Integrating sheath and radiation-based acceleration using scaling coefficients for tailoring radiation dominant hybrid acceleration.

An optimal target condition for generating GeV-energy ions with linearly polarized laser pulse is revealed by a hybrid acceleration theory based on the fractional contributions of the target normal sheath acceleration (TNSA) and the radiation pressure acceleration (RPA) mechanisms in the RPA-dominant regime. The theory is established with two scaling coefficients, which scale the TNSA and RPA velocities, and are sophisticated through two-dimensional particle-in-cell simulations where GeV-energy ions are obtained by RPA-dominant hybrid acceleration. By imposing limits on the scaling coefficients, three separate acceleration regions are obtained including a RPA-dominant acceleration region, which is optimal to generate GeV-energy ions. The past experiment/simulation results are in good agreement with the acceleration regions obtained. This RPA-dominant region is narrower than previously reported, and this region becomes even narrower with increasing material density.

Oral infection with Porphyromonas gingivalis augmented gingival epithelial barrier molecules alteration with aging.

OBJECTIVE: Disruption of the gingival epithelial barrier is often mediated by aging or the pathogen Porphyromonas gingivalis. This study examined the combined effects of aging and P. gingivalis exposure on gingival epithelial barrier molecules. METHODS: In vitro experiments involved treating young- and senescence-induced primary human gingival epithelial progenitor cells (HGEPp) with P. gingivalis lipopolysaccharide (LPS). Transepithelial electrical resistance (TER) and paracellular permeability were measured. In vivo, male C57BL/6J mice aged 10 (young) and 80 (old) weeks were divided into four groups: young, old, young with P. gingivalis (Pg-Young) inoculation, and old with P. gingivalis (Pg-Old) inoculation. P. gingivalis was inoculated orally thrice a week for 5 weeks. The mice were sacrificed 30 days after the last inoculation, and samples were collected for further procedures. The junctional molecules (Claudin-1, Claudin-2, E-cadherin, and Connexin) were analyzed for mRNA expression using qRT-PCR and protein production using western blotting and immunohistochemistry. The alveolar bone loss and inflammatory cytokine levels in gingival tissues were also assessed. RESULTS: LPS-treated senescent cells exhibited a pronounced reduction in TER, increased permeability to albumin protein, significant upregulation of Claudin-1 and Claudin-2, and significant downregulation of E-cadherin and Connexin. Furthermore, the Pg-Old group showed identical results with aging in addition to an increase in alveolar bone loss, significantly higher than that in the other groups. CONCLUSION: In conclusion, the host susceptibility to periodontal pathogens increases with age through changes in the gingival epithelial barrier molecules.

Transcriptomic analysis of the submandibular gland under psychological stress condition.

BACKGROUND: Psychological stress is associated with changes in salivary flow and composition. However, studies to show the effect of psychological stress on the transcriptome of the salivary gland are limited. This study aims to perform a transcriptomic analysis of the submandibular gland under psychological stress using a chronic restraint stress model of rats. METHODS: Sprague-Dawley rats were divided into stress groups and control groups. Psychological stress was induced in the stress group rats by enclosing them in a plastic tube for 4 h daily over 6 weeks. RNA sequencing was performed on RNA extracted from the submandibular gland. The differentially expressed genes were identified, and the genes of interest were further validated using qRT-PCR, immunofluorescence, and western blot. RESULTS: A comparison between control and stress groups showed 45 differentially expressed genes. The top five altered genes in RNA sequencing data showed similar gene expression in qRT-PCR validation. The most downregulated gene in the stress group, FosB, was a gene of interest and was further validated for its protein-level expression using immunofluorescence and western blot. The genesets for gene ontology cellular component, molecular function, and KEGG showed that pathways related to ribosome biosynthesis and function were downregulated in the stress group compared to the control. CONCLUSION: Psychological stress showed transcriptomic alteration in the submandibular gland. The findings may be important in understanding stress-related oral diseases.

Down-regulating Effect of a Standardized Extract of Cultured Lentinula edodes mycelia on Cortactin in Prostate Cancer Cells Is Dependent on Malignant Potential.

BACKGROUND/AIM: The incidence and mortality rates of prostate cancer have been increasing worldwide. Although prostate cancer cells grow slowly in the local original site, once the cancer cells spread to distant organs they grow rapidly and show very aggressive features. Cortactin is a protein that regulates the actin cytoskeleton and plays crucial roles in cancer metastasis. Up-regulated cortactin is correlated with the metastatic capacity of prostate cancer cells. AHCC®, a standardized extract of cultured Lentinula edodes mycelia, has been previously reported to have cortactin-down-regulating effects on human pancreatic cancer cells. In the present study, the effects of AHCC® treatment on cortactin levels in prostate cancer cells was evaluated. MATERIALS AND METHODS: LNCaP.FGC, DU145, and PC-3 are human prostate cancer cell lines. LNCaP.FGC is well differentiated, androgen-dependent, and poorly metastatic. DU145 is less differentiated, androgen-independent, and moderate metastatic. PC-3 is less differentiated, androgen-independent, and highly metastatic. The effects of AHCC® treatment on cortactin levels in prostate cancer cells was evaluated by western blot. RESULTS: In vitro AHCC® treatment decreased cortactin levels in LNCaP.FGC and DU145 cells but did not change those in PC-3 cells. CONCLUSION: AHCC® treatment down-regulated cortactin expression in poor and moderate metastatic LNCaP.FGC and DU145 cells but showed no effect on cortactin expression in the highly metastatic PC-3 cells. Further studies are required to elucidate the mechanism of the resistance to AHCC® treatment in highly metastatic PC-3 cells.

A Standardized Extract of Cultured Lentinula edodes mycelia Up-regulates COX-2 in Inflammation-related Malignant Progressive Fibrosarcoma Cell Clone QRsP-11.

BACKGROUND/AIM: Cyclooxygenase is an enzyme that transforms arachidonic acid to prostaglandins. Cyclooxygenase-2 (COX-2) is an isoform of cyclooxygenase. There exist many reports on the expression levels of COX-2 in cancer tissues, and prognosis of cancer patients has been reported to be related to COX-2 up-regulation. In the present study we assessed the suppressive effect of AHCC® on the expression of COX-2 in QRsP-11cells. MATERIALS AND METHODS: QR-32 is a clone which was derived from murine fibrosarcoma BMT-11 cells by treatment with quercetin. These clone cells regress spontaneously after injection into C57BL/6 mice. QRsP-11 is a clone derived from QR-32, showing very aggressive tumorigenicity. AHCC® is a standardized extract of cultured Lentinula edodes mycelia and has been reported to exert suppressive effects on various tumor-associated proteins including HSP27. The protein levels of COX-2 in QR-32 and QRsP-11 cells were compared by using western blotting. Furthermore, the expression levels of COX-2 were assessed in QRsP-11 cells after AHCC®-treatment. RESULTS: Western blot analysis showed a significant up-regulation of COX-2 in QRsP-11 cells compared to QR-32 cells. In vitro AHCC®-treatment increased COX-2 expression levels in QRsP-11 cells contrary to expectations. CONCLUSION: When using AHCC® in cancer treatment, it might be important to decrease COX-2 expression by means of non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin. Further studies are required to clarify the mechanism of up-regulation of COX-2 through AHCC®-treatment.

Systemic Administration of Lipopolysaccharide from Porphyromonas gingivalis Decreases Neprilysin Expression in the Mouse Hippocampus.

BACKGROUND/AIM: Alzheimer's disease is the most common type of neurodegenerative disorder in elderly individuals worldwide. Increasing evidence suggests that periodontal diseases are involved in the pathogenesis of Alzheimer's disease, and an association between periodontitis and amyloid-ß deposition in elderly individuals has been demonstrated. The aim of the present study was to examine the effects of systemic administration of Porphyromonas gingivalis-derived lipopolysaccharide (PG-LPS) on neprilysin expression in the hippocampus of adult and senescence-accelerated mice. MATERIALS AND METHODS: PG-LPS diluted in saline was intraperitoneally administered to male C57BL/6J and senescence-accelerated mouse prone 8 (SAMP8) mice at a dose of 5 mg/kg every 3 days for 3 months. Both C57BL/6J and SAMP8 mice administered saline without PG-LPS comprised the control group. The mRNA expression levels of neprilysin and interleukin (IL)-10 were evaluated using the quantitative reverse transcriptase-polymerase chain reaction. The protein levels of neprilysin were assessed using western blotting. Sections of the brain tissues were immunohistochemically stained. RESULTS: The serum IL-10 concentration significantly increased in both mouse strains after stimulation with PG-LPS. Neprilysin expression at both mRNA and protein levels was significantly lower in the SAMP8 PG-LPS group than those in the SAMP8 control group; however, they did not differ in PG-LPS-treated or non-treated C57BL/6J mice. Additionally, the immunofluorescence intensity of neprilysin in the CA3 region of the hippocampus in PG-LPS-treated SAMP8 mice was significantly lower than that in control SAMP8 mice. CONCLUSION: Porphyromonas gingivalis may reduce the expression of neprilysin in elderly individuals and thus increase amyloid-ß deposition.

Effect of psychological stress on the oral-gut microbiota and the potential oral-gut-brain axis.

Psychological stress in a chronic course is implicated in various diseases, such as coronary artery disease, diabetes, ulcerative colitis, and psychosomatic pain disorders. Commensal microbiota in the host tissues interact with each other and maintain overall health. Oral and gut microbiomes are considered as the most ecologically rich and taxonomically diverse microbiota communities in humans. The effects of psychological stress on the gut microbiome have been well documented, and the interaction is commonly referred as the microbiota-gut-brain axis. Like the gut microbiome, the oral microbiome contributes to maintaining both local and systemic health. Although the effects of psychological stress on the oral microbiome have been studied, comprehensive knowledge about the oral-brain axis is lacking. The oral cavity and gut can communicate with each other through the microbiota. Three-way interactions within the oral-gut-brain microbiota might exist in patients with psychological stress and disorders. The effect of psychological stress on the gut and oral microbiomes, and the potential interactions within the oral-gut-brain axis are discussed in this review.

AT-hook DNA-binding motif-containing protein one knockdown downregulates EWS-FLI1 transcriptional activity in Ewing's sarcoma cells.

Ewing's sarcoma is the second most common bone malignancy in children or young adults and is caused by an oncogenic transcription factor by a chromosomal translocation between the EWSR1 gene and the ETS transcription factor family. However, the transcriptional mechanism of EWS-ETS fusion proteins is still unclear. To identify the transcriptional complexes of EWS-ETS fusion transcription factors, we applied a proximal labeling system called BioID in Ewing's sarcoma cells. We identified AHDC1 as a proximal protein of EWS-ETS fusion proteins. AHDC1 knockdown showed a reduced cell growth and transcriptional activity of EWS-FLI1. AHDC1 knockdown also reduced BRD4 and BRG1 protein levels, both known as interacting proteins of EWS-FLI1. Our results suggest that AHDC1 supports cell growth through EWS-FLI1.

Proteomic and microbiota analyses of the oral cavity during psychological stress.

Psychological stress is associated with various oral diseases such as aphthous stomatitis, oral lichen planus, taste disturbances and glossodynia. However, the underlying mechanism is still unknown. The aim of this study was to determine the effect of psychological stress on salivary proteins and the oral microbiota in a rat model of chronic restraint stress. Six-week-old Sprague Dawley rats were subjected to restraint stress for four hours daily for 1 month. The behavior, weights of the adrenal glands, and serum corticosterone levels were evaluated as stress markers. Proteomic analysis of the saliva was performed using two-dimensional gel electrophoresis followed by mass spectrometry and Western blotting. Analysis of the oral microbiota was performed via 16S rRNA next-generation sequencing. The low mean body weights, lower number of entries and time spent in the open arm of elevated plus maze, high adrenal gland/body weight ratios, and high serum corticosterone levels confirmed the high levels of stress in the stress group of rats compared to the controls. Thirty-three protein spots were found to be significantly altered between the two groups. After silver staining, seven visible spots were subjected for mass spectrometry, and the expression levels of the two most significantly altered proteins, BPI fold containing family A member 2 and von Ebner's gland protein, were confirmed by Western blotting. 16S rRNA sequencing analysis revealed a significant reduction in alpha diversity in the stress group compared to the controls. The abundances of oral bacteria, such as Facklamia and Corynebacterium, were significantly altered between the two groups. Additionally, analysis with PICRUSt2 software predicted 37 different functional pathways to be altered between the groups. In conclusion, the present study identified altered salivary proteins and oral microbiota due to psychological stress. These findings might aid in understanding the pathogenesis of stress-related oral diseases.

An in vitro senescence model of gingival epithelial cell induced by hydrogen peroxide treatment.

Gingival tissue shows progressive changes with aging and an in vitro model of gingival tissue could be useful in understanding age-associated oral diseases. The present study aims to establish a hydrogen peroxide (H2O2) treatment model to induce aging in human gingival epithelial cells. In addition, fisetin, a flavonoid component studied for the anti-aging property is used to examine if it could reverse the induced senescence. Primary human gingival epithelial progenitor (HGEPp) cells were cultured and treated with different concentrations of H2O2. A cell vitality and morphology, senescence-associated beta-galactosidase (SA-ß-gal) staining, mRNA and protein expression analysis of known senescence markers p16, p21, and p53, and cell cycle assay were performed. The cells showed dose-dependent changes in vitality and morphology, SA-ß-gal staining, relative mRNA and protein expression, and cell cycle assay after H2O2 treatment. Based on these results, 400 µM H2O2 was considered as an optimal concentration to induce senescence. Treatment of senescence-induced cells with fisetin downregulated all the senescence markers used in this study. In conclusion, a senescence model of gingival epithelial cells induced by hydrogen peroxide treatment was established which could be employed to study age-related periodontal diseases.

A Marine Carotenoid of Fucoxanthinol Accelerates the Growth of Human Pancreatic Cancer PANC-1 Cells.

Fucoxanthin and its metabolite fucoxanthinol (FxOH), highly polar xanthophylls, exert strong anticancer effects against many cancer cell types. However, the effects of Fx and FxOH on pancreatic cancer, a high mortality cancer, remain unclear. We herein investigated whether FxOH induces apoptosis in human pancreatic cancer cells. FxOH (5.0 µmol/L) significantly promoted the growth of human pancreatic cancer PANC-1 cells, but induced apoptosis in human colorectal cancer DLD-1 cells. A microarray-based gene analysis revealed that the gene sets of cell cycle, adhesion, PI3K/AKT, MAPK, NRF2, adipogenesis, TGF-ß, STAT, and Wnt signals in PANC-1 cells were markedly altered by FxOH. A western blot analysis showed that FxOH up-regulated the expression of integrin ß1 and PPARγ as well as the activation of pFAK(Tyr397), pPaxillin(Tyr31), and pAKT(Ser473) in PANC-1 cells, but exerted the opposite effects in DLD-1 cells. Moreover, the expression of FYN, a downstream target of integrin subunits, was up-regulated (7.4-fold by qPCR) in FxOH-treated PANC-1 cells. These results suggest that FxOH accelerates the growth of PANC-1 cells by up-regulating the expression of integrin ß1, FAK, Paxillin, FYN, AKT, and PPARγ.

Exploring the mechanical properties of nanometer-thick elastic films through micro-drop impinging on large-area suspended graphene.

In this work, the dependence of effective Young's modulus on the thickness of suspended graphene was confirmed through a drop impingement method. Large area suspended graphene (LSG) layers with a diameter of up to 400 µm and a nanometer thickness were prepared through transferring chemical vapor deposition grown graphene from copper substrates. 4, 8, and 12-layer LSG samples were found to be crumpled yet defect-free. The mechanical properties of LSG were first studied by observing its interaction with impinging droplets from an ink-jet nozzle. First, the effective Young's modulus was calculated by fitting the instant deformation captured by high speed photography within microseconds. Next, droplets deposited on LSG caused deformation and generated wrinkles and the effective Young's modulus was calculated from the number of wrinkles. The above methods yielded effective Young's modulus values ranging from 0.3 to 3.4 TPa. The results from these methods all indicated that the effective Young's modulus increases with the decreasing thickness or size of suspended graphene layers. Moreover, the crumpled LSG yields higher effective Young's modulus than ideal flat graphene. These comprehensive results from complementary methodologies with precise LSG thickness control down to the nanometer scale provide good evidence to resolve the debate on the thickness dependence of mechanical strength for LSG.

The Expression Levels of Vinculin in Pancreatic Cancer Tissues Significantly Correlates With Patient Survival.

BACKGROUND/AIM: Proteomics is an approach that can detect differentially expressed proteins between cancerous and non-cancerous tissue samples. Previously, we found that vinculin was predominantly expressed in pancreatic cancerous tissues compared to adjacent non-cancerous tissues by performing proteomic differential display analysis. However, the clinicopathological significance of vinculin in pancreatic cancer has not yet been documented. MATERIALS AND METHODS: The GEPIA2 and the Human Protein Atlas databases were used to analyze vinculin expression levels in cancerous tissue samples and investigate whether its expression level is clinically associated with patient survival. RESULTS: Vinculin mRNA expression levels were solely increased in pancreatic cancer tissues, and increased expression was inversely related to patient survival. Higher levels of vinculin protein were found in pancreatic cancer tissues. In contrast, faint staining of vinculin was observed throughout the normal pancreatic tissues. CONCLUSION: Vinculin may be an unfavorable prognostic indicator for patients with pancreatic cancer.

Nine Cases of SARS-CoV-2-PCR-positive Samples Showed No Increase of Antibodies Against SARS-CoV-2.

BACKGROUND/AIM: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been affecting Hokkaido, Japan since late February 2020 until present. The aim of this study was to report the relationship between anti-SARS-CoV-2 antibody-positive and SARS-CoV-2 PCR-positive cases by analyzing anti-SARS-CoV-2 antibodies (IgG and total-Ig). PATIENTS AND METHODS: Serum samples were collected from care workers and nurses in two nursing homes and two hospitals which underwent virus outbreak. All people were confirmed to be SARS-CoV-2-positive by RT-qPCR and their sera was analyzed for anti-SARS-CoV-2 antibodies (IgG and total-Ig). RESULTS: Although 34 out of 43 samples (79.1%) showed enough amount of anti-SARS-CoV-2 antibodies, 9 RT-qPCR -positive samples (20.9%) showed absence of anti-SARS-CoV-2 antibodies in their sera. CONCLUSION: The results that 20.9% of RT-qPCR-positive samples with SARS-CoV-2 showed absence of anti-SARS-CoV-2 antibodies provides a possibility that the innate immune reaction could eliminate the virus without activating adaptive immune reaction.

A standardized extract of cultured Lentinula edodes mycelia downregulates cortactin in gemcitabine-resistant pancreatic cancer cells.

AHCC®, a standardized extract of cultured Lentinula edodes mycelia, enhances the therapeutic effects and reduces the adverse effects of chemotherapy. Our previous study reported that treatment with AHCC® downregulated the expression levels of tumor-associated proteins in the gemcitabine-resistant pancreatic cancer cell line, KLM1-R. However, to the best of our knowledge, the role of AHCC® in the inhibition of cell migration remains unexplored. Cortactin (CTTN), an actin nucleation-promoting factor, has been reported to be upregulated and correlated with migration, invasion and metastasis in pancreatic cancer cells. The present study aimed to investigate the effects of AHCC® on cell migration and the protein expression level of CTTN in KLM1-R cells. The Gene Expression Profiling Interactive Analysis (GEPIA2), an online bioinformatics platform, was used to analyze CTTN mRNA expression levels in pancreatic cancer tissues compared with normal pancreatic tissues. CTTN mRNA expression and its association with clinicopathological characteristics were assessed by using the GEPIA2 platform. Next, the effects of AHCC® on KLM1-R cell migration were investigated by in vitro wound-healing assay. The KLM1-R cells were treated with AHCC® at a concentration of 10 mg/ml for 48 h. Western blotting was performed on of cell lysates with anti-CTTN or anti-actin antibodies to assess the protein expression levels of CTTN. Bioinformatics analysis indicated that the mRNA expression level of CTTN increased in pancreatic cancer tissues. The increased mRNA expression levels of CTTN were inversely associated with clinicopathological characteristics, including disease stages and prolonged patient survival times. The administration of 10 mg/ml AHCC® significantly inhibited KLM1-R cells migration compared with controls. The protein expression levels of CTTN were significantly reduced in AHCC®-treated KLM1-R cells, whereas actin expression was not affected. The downregulation of CTTN indicated the anti-metastatic potential of AHCC® in pancreatic cancer cells. Overall, AHCC® may have the potential to be a complementary and alternative therapeutic approach in treating pancreatic cancer.

Discriminative detection of laser-accelerated multi-MeV carbon ions utilizing solid state nuclear track detectors.

A new diagnosis method for the discriminative detection of laser-accelerated multi-MeV carbon ions from background oxygen ions utilizing solid-state nuclear track detectors (SSNTDs) is proposed. The idea is to combine two kinds of SSNTDs having different track registration sensitivities: Bisphenol A polycarbonate detects carbon and the heavier ions, and polyethylene terephthalate detects oxygen and the heavier ions. The method is calibrated with mono-energetic carbon and oxygen ion beams from the heavy ion accelerator. Based on the calibration data, the method is applied to identify carbon ions accelerated from multilayered graphene targets irradiated by a high-power laser, where the generation of high-energy high-purity carbon ions is expected. It is found that 93 ± 1% of the accelerated heavy ions with energies larger than 14 MeV are carbons. The results thus obtained support that carbon-rich heavy ion acceleration is achieved.

ITGA2, LAMB3, and LAMC2 may be the potential therapeutic targets in pancreatic ductal adenocarcinoma: an integrated bioinformatics analysis.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer with an abysmal prognosis rate over the last few decades. Early diagnosis and prevention could effectively combat this malignancy. Therefore, it is crucial to discover potential biomarkers to identify asymptomatic premalignant or early malignant tumors of PDAC. Gene expression analysis is a powerful technique to identify candidate biomarkers involved in disease progression. In the present study, five independent gene expression datasets, including 321 PDAC tissues and 208 adjacent non-cancerous tissue samples, were subjected to statistical and bioinformatics analysis. A total of 20 differentially expressed genes (DEGs) were identified in PDAC tissues compared to non-cancerous tissue samples. Gene ontology and pathway enrichment analysis showed that DEGs were mainly enriched in extracellular matrix (ECM), cell adhesion, ECM-receptor interaction, and focal adhesion signaling. The protein-protein interaction network was constructed, and the hub genes were evaluated. Collagen type XII alpha 1 chain (COL12A1), fibronectin 1 (FN1), integrin subunit alpha 2 (ITGA2), laminin subunit beta 3 (LAMB3), laminin subunit gamma 2 (LAMC2), thrombospondin 2 (THBS2), and versican (VCAN) were identified as hub genes. The correlation analysis revealed that identified hub genes were significantly interconnected. Wherein COL12A1, FN1, ITGA2, LAMB3, LAMC2, and THBS2 were significantly associated with PDAC pathological stages. The Kaplan-Meier survival plots revealed that ITGA2, LAMB3, and LAMC2 expression were inversely correlated with a prolonged patient survival period. Furthermore, the Human Protein Atlas database was used to validate the expression and cellular origins of hub genes encoded proteins. The protein expression of hub genes was higher in pancreatic cancer tissue than in normal pancreatic tissue samples, wherein ITGA2, LAMB3, and LAMC2 were exclusively expressed in pancreatic cancer cells. Pancreatic cancer cell-specific expression of these three proteins may play pleiotropic roles in cancer progression. Our results collectively suggest that ITGA2, LAMB3, and LAMC2 could provide deep insights into pancreatic carcinogenesis molecular mechanisms and provide attractive therapeutic targets.

Proteomic Analysis of Hepatocellular Carcinoma Tissues With Encapsulation Shows Up-regulation of Leucine Aminopeptidase 3 and Phosphoenolpyruvate Carboxykinase 2.

BACKGROUND/AIM: Cancer is the most fatal disease worldwide whose most lethal characteristics are invasion and metastasis. Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. HCC often shows encapsulation, which is related to better prognosis. In this study, proteomic analysis of HCC tissues with and without encapsulation was performed, in order to elucidate the factors which play important roles in encapsulation. MATERIALS AND METHODS: Five HCC tissues surrounded by a capsule and five HCC tissues which broke the capsule were obtained from patients diagnosed with HCC who underwent surgical liver resection. Protein samples from these tissues were separated by two-dimensional gel electrophoresis (2-DE), and the protein spots whose expression was different between encapsulated and non-encapsulated HCC tissues were identified through gel imaging analysis software. The selected protein spots were analyzed and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Two-DE analysis showed 14 spots whose expression was different between encapsulated and non-encapsulated HCC tissues. Of these, 9 were up-regulated and 5 were down-regulated in HCC tissues without encapsulation. The validation by Western blot confirmed that leucine aminopeptidase 3 (LAP3) and phosphoenolpyruvate carboxykinase mitochondrial (PCK2) were up-regulated significantly in HCC tissues with a capsule, compared to HCC tissues that broke the capsule. CONCLUSION: These findings suggest that LAP3 and PCK2 could be factors responsible for the maintenance of encapsulation in HCC tissues.