• Molecules and Cells
• /
• v.44 no.9
• /
• pp.627-636
• /
• 2021

The three-dimensional organization of chromatin and its time-dependent changes greatly affect virtually every cellular function, especially DNA replication, genome maintenance, transcription regulation, and cell differentiation. Sequencing-based techniques such as ChIP-seq, ATAC-seq, and Hi-C provide abundant information on how genomic elements are coupled with regulatory proteins and functionally organized into hierarchical domains through their interactions. However, visualizing the time-dependent changes of such organization in individual cells remains challenging. Recent developments of CRISPR systems for site-specific fluorescent labeling of genomic loci have provided promising strategies for visualizing chromatin dynamics in live cells. However, there are several limiting factors, including background signals, off-target binding of CRISPR, and rapid photobleaching of the fluorophores, requiring a large number of target-bound CRISPR complexes to reliably distinguish the target-specific foci from the background. Various modifications have been engineered into the CRISPR system to enhance the signal-to-background ratio and signal longevity to detect target foci more reliably and efficiently, and to reduce the required target size. In this review, we comprehensively compare the performances of recently developed CRISPR designs for improved visualization of genomic loci in terms of the reliability of target detection, the ability to detect small repeat loci, and the allowed time of live tracking. Longer observation of genomic loci allows the detailed identification of the dynamic characteristics of chromatin. The diffusion properties of chromatin found in recent studies are reviewed, which provide suggestions for the underlying biological processes.

• Nano
• /
• v.13 no.11
• /
• pp.1850131.1-1850131.19
• /
• 2018

In this paper, an electrochemical sensor for epinephrine (EP), a neurotransmitter was developed by anchoring molecularly imprinted polymeric matrix (MIP) on the surface of gold-coated quartz crystal electrode of electrochemical quartz crystal microbalance (EQCM) using starch nanoparticles (Starch NP) - reduced graphene oxide (RGO) nanocomposite as polymeric format for the first time. Use of EP in therapeutic treatment requires proper dose and route of administration. Proper follow-up of neurological disorders and timely diagnosis of them has been found to depend on EP level. The MIP sensor was developed by electrodeposition of starch NP-RGO composite on EQCM electrode in presence of template EP. As the imprinted sites are located on the surface, high specific surface area enables good accessibility and high binding affinity to template molecule. Differential pulse voltammetry (DPV) and piezoelectrogravimmetry were used for monitoring binding/release, rebinding of template to imprinted cavities. MIP-coated EQCM electrode were characterized by contact angle measurements, AFM images, piezoelectric responses including viscoelasticity of imprinted films, and other voltammetric measurements including direct (DPV) and indirect (using a redox probe) measurements. Selectivity was assessed by imprinting factor (IF) as high as 3.26 (DPV) and 3.88 (EQCM). Sensor was rigorously checked for selectivity in presence of other structurally close analogues, real matrix (blood plasma), reproducibility, repeatability, etc. Under optimized conditions, the EQCM-MIP sensor showed linear dynamic ranges ($1-10{\mu}M$). The limit of detection 40 ppb (DPV) and 290 ppb (EQCM) was achieved without any cross reactivity and matrix effect indicating high sensitivity and selectivity for EP. Hence, an eco-friendly MIP-sensor with high sensitivity and good selectivity was fabricated which could be applied in "real" matrices in a facile manner.

• Molecules and Cells
• /
• v.42 no.5
• /
• pp.418-425
• /
• 2019

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.

• Applied Microscopy
• /
• v.51
• /
• pp.18.1-18.12
• /
• 2021

Hair, having distinct stages of growth, is a dynamic component of the integumentary system. Nonetheless, derangement in its structure and growth pattern often provides vital clues for the diagnosis of systemic diseases. Assessment of the hair structure by various microscopy techniques is, hence, a valuable tool for the diagnosis of several systemic and cutaneous disorders. Systemic illnesses like Comel-Netherton syndrome, Griscelli syndrome, Chediak Higashi syndrome, and Menkes disease display pathognomonic findings on hair microscopy which, consequently, provide crucial evidence for disease diagnosis. With minimal training, light microscopy of the hair can easily be performed even by clinicians and other health care providers which can, thus, serve as a useful tool for disease diagnosis at the patient's bedside. This is especially true for resource-constrained settings where access and availability of advanced investigations (like molecular diagnostics) is a major constraint. Despite its immense clinical utility and non-invasive nature, hair microscopy seems to be an underutilized diagnostic modality. Lack of awareness regarding the important findings on hair microscopy may be one of the crucial reasons for its underutilization. Herein, we, therefore, present a comprehensive overview of the available methods for hair microscopy and the pertinent findings that can be observed in various diseases.

• Molecules and Cells
• /
• v.46 no.6
• /
• pp.374-386
• /
• 2023

Thermal stress induces dynamic changes in nuclear proteins and relevant physiology as a part of the heat shock response (HSR). However, how the nuclear HSR is fine-tuned for cellular homeostasis remains elusive. Here, we show that mitochondrial activity plays an important role in nuclear proteostasis and genome stability through two distinct HSR pathways. Mitochondrial ribosomal protein (MRP) depletion enhanced the nucleolar granule formation of HSP70 and ubiquitin during HSR while facilitating the recovery of damaged nuclear proteins and impaired nucleocytoplasmic transport. Treatment of the mitochondrial proton gradient uncoupler masked MRP-depletion effects, implicating oxidative phosphorylation in these nuclear HSRs. On the other hand, MRP depletion and a reactive oxygen species (ROS) scavenger non-additively decreased mitochondrial ROS generation during HSR, thereby protecting the nuclear genome from DNA damage. These results suggest that suboptimal mitochondrial activity sustains nuclear homeostasis under cellular stress, providing plausible evidence for optimal endosymbiotic evolution via mitochondria-to-nuclear communication.

• Genomics & Informatics
• /
• v.21 no.3
• /
• pp.30.1-30.13
• /
• 2023

Ephs belong to the largest family of receptor tyrosine kinase and are highly conserved both sequentially and structurally. The structural organization of Eph is similar to other receptor tyrosine kinases; constituting the extracellular ligand binding domain, a fibronectin domain followed by intracellular juxtamembrane kinase, and SAM domain. Eph binds to respective ephrin ligand, through the ligand binding domain and forms a tetrameric complex to activate the kinase domain. Eph-ephrin regulates many downstream pathways that lead to physiological events such as cell migration, proliferation, and growth. Therefore, considering the importance of Eph-ephrin class of protein in tumorigenesis, 7,620 clinically reported missense mutations belonging to the class of variables of unknown significance were retrieved from cBioPortal and evaluated for pathogenicity. Thirty-two mutations predicted to be pathogenic using SIFT, Polyphen-2, PROVEAN, SNPs&GO, PMut, iSTABLE, and PremPS in-silico tools were found located either in critical functional regions or encompassing interactions at the binding interface of Eph-ephrin. However, seven were reported in nonsmall cell lung cancer (NSCLC). Considering the relevance of receptor tyrosine kinases and Eph in NSCLC, these seven mutations were assessed for change in the folding pattern using molecular dynamic simulation. Structural alterations, stability, flexibility, compactness, and solvent-exposed area was observed in EphA3 Trp790Cys, EphA7 Leu749Phe, EphB1 Gly685Cys, EphB4 Val748Ala, and Ephrin A2 Trp112Cys. Hence, it can be concluded that the evaluated mutations have potential to alter the folding pattern and thus can be further validated by in-vitro, structural and in-vivo studies for clinical management.

• Molecules and Cells
• /
• v.46 no.9
• /
• pp.527-534
• /
• 2023

Liver ischemia-reperfusion injury (IRI) is the main cause of organ dysfunction and failure after liver surgeries including organ transplantation. The mechanism of liver IRI is complex and numerous signals are involved but cellular metabolic disturbances, oxidative stress, and inflammation are considered the major contributors to liver IRI. In addition, the activation of inflammatory signals exacerbates liver IRI by recruiting macrophages, dendritic cells, and neutrophils, and activating NK cells, NKT cells, and cytotoxic T cells. Technological advances enable us to understand the role of specific immune cells during liver IRI. Accordingly, therapeutic strategies to prevent or treat liver IRI have been proposed but no definitive and effective therapies exist yet. This review summarizes the current update on the immune cell functions and discusses therapeutic potentials in liver IRI. A better understanding of this complex and highly dynamic process may allow for the development of innovative therapeutic approaches and optimize patient outcomes.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.19 no.1
• /
• pp.537-545
• /
• 2018

In this study, transcriptome analysis was conducted to collect various information from Fagopyrum esculentum and Fagopyrum tataricum during the early germination stage. Total RNA was extracted from the seeds and at 12, 24, and 36 hrs after germination of Jeju native Fagopyrum esculentum and Fagopyrum tataricum and sequenced using the Illumina Hiseq 2000 platform. Raw data analysis was conducted using the Dynamic Trim and Lengths ORT programs in the SolexaQA package, and assembly and annotation were performed. Based on RNA-seq raw data, we obtained 16.5 Gb and 16.2 Gb of transcriptome data corresponding to about 84.2% and 81.5% of raw data, respectively. De novo assembly and annotation revealed 43,494 representative transcripts corresponding to 47.5Mb. Among them, 23,165 sequences were shown to have similar sequences with annotation DB. Moreover, Gene Ontology (GO) analysis of buckwheat representative transcripts confirmed that the gene is involved in metabolic processes (49.49%) of biological processes, as well as cell function (46.12%) in metabolic process, and catalytic activity (80.43%) in molecular function In the case of gibberellin receptor GID1C, which is related to germination of seeds, the expression levels increased with time after germination in both F. esculentum and F. tataricum. The expression levels of gibberellin 20-oxidase 1 were increased within 12 hrs of gemination in F. esculentum but continuously until 36 hrs in F. tataricum. This buckwheat transcriptome profile analysis of the early germination stage will help to identify the mechanism causing functional and morphological differences between species.

• Nuclear Medicine and Molecular Imaging
• /
• v.41 no.3
• /
• pp.194-200
• /
• 2007

Purpose: $^{13}N$-ammonia is a well known radiopharmaceutical for the measurement of a myocardial blood flow (MBF) non-invasively using PET-CT. In this study, we investigated a correlation between MBF obtained from dynamic imaging and myocardial perfusion score (MPS) obtained from static imaging for usefulness of cardiac PET study. Methods: Twelve patients (11 males, 1 female, $57.9{\pm}8.6$ years old) with suspicious coronary artery disease underwent PET-CT scan. Dynamic scans (6 min: $5\;sec\;{\times}\;12,\;10\;sec\;{\times}\;6,\;20\;sec\;{\times}\;3,\;and\;30\;sec\;{\times}\;6$) were initiated simultaneously with bolus injection of 11 MBq/kg $^{13}N-ammonia$ to acquire rest and stress image. Gating image was acquired during 13 minutes continuously. Nine-segment model (4 basal walls, 4 mid walls, and apex) was used for a measurement of MBF. Time activity curve of input function and myocardium was extracted from ROI methods in 9 regions for quantification. The MPS were evaluated using quantitative analysis software. To compare between 20-segment model and 9-segment model, 6 basal segments were excluded and averaged segmental scores were used. Results: There are weak correlation between MBF (rest, 0.18-2.38 ml/min/g; stress, 0.40-4.95 ml/min/g) and MPS (rest 22-91%, stress, 14-90%), however the correlation coefficient between corrected MBF and MPS in rest state was higher than stress state (rest r=0.59; stress r=0.80). As a thickening increased, correlation between MBF and MPS also showed good correlation at each segments. Conclusions: Corrected and translated MPS as its characteristics using $^{13}N$-ammonia showed good correlation with absolute MBF measured by dynamic image in this study. Therefore, we showed MPS is one of good indices which reflect MBF. We anticipate PET-CT could be used as useful tool for evaluation of myocardial function in nuclear cardiac study.

• Korean Chemical Engineering Research
• /
• v.54 no.1
• /
• pp.120-126
• /
• 2016

Eco-friendly acrylic resin/clay nanocomposites containing pristine montmorillonite (PM) or modified clays (30B and 25A) were prepared from acrylic and styrenic monomers using non-aqueous dispersion (NAD) polymerization. Effect of nanoclays on physical properties of polymerization product and resulting nanocomposites was investigated. In view of NAD particle stability, addition of nanoclay at the beginning of polymerization is proved to be good. Results of gel fraction, acid value and viscosity of the NAD product showed that nanocomposites containing clay 25A showed better physical properties than the ones with other clays. GPC results exhibit the increase in molecular weight and decrease in polydispersity index for the 25A nanocomposite. Increase in layer distance confirmed from XRD analysis showed good dispersion of 25A in the nanocomposite. Thermal and dynamic mechanical analysis showed that highest glass transition temperature and storage modulus for 25A nanocomposites. These results indicate that 25A nanoclay gives the best properties in the process of non-aqueous dispersion polymerization of acrylic resin/nanoclay nanocomposites.