Cardiotoxicity Assessment through a Polymer-Based Cantilever Platform: An Integrated Electro-Mechanical Screening Approach.

Preclinical drug screening for cardiac toxicity has traditionally relied on observing changes in cardiomyocytes' electrical activity, primarily through invasive patch clamp techniques or non-invasive microelectrode arrays (MEA). However, relying solely on field potential duration (FPD) measurements for electrophysiological assessment can miss the full spectrum of drug-induced toxicity, as different drugs affect cardiomyocytes through various mechanisms. A more comprehensive approach, combining field potential and contractility measurements, is essential for accurate toxicity profiling, particularly for drugs targeting contractile proteins without affecting electrophysiology. However, previously proposed platform has significant limitations in terms of simultaneous measurement. The novel platform addresses these issues, offering enhanced, non-invasive evaluation of drug-induced cardiotoxicity. It features eight cantilevers with patterned strain sensors and MEA, enabling real-time monitoring of both cardiomyocyte contraction force and field potential. This system can detect minimum cardiac contraction force of ≈2 µN and field potential signals with 50 µm MEA diameter, using the same cardiomyocytes in measurements of two parameters. Testing with six drugs of varied mechanisms of action, the platform successfully identifies these mechanisms and accurately assesses toxicity profiles, including drugs not inhibiting potassium channels. This innovative approach presents a comprehensive, non-invasive method for cardiac function assessment, poised to revolutionize preclinical cardiotoxicity screening.

Quantitative assessment of cardiomyocyte mechanobiology through high-throughput cantilever-based functional well plate systems.

Proper regulation of the in vitro cell culture environment is essential for disease modelling and drug toxicity screening. The main limitation of well plates used for cell culture is that they cannot accurately maintain energy sources and compounds needed during cell growth. Herein, to understand the importance of perfusion in cardiomyocyte culture, changes in contractile force and heart rate during cardiomyocyte growth are systematically investigated, and the results are compared with those of a perfusion-free system. The proposed perfusion system consists of a Peltier refrigerator, a peristaltic pump, and a functional well plate. A functional well plate with 12 wells is made through injection moulding, with two tubes integrated in the cover for each well to continuously circulate the culture medium. The contractile force of cardiomyocytes growing on the cantilever surface is analysed through changes in cantilever displacement. The maturation of cardiomyocytes is evaluated through fluorescence staining and western blot; cardiomyocytes cultured in the perfusion system show greater maturity than those cultured in a manually replaced culture medium. The pH of the culture medium manually replaced at intervals of 3 days decreases to 6.8, resulting in an abnormal heartbeat, while cardiomyocytes cultured in the perfusion system maintained at pH 7.4 show improved contractility and a uniform heart rate. Two well-known ion channel blockers, verapamil and quinidine, are used to measure changes in the contractile force of cardiomyocytes from the two systems. Cardiomyocytes in the perfusion system show greater stability during drug toxicity screening, proving that the perfusion system provides a better environment for cell growth.

Correction: Quantitative assessment of cardiomyocyte mechanobiology through high-throughput cantilever-based functional well plate systems.

Correction for 'Quantitative assessment of cardiomyocyte mechanobiology through high-throughput cantilever-based functional well plate systems' by Jongyun Kim et al., Analyst, 2023, 148, 5133-5143, https://doi.org/10.1039/D3AN01286G.

Surface Charge-Dependent Cytotoxicity of Plastic Nanoparticles in Alveolar Cells under Cyclic Stretches.

Polystyrene nanoparticles (PS-NPs) derived from both environmental and occupational sources are an important class of ultrafine particles associated with human pulmonary disorders. The effects of surface charges of particle internalization and toxicity to alveolar cells, especially under conditions comparable to those found during breathing, have not been examined. Here, we applied cyclic stretches (CS) to human alveolar cells during nanoparticle exposure and show an enhanced accumulation of positively charged polystyrene nanoparticles as compared to similar negatively charged particles. The cellular uptake of the positive particles into live cells was visualized with three-dimensional optical diffraction tomography (3-D ODT). The simultaneous application of both periodic stretching as well as positively charged nanoparticles led to blebbing morphology and activation of apoptotic signaling compared to control cells. Our findings provide a better understanding of how surface charge mediates the uptake and toxicity of nanoplastics under the dynamical mechanical conditions relevant for breathing exposures.

Numerical analysis of intracellular amino acid profiles of breast cancer cells with K-Ras or PI3K mutation in response to kinase inhibitors.

BACKGROUND: Various efforts to understand the relationship between biological information and disease have been done using many different types of highthroughput data such as genomics and metabolomics. However, information obtained from previous studies was not satisfactory, implying that new direction of studies is in need. Thus, we have tried profiling intracellular free amino acids in normal and cancerous cells to extract some information about such relationship by way of the change in IFAA levels in response to the treatment of three kinase inhibitors. We define two measures such as relative susceptibility (RS) and relative efficacy (RE) to numerically quantify susceptibility of cell line to treatment and efficacy of treatment on cell line, respectively. METHODS: We applied principal component analysis (PCA) to the intracellular free amino acids (IFAAs) of isogenic breast cells with oncogenic mutation in K-Ras or PI3K genes to investigate the change in IFAA levels in response to the treatment of three kinase inhibitors. Two-dimensional plot, which was graphically represented by using the first two principal components (PCs), enabled us to evaluate the treatment efficacy in cancerous cells in terms of the quantitative distance of two IFAA profiles from cancerous and normal cells with the same treatment condition. RESULTS: The biggest change in metabolic states in K-Ras mutant cell was caused by REGO for both treatment time (RS=2.31 (24 h) and 1.64 (48 h)). Regarding RE, REGO was the most effective on K-Ras/PI3K mutant cell line for treatment time 24h (RE=1.28) while PI3K inhibitor had good effect on K-Ras mutant cell line for 48h (RE=1.1). CONCLUSIONS: Numerical study on the link between amino acid profile and cancer has been done in two different dimensions. We then summarized such link in terms of two new metrics such as RS and RE, which we first define in this work. Although our study based on those metrics seems to work, we think that the usefulness of the metrics in cancer study of this kind need to be further investigated.

Biomechanical Characterization of Cardiomyocyte Using PDMS Pillar with Microgrooves.

This paper describes the surface-patterned polydimethylsiloxane (PDMS) pillar arrays for enhancing cell alignment and contraction force in cardiomyocytes. The PDMS micropillar (µpillar) arrays with microgrooves (µgrooves) were fabricated using a unique micro-mold made using SU-8 double layer processes. The spring constant of the µpillar arrays was experimentally confirmed using atomic force microscopy (AFM). After culturing cardiac cells on the two different types of µpillar arrays, with and without grooves on the top of µpillar, the characteristics of the cardiomyocytes were analyzed using a custom-made image analysis system. The alignment of the cardiomyocytes on the µgrooves of the µpillars was clearly observed using a DAPI staining process. The mechanical force generated by the contraction force of the cardiomyocytes was derived from the displacement of the µpillar arrays. The contraction force of the cardiomyocytes aligned on the µgrooves was 20% higher than that of the µpillar arrays without µgrooves. The experimental results prove that applied geometrical stimulus is an effective method for aligning and improving the contraction force of cardiomyocytes.

Comparative interactomes of SIRT6 and SIRT7: Implication of functional links to aging.

Sirtuins are NAD(+) -dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 that are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6- and SIRT7-interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming seven and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly, and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co-immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins. All MS data have been deposited in the ProteomeXchange with identifiers PXD000159 and PXD000850 (http://proteomecentral.proteomexchange.org/dataset/PXD000159, http://proteomecentral.proteomexchange.org/dataset/PXD000850).

Reading single DNA with DNA polymerase followed by atomic force microscopy.

The importance of DNA sequencing in the life sciences and personalized medicine is continually increasing. Single-molecule sequencing methods have been developed to analyze DNA directly without the need for amplification. Here, we present a new approach to sequencing single DNA molecules using atomic force microscopy (AFM). In our approach, four surface-conjugated nucleotides were examined sequentially with a DNA polymerase-immobilized AFM tip. By observing the specific rupture events upon examination of a matching nucleotide, we could determine the template base bound in the polymerase's active site. The subsequent incorporation of the complementary base in solution enabled the next base to be read. Additionally, we observed that the DNA polymerase could incorporate the surface-conjugated dGTP when the applied force was controlled by employing the force-clamp mode.

Microplastics as an emerging threat to amphibians: Current status and future perspectives.

Given their pervasiveness in the environment, particularly in aquatic ecosystems, plastics are posing a growing concern worldwide. Many vertebrates and invertebrates in marine, freshwater, and terrestrial ecosystems exhibit microplastic (MP) uptake and accumulation. Some studies have indicated the fatal impacts of MPs on animals and their possible transfer through food chains. Thus, it is crucial to study MP pollution and its impacts on environment-sensitive and globally threatened animal groups, such as amphibians, which also play an important role in the energy transfer between ecosystems. Unfortunately, research in this field is lacking and sources of organized information are also scarce. Hence, we systematically reviewed published literature on MPs in amphibians to fill the existing knowledge gap. Our review revealed that most of the previous studies have focused on MP bioaccumulation in amphibians, whereas, only a few research highlighted its impacts. We found that more than 80% of the studied species exhibited MP accumulation. MPs were reported to persist in different organs for a long time and get transferred to other trophic levels. They can also exhibit cytotoxic and mutagenic effects and may have fatal impacts. Moreover, they can increase the disease susceptibility of amphibians. Our study concludes the MPs as a potential threat to amphibians and urges increasing the scope and frequency of research on MP pollution and its impacts on this vulnerable animal group. We also provide a generalized method for studying MPs in amphibians with future perspectives and research directions. Our study is significant for extending the knowledge of MPs and their impacts on amphibians and guiding prospective research.

Dynamic mucus secretion in ventral surfaces of toe pads of the tree frog (Dryophytes japonica).

The tree frog is a prominent amphibian among terrestrial vertebrates known for its ability to adhere to various surfaces through the capillary forces of water in the microchannels between micropillars on its disc-shaped toe pads, a phenomenon known as wet adhesion. However, the secretion pattern of mucus on the attachment surface of living tree frog toe pads and the distribution of active mucus pores (AMPs) have not yet been fully elucidated. In this study, we utilized synchrotron X-ray micro-computed tomography and interference reflection microscopy to obtain the spatial distribution of the entire population of ventral mucus glands on the toe pads of living tree frogs and the real-time mucus secretion patterns from the ventral mucus pores on the contact surface under different environmental conditions. We observed that the number and secretion frequency of AMPs on the toe pad are regulated according to environmental conditions. Such dynamic mucus secretion on the tree frog's toe pad could contribute to the understanding of capillary force regulation for wet adhesion and the development of adhesive surfaces by mimicking the mucus-secreting toe pad.

REST-dependent expression of TRF2 renders non-neuronal cancer cells resistant to DNA damage during oxidative stress.

REST is a neuronal gene silencing factor ubiquitously expressed in non-neuronal tissues. REST is additionally believed to serve as a tumor suppressor in non-neuronal cancers. Conversely, recent findings on REST-dependent tumorigenesis in non-neuronal cells consistently suggest a potential role of REST as a tumor promoter. Here, we have uncovered for the first time the mechanism by which REST contributes to cancer cell survival in non-neuronal cancers. We observed abundant expression of REST in various types of non-neuronal cancer cells compared to normal tissues. The delicate roles of REST were further evaluated in HCT116 and HeLa, non-neuronal cancer cell lines expressing REST. REST silencing resulted in decreased cell survival and activation of the DNA damage response (DDR) through a decrease in the level of TRF2, a telomere-binding protein. These responses were correlated with reduced colony formation ability and accelerated telomere shortening in cancer cells upon the stable knockdown of REST. Interestingly, REST was down-regulated under oxidative stress conditions via ubiquitin proteasome system, suggesting that sustainability of REST expression is critical to determine cell survival during oxidative stress in a tumor microenvironment. Our results collectively indicate that REST-dependent TRF2 expression renders cancer cells resistant to DNA damage during oxidative stress, and mechanisms to overcome oxidative stress, such as high levels of REST or the stress-resistant REST mutants found in specific human cancers, may account for REST-dependent tumorigenesis.

Kinetic characterization of on-chip DNA ligation on dendron-coated surfaces with nanoscaled lateral spacings.

We analyzed the enzymatic profiles of on-chip DNA ligation as we controlled the lateral spacing of surface-immobilized DNA substrates using dendron molecules with different sizes at the nanoscale. Enzymatic on-chip DNA ligation was performed on the dendron-coated surface within 20 min with no need for post-ligation gel electrophoresis. The enzymatic DNA repair was assessed by the fluorescence intensity at the repaired DNA duplex after thermally dissociating the unligated Cy3-labeled DNA from the DNA duplex, in which the Cy3-labeled DNA was hybridized prior to the on-chip DNA ligation. The rate of the nick-sealing reaction on the 27-acid dendron surface was 3-fold higher than that on the 9-acid dendron surface, suggesting that the wider lateral spacing determined by the larger dendron molecule could facilitate the access of DNA ligase to the nick site. The performance of on-chip DNA ligation was dropped to 10% and 3% when the nick was replaced by one- and two-nucleotide-long gaps, respectively. The 5' terminal phosphorylation of DNA strands by polynucleotide kinase and the on-chip DNA cleavage by endonucleases were also quantitatively monitored throughout the on-chip DNA ligation on the dendron-coated surface. A better understanding of the enzymatic kinetics of on-chip DNA ligation will contribute to a more reliable performance of various on-chip DNA ligation-based assays.

Enhanced cardiomyocyte structural and functional anisotropy through synergetic combination of topographical, conductive, and mechanical stimulation.

Drug-induced cardiotoxicity, a significant concern in the pharmaceutical industry, often results in the withdrawal of drugs from the market. The main cause of drug-induced cardiotoxicity is the use of immature cardiomyocytes during in vitro drug screening procedures. Over time, several methods such as topographical, conductive, and mechanical stimulation have been proposed to enhance both maturation and contractile properties of these cardiomyocytes. However, the synergistic effects of integrating topographical, conductive, and mechanical stimulation for cardiomyocyte maturation remain underexplored and poorly understood. To address this limitation, herein, we propose a grooved polydimethylsiloxane (PDMS) membrane embedded with silver nanowires (AgNWs-E-PDMS). The proposed AgNWs-E-PDMS membrane enhances the maturation of cardiomyocytes and provides a more accurate evaluation of drug-induced cardiotoxicity. When subjected to 10% tensile stress on the AgNWs-E-PDMS membrane, cardiomyocytes displayed substantial enhancements. Specifically, the contraction force, sarcomere length, and connexin-43 (Cx43) expression are increased by 2.0-, 1.5-, and 2.4-times, respectively, compared to the control state. The practical feasibility of the proposed device as a drug screening platform is demonstrated by assessing the adverse effects of lidocaine on cardiomyocytes. The contraction force and beat rate of lidocaine treated cardiomyocytes cultured on the AgNWs-E-PDMS membrane under mechanical stimulation decreased to 0.9 and 0.64 times their initial values respectively, compared to 0.6 and 0.51 times in the control state. These less pronounced changes in the contraction force and beat rate signify the superior drug response in the cardiomyocytes, a result of their enhanced maturation and growth on the AgNWs-E-PDMS membrane combined with mechanical stimulation.

Correction: Enhanced cardiomyocyte structural and functional anisotropy through synergetic combination of topographical, conductive, and mechanical stimulation.

Correction for 'Enhanced cardiomyocyte structural and functional anisotropy through synergetic combination of topographical, conductive, and mechanical stimulation' by Jongyun Kim et al., Lab Chip, 2023, 23, 4540-4551, https://doi.org/10.1039/D3LC00451A.

Overexpression of renal tumor antigen is associated with tumor invasion and poor prognosis of hepatocellular carcinoma.

PURPOSE: The aim of this study was to investigate the roles of renal tumor antigen (RAGE) in the progression and clinical outcome of hepatocellular carcinoma (HCC). METHODS: RAGE mRNA levels in 350 cases of HCC were investigated by quantitative real-time reverse transcription polymerase chain reaction. We analyzed the relationship of RAGE mRNA level with clinicopathologic parameters and clinical outcome. To identify the possible role of RAGE on cellular invasion, we performed in vitro analyses using small interfering RNAs (siRNAs). RESULTS: RAGE mRNA level was significantly higher in HCC than in noncancerous hepatic tissues (P < 0.001). Overexpression of RAGE was significantly correlated with the presence of multiple tumors (P = 0.021), high alfa-fetoprotein level (P = 0.042), and advanced tumor stage (P = 0.016). Higher levels of RAGE expression were associated with significantly shorter overall survival time (P = 0.029). Knockdown of RAGE expression by siRNAs suppressed the invasive ability of HCC cells and the expression and secretion of matrix metalloproteinase-9 (MMP-9). We found that RAGE and MMP-9 expressions were correlated in HCCs, and furthermore, the combination of RAGE and MMP-9 expression was associated with the survival of patients (P = 0.0066). CONCLUSIONS: Our results suggest that RAGE may be important in tumor invasion and could be a potential predictor for the prognosis of HCC patients.

Synergistic effect of orientation and lateral spacing of protein G on an on-chip immunoassay.

The proper orientation and lateral spacing of antibody molecules are a crucial element for an on-chip immunoassay in which the antibody or its antigen-binding fragments are immobilized on a solid surface. We covalently immobilized a modified protein G (Cys-protein G: protein G with only an N-terminal cysteine) on a dendron-coated surface to control its orientation and lateral spacing simultaneously. The cysteine-specific immobilization of Cys-protein G through the N-terminal cysteine resulted in 2.2-fold higher binding efficiency of Cys-protein G to IgG(2a) capture antibody than its random immobilization via lysine residues. The lateral spacing of 3.2 nm due to the surface modification with the 9-acid dendron molecule contributed to a 1.5-fold increase in the antibody-binding ability of Cys-protein G. Topographic images of atomic force microscopy exhibited a uniform coverage of Cys-protein G molecules immobilized on the thiol-reactive 9-acid dendron surface and homogeneous distribution of antibody bound to Cys-protein G. In the sandwich immunoassay, the control of the orientation of Cys-protein G led to 10-fold higher detection capability for rIL-2 compared with the randomly oriented protein G. The synergistic advantage of the unidirectional orientation and homogeneous lateral spacing of Cys-protein Gs on the dendron-coated surface can be applied to the development of more sensitive and reproducible antibody microarrays.

MEA-integrated cantilever platform for comparison of real-time change in electrophysiology and contractility of cardiomyocytes to drugs.

Drug-induced cardiotoxicity is a potentially severe side effect that can alter the contractility and electrophysiology of the cardiomyocytes. Cardiotoxicity is generally assessed through animal models using conventional drug screening platforms. Despite significant developments in drug screening platforms, the difficulty in measuring electrophysiology and contractile profile together affects the investigation of cardiotoxicity in potential drugs. Some drugs can prove to be more toxic to contractility than electrophysiology, which demands the need for a reliable, dual, and simultaneous drug screening platform. Herein, we propose the microelectrode array integrated SU-8 cantilever for dual and simultaneous measurement of electrophysiology and contractility of cardiomyocytes. The SU-8 cantilever is integrated with microelectrode array (C-MEA) using conventional photolithographic techniques. Drug tests are conducted to verify the feasibility of the C-MEA platform using three cardiovascular drugs. Clinically recognized drugs, quinidine and verapamil, are used to activate both the hERG channel and the contractile characteristics of cardiomyocytes. The effect of ion channel blockers on the field potential duration (FPD) of the cardiomyocytes is compared with several contractility-based parameters. The contraction-relaxation duration (CRD) profile is relatively close to that of FPD in tested drugs (half-maximal (IC50) toxicities are 1.093 µM (FPD) and 1.924 µM (CRD) for quinidine and 166.2 nM (FPD) and 459.4 nM (CRD) for verapamil). Blebbistatin, a known myosin II inhibitor, primarily affects the contractile profile of cardiomyocytes but not their field potential, with no evident correlation between contractility and field potential profiles. The proposed cantilever-based mechano-electrophysiology measurements platform provides a promising and accurate means to assess cardiotoxicity.

The effect of topographical and mechanical stimulation on the structural and functional anisotropy of cardiomyocytes grown on a circular PDMS diaphragm.

Due to their immature morphology and functional immaturity, cardiomyocytes have limited use as an in vitro disease model of the native heart. Mechanical stimulation induces structural growth in cardiomyocytes in vitro by addressing the electrical-mechanical interactions between the tissues. However, current in vitro models are restricted in their capacity to replicate the milieu observed in natural myocardium. Herein, we proposed a Galinstan strain sensor integrated nanogrooved circular PDMS diaphragm to mimic the native cardiac tissues. The impact of combined topographical and mechanical stimulation on cultured cardiomyocytes at various strain areas on a circular PDMS diaphragm is studied in detail. An inverted microscope is used to image live cells and video acquisition to study the contractility of cultured cardiomyocytes. The structural changes of the cultured cardiomyocytes are investigated by its sarcomere length and connexin-43 (Cx43) expression using immunocytochemistry analysis. Cyclic strain is found to promote structural development in cultured cardiomyocytes, and diaphragms with nano-groove patterns displayed increased contractile activity and gene expression (sarcomere length ∼1.97 ± 0.03 µm and normalized Cx43-1.57) as compared to flat diaphragms (sarcomere length ∼1.82 ± 0.02 µm and normalized Cx43-1.32). The nanogrooved circular diaphragm exhibited distinct stretching mechanisms at various places, with the equibi-axial stretching regions providing the optimal structural growth and formation of natural myocardium at the diaphragm's center. Cardiomyocytes that are more mature have the potential to produce a more realistic in vitro cardiac model for disease modeling and medication development.

Covalent Positioning of Single DNA Molecules for Nanopatterning.

Bottom-up micropatterning or nanopatterning can be viewed as the localization of target molecules to the desired area of a surface. A majority of these processes rely on the physical adsorption of ink-like molecules to the paper-like surface, resulting in unstable immobilization of the target molecules owing to their noncovalent linkage to the surface. Herein, successive single nick-sealing facilitated the covalent immobilization of individual DNA molecules at defined positions on a dendron-coated silicon surface using atomic force microscopy. The covalently-patterned ssDNA was visualized when the streptavidin-coated gold nanoparticles bound to the biotinylated DNA. The successive covalent positioning of the target DNA under ambient conditions may facilitate the bottom-up construction of DNA-based durable nanostructures, nanorobots, or memory system.

64 PI/PDMS hybrid cantilever arrays with an integrated strain sensor for a high-throughput drug toxicity screening application.

Herein, we propose a novel biosensing platform involving an array of 64 hybrid cantilevers and integrated strain sensors to measure the real-time contractility of the drug-treated cardiomyocytes (CMs). The strain sensor is integrated on the polyimide (PI) cantilever. To improve the strain sensor reliability and construct the engineered cardiac tissue, the nanogroove-patterned polydimethylsiloxane (PDMS) encapsulation layer is bonded on the PI cantilever. The preliminary sensing characteristics demonstrate the superior structural integrity, robustness, enhanced sensitivity, and repeatability of the proposed devices. The long-term durability and biocompatibility of the PI/PDMS hybrid cantilever is verified by evaluating the cell viability and contractility. We also validate the proposed biosensing platform for cardiotoxicity measurement by applying it to two specific cardiovascular drugs: quinidine and verapamil. In response to quinidine and verapamil, the engineered CMs exhibited negative inotropic and chronotropic effects. The fabricated cantilever device successfully detected the quinidine-induced adverse effects in CMs such as early after depolarization (EADs) and Torsade de points (TdP) in real-time. The array of hybrid cantilevers with integrated strain sensors has the potential to satisfy the need for innovative analytic platforms owing to its high throughput and simplified data analysis.