Novel Plasmids and Resistance Phenotypes in Yersinia pestis: Unique Plasmid Inventory of Strain Java 9 Mediates High Levels of Arsenic Resistance

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium

Identification of the Transcriptional Regulator NcrB in the Nickel Resistance Determinant of Leptospirillum ferriphilum UBK03

The nickel resistance determinant ncrABCY was identified in Leptospirillum ferriphilum UBK03. Within this operon, ncrA and ncrC encode two membrane proteins that form an efflux system, and ncrB encodes NcrB, which belongs to an uncharacterized family (DUF156) of proteins. How this determinant is regulated remains unknown. Our data indicate that expression of the nickel resistance determinant is induced by nickel. The promoter of ncrA, designated pncrA, was cloned into the promoter probe vector pPR9TT, and co-transformed with either a wild-type or mutant nickel resistance determinant. The results revealed that ncrB encoded a transcriptional regulator that could regulate the expression of ncrA, ncrB, and ncrC. A GC-rich inverted repeat sequence was identified in the promoter pncrA. Electrophoretic mobility shift assays (EMSAs) and footprinting assays showed that purified NcrB could specifically bind to the inverted repeat sequence of pncrA in vitro; this was confirmed by bacterial one-hybrid analysis. Moreover, this binding was inhibited in the presence of nickel ions. Thus, we classified NcrB as a transcriptional regulator that recognizes the inverted repeat sequence binding motif to regulate the expression of the key nickel resistance gene, ncrA

Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

<p>Abstract</p> <p>Background</p> <p>Mifepristone (MF) has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR). The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR.</p> <p>Methods</p> <p>Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored <it>in vitro </it>by the capacity of Cdk2 to phosphorylate histone H1.</p> <p>Results</p> <p>MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR.</p> <p>Conclusions</p> <p>Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and apoptotic lethality at higher doses in association with increased hypodiploid DNA content. Contrary to common opinion, growth inhibition of cancer cells by antiprogestin MF is not dependent upon expression of classical, nuclear PR.</p

Solution fibre spinning technique for the fabrication of tuneable decellularised matrix-laden fibres and fibrous micromembranes

Recreating tissue-specific microenvironments of the extracellular matrix (ECM) in vitro is of broad interest for the fields of tissue engineering and organ-on-a-chip. Here, we present biofunctional ECM protein fibres and suspended membranes, with tuneable biochemical, mechanical and topographical properties. This soft and entirely biologic membrane scaffold, formed by micro-nano-fibres using low voltage electrospinning, displays three unique characteristics for potential cell culture applications: high-content of key ECM proteins, single-layered mesh membrane, and flexibility for in situ integration into a range of device setups. Extracellular matrix (ECM) powder derived from urinary bladder, was used to fabricate the ECM-laden fibres and membranes. The highest ECM concentration in the dry protein fibre was 50 wt%, with the rest consisting of gelatin. Key ECM proteins, including collagen IV, laminin, and fibronectin, were shown to be preserved post the biofabrication process. The single fibre tensile Young's modulus can be tuned for over two orders of magnitude between ∼600 kPa and 50 MPa depending on the ECM content. Combining the fibre mesh printing with 3D printed or microfabricated structures, culture devices were constructed for endothelial layer formation, and a trans-membrane co-culture formed by glomerular cell types of podocytes and glomerular endothelial cells, demonstrating feasibility of the membrane culture. Our cell culture observation points to the importance of membrane mechanical property and re-modelling ability as a factor for soft membrane-based cell cultures. The ECM-laden fibres and membranes presented here would see potential applications in in vitro assays, and tailoring structure and biological functions of tissue engineering scaffolds. Statement of Significance: Recreating tissue-specific microenvironments of the extracellular matrix (ECM) is of broad interest for the fields of tissue engineering and organ-on-a-chip. Both the biochemical and biophysical signatures of the engineered ECM interplay to affect cell response. Currently, there are limited biomaterials processing methods which allow to design ECM membrane properties flexibly and rapidly. Solvents and additives used in many existing processes also induced unwanted ECM protein degradation and toxic residues. This paper presents a solution fibre spinning technique, where careful selection of the solution combination led to well-preserved ECM proteins with tuneable composition. This technique also provides a highly versatile approach to fabricate ECM fibres and membranes, leading to designable fibre Young's modulus for over two orders of magnitude

Extreme arsenic resistance by the acidophilic archaeon 'Ferroplasma acidarmanus' Fer1

‘Ferroplasma acidarmanus’ Fer1 is an arsenic-hypertolerant acidophilic archaeon isolated from the Iron Mountain mine, California; a site characterized by heavy metals contamination. The presence of up to 10 g arsenate per litre [As(V); 133 mM] did not significantly reduce growth yields, whereas between 5 and 10 g arsenite per litre [As(III); 67–133 mM] significantly reduced the yield. Previous bioinformatic analysis indicates that ‘F. acidarmanus’ Fer1 has only two predicted genes involved in arsenic resistance and lacks a recognizable gene for an arsenate reductase. Biochemical analysis suggests that ‘F. acidarmanus’ Fer1 does not reduce arsenate indicating that ‘F. acidarmanus’ Fer1 has an alternative resistance mechanism to arsenate other than reduction to arsenite and efflux. Primer extension analysis of the putative ars transcriptional regulator (arsR) and efflux pump (arsB) demonstrated that these genes are co-transcribed, and expressed in response to arsenite, but not arsenate. Two-dimensional polyacrylamide gel electrophoresis analysis of ‘F. acidarmanus’ Fer1 cells exposed to arsenite revealed enhanced expression of proteins associated with protein refolding, including the thermosome Group II HSP60 family chaperonin and HSP70 DnaK type heat shock proteins. This report represents the first molecular and proteomic study of arsenic resistance in an acidophilic archaeon

Molecular Bridging of Silicon Nanogaps

The highly doped electrodes of a vertical silicon nanogap device have been bridged by a 5.85 nm long molecular wire, which was synthesized in situ by grafting 4-ethynylbenzaldehyde via C-Si links to the top and bottom electrodes and thereafter by coupling an amino-terminated fluorene unit to the aldehyde groups of the activated electrode surfaces. The number of bridging molecules is constrained by relying on surface roughness to match the 5.85 nm length with an electrode gap that is nominally 1 nm wider and may be controlled by varying the reaction time: the device current increases from <= 1 pA at 1 V following the initial grafting step to 10-100 nA at 1 V when reacted for 5-15 min with the amino-terminated linker and 10 mu A when reacted for 16-53 h. It is the first time that both ends of a molecular wire have been directly grafted to silicon electrodes, and these molecule-Induced changes are reversible. The bridges detach when the device Is rinsed with dilute add solution, which breaks the imine links of the in situ formed wire and causes the current to revert to the subpicoampere leakage value of the 4-ethynylbenzaldehyde-grafted nanogap structure