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Human retinal organoids (ROs) have emerged as valuable tools for studying retinal development, modeling human retinal diseases, and screening drugs. However, their application is limited primarily due to time-intensive generation, high costs, and low reproducibility. Quality assessment of RO differentiation is crucial for their application in research. However, traditional methods such as morphological evaluation and immunohistochemical analysis have limitations due to their lack of precision and invasiveness, respectively. This study aims to identify non-invasive biomarkers for RO differentiation quality using exosomal microRNAs (miRNAs), which are known to reflect cell-specific functions and development in the retina. We differentiated ROs from human induced pluripotent stem cells (hiPSCs) and classified them into 'superior' and 'inferior' groups based on morphological and immunohistochemical criteria. Exosomes from the conditioned media were isolated and analyzed for miRNA content. Our findings revealed distinct miRNA profiles between superior and inferior ROs, with superior ROs exhibiting higher miRNA diversity and specifically up- or down-regulated miRNAs. Gene ontology and pathway enrichment analyses indicated that the target genes of these miRNAs are involved in neuron proliferation and differentiation. The study suggests the potential of exosomal hsa-miR-654-3p and hsa-miR-451a as non-invasive biomarkers for real-time monitoring of RO quality, facilitating the development of standardized, efficient, and cost-effective culture methods.
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Biomarcadores , Diferenciación Celular , Exosomas , Células Madre Pluripotentes Inducidas , MicroARNs , Organoides , Retina , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Organoides/metabolismo , Organoides/citología , Diferenciación Celular/genética , Retina/citología , Retina/metabolismo , Biomarcadores/metabolismo , Exosomas/metabolismo , Exosomas/genética , Células CultivadasRESUMEN
Increasing evidence suggests that exosomes are involved in retinal cell degeneration, including their insufficient release; hence, they have become important indicators of retinopathies. The exosomal microRNA (miRNA), in particular, play important roles in regulating ocular and retinal cell functions, including photoreceptor maturation, maintenance, and visual function. Here, we generated retinal organoids (ROs) from human induced pluripotent stem cells that differentiated in a conditioned medium for 60 days, after which exosomes were extracted from ROs (Exo-ROs). Subsequently, we intravitreally injected the Exo-RO solution into the eyes of the Royal College of Surgeons (RCS) rats. Intravitreal Exo-RO administration reduced photoreceptor apoptosis, prevented outer nuclear layer thinning, and preserved visual function in RCS rats. RNA sequencing and miRNA profiling showed that exosomal miRNAs are mainly involved in the mitogen-activated protein kinase (MAPK) signaling pathway. In addition, the expression of MAPK-related genes and proteins was significantly decreased in the Exo-RO-treated group. These results suggest that Exo-ROs may be a potentially novel strategy for delaying retinal degeneration by targeting the MAPK signaling pathway.
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Exosomas , Células Madre Pluripotentes Inducidas , MicroARNs , Degeneración Retiniana , Cirujanos , Ratas , Humanos , Animales , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Exosomas/metabolismo , Especies Reactivas de Oxígeno , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
Methyltransferases transfer a methyl group to a diverse group of natural products, thus providing structural diversity, stability, and altered pharmacological properties to the molecules. A limited number of regiospecific sugar-O-methyltransferases are functionally characterized. Thus, discovery of such an enzyme could solve the difficulties of biological production of methoxy derivatives of glycosylated molecules. In the current study, a regiospecific sugar-O-methyltransferase, ThnM1, belonging to the biosynthetic gene cluster (BGC) of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene produced by Nocardia sp. strain CS682, was analyzed and functionally characterized. ThnM1 demonstrated promiscuity to diverse chemical structures such as rhamnose-containing anthraquinones and flavonoids with regiospecific methylation at the 2'-hydroxyl group of the sugar moiety. Compared with other compounds, anthraquinone rhamnosides were found to be the preferred substrates for methylation. Thus, the enzyme was further employed for whole-cell biotransformation using engineered Escherichia coli to produce a methoxy-rhamnosyl derivative of quinizarin, an anthraquinone derivative. The structure of the newly generated derivative from Escherichia coli fermentation was elucidated by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopic analyses and identified as quinizarin-4-O-α-l-2-O-methylrhamnoside (QRM). Further, the biological impact of methylation was studied by comparing the cytotoxicity of QRM with that of quinizarin against the U87MG, SNU-1, and A375SM cancer cell lines. IMPORTANCE ThnM1 is a putative sugar-O-methyltransferase produced by the Nocardia sp. strain CS682 and is encoded by a gene belonging to the biosynthetic gene cluster (BGC) of 1-(α-l-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene. We demonstrated that ThnM1 is a promiscuous enzyme with regiospecific activity at the 2'-OH of rhamnose. As regiospecific methylation of sugars by chemical synthesis is a challenging step, ThnM1 may fill the gap in the potential diversification of natural products by methylating the rhamnose moiety attached to them.
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Productos Biológicos , Nocardia , Productos Biológicos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Metiltransferasas/metabolismo , Nocardia/genética , Nocardia/metabolismo , Ramnosa/metabolismo , Azúcares/metabolismoRESUMEN
BACKGROUND: To investigate the clinical findings of choroideremia patients and perform genetic analysis by whole-exome sequencing (WES). METHODS: A total of 94 patients initially diagnosed with retinitis pigmentosa (RP) at another hospital, and who visited our hospital for genetic analysis by WES, were included in the study, along with 64 family members. All subjects underwent comprehensive ophthalmic evaluation, including best-corrected visual acuity, slit lamp examination, fundus photography, fundus autofluorescence (FAF), fluorescein angiography (FAG), visual field (VF), electroretinogram (ERG), and optical coherence tomography (OCT). RESULTS: In six male patients with suspected choroideremia, extensive retinal pigment epithelium (RPE) and severe loss of choroid were observed in the fundus, but not in the macula. CHM gene mutation was confirmed in five patients. A novel single nucleotide variant at a splice site was observed in one patient. OCT showed marked thinning of the outernuclear layer and choroid, except in the macula. FAF showed a small area of hyperfluorescence in the posterior pole. In addition, characteristic interlaminar bridges were observed in four patients. On FAG, hypofluorescence was seen up to the far-peripheral retina in five patients. CONCLUSION: Of the 94 patients initially diagnosed with RP, CHM mutation was identified in five (5.3%) by WES. Choroideremia should be considered as a differential diagnosis of RP. WES would be useful for identifying the causes of hereditary retinal disease.
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Coroideremia/fisiopatología , Pruebas Genéticas/estadística & datos numéricos , Retinitis Pigmentosa/genética , Adulto , Coroideremia/epidemiología , Coroideremia/genética , Electrorretinografía/métodos , Electrorretinografía/estadística & datos numéricos , Femenino , Angiografía con Fluoresceína/métodos , Angiografía con Fluoresceína/estadística & datos numéricos , Pruebas Genéticas/métodos , Humanos , Masculino , Persona de Mediana Edad , República de Corea/epidemiología , Retinitis Pigmentosa/epidemiología , Retinitis Pigmentosa/etiología , Secuenciación del Exoma/métodosRESUMEN
Hepatocellular carcinoma (HCC) is a malignant type of primary liver cancer with high incidence and mortality, worldwide. A major challenge in the treatment of HCC is chemotherapeutic resistance. It is therefore necessary to develop novel anticancer drugs for suppressing the growth of HCC cells and overcoming drug resistance for improving the treatment of HCC. Violacein is a deep violet-colored indole derivative that is produced by several bacterial strains, including Chromobacterium violaceum, and it possesses numerous pharmacological properties, including antitumor activity. However, the therapeutic effects of violacein and the mechanism underlying its antitumor effect against HCC remain to be elucidated. This study is the first to demonstrate that violacein inhibits the proliferation and stemness of Huh7 and Hep3B HCC cells. The antiproliferative effect of violacein was attributed to cell cycle arrest at the sub-G1 phase and the induction of apoptotic cell death. Violacein induced nuclear condensation, dissipated mitochondrial membrane potential (MMP), increased generation of reactive oxygen species (ROS), activated the caspase cascade, and upregulated p53 and p21. The anticancer effect of violacein on HCC cells was also associated with the downregulation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2 signaling. Violacein not only suppressed the proliferation and formation of tumorspheres of Huh7 and Hep3B cancer stem-like cells but also reduced the expression of key markers of cancer stemness, including CD133, Sox2, Oct4, and Nanog, by inhibiting the signal transducer and activator of transcription 3 (STAT3)/AKT/ERK pathways. These results suggest the therapeutic potential of violacein in effectively suppressing HCC by targeting the proliferation and stemness of HCC cells.
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Antineoplásicos/farmacología , Productos Biológicos/farmacología , Autorrenovación de las Células/efectos de los fármacos , Indoles/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Productos Biológicos/química , Carcinoma Hepatocelular , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Indoles/química , Neoplasias Hepáticas , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Increasing studies have reported that cancer stem cells (CSCs) play critical roles in therapeutic resistance, recurrence, and metastasis of tumors, including cervical cancer. Pterostilbene, a dimethylated derivative of resveratrol, is a plant polyphenol compound with potential chemopreventive activity. However, the therapeutic effect of pterostilbene against cervical CSCs remains unclear. In this study, we compared the anticancer effects of resveratrol and pterostilbene using both HeLa cervical cancer adherent and stem-like cells. Pterostilbene more effectively inhibited the growth and clonogenic survival, as well as metastatic ability of HeLa adherent cells than those of resveratrol. Moreover, the superior inhibitory effects of pterostilbene compared to resveratrol were associated with the enhanced activation of multiple mechanisms, including cell cycle arrest at S and G2/M phases, induction of ROS-mediated caspase-dependent apoptosis, and inhibition of matrix metalloproteinase (MMP)-2/-9 expression. Notably, pterostilbene exhibited a greater inhibitory effect on the tumorsphere-forming and migration abilities of HeLa cancer stem-like cells compared to resveratrol. This greater effect was achieved through more potent inhibition of the expression levels of stemness markers, such as CD133, Oct4, Sox2, and Nanog, as well as signal transducer and activator of transcription 3 signaling. These results suggest that pterostilbene might be a potential anticancer agent targeting both cancer cells and cancer stem-like cells of cervical cancer via the superior bioavailability to resveratrol.
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Antineoplásicos Fitogénicos/farmacocinética , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Resveratrol/farmacocinética , Estilbenos/administración & dosificación , Estilbenos/farmacocinética , Neoplasias del Cuello Uterino/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Disponibilidad Biológica , Biomarcadores , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Femenino , Expresión Génica , Humanos , Estructura Molecular , Resveratrol/química , Estilbenos/química , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/patologíaRESUMEN
Melanoma is the most serious type of skin cancer and remains highly drug-resistant. Therefore, the discovery of novel effective agents against melanoma is in high demand. Herein, we investigated the cytotoxic activities in melanoma cells and underlying molecular mechanisms of beauvericin (BEA) and its analogue beauvericin G1 (BEA G1), which are cyclohexadepsipeptides isolated from fungi. BEA and BEA G1 significantly suppressed the growth, clonogenicity, migration, and invasion of A375SM human melanoma cells and promoted caspase-dependent apoptosis through upregulation of death receptors, as well as modulation of pro- and anti-apoptotic Bcl-2 family members. Furthermore, the effects of BEA and BEA G1 were associated with the suppression of multiple molecular targets that play crucial roles in melanoma oncogenesis, including ERK, JNK, p38, NF-κB, STAT3, and MITF. Notably, the cytotoxic efficacy of BEA G1 against A375SM cells was stronger than that of BEA. These findings suggest that BEA and BEA G1 can be further investigated as potent cytotoxic natural compounds for the suppression of melanoma progression.
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Antineoplásicos/química , Antineoplásicos/farmacología , Depsipéptidos/química , Depsipéptidos/farmacología , Apoptosis/efectos de los fármacos , Productos Biológicos/química , Productos Biológicos/farmacología , Biomarcadores , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Estructura Molecular , Transducción de SeñalRESUMEN
Glioblastoma multiforme (GBM) is the most aggressive and common type of human primary brain tumor. Glioblastoma stem-like cells (GSCs) have been proposed to contribute to tumor initiation, progression, recurrence, and therapeutic resistance of GBM. Therefore, targeting GSCs could be a promising strategy to treat this refractory cancer. Calmodulin (CaM), a major regulator of Ca2+ -dependent signaling, controls various cellular functions via interaction with multiple target proteins. Here, we investigated the anticancer effect of hydrazinobenzoylcurcumin (HBC), a Ca 2+ /CaM antagonist, against GSCs derived from U87MG and U373MG cells. HBC significantly inhibited not only the self-renewal capacity, such as cell growth and neurosphere formation but also the metastasis-promoting ability, such as migration and invasion of GSCs. HBC induced apoptosis of GSCs in a caspase-dependent manner. Notably, HBC repressed the phosphorylation of Ca 2+ /CaM-dependent protein kinase II (CaMKII), c-Met, and its downstream signal transduction mediators, thereby reducing the expression levels of GSC markers, such as CD133, Nanog, Sox2, and Oct4. In addition, the knockdown of CaMKIIγ remarkably decreased the cancer stem cell-like phenotypes as well as the expression of stemness markers by blocking c-Met signaling pathway in U87MG GSCs. These results suggest that HBC suppresses the stem-like features of GBM cells via downregulation of CaM/CaMKII/c-Met axis and therefore CaMKII may be a novel therapeutic target to eliminate GSCs.
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Biomarcadores de Tumor/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Curcumina/análogos & derivados , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Pirazoles/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Movimiento Celular , Proliferación Celular , Curcumina/farmacología , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Pronóstico , Células Tumorales CultivadasRESUMEN
A fused pyrrolopyridine core having substituents on the nitrogen atom instead of the carbon atom of the indoloindole unit is developed as a new donor unit for organic electronics. The new donor-acceptor copolymers, PDHPHBT, PDHPFBT, and PDHP2FBT, are synthesized using the new donor unit, well-known benzothiadiazole derivatives containing fluorine atoms as the acceptor. The thermal, optical, and electrochemical properties of these novel copolymers are reported. A solar cell using PDHPFBT with diphenyl ether has an open-circuit voltage, short-circuit current, fill factor, and power conversion efficiency of 0.86 V, 11.32 mA cm-2 , 0.59%, and 5.68%, respectively, under AM 1.5G illumination (100 mW cm-2 ) in the absence of annealing.
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Suministros de Energía Eléctrica , Polímeros/química , Pirimidinas/química , Pirroles/química , Energía Solar , Estructura Molecular , Polímeros/síntesis químicaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0269937.].
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Since the introduction of the positive list system (PLS) for agricultural products in the Republic of Korea, the demand for a quick, easy multi-residue analysis method increased continuously. Herein, the quick, easy, cheap, effective, rugged, and safe (QuEChERS) technique combined with liquid chromatography−tandem mass spectrometry was employed to optimize a method for the multi-residue analysis of 287 pesticide residues in mandarin orange and grapefruit. Method validation was conducted in terms of selectivity, limit of quantitation (LOQ), linearity, accuracy, precision, and matrix effect. All the compounds at low spiking levels (1, 2.5, 5, or 10 mg/kg) could be quantified at LOQs lower than 0.01 mg/kg (PLS level). The linearity of the matrix-matched calibration curve for each compound is in the range 0.5−50 µg/L, and its coefficient of determination (R2) is >0.990. Satisfactory recovery values of 70−120% with a relative standard deviation of ≤20% are obtained for all compounds in the mandarin orange and grapefruit samples. A negligible matrix effect (−20−20%) is observed for more than 94.8% and 85.4% of the pesticides in mandarin orange and grapefruit, respectively. Therefore, this analytical method can contribute to pesticide residue analyses of citrus fruits for routine laboratory testing.
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Choroidal neovascularization (CNV) is a defining characteristic feature of neovascular age-related macular degeneration (nAMD) that frequently results in irreversible vision loss. The current strategies for the treatment of nAMD are mainly based on neutralizing vascular endothelial growth factor (VEGF). However, anti-VEGF therapies are often associated with subretinal fibrosis that eventually leads to damages in macula. In this study, we tested whether an anti-fibrotic and anti-angiogenic protein CCN5 can potentially be an effective and safe therapeutic modality in a mouse model of CNV. Laser photocoagulation was utilized to induce CNV, which was followed by intravitreal injection of recombinant adeno-associated virus serotype 2 encoding CCN5 (rAAV2-CCN5). Our data demonstrated that rAAV2-CCN5, but not a control viral vector, rAAV2-VLP, prominently attenuated both CNV lesions and angiogenesis. Aflibercept, which was utilized as a positive control, exhibited similar effects on CNV lesions and angiogenesis in our experimental settings. Upon laser photocoagulation, retinal pigmented epithelium (RPE) cells underwent significant morphological changes including cellular enlargement and loss of hexagonality. rAAV2-CCN5 significantly normalized these morphological defects. Laser photocoagulation also led to fibrotic deformation in RPE cells through inducing epithelial-mesenchymal transition (EMT), which was completely blocked by rAAV2-CCN5. In a striking contrast, aflibercept as well as rAAV2-VLP failed to exhibit any effects on EMT. Collectively, this study suggest that CCN5 might provide a potential novel strategy for the treatment of nAMD with a capability to inhibit CNV and fibrosis simaultaneously.
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Neovascularización Coroidal , Parvovirinae , Animales , Neovascularización Coroidal/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Epitelio/metabolismo , Fibrosis , Ratones , Ratones Endogámicos C57BL , Parvovirinae/genética , Factor A de Crecimiento Endotelial VascularRESUMEN
The choice of the chlorine (Cl) atom as an electron-withdrawing substituent in conjugated polymers leads to a higher potential in the commercialization of polymer solar cells than its fluorine counterpart because of the versatility and cost-effectiveness of the chlorination process. In addition, the population and location of Cl substituents can significantly influence the photovoltaic characteristics of polymers. In this study, three chlorinated quinoxaline-based polymers were invented to examine the numerical and positioning effects of the Cl atom on their photovoltaic characteristics. The number of Cl substituents in the reference polymer, PBCl-Qx, was adjusted to three: two Cl atoms in the benzodithiophene-type D unit and one Cl atom in the quinoxaline-type A unit. Subsequently, two more Cl atoms were selectively introduced at the 4- and 5-positions of the alkylated thiophene moieties at the 2,3-positions of the quinoxaline moiety in PBCl-Qx to obtain the additional polymers PBCl-Qx4Cl and PBCl-Qx5Cl, respectively. The conventional PBCl-Qx4Cl device exhibited a better power conversion efficiency (PCE) of 12.95% as compared to those of PBCl-Qx (12.44%) and PBCl-Qx5Cl (11.82%) devices. The highest PCE of the device with PBCl-Qx4Cl was ascribed to an enhancement in the open-circuit voltage and fill factor induced by the deeper energy level of the highest occupied molecular orbital and the favorable morphological features in its blended film with Y6.
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BACKGROUND AND AIMS: Pathologic complete remission (CR) of rectal cancer after neoadjuvant chemoradiation therapy (CRT) is generally confirmed by routine hematoxylin and eosin (H&E) staining. The aim of this study was to identify residual rectal cancer cells in primary lesions of patients with pathologic CR by immunohistochemical staining for cytokeratin. PATIENTS AND METHODS: The medical records of 358 rectal cancer patients who underwent neoadjuvant CRT prior to radical surgery between October 2002 and August 2007 were reviewed. The authors stained sections of resected specimens of 58 patients (15.9%; 42 males; mean age 54 years) who achieved pathologic CR (as determined originally by H&E staining) with H&E and performed immunohistochemistry (IHC) using monoclonal anti-cytokeratin antibody. These stained sections were reviewed for residual rectal cancer cells by a pathologist. RESULTS: Of the 58 patients that achieved CR by initial pathologic examinations, eight (13.8%) were found to contain tumor by cytokeratin IHC. H&E staining revealed that six of these were positive for cancer cells, but the remaining two were negative for residual rectal cancer cells. CONCLUSION: Through better identification of residual rectal cancer cells, cytokeratin IHC offers a means of improving staging accuracy and thus provides useful information for prognosis and treatment decisions for patients with rectal cancer who had a clinical CR after CRT.
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Queratinas/metabolismo , Neoplasias del Recto/tratamiento farmacológico , Neoplasias del Recto/radioterapia , Coloración y Etiquetado/métodos , Demografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasia Residual/patología , Neoplasias del Recto/metabolismo , Neoplasias del Recto/patología , Inducción de RemisiónRESUMEN
To evaluate the feasibility of low-concentration contrast medium (CM) for vascular enhancement, image quality, and radiation dose on computed tomography aortography (CTA) using a combined low-tube-voltage and iterative reconstruction (IR) technique. Ninety subjects underwent dual-source CT (DSCT) operating in dual-source, high-pitch mode. DSCT scans were performed using both high-concentration CM (Group A, n = 50; Iomeprol 400) and low-concentration CM (Group B, n = 40; Iodixanol 270). Group A was scanned using a reference tube potential of 120 kVp and 120 reference mAs under automatic exposure control with IR. Group B was scanned using low-tube-voltage (80 or 100 kVp if body mass index ≥25 kg/m(2)) at a fixed current of 150 mAs, along with IR. Images of the two groups were compared regarding attenuation, image noise, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), iodine load, and radiation dose in various locations of the CTA. In comparison between Group A and Group B, the average mean attenuation (454.73 ± 86.66 vs. 515.96 ± 101.55 HU), SNR (25.28 ± 4.34 vs. 31.29 ± 4.58), and CNR (21.83 ± 4.20 vs. 27.55 ± 4.81) on CTA in Group B showed significantly greater values and significantly lower image noise values (18.76 ± 2.19 vs. 17.48 ± 3.34) than those in Group A (all Ps < 0.05). Homogeneous contrast enhancement from the ascending thoracic aorta to the infrarenal abdominal aorta was significantly superior in Group B (P < 0.05). Low-concentration CM and a low-tube-voltage combination technique using IR is a feasible method, showing sufficient contrast enhancement and image quality.