Expected resolution limits of x-ray free-electron laser single-particle imaging for realistic source and detector properties.

The unprecedented intensity of x-ray free-electron laser sources has enabled single-particle x-ray diffraction imaging (SPI) of various biological specimens in both two-dimensional projection and three dimensions (3D). The potential of studying protein dynamics in their native conditions, without crystallization or chemical staining, has encouraged researchers to aim for increasingly higher resolutions with this technique. The currently achievable resolution of SPI is limited to the sub-10 nanometer range, mainly due to background effects, such as instrumental noise and parasitic scattering from the carrier gas used for sample delivery. Recent theoretical studies have quantified the effects of x-ray pulse parameters, as well as the required number of diffraction patterns to achieve a certain resolution, in a 3D reconstruction, although the effects of detector noise and the random particle orientation in each diffraction snapshot were not taken into account. In this work, we show these shortcomings and address limitations on achievable image resolution imposed by the adaptive gain integrating pixel detector noise.

Time-Resolved XUV Opacity Measurements of Warm Dense Aluminum.

The free-free opacity in plasmas is fundamental to our understanding of energy transport in stellar interiors and for inertial confinement fusion research. However, theoretical predictions in the challenging dense plasma regime are conflicting and there is a dearth of accurate experimental data to allow for direct model validation. Here we present time-resolved transmission measurements in solid-density Al heated by an XUV free-electron laser. We use a novel functional optimization approach to extract the temperature-dependent absorption coefficient directly from an oversampled pool of single-shot measurements, and find a pronounced enhancement of the opacity as the plasma is heated to temperatures of order of the Fermi energy. Plasma heating and opacity enhancement are observed on ultrafast timescales, within the duration of the femtosecond XUV pulse. We attribute further rises in the opacity on ps timescales to melt and the formation of warm dense matter.

A versatile liquid-jet setup for the European XFEL.

The SPB/SFX instrument of the European XFEL provides unique possibilities for high-throughput serial femtosecond crystallography. This publication presents the liquid-jet sample delivery setup of this instrument. The setup is compatible with state-of-the-art gas dynamic virtual nozzle systems as well as high-viscosity extruders and provides space and flexibility for other liquid injection devices and future upgrades. The liquid jets are confined in a differentially pumped catcher assembly and can be replaced within a couple of minutes through a load-lock. A two-microscope imaging system allows visual control of the jets from two perspectives.

Transferring the entatic-state principle to copper photochemistry.

The entatic state denotes a distorted coordination geometry of a complex from its typical arrangement that generates an improvement to its function. The entatic-state principle has been observed to apply to copper electron-transfer proteins and it results in a lowering of the reorganization energy of the electron-transfer process. It is thus crucial for a multitude of biochemical processes, but its importance to photoactive complexes is unexplored. Here we study a copper complex-with a specifically designed constraining ligand geometry-that exhibits metal-to-ligand charge-transfer state lifetimes that are very short. The guanidine-quinoline ligand used here acts on the bis(chelated) copper(I) centre, allowing only small structural changes after photoexcitation that result in very fast structural dynamics. The data were collected using a multimethod approach that featured time-resolved ultraviolet-visible, infrared and X-ray absorption and optical emission spectroscopy. Through supporting density functional calculations, we deliver a detailed picture of the structural dynamics in the picosecond-to-nanosecond time range.

EXPERIMENTAL AND MONTE CARLO INVESTIGATIONS OF BCF-12 SMALL­AREA PLASTIC SCINTILLATION DETECTORS FOR NEUTRON PINHOLE CAMERA.

Plastic organic scintillators such as the blue-emitting BCF-12 are versatile and inexpensive tools. Recently, BCF-12 scintillators have been foreseen for investigation of the spatial distribution of neutrons emitted from dense magnetized plasma. For this purpose, small-area (5 mm × 5 mm) detectors based on BCF-12 scintillation rods and Hamamatsu photomultiplier tubes were designed and constructed at the Institute of Nuclear Physics. They will be located inside the neutron pinhole camera of the PF-24 plasma focus device. Two different geometrical layouts and approaches to the construction of the scintillation element were tested. The aim of this work was to determine the efficiency of the detectors. For this purpose, the experimental investigations using a neutron generator and a Pu-Be source were combined with Monte Carlo computations using the Geant4 code.

Estimating chronic hepatitis C prognosis using transient elastography-based liver stiffness: A systematic review and meta-analysis.

Chronic hepatitis C (CHC) is a leading cause of hepatic fibrosis and cirrhosis. The level of fibrosis is traditionally established by histology, and prognosis is estimated using fibrosis progression rates (FPRs; annual probability of progressing across histological stages). However, newer noninvasive alternatives are quickly replacing biopsy. One alternative, transient elastography (TE), quantifies fibrosis by measuring liver stiffness (LSM). Given these developments, the purpose of this study was (i) to estimate prognosis in treatment-naïve CHC patients using TE-based liver stiffness progression rates (LSPR) as an alternative to FPRs and (ii) to compare consistency between LSPRs and FPRs. A systematic literature search was performed using multiple databases (January 1990 to February 2016). LSPRs were calculated using either a direct method (given the difference in serial LSMs and time elapsed) or an indirect method given a single LSM and the estimated duration of infection and pooled using random-effects meta-analyses. For validation purposes, FPRs were also estimated. Heterogeneity was explored by random-effects meta-regression. Twenty-seven studies reporting on 39 groups of patients (N = 5874) were identified with 35 groups allowing for indirect and 8 for direct estimation of LSPR. The majority (~58%) of patients were HIV/HCV-coinfected. The estimated time-to-cirrhosis based on TE vs biopsy was 39 and 38 years, respectively. In univariate meta-regressions, male sex and HIV were positively and age at assessment, negatively associated with LSPRs. Noninvasive prognosis of HCV is consistent with FPRs in predicting time-to-cirrhosis, but more longitudinal studies of liver stiffness are needed to obtain refined estimates.

Phillips-Tikhonov regularization with a priori information for neutron emission tomographic reconstruction on Joint European Torus.

A method of tomographic reconstruction of the neutron emissivity in the poloidal cross section of the Joint European Torus (JET, Culham, UK) tokamak was developed. Due to very limited data set (two projection angles, 19 lines of sight only) provided by the neutron emission profile monitor (KN3 neutron camera), the reconstruction is an ill-posed inverse problem. The aim of this work consists in making a contribution to the development of reliable plasma tomography reconstruction methods that could be routinely used at JET tokamak. The proposed method is based on Phillips-Tikhonov regularization and incorporates a priori knowledge of the shape of normalized neutron emissivity profile. For the purpose of the optimal selection of the regularization parameters, the shape of normalized neutron emissivity profile is approximated by the shape of normalized electron density profile measured by LIDAR or high resolution Thomson scattering JET diagnostics. In contrast with some previously developed methods of ill-posed plasma tomography reconstruction problem, the developed algorithms do not include any post-processing of the obtained solution and the physical constrains on the solution are imposed during the regularization process. The accuracy of the method is at first evaluated by several tests with synthetic data based on various plasma neutron emissivity models (phantoms). Then, the method is applied to the neutron emissivity reconstruction for JET D plasma discharge #85100. It is demonstrated that this method shows good performance and reliability and it can be routinely used for plasma neutron emissivity reconstruction on JET.

Human lymphocytic B-leukemia cell line treatment with the bacterial toxin listeriolysin O and rituximab (anti-CD20 antibody): Effects of similar localization of their receptors.

Small B-cell lymphocytic lymphoma/chronic lymphocytic leukemia, which typically affects elderly people, is a group of conditions that are not clinically uniform. It has been suggested that using the combined activity of the monoclonal antibody anti-CD20 (rituximab) and Listeria monocytogenes toxin listeriolysin O (LLO) for this condition could produce an enhanced treatment effect. Here, we tested the effect of the joint activity of rituximab and LLO, which is a cell membrane toxin, in human leukemia cell lines. The human B-leukemia Raji cell line, which expresses CD20, and the T-cell Jurkat cell line, which does not express CD20, for comparison were used in model tests. Cell cytotoxicity of rituximab or LLO and both applied jointly to the cell lines was compared in the presence of human plasma complement. Optimal cytotoxic effects dependent on rituximab or LLO concentration were tested separately. LD50 values were determined and used for optimal application of a mixture of the two factors. The cytotoxic effect on Raji cells of both rituximab and LLO was more than 2.5 times that of LLO alone and 1.5 times that of rituximab alone. At the highest tested concentrations, a mixture of the tested factors had a non-specific cytotoxic effect on the Jurkat cell line, as well. The rituximab and LLO binding sites appear to be in a similar region of the Raji leukemia cell membrane, suggesting an effective interaction of both factors. The joint interaction of these compounds in cell membrane pore formation suggests an explanation for the more effective cytotoxic activity that their combination was observed in this experiment.

The targeting nanothin polyelectrolyte shells in system with immobilized bacterial cells for antitumor factor production.

Development of anticancer treatment strategies is ongoing considering still inadequate efficiency of existing anticancer therapeutics. Moreover, the lack of therapeutic agents selectivity against the tumor cells requires further investigations into novel anticancer strategies. The use of pathogenic microorganisms producing an oncolytic agent may be an approach for apoptotic therapy in cancer treatment. The purpose of this study was to investigate the targeting efficiency of Bacillus subtilis bacterial cells coated with modified polyelectrolyte shells applied to protect the bacterial cells from potential host immune response as well as to enhance the tumor-targeting efficiency. The shells were modified with transferrin to increase affinity toward the target tumor cells. The impact of bacterial cells coated with unmodified or modified nanothin shells on human leukemia cells was evaluated in vitro. It was observed that the bacterial cells coated with modified shells with incorporated transferrin exhibited stronger lethal impact on leukemia cells as compared to bacterial cells with unmodified shell coating. Applied modified membrane conformation allowing for functioning of encapsulated microorganisms may find potential use in local antitumor treatment purposes.

Cytotoxicity of bacterial metabolic products, including listeriolysin O, on leukocyte targets.

Bacterial toxins can exhibit anticancer activities. Here we investigated the anticancer effects of the listeriolysin O toxin produced by Listeria monocytogenes. We found that supernatants of Listeria monocytogenes strains (wild type, 1189, and 1190) were cytotoxic to the Jurkat cell line and human peripheral blood mononuclear cells (PBMC) in a concentration-dependent manner. The supernatant of strain 1044, not producing listeriolysin O, was inactive. The supernatants of Listeria strains were also cytotoxic toward B cells of chronic leukemia patients, with no significant differences in activities between strains. We also tested supernatants of Bacillus subtilis strains BR1-90, BR1-S, and BR1-89 producing listeriolysin O. BR1-S and BR1-89 were cytotoxic to PBMC and to Jurkat cells, the latter being more sensitive to the supernatants. BR1-90 was not hemolytic or cytotoxic to PBMC, but was cytotoxic to Jurkat cells in the concentration range of 10-30%, suggesting that listeriolysin O is selectively effective against T cells. Overall, the response of human peripheral blood mononuclear and human leukemia cell lines to bacteria supernatants containing listeriolysin O depended on the bacteria strain, target cell type, and supernatant concentration.

Opposite patterns of diurnal activity in the box jellyfish Tripedalia cystophora and Copula sivickisi.

Cubozoan medusae have a stereotypic set of 24 eyes, some of which are structurally similar to vertebrate and cephalopod eyes. Across the approximately 25 described species, this set of eyes varies surprisingly little, suggesting that they are involved in an equally stereotypic set of visual tasks. During the day Tripedalia cystophora is found at the edge of mangrove lagoons where it accumulates close to the surface in sun-lit patches between the prop roots. Copula sivickisi (formerly named Carybdea sivickisi) is associated with coral reefs and has been observed to be active at night. At least superficially, the eyes of the two species are close to identical. We studied the diurnal activity pattern of these two species both in the wild and under controlled conditions in laboratory experiments. Despite the very similar visual systems, we found that they display opposite patterns of diurnal activity. T. cystophora is active exclusively during the day, whereas C. sivickisi is actively swimming at night, when it forages and mates. At night T. cystophora is found on the muddy bottom of the mangrove lagoon. C. sivickisi spends the day attached to structures such as the underside of stones and coral skeletons. This species difference seems to have evolved to optimize foraging, since the patterns of activity follow those of the available prey items in their respective habitats.

Cytotoxicity of listeriolysin O produced by membrane-encapsulated Bacillus subtilis on leukemia cells.

Encapsulation of biological material in the permiselective membrane allows to construct a system separating cells from their products, which may find biotechnological as well as biomedical applications in biological processes regulation. Application of a permiselective membrane allows avoiding an attack of the implanted microorganisms on the host. Our aim was to evaluate the performance of Bacillus subtilis encapsulated in an elaborate membrane system producing listeriolysin O, a cytolysin from Listeria monocytogenes, with chosen eukaryotic cells for future application in anticancer treatment. The system of encapsulating in membrane live Bacillus subtilis BR1-S secreting listeriolysin O was proven to exert the effective cytotoxic activity on eukaryotic cells. Interestingly, listeriolysin O showed selective cytotoxic activity on eukaryotic cells: more human leukemia Jurkat T cells were killed than human chronic lymphocytic B cells leukemia at similar conditions in vitro. This system of encapsulated B. subtilis, continuously releasing bacterial products, may affect selectively different types of cells and may have future application in local anticancer treatment.

Modulation of inv gene expression by the OmpR two-component response regulator protein of Yersinia enterocolitica.

To elucidate the physiological meaning of OmpR-dependent expression of invasin gene (inv) inhibition in Yersinia enterocolitica, the function of the EnvZ/OmpR regulatory pathway in osmoregulation of inv expression was analyzed in detail. The osmoregulation of inv expression was found to be a multifaceted process involving both OmpR-dependent and -independent mechanisms. Analysis of inv transcription in strains lacking OmpR or EnvZ proteins indicated that kinase EnvZ is not the only regulator of OmpR phosphorylation. Using the transcriptional inv::lacZ fusion in a heterologous system (Escherichia coli) we tried to clarify the role of OmpR in the inv regulatory circuit composed of negative (H-NS) and positive (RovA) regulators of inv gene transcription. We were able to show a significant increase in inv expression in E. coli ompR background under H-NS( Ecoli )-repressed condition. Moreover, H-NS-mediated inv repression was relieved when RovA of Y. enterocolitica was expressed from a plasmid. Furthermore, we showed that RovA may activate inv expression irrespective on the presence of H-NS protein. Using this strategy we showed that OmpR of Y. enterocolitica decrease RovA-mediated inv activation.

Swim pacemakers in box jellyfish are modulated by the visual input.

A major part of the cubozoan central nervous system is situated in the eye-bearing rhopalia. One of the neuronal output channels from the rhopalia carries a swim pacemaker signal, which has a one-to-one relation with the swim contractions of the bell shaped body. Given the advanced visual system of box jellyfish and that the pacemaker signal originates in the vicinity of these eyes, it seems logical to assume that the pacemakers are modified by the visual input. Here, the firing frequency and distribution of inter-signal intervals (ISIs) of single pacemakers are examined in the Caribbean box jellyfish, Tripedalia cystophora. It is shown that the absolute ambient light intensity, if kept constant, has no influence on the signal, but if the intensity changes, it has a major impact on both frequency and ISIs. If the intensity suddenly drops there is an increase in firing frequency, and the ISIs become more homogeneously distributed. A rise in intensity, on the other hand, produces a steep decline in the frequency and makes the ISIs highly variable. These electrophysiological data are correlated with behavioral observations from the natural habitat of the medusae.

[MRI-based diagnosis of giant cell arteritis]. / Differentialdiagnose eines un- spezifischen Beschwerdebildes: Riesenzellarteriitis (Morbus Horton).

The giant cell arteritis and its symptoms are usually non-specific and accompanied with symptoms of polymyalgia rheumatica. As complications of the giant cell arteritis ischemia, infarction or rupture of the damaged vessel can occur. We report on a 56-year-old female patient, who suffered for one year about weight loss, tiredness and intolerance as well as symptoms of polymyalgia rheumatica. Gastroscopy and colonoscopy showed normal findings. In the context of the malignancy search we made a computer tomography and magnet resonance tomography. The data showed an enlargement and an enhancement of the aorta, which led us to the suspicion of a giant cell arteritis. We started immediately with a medical treatment. The biopsy of the arteries temporales supported histological the diagnosis.

Listeria monocytogenes protein p60 affects hemolytic activity and uptake of bacteria by macrophages.

Bacillus subtilis strains expressing listeriolysin O (LLO) and simultaneously LLO and p60 protein were constructed. The effect of p60 protein on hemolytic activity and on the invasion of professional phagocytes was demonstrated in the absence of other virulence factors of L. monocytogenes. The hemolytic activity of LLO in the presence of p60 protein decreased which indicates that p60 promoted adhesion and subsequent invasion of professional phagocytes.

Contribution of hemolysin and phospholipase activity to cytolytic properties and viability of Listeria monocytogenes.

The life cycle of Listeria monocytogenes strongly depends on cytolysin which facilitates the pathogen's escape from the endosome. This process is very ineffective in the absence of phospholipases. In this study we investigated the molecular mechanism of cooperation between hemolysin and phospholipases. We propose a model involving interactions between proteins that increase the affinity of cytolysins for the membrane as well direct enzymatic enhancement of lysis.

Insertional knock-out of protein translocation systems common for Yersinia enterocolitica and Listeria monocytogenes.

To carry out efficient insertional mutagenesis in Listeria monocytogenes and to facilitate the characterisation of disrupted genes, a novel derivative of plasmid pACYC 184 was constructed, pLIV virA3, carrying a fragment from the virA region of the of Y. enterocolitica plasmid pYVe 0:9. After transformation of this plasmid into L. monocytogenes it was possible to select for its integration into the host DNA at 42 degrees C. Insertional mutants of L. monocytogenes obtained by using pLIV vector containing plasmid DNA fragments from Y. enterocolitica were constructed and are described.

Psychopharmacology and mental retardation: a 10 year review (1990-1999).

We conducted a 10 year review of the literature pertaining to psychopharmacology and mental retardation. Studies were included or excluded from the review based on meeting one or more of the methodological criteria normally considered fundamental for sound scientific research. The vast majority of studies conducted in the last 10 years in this area had major methodological flaws. While a large number of medications were prescribed for various psychological disorders and behavior problems, most drug administrations were not based in science, were not evaluated appropriately, and generally did not follow best practices for treatment of persons with mental retardation. Very few medications prescribed were behavior or psychiatric symptom specific; that is, most medications were given to suppress a myriad of aberrant behaviors thus chemically restraining the individual in question. Practices such as these present serious problems for service providers due to the deleterious side effects of many psychotropic medications and the federal government's intervention into the care-provision practices of developmental centers, community homes, and other living arrangements for persons with mental retardation. Implications of our review are discussed.

[Sulfasalazine-induced pulmonary infiltrates and Legionella pneumonia]. / Salazopyrin-induzierte Lungeninfiltrate und Legionellenpneumonie.

After 2 weeks' treatment with sulfasalazine (SASP) and mesalazine enema, a 32-year-old female with recently diagnosed ulcerative colitis developed bilateral pulmonary infiltrates with peripheral eosinophilia. Both drugs were discontinued. In view of a high-positive antibody titre (1:4096) against Legionella pneumophila serogroups 1-5, legionnaires' disease was assumed and empirical antilegionella therapy with macrolid antibiotic was started. The patient's condition improved within days. Three months later SASP was given again in view of exacerbation of the inflammatory bowel disease. Three days after initiation of therapy acute pulmonary symptoms again developed with bilateral, confluent opacities and blood eosinophilia. The abnormalities resolved completely after the drug was discontinued and prophylactic antibiotic therapy was given. Peripheral lung infiltrates with blood eosinophilia are a rare side effect of SASP therapy. The prognosis of the illness after the drug has been discontinued is generally good, usually with complete recovery of pulmonary function. The risk factors for sulfasalazine pulmonary toxicity are not well known. We describe the first case of SASP-induced hypersensitivity lung disease with simultaneous Legionella pneumophila infection.