Conservation of Genomic Localization and Sequence Content of Sau3AI-Like Restriction-Modification Gene Cassettes among Listeria monocytogenes Epidemic Clone I and Selected Strains of Serotype 1/2a

Listeria monocytogenes is a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported among L. monocytogenes isolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.Listeria monocytogenes is a Gram-positive, facultative intracellular food-borne pathogen capable of causing severe disease (listeriosis) in animals and humans. Listeriosis most often affects pregnant women and their fetuses, neonates, the elderly, and immunocompromised individuals. The disease is predominantly transmitted via the consumption of contaminated foods and has a ca. 20% fatality rate (12, 27). Application of numerous genotyping methods has consistently shown that the organism has a clonal population structure with three major phylogenetic lineages: lineage I consists of strains of serotypes 1/2b, 3b, and 4b, while those of serotypes 1/2a, 1/2c, 3a, and 3c are clustered in lineage II; strains of serotypes 4a and 4c, along with certain serotype 4b strains, constitute lineage III (37, 38).Most epidemics of human listeriosis have involved a small number of closely related strains (epidemic clones), predominantly of serotype 4b (7, 35). The earliest identified clone, epidemic clone I (ECI), has been responsible for several major outbreaks in Europe and North America. In addition, strains of this clonal group are frequently encountered in sporadic illness (10, 28, 29). ECI strains have also been found to comprise a significant portion of the serotype 4b strains from foods and from the environments of food processing plants (10, 11, 40).Genomic DNA of ECI strains has been long known to resist digestion with Sau3AI, suggesting methylation of cytosine at GATC sites (41). Genome sequencing of the ECI strain F2365, implicated in the 1985 California outbreak of listeriosis, revealed a putative restriction-modification (RM) gene cassette with specificity for GATC sites (25). This RM cassette was harbored by all tested serotype 4b strains with Sau3AI-resistant DNA and was absent from those with DNA that could be digested with Sau3AI (40). These findings were in agreement with previous evidence that a fragment of the putative methyltransferase gene was specific to ECI and absent from other strains (14).In spite of extensive documentation for the presence of this putative RM cassette in ECI strains, and its apparent absence among other serotype 4b strains, limited information is available about the possible presence of the cassette among other lineages of L. monocytogenes. Furthermore, conclusive evidence for involvement of the cassette in the resistance of the DNA of ECI strains to Sau3AI digestion has been lacking. In this study, we investigated a panel of food-derived serotype 1/2a strains with Sau3AI-resistant DNA and characterized the genetic content and genomic localization of the RM cassette harbored by these strains. Furthermore, we employed deletion mutagenesis to assess the involvement of the RM cassette in Sau3AI resistance of the DNA of the ECI strain F2365, as well as of a serotype 1/2a strain harboring the cassette.     

Chemometric methods for simultaneous quantification of lactic,malic and fumaric acids

Lactic, fumaric and malic acids are commonly used in food and pharmaceutical industries. During microbial production of these compounds, it is important to determine their concentrations in the fermentation broth with a rapid and sensitive method. Spectrophotometry is commonly used. However, UV‐spectral overlap between these organic acids makes it difficult to determine each of them individually from the mixture. In order to overcome this problem, statistical methods, namely principal component regression (PCR) and partial least squares‐1 methods, were tested and compared with conventional HPLC techniques. The absorbance data matrix was obtained by measuring the absorbances of 21 ternary mixtures of lactic, fumaric and malic acids in a wavelength range of 210–260 nm. Calibration and validation were performed by using the data obtained in a mixture of these organic acids. The prediction abilities of the methods were tested by applying them to fermentation broths. The precision of the PCR method was better than that of the partial least squares‐1 method. In the PCR method, the correlation coefficients between actual and predicted concentrations of the organic acids were calculated as 0.970 for lactic acid and 0.996 for fumaric acid in fermentation broths. The concentration of malic acid was not detected due to its low concentration in samples. These results show that the PCR method can be applied for simultaneous determination of lactic, fumaric and malic acids in fermentation broths.     

Interaction of magnetite-based receptors in the beak with the visual system underlying 'fixed direction' responses in birds

Background

European robins, Erithacus rubecula, show two types of directional responses to the magnetic field: (1) compass orientation that is based on radical pair processes and lateralized in favor of the right eye and (2) so-called 'fixed direction' responses that originate in the magnetite-based receptors in the upper beak. Both responses are light-dependent. Lateralization of the 'fixed direction' responses would suggest an interaction between the two magnetoreception systems.

Results

Robins were tested with either the right or the left eye covered or with both eyes uncovered for their orientation under different light conditions. With 502 nm turquoise light, the birds showed normal compass orientation, whereas they displayed an easterly 'fixed direction' response under a combination of 502 nm turquoise with 590 nm yellow light. Monocularly right-eyed birds with their left eye covered were oriented just as they were binocularly as controls: under turquoise in their northerly migratory direction, under turquoise-and-yellow towards east. The response of monocularly left-eyed birds differed: under turquoise light, they were disoriented, reflecting a lateralization of the magnetic compass system in favor of the right eye, whereas they continued to head eastward under turquoise-and-yellow light.

Conclusion

'Fixed direction' responses are not lateralized. Hence the interactions between the magnetite-receptors in the beak and the visual system do not seem to involve the magnetoreception system based on radical pair processes, but rather other, non-lateralized components of the visual system.     

Characterization of the fecal microbiome from non-human wild primates reveals species specific microbial communities

Background

Host-associated microbes comprise an integral part of animal digestive systems and these interactions have a long evolutionary history. It has been hypothesized that the gastrointestinal microbiome of humans and other non-human primates may have played significant roles in host evolution by facilitating a range of dietary adaptations. We have undertaken a comparative sequencing survey of the gastrointestinal microbiomes of several non-human primate species, with the goal of better understanding how these microbiomes relate to the evolution of non-human primate diversity. Here we present a comparative analysis of gastrointestinal microbial communities from three different species of Old World wild monkeys.

Methodology/Principal Findings

We analyzed fecal samples from three different wild non-human primate species (black-and-white colobus [Colubus guereza], red colobus [Piliocolobus tephrosceles], and red-tailed guenon [Cercopithecus ascanius]). Three samples from each species were subjected to small subunit rRNA tag pyrosequencing. Firmicutes comprised the vast majority of the phyla in each sample. Other phyla represented were Bacterioidetes, Proteobacteria, Spirochaetes, Actinobacteria, Verrucomicrobia, Lentisphaerae, Tenericutes, Planctomycetes, Fibrobacateres, and TM7. Bray-Curtis similarity analysis of these microbiomes indicated that microbial community composition within the same primate species are more similar to each other than to those of different primate species. Comparison of fecal microbiota from non-human primates with microbiota of human stool samples obtained in previous studies revealed that the gut microbiota of these primates are distinct and reflect host phylogeny.

Conclusion/Significance

Our analysis provides evidence that the fecal microbiomes of wild primates co-vary with their hosts, and that this is manifested in higher intraspecies similarity among wild primate species, perhaps reflecting species specificity of the microbiome in addition to dietary influences. These results contribute to the limited body of primate microbiome studies and provide a framework for comparative microbiome analysis between human and non-human primates as well as a comparative evolutionary understanding of the human microbiome.     

Ectrodactyly, ectodermal dysplasia, macular degeneration syndrome: a further contribution

EEM syndrome is a rare condition characterised by ectodermal dysplasia, ectrodactyly and macular dystrophy. Additional abnormalities such as alopecia, cataract, absent eyebrows, and oligodontia may occur. We report two brothers and a sister born to consanguineous parents with EEM syndrome. EEM syndrome differs from other ectrodactly syndromes by the characteristic findings in the ocular fundus showing extensive retinochoroidal atrophy with diffuse retinal pigmentation and mild arteriolar attenuation at the posterior pole. In contrast to other ectrodactyly syndromes autosomal recessive inheritance is most likely.     

Sequential occurrence of mitochondrial and plasma membrane alterations, fluctuations in cellular Ca2+ and pH during initial and later phases of cell death

The sequential occurrence of plasma and mitochondrial membrane alterations, intra-cellular pH shifts and changes in intracellular Ca²⁺ concentration after induction of cell death was monitored by flow cytometry in Jurkat and HSB2-cells. Cell death was induced by treatment with anti-Fas antibodies or by irradiation. Phosphatidylserine (PS) exposure and plasma membrane integrity were measured with FITC-Annexin V adhesion and by Propidium Iodide exclusion. Transition of the mitochondrial membrane potential was monitored by the occurrence of decay of DiOC₆ fluorescence. Intracellular pH shifts were monitored by changes in the ratio of fluorescence at 575 nm and at 635 nm of SNARF-1-AM. Fluctuations in intracellular Ca²⁺ concentration were established by changes in Fura red quenching.The Jurkat cells were sensitive to anti-Fas treatment, while HSB-2 cells were not. HSB-2 cells appeared more sensitive to radiation damage than Jurkat cells.In all experiments the transition of mitochondrial membrane potential occurred first, almost immediately followed by PS exposure. Fluctuations in intracellular Ca²⁺ concentration occurred later and were less outspoken. A decrease in intracellular pH occurred not earlier than 24 hours after anti-Fas treatment. Chelation of intracellular Ca²⁺ concentration with BAPTA-AM had no effect on the time sequence of cell death related events.     

The role of prednisolone and epinephrine on gastric tissue and erythrocyte antioxidant status in adrenalectomized rats.

It has been believed that overproduction of free radicals and/or deficiency of antioxidant systems, and stress hormones may play a role in etiopathogenesis of many diseases, including gastric ulcer. This study evaluated whether there was an effect of adrenalectomy on lipid peroxidation [malondialdehyde (MDA)] and antioxidant [superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione (GSH) levels] systems in gastric tissue and erythrocyte in rats. As well, the impacts of administration of prednisolone and epinephrine on these systems in adrenalectomized rats were investigated. Thirty-three rats were randomly grouped as sham-operated (group I), adrenalectomized (group II), adrenalectomized + prednisolone (group III) and adrenalectomized + epinephrine (group IV). After experimental procedures, blood and gastric tissues samples were taken from each animal in all groups. Colorimetric assays were employed to determine gastric tissue and erythrocyte levels of MDA and GSH, and SOD and GPX activities. Adrenalectomy in group II rats caused a marked decrease of SOD and GPX activities and MDA levels, and an increase of GSH levels in gastric tissue and erythrocyte, when compared to sham-operated rats. However, especially epinephrine injection after adrenalectomy resulted in a significantly increase of measured antioxidant enzyme activities and GSH levels in both gastric tissue and erythrocyte. These results indicate that adrenalectomy appeared to alter the levels of antioxidants and lipid peroxidation product in gastric tissue and erythrocyte. Thus, the present study provides a physiological regulatory role of adrenal gland in the maintenance of oxidant/antioxidant balance in gastric tissue and erythrocyte.     

Marker assisted genetic analysis of non-brittle rachis trait in barley

Brittle rachis is a head shattering mechanism of barley. Two tightly linked complementary genes, btr1 and btr2, were believed to control the non-brittle rachis trait. Position of non-brittle rachis loci btr1btr2 on the short arm of Chromosome 3 was investigated using RFLP markers. Two approaches were employed. First, a Hordeum vulgare subsp. spontaneum fragment that confers brittleness in a cv. Bowman near isogenic line was detected. This fragment is 18-33 cM in length and contains MWG798B, ABG057, MWG014, BCD706 and KFP216 markers of the short arm of Chromosome 3. In the second approach, position of btr1 locus in a H. vulgare subsp. spontaneum (Wadi Qilt 23-38)xH. vulgare subsp. vulgare (cv. Harrington) cross was detected using a selective genotyping approach in BC2F1 generation. F-tests and analysis of genotypic compositions of BC2F1 lines showed that btr1 locus, and supposedly the tightly linked btr2 locus, is in 4.3 cM KFP216-RisP114 interval of short arm of Chromosome 3. Results also yielded clues for the presence of at least two additional loci that affect the non-brittle rachis trait. Allelism tests using genotypes with known non-brittle rachis gene compositions provided additional evidence for presence of such loci.     

Anterolateral thigh flap in the treatment of postburn flexion contractures of the knee

The use of the anterolateral thigh fasciocutaneous flap in the reconstruction of soft-tissue defects around the knee among burn patients is described. The anterolateral thigh fasciocutaneous flap was used for eight patients (all male; mean age, 45 years; age range, 32 to 60 years). Flexion contracture was observed for seven patients with unhealed wounds and one patient with a healed burn wound. The anterolateral thigh flap was used as a free flap for six patients and as a distally based island flap for two patients. The flaps ranged from 8 to 17 cm in width and from 12 to 30 cm in length. Seven flaps were based on a musculocutaneous perforator, and two of them were thinned before transfer to the defect. A true septocutaneous perforator was observed in only one case. The mean follow-up period was 12.5 months (range, 3 to 23 months). Only one flap exhibited distal superficial necrosis, which did not compromise the final result. All patients returned to ambulatory status in 15 to 22 days. Extensor splints were applied to prevent mobilization of the skin graft at the flap donor site for only 7 days. The anterolateral thigh flap has many advantages for the reconstruction of postburn flexion contracture of the knee, as follows: (1) very large thin flaps can be elevated, (2) the two-team approach is possible, (3) color and texture matches are good, (4) the donor-site scar can be easily hidden, and (5) the technique allows early mobilization and patients can return to normal daily activity in a short time. Free or distally based anterolateral thigh flaps are a good choice, both aesthetically and functionally, for the reconstruction of soft-tissue defects of the knee region.