In Vivo Random Mutagenesis of Bacillus subtilis by Use of TnYLB-1, a mariner-Based Transposon

This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either σ^A- or σ^B-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kan^r cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Erm^r gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10⁻²) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a “TA” dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo⁻ clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.     

How social network structure affects decision-making in Drosophila melanogaster

Animals use a number of different mechanisms to acquire crucial information. During social encounters, animals can pass information from one to another but, ideally, they would only use information that benefits survival and reproduction. Therefore, individuals need to be able to determine the value of the information they receive. One cue can come from the behaviour of other individuals that are already using the information. Using a previous extended dataset, we studied how individual decision-making is influenced by the behaviour of conspecifics in Drosophila melanogaster. We analysed how uninformed flies acquire and later use information about oviposition site choice they learn from informed flies. Our results suggest that uninformed flies adjust their future choices based on how coordinated the behaviours of the informed individuals they encounter are. Following social interaction, uninformed flies tended either to collectively follow the choice of the informed flies or to avoid it. Using social network analysis, we show that this selective information use seems to be based on the level of homogeneity of the social network. In particular, we found that the variance of individual centrality parameters among informed flies was lower in the case of a ‘follow’ outcome compared with the case of an ‘avoid’ outcome.     

Capping and dynamic relation between domains 1 and 2 of gelsolin

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N-terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2–3) to the side of the filaments to position the N-terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

Design and synthesis of a peptide derived from positions 195–244 of human cdc25C phosphatase

We have designed, synthesized and purified a 51 amino acid peptide derived from an essential domain of human cdc25C phosphatase. In vivo, differential phosphorylation of this domain regulates either the induction of mitotic processes, or the checkpoint arrest of eukaryotic cells in response to DNA damage. Peptide synthesis was achieved using the stepwise Fmoc strategy and resulted in an important yield of highly pure peptide. The final peptide was identified by amino acid analysis, electrospray mass spectrometry and nuclear magnetic resonance, which revealed that one of the two methionines within the peptide was oxidized into its sulphoxide derivative We investigated whether this 51 amino acid peptide folded into secondary structures in solution by circular dichroism and observed the formation of alpha helices in TFE. Finally, we verified that this peptide could bind to its biologically relevant 14‐3‐3 partner in vitro by fluorescence spectroscopy. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Étude biostratigraphique et paléoenvironnementale des diatomées pyritisées de l'intervalle Paléocène supérieur-Éocène inférieur du sondage de Saint-Josse (France)

This paper presents the results of a biostratigraphic analysis of pyritized diatom assemblages in the Saint-Josse core (CC82) located in the northern part of the Paris basin (coordinates: X =555?000, Y =307?324 and Z =56.9 m). During the Paleocene-Eocene this locality was situated in a small bight of the North Sea Basin. The results are obtained from sediments of the Thanet and Mont-Bernon group equivalents belonging to the Paleocene-Eocene interval. Thirteen samples were studied and eleven contain pyritized diatoms. By epigenesis, the siliceous frustule of the diatom is changed into a pyritized skeleton with all the ornamentation very well preserved such as the velum, the areolae, the labiate processes, etc. Dispersive X-Ray analyses show this complete replacement of silica by pyrite. Another form of pyritization is crystallization of pyrite (in cubes, pyritohedrons, octahedrons, framboids) in internal cavities of the diatoms to produce internal moulds, also called steinkerns, which preserve the external morphology of the diatoms and some details such as the girdle. Despite pyritization, diatoms can often be recognized at the species level; brackish and marine species were observed but no fresh water specimens were encountered. Pyrite is easily altered into iron oxides. In outcrops, pyritized diatoms can be obscured or destroyed by oxidation. We have chosen borehole samples for this study to avoid the affects of weathering. Deposits from the Sorrus Unit (of the Thanet Group) have low abundances of pyritized diatoms (3 to 25 specimens per sample). This unit contains allochtonous diatom assemblages comprising Actinoptychussenarius, Coscinodiscus morsianus var. morsianus, and Coscinodiscus morsianus var. moelleri. Sample 34.31-34.45 from this unit is particularly interesting. It contains three specimens of Paralia siberica var. laevis, which is typical of an estuarine or littoral paleoenvironment, and corresponds to the more continental deposit of the Sorrus Unit. Seven samples, taken in the Argile de Saint-Aubin Unit (from the Mont-Bernon Group), contain more than 200 pyritized diatoms and between 9 to 16 different species per sample. A brackish assemblage (Actinoptychus senarius, Coscinodiscus commutatus and Stellarima microtrias) and a marine assemblage (C. morsianus var. morsianus, Coscinodiscus var. moelleri, Fenestrella antiqua, Odontellaheibergii, Rhizosolenia sp. 1 and Trinacria regina) are observed. The brackish assemblage expands in the upper part of the Saint-Aubin Unit indicating more widespread brackish paleoenvironmental conditions. Pyritized diatoms are important fossil markers in the Paris basin mainly when siliceous fossils (silicoflagellates or radiolarians) and others microfossils (foraminifers and dinoflagellates) are very poor or absent. Two diatom assemblages have been identified in the Saint-Josse borehole. The first assemblage D1, comprising C. morsianus var. moelleri (high occurrence), C. morsianus (a few) and T. regina (rare), is recorded in the Sorrus, La Calotterie, Bois Gorguette, Le Goulet and lower Saint-Aubin Units. The second assemblage D2, characterized by great abundance of F. antiqua and decreasing numbers of C. morsianus species, is found in the upper part of the Saint-Aubin Unit. These diatom assemblages can be correlated with other biozonations established in the North Sea basin and based on nannoplankton and diatoms (King, 1983), diatoms (Mitlehner, 1996) and calcareous nannoplankton (Martini, 1971). F. antiqua and C. morsianus var. moelleri are the more important diatom species encountered. F. antiqua (previously named Coscinodiscus sp. 1, King, 1983) is the fossil marker used by King to define his NSP4 biozone. The base of NSP4 corresponds to the lowest occurrence of F. antiqua and the top is marked by the highest occurrence of this species. The Sorrus, La Calotterie, Bois Gorguette, Le Goulet and Saint-Aubin (“Sparnacian” stage) Units, the last one comprising F. antiqua (increasing to acme), correspond to the NSP4 zone of King (1983). The Mont-Hulin and Château de la Bruyère Units correspond probably to the NSP5 zone of King (1983). F. antiqua and C. morsianus var. moelleri were also used by Mitlehner (1996) to differentiate the NSP4a zone (low abundance of F. antiqua and abundance of C. morsianus var. moelleri) from the NSP4b zone (great abundance and acme of F. antiqua). The abundance of C. morsianus var. moelleri is recorded in the lower part of Saint-Aubin Unit and the abundance of F. antiqua (high peak at 46% in the sample 17.87) is found in the upper part of Saint-Aubin Unit. Therefore, Sorrus, La Calotterie, Bois Gorguette, Le Goulet and the lower part of Saint-Aubin Units are correlated with the NSP4a zone and the upper part of Saint-Aubin Unit with the NSP4b zone. By correlating the calcareous nannoplankton biozonation (NP) of Martini (1971) with the biozonation of King (1983), adapted by Mitlehner (1996), the boundary between the NP9 and NP10 zones could be located in the 18.75-17.87 interval and the NP10-NP11 boundary at the top of Saint-Aubin Unit. Lithostratigraphic correlations between the units of the Saint-Josse borehole (Paris basin) and the formations of the North Sea basin can be established. The great abundance of C. morsianus var. moelleri and the great abundance (including acme) of F. antiqua are respectively recorded in the Sele and Balder Formations. Therefore, the interval Sorrus/lower Saint-Aubin Units is correlated with the Sele Formation and upper part of Saint-Aubin Unit with the Balder Formation.     

The ongoing saga of Agrobacterium-host interactions

Infection of plant cells by Agrobacterium leads to activation of specific mitogen-activated protein kinase (MAPK). In a recent paper, Djamei et al. (2007) showed that MAPK-mediated phosphorylation of VirE2-interacting protein 1 (VIP1) is required for its translocation into the host-cell nucleus and for activation of a pathogenesis-related gene, and that Agrobacterium uses the phosphorylated VIP1 to deliver its transfer-DNA molecule into the host cell. These findings join a long line of evidence showing how this clever bacterium has developed ways of using and abusing host biological systems for its own needs.     

Total fluid and specific beverage intake and risk of renal cell carcinoma in Canada

Background: Intake of total fluids and specific beverages may be associated with the risk of renal cell carcinoma (RCC) through a diluting effect of carcinogens. However, total fluid consumption and RCC risk has not received sufficient examination. In this study, we assessed the roles of total fluid intake and type of beverage intake in the risk of RCC. Methods: Mailed questionnaires were completed by 1138 newly diagnosed, histologically confirmed RCC cases and 5039 population controls between 1994 and 1997 in 8 Canadian provinces. Data collection included information on socio-economic status, physical activity, smoking habits, alcoholic and non-alcoholic beverage use, diet, residential history and occupational history. Odds ratios (ORs) and 95% confidence intervals (CIs) were derived through unconditional logistic regression. Results: Higher total fluid intake was associated with risk of RCC; the OR for the highest versus the lowest quartile was 1.49 (95% CI 1.20–1.85). Intake of total juices and coffee was also related to the risk of RCC; for the highest versus the lowest quartile, the ORs were 1.53 (95% CI 1.18–1.99) and 1.33 (95% CI 1.07–1.66), respectively. These positive associations were stronger in men, but not in women. Higher coffee intake was more strongly associated with RCC in normal weight subjects. In contrast, total intake of alcohol was inversely associated with the risk of RCC. Intake of tap water (not in coffee or tea), bottled water, tea, soft drinks and milk was not related to RCC. Conclusions: The risk of RCC for higher intake of total fluids, coffee and juices might involve gender differences.     

Cost-effectiveness of primary prophylaxis of AIDS associated cryptococcosis in Cambodia

Background

Cryptococcal infection is a frequent cause of mortality in Cambodian HIV-infected patients with CD4+ count ≤100 cells/µl. This study assessed the cost-effectiveness of three strategies for cryptococcosis prevention in HIV-infected patients.

Methods

A Markov decision tree was used to compare the following strategies at the time of HIV diagnosis: no intervention, one time systematic serum cryptococcal antigen (CRAG) screening and treatment of positive patients, and systematic primary prophylaxis with fluconazole. The trajectory of a hypothetical cohort of HIV-infected patients with CD4+ count ≤100 cells/µl initiating care was simulated over a 1-year period (cotrimoxazole initiation at enrollment; antiretroviral therapy within 3 months). Natural history and cost data (US$ 2009) were from Cambodia. Efficacy data were from international literature.

Results

In a population in which 81% of patients had a CD4+ count ≤50 cells/ µl and 19% a CD4+ count between 51–100 cells/µl, the proportion alive 1 year after enrolment was 61% (cost $ 472) with no intervention, 70% (cost $ 483) with screening, and 72% (cost $ 492) with prophylaxis. After one year of follow-up, the cost-effectiveness of screening vs. no intervention was US$ 180/life year gained (LYG). The cost-effectiveness of prophylaxis vs. screening was $ 511/LYG. The cost-effectiveness of prophylaxis vs. screening was estimated at $1538/LYG if the proportion of patients with CD4+ count ≤50 cells/µl decreased by 75%.

Conclusion

In a high endemic area of cryptococcosis and HIV infection, serum CRAG screening and prophylaxis are two cost effective strategies to prevent AIDS associated cryptococcosis in patients with CD4+ count ≤100 cells/µl, at a short-term horizon, screening being more cost-effective but less effective than prophylaxis. Systematic primary prophylaxis may be preferred in patients with CD4+ below 50 cells/µl while systematic serum CRAG screening for early targeted treatment may be preferred in patients with CD4+ between 51–100 cells/µl.     

Combined Therapy Against Murine-Disseminated Infection by <Emphasis Type="Italic">Fusarium verticillioides</Emphasis>

Using a murine model of disseminated infection by two strains of Fusarium verticillioides, we have evaluated the efficacy of high doses of amphotericin B (AMB) (3 mg/kg of body weight/day), voriconazole (VRC) (60 mg/kg of body weight/day), posaconazole (PSC) (100 mg/kg of body weight/day), and the combinations of AMB plus VRC or PSC. In general, our results were very modest. Neither combination was superior to the respective monotherapies. VRC alone and in combination with AMB was able to prolong survival but not to reduce tissue burden, and AMB plus PSC was able to reduce fungal load in organs but not to prolong survival.