This was accomplished by measuring the incorporation of [3H]aceta

This was accomplished by measuring the incorporation of [3H]acetate into TG (Fig. 2A), and in vivo hepatic TG secretion following inhibition of VLDL metabolism with poloxamer (P-407) (Fig. 2B). We also determined ketone bodies in serum (Fig. 2C) and the in vitro secretion of acid-soluble metabolites (Krebs cycle metabolites and ketones) (Fig. 2D), as a measure of FA β-oxidation. Whereas lipogenesis and FA β-oxidation were barely altered in hepatocytes from Gnmt−/− mice (Fig. 2A,C,D), the hepatic TG secretion rate in GNMT-depleted livers was elevated compared to livers from WT animals (Fig. 2B). Consistent with these studies, a comprehensive gene expression analysis showed that, overall, the expression

of genes that supply NADPH and acyl-CoA find more for lipid synthesis was unaltered in mice FK506 solubility dmso without GNMT (Fig. 2E). Despite the marked hepatic steatosis, mice without GNMT did not show insulin resistance or changes in serum FA concentrations (Supporting Fig. 1a,b). Depletion of GNMT in mice did not alter food intake or body weight (Supporting Fig. 1c,d). The greater liver weight in Gnmt−/− mice was not accompanied by differences in body weight, which may be explained by the reduced mass of the white adipose tissue in these animals (Supporting Fig. 1d-f). Based on the results depicted in Fig. 2, which demonstrate that Gnmt−/− mice have increased lipid secretion without affecting lipid synthesis or

oxidation, it is not obvious how to explain the presence of fatty livers in these animals. We reasoned that an elevation of SAMe in Gnmt−/− mice

would activate the flux from PE to PC via PEMT, which would lead to increased PC catabolism and the corresponding augmentation of hepatic DG and TG production (Fig. 2). To confirm this hypothesis, we measured the incorporation of [3H]ethanolamine into PE and PC MCE in hepatocytes isolated from 3-month-old Gnmt−/− mice and calculated the radioactivity incorporated into PC as a percentage of the radiolabel incorporated into PC+PE (Fig. 3A). Because PC formed via PEMT primarily contains long-chain polyunsaturated FA (PUFA), such as docosahexaenoic acid (22:6n-3), whereas PC synthesized by the CDP-choline route do not, we also determined the PC(22:6n-3) to total PC ratio in GNMT-depleted and WT livers as a marker of hepatic PEMT activity.[21] Given that PEMT activity is primarily located in the endoplasmic reticulum,[22] we measured the content of PE and PC in whole liver microsomes (Fig. 3B,C). As shown in Fig. 3, high SAMe levels in Gnmt−/− hepatocytes associated with a 2.5-fold increase in the flux from PE to PC (P < 0.001) (Fig. 3A), and an increase in the PC(22:6)/PC ratio (from 0.18 ± 0.005 in WT to 0.25 ± 0.005 in GNMT-depleted livers, P = 3.23E-06). Also as predicted, the content of PE was reduced ∼2-fold in microsomes isolated from GNMT-depleted livers (P < 0.05), whereas the amount of PC was increased 2-fold (P < 0.05) (Fig. 3B,C).

Moreover, Trpv1 depletion markedly blunted EtOH-me-diated inducti

Moreover, Trpv1 depletion markedly blunted EtOH-me-diated induction of plasminogen activator inhibitor-1

(Pai-1), an important mediator of alcohol-induced hepatic inflammation, via fibrin accumulation. EtOH-induced DMXAA in vitro Pai-1 up-regulation in WT but not in Trpv1−/− animals was in parallel with the activation of hepatic ERK. Exposure of hepatocytes to 9-HODE and 13-HODE in vitro resulted in activation of TRPV1 signal trans-duction with increased intracellular Ca2+ levels, suggesting that OXLAM/TRPV1/Ca2+ signaling may be a potentially relevant pathway contributing to ALD. Conclusions: This study indicates for the first time that the TRPV1 receptor pathway may be involved in the hepatic inflammatory response in an experimental animal model of ALD. TRPV1-OXLAM interactions appear to play a significant role in hepatic inflammation/injury, further supporting an important role for dietary lipids in ALD. Disclosures: Craig J. McClain – Consulting:

Vertex, Gilead, Baxter, Celgene, Nestle, Danisco, Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline; Speaking and Teaching: Roche The following people have nothing to disclose: Irina Kirpich, Keith C. Falkner, Juliane http://www.selleckchem.com/products/BIBW2992.html I. Beier, Gavin E. Arteel, Christopher Ramsden, Ariel E. Feldstein Background/Aim: Steatosis is an early pathogenic lesion in the spectrum of alcoholic liver disease. Neuropilin-1 (NRP) is a growth factor co-receptor implicated in hepatic stellate cell (HSC) activation. Recent studies have suggested that HSC may regulate parenchymal cell injury and inflammation that precedes liver fibrosis. Therefore, we sought to test the hypothesis that NRP in HSC may regulate steatosis in response to alcohol feeding in mice. Methods: NRP floxed mice (NRP-1loxP) were crossed with Collagen 1a Cre mice (ColCre) to generate mice with HSC selective deletion of NRP (ColCre/NRPloxP). Col-Cre/NRPloxP or pairfed wildtype mice were fed control or Lieber-deCarli diet for 10 days followed by alcohol

gavage (chronic/binge alcohol feeding model). Steatosis was measured and quantified by Oil Red staining, BODIPY staining, and triglyceride measurements from frozen liver tissues. Inflammation was assessed by real-time PCR for tumor necrosis factor-alpha (TNF-alpha) and Interleukin-1beta (IL-1beta) mRNA from liver 上海皓元 lysates. Results: Hepatic steatosis was 90% lower in ColCre/NRPloxP mice in response to alcohol feeding compared to wildtype animals (n=5-7; p<0.05) as assessed by Oil Red staining. This finding was confirmed by BODIPY staining (n=6-10; p<0.05). ColCre/NRPloxP mice also demonstrated a 50% reduction in hepatic triglyceride content after alcohol feeding compared to wildtype controls (p<0.05). TNF-alpha and IL-1beta mRNA expression increased 2 and 3 fold, respectively, in wild-type mice in response to alcohol feeding but not in ColCre/NRPloxP mice (n=6-10; p<0.05).

5) recieved initially a combination therapy of EVR with very-low

5) recieved initially a combination therapy of EVR with very-low dose CSA (81.8%) or tacrolimus (18.2%).

EVR treatment was started on post op day 1, 2 and 3 in 23, 8 and 2 patients, respectively. The EVR, CSA and TAC doses were adjusted to aim at a trough target level between 3-8 ng/ml, 50-80 ng/ml and 3-5 ng/ml, respectively. Other concommittant initial immu-nosuppressive therapy included basiliximab in 13 patients (39.4%), and prednisolone in all patients. Mean follow-up was 883 days. Indications for early treatment with EVR were renal dysfunction (39.4%), prophylaxis for recurrent HCC 36.4%), neurological problems (6%), other preexisting malignancies (3%) or combined check details OLT/kidney transplantation (15%). During follow-up CNI was stopped in 4/33 patients (12.1%). Altogether only 2/33 patients (6%) experienced a mild episode of BPAR (BANF score 4 and 5) 20 and 80 weeks post OLT. Both patients responded well to steroids. No patient required a retransplantation. No patient developed hepatic artery thrombosis. Impaired wound healing was an uncommon complication (9%).

The 1- and 2- year patient survival rate was 90.9% and 81.8%, respectively. HCC recurred in 2/12 patients. Post-operatively 8/27 (29.6%) OLT recipients not undergoing click here kidney transplantation required dialysis. At last follow-up only 1 of these patients had terminal renal insufficiency. In 8 (24.2%) patients EVR treatment was stopped after a mean treatment duration of 311.5 days for hematological side effects (9%), infections (6%), dermatological side effects (6%), polyarthral-gia

(3%). Other side effects included hypercholesterolemia (51.5%), anemia (12.1%), leukopenia (6%), edema (3%), proteinuria (9%). During follow-up incisional hernias occurred frequently (45.4%), but rarely required surgical repair (21.2%). Conclusion: Everolimus treatment start directly post operative in combination with medchemexpress very low-dose CNI is effective and safe post OLT resulting in a low rejection rate. Disclosures: Martina Koch – Grant/Research Support: Novartis Lutz Fischer – Advisory Committees or Review Panels: Novartis, Gilead; Grant/ Research Support: Astellas; Speaking and Teaching: Novartis Bjoern Nashan – Advisory Committees or Review Panels: Novartis, Bristol-Myer Squibb; Speaking and Teaching: Novartis, Bristol-Myer Squibb The following people have nothing to disclose: Martina Sterneck, Antonio Galante, Gesa Pamperin, Jun Li Introduction: Young people with liver disease, aged 12-25 years, are a unique population that requires special attention with respect to adherence to treatment and their subsequent transition to adult services. Reports on long-term survival following liver transplantation (LT) show decreased patient and graft survival in young adults with non-adherence (NA) as one of the main contributory factors.

5) recieved initially a combination therapy of EVR with very-low

5) recieved initially a combination therapy of EVR with very-low dose CSA (81.8%) or tacrolimus (18.2%).

EVR treatment was started on post op day 1, 2 and 3 in 23, 8 and 2 patients, respectively. The EVR, CSA and TAC doses were adjusted to aim at a trough target level between 3-8 ng/ml, 50-80 ng/ml and 3-5 ng/ml, respectively. Other concommittant initial immu-nosuppressive therapy included basiliximab in 13 patients (39.4%), and prednisolone in all patients. Mean follow-up was 883 days. Indications for early treatment with EVR were renal dysfunction (39.4%), prophylaxis for recurrent HCC 36.4%), neurological problems (6%), other preexisting malignancies (3%) or combined NVP-BKM120 mouse OLT/kidney transplantation (15%). During follow-up CNI was stopped in 4/33 patients (12.1%). Altogether only 2/33 patients (6%) experienced a mild episode of BPAR (BANF score 4 and 5) 20 and 80 weeks post OLT. Both patients responded well to steroids. No patient required a retransplantation. No patient developed hepatic artery thrombosis. Impaired wound healing was an uncommon complication (9%).

The 1- and 2- year patient survival rate was 90.9% and 81.8%, respectively. HCC recurred in 2/12 patients. Post-operatively 8/27 (29.6%) OLT recipients not undergoing see more kidney transplantation required dialysis. At last follow-up only 1 of these patients had terminal renal insufficiency. In 8 (24.2%) patients EVR treatment was stopped after a mean treatment duration of 311.5 days for hematological side effects (9%), infections (6%), dermatological side effects (6%), polyarthral-gia

(3%). Other side effects included hypercholesterolemia (51.5%), anemia (12.1%), leukopenia (6%), edema (3%), proteinuria (9%). During follow-up incisional hernias occurred frequently (45.4%), but rarely required surgical repair (21.2%). Conclusion: Everolimus treatment start directly post operative in combination with 上海皓元 very low-dose CNI is effective and safe post OLT resulting in a low rejection rate. Disclosures: Martina Koch – Grant/Research Support: Novartis Lutz Fischer – Advisory Committees or Review Panels: Novartis, Gilead; Grant/ Research Support: Astellas; Speaking and Teaching: Novartis Bjoern Nashan – Advisory Committees or Review Panels: Novartis, Bristol-Myer Squibb; Speaking and Teaching: Novartis, Bristol-Myer Squibb The following people have nothing to disclose: Martina Sterneck, Antonio Galante, Gesa Pamperin, Jun Li Introduction: Young people with liver disease, aged 12-25 years, are a unique population that requires special attention with respect to adherence to treatment and their subsequent transition to adult services. Reports on long-term survival following liver transplantation (LT) show decreased patient and graft survival in young adults with non-adherence (NA) as one of the main contributory factors.

Third, evidence suggests that antigen-naïve B cells exert anti-in

Third, evidence suggests that antigen-naïve B cells exert anti-inflammatory properties,48 which may inhibit APC maturation and proinflammatory differentiation49; in this regard, it has been demonstrated

that dendritic cells from B cell–deficient mice produce higher levels of IL-12 and promote proinflammatory T cell differentiation.8 Thus, our findings suggest that B cell depletion therapy may be contraindicated in PBC. Indeed, we know that B cell depletion in dnTGF-βRII mice exacerbates inflammation of bile ducts.24 Although a biochemical benefit of anti-CD20 therapy in PBC patients refractory to ursodeoxycholic acid has been reported,50 we suggest that additional data regarding the role of B cells find more in human mucosal autoimmune diseases such as PBC are needed. Finally, our findings underscore the fact that unanticipated problematic issues can arise from the use of biologics in humans and thus the importance

of trials in murine models and rigorous post marketing surveillance of such agents in humans. “
“To investigate the impact of hospital-acquired Clostridium difficile infection (CDI) on hospital costs and patient length of stay. Data from the 2007–2008 New York State Department of Health’s Statewide Planning and Research Cooperative System (SPARCS) database was analyzed using regression analysis and descriptive statistics. After analysis of 4 853 800 patient discharges, the incidence rate of hospital-acquired CDI was 0.8 cases per 1000 discharges. The estimated marginal cost associated with each hospital

infection was approximately $29 000. The estimated Wnt drug annual cost of CDI in New York State was approximately $55 million with nearly 23 000 additional hospital days. The development of hospital-acquired CDI is associated with a significant increase in hospital costs and patient length of stay. Extrapolation of MCE公司 these estimates to all US hospitals suggests this condition represents a major burden to the US healthcare system. Our findings may help hospitals understand the impact of these infections, as well as potential implications if deemed preventable by Centers for Medicare & Medicaid Services and/or private payers. Additionally, this information may benefit hospitals or health care systems transitioning to alternative payment models, such as episode-based payments or accountable care. Healthcare providers and hospitals would benefit from better understanding the impact and frequency of these infections in order to best target preventive strategies. “
“Aim:  Nuclear factor-κB (NF-κB) is a critical signaling mediator in inflammation, apoptosis resistance and oncogenesis. It has been reported that NF-κB is activated in several cancers, including hepatocellular carcinoma (HCC). Studies of genetic disruptions in mice also suggest that NF-κB plays critical roles in hepatocarcinogenesis. The aim of the present study is to characterize NF-κB activation and correlate it with the degree of malignancy in HCC.

Physicians taking care of patients with advanced HCC after a VB e

Physicians taking care of patients with advanced HCC after a VB episode should individualize therapies according to clinical practice, common sense, and patient needs. Some may judge that the survival benefit in these BCLC C and D patients who received secondary prophylaxis is not clinically relevant Z-VAD-FMK concentration (average, 3 months) and that more-interventional therapies (banding ligation) should be avoided, taking into account the possible adverse effects. Nevertheless, this survival benefit is similar to the survival benefit offered with sorafenib treatment in BCLC C patients,

which also has side effects, which may affect quality of life. The present study, showing a global survival effect of prophylaxis patients with advanced HCC, provides further evidence to indicate prophylaxis in this subgroup of patients as long as their clinical condition

allows them to do so. There are several setbacks to the study. Some patients with very advanced HCC and UGI bleeding were not included in the study because no endoscopy was performed. This could lead to some bias in the results, because it is probable that these patients who were not included would be the ones who would be most likely to die. However, the decision to exclude these patients from the study was based on several Ulixertinib datasheet reasons. First, although suspected, the cause of the bleeding was not proven because endoscopy was not performed. It is well established that approximately one third of UGI bleeding

episodes 上海皓元医药股份有限公司 in patients with cirrhosis are the result of other causes, rather than esophageal varices.[40, 41] Second, most likely, the patients who would not receive endoscopy would probably be the sickest ones and therefore with the most dismal outcome. Therefore, inclusion of these patients in the analysis might further enhance the differences in the outcomes of VB in patients with and without HCC. Furthermore, and although it seems that patients with HCC without secondary prophylaxis were more sick than the ones who received secondary prophylaxis, which may have influenced the physician’s opinion, it could be that there are other factors that influenced this decision that are not included in the analysis. Unfortunately, the study design does not allow analysis of the effect of sorafenib treatment on variceal bleeding. It has been established, both in animal and human studies, that sorafenib has a portal hypotensive effect, perhaps through an inhibition of angiogenesis.[42, 43] Therefore, there could be an effect of the administration of this drug on the outcomes. In the present study, sorafenib was administered exclusively to patients with advanced HCC; therefore, it is logical to speculate that lack of sorafenib could further worsen the outcome of these patients, who already have a dismal prognosis. Another limitation of the study is the uneven distribution of the etiologies among patients with and without HCC.

Our results suggest the lack of evolutionary adaptation of differ

Our results suggest the lack of evolutionary adaptation of different cuckoo gentes to their

corresponding hosts in terms of egg find more shape. However, our analyses revealed that cuckoo eggs showed a geographical difference in egg shape. “
“Fever is part of an acute phase response that organisms launch to defend themselves against an invasion by microbial pathogens such as bacteria and viruses. The elevation of an individual’s body temperature necessary to achieve a fever is considered energetically costly, and variation in the expression of the febrile response has been reported with respect to season, sex and the reproductive status of an animal. The effects of these parameters on fever responses are well characterized for laboratory rodents, but comparable data from wild rodents are currently lacking. We evaluated the febrile response of wild highveld mole-rats Cryptomys hottentotus pretoriae to lipopolysaccharide (LPS) during winter and summer. This social rodent retains its breeding potential throughout the year and exhibits a reproductive division of labour. Highveld mole-rats increased their body temperature to a greater degree in response to a dose of 1 mg kg−1 LPS than to saline or handling alone. The fever response did not differ between seasons, while the stress-induced

BI-2536 hyperthermia 上海皓元 in response to handling was greater in summer compared with winter. In contrast, males and breeders exhibited larger changes in body temperature following LPS administration than females and non-breeders, respectively. These findings are in accordance with those reported for laboratory species and suggest that general principles govern the modulation of innate immune responses such as fever among small

mammals. “
“The systematics of Peripatopsis moseleyi (Wood-Mason, 1879), a widely distributed South Africa velvet worm species, was examined to test the occurrence of cryptic lineages within this taxon. A total of 81 specimens of P. moseleyi were collected from 12 localities throughout its known distribution in the Eastern Cape and KwaZulu-Natal provinces of South Africa. All specimens were sequenced for a 631 bp fragment of the mitochondrial cytochrome oxidase one subunit (COI) locus, while a 717 bp pair fragment of the 18S rDNA locus was sequenced for a single sample for each of the clades evident from the COI topology. DNA sequence data were analysed using maximum parsimony and Bayesian inferences, while a haplotype network was constructed and an analysis of molecular variation was conducted. Gross morphological characteristics, such as the number of pre-genital leg pairs, the genital areas and colour variation in each sample locality were examined.

[159, 161, 166] Reported factors related to HBeAg negative conver

[159, 161, 166] Reported factors related to HBeAg negative conversion were ALT level (high), and the history of IFN therapy in the past.[159, 166] If hepatitis associated with lamivudine-resistant HBV occurs, adefovir resistance develops if therapy is changed from lamivudine to adfovir, but if lamivudine+adefovir combination therapy is administered, Abiraterone manufacturer the reported incidence of HBV resistant to both agents is low.[191] Entecavir therapy is also administered to patients with lamivudine-resistant HBV (including cases unresponsive to lamivudine). The short-term results for entecavir therapy are good, and in some USA studies reported an HBV DNA negative

conversion rate of 21% at 1 year, and 34–40% at 2 years, and an ALT normalization rate of 65% at 1 year, and 81% at 2 years.[192, 193] However, the appearance of entecavir-resistant HBV associated with long term administration of entecavir has been confirmed. The incidence of entecavir-resistant HBV was 6% at 1 year and 8–13% at 2 years, and rebound of the HBV DNA load due to entecavir-resistant

HBV was 1% at 1 year and 9% at 2 years. A Japanese study reported favorable results with a HBV DNA negative conversion rate of 16% at 6 months and 33% at 1 year, and ALT normalization rate of 78% at 6 months and 81% at 1 year,[194-196] although entecavir-resistant HBV was detected in 26% STI571 cost of cases up to year 3, in whom hepatitis rebounded in 40%.[196] In this way, entecavir therapy for lamivudine-resistant (or unresponsive) HBV may also produce viral strains resistant to entecavir. Recommendations Lamivudine+adefovir combination therapy is recommended for treatment of lamivudine-resistant HBV. Entecavir therapy of lamivudine-resistant HBV may also produce viral strains resistant to entecavir. Reported adefovir-resistant

mutations include rtA181V/T, rtI233V and rtN236T in the HBV polymerase reverse transcriptase (rt) region. Of these mutations, in vitro and in vivo testing has demonstrated sensitivity to both lamivudine and entecavir for the rtN236T mutation, but lamivudine resistance for the rtA181V mutation.[7, 197] 上海皓元医药股份有限公司 In 132 patients with lamivudine-resistant HBV treated with lamivudine+adefovir combination therapy, multiple resistant strains were seen in 3 cases before the commencement of adefovir therapy, and in 2 further cases after therapy commenced (overall incidence 4%).[168] Entecavir+adefovir combination therapy is administered to patients with HBV resistant to both lamivudine and adefovir, with undetermined results. On the other hand, in reports from Europe, in cases with resistance to lamivudine or adefovir monotherapy, or resistant/unresponsive to lamivudine+adefovir combination therapy, administration of the new agent tenofovir (median treatment period 23 months) yielded HBV DNA negative conversion in 79% of cases, HBeAg negative conversion in 24%, and HBsAg negative conversion in 3%.

And wherever possible, we identified the proteins and signaling p

And wherever possible, we identified the proteins and signaling pathways responsible for these activities. Our strategy was always to first understand normal cholangiocyte function in order to allow the generation of hypotheses relevant to disease.42–66 So, what’s the lesson here? Well, there are several, and they’re all important. First, physician-scientists develop the questions they study in the laboratory from the patients they see in the clinic. Second, don’t always Opaganib mouse listen to your senior colleagues (sorry Jurgen). Third, propose the questions and then make sure you develop the necessary techniques rather than

the other way around (avoid the “have technique, looking for a question” approach). Finally, make sure you’re having fun; insights require enthusiasm. I never purposefully aspired to administrative leadership positions

within academic medicine. Indeed, for the first 10 years of my career, I focused entirely on developing my laboratory and a focused clinical practice, and turned down multiple job opportunities GDC-0068 in vitro as Division Chief at other institutions. However, in the late 1980s, Bob Frye, a world-renowned cardiologist, became the fourth Chair of the Department of Medicine (DOM) at the Mayo Clinic, and asked me to be his Vice-Chair for Research. By then, my laboratory was established and progressing well. In addition, I greatly admired Bob and felt strongly that under his leadership the DOM had the capacity to expand its research enterprise, and so I accepted the position.

I found that I liked medical administration. I enjoyed both developing strategy and executing tactics, and found building something new was professionally rewarding (not unlike what one does in the laboratory). Indeed, colleagues have described my management style as one of “visionary pragmatism”; I like to decide where to go and then execute in getting there. When the position of Chief of GI at the Mayo Clinic became open, I was offered the opportunity and accepted it with enthusiasm. I spent 9 years as Chief of GI and consider my major contributions to be doubling its size by recruiting outstanding individuals, expanding 上海皓元 the research enterprise, and restructuring the division into interest groups and focused clinics. Because of the disciplined approach that I learned from the Jesuits (i.e., “age quod agis”, that is, “do what you’re doing”), as well as my willingness to delegate to the outstanding individuals who helped me lead the division, especially Keith Lindor, I found that I could continue to expand my research program, maintain a focused clinical practice, and lead what ultimately evolved into the largest (some would say the best) division of gastroenterology, all at the same time.

We set a 2-fold threshold for changes in gene expression per indi

We set a 2-fold threshold for changes in gene expression per individual patient, i.e., changes lower than 0.5-fold were considered down-regulation and higher than 2-fold were considered up-regulation. Statistical analysis was performed using a two-tailed paired

t test and Wilcoxon matched-pairs test and differences were considered significant when P < 0.05. For miRNA data analysis an additional manual screen was performed in order to check that the control group had consistent Ct values (Ct obtained for all three HL samples and less than 1.5 Ct variation between all three). All miRNAs that had deviating Ct values between the three HL samples were excluded from the analysis. Statistical analysis was performed using a two-tailed t test and differences were considered significant when P < 0.05. Softwares TargetScan24 and PicTar23 were used for ABC 3′untranslated region (UTR) target prediction of cellular miRNAs. Additionally,

PS-341 in vitro 3′UTR sequences were manually screened for miRNA seed-matching sequences. Predictions are presented in Supporting Tables S1, S2. Luciferase reporters were made selleck by cloning of ABC 3′UTR sequences (Tables S3, S4), in the renilla luciferase gene in the psiCheck-2 vector (Promega, Madison, WI). Constructs with mutated miRNA seed sequence in the ABC genes (nt 2 to 6) were synthesized by IDT (Coralville, IA). Primary miRNA (pri-miRNA) sequences were amplified (primer sequences, Table S3) from human adult normal breast tissue genomic DNA (Biochain, Hayward, CA). miRNA expression plasmids were made by cloning of the pri-miRNAs in the pcDNA6.2 vector medchemexpress (Invitrogen). All constructs were verified by sequencing (Macrogen, Seoul, Korea). Human embryonic kidney (HEK) 293T cells were cultured according to the American Tissue Culture Collection (ATCC) instructions.

Cells were plated in 6-, 24-, or 96-well plates 1 day prior to transfection. Transfections were performed with Lipofectamine 2000 or LTX reagent (Invitrogen) according to the manufacturer’s instructions. For luciferase assays, HEK293T cells were cotransfected with 5 ng of Luc-ABC reporter that contains both firefly and renilla luciferase genes and 150 ng of the corresponding miRNA expression constructs. Expression values when the miR-Control (miR-Ctrl) was transfected were set at 1. Transfected cells were assayed at 72 hours posttransfection and firefly and renilla luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The relative luciferase activity was calculated as the ratio between the renilla and firefly luciferase activities. In order to perform ABC gene and miRNA expression profiling, tissues were sampled from HCC and AHL from 19 patients. Three patients received chemotherapy (FR06, FR16, and FR17) prior to sampling, whereas 16 were untreated.