Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes

Regiospecific analysis of Mono and Diglycerides in Glycerolysis products by GC x GC TOF-MS.

Comprehensive bidimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOF-MS) was used for the characterization of regiospecific mono- and diglycerides (MG-DG) content in the glycerolysis products derived from five different lipids included lard (LA), sun flower seed oil (SF), corn oil (CO), butter (BU), and palm oil (PA). The combination of fast and high temperature non-orthogonal column set namely DB17ht (6 m × 0.10 mm × 0.10 μm) as the primary column and SLB-5 ms (60 cm × 0.10 mm × 0.10 μm) as the secondary column was applied in this work. System configuration involved high oven ramp temperature to obtain precise mass spectral identification and highest effluent’s resolution. 3-Monopalmitoyl-sn-glycerol (MG 3-C16) was the highest concentration in LA, BU and PA while monostearoyl-sn-glycerol (MG C18) in CO and 1,3-dilinoleol-rac-glycerol (DG C18:2c) in SF. Principal component analysis accounted 82% of variance using combination of PC1 and PC2. The presence of monostearoyl-sn-glycerol (MG C18), 3-Monopalmitoyl-sn-glycerol (MG 3-C16), 1,3-dilinoleol-rac-glycerol (DG C18:2c), 1,3-dipalmitoyl-glycerol (DG 1,3-C16), and 1,3-dielaidin (DG C18:1t) caused differentiation of the samples tested

Population dynamics of an RNA virus and its defective interfering particles in passage cultures

<p>Abstract</p> <p>Background</p> <p>Viruses can fall prey to their defective interfering (DI) particles. When viruses are cultured by serial passage on susceptible host cells, the presence of virus-like DI particles can cause virus populations to rise and fall, reflecting predator-prey interactions between DI and virus particles. The levels of virus and DI particles in each population passage can be determined experimentally by plaque and yield-reduction assays, respectively.</p> <p>Results</p> <p>To better understand DI and virus particle interactions we measured vesicular stomatitis virus and DI particle production during serial-passage culture on BHK cells. When the multiplicity of infection (MOI, or ratio of infectious virus particles to cells) was fixed, virus yields followed a pattern of progressive decline, with higher MOI driving earlier and faster drops in virus level. These patterns of virus decline were consistent with predictions from a mathematical model based on single-passage behavior of cells co-infected with virus and DI particles. By contrast, the production of virus during fixed-volume passages exhibited irregular fluctuations that could not be described by either the steady-state or regular oscillatory dynamics of the model. However, these irregularities were, to a significant degree, reproduced when measured host-cell levels were incorporated into the model, revealing a high sensitivity of virus and DI particle populations to fluctuations in available cell resources.</p> <p>Conclusions</p> <p>This study shows how the development of mathematical models, when guided by quantitative experiments, can provide new insight into the dynamic behavior of virus populations.</p

Negative Staining and Image Classification – Powerful Tools in Modern Electron Microscopy

Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image contrast, much smaller molecules can be visualized by negative staining. Also, while molecules prepared by vitrification usually adopt random orientations in the amorphous ice layer, negative staining tends to induce preferred orientations of the molecules on the carbon support film. Combining negative staining with image classification techniques makes it possible to work with very heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens

Viral epidemics in a cell culture: novel high resolution data and their interpretation by a percolation theory based model

Because of its relevance to everyday life, the spreading of viral infections has been of central interest in a variety of scientific communities involved in fighting, preventing and theoretically interpreting epidemic processes. Recent large scale observations have resulted in major discoveries concerning the overall features of the spreading process in systems with highly mobile susceptible units, but virtually no data are available about observations of infection spreading for a very large number of immobile units. Here we present the first detailed quantitative documentation of percolation-type viral epidemics in a highly reproducible in vitro system consisting of tens of thousands of virtually motionless cells. We use a confluent astroglial monolayer in a Petri dish and induce productive infection in a limited number of cells with a genetically modified herpesvirus strain. This approach allows extreme high resolution tracking of the spatio-temporal development of the epidemic. We show that a simple model is capable of reproducing the basic features of our observations, i.e., the observed behaviour is likely to be applicable to many different kinds of systems. Statistical physics inspired approaches to our data, such as fractal dimension of the infected clusters as well as their size distribution, seem to fit into a percolation theory based interpretation. We suggest that our observations may be used to model epidemics in more complex systems, which are difficult to study in isolation.Comment: To appear in PLoS ONE. Supporting material can be downloaded from http://amur.elte.hu/BDGVirus

Time to discontinuation of atypical versus typical antipsychotics in the naturalistic treatment of schizophrenia

BACKGROUND: There is an ongoing debate over whether atypical antipsychotics are more effective than typical antipsychotics in the treatment of schizophrenia. This naturalistic study compares atypical and typical antipsychotics on time to all-cause medication discontinuation, a recognized index of medication effectiveness in the treatment of schizophrenia. METHODS: We used data from a large, 3-year, observational, non-randomized, multisite study of schizophrenia, conducted in the U.S. between 7/1997 and 9/2003. Patients who were initiated on oral atypical antipsychotics (clozapine, olanzapine, risperidone, quetiapine, or ziprasidone) or oral typical antipsychotics (low, medium, or high potency) were compared on time to all-cause medication discontinuation for 1 year following initiation. Treatment group comparisons were based on treatment episodes using 3 statistical approaches (Kaplan-Meier survival analysis, Cox Proportional Hazards regression model, and propensity score-adjusted bootstrap resampling methods). To further assess the robustness of the findings, sensitivity analyses were performed, including the use of (a) only 1 medication episode for each patient, the one with which the patient was treated first, and (b) all medication episodes, including those simultaneously initiated on more than 1 antipsychotic. RESULTS: Mean time to all-cause medication discontinuation was longer on atypical (N = 1132, 256.3 days) compared to typical antipsychotics (N = 534, 197.2 days; p < .01), and longer on atypicals compared to typicals of high potency (N = 320, 187.5 days; p < .01), medium potency (N = 140, 213.5 days; p < .01), and low potency (N = 74, 208.7 days; p < .01). Among the atypicals, only clozapine, olanzapine, and risperidone had significantly longer time to all-cause medication discontinuation compared to typicals, regardless of potency level, and compared to haloperidol with prophylactic anticholinergic treatment. When compared to perphenazine, a medium-potency typical antipsychotic, only clozapine and olanzapine had a consistently and significantly longer time to all-cause medication discontinuation. Results were confirmed by sensitivity analyses. CONCLUSION: In the usual care of schizophrenia patients, time to medication discontinuation for any cause appears significantly longer for atypical than typical antipsychotics regardless of the typical antipsychotic potency level. Findings were primarily driven by clozapine and olanzapine, and to a lesser extent by risperidone. Furthermore, only clozapine and olanzapine therapy showed consistently and significantly longer treatment duration compared to perphenazine, a medium-potency typical antipsychotic

Predictors and correlates for weight changes in patients co-treated with olanzapine and weight mitigating agents; a post-hoc analysis

<p>Abstract</p> <p>Background</p> <p>This study focuses on exploring the relationship between changes in appetite or eating behaviors and subsequent weight change for adult patients with schizophrenia or bipolar disorder treated with olanzapine and adjunctive potential weight mitigating pharmacotherapy. The aim is not to compare different weight mitigating agents, but to evaluate patients' characteristics and changes in their eating behaviors during treatment. Identification of patient subgroups with different degrees of susceptibility to the effect of weight mitigating agents during olanzapine treatment may aid clinicians in treatment decisions.</p> <p>Methods</p> <p>Data were obtained from 3 randomized, double-blind, placebo-controlled, 16-week clinical trials. Included were 158 patients with schizophrenia or bipolar disorder and a body mass index (BMI) ≥ 25 kg/m²who had received olanzapine treatment in combination with nizatidine (n = 68), sibutramine (n = 42), or amantadine (n = 48). Individual patients were analyzed for categorical weight loss ≥ 2 kg and weight gain ≥ 1 kg. Variables that were evaluated as potential predictors of weight outcomes included baseline patient characteristics, factors of the Eating Inventory, individual items of the Eating Behavior Assessment, and the Visual Analog Scale.</p> <p>Results</p> <p>Predictors/correlates of weight loss ≥ 2 kg included: high baseline BMI, low baseline interest in food, and a decrease from baseline to endpoint in appetite, hunger, or cravings for carbohydrates. Reduced cognitive restraint, increase in hunger, and increased overeating were associated with a higher probability of weight gain ≥ 1 kg.</p> <p>Conclusion</p> <p>The association between weight gain and lack of cognitive restraint in the presence of increased appetite suggests potential benefit of psychoeducational counseling in conjunction with adjunctive pharmacotherapeutic agents in limiting weight gain during antipsychotic drug therapy.</p> <p>Trial Registration</p> <p>This analysis was not a clinical trial and did not involve any medical intervention.</p

Complex dynamics of defective interfering baculoviruses during serial passage in insect cells

Defective interfering (DI) viruses are thought to cause oscillations in virus levels, known as the 'Von Magnus effect'. Interference by DI viruses has been proposed to underlie these dynamics, although experimental tests of this idea have not been forthcoming. For the baculoviruses, insect viruses commonly used for the expression of heterologous proteins in insect cells, the molecular mechanisms underlying DI generation have been investigated. However, the dynamics of baculovirus populations harboring DIs have not been studied in detail. In order to address this issue, we used quantitative real-time PCR to determine the levels of helper and DI viruses during 50 serial passages of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in Sf21 cells. Unexpectedly, the helper and DI viruses changed levels largely in phase, and oscillations were highly irregular, suggesting the presence of chaos. We therefore developed a simple mathematical model of baculovirus-DI dynamics. This theoretical model reproduced patterns qualitatively similar to the experimental data. Although we cannot exclude that experimental variation (noise) plays an important role in generating the observed patterns, the presence of chaos in the model dynamics was confirmed with the computation of the maximal Lyapunov exponent, and a Ruelle-Takens-Newhouse route to chaos was identified at decreasing production of DI viruses, using mutation as a control parameter. Our results contribute to a better understanding of the dynamics of DI baculoviruses, and suggest that changes in virus levels over passages may exhibit chaos.The authors thank Javier Carrera, Just Vlak and Lia Hemerik for helpful discussion. MPZ was supported by a Rubicon Grant from the Netherlands Organization for Scientific Research (NWO, www.nwo.nl) and a 'Juan de la Cierva' postdoctoral contract (JCI-2011-10379) from the Spanish 'Secretaria de Estado de Investigacion, Desarrollo e Innovacion'. JS was supported by the Botin Foundation. SFE was supported by grant BFU2012-30805, also from the Spanish 'Secretaria de Estado de Investigacion, Desarrollo e Innovacion'.Zwart, MP.; Pijlman, G.; Sardanyes Cayuela, J.; Duarte, J.; Januario, C.; Elena Fito, SF. (2013). Complex dynamics of defective interfering baculoviruses during serial passage in insect cells. Journal of Biological Physics. 39(2):327-342. doi:10.1007/s10867-013-9317-9S327342392Von Magnus, P.: Incomplete forms of influenza virus. Adv. Virus. Res. 2, 59–79 (1954)Huang, A.S.: Defective interfering viruses. Annu. Rev. Microbiol. 27, 101–117 (1973)Kool, M., Voncken, J.W., Vanlier, F.L.J., Tramper, J., Vlak, J.M.: Detection and analysis of Autographa californica nuclear polyhedrosis-virus mutants with defective interfering properties. Virology 183, 739–746 (1991)Wickham, T.J., Davis, T., Granados, R.R., Hammer, D.A., Shuler, M.L., Wood, H.A.: Baculovirus defective interfering particles are responsible for variations in recombinant protein-production as a function of multiplicity of infection. Biotechnol. Lett. 13, 483–488 (1991)Pijlman, G.P., van den Born, E., Martens, D.E., Vlak, J.M.: Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells. Virology 283, 132–138 (2001)Giri, L., Feiss, M.G., Bonning, B.C., Murhammer, D.W.: Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k. J. Gen. Virol. 93, 389–399 (2012)King, L.A., Possee, R.D.: The Baculovirus Expression System. University Press, Cambridge (1992)Lee, H.Y., Krell, P.J.: Reiterated DNA fragments in defective genomes of Autographa californica nuclear polyhedrosis virus are competent for AcMNPV-dependent DNA replication. Virology 202, 418–429 (1994)Pijlman, G.P., Dortmans, J., Vermeesch, A.M.G., Yang, K., Martens, D.E., Goldbach, R.W., Vlak, J.M.: Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses. J. Virol. 76, 5605–5611 (2002)Pijlman, G.P., van Schijndel, J.E., Vlak, J.M.: Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells. J. Gen. Virol. 84, 2669–2678 (2003)Pijlman, G.P., Vermeesch, A.M.G., Vlak, J.M.: Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells. J. Invertebr. Pathol. 84, 214–219 (2003)Roux, L., Simon, A.E., Holland, J.J.: Effects of defective interfering viruses on virus-replication and pathogenesis in vitro and in vivo. Adv. Virus. Res. 40, 181–211 (1991)Grabau, E.A., Holland, J.J.: Analysis of viral and defective-interfering nucleocapsids in acute and persistent infection by Rhadoviruses. J. Gen. Virol. 60, 87–97 (1982)Kawai, A., Matsumoto, S., Tanabe, K.: Characterization of Rabies viruses recovered from persistently infected BHK cells. Virology 67, 520–533 (1975)Roux, L., Holland, J.J.: Viral genome synthesis in BHK-21 cells persistently infected with Sendai virus. Virology 100, 53–64 (1980)Palma, E.L., Huang, A.: Cyclic production of vesicular stomatitis virus cause by defective interfering particles. J. Infect. Dis. 129, 402–410 (1974).Stauffer Thompson, K.A., Yin, J.: Population dynamics of an RNA virus and its defective interfering particles in passage cultures. Virol. J. 7, 257–266 (2010)Szathmáry, E.: Cooperation and defection – playing the field in virus dynamics. J. Theor. Biol. 165, 341–356 (1993)Bangham, C.R.M., Kirkwood, T.B.L.: Defective interfering particles – effects in modulating virus growth and persistence. Virology 179, 821–826 (1990)Kirkwood, T.B.L., Bangham, C.R.M.: Cycles, chaos, and evolution in virus cultures – a model of defective interfering particles. Proc. Natl. Acad. Sci. USA 91, 8685–8689 (1994)De Gooijer, C.D., Koken, R.H.M., van Lier, F.L.J., Kool, M., Vlak, J.M., Tramper, J.: A structured dynamic model for the baculovirus infection process in insect-cell reactor configurations. Biotech. Bioeng. 40, 537–548 (1992)Van Lier, F.L.J., van der Meijs, W.C.J., Grobben, N.G., Olie, R.A., Vlak, J.M., Tramper, J.: Continuous beta-galactosidase production with a recombinant baculovirus insect-cell system in bioreactors. J. Biotechnol. 22, 291–298 (1992)Van Lier, F.L.J., van den Hombergh, J., de Gooijer, C.D., den Boer, M.M., Vlak, J.M., Tramper, J.: Long-term semi-continuous production of recombinant baculovirus protein in a repeated (fed-)batch two-stage reactor system. Enzyme Microb. Technol. 18, 460–466 (1996)Zwart, M.P., Erro, E., van Oers, M.M., de Visser, J.A.G.M., Vlak, J.M.: Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants. J. Gen. Virol. 89, 1220–1224 (2008)Luckow, V.A., Lee, S.C., Barry, G.F., Olins, P.O.: Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J. Virol. 67, 4566–4579 (1993)Vaughn, J.L., Goodwin, R.H., Tompkins, G.J., McCawley, P.: Establishment of 2 cell lines from insect Spodoptera frugiperda (Lepidoptera, Noctuidae). In Vitro 13, 213–217 (1977)Zwart, M.P., van Oers, M.M., Cory, J.S., van Lent, J.W.M., van der Werf, W., Vlak, J.M.: Development of a quantitative real-time PCR for determination of genotype frequencies for studies in baculovirus population biology. J. Virol. Meth. 148, 146–154 (2008)Zwart, M.P., Hemerik, L., Cory, J.S., de Visser, J.A.G.M., Bianchi, F.J.J.A., van Oers, M.M., Vlak, J.M., Hoekstra, R.F., van der Werf, W.: An experimental test of the independent action hypothesis in virus-insect pathosystems. Proc. R. Soc. B 276, 2233–2242 (2009)Olkin, I., Gleser, L.J., Derman, C.: Probability Models and Applications. Macmillan, New York (1994)Parker, T., Chua, L.: Practical Numerical Algorithms for Chaotic Systems. Springer-Verlag, Berlin (1989)Dieci, L., van Vleck, E.S.: Computation of a few Lyapunov exponents for continuous and discrete dynamical systems. J. Appl. Numer. Math. 17, 275–291 (1995)Matsumoto, T., Chua, L.O., Komuro, M.: The double scroll. IEEE Trans. Circuits Syst. 32, 797–818 (1985)Chua, L.O., Komuro, M., Matsumoto, T.: The double scroll family: rigorous proof of chaos. IEEE Trans. Circuits Syst. 33, 1072–1097 (1986)Ramasubramanian, K., Sriram, M.S.: A comparative study of computation of Lyapunov spectra with different algorithms. Phys. D: Nonlin. Phenom. 139, 72–86 (2000)Lee, H.Y., Krell, P.J.: Generation and analysis of defective genomes of Autographa californica nuclear polyhedrosis virus. J. Virol. 66, 4339–4347 (1992)Kovacs, G.R., Choi, J., Guarino, L.A., Summers MD: Functional dissection of the Autographa californica nuclear polyhedrosis virus immediate early 1 transcriptional regulatory protein. J. Virol. 66, 7429–7437 (1992)Legendre, P., Legendre, L.: Numerical Ecology. Elsevier, Amsterdam (1998)Schuster, H.G.: Deterministic Chaos: An Introduction. Wiley-VCH Verlag GmbH & Co. KGaA, Wienheim (2005)Strogatz, S.H.: Nonlinear Dynamics and Chaos: With Applications to Physics, Biology, Chemistry and Engineering. Westview Press, Cambridge (1994)Dennis, B., Desharnais, R.A., Cushing, J.M., Henson, S.M., Constantino, R.F.: Can noise induce chaos? Oikos 102, 329–339 (2003)Crutchfield, J.P., Huberman, B.A.: Fluctuations and the onset of chaos. Phys. Lett. A 77, 407–410 (1980)Crutchfield, J.P., Farmer, J.D.: Fluctuations and simple chaotic dynamics. Phys. Rep. 92, 45–82 (1982

Health-related quality of life of patients following selected types of lumbar spinal surgery: A pilot study

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Genetic diversity and local connectivity in the mediterranean red gorgonian coral after mass mortality events

Estimating the patterns of connectivity in marine taxa with planktonic dispersive stages is a challenging but crucial task because of its conservation implications. The red gorgonian Paramuricea clavata is a habitat forming species, characterized by short larval dispersal and high reproductive output, but low recruitment. In the recent past, the species was impacted by mass mortality events caused by increased water temperatures in summer. In the present study, we used 9 microsatellites to investigate the genetic structure and connectivity in the highly threatened populations from the Ligurian Sea (NW Mediterranean). No evidence for a recent bottleneck neither decreased genetic diversity in sites impacted by mass mortality events were found. Significant IBD pattern and high global F-ST confirmed low larval dispersal capability in the red gorgonian. The maximum dispersal distance was estimated at 20-60 km. Larval exchange between sites separated by hundreds of meters and between different depths was detected at each site, supporting the hypothesis that deeper subpopulations unaffected by surface warming peaks may provide larvae for shallower ones, enabling recovery after climatically induced mortality events