Identification of Novel Kinases of Tau Using Fluorescence Complementation Mass Spectrometry (FCMS).

Hyperphosphorylation of the microtubule-associated protein Tau is a major hallmark of Alzheimer's disease and other tauopathies. Understanding the protein kinases that phosphorylate Tau is critical for the development of new drugs that target Tau phosphorylation. At present, the repertoire of the Tau kinases remains incomplete, and methods to uncover novel upstream protein kinases are still limited. Here, we apply our newly developed proteomic strategy, fluorescence complementation mass spectrometry, to identify novel kinase candidates of Tau. By constructing Tau- and kinase-fluorescent fragment library, we detected 59 Tau-associated kinases, including 23 known kinases of Tau and 36 novel candidate kinases. In the validation phase using in vitro phosphorylation, among 15 candidate kinases we attempted to purify and test, four candidate kinases, OXSR1 (oxidative-stress responsive gene 1), DAPK2 (death-associated protein kinase 2), CSK (C-terminal SRC kinase), and ZAP70 (zeta chain of T-cell receptor-associated protein kinase 70), displayed the ability to phosphorylate Tau in time-course experiments. Furthermore, coexpression of these four kinases along with Tau increased the phosphorylation of Tau in human neuroglioma H4 cells. We demonstrate that fluorescence complementation mass spectrometry is a powerful proteomic strategy to systematically identify potential kinases that can phosphorylate Tau in cells. Our discovery of new candidate kinases of Tau can present new opportunities for developing Alzheimer's disease therapeutic strategies.

Phosphoproteomic Approaches for Identifying Phosphatase and Kinase Substrates.

Protein phosphorylation is a ubiquitous post-translational modification controlled by the opposing activities of protein kinases and phosphatases, which regulate diverse biological processes in all kingdoms of life. One of the key challenges to a complete understanding of phosphoregulatory networks is the unambiguous identification of kinase and phosphatase substrates. Liquid chromatography-coupled mass spectrometry (LC-MS/MS) and associated phosphoproteomic tools enable global surveys of phosphoproteome changes in response to signaling events or perturbation of phosphoregulatory network components. Despite the power of LC-MS/MS, it is still challenging to directly link kinases and phosphatases to specific substrate phosphorylation sites in many experiments. Here, we survey common LC-MS/MS-based phosphoproteomic workflows for identifying protein kinase and phosphatase substrates, noting key advantages and limitations of each. We conclude by discussing the value of inducible degradation technologies coupled with phosphoproteomics as a new approach that overcomes some limitations of current methods for substrate identification of kinases, phosphatases, and other regulatory enzymes.

Online Science Instruction Can Promote Adolescents' Autonomy Need Satisfaction: a Latent Growth Curve Analysis.

This research examined the differential motivational effects of a pre-college science enrichment program delivered in both online and in-person learning formats. Using self-determination theory as a guiding framework, we hypothesized that (a) students would exhibit growth in their perceived satisfaction of needs for autonomy, competence, and relatedness, (b) online learning would be associated with greater growth in autonomy, and (c) in-person learning would be associated with greater growth in both competence and relatedness. Using a sample of 598 adolescent participants, results of latent growth curve modeling indicated that satisfaction of the three needs grew unconditionally over the course of the program. However, format type was unrelated to growth in need satisfaction. Rather, this effect was found to be conditional upon the type of science project undertaken by students: astrophysics students exhibited significantly greater autonomy growth when receiving online instruction than did biochemistry students. Our findings suggest that online science learning can be just as effective in motivating students as in-person learning provided that the learning tasks are conducive to remote instruction.

The Cdk/cDc14 module controls activation of the Yen1 holliday junction resolvase to promote genome stability.

Faithful genome transmission during cell division requires precise, coordinated action of DNA metabolic enzymes, including proteins responsible for DNA damage detection and repair. Dynamic phosphorylation plays an important role in controlling repair enzymes during the DNA damage response (DDR). Cdc14 phosphatases oppose cyclin-dependent kinase (Cdk) phosphorylation and have been implicated in the DDR in several model systems. Here, we have refined the substrate specificity of budding yeast Cdc14 and, using this insight, identified the Holliday junction resolvase Yen1 as a DNA repair target of Cdc14. Cdc14 activation at anaphase triggers nuclear accumulation and enzymatic activation of Yen1, likely to resolve persistent recombinational repair intermediates. Consistent with this, expression of a phosphomimetic Yen1 mutant increased sister chromatid nondisjunction. In contrast, lack of Cdk phosphorylation resulted in constitutive activity and elevated crossover-associated repair. The precise timing of Yen1 activation, governed by core cell-cycle regulators, helps coordinate DNA repair with chromosome segregation and safeguards against genome destabilization.

Discovering the N-Terminal Methylome by Repurposing of Proteomic Datasets.

Protein α-N-methylation is an underexplored post-translational modification involving the covalent addition of methyl groups to the free α-amino group at protein N-termini. To systematically explore the extent of α-N-terminal methylation in yeast and humans, we reanalyzed publicly accessible proteomic datasets to identify N-terminal peptides contributing to the α-N-terminal methylome. This repurposing approach found evidence of α-N-methylation of established and novel protein substrates with canonical N-terminal motifs of established α-N-terminal methyltransferases, including human NTMT1/2 and yeast Tae1. NTMT1/2 are implicated in cancer and aging processes but have unclear and context-dependent roles. Moreover, α-N-methylation of noncanonical sequences was surprisingly prevalent, suggesting unappreciated and cryptic methylation events. Analysis of the amino acid frequencies of α-N-methylated peptides revealed a [S]1-[S/A/Q]2 pattern in yeast and [A/N/G]1-[A/S/V]2-[A/G]3 in humans, which differs from the canonical motif. We delineated the distribution of the two types of prevalent N-terminal modifications, acetylation and methylation, on amino acids at the first position. We tested three potentially methylated proteins and confirmed the α-N-terminal methylation of Hsp31 by additional proteomic analysis and immunoblotting. The other two proteins, Vma1 and Ssa3, were found to be predominantly acetylated, indicating that proteomic searching for α-N-terminal methylation requires careful consideration of mass spectra. This study demonstrates the feasibility of reprocessing proteomic data for global α-N-terminal methylome investigations.

Relationship between sodium channel function and clinical phenotype in SCN5A variants associated with Brugada syndrome.

The identification of a pathogenic SCN5A variant confers an increased risk of conduction defects and ventricular arrhythmias (VA) in Brugada syndrome (BrS). However, specific aspects of sodium channel function that influence clinical phenotype have not been defined. A systematic literature search identified SCN5A variants associated with BrS. Sodium current (INa ) functional parameters (peak current, decay, steady-state activation and inactivation, and recovery from inactivation) and clinical features (conduction abnormalities [CA], spontaneous VA or family history of sudden cardiac death [SCD], and spontaneous BrS electrocardiogram [ECG]) were extracted. A total of 561 SCN5A variants associated with BrS were identified, for which data on channel function and clinical phenotype were available in 142. In the primary analysis, no relationship was found between any aspect of channel function and CA, VA/SCD, or spontaneous BrS ECG pattern. Sensitivity analyses including only variants graded pathogenic or likely pathogenic suggested that reduction in peak current and positive shift in steady-state activation were weakly associated with CA and VA/SCD, although sensitivity and specificity remained low. The relationship between in vitro assessment of channel function and BrS clinical phenotype is weak. The assessment of channel function does not enhance risk stratification. Caution is needed when extrapolating functional testing to the likelihood of variant pathogenicity.

The pseudosubstrate inhibitor Acm1 inhibits the anaphase-promoting complex/cyclosome by combining high-affinity activator binding with disruption of Doc1/Apc10 function.

The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif-dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.

Re-examining the role of Cdc14 phosphatase in reversal of Cdk phosphorylation during mitotic exit.

Inactivation of cyclin-dependent kinase (Cdk) and reversal of Cdk phosphorylation are universally required for mitotic exit. In budding yeast (Saccharomyces cerevisiae), Cdc14 is essential for both and thought to be the major Cdk-counteracting phosphatase. However, Cdc14 is not required for mitotic exit in many eukaryotes, despite highly conserved biochemical properties. The question of how similar enzymes could have such disparate influences on mitotic exit prompted us to re-examine the contribution of budding yeast Cdc14. By using an auxin-inducible degron, we show that severe Cdc14 depletion has no effect on the kinetics of mitotic exit and bulk Cdk substrate dephosphorylation, but causes a cell separation defect and is ultimately lethal. Phosphoproteomic analysis revealed that Cdc14 is highly selective for distinct Cdk sites in vivo and does not catalyze widespread Cdk substrate dephosphorylation. We conclude that additional phosphatases likely contribute substantially to Cdk substrate dephosphorylation and coordination of mitotic exit in budding yeast, similar to in other eukaryotes, and the critical mitotic exit functions of Cdc14 require trace amounts of enzyme. We propose that Cdc14 plays very specific, and often different, roles in counteracting Cdk phosphorylation in all species.

Early experience of thoracoscopic vs. catheter ablation for atrial fibrillation.

AIMS: Video-assisted thoracoscopic surgery (VATS) ablation has been advocated as a treatment option for non-paroxysmal atrial fibrillation (AF) in recent guidelines. Real-life data on its safety and efficacy during a centre's early experience are sparse. METHODS AND RESULTS: Thirty patients (28 persistent/longstanding persistent AF) underwent standalone VATS ablation for AF by an experienced thoracoscopic surgeon, with the first 20 cases proctored by external surgeons. Procedural and follow-up outcomes were collected prospectively, and compared with 90 propensity-matched patients undergoing contemporaneous catheter ablation (CA). Six (20.0%) patients undergoing VATS ablation experienced ≥1 major complication (death n = 1, stroke n = 2, conversion to sternotomy n = 3, and phrenic nerve injury n = 2). This was significantly higher than the 1.1% major complication rate (tamponade requiring drainage n = 1) seen with CA (P < 0.001). Twelve-month single procedure arrhythmia-free survival rates without antiarrhythmic drugs were 56% in the VATS and 57% in the CA cohorts (P = 0.22), and 78% and 80%, respectively given an additional CA and antiarrhythmic drugs (P = 0.32). CONCLUSION: During a centre's early experience, VATS ablation may have similar success rates to those from an established CA service, but carry a greater risk of major complications. Those embarking on a programme of VATS AF ablation should be aware that complication and success rates may differ from those reported by selected high-volume centres.

Substrate Recognition by the Cdh1 Destruction Box Receptor Is a General Requirement for APC/CCdh1-mediated Proteolysis.

The anaphase-promoting complex, or cyclosome (APC/C), is a ubiquitin ligase that selectively targets proteins for degradation in mitosis and the G1 phase and is an important component of the eukaryotic cell cycle control system. How the APC/C specifically recognizes its substrates is not fully understood. Although well characterized degron motifs such as the destruction box (D-box) and KEN-box are commonly found in APC/C substrates, many substrates apparently lack these motifs. A variety of alternative APC/C degrons have been reported, suggesting either that multiple modes of substrate recognition are possible or that our definitions of degron structure are incomplete. We used an in vivo yeast assay to compare the G1 degradation rate of 15 known substrates of the APC/C co-activator Cdh1 under normal conditions and conditions that impair binding of D-box, KEN-box, and the recently identified ABBA motif degrons to Cdh1. The D-box receptor was required for efficient proteolysis of all Cdh1 substrates, despite the absence of canonical D-boxes in many. In contrast, the KEN-box receptor was only required for normal proteolysis of a subset of substrates and the ABBA motif receptor for a single substrate in our system. Our results suggest that binding to the D-box receptor may be a shared requirement for recognition and processing of all Cdh1 substrates.

A proteomic strategy for global analysis of plant protein complexes.

Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.

Ablation index, a novel marker of ablation lesion quality: prediction of pulmonary vein reconnection at repeat electrophysiology study and regional differences in target values.

AIMS: Force-Time Integral (FTI) is commonly used as a marker of ablation lesion quality during pulmonary vein isolation (PVI), but does not incorporate power. Ablation Index (AI) is a novel lesion quality marker that utilizes contact force, time, and power in a weighted formula. Furthermore, only a single FTI target value has been suggested despite regional variation in left atrial wall thickness. We aimed to study AI's and FTI's relationships with PV reconnection at repeat electrophysiology study, and regional threshold values that predicted no reconnection. METHODS AND RESULTS: Forty paroxysmal atrial fibrillation patients underwent contact force-guided PVI, and the minimum and mean AI and FTI values for each segment were identified according to a 12-segment model. All patients underwent repeat electrophysiology study at 2 months, regardless of symptoms, to identify sites of PV reconnection. Late PV reconnection was seen in 53 (11%) segments in 25 (62%) patients. Reconnected segments had significantly lower minimum AI [308 (252-336) vs. 373 (323-423), P < 0.0001] and FTI [137 (92-182) vs. 228 (157-334), P < 0.0001] compared with non-reconnected segments. Minimum AI and FTI were both independently predictive, but AI had a smaller P value. Higher minimum AI and FTI values were required to avoid reconnection in anterior/roof segments than for posterior/inferior segments (P < 0.0001). No reconnection was seen where the minimum AI value was ≥370 for posterior/inferior segments and ≥480 for anterior/roof segments. CONCLUSION: The minimum AI value in a PVI segment is independently predictive of reconnection of that segment at repeat electrophysiology study. Higher AI and FTI values are required for anterior/roof segments than for posterior/inferior segments to prevent reconnection.

FgCDC14 regulates cytokinesis, morphogenesis, and pathogenesis in Fusarium graminearum.

Members of Cdc14 phosphatases are common in animals and fungi, but absent in plants. Although its orthologs are conserved in plant pathogenic fungi, their functions during infection are not clear. In this study, we showed that the CDC14 ortholog is important for pathogenesis and morphogenesis in Fusarium graminearum. FgCDC14 is required for normal cell division and septum formation and FgCdc14 possesses phosphatase activity with specificity for a subset of Cdk-type phosphorylation sites. The Fgcdc14 mutant was reduced in growth, conidiation, and ascospore formation. It was defective in ascosporogenesis and pathogenesis. Septation in Fgcdc14 was reduced and hyphal compartments contained multiple nuclei, indicating defects in the coordination between nuclear division and cytokinesis. Interestingly, foot cells of mutant conidia often differentiated into conidiogenous cells, resulting in the production of inter-connected conidia. In the interphase, FgCdc14-GFP localized to the nucleus and spindle-pole-body. Taken together, our results indicate that Cdc14 phosphatase functions in cell division and septum formation in F. graminearum, likely by counteracting Cdk phosphorylation, and is required for plant infection.

Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti(IV) Functionalized Nanopolymers.

Accurate protein phosphorylation analysis reveals dynamic cellular signaling events not evident from protein expression levels. The most dominant biochemical assay, western blotting, suffers from the inadequate availability and poor quality of phospho-specific antibodies for phosphorylated proteins. Furthermore, multiplexed assays based on antibodies are limited by steric interference between the antibodies. Here we introduce a multifunctionalized nanopolymer for the universal detection of phosphoproteins that, in combination with regular antibodies, allows multiplexed imaging and accurate determination of protein phosphorylation on membranes.

Reablated Sites of Acute Reconnection After Pulmonary Vein Isolation Do Not Predict Sites of Late Reconnection at Repeat Electrophysiology Study.

INTRODUCTION: Acute reconnection of pulmonary veins (PVs) is frequently seen in the waiting period following pulmonary vein isolation (PVI). There are concerns that reablation at these sites may not be durably effective due to tissue edema caused by the initial ablation. We aimed to prospectively study the relationship between acute and late reconnection. METHODS AND RESULTS: Wide-area circumferential PVI was performed in 40 paroxysmal AF patients. Spontaneous reconnection was assessed after a minimum 20-minute waiting period, with adenosine administered to unmask dormant reconnection. All sites of acute reconnection were ablated to reisolate the PV. All 40 patients then underwent repeat electrophysiology study after 2 months, regardless of symptoms, to identify late reconnection. Sites of acute and late reconnection were compared according to a 12-segment PVI model. Acute reconnection was seen in 28 (6%) PVI segments in 20 (50%) patients, affecting 24/160 (15%) PVs. All were successfully reisolated. At repeat electrophysiology study, 51 (11%) PVI segments were reconnected in 25 (62%) patients, affecting 41 (25%) PVs. The proportion of PVI segments with and without acute reconnection exhibiting late reconnection at repeat study was no different (14% vs. 10%, P = 0.524). There was also no difference in late reconnection between PVI circles or patients with and without acute reconnection. CONCLUSION: Most PVI segments that undergo further ablation for acute reconnection show persistent isolation at repeat electrophysiology study, and the rate of late reconnection for these segments is no different to that for segments that did not acutely reconnect. This implies that effective reablation is delivered at these sites.

Radiofrequency ablation of the interventricular septum to treat outflow tract gradients in hypertrophic obstructive cardiomyopathy: a novel use of CARTOSound® technology to guide ablation.

AIMS: Septal reduction is needed for hypertrophic obstructive cardiomyopathy (HOCM) patients with severe left ventricular outflow tract (LVOT) gradients and symptoms despite medication. Myectomy cannot be performed in all. Alcohol septal ablation cannot be performed in 5-15% due to technical difficulties. A method of delivering percutaneous tissue damage to the septum that is not reliant on coronary anatomy is desirable. To directly ablate the interventricular septum at the mitral valve (MV) systolic anterior motion (SAM)-septal contact point using radiofrequency (RF) energy guided by CARTOSound. METHODS AND RESULTS: Five patients underwent RF ablation (RFA); we describe follow-up at 6 months in four patients. Intracardiac echocardiography (ICE) images are merged with CARTO to create a shell of the cardiac chambers. The SAM-septal contact area is marked from ICE images and mapped on to the CARTO shell; this becomes the target for RF delivery. Conduction tissue is mapped and avoided where possible. Twenty-eight to 42 min of RF energy was delivered to the target area using retrograde aortic access and SmartTouch catheters. Resting LVOT gradient improved from 64.2 (±50.6) to 12.3 (±2.5) mmHg. Valsalva/exercise-induced gradient reduced from 93.5 (±30.9) to 23.3 (±8.3) mmHg. Three patients improved New York Heart Association status from III to II, one patient improved from class III to I. Exercise time on bicycle ergometer increased from 612 to 730 s. Cardiac magnetic resonance shows late gadolinium enhancement up to 8 mm depth at LV target myocardium. One patient died following a significant retroperitoneal haemorrhage. CONCLUSION: Radiofrequency ablation using CARTOSound(®) guidance is accurate and effective in treating LVOT gradients in HOCM in this preliminary group of patients.

Perturbed atrial calcium handling in an ovine model of heart failure: potential roles for reductions in the L-type calcium current.

Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during ß-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca(2+) and the response to ß-adrenergic (ß-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca(2+) concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca(2+) transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca(2+) removal (kSR, by 32%), L-type Ca(2+) current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca(2+) content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca(2+) current (ICa-L) in control cells reproduced both the decrease in Ca(2+) transient amplitude and increase of SR Ca(2+) content observed in voltage-clamped HF cells. During ß-AR stimulation Ca(2+) transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca(2+) content was highest in HF cells during ß-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca(2+) transient amplitude and increased SR Ca(2+) content observed in voltage-clamped cells.

A novel marker to predict early recurrence after atrial fibrillation ablation: the ablation effectiveness quotient.

INTRODUCTION: Inability to predict clinical outcome despite acutely successful pulmonary vein isolation (PVI) remains the Achilles' heel of atrial fibrillation ablation (AFA). Arrhythmia recurrence is frequently due to recovery of radiofrequency (RF) ablation lesions believed to be complete at the original procedure. OBJECTIVES: We hypothesized that a high ratio between post-AFA levels of serum high sensitivity cardiac troponin T (HScTnT), a highly specific marker of acute myocardial injury, and duration of RF application (the ablation effectiveness quotient, AEQ) would indicate effective ablation and correlate with early clinical success. METHODS: We prospectively measured HScTnT levels in 60 patients (42 [70%] male, 22 [37%] with paroxysmal AF [PAF], mean age 62.5 ± 10.6 years) 12-18 hours after AFA and calculated the AEQ for each. Patients were followed-up with ECGs and Holter monitors for recurrence of atrial tachyarrhythmia (AT). RESULTS: Early recurrence of AT within 6 months occurred in 22 (37%). AT recurrence was not significantly related to left atrial size or comorbidities, nor to RF time or HScTnT level. Mean AEQ was significantly lower in those with recurrence than those without (0.35 ± 0.14 ng/L/s vs. 0.45 ± 0.18 ng/L/s), P = 0.02. Subgroup analysis showed this finding was due to patients with PAF in whom early significance was maintained to one year, with an AEQ >0.4 ng/L/s having 75% sensitivity and 90% specificity in predicting freedom from AT. CONCLUSION: A high AEQ correlates well with freedom from AT in patients with PAF in both the short and medium term. If confirmed in further studies, AEQ may become a useful marker of risk of AT post-AFA.

Drosophila activated Cdc42 kinase has an anti-apoptotic function.

Activated Cdc42 kinases (Acks) are evolutionarily conserved non-receptor tyrosine kinases. Activating somatic mutations and increased ACK1 protein levels have been found in many types of human cancers and correlate with a poor prognosis. ACK1 is activated by epidermal growth factor (EGF) receptor signaling and functions to regulate EGF receptor turnover. ACK1 has additionally been found to propagate downstream signals through the phosphorylation of cancer relevant substrates. Using Drosophila as a model organism, we have determined that Drosophila Ack possesses potent anti-apoptotic activity that is dependent on Ack kinase activity and is further activated by EGF receptor/Ras signaling. Ack anti-apoptotic signaling does not function through enhancement of EGF stimulated MAP kinase signaling, suggesting that it must function through phosphorylation of some unknown effector. We isolated several putative Drosophila Ack interacting proteins, many being orthologs of previously identified human ACK1 interacting proteins. Two of these interacting proteins, Drk and yorkie, were found to influence Ack signaling. Drk is the Drosophila homolog of GRB2, which is required to couple ACK1 binding to receptor tyrosine kinases. Drk knockdown blocks Ack survival activity, suggesting that Ack localization is important for its pro-survival activity. Yorkie is a transcriptional co-activator that is downstream of the Salvador-Hippo-Warts pathway and promotes transcription of proliferative and anti-apoptotic genes. We find that yorkie and Ack synergistically interact to produce tissue overgrowth and that yorkie loss of function interferes with Ack anti-apoptotic signaling. Our results demonstrate how increased Ack signaling could contribute to cancer when coupled to proliferative signals.

Improving safety in catheter ablation for atrial fibrillation: a prospective study of the use of ultrasound to guide vascular access.

INTRODUCTION: The most frequent complications of AF ablation (AFA) are related to vascular access, but there is little evidence as to how these can be minimized. METHODS: Consecutive patients undergoing AFA at a high-volume center received either standard care (Group S) or routine ultrasound-guided vascular access (Group U). Vascular complications were assessed before hospital discharge and by means of postal questionnaire 1 month later. Outcome measures were BARC 2+ bleeding complications, postprocedural pain, and prolonged bruising. RESULTS: Patients in Group S (n = 146) and U (n = 163) were well matched at baseline. Follow-up questionnaires were received from 92.6%. Patients in Group U were significantly less likely to have a BARC 2+ bleed, 10.4% versus 19.9% P = 0.02, were less likely to suffer groin pain after discharge (27.1% vs. 42.8%; P = 0.006) and were less likely to experience prolonged local bruising (21.5% vs. 40.4%; P = 0.001). Multivariable logistic regression analysis revealed a significant association of vascular complications with nonultrasound guided access (OR 3.12 95%CI 1.54-5.34; P = 0.003) and increasing age (OR 1.05 95%CI 1.01-1.09; P = 0.02). CONCLUSION: Routine use of ultrasound-guided vascular access for AFA is associated with a significant reduction in bleeding complications, postprocedural pain, and prolonged bruising when compared to standard care.