The Effects of Mary Rose Conservation Treatment on Iron Oxidation Processes and Microbial Communities Contributing to Acid Production in Marine Archaeological Timbers

The Tudor warship the Mary Rose has reached an important transition point in her conservation. The 19 year long process of spraying with polyethylene glycol (PEG) has been completed (April 29th 2013) and the hull is air drying under tightly controlled conditions. Acidophilic bacteria capable of oxidising iron and sulfur have been previously identified and enriched from unpreserved timbers of the Mary Rose, demonstrating that biological pathways of iron and sulfur oxidization existed potentially in this wood, before preservation with PEG. This study was designed to establish if the recycled PEG spray system was a reservoir of microorganisms capable of iron and sulfur oxidization during preservation of the Mary Rose. Microbial enrichments derived from PEG impregnated biofilm collected from underneath the Mary Rose hull, were examined to better understand the processes of cycling of iron. X-ray absorption spectroscopy was utilised to demonstrate the biological contribution to production of sulfuric acid in the wood. Using molecular microbiological techniques to examine these enrichment cultures, PEG was found to mediate a shift in the microbial community from a co-culture of Stenotrophomonas and Brevunidimonas sp, to a co-culture of Stenotrophomonas and the iron oxidising Alicyclobacillus sp. Evidence is presented that PEG is not an inert substance in relation to the redox cycling of iron. This is the first demonstration that solutions of PEG used in the conservation of the Mary Rose are promoting the oxidation of ferrous iron in acidic solutions, in which spontaneous abiotic oxidation does not occur in water. Critically, these results suggest PEG mediated redox cycling of iron between valence states in solutions of 75% PEG 200 and 50% PEG 2000 (v/v) at pH 3.0, with serious implications for the future use of PEG as a conservation material of iron rich wooden archaeological artefacts

Lawsonia intracellularis exploits β-catenin/Wnt and Notch signalling pathways during infection of intestinal crypt to alter cell homeostasis and promote cell proliferation

Lawsonia intracellularis is an obligate intracellular bacterial pathogen that causes proliferative enteropathy (PE) in pigs. L. intracellularis infection causes extensive intestinal crypt cell proliferation and inhibits secretory and absorptive cell differentiation. However, the affected host upstream cellular pathways leading to PE are still unknown. β-catenin/Wnt signalling is essential in maintaining intestinal stem cell (ISC) proliferation and self-renewal capacity, while Notch signalling governs differentiation of secretory and absorptive lineage specification. Therefore, in this report we used immunofluorescence (IF) and quantitative reverse transcriptase PCR (RTqPCR) to examine β-catenin/Wnt and Notch-1 signalling levels in uninfected and L. intracellularis infected pig ileums at 3, 7, 14, 21 and 28 days post challenge (dpc). We found that while the significant increase in Ki67+ nuclei in crypts at the peak of L. intracellularis infection suggested enhanced cell proliferation, the expression of c-MYC and ASCL2, promoters of cell growth and ISC proliferation respectively, was down-regulated. Peak infection also coincided with enhanced cytosolic and membrane-associated β-catenin staining and induction of AXIN2 and SOX9 transcripts, both encoding negative regulators of β-catenin/Wnt signalling and suggesting a potential alteration to β-catenin/Wnt signalling levels, with differential regulation of the expression of its target genes. We found that induction of HES1 and OLFM4 and the down-regulation of ATOH1 transcript levels was consistent with the increased Notch-1 signalling in crypts at the peak of infection. Interestingly, the significant down-regulation of ATOH1 transcript levels coincided with the depletion of MUC2 expression at 14 dpc, consistent with the role of ATOH1 in promoting goblet cell maturation. The lack of significant change to LGR5 transcript levels at the peak of infection suggested that the crypt hyperplasia was not due to the expansion of ISC population. Overall, simultaneous induction of Notch-1 signalling and the attenuation of β-catenin/Wnt pathway appear to be associated with the inhibition of goblet cell maturation and enhanced crypt cell proliferation at the peak of L. intracellularis infection. Moreover, the apparent differential regulation of apoptosis between crypt and lumen cells together with the strong induction of Notch-1 signalling and the enhanced SOX9 expression along crypts 14 dpc suggest an expansion of actively dividing transit amplifying and/or absorptive progenitor cells and provide a potential basis for understanding the development and maintenance of PE