An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells

RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.     

Genetic polymorphisms and plasma levels of transforming growth factor-beta(1) in Chinese patients with tuberculosis in Hong Kong

Susceptibility to tuberculosis (TB) may be affected by host genetic factors. Elevated levels of transforming growth factor-beta 1 (TGF-β₁) were found in plasma of patients with active TB compared with those of healthy contacts. To investigate the association of TGF-β₁ gene polymorphisms (C-509T and T869C) and plasma levels with the risk of TB in Hong Kong Chinese adults, a case-control study was carried out on 174 active TB patients and 174 healthy controls matched for age, gender and smoking. Blood samples from 180 blood donors served as another control group. Genotyping was carried out on genomic DNA using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma TGF-β₁ was measured by commercially available ELISA kit. We found no differences in the distribution of genotypes or alleles of TGF-β₁ gene polymorphisms at C-509T and T869C between patients and either group of healthy controls. Patients with TB had elevated plasma TGF-β₁ levels compared with healthy controls irrespective of their genotypes (p < 0.001). In conclusion, TGF-β₁ gene polymorphism at C-509T and T869C is not associated with TB susceptibility in Hong Kong Chinese adults, but elevated plasma TGF-β₁ levels suggests that this cytokine may play a role in the pathogenesis of tuberculosis.     

Opportunities and limitations of SELDI-TOF-MS in biomedical research: practical advices

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, or surface-enhanced laser desorption/ionization ProteinChip technology, has been widely used in obtaining the quantitative profiles of tissue proteomes, particularly plasma proteomes. Its high-throughput nature and simplicity in its experimental procedures have allowed this technology to become a popular research tool for biomarker discovery in the past 5 years. After accumulating more research experiences, researchers now have a better understanding of the characteristics and limitations of this technology, as well as the pitfalls in biomarker research, by undertaking a comparative proteomic approach. This review provides an overview of the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, discusses its limitations and provides some possible solutions to help apply this technology to biomarker research.     

A comparison of the LDL-cholesterol lowering efficacy of plant stanols and plant sterols over a continuous dose range: results of a meta-analysis of randomized, placebo-controlled trials

Purpose

To determine if plant stanols and plant sterols differ with respect to their low-density lipoprotein cholesterol (LDL-CH) lowering efficacies across a continuous dose range.

Methods

Dose-response relationships were evaluated separately for plant stanols and plant sterols and reductions in LDL-CH, using a first-order elimination function.

Results

Altogether, 113 publications and 1 unpublished study report (representing 182 strata) complied with the pre-defined inclusion and exclusion criteria and were included in the assessment. The maximal LDL-CH reductions for plant stanols (16.4%) and plant stanol ester (17.1%) were significantly greater than the maximal LDL-CH reductions for plant sterols (8.3%) and plant sterol ester (8.4%). These findings persisted in several additional analyses.

Discussion and conclusions

Intakes of plant stanols in excess of the recommended 2 g/day dose are associated with additional and dose-dependent reductions in LDL-CH, possibly resulting in further reductions in the risk of coronary heart disease (CHD).     

Addition of ammonia and/or oxygen to an ionic liquid for delignification of miscanthus

Ammonia and/or oxygen were used to enhance the delignification of miscanthus dissolved in 1-ethyl-3-methylimidazolium acetate at 140 °C. After dissolution of the gas at 9 bar, water was added as antisolvent to regenerate the dissolved biomass. In a next step, an acetone/water mixture was used to remove carbohydrate-free lignin from the regenerated biomass. The lignin content in the final product was around 10%, much lower than the ca. 23% lignin content of the raw dry miscanthus. This lignin reduction is achieved without diminution of cellulose or of total carbohydrates recovered, relative to the recovery achieved with the ionic liquid pretreatment in contact with air or nitrogen.     

37-kDa laminin receptor precursor mediates GnRH-II-induced MMP-2 expression and invasiveness in ovarian cancer cells

GnRH-II enhances ovarian cancer cell invasion in an autocrine manner. We have now found that GnRH-II increases 37-kDa laminin receptor precursor (LRP) production in GnRH receptor (GnRHR)-positive OVCAR-3 and CaOV-3 ovarian cancer cells, while small interfering RNA (siRNA)-mediated depletion of GnRH-II or GnRHR mRNA abrogates this. The invasiveness of ovarian cancer cells is also reduced >85% by siRNA-mediated knockdown of LRP levels and >50% by pretreatment of Matrigel with a synthetic peptide that blocks interactions between laminin and the 67-kDa nonintegrin laminin receptor which comprises two LRP subunits. Conversely, overexpressing LRP in CaOV-3 cells increases their invasiveness 5-fold, while overexpressing LRP with a nonfunctional laminin-binding site does not. Depletion of LRP by siRNA treatment reduces CaOV-3 cell attachment to laminin-coated plates by ～80% but only reduces their binding to Matrigel by ～20%. Thus, while LRP influences CaOV-3 cell adhesion to laminin, LRP must act in other ways to enhance invasion. Matrix metalloproteinases (MMPs) are key mediators of invasion, and LRP siRNA treatment of OVCAR-3 and CaOV-3 cells inhibits MMP-2 but not MMP-9 mRNA levels. Overexpressing LRP in these cells increases MMP-2 production specifically, while a laminin-binding deficient LRP does not. Importantly, LRP siRNA treatment abolishes GnRH-II-induced MMP-2 production, and invasion in OVCAR-3 and CaOV-3 cells, which was also seen after MMP-2 siRNA treatment. These results suggest that GnRH-II-induced LRP expression increases the amount of the 67-kDa nonintegrin laminin receptor, which appears to interact with laminin in the extracellular matrix to promote MMP-2 expression and enhance ovarian cancer cell invasion.