Impact of biatrial defragmentation in patients with paroxysmal atrial fibrillation: Results from a randomized prospective study

Background Single procedure success rates of pulmonary vein isolation (PVI) in patients with paroxysmal atrial fibrillation (PAF) are still unsatisfactory. In patients with persistent atrial fibrillation (AF), ablation of complex fractionated atrial electrograms (CFAEs) after PVI results in improved outcomes. Objective We aimed to investigate if PAF-patients with intraprocedurally sustained AF after PVI might benefit from additional CFAE ablation. Methods A total of 1134 consecutive patients underwent a first catheter ablation procedure of PAF between June 2008 and December 2012. In most patients, AF was either not inducible or terminated during PVI. In 68 patients (6%), AF sustained after successful PVI. These patients were randomized to either cardioversion (PVI-alone group; n = 33) or additional CFAE ablation (PVI+CFAE group; n = 35) and followed up every 1–3 months and serial Holter recordings were also obtained. The primary end point was the recurrence of AF/atrial tachycardia (AT) after a blanking period of 3 months. Results Procedure duration (127 ± 6 minutes vs 174 ± 10 minutes), radiofrequency application time (44 ± 3 minutes vs 74 ± 5 minutes), and fluoroscopy time (26 ± 2 minutes vs 41 ± 3 minutes) were longer in the PVI+CFAE group (all P < .001). In 30 of 35 patients (86%) in the PVI+CFAE group, ablation terminated AF. There was no significant group difference with respect to freedom from AF/AT (22 of 33 [67%] vs 22 of 35 [63%]; P = .66). Subsequently, 10 of 11 patients in the PVI-alone group (91%) and 11 of 13 patients in PVI+CFAE group (85%) underwent repeat ablation (P = 1.00). Overall, 29 of 33 [88%] vs 30 of 35 [86%] patients (P = 1.00) were free from AF/AT after 1.4 ± 0.1 vs 1.4 ± 0.2 (P = .87) procedures. Conclusion Patients with sustained AF after PVI in a PAF cohort are rare. Regarding AF/AT recurrence, these patients did not benefit from further CFAE ablation compared to PVI alone, but are exposed to longer procedure duration, fluoroscopy time, and radiofrequency application time.

Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.

New LA-ICP-MS cryocell and calibration technique for sub-millimeter analysis of ice cores

Ice cores provide a robust reconstruction of past climate. However, development of timescales by annual-layer counting, essential to detailed climate reconstruction and interpretation, on ice cores collected at low-accumulation sites or in regions of compressed ice, is problematic due to closely spaced layers. Ice-core analysis by laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS) provides sub-millimeter-scale sampling resolution (on the order of 100μm in this study) and the low detection limits (ng L–1) necessary to measure the chemical constituents preserved in ice cores. We present a newly developed cryocell that can hold a 1m long section of ice core, and an alternative strategy for calibration. Using ice-core samples from central Greenland, we demonstrate the repeatability of multiple ablation passes, highlight the improved sampling resolution, verify the calibration technique and identify annual layers in the chemical profile in a deep section of an ice core where annual layers have not previously been identified using chemistry. In addition, using sections of cores from the Swiss/Italian Alps we illustrate the relationship between Ca, Na and Fe and particle concentration and conductivity, and validate the LA-ICP-MS Ca profile through a direct comparison with continuous flow analysis results.

Characterisation of a Natural Quartz Crystal as a Reference Material for Microanalytical Determination of Ti, Al, Li, Fe, Mn, Ga and Ge

A natural smoky quartz crystal from Shandong province, China, was characterised by laser ablation ICP-MS, electron probe microanalysis (EPMA) and solution ICP-MS to determine the concentration of twenty-four trace and ultra trace elements. Our main focus was on Ti quantification because of the increased use of this element for titanium in- quartz (TitaniQ) thermobarometry. Pieces of a uniform growth zone of 9 mm thickness within the quartz crystal were analysed in four different LA-ICP-MS laboratories, three EPMA laboratories and one solution-ICP-MS laboratory. The results reveal reproducible concentrations of Ti (57 ± 4 lg g-1),Al (154 ± 15 lg g-1), Li (30 ± 2 lg g-1), Fe (2.2 ± 0.3 lg g-1), Mn (0.34 ± 0.04 lg g-1), Ge (1.7 ± 0.2 lg g-1) and Ga (0.020 ± 0.002 lg g-1) and detectable, but less reproducible, concentrations of Be, B, Na, Cu, Zr, Sn and Pb. oncentrations of K, Ca, Sr, Mo, Ag, Sb, Ba and Au were below the limits of detection of all three techniques. The uncertainties on the average concentration determinations by multiple techniques and laboratories for Ti, Al, Li, Fe, Mn, Ga and Ge are low; hence, this quartz can serve as a reference material or a secondary reference material for microanalytical applications involving the quantification of trace elements in quartz.

Phyllotaxis involves auxin drainage through leaf primordia

The spatial arrangement of leaves and flowers around the stem, known as phyllotaxis, is controlled by an auxin-dependent reiterative mechanism that leads to regular spacing of the organs and thereby to remarkably precise phyllotactic patterns. The mechanism is based on the active cellular transport of the phytohormone auxin by cellular influx and efflux carriers, such as AUX1 and PIN1. Their important role in phyllotaxis is evident from mutant phenotypes, but their exact roles in space and time are difficult to address due to the strong pleiotropic phenotypes of most mutants in phyllotaxis. Models of phyllotaxis invoke the accumulation of auxin at leaf initials and removal of auxin through their developing vascular strand, the midvein. We have developed a precise microsurgical tool to ablate the midvein at high spatial and temporal resolution in order to test its function in leaf formation and phyllotaxis. Using amplified femtosecond laser pulses, we ablated the internal tissues in young leaf primordia of tomato (Solanum lycopersicum) without damaging the overlying L1 and L2 layers. Our results show that ablation of the future midvein leads to a transient accumulation of auxin in the primordia and to an increase in their width. Phyllotaxis was transiently affected after midvein ablations, but readjusted after two plastochrons. These results indicate that the developing midvein is involved in the basipetal transport of auxin through young primordia, which contributes to phyllotactic spacing and stability.

A component of the mitochondrial outer membrane proteome of T. brucei probably contains covalent bound fatty acids

A subclass of eukaryotic proteins is subject to modification with fatty acids, the most common of which are palmitic and myristic acid. Protein acylation allows association with cellular membranes in the absence of transmembrane domains. Here we examine POMP39, a protein previously described to be present in the outer mitochondrial membrane proteome (POMP) of the protozoan parasite Trypanosoma brucei. POMP39 lacks canonical transmembrane domains, but is likely both myristoylated and palmitoylated on its N-terminus. Interestingly, the protein is also dually localized on the surface of the mitochondrion as well as in the flagellum of both insect-stage and the bloodstream form of the parasites. Upon abolishing of global protein acylation or mutation of the myristoylation site, POMP39 relocates to the cytosol. RNAi-mediated ablation of the protein neither causes a growth phenotype in insect-stage nor bloodstream form trypanosomes.

IL-33 signaling contributes to the pathogenesis of myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are characterized by the clonal expansion of one or more myeloid cell lineage. In most cases, proliferation of the malignant clone is ascribed to defined genetic alterations. MPNs are also associated with aberrant expression and activity of multiple cytokines; however, the mechanisms by which these cytokines contribute to disease pathogenesis are poorly understood. Here, we reveal a non-redundant role for steady-state IL-33 in supporting dysregulated myelopoiesis in a murine model of MPN. Genetic ablation of the IL-33 signaling pathway was sufficient and necessary to restore normal hematopoiesis and abrogate MPN-like disease in animals lacking the inositol phosphatase SHIP. Stromal cell-derived IL-33 stimulated the secretion of cytokines and growth factors by myeloid and non-hematopoietic cells of the BM, resulting in myeloproliferation in SHIP-deficient animals. Additionally, in the transgenic JAK2V617F model, the onset of MPN was delayed in animals lacking IL-33 in radio-resistant cells. In human BM, we detected increased numbers of IL-33-expressing cells, specifically in biopsies from MPN patients. Exogenous IL-33 promoted cytokine production and colony formation by primary CD34+ MPN stem/progenitor cells from patients. Moreover, IL-33 improved the survival of JAK2V617F-positive cell lines. Together, these data indicate a central role for IL-33 signaling in the pathogenesis of MPNs.

Magnetic anisotropy in natural amphibole crystals

Anisotropy of magnetic susceptibility (AMS) is often used as a proxy for mineral fabric in deformed rocks. To do so quantitatively, it is necessary to quantify the intrinsic magnetic anisotropy of single crystals of rock-forming minerals. Amphiboles are common in mafic igneous and metamorphic rocks and often define rock texture due to their general prismatic crystal habits. Amphiboles may dominate the magnetic anisotropy in intermediate to felsic igneous rocks and in some metamorphic rock types, because they have a high Fe concentration and they can develop a strong crystallographic preferred orientation. In this study, the AMS is characterized in 28 single crystals and I crystal aggregate of compositionally diverse clino- and ortho-amphiboles. High-field methods were used to isolate the paramagnetic component of the anisotropy, which is unaffected by ferromagnetic inclusions that often occur in amphibole crystals. Laue imaging, laser ablation-inductively coupled plasma-mass spectrometry, and Mossbauer spectroscopy were performed to relate the magnetic anisotropy to crystal structure and Fe concentration. The minimum susceptibility is parallel to the crystallographic a*-axis and the maximum susceptibility is generally parallel to the crystallographic b-axis in tremolite, actinolite, and hornblende. Gedrite has its minimum susceptibility along the a-axis, and maximum susceptibility aligned with c. In richterite, however, the intermediate susceptibility is parallel to the b-axis and the minimum and maximum susceptibility directions are distributed in the a-c plane. The degree of anisotropy, k', increases generally with Fe concentration, following a linear trend: k' = 1.61 x 10(-9) Fe - 1.17 x 10(-9) m(3)/kg. Additionally, it may depend on the Fe2+/Fe3+ ratio. For most samples, the degree of anisotropy increases by a factor of approximately 8 upon cooling from room temperature to 77 K. Fen-oactinolite, one pargasite crystal and riebeckite show a larger increase, which is related to the onset of local ferromagnetic (s.l.) interactions below about 100 K. This comprehensive data set increases our understanding of the magnetic structure of amphiboles, and it is central to interpreting magnetic fabrics of rocks whose AMS is controlled by amphibole minerals.

Fluid-related inclusions in Alpine high-pressure peridotite reveal trace element recycling during subduction-zone dehydration of serpentinized mantle (Cima di Gagnone, Swiss Alps).

Serpentinites release at sub-arc depths volatiles and several fluid-mobile trace elements found in arc magmas. Constraining element uptake in these rocks and defining the trace element composition of fluids released upon serpentinite dehydration can improve our understanding of mass transfer across subduction zones and to volcanic arcs. The eclogite-facies garnet metaperidotite and chlorite harzburgite bodies embedded in paragneiss of the subduction melange from Cima di Gagnone derive from serpentinized peridotite protoliths and are unique examples of ultramafic rocks that experienced subduction metasomatism and devolatilization. In these rocks, metamorphic olivine and garnet trap polyphase inclusions representing the fluid released during high-pressure breakdown of antigorite and chlorite. Combining major element mapping and laser-ablation ICP-MS bulk inclusion analysis, we characterize the mineral content of polyphase inclusions and quantify the fluid composition. Silicates, Cl-bearing phases, sulphides, carbonates, and oxides document post-entrapment mineral growth in the inclusions starting immediately after fluid entrapment. Compositional data reveal the presence of two different fluid types. The first (type A) records a fluid prominently enriched in fluid-mobile elements, with Cl, Cs, Pb, As, Sb concentrations up to 10(3) PM (primitive mantle), similar to 10(2) PM Tit Ba, while Rb, B, Sr, Li, U concentrations are of the order of 10(1) PM, and alkalis are similar to 2 PM. The second fluid (type B) has considerably lower fluid-mobile element enrichments, but its enrichment patterns are comparable to type A fluid. Our data reveal multistage fluid uptake in these peridotite bodies, including selective element enrichment during seafloor alteration, followed by fluid-rock interaction along with subduction metamorphism in the plate interface melange. Here, infiltration of sediment-equilibrated fluid produced significant enrichment of the serpentinites in As, Sb, B, Pb, an enriched trace element pattern that was then transferred to the fluid released at greater depth upon serpentine dehydration (type A fluid). The type B fluid hosted by garnet may record the composition of the chlorite breakdown fluid released at even greater depth. The Gagnone study-case demonstrates that serpentinized peridotites acquire water and fluid-mobile elements during ocean floor hydration and through exchange with sediment-equilibrated fluids in the early subduction stages. Subsequent antigorite devolatilization at subarc depths delivers aqueous fluids to the mantle wedge that can be prominently enriched in sediment-derived components, potentially triggering arc magmatism without the need of concomitant dehydration/melting of metasediments or altered oceanic crust.

Dystrophic cardiomyopathy: role of TRPV2 channels in stretch-induced cell damage

Aims Duchenne muscular dystrophy (DMD), a degenerative pathology of skeletal muscle, also induces cardiac failure and arrhythmias due to a mutation leading to the lack of the protein dystrophin. In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The absence of dystrophin results in an elevated stress-induced Ca2+ influx due to the inadequate functioning of several proteins, such as stretch-activated channels (SACs). Our aim was to investigate whether transient receptor potential vanilloid channels type 2 (TRPV2) form subunits of the dysregulated SACs in cardiac dystrophy. Methods and results We defined the role of TRPV2 channels in the abnormal Ca2+ influx of cardiomyocytes isolated from dystrophic mdx mice, an established animal model for DMD. In dystrophic cells, western blotting showed that TRPV2 was two-fold overexpressed. While normally localized intracellularly, in myocytes from mdx mice TRPV2 channels were translocated to the sarcolemma and were prominent along the T-tubules, as indicated by immunocytochemistry. Membrane localization was confirmed by biotinylation assays. Furthermore, in mdx myocytes pharmacological modulators suggested an abnormal activity of TRPV2, which has a unique pharmacological profile among TRP channels. Confocal imaging showed that these compounds protected the cells from stress-induced abnormal Ca2+ signals. The involvement of TRPV2 in these signals was confirmed by specific pore-blocking antibodies and by small-interfering RNA ablation of TRPV2. Conclusion Together, these results establish the involvement of TRPV2 in a stretch-activated calcium influx pathway in dystrophic cardiomyopathy, contributing to the defective cellular Ca2+ handling in this disease.

TimX, a novel player in protein import across the inner mitochondrial membrane of T. brucei

Mitochondrial protein import is an essential function of the unique mitochondrion in T. brucei as roughly 1000 different nuclear encoded proteins need to be correctly localized to their mitochondrial subcompartment. For this reason the responsible import machinery is expected to be similarly complex as in other Eukaryotes. This was recently demonstrated for the translocation machinery in the outer mitochondrial membrane. In contrast, the composition of the inner membrane import machinery and the exact molecular pathway(s) taken by various substrates are still ill-defined. To elucidate this further, we performed a pulldown analysis of epitope tagged TbTim17 in combination with quantitative mass spectrometry. By this we identified novel components of the mitochondrial import machinery in trypanosomes. One of these, TimX, is an essential mitochondrial membrane protein of 42 kDa that is unique to kinetoplastids. This protein migrates on Blue Native PAGE in a high molecular weight complex similar to TbTim17. Ablation of either of the two proteins leads to a destabilization of the complex containing the other protein. Furthermore, its involvement in protein import could be demonstrated by in vivo and in vitro protein import assays. This corroborates that TimX together with TbTim17 forms a protein import complex in the inner mitochondrial membrane. As TbTim17 the TimX protein was subjected to pulldown analysis in combination with quantitative mass spectrometry. The overlap of candidates defined by these two sets of IPs likely defines further components of the inner membrane translocase which are presently being analyzed. In summary our study on novel components of the trypanosome mitochondrial protein import system gives us fascinating new insights into evolution of the mitochondrion.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Their protein import systems are of similar complexity yet different composition than those in other eukaryotes. To elucidate the composition of the trypanosomal translocase of the inner mitochondrial membrane (TIM) we performed CoIPs of epitope-tagged TbTim17 and two other candidates in combination with SILAC-based quantitative mass spectrometry. This led to the identification of ten candidates for core TIM subunits. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a trypanosomatid-specific 42 kDa protein (Tim42) and two putative orthologues of inactive rhomboid proteases were analyzed further. All three proteins are essential in both life cycle stages and their ablation results in a strong protein import defect in vivo and in vitro. Blue native PAGE revealed their presence in a high molecular weight complex. Unlike anticipated, trypanosomes have a highly complex TIM translocase that has extensively been redesigned. None of the three novel TIM subunits has ever been associated with mitochondrial protein import. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. This suggests an exciting analogy between protein translocases of mitochondria and the ER.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is often considered as one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Its protein import systems are therefore of great interest. Recently, it was shown that the outer mitochondrial membrane protein translocase is of similar complexity yet different composition than in other eukaryotes (1). In the inner membrane however, only a single orthologue of the pore forming Tim17/22/23 protein family was identified and termed TbTim17. Based on this finding it has been suggested that, instead of separate TIM22 and TIM23 complexes as in other eukaryotes, trypanosomes may have a single multifunctional translocase of the inner mitochondrial membrane (TIM) of reduced complexity. To elucidate the composition of the trypanosomal TIM complex we performed co-immunoprecipitations (CoIP) of epitope-tagged TbTim17 in combination with SILAC-based quantitative mass spectrometry. This led to the identification of 22 highly enriched TbTim17-interacting proteins. We tagged two of the top-scoring proteins for reciprocal CoIP analyses and recovered a set of ten proteins that are highly enriched in all three CoIPs. These proteins are excellent candidates for core subunits of the trypanosomal TIM complex. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a novel trypanosomatid-specific 42 kDa protein, termed Tim42, and two putative orthologues of probably inactive rhomboid proteases were chosen for further analysis. All three proteins are essential in both life cycle stages and in a cell line that can grow in the absence of mitochondrial DNA. Additionally, their ablation by RNAi results in a strong protein import defect both in vivo and in vitro. Blue native PAGE reveals that Tim42, like TbTim17 is present in a high molecular weight complex. Moreover, ablation of either Tim42 or TbTim17 leads to a destabilization of the complex containing the other protein, suggesting a tight interaction of the two proteins. In summary our study shows that unlike anticipated trypanosomes have a highly complex TIM translocase that has extensively been redesigned. We have characterized three novel TIM subunits that have never been associated with mitochondrial protein import before. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. Our study provides insight into mitochondrial evolution over large phylogenetic distances and suggests an exciting analogy between protein translocation systems of mitochondria and the ER.

What can a unicellular parasite tell us about mitochondrial biogenesis?

In the unicellular parasite Trypanosoma brucei, as in other eukaryotes, more than 95% of all mitochondrial proteins are imported from the cytosol. The recently characterized multisubunit ATOM complex, the functional analogue of the TOM complex of yeast, mediates import of essentially all proteins across the outer mitochondrial membrane in T. brucei. Moreover, an additional protein termed pATOM36, which is loosely associated with the ATOM complex, has been implicated in the import of only a subset of mitochondrial proteins. Here we have investigated more precisely which role pATOM36 plays in mitochondrial protein import. RNAi mediated ablation of pATOM36 specifically depletes a subset of outer mitochondrial membrane proteins including ATOM complex subunits and as a consequence results in the collapse of the ATOM complex as shown by Blue native PAGE. In addition, a SILAC-based global proteomic analysis of uninduced and induced pATOM36 RNAi cells together with in vitro import experiments suggest that pATOM36 might be a novel protein import factor acting on a subset of alpha-helically anchored mitochondrial outer membrane proteins. Identification of pATOM36 interaction partners by co-immunoprecipitation together with immunofluorescence analysis shows that unexpectedly a fraction of the protein is associated with the tripartite attachment complex (TAC). This complex is essential for proper inheritance of the mitochondrial DNA in T. brucei. It forms a physical connection between the single unit mitochondrial DNA and the basal body of the flagellum that is stable throughout the cell cycle. Thus, pATOM36 simultaneously mediates ATOM assembly, and thus protein import, as well as mitochondrial DNA inheritance since it is an essential component of the TAC.

Pulmonary Vein Isolation Versus Defragmentation: The CHASE-AF Clinical Trial

BACKGROUND Long-term success rates using ablation for persistent atrial fibrillation (AF) are disappointing and usually do not exceed 60%. OBJECTIVES This study sought to compare arrhythmia-free survival between pulmonary vein isolation (PVI) and a stepwise approach (full defrag) consisting of PVI, ablation of complex fractionated electrograms, and additional linear ablation lines in the setting of atrial tachycardias (AT) in patients with persistent AF after PVI. METHODS From November 2010 to February 2013, 205 patients (151 men; 61.7 ± 10.2 years of age) underwent de novo ablation for persistent AF. Subsequently, patients were prospectively randomized to either PVI alone (n = 78) or full defrag (n = 75), with 52 patients not randomized due to AF termination with the original PVI. The primary endpoint was recurrence of any AT after a blanking period of 3 months. RESULTS During the entire study, 241 ablations were performed (mean: 1.59 in the PVI-alone group, 1.55 in the full-defrag group). With the stepwise approach, termination of AF occurred in 45 (60%) patients. However, arrhythmia-free survival did not differ whether patients underwent single or multiple procedures (p = 0.468). Procedure duration, fluoroscopy time, and radiofrequency duration were significantly longer in the full-defrag group (all p < 0.001). CONCLUSIONS A stepwise approach aimed at AF termination does not seem to provide additional benefit over PVI alone in patients with persistent AF, but it is associated with significantly longer procedural and fluoroscopic duration as well as radiofrequency application time. (The Randomized Catheter Ablation of Persist End Atrial Fibrillation Study [CHASE-AF]; NCT01580124).