Use of flow cytometry to rapidly optimize the transfection of animal cells

Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.     

Introduced brown algae in the North East Atlantic, with particular respect toUndaria pinnatifida (Harvey) suringar

The recent introduction of the macroalgaUndaria pinnatifida (Harvey) Suringar into the North Atlantic is the latest of a large number of introductions, which have occurred over many years. Some have been deliberate introductions for mariculture or research, while most have been accidental, via vectors such as shipping and shellfish imports. Not all newly recorded species are introductions; some are thought to be merely extensions of distribution, e.g.Laminaria ochroleuca, while others may have been overlooked previously, e.g.Scytosiphon dotyi. Subsequent to its accidental introduction into the waters around the Mediterranean French coast at Sete, most likely with imported oysters,Undaria was deliberately introduced into the North Atlantic, to Brittany, in 1983 by IFREMER for commercial exploitation.Undaria has since spread from the original sites in Brittany, and is now established at several sites on the south coast of England. This paper discusses the introduced brown algae in the North Atlantic and outlines the establishment ofUndaria in the UK.     

Ethanol production at 45 °C by Kluyveromyces marxianus IMB3 during growth on molasses pre-treated with Amberlite® and non-living biomass

The use of high concentrations of molasses as a fermentation feed-stock for ethanol production is normally precluded by the presence of inhibitory compounds. Use of the thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 in fermentations containing high concentrations of molasses resulted in sub-optimal production of ethanol. The results suggested that this was caused by the presence of inhibitory materials rather than an intolerance to increased concentrations of ethanol. In the current study we describe the pretreatment of molasses preparations with either an Amberlite^® monobed mixed ion-exchange resin or non-living microbial biomass from a local distillery. In the study molasses samples diluted to yield a final sugar concentration of 160?g/l were used as the substrate. Control fermentations using the untreated molasses dilutions yielded a maximum ethanol concentration of 40?g/l, representing 49% of the maximum theoretical yield. Fermentations using molasses samples pre-treated with Amberlite^® or non-living biomass yielded maximum ethanol concentrations of 58 and 54?g/l, representing 71 and 66% of the maximum theoretical yield, respectively. The results suggest that pre-treatment brings about removal of toxic or inhibitory materials from the fermentation feed-stock and we believe that such pre-treatments, particularly using the less expensive non-living biomass preparations may find a role in processes concerned with the commercial production of ethanol from molasses using this microorganism.     

Environmental Effects on Disulfide Bonding Patterns of Bovine κ-Casein

Bovine -casein, the stabilizing protein of the colloidal milk protein complex, has a unique disulfide bonding pattern. The protein exhibits varying molecular sizes on SDS-PAGE ranging from monomer to octamer and above in the absence of reducing agents. Heating the samples with SDS prior to electrophoresis caused an apparent decrease in polymeric distribution: up to 60% monomer after 30min at 90°C as estimated by densitometry of SDS-PAGE. In contrast, heating the samples without detergent at 90 or 37°C caused a significant increase in high-molecular-weight polymers as judged by electrophoresis and analytical ultracentrifugation. In 6 M urea, the protein could be completely reduced, but upon dialysis, varying degrees of polymer reformation occurred depending on the dialysis conditions. Spontaneous reoxidation to polymeric forms is favored at low pH (<5.15) and low ionic strength. The results are discussed with respect to the influence of the method of preparation on the polymer size of -caseins and on their resultant physical chemical properties.     

Coronary vascular volume remodelling in rainbow trout Oncorhynchus mykiss

The 34% increase in relative ventricular mass (M_rv) resulting from chronic anaemia (induced by an intraperitoneal injection of phenylhydrazine hydrochloride) was accompanied by a 117% increase in coronary vascular volume of diploid rainbow trout Oncorhynchus mykiss. Coronary vascular volume of normocythemic triploid fish was similar to that of normocythemic diploid fish despite a larger M_rv. These observations, in combination with previous studies, suggest that the vascularity of compact myocardium in O. mykiss can vary independently of M_rv.     

Critical evaluation of municipal solid waste composting and potential compost markets

Mechanical biological treatment (MBT) of mixed waste streams is becoming increasingly popular as a method for treating municipal solid waste (MSW). Whilst this process can separate many recyclates from mixed waste, the resultant organic residue can contain high levels of heavy metals and physical and biological contaminants. This review assesses the potential end uses and sustainable markets for this organic residue. Critical evaluation reveals that the best option for using this organic resource is in land remediation and restoration schemes. For example, application of MSW-derived composts at acidic heavy metal contaminated sites has ameliorated soil pollution with minimal risk. We conclude that although MSW-derived composts are of low value, they still represent a valuable resource particularly for use in post-industrial environments. A holistic view should be taken when regulating the use of such composts, taking into account the specific situation of application and the environmental pitfalls of alternative disposal routes.     

RUNX3-mediated repression of RUNX1 in B cells

Are there three different RUNX genes in the human genome just to allow differential expression in different cell types or are the RUNX proteins functionally different? Much previous evidence has suggested that the RUNX proteins were functionally redundant but, in human B cells, RUNX1 and RUNX3 have clearly been shown to have different functions at a key developmental stage. RUNX1 is expressed in mature resting B cells but proliferating B cell lines express RUNX3. Repression of RUNX1 by RUNX3 is required to allow the cell proliferation and this provides an experimental system to investigate the mechanism of the different activities of RUNX1 and RUNX3 in these cells. J. Cell. Physiol. 221: 283–287, 2009. © 2009 Wiley-Liss, Inc.     

Methylation of HOXA9 and ISL1 Predicts Patient Outcome in High-Grade Non-Invasive Bladder Cancer

Introduction

Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis.

Methods

Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases.

Results

The ISL1 genes’ promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM).

Conclusions

In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.