scholarly journals Cardiac-targeting magnetic lipoplex delivery of SH-IGF1R plasmid attenuate norepinephrine-induced cardiac hypertrophy in murine heart

2014 ◽  
Vol 34 (5) ◽  
Author(s):  
Yiping Xu ◽  
Xuebiao Li ◽  
Minjian Kong ◽  
Daming Jiang ◽  
Aiqiang Dong ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Molecular Mechanisms ◽  
High Efficiency ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Pressure Overload ◽  
Posterior Wall ◽  
Promising Method ◽  
Heart Weight

Recent studies have demonstrated a number of molecular mechanisms contributing to the initiation of cardiac hypertrophy response to pressure overload. IGF1R (insulin-like growth factor-1 receptor), an important oncogene, is overexpressed in hypertrophic heart and mediates the hypertrophic pathology process. In this study, we applied with liposomal magnetofection that potentiated gene transfection by applying an external magnetic field to enhance its transfection efficiency. Liposomal magnetofection provided high efficiency in transgene expression in vivo. In vivo, IGF1R-specific-shRNA (small-hairpin RNA) by magnetofection inhibited IGF1R protein expression by 72.2±6.8, 80.7±9.6 and 84.5±5.6%, at 24, 48 and 72 h, respectively, after pGFPshIGF1R injection, indicating that liposomal magnetofection is a promising method that allows the targeting of gene therapy for heart failure. Furthermore, we found that the treated animals (liposomal magnetofection with shIGF1R) showed reduced septal and posterior wall thickness, reduced HW:BWs (heart weight-to-body weights) compared with controls. Moreover, we also found that liposomal magnetofection-based shIGF1R transfection decreased the expression level of p-ERK (phosphorylated extracellular-signal-regulated kinase)1/2, p-AKT1 (phosphorylated protein kinase B1) compared with untreated hearts. These results suggested that liposomal magnetofection-mediated IGF1R-specific-shRNA may be a promising method, and suppression the IGF1R expression inhibited norepinephrine-induced cardiac hypertrophic process via inhibiting PI3K (phosphoinositide 3-kinase)/AKT pathway.

Abstract 080: Eya4 Induces Hypertrophy Via Regulation Of p27kip1

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Tatjana Williams ◽  
Moritz Hundertmark ◽  
Peter Nordbeck ◽  
Sabine Voll ◽  
Melanie Muehlfelder ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cardiac Hypertrophy ◽  
Molecular Mechanisms ◽  
Pressure Overload ◽  
Dominant Negative ◽  
Heart Weight ◽  
Dominant Negative Effect ◽  
Mutant Form ◽  
Cardiac Phenotype ◽  
Truncating Mutation

Introduction: E193, a truncating mutation in the transcription cofactor Eyes absent 4 (Eya4) causes hearing impairment followed by heart failure. Here we identified the Eya4 dependent molecular mechanisms leading to the cardiac phenotype in the E193 mutation. Methods and Results: First we showed in vitro that the cyclin-dependent kinase inhibitor protein p27kip1 is a direct target of Eya4/Six1 and is suppressed upon Eya4 overexpression, whereas E193 has a dominant negative effect, releasing Eya4 mediated suppression of p27. We next generated transgenic mice with cardiac specific constitutive overexpression of full-length Eya4 or the mutant form E193. While E193 transgenic mice developed age-dependent DCM, Eya4 mice displayed cardiac hypertrophy already under basal conditions as judged by increases in heart weight and cardiomyocyte cross-sectional areas along with increases in myocardial dimension and mass. These two distinct cardiac phenotypes were even more aggravated upon pressure overload suggesting Eya4 is a regulator of cardiac hypertrophy. We also observed that the activity of Casein Kinase 2-α and the phosphorylation status of HDAC2 were significantly upregulated in the Eya4 transgenic mice, while they were significantly reduced in E193 mice, under baseline conditions and pressure overload. We were also able to identify a new human mutation (E215) with a phenotype comparable to the one seen in E193 patients. Conclusion: Our results implicate that Eya4/Six1 regulates cardiac hypertrophic reactions via p27/CK2-α/HDAC2 and indicate that truncating mutations in Eya4 interfere with this newly established signalling pathway.

Abstract 159: Cardiac Hypertrophy and Sudden Death in a Mouse Model of STIM1 Overexpression

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Sanjeewa A Goonasekera ◽  
Jop van Berlo ◽  
Adam R Burr ◽  
Robert N Correll ◽  
Allen J York ◽  
...  
Keyword(s):  
Mouse Model ◽  
Cardiac Hypertrophy ◽  
Molecular Mechanisms ◽  
Ventricular Myocytes ◽  
Interstitial Fibrosis ◽  
Heart Weight ◽  
Activated T Cells ◽  
Lung Weight ◽  
Tibia Length

Background: STIM1, an ER/SR resident Ca 2+ sensing protein regulates Ca 2+ entry following internal Ca 2+ store depletion in a broad range of tissues and cell types. However their putative roles in excitable tissue such as cardiac myocytes is uncertain. Results: Here we generated a mouse model of STIM1 overexpression in cardiac and skeletal muscle. Western blot analysis suggested approximately 4-6 fold STIM1 overexpression in Tg mouse hearts compared to Ntg littermates. Immunocytochemistry carried out in ventricular myocytes revealed that STIM1 and the cardiac ryanodine receptor (RyR2) co-localize. Functionally, the amplitude of Ca 2+ entry following SR Ca 2+ depletion was 2-fold greater in myocytes isolated from STIM1 Tg mice compared to NTg littermates. Echocardiographic analysis in STIM1 Tg mice showed age dependent remodeling of the myocardium with a significant decrease in fractional shortening at 16 weeks of age (14.4.5±3.8 in STIM1 Tg vs. 36.9±1.5 in Ntg). These changes were accompanied by a significant increase in heart weight to tibia length (13.6 +/- 1.4 vs 6.5 +/- 0.24) and increased lung weight to tibia length ratio (11.6+/- 2.1 vs 8.1 +/- 0.38) in STIM1 Tg mice compared to Ntg littermates. Photometry experiments in isolated ventricular myocytes demonstrated significantly increased Ca 2+ transient amplitude with an unexpected decrease in the SR Ca 2+ load associated with STIM1 overexpression. In addition transgenic mice showed increased calcineurin-nuclear factor of activated T cells (NFAT) activation in vivo, increased CaMKII activity, interstitial fibrosis and exaggerated hypertrophy following two weeks of neuroendocrine agonist or pressure overload stimulation. Conclusion: Our observations suggest that STIM1 overexpression by itself can lead to cardiac hypertrophy and contribute to pathological cardiac remodeling and possibly sudden cardiac death. The molecular mechanisms underlying these phenomena are currently under investigation.

Blockade of MyD88 attenuates cardiac hypertrophy and decreases cardiac myocyte apoptosis in pressure overload-induced cardiac hypertrophy in vivo

2006 ◽  
Vol 290 (3) ◽  
pp. H985-H994 ◽  
Author(s):  
Tuanzhu Ha ◽  
Fang Hua ◽  
Yuehua Li ◽  
Jing Ma ◽  
Xiang Gao ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Cardiac Function ◽  
Cardiac Myocyte ◽  
Pressure Overload ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Heart Weight ◽  
Aortic Banding ◽  
Myocyte Apoptosis ◽  
Cardiac Myocyte Apoptosis

In this study, we evaluated whether blocking myeloid differentiation factor-88 (MyD88) could decrease cardiac myocyte apoptosis following pressure overload. Adenovirus expressing dominant negative MyD88 (Ad5-dnMyD88) or Ad5-green fluorescent protein (GFP) (Ad5-GFP) was transfected into rat hearts ( n = 8/group) immediately followed by aortic banding for 3 wk. One group of rats ( n = 8) was subjected to aortic banding for 3 wk without transfection. Sham surgical operation ( n = 8) served as control. The ratios of heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) were calculated. Cardiomyocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. Cardiac myocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and myocardial interstitial fibrosis was examined by Masson's Trichrome staining. Aortic banding significantly increased the HW/BW by 41.0% (0.44 ± 0.013 vs. 0.31 ± 0.008), HW/TL by 47.2% (42.7 ± 1.30 vs. 29.0 ± 0.69), cardiac myocyte size by 49.6%, and cardiac myocyte apoptosis by 11.5%, and myocardial fibrosis and decreased cardiac function compared with sham controls. Transfection of Ad5-dnMyD88 significantly reduced the HW/BW by 18.2% (0.36 ± 0.006 vs. 0.44 ± 0.013) and HW/TL by 22.3% (33.2 ± 0.95 vs. 42.7 ± 1.30) and decreased cardiomyocyte size by 56.8%, cardiac myocyte apoptosis by 76.2%, as well as fibrosis, and improved cardiac function compared with aortic-banded group. Our results suggest that MyD88 is an important component in the Toll-like receptor-4-mediated nuclear factor-κB activation pathway that contributes to the development of cardiac hypertrophy. Blockade of MyD88 significantly reduced cardiac hypertrophy, cardiac myocyte apoptosis, and improved cardiac function in vivo.

Efficiency and capacity of protein synthesis are increased in pressure overload cardiac hypertrophy

1988 ◽  
Vol 255 (2) ◽  
pp. H325-H328 ◽  
Author(s):  
R. Nagai ◽  
R. B. Low ◽  
W. S. Stirewalt ◽  
N. R. Alpert ◽  
R. Z. Litten
Keyword(s):  
Protein Synthesis ◽  
Pulmonary Artery ◽  
Cardiac Hypertrophy ◽  
Right Ventricle ◽  
Pressure Overload ◽  
Heart Weight ◽  
Rna Content ◽  
The Right ◽  
Pulmonary Artery Constriction

We measured the rate of protein synthesis and total RNA content in the right ventricle (RV) at day 2 and day 4 after pulmonary artery constriction to determine the contributions of changes in capacity and efficiency of in vivo protein synthesis to pressure overload (PO) cardiac hypertrophy. A significant increase in the proportion of RV weight to total heart weight was observed at day 2 and day 4 when compared with untreated controls. The rate of protein synthesis was significantly higher at day 2 post-PO (0.31 +/- 0.06 day-1 or 30 +/- 5 mg.g RV-1.day-1, means +/- SD, P less than 0.05) as well as at day 4 (0.25 +/- 0.05 day-1 or 28 +/- 9 mg.g RV-1.day-1, P less than 0.05) than for untreated rabbits (0.15 +/- 0.03 day-1 or 17 +/- 4 mg.g RV-1.day-1). RNA content was significantly higher at day 2 (1.47 +/- 0.17 mg/g RV, P less than 0.05) than in controls (1.16 +/- 0.14 mg/g RV), whereas there was a slight but nonsignificant increase at day 4 (1.36 +/- 0.21 mg/g RV, P less than 0.1). The efficiency of protein synthesis (synthesis/RNA) per gram RV was significantly increased both at day 2 (20.5 +/- 2.2 g protein.g RNA-1.day-1, P less than 0.05) and day 4 (19.8 +/- 3.5 g protein.g RNA-1.day-1, P less than 0.05) compared with control (14.6 +/- 2.3 g protein.g RNA-1.day-1). The increase in efficiency appeared to be caused by pressure overload itself based on a comparison of 0-4 day data vs. data obtained from sham animals (P less than 0.05).

Abstract 289: Retinoblastoma Gene Deletion Promotes Cardiac Dysfunction, Fibrosis and Apoptosis in Response to Pressure Overload

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Paul B Ammann ◽  
Takanobu Yamamoto ◽  
Peiyong Zhai ◽  
Junichi Sadoshima
Keyword(s):  
Cardiac Hypertrophy ◽  
Cardiac Dysfunction ◽  
Pressure Overload ◽  
Left Ventricular ◽  
Heart Weight ◽  
Tunel Staining ◽  
Pressure Gradients ◽  
Lung Weight ◽  
Rb Protein

The retinoblastoma (Rb) protein is a universal cell cycle regulator in mammals. When the Rb protein is phosphorylated by Cyclins/Cdks, it dissociates from E2F, and Rb-dependent E2F repression is subsequently inactivated. Furthermore, the Rb protein has also been implicated in the regulation of cardiac hypertrophy and apoptosis in cardiomyocytes (CMs). To elucidate the role of Rb in response to mechanical stress, we conducted transverse aortic constriction (TAC) in cardiac-specific Rb knockout mice (cRb-KO) in vivo (C57BL/6J). Cardiac-specific deletion of Rb was achieved by crossing Rb flox/flox mice with αMHC-Cre mice. Under basal conditions, 3- to 5-month-old cRb-KO mice showed increased heart weight (HW) (left ventricular weight/ tibial length (TL): 5.93 ± 029 vs. 4.76 ± 0.14, p< 0.01), increased apoptosis as determined by TUNEL staining (0.12% vs. 0.02%, p< 0.05) and a trend towards cardiac dysfunction (-dP/dt: 4320 ± 388 vs. 5933 ± 489 mmHg/sec, p < 0.05) compared to control mice (Rb flox/flox) Following 2 weeks of TAC, cRb-KO mice showed increased heart weight (HW/TL: 8.58 ± 0.35 vs. 7.50 ± 0.24, p < 0.05), cardiac dysfunction (ejection fraction (EF): 51.1% ± 4.0 vs. 74.3% ± 0.9, p < 0.01) , increased apoptosis as determined by TUNEL staining (0.48% vs. 0.05%, p < 0.01) and increased fibrosis as determined by Masson’s Trichrome staining (1.84% vs. 1.03%, p < 0.05) compared to Rb flox/flox mice after TAC. In response to 4 weeks of TAC, cRb-KO mice showed increased heart weight (HW/TL: 12.93 ± 085 vs. 9.32 ± 0.34, p < 0.01), lung weight (LW) (LW/TL: 18.35 ± 2.66 vs. 10.21 ± 1.93, p < 0.01), cardiac dysfunction (EF: 34.5% ± 8.3 vs. 64.3% ± 8.9, p < 0.01), increased apoptosis as determined by TUNEL staining (0,42% vs. 0,18%, p < 0.05) and increased fibrosis as determined by Masson’s Trichrome staining (4.2 % vs. 1.1 %, p < 0.05) compared to Rb flox/flox mice after TAC. Pressure gradients were similar between the cRb-KO mice submitted to 2 and 4 weeks of TAC and their respective controls. In conclusion, our results suggest that endogenous Rb plays an important role in mediating cell survival in CMs and negatively regulates cardiac hypertrophy at baseline. Furthermore, we showed that the Rb protein is important for the maintenance of cardiac function in response to pressure overload.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

Retracted Article: A multifunctional self-dissociative polyethyleneimine derivative coating polymer for enhancing the gene transfection efficiency of DNA/polyethyleneimine polyplexes in vitro and in vivo

2015 ◽  
Vol 6 (5) ◽  
pp. 780-796 ◽  
Author(s):  
Cheng Wang ◽  
Xiuli Bao ◽  
Xuefang Ding ◽  
Yang Ding ◽  
Sarra Abbad ◽  
...  
Keyword(s):  
Transfection Efficiency ◽  
Gene Transfection ◽  
Retracted Article ◽  
First Time

A novel coating polymer LPHF is developed for the first time to elevate the transfection efficiency of DP binary polyplexes in vitro and in vivo.

Abstract 292: TRPA1 Promoting Myofibroblast Differentiation Mediate Pressure Overload-Induced Cardiac Fibrosis

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_2) ◽  
Author(s):  
Shuang Li ◽  
Dong Han ◽  
Dachun Yang
Keyword(s):  
Knockout Mice ◽  
Molecular Mechanisms ◽  
Cardiac Fibrosis ◽  
Transient Receptor Potential ◽  
Transforming Growth Factor ◽  
Ventricular Remodeling ◽  
Cardiac Fibroblasts ◽  
Gene Transfection ◽  
Pressure Overload

Background: Hypertensive ventricular remodeling is a common cause of heart failure. Activation and accumulation of cardiac fibroblasts is the key contributors to this progression. Our previous studies indicate that transient receptor potential ankyrin 1 (TRPA1), a Ca 2+ channel necessary and sufficient, play a prominent role in ventricular remodeling. However, the molecular mechanisms regulating remain poorly understood. Methods: We used TRPA1 agonists cinnamaldehyde (CA) pretreatment and TRPA1 knockout mice to understand the role of TRPA1 in ventricular remodeling of hypertensive heart. We also examine the mechanisms through gene transfection and in vitro experiments. Results: TRPA1 overexpression fully activated myofibroblast transformation, while fibroblasts lacking TRPA1 were refractory to transforming growth factor β (TGF-β) -induced transdifferentiation. TRPA1 knockout mice showed hypertensive ventricular remodeling reversal following pressure overload. We found that the TGF-β induced TRPA1 expression through calcineurin-NFAT-Dyrk1A signaling pathway via the TRPA1 promoter. Once induced, TRPA1 activates the Ca 2+ -responsive protein phosphatase calcineurin, which itself induced myofibroblast transdifferentiation. Moreover, inhibition of calcineurin prevented TRPA1-dependent transdifferentiation. Conclusion: Our study provides the first evidence that TRPA1 regulation in cardiac fibroblasts transformation in response to hypertensive stimulation. The results suggesting a comprehensive pathway for myofibroblast formation in conjunction with TGF-β, Calcineurin, NFAT and Dyrk1A. Furthermore, these data indicate that negative modulation of cardiac fibroblast TRPA1 may represent a therapeutic strategy against hypertensive cardiac remodeling.

Abstract 47: Loss of the Cardio Protection Inferred by S-nitrosylation of GRK2 At Cysteine 340 Leads to Decreased Cardiac Performance and a Hypertensive Phenotype With Aging

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Christopher J Traynham ◽  
Ancai Yuan ◽  
Erhe Gao ◽  
Walter Koch
Keyword(s):  
Cardiac Hypertrophy ◽  
Cardiac Function ◽  
Ischemia Reperfusion ◽  
Cardiac Performance ◽  
Heart Weight ◽  
Cardiac Phenotype ◽  
Cardio Protection ◽  
G Protein Coupled ◽  
Global Population

In the next 35 years, the global population of individuals above 60 years of age will double to approximately 2 billion. In the aged population, cardiovascular diseases are known to occur at a higher prevalence ultimately leading to increased mortality. G protein-coupled receptors (GPCRs) have been identified as vital regulators of cardiac function. GPCR kinases (GRKs) are important in cardiac GPCR regulation through desensitization of these receptors. GRK2 is highly expressed in the heart, and has been widely characterized due to its upregulation in heart failure. Studies from our lab have shown that elevated GRK2 levels in ischemia-reperfusion (I/R) injury result in a pro-death phenotype. Interestingly, cardio-protection can be inferred via S-nitrosylation of GRK2 at cysteine 340. Further, we have generated a knock-in GRK2 340S mouse, in which cysteine 340 was mutated to block dynamic GRK2 S-nitrosylation. GRK2 340S mice are more susceptible to I/R injury. Given that GRK2 340S mice are more susceptible to oxidative stress, and there is a nitroso-redox imbalance in senescence, it is possible that these mice are more likely to exhibit decreased cardiac performance as they age. Therefore, we hypothesize that with age GRK2 340S knockin mice will develop an overall worsened cardiac phenotype compared to control wild-type (WT) mice. To test this hypothesis, 340S and WT mice were aged for a year, and cardiac function was evaluated via echocardiography. Aged 340S mice exhibited significantly decreased ejection fraction and fraction shortening relative to aged WT controls. Prior to tissue harvesting, in-vivo hemodynamics was conducted via Millar catheterization. At baseline, aged 340S mice exhibited increased systolic blood pressure compared to aged WT mice. At the conclusion of this protocol, mice were sacrificed and heart weight (HW), body weight (BW), and tibia length (TL) measured to evaluate cardiac hypertrophy. Aged 340S mice exhibited significantly increased HW/BW and HW/TL ratios, indicative of cardiac hypertrophy, relative to aged WT controls. Taken together, these data suggest that with age, loss of the cardio protection inferred by S-nitrosylation of GRK2 at leads to decreased cardiac performance, and an overall worsened cardiac phenotype.

Abstract 791: A Critical Role for Bmper (BMP-binding Endothelial cell precursor-derived Regulator) in Cardiac Muscle Mass Regulation during Development and Pressure Overload Induced Hypertrophy

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Monte Willis ◽  
Rongqin Ren ◽  
Cam Patterson
Keyword(s):  
Cardiac Function ◽  
Cardiac Muscle ◽  
Muscle Mass ◽  
Cardiac Development ◽  
Critical Role ◽  
Pressure Overload ◽  
Posterior Wall ◽  
Fractional Shortening

Bone morphogenetic proteins (BMPs) of the TGF-beta superfamily, have been implicated in multiple processes during cardiac development. Our laboratory recently described an unprecedented role for Bmper in antagonizing BMP-2, BMP-4, and BMP-6. To determine the role of Bmper on cardiac development in vivo, we created Bmper null (Bmper −/−) mice by replacing exons 1 and 2 with GFP. Since Bmper −/− mice are perinatally lethal, we determined pre-natal cardiac function of Bmper −/− mice in utero just before birth. By echocardiography, E18.5 Bmper −/− embryos had decreased cardiac function (24.2 +/− 8.1% fractional shortening) compared to Bmper +/− and Bmper +/+ siblings (52.2 +/− 1.6% fractional shortening) (N=4/group). To further characterize the role of Bmper on cardiac function in adult mice, we performed echocardiography on 8-week old male and female Bmper +/− and littermate control Bmper +/+. Bmper +/− mice had an approximately 15% decrease in anterior and posterior wall thickness compared to sibling Bmper +/+ mice at baseline (n=10/group). Cross-sectional areas of Bmper +/− cardiomyocytes were approximately 20% less than wild type controls, indicating cardiomyocyte hypoplasia in adult Bmper +/− mice at baseline. Histologically, no significant differences were identified in representative H&E and trichrome stained adult Bmper +/− and Bmper +/+ cardiac sections at baseline. To determine the effects of Bmper expression on the development of cardiac hypertrophy, both Bmper +/− and Bmper +/+ sibling controls underwent transaortic constriction (TAC), followed by weekly echocardiography. While a deficit was identified in Bmper +/− mice at baseline, both anterior and posterior wall thicknesses increased after TAC, such that identical wall thicknesses were identified in Bmper +/− and Bmper +/+ mice 1–4 weeks after TAC. Notably, cardiac function (fractional shortening %) and histological evaluation revealed no differences between Bmper +/− and Bmper +/+ any time after TAC. These studies identify for the first time that Bmper expression plays a critical role in regulating cardiac muscle mass during development, and that Bmper regulates the development of hypertrophy in response to pressure overload in vivo.

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