Applications of the Environmental Scanning Electron Microscope to Conservation Science

The environmental scanning electron microscope (E-SEM) provides electron imaging at relatively high sample pressure, with imaging and analysis capabilities comparable to those of traditional high vacuum SEM. Several case studies demonstrate the advantages and research potential of this new technology as applied to conservation science: 1) dynamic study of wetting and drying of consolidated and unconsolidated adobe samples; 2) semi-dynamic study of lead corrosion as a result of exposure to formaldehyde; 3) electron imaging of outgassing samples-parchment; 4) study of uncoated, non-conductive samples-swabs from Sistine Chapel cleaning; 5) X-ray analysis of uncoated insulators-gold and garnet jewelry. The environmental scanning electron microscope offers unique capabilities for dynamic experiments, imaging of outgassing samples and insulators, which may be applied to the study of deterioration mechanisms, material treatments, and ancient materials and technologies

Identification of two substrates of FTS_1067 protein – An essential virulence factor of Francisella tularensis

Francisella tularensis is a highly virulent intracellular pathogen with the capacity to infect a variety of hosts including humans. One of the most important proteins involved in F. tularensis virulence and pathogenesis is the protein DsbA. This protein is annotated as a lipoprotein with disulfide oxidoreductase/isomerase activity. Therefore, its interactions with different substrates, including probable virulence factors, to assist in their proper folding are anticipated. We aimed to use the immunopurification approach to find DsbA (gene locus FTS_1067) interacting partners in F. tularensis subsp. holarctica strain FSC200 and compare the identified substrates with proteins which were found in our previous comparative proteome analysis. As a result of our work two FTS_1067 substrates, D-alanyl-D-alanine carboxypeptidase family protein and HlyD family secretion protein, were identified. Bacterial two-hybrid systems were further used to test their relevance in confirming FTS_1067 protein interactions

Development of human monoclonal antibodies against conserved antigens from Streptococcus pneumoniae

Streptococcus pneumoniae ist ein weitverbreiteter Erreger verschiedenster lebensbedrohlicher Krankheiten wie Pneumonien, Bakteriämien oder Meningitis. Obgleich lizensierte Impfstoffe und Antibiotika zur Verfügung stehen, bleibt die Morbidität und Mortalität, nicht nur in Entwicklungsländern, sondern auch in Industrieländern auf einem hohen Niveau. Aufgrund zunehmender Antibiotikaresistenzen und der suboptimalen Wirksamkeit verfügbarer Impfstoffe in Risikogruppen, steigt der Bedarf für neue Therapien, wie beispielsweise einer passiven Immunisierung mit monoklonalen Antikörpern. In dieser Arbeit wurden drei konservierte Antigene von S. pneumoniae – Lipoteichonsäure (LTA), pneumococcal surface protein A (PspA) und Pneumolysin (PLY) – auf ihre Eignung als Zielmoleküle für die Entwicklung einer anti-infektiösen Antikörper-Therapie hin untersucht. Obwohl die Isolierung und Reinigung nativer LTA für den Erreger Streptococcus pyogenes erfolgreich optimiert wurde, konnte LTA nicht in ausreichender Menge und Reinheit aus S. pneumoniae gewonnen und somit keine weiterführenden Studien mit diesem Antigen durchgeführt werden. Verschiedenste Konstrukte von PspA und PLY wurden rekombinant exprimiert, gereinigt und nachfolgend deren immunogene Wirkung in naïven C3H/HeN Mäusen demonstriert. Die gewonnenen Antiseren wurden in Folge genauestens in vitro analysiert. Oberflächenfärbungen lebender Pneumokokken zeigten, dass PspA spezifische Antikörper nicht alle PspA-Varianten detektieren konnten, sondern nur solche des selben PspA-Typs. Polyklonale Antikörper gegen Typ 1 PspA reagierten auch mit anderen Prolin-reichen Oberflächenproteinen, vermutlich PspC, jedoch nicht mit PspA Typ 2. Des weiteren wurden die generierten Antiseren in zuvor etablierten Mausmodellen durch passiven Transfer und nachfolgeneder letaler Infektion mit verschiedenen S. pneumoniae Stämmen auf ihre Wirksamkeit getestet. Die Ergebnisse korrelierten mit den Daten der Oberflächenfärbung: Anti-PspA Hyperimmunseren induzierten eine Protektion gegen Pneumokokken mit homologen jedoch nicht heterologen PspA. Anti-PLY Seren hingegen konnten keine vollständig protektive Wirkung erzielen, bewirkten jedoch eine erhöhte Überlebensrate. Die beobachtete Protektion korrelierte mit der Stärke der durch die Infektion induzierte inflammatorischen IL-6 Reaktion in den Tieren. Im Hinblick auf die Wirkungsweise von PLY wurden zwei in vitro Analysen zur Antikörper-Charakterisierung entwickelt: ein Hämolyse-Inhibitions Assay und ein hTLR4-Reporter Assay. Pneumolysin spezifische murine poly- und monoklonale Antikörper bewirkten eine Reduktion der hämolytischen Aktivität von PLY auf Erythrozyten und interferierten auch mit der Aktivierung von TLR4 durch PLY. Humane monoklonale Antikörper sollen in weiterer Folge aus humanen B-Zellen basierend auf der „Sindbis Virus Based Mammalian Cell Surface Display“ Technologie generiert werden. Zu diesem Zweck wurden gesunde Spender aufgrund ihres Antigen-spezifischen Titers im ELISA identifiziert. Zudem wurden die für die B-Zell-Selektion notwendigen PBMC-Färbetechniken optimiert. Zusammenfassend demonstriert diese Arbeit einen vertiefenden Einblick in die Wirkungsweise von PspA und PLY-spezifischen Antikörpern sowie die Entwicklung von für die Selektion und Validierung humaner monoklonaler Antikörper notwendigen Analyseverfahren. Somit konnte die Grundlage für eine Antikörper-basierende Therapie zur Prävention und Behandlung von lebensbedrohlichen Pneumokokkeninfektionen geschaffen werden.Streptococcus pneumoniae is a common human pathogen that causes a variety of life-threatening invasive diseases such as pneumonia, bacteremia and meningitis. Despite the availability of licensed vaccines and antibiotic treatments, morbidity and mortality attributed to this bacterium remain significant in developing and developed countries. Due to increasing antibiotic resistance and limited efficacy of existing vaccines in at-risk populations, there is a need for new treatment strategies such as passive immunotherapy using human monoclonal antibodies (mAbs). In this study, three conserved antigens of S. pneumoniae – lipoteichoic acid (LTA), pneumococcal surface protein A (PspA) and pneumolysin (PLY) – were characterized for their suitability as targets for a mAb-based anti-infective therapy. Although isolation and purification procedures could be optimized for LTA from Streptococcus pyogenes, native LTA could not be extracted from S. pneumoniae in sufficient quantity and quality, thus limiting more in-depth studies of this antigen. Recombinant full-length PspA and PLY as well as domains thereof were expressed, purified and subsequently proven to be highly immunogenic in naïve C3H/HeN mice. These antisera were characterized in-depth in vitro: in surface staining and in ELISA, antibodies were shown to recognize PspA in a clade-specific manner. Polyclonal antibodies against Family 1 PspA also reacted with other Proline-rich cell-surface proteins – presumably PspC – but not with Family 2 PspA. Consequently these antisera were tested in vivo by passive transfer and subsequent lethal challenge with different S. pneumoniae strains in mice. The results correlated with surface staining data: anti-PspA hyperimmune sera were only effective against pneumococci expressing homologous PspA but not against those with a heterologous variant. Anti PLY sera were not fully protective although conferring prolonged survival. Interestingly the observed protection correlated with the level of inflammatory IL-6, induced in mice. Two in vitro assays exploiting the function of PLY were set up to allow a detailed characterization of selected antibodies: a Hemolysis-Inhibition Assay and an hTLR4-Reporter Assay. PLY-specific murine polyclonal and monoclonal antibodies reduced the hemolytic activity of PLY on erythrocytes and interfered with the activation of TLR4 through PLY. Since human mAbs against PLY will be generated from B-cells based on the “Sindbis Virus Based Mammalian Cell Surface Display” technology, healthy human donors were identified based on their antibody titers in ELISA. In addition PMBC staining conditions that are required for the selection of antigen-specific memory B cells were optimized. In conclusion, a deeper insight into the mode of action of PspA- and PLY-specific antibodies could be gained with this work and analytical methods that are required for the selection and validation of human mAbs were developed. This way a basis for the development of a mAb-based therapy for the prevention and treatment of life-threatening pneumococcal diseases was established

Nucleoid-Associated Protein HU: A Lilliputian in Gene Regulation of Bacterial Virulence

Nucleoid-associated proteins belong to a group of small but abundant proteins in bacterial cells. These transcription regulators are responsible for many important cellular processes and also are involved in pathogenesis of bacteria. The best-known nucleoid-associated proteins, such as HU, FIS, H-NS, and IHF, are often discussed. The most important findings in research concerning HU protein are described in this mini review. Its roles in DNA compaction, shape modulation, and negative supercoiling induction have been studied intensively. HU protein regulates bacteria survival, growth, SOS response, virulence genes expression, cell division, and many other cell processes. Elucidating the mechanism of HU protein action has been the subject of many research projects. This mini review provides a comprehensive overview of the HU protein

The use of microelectrodes with AGNES

Absence of gradients and nernstian equilibrium stripping (AGNES) is a new electroanalytical technique designed to determine free heavy metal ion concentrations in solutions. AGNES had been applied, up to date, with conventional equipment such as the hanging mercury drop electrode (HMDE). Due to their much smaller volume, microelectrodes can reach a given preconcentration factor within a much shorter deposition time, so their use for AGNES has been evaluated in this work. For the particular case of the mercury microelectrode deposited onto an Ir disk (radius around 5 lm), AGNES has been successfully used for speciation purposes in the system Pb + PDCA (pyridinedicarboxylic acid). However, due to a relatively large capacitive current, which decays slowly, the limit of quantification for such microelectrodes has only been reduced by one half with respect to that of the HMDE

Incidence of Vascular Complications Arising from Anterior Spinal Surgery in the Thoraco-Lumbar Spine

Study DesignModern biomaterials and instrumentation have popularised surgery of the thoraco-lumbar spine through an anterior route. The advantage of anterior surgery is that it allows for a direct decompression of the compromised spinal canal. However, the potential for devastating long-term sequelae as a result of complications is high.PurposeThe aim of this study was to give a general overview and identify the incidence of vascular complications.Overview of LiteratureThere is limited literature describing the overall incidence and complications of anterior spinal surgery.MethodsA retrospective review of a prospective database of 1,262 consecutive patients with anterior surgery over a twelve-year period.ResultsIn our study, 1.58% (n=20) of patients suffered complications. Injury to a major vessel was encountered in 14 (1.11%) cases, of which nine involved an injury to the common iliac vein. In six cases, the original procedure was abandoned due to a life-threatening vascular injury (n=3) and unfavourable anatomy (n=3).ConclusionsThe incidence of vascular and other complications in our study was relatively low. Nevertheless, the potential for devastating long-term sequelae as a result of complications remains high. A thorough knowledge and awareness of normal and abnormal anatomy should be gained before attempting such a procedure, and a vascular surgical assistance especially should be readily accessible. We believe use of access surgeons is mandatory in cases with difficult or aberrant anatomy

Micro-drilling of polymer tubular ultramicroelectrode arrays for electrochemical sensors

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Spectroscopic techniques and the conservation of artists’ acrylic emulsion paints

Artists’ acrylic emulsion paints are used in many contexts such as paintings, murals, sculptures, works on paper and mixed media; and are forming increasing proportions of modern and contemporary art collections. Although acrylic emulsion paints have been the focus of museum-led research over the past decade, the impact of artists’ technique and conservation treatment on the upper-most surface of these paints remains essentially unexplored ; This paper summarises previous studies using vibrational (FTIR) spectroscopy and presents initial assessments of paint surfaces using X-ray spectroscopies (XPS and NEXAFS) aimed at characterising artists’ acrylic paint film surfaces after natural ageing and wet surface cleaning treatment. Both techniques were found to be well suited for surface-sensitive investigations of the organic materials associated with artists’ acrylic paints, including explorations into: (A) cleaning system residues, (B) surfactant extraction from paint surfaces, (C) the identification of migrated surfactant, and (D) monitoring pigment changes at the paint/air interface of paint films ; It has been shown is that these X-ray spectroscopic techniques can be used for the analysis of almost purely organic materials in a way that complements mass spectroscopic techniques, FTIR and XRF. This investigation forms part of broader, currently ongoing, multi-technique investigation into the properties of artists’ acrylic paints and development of conservation treatments for works-of-art made with these materials

Modified activities of macrophages’ deubiquitinating enzymes after Francisella infection

Francisella tularensis influences several host molecular/signaling pathways during infection. Ubiquitination and deubiquitination are among the most important regulatory mechanisms and respectively occur through attachment or removal of the ubiquitin molecule. The process is necessary not only to mark molecules for degradation, but also, for example, to the activation of signaling pathways leading to pro-inflammatory host response. Many intracellular pathogens, including Francisella tularensis, have evolved mechanisms of modifying such host immune responses to escape degradation. Here, we describe that F. tularensis interferes with the host’s ubiquitination system. We show increased total activity of deubiquitinating enzymes (DUBs) in human macrophages after infection, while confirm reduced enzymatic activities of two specific DUBs (USP10 and UCH-L5), and demonstrate increased activity of USP25. We further reveal the enrichment of these three enzymes in exosomes derived from F. tularensis-infected cells. The obtained results show the regulatory effect on ubiquitination mechanism in macrophages during F. tularensis infection

Transient study of the oxygen reduction reaction on reduced Pt and Pt alloys microelectrodes: evidence for the reduction of pre-adsorbed oxygen species linked to dissolved oxygen

Using chronoamperometry at preconditioned oxide-free Pt microdisc electrodes in aqueous media, we investigated the oxygen reduction reaction (ORR) on the millisecond timescale and obtained results consistent with the reduction of oxygen species which adsorb on the electrode before the ORR is electrochemically driven. Furthermore these adsorbed species are clearly linked to oxygen in solution. At long times, the amperometric response is solely controlled by the diffusion of dissolved oxygen towards the microelectrode. However, at short times, typically below 50 ms, the reduction of pre-adsorbed oxygen produces a large extra current whose magnitude depends on the oxygen concentration in solution, deliberate electrode poisoning and the rest time before the potential step. Using sampled current voltammetry we show that this extra current affects the entire potential range of the ORR. Using microdisc electrodes made with Pt alloys we find that the amperometric response is sufficiently sensitive to distinguish oxygen coverage differences between Pt, Pt0.9Rh0.1 and Pt0.9Ir0.1 microdiscs. These unexpected and, to our knowledge, never previously reported results provide new insight into the oxygen reduction reaction on Pt. The existence over a wide potential range of irreversibly adsorbed oxygen species arising from dissolved oxygen and different from Pt oxide is particularly relevant to the development of oxygen reduction catalysts for low temperature fuel cells