Epigenetic modification of gene expression in cancer cells by terahertz demethylation.

Terahertz (THz) radiation can affect the degree of DNA methylation, the spectral characteristics of which exist in the terahertz region. DNA methylation is an epigenetic modification in which a methyl (CH3) group is attached to cytosine, a nucleobase in human DNA. Appropriately controlled DNA methylation leads to proper regulation of gene expression. However, abnormal gene expression that departs from controlled genetic transcription through aberrant DNA methylation may occur in cancer or other diseases. In this study, we demonstrate the modification of gene expression in cells by THz demethylation using resonant THz radiation. Using an enzyme-linked immunosorbent assay, we observed changes in the degree of global DNA methylation in the SK-MEL-3 melanoma cell line under irradiation with 1.6-THz radiation with limited spectral bandwidth. Resonant THz radiation demethylated living melanoma cells by 19%, with no significant occurrence of apurinic/apyrimidinic sites, and the demethylation ratio was linearly proportional to the power of THz radiation. THz demethylation downregulates FOS, JUN, and CXCL8 genes, which are involved in cancer and apoptosis pathways. Our results show that THz demethylation has the potential to be a gene expression modifier with promising applications in cancer treatment.

Numerical Model for Cerebrovascular Hemodynamics with Indocyanine Green Fluorescence Videoangiography.

OBJECTIVE: The use of indocyanine green videoangiography (ICG-VA) to assess blood flow in the brain during cerebrovascular surgery has been increasing. Clinical studies on ICG-VA have predominantly focused on qualitative analysis. However, quantitative analysis numerical modelling for time profiling enables a more accurate evaluation of blood flow kinetics. In this study, we established a multiple exponential modified Gaussian (multi-EMG) model for quantitative ICG-VA to understand accurately the status of cerebral hemodynamics. METHODS: We obtained clinical data of cerebral blood flow acquired the quantitative analysis ICG-VA during cerebrovascular surgery. Varied asymmetric peak functions were compared to find the most matching function form with clinical data by using a nonlinear regression algorithm. To verify the result of the nonlinear regression, the mode function was applied to various types of data. RESULTS: The proposed multi-EMG model is well fitted to the clinical data. Because the primary parameters-growth and decay rates, and peak center and heights-of the model are characteristics of model function, they provide accurate reference values for assessing cerebral hemodynamics in various conditions. In addition, the primary parameters can be estimated on the curves with partially missed data. The accuracy of the model estimation was verified by a repeated curve fitting method using manipulation of missing data. CONCLUSION: The multi-EMG model can possibly serve as a universal model for cerebral hemodynamics in a comparison with other asymmetric peak functions. According to the results, the model can be helpful for clinical research assessment of cerebrovascular hemodynamics in a clinical setting.

Improving signal-to-noise ratio of a terahertz signal using a WaveNet-based neural network.

When acquiring a terahertz signal from a time-domain spectroscopy system, the signal is degraded by measurement noise and the information embedded in the signal is distorted. For high-performing terahertz applications, this study proposes a method for enhancing such a noise-degraded terahertz signal using machine learning that is applied to the raw signal after acquisition. The proposed method learns a function that maps the degraded signal to the clean signal using a WaveNet-based neural network that performs multiple layers of dilated convolutions. It also includes learnable pre- and post-processing modules that automatically transform the time domain where the enhancement process operates. When training the neural network, a data augmentation scheme is adopted to tackle the issue of insufficient training data. The comparative evaluation confirms that the proposed method outperforms other baseline neural networks in terms of signal-to-noise ratio. The proposed method also performs significantly better than the averaging of multiple signals, thereby facilitating the procurement of an enhanced signal without increasing the measurement time.

Machine Learning Techniques for THz Imaging and Time-Domain Spectroscopy.

Terahertz imaging and time-domain spectroscopy have been widely used to characterize the properties of test samples in various biomedical and engineering fields. Many of these tasks require the analysis of acquired terahertz signals to extract embedded information, which can be achieved using machine learning. Recently, machine learning techniques have developed rapidly, and many new learning models and learning algorithms have been investigated. Therefore, combined with state-of-the-art machine learning techniques, terahertz applications can be performed with high performance that cannot be achieved using modeling techniques that precede the machine learning era. In this review, we introduce the concept of machine learning and basic machine learning techniques and examine the methods for performance evaluation. We then summarize representative examples of terahertz imaging and time-domain spectroscopy that are conducted using machine learning.

Transformation of terahertz vibrational modes of cytosine under hydration.

Cytosine and cytosine monohydrate are representative biomolecules for investigating the effect of hydrogen bonds in deoxyribonucleic acid. To better understand intermolecular interactions, such as hydrogen bonds, between nucleobases it is necessary to identify the low-frequency vibrational modes associated with intermolecular interactions and crystalline structures. In this study, we investigated the characteristic low-frequency vibrational modes of cytosine and cytosine monohydrate using terahertz time-domain spectroscopy (THz-TDS). The crystal geometry was obtained by the powder X-ray diffraction technique. The optimized atomic positions and the normal modes in the terahertz region were calculated using density functional theory (DFT), which agreed well with the experimental results. We found that overall terahertz absorption peaks of cytosine and cytosine monohydrate consist of collective vibrations mixed with intermolecular and intramolecular vibrations in mode character analysis, and that the most intense peaks of both samples involve remarkable intermolecular translational vibration. These results indicate that THz-TDS combined with DFT calculations including mode character analysis can be an effective method for understanding how water molecules contribute to the characteristics of the low-frequency vibrational modes by intermolecular vibrations with hydrogen bonding in biological and biomedical applications.

Determining terahertz resonant peaks of biomolecules in aqueous environment.

The resonant peaks of biomolecules provide information on the molecules' physical and chemical properties. Although many biomolecules have resonant peaks in the terahertz region, it is difficult to observe their specific signals in aqueous environments. Hence, this paper proposes a method for determining these peaks. We found the specific resonant peaks of a modified nucleoside, 5-methlycytidine and modified HEK293T DNA in an aqueous solution through baseline correction. We evaluated the consistency of various fitting functions used for determining the peaks with various parameters. We separated two resonance peaks of 5-methlycytidine at 1.59 and 1.97 THz and for artificially methylated HEK293T DNA at 1.64 and 2.0 THz.

Effective demethylation of melanoma cells using terahertz radiation.

Terahertz (THz) demethylation is a photomedical technique applied to dissociate methyl-DNA bonds and reduce global DNA methylation using resonant THz radiation. We evaluated the performance of THz demethylation and investigated the DNA damage caused by THz irradiation. The demethylation rate in M-293T DNA increased linearly with the irradiation power up to 48%. The degree of demethylation increased with exposure to THz radiation, saturating after 10 min. Although THz demethylation occurred globally, most of the demethylation occurred within the partial genes in the CpG islands. Subsequently, we performed THz demethylation of melanoma cells. The degree of methylation in the melanoma cell pellets decreased by approximately 10-15%, inducing ∼5-8 abasic sites per 105 bp; this was considerably less than the damaged DNA irradiated by the high-power infrared laser beam used for generating THz pulses. These results provide initial data for THz demethylation and demonstrate the applicability of this technique in advanced cancer cell research. THz demethylation has the potential to develop into a therapeutic procedure for cancer, similar to that involving chemical demethylating agents.

Highly Sensitive and Selective Detection of Steroid Hormones Using Terahertz Molecule-Specific Sensors.

Discrimination and quantification of trace amounts of steroid hormones in biological specimens are needed to elucidate their changing expression because their biological functions are responsible for the development and prevention of endocrine disorders. Although mass-spectrometry-based assays are most commonly recommended, development of a new type of highly sensitive and selective detection methods in clinical practices is needed. Here, we introduce a label-free type of terahertz molecule sensor capable of sensing and identifying progesterone and 17α-OH-progesterone selectively. Nanoslot-array-based sensing chips were used as launching pads for absorption cross-section enhancement of molecules at a reliable terahertz frequency. With use of nanoslots with resonances at 1.17 THz corresponding to intrinsic THz absorption resonance mode for progesterone and at 1.51 THz for 17α-OH-progesterone, respectively, each steroid shows prominent transmittance change in terms of its amount. In particular, the sensing performance has been much improved by controlling evaporation speed, in turn resulting in an efficient, homogeneous distribution of the molecules onto a sensing hot spot.

Detection and manipulation of methylation in blood cancer DNA using terahertz radiation.

DNA methylation is a pivotal epigenetic modification of DNA that regulates gene expression. Abnormal regulation of gene expression is closely related to carcinogenesis, which is why the assessment of DNA methylation is a key factor in cancer research. Terahertz radiation may play an important role in active demethylation for cancer therapy because the characteristic frequency of the methylated DNA exists in the terahertz region. Here, we present a novel technique for the detection and manipulation of DNA methylation using terahertz radiation in blood cancer cell lines. We observed the degree of DNA methylation in blood cancer at the characteristic resonance of approximately 1.7 THz using terahertz time-domain spectroscopy. The terahertz results were cross-checked with global DNA methylation quantification using an enzyme-linked immunosorbent assay. We also achieved the demethylation of cancer DNA using high-power terahertz radiation at the 1.7-THz resonance. The demethylation degrees ranged from 10% to 70%, depending on the type of cancer cell line. Our results show the detection of DNA methylation based on the terahertz molecular resonance and the manipulation of global DNA methylation using high-power terahertz radiation. Terahertz radiation may have potential applications as an epigenetic inhibitor in cancer treatment, by virtue of its ability to induce DNA demethylation, similarly to decitabine.

Unsaturated Drift Velocity of Monolayer Graphene.

We observe that carriers in graphene can be accelerated to the Fermi velocity without heating the lattice. At large Fermi energy | EF| > 110 meV, electrons excited by a high-power terahertz pulse ETHz relax by emitting optical phonons, resulting in heating of the graphene lattice and optical-phonon generation. This is owing to enhanced electron-phonon scattering at large Fermi energy, at which the large phase space is available for hot electrons. The emitted optical phonons cause carrier scattering, reducing the drift velocity or carrier mobility. However, for | EF| ≤ 110 meV, electron-phonon scattering rate is suppressed owing to the diminishing density of states near the Dirac point. Therefore, ETHz continues to accelerate carriers without them losing energy to optical phonons, allowing the carriers to travel at the Fermi velocity. The exotic carrier dynamics does not result from the massless nature, but the electron-optical-phonon scattering rate depends on Fermi level in the graphene. Our observations provide insight into the application of graphene for high-speed electronics without degrading carrier mobility.

Transient Carrier Cooling Enhanced by Grain Boundaries in Graphene Monolayer.

Using a high terahertz (THz) electric field (ETHz), the carrier scattering in graphene was studied with an electric field of up to 282 kV/cm. When the grain size of graphene monolayers varies from small (5 µm) and medium (70 µm) to large grains (500 µm), the dominant carrier scattering source in large- and small-grained graphene differs at high THz field, i.e., there is optical phonon scattering for large grains and defect scattering for small grains. Although the electron-optical phonon coupling strength is the same for all grain sizes in our study, the enhanced optical phonon scattering in the high THz field from the large-grained graphene is caused by a higher optical phonon temperature, originating from the slow relaxation of accelerated electrons. Unlike the large-grained graphene, lower electron and optical phonon temperatures are found in the small-grained graphene monolayer, resulting from the effective carrier cooling through the defects, called supercollisions. Our results indicate that the carrier mobility in the high-crystalline graphene is easily vulnerable to scattering by the optical phonons. Thus, controlling the population of defect sites, as a means for carrier cooling, can enhance the carrier mobility at high electric fields in graphene electronics by suppressing the heating of optical phonons.

Terahertz transmission control using polarization-independent metamaterials.

We present terahertz (THz) transmission control by several uniquely designed patterns of nano-slot antenna array. Collinearly aligned slot antenna arrays have been usually applied to THz filters with frequency band tunability by their geometry. Normally the amplitude in transmission (reflection) in the collinear alignment case can be varied via rotating the azimuthal angle with a sinusoidal trend, which can limit their utilization and performance only at fixed angle between the alignment of the resonant antennas and incident beam polarization. To pursue a variety of metamaterial uses, here, we present polarization-independent THz filters using variously aligned antenna array (asterisk, chlorophyll, and honeycomb patterns) in such counter-intuitive aspects. Besides, unprecedented multi resonance behaviors were observed in chlorophyll and honeycomb patterns, which can be explained with interferences by adjacent structures. The measured spectra were analyzed by harmonic oscillator model with simplified coupling between slots and their adjacent.

Terahertz imaging of metastatic lymph nodes using spectroscopic integration technique.

Terahertz (THz) imaging was used to differentiate the metastatic states of frozen lymph nodes (LNs) by using spectroscopic integration technique (SIT). The metastatic states were classified into three groups: healthy LNs, completely metastatic LNs, and partially metastatic LNs, which were obtained from three mice without infection and six mice infected with murine melanoma cells for 30 days and 15 days, respectively. Under histological examination, the healthy LNs and completely metastatic LNs were found to have a homogeneous cellular structure but the partially metastatic LNs had interfaces of the melanoma and healthy tissue. THz signals between the experimental groups were not distinguished at room temperature due to high attenuation by water in the tissues. However, a signal gap between the healthy and completely metastatic LNs was detected at freezing temperature. The signal gap could be enhanced by using SIT that is a signal processing method dichotomizing the signal difference between the healthy cells and melanoma cells with their normalized spectral integration. This technique clearly imaged the interfaces in the partially metastatic LNs, which could not be achieved by existing methods using a peak point or spectral value. The image resolution was high enough to recognize a metastatic area of about 0.7 mm size in the partially metastatic LNs. Therefore, this pilot study demonstrated that THz imaging of the frozen specimen using SIT can be used to diagnose the metastatic state of LNs for clinical application.

Terahertz molecular resonance of cancer DNA.

Carcinogenesis involves the chemical and structural alteration of biomolecules in cells. Aberrant methylation of DNA is a well-known carcinogenic mechanism and a common chemical modification of DNA. Terahertz waves can directly observe changes in DNA because the characteristic energies lie in the same frequency region. In addition, terahertz energy levels are not high enough to damage DNA by ionization. Here, we present terahertz molecular resonance fingerprints of DNA methylation in cancer DNA. Methylated cytidine, a nucleoside, has terahertz characteristic energies that give rise to the molecular resonance of methylation in DNA. Molecular resonance is monitored in aqueous solutions of genomic DNA from cancer cell lines using a terahertz time-domain spectroscopic technique. Resonance signals can be quantified to identify the types of cancer cells with a certain degree of DNA methylation. These measurements reveal the existence of molecular resonance fingerprints of cancer DNAs in the terahertz region, which can be utilized for the early diagnosis of cancer cells at the molecular level.

Enhancement of terahertz reflection tomographic imaging by interference cancellation between layers.

This paper proposes a method to enhance terahertz reflection tomographic imaging by interference cancellation between layers. When the gap between layers is small, the signal reflected on the upper layer interferes with that on the lower layer, which degrades the quality of the reconstructed tomographic image in the lower layer. The proposed method estimates the upper-layer reflection signal by system modeling, which is then eliminated from the acquired signal. In this way, it can provide the correct lower-layer reflection signal, thereby improving the quality of the lower-layer tomographic image. The performance of the proposed method was confirmed using computer simulation data and real terahertz reflection data.

Highly sensitive and selective sugar detection by terahertz nano-antennas.

Molecular recognition and discrimination of carbohydrates are important because carbohydrates perform essential roles in most living organisms for energy metabolism and cell-to-cell communication. Nevertheless, it is difficult to identify or distinguish various carbohydrate molecules owing to the lack of a significant distinction in the physical or chemical characteristics. Although there has been considerable effort to develop a sensing platform for individual carbohydrates selectively using chemical receptors or an ensemble array, their detection and discrimination limits have been as high in the millimolar concentration range. Here we show a highly sensitive and selective detection method for the discrimination of carbohydrate molecules using nano-slot-antenna array-based sensing chips which operate in the terahertz (THz) frequency range (0.5-2.5 THz). This THz metamaterial sensing tool recognizes various types of carbohydrate molecules over a wide range of molecular concentrations. Strongly localized and enhanced terahertz transmission by nano-antennas can effectively increase the molecular absorption cross sections, thereby enabling the detection of these molecules even at low concentrations. We verified the performance of nano-antenna sensing chip by both THz spectra and images of transmittance. Screening and identification of various carbohydrates can be applied to test even real market beverages with a high sensitivity and selectivity.

Feasibility of terahertz reflectometry for discrimination of human early gastric cancers.

We have investigated the feasibility of THz time-domain reflectometry for the discrimination of human early gastric cancer (EGC) from the normal gastric region. Eight fresh EGC tissues, which were resected by endoscopic submucosal dissection, were studied. Of them, six lesions were well discriminated on THz images and the regions well correlated with tumor regions on pathologically mapped images. Four THz parameters could be suggested for quantitative discrimination of EGCs.

Study of freshly excised brain tissues using terahertz imaging.

We demonstrated that tumors in freshly excised whole brain tissue could be differentiated clearly from normal brain tissue using a reflection-type terahertz (THz) imaging system. THz binary images of brain tissues with tumors indicated that the tumor boundaries in the THz images corresponded well to those in visible images. Grey and white-matter regions were distinguishable owing to the different distribution of myelin in the brain tissue. THz images corresponded closely with magnetic resonance imaging (MRI) results. The MRI and hematoxylin and eosin-stained microscopic images were investigated to account for the intensity differences in the THz images for fresh and paraffin-embedded brain tissue. Our results indicated that the THz signals corresponded to the cell density when water was removed. Thus, THz imaging could be used as a tool for label-free and real-time imaging of brain tumors, which would be helpful for physicians to determine tumor margins during brain surgery.

Terahertz spectroscopic imaging and properties of gastrointestinal tract in a rat model.

We have investigated basic properties of normal gastrointestinal (GI) tract tissues, including glandular stomach (GS), fore stomach (FS), large intestine (LI), small intestine (SI), and esophagus (ESO), from a rat model using terahertz (THz) reflection imaging and spectroscopy. The THz images collected from stratified squamous epithelia (SSE) of FS and ESO show a lower peak-to-peak value compared to those from columnar epithelia (CE) of GS, LI, or SI because the SSE contains less water than CE. The refractive index and absorption coefficient of FS were less than those of GS or LI, both having values similar to those of water. Additionally, we report internal reflection THz signals from ESO, although we were unable to determine the exact interface for this internal reflection.

Measurement depth enhancement in terahertz imaging of biological tissues.

We demonstrate the use of a THz penetration-enhancing agent (THz-PEA) to enhance the terahertz (THz) wave penetration depth in tissues. The THz-PEA is a biocompatible material having absorption lower than that of water, and it is easily absorbed into tissues. When using glycerol as a THz-PEA, the peak value of the THz signal which was transmitted through the fresh tissue and reflected by a metal target, was almost doubled compared to that of tissue without glycerol. THz time-of-flight imaging (B-scan) was used to display the sequential glycerol delivery images. Enhancement of the penetration depth was confirmed after an artificial tumor was located below fresh skin. We thus concluded that the THz-PEA technique can potentially be employed to enhance the image contrast of the abnormal lesions below the skin.