Interaction between a J-domain co-chaperone and a specific Argonaute protein contributes to microRNA function in animals.

MicroRNAs (miRNAs) are essential regulators of several biological processes. They are loaded onto Argonaute (AGO) proteins to achieve their repressive function, forming the microRNA-Induced Silencing Complex known as miRISC. While several AGO proteins are expressed in plants and animals, it is still unclear why specific AGOs are strictly binding miRNAs. Here, we identified the co-chaperone DNJ-12 as a new interactor of ALG-1, one of the two major miRNA-specific AGOs in Caenorhabditis elegans. DNJ-12 does not interact with ALG-2, the other major miRNA-specific AGO, and PRG-1 and RDE-1, two AGOs involved in other small RNA pathways, making it a specific actor in ALG-1-dependent miRNA-mediated gene silencing. The loss of DNJ-12 causes developmental defects associated with defective miRNA function. Using the Auxin Inducible Degron system, a powerful tool to acutely degrade proteins in specific tissues, we show that DNJ-12 depletion hampers ALG-1 interaction with HSP70, a chaperone required for miRISC loading in vitro. Moreover, DNJ-12 depletion leads to the decrease of several miRNAs and prevents their loading onto ALG-1. This study uncovers the importance of a co-chaperone for the miRNA function in vivo and provides insights to explain how different small RNAs associate with specific AGO in animals.

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans.

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.

DEFI 2005: a randomized controlled trial of the effect of automated external defibrillator cardiopulmonary resuscitation protocol on outcome from out-of-hospital cardiac arrest.

BACKGROUND: Using automated external defibrillators (AEDs) that implement the Guidelines 2000 resuscitation protocol constrains administration of cardiopulmonary resuscitation (CPR) to <50% of AED connection time. We tested a different AED protocol aimed at increasing the CPR administered to patients with out-of-hospital cardiac arrest. METHODS AND RESULTS: In a randomized controlled trial, patients with out-of-hospital cardiac arrest requiring defibrillation were treated with 1 of 2 AED protocols. In the control protocol, based on Guidelines 2000, sequences of up to 3 stacked countershocks were delivered, with rhythm analyses initially and after the first and second shocks. The study protocol featured 1 minute of CPR before the first shock, shorter CPR interruptions before and after each shock, and no stacked shocks. The primary end point was survival to hospital admission. Of 5107 out-of-hospital cardiac arrest patients connected to an AED, 1238 required defibrillation, and 845 were included in the final analysis. Study patients (n=421) had shorter preshock pauses (9 versus 19 seconds; P<0.001), had shorter postshock pauses (11 versus 33 seconds; P<0.001), and received more CPR (61% versus 48%; P<0.001) and fewer shocks (2.5 versus 2.9; P<0.001) than control patients (n=424). Similar proportions survived to hospital admission (43.2% versus 42.7%; P=0.87), survived to hospital discharge (13.3% versus 10.6%; P=0.19), achieved return of spontaneous circulation before physician arrival (47.0% versus 48.6%; P=0.65), and survived to 1 year (P=0.77). CONCLUSIONS: Following prompts from AEDs programmed with a protocol similar to Guidelines 2005, firefighters shortened pauses in CPR and improved overall hands-on time, but survival to hospital admission of patients with ventricular fibrillation out-of-hospital cardiac arrest did not improve. Clinical Trial Registration- http://www.clinicaltrials.gov. Unique identifier: NCT00139542.

Two molecular features contribute to the Argonaute specificity for the microRNA and RNAi pathways in C. elegans.

In Caenorhabditis elegans, specific Argonaute proteins are dedicated to the RNAi and microRNA pathways. To uncover how the precise Argonaute selection occurs, we designed dsRNA triggers containing both miRNA and siRNA sequences. While dsRNA carrying nucleotides mismatches can only enter the miRNA pathway, a fully complementary dsRNA successfully rescues let-7 miRNA function and initiates silencing by RNAi. We demonstrated that RDE-1 is essential for RNAi induced by the perfectly paired trigger, yet is not required for silencing by the let-7 miRNA. In contrast, ALG-1/ALG-2 are required for the miRNA function, but not for the siRNA-directed gene silencing. Finally, a dsRNA containing a bulged miRNA and a perfectly paired siRNA can enter both pathways suggesting that the sorting of small RNAs occurs after that the dsRNA trigger has been processed by Dicer. Thus, our data suggest that the selection of Argonaute proteins is affected by two molecular features: (1) the structure of the small RNA duplex; and (2) the Argonautes specific characteristics.

Effect of timing and duration of a single chest compression pause on short-term survival following prolonged ventricular fibrillation.

BACKGROUND: Pauses during chest compressions are thought to have a detrimental effect on resuscitation outcome. The Guidelines 2005 have recently eliminated the post-defibrillation pause. Previous animal studies have shown that multiple pauses of increasing duration decrease resuscitation success. We investigated the effect of varying the characteristics of a single pause near defibrillation on resuscitation outcome. METHODS: Part A: 48 swine were anesthetized, fibrillated for 7min and randomized. Chest compressions were initiated for 90s followed by defibrillation and then resumption of chest compressions. Four groups were studied-G2000: 40s pause beginning 20s before, and ending 20s after defibrillation, A1: a 20s pause just before defibrillation, A2: a 20s pause ending 30s prior to defibrillation, and group A3: a 10s pause ending 30s prior to defibrillation. Part B: 12 swine (Group B) were studied with a protocol identical to Part A but with no pause in chest compressions. Primary endpoint was survival to 4h. RESULTS: The survival rate was significantly higher for groups A1, A2, A3, and B (5/12, 7/12, 5/12, and 5/12 survived) than for the G2000 group (0/12, p<0.05). Survival did not differ significantly among groups A1, A2, A3, and B. CONCLUSIONS: These results suggest that the Guidelines 2005 recommendation to omit the post-shock pulse check and immediately resume chest compressions may be an important resuscitation protocol change. However, these results also suggest that clinical maneuvers further altering a single pre-shock chest compression pause provide no additional benefit.

Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments.

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.

Attenuating the defibrillation dosage decreases postresuscitation myocardial dysfunction in a swine model of pediatric ventricular fibrillation.

OBJECTIVE: The optimal biphasic defibrillation dose for children is unknown. Postresuscitation myocardial dysfunction is common and may be worsened by higher defibrillation doses. Adult-dose automated external defibrillators are commonly available; pediatric doses can be delivered by attenuating the adult defibrillation dose through a pediatric pads/cable system. The objective was to investigate whether unattenuated (adult) dose biphasic defibrillation results in greater postresuscitation myocardial dysfunction and damage than attenuated (pediatric) defibrillation. DESIGN: Laboratory animal experiment. SETTING: University animal laboratory. SUBJECTS: Domestic swine weighing 19 +/- 3.6 kg. INTERVENTIONS: Fifty-two piglets were randomized to receive biphasic defibrillation using either adult-dose shocks of 200, 300, and 360 J or pediatric-dose shocks of approximately 50, 75, and 85 J after 7 mins of untreated ventricular fibrillation. Contrast left ventriculograms were obtained at baseline and then at 1, 2, 3, and 4 hrs postresuscitation. Postresuscitation left ventricular ejection fraction and cardiac troponins were evaluated. MEASUREMENTS AND MAIN RESULTS: By design, piglets in the adult-dose group received shocks with more energy (261 +/- 65 J vs. 72 +/- 12 J, p < .001) and higher peak current (37 +/- 8 A vs. 13 +/- 2 A, p < .001) at the largest defibrillation dose needed. In both groups, left ventricular ejection fraction was reduced significantly at 1, 2, and 4 hrs from baseline and improved during the 4 hrs postresuscitation. The decrease in left ventricular ejection fraction from baseline was greater after adult-dose defibrillation. Plasma cardiac troponin levels were elevated 4 hrs postresuscitation in 11 of 19 adult-dose piglets vs. four of 20 pediatric-dose piglets (p = .02). CONCLUSIONS: Unattenuated adult-dose defibrillation results in a greater frequency of myocardial damage and worse postresuscitation myocardial function than pediatric doses in a swine model of prolonged out-of-hospital pediatric ventricular fibrillation cardiac arrest. These data support the use of pediatric attenuating electrodes with adult biphasic automated external defibrillators to defibrillate children.

Better outcome after pediatric defibrillation dosage than adult dosage in a swine model of pediatric ventricular fibrillation.

OBJECTIVES: This study was designed to compare outcome after adult defibrillation dosing versus pediatric dosing in a piglet model of prolonged prehospital ventricular fibrillation (VF). BACKGROUND: Weight-based 2 to 4 J/kg monophasic defibrillation dosing is recommended for children in VF, but impractical for automated external defibrillator (AED) use. Present AEDs can only provide adult shock doses or newly developed attenuated adult doses intended for children. A single escalating energy sequence (50/75/86 J) of attenuated adult-dose biphasic shocks (pediatric dosing) is at least as effective as escalating monophasic weight-based dosing for prolonged VF in piglets, but this approach has not been compared to standard adult biphasic dosing. METHODS: Following 7 min of untreated VF, piglets weighing 13 to 26 kg (19 +/- 1 kg) received either biphasic 50/75/86 J (pediatric dose) or biphasic 200/300/360 J (adult dose) therapies during simulated prehospital life support. RESULTS: Return of spontaneous circulation was attained in 15 of 16 pediatric-dose piglets and 14 of 16 adult-dose piglets. Four hours postresuscitation, pediatric dosing resulted in fewer elevations of cardiac troponin T (0 of 12 piglets vs. 6 of 11 piglets, p = 0.005) and less depression of left ventricular ejection fraction (p < 0.05). Most importantly, more piglets survived to 24 h with good neurologic scores after pediatric shocks than adult shocks (13 of 16 piglets vs. 4 of 16 piglets, p = 0.004). CONCLUSIONS: In this model, pediatric shocks resulted in superior outcome compared with adult shocks. These data suggest that adult defibrillation dosing may be harmful to pediatric patients with VF and support the use of attenuating electrodes with adult biphasic AEDs to defibrillate children.

Effects of shock strengths on ventricular defibrillation failure.

BACKGROUND: The mechanism of defibrillation is controversial. Reentry appearing immediately after the shock has been shown to be responsible for defibrillation failure in some studies while other studies have demonstrated that a rapid train of focal activations with the first focus appearing >50 ms after the shock is responsible for failed defibrillation. We tested the hypothesis that both patterns can occur, but at different shock strengths. METHODS AND RESULTS: Biphasic 6/4 ms shocks of 100-900 V in 100-V increments were given after 10 s of ventricular fibrillation from electrodes in right ventricular apex and right atrium in five isolated pig hearts. Transmembrane activity was optically mapped from the anterior and posterior epicardium using two CCD cameras. The defibrillation threshold (DFT) was 786+/-199 V. The interval from the shock to the earliest post-shock activation was zero for shocks <400 V but increased with increasing shock voltage to 62+/-6 ms at 800 V. The number of post-shock phase singularities, which is related to reentry incidence, decreased continuously from pre-shock values for 100-V shocks to zero as the shock strength increased to 600 V. Focal activations were observed after shocks >600 V with no epicardial reentry present. CONCLUSION: Reentry is responsible for defibrillation failure for low-strength shocks. As the shock strength approaches the DFT, a focal epicardial activation pattern becomes responsible for failed defibrillation. Thus, the mechanism of defibrillation failure depends on shock strength, with focal activation as the mechanism for the clinically important near-DFT strength shocks.

Mechanical chest compressions improved aspects of CPR in the LINC trial.

AIM: We studied resuscitation process metrics in patients with out-of-hospital cardiac arrest enrolled in a randomized trial comparing one protocol designed to best use a mechanical CPR device, with another based on the 2005 European Resuscitation Council guidelines for manual CPR. METHODS: We analyzed clinical data, ECG signals, and transthoracic impedance signals for a subset of the patients in the LUCAS in Cardiac Arrest (LINC) trial, including 124 patients randomized to mechanical and 82 to manual CPR. Chest compression fraction (CCF) was defined as the fraction of time during cardiac arrest that chest compressions were administered. RESULTS: Patients in the mechanical CPR group had a higher CCF than those in the manual CPR group [0.84 (0.78, 0.91) vs. 0.79 (0.70, 0.86), p < 0.001]. The median duration of their pauses for defibrillation was also shorter [0 s (0, 6.0) vs. 10.0 s (7.0, 14.3), p < 0.001]. Compressions were interrupted for a median of 36.0 s to apply the compression device. There was no difference between groups in duration of the longest pause in compressions [32.5s vs. 26.0 s, p = 0.24], number of compressions received per minute [86.5 vs. 88.3, p = 0.47], defibrillation success rate [73.2% vs. 81.0%, p = 0.15], or refibrillation rate [74% vs. 77%, p = 0.79]. CONCLUSIONS: A protocol using mechanical chest compression devices reduced interruptions in chest compressions, and enabled defibrillation during ongoing compressions, without adversely affecting other resuscitation process metrics. Future emphasis on optimizing device deployment may be beneficial.

Attenuated adult biphasic shocks compared with weight-based monophasic shocks in a swine model of prolonged pediatric ventricular fibrillation.

AIM: To compare the safety and efficacy of attenuated adult biphasic shocks with standard monophasic weight-based shocks in a piglet model of prolonged prehospital ventricular fibrillation (VF). BACKGROUND: If attenuated adult shocks are safe and effective for prehospital pediatric VF, automated external defibrillators (AEDs) can be easily adapted for pediatric use. METHODS: After 7 min of untreated VF, piglets were randomized to treatment with attenuated adult biphasic shocks or weight-based monophasic shocks. The attenuated adult biphasic group received 200/300/360 J shocks, attenuated by specialized pediatric electrodes to 51/78/81 J and the monophasic weight-based control group received 2/4/4 J/kg shocks. Forty-eight female piglets were studied, 16 in each of three weight categories: 4 kg (neonatal), 14 kg (younger child) and 24 kg (older child). The primary outcome measures of efficacy and safety were 24h survival with good neurological outcome and post-resuscitation left ventricular ejection fraction (LVEF), respectively. RESULTS: For the 24 kg piglets, attenuated adult biphasic shocks resulted in superior 24 h survival with good neurological outcome (6/8 versus 0/8, P < 0.001) and greater LVEF 4 h post-resuscitation (34 +/- 4% versus 18 +/- 5%, P < 0.05). For the 14 and 4 kg piglets, 24 h survival with good neurological outcome occurred in 7/8 versus 5/8 and 7/8 versus 3/8, respectively, and LVEF 4 h post-resuscitation was 30 +/- 3% versus 36 +/- 6% and 30 +/- 3% versus 22 +/- 4%, respectively. CONCLUSIONS: The escalating attenuated adult biphasic dosage strategy was at least as safe and effective as the standard weight-based monophasic dose over a wide range of weights in this piglet model of prehospital VF. This work supports the concept of using an attenuated adult biphasic dosage in children.

Effect of action potential duration and conduction velocity restitution and their spatial dispersion on alternans and the stability of arrhythmias.

INTRODUCTION: The slope of the action potential duration (APD) restitution curve has been used to explain wavebreaks during arrhythmia initiation and maintenance. This hypothesis remains incomplete to fully describe the experimental data. Other factors contributing to wavebreaks must be studied to further understand arrhythmia dynamics. METHODS AND RESULTS: Control APDs were measured from isolated rabbit hearts using a monophasic action potential probe. APD and conduction velocity (CV) restitution were quantified over the heart surface for two drugs, diacetyl monoxime (DAM) and cytochalasin D (CytoD), using a dual camera video imaging system. For all pacing intervals: (1) control APDs were shorter than for CytoD but longer than for DAM; and (2) CV was greater for CytoD compared with DAM. APD dispersion increased as pacing interval decreased for both drugs. For DAM, increased dispersion was due to a difference in APD restitution between the right and left ventricle. For CytoD, increased dispersion was due to discordant alternans, with no significant spatial variation in restitution. Fibrillation was sustained only in the control hearts; with DAM, stable reentry was sustained with shorter APD and cycle length compared with CytoD for which only nonsustained unstable reentry occurred. CONCLUSION: Alternans and arrhythmia dynamics are affected by the spatial dispersion of APD restitution as well as CV restitution, not simply the slope of APD restitution. Therefore, a direct link of the APD restitution slope to alternans and arrhythmia dynamics in rabbit heart does not exist. Designing antiarrhythmic drugs to alter only the restitution slope may not be appropriate.

Restitution dynamics during pacing and arrhythmias in isolated pig hearts.

INTRODUCTION: The dependence of action potential duration (APD) on the preceding diastolic interval (DI), i.e., restitution, has been purported to predict the development of alternans and reentrant arrhythmias. However, restitution depends on the history of activation (i.e., memory), and its relevance to arrhythmia induction and maintenance is unknown. METHODS AND RESULTS: Using a dual-camera video imaging system, we recorded action potentials from thousands of sites on the surface of the isolated pig heart. A steady-state pacing (SSP) protocol was performed to generate the SSP APD restitution curve. During SSP, the minimum DI and APD were 57 +/- 6 ms and 107 +/- 6 ms, respectively. The restitution slope was >1 for DIs <85 +/- 5 ms; however, alternans were not observed. Abrupt decreases in cycle length (CL) resulted in a rapid (<5 beats) decrease in APD followed by a slower decrease to "steady state." DI, APD pairs for the initial beats following these rate changes were significantly above the SSP restitution curve. DI, APD pairs measured during sustained ventricular fibrillation clustered significantly below the SSP restitution curve, at significantly shorter APDs (57 +/- 4 ms) and DIs (49 +/- 6 ms) than could be achieved during SSP. In addition, abrupt increases in CL following SSP resulted in APDs significantly shorter than those predicted from the SSP restitution curve. CONCLUSION: Our results indicate that the responses of APD and DI to sudden rate changes and during arrhythmias are not predicted by the SSP restitution relationship. Acute dynamics act to damp out the proarrhythmic oscillations predicted from the SSP restitution curve.

Efficacy of lower-energy biphasic shocks for transthoracic defibrillation: a follow-up clinical study.

OBJECTIVE: This clinical study prospectively evaluated the first-shock defibrillation efficacy of 150-joule impedance-compensated, 200-microF biphasic truncated exponential (BTE) shocks in patients with electrically-induced ventricular fibrillation (VF), and compared it with a historical control group treated with 200-J monophasic damped sine (MDS) shocks. METHODS: Ventricular tachyarrhythmias were induced in patients undergoing electrophysiologic (EP) testing for ventricular arrhythmias or testing of an implantable cardioverter-defibrillator (ICD). A 150-J shock was delivered as the primary therapy to terminate induced arrhythmias in the EP group, and as a "rescue" shock when a single ICD shock failed to terminate the arrhythmias in the ICD group. RESULTS: Ninety-six patients received study shocks. The preshock rhythm was classified as VF in 77 patients and as ventricular tachycardia (VT) in 19 patients. First-shock success rates for VF and VT were 75 out of 77 (97.4%) and 19 out of 19 (100%) for the 150-J BTE compared with the historical control rates of 61 out of 68 (89.7%) and 29 out of 31 (94%) for 200-J MDS. The first-shock success rate for VF treated with 150-J BTE was technically equivalent to that of 200-J MDS (p=0.001). The transthoracic impedance did not vary between groups, yet the peak current delivered by the 150-J BTE shock was about 50% lower. CONCLUSIONS: This study demonstrated that 150-J shocks of this impedance-compensated, 200-microF BTE waveform provided very high efficacy for defibrillation of short duration, electrically-induced VF. These lower-energy biphasic shocks had a success rate equivalent to that of 200-J MDS shocks, and they provided this efficacy while exposing patients to much less current than the monophasic shocks.