Construction of targeted insertion mutations in Francisella tularensis subsp. novicida

Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast and efficient construction of targeted insertion mutations in F. tularensis subsp. novicida that have characteristics of nonpolar mutations; optimized targeted mutagenesis strategies will promote the study of this mysterious bacterium and facilitate vaccine development against tularemia. Moreover the general strategy of gene disruption by PCR-based antibiotic resistance cassette insertion is broadly applicable to many bacterial species.     

Untersuchungen über gerbstoffführende Pflanzen

Ohne Zusammenfassung     

Beitrag zur Phanerogamenflora der Bukowina und des angrenzenden Theiles von Siebenbürgen

Ohne Zusammenfassung     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Genetic characterization of clinical and agri-food isolates of multi drug resistant <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Heidelberg from Canada

Background  

Salmonella enterica serovar Heidelberg ranks amongst the most prevalent causes of human salmonellosis in Canada and an increase in resistance to extended spectrum cephalosporins (ESC) has been observed by the Canadian Integrated Program for Antimicrobial Resistance Surveillance. This study examined the genetic relationship between S. Heidelberg isolates from livestock, abattoir, retail meat, and clinical human specimens to determine whether there was a link between the emergence of MDR S. Heidelberg in chicken agri-food sources and the simultaneous increase of MDR S. Heidelberg in human clinical samples.     

The 3,4-oxide is responsible for the DNA binding of benzo[ghi]perylene, a polycyclic aromatic hydrocarbon without a "classic" bay-region

The polycyclic aromatic hydrocarbon (PAH) benzo[ghi]perylene (BghiP) lacks a "classic" bay-region and is therefore unable to form vicinal dihydrodiol epoxides thought to be responsible for the genotoxicity of carcinogenic PAHs like benzo[a]pyrene. The bacterial mutagenicity of BghiP increases considerably after inhibition of the microsomal epoxide hydrolase (mEH) indicating arene oxides as genotoxic metabolites. Two K-region epoxides of BghiP, 3,4-epoxy-3,4-dihydro-BghiP (3,4-oxide) and 3,4,11,12-bisepoxy-3,4,11,12-tetrahydro-BghiP (3,4,11,12-bisoxide) identified in microsomal incubations of BghiP are weak bacterial mutagens in strain TA98 of Salmonella typhimurium with 5.5 and 1.5 his+-revertant colonies/nmol, respectively. After microsomal activation of BghiP in the presence of calf thymus DNA three DNA adducts were detected using 32P-postlabeling. The total DNA binding of 2.1 fmol/microg DNA, representing 7 adducts in 10(7) nucleotides, was raised 3.6-fold when mEH was inhibited indicating arene oxides as DNA binding metabolites. Co-chromatography revealed the identity between the main adduct of metabolically activated BghiP and the main adduct of the 3,4-oxide. DNA adducts of BghiP originating from the 3,4,11,12-bisoxide were not found. Therefore, a K-region epoxide is proposed to be responsible for the genotoxicity of BghiP and possibly of other PAHs without a "classic" bay-region.     

Binding synergy and cooperativity in dihydrodipicolinate reductase: implications for mechanism and the design of biligand inhibitors

Dihydrodipicolinate reductase (DHPR) is a homotetramer that catalyzes reduction of dihydrodipicolinate (DHP). We recently reported a biligand inhibitor ( K i = 100 nM) of DHPR, comprised of fragments that bind in the NADH (CRAA = catechol rhodanine acetic acid) and DHP (PDC = pyridine dicarboxylate) binding sites. Herein, we characterize binding synergy and cooperativity for ligand binding to Escherichia coli DHPR: NADH or CRAA and PDC (stable analog of DHP). While K d values indicate little synergy between NADH and PDC, (1)H- (15)N HSQC chemical shift perturbation and saturation transfer difference (STD) titrations indicate that PDC induces a more dramatic conformational change than NADH, consistent with a role in domain closure. PDC binds cooperatively (Hill coefficient = 2), while NADH does not, based on STD titrations that monitor only fast exchange processes. However, HSQC titrations monitoring Trp253 (located between monomers) indicate that NADH binds in two steps, with high affinity binding to only one of the monomers. Therefore, DHPR binds cofactor via a sequential model, with negative cooperativity. These results, interpreted in light of steady-state data, suggest that DHPR activity requires NADH binding at only one of the four monomers. Implications of our results for fragment assembly are discussed, using CRAA tethering to PDC as a model biligand: (a) if one fragment (ex. PDC) must induce a large structural change before the other fragment is brought proximal, this must be screened for upfront, and (b) cooperative or synergistic interactions between binding sites can lead to unexpected and misleading effects in NMR-based screening.     

Linking exponential components to kinetic states in Markov models for single-channel gating

Discrete state Markov models have proven useful for describing the gating of single ion channels. Such models predict that the dwell-time distributions of open and closed interval durations are described by mixtures of exponential components, with the number of exponential components equal to the number of states in the kinetic gating mechanism. Although the exponential components are readily calculated (Colquhoun and Hawkes, 1982, Phil. Trans. R. Soc. Lond. B. 300:1-59), there is little practical understanding of the relationship between components and states, as every rate constant in the gating mechanism contributes to each exponential component. We now resolve this problem for simple models. As a tutorial we first illustrate how the dwell-time distribution of all closed intervals arises from the sum of constituent distributions, each arising from a specific gating sequence. The contribution of constituent distributions to the exponential components is then determined, giving the relationship between components and states. Finally, the relationship between components and states is quantified by defining and calculating the linkage of components to states. The relationship between components and states is found to be both intuitive and paradoxical, depending on the ratios of the state lifetimes. Nevertheless, both the intuitive and paradoxical observations can be described within a consistent framework. The approach used here allows the exponential components to be interpreted in terms of underlying states for all possible values of the rate constants, something not previously possible.     

Early life environment and snoring in adulthood

Background

To our knowledge, no studies of the possible association of early life environment with snoring in adulthood have been published. We aimed to investigate whether early life environment is associated with snoring later in life.

Methods

A questionnaire including snoring frequency in adulthood and environmental factors in early life was obtained from 16,190 randomly selected men and women, aged 25–54 years, in Sweden, Norway, Iceland, Denmark and Estonia (response rate 74%).

Results

A total of 15,556 subjects answered the questions on snoring. Habitual snoring, defined as loud and disturbing snoring at least 3 nights a week, was reported by 18%. Being hospitalized for a respiratory infection before the age of two years (adjusted odds ratio (OR) = 1.27; 95% confidence interval (CI) 1.01–1.59), suffering from recurrent otitis as a child (OR = 1.18; 95%CI 1.05–1.33), growing up in a large family (OR = 1.04; 95%CI 1.002–1.07) and being exposed to a dog at home as a newborn (OR = 1.26; 95%CI 1.12–1.42) were independently related to snoring later in life and independent of a number of possible confounders in adulthood. The same childhood environmental factors except household size were also related with snoring and daytime sleepiness combined.

Conclusion

The predisposition for adult snoring may be partly established early in life. Having had severe airway infections or recurrent otitis in childhood, being exposed to a dog as a newborn and growing up in a large family are environmental factors associated with snoring in adulthood.