The Effect of Regulatory Sequence Elements upon the Initiation of DNA Replication of the Minute Virus of Mice

AbstractThe minute virus of mice (MVM) genome is a linear single-stranded length of approximately 5000 nucleotides of DNA with unique terminal palindromic sequences at both ends. The left (3′) hairpin is used to prime the initiation of DNA synthesis on parental single-strand DNA while the right (5′) hairpin or stem-plus-arms structure can also prime the initiation of DNA synthesis during synthesis of dimer and higher oligomers as well as synthesis of progeny single strands. Previous studies have shown that if viral duplex DNA was input into anin vitroDNA replication system using extracts from uninfected HeLa cells, the 5′ end of the molecule was able to form a hairpin and initiate DNA synthesis by DNA polymerase δ (Cossonset al.(1996),Virology216, 258–264). In this study, the effect of the deletion of knowncis-acting genetic elements upon the initiation of DNA replication was studied using a series of MVM mutants with deletions within the 5′ terminal region. Mutants containing deletions of elements A (nucleotides 4489–4636), B (nucleotides 4636–4695), and either one or both of the 65-bp repeats (nucleotides 4720–4785 and 4785–4849) were used as template in thein vitroDNA replication system. When element A was deleted, the efficiency of initiation decreased significantly. Subsequent removal of element B, leaving just the two 65-bp repeats, restored levels of initiation back to those seen in the wild-type genome. In the absence of either A or B both 65-bp repeats were necessary for efficient initiation, and removal of one of these repeats caused a decrease in efficiency. Thus, element B appeared to have a negative regulatory effect (in the absence of element A), and element A appeared to have a positive regulatory effect, at least in the presence of element B. These data demonstrate, for the first time, a complex interaction between thesecis-acting regulatory elements which can function as both positive or negative regulators in the initiation of MVM DNA replication

Distribution and Activity of Bacteria in the Headwaters of the Rhode River Estuary, Maryland, USA

Abstract: A transect along the axis of the headwaters of a tidal estuary was sampled for microbial, nutrient, and physical parameters. Chlorophyll a averaged 42 μg 1 -1 and phytoplankton comprised an estimated 80% of the total microbial biomass as determined by adenosine triphosphate (ATP). Bacterial concentrations ranged from 0.3-53.9 × 10 6 cells ml -1 and comprised about 4% of the total living microbial biomass. Bacterial production, determined by 3H-methyl-thymidine incorporation was about 0.05-2.09 × 10 9 cells 1 -1 h -1 , with specific growth rates of 0.26-1.69 d -1 . Most bacterial production was retained on 0.2 μm pore size filters, but passed through 1.0 μm filters. Significant positive correlations were found between all biomass measures and most nutrient measures with the exception of dissolved inorganic nitrogen nutrients where correlations were negative. Seasonal variability was evident in all parameters and variability among the stations was evident in most. The results suggest that bacterial production requires a significant carbon input, likely derived from autotrophic production, and that microbial trophic interactions are important. Article: Introduction Coastal and estuarine systems are sites where competition for nutrients by microorganisms and remineralization of photosynthetically fixed carbon are important processes. High rates of bacterial activity have been found in such systems for both inorganic nutrients and organic compounds Utilization of carbon fixed by primary production is a primary role of heterotrophic microorganisms, and recent studies suggest that the magnitude of this role has been underestimated Estuarine systems may exhibit even greater dependence upon trophic links through microheterotrophs tha

Chlorhexidine gluconate usage is associated with antiseptic tolerance in staphylococci from the neonatal intensive care unit

Background: Intravascular catheters are essential for care in Neonatal Intensive Care Units (NICUs) but predispose infants to catheter-associated infections including late-onset sepsis, commonly caused by CoNS. Antiseptics are applied to prevent infection with chlorhexidine (CHG) and octenidine (OCT) the most common agents used. Objectives: To investigate the association between antiseptic use and bacterial susceptibility. Methods: CoNS isolates were collected from two NICUs with differing antiseptic regimens: Norwich, UK (using CHG) and Lubeck, Germany (using OCT). CoNS were isolated from different body sites of babies upon admission, and weekly thereafter. Antiseptic susceptibility testing was performed, and a selection underwent genome sequencing. Results: A total of 1274 isolates were collected. UK isolates (n = 863) were significantly less susceptible than German isolates (n = 411) to both CHG (mean MIC: 20.1 mg/L versus 8.9 mg/L) and OCT (mean MIC: 2.3 mg/L versus 1.6 mg/L). UK isolates taken on admission were more susceptible to CHG than subsequent isolates. No cross-resistance between the agents was seen. Genome sequencing of 122 CoNS showed the most common species to be Staphylococcus epidermidis and Staphylococcus haemolyticus and phylogenetic analysis suggested antiseptic tolerance evolved multiple times in independent lineages. There was no evidence of dominant antiseptic tolerant clones and carriage of genes previously implicated in antimicrobial susceptibility (qac, smr, norA/B), did not correlate with CHG or OCT susceptibility. Conclusions: Long-term CHG use may select for CHG and OCT tolerance in CoNS. This highlights the different potential for separate antiseptic regimens to select for resistance development. This could be an important factor in developing future infection control policies

Bose-Einstein Correlations of Three Charged Pions in Hadronic Z^0 Decays

Bose-Einstein Correlations (BEC) of three identical charged pions were studied in 4 x 10^6 hadronic Z^0 decays recorded with the OPAL detector at LEP. The genuine three-pion correlations, corrected for the Coulomb effect, were separated from the known two-pion correlations by a new subtraction procedure. A significant genuine three-pion BEC enhancement near threshold was observed having an emitter source radius of r_3 = 0.580 +/- 0.004 (stat.) +/- 0.029 (syst.) fm and a strength of \lambda_3 = 0.504 +/- 0.010 (stat.) +/- 0.041 (syst.). The Coulomb correction was found to increase the \lambda_3 value by \~9% and to reduce r_3 by ~6%. The measured \lambda_3 corresponds to a value of 0.707 +/- 0.014 (stat.) +/- 0.078 (syst.) when one takes into account the three-pion sample purity. A relation between the two-pion and the three-pion source parameters is discussed.Comment: 19 pages, LaTeX, 5 eps figures included, accepted by Eur. Phys. J.

With or without force? : European public opinion on democracy promotion

A Large part of the education provided at colleges and universities of today requires for thestudent to be more independent in their studies. This demands that the physical space,where the students choose to study, is designed in a way that can encourage and supportlearning. It seems as though that many of the learning spaces of today don’t always meetthe students’ needs. The university library at the University of Umeå is currently planningto design new learning spaces for the students. The aim of this study is to examine how thephysical learning space can be designed to engage and encourage the students in theirlearning process. Based on literature describing learning spaces we have initially identified three mainareas to examine- Learning, Information Technology and Learning space design. Theseareas are all important features in the design of new learning spaces. With informationdrawn from that literature we conducted an empirical study at the library of the Universityof Umeå. The empirical study was carried out through observations and focus groupinterviews. To give us more insight about the students’ thoughts about the learning spacewe also compared our findings with a survey conducted by the library personnel in 2008and 2010. The result of our study shows that there are some areas to be improved in theexisting learning space. The students are working more collaboratively which requiresmore group areas. Our study also shows that flexibility, more student interaction and asocial and engaging environment are all important features in the design of new learningspaces

Real-Time Imaging Reveals the Dynamics of Leukocyte Behaviour during Experimental Cerebral Malaria Pathogenesis

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6Chi monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8+ T lymphocytes. A critical number of CD8+ T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8+ T lymphocytes in regulating vascular pathology in this disease. © 2014 Pai et al

Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features

Concurrent Helminthic Infection Protects Schoolchildren with Plasmodium vivax from Anemia

malaria in rural areas in the municipality of Careiro, in the Western Brazilian Amazon. (n = 9). In children without intestinal helminthes, a significant decrease in the hemoglobin during the malarial attack was seen as compared to the baseline concentration. In the survival analysis, no difference was seen in the time (in days) from the baseline cross-sectional to the first malarial infection, between parasitized and non-parasitized children.