Molecular Readout Research Articles

Single-molecule imaging of pore-forming toxin dynamics in droplet interface bilayers.

Single-channel recording from pore-forming toxins (PFTs) provides a clear and direct molecular readout of toxin action. However to complete any mechanistic understanding of PFT behavior, this functional kinetic readout must be linked to the underlying changes in toxin structure, binding, conformation, or stoichiometry. Here we review how single-molecule imaging methods might be used to further our understanding of PFTs, and provide detailed practical guidance on the use of droplet interface bilayers as a method capable of examining both single-molecule fluorescence and single-channel electrical signals from PFTs.

Dynamic Bio-Barcode Assay Enables Electrochemical Detection of a Cancer Biomarker in Undiluted Human Plasma: A Sample-In-Answer-Out Approach.

There is a need for biosensing systems that can be operated at the point-of-care (POC) for disease screening and diagnostics and health monitoring. In spite of this, simple to operate systems with the required analytical sensitivity and specificity in clinical samples, using a sample-in-answer-out approach, remain elusive. Reported here is an electrochemical bio-barcode assay (e-biobarcode assay) that integrates biorecognition with signal transduction using molecular (DNA/protein) machines and signal readout using nanostructured electrodes. The e-biobarcode assay eliminates multistep processing and uses a single step for analysis following sample collection into the reagent tube. A clinically relevant performance for the analysis of prostate specific antigen (PSA) in undiluted and unprocessed human plasma: a log-linear range of 1 ng mL-1 -200 ng mL-1 and a LOD of 0.4 ng mL-1 , was achieved. The e-biobarcode assay offers a realistic solution for biomarker analysis at the POC.

Dynamic Bio‐Barcode Assay Enables Electrochemical Detection of a Cancer Biomarker in Undiluted Human Plasma: A Sample‐In‐Answer‐Out Approach

AbstractThere is a need for biosensing systems that can be operated at the point‐of‐care (POC) for disease screening and diagnostics and health monitoring. In spite of this, simple to operate systems with the required analytical sensitivity and specificity in clinical samples, using a sample‐in‐answer‐out approach, remain elusive. Reported here is an electrochemical bio‐barcode assay (e‐biobarcode assay) that integrates biorecognition with signal transduction using molecular (DNA/protein) machines and signal readout using nanostructured electrodes. The e‐biobarcode assay eliminates multistep processing and uses a single step for analysis following sample collection into the reagent tube. A clinically relevant performance for the analysis of prostate specific antigen (PSA) in undiluted and unprocessed human plasma: a log‐linear range of 1 ng mL−1–200 ng mL−1 and a LOD of 0.4 ng mL−1, was achieved. The e‐biobarcode assay offers a realistic solution for biomarker analysis at the POC.

Solid-state nanopore sensors

Nanopore-based sensors have established themselves as a prominent tool for solution-based, single-molecule analysis of the key building blocks of life, including nucleic acids, proteins, glycans and a large pool of biomolecules that have an essential role in life and healthcare. The predominant molecular readout method is based on measuring the temporal fluctuations in the ionic current through the pore. Recent advances in materials science and surface chemistries have not only enabled more robust and sensitive devices but also facilitated alternative detection modalities based on field-effect transistors, quantum tunnelling and optical methods such as fluorescence and plasmonic sensing. In this Review, we discuss recent advances in nanopore fabrication and sensing strategies that endow nanopores not only with sensitivity but also with selectivity and high throughput, and highlight some of the challenges that still need to be addressed. Nanopore sensors enable the solution-based analysis of nucleic acids, proteins and other biomolecules at the single-molecule level. This Review discusses new fabrication and sensing strategies — including field-effect transistors, quantum tunnelling and optical methods — that enhance the sensitivity and selectivity of nanopores.

DNA methylation modules associate with incident cardiovascular disease and cumulative risk factor exposure

BackgroundEpigenome-wide association studies using DNA methylation have the potential to uncover novel biomarkers and mechanisms of cardiovascular disease (CVD) risk. However, the direction of causation for these associations is not always clear, and investigations to-date have often failed to replicate at the level of individual loci.MethodsHere, we undertook module- and region-based DNA methylation analyses of incident CVD in the Women’s Health Initiative (WHI) and Framingham Heart Study Offspring Cohort (FHS) in order to find more robust epigenetic biomarkers for cardiovascular risk. We applied weighted gene correlation network analysis (WGCNA) and the Comb-p algorithm to find methylation modules and regions associated with incident CVD in the WHI dataset.ResultsWe discovered two modules whose activation correlated with CVD risk and replicated across cohorts. One of these modules was enriched for development-related processes and overlaps strongly with epigenetic aging sites. For the other, we showed preliminary evidence for monocyte-specific effects and statistical links to cumulative exposure to traditional cardiovascular risk factors. Additionally, we found three regions (associated with the genes SLC9A1, SLC1A5, and TNRC6C) whose methylation associates with CVD risk.ConclusionsIn sum, we present several epigenetic associations with incident CVD which reveal disease mechanisms related to development and monocyte biology. Furthermore, we show that epigenetic modules may act as a molecular readout of cumulative cardiovascular risk factor exposure, with implications for the improvement of clinical risk prediction.

Plasma Proteome Signature of Sepsis: a Functionally Connected Protein Network.

Sepsis is an extreme host response to infection that leads to loss of organ function and cardiovascular integrity. Mortality from sepsis is on the rise. Despite more than three decades of research and clinical trials, specific diagnostic and therapeutic strategies for sepsis are still absent. The use of LFQ- and TMT-based quantitative proteomics is reported here to study the plasma proteome in five mouse models of sepsis. A knowledge-based interpretation of the data reveals a protein network with extensive connectivity through documented functional or physical interactions. The individual proteins in the network all have a documented role in sepsis and are known to be extracellular. The changes in protein abundance observed in the mouse models of sepsis have for the most part the same directionality (increased or decreased abundance) as reported in the literature for human sepsis. This network has been named the Plasma Proteome Signature of Sepsis (PPSS). The PPSS is a quantifiable molecular readout that can supplant the current symptom-based approach used to diagnose sepsis. This type of molecular interpretation of sepsis, its progression, and its response to therapeutic intervention are an important step in advancing our understanding of sepsis, and for discovering and evaluating new therapeutic strategies.

Microtubule dynamics modulate sensing during cold acclimation in grapevine suspension cells

Cold acclimation is of practical relevance, since it can avoid cold-induced damage in various crops. To efficiently activate cold acclimation requires that the chilling stress is perceived and processed efficiently. In the current work, we use a transgenic cell line of V. rupestris expressing a GFP-labelled tubulin to follow the effect of cold acclimation and the relation between microtubules and the expression of the transcription factor Cold Box Factor 4 (CBF4) as molecular readout for adaptive responses to cold stress. We find that chilling induced cold tolerance correlated with increased CBF4 expression. We show that cold acclimation can be achieved through stabilisation of microtubules by taxol, as well as through transient elimination of microtubules by pronamide in the absence of cold stress. Furthermore, results from inhibitor studies indicate that transcriptional activation of CBF4 appears to be under control of calcium influx. We screened a population of the ancestor of V. sylvestris and could identify different clades with strong induction of CBF4, indicative of genetic variation in cold adaptability that can be used for breeding. We summarize our findings into a working model where microtubule dynamics controls the sensitivity of cold induced calcium influx mediating the induction of CBF4 culminating in cold hardening.

Comparative biochemical analysis of UHRF proteins reveals molecular mechanisms that uncouple UHRF2 from DNA methylation maintenance.

UHRF1 is a histone- and DNA-binding E3 ubiquitin ligase that functions with DNMT1 to maintain mammalian DNA methylation. UHRF1 facilitates DNMT1 recruitment to replicating chromatin through a coordinated mechanism involving histone and DNA recognition and histone ubiquitination. UHRF2 shares structural homology with UHRF1, but surprisingly lacks functional redundancy to facilitate DNA methylation maintenance. Molecular mechanisms uncoupling UHRF2 from DNA methylation maintenance are poorly defined. Through comprehensive and comparative biochemical analysis of recombinant human UHRF1 and UHRF2 reader and writer activities, we reveal conserved modes of histone PTM recognition but divergent DNA binding properties. While UHRF1 and UHRF2 diverge in their affinities toward hemi-methylated DNA, we surprisingly show that both hemi-methylated and hemi-hydroxymethylated DNA oligonucleotides stimulate UHRF2 ubiquitin ligase activity toward histone H3 peptide substrates. This is the first example of an E3 ligase allosterically regulated by DNA hydroxymethylation. However, UHRF2 is not a productive histone E3 ligase toward purified mononucleosomes, suggesting UHRF2 has an intra-domain architecture distinct from UHRF1 that is conformationally constrained when bound to chromatin. Collectively, our studies reveal that uncoupling of UHRF2 from the DNA methylation maintenance program is linked to differences in the molecular readout of chromatin signatures that connect UHRF1 to ubiquitination of histone H3.

Ageing-associated DNA methylation dynamics are a molecular readout of lifespan variation among mammalian species

BackgroundMammalian species exhibit a wide range of lifespans. To date, a robust and dynamic molecular readout of these lifespan differences has not yet been identified. Recent studies have established the existence of ageing-associated differentially methylated positions (aDMPs) in human and mouse. These are CpG sites at which DNA methylation dynamics show significant correlations with age. We hypothesise that aDMPs are pan-mammalian and are a dynamic molecular readout of lifespan variation among different mammalian species.ResultsA large-scale integrated analysis of aDMPs in six different mammals reveals a strong negative relationship between rate of change of methylation levels at aDMPs and lifespan. This relationship also holds when comparing two different dog breeds with known differences in lifespans. In an ageing cohort of aneuploid mice carrying a complete copy of human chromosome 21, aDMPs accumulate far more rapidly than is seen in human tissues, revealing that DNA methylation at aDMP sites is largely shaped by the nuclear trans-environment and represents a robust molecular readout of the ageing cellular milieu.ConclusionsOverall, we define the first dynamic molecular readout of lifespan differences among mammalian species and propose that aDMPs will be an invaluable molecular tool for future evolutionary and mechanistic studies aimed at understanding the biological factors that determine lifespan in mammals.

A DNA Tracer System for Hydrological Environment Investigations.

To monitor and manage hydrological pollution effectively, tracing sources of pollutants is of great importance and also is in urgent need. A variety of tracers have been developed such as isotopes, silica, bromide, and dyes; however, practical limitations of these traditional tracers still exist such as lack of multiplexed, multipoint tracing and interference of background noise. To overcome these limitations, a new tracing system based on DNA nanomaterials, namely DNA tracer, has already been developed. DNA tracers possess remarkable advantages including sufficient species, specificity, environmental friendly, stable migration, and high sensitivity as well as allowing for multipoints tracing. In this review article, we introduce the molecular design, synthesis, protection and signal readout strategies of DNA tracers, compare the advantages and disadvantages of DNA tracer with traditional tracers, and summarize the-state-of-art applications in hydrological environment investigations. In the end, we provide our perspective on the future development of DNA tracers.

Computational Network Analysis for Drug Toxicity Prediction.

The computational prediction of compound effects from molecular data is an important task in hazard and risk assessment and pivotal for judging the safety of any drug, chemical or cosmetic compound. In particular, the identification of such compound effects at the level of molecular interaction networks can be helpful for the construction of adverse outcome pathways (AOPs). AOPs emerged as a guiding concept for toxicity prediction, because of the inherent mechanistic information of such networks. In fact, integrating molecular interactions in transcriptome analysis and observing expression changes in closely interacting genes might allow identifying the key molecular initiating events of compound toxicity.In this work we describe a computational approach that is suitable for the identification of such network modules from transcriptomics data, which is the major molecular readout of toxicogenomics studies. The approach is composed of different tools (1) for primary data analysis, i.e., the biostatistical quantification of the gene expression changes, (2) for functional annotation and prioritization of genes using literature mining, as well as (3) for the construction of an interaction network that consists of interactions with high confidence and the identification of predictive modules from these networks. We describe the different steps of the approach and demonstrate its performance with public data on drugs that induce hepatic and cardiac toxicity.

Histone H3 Threonine 11 Phosphorylation Is Catalyzed Directly by the Meiosis-Specific Kinase Mek1 and Provides a Molecular Readout of Mek1 Activity in Vivo.

Saccharomyces cerevisiae Mek1 is a CHK2/Rad53-family kinase that regulates meiotic recombination and progression upon its activation in response to DNA double-strand breaks (DSBs). The full catalog of direct Mek1 phosphorylation targets remains unknown. Here, we show that phosphorylation of histone H3 on threonine 11 (H3 T11ph) is induced by meiotic DSBs in S. cerevisiae and Schizosaccharomyces pombe Molecular genetic experiments in S. cerevisiae confirmed that Mek1 is required for H3 T11ph and revealed that phosphorylation is rapidly reversed when Mek1 kinase is no longer active. Reconstituting histone phosphorylation in vitro with recombinant proteins demonstrated that Mek1 directly catalyzes H3 T11 phosphorylation. Mutating H3 T11 to nonphosphorylatable residues conferred no detectable defects in otherwise unperturbed meiosis, although the mutations modestly reduced spore viability in certain strains where Rad51 is used for strand exchange in place of Dmc1. H3 T11ph is therefore mostly dispensable for Mek1 function. However, H3 T11ph provides an excellent marker of ongoing Mek1 kinase activity in vivo Anti-H3 T11ph chromatin immunoprecipitation followed by deep sequencing demonstrated that H3 T11ph was highly enriched at presumed sites of attachment of chromatin to chromosome axes, gave a more modest signal along chromatin loops, and was present at still lower levels immediately adjacent to DSB hotspots. These localization patterns closely tracked the distribution of Red1 and Hop1, axis proteins required for Mek1 activation. These findings provide insight into the spatial disposition of Mek1 kinase activity and the higher order organization of recombining meiotic chromosomes.

Abstract 4337: PDGFR-α induced stiffness abrogates mammary ductal development and enhances tumorigenesis in vivo

Abstract Breast cancer is a leading cause of mortality in women worldwide, in part due to the tumor microenvironment which increases tumor heterogeneity and abets tumor growth. Fibroblasts are cells of mesenchymal origin that are an important component of normal and tumor stroma. Genetic alterations in these cells were shown by several groups including our own to cause fibroblast activation and fuel tumor progression giving rise to more aggressive disease. Platelet-Derived Growth Factor Receptor (PDGFR) alpha is a receptor tyrosine kinase that is chiefly expressed in mesenchymal cells such as fibroblasts. Ligand binding (PDGFAA) activates this receptor. PDGFRα signaling plays critical roles in development and aberrant signaling is seen in several types of cancer, such as lung, pancreas, GI and brain. The central goal of this study was to elucidate the role of stromal PDGFRα in breast cancer development and metastasis, where its role remains largely unknown. To address this goal, we developed a genetic mouse model of stromal activation of PDGFRα in the mammary gland by crossing an auto-activating Pdgfra mutant allele with a mesenchymal specific Cre recombinase. We found that stromal PDGFRα activation completely abrogated postnatal mammary gland ductal formation, with significantly reduced terminal end bud formation. PDGFRα activation also led to progressive fibrosis in the mouse mammary fat pad. As early as four weeks of age, mammary collagen (trichrome staining; second harmonic generation) and hyaluronan deposition (Alcian Blue) was greatly increased in vivo. In fact, this increase in collagen and hyaluronan deposition in mutant animals is believed to be responsible for the observed increased in stiffness of mutant mammary tissue (atomic force microscopy {AFM}). pFAK, which can be activated due to mechanical stress, was increased in mammary epithelia of the mutant mice in vivo corroborating the AFM results. Further, when tumor cells were injected into the mammary glands of the PDGFRα mutants, tumors grew faster as compared to controls. Importantly, we found that mRNA expression of PDGFRA correlates with worsened patient outcomes in HER2+ disease, while expression of both the ligand (PDGFA) and the receptor were found to correlate with increased incidence of lung metastases. We further discovered that in HER2+ patients, PDGFRA levels correlate with breast density. Breast density is the third strongest risk factor for breast cancer, and is directly related to collagen deposition and breast stiffness, thus suggesting a novel predictive role of PDGFRA as a molecular readout of stiffness and density. Studies are underway to utilize mouse models of HER2+ breast cancer to study both primary tumor growth and metastases. Taken together, our mouse studies and paralleling human data analyses suggest that the stromal PDGFRα signaling provides a novel theranostic window in breast cancer treatment and prognosis. Citation Format: Anisha Mathur Hammer, Gina M. Sizemore, Vasudha Shukla, Steven T. Sizemore, Maria Cuitino, Cynthia J. Timmers, Quinn Verfurth, Arnab Chakravarti, Gustavo W. Leone, Samir N. Ghadiali, Michael C. Ostrowski. PDGFR-α induced stiffness abrogates mammary ductal development and enhances tumorigenesis in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4337. doi:10.1158/1538-7445.AM2017-4337

SERS- and Electrochemically Active 3D Plasmonic Liquid Marbles for Molecular-Level Spectroelectrochemical Investigation of Microliter Reactions.

Liquid marbles are emergent microreactors owing to their isolated environment and the flexibility of materials used. Plasmonic liquid marbles (PLMs) are demonstrated as the smallest spectroelectrochemical microliter-scale reactor for concurrent spectro- and electrochemical analyses. The three-dimensional Ag shell of PLMs are exploited as a bifunctional surface-enhanced Raman scattering (SERS) platform and working electrode for redox process modulation. The combination of SERS and electrochemistry (EC) capabilities enables in situ molecular read-out of transient electrochemical species, and elucidate the potential-dependent and multi-step reaction dynamics. The 3D configuration of our PLM-based EC-SERS system exhibits 2-fold and 10-fold superior electrochemical and SERS performance than conventional 2D platforms. The rich molecular-level electrochemical insights and excellent EC-SERS capabilities offered by our 3D spectroelectrochemical system are pertinent in charge transfer processes.

New Molecular Surrogate Assay for Genotoxicity Assessment of Gene Therapy Vectors (SAGA)

The variety of gene therapy vectors for a multitude of different diseases has increased tremendously over the years. However, a number of patients that underwent gene therapy in different trials developed hematological malignancy caused by integration of the provirus in the vicinity of proto-oncogenes. These severe adverse advents prompted intense research efforts towards safer gene therapy, leading to the removal of the long terminal repeat enhancer elements and the use of internal promoters in retroviral vectors. Still, a bottleneck of transition from basic research to clinical application is the test for safety of integrating retro- and lentiviral vectors.Instead of laborious in vivo models with limited predictive value, in vitro assays to screen for insertional mutagenesis are strongly desirable. A decade ago, our lab developed the in vitro immortalization (IVIM) assay to quantify the genotoxic potential of viral vectors, which has been widely used to complete preclinical safety documentation of newly developed integrating vector systems. Despite general acceptance in the field of hematopoietic gene therapy, bias for insertional mutants of the myeloid lineage, a low sensitivity and a long assay run time are clear limitations.We now developed the molecular surrogate assay for genotoxicity assessment (SAGA). The new test is more robust, sensitive and biologically informative. As input we used murine lineage-negative hematopoietic stem and progenitor cells (HSPC) that were cultured as described for the IVIM assay. The murine HSPC were transduced with a number of different gammaretro- and lentiviral vectors, including vectors that have been employed in clinical trials for X-SCID and Wiskott-Aldrich Syndrome. After 14 days, whole mRNA was isolated from transduced and non-transduced samples and analyzed by Agilent custom microarrays (n=86) and qPCR from nine independent SAGA assays. We applied several Machine Learning algorithms to derive a core set of genes which distinguishes transformed from non-transformed samples in each individual SAGA assay. This set of genes from the individual analysis was further analyzed to derive a core set of genes that is able to robustly separate transformed from non-transformed samples in all assays performed. In order to account for platform-specific effects we validated all microarray results by conventional qPCR-methodology. The SAGA gene set was then cross-validated in an independent validation cohort of SAGA-assays that were not part of the SAGA-training set from which the signature was derived from.The SAGA assay was used to quantify the mutagenic potential of several benchmark vectors. It correctly assigned a high mutagenic potential to vectors (MFG.yc and CMMP.WASP) which led to serious adverse events (SAEs) in clinical trials. Most importantly, the SAGA assay reliably scored high for mutagenic vectors, even when the vector did not transform in IVIM-assays conducted in parallel, demonstrating the higher sensitivity of the SAGA-principle. In contrast, SIN lentiviral vectors with weaker internal promoters (LV.EFS.yc and LV.EFS.ADA) showed no enrichment of the SAGA-core signature and hence scored much safer in the SAGA test. We present the results for these vectors side-by-side either using IVIM or SAGA. In summary, we generated an advanced version of the currently used in vitro insertional mutagenesis screening system by integrating a molecular read-out which enhances reproducibility, sensitivity and reduces assay duration, paving the way for a better preclinical risk assessment of gene therapy vectors. DisclosuresNo relevant conflicts of interest to declare.

The Mouse Isolated Perfused Kidney Technique.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy. This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney's preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

Tau PET: the next frontier in molecular imaging of dementia.

We have arrived at an exciting juncture in dementia research: the second major pathological hallmark of Alzheimer's disease (AD)-tau-can now be seen for the first time in the living human brain. The major proteinopathies in AD include amyloid-β plaques and neurofibrillary tangles (NFTs) made of hyperphosphorylated paired helical filament (PHF) tau. Since its advent more than a decade ago, amyloid PET imaging has revolutionized the field of dementia research, enabling more confident diagnosis of the likely pathology in patients with a variety of clinical dementia syndromes, paving the way for the identification of people with preclinical or prodromal AD pathology, and serving as a minimally invasive molecular readout in clinical trials of putative disease-modifying interventions. Now that we are on the brink of a second revolution in molecular imaging in dementia, it is worth considering the likely potential impact of this development on the field.

Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons.

Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of invivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the invitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays.

In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.

BackgroundChronic inflammation of the airways is a central component in lung diseases and is frequently associated with bacterial infections. Monitoring the pro-inflammatory capability of bacterial virulence factors in vivo is challenging and usually requires invasive methods.MethodsLung inflammation was induced using the culture supernatants from two Pseudomonas aeruginosa clinical strains, VR1 and VR2, isolated from patients affected by cystic fibrosis and showing different phenotypes in terms of motility, colony characteristics and biofilm production as well as pyoverdine and pyocyanine release. More interesting, the strains differ also for the presence in supernatants of metalloproteases, a family of virulence factors with known pro-inflammatory activity. We have evaluated the benefit of using a mouse model, transiently expressing the luciferase reporter gene under the control of an heterologous IL-8 bovine promoter, to detect and monitoring lung inflammation.ResultsIn vivo imaging indicated that VR1 strain, releasing in its culture supernatant metalloproteases and other virulence factors, induced lung inflammation while the VR2 strain presented with a severely reduced pro-inflammatory activity. The bioluminescence signal was detectable from 4 to 48 h after supernatant instillation. The animal model was also used to test the anti-inflammatory activity of azithromycin (AZM), an antibiotic with demonstrated inhibitory effect on the synthesis of bacterial exoproducts. The inflammation signal in mice was in fact significantly reduced when bacteria grew in the presence of a sub-lethal dose of AZM causing inhibition of the synthesis of metalloproteases and other bacterial elements. The in vivo data were further supported by quantification of immune cells and cytokine expression in mouse broncho-alveolar lavage samples.ConclusionsThis experimental animal model is based on the transient transduction of the bovine IL-8 promoter, a gene representing a major player during inflammation, essential for leukocytes recruitment to the inflamed tissue. It appears to be an appropriate molecular read-out for monitoring the activation of inflammatory pathways caused by bacterial virulence factors. The data presented indicate that the model is suitable to functionally monitor in real time the lung inflammatory response facilitating the identification of bacterial factors with pro-inflammatory activity and the evaluation of the anti-inflammatory activity of old and new molecules for therapeutic use.

On-chip purification and detection of hepatitis C virus RNA from human plasma

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease worldwide. The diagnosis and monitoring of HCV infection is a crucial need in the clinical management. The conventional diagnostic technologies are challenged when trying to address molecular diagnostics, especially because they require a complex and time-consuming sample preparation phase. Here, a new concept based on surface functionalization was applied to viral RNA purification: first of all polydimethylsiloxane (PDMS) flat surfaces were modified to hold RNA adsorption. After a careful chemical and morphological analysis of the modified surfaces, the functionalization protocols giving the best RNA adsorbing surfaces were applied to PDMS microdevices. The functionalized microdevices were then used for RNA purification from HCV infected human plasma samples. RNA purification and RT were successfully performed in the same microdevice chamber, saving time of analysis, reagents, and labor. The PCR protocol for HCV cDNA amplification was also implemented in the microdevice, demonstrating that the entire process of HCV analysis, from plasma to molecular readout, could be performed on-chip. Not only HCV but also other microdevice-based viral RNA detection could therefore result in a successful Point-of-Care (POC) diagnostics for resource-limited settings.