Hopanoid Production Is Required for Low-pH Tolerance, Antimicrobial Resistance, and Motility in Burkholderia cenocepacia

Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize membranes and to regulate their fluidity and permeability. To date, very few studies have evaluated the role of hopanoids in bacterial physiology. The synthesis of hopanoids depends on the enzyme squalene-hopene cyclase (Shc), which converts the linear squalene into the basic hopene structure. Deletion of the 2 genes encoding Shc enzymes in Burkholderia cenocepacia K56-2, BCAM2831 and BCAS0167, resulted in a strain that was unable to produce hopanoids, as demonstrated by gas chromatography and mass spectrometry. Complementation of the Delta shc mutant with only BCAM2831 was sufficient to restore hopanoid production to wild-type levels, while introducing a copy of BCAS0167 alone into the Delta shc mutant produced only very small amounts of the hopanoid peak. The Delta shc mutant grew as well as the wild type in medium buffered to pH 7 and demonstrated no defect in its ability to survive and replicate within macrophages, despite transmission electron microscopy (TEM) revealing defects in the organization of the cell envelope. The Delta shc mutant displayed increased sensitivity to low pH, detergent, and various antibiotics, including polymyxin B and erythromycin. Loss of hopanoid production also resulted in severe defects in both swimming and swarming motility. This suggests that hopanoid production plays an important role in the physiology of B. cenocepacia.

Interplay of the Wzx translocase and the corresponding polymerase and chain length regulator proteins in the translocation and periplasmic assembly of lipopolysaccharide o antigen

Genetic evidence suggests that a family of bacterial and eukaryotic integral membrane proteins (referred to as Wzx and Rft1, respectively) mediates the transbilayer movement of isoprenoid lipid-linked glycans. Recent work in our laboratory has shown that Wzx proteins involved in O-antigen lipopolysaccharide (LPS) assembly have relaxed specificity for the carbohydrate structure of the O-antigen subunit. Furthermore, the proximal sugar bound to the isoprenoid lipid carrier, undecaprenyl-phosphate (Und-P), is the minimal structure required for translocation. In Escherichia coli K-12, N-acetylglucosamine (GlcNAc) is the proximal sugar of the O16 and enterobacterial common antigen (ECA) subunits. Both O16 and ECA systems have their respective translocases, WzxO16 and WzxE, and also corresponding polymerases (WzyO16 and WzyE) and O-antigen chain-length regulators (WzzO16 and WzzE), respectively. In this study, we show that the E. coli wzxE gene can fully complement a wzxO16 translocase deletion mutant only if the majority of the ECA gene cluster is deleted. In addition, we demonstrate that introduction of plasmids expressing either the WzyE polymerase or the WzzE chain-length regulator proteins drastically reduces the O16 LPS-complementing activity of WzxE. We also show that this property is not unique to WzxE, since WzxO16 and WzxO7 can cross-complement translocase defects in the O16 and O7 antigen clusters only in the absence of their corresponding Wzz and Wzy proteins. These genetic data are consistent with the notion that the translocation of O-antigen and ECA subunits across the plasma membrane and the subsequent assembly of periplasmic O-antigen and ECA Und-PP-linked polymers depend on interactions among Wzx, Wzz, and Wzy, which presumably form a multiprotein complex.

Nonlinear electrostatic excitations of charged dust in degenerate ultra-dense quantum dusty plasmas

The linear and nonlinear properties of low-frequency electrostatic excitations of charged dust particles (or defects) in a dense collisionless, unmagnetized Thomas-Fermi plasma are investigated. A fully ionized three-component model plasma consisting of electrons, ions, and negatively charged massive dust grains is considered. Electrons and ions are assumed to be in a degenerate quantum state, obeying the Thomas-Fermi density distribution, whereas the inertial dust component is described by a set of classical fluid equations. Considering large-amplitude stationary profile travelling-waves in a moving reference frame, the fluid evolution equations are reduced to a pseudo-energy-balance equation, involving a Sagdeev-type potential function. The analysis describes the dynamics of supersonic dust-acoustic solitary waves in Thomas-Fermi plasmas, and provides exact predictions for their dynamical characteristics, whose dependence on relevant parameters (namely, the ion-to-electron Fermi temperature ratio, and the dust concentration) is investigated. An alternative route is also adopted, by assuming weakly varying small-amplitude disturbances off equilibrium, and then adopting a multiscale perturbation technique to derive a Korteweg–de Vries equation for the electrostatic potential, and finally solving in terms for electric potential pulses (electrostatic solitons). A critical comparison between the two methods reveals that they agree exactly in the small-amplitude, weakly superacoustic limit. The dust concentration (Havnes) parameter h = Zd0nd0/ne0 affects the propagation characteristics by modifying the phase speed, as well as the electron/ion Fermi temperatures. Our results aim at elucidating the characteristics of electrostatic excitations in dust-contaminated dense plasmas, e.g., in metallic electronic devices, and also arguably in supernova environments, where charged dust defects may occur in the quantum plasma regime.

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint.

Antineutrophil cytoplasmic antibodies in myelodysplasia

Antibodies to neutrophil cytoplasmic antigens (ANCA) are good serological markers for patients with mainly vasculitic conditions. Two main types of ANCAs have been detected, the first termed cytoplasmic antineutrophil cytoplasmic antibody (cANCA) are mainly associated with patients with Wegener's granulomatosis, the other termed perinuclear antineutrophil cytoplasmic antibody (pANCA) are mainly associated with patients with renal vasculitis, rheumatic and collagen disorders. These antibodies are against various constituents of neutrophil granules. In patients with myelodysplasia, defects in normal granulocyte development are seen. We report a series of twelve patients with myelodysplasia of whom at least four showed a low titre and one a high titre of pANCA. Two of these patients also had demonstrable activity against myeloperoxidase (MPO). None of these patients had any evidence of systemic or cutaneous vasculitis or of any autoimmune disorder. There was no pANCA positivity in an age matched control group.

Lorentz TEM investigation of the switching behavior in artificial spin ice building blocks

Frustration – the inability to simultaneously satisfy all interactions – occurs in a wide range of systems including neural networks, water ice and magnetic systems. An example of the latter is the so called spin-ice in pyrochlore materials [1] which have attracted a lot of interest not least due to the emergence of magnetic monopole defects when the ‘ice rules’ governing the local ordering breaks down [2]. However it is not possible to directly measure the frustrated property – the direction of the magnetic moments – in such spin ice systems with current experimental techniques. This problem can be solved by instead studying artificial spin-ice systems where the molecular magnetic moments are replaced by nanoscale ferromagnetic islands [3-8]. Two different arrangements of the ferromagnetic islands have been shown to exhibit spin ice behaviour: a square lattice maintaining four moments at each vertex [3,8] and the Kagome lattice which has only three moments per vertex but equivalent interactions between them [4-7]. Magnetic monopole defects have been observed in both types of lattices [7-8]. One of the challenges when studying these artificial spin-ice systems is that it is difficult to arrive at the fully demagnetised ground-state [6-8].
Here we present a study of the switching behaviour of building blocks of the Kagome lattice influenced by the termination of the lattice. Ferromagnetic islands of nominal size 1000 nm by 100 nm were fabricated in five island blocks using electron-beam lithography and lift-off techniques of evaporated 18 nm Permalloy (Ni80Fe20) films. Each block consists of a central island with four arms terminated by a different number and placement of ‘injection pads’, see Figure 1. The islands are single domain and magnetised along their long axis. The structures were grown on a 50 nm thick electron transparent silicon nitride membrane to allow TEM observation, which was back-coated with a 5 nm film of Au to prevent charge build-up during the TEM experiments.
To study the switching behaviour the sample was subjected to a magnetic field strong enough to magnetise all the blocks in one direction, see Figure 1. Each block obeys the Kagome lattice ‘ice-rules’ of “2-in, 1-out” or “1-in, 2-out” in this fully magnetised state. Fresnel mode Lorentz TEM images of the sample were then recorded as a magnetic field of increasing magnitude was applied in the opposite direction. While the Fresnel mode is normally used to image magnetic domain structures [9] for these types of samples it is possible to deduce the direction of the magnetisation from the Lorentz contrast [5]. All images were recorded at the same over-focus judged to give good Lorentz contrast.
The magnetisation was found to switch at different magnitudes of the applied field for nominally identical blocks. However, trends could still be identified: all the blocks with any injection pads, regardless of placement and number, switched the direction of the magnetisation of their central island at significantly smaller magnitudes of the applied magnetic field than the blocks without injection pads. It can therefore be concluded that the addition of an injection pad lowers the energy barrier to switching the connected island, acting as a nucleation site for monopole defects. In these five island blocks the defects immediately propagate through to the other side, but in a larger lattice the monopoles could potentially become trapped at a vertex and observed [10].
References

[1] M J Harris et al, Phys Rev Lett 79 (1997) p.2554.
[2] C Castelnovo, R Moessner and S L Sondhi, Nature 451 (2008) p. 42.
[3] R F Wang et al, Nature 439 (2006) 303.
[4] M Tanaka et al, Phys Rev B 73 (2006) 052411.
[5] Y Qi, T Brintlinger and J Cumings, Phys Rev B 77 (2008) 094418.
[6] E Mengotti et al, Phys Rev B 78 (2008) 144402.
[7] S Ladak et al, Nature Phys 6 (2010) 359.
[8] C Phatak et al, Phys Rev B 83 (2011) 174431.
[9] J N Chapman, J Phys D 17 (1984) 623.
[10] The authors gratefully acknowledge funding from the EPSRC under grant number EP/D063329/1.

Interstitial Nitrogen in Diamond – Optical and EPR Studies

Nitrogen is one of the most common impurities in diamond. On a substitutional site it acts as a deep donor, approximately 1.7 eV below the conduction band. Irradiation of nitrogen containing diamond and subsequent annealing creates the nitrogen vacancy centre, which has recently attracted much attention for quantum information processing application. Another possible product of irradiation and annealing of nitrogen containing diamond is interstitial nitrogen. Presumably, a mobile carbon interstitial migrates to a substitutional nitrogen to produce an interstitial nitrogen complex which may or may not be mobile. The configuration(s) of interstitial nitrogen related defects (e.g. bond centred, [001]-split) are not known. An infra-red (IR) absorption peak at 1450 cm-1 labelled H1a has been associated with an nitrogen interstitial complex. [1] Theoretical modelling suggested that this IR local mode is due to a bond centred nitrogen interstitial [2]. However, more recent modelling [3] suggests that this defect is mobile at temperatures were H1a is stable and instead assign H1a to two nitrogen atoms occupying a single lattice site in a [001]-split configuration. To date no electron paramagnetic resonance (EPR) spectra have been conclusively associated with an interstitial nitrogen defect.

In this study we present data from new EPR and optical absorption studies in combination with uniaxial stress of nitrogen interstitial related defects in electron irradiated and annealed nitrogen doped diamond. These measurements yield symmetry information about the defects allowing us to determine which of the proposed models are possible. EPR spectra of nitrogen interstitial related defects in samples isotopically enriched with 15N are reported and we show that these explain the lack of previous EPR data for these defects. Correlations between the IR absorbance and the integrated intensity of the new EPR defects are studied for varying irradiation doses and annealing temperatures.

Evaluation of corneal endothelium and keratic precipitates by specular microscopy in anterior uveitis

Background - The study of corneal endothelium, by specular microscopy, in patients with anterior uveitis has largely been restricted to observations on the endothelial cells. In this prospective study 'keratic precipitates' (KP) in different types of uveitis were examined in different stages of the disease process and the endothelial changes occurring in the vicinity of the KP were evaluated in comparison with the endothelium of the uninvolved eye. Methods - 13 patients with active unilateral uveitis were recruited. The mean age was 42.9 years (range 20-76 years). A Tomey-1100 contact wide field specular (x10) microscope was used to capture endothelial images and KP until the resolution of uveitis. Data regarding type of uveitis, number, size, and nature of KP were recorded. Automated morphometric analysis was done for cell size, cell density and coefficient of variation, and statistical comparisons of cell size and cell density were made (Student's t test) between the endothelium in the vicinity of fresh and resolving KP, fresh KP and normal endothelium, and resolving KP and normal endothelium. Results - On specular microscopy, fresh KP were seen as dense, white glistening deposits occupying 5-10 endothelial cells in diameter and fine KP were widely distributed and were one or two endothelial cells in diameter. The KP in Posner-Schlossman syndrome had a distinct and different morphology. With clinical remission of uveitis, the KP were observed to undergo characteristic morphological changes and old KP demonstrated a large, dark halo surrounding a central white deposit and occasionally a dark shadow or a 'lacuna' replaced the site of the original KP. Endothelial blebs were noted as dark shadows or defects in the endothelial mosaic in patients with recurrent uveitis. There was significant statistical difference in the mean cell size and cell density of endothelial cells in the vicinity of fresh KP compared with normal endothelium of the opposite eye. Conclusion - This study elucidated the different specular microscopic features of KP in anterior uveitis. Distinct morphological features of large and fine KP were noted. These features underwent dramatic changes on resolution of uveitis. The endothelium was abnormal in the vicinity of KP, which returned to near normal values on resolution of uveitis.

Putrescine Reduces Antibiotic-Induced Oxidative Stress as a Mechanism of Modulation of Antibiotic Resistance in Burkholderia cenocepacia

Communication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic resistant bacterium Burkholderia cenocepacia protects less resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure of B. cenocepacia to sub-lethal concentrations of PmB and other bactericidal antibiotics induce reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS such as superoxide ion and hydrogen peroxide was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-lux reporter system fusions. We evaluated wild type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sub-lethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation, and a parallel increased sensitivity to PmB. Of the 3 B. cenocepacia putrescine synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study exposes BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance, and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.

Role of capsular modified heptose in the virulence of Campylobacter jejuni

The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.

DNA mismatch repair and the transition to hormone independence in breast and prostate cancer

The molecular basis for the progression of breast and prostate cancer from hormone dependent to hormone independent disease remains a critical issue in the management of these two cancers. The DNA mismatch repair system is integral to the maintenance of genomic stability and suppression of tumorigenesis. No firm consensus exists regarding the implications of mismatch repair (MMR) deficiencies in the development of breast or prostate cancer. However, recent studies have reported an association between mismatch repair deficiency and loss of specific hormone receptors, inferring a potential role for mismatch repair deficiency in this transition. An updated review of the experimental data supporting or contradicting the involvement of MMR defects in the development and progression of breast and prostate cancer will be provided with particular emphasis on their implications in the transition to hormone independence.

The emergence of DNA methylation as a key modulator of aberrant cell death in prostate cancer

It is now well established that cancer cells exhibit a number of genetic defects in the machinery that governs programmed cell death and that sabotage of apoptosis is one of the principal factors aiding in the evolution of the carcinogenic phenotype. A number of studies have implicated aberrant DNA methylation as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many processes including apoptosis. To date, studies on the methylation profile of apoptotic genes have largely focused on cancers of the breast, colon and stomach, with only limited data available on prostate cancer. Here we discuss the major developments in the field of DNA methylation and its role in the regulation of aberrant apoptosis in prostate cancer. The most significant advances have involved the discovery of apoptotic gene targets of methylation, including XAF1, (fragile histidine triad (FHIT ), cellular retinol binding protein 1 (CRBP1), decoy receptor 1(DCR1), decoy receptor 2 (DCR2 ), target of methylation-induced silenceing 1 (TMS1), TNF receptor superfamily, member 6 (FAS), Reprimo (RPRM) and GLI pathogenesis-related 1 (GLIPR1). These genes are reported to be hypermethylated in prostate cancer and some offer potential as diagnostic and prognostic markers. We also introduce the concept of an 'apoptotic methylation signature' for prostate cancer and evaluate its potential in a diagnostic, prognostic and therapeutic setting.

Seed dormancy in Arabidopsis thaliana is controlled by alternative polyadenylation of DOG1

DOG1 is a key regulator of seed dormancy in Arabidopsis and other plants. Interestingly, the C-terminus of DOG1 is either absent or not conserved in many plant species. Here, we show that in Arabidopsis DOG1 transcript is subject to alternative polyadenylation. In line with this, mutants in RNA 3' processing complex display weakened seed dormancy in parallel with defects in DOG1 proximal polyadenylation site selection, suggesting that the short DOG1 transcript, is functional. This is corroborated by the finding that the proximally polyadenylated short DOG1 mRNA is translated in vivo and complements the dog1 mutation. In summary, our findings indicate that the short DOG1 protein isoform produced from the proximally polyadenylated DOG1 mRNA is a key player in the establishment of seed dormancy in Arabidopsis and characterize a set of mutants in RNA 3' processing complex required for production of proximally polyadenylated functional DOG1 transcript.

Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.

Clinical Application of Poly(ADP-Ribose) Polymerase Inhibitors in High-Grade Serous Ovarian Cancer

: High-grade serous ovarian cancer is characterized by genomic instability, with one half of all tumors displaying defects in the important DNA repair pathway of homologous recombination. Given the action of poly(ADP-ribose) polymerase (PARP) inhibitors in targeting tumors with deficiencies in this repair pathway by loss of BRCA1/2, ovarian tumors could be an attractive population for clinical application of this therapy. PARP inhibitors have moved into clinical practice in the past few years, with approval from the Food and Drug Administration (FDA) and European Medicines Agency (EMA) within the past 2 years. The U.S. FDA approval of olaparib applies to fourth line treatment in germline BRCA-mutant ovarian cancer, and European EMA approval to olaparib maintenance in both germline and somatic BRCA-mutant platinum-sensitive ovarian cancer. In order to widen the ovarian cancer patient population that would benefit from PARP inhibitors, predictive biomarkers based on a clear understanding of the mechanism of action are required. Additionally, a better understanding of the toxicity profile is needed if PARP inhibitors are to be used in the curative, rather than the palliative, setting. We reviewed the development of PARP inhibitors in phase I-III clinical trials, including combination trials of PARP inhibitors and chemotherapy/antiangiogenics, the approval for these agents, the mechanisms of resistance, and the outstanding issues, including the development of biomarkers and the rate of long-term hematologic toxicities with these agents.

IMPLICATIONS FOR PRACTICE: The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib has recently received approval from the Food and Drug Administration (FDA) and European Medicines Agency (EMA), with a second agent (rucaparib) likely to be approved in the near future. However, the patient population with potential benefit from PARP inhibitors is likely wider than that of germline BRCA mutation-associated disease, and biomarkers are in development to enable the selection of patients with the potential for clinical benefit from these agents. Questions remain regarding the toxicities of PARP inhibitors, limiting the use of these agents in the prophylactic or adjuvant setting until more information is available. The indications for olaparib as indicated by the FDA and EMA are reviewed.