Impact of video on the understanding and satisfaction of patients receiving informed consent before elective inpatient coronary angiography: A randomized trial.

BACKGROUND: Appropriate information about the benefits and risks of invasive procedures is crucial, but limited data is available in this field. The aim of this study was to evaluate the incremental value of a short video about coronary angiography compared with standard information, in terms of patient understanding, satisfaction and anxiety. METHODS: This prospective multicenter study included patients admitted for scheduled coronary angiography, who were randomized to receive either standard information or video information by watching a three-dimensional educational video. After information was delivered, patients were asked to complete a dedicated 16-point information questionnaire, as well as satisfaction and anxiety scales. RESULTS: From 21 September to 4 October 2015, 821 consecutive patients were randomized to receive either standard information (n=415) or standard information with an added educational video (n=406). The information score was higher in the video information group than in the standard group (11.8±2.8 vs 9.5±3.1; P<.001). This result was consistent across age and education level subgroups. Self-reported satisfaction was also higher in the video information group (8.4±1.9 vs. 7.7±2.3; P<.001), while anxiety level did not differ between groups. The variables associated with a higher information score were the use of the educational video, younger age, higher level of education, previous follow-up by a cardiologist, prior information about coronary angiography and previous coronary angiography. CONCLUSIONS: In comparison with standard information, viewing a dedicated educational video improved patients' understanding and satisfaction before scheduled coronary angiography. These results are in favor of widespread use of this incremental information tool.

Incremental predictive value of mean platelet volume/platelet count ratio in in-hospital stroke after acute myocardial infarction.

Stroke is a serious complication after acute myocardial infarction (AMI) and is associated with an increased risk of death. Though the pathophysiological mechanisms are not exactly known, increased inflammation and platelet reactivity could play an important role in the occurrence of stroke during AMI. We aimed to investigate the relationship between both mean platelet volume (MPV), a parameter of platelet function, and C-reactive protein (CRP) and the occurrence of in-hospital ischemic stroke (IHS) after AMI. Data were obtained from a French regional survey for AMI that included 5976 patients admitted to an intensive care unit (ICU) between 2001 and 2010. Patients were divided into two groups according to the occurrence of IHS. MPV, platelet count (PC), and CRP were routinely measured at admission to the ICU; 99 (1.6%) IHSs were recorded during hospitalization after admission for AMI. In multivariate analysis, IHS was independently associated with a history of stroke (OR: 1.99%, CI: 1.1-3.49, p = 0.01), impaired left ventricular ejection fraction <40% (OR: 1.88, 95% CI: 1.20-2.94, p = 0.006), impaired renal function (OR: 1.94, 95% CI: 1.27-2.95, p = 0.002), CRP > 10 mg/l (OR: 2.19, 95% CI: 1.44-3.33, p < 0.001), and MPV/PC ratio (OR: 1.04, 95% CI: 1.01-1.08, p = 0.023). Compared with the first to fourth quintiles, the last quintile of the MPV/PC ratio was associated with higher rates of IHS on survival curve analysis (p = 0.014). At hospital admission, a high MPV/PC ratio and a high level of CRP might help to identify patients at increased risk of IHS. Moreover, these results provide new insights into the potential role played by increased inflammation and platelet reactivity in the occurrence of stroke after AMI.

Short-term moderate diet restriction in adulthood can reverse oxidative, cardiovascular and metabolic alterations induced by postnatal overfeeding in mice.

We aimed to determine whether moderate diet restriction could restore cardiac, oxidative and metabolic alterations induced by postnatal overfeeding (PNOF). Litters of C57BL/6 male mice were either maintained at 9 (normal litter, NL), or reduced to 3 (small litter, SL) in order to induce PNOF. At 6 months, half of the NL and SL mice were subjected to 20% calorie-restriction (CR: NLCR, SLCR) for one month, while the other half continued to eat ad libitum (AL: NLAL, SLAL). Six-month old SL mice presented overweight, fat accumulation, hyperleptinemia, glucose intolerance, insulin resistance, increased cardiac ROS production and decreased left ventricular ejection fraction (LVEF). After CR, SL mice body weight was normalized; however, their fat mass and leptinemia were not decreased, glucose metabolism was improved and LVEF was increased. In SL mice, CR increased the cardiac mitochondrial respiratory rate and decreased cardiac ROS production. Hearts from SLCR mice showed better recovery and smaller postischemic infarct size. Intriguingly, no difference was observed between NLAL and NLCR mice for most of the parameters investigated. Short-term moderate CR not only normalized body weight in SL mice but also improved metabolic programming and reversed oxidative and cardiac dysfunction induced by PNOF.

Overweight in mice, induced by perinatal programming, exacerbates doxorubicin and trastuzumab cardiotoxicity.

PURPOSE: Trastuzumab (TRZ) is believed to potentiate doxorubicin (DOX) cardiotoxicity, resulting in left ventricular dysfunction. There is some evidence that overweight could influence anticancer drug-induced cardiotoxicity, though no study has evaluated the impact of moderate overweight, induced by postnatal nutritional programming, on the cardiotoxic effects of DOX alone or in combination with TRZ. METHODS: Immediately after birth, litters of C57BL/6 mice were either maintained at 9 pups (normal litter, NL) or reduced to 3 (small litter, SL) in order to induce programming of ~15 % overweight through postnatal overfeeding. At 4 months, NL and SL mice received a single intra-peritoneal injection of either saline, DOX (6 mg/kg), TRZ (10 mg/kg) or both (DOX-TRZ). Transthoracic echocardiography was performed 24 h before as well as 10 and 20 days after treatments. RESULTS: Twenty days after DOX administration, systolic dysfunction was observed only in the overweight SL group, while NL mice group had a normal left ventricular ejection fraction. However, in the NL group, functional impairment appeared when TRZ was co-administered. Forty-eight hours after drug administration, gene expression of natriuretic peptides (ANP, BNP) appeared to be potentiated in DOX-TRZ mice of both the NL and SL group, whereas the expression of ß-MHC increased significantly in overweight SL mice only. CONCLUSIONS: In an acute model of DOX cardiotoxicity, moderately overweight adult mice were more sensitive to cardiac systolic impairment. Moreover, our results confirm the potentiating action of TRZ on DOX-induced cardiotoxicity in lean mice.

Clinical effectiveness of the systematic use of the GRACE scoring system (in addition to clinical assessment) for ischaemic outcomes and bleeding complications in the management of NSTEMI compared with clinical assessment alone: a prospective study.

UNLABELLED: We assessed the interest of systematically using the GRACE scoring system (in addition to clinical assessment) for in- hospital outcomes and bleeding complications in the management of NSTEMI compared with clinical assessments alone. Multicentre, randomized study that included 572 consecutive NSTEMI patients, randomized 1:1, into group A: clinical stratification alone and group B: clinical+ GRACE score stratification. MAIN OUTCOME MEASURES: in-hospital outcomes and bleeding complications. There was no significant difference between the two groups for baseline data or for in-hospital MACE. In multivariate analysis, only a GRACE >140 (OR: 3.5, 95 % CI: 1.8-6.6, p < 0.001) and PCI (OR: 0.55, 95 % CI: 0.3-1.0; p = 0.05) were independent predictors of in-hospital MACE. The sub-analysis of group B showed that 56 patients (20 %) were given a compliance score of 0, showing that diagnostic angiography was performed later than as recommended by the guidelines. Interestingly, 91 % had a GRACE score >140, and these patients were significantly older, and were more likely to have a history of diabetes, stroke and renal failure, together with symptoms of heart failure. After multivariate analysis, the independent predictors of a lack of compliance with guideline delays were a GRACE score >140 (OR: 9.2; CI: 4.2-20.3, p < 0.001) and secondary referral from a non-PCI cardiology department (OR: 2.7; CI: 1.4-5.2, p = 0.003). In a real-world setting of patients admitted with NSTEMI, the systematic use of the GRACE scoring system at admission in the PCI centre does not improve in-hospital outcomes and bleeding complications.

Pom1 gradient buffering through intermolecular auto-phosphorylation.

Concentration gradients provide spatial information for tissue patterning and cell organization, and their robustness under natural fluctuations is an evolutionary advantage. In rod-shaped Schizosaccharomyces pombe cells, the DYRK-family kinase Pom1 gradients control cell division timing and placement. Upon dephosphorylation by a Tea4-phosphatase complex, Pom1 associates with the plasma membrane at cell poles, where it diffuses and detaches upon auto-phosphorylation. Here, we demonstrate that Pom1 auto-phosphorylates intermolecularly, both in vitro and in vivo, which confers robustness to the gradient. Quantitative imaging reveals this robustness through two system's properties: The Pom1 gradient amplitude is inversely correlated with its decay length and is buffered against fluctuations in Tea4 levels. A theoretical model of Pom1 gradient formation through intermolecular auto-phosphorylation predicts both properties qualitatively and quantitatively. This provides a telling example where gradient robustness through super-linear decay, a principle hypothesized a decade ago, is achieved through autocatalysis. Concentration-dependent autocatalysis may be a widely used simple feedback to buffer biological activities.

Pre-operative growth differentiation factor 15 as a novel biomarker of acute kidney injury after cardiac bypass surgery.

BACKGROUND: Pre-operative GDF-15 plasma levels significantly improve the prognostic value of the EuroSCORE for mortality after cardiac surgery. However, despite the strong correlation between GDF-15 and renal function, no data are available regarding the potential interest of pre-operative GDF-15 levels to improve the prediction of acute kidney injury (AKI) after cardiac artery bypass graft (CABG) surgery. METHODS: All patients operated on by 2 surgeons for CABG surgery at our university hospital from September 2011 to March 2013 were screened for participation in this prospective, observational study. EXCLUSION CRITERIA: age <18years or >80years, previous atrial fibrillation/flutter, previous severe renal failure, previous cardiac surgery, emergency surgery. AKI was defined according to KDIGO criteria. GDF-15 levels in plasma were measured before induction and 12h after surgery. RESULTS: 134 patients were included in this study. 42 (31%) developed post-operative AKI. AKI patients had a significantly higher pre-operative log-GDF-15 level (OR=3.64; 95% CI=1.41-9.40, p=0.008), a lower pre-operative eGFR (OR=0.98; 95% CI=0.96-0.99; p=0.026), and most often underwent on-pump surgery (OR=2.60; 95% CI=1.14-5.96, p=0.024). On ROC curves, GDF-15 before induction was found to be the best pre-operative biomarker to predict AKI (AUC=0.83; CI=0.75-0.89), compared with eGFR (AUC=0.67; 95% CI=0.59-0.75), p=0.003 and NT-proBNP (AUC=0.62; CI=0.51-0.72), p<0.001. Pre-operative GDF-15 was also significantly better than the EuroSCORE in predicting AKI (AUC 0.62, 95% CI=0.54-0.70), p<0.001. CONCLUSIONS: Pre-operative GDF-15 plasma levels are associated with post-operative AKI in CABG patients. If confirmed in larger cohorts, pre-operative GDF-15 may be of value to improve pre-operative risk stratification among candidates for surgery.

Anthracyclines/trastuzumab: new aspects of cardiotoxicity and molecular mechanisms.

Anticancer drugs continue to cause significant reductions in left ventricular ejection fraction resulting in congestive heart failure. The best-known cardiotoxic agents are anthracyclines (ANTHs) such as doxorubicin (DOX). For several decades cardiotoxicity was almost exclusively associated with ANTHs, for which cumulative dose-related cardiac damage was the use-limiting step. Human epidermal growth factor (EGF) receptor 2 (HER2; ErbB2) has been identified as an important target for breast cancer. Trastuzumab (TRZ), a humanized anti-HER2 monoclonal antibody, is currently recommended as first-line treatment for patients with metastatic HER2(+) tumors. The use of TRZ may be limited by the development of drug intolerance, such as cardiac dysfunction. Cardiotoxicity has been attributed to free-iron-based, radical-induced oxidative stress. Many approaches have been promoted to minimize these serious side effects, but they are still clinically problematic. A new approach to personalized medicine for cancer that involves molecular screening for clinically relevant genomic alterations and genotype-targeted treatments is emerging.

Paradoxically, iron overload does not potentiate doxorubicin-induced cardiotoxicity in vitro in cardiomyocytes and in vivo in mice.

Doxorubicin (DOX) is known to induce serious cardiotoxicity, which is believed to be mediated by oxidative stress and complex interactions with iron. However, the relationship between iron and DOX-induced cardiotoxicity remains controversial and the role of iron chelation therapy to prevent cardiotoxicity is called into question. Firstly, we evaluated in vitro the effects of DOX in combination with dextran-iron on cell viability in cultured H9c2 cardiomyocytes and EMT-6 cancer cells. Secondly, we used an in vivo murine model of iron overloading (IO) in which male C57BL/6 mice received a daily intra-peritoneal injection of dextran-iron (15mg/kg) for 3weeks (D0-D20) and then (D21) a single sub-lethal intra-peritoneal injection of 6mg/kg of DOX. While DOX significantly decreased cell viability in EMT-6 and H9c2, pretreatment with dextran-iron (125-1000µg/mL) in combination with DOX, paradoxically limited cytotoxicity in H9c2 and increased it in EMT-6. In mice, IO alone resulted in cardiac hypertrophy (+22%) and up-regulation of brain natriuretic peptide and ß-myosin heavy-chain (ß-MHC) expression, as well as an increase in cardiac nitro-oxidative stress revealed by electron spin resonance spectroscopy. In DOX-treated mice, there was a significant decrease in left-ventricular ejection fraction (LVEF) and an up-regulation of cardiac ß-MHC and atrial natriuretic peptide (ANP) expression. However, prior IO did not exacerbate the DOX-induced fall in LVEF and there was no increase in ANP expression. IO did not impair the capacity of DOX to decrease cancer cell viability and could even prevent some aspects of DOX cardiotoxicity in cardiomyocytes and in mice.

Frequency and predictors of stroke after acute myocardial infarction: specific aspects of in-hospital and postdischarge events.

BACKGROUND AND PURPOSE: Stroke is a serious complication after acute myocardial infarction (AMI) and is closely associated with decreased survival. This study aimed to investigate the frequency, characteristics, and factors associated with in-hospital and postdischarge stroke in patients with AMI. METHODS: Eight thousand four hundred eighty-five consecutive patients admitted to a cardiology intensive care unit for AMI, between January 2001 and July 2010. Stroke/transient ischemic attack were collected during 1-year follow-up. RESULTS: One hundred twenty-three in-hospital strokes were recorded: 65 (52.8%) occurred on the first day after admission for AMI, and 108 (87%) within the first 5 days. One hundred six patients (86.2%-incidence rate 1.25%) experienced in-hospital ischemic stroke, and 14 patients (11.4%-incidence rate 0.16%) were diagnosed with an in-hospital hemorrhagic stroke. In-hospital ischemic stroke subtypes according to the Trial of Org 10 172 in Acute Stroke Treatment (TOAST) classification showed that only 2 types of stroke were identified more frequently. As expected, the leading subtype of in-hospital ischemic stroke was cardioembolic stroke (n=64, 60%), the second was stroke of undetermined pathogenesis (n=38, 36%). After multivariable backward regression analysis, female sex, previous transient ischemic attack (TIA)/stroke, new-onset atrial fibrillation, left ventricular ejection fraction (odds ratio per point of left ventricular ejection fraction), and C-reactive protein were independently associated with in-hospital ischemic stroke. When antiplatelet and anticoagulation therapy within the first 48 hours was introduced into the multivariable model, we found that implementing these treatments (≥1) was an independent protective factor of in-hospital stroke. In-hospital hemorrhagic stroke was dramatically increased (5-fold) when thrombolysis was prescribed as the reperfusion treatment. However, the different parenteral anticoagulants were not predictors of risk in univariable analysis. Finally, only 45 postdischarge strokes were recorded. Postdischarge stroke subtypes showed a more heterogeneous distribution of mechanisms. The annual rate of stroke post-AMI remained stable throughout the 10-year study period. CONCLUSIONS: The present study describes specific predictors of in-hospital and postdischarge stroke in patients with AMI. It showed a marked increase in the risk of death, both during hospitalization and in the year after AMI. After hospital discharge, stroke remains a rare event and is mostly associated with high cardiovascular risk.

Distinct levels in Pom1 gradients limit Cdr2 activity and localization to time and position division.

Where and when cells divide are fundamental questions. In rod-shaped fission yeast cells, the DYRK-family kinase Pom1 is organized in concentration gradients from cell poles and controls cell division timing and positioning. Pom1 gradients restrict to mid-cell the SAD-like kinase Cdr2, which recruits Mid1/Anillin for medial division. Pom1 also delays mitotic commitment through Cdr2, which inhibits Wee1. Here, we describe quantitatively the distributions of cortical Pom1 and Cdr2. These reveal low profile overlap contrasting with previous whole-cell measurements and Cdr2 levels increase with cell elongation, raising the possibility that Pom1 regulates mitotic commitment by controlling Cdr2 medial levels. However, we show that distinct thresholds of Pom1 activity define the timing and positioning of division. Three conditions-a separation-of-function Pom1 allele, partial downregulation of Pom1 activity, and haploinsufficiency in diploid cells-yield cells that divide early, similar to pom1 deletion, but medially, like wild-type cells. In these cells, Cdr2 is localized correctly at mid-cell. Further, Cdr2 overexpression promotes precocious mitosis only in absence of Pom1. Thus, Pom1 inhibits Cdr2 for mitotic commitment independently of regulating its localization or cortical levels. Indeed, we show Pom1 restricts Cdr2 activity through phosphorylation of a C-terminal self-inhibitory tail. In summary, our results demonstrate that distinct levels in Pom1 gradients delineate a medial Cdr2 domain, for cell division placement, and control its activity, for mitotic commitment.

Prognostic value of fragmented QRS on a 12-lead ECG in patients with acute myocardial infarction.

OBJECTIVE: To investigate the determinants and the prognostic value of fragmented QRS (fQRS) after AMI. PATIENTS AND METHODS: Prospective cohort of 307 consecutive patients with AMI. MAIN OUTCOMES MEASURED: MACE (death plus non-fatal recurrent MI), hospitalization for an episode of heart failure, ventricular arrhythmia (VT or VF) at two years follow-up. RESULTS: On the serial 12-lead ECG recorded during the in-hospital stay, 162 (53%) had no fQRS (no fQRS group). 145 (47%) presented an fQRS, which was persistent in 108 (34%) patients (persistent fQRS group) and transient in 37 (12%) patients (transient fQRS group). Patients with a fragmented QRS (transient or persistent) were older, more likely to be hypertensive and less likely to be smokers than were patients without fQRS. By multivariate logistic regression analysis, only hypertension (OR (95% CI): 1.66 (1.00-2.74); p = 0.047) was associated with an fQRS. During a mean follow-up of 846 ± 297 days, there were 82 MACE recorded: 17 patients died from a CV cause (10% event rate) among patients without fQRS, 22 (20% event rate) among patients with persistent fQRS and 3 (8% event rate) among patients with transient fQRS. Similarly, non-fatal recurrent MI occurred more frequently in patients with fQRS (18 (16%) and 10 (27%)) for persistent and transient fQRS, respectively, vs. 16 (10%) in the no fQRS group (p = 0.019). However, the occurrence of heart failure symptoms and ventricular arrhythmia was not significantly different (p = 0.162 and p = 0.242, respectively). Survival analysis by the Kaplan-Meier method showed a significant difference (log rank p = 0.026) between groups, and only persistent fQRS was associated with decreased survival. In multivariate cox regression analysis, the GRACE score, blood glucose on admission, and B-blockers in the acute phase were independent predictors of MACE at two years. fQRS was not a significant independent predictor of MACE (HR (95% CI): 1.57 (0.95-2.60); p = 0.08). Moreover, fQRS was not a predictor of heart failure or ventricular arrhythmia in univariate analysis. CONCLUSIONS: Persistent fQRS on a 12-lead ECG is a marker of decreased survival after AMI, whereas transient fQRS correlates with recurrent MI.

Fission yeast: in shape to divide.

How are cell morphogenesis and cell cycle coordinated? The fission yeast is a rod-shaped unicellular organism widely used to study how a cell self-organizes in space and time. Here, we discuss recent advances in understanding how the cell acquires and maintains its regular rod shape and uses it to control cell division. The cellular body plan is established by microtubules, which mark antipodal growth zones and medial division. In turn, cellular dimensions are defined by the small GTPase Cdc42 and downstream regulators of vesicle trafficking. Yeast cells then repetitively use their simple rod shape to orchestrate the position and timing of cell division.

A phosphorylation cycle shapes gradients of the DYRK family kinase Pom1 at the plasma membrane.

Concentration gradients regulate many cell biological and developmental processes. In rod-shaped fission yeast cells, polar cortical gradients of the DYRK family kinase Pom1 couple cell length with mitotic commitment by inhibiting a mitotic inducer positioned at midcell. However, how Pom1 gradients are established is unknown. Here, we show that Tea4, which is normally deposited at cell tips by microtubules, is both necessary and, upon ectopic cortical localization, sufficient to recruit Pom1 to the cell cortex. Pom1 then moves laterally at the plasma membrane, which it binds through a basic region exhibiting direct lipid interaction. Pom1 autophosphorylates in this region to lower lipid affinity and promote membrane release. Tea4 triggers Pom1 plasma membrane association by promoting its dephosphorylation through the protein phosphatase 1 Dis2. We propose that local dephosphorylation induces Pom1 membrane association and nucleates a gradient shaped by the opposing actions of lateral diffusion and autophosphorylation-dependent membrane detachment.

Mid1p/anillin and the septation initiation network orchestrate contractile ring assembly for cytokinesis.

In both animal cells and fungi, cytokinesis proceeds via a contractile actomyosin ring (CAR). Many CAR components and regulators are evolutionarily conserved. In Schizosaccharomyces pombe, the spatial cue for cytokinesis is provided by Mid1p/Anillin, whereas temporal coordination is ensured by the septation initiation network (SIN). However, neither Mid1p nor the SIN is considered to be essential for CAR assembly per se. Here, using 4D imaging, we reveal an unanticipated, novel role for the SIN in CAR assembly. We demonstrate that CAR assembly involves three, genetically separable steps: establishment of a cortical network of CAR proteins, its lateral condensation, and finally, the formation of a homogeneous CAR. We show that SIN mutants fail to form a homogeneous CAR; we identify hypophosphorylation and recruitment of the conserved PCH-family protein Cdc15p to the CAR as critical steps requiring SIN function. Furthermore, we show that in the absence of Mid1p, CAR assembly proceeds via an actomyosin filament, rather than a cortical network of CAR proteins. This mode of assembly is totally dependent on SIN signaling, thereby demonstrating a direct role for the SIN in CAR formation. Taken together, these data establish that Mid1p and the SIN are the key regulators that orchestrate CAR assembly.

A translation-independent role of oskar RNA in early Drosophila oogenesis.

The Drosophila maternal effect gene oskar encodes the posterior determinant responsible for the formation of the posterior pole plasm in the egg, and thus of the abdomen and germline of the future fly. Previously identified oskar mutants give rise to offspring that lack both abdominal segments and a germline, thus defining the ;posterior group phenotype'. Common to these classical oskar alleles is that they all produce significant amounts of oskar mRNA. By contrast, two new oskar mutants in which oskar RNA levels are strongly reduced or undetectable are sterile, because of an early arrest of oogenesis. This egg-less phenotype is complemented by oskar nonsense mutant alleles, as well as by oskar transgenes, the protein-coding capacities of which have been annulled. Moreover, we show that expression of the oskar 3' untranslated region (3'UTR) is sufficient to rescue the egg-less defect of the RNA null mutant. Our analysis thus reveals an unexpected role for oskar RNA during early oogenesis, independent of Oskar protein. These findings indicate that oskar RNA acts as a scaffold or regulatory RNA essential for development of the oocyte.

Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization.

oskar messenger RNA localization at the posterior pole of the Drosophila oocyte is essential for germline and abdomen formation in the future embryo. The nuclear shuttling proteins Y14/Tsunagi and Mago nashi are required for oskar mRNA localization, and they co-localize with oskar mRNA at the posterior pole of the oocyte. Their human homologues, Y14/RBM8 and Magoh, are core components of the exon-exon junction complex (EJC). The EJC is deposited on mRNAs in a splicing-dependent manner, 20-24 nucleotides upstream of exon-exon junctions, independently of the RNA sequence. This indicates a possible role of splicing in oskar mRNA localization, challenging the established notion that the oskar 3' untranslated region (3'UTR) is sufficient for this process. Here we show that splicing at the first exon-exon junction of oskar RNA is essential for oskar mRNA localization at the posterior pole. We revisit the issue of sufficiency of the oskar 3'UTR for posterior localization and show that the localization of unrelated transcripts bearing the oskar 3'UTR is mediated by endogenous oskar mRNA. Our results reveal an important new function for splicing: regulation of messenger ribonucleoprotein complex assembly and organization for mRNA cytoplasmic localization.