Treatment with αvß3-integrin-specific 29P attenuates pressure-overload induced cardiac remodelling after transverse aortic constriction in mice.

Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin targeting has been used in the treatment of ocular disease and cancer, but little is known about its utility in the treatment of heart failure. Here we sought to determine whether the second generation orally available, αvß3-specific RGD-mimetic, 29P , was cardioprotective. Male mice were subjected to transverse aortic constriction (TAC) and treated with 50 µg/kg 29P or volume-matched saline as Vehicle control. At 3 weeks post-TAC, echocardiography showed that 29P treatment significantly restored cardiac function and structure indicating the protective effect of 29P treatment in this model of heart failure. Importantly, 29P treatment improved cardiac function giving improved fractional shortening, ejection fraction, heart weight and lung weight to tibia length fractions, together with partial restoration of Ace and Mme levels, as markers of the TAC insult. At a tissue level, 29P reduced cardiomyocyte hypertrophy and interstitial fibrosis, both of which are major clinical features of heart failure. RNA sequencing identified that, mechanistically, this occurred with concomitant alterations to genes involved molecular pathways associated with these processes such as metabolism, hypertrophy and basement membrane formation. Overall, targeting αvß3 with 29P provides a novel strategy to attenuate pressure-overload induced cardiac hypertrophy and fibrosis, providing a possible new approach to heart failure treatment.

Genetic and Pharmacological YAP Activation Induces Proliferation and Improves Survival in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiomyocyte loss following myocardial infarction cannot be addressed with current clinical therapies. Cell therapy with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a potential approach to replace cardiomyocyte loss. However, engraftment rates in pre-clinical studies have been low, highlighting a need to refine current iPSC-CM technology. In this study, we demonstrated that inducing Yes-associated protein (YAP) by genetic and pharmacological approaches resulted in increased iPSC-CM proliferation and reduced apoptosis in response to oxidative stress. Interestingly, iPSC-CM maturation was differently affected by each strategy, with genetic activation of YAP resulting in a more immature cardiomyocyte-like phenotype not witnessed upon pharmacological YAP activation. Overall, we conclude that YAP activation in iPSC-CMs enhances cell survival and proliferative capacity. Therefore, strategies targeting YAP, or its upstream regulator the Hippo signalling pathway, could potentially be used to improve the efficacy of iPSC-CM technology for use as a future regenerative therapy in myocardial infarction.

Micro RNA-411 Expression Improves Cardiac Phenotype Following Myocardial Infarction in Mice.

Induction of endogenous regenerative capacity has emerged as one promising approach to repair damaged hearts following myocardial infarction (MI). Re-expression of factors that are exclusively expressed during embryonic development may reactivate the ability of adult cardiomyocytes to regenerate. Here, we identified miR-411 as a potent inducer of cardiomyocyte proliferation. Overexpression of miR-411 in the heart significantly increased cardiomyocyte proliferation and survival in a model MI. We found that miR-411 enhances the activity of YAP, the main downstream effector of the Hippo pathway, in cardiomyocytes. In conclusion, miR-411 induces cardiomyocyte regeneration and improves cardiac function post-MI likely by modulating the Hippo/YAP pathway.

Cardiomyocyte-specific loss of plasma membrane calcium ATPase 1 impacts cardiac rhythm and is associated with ventricular repolarisation dysfunction.

Plasma membrane calcium ATPase 1 (PMCA1, Atp2b1) is emerging as a key contributor to cardiac physiology, involved in calcium handling and myocardial signalling. In addition, genome wide association studies have associated PMCA1 in several areas of cardiovascular disease including hypertension and myocardial infarction. Here, we investigated the role of PMCA1 in basal cardiac function and heart rhythm stability. Cardiac structure, heart rhythm and arrhythmia susceptibility were assessed in a cardiomyocyte-specific PMCA1 deletion (PMCA1CKO) mouse model. PMCA1CKO mice developed abnormal heart rhythms related to ventricular repolarisation dysfunction and displayed an increased susceptibility to ventricular arrhythmias. We further assessed the levels of cardiac ion channels using qPCR and found a downregulation of the voltage-dependent potassium channels, Kv4.2, with a corresponding reduction in the transient outward potassium current which underlies ventricular repolarisation in the murine heart. The changes in heart rhythm were found to occur in the absence of any structural cardiomyopathy. To further assess the molecular changes occurring in PMCA1CKO hearts, we performed proteomic analysis. Functional characterisation of differentially expressed proteins suggested changes in pathways related to metabolism, protein-binding, and pathways associated cardiac function including ß-adrenergic signalling. Together, these data suggest an important role for PMCA1 in basal cardiac function in relation to heart rhythm control, with reduced cardiac PMCA1 expression resulting in an increased risk of arrhythmia development.

The Feasibility and Applicability of Percutaneous Deep Vein Arterialization in Peripheral Artery Disease.

Peripheral artery disease (PAD) can often present with chronic limb threatening ischemia (CLTI), including ischemic rest pain and severe tissue loss. Progression of PAD can lead to "no option" or end-stage disease in which there are no traditional open or endovascular interventions available for revascularization. This cohort of patients have a poor prognosis, with a major amputation rate of 40% and mortality of up to 20% at six months. For this patient population, surgical deep vein arterialization (DVA) is offered as an attempt to provide blood flow to the distal preserved venous bed and reverse the ischemic process. Surgical DVA has traditionally been offered as an option and was pioneered by Herb Dardik. The evolution of endovascular technology has allowed for percutaneous DVA (pDVA). Using ultrasound and fluoroscopic guidance, an arteriovenous channel is created between a tibial artery and vein and reinforced with covered stent grafts to increase distal limb perfusion with the goals of improving wound healing and amputation-free survival. Lysis of venous valves with a valvulotome also aids with reversal of flow into the distal venous system. Investigations of percutaneous deep vein arterialization are underway with one device, the LimFlow System (LimFlow SA, Paris, France), which is undergoing feasibility trials. Here we present the current clinical indications, feasibility, results, and our institutional experience with the use of percutaneous deep vein arterialization.

Chiral α-Stereogenic Oxetanols and Azetidinols via Alcohol-Mediated Reductive Coupling of Allylic Acetates: Enantiotopic π-Facial Selection in Symmetric Ketone Addition.

Iridium-tol-BINAP-catalyzed reductive coupling of allylic acetates with oxetanones and azetidinones mediated by 2-propanol provides chiral α-stereogenic oxetanols and azetidinols. As illustrated in 50 examples, complex, nitrogen-rich substituents that incorporate the top 10 N-heterocycles found in FDA-approved drugs are tolerated. In addition to 2-propanol-mediated reductive couplings, oxetanols and azetidinols may serve dually as reductant and ketone proelectrophiles in redox-neutral C-C couplings via hydrogen auto-transfer, as demonstrated by the conversion of dihydro-1a and dihydro-1b to adducts 3a and 4a, respectively. The present method delivers hitherto inaccessible chiral oxetanols and azetidinols, which are important bioisosteres.

The plasma membrane calcium ATPase 4 does not influence parasite levels but partially promotes experimental cerebral malaria during murine blood stage malaria.

BACKGROUND: Recent genome wide analysis studies have identified a strong association between single nucleotide variations within the human ATP2B4 gene and susceptibility to severe malaria. The ATP2B4 gene encodes the plasma membrane calcium ATPase 4 (PMCA4), which is responsible for controlling the physiological level of intracellular calcium in many cell types, including red blood cells (RBCs). It is, therefore, postulated that genetic differences in the activity or expression level of PMCA4 alters intracellular Ca2+ levels and affects RBC hydration, modulating the invasion and growth of the Plasmodium parasite within its target host cell. METHODS: In this study the course of three different Plasmodium spp. infections were examined in mice with systemic knockout of Pmca4 expression. RESULTS: Ablation of PMCA4 reduced the size of RBCs and their haemoglobin content but did not affect RBC maturation and reticulocyte count. Surprisingly, knockout of PMCA4 did not significantly alter peripheral parasite burdens or the dynamics of blood stage Plasmodium chabaudi infection or reticulocyte-restricted Plasmodium yoelii infection. Interestingly, although ablation of PMCA4 did not affect peripheral parasite levels during Plasmodium berghei infection, it did promote slight protection against experimental cerebral malaria, associated with a minor reduction in antigen-experienced T cell accumulation in the brain. CONCLUSIONS: The finding suggests that PMCA4 may play a minor role in the development of severe malarial complications, but that this appears independent of direct effects on parasite invasion, growth or survival within RBCs.

Ethanol: Unlocking an Abundant Renewable C2 -Feedstock for Catalytic Enantioselective C-C Coupling.

With annual production at >85 million tons/year, ethanol is the world's largest-volume renewable small molecule carbon source, yet its use as a C2 -feedstock in enantioselective C-C coupling is unknown. Here, the first catalytic enantioselective C-C couplings of ethanol are demonstrated in reactions with structurally complex, nitrogen-rich allylic acetates incorporating the top 10 N-heterocycles found in FDA-approved drugs.

Ultrasound-guided cannulation for medical students.

BACKGROUND: There is growing recommendation to integrate ultrasound training into undergraduate medical curricula; with limited evidence of its implementation in the United Kingdom. Peripheral intravenous cannulation (PIVC) often has high failure rates-particularly for patients with difficult vascular access. Ultrasound-guided PIVC (US-PIVC) has been shown to increase the rates of success and may reduce dependence on seniors. Teaching US-PIVC to senior medical students may prove a clinically valuable and feasible means of introducing ultrasound into medical programmes. METHODS: We initially surveyed 18 doctors to assess their perceptions and experience of ultrasound. Thirty-five final-year medical students took part in a novel US-PIVC course. Students' competence was assessed at the end of the session using an objective assessment. Students' pre- and post-course attitudes and confidence were evaluated using questionnaires. RESULTS: All doctors surveyed reported difficulty performing PIVC since starting work: 66% on a monthly basis. None of the students (0%) and 11% of the doctors had previously received ultrasound training. By the end of the course, all students 'agreed' or 'strongly agreed' that the session was enjoyable and that they felt confident performing US-PIVC. Thirty-four of 35 participants attained the minimum competence standard. Students' confidence performing PIVC, regardless of ultrasound guidance, increased significantly (p = 0.002) following the session. DISCUSSION: As ultrasound becomes a more readily available point-of-care technology, it should be considered for inclusion in the undergraduate medical curriculum. Our course appears to be an effective model of teaching US-PIVC to final-year medical students. Similar courses could act as a pragmatic and clinically rewarding means of implementing US-PIVC teaching into the latter years of undergraduate curricula.

PMCA4 inhibition does not affect cardiac remodelling following myocardial infarction, but may reduce susceptibility to arrhythmia.

Ischaemic heart disease is the world's leading cause of mortality. Survival rates from acute myocardial infarction (MI) have improved in recent years; however, this has led to an increase in the prevalence of heart failure (HF) due to chronic remodelling of the infarcted myocardium, for which treatment options remain poor. We have previously shown that inhibition of isoform 4 of the plasma membrane calcium ATPase (PMCA4) prevents chronic remodelling and HF development during pressure overload, through fibroblast mediated Wnt signalling modulation. Given that Wnt signalling also plays a prominent role during remodelling of the infarcted heart, this study investigated the effect of genetic and functional loss of PMCA4 on cardiac outcomes following MI. Neither genetic deletion nor pharmacological inhibition of PMCA4 affected chronic remodelling of the post-MI myocardium. This was the case when PMCA4 was deleted globally, or specifically from cardiomyocytes or fibroblasts. PMCA4-ablated hearts were however less prone to acute arrhythmic events, which may offer a slight survival benefit. Overall, this study demonstrates that PMCA4 inhibition does not affect chronic outcomes following MI.

Signaling via the Interleukin-10 Receptor Attenuates Cardiac Hypertrophy in Mice During Pressure Overload, but not Isoproterenol Infusion.

Inflammation plays a key role during cardiac hypertrophy and the development of heart failure. Interleukin-10 (IL-10) is a major anti-inflammatory cytokine that is expressed in the heart and may play a crucial role in cardiac remodeling. Based on the evidence that IL-10 potentially reduces pathological hypertrophy, it was hypothesized that signaling via the IL-10 receptor (IL10R) in the heart produces a protective role in reducing cardiac hypertrophy. The aim of this study was to investigate the effects of the ablation of Il-10-r1 gene during pathological cardiac hypertrophy in mice. We found that IL-10R1 gene silencing in cultured cardiomyocytes diminished the anti-hypertrophic effect of Il-10 in TNF-α induced hypertrophy model. We then analyzed mice deficient in the Il-10-r1 gene (IL-10R1-/- mice) and subjected them to transverse aortic constriction or isoproterenol infusion to induce pathological hypertrophy. In response to transverse aortic constriction for 2 weeks, IL-10R1-/- mice displayed a significant increase in the hypertrophic response as indicated by heart weight/body weight ratio, which was accompanied by significant increases in cardiomyocyte surface area and interstitial fibrosis. In contrast, there was no difference in hypertrophic response to isoproterenol infusion (10 days) between the knockout and control groups. Analysis of cardiac function using echocardiography and invasive hemodynamic studies did not show any difference between the WT and IL-10R1-/- groups, most likely due to the short term nature of the models. In conclusion, our data shows that signaling via the IL-10 receptor may produce protective effects against pressure overload-induced hypertrophy but not against ß-adrenergic stimuli in the heart. Our data supports previous evidence that signaling modulated by IL-10 and its receptor may become a potential target to control pathological cardiac hypertrophy.

Pharmacological inhibition of Hippo pathway, with the novel kinase inhibitor XMU-MP-1, protects the heart against adverse effects during pressure overload.

BACKGROUND AND PURPOSE: The Hippo pathway has emerged as a potential therapeutic target to control pathological cardiac remodelling. The core components of the Hippo pathway, mammalian Ste-20 like kinase 1 (Mst1) and mammalian Ste-20 like kinase 2 (Mst2), modulate cardiac hypertrophy, apoptosis, and fibrosis. Here, we study the effects of pharmacological inhibition of Mst1/2 using a novel inhibitor XMU-MP-1 in controlling the adverse effects of pressure overload-induced hypertrophy. EXPERIMENTAL APPROACH: We used cultured neonatal rat cardiomyocytes (NRCM) and C57Bl/6 mice with transverse aortic constriction (TAC) as in vitro and in vivo models, respectively, to test the effects of XMU-MP-1 treatment. We used luciferase reporter assays, western blots and immunofluorescence assays in vitro, with echocardiography, qRT-PCR and immunohistochemical methods in vivo. KEY RESULTS: XMU-MP-1 treatment significantly increased activity of the Hippo pathway effector yes-associated protein and inhibited phenylephrine-induced hypertrophy in NRCM. XMU-MP-1 improved cardiomyocyte survival and reduced apoptosis following oxidative stress. In vivo, mice 3 weeks after TAC, were treated with XMU-MP-1 (1 mg·kg-1 ) every alternate day for 10 further days. XMU-MP-1-treated mice showed better cardiac contractility than vehicle-treated mice. Cardiomyocyte cross-sectional size and expression of the hypertrophic marker, brain natriuretic peptide, were reduced in XMU-MP-1-treated mice. Improved heart function in XMU-MP-1-treated mice with TAC, was accompanied by fewer TUNEL positive cardiomyocytes and lower levels of fibrosis, suggesting inhibition of cardiomyocyte apoptosis and decreased fibrosis. CONCLUSIONS AND IMPLICATIONS: The Hippo pathway inhibitor, XMU-MP-1, reduced cellular hypertrophy and improved survival in cultured cardiomyocytes and, in vivo, preserved cardiac function following pressure overload.

The Cross-Talk Between the TNF-α and RASSF-Hippo Signalling Pathways.

The regulation of cell death through apoptosis is essential to a number of physiological processes. Defective apoptosis regulation is associated with many abnormalities including anomalies in organ development, altered immune response and the development of cancer. Several signalling pathways are known to regulate apoptosis including the Tumour Necrosis Factor-α (TNF-α) and Hippo signalling pathways. In this paper we review the cross-talk between the TNF-α pathway and the Hippo signalling pathway. Several molecules that tightly regulate the Hippo pathway, such as members of the Ras-association domain family member (RASSF) family proteins, interact and modulate some key proteins within the TNF-α pathway. Meanwhile, TNF-α stimulation also affects the expression and activation of core components of the Hippo pathway. This implies the crucial role of signal integration between these two major pathways in regulating apoptosis.

Cardiac hypertrophy or failure? - A systematic evaluation of the transverse aortic constriction model in C57BL/6NTac and C57BL/6J substrains.

BACKGROUND: The mouse model of transverse aortic constriction (TAC) has been widely used as a cardiac stress in the investigation of the molecular mechanisms of cardiac hypertrophy. Recently, the International Knockout Mouse Consortium has selected the C57BL/6NTac (BL/6N) mouse strain to generate null alleles for all mouse genes; however, a range of genetic and cardiac phenotypic differences have been reported between this substrain and the commonly used C57BL/6J (BL/6J) substrain. It has been reported by Garcia-Menendez and colleagues that 12-week C57BL/6NTac mice are susceptible to heart failure but little is known about the cardiac remodeling in this substrain as cardiac function progresses from compensation to decompensation. METHODS: BL/6J and BL/6N mice were subjected to pressure overload via TAC. The impact of both age and duration of cardiac pressure overload induced by TAC on cardiac remodelling were systematically assessed. RESULTS: Our data showed that BL/6N mice developed eccentric hypertrophy with age- and time-dependent deterioration in cardiac function, accompanied by considerable interstitial fibrosis. In contrast, BL/6J mice were more resilient to TAC-induced cardiac stress and developed variable cardiac phenotypes independent of age and the duration of pressure overload. This was likely due to the greater variability in pre-TAC aortic arch dimension as measured by echocardiography. In addition to increased expression of brain natriuretic peptide and collagen gene type 1 and 3, BL/6N mice also had greater angiotensin II type 2 receptor (AT2R) gene expression than BL/6J counterparts at baseline and after 2-weeks TAC, which may contribute to the exacerbated interstitial fibrosis. CONCLUSIONS: BL/6N and BL/6J mice have very different responses to TAC stimulation and these differences should be taken into consideration when using the substrains to investigate the mechanisms of hypertrophy and heart failure.

Advanced glycation end products reduce the calcium transient in cardiomyocytes by increasing production of reactive oxygen species and nitric oxide.

Advanced glycation end products (AGE) are central to the development of cardiovascular complications associated with diabetes mellitus. AGE may alter cellular function through cross-linking of cellular proteins or by activating the AGE receptor (RAGE). However, the signalling molecules involved during AGE stimulation in cardiomyocytes remain unclear. Here, we investigated the effects of AGE treatment on intracellular calcium homeostasis of isolated cardiomyocytes and studied the activation of signalling molecules involved in this process. Treatment of cardiomyocytes with AGE for 24 h resulted in a dose-dependent reduction in calcium transient amplitude, reaching a maximum 50% reduction at a dose of 1 mg·mL-1. This was accompanied with a 32% reduction in sarcoplasmic reticulum calcium content but without any detectable changes in the expression of major calcium channels. Mechanistically, we observed a significant increase in the production of reactive oxygen species (ROS) in AGE-treated cardiomyocytes and enhancement of NADPH oxidase activity. This was accompanied with activation of p38 kinase and nuclear translocation of NF-κB, and subsequently induction of inducible NO synthase (iNOS) expression, leading to excessive nitric oxide production. Overall, our data reveal the molecular signalling that may underlie the alteration of intracellular calcium homeostasis in cardiac myocytes due to AGE stimulation. This may provide new insights into the pathophysiological mechanisms of the development of diabetic cardiomyopathy.

Measuring the response of human head and neck squamous cell carcinoma to irradiation in a microfluidic model allowing customized therapy.

Radiotherapy is the standard treatment for head and neck squamous cell carcinoma (HNSCC), however, radioresistance remains a major clinical problem despite significant improvements in treatment protocols. Therapeutic outcome could potentially be improved if a patient's tumour response to irradiation could be predicted ex vivo before clinical application. The present study employed a bespoke microfluidic device to maintain HNSCC tissue whilst subjecting it to external beam irradiation and measured the responses using a panel of cell death and proliferation markers. HNSCC biopsies from five newly-presenting patients [2 lymph node (LN); 3 primary tumour (PT)] were divided into parallel microfluidic devices and replicates of each tumour were subjected to single-dose irradiation (0, 5, 10, 15 and 20 Gy). Lactate dehydrogenase (LDH) release was measured and tissue sections were stained for cytokeratin (CK), cleaved-CK18 (cCK18), phosphorylated-H2AX (γH2AX) and Ki­67 by immunohistochemistry. In addition, fragmented DNA was detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Compared with non­irradiated controls, higher irradiation doses resulted in elevated CK18-labelling index in two lymph nodes [15 Gy; 34.8% on LN1 and 31.7% on LN2 (p=0.006)] and a single laryngeal primary tumour (20 Gy; 31.5%; p=0.014). Significantly higher levels of DNA fragmentation were also detected in both lymph node samples and one primary tumour but at varying doses of irradiation, i.e., LN1 (20 Gy; 27.6%; p=0.047), LN2 (15 Gy; 15.3%; p=0.038) and PT3 (10 Gy; 35.2%; p=0.01). The γH2AX expression was raised but not significantly in the majority of samples. The percentage of Ki­67 positive nuclei reduced dose-dependently following irradiation. In contrast no significant difference in LDH release was observed between irradiated groups and controls. There is clear inter- and intra-patient variability in response to irradiation when measuring a variety of parameters, which offers the potential for the approach to provide clinically valuable information.

Maintenance of head and neck tumor on-chip: gateway to personalized treatment?

AIM: Head and neck squamous cell carcinomas (HNSCC) are solid tumors with low overall survival (40-60%). In a move toward personalized medicine, maintenance of tumor biopsies in microfluidic tissue culture devices is being developed. METHODOLOGY/RESULTS: HNSCC (n = 15) was dissected (5-10 mg) and either analyzed immediately or cultured in a microfluidic device (37°C) for 48 h. No difference was observed in morphology between pre- and postculture specimens. Dissociated samples were analyzed using trypan blue exclusion (viability), propidium iodide flow cytometry (death) and MTS assay (proliferation) with no significant difference observed highlighting tissue maintenance. Computational fluid dynamics showed laminar flow within the system. CONCLUSION: The microfluidic culture system successfully maintained HNSCC for 48 h, the culture system will allow testing of different treatment modalities with response monitoring.

The Plasma Membrane Calcium ATPases and Their Role as Major New Players in Human Disease.

The Ca2+ extrusion function of the four mammalian isoforms of the plasma membrane calcium ATPases (PMCAs) is well established. There is also ever-increasing detail known of their roles in global and local Ca2+ homeostasis and intracellular Ca2+ signaling in a wide variety of cell types and tissues. It is becoming clear that the spatiotemporal patterns of expression of the PMCAs and the fact that their abundances and relative expression levels vary from cell type to cell type both reflect and impact on their specific functions in these cells. Over recent years it has become increasingly apparent that these genes have potentially significant roles in human health and disease, with PMCAs1-4 being associated with cardiovascular diseases, deafness, autism, ataxia, adenoma, and malarial resistance. This review will bring together evidence of the variety of tissue-specific functions of PMCAs and will highlight the roles these genes play in regulating normal physiological functions and the considerable impact the genes have on human disease.

Genetic ablation of the mammalian sterile-20 like kinase 1 (Mst1) improves cell reprogramming efficiency and increases induced pluripotent stem cell proliferation and survival.

Adult fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSC) for use in various applications. However, there are challenges in iPSC generation including low reprogramming efficiency, yield, cell survival and viability. Since the Hippo signalling pathway is a key pathway involved in regulating cell proliferation and survival, we here test whether modification of the Hippo pathway will enhance the efficiency of iPSC generation and improve their survival. The Hippo pathway was modified by genetic ablation of the mammalian sterile-20 like kinase 1 (Mst1), a major component of the pathway. Using adult skin fibroblasts isolated from Mst1 knockout mice (Mst1-/-) as a source of iPSC we found that genetic ablation of Mst1 leads to significantly increased reprogramming efficiency by 43.8%. Moreover, Mst1-/- iPSC displayed increase proliferation by 12% as well as an increase in cell viability by 20% when treated with a chemical hypoxic inducer. Mechanistically, we found higher activity of YAP, the main downstream effector of the Hippo pathway, in iPSC lacking Mst1. In conclusion, our data suggests that Mst1 can be targeted to improve the efficiency of adult somatic cell reprogramming as well as to enhance iPSC proliferation and survival.