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Mutations in the nuclear envelope (NE) protein lamin A/C (encoded by LMNA), cause a severe form of dilated cardiomyopathy (DCM) with early-onset life-threatening arrhythmias. However, molecular mechanisms underlying increased arrhythmogenesis in LMNA-related DCM (LMNA-DCM) remain largely unknown. Here we show that a frameshift mutation in LMNA causes abnormal Ca 2+ handling, arrhythmias and disformed NE in LMNA-DCM patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). Mechanistically, lamin A interacts with sirtuin 1 (SIRT1) where mutant lamin A/C accelerates degradation of SIRT1, leading to mitochondrial dysfunction and oxidative stress. Elevated reactive oxygen species (ROS) then activates the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII)-ryanodine receptor 2 (RYR2) pathway and aggravates the accumulation of SUN1 in mutant iPSC-CMs, contributing to arrhythmias and NE deformation, respectively. Taken together, the lamin A/C deficiency-mediated ROS disorder is revealed as central to LMNA-DCM development. Manipulation of impaired SIRT1 activity and excessive oxidative stress is a potential future therapeutic strategy for LMNA-DCM., (© 2024. The Author(s).)

Hypertrophic cardiomyopathy (HCM), characterized by left ventricular hypertrophy and preserved or increased left ventricular ejection fraction, is the most common autosomal dominant inherited cardiovascular disease. We generated a human induced pluripotent stem cell (hiPSC) line derived from a HCM patient who carried a heterozygous missense mutation in the myosin heavy chain 6 (MYH6) gene. With a non-integrated Sendai viral method, the patient-specific hiPSCs were generated from skin fibroblasts. We confirmed the stemness of the hiPSCs and its capability of differentiating into three germ layers. Meanwhile, the generated hiPSCs showed human embryonic stem cell-like morphology and normal karyotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Learning better representations is essential in medical image analysis for computer-aided diagnosis. However, learning discriminative semantic features is a major challenge due to the lack of large-scale well-annotated datasets. Thus, how can we learn a well-structured categorizable embedding space in limited-scale and unlabeled datasets? In this paper, we proposed a novel clustering-guided twin-contrastive learning framework (CTCL) that learns the discriminative representations of probe-based confocal laser endomicroscopy (pCLE) images for gastrointestinal (GI) tumor classification. Compared with traditional contrastive learning, in which only two randomly augmented views of the same instance are considered, the proposed CTCL aligns more semantically related and class-consistent samples by clustering, which improved intra-class tightness and inter-class variability to produce more informative representations. Furthermore, based on the inherent properties of CLE (geometric invariance and intrinsic noise), we proposed to regard CLE images with any angle rotation and CLE images with different noises as the same instance, respectively, for increased variability and diversity of samples. By optimizing CTCL in an end-to-end expectation-maximization framework, comprehensive experimental results demonstrated that CTCL-based visual representations achieved competitive performance on each downstream task as well as more robustness and transferability compared with existing state-of-the-art SSL and supervised methods. Notably, CTCL achieved 75.60%/78.45% and 64.12%/77.37% top-1 accuracy on the linear evaluation protocol and few-shot classification downstream tasks, respectively, which outperformed the previous best results by 1.27%/1.63% and 0.5%/3%, respectively. The proposed method holds great potential to assist pathologists in achieving an automated, fast, and high-precision diagnosis of GI tumors and accurately determining different stages of tumor development based on CLE images.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with colonic crypts (CC) being crucial in its development. Accurate segmentation of CC is essential for decisions CRC and developing diagnostic strategies. However, colonic crypts' blurred boundaries and morphological diversity bring substantial challenges for automatic segmentation. To mitigate this problem, we proposed the Dual-Branch Asymmetric Encoder-Decoder Segmentation Network (DAUNet), a novel and efficient model tailored for confocal laser endomicroscopy (CLE) CC images. In DAUNet, we crafted a dual-branch feature extraction module (DFEM), employing Focus operations and dense depth-wise separable convolution (DDSC) to extract multiscale features, boosting semantic understanding and coping with the morphological diversity of CC. We also introduced the feature fusion guided module (FFGM) to adaptively combine features from both branches using cross-group spatial and channel attention to improve the model representation in focusing on specific lesion features. These modules are seamlessly integrated into the encoder for effective multiscale information extraction and fusion, and DDSC is further introduced in the decoder to provide rich representations for precise segmentation. Moreover, the local multi-layer perceptron (LMLP) module is designed to decouple and recalibrate features through a local linear transformation that filters out the noise and refines features to provide edge-enriched representation. Experimental evaluations on two datasets demonstrate that the proposed method achieves Intersection over Union (IoU) scores of 81.54% and 84.83%, respectively, which are on par with state-of-the-art methods, exhibiting its effectiveness for CC segmentation. The proposed method holds great potential in assisting physicians with precise lesion localization and region analysis, thereby improving the diagnostic accuracy of CRC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objective. As an emerging diagnosis technology for gastrointestinal diseases, confocal laser endomicroscopy (CLE) is limited by the physical structure of the fiber bundle, leading to the inevitable production of various forms of noise during the imaging process. However, existing denoising methods based on hand-crafted features inefficiently deal with realistic noise in CLE images. To alleviate this challenge, we proposed context-aware kernel estimation and multi-scale dynamic fusion modules to remove realistic noise in CLE images, including multiplicative and additive white noise. Approach. Specifically, a realistic noise statistics model with random noise specific to CLE data is constructed and further used to develop a self-supervised denoised model without the participation of clean images. Secondly, context-aware kernel estimation, which improves the representation of features by similar learnable region weights, addresses the problem of the non-uniform distribution of noises in CLE images and proposes a lightweight denoised model (CLENet). Thirdly, we have developed a multi-scale dynamic fusion module that decouples and recalibrates features, providing a precise and contextually enriched representation of features. Finally, we integrated two developed modules into a U-shaped backbone to build an efficient denoising network named U-CLENet. Main Results. Both proposed methods achieve comparable or better performance with low computational complexity on two gastrointestinal disease CLE image datasets using the same training benchmark. Significance. The proposed approaches improve the visual quality of unclear CLE images for various stages of tumor development, helping to reduce the rate of misdiagnosis in clinical decision-making and achieve computer graphics-assisted diagnosis., (© 2023 Institute of Physics and Engineering in Medicine.)

Background: Mutations in the cardiac sodium channel gene SCN5A cause Brugada syndrome (BrS), an arrhythmic disorder that is a leading cause of sudden death and lacks effective treatment. An association between SCN5A and Wnt/β-catenin signaling has been recently established. However, the role of Wnt/β-catenin signaling in BrS and underlying mechanisms remains unknown., Methods: Three healthy control subjects and one BrS patient carrying a novel frameshift mutation (T1788fs) in the SCN5A gene were recruited in this study. Control and BrS patient-specific induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts using nonintegrated Sendai virus. All iPSCs were differentiated into cardiomyocytes using monolayer-based differentiation protocol. Action potentials and sodium currents were recorded from control and BrS iPSC-derived cardiomyocytes (iPSC-CMs) by single-cell patch clamp., Results: BrS iPSC-CMs exhibited increased burden of arrhythmias and abnormal action potential profile featured by slower depolarization, decreased action potential amplitude, and increased beating interval variation. Moreover, BrS iPSC-CMs showed cardiac sodium channel (Na v 1.5) loss-of-function as compared to control iPSC-CMs. Interestingly, the electrophysiological abnormalities and Na v 1.5 loss-of-function observed in BrS iPSC-CMs were accompanied by aberrant activation of Wnt/β-catenin signaling. Notably, inhibition of Wnt/β-catenin significantly rescued Na v 1.5 defects and arrhythmic phenotype in BrS iPSC-CMs. Mechanistically, SCN5A-encoded Na v 1.5 interacts with β-catenin, and reduced expression of Na v 1.5 leads to re-localization of β-catenin in BrS iPSC-CMs, which aberrantly activates Wnt/β-catenin signaling to suppress SCN5A transcription., Conclusions: Our findings suggest that aberrant activation of Wnt/β-catenin signaling contributes to the pathogenesis of SCN5A-related BrS and point to Wnt/β-catenin as a potential therapeutic target., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Perilipin 5 (Plin5) is well known to maintain the stability of intracellular lipid droplets (LDs) and regulate fatty acid metabolism in oxidative tissues. It is highly expressed in the heart, but its roles have yet to be fully elucidated., Methods: Plin5-deficient mice and Plin5/leptin-double-knockout mice were produced, and their histological structures and myocardial functions were observed. Critical proteins related to fatty acid and glucose metabolism were measured in heart tissues, neonatal mouse cardiomyocytes and Plin5-overexpressing H9C2 cells. 2-NBDG was employed to detect glucose uptake. The mitochondria and lipid contents were observed by MitoTracker and BODIPY 493/503 staining in neonatal mouse cardiomyocytes., Results: Plin5 deficiency impaired glucose utilization and caused insulin resistance in mouse cardiomyocytes, particularly in the presence of fatty acids (FAs). Additionally, Plin5 deficiency increased the NADH content and elevated the expression of lactate dehydrogenase (LDHA) in cardiomyocytes, which resulted in increased lactate production. Moreover, when fatty acid oxidation was blocked by etomoxir or LDHA was inhibited by GSK2837808A in Plin5-deficient cardiomyocytes, glucose utilization was improved. Leptin-deficient mice exhibited myocardial hypertrophy, insulin resistance and altered substrate utilization, and Plin5 deficiency exacerbated myocardial hypertrophy in leptin-deficient mice., Conclusion: Our results demonstrated that Plin5 plays a critical role in coordinating fatty acid and glucose oxidation in cardiomyocytes, providing a potential target for the treatment of metabolic disorders in the heart., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Although human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel K ir 2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking., Methods: iPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs)., Results: We showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of K ir 2.1 and current density of K ir 2.1-encoded I K1 . The larger I K1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by I K1 blocker BaCl 2 . KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca 2+ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction., Conclusions: Overexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca 2+ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine., (© 2023. The Author(s).)

As an emerging early diagnostic technology for gastrointestinal diseases, confocal laser endomicroscopy lacks large-scale perfect annotated data, leading to a major challenge in learning discriminative semantic features. So, how should we learn representations without labels or a few labels? In this paper, we proposed a feature-level MixSiam method based on the traditional Siamese network that learns the discriminative features of probe-based confocal laser endomicroscopy (pCLE) images for gastrointestinal (GI) tumor classification. The proposed method is divided into two stages: self-supervised learning (SSL) and few-shot learning (FS). First, in the self-supervised learning stage, the novel feature-level-based feature mixing approach introduced more task-relevant information via regularization, facilitating the traditional Siamese structure can adapt to the large intra-class variance of the pCLE dataset. Then, in the few-shot learning stage, we adopted the pre-trained model obtained through self-supervised learning as the base learner in the few-shot learning pipeline, enabling the feature extractor to learn richer and more transferable visual representations for rapid generalization to other pCLE classification tasks when labeled data are limited. On two disjoint pCLE gastrointestinal image datasets, the proposed method is evaluated. With the linear evaluation protocol, feature-level MixSiam outperforms the baseline by 6% (Top-1) and the supervised model by 2% (Top1), which demonstrates the effectiveness of the proposed feature-level-based feature mixing method. Furthermore, the proposed method outperforms the previous baseline method for the few-shot classification task, which can help improve the classification of pCLE images lacking large-scale annotated data for different stages of tumor development., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disease characterized by left ventricular hypertrophy and a high risk of sudden death. In this study, a skin biopsy was obtained from a HCM patient harboring a heterozygous missense mutation (c.3764C>A; p.A1225D) in the myosin binding protein C3 (MYBPC3) gene. The isolated fibroblasts were reprogrammed using non-integrated Sendai viral method to establish the patient-specific induced pluripotent stem cell (iPSC) line. The established iPSC line displayed normal morphology and karyotype, expressed pluripotency markers, and can differentiate into three germ layers in vivo., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant inherited cardiovascular disease characterized by left ventricular hypertrophy and cardiomyocyte disarray. In this study, a skin biopsy was obtained from a HCM patient, who carried a missense mutation (c.4384G > A; p.E1462K) in the myosin heavy chain 7 (MYH7) gene. The skin fibroblasts were subsequently reprogrammed with a non-integrated Sendai viral method to generate a patient-specific induced pluripotent stem cell (iPSC) line. The generated iPSC line showed typical morphology and normal karyotype, expressed pluripotency markers, and was capable to differentiate into three germ layers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Long QT syndrome (LQT) is an inherited primary arrhythmic disorder characterized by prolonged QT interval on the surface electrocardiogram and life-threatening arrhythmia. In this study, a skin biopsy was obtained from an LQT type 2 (LQT2) patient, who carried a nonsense mutation (c.1956C > A; p.Y652X) in the potassium voltage-gated channel subfamily H member 2 (KCNH2) gene. The skin fibroblasts were reprogrammed by non-integrated Sendai viral method to generate a patient-specific induced pluripotent stem cell (iPSC) line. The generated iPSC line showed typical embryonic stem cell-like morphology, exhibited normal karyotype, expressed pluripotency markers, and was capable to differentiate into three germ layers., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Diabetes mellitus is a serious metabolic condition associated with a multitude of cardiovascular complications. Moreover, the prevalence of diabetes in heart failure populations is higher than that in control populations. However, the role of cardiomyocyte alterations in type 2 diabetes mellitus (T2DM) has not been well characterized and the underlying mechanisms remain elusive. In this study, two patients who were diagnosed as T2DM were recruited and patient-specific induced pluripotent stem cells (iPSCs) were generated from urine epithelial cells using nonintegrated Sendai virus. The iPSC lines derived from five healthy subjects were used as controls. All iPSCs were differentiated into cardiomyocytes (iPSC-CMs) using the monolayer-based differentiation protocol. T2DM iPSC-CMs exhibited various disease phenotypes, including cellular hypertrophy and lipid accumulation. Moreover, T2DM iPSC-CMs exhibited higher susceptibility to high-glucose/high-lipid challenge than control iPSC-CMs, manifesting an increase in apoptosis. RNA-Sequencing analysis revealed a differential transcriptome profile and abnormal activation of TGFβ signaling pathway in T2DM iPSC-CMs. We went on to show that inhibition of TGFβ significantly rescued the hypertrophic phenotype in T2DM iPSC-CMs. In conclusion, we demonstrate that the iPSC-CM model is able to recapitulate cellular phenotype of T2DM. Our results indicate that iPSC-CMs can therefore serve as a suitable model for investigating molecular mechanisms underlying diabetic cardiomyopathies and for screening therapeutic drugs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Opioid and its receptors play important roles in glucose homeostasis. However, few reports were available for the study of κ-opioid receptor in glucose regulation. In our study, we found that the blood glucose of diabetic mice dropped significantly following the treatment with U50,488H (a selective κ-opioid receptor agonist). This phenomenon was time-dependent and associated with the coincident alteration of Glut4 translocation in the skeleton muscles. U50,488H increased the serum adiponectin, but not serum insulin in diabetic mice. U50,488H increased the AdipoR1 expression at both mRNA and protein levels. It also promoted AMPK phosphorylation and Glut4 translocation. All these effects were abolished by nor-BNI (a selective κ-opioid receptor antagonist). These findings suggest that activation of κ-opioid receptor reduces hyperglycemia in streptozotocin-induced diabetic mice. This effect is associated with the translocation of Glut4 and might be relevant to increased adiponectin, AdipoR1, and AMPK phosphorylation., (© 2014 Wiley Periodicals, Inc.)

The adipocyte-secreted hormone adiponectin (APN) exerts protective effects on the heart under stress conditions. Recent studies have demonstrated that APN induces a marked Ca(2+) influx in skeletal muscle. However, whether APN modulates [Ca(2+)]i activity, especially [Ca(2+)]i transients in cardiomyocytes, is still unknown. This study was designed to determine whether APN modulates [Ca(2+)]i transients in cardiomyocytes. Adult male wild-type (WT) and APN knockout (APN KO) mice were subjected to myocardial ischemia/reperfusion (I/R, 30min/30min) injury. CaMKII-PLB phosphorylation and SR Ca(2+)-ATPase (SERCA2) activity were downregulated in I/R hearts of WT mice and further decreased in those of APN KO mice. Both the globular domain of APN and full-length APN significantly reversed the decrease in CaMKII-PLB phosphorylation and SERCA2 activity in WT and APN KO mice. Interestingly, compared with WT littermates, single myocytes isolated from APN KO mice had remarkably decreased [Ca(2+)]i transients, cell shortening, and a prolonged Ca(2+) decay rate. Further examination revealed that APN enhances SERCA2 activity via CaMKII-PLB signaling. In in vivo and in vitro experiments, both APN receptor 1/2 and S1P were necessary for the APN-stimulated CaMKII-PLB-SERCA2 activation. In addition, S1P activated CaMKII-PLB signaling in neonatal cardiomyocytes in a dose dependent manner and improved [Ca(2+)]i transients in APN KO myocytes via the S1P receptor (S1PR1/3). Further in vivo experiments revealed that pharmacological inhibition of S1PR1/3 and SERCA2 siRNA suppressed APN-mediated cardioprotection during I/R. These data demonstrate that S1P is a novel regulator of SERCA2 that activates CaMKII-PLB signaling and mediates APN-induced cardioprotection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Myocardial ischemia/reperfusion injury (I/RI) is the principal cause of mortality and morbidity in diabetic patients undergoing cardiac surgery. However, there is no specific measure available to protect diabetic hearts in this clinical setting. Our clinical studies showed that adenosine pre-treatment or post-treatment and adding adenosine to cardioplegia solution had significant myocardial protective effects in patients undergoing cardiac surgery. However, the specific protective effects and mechanisms of adenosine in diabetic myocardial I/RI are not clear. Calpain is an important proteolytic enzyme in the myocardium. Studies show that the activation of calpain is an injury factor in not only the diabetic myocardium but also myocardial I/RI progression. We therefore hypothesize that adenosine play a protective role in diabetic myocardial I/RI through the inhibition of calpain., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study, we evaluated the effect of curcumin (Cur) post-treatment on isolated perfused rat hearts that had been subjected to a protocol of ischemia and reperfusion injury. We also examined whether the Janus kinase 2 and signal transducer and activator 3 of transcription (JAK2/STAT3) signaling pathway plays a role in the cardioprotective effects of Cur post-treatment. Isolated perfused rat hearts were subjected to 60 min of ischemia, followed by 60 min of reperfusion. The hearts were exposed to 1-μM Cur during the first 10 min of reperfusion in the absence or presence of the JAK kinase-specific inhibitor AG490 (AG, 1 μM). The Cur treatment conferred a cardioprotective effect, and the treated hearts demonstrated an improved post-ischemic cardiac functional recovery, a decreased myocardial infarct size and decreased lactate dehydrogenase release in the coronary flow, a reduced number of apoptotic cardiomyocytes, up-regulation of the anti-apoptotic protein Bcl2 and down-regulation of the pro-apoptotic protein Caspase3. AG blocked the Cur-mediated cardioprotection by inhibiting the JAK2/STAT3 signaling pathway, as reflected by the abrogation of the Cur-induced up-regulation of Bcl2 and down-regulation of Caspase3. The results suggest that Cur post-treatment can attenuate IR injury through the activation of the JAK2/STAT3 signaling pathway, which transmits a survival signal to the myocardium.

Background: One of the most important factors restricting heart transplantation is the limited myocardial ischemia time. This study investigated the effects of urethane on the hypothermic preservation of donor rat hearts., Materials and Methods: Hearts isolated from rats were divided into 2 groups (n = 8), a control group with histidine-tryptophan-ketoglutarate (HTK) solution alone and an experimental group with HTK solution plus 30 mM urethane. Hearts were mounted on a Langendorff apparatus to estimate the baseline cardiac function; the hearts were then arrested and stored in one of the 2 solutions for 6 hours and 18 hours at 4 degrees C. After preservation, the hearts were reperfused, and cardiac function was evaluated. Lactate dehydrogenase (LDH) release, adenosine triphosphate (ATP) content, cardiomyocyte apoptosis, and myocardial ultrastructure were examined., Results: Compared with the control group, the experimental group showed a significantly higher recovery of cardiac function for both 6 hours and 18 hours of preservation and demonstrated a lower rate of cardiomyocyte apoptosis (8.5% + or - 1.2% versus 12.2% + or - 1.8% for 6 hours; 14.1% + or - 2.1% versus 31.4% + or - 2.7% for 18 hours). ATP content was significantly higher in the experimental group than in the control group after 18 hours of preservation (229.4 + or - 29.7 microg/g versus 153.2 + or - 21.1 microg/g). The experimental group also showed lower levels of LDH release after 18 hours of preservation. Electron microscopy studies demonstrated better cardiomyocyte structure in the experimental group for both 6 hours and 18 hours of preservation., Conclusions: Use of urethane improved cardiac functional recovery and led to significant protective effects on rat hearts placed in a hypothermic preservation solution for a prolonged period.