The use of <Emphasis Type="Italic">spa</Emphasis> and phage typing for characterization of clinical isolates of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in the University Clinical Center in Gdańsk,Poland

The emergence of spa types and spa–clonal complexes (CC) among clinical methicillin-resistant Staphylococcus aureus isolates collected from the University Clinical Center in Gdańsk between 2008 and 2009 were investigated. Phage typing was used as the initial screening in the study. The basic set of phages and the additional set of phages were used. Most of the isolates (56 %) belonged to the phage group III. With the additional set of phages, eight types were found, with predominant one MR8 (50 %). Sixteen distinct spa types were observed. The most frequent were t003 (22 %), t151 (16 %), and t008 (12 %). The spa types were clustered into two spa-CC and eight singletons. The predominant CC010 (50 %) consisted of six types, with the most common t003 (36.7 %) and t151(26.7 %), and in 80 % was identified as staphylococcal chromosomal casette mec (SCCmec) type II. The second cluster has no founder (12 %) with only two spa types: t037 belonging to SCCmec type III and t029. In the most frequent singleton, spa type t008 alone was clustered in 12 % of the isolates. All singletons correspond to SCCmec type IV. The CC010 was distributed in most of the hospital wards, corresponded to Multilocus sequence typing type ST5/ST225 and was constantly present throughout the observed period. The isolates of CC010 generally belonged to the phage group III, and most of them (53.3 %) were resistant to erythromycin, clindamycin, and ciprofloxacin. The concordance between spa-clone and phage type was very high, but the same phage type MR8 was observed within different spa types of the predominant clone.     

Thermally enhanced radioresponse of cultured chinese hamster cells: inhibition of repair of sublethal damage and enhancement of lethal damage

X-irradiation of Chinese hamster cells at temperatures above 37°C results in enhanced killing response. The magnitude of this thermal effect increases with increasing temperature and varies inversely with dose rate during the exposure of the cells to the combined effects of elevated temperature and ionizing radiation. Postirradiation incubation at an elevated temperature is also effective in enhancing the response but not preirradiation hyperthermia. Split-dose experiments demonstrate that hyperthermia also inhibits the repair of sublethal damage for temperatures up to ～41°C. Above 41°C, lethal damage expression is enhanced as well. Fluctuations in the age-response structure of cells x-irradiated at 42°C are reduced, a result consistent with a reduced capacity for sublethal damage when cells are hyperthermic during irradiation.     

Τesting models for the beginnings of the Aurignacian and the advent of figurative art and music: the radiocarbon chronology of Geißenklösterle

The German site of Geißenklösterle is crucial to debates concerning the European Middle to Upper Palaeolithic transition and the origins of the Aurignacian in Europe. Previous dates from the site are central to an important hypothesis, the Kulturpumpe model, which posits that the Swabian Jura was an area where crucial behavioural developments took place and then spread to other parts of Europe. The previous chronology (critical to the model), is based mainly on radiocarbon dating, but remains poorly constrained due to the dating resolution and the variability of dates. The cause of these problems is disputed, but two principal explanations have been proposed: a) larger than expected variations in the production of atmospheric radiocarbon, and b) taphonomic influences in the site mixing the bones that were dated into different parts of the site. We reinvestigate the chronology using a new series of radiocarbon determinations obtained from the Mousterian, Aurignacian and Gravettian levels. The results strongly imply that the previous dates were affected by insufficient decontamination of the bone collagen prior to dating. Using an ultrafiltration protocol the chronometric picture becomes much clearer. Comparison of the results against other recently dated sites in other parts of Europe suggests the Early Aurignacian levels are earlier than other sites in the south of France and Italy, but not as early as recently dated sites which suggest a pre-Aurignacian dispersal of modern humans to Italy by ∼45000 cal BP. They are consistent with the importance of the Danube Corridor as a key route for the movement of people and ideas. The new dates fail to refute the Kulturpumpe model and suggest that Swabian Jura is a region that contributed significantly to the evolution of symbolic behaviour as indicated by early evidence for figurative art, music and mythical imagery.     

Thermal enhancement of DNA damage by an alkylating agent in human cells

Human skin cells were incubated at various temperatures during and after treatment with methyl methanesulfonate and the number of single-strand breaks introduced into the cellular DNA then estimated by alkaline sucrose sedimentation. Elevation of temperature above 37° greatly enhanced damage to the DNA caused by methyl methanesulfonate. Inactivation of an essential step in the repair of DNA was indicated by the observation that rejoining of breaks in the DNA was halted above a critical temperature (about 41.5°). Enhancement of damage to DNA increased with temperature, especially above 42°. Similar results were obtained for Chinese hamster cells. A correlation of these results with cell viability is discussed.     

Cooperative Regulation of Cellular Proliferation by Intercellular Diffusion

A theoretical model for the cooperative control of cellular kinetics is investigated. A critical substance A is produced by the cells whose concentration in a given cell determines whether that cell can divide. The substance A can leak out of the cells into the surrounding medium as well as be reabsorbed by the cells. This feature then implies communication between the cells since all concentrations will be functions of the population density. The substance A also has a lifetime, i.e. decays, for example, by denaturation. This system can be described by three coupled nonlinear differential equations which can be solved analytically in certain limiting cases and can, of course, be studied in detail by computer techniques. Our investigations have shown that (a) there is a critical initial cell population density below which cell proliferation will not occur, (b) cell proliferation can be stimulated by supplying substance A to the medium and there is a critical initial concentration in the medium for initiating proliferation when the cell population density is subcritical, and (c) a well-defined induction period prior to exponential growth may exist whose length depends on the system parameters and initial conditions.     

A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma

Herein, we have identified cross-talk between the Hippo and fibroblast growth factor receptor (FGFR) oncogenic signaling pathways in cholangiocarcinoma (CCA). Yes-associated protein (YAP) nuclear localization and up-regulation of canonical target genes was observed in CCA cell lines and a patient-derived xenograft (PDX). Expression of FGFR1, -2, and -4 was identified in human CCA cell lines, driven, in part, by YAP coactivation of TBX5. In turn, FGFR signaling in a cell line with minimal basal YAP expression induced its cellular protein expression and nuclear localization. Treatment of YAP-positive CCA cell lines with BGJ398, a pan-FGFR inhibitor, resulted in a decrease in YAP activation. FGFR activation of YAP appears to be driven largely by FGF5 activation of FGFR2, as siRNA silencing of this ligand or receptor, respectively, inhibited YAP nuclear localization. BGJ398 treatment of YAP-expressing cells induced cell death due to Mcl-1 depletion. In a YAP-associated mouse model of CCA, expression of FGFR 1, 2, and 4 was also significantly increased. Accordingly, BGJ398 treatment was tumor-suppressive in this model and in a YAP-positive PDX model. These preclinical data suggest not only that the YAP and Hippo signaling pathways culminate in an Mcl-1-regulated tumor survival pathway but also that nuclear YAP expression may be a biomarker to employ in FGFR-directed therapy.     

Use of Inorganic and Organic Nitrogen by Synechococcus spp. and Diatoms on the West Florida Shelf as Measured Using Stable Isotope Probing

The marine nitrogen (N) cycle is a complex network of biological transformations in different N pools. The linkages among these different reservoirs are often poorly understood. Traditional methods for measuring N uptake rely on bulk community properties and cannot provide taxonomic information. ¹⁵N-based stable isotope probing (SIP), however, is a technique that allows detection of uptake of individual N sources by specific microorganisms. In this study we used ¹⁵N SIP methodology to assess the use of different nitrogen substrates by Synechococcus spp. and diatoms on the west Florida shelf. Seawater was incubated in the presence of ¹⁵N-labeled ammonium, nitrate, urea, glutamic acid, and a mixture of 16 amino acids. DNA was extracted and fractionated using CsCl density gradient centrifugation. Quantitative PCR was used to quantify the amounts of Synechococcus and diatom DNA as a function of density, and ¹⁵N tracer techniques were used to measure rates of N uptake by the microbial community. The ammonium, nitrate, urea, and dissolved primary amine uptake rates were 0.077, 0.065, 0.013, and 0.055 μmol N liter⁻¹ h⁻¹, respectively. SIP data indicated that diatoms and Synechococcus spp. actively incorporated N from [¹⁵N]nitrate, [¹⁵N]ammonium, and [¹⁵N]urea. Synechococcus also incorporated nitrogen from [¹⁵N]glutamate and ¹⁵N-amino acids, but no evidence indicating uptake of labeled amino acids by diatoms was detected. These data suggest that N flow in communities containing Synechococcus spp. and diatoms has more plasticity than the new-versus-recycled production paradigm suggests and that these phytoplankters should not be viewed strictly as recycled and new producers, respectively.The marine nitrogen (N) cycle is a complex network of biological transformations in different inorganic and organic N reservoirs (58). Processes related to the N cycle can at times limit productivity in marine systems (47) and influence the rate at which carbon (C) is exported from the euphotic zone to the deep ocean and marine sediments, where it can be sequestered (21). The historical paradigm with respect to the marine N and C cycles is deeply interwoven with the concepts of new and regenerated primary production in the euphotic zone (17, 20). New and export production have traditionally been equated with large nutrient influxes, particularly influxes of nitrate, which lead to diatom productivity. When high levels of nitrate are present, diatoms often dominate and exhibit high sinking rates due to aggregation and/or packaging into fecal pellets (18, 48). By contrast, the subtropical and tropical oligotrophic surface oceans have been viewed primarily as areas where recycled productivity dominates.In recent years, however, our view of the linkages between the marine N and C cycles has become increasingly complex (58). For example, geochemical rate estimates have suggested that N fixation rates in surface waters of the tropical and subtropical oceans may be many times greater than previously thought (13, 34). The divergence between in situ observations and data obtained using the geochemical mass balance approach is attributed, among other things, to meso-scale physical forcing (38) and to diazotrophic activity of planktonic cyanobacteria (14, 57). Furthermore, it is now appreciated that the ability to use nitrate is more widely distributed among the marine bacteria than previously thought and that bacteria are capable of competing with phytoplankton for both ammonium and nitrate (29, 31). Despite these advances, direct measurements of uptake of specific forms of N by individual populations of phyto- and bacterioplankton are scarce. This is primarily due to the fact that most measurements of N uptake are made using glass fiber filters that collect autotrophs and some variable fraction of heterotrophic bacteria (3). Uptake rates thus represent bulk uptake by hundreds of different phytoplankton and bacterial species. Methods that could be used to investigate uptake of N by specific species (e.g., Synechococcus spp.) or groups of species (e.g., diatoms) would therefore greatly improve our ability to elucidate N fluxes in marine systems.DNA stable isotope probing (SIP) is a technique that is based on the observation that DNA molecules with different densities can be separated by ultracentrifugation in a concentrated solution of cesium chloride (CsCl). CsCl density gradient centrifugation has a long history in biological research and was first used to demonstrate the semiconservative nature of DNA replication (39). In their experiments, Meselson and Stahl grew Escherichia coli in medium in which all available forms of N contained the heavy, stable isotope ¹⁵N. Fully labeled with ¹⁵N, DNA has an average density of 1.722 g cm⁻³, whereas ¹⁴N-containing DNA has an average density of 1.700 g cm⁻³ (10, 11). This small, yet significant difference in density is enough to allow separation of ¹⁴N-containing DNA from ¹⁵N-containing DNA. DNA SIP has been used to study the dynamics of microbial communities (46). Radajewski et al. (46) used DNA SIP to identify the microbial species involved in the biotransformation of specific ¹³C-labeled substrates among the large pool of bacterial species that typically are present in environmental communities. These authors demonstrated that ¹³C-labeled DNA could be recovered from microbial populations after incubation. ¹³C-labeled DNA was then taxonomically characterized using routine molecular ecology methods to identify active community members, demonstrating that SIP can be a powerful technique for taxonomic identification of microbes performing specific metabolic processes under in situ conditions. A series of studies have since been performed using the ¹³C-based technique to examine microbial communities in different environments (for reviews, see references 19 and 40).More recently, ¹⁵N-based SIP techniques have been developed to facilitate identification of the free-living diazotrophs responsible for in situ N fixation in soil (10). This work demonstrated that ¹⁵N-based SIP techniques could be used to study N flow in environmental communities. In the present study, ¹⁵N-based SIP techniques (10, 11) were employed to assess the use of a suite of inorganic and organic nitrogen substrates by Synechococcus spp. and diatoms in a coastal marine system. Our goal was to investigate the traditional characterization of Synechococcus spp. as recycled producers (mainly ammonium uptake) and diatoms as new producers (nitrate uptake). To do this, seawater was incubated with a series of ¹⁵N-labeled N substrates. DNA was then extracted at the end of the incubation period, and quantitative PCR (qPCR) was used to determine the amounts of Synechococcus and diatom DNA as a function of density in fractionated gradients. Shifts in the densities of Synechococcus and diatom DNA as the result of incubation with ¹⁵N-labeled N substrates were interpreted as evidence of uptake. Our data indicate that Synechococcus spp. and diatoms both actively incorporated [¹⁵N]ammonium, [¹⁵N]nitrate, and [¹⁵N]urea. Synechococcus spp. appeared to also incorporate N from [¹⁵N]glutamate and ¹⁵N-amino acids. These data suggest that N flow in communities containing Synechococcus spp. and diatoms has more plasticity than the new-versus-regenerated production paradigm suggests and that these two types of phytoplankton should not be viewed strictly as recycled and new producers, respectively.     

Cellular inhibitor of apoptosis 1 (cIAP-1) degradation by caspase 8 during TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent with high selectivity for malignant cells. Many tumors, however, are resistant to TRAIL cytotoxicity. Although cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and -2) are often over-expressed in cancers, their role in mediating TRAIL resistance remains unclear. Here, we demonstrate that TRAIL-induced apoptosis of liver cancer cells is associated with degradation of cIAP-1 and X-linked IAP (XIAP), whereas cIAP-2 remains unchanged. Lower concentrations of TRAIL causing minimal or no apoptosis do not alter cIAP-1 or XIAP protein levels. Silencing of cIAP-1 expression, but not XIAP or cIAP-2, as well as co-treatment with a second mitochondrial activator of caspases (SMAC) mimetic (which results in rapid depletion of cIAP-1), sensitizes the cells to TRAIL. TRAIL-induced loss of cIAP-1 and XIAP requires caspase activity. In particular, caspase 8 knockdown stabilizes both cIAP-1 and XIAP, while caspase 9 knockdown prevents XIAP, but not cIAP-1 degradation. Cell-free experiments confirmed cIAP-1 is a substrate for caspase 8, with likely multiple cleavage sites. These results suggest that TRAIL-mediated apoptosis proceeds through caspase 8-dependent degradation of cIAP-1. Targeted depletion of cIAP-1 by SMAC mimetics in conjunction with TRAIL may be beneficial for the treatment of human hepatobiliary malignancies.     

Decreasing Mitochondrial Fission Prevents Cholestatic Liver Injury

Mitochondria frequently change their shape through fission and fusion in response to physiological stimuli as well as pathological insults. Disrupted mitochondrial morphology has been observed in cholestatic liver disease. However, the role of mitochondrial shape change in cholestasis is not defined. In this study, using in vitro and in vivo models of bile acid-induced liver injury, we investigated the contribution of mitochondrial morphology to the pathogenesis of cholestatic liver disease. We found that the toxic bile salt glycochenodeoxycholate (GCDC) rapidly fragmented mitochondria, both in primary mouse hepatocytes and in the bile transporter-expressing hepatic cell line McNtcp.24, leading to a significant increase in cell death. GCDC-induced mitochondrial fragmentation was associated with an increase in reactive oxygen species (ROS) levels. We found that preventing mitochondrial fragmentation in GCDC by inhibiting mitochondrial fission significantly decreased not only ROS levels but also cell death. We also induced cholestasis in mouse livers via common bile duct ligation. Using a transgenic mouse model inducibly expressing a dominant-negative fission mutant specifically in the liver, we demonstrated that decreasing mitochondrial fission substantially diminished ROS levels, liver injury, and fibrosis under cholestatic conditions. Taken together, our results provide new evidence that controlling mitochondrial fission is an effective strategy for ameliorating cholestatic liver injury.