Overlapping genes in a yeast double-stranded RNA virus.

The Saccharomyces cerevisiae viruses have a large viral double-stranded RNA which encodes the major viral capsid polypeptide. We have previously shown that this RNA (L1) also encodes a putative viral RNA-dependent RNA polymerase (D. F. Pietras, M. E. Diamond, and J. A. Bruenn, Nucleic Acids Res., 16:6226, 1988). The organization and expression of the viral genome is similar to that of the gag-pol region of the retroviruses. The complete sequence of L1 demonstrates two large open reading frames on the plus strand which overlap by 129 bases. The first is the gene for the capsid polypeptide, and the second is the gene for the putative RNA polymerase. One of the products of in vitro translation of the denatured viral double-stranded RNA is a polypeptide of the size expected of a capsid-polymerase fusion protein, resulting from a -1 frameshift within the overlapping region. A polypeptide of the size expected for a capsid-polymerase fusion product was found in virions, and it was recognized in Western blots (immunoblots) by antibodies to a synthetic peptide derived from the predicted polymerase sequence.     

Evolution of defective-interfering double-stranded RNAs of the yeast killer virus.

We have characterized by T1 fingerprint analysis several defective interfering (DI) double-stranded RNAs of the simple yeast virus ScV. A common sequence of about 0.5 to 0.6 kilobase pairs, including both 3' termini of the parental RNA, was present in each DI RNA. Several DI RNAs had novel T1 oligonucleotides not present in their parental RNA.     

Is embryo abortion a post‐zygotic barrier to gene flow between Littorina ecotypes?

Genetic incompatibilities contribute to reproductive isolation between many diverging populations, but it is still unclear to what extent they play a role if divergence happens with gene flow. In contact zones between the "Crab" and "Wave" ecotypes of the snail Littorina saxatilis, divergent selection forms strong barriers to gene flow, while the role of post‐zygotic barriers due to selection against hybrids remains unclear. High embryo abortion rates in this species could indicate the presence of such barriers. Post‐zygotic barriers might include genetic incompatibilities (e.g. Dobzhansky–Muller incompatibilities) but also maladaptation, both expected to be most pronounced in contact zones. In addition, embryo abortion might reflect physiological stress on females and embryos independent of any genetic stress. We examined all embryos of >500 females sampled outside and inside contact zones of three populations in Sweden. Females' clutch size ranged from 0 to 1,011 embryos (mean 130 ± 123), and abortion rates varied between 0% and 100% (mean 12%). We described female genotypes by using a hybrid index based on hundreds of SNPs differentiated between ecotypes with which we characterized female genotypes. We also calculated female SNP heterozygosity and inversion karyotype. Clutch size did not vary with female hybrid index, and abortion rates were only weakly related to hybrid index in two sites but not at all in a third site. No additional variation in abortion rate was explained by female SNP heterozygosity, but increased female inversion heterozygosity added slightly to increased abortion. Our results show only weak and probably biologically insignificant post‐zygotic barriers contributing to ecotype divergence, and the high and variable abortion rates were marginally, if at all, explained by hybrid index of females.     

The evolutionary trade-offs in phage-resistant Klebsiella pneumoniae entail cross-phage sensitization and loss of multidrug resistance

Bacteriophage therapy is currently being evaluated as a critical complement to traditional antibiotic treatment. However, the emergence of phage resistance is perceived as a major hurdle to the sustainable implementation of this antimicrobial strategy. By combining comprehensive genomics and microbiological assessment, we show that the receptor-modification resistance to capsule-targeting phages involves either escape mutation(s) in the capsule biosynthesis cluster or qualitative changes in exopolysaccharides, converting clones to mucoid variants. These variants introduce cross-resistance to phages specific to the same receptor yet sensitize to phages utilizing alternative ones. The loss/modification of capsule, the main Klebsiella pneumoniae virulence factor, did not dramatically impact population fitness, nor the ability to protect bacteria against the innate immune response. Nevertheless, the introduction of phage drives bacteria to expel multidrug resistance clusters, as observed by the large deletion in K. pneumoniae 77 plasmid containing bla_CTX-M, ant(3″), sul2, folA, mph(E)/mph(G) genes. The emerging bacterial resistance to viral infection steers evolution towards desired population attributes and highlights the synergistic potential for combined antibiotic-phage therapy against K. pneumoniae.     

AlkB Dioxygenase Preferentially Repairs Protonated Substrates: SPECIFICITY AGAINST EXOCYCLIC ADDUCTS AND MOLECULAR MECHANISM OF ACTION*

Efficient repair by Escherichia coli AlkB dioxygenase of exocyclic DNA adducts 3,N⁴-ethenocytosine, 1,N⁶-ethenoadenine, 3,N⁴-α-hydroxyethanocytosine, and reported here for the first time 3,N⁴-α-hydroxypropanocytosine requires higher Fe(II) concentration than the reference 3-methylcytosine. The pH optimum for the repair follows the order of pK_a values for protonation of the adduct, suggesting that positively charged substrates favorably interact with the negatively charged carboxylic group of Asp-135 side chain in the enzyme active center. This interaction is supported by molecular modeling, indicating that 1,N⁶-ethenoadenine and 3,N⁴-ethenocytosine are bound to AlkB more favorably in their protonated cationic forms. An analysis of the pattern of intermolecular interactions that stabilize the location of the ligand points to a role of Asp-135 in recognition of the adduct in its protonated form. Moreover, ab initio calculations also underline the role of substrate protonation in lowering the free energy barrier of the transition state of epoxidation of the etheno adducts studied. The observed time courses of repair of mixtures of stereoisomers of 3,N⁴-α-hydroxyethanocytosine or 3,N⁴-α-hydroxypropanocytosine are unequivocally two-exponential curves, indicating that the respective isomers are repaired by AlkB with different efficiencies. Molecular modeling of these adducts bound by AlkB allowed evaluation of the participation of their possible conformational states in the enzymatic reaction.     

Laser interferometry analysis of ciprofloxacin and ampicillin diffusion from liposomal solutions to water phase

The paper presents experimental investigations of diffusion of antibiotics (ciprofloxacin or ampicillin) into the water phase from mixtures of neutral or negatively charged liposomes, and antibiotic–liposome interactions. Using the laser interferometry technique, the amounts and fluxes of released antibiotics, concentration field evolution, and the velocity of the concentration boundary layer’s “growth” were determined. To avoid the limitations of membranes, a measurement system without the artificial boundary of phases with a free water–solution interface has been proposed. It was found that the diffusion of anionic and neutral liposomes into the water phase was insignificant and mainly the diffusion of antibiotics was measured. Differences in the diffusion kinetics of ciprofloxacin and ampicillin from liposomal solutions to the water phase were observed. Ampicillin diffused more efficiently than ciprofloxacin regardless of the liposomal solution type. Moreover, the amount of ampicillin and ciprofloxacin released from the anionic liposomal phase was higher than that from the neutral one. Our results confirm that ciprofloxacin at neutral pH shows little tendency to bind neutral liposomes. Additionally, it was also observed that ciprofloxacin disrupts negatively charged liposomes as a final effect of antibiotic–lipid interactions.     

TLR9 -1486T/C and 2848C/T SNPs Are Associated with Human Cytomegalovirus Infection in Infants

Toll-like receptor 9 (TLR9) recognizes non-methylated viral CpG-containing DNA and serves as a pattern recognition receptor that signals the presence of human cytomegalovirus (HCMV). Here, we present the genotype distribution of single-nucleotide polymorphisms (SNPs) of the TLR9 gene in infants and the relationship between TLR9 polymorphisms and HCMV infection. Four polymorphisms (-1237T/C, rs5743836; -1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) in the TLR9 gene were genotyped in 72 infants with symptomatic HCMV infection and 70 healthy individuals. SNP genotyping was performed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digested fragments were separated and identified by capillary electrophoresis. The HCMV DNA copy number was measured by a quantitative real-time PCR assay. We found an increased frequency of heterozygous genotypes TLR9 -1486T/C and 2848C/T in infants with HCMV infection compared with uninfected cases. Heterozygous variants of these two SNPs increased the risk of HCMV disease in children (P = 0.044 and P = 0.029, respectively). In infants with a mutation present in at least one allele of -1486T/C and 2848C/T SNPs, a trend towards increased risk of cytomegaly was confirmed after Bonferroni’s correction for multiple testing (Pc = 0.063). The rs352139 GG genotype showed a significantly reduced relative risk for HCMV infection (Pc = 0.006). In contrast, the -1237T/C SNP was not related to viral infection. We found no evidence for linkage disequilibrium with the four examined TLR9 SNPs. The findings suggest that the TLR9 -1486T/C and 2848C/T polymorphisms could be a genetic risk factor for the development of HCMV disease.     

Endurance Exercise Enhances the Effect of Strength Training on Muscle Fiber Size and Protein Expression of Akt and mTOR

Reports concerning the effect of endurance exercise on the anabolic response to strength training have been contradictory. This study re-investigated this issue, focusing on training effects on indicators of protein synthesis and degradation. Two groups of male subjects performed 7 weeks of resistance exercise alone (R; n = 7) or in combination with preceding endurance exercise, including both continuous and interval cycling (ER; n = 9). Muscle biopsies were taken before and after the training period. Similar increases in leg-press 1 repetition maximum (30%; P<0.05) were observed in both groups, whereas maximal oxygen uptake was elevated (8%; P<0.05) only in the ER group. The ER training enlarged the areas of both type I and type II fibers, whereas the R protocol increased only the type II fibers. The mean fiber area increased by 28% (P<0.05) in the ER group, whereas no significant increase was observed in the R group. Moreover, expression of Akt and mTOR protein was enhanced in the ER group, whereas only the level of mTOR was elevated following R training. Training-induced alterations in the levels of both Akt and mTOR protein were correlated to changes in type I fiber area (r = 0.55–0.61, P<0.05), as well as mean fiber area (r = 0.55–0.61, P<0.05), reflecting the important role played by these proteins in connection with muscle hypertrophy. Both training regimes reduced the level of MAFbx protein (P<0.05) and tended to elevate that of MuRF-1. The present findings indicate that the larger hypertrophy observed in the ER group is due more to pronounced stimulation of anabolic rather than inhibition of catabolic processes.     

The relationship between chronotype and personality among patients with alcohol dependence syndrome: Pilot study

The study investigates the distribution of chronotypes among alcohol-dependent subjects and the relationship between personality and chronotype. Fifty-eight alcohol-dependent patients and 29 age-matched healthy controls were studied using Ogińska’s Chronotype Questionnaire (ChQ), Eysenck’s Personality Questionnaire – Revised (EPQ-R), Selzer’s Michigan Alcoholism Screening Test (MAST) and a sociodemographic status questionnaire designed by the authors. The alcohol-dependent patients tended to be morning type, based on the morningness–eveningness ChQ scale, with a weakly marked rhythm, based on the distinctness ChQ scale. Preference towards morningness was associated with older age, but no relation between chronotype and severity of alcohol dependence was found. A high amplitude of the rhythm was associated with higher neuroticism. Therefore, despite being in the minority, patients with a distinct circadian rhythm (i.e. with a high amplitude) are at greater risk of mood and anxiety disorders and hence should be given special consideration.     

New semidominant mutations that affect mouse development

Dominantly acting mutations that produce visible phenotypes are frequently recovered, either during routine maintenance of colonies or from mutagenesis experiments. We have studied 12 dominant mouse mutations that cause a tail dysmorphology, a coat spotting phenotype, or a combination of these. The majority of these mutations act in a semidominant manner with the homozygous state associated with embryonic lethality and a visible phenotype at or before midgestation. The homozygous phenotypes include axis truncation and neural crest cell defects, as may be expected from the heterozygous phenotypes. The majority of mutations, however, also produced other phenotypes that include neural tube closure defects and aberrant heart looping. In one coat spotting mutant the homozygous condition is lethal before neural crest cell production commences. The mutated genes often function in processes additional to those alluded to by the heterozygous phenotype.