Reporter assays for studying quadruplex nucleic acids.

DNA and RNA G-quadruplexes have gained increasing attention due to their potential role in a wide range of biological functions. The majority of functional studies characterize the influence of quadruplexes in gene expression including transcription and translation. Many of these studies have used reporter assays to elucidate the effect of quadruplexes at certain positions in promoters and untranslated mRNA regions (UTRs). Reporter assays are the preferred method to ascertain the biological function of DNA or RNA G-quadruplexes intracellularly due to their ready availability, fast cloning and experimental setup and reproducibility. Moreover, these reporter assays are also helpful to compare or screen for selectivity and efficacy of small molecules that target DNA and RNA G-quadruplexes in the cellular context. Here we briefly discuss various aspects of reporter assays followed by a review of available studies using reporter assays to understand the role and functions of DNA and RNA quadruplexes in gene expression.

Comparative investigation of the genomic regions involved in antigenic variation of the TprK antigen among treponemal species, subspecies, and strains.

Although the three Treponema pallidum subspecies (T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum), Treponema paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme cause clinically distinct diseases, these pathogens are genetically and antigenically highly related and are able to cause persistent infection. Recent evidence suggests that the putative surface-exposed variable antigen TprK plays an important role in both treponemal immune evasion and persistence. tprK heterogeneity is generated by nonreciprocal gene conversion between the tprK expression site and donor sites. Although each of the above-mentioned species and subspecies has a functional tprK antigenic variation system, it is still unclear why the level of expression and the rate at which tprK diversifies during infection can differ significantly among isolates. To identify genomic differences that might affect the generation and expression of TprK variants among these pathogens, we performed comparative sequence analysis of the donor sites, as well as the tprK expression sites, among eight T. pallidum subsp. pallidum isolates (Nichols Gen, Nichols Sea, Chicago, Sea81-4, Dal-1, Street14, UW104, and UW126), three T. pallidum subsp. pertenue isolates (Gauthier, CDC2, and Samoa D), one T. pallidum subsp. endemicum isolate (Iraq B), the unclassified Fribourg-Blanc isolate, and the Cuniculi A strain of T. paraluiscuniculi. Synteny and sequence conservation, as well as deletions and insertions, were found in the regions harboring the donor sites. These data suggest that the tprK recombination system is harbored within dynamic genomic regions and that genomic differences might be an important key to explain discrepancies in generation and expression of tprK variants among these Treponema isolates.

"Insulin-like" effects of palmitate compromise insulin signalling in hypothalamic neurons.

Saturated fatty acids are implicated in the development of metabolic diseases, including obesity and type 2 diabetes. There is evidence, however, that polyunsaturated fatty acids can counteract the pathogenic effects of saturated fatty acids. To gain insight into the early molecular mechanisms by which fatty acids influence hypothalamic inflammation and insulin signalling, we performed time-course experiments in a hypothalamic cell line, using different durations of treatment with the saturated fatty acid palmitate, and the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA). Western blot analysis revealed that palmitate elevated the protein levels of phospho(p)AKT in a time-dependent manner. This effect is involved in the pathogenicity of palmitate, as temporary inhibition of the PI3K/AKT pathway by selective PI3K inhibitors prevented the palmitate-induced attenuation of insulin signalling. Similar to palmitate, DHA also increased levels of pAKT, but to a weaker extent. Co-administration of DHA with palmitate decreased pAKT close to the basal level after 8 h, and prevented the palmitate-induced reduction of insulin signalling after 12 h. The monounsaturated fatty acid oleate had a similar effect on the palmitate-induced attenuation of insulin signalling, the polyunsaturated fatty acid linoleate had no effect. Measurement of the inflammatory markers pJNK and pNFκB-p65 revealed tonic elevation of both markers in the presence of palmitate alone. DHA alone transiently induced elevation of pJNK, returning to basal levels by 12 h treatment. Co-administration of DHA with palmitate prevented palmitate-induced inflammation after 12 h, but not at earlier timepoints.