The majority of HCC patients (>80% in highly industrialized countries) have chronic hepatitis or cirrhosis, which influences disease progression and may severely restrict patient prognosis, therapeutic options, and design of clinical trials.1

The morphological sequence of premalignant and early malignant changes is well defined, but the lesions are hardly accessible by diagnostic means,2 which constitutes a significant difference from colon, breast, or skin cancer. Well-characterized model systems are available for mechanistic as well as preclinical analyses,3 and HCC cell lines have been workhorses for biochemical as well as molecular biological Torin 1 and recently even systems biological analyses, providing a wealth of basic research data for current translational approaches. Because of the obstacles characterized above, HCC has long been an orphan tumor disease with

regard to translational research efforts, clinical trial perspectives, and therapeutic options, which stands in sharp contrast to its enormous clinical relevance. HCC is the sixth most frequent cancer and the third most frequent cause of cancer-related death, and numbers of cases are rising, even in industrialized countries.4 Nevertheless, knowledge about molecular pathogenesis of HCC is lagging behind other major tumor diseases, such as breast and colon cancer, where multiple systemic treatment options are starting to convert in many cases previously untreatable metastatic tumors into a chronic disease. LDE225 cell line Recently, this picture has started to change for HCC and has gained some momentum: The growing Chinese economy has fueled the industry’s interest and improved options for clinical trials and novel therapeutics. The successful SHARP (Sorafenib HCC Assessment Randomized Protocol) trial established sorafenib as the first effective and approved systemic treatment for HCC and proved that despite

all obstacles, trials employing systemic treatments can be successful.5 Other treatment options such as radioembolization6 and oncolytic approaches7 have entered the field. Meanwhile, more than 150 phase 1 to 3 trials are ongoing, but only some of them are based on rational approaches using knowledge about molecular pathogenesis of human HCC. Comprehensive, large-scale selleck profiling approaches on representative collectives are missing so far, but numerous analyses have been performed at the genomic, epigenetic, and expression level, providing insight into relevant mechanisms, targets, as well as markers, suggesting future strategies for systemic HCC treatment. CGH, comparative genomic hybridization; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; miRNA, microRNA. Genomic, microRNA (miRNA), and some protein-based assays are more robust and less vulnerable to influences imposed by imperfect sample conditions.

4 This is unlike treatment with directly targeting antivirals, such as those for HCV or HIV, where treatment failure is often associated with drug resistance that can affect responsiveness to subsequent courses of treatment. Other examples where bridging analyses have impacted regulatory decision making include oxcarbazepine, topiramate, clevidipine,

and levofloxacin, to name a few.5-8 Bridging knowledge to provide clinical evidence of effectiveness and to support dosing recommendations not only is acceptable from a regulatory perspective, but when scientifically supported and warranted it is also encouraged to increase the efficiency by which new drugs and optimal dosing recommendations are made available to patients in need.9 This report summarizes the rationale to support the BOC dosing recommendations in prior P/R-null responders and treatment-naïve BOC late responders.10 BOC, boceprevir; FDA, U.S. Food selleck chemicals llc and Drug Administration; HCV, hepatitis C virus; P/R, peginterferon alpha and ribavirin; RGT, response-guided therapy; SVR, sustained virologic response. The SPRINT-II and RESPOND-II

trial designs are illustrated selleck compound in Fig. 1. The primary endpoint for both SPRINT-II and RESPOND-II was the proportion of subjects with SVR (sustained virologic response), defined as HCV RNA undetected at 24 weeks after the last dose of the study drug. Both trials had three treatment arms: (1) P/R for 48 weeks (Arm 1, P/R); (2) a response-guided therapy (RGT) arm with P/R lead-in for 4 weeks, followed by P/R with BOC for 24 weeks in SPRINT-II and 32 weeks in RESPOND-II (Arm 2, BOC-RGT); selleck screening library and (3) P/R lead-in for 4 weeks, followed by BOC with P/R for 44 weeks (BOC44) (Arm 3, BOC44). In SPRINT-II subjects randomized to the RGT arm who had HCV RNA undetected at weeks 8 through week 24 stopped all treatment

at week 28 and were classified as BOC treatment-naïve early responders. The remaining subjects in the RGT arm, who had HCV RNA detected at treatment week 8 or any subsequent week, but who had HCV RNA undetected at week 24, stopped BOC at week 28 but received P/R through week 48. These subjects are referred to as BOC treatment-naïve late responders. In the RGT arm of RESPOND-II treatment-experienced early responders (subjects with HCV RNA undetected at weeks 8 through 12) stopped all treatment at week 36, whereas treatment-experienced late responders (HCV RNA detected at week 8 but HCV RNA undetected at week 12) stopped BOC at week 36 but continued P/R through week 48. Although prior P/R null responders were excluded from RESPOND-II, the sponsor proposed that data from treatment-naïve subjects could be used to estimate the treatment effect in prior null responders because included among treatment-naïve subjects are a subset of subjects who are intrinsically poor responders to interferon.

The lifetime risk of HBV carriers to develop cirrhosis, liver failure, or HCC may be as high as 15% to 40%.[1-3] The identification of risk factors for the development of advanced liver disease, including cirrhosis and HCC, in HBV carriers is important for implementing effective PARP inhibitor treatment. Recently, several qualitative and quantitative hepatitis B viral factors affecting the prognosis of HBV carriers have been identified.[3, 4] Among these viral factors, baseline serum HBV-DNA level is the main driving force for cirrhosis and HCC development in adult HBV

carriers.[5, 6] Recently, quantitative HBsAg (qHBsAg) has been increasingly recognized to be a promising biomarker to predict both favorable and adverse outcomes of HBV carriers. Based on the weight of each risk factor associated with HBV-related HCC and through a stratification process, it is possible to identify HBV carriers who are at an increased risk of disease progression and HCC development (Table 1). In this article, hepatitis B viral factors

leading to disease progression Selleck KU 57788 and the risk stratification for HBV-related HCC will be reviewed and discussed. Low serum level of HBV-DNA Low serum level of HBsAg HBV genotype C/D BCP A1762T/G1764A mutation Pre-S deletion High serum level of HBV-DNA High serum level of HBsAg According to the divergence in the entire HBV genomic sequences, at least 10 HBV genotypes (A to J) have been defined.[7-9] Several studies suggested that HBV genotype can influence the long-term outcomes of HBV infection. For

example, HBV genotype C and D patients, compared with genotype A and B patients, have late or absent HBeAg seroconversion after multiple hepatitis flares that accelerate the progression of chronic hepatitis.[10-12] Most case–control this website studies and community-based prospective cohort study indicated that patients with genotype C HBV infection have a higher risk of cirrhosis and HCC than those with genotype B infection.[13-17] In addition, several reports have also shown that HBV genotype B was associated with HCC development in young non-cirrhotic patients. Whereas genotype C was associated with HCC development in old cirrhotic patients.[13, 18, 19] Due to the spontaneous error of viral reverse transcription, HBV mutant strains occur during the natural course of infection as well as with antiviral therapy. Mutations in precore, core promoter, and deletion mutation in pre-S/S genes have been reported to be associated with the progression of liver disease, including cirrhosis and HCC. Previous studies revealed that dual mutations in basal core promoter (BCP) A1762T/G1764A were strongly associated with the risk of HCC development.

Hence, separation of the pulmonary and systemic circulation is desirable. The Fontan operation allows systemic

venous return to the pulmonary arteries bypassing the right ventricle. The Fontan operation (Fig. 1) may be accomplished by either creating a direct cavopulmonary (sometimes in a staged manner) or atriopulmonary anastomosis. In the first stage (i.e., superior caval-pulmonary anastomosis or bidirectional Glenn procedure), the superior vena cava is connected to the pulmonary arteries. Eventually, the inferior vena cava is also connected to the pulmonary arteries completing the circulation (Fontan completion). In earlier iterations of the Fontan, an atriopulmonary anastomosis was created with the hope that a hypertrophied right atrium would serve as a functional pump. However, it was associated

with a risk check details of atrial dysrhythmias and atrial thrombi.10 More commonly, a cavopulmonary anastomosis is achieved by the use of an intra-atrial tunnel or patch or by utilizing an extracardiac conduit to connect the vena cavae to the pulmonary arteries (Fig. 1). As a consequence of surgical palliation, significant liver disease can develop as a result of the interplay of central venous hypertension/passive congestion and hypoxia resulting from left ventricular Napabucasin dysfunction. Development of significant hepatic injury after a Fontan procedure is multifactorial. The determinants of cardiac output are central venous pressure, pulmonary vascular resistance, and systemic ventricular see more end-diastolic pressure. Over time, a “failure of Fontan physiology” is common. Failure of the Fontan circuit may result from elevated pulmonary vascular resistance, pulmonary thrombi, narrowing and scarring in the Fontan pathway or pulmonary arteries, and failure of the systemic ventricle, which results in elevated pressure in the pulmonary venous atrium. Chronic elevation of central venous pressure is common, and reduced cardiac output from the functioning single ventricle

is frequent, particularly as diastolic and systolic dysfunction ensues. The former results from the absence of a subpulmonic pump.11 There is impaired coupling between the ventricles and the arterial system with late ventricular dysfunction.12 Atrial arrhythmias may contribute to this decline with relative hypotension and desaturation. The development of pulmonary venovenous collaterals as pressure “pop-offs” are not uncommon in the adult population and further contribute to hypoxemia. Pulmonary arteriovenous malformations, most often observed after a classic Glenn procedure, also contribute to hypoxemia. Chronic hypoxia resulting from a depressed cardiac output, in addition to the aforementioned changes, may also lead to hepatic injury.

2. According to patients’ reaction to glucocorticoid treatment, we can divide them into Hormone effective group and Hormone refractory group, compare the clinical data of two groups and at the same time analyze from parallel single factor and multi-factor logistic regression. 3. Analyzing the surgical patients’ clinical data through single factor and multi-factor logistic regression, counting and analyzing the clinical high risk factor for excision. Results: The clinical feature analysis of SUC: among 106 patients

with SUC, there were 72 cases of men and 34 cases of female, LEE011 cost male: female = 2.09: 1; aged from 15 to 73 years old (39.52 ± 13.8), the highest rate of 50% (86/106) is of the group aged from 25 to 45 years old; The rate of the group check details in which main lesion type is the chronic recurrent type is 89.6% (95/106); The rate of the group in which the main clinical manifestation is stomachache, diarrhea, mucopurulent bloody stool, the rate of Moderately severe

stomachache is 83% (88/106), the defecation times >6 times/day 73.6% (78/106), >10 times/day 19.8% (21/106), the rate for Moderately severe hemafecia is 84.9% (90/106); the rate of extensive colonic lesions among Colon lesions is 83% (88/106); the main grade of Endoscopic mucosal inflammation is 2–3 points 87.7% (93/106); anemia 65.1% (69/106), Moderately severe anemia 34.9% (37/106); platelet increased to 48.1% (51/106); CRP increased to 88.7% (94/106), CRP ≥ 40 mg/l 50% (53/106), CRP > 70 mg/l 14.2% (15/106); hypoproteinemia 42.2% (45/106). The clinical high-risk feature analysis of SUC: among the 106 patients with SUC, 89.6% (95/106) accepted

the treatment of Glucocorticoid, Refractory group and Effective group respectively took up 35.8% (34/95) and 64.2% (61/95). Refractory group differ from Effective group’s because their diarrhea 10 ≥ time/day, Severe mucous purulent find more blood, Severe anemia, soterocyte increased, CRP > 70 mg/l, Albumin < 25 g/l difference was statistically significant (P < 0.05), difference wasn't statistically significant in sex, age, other clinical features and laboratory indicators (P > 0.05), but through the logistic regression analysis of multi-factor, severe anemia (OR = 6.750, 95% CI = 2.656–17.152, P = 0.000), thrombocythemia (OR = 4.032, 95% CI = 1.226 -13.261, P = 0.015), Albumin < 25 g/l (OR = 3.022, 95% CI = 1.236–7.390, P = 0.022) are independent predictors of the refractoriness of hormone.

3 To better put into perspective what will happen to our patients with advanced NASH, it is logical to compare it to HCV, a disease with a well-established natural history. In a small prospective cohort study of Australian patients published in HEPATOLOGY nearly a decade ago, Hui and colleagues compared 23 patients with NASH-derived cirrhosis to 23 patients with untreated HCV-derived cirrhosis and 23 nonresponders with HCV-derived cirrhosis. The authors found that patients with NASH

cirrhosis experienced less hepatic decompensation, but a similar mortality to their HCV cirrhosis counterparts.4 In this issue of HEPATOLOGY, Bhala et al. extend their findings to a multinational prospective cohort study that includes patients from Italy, the United States, the United Kingdom, and Australia. They investigated the long-term outcome of patients with NASH or HCV and advanced fibrosis (stage 3 or 4). mTOR inhibitor They compared 247 patients with NASH to 264 patients Peptide 17 research buy with HCV (nonresponders or untreated) in the analysis and followed them for a mean of 85.6 and 74.9 months, respectively. The findings demonstrate

that whereas the HCV cohort had more liver-related morbidity and incident HCC than the NASH cohort, rates of CV events and overall mortality were no different. Importantly, the current study differs from prior work in that it included patients with stage 3 fibrosis, in addition to those with compensated

cirrhosis. This is a timely study that sheds light on some aspects of the natural history of NAFLD. However, it has limitations that should be considered when interpreting the results. Given the heterogeneity of what we currently refer to as NASH, it is unlikely that any study would be generalizable to the entire NASH population. Ethnic differences in the susceptibility to develop NAFLD or progressive liver injury are well documented.5 For example, Hispanics and Asians (particularly the Indian subcontinent and southeast Asia), are at increased risk for advanced NASH, whereas African check details Americans are relatively protected despite the presence of similar metabolic risk factors.6-8 Although the current study is a multinational study from four countries (Australia, Italy, the United States, and the United Kingdom), 92% of the patients with NASH were Caucasian. Thus, as the authors concede, it is not clear whether these findings are applicable to other races. Although this is a potential weakness of the study, one could argue that given the heterogeneity of the NASH population at large, studies of ethnic-specific cohorts are important. It is known that HCV treatment response deters the rate of decompensation and the development of HCC.9 Thus, the natural history of the HCV group chosen by Bhala et al. was at less risk of being influenced by external factors such as viral clearance.

After that patients were screened for depression using the NICE clinical guideline initial depression screening tool. Data was analyzed in SPSS version 17 using descriptive statistics and Univariate analysis. Results: Out of 246 patients 56.9% were male and 43.1% were female. Mean age was 35.84 years while mean duration of disease was 2.33 years. Out of all patients 28.5% of the patients belong to postprandial distress syndrome, 28.9% belong to epigastric pain syndrome while 42.7% belong to both groups.

Frequency of depression was 75.6% among patients screened for depression selleck products with 19% of the patients saying that they had thought of death in the last month. Female sex was significantly associated with depression in univariate analysis (OR 2.32, p value 0.01) while dyspepsia group or duration of the disease were not. Conclusion: Keeping in view the high prevalence of depression in functional dyspepsia all patients with functional dyspepsia must be screened for depression. Key Word(s): 1. GI Gastroenterology; 2. Rome III; Presenting Author: selleck screening library MARIE

ANTOINETTEDE CASTRO LONTOK Additional Authors: ROMMELPARULAN ROMANO, JOSEDECENA SOLLANO Corresponding Author: MARIE ANTOINETTEDE CASTRO LONTOK, ROMMELPARULAN ROMANO Affiliations: Asian Hospital and Medical Center; University of Santo Tomas Hospital Objective: The diagnosis of gastroesophageal reflux disease (GERD) in the community is largely based on the clinicians’ assessment of the symptom presentation by patients. The locally-validated

Filipino version of the Frequency Scale of Symptoms of GERD (FSSG) was used to investigate the most common symptoms associated with reflux in the primary care setting in the Philippines, as well as, determine response to PPI treatment. Methods: Patients presenting with selleck chemicals llc reflux symptoms seen by primary care physicians were recruited. The FIlipino version of the FSSG questionnaire were administered before and after completion of PPI treatment. Patients were given 4 weeks of Rabeprazole 20 mg once a day. Pre and post treatment F test scores were computed. Outcome measured was resolution or non-resolution of symptom/s after PPI treatment. Data were collated and statistical analysis done using SPSS v20. Results: A total of 1,578 subjects were enrolled and analyzed in this study. The most common symptom presented was a sensation of heartburn present in 1,359 (86.12%) of the subjects, followed by bloatedness (83.52%). Evaluating response to treatment, there was a statistically significant difference between the pre- and post-treatment F test scores (p < 0.001). The highest positive symptom response was seen pertaining to sour taste in the mouth (90.3%) and the symptom least responsive to PPI therapy is feeling ill after a binge meal (81.9%). Conclusion: Using the locally validated FSSG questionnaire, the most common clinical presentation of GERD patients include heartburn and bloatedness.

After that patients were screened for depression using the NICE clinical guideline initial depression screening tool. Data was analyzed in SPSS version 17 using descriptive statistics and Univariate analysis. Results: Out of 246 patients 56.9% were male and 43.1% were female. Mean age was 35.84 years while mean duration of disease was 2.33 years. Out of all patients 28.5% of the patients belong to postprandial distress syndrome, 28.9% belong to epigastric pain syndrome while 42.7% belong to both groups.

Frequency of depression was 75.6% among patients screened for depression Metformin research buy with 19% of the patients saying that they had thought of death in the last month. Female sex was significantly associated with depression in univariate analysis (OR 2.32, p value 0.01) while dyspepsia group or duration of the disease were not. Conclusion: Keeping in view the high prevalence of depression in functional dyspepsia all patients with functional dyspepsia must be screened for depression. Key Word(s): 1. GI Gastroenterology; 2. Rome III; Presenting Author: Sirolimus in vivo MARIE

ANTOINETTEDE CASTRO LONTOK Additional Authors: ROMMELPARULAN ROMANO, JOSEDECENA SOLLANO Corresponding Author: MARIE ANTOINETTEDE CASTRO LONTOK, ROMMELPARULAN ROMANO Affiliations: Asian Hospital and Medical Center; University of Santo Tomas Hospital Objective: The diagnosis of gastroesophageal reflux disease (GERD) in the community is largely based on the clinicians’ assessment of the symptom presentation by patients. The locally-validated

Filipino version of the Frequency Scale of Symptoms of GERD (FSSG) was used to investigate the most common symptoms associated with reflux in the primary care setting in the Philippines, as well as, determine response to PPI treatment. Methods: Patients presenting with this website reflux symptoms seen by primary care physicians were recruited. The FIlipino version of the FSSG questionnaire were administered before and after completion of PPI treatment. Patients were given 4 weeks of Rabeprazole 20 mg once a day. Pre and post treatment F test scores were computed. Outcome measured was resolution or non-resolution of symptom/s after PPI treatment. Data were collated and statistical analysis done using SPSS v20. Results: A total of 1,578 subjects were enrolled and analyzed in this study. The most common symptom presented was a sensation of heartburn present in 1,359 (86.12%) of the subjects, followed by bloatedness (83.52%). Evaluating response to treatment, there was a statistically significant difference between the pre- and post-treatment F test scores (p < 0.001). The highest positive symptom response was seen pertaining to sour taste in the mouth (90.3%) and the symptom least responsive to PPI therapy is feeling ill after a binge meal (81.9%). Conclusion: Using the locally validated FSSG questionnaire, the most common clinical presentation of GERD patients include heartburn and bloatedness.

Increased iNOS protein and mRNA expression was found in ammonia-treated find more cultured rat astrocytes6, 20 and in brains of portocaval-shunted rats in vivo.21 However, iNOS mRNA expression was significantly reduced by approximately 50%

in ammonia (5 mmol/L, 6 hours)-treated microglia. As for control, lipopolysaccharide (LPS, 1 ng/mL, 6 hours) strongly increased iNOS mRNA expression (Supporting Information Fig. 1A). In addition, NH4Cl (5 mmol/L) exposure for 20 hours had no effect on iNOS protein expression as detected by immunofluorescence (data not shown). Activated microglia can express cyclooxygenase-2 (COX-2) and can be a powerful source of proinflammatory prostanoids, such as PGE2. However, NH4Cl (5 mmol/L) had no significant effect on COX-2 mRNA expression in microglia and astrocytes (Supporting Information Fig. 1B) and

even lowered the PGE2 and 6-keto prostaglandin F1α (PGF1α) content of microglial supernatants after 6 hours and 20 hours of ammonia exposure (Fig. 4B). On the other hand, and in contrast to microglia, NH4Cl (5 mmol/L) stimulated the release of PGE2 and 6-keto PGF1α from cultured astrocytes (Fig. 4A). LPS treatment (1 ng/mL, 6 hours or 20 hours), which served as a positive control, increased PGE2 and 6-keto PGF1α release from both buy OSI-906 cultured microglia and astrocytes (Fig. 4A,B). Extracellular glutamate can promote neuroinflammation by overactivation of N-methyl-D-aspartate receptors.22 In order to analyze the effect of ammonia on glutamate release, cultured microglia and astrocytes were incubated with NH4Cl (5 mmol/L) for 6 hours and 20 hours, and the glutamate content was measured in the supernatant. As shown in Supporting Information Fig. 2, no increase in extracellular glutamate was detected in the supernatant of cultured microglia exposed to NH4Cl (5 mmol/L) for 6 hours or 20 hours, whereas ammonia triggered glutamate release from cultured astrocytes as described recently.5 Activated microglia can promote neuroinflammation through the release of proinflammatory cytokines,23

which may play a role in the pathobiology of HE.1, 10, 11, 24 As shown in Fig. 5, treatment of cultured microglia or astrocytes with NH4Cl did not significantly change tumor necrosis factor α (TNF-α), selleck compound interleukin (IL)-1α, or IL-6 mRNA expression. Interleukin-1β mRNA expression was reduced by NH4Cl treatment in microglia, but increased in astrocytes. LPS, which served as a positive control, strongly increased cytokine mRNA expression (Fig. 5) and prostanoid synthesis (Fig. 4) in both cell types. As shown by immunofluorescence (Fig. 6A) and western blot analysis (Fig. 6B,C), intraperitoneal injection of ammonium acetate (4.5 mmol/kg) increased Iba-1 expression in the cerebral cortex within 6 hours, suggestive for in vivo microglia activation after ammonia intoxication.

All samples were measured for their individual levels, and each sample was analyzed in triplicate manner, taking the mean of the three determinations. For PAS staining, histochemical staining of glycoconjugates was carried out as per the method of Pandurangan et al.,[14] using 2% PAS’ reagent

in dark for 20 min. Apoptotic cells in the LEE011 purchase gastric mucosa were detected using the In Situ Cell Apoptosis Detection kit (Promega, Madison, WI, USA), with at least three replicates for each group. Immunohistochemistry was performed on replicate sections of mouse colon tissues. Sections fixed in 10% buffered formalin and embedded in paraffin were deparaffinized, rehydrated, and boiled three times in 100 mM Tris-buffered saline (pH 7.6) with 5% urea in an 850 W microwave oven for 5 min each. Sections were also incubated with F4/80 and CD31 antibody in the presence of 1.0% bovine serum albumin and finally incubated for 16 h at 4°C. The sections were counterstained with hematoxylin. Various concentrations of SAC were added to a Dabrafenib in vivo total volume of 200 μl containing 0.05 mM FeSO4, 1 mM H2O2, 1 mM 5,5-dimethylpyrroline-N-oxide (DMPO, Sigma), 5-tert-Butoxycarbonyl-5-methyl-1-pyrroline-N-oxide(BMPO, Enzo, Plymouth Meeting, PA, USA), and 50 mM, sodium phosphate at pH 7.4 at room temperature. Reactions were initiated by adding H2O2.

After incubation for 1 min, aliquots of the reactions were transferred to a quartz cell, and the spectrum of DMPO-OH and BMPO-OH was examined using an ESR spectrophotometer

(JES-TE300, JEOL, Tokyo, Japan), under the following conditions: magnetic field, 338.0 ± 5.0 mT; microwave power, 4.95 mW; frequency, 9.421700 GHz; modulation amplitude, 5 mT; sweep time, 0.5 min; and time constant, 0.03 s. The rat gastric mucosal cells, RGM1, were kindly given by Prof. Hirofumi Matsui (University of Tsukuba, Japan) and were maintained at 37°C in selleck kinase inhibitor a humidified atmosphere containing 5% CO2 and cultured in Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal bovine serum and 100 U/mL penicillin. RGM1 cells were seeded in a 100-mm dish and grown to 80% confluence in the complete growth medium. The cells were treated with SAC. After 6 h, the cells were further treated with TNF-α for 3 h (nuclear extracts), 6 h (RNA), and 24 h (whole cell lysates). The cells were then washed with PBS and lysed, and the cells were treated with several inhibitors. After 1 h, the cells were further treated with TNF-α for 1 h and for 24 h. The cells were then washed with PBS and lysed. After incubation, media was removed by suction and cells were washed with PBS twice. RiboEX (500 μL; GeneAll, Seoul, Korea) was added to plates, which were then incubated for 10 min at 4°C. RiboEX was harvested and placed in a 1.5-mL tube, and 100 μL of chloroform was added and gently mixed. After incubation for 10 min in ice, samples were centrifuged at 10 000 × g for 30 min.