Machine learning-based clinical decision support system for treatment recommendation and overall survival prediction of hepatocellular carcinoma: a multi-center study.

The treatment decisions for patients with hepatocellular carcinoma are determined by a wide range of factors, and there is a significant difference between the recommendations of widely used staging systems and the actual initial treatment choices. Herein, we propose a machine learning-based clinical decision support system suitable for use in multi-center settings. We collected data from nine institutions in South Korea for training and validation datasets. The internal and external datasets included 935 and 1750 patients, respectively. We developed a model with 20 clinical variables consisting of two stages: the first stage which recommends initial treatment using an ensemble voting machine, and the second stage, which predicts post-treatment survival using a random survival forest algorithm. We derived the first and second treatment options from the results with the highest and the second-highest probabilities given by the ensemble model and predicted their post-treatment survival. When only the first treatment option was accepted, the mean accuracy of treatment recommendation in the internal and external datasets was 67.27% and 55.34%, respectively. The accuracy increased to 87.27% and 86.06%, respectively, when the second option was included as the correct answer. Harrell's C index, integrated time-dependent AUC curve, and integrated Brier score of survival prediction in the internal and external datasets were 0.8381 and 0.7767, 91.89 and 86.48, 0.12, and 0.14, respectively. The proposed system can assist physicians by providing data-driven predictions for reference from other larger institutions or other physicians within the same institution when making treatment decisions.

Time-sequential fibroblast-to-myofibroblast transition in elastin-variable 3D hydrogel environments by collagen networks.

BACKGROUND: Fibrosis plays an important role in both normal physiological and pathological phenomena as fibroblasts differentiate to myofibroblasts. The activation of fibroblasts is determined through interactions with the surrounding extracellular matrix (ECM). However, how this fibroblast-to-myofibroblast transition (FMT) is regulated and affected by elastin concentration in a three-dimensional (3D) microenvironment has not been investigated. METHODS: We developed an insoluble elastin-gradient 3D hydrogel system for long-lasting cell culture and studied the molecular mechanisms of the FMT in embedded cells by nanoflow LC-MS/MS analysis along with validation through real-time PCR and immunofluorescence staining. RESULTS: By optimizing pH and temperature, four 3D hydrogels containing fibroblasts were successfully fabricated having elastin concentrations of 0, 20, 50, and 80% in collagen. At the low elastin level (20%), fibroblast proliferation was significantly increased compared to others, and in particular, the FMT was clearly observed in this condition. Moreover, through mass spectrometry of the hydrogel environment, it was confirmed that differentiation proceeded in two stages. In the early stage, calcium-dependent proteins including calmodulin and S100A4 were highly associated. On the other hand, in the late stage after several passages of cells, distinct markers of myofibroblasts were presented such as morphological changes, increased production of ECM, and increased α-SMA expression. We also demonstrated that the low level of elastin concentration induced some cancer-associated fibroblast (CAF) markers, including PDGFR-ß, and fibrosis-related disease markers, including THY-1. CONCLUSION: Using our developed 3D elastin-gradient hydrogel system, we evaluated the effect of different elastin concentrations on the FMT. The FMT was induced even at a low concentration of elastin with increasing CAF level via calcium signaling. With this system, we were able to analyze varying protein expressions in the overall FMT process over several cellular passages. Our results suggest that the elastin-gradient system employing nonlinear optics imaging provides a good platform to study activated fibroblasts interacting with the microenvironment, where the ECM plays a pivotal role.

Particulate Matter Promotes Melanin Production through Endoplasmic Reticulum Stress‒Mediated IRE1α Signaling.

Particulate matter (PM) is believed to be related to cardiovascular and respiratory diseases. The skin is also known to be affected by PM exposure as a result of skin barrier dysfunction, cutaneous inflammation, and apoptotic cell death. Epidemiological studies have suggested that PM is related to pigment spots. Recently, diesel exhaust particles are reported to cause a tanning response mediated by oxidative stress. However, the direct effects of PM on melanogenesis and the related mechanisms have not yet been clarified. Our study showed that PM can increase melanin production in melanocyte, mouse skin, and human skin models. RNA-sequencing analyses of melanocytes revealed that the expressions of unfolded protein response molecules were increased after PM exposure. In particular, IRE1α signaling pathway, which was consistently upregulated, was related to PM-triggered melanogenesis. In addition, PM-induced melanogenesis was abrogated by an IRE1α inhibitor. Therefore, our findings corroborate previous findings in melanocytes and in mouse and human models and also illuminate the involvement of the IRE1α pathway as a mechanism of PM-induced melanogenesis.

Molecule-Resolved Visualization of Particulate Matter on Human Skin Using Multimodal Nonlinear Optical Imaging.

Precise measurement of particulate matter (PM) on skin is important for managing and preventing PM-related skin diseases. This study aims to directly visualize the deposition and penetration of PM into human skin using a multimodal nonlinear optical (MNLO) imaging system. We successfully obtained PM particle signals by merging two different sources, C-C vibrational frequency and autofluorescence, while simultaneously visualizing the anatomical features of the skin via keratin, collagen, and elastin. As a result, we found morphologically dependent PM deposition, as well as increased deposition following disruption of the skin barrier via tape-stripping. Furthermore, PM penetrated more and deeper into the skin with an increase in the number of tape-strippings, causing a significant increase in the secretion of pro-inflammatory cytokines. Our results suggest that MNLO imaging could be a useful technique for visualizing and quantifying the spatial distribution of PM in ex vivo human skin tissues.

Early Normalization of Alanine Aminotransferase during Antiviral Therapy Reduces Risk of Hepatocellular Carcinoma in HBV Patients.

Potent antiviral agents effectively reduce liver-related events in patients with chronic hepatitis B. This study aimed to determine whether alanine aminotransferase normalization using potent antiviral agents was related to hepatocellular carcinoma development. From 2007 to 2017, we included 610 patients with chronic hepatitis B who received entecavir or tenofovir disoproxil fumarate. The patients were divided into the alanine aminotransferase normalization group (Gr.1) and non-normalization group (Gr.2) within a year of potent antiviral treatment. Liver-related events included hepatic encephalopathy, variceal bleeding, and ascites. The mortality rate and hepatocellular carcinoma incidence were investigated for each group. The patients who showed ALT normalization at 1 year of treatment were 397 (65.1%) of 610. During a median follow-up period of 86 months, 65 (10.7%) patients developed hepatocellular carcinoma. The cumulative incidence of hepatocellular carcinoma was significantly lower in Gr.1 than in Gr.2 (p < 0.001). Risk factors for alanine aminotransferase non-normalization were body mass index, cholesterol, and liver cirrhosis at baseline. Male sex, age, platelet level, alcohol use, presence of cirrhosis at baseline, and non-normalization after 1 year of treatment were independent risk factors for hepatocellular carcinoma. Alanine aminotransferase normalization within 1 year of initiating antiviral agents reduces the risk of hepatocellular carcinoma development.

Length difference of multi-walled carbon nanotubes generates differential cytotoxic responses.

Carbon nanotubes have recently been rated as an effective biomaterial owing to their functionalization ability. However, the safety of multi-walled carbon nanotubes (MWCNTs) has yet to be clearly understood. To investigate how cells differentially react to minor geometric differences, we prepared well-dispersed and stable long and short MWCNTs showing an approximately 100-nm length difference in an in vitro system. Through an optimal combination of bovine serum albumin (BSA) and fetal bovine serum (FBS) biosurfactants and ultrasonication, we first confirmed that the MWCNTs were maintained without aggregation throughout the experiments. Internalized MWCNTs in human coronary artery smooth muscle cells were then quantified in a label-free manner using coherent anti-Stokes Raman scattering, followed by an analysis of their localization via two-photon excitation fluorescence. Intracellular MWCNTs were found to primarily localize in mitochondria with abnormal morphologies. Mitochondrial dysfunction, which was found to result from early stages of oxidative stress that consequently lead to cell death, was then proved via decreasing mitochondrial membrane potentials, with short MWCNTs showing significantly greater cytotoxicity than long MWCNTs. Our results suggest that even small length differences of MWCNTs may lead to differential responses in cells.

Role of tenofovir disoproxil fumarate in prevention of perinatal transmission of hepatitis B virus from mother to child: a systematic review and meta-analysis.

BACKGROUND/AIMS: To prevent the perinatal transmission of hepatitis B virus (HBV) from mother to child, administration of an antiviral agent during pregnancy has been attempted in women who are either hepatitis B e antigen positive or have a high viral load. In this systematic review and meta-analysis with randomized controlled trials, we analyzed the efficacy and safety of tenofovir disoproxil fumarate (TDF) in preventing the perinatal transmission of HBV in pregnant women who have high HBV DNA titers. METHODS: Multiple comprehensive databases (PubMed, EMBASE, and Cochrane databases) were searched for studies evaluating the efficacy of TDF for the prevention of perinatal transmission of HBV. RESULTS: Two studies (one open label study and one double blind study) were included and analyzed. Intention-to-treat analysis (527 pregnancies) showed that the preventive effect of TDF was not significant (odds ratio [OR], 0.53; 95% confidence interval [CI], 0.13 to 2.17; p = 0.38, I2 = 81%). However, the per-protocol analysis showed that TDF significantly reduced perinatal transmission (OR, 0.10; 95% CI, 0.01 to 0.77; p = 0.03, I2 = 0%). There was no significant difference between the TDF group and the control group with respect to maternal and fetal safety outcomes. CONCLUSION: In pregnant women who have high HBV DNA titers, TDF can reduce the perinatal transmission from mother to child without significant adverse events.

Synthetic Retinoid Seletinoid G Improves Skin Barrier Function through Wound Healing and Collagen Realignment in Human Skin Equivalents.

The outer epidermal skin is a primary barrier that protects the body from extrinsic factors, such as ultraviolet (UV) radiation, chemicals and pollutants. The complete epithelialization of a wound by keratinocytes is essential for restoring the barrier function of the skin. However, age-related alterations predispose the elderly to impaired wound healing. Therefore, wound-healing efficacy could be also considered as a potent function of an anti-aging reagent. Here, we examine the epidermal wound-healing efficacy of the fourth-generation retinoid, seletinoid G, using HaCaT keratinocytes and skin tissues. We found that seletinoid G promoted the proliferation and migration of keratinocytes in scratch assays and time-lapse imaging. It also increased the gene expression levels of several keratinocyte proliferation-regulating factors. In human skin equivalents, seletinoid G accelerated epidermal wound closure, as assessed using optical coherence tomography (OCT) imaging. Moreover, second harmonic generation (SHG) imaging revealed that seletinoid G recovered the reduced dermal collagen deposition seen in ultraviolet B (UVB)-irradiated human skin equivalents. Taken together, these results indicate that seletinoid G protects the skin barrier by accelerating wound healing in the epidermis and by repairing collagen deficiency in the dermis. Thus, seletinoid G could be a potent anti-aging agent for protecting the skin barrier.

Site-specific impairment of perivascular adipose tissue on advanced atherosclerotic plaques using multimodal nonlinear optical imaging.

Perivascular adipose tissue (PVAT), as a mechanical support, has been reported to systemically regulate vascular physiology by secreting adipokines and cytokines. How PVAT spatially and locally changes as atherosclerosis progresses is not known, however. We aimed to reveal the molecular changes in PVAT in advanced atherosclerosis based on multimodal nonlinear optical (MNLO) imaging. First, using an atherogenic apolipoprotein E knockout mouse model, we precisely assessed the browning level of thoracic PVAT via a correlative analysis between the size and number of lipid droplets (LDs) of label-free MNLO images. We also biochemically demonstrated the increased level of brown fat markers in the PVAT of atherosclerosis. In the initial stage of atherosclerosis, the PVAT showed a highly activated brown fat feature due to the increased energy expenditure; however, in the advanced stage, only the PVAT in the regions of the atherosclerotic plaques, not that in the nonplaque regions, showed site-specific changes. We found that p-smad2/3 and TGF-ß signaling enhanced the increase in collagen to penetrate the PVAT and the agglomeration of LDs only at the sites of atherosclerotic plaques. Moreover, atherosclerotic thoracic PVAT (tPVAT) was an increased inflammatory response. Taken together, our findings show that PVAT changes differentially from the initial stages to advanced stages of atherosclerosis and undergoes spatial impairment focused on atherosclerotic plaques. Our study may provide insight into the local control of PVAT as a therapeutic target.

Evidence that 6q25.1 variant rs6931104 confers susceptibility to chronic myeloid leukemia through RMND1 regulation.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder. Our previous study reported novel loci as genetic markers associated with increased susceptibility to CML. The present study conducted an expression quantitative trait loci (eQTL) analysis to confirm that the single nucleotide polymorphisms (SNPs) at these loci affect the expression of candidate CML-susceptible genes. We identified that three SNPs (rs963193, rs6931104, and rs9371517) were related to the gene expression pattern of RMND1 (Required For Meiotic Nuclear Division 1 Homolog) in both granulocytes and mononuclear cells from 83 healthy donors. Furthermore, reduced expression of RMND1 expression was noted in CML patients compared with that in healthy individuals. We used the eQTL browsing tool to assess the regulatory information on the three associated significant SNPs, out of which rs6931104 showed strong evidence of regulatory effects. Chromatin immunoprecipitation (ChIP) assays demonstrated that A alleles of rs6931104 could significantly change the binding affinity of transcription factor (TF) RFX3 compared to the G alleles. Then, we performed in vitro experiments on BCR-ABL1-positive (BCR-ABL1+) cell lines. We found that expression of the CML-susceptible gene RMND1 is affected by the binding affinity of TF RFX3, suggesting that RFX3 plays a role in RMND1 expression. Our findings suggest potential target genes for associations of genetic susceptibility risk loci and provide further insights into the pathogenesis and mechanism of CML.

Dehydroabietic Acid Induces Regeneration of Collagen Fibers in Ultraviolet B-Irradiated Human Dermal Fibroblasts and Skin Equivalents.

BACKGROUND/AIMS: Dehydroabietic acid (DAA) is a natural phytochemical found in red pine trees and herbal plants. While DAA and its derivatives are known for improving diabetes and hyperlipidemia, the antiaging effect and its underlying mechanisms of DAA on skin have not been fully examined. Here, we assessed the antiaging effects of DAA on human dermal fibroblasts and skin equivalents. METHODS: We investigated the effect of DAA on the secretion of type I procollagen and matrix metalloproteinase-1 (MMP-1) in ultraviolet B (UVB)-irradiated neonatal normal human dermal fibroblasts (NHDFn). Using nonlinear optical imaging techniques, we visualized quantitative and qualitative changes of collagen fibers by DAA treatment in human skin equivalent models. RESULTS: DAA induces increases in type I procollagen secretion when treated on UVB-irradiated NHDFn. DAA also downregulates secretion of MMP-1 through the inhibition of the JNK signaling pathway. In human skin equivalent models, we successfully visualized the spatial distribution of collagen fibers in the dermis and found that quantity, diameter, and arrangement of collagen fibers in the dermis were significantly improved by DAA treatment. CONCLUSION: Our results suggest that DAA could be a useful agent for improving skin photoaging through the protection and regeneration of collagen fibers in skin.

Realization of an ultrathin acoustic lens for subwavelength focusing in the megasonic range.

In this study, we report the first experimental realization of an ultrathin (0.14λ, λ = 1.482 mm means wavelength at 1 MHz in the water medium) subwavelength focusing acoustic lens that can surpass the Rayleigh diffraction limit (0.61λ/NA, NA means numerical aperture). It is termed a Super-Oscillatory Acoustic Lens (SOAL), and it operates in the megasonic range. The SOAL represents an interesting feature allowing the achievement of subwavelength focusing without the need to operate in close proximity to the object to be imaged. The optimal layout of the SOAL is obtained by utilizing a systematic design approach, referred to here as topology optimization. To this end, the optimization formulation is newly defined. The optimized SOAL is fabricated using a photo-etching process and its subwavelength focusing performance is verified experimentally via an acoustic intensity measurement system. From these measurements, we found that the proposed optimized SOAL can achieve superior focusing features with a Full Width at Half Maximum (FWHM) of ~0.40λ/NA ≃ 0.84 mm (for our SOAL, NA = 0.707) with the transmission efficiency of 26.5%.

Simultaneous Evaluation of Thermal and Non-Thermal Effects of High-Intensity Focused Ultrasound on a Tissue-Mimicking Phantom.

Physiologically relevant phantoms with high reliability are essential for extending the therapeutic applications of high-intensity therapeutic ultrasound. Here we describe a tissue-mimicking phantom capable of quantifying temperature changes and observing non-thermal phenomena by high-intensity therapeutic ultrasound. Using polydiacetylene liposomes, we fabricated agar-based polydiacetylene hydrogel phantoms (PHPs) that not only respond to temperature, but also have acoustic properties similar to those of human liver tissue. The color of PHPs changed from blue to red depending on the temperature in the range 40°C-70°C, where the red/blue ratio of PHP had a good linearity of 99.06% for the temperature changes. Furthermore, repeated high-intensity focused ultrasound led to histotripsy on the PHP with liquefied and damaged areas measuring 0.7 and 4.0 cm2, respectively, at the signal generator amplitude setting voltage of 80 mV. Our results indicate not only the usability of the thermochromic phantom, but also its potential for evaluating non-thermal phenomena in various high-intensity focused ultrasound therapies.

Characterization and application of porous gold nanoparticles as 2-photon luminescence imaging agents: 20-fold brighter than gold nanorods.

Two-photon nonlinear microscopy with the aid of plasmonic contrast agents is an attractive bioimaging technique capable of generating high-resolution images in 3 dimensions and facilitating targeted imaging with deep tissue penetration. In this work, physically synthesized gold nanoparticles containing multiple nanopores are used as 2-photon contrast agents and are reported to emit a 20-fold brighter 2-photon luminescence as compared to typical contrast agents, that is, gold nanorods. A successful application of our porous gold nanoparticles is experimentally demonstrated by in vitro nonlinear optical imaging of adipocytes at subcellular level.

Thiophene bridged aldehydes (TBAs) image ALDH activity in cells via modulation of intramolecular charge transfer.

Aldehyde dehydrogenases (ALDHs) catalyze the oxidation of an aldehyde to a carboxylic acid and are implicated in the etiology of numerous diseases. However, despite their importance, imaging ALDH activity in cells is challenging due to a lack of fluorescent imaging probes. In this report, we present a new family of fluorescent probes composed of an oligothiophene flanked by an aldehyde and an electron donor, termed thiophene-bridged aldehydes (TBAs), which can image ALDH activity in cells. The TBAs image ALDH activity via a fluorescence sensing mechanism based on the modulation of intramolecular charge transfer (ICT) and this enables the TBAs and their ALDH-mediated oxidized products, thiophene-bridged carboxylates (TBCs), to have distinguishable fluorescence spectra. Herein, we show that the TBAs can image ALDH activity in cells via fluorescence microscopy, flow cytometry, and in a plate reader. Using TBA we were able to develop a cell-based high throughput assay for ALDH inhibitors, for the first time, and screened a large, 1460-entry electrophile library against A549 cells. We identified α,ß-substituted acrylamides as potent electrophile fragments that can inhibit ALDH activity in cells. These inhibitors sensitized drug-resistant glioblastoma cells to the FDA approved anti-cancer drug, temozolomide. The TBAs have the potential to make the analysis of ALDH activity in cells routinely possible given their ability to spectrally distinguish between an aldehyde and a carboxylic acid.

Antimelanogenic Efficacy of Melasolv (3,4,5-Trimethoxycinnamate Thymol Ester) in Melanocytes and Three-Dimensional Human Skin Equivalent.

BACKGROUND/AIMS: Excessive melanogenesis often causes unaesthetic hyperpigmentation. In a previous report, our group introduced a newly synthesized depigmentary agent, Melasolv™ (3,4,5-trimethoxycinnamate thymol ester). In this study, we demonstrated the significant whitening efficacy of Melasolv using various melanocytes and human skin equivalents as in vitro experimental systems. METHODS: The depigmentary effect of Melasolv was tested in melan-a cells (immortalized normal murine melanocytes), α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 murine melanoma cells, primary normal human melanocytes (NHMs), and human skin equivalent (MelanoDerm). The whitening efficacy of Melasolv was further demonstrated by photography, time-lapse microscopy, Fontana-Masson (F&M) staining, and 2-photon microscopy. RESULTS: Melasolv significantly inhibited melanogenesis in the melan-a and α-MSH-stimulated B16 cells. In human systems, Melasolv also clearly showed a whitening effect in NHMs and human skin equivalent, reflecting a decrease in melanin content. F&M staining and 2-photon microscopy revealed that Melasolv suppressed melanin transfer into multiple epidermal layers from melanocytes as well as melanin synthesis in human skin equivalent. CONCLUSION: Our study showed that Melasolv clearly exerts a whitening effect on various melanocytes and human skin equivalent. These results suggest the possibility that Melasolv can be used as a depigmentary agent to treat pigmentary disorders as well as an active ingredient in cosmetics to increase whitening efficacy.

Fabrication of size-controlled linoleic acid particles and evaluation of their in-vitro lipotoxicity.

The biological activities of fatty acids (FAs) can differ with size even for lipids of similar compositions. The aim of this study was to develop size-controlled FA particles and to evaluate their toxicity as a function of size. Well-stabilized nano- and microscale linoleic acid (LA) were fabricated based on specific physical factors. Then, resulting LAs were characterized by size distribution, surface charge, assembly structure, composition, and serum effects. The sizes of the nano- (LAnano) and microscale (LAmicro) LAs, determined by electron microscopy, were 109 nm and 12 µm, respectively. LAnano, a multilamellar structure as determined by cryo-electron microscopy, was rapidly internalized into cells via free fatty acid receptor 3. After internalization, LAnano, but not LAmicro, induced nuclear translocation of fatty acid binding protein 4 (FABP4). Translocation of FABP4 into the nucleus then induced expression of the FA metabolism-related genes InsR and AdipoR1. Their expression was significantly increased in the presence of only LAnano. Cytotoxicity was also significantly increased in cells treated with LAnano, but not LAmicro, as indicated by the endoplasmic reticulum stress markers CHOP and GRP78. Therefore, our results demonstrated that FAs with the same composition but varying in size can cause different cellular responses.

Severe Endobronchial Inflammation Induced by Aspiration of a Ferrous Sulfate Tablet.

Iron supplements such as ferrous sulfate tablets are usually used to treat iron-deficiency anemia in some elderly patients with primary neurologic disorders or decreased gag reflexes due to stroke, senile dementia, or parkinsonism. While the aspiration of ferrous sulfate is rarely reported, it is a potentially life-threatening condition that can lead to airway necrosis and bronchial stenosis. A detailed history and high suspicion of aspiration are required to avoid delays in diagnosis and treatment. The diagnosis can be confirmed by bronchoscopic examination and a tissue biopsy. Early removal of the aspirated tablet prevents acute complications, such as bronchial necrosis, hemoptysis, and lobar consolidation. Tablet removal is also necessary to prevent late bronchial stenosis. We presented the first case in Korea of a ferrous sulfate tablet aspiration that induced severe endobronchial inflammation.

Real-time GPU-accelerated processing and volumetric display for wide-field laser-scanning optical-resolution photoacoustic microscopy.

Fast signal processing and real-time displays are essential for practical imaging modality in various fields of applications. However, the imaging speed in optical-resolution photoacoustic microscopy (OR-PAM), in particular, depends on factors such as the pulse repetition rate of the laser, scanning method, field of view (FOV), and signal processing time. In the past, efforts to increase acquisition speed either focused on developing new scanning methods or using lasers with higher pulse repetition rates. However, high-speed signal processing is also important for real-time volumetric display in OR-PAM. In this study, we carried out parallel signal processing using a graphics processing unit (GPU) to enable fast signal processing and wide-field real-time displays in laser-scanning OR-PAM. The average total GPU processing time for a B-mode PAM image was approximately 1.35 ms at a display speed of 480 fps when the data samples were acquired with 736 (axial) × 500 (lateral) points/B-mode-frame at a pulse repetition rate of 300 kHz. In addition, we successfully displayed maximum amplitude projection images of a mouse's ear as volumetric images with an FOV of 3 mm × 3 mm (500 × 500 pixels) at 1.02 s, corresponding to 0.98 fps.