Investigating the physiology of viable but non-culturable bacteria by microfluidics and time-lapse microscopy.

This is the final version of the article. Available from BioMed Central via the DOI in this record.BACKGROUND: Clonal microbial populations often harbor rare phenotypic variants that are typically hidden within the majority of the remaining cells, but are crucial for the population's resilience to external perturbations. Persister and viable but non-culturable (VBNC) cells are two important clonal bacterial subpopulations that can survive antibiotic treatment. Both persister and VBNC cells pose a serious threat to human health. However, unlike persister cells, which quickly resume growth following drug removal, VBNC cells can remain non-growing for prolonged periods of time, thus eluding detection via traditional microbiological assays. Therefore, understanding the molecular mechanisms underlying the formation of VBNC cells requires the characterization of the clonal population with single-cell resolution. A combination of microfluidics, time-lapse microscopy, and fluorescent reporter strains offers the perfect platform for investigating individual cells while manipulating their environment. METHODS: Here, we report a novel single-cell approach to investigate VBNC cells. We perform drug treatment, bacterial culturing, and live/dead staining in series by using transcriptional reporter strains and novel adaptations to the mother machine technology. Since we track each cell throughout the experiment, we are able to quantify the size, morphology and fluorescence that each VBNC cell displayed before, during and after drug treatment. RESULTS: We show that VBNC cells are not dead or dying cells but share similar phenotypic features with persister cells, suggesting a link between these two subpopulations, at least in the Escherichia coli strain under investigation. We strengthen this link by demonstrating that, before drug treatment, both persister and VBNC cells can be distinguished from the remainder of the population by their lower fluorescence when using a reporter strain for tnaC, encoding the leader peptide of the tnaCAB operon responsible for tryptophan metabolism. CONCLUSION: Our data demonstrates the suitability of our approach for studying the physiology of non-growing cells in response to external perturbations. Our approach will allow the identification of novel biomarkers for the isolation of VBNC and persister cells and will open new opportunities to map the detailed biochemical makeup of these clonal subpopulations.This work was supported by a Royal Society Research Grant (RG140203), a Welcome Trust ISSF (WT097835/Z/11/Z) and a Start up Grant from the University of Exeter awarded to SP. AS acknowledges support from the BBSRC through a SWBio-DTP studentship (BB/M009122/1). JM was generously supported by a Wellcome Trust Institutional Strategic Support Award (WT097835MF). This work was partly supported by BBSRC award BB/1024631/1 to RWT

A framework for optimization of diffusion-weighted MRI protocols for large field-of-view abdominal-pelvic imaging in multicenter studies.

PURPOSE: To develop methods for optimization of diffusion-weighted MRI (DW-MRI) in the abdomen and pelvis on 1.5 T MR scanners from three manufacturers and assess repeatability of apparent diffusion coefficient (ADC) estimates in a temperature-controlled phantom and abdominal and pelvic organs in healthy volunteers. METHODS: Geometric distortion, ghosting, fat suppression, and repeatability and homogeneity of ADC estimates were assessed using phantoms and volunteers. Healthy volunteers (ten per scanner) were each scanned twice on the same scanner. One volunteer traveled to all three institutions in order to provide images for qualitative comparison. The common volunteer was excluded from quantitative analysis of the data from scanners 2 and 3 in order to ensure statistical independence, giving n = 10 on scanner 1 and n = 9 on scanners 2 and 3 for quantitative analysis. Repeatability and interscanner variation of ADC estimates in kidneys, liver, spleen, and uterus were assessed using within-patient coefficient of variation (wCV) and Kruskal-Wallis tests, respectively. RESULTS: The coefficient of variation of ADC estimates in the temperature-controlled phantom was 1%-4% for all scanners. Images of healthy volunteers from all scanners showed homogeneous fat suppression and no marked ghosting or geometric distortion. The wCV of ADC estimates was 2%-4% for kidneys, 3%-7% for liver, 6%-9% for spleen, and 7%-10% for uterus. ADC estimates in kidneys, spleen, and uterus showed no significant difference between scanners but a significant difference was observed in liver (p < 0.05). CONCLUSIONS: DW-MRI protocols can be optimized using simple phantom measurements to produce good quality images in the abdomen and pelvis at 1.5 T with repeatable quantitative measurements in a multicenter study

Lentiviral Engineered Fibroblasts Expressing Codon Optimized COL7A1 Restore Anchoring Fibrils in RDEB

Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen (C7), a protein essential for anchoring fibril formation at the dermal-epidermal junction (DEJ). Whilst allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of C7 at the DEJ. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of C7 was confirmed, with transduced cells exhibiting supra-normal levels of protein expression and ex vivo migration of fibroblasts was restored in functional assays. Gene modified RDEB fibroblasts also deposited C7 at the DEJ of human RDEB skin xenografts placed on NOD-scid IL2Rgamma(null) recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the DEJ. Fibroblast mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man

Ploidy elicits a whole-genome dosage effect: growth of triploid Atlantic salmon is linked to the genetic origin of the second maternal chromosome set

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Factors associated with quality of services for marginalized groups with mental health problems in 14 European countries

This research was financially supported by DG-Sanco (contract: 800197; 2007-2010). The authors would like to thank all of the professionals and services who participated in the PROMO assessment of services. A PhD grant from Fundação para a Ciência e Tecnologia–Portugal (SFRH/BD/66388/2009) to the first author is acknowledged

Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair

Eukaryotic cells possess a universal repair machinery that ensures rapid resealing of plasma membrane disruptions. Before resealing, the torn membrane is submitted to considerable tension, which functions to expand the disruption. Here we show that annexin-A5 (AnxA5), a protein that self-assembles into two-dimensional (2D) arrays on membranes upon Ca2+ activation, promotes membrane repair. Compared with wild-type mouse perivascular cells, AnxA5-null cells exhibit a severe membrane repair defect. Membrane repair in AnxA5-null cells is rescued by addition of AnxA5, which binds exclusively to disrupted membrane areas. In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair. We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca2+, AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing

A comparison of SNP and STR loci for delineating population structure and performing individual genetic assignment

<p>Abstract</p> <p>Background</p> <p>Technological advances have lead to the rapid increase in availability of single nucleotide polymorphisms (SNPs) in a range of organisms, and there is a general optimism that SNPs will become the marker of choice for a range of evolutionary applications. Here, comparisons between 300 polymorphic SNPs and 14 short tandem repeats (STRs) were conducted on a data set consisting of approximately 500 Atlantic salmon arranged in 10 samples/populations.</p> <p>Results</p> <p>Global F_STranged from 0.033-0.115 and -0.002-0.316 for the 14 STR and 300 SNP loci respectively. Global F_STwas similar among 28 linkage groups when averaging data from mapped SNPs. With the exception of selecting a panel of SNPs taking the locus displaying the highest global F_STfor each of the 28 linkage groups, which inflated estimation of genetic differentiation among the samples, inferred genetic relationships were highly similar between SNP and STR data sets and variants thereof. The best 15 SNPs (30 alleles) gave a similar level of self-assignment to the best 4 STR loci (83 alleles), however, addition of further STR loci did not lead to a notable increase assignment whereas addition of up to 100 SNP loci increased assignment.</p> <p>Conclusion</p> <p>Whilst the optimal combinations of SNPs identified in this study are linked to the samples from which they were selected, this study demonstrates that identification of highly informative SNP loci from larger panels will provide researchers with a powerful approach to delineate genetic relationships at the individual and population levels.</p

Extraordinarily high biomass benthic community on Southern Ocean seamounts

We describe a previously unknown assemblage of seamount-associated megabenthos that has by far the highest peak biomass reported in the deep-sea outside of vent communities. The assemblage was found at depths of 2–2.5 km on rocky geomorphic features off the southeast coast of Australia, in an area near the Sub-Antarctic Zone characterised by high rates of surface productivity and carbon export to the deep-ocean. These conditions, and the taxa in the assemblage, are widely distributed around the Southern mid-latitudes, suggesting the high-biomass assemblage is also likely to be widespread. The role of this assemblage in regional ecosystem and carbon dynamics and its sensitivities to anthropogenic impacts are unknown. The discovery highlights the lack of information on deep-sea biota worldwide and the potential for unanticipated impacts of deep-sea exploitation

Small but crucial : the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans

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