Drp1 regulates transcription of ribosomal protein genes in embryonic hearts.

Mitochondrial dysfunction causes severe congenital cardiac abnormalities and prenatal/neonatal lethality. The lack of sufficient knowledge regarding how mitochondrial abnormalities affect cardiogenesis poses a major barrier for the development of clinical applications that target mitochondrial deficiency-induced inborn cardiomyopathies. Mitochondrial morphology, which is regulated by fission and fusion, plays a key role in determining mitochondrial activity. Dnm1l encodes a dynamin-related GTPase, Drp1, which is required for mitochondrial fission. To investigate the role of Drp1 in cardiogenesis during the embryonic metabolic shift period, we specifically inactivated Dnm1l in second heart field-derived structures. Mutant cardiomyocytes in the right ventricle (RV) displayed severe defects in mitochondrial morphology, ultrastructure and activity. These defects caused increased cell death, decreased cell survival, disorganized cardiomyocytes and embryonic lethality. By characterizing this model, we reveal an AMPK-SIRT7-GABPB axis that relays the reduced cellular energy level to decrease transcription of ribosomal protein genes in cardiomyocytes. We therefore provide the first genetic evidence in mouse that Drp1 is essential for RV development. Our research provides further mechanistic insight into how mitochondrial dysfunction causes pathological molecular and cellular alterations during cardiogenesis.

Research Progress on Aortic Root Aneurysms.

Aortic root aneurysms are one of the most common aortic root diseases, involving the aortic valve, aortic sinus, bilateral coronary arteries, and part of the ascending aorta. It is a life-threatening aortic disease with a high mortality rate of approximately 90%, due to aortic aneurysm rupture. Aortic valve insufficiency is one of the most common complications of aortic root aneurysms that can lead to acute left heart failure. The etiology of aortic root aneurysms is not yet completely clear and is mainly related to genetic diseases, such as Marfan syndrome and atherosclerosis. It can also occur secondary to aortic valve stenosis or a bivalve deformity. Surgery is the primary treatment for aortic root aneurysms, and aortic root replacement is a classic surgical method. However, the incidences of perioperative complications and mortality are relatively high, particularly in high-risk patients. In recent years, the anatomical structure of the aortic root has been gradually refined, and an in-depth understanding of root aneurysms has led to individualized treatment methods. Conservative drug therapy (ß-receptor blockers, angiotensin-converting enzyme inhibitors, or angiotensin receptor blockers), Bentall and modified Bentall surgeries (Button technology, Cabrol surgery, and modified Cabrol surgery), valve-sparing aortic root replacement (David and Yacoub), personalized external aortic root support, and endovascular intervention therapy have significantly improved the perioperative and long-term survival rates of patients with aortic root aneurysms. However, different treatment methods have their own advantages and disadvantages. This review aimed to summarize the current research progress and treatment of aortic root aneurysms.

A comfort analysis of AR glasses on physical load during long-term wearing.

The present study investigated the effect of the physical load of augmented reality (AR) glasses on subjective assessments for an extended duration of a video viewing task. Ninety-six subjects were recruited for this test and were divided by spectacle use, sex, age, and body mass index (BMI). Four glasses frame weights were assessed. To investigate their effectiveness, a novel prototype adopting three design interventions, (1) adjustable frame width, (2) ergonomic temples, and (3) fixed centre of gravity, was designed with regard to subjective discomfort ratings (nose, ear, and overall). Subjective discomfort in all regions was significantly increased with increasing physical load on the nose. In addition, non-spectacle users, women, older users, and participants in the middle BMI category reported higher discomfort than other groups. This finding could have important implications for the ergonomic design of AR glasses and could help to identify design considerations relevant to the emerging wearable display industry. Practitioner summary: This research aims to explore the influence of the physical load of augmented reality (AR) glasses. It found that discomfort was increased with added nose load. Non-spectacle users, women, older users, and participants in the middle BMI category were more sensitive to discomfort. The results have important implications for glasses-type wearables' design.

In-ear earphone design-oriented pressure sensitivity evaluation on the external ear.

This study aimed to explore the pressure sensitivity of the external ear that can be the basis for adapting the pressure distribution on the concha for in-ear earphone design. Overall, 30 participants were included in this study, where an electronic mechanical algometer with a stepping motor was used to apply constant pressure. Before the experiment, the customised concha shell models of the participants were positioned in the ear perpendicular to the concha surface. Furthermore, the pressure discomfort threshold (PDT), moderate pressure discomfort (MPD), and maximum pressure threshold (MPT) in eight regions of the ear were recorded. This study's results indicate that the four regions of the external ear are less sensitive to pressure than those of the other regions. Additionally, women had higher pressure sensitivity values in the external ear. Therefore, this study's findings could have important implications for earphone designs and evaluating discomfort conditions in the external ear. Practitioner summary: This study explores the pressure sensitivity threshold (PDT, MPD, and MPT) on the external ear and the relevant implications for in-ear earphone design. Interestingly, regions closer to the bone structure were less sensitive to pressure, and men could tolerate greater pressure on the external ear than women.

Ex Vivo Hydrostatic Pressure Loading of Atrial Tissues Activates Profibrotic Transcription via TGF-ß Signal Pathway.

Excessive mechanical stress causes fibrosis-related atrial arrhythmia. Herein, we tried to investigate the mechanism of atrial fibrogenesis in response to mechanical stress by ex vivo approach. We collected atrial tissues from mice and then cultured them as "explants" under atmospheric pressure (AP group) or 50 mmHg hydrostatic pressure loading (HP group) conditions. Pathway-specific PCR array analysis on the expression of fibrosis-related genes indicated that the loading of atrial tissues to 50 mmHg for 24 hours extensively upregulated a series of profibrotic genes. qRT-PCR data also showed that loading atrial tissues to 50 mmHg enhanced Rhoa, Rock2, and Thbs1 expression at different time points. Interestingly, the enhanced expression of Thbs1 at 1 hour declined at 6-24 hours and then increased again at 72 hours. In contrast, an enhanced expression of Tgfb1 was observed at 72 hours. In contrast, daily loading to 50 mmHg for 3 hours significantly accelerated the outgrowth of mesenchymal stem-like stromal cells from atrial tissues; however, we did not observe significant phenotypic changes in these outgrowing cells. Our ex vivo experimental data clearly show the induction of profibrotic transcription of atrial tissues by HP loading, which confirms the common pathological feature of atrial fibrosis following pressure overload.

Circular RNA Foxo3 in cardiac ischemia-reperfusion injury in heart transplantation: A new regulator and target.

Ischemia-reperfusion (I/R) injury occurring in heart transplantation (HT) remains as a leading cause of transplant heart graft failure. Circular RNAs (circRNAs) play important roles in gene regulation and diseases. However, the impact of circRNAs on I/R injury during HT remains unknown. This study aims to investigate the role of circular RNA Foxo3 (circFoxo3) in I/R injury in HT. Using an in vivo mouse HT model and an in vitro cardiomyocyte culture model, we demonstrated that circFoxo3 is significantly upregulated in I/R-injured hearts and hypoxia/reoxygenation (H/R)-damaged cardiomyocytes. Knockdown of circFoxo3 using siRNA not only reduces cell apoptosis and death, mitochondrial damage, and expression of apoptosis/death-related genes in vitro, but also protects heart grafts from prolonged cold I/R injury in HT. We also show that circFoxo3 interacts with Foxo3 proteins and inhibits the phosphorylation of Foxo3 and that it indirectly affects the expression of miR-433 and miR-136. In conclusion, circRNA is involved in I/R injury in HT and knockdown of circFoxo3 with siRNA can reduce I/R injury and improve heart graft function through interaction with Foxo3. This study highlights that circRNA is a new type of molecular regulator and a potential target for preventing I/R injury in HT.

SEMA6D regulates perinatal cardiomyocyte proliferation and maturation in mice.

Cardiomyocytes undergo dramatic changes during the fetal to neonatal transition stage to adapt to the new environment. The molecular and genetic mechanisms regulating these changes remain elusive. In this study, we showed Sema6D as a novel signaling molecule regulating perinatal cardiomyocyte proliferation and maturation. SEMA6D is a member of the Semaphorin family of signaling molecules. To reveal its function during cardiogenesis, we specifically inactivated Sema6D in embryonic cardiomyocytes using a conditional gene deletion approach. All mutant animals showed hypoplastic myocardial walls in neonatal hearts due to reduced cell proliferation. We further revealed that expression of MYCN and its downstream cell cycle regulators is impaired in late fetal hearts in which Sema6D is deleted, suggesting that SEMA6D acts through MYCN to regulate cardiomyocyte proliferation. In early postnatal mutant hearts, expression of adult forms of sarcomeric proteins is increased, while expression of embryonic forms is decreased. These data collectively suggest that SEMA6D is required to maintain late fetal/early neonatal cardiomyocytes at a proliferative and less mature status. Deletion of Sema6D in cardiomyocytes led to reduced proliferation and accelerated maturation. We further examined the consequence of these defects through echocardiographic analysis. Embryonic heart deletion of Sema6D significantly impaired the cardiac contraction of male adult hearts, while having a minor effect on female mutant hearts, suggesting that the effect of Sema6D-deletion in adult hearts is sex dependent.

A New Aortic Arch Inclusion Technique With Frozen Elephant Trunk for Type A Aortic Dissection.

OBJECTIVE: Our aims were to describe a new surgical technique for the treatment of type A aortic dissection (TAAD) and to report the operative outcomes of 154 patients. SUMMARY BACKGROUND DATA: Surgical treatment of TAAD is complicated and carries a high mortality risk. To lower this risk, we developed a simplified procedure in which a stent graft was implanted as frozen elephant trunk (FET), and the proximally trimmed vascular graft was sutured from the inside of the aortic arch using the inclusion technique under moderate hypothermic circulatory arrest and antegrade selective cerebral perfusion. METHODS: We conducted a retrospective analysis of 154 cases of TAAD treated with our novel technique (93 men and 61 women, 52.5 ±â€Š11.4 years). Computed tomography angiography was performed before discharge and at 6 months postoperatively. RESULTS: In-hospital mortality rate was 5.19%, with paraplegia occurring in 2 patients (1.3%) and stroke in 6 (3.9%). The rate of closure of the aortic arch false lumen was 77.8%, with a 69.2% rate of descending thoracic aorta thrombosis at discharge. The survival rate was 91.1% at a mean follow-up of 21 ±â€Š10 months, with rates of aortic arch false lumen closure of 92.4% and descending thoracic aorta thrombosis of 74.3% at 6 months postoperatively. CONCLUSIONS: The aortic arch inclusion technique with FET provides a safe alternative for TAAD treatment, with satisfactory operative results. Short-term follow-up results are encouraging, and long-term outcomes need further evaluation.

Nicaraven Attenuates Postoperative Systemic Inflammatory Responses-Induced Tumor Metastasis.

BACKGROUND: Inflammation has been demonstrated to promote cancer metastasis. Due to the well-known systemic inflammatory responses (SIR) after major surgery, it is critical to investigate and attenuate SIR-induced tumor metastasis of cancer patients suffering surgical procedures. METHODS: C57BL/6 mice were intravenously injected with Lewis lung cancer cells at 6, 24, and 72 h after the induction of intestinal ischemia/reperfusion (I/R) injury. We found that the number of tumor nodules significantly increased in lungs of mice injected with cancer cells at 6 h but not at 24 and 72 h after I/R injury. The administration of nicaraven 30 min before and 24 h after I/R injury effectively attenuated the enhanced tumor metastasis to lungs. Protein array showed the increase of various cytokines in plasma of mice at 6 h after I/R injury, but many of them were attenuated by the administration of nicaraven. Immunostaining indicated the increase of Ly6g-, CD206-, and CD11c-positive inflammatory cells in the lungs, but it was also attenuated by nicaraven administration. CONCLUSIONS: Postoperative SIR-induced tumor metastasis have been clearly evidenced in our experimental model, and the administration of nicaraven may ameliorate the SIR-induced tumor metastasis by suppressing inflammatory responses.

The role of miR-711 in cardiac cells in response to oxidative stress and its biogenesis: a study on H9C2 cells.

BACKGROUND: Oxidative stress results in cell apoptosis/death and plays a detrimental role in disease development and progression. Stressors alter the miRNA expression profile and miRNAs play a role in the cell response to stress. We previously showed that miR-711 is significantly over-expressed in extended cold ischemia reperfusion injured hearts in heart transplant. In this study, we aimed to investigate the role of miR-711 in cardiac cell damage in response to oxidative stress and how miR-711 is regulated. METHODS: Rat cardiac cell line H9c2 cells were cultured and exposed to oxidative conditions (Antimycin A (AA), H2O2, CoCl2, or cold hypoxia/reoxygenation (H/R)) in vitro. H9c2 cells were transfected with miR-711 mimics, miR-711 inhibitors, or small interference RNA, using transfection reagents. The expression of miR-711 was measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cell apoptosis/death was detected by flow cytometry and an IncuCyte system. Mitochondrial damage was detected by measuring the mitochondria membrane potential by flow cytometry. Gene expression was detected by qRT-PCR at the mRNA level and Western blotting and immunocytochemistry staining at the protein level. RESULTS: We found that miR-711 was significantly up-regulated in cells treated with H2O2, AA, CoCl2, and cold H/R. Over-expression of miR-711 increased cell apoptosis/death induced by AA and H/R whereas cell death was reduced by miR-711 inhibitors. MiR-711 induced cell death through negative regulation of angiopoietin 1 (Ang-1), fibroblast growth factor 14 (FGF14) and calcium voltage-gated channel subunit alpha1C (Cacna1c) genes. Both knockdown of hypoxia inducible factor 1α (HIF-1α) and inactivation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFКB) pathway inhibited over-expression of miR-711. CONCLUSION: Oxidative stress increases the expression of miR-711. Over-expression of miR-711 induces cell apoptosis/death. HIF-1α and NFКB regulate miR-711 in H9c2 cells during oxidative stress. miR-711 is a new target for preventing oxidative stress.

A Long-Term Complication Occurred After Transcatheter Closure of Large Atrial Septal Defect.

Transcatheter closure of ostium secondum atrial septal defect has become an alternative method to surgical closure. However, the incidence of complications and long-term results of using large size (> 40 mm) Amplatzer septal occluders are unknown. This case reported a 59 years old woman, whom received transcatheter closure of atrial septal defect (36 mm) with a 40 mm Amplatzer septal occluder 10 years ago and was diagnosed with heart failure. Transthroacic echocardiography showed severe mitral valve regurgitation. Intra-operatively, we confirmed and removed the large device, but we found that the mitral annulus was badly damaged. Mitral valve replacement was performed. We believe large size devices need to be implanted cautiously, especially for the large defect with insufficient rims, and also routinely follow-up is necessary.

A New Aortic Arch Inclusion Technique with Frozen Elephant Trunk for Aortic Arch Aneurysm Treatment.

Various surgical techniques have been proposed for treating aortic arch aneurysm (AAA); however, the optimal treatment has not been well defined. This study introduces a new aortic arch inclusion technique with frozen elephant trunk (FET) for AAA treatment.A retrospective analysis was performed among 22 patients for AAA surgical treatment between March 2010 and March 2019. Patients were classified into Z1, Z2, and Z3 groups based on the origins of aneurysms. A stent graft with a 10 cm stented graft and 5-9 cm proximal vascular prosthesis was released into the descending thoracic aorta as FET through an incision in the aortic arch. The proximal vascular prosthesis was retracted into the aortic arch, trimmed to expose the orifices of the brachiocephalic vessels, and sutured inside the aortic arch using the inclusion technique. The proximal sealing location of the vascular graft was tailored to cover the origins of aneurysms.There was no 30-day mortality. No patient had postoperative stroke or paraplegia. Complete aneurysm thrombosis was achieved in all patients. One patient died of severe respiratory tract stenosis 3 months postoperatively. All other 21 patients were alive during 53.3 ± 36.5-month follow-up. Computed tomography angiography was obtained in 15 patients during follow-up. Endoleak was observed in one patient, and the other 14 patients were free from aneurysm-related or graft-related complications during follow-up.The aortic arch inclusion technique with FET provides an alternative technique in treating AAA with satisfactory mid-term follow-up results. A larger patient population with long-term follow-up results is warranted.

Circular RNA in cardiovascular disease.

Circular RNA (circRNA) are endogenous transcripts that display differential expression across species, developmental stages, and pathologies. Their lack of free ends confers increased stability when compared with linear transcripts, making them ideal candidates for future diagnostic biomarkers and therapeutic interventions. Increasing evidence has implicated circRNA in the pathogenesis of multiple cardiovascular diseases. In this paper, we summarize current understanding of circRNA biogenesis, properties, expression profiles, detection methods, functions, and their implication in cardiac pathologies including/ischemia reperfusion injury, myocardial infarction, cardiac senescence, cardiac fibrosis, cardiomyopathy, cardiac hypertrophy and heart failure, atherosclerosis, coronary artery disease, and aneurysm.

Supplementing preservation solution with mitochondria-targeted H2 S donor AP39 protects cardiac grafts from prolonged cold ischemia-reperfusion injury in heart transplantation.

Heart transplant has been accepted as the standard treatment for end-stage heart failure. Because of its susceptibility to ischemia-reperfusion injury, the heart can be preserved for only 4 to 6 hours in cold static preservation solutions. Prolonged ischemia time is adversely associated with primary graft function and long-term survival. New strategies to preserve donor hearts are urgently needed. We demonstrate that AP39, a mitochondria-targeting hydrogen sulfide donor, significantly increases cardiomyocyte viability and reduces cell apoptosis/death after cold hypoxia/reoxygenation in vitro. It also decreases gene expression of proinflammatory cytokines and preserves mitochondria function. Using an in vivo murine heart transplant model, we show that preserving donor hearts with AP39-supplemented University of Wisconsin solution (n = 7) significantly protects heart graft function, measured by quantitative ultrasound scan, against prolonged cold ischemia-reperfusion injury (24 hours at 4°C), along with reducing tissue injury and fibrosis. Our study demonstrates that supplementing preservation solution with AP39 protects cardiac grafts from prolonged ischemia, highlighting its therapeutic potential in preventing ischemia-reperfusion injury in heart transplant.

Extracorporeal Membrane Oxygenation in Stanford Type A Aortic Dissection.

The aim of this study was to summarize the clinical experience of postoperative extracorporeal membrane oxygenation (ECMO) support in Stanford type A aortic dissection (STAAD) patients.We retrospectively reviewed 246 consecutive acute STAAD patients undergoing operations at our institution from January 2012 to December 2016. Postoperative ECMO was used in 7 patients. There were 5 males and 2 females with a mean age of 43.1 ± 9.3 years. All 7 patients with acute STAAD underwent ascending aorta replacement and total arch repair with a self-designed stent graft (Micropart Corp, Shanghai, China). Concomitant procedures were aortic root replacement in 1 patient and coronary artery bypass grafting (CABG) in 2 patients. All patients received veno-arterial ECMO through the femoral artery and vein. Five patients were extubated before being removed from ECMO. The mean ECMO supporting time was 244.5 ± 57.8 hours. All 7 patients were successfully weaned from ECMO support, and 6 (85.7%) patients survived to discharge. The average hospital time was 26.3 ± 8.8 days. One patient died of cardiac arrest after being weaned from ECMO. Two patients underwent reoperation for bleeding and 1 patient showed transient delirium. The remaining patients all survived during a median follow-up of 19 months.ECMO provides a good temporary cardiopulmonary support in STAAD patients with refractory cardiogenic shock after surgery for aortic dissection. The early use of ECMO and preventing its complications actively can improve the patient survival rate.

A New Aortic Root Reinforcement Technique for Acute Type A Aortic Dissection Surgery.

Reinforcing the dissected and fragile aortic root is critical in acute type A aortic dissection (ATAAD) surgery. This study introduces our new aortic root reinforcement technique and reports our early operative results and midterm follow-up results.A retrospective analysis study was performed on 235 patients (aged 53.2 ±15.5 years) who were admitted to our hospital for ATAAD surgery and underwent the procedure with our new technique between October 2011 and June 2016. Two vascular graft rings were placed inside and outside aortic root, followed by a running horizontal mattress suture, placed just above the coronary artery ostiums and aortic valve commissures, with another horizontal suture at distal end of the aortic root stump, to reinforce the inner vascular graft, aortic wall, and outside vascular graft. Then additional 3-5 vertical mattress sutures were placed for further reinforcement within the reconstructed aortic root. Computed tomography angiography was performed at discharge and annually during follow-up.The patients' 30-day mortality was 5.1% (12/235). There was no uncontrollable intraoperative bleeding from the aortic root, and re-exploration for bleeding occurred in 0.79% (2/235). The survival rate was 90.2% during follow-up of 4.2 ± 2.1 years. There were no requests for aortic root reoperations during follow-up. All patients were free from aortic root disruptions, proximal anastomosis complications, and re-dissections of the reconstructed aortic root.Our new aortic root reinforcement technique provides a safe and effective technique for aortic root in ATAAD surgery, by reinforcing friable aortic root tissues and minimizing aortic root complications.

Aorto-Cutaneous Fistula after Surgical Treatment of Stanford Type A Aortic Dissection.

An aorto-cutaneous fistula is a rare complication that occurs after aortic surgery. Due to its rarity, postoperative complications are not normally highlighted in most standard teaching. We report here a case of aorto-cutaneous fistula after surgical treatment of a Stanford type A aortic dissection (AD) in a 67-year-old Chinese male. The patient presented with severe right heart dysfunction and a mass was found in the upper-middle of his chest, which started bleeding in the next years. On admission, preoperative aortic computed tomography angiography (CTA) showed a huge hematoma located in the anterior superior mediastinum and a shunt between the embedding cavity of the aortic root and right atrium. An emergent procedure was performed. Intraoperatively, we found two leaks approximately 2 mm from the anastomosis of the greater curvature of the ascending aortic graft and stented graft after the hematoma was cleared and we confirmed the shunt had a large amount of blood flow after a right atrium incision. After the surgery, the patient was diagnosed with a cerebral hemorrhage, and his family decided to refuse therapy on the third postoperative day (p.o.d.).

A Vaginal Stillbirth after Aortic Surgery of Type B Aortic Dissection in a Pregnant Woman.

Acute aortic dissection occurring during pregnancy poses great danger to both the mother and fetus. Cesareans are usually performed before or after the aortic repair depending on the conditions of the mother and fetus. Here we report our experience in treating a 32-week pregnant woman with a type B aortic dissection, whose baby had died before admission. A cesarean section was initially arranged after emergency aortic repair. However, the patient started to deliver the fetus vaginally after the aortic surgery and the stillborn baby was delivered vaginally. This case report provides new insight into the method of delivery in a pregnant woman with an aortic dissection.

Pioglitazone Suppresses CXCR7 Expression To Inhibit Human Macrophage Chemotaxis through Peroxisome Proliferator-Activated Receptor γ.

Cardiovascular disease is the leading cause of morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). Pioglitazone, the widely used thiazolidinedione, is shown to be efficient in the prevention of cardiovascular complications of T2DM. In this study, we report that pioglitazone inhibits CXCR7 expression and thus blocks chemotaxis in differentiated macrophage without perturbing cell viability or macrophage differentiation. In addition, pioglitazone-mediated CXCR7 suppression and chemotaxis inhibition occur via activating peroxisome proliferator-activated receptor γ (PPARγ) but not PPARα in differentiated macrophage. More importantly, pioglitazone therapy-induced PPARγ activation suppresses CXCR7 expression in human carotid atherosclerotic lesions. Collectively, our data demonstrate that pioglitazone suppresses CXCR7 expression to inhibit human macrophage chemotaxis through PPARγ.

Liquid chromatography-tandem mass spectrometric assay for the determination of vaccarin in rat plasma: application to a pharmacokinetic study.

Vaccarin, a flavonoid glycoside, is considered one of the major active constituents of Vaccaria segetalis. A simple and specific liquid chromatography-tandem mass spectrometric method was developed and validated for quantifying vaccarin in rat plasma following intravenous dosing. Plasma samples were precipitated with methanol and separated on a Venusil-C18 analytical column (2.1 × 50 mm, 5 µm particles) with gradient elution consisting of methanol and 0.1% (v/v) formic acid as the mobile phase. The detection was performed on an Agilent Triple Quad LC/MS with electrospray ionization inlet in the positive multiple reaction monitoring mode. Good linearity was achieved over the concentration range of 12.5-25,000 ng/mL (r(2) > 0.99). Intra- and inter-day precisions were <9.1%, and accuracy ranged from -2.8 to 8.7%. The lower limit of quantification for vaccarin was 12.5 ng/mL, and the analyte was stable under various storage conditions. This validated method was successfully applied to the preliminary pharmacokinetic studies of vaccarin following intravenous administrations of 1.21, 2.41, and 4.82 mg/kg vaccarin in rats.