunits. The structural data also validate predictions of weakly stable regions in the transmembrane ß-barrel domains of the protein-conducting subunit Tom40, which signal the existence of ß-strands located in interfaces of protein-protein interactions.

Anomalous behavior of water inside the SecY translocon.

The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359-E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a "primed" open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182-17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple water-filled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications.

Engineering Dirac electrons emergent on the surface of a topological insulator.

The concept of the topological insulator (TI) has introduced a new point of view to condensed-matter physics, relating a priori unrelated subfields such as quantum (spin, anomalous) Hall effects, spin-orbit coupled materials, some classes of nodal superconductors, superfluid 3He, etc. From a technological point of view, TIs are expected to serve as platforms for realizing dissipationless transport in a non-superconducting context. The TI exhibits a gapless surface state with a characteristic conic dispersion (a surface Dirac cone). Here, we review peculiar finite-size effects applicable to such surface states in TI nanostructures. We highlight the specific electronic properties of TI nanowires and nanoparticles, and in this context we contrast the cases of weak and strong TIs. We study the robustness of the surface and the bulk of TIs against disorder, addressing the physics of Dirac and Weyl semimetals as a new research perspective in the field.

Comprehensive exploration approach of coal mine water-conducting channels in urban environment: a case study in Xintai, China.

In the process of coal mining, prevention and control of water hazard is essential. It is the precondition for water hazard control to detect and determine the distribution of underground water-conducting channels. In urban environments, traditional methods such as active source seismic exploration and transient electromagnetic exploration commonly used in the field are difficult to carry out effectively due to various factors. In this paper, the microtremor survey method (MSM) and the opposing coils transient electromagnetic method (OCTEM) are adapted to conduct the surface exploration of the coal mine water-conducting channels in the urban environment. Combined with the detection results of the low-velocity area and the low-resistivity area, the distribution of water-conducting channels is preliminarily analyzed and determined, which is basically consistent with the drilling and coring results. It verifies the feasibility and accuracy of the comprehensive exploration method used in this paper.

Specificity of SecYEG for PhoA precursors and SecA homologs on SecA protein-conducting channels.

Previous studies showed that Escherichia coli membranes depleted of SecYEG are capable of translocating certain precursor proteins, but not other precursors such as pPhoA, indicating a differential requirement for SecYEG. In this study, we examined the role of SecYEG in pPhoA translocation using a purified reconstituted SecA-liposomes system. We found that translocation of pPhoA, in contrast to that of pOmpA, requires the presence of purified SecYEG. A differential specificity of the SecYEG was also revealed in its interaction with SecA: EcSecYEG did not enhance SecA-mediated pOmpA translocation by purified SecA either from Pseudomonas aeruginosa or Bacillus subtilis. Neither was SecYEG required for eliciting ion channel activity, which could be opened by unfolded pPhoA or unfolded PhoA. Addition of the SecYEG complex did restore the specificity of signal peptide recognition in the ion-channel activity. We concluded that SecYEG confers specificity in interacting with protein precursors and SecAs.

Proton transfer pathway in anion channelrhodopsin-1.

Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here, we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was also analyzed using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.

Personalized Cardiac Computational Models: From Clinical Data to Simulation of Infarct-Related Ventricular Tachycardia.

In the chronic stage of myocardial infarction, a significant number of patients develop life-threatening ventricular tachycardias (VT) due to the arrhythmogenic nature of the remodeled myocardium. Radiofrequency ablation (RFA) is a common procedure to isolate reentry pathways across the infarct scar that are responsible for VT. Unfortunately, this strategy show relatively low success rates; up to 50% of patients experience recurrent VT after the procedure. In the last decade, intensive research in the field of computational cardiac electrophysiology (EP) has demonstrated the ability of three-dimensional (3D) cardiac computational models to perform in-silico EP studies. However, the personalization and modeling of certain key components remain challenging, particularly in the case of the infarct border zone (BZ). In this study, we used a clinical dataset from a patient with a history of infarct-related VT to build an image-based 3D ventricular model aimed at computational simulation of cardiac EP, including detailed patient-specific cardiac anatomy and infarct scar geometry. We modeled the BZ in eight different ways by combining the presence or absence of electrical remodeling with four different levels of image-based patchy fibrosis (0, 10, 20, and 30%). A 3D torso model was also constructed to compute the ECG. Patient-specific sinus activation patterns were simulated and validated against the patient's ECG. Subsequently, the pacing protocol used to induce reentrant VTs in the EP laboratory was reproduced in-silico. The clinical VT was induced with different versions of the model and from different pacing points, thus identifying the slow conducting channel responsible for such VT. Finally, the real patient's ECG recorded during VT episodes was used to validate our simulation results and to assess different strategies to model the BZ. Our study showed that reduced conduction velocities and heterogeneity in action potential duration in the BZ are the main factors in promoting reentrant activity. Either electrical remodeling or fibrosis in a degree of at least 30% in the BZ were required to initiate VT. Moreover, this proof-of-concept study confirms the feasibility of developing 3D computational models for cardiac EP able to reproduce cardiac activation in sinus rhythm and during VT, using exclusively non-invasive clinical data.

Evolutionary well-conserved region in the signal peptide of parathyroid hormone-related protein is critical for its dual localization through the regulation of ER translocation.

Parathyroid hormone-related protein (PTHrP) has two different targeting signals: an N-terminal signal peptide for the endoplasmic reticulum (ER) targeting and an internal nuclear localization signal. The protein not only functions as a secretory protein, but is also found in the nucleus and/or nucleolus under certain conditions. PTHrP signal peptide is less hydrophobic than most signal peptides mainly due to its evolutionarily well-conserved region (QQWS). The substitution of four tandem leucine residues for this conserved region resulted in a significant inhibition of the signal peptide cleavage. At the same time, proportion of nuclear and/or nucleolar localization decreased, probably due to tethering of the protein to the ER membrane by the uncleaved mutant signal peptide. Almost complete cleavage of the signal peptide accompanied by a lack of nuclear/nucleolar localization was achieved by combining the hydrophobic h-region and an optimized sequence of the cleavage site. In addition, mutational modifications of the distribution of charged residues in and around the signal peptide affect its cleavage and/or nuclear/nucleolar localization of the protein. These results indicate that the well-conserved region in the signal peptide plays an essential role in the dual localization of PTHrP through ER targeting and/or the membrane translocation.

N-Terminal Signal Peptides of G Protein-Coupled Receptors: Significance for Receptor Biosynthesis, Trafficking, and Signal Transduction.

Signal sequences play a key role during the first steps of the intracellular transport of G protein-coupled receptors (GPCRs). They are involved in targeting of the nascent chains to the membrane of the endoplasmic reticulum (ER) and initiate integration of the newly synthesized receptors into this compartment. Two classes of signal sequences are known: N-terminal signal peptides, which are usually cleaved-off following ER insertion and internal signal sequences, the so-called signal anchor sequences, which form part of the mature proteins. About 5-10% of the GPCRs contain N-terminal signal peptides; the vast majority possesses signal anchor sequences. The reason why only a subset of GPCRs require signal peptides for ER targeting/insertion was addressed in the past decade by a limited number of studies indicating that the presence of signal peptides facilitates N-tail translocation at the ER membrane. Interestingly, recent work showed that signal peptides of GPCRs do not only serve "classical" functions in the early secretory pathway. Uncleaved pseudo signal peptides may regulate receptor densities in the plasma membrane, receptor dimerization, and G protein coupling selectivity. On the other hand, even cleaved and released peptides may have post-ER functions. In this review, we summarize the current knowledge about cleavable signal peptides of GPCRs and address also the question whether these sequences may serve as future drug targets in pharmacology.

ACEMBLing a multiprotein transmembrane complex: the functional SecYEG-SecDF-YajC-YidC Holotranslocon protein secretase/insertase.

Membrane proteins constitute about one third of the proteome. The ubiquitous Sec machinery facilitates protein movement across or integration of proteins into the cytoplasmic membrane. In Escherichia coli post- and co-translational targeting pathways converge at the protein-conducting channel, consisting of a central pore, SecYEG, which can recruit accessory domains SecDF-YajC and YidC, to form the holotranslocon (HTL) supercomplex. Detailed analysis of HTL function and architecture remained elusive until recently, largely due to the lack of a purified, recombinant complex. ACEMBL is an advanced DNA recombineering-based expression vector system we developed for producing challenging multiprotein complexes. ACEMBL affords the means to combine multiple expression elements including promoter DNAs, tags, genes of interest, and terminators in a combinatorial manner until optimal multigene expression plasmids are constructed that yield correctly assembled, homogenous, and active multiprotein complex specimens. We utilized ACEMBL for recombinant HTL overproduction. We developed protocols for detergent solubilizing and purifying the HTL. Highly purified complex was then used to reveal HTL function and the interactions between its constituents. HTL activity in protein secretion and membrane protein insertion was analyzed in both the presence and absence of the proton-motive force. Setting up ACEMBL for the assembly of multigene expression constructs that achieve high yields of functional multisubunit membrane protein complex is straightforward. Here, we used ACEMBL for obtaining active HTL supercomplex in high quality and quantity. The concept can likewise be applied to obtain many other assemblies of similar complexity, by overexpression in prokaryotic, and also eukaryotic hosts.