Franck Molina

Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates

With the use of digital genetic amplifiers and logic gates, prototype whole-cell biosensors can be engineered to detect diagnostic biomarkers in complex human clinical samples. A little help from our (little) friends It’s only logical: Translation of diagnostics to home health care or remote settings requires simple methods for measuring markers in complex clinical samples. And living cells—with their ability to detect biomolecules, process the signal, and respond—are logical choices as biosensing devices. The recent buzz on the human microbiota has expanded our view of bacteria beyond infectious enemies to metabolic buddies. Now, Courbet et al. refine that view further by engineering bacteria to serve as whole-cell diagnostic biosensors in human biological samples. Although whole-cell biosensors have been shown to serve as analytical tools, their quirky operation and low signal-to-noise ratio in complex clinical samples have limited their use as diagnostic devices in the clinic. The authors engineered bacterial biosensors capable of signal digitization and amplification, multiplexed signal processing (with the use of Boolean logic gates), and months-long data storage. As a proof of concept, the “bactosensors” detected pathological levels of glucose in urine from diabetic patients, providing a framework for the design of sensor modules that detect diverse biomarkers for diagnostics. Whole-cell biosensors have several advantages for the detection of biological substances and have proven to be useful analytical tools. However, several hurdles have limited whole-cell biosensor application in the clinic, primarily their unreliable operation in complex media and low signal-to-noise ratio. We report that bacterial biosensors with genetically encoded digital amplifying genetic switches can detect clinically relevant biomarkers in human urine and serum. These bactosensors perform signal digitization and amplification, multiplexed signal processing with the use of Boolean logic gates, and data storage. In addition, we provide a framework with which to quantify whole-cell biosensor robustness in clinical samples together with a method for easily reprogramming the sensor module for distinct medical detection agendas. Last, we demonstrate that bactosensors can be used to detect pathological glycosuria in urine from diabetic patients. These next-generation whole-cell biosensors with improved computing and amplification capacity could meet clinical requirements and should enable new approaches for medical diagnosis.

Hypothesis: bacteria control host appetites

To help investigate the relationship between inflammatory and other diseases and the composition of the gut microbiota, we propose that a positive-feedback loop exists between the preferences of the host for a particular dietary regimen, the composition of the gut microbiota that depends on this regimen, and the preferences of the host as influenced by the gut microbiota. We cite evidence in support of this hypothesis and make testable predictions.

Lipoplex nanostructures reveal a general self-organization of nucleic acids.

BACKGROUNDnLipid and plasmid DNA complexes (Lx) were designed for gene transfer and were studied comprehensibly to elucidate their formation and ultrastructure.nnnMETHODSnWe compared supramolecular self-assembly into stable Lx containing nucleic acids of various types and lengths using Cryo-Electron Microscopy, Small Angle X-ray Scattering and dynamic light scattering.nnnRESULTSnAnalysis of these complexes showed that they reproducibly formed monodisperse and spherical multilamellar particles. The same concentric and lamellar structure with different packing regimes was produced by circular double-stranded DNA, linear double-stranded DNA, single-stranded DNA, oligodeoxynucleotides or RNA. Strikingly, thousands of oligonucleotide molecules seem to align with one another and to behave as longer nucleic acid molecules in forming structurally similar particles. Neither excess cationic lipids nor excess DNA of different forms changed significantly the mean diameter and the size distribution of Lx particles. This suggests a role for Lx formation of steric size in addition to the conventional thermodynamic mechanism. The Lx ultrastructure is highly ordered and crystalline and is in a lamellar and/or hexagonal phase. Increasing NA size led to an increased proportion of Lx in a hexagonal structure phase as in the case of T4 phage virus DNA. These observations were made using two Lx made from different lipids exhibiting negative and positive charged surface. We also demonstrated structural similarities between the supramolecular auto-organization of Lx and that found in some viruses. In particular, both synthetic and viral particles have an ultrastructure that exhibits a phase transition between lamellar and hexagonal phases.nnnGENERAL SIGNIFICANCEnTaken together, our data point towards the possible existence of a ubiquitous organization of genetic materials, at least with cationic lipids, that has implications for both therapy and the origins of life.

TonB induces conformational changes in surface-exposed loops of FhuA, outer membrane receptor of Escherichia coli.

FhuA, outer membrane receptor of Escherichia coli, transports hydroxamate‐type siderophores into the periplasm. Cytoplasmic membrane–anchored TonB transduces energy to FhuA to facilitate siderophore transport. Because the N‐terminal cork domain of FhuA occludes the C‐terminal β‐barrel lumen, conformational changes must occur to enable siderophore passage. To localize conformational changes at an early stage of the siderophore transport cycle, four anti‐FhuA monoclonal antibodies (mAbs) were purified to homogeneity, and the epitopes that they recognize were determined by phage display. We mapped continuous and discontinuous epitopes to outer surface‐exposed loops 3, 4, and 5 and to β‐barrel strand 14. To probe for conformational changes of FhuA, surface plasmon resonance measured mAb binding to FhuA in its apo‐ and siderophore‐bound states. Changes in binding kinetics were observed for mAbs whose epitopes were mapped to outer surface‐exposed loops. Further, we measured mAb binding in the absence and presence of TonB. After forming immobilized FhuA–TonB complexes, changes in kinetics of mAb binding to FhuA were even more pronounced compared with kinetics of binding in the absence of TonB. Measurement of extrinsic fluorescence of the dye MDCC conjugated to residue 336 in outer surface‐exposed loop 4 revealed 33% fluorescence quenching upon ferricrocin binding and up to 56% quenching upon TonB binding. Binding of mAbs to apo‐ and ferricrocin‐bound FhuA complemented by fluorescence spectroscopy studies showed that their cognate epitopes on loops 3, 4, and 5 undergo conformational changes upon siderophore binding. Further, our data demonstrate that TonB binding promotes conformational changes in outer surface‐exposed loops of FhuA.

Identification of New Sphingomyelinases D in Pathogenic Fungi and Other Pathogenic Organisms

Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.

Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome

The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a public urinary proteome, common to healthy individuals, seems to coexist with a private urinary proteome, which is more specific to each individual.

Tmprss3 loss of function impairs cochlear inner hair cell Kcnma1 channel membrane expression

Before acquiring their mature state, cochlear hair cells undergo a series of changes in expression of ion channels. How this complex mechanism is achieved is not fully understood. Tmprss3, a type II serine protease expressed in hair cells, is required for their proper functioning at the onset of hearing. To unravel the role of Tmprss3 in the acquisition of mature K(+) currents, we compared their function by patch-clamp technique in wild-type Tmprss3(WT) and Tmprss3(Y260X)-mutant mice. Interestingly, only outward K(+) currents were altered in Tmprss3(Y260X)-mutant mice. To determine by which mechanism this occurred, we compared the protein network of Tmprss3(WT) and Tmprss3(Y260X)-mutant mice using proteomic analysis. This led to the identification of a pathway related to potassium Kcnma1 channels. This pathway was validated by immunohistochemistry, focusing on the most downregulated protein that was identified as a cochlear Kcnma1-associated protein, APOA1. Finally, we show that, in contrast to Tmprss3(WT), Kcnma1 channels were absent at the neck of inner hair cells (IHCs) in Tmprss3(Y260X)-mutant mice. In conclusion, our data suggest that lack of Tmprss3 leads to a decrease in Kcnma1 potassium channels expression in (IHCs).

In vitro screening for drug-induced depression and/or suicidal adverse effects: a new toxicogenomic assay based on CE-SSCP analysis of HTR2C mRNA editing in SH-SY5Y cells.

Many drugs in clinical trials, or already on the market, can have psychiatric side effects, independently of their therapeutic indication (e.g., Acomplia, Taranabant, and Roaccutane). There is currently no in vitro or in vivo approved test for the detection/prediction of such adverse effects, and the Food and Drugs Administration (FDA) can only issue general alerts on specific therapeutic classes. The development of a screening assay is therefore of real interest. The anti-viral and anti-tumor action of human interferon-alpha (hIFNα) is associated with a variety of neuropsychiatric side effects, including major depression, suicidal ideation and suicide. RNA editing of the serotonin 2C receptor (HTR2C) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification, the regulation of which is altered in depressed suicide victims. In this study, we show that in the SH-SY5Y neuroblastoma cell line, hIFNα specifically activates the ADAR1a isoform and thereby modifies the HTR2C mRNA editing profile. As this hIFNα-induced altered profile partly overlaps with that observed in the brain of depressed suicide victims, we investigated whether it could be used as a signature to identify drugs with depression and/or suicidal side effects. By means of the Biocortech proprietary screening assay, which allows the relative quantification of all the edited HTR2C isoforms in a sample, we blind-tested the effect of 50 marketed molecules on HTR2C mRNA editing in SH-SY5Y cells and identified 17 compounds with an IFN-like editing profile. This new toxicogenomic assay can identify compounds with potential psychiatric adverse events with a positive predictive value of 90xa0%.

A new method for 2D gel spot alignment: application to the analysis of large sample sets in clinical proteomics

BackgroundIn current comparative proteomics studies, the large number of images generated by 2D gels is currently compared using spot matching algorithms. Unfortunately, differences in gel migration and sample variability make efficient spot alignment very difficult to obtain, and, as consequence most of the software alignments return noisy gel matching which needs to be manually adjusted by the user.ResultsWe present Sili2DGel an algorithm for automatic spot alignment that uses data from recursive gel matching and returns meaningful Spot Alignment Positions (SAP) for a given set of gels. In the algorithm, the data are represented by a graph and SAP by specific subgraphs. The results are returned under various forms (clickable synthetic gel, text file, etc.). We have applied Sili2DGel to study the variability of the urinary proteome from 20 healthy subjects.ConclusionSili2DGel performs noiseless automatic spot alignment for variability studies (as well as classical differential expression studies) of biological samples. It is very useful for typical clinical proteomic studies with large number of experiments.

Region-specific alterations of A-to-I RNA editing of serotonin 2c receptor in the cortex of suicides with major depression

Brain region-specific abnormalities in serotonergic transmission appear to underlie suicidal behavior. Alterations of RNA editing on the serotonin receptor 2C (HTR2C) pre-mRNA in the brain of suicides produce transcripts that attenuate 5-HT2CR signaling by impairing intracellular G-protein coupling and subsequent intracellular signal transduction. In brain, the distribution of RNA-editing enzymes catalyzing deamination (A-to-I modification) shows regional variation, including within the cerebral cortex. We tested the hypothesis that altered pre-mRNA 5-HT2CR receptor editing in suicide is region-specific. To this end, we investigated the complete 5-HT2CR mRNA-editing profile in two architectonically distinct cortical areas involved in mood regulation and decision-making in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicides. By using an original biochemical detection method, that is, capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), we corroborated the 5-HT2CR mRNA-editing profile previously described in the dorsolateral prefrontal cortex (Brodmann area 9 (BA9)). Editing of 5-HT2CR mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Compared with non-psychiatric control individuals, alterations of editing levels of 5-HT2CR mRNA were detected in both cortical areas of depressed suicides. A marked increase in editing on 5-HT2CR was especially observed in the anterior cingulate cortex in suicides, implicating this cortical area in suicide risk. The results suggest that region-specific changes in RNA editing of 5-HT2CR mRNA and deficient receptor function likely contribute to the etiology of major depressive disorder or suicide.