Augmenter of liver regeneration enhances cell proliferation through the microRNA-26a/Akt/cyclin D1 pathway in hepatic cells.

AIM: Hepatocytes can proliferate and regenerate when injured by toxins, viral infections, and so on. Augmenter of liver regeneration (ALR) is a key regulator of liver regeneration, but the mechanism is unknown. The role of ALR in other cell types is not known. In the present study, we investigated the relationship between microRNA (miRNA)-26a and ALR in the Huh7 cell line and adipose tissue-derived mesenchymal cells from chronic liver disease patients and healthy individuals. METHODS: Huh7 cells were transfected independently with ALR and miRNA-26a expression vectors, and their effects on cell proliferation, the expression of miRNA-26a, and activation of the phosphatase and tensin homolog and Akt signaling pathways were determined. The experiments were repeated on mesenchymal stem cells derived from healthy individuals and chronic liver disease patients to see whether the observations can be replicated in primary cells. RESULTS: Overexpression of ALR or miRNA-26a resulted in an increase of the phosphorylation of Akt and cyclin D1 expression, whereas it resulted in decreased levels of p-GSK-3ß and phosphatase and tensin homolog in Huh7 cells. The inhibition of ALR expression by ALR siRNA or anti-miR-26a decreased the Akt/cyclin D1 signaling pathway, leading to decreased proliferation. Mesenchymal stem cells isolated from the chronic liver disease patients had a higher ALR expression, while the mesenchymal stem cells isolated from healthy volunteers responded to the growth factor treatments for increased ALR expression. It was found that there was a significant increase in miRNA-26a expression and proliferation. CONCLUSIONS: These data clearly showed that ALR induced the expression of miRNA-26a, which downregulated phosphatase and tensin homolog, resulting in an increased p-Akt/cyclin D1 pathway and enhanced proliferation in hepatic cells.

Characterization of liver specific promoters in a foamy viral vector pMD09.

Foamy viruses (FVs) or spumaviruses are retroviruses that are explored as vectors for gene therapy. The good feature of foamy viruses is its broad tropism; however, their infections result in non-targeted gene expression. Here, we attempted to design the liver targeted viral gene delivery by employing liver specific gene promoters like albumin (ALB), transthyretin (TTR) and hepatitis B virus (HBV) promoters. We compared the relative gene expression of liver specific promoters versus the U3 promoter in liver cell line (HepG2) and non-liver cell lines: human fibrosarcoma cell line (HT1080), baby hamster kidney cell line (BHK), human embryonic kidney cell line (HEK 293T) and cervical cancer cell line (HeLa). We have found that the promoter exchange didn't affect viral assembly. The ability to drive gene expression was best with TTR promoter which was followed by HBV and ALB promoter. The use of TTR, HBV and ALB promoters are helpful in achieving liver specific gene expression. Keywords: foamy virus; gene therapy; liver; albumin; transthyretin promoter; HBV promoter.

TGF-ß induces liver fibrosis via miRNA-181a-mediated down regulation of augmenter of liver regeneration in hepatic stellate cells.

OBJECTIVE: To study the role of miRNA-181a and augmenter of liver regeneration in TGF-ß-induced fibrosis in hepatic stellate cells. METHODS: LX2 cells were treated with 20 ng/ml TGF-ß for 24 h. miRNA-181a, ALR plasmid and empty vectors were transfected using siPORT NeoFx reagent. Cells were harvested after 48 h or 72 h of transfection for protein or RNA analysis. Western blotting was performed for ALR, TGF-ß receptor II (TGFß-RII), collagen 1A1 (COLL1A1), alpha-smooth muscle cell actin (α-SMA), rac1, E-cadherin and ß-actin. Quantitative RT-PCR was performed for ALR, GAPDH, miRNA-181a or 5S rRNA. RESULTS: TGF-ß induced the expression of miRNA-181a, which in turn down-regulated ALR thereby induced the fibrosis markers, such as COLL1A1, α-SMA and rac1 in hepatic stellate cells. Over-expression of miRNA-181a down-regulated expression of ALR and up-regulated expression of fibrosis markers. On the other hand, ALR over-expression resulted in a decrease in miRNA-181a expression and fibrosis markers. Over-expression of ALR also inhibited the expression of TGFß-RII and increased expression E-cadherin. CONCLUSION: TGF-ß induced miRNA-181a, which in turn induced fibrosis, at least in part, by inhibiting ALR. ALR inhibited TGF-ß action by decreasing the expression of TGFß-RII, thereby inhibiting miRNA-181a expression and fibrosis markers. ALR could serve as a potential molecule to inhibit liver fibrosis.

Surgical treatment pattern and outcomes in epithelial ovarian cancer patients from a cancer institute in Kerala, India.

OBJECTIVE: To evaluate the treatment and survival pattern of patients with advanced epithelial ovarian cancer. METHODS AND RESULTS: Retrospective study of all advanced epithelial ovarian cancer patients treated in the department of gynaecologic oncology from an academic centre, in a four year period from 1 January 2008-31 December 2011. SELECTION CRITERIA: All patients with advanced epithelial ovarian cancer (stage III and IV) who underwent surgery from 2008-2011and had a follow-up of at least three months after completion of treatment were included. The decision on whether primary surgery or neoadjuvant chemotherapy (NACT) in advanced ovarian cancer was based on age, performance status, clinical and imaging findings. RESULTS: A total of 178 cases of epithelial ovarian cancer were operated on during this four year period. Among them 28 patients were recurrent cases, 22 had early stages of ovarian cancer, and the rest 128 had stage III and IV ovarian cancer. In these 128 patients, 50(39.1%) underwent primary surgery and 78(60.9%) had NACT followed by surgery. In the primary surgery group 36(72.0%) patients had optimal debulking while in the NACT group 59(75.6%) patient had optimal debulking. With a median follow-up of 34 months, the median overall survival (OS) and progression free survival (PFS) was 53 and 49 months respectively. Patients who underwent primary surgery had better median PFS than patients who had NACT (56 months versus 39 months, p = 0.002). In stage III C the difference median PFS was significant for those treated with primary surgery when compared with NACT (55 months versus 39 months, p = 0.012). In patients who had optimal debulking to no residual disease (n = 90), primary surgery gave a significant improved PFS (59 months versus 38 months, p = 0.001) when compared with NACT. In univariate analysis, NACT was associated with increased risk of death (HR: 0.350; CI: 0.177-0.693). CONCLUSION: In advanced epithelial ovarian cancer, primary surgery seems to have a definite survival advantage over NACT in patients who can be optimally debulked to no residual disease.

Nonalcoholic fatty liver disease: the concept and confusion.

Nonalcoholic fatty liver disease (NAFLD) is generally considered as a disease associated with diabetes mellitus type 2. But on a closer evaluation we realize a host of confusion associated with this from the nomenclature, diagnosis to pathogenesis. The term refers to a spectrum ranging from steatosis to steatosis with inflammation (NASH) to cirrhosis in the absence of alcohol abuse. But in fact NAFLD is a vague term for a spectrum of diseases which differ not only in the clinical presentation but also in the etiology. NAFLD is loose to incorporate so many etiologies excluding alcoholism and few other "known" etiologies, presenting as fat in liver. Considering the diverse etiologies there is a need for personalized management in NAFLD, which at present is difficult. Currently fatty liver disease could be considered as an added Hepato-cardiovascular-renal and cancer risk factor rather than a specific diagnosis.

Conjoined epigastric heteropagus twins: excision of a parasitic twin from the anterior abdominal wall of her sibling.

Epigastric heteropagus twins (EHT) are an exceedingly rare form of asymmetric conjoined twins in whom the dependent twin (parasite) is attached to the right or left upper abdomen of the dominant part (autosite). Such a case observed at our institution with 34 month follow-up is presented here and the surgical technique described. A magnetic resonance imaging (MRI)-supported surgical separation of the parasite with successful closure of the abdominal wall defect of the autosite was performed. Follow-up studies showed an autosite which was alive and in optimal health. A comprehensive review including data from English and non-English literature is presented.

Application of positive ion chemical ionisation and tandem mass spectrometry combined with gas chromatography to the trace level analysis of ethyl carbamate in bread.

A rapid, sensitive and selective method has been developed and validated for the analysis of the contaminant ethyl carbamate (EC) in bread products at the part-per-billion level. The new procedure uses positive ion chemical ionisation (PICI) and tandem mass spectrometry (MS/MS), combined with gas chromatography (GC), on a 'bench-top' triple-quadrupole mass spectrometer. Ammonia was the PICI reagent gas of choice because of its ability to produce abundant [M+H]+ and [M+NH4]+ ions from EC and deuterium-labelled EC (LEC) used as an internal standard. For identification and quantification, selected reaction monitoring (SRM) was used to follow the precursor-to-product ion transitions of m/z 107 --> 90, m/z 107 --> 62 and m/z 90 --> 62 for EC, as well as m/z 112 --> 63 for the LEC internal standard. The limits of detection and quantification were 0.6 and 1.2 microg kg(-1), respectively, and the recovery of the method was 101 +/- 10% at 10 microg kg(-1) and 98 +/- 5% at 100 microg kg(-1). The precision of the method, established under conditions of intermediate reproducibility, did not exceed a relative standard deviation of 7%. The quantitative performance of the new GC/PICI-SRM procedure compared favourably with that of a reference method based on GC/MS and selected ion monitoring (correlation coefficient, r = 0.997). However, the new method had the advantages of reduced sample preparation time, improved sensitivity and unambiguous identification of EC at all concentrations. Application of the new method to the analysis of 50 UK breads showed that levels of EC ranged from 0.6 to 2.3 microg kg(-1) in retail products and from 3.1 to 12.2 microg kg(-1) for breads prepared using domestic breadmaking machines (dry weight basis). Toasting bread in a domestic toaster led to increases of between two- and three-fold in mean EC concentrations.

Results of the Mutaf procedure in patients with gastroesophageal reflux disease.

Operations to treat complicated gastroesophageal reflux disease (GERD) have become the third most commonly performed general surgical procedure in large pediatric centers, and many surgical techniques have come into use to prevent gastroesophageal reflux. This paper presents our experience with gastric tube cardioplasty, a new procedure described by Oktay Mutaf (Mutaf procedure). Eleven children with GERD were successfully operated on. The postoperative period was uneventful, and all patients were symptom-free.

Fetus in fetu and cystic rectal duplication in a newborn.

A case of sacrococcygeal teratoma is presented with characteristics of fetus in fetu in association with rectal duplication and bilateral cryptorchidism.

Morphological and physiological findings of fetal adrenal grafts in rats who underwent bilateral staged adrenalectomy.

Earlier studies have shown successful transplantation of fetal grafts into the greater omentum of rats. This report evaluates the effects of fetal adrenal glands in adult rats, who underwent bilateral staged adrenalectomy. At first we carried out a preliminary study to investigate the outcome of bilateral adrenalectomy: There were three groups each consisting of 10 rats. In Group 1 we performed bilateral adrenalectomy, and all rats died postoperatively within 8 hours. In Group 2 the rats underwent sham procedure, and only one rat died. Group 3 was for control and all rats survived. After the preliminary study we constructed a 4th group (Tx group) with 20 rats. We performed a left adrenalectomy and immediately transplanted fetal adrenal glands into the greater omentum (stage 1). All animals survived the operation. After 6 weeks we did a second laparotomy and excised the right adrenal gland (stage 2). All of rats from Group 4 compared to Group 1 did not die postoperatively within 8 hours. 6 rats survived staged bilateral adrenalectomy with fetal grafts. Histologic investigation of fetal grafts revealed well developed cortex containing all three layers, but we could not find any medullary cells. In view of these findings we speculate that the fetal adrenal grafts have functioned in this model.

Does fibrin glue cause foreign body reactions?

The study was performed in 20 Wistar rats. Approximately 4 mm perforations were created in the left colon of ten rats to which fibrin glue was applied (Group D). The control group had the same injury without any therapeutic or surgical measures (Group K). Of the 20 rats in both groups none died during the postoperative period. All animals sacrificed on the 11th postoperative day. All ten rats of Group D displayed inflammatory foreign body reactions. The control group showed no such reaction.