Unique reporter-based sensor platforms to monitor signalling in cells

Introduction: In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. <p/>Methods: Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. <p/>Findings: We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. <p/>Conclusions: We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein

Molecular Networking Reveals Two Distinct Chemotypes in Pyrroloiminoquinone-Producing Tsitsikamma favus Sponges

The temperate marine sponge, Tsitsikamma favus, produces pyrroloiminoquinone alkaloids with potential as anticancer drug leads. We profiled the secondary metabolite reservoir of T. favus sponges using HR-ESI-LC-MS/MS-based molecular networking analysis followed by preparative purification efforts to map the diversity of new and known pyrroloiminoquinones and related compounds in extracts of seven specimens. Molecular taxonomic identification confirmed all sponges as T. favus and five specimens (chemotype I) were found to produce mainly discorhabdins and tsitsikammamines. Remarkably, however, two specimens (chemotype II) exhibited distinct morphological and chemical characteristics: the absence of discorhabdins, only trace levels of tsitsikammamines and, instead, an abundance of unbranched and halogenated makaluvamines. Targeted chromatographic isolation provided the new makaluvamine Q, the known makaluvamines A and I, tsitsikammamine B, 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C, and the related pyrrolo-ortho-quinones makaluvamine O and makaluvone. Purified compounds displayed different activity profiles in assays for topoisomerase I inhibition, DNA intercalation and antimetabolic activity against human cell lines. This is the first report of makaluvamines from a Tsitsikamma sponge species, and the first description of distinct chemotypes within a species of the Latrunculiidae family. This study sheds new light on the putative pyrroloiminoquinone biosynthetic pathway of latrunculid sponge

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. © 2014 Jiwaji et al

Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies

Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-on-a-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single hand-held system

Levels of hMT-IIa in HeLa cells transfected with increasing concentrations of hMT-IIa or control ssDNA.

<p>Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. All samples were induced samples with 12.5 µM CdCl₂. Total hMT-IIa activity relative to <i>B2M</i> in cells transfected with varying levels of ssDNA was normalized to hMT-IIa-<i>B2M</i> expression in cells transfected with 0 nM ssDNA giving us a value for hMT-IIa activity in the presence of x nM ssDNA. The levels of hMT-IIa transcript in cells transfected with control ssDNA (C) was set to 1.0. The level of hMT-IIa gene expression (MT) in cells transfected with hMT-IIa ssDNA was compared to the levels of hMT-IIa in cells transfected with control ssDNA at each concentration providing a measure of the reduction in hMT-IIa activity in hMT-IIa ssDNA transfected compared to those transfected with control ssDNA. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. All samples were induced with 12.5 µM CdCl₂. HeLa cells were transfected with hMT-IIa-specific ssDNA (ssDNA). Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099458#pone.0099458.s008" target="_blank">Table S2B</a>.</p

Quantification of the levels of hMT-IIa transcript in HeLa cells after treatment with ssDNA-functionalized nanoparticles.

<p>Induced samples were treated with 12.5 µM CdCl₂ (+Cd). HeLa cells were transfected with 5 nM unfunctionalized (−), 5 nM control (C) or 5 nM hMTIIa (MT)-specific ssDNA functionalized gold nanoparticles. The level of hMTIIa gene expression (normalized to <i>B2M</i>) in HeLa cells transfected with unfunctionalized (−)gold nanoparticles was normalized to 100% and all other fold inductions were expressed relative to this. The error bars were calculated as 1 standard error of the mean each way and represented as a percentage of the activity.</p