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DNA and RNA biomarkers have not progressed beyond the automated specialized clinic due to failure in the reproducibility necessary to standardize robust and rapid nucleic acid detection at the point of care, where health outcomes can be most improved by early-stage diagnosis and precise monitoring of therapy and disease prognosis. We demonstrate here a new analytical platform to meet this challenge using functional 3D hydrogels engineered from peptide and oligonucleotide build-ing blocks to provide sequence-specific, PCR-free fluorescent detection of un-labelled nucleic acid sequences. We discrimi-nated at picomolar detection limits (

Background: DNA methylation is an epigenetic mechanism involved in the pathogenicity of severalmajor bacterial pathogens. It can decrease the affinity of some transcriptional regulators to theirbinding site, leading to sub‐populations expressing or not various genes, depending on the DNAmethylation state. Dam DNA methyltransferase is widespread in Gammaproteobacteria andmethylates the adenine of GATC sites.Objectives: The role of Dam was investigated in Photorhabdus luminescens during its symbiosis witha soil nematode and during its pathogenic stage in insects.Methods: SMRT sequencing (PacBio), which allows identification of the DNA methylation of thewhole genome (methylome), RNAseq and phenotypic analysis were performed in a P. luminescensstrain overexpressing Dam.Results: Dam overexpression caused a decrease in motility whereas it increased biofilm formation.While symbiosis ability of the Dam overexpressing strain was not significantly different from that of acontrol strain, the nemato‐bacterial complex displayed an impaired pathogenicity in insect, as alsoobserved after direct insect injection of the bacteria alone. Transcriptomic analysis revealed that theobserved phenotypes were related to differences at the transcriptional level. More than 99% of theGATC sites of the genome were found methylated and DNA methylation levels did not change overgrowth kinetics. However, the Dam‐overexpressing strain displayed more methylated GATC sitesthan the control and most of these sites were located in promoter regions. These sites may beinvolved in the observed differences in phenotypes and gene expression and provide

Dam DNA methylation is an epigenetic mechanism as it can regulate genes expression by reducing the affinity of transcriptional regulators for their binding site (Casadesus and Low, 2006). This DamDNA methylation has been shown to be involved in pathogenicity and phenotypic heterogeneity of several bacteria (Casadesus and Low, 2013).Photorhabdus luminescens is an entomopathogenic bacteria switching from symbiosis, with a nematode, to pathogenicity, in the insect (Boemare et al., 1993). This bacterium displays phenotypic heterogeneity. Because a Dam MTase has been identified in its genome sequence, we investigated its role in the life-cycle of P. luminescens. Methylome analysis revealed that 99% of GATC sites in the genome are methylated in all tested growth conditions. Overexpressing Dam methylates most of the unmethylated sites and causes a decrease in motility and pathogenicity of the bacteria whereas it increases biofilms formation. It does not impair the bacterial ability to perform a mutualistic association with the nematode. Transcriptomic analysis revealed that the observed phenotypes are related to differences at the transcriptional level.Coupling phenotypic, transcriptomic and methylomic analysis provides clues to identify genes transcriptionally regulated by DNA methylation and to understand Dam DNA methylation involvement in P. luminescens life-cycle.

Whole Genome DNA Methylation (Methylome) Analysis and Role of Dam DNA Methyltransferase in the Entomopathogenic Bacterium Photorhabdus luminescens. MicrobiOccitanie 2019- Rencontre des Microbiologistes Région Occitanie

Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that the P. luminescens Dam plays a role in the pathogenicity of the nemato-bacterial complex.

Fourier transform mass spectrometry (FTMS) is widely used to characterise the chemical complexity of mixtures, such as natural organic matter (NOM), petroleum, and agri-food products (including Scotch Whisky). Although electrospray ionisation (ESI) is by far the most widely used ionisation source in these studies, other ionisation techniques are available and may offer complimentary information. In a recent study, we found matrix free laser desorption/ionisation (LDI) to be effective for the analysis of Suwannee River fulvic acid (SRFA), and to provide complementary chemical insights. In this study, LDI along with Atmospheric Pressure Photoionisation (APPI) and Atmospheric Pressure Chemical Ionisation (APCI) were compared to ESI for the analysis of Scotch Whisky. High mass accuracy (54 ppb, mean) allowed for assignment of 86% of peaks, with 3993 unique molecular formulas identified from four representative samples analysed. All four ionisation techniques, performed in negative mode, identified thousands of formulas. Many were unique to each ionisation source, whilst 699 formulas were common to all techniques. Ions were identified in both deprotonated and radical anion forms. Our study highlights the importance of a multi ionisation source approach; we recommend that analysis of complex mixtures, especially novel ones, should not be limited solely to ESI.

Supplemental material, Supplementary_file_I for Why do stroke survivors not receive recommended amounts of active therapy? Findings from the ReAcT study, a mixed-methods case-study evaluation in eight stroke units by David J Clarke, Louisa-Jane Burton, Sarah F Tyson, Helen Rodgers, Avril Drummond, Rebecca Palmer, Alex Hoffman, Matthew Prescott, Pippa Tyrrell, Lianne Brkic, Katie Grenfell and Anne Forster in Clinical Rehabilitation