Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Development of novel galactosylation method for the expression of recombinant human transferrin in insect cell culture

The objective of this research is to develop a novel galactosylation method for the expression of recombinant human transferrin (hTf) with better N-glycan quality. The baculovirus-insect cell system, consisting of hTf as the model protein, 1,4-galactosyltransferase (1,4-GalT) as the enzyme, and uridine-diphosphogalactose (UDP-Gal) as the sugar nucleotide, has been successfully established. In the early part of the study, fundamental works were carried out to optimize Spodoptera frugiperda (Sf-9) cells growth and mock infection. Serum concentration, different type of media, cell subculturing condition, initial cell density and spent medium carry over had been found to significantly influence the growth kinetics of Sf-9 cells. Multiplicity of infection (MOI) and spent medium carry over were found to have direct impact on viral infectivity. The optimized parameters were then used to evaluate the expression of recombinant hTf and 1,4-GalT in Sf-9 cells. Subsequently, native UDP-Gal levels at normal and upon baculovirus infection produced in Sf-9 cells were monitored using Reverse Phase High Performance Liquid Chromatography. UDP-Gal concentration was discovered to decrease gradually once infected with the recombinant baculovirus. Finally, baculovirus coinfection study was carried out to evaluate the recombinant glycoprotein quality. However, lectin binding analysis using Ricinus communis agglutinin-I, revealed that coexpression between rhTf and -1,4GalT (in vivo) did not show encouraging result due to the reduction of UDP-Gal upon baculovirus infection. This finding suggested that the introduction of -1,4GalT alone was not sufficient for successful galactosylation. However, another strategy was used to overcome the problem. Commercial GalT and UDP-Gal were introduced artificially to the rhTf after it was secreted from cell culture. It was found that the in vitro strategy promoted better Nglycan quality in insect cell

Baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture

Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyze the galactosylation process by the introduction of �- 1,4galactosyltransferase (�-1,4GalT) as the glycosyltransferase of interest, and uridine-5’- diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of �-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better Nglycan quality. Recombinant �-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine �-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal�14GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and �-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of �-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality

UV PROTECTION PROPERTIES OF TOCOTRIENOL IN PHOTOAGING AND SKIN CANCER

Ph.DDOCTOR OF PHILOSOPH

Cell Culture Optmization for the Baculovirus Expression Vector System (BEVS)

An experiment was carried out to study the fundamental factors that affect the growth of Spodoptera frugiperda Sf-9 insect cells. Initial cell density, spent medium carry over and inoculum phase withdrawal significantly influenced the growth kinetics of Sf-9 cells. The percentage of cells infected with Autographa californica multiply-enveloped Nuclear Polyhedrosis Virus (AcMNPV) was obviously affected by spent medium carry over. On the other hand, initial cell density and Multiplicities of Infection (MOI) have minimal influence on infectivity percentage

Baculovirus infection reduced UDP-galactose level in spodoptera frugiperda insect cells

Incomplete glycosylation is always an issue in the expression of recombinant glycoprotein in Baculovirus-Insect Cell Expression System (BEVS). The factors that ensure successful glycosylations are in the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substance donor and the recombinant protein as the substrate acceptor. Insect cell lines have been reported to produce small amount of β-1,4galactosyltransferase (β-1,4GaIT) and thus insufficient for effective galactosylation. In our approach, recombinant β-1, 4GalT is being introduced during protein expression by the co-infection with baculovirus carrying, bovine β-1, 4GalT. In this paper, we report our finding on native UDP-Galactose level at normal and upon baculovirus infection in Spodoptera frugiperda (Sf-9) insect cells. We established and monitored native UDP-Galactose content in Sf-9 insect cells using Reverse Phase High Performance Liquid Chromatography. It was found that UDP-Galactose concentration decreased gradually once infected with the recombinant baculovirus. Although UDP-Galactose content was at 0.009 mg/ml prior infection, the level dropped to almost zero upon five days of infection and thus insufficient for effective galactosylation. This interesting finding suggests that the introduction of β-1,4GalT alone is not sufficient for successful galactosylation

Evidence of (gamma)-Tocotrienol as an apoptosis-inducing, invasion-suppressing, and chemotherapy drug-sensitizing agent in human melanoma cells

To date, the most effective cure for metastatic melanoma remains the surgical resection of the primary tumor. Recently, tocotrienol-rich-fraction has shown antiproliferative effect on cancer cells. To elucidate this anticancer property in malignant melanoma, this study aimed, first, to identify the most potent isomer for eliminating melanoma cells and second to decipher the molecular pathway responsible for its activity. Results showed that the inhibitory effect of gamma-tocotrienol was most potent, which resulted in induction of apoptosis as evidenced by activation of procaspases and the accumulation of sub-G1 cell population. Examination of the prosurvival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of NF-kappaB, EGF-R, and Id family proteins. Meanwhile, gamma-tocotrienol treatment also resulted in induction of JNK signaling pathway, and inhibition of JNK activity by selective inhibitor was able to partially block the effect of gamma-tocotrienol. Interestingly, gamma-tocotrienol treatment led to suppression of mesenchymal markers and the restoration of E-cadherin and gamma-catenin expression, which was associated with suppression of cell invasion capability. Furthermore, synergistic effect was observed when cells were cotreated with gamma-tocotrienol and chemotherapy drugs. Together, our results demonstrated for the first time the anti-invasion and chemonsensitization effect of gamma-tocotrienol against human malignant melanoma cells.link_to_subscribed_fulltex

Anti-inflammatory γ- and δ-tocotrienols improve cardiovascular, liver and metabolic function in diet-induced obese rats

Purpose This study tested the hypothesis that γ- and δ-tocotrienols are more effective than α-tocotrienol and α-tocopherol in attenuating the signs of diet-induced metabolic syndrome in rats. Methods Five groups of rats were fed a corn starch-rich (C) diet containing 68%carbohydrates as polysaccharides, while the other five groups were fed a diet (H) high in simple carbohydrates (fructose and sucrose in food, 25 % fructose in drinking water, total 68 %) and fats (beef tallow, total 24 %) for 16 weeks. Separate groups from each diet were supplemented with either α-, γ-, δ-tocotrienol or α-tocopherol (85 mg/kg/day) for the final 8 of the 16 weeks. Results H rats developed visceral obesity, hypertension, insulin resistance, cardiovascular remodelling and fatty liver. α-Tocopherol, α-, γ- and δ-tocotrienols reduced collagen deposition and inflammatory cell infiltration in the heart. Only γ- and δ-tocotrienols improved cardiovascular function and normalised systolic blood pressure compared to H rats. Further, δ-tocotrienol improved glucose tolerance, insulin sensitivity, lipid profile and abdominal adiposity. In the liver, these interventions reduced lipid accumulation, inflammatory infiltrates and plasma liver enzyme activities. Tocotrienols were measured in heart, liver and adipose tissue showing that chronic oral dosage delivered tocotrienols to these organs despite low or no detection of tocotrienols in plasma. Conclusion In rats, δ-tocotrienol improved inflammation, heart structure and function, and liver structure and function, while γ-tocotrienol produced more modest improvements, with minimal changes with α-tocotrienol and α-tocopherol. The most important mechanism of action is likely to be reduction in organ inflammation

Id1, inhibitor of differentiation, is a key protein mediating anti-tumor responses of gamma-tocotrienol in breast cancer cells

Gamma-tocotrienol has demonstrated anti-proliferative effect on breast cancer (BCa) cells, but mechanisms involved are largely unknown. This study aimed at deciphering the molecular pathways responsible for its activity. Our results showed that treatment of BCa cells with gamma-tocotrienol resulted in induction of apoptosis as evidenced by activation of pro-caspases, accumulation of sub-G1 cells and DNA fragmentations. Examination of the pro-survival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of Id1 and NF-κB through modulation of their upstream regulators (Src, Smad1/5/8, Fak and LOX). Meanwhile, gamma-tocotrienol treatment also resulted in the induction of JNK signaling pathway and inhibition of JNK activity by specific inhibitor partially blocked the effect of gamma-tocotrienol. Furthermore, synergistic effect was observed when cells were co-treated with gamma-tocotrienol and Docetaxel. Interestingly, in cells that treated with gamma-tocotrienol, alpha-tocopherol or β-aminoproprionitrile were found to partially restore Id1 expression. Meanwhile, this restoration of Id1 was found to protect the cells from gamma-tocotrienol induced apoptosis. Consistent outcome was observed in cells ectopically transfected with the Id-1 gene. Our results suggested that the anti-proliferative and chemosensitization effect of gamma-tocotrienol on BCa cells may be mediated through downregulation of Id1 protein. © 2009 Elsevier Ireland Ltd. All rights reserved.link_to_subscribed_fulltex