Potentiation of concurrent expression of lipogenic genes by novel strong promoters in the oleaginous microalga Phaeodactylum tricornutum.

There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes. Regulatory role of the promoters was preliminarily assessed by using GUS reporter which demonstrated strong GUS expression. Thereafter, two key lipogenic genes including malic enzyme (PtME) and 5-desaturase (PtD5b), were overexpressed by the two promoters Pt202 and Pt667, respectively, in P. tricornutum. Combinatorial gene overexpression did not impair general physiological performance, meanwhile neutral lipid content was remarkably increased by 2.4-fold. GC-MS analysis of fatty acid methyl esters revealed that eicosapentaenoic acid (EPA; C20:5) was increased significantly. The findings augment a crucial kit to microalgal genetic tools that could facilitate the multiple-gene expression driven by various promoters, and promote microalgae for industrial bioproduction.

Constitutive and Chloroplast Targeted Expression of Acetyl-CoA Carboxylase in Oleaginous Microalgae Elevates Fatty Acid Biosynthesis.

Photosynthetic microalgae are of burgeoning interest in the generation of commercial bioproducts. Microalgae accumulate high lipid content under adverse conditions, which in turn compromise their growth and hinder their commercial potential. Hence, it is necessary to engineer microalgae to mitigate elevated lipid accumulation and biomass. In this study, we identified acetyl-CoA carboxylase (ACCase) in oleaginous microalga Phaeodactylum tricornutum (PtACC2) and expressed constitutively in the chloroplast to demonstrate the potential of chloroplast engineering. Molecular characterization of transplastomic microalgae revealed that PtACC2 was integrated, transcribed and expressed successfully, and localized in the chloroplast. Enzymatic activity of ACCase was elevated by 3.3-fold, and the relative neutral lipid content increased substantially by 1.77-fold, and lipid content reached up to 40.8% of dry weight. Accordingly, the number and size of oil bodies markedly increased. Fatty acid profiling showed that the content of monounsaturated fatty acids increased, while polyunsaturated fatty acids decreased. This method provides a valuable genetic engineering toolbox for microalgal bioreactors with industrial significance.

Establishment of a colorectal cancer nude mouse visualization model of HIF-1α overexpression.

The aim of the present study was to establish a model of tumor cell growth and visualize HIF-1α overexpression in a nude mouse xenograft model of colorectal cancer (CRC). In the study, HIF-1α lentiviral vector and helper plasmid were co-transfected into 293T packaging cells using a liposome method, and the virus was collected following transfection and used to infect CRC SW480, SW620, LoVo and HCT116 cells. Puromycin was used for the selection and large-scale amplification of the stable HIF-1α expression of green fluorescent protein (GFP)-positive cells. HIF-1α-expressing cells were injected intraperitoneally into a nude mouse xenograft model, and resulting tumor nodules was separated and confirmed using an inverted fluorescence microscope. The results demonstrated that HIF-1α was not expressed in CRC cells in normoxic conditions. When treated with CoCl2, the expression of HIF-1α could be induced in all the cancer cell lines, except SW480. HIF-1α was highly expressed following infection with lentiviral particles. Stable expression of HIF-1α promoted migration in the SW480 cells. Following intraperitoneal injection of nude mice with SW480-HIF-1α, a significant number of tumor nodules formed in the intestinal wall compared with the controls (P<0.05). The successful construction of the dual expression HIF-1α and GFP visualization xenograft model provides a good foundation for the screening of HIF-1α-related functions and for investigating the therapeutic potential of drugs that target HIF-1α.

In vivo induction of CYP in mice by carbamazepine is independent on PXR.

BACKGROUND: The antiepileptic drug carbamazepine (CBZ) is a typical inducer of cytochrome P450 (CYP) 3A and 2C in the clinic. It is considered a strong constitutive androstane receptor activator, however both CBZ and its main metabolite CBZ 10, 11-epoxide have been reported to be pregnane X receptor (PXR) activators whose maximal efficacy and potency are comparable with the human PXR ligand rifampicin. It is unknown whether or not PXR plays a substantially important role in in vivo induction of CYP by CBZ administration. METHODS: In this study, wild type and Pxr-/- mice were administered with CBZ for 5 days. Serum and liver samples were collected and subjected to hepatotoxicity assessment and CYP induction analysis. RESULTS: CYP2b, 2c and 3a were induced similarly in terms of transcription level, enzyme activity and protein abundance in both wild type and Pxr-/- mice. Inductive profile of CYPs in mice by CBZ administration accorded with those reported in rats, but differed from clinically reported data. CONCLUSIONS: These data suggest that in vivo induction of CYP in mice by multiple administration of CBZ is independent of PXR. Knowledge of the featured CYP induction profile in mice helps us understand species related CYP induction profiles among rodents and humans resulting from administration of CBZ.

Upregulation of Acetylcholinesterase Mediated by p53 Contributes to Cisplatin-Induced Apoptosis in Human Breast Cancer Cell.

BACKGROUND: The expression of acetylcholinesterase (AChE) could be induced during apoptosis in various cell types. And reduced AChE expression either by siRNA could prevent apoptosis. However, the detailed mechanisms underlying the AChE regulation are largely unknown in human breast cancer cell. MATERIAL AND METHODS: MCF-7 cells were cultured and treated by cisplatin in the absence or presence of p53 siRNA. RESULTS: In this study, the regulation of AChE expression during apoptosis induced by cisplatin, a current used anticancer drug, was investigated in human breast cancer cell line MCF-7. Exposure of MCF-7 cells to cisplatin resulted in apoptosis in a time- and concentration-dependent manner. Meanwhile, the upregulated AChE and p53 were also observed during apoptosis. Silencing interfering RNA directed against p53 blocked the expression of AChE. CONCLUSION: Taken together, these results suggested that AChE expression could be upregulated by the activation of p53 during apoptosis induced by cisplatin in MCF-7 cells.

The early diagnosis of pancreatic cancer and diabetes: what's the relationship?

Pancreatic cancer (PC) has a dismal prognosis as cancer-specific symptoms occur only at an advanced stage. If the cancer is to be discovered early, it will have to be done in asymptomatic individuals. Since the incidence of PC is low, screening for asymptomatic cancer in the general population will not be feasible. Screening will have to be restricted to subjects at high risk for PC. The proportion of PC patients who also have hyperglycemia or diabetes has previously been under appreciated; new data show that up to 80% are either hyperglycemic or diabetic and this can be evident in the pre-symptomatic phase. Diabetes improves following PC resection suggesting that diabetes is caused by the cancer. Conversely, older subjects with new-onset diabetes have an approximately eight fold higher risk of having PC compared to the general population. Recognition of new-onset diabetes as an early manifestation of PC could lead to diagnosis of asymptomatic, early stage PC. However, primary type 2 diabetes is common and PC is relatively uncommon in the general population and the two forms of diabetes are clinically indistinguishable. The success of the strategy to use new-onset hyperglycemia and diabetes as a screening tool to identify subjects with a high likelihood of having asymptomatic PC will depend largely on our ability to differentiate PC-associated diabetes from the more common type 2 diabetes using a (serologic) biomarker.

Distal bile duct cancers complicated with cholangiobronchopleural fistula after ERCP: A case report.

Distal (lower) bile duct cancers arise in the lower half of the biliary tree closer to the small intestine. Biliary disease complicated with cholangiobronchopleural fistula, which may occur in cases of multiple hepatobiliary stones or biliary ascariasis-associated severe infection, has rarely been reported in the literature, particularly following endoscopic retrograde cholangiopancreatography (ERCP). The present study describes the case of a 60-year-old female with distal cholangiocarcinoma complicated with cholangiobronchopleural fistula after ERCP for this rare disease. This complication was likely due to the inability to control retrograde infection following ERCP and, thus, the infection was disseminated. This resulted in mixed infection involving the diaphragm and pleura, and further penetrating the bronchus. The patient was managed with pancreatoduodenectomy and has since remained in good health.

Graft versus host disease following liver transplantation: A case report.

Graft versus host disease (GVHD) is an uncommon complication following liver transplantation. In the present case report, a 53-year-old male hepatitis B virus carrier was diagnosed with primary liver cancer with post-hepatitis cirrhosis. Preoperative cytomegalovirus (CMV), Epstein-Barr virus, coxsackievirus, herpes simplex virus and autoimmune antibody series were negative. Preoperative human leukocyte antigen type was also negative. Following classic orthotropic liver transplantation, postoperative treatment included immunosuppression therapy, infection protection, anti-human immunodeficiency virus therapy and CMV infection protection therapy. Chemotherapy was initiated at day 16 following surgery. At day 26 following the transplantation, the patient developed a fever of unknown cause, and a scattered red rash was observed behind the left ear and on the neck. The patient presented with a fever of unknown cause, rash, symptoms of the digestive tract, leukocytopenia and pancytopenia. A diagnosis of GVHD was confirmed following a skin biopsy. Symptomatic therapies, including antivirals, anti-anaphylaxis drugs and steroids were administered. However, the patient succumbed to infection, acute respiratory distress syndrome and multiple organ failure at day 46 following surgery. Therefore, an effective therapeutic strategy for the treatment of GVHD following liver transplantation is yet to be established, and further research is required prior to such a regimen being developed.

Silibinin inhibits proliferation, induces apoptosis and causes cell cycle arrest in human gastric cancer MGC803 cells via STAT3 pathway inhibition.

BACKGROUND: To investigate the effect of silibinin on proliferation and apoptosis in human gastric cancer cell line MGC803 and its possible mechanisms. MATERIALS AND METHODS: Human gastric cancer cell line MGC803 cells were treated with various concentration of silibinin. Cellular viability was assessed by CCK-8 assay and apoptosis and cell cycle distribution by flow cytometry. Protein expression and mRNA of STAT3, and cell cycle and apoptosis regulated genes were detected by Western blotting and real-time polymerase chain reaction, respectively. RESULTS: Silibinin inhibits growth of MGC803 cells in a dose- and time-dependent manner. Silibinin effectively induces apoptosis of MGC803 cells and arrests MGC803 cells in the G2/M phase of the cell cycle, while decreasing the protein expression of p-STAT3, and of STAT3 downstream target genes including Mcl-1, Bcl-xL, survivin at both protein and mRNA levels. In addition, silibinin caused an increase in caspase 3 and caspase 9 protein as well as mRNA levels. Silibinin caused G2/M phage arrest accompanied by a decrease in CDK1 and Cyclin B1 at protein and mRNA levels.. CONCLUSIONS: These results suggest that silibinin inhibits the proliferation of MGC803 cells, and it induces apoptosis and causes cell cycle arrest by down-regulating CDK1, cyclinB1, survivin, Bcl-xl, Mcl-1 and activating caspase 3 and caspase 9, potentially via the STAT3 pathway.

Expression of vascular endothelial growth factor and basic fibroblast growth factor in acute rejection reaction following rat orthotopic liver transplantation.

The aim of the present study was to investigate the expression levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in acute rejection reaction (ARR) following orthotopic liver transplantation in a rat model. Serum VEGF and bFGF levels were detected using ELISA, and their expression levels in liver and spleen tissues were determined using immunohistochemistry. The mRNA expression levels of VEGF and bFGF were detected by conducting a quantitative polymerase chain reaction during the ARR following orthotopic liver transplantation. The expression levels of VEGF and bFGF in the serum 3 days following liver transplantation were significantly higher compared with those in the other groups (1 and 7 days following transplantation; P<0.01). In addition, the numbers of cells in the liver tissue that were shown to be positive for the expression VEGF and bFGF using immunohistochemistry were significantly higher 3 days following transplantation than at the other time points (P<0.0001). Furthermore, the numbers of cells positive for VEGF and bFGF expression in the spleen detected 3 days following the transplantation surgery were also significantly higher compared with those at the other time points (P<0.01). VEGF and bFGF mRNA expression levels were also increased from 1 day following the surgery and reached a peak at day 3, prior to declining gradually and remaining at a relatively high level. VEGF and bFGF mRNA expression levels changed dynamically, by peaking and then declining, in ARR following orthotopic liver transplantation. These changes may have an important impact on angiogenesis and the inflammatory reaction, and the identification of these changes increases the current understanding of ARR following orthotopic liver transplantation.

Promoter methylation­associated silencing of p27kip1 gene with metastasis in esophageal squamous cell carcinoma.

The aim of the present study was to determine the frequency of p27kip1 promoter methylation in esophageal squamous cell carcinoma (ESCC). The methylation status of the p27kip1 promoter was analyzed by methylation­specific polymerase chain reaction (MSP) in 50 ESCC and matched non­tumor tissues. Cell lines were treated with the demethylation agent 5­aza­2'­deoxycytidine (5­Aza­CdR) and p27kip1 mRNA expression was detected by quantitative polymerase chain reaction. p27kip1 methylation was found in 36% (18/50) of ESCC patients, but only in 12% (6/50) of the corresponding non­tumor tissues (P=0.005). There were statistically significant associations between the presence of methylation and tumor metastasis (P=0.002). The p27kip1 mRNA was lower in ESCC compared with non­tumor tissues (mean ± standard deviation, ­0.886±3.298 vs. 0.988±0.257; P=0.0033). Furthermore, a significant association was identified between the methylation status of the p27kip1 promoter and p27kip1 mRNA expression in the tissue (P<0.01). Thus, demethylation by 5­Aza­CdR was capable of inducing p27kip1 mRNA expression in esophageal cancer cell lines. The high promoter methylation of p27kip1 is a common phenomenon in ESCC, which may be an important mechanism of silencing p27kip1 mRNA expression.

Roles of vascular endothelial growth factor in acute rejection reaction following liver transplantation.

The presently known cytokines that participate in acute rejection of organ transplantation include four categories by order of function: inflammatory cytokines, immunospecific cytokines, inflammatory cell activating cytokines and growth cytokines. Of them, growth cytokines that directly induce division, proliferation and migration of endothelial cells mainly include the vascular endothelial growth factor (VEGF) family and the fibroblast growth factor (FGF) family [1]. Recent studies [2] showed that interactions and time overlap of inflammatory cell infiltration and angiogenesis are the main mechanisms that induce acute rejection (AR) following organ transplantation, which has been demonstrated by the clinical fact that AR symptoms after liver transplantation could only be relieved by combination use of drugs for improving micro vessels and those for improving micro bile ducts. This article is a review of VEGF that mediates inflammatory cell infiltration and angiogenesis in the portal area [3].