Transfer of the Pheromone-Inducible Plasmid pCF10 among Enterococcus faecalis Microorganisms Colonizing the Intestine of Mini-Pigs

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10⁶ and 10⁷ CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10⁶ CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10³ and 10⁴ CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.     

Differences in the domain forming properties of N-palmitoylated neutral glycosphingolipids in bilayer membranes

We have compared the domain forming properties of three neutral acyl chain defined glycosphingolipids differing in their head group structures. The aim of the study was to explore if glycosphingolipids and sterols exist in the same lateral domains in bilayer membranes and how the structure of the head group influences the capacity of the glycosphingolipids to colocalize with cholesterol. The glycosphingolipids used in the study were galactosyl-, glucosyl- and lactosylceramides with a palmitic acid in the N-linked position. Domain formation in mixed bilayer vesicles was examined using fluorescent reporter molecules associating with ordered domains, together with a fluorescence quencher lipid in the disordered membrane phase. Our results show that the glycosphingolipids studied were poor in forming sterol-enriched domains compared to palmitoyl-sphingomyelin as detected by cholestatrienol quenching. However, the tendency to associate with cholesterol was clearly dependent on the carbohydrate structure of the glycosphingolipids, also when two glycosphingolipids with different head groups were mixed in the bilayer. All palmitoylated glycosphingolipids associated with palmitoyl-sphingomyelin/cholesterol domains. Our results show that the head group structures of neutral glycosphingolipids markedly affect their domain forming properties in bilayers both with and without cholesterol. The most striking observation being that large differences in domain forming properties were seen even between glucosylceramide and galactosylceramide, which differ only in the stereochemistry of one hydroxyl group in the carbohydrate head group.     

The impact of changing the season in which cereals are sown on the diversity of the weed flora in rotational fields in Denmark

1. Surveys have shown that there has been a dramatic decrease in the weed flora of fields under rotational cultivation during the last 30 years. This trend has been particularly noticeable in winter cereals, a crop of increasing importance in the landscape.
2. The weed flora of spring and winter cereals was compared in 19 unsprayed fields during a 5-year study to test the hypothesis that cereal type exerts no effect on the flora or on the absolute and relative abundance of single species.
3. Plant and species densities, and accumulated species richness, were lower in winter than in spring cereals.
4. Floristic similarity was greater among spring cereal fields and between spring and winter cereals within the same fields than among winter cereal fields.
5. Species that occurred with unequal density in spring and winter cereals occurred at higher densities in the spring cereals; these species germinated mainly in the spring. However, for a few species the relative plant abundance was highest in winter cereals; these species were able to germinate both in the spring and autumn.
6. Some species – on the relative scale – occurred indifferently of season of sowing; all but one of these species were able to germinate both in the spring and autumn.
7. Plant species and taxa that are important food resources for arthropod herbivores occurred at greater densities in spring than in winter cereals and, in addition, occurred with the highest relative abundance in spring cereals.
8. Change in land use from spring to winter cereals involves not only an immediate reduction of more than 25% in the density of plants and species, but also a change and increased uncertainty in the composition of the weed flora. These findings may have serious implications for the ecology of wildlife in the agricultural landscape.     

Every plant for himself; the effect of a phenolic monoterpene on germination and biomass of Thymus pulegioides and T. serpyllum

Thyme plants are known for their production of aromatic oils, whose main component is terpenes. The plants leach terpenes to their surroundings and thereby affect the seed germination and biomass of associated plants, but also potentially themselves. A variation in the dominant terpenes produced by thyme plants is found both within and among species. In Denmark two thyme species (Thymus pulegioides and T. serpyllum) are naturally occurring. The essential oil of T. pulegioides in Denmark is mainly dominated by one monoterpene; ‘carvacrol’. In contrast, the essential oil of T. serpyllum constitutes a mix of two–four different types of terpenes, both mono‐ and sesqui‐terpenes. As the effects of terpenes on plant performance can vary with the type of terpene, and in order to study species‐specific responses, we examined how the dominating T. pulegioides monoterpene ‘carvacrol’ affected germination and growth of both T. pulegioides and T. serpyllum. We compared the performance of seeds and seedlings of both thyme species on soil treated with carvacrol versus control soil. We found no effect of treatment on germination, but we detected a highly significant effect of treatment on seedling biomass. For both thyme species, seedling biomass was significantly higher on terpene soil compared to control soil, suggesting a general adaptation to the presence of terpenes in the soil for both thyme species. Moreover, while no difference in seedling biomass between species on control soil was found, T. pulegiodes seedlings were significantly larger than T. serpyllum when grown on soil treated with its ‘home’ terpene, suggesting an additional species specific response. Dividing the biomass into aboveground and root biomass showed that the increased biomass on terpene‐soil was due to increased aboveground biomass, whereas no difference in root biomass was detected among treatments and species. We discuss whether this response may be caused by an adaptation to a predictable terpene‐mediated alteration in nitrogen‐availability.     

How the molecular features of glycosphingolipids affect domain formation in fluid membranes

Glycosphingolipids, sphingomyelin and cholesterol are often all found in the detergent resistant fraction of biological membranes and are therefore recognized as raft components, but they do not necessarily co-localize in the same lateral domains. From cell biological studies it is evident that different sphingolipid species can be found in different lateral regions within the same cellular membrane. Biophysical studies have shown that their tendency to co-localize with each other and with other membrane components is largely governed by structural features of all lipids present. Glycosphingolipids form gel-phase like domains in fluid lipid bilayers. Sphingomyelin readily associates with cholesterol, forming liquid-ordered phase domains, but glycosphingolipids do not readily form cholesterol-enriched domains by themselves. However, mixed sphingomyelin- and glycosphingolipid-rich domains appear to incorporate cholesterol. Recent studies indicate that the ceramide backbone structure as well as the number of sugar units and presence of charge in the glycosphingolipid head group will influence the partitioning of these lipids between lateral membrane domains. The properties of the domains will be largely influenced by the presence of glycosphingolipids, which have very high melting temperatures. The lateral partitioning of glycosphingolipid molecular species has only recently been studied more intensively, and a lot remains to be done in this field of research.     

Thermotropic behavior and lateral distribution of very long chain sphingolipids

Sphingolipids containing very long acyl chains are abundant in certain specialized tissues and minor components of plasma membranes in most mammalian cells. There are cellular processes in which these sphingolipids are required, and the function seems to be mediated through sphingolipid-rich membrane domains. This study was conducted to explore how very long acyl chains of sphingolipids influence their lateral distribution in membranes. Differential scanning calorimetry showed that 24:0- and 24:1-sphingomyelins, galactosylceramides and glucosylceramides exhibited complex thermotropic behavior and partial miscibility with palmitoyl sphingomyelin. The T_m was decreased by about 20 °C for all 24:1-sphingolipids compared to the corresponding 24:0-sphingolipids. The ability to pack tightly with ordered and extended acyl chains is a necessity for membrane lipids to partition into ordered domains in membranes and thus the 24:1-sphingolipids appeared less likely to do so. Fluorescence quenching measurements showed that the 24:0-sphingolipids formed ordered domains in multicomponent membranes, both as the only sphingolipid and mixed with palmitoyl sphingomyelin. These domains had a high packing density which appeared to hinder the partitioning of sterols into them, as reported by the fluorescent cholesterol analog cholestatrienol. 24:0-SM was, however, better able to accommodate sterol than the glycosphingolipids. The 24:1-sphingolipids could, depending on head group structure, either stabilize or disrupt ordered sphingolipid/cholesterol domains. We conclude that very long chain sphingolipids, when present in biological membranes, may affect the physical properties of or the distribution of sterols between lateral domains. It was also evident that not only the very long acyl chain but also the specific molecular structure of the sphingolipids was of importance for their membrane properties.     

Differential ability of cholesterol-enriched and gel phase domains to resist benzyl alcohol-induced fluidization in multilamellar lipid vesicles

Benzyl alcohol (BA) has a well-known fluidizing effect on both artificial and cellular membranes. BA is also likely to modulate the activities of certain membrane proteins by decreasing the membrane order. This phenomenon is presumably related to the ability of BA to interrupt interactions between membrane proteins and the surrounding lipids by fluidizing the lipid bilayer. The components of biological membranes are laterally diversified into transient assemblies of varying content and order, and many proteins are suggested to be activated or inactivated by their localization in or out of membrane domains displaying different physical phases. We studied the ability of BA to fluidize artificial bilayer membranes representing liquid-disordered, cholesterol-enriched and gel phases. Multilamellar vesicles were studied by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid, which display different phase partitioning. Domains of different degree of order and thermal stability showed varying abilities to resist fluidization by BA. In bilayers composed of mixtures of an unsaturated phosphatidylcholine, a saturated high melting temperature lipid (sphingomyelin or phosphatidylcholine) and cholesterol, BA fluidized and lowered the melting temperature of the ordered and gel phase domains. In general, cholesterol-enriched domains were more resistant to BA than pure gel phase domains. In contrast, bilayers containing high melting temperature gel phase domains containing a ceramide or a galactosylceramide proved to be the most effective in resisting fluidization. The results of our study suggest that the ability of BA to affect the fluidity and lateral organization of the membranes was dependent on the characteristic features of the membrane compositions studied and related to the intermolecular cohesion in the domains.     

Analysis of rabbit doe longevity using a semiparametric log-Normal animal frailty model with time-dependent covariates

Data on doe longevity in a rabbit population were analysed using a semiparametric log-Normal animal frailty model. Longevity was defined as the time from the first positive pregnancy test to death or culling due to pathological problems. Does culled for other reasons had right censored records of longevity. The model included time dependent covariates associated with year by season, the interaction between physiological state and the number of young born alive, and between order of positive pregnancy test and physiological state. The model also included an additive genetic effect and a residual in log frailty. Properties of marginal posterior distributions of specific parameters were inferred from a full Bayesian analysis using Gibbs sampling. All of the fully conditional posterior distributions defining a Gibbs sampler were easy to sample from, either directly or using adaptive rejection sampling. The marginal posterior mean estimates of the additive genetic variance and of the residual variance in log frailty were 0.247 and 0.690.     

Infections in temporal proximity to HPV vaccination and adverse effects following vaccination in Denmark: A nationwide register-based cohort study and case-crossover analysis

BackgroundPublic trust in the human papilloma virus (HPV) vaccination programme has been challenged by reports of potential severe adverse effects. The reported adverse symptoms were heterogeneous and overlapping with those characterised as chronic fatigue syndrome (CFS) and have been described as CFS-like symptoms. Evidence suggests that CFS is often precipitated by an infection. The aim of the study was to examine if an infection in temporal proximity to HPV vaccination is a risk factor for suspected adverse effects following HPV vaccination.Methods and findingsThe study was a nationwide register-based cohort study and case-crossover analysis. The study population consisted of all HPV vaccinated females living in Denmark, born between 1974 and 2006, and vaccinated between January 1, 2006 and December 31, 2017. The exposure was any infection in the period ± 1 month around time of first HPV vaccination and was defined as (1) hospital-treated infection; (2) redemption of anti-infective medication; or (3) having a rapid streptococcal test done at the general practitioner. The outcome was referral to a specialised hospital setting (5 national HPV centres opened June 1, 2015) due to suspected adverse effects following HPV vaccination. Multivariable logistic regression was used to estimate the association between infection and later HPV centre referral. The participants were 600,400 HPV-vaccinated females aged 11 to 44 years. Of these, 48,361 (9.7%) females had a hospital-treated infection, redeemed anti-infective medication, or had a rapid streptococcal test ± 1 month around time of first HPV vaccination. A total of 1,755 (0.3%) females were referred to an HPV centre. Having a hospital-treated infection in temporal proximity to vaccination was associated with significantly elevated risk of later referral to an HPV centre (odds ratio (OR) 2.75, 95% confidence interval (CI) 1.72 to 4.40; P < 0.001). Increased risk was also observed among females who redeemed anti-infective medication (OR 1.56, 95% CI 1.33 to 1.83; P < 0.001) or had a rapid streptococcal test (OR 1.45, 95% CI 1.10 to 1.93; P = 0.010). Results from a case-crossover analysis, which was performed to adjust for potential unmeasured confounding, supported the findings. A key limitation of the study is that the HPV centres did not open until June 1, 2015, which may have led to an underestimation of the risk of suspected adverse effects, but stratified analyses by year of vaccination yielded similar results.ConclusionsTreated infection in temporal proximity to HPV vaccination is associated with increased risk for later referral with suspected adverse vaccine effects. Thus, the infection could potentially be a trigger of the CFS-like symptoms in a subset of the referred females. To our knowledge, the study is the first to investigate the role of infection in the development of suspected adverse effects after HPV vaccination and replication of these findings are needed in other studies.

Lene Wulff Krogsgaard and co-workers study temporal associations between infections and HPV vaccination.     

Uracil-DNA glycosylase in base excision repair and adaptive immunity: species differences between man and mouse

Genomic uracil is a DNA lesion but also an essential key intermediate in adaptive immunity. In B cells, activation-induced cytidine deaminase deaminates cytosine to uracil (U:G mispairs) in Ig genes to initiate antibody maturation. Uracil-DNA glycosylases (UDGs) such as uracil N-glycosylase (UNG), single strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and thymine-DNA glycosylase remove uracil from DNA. Gene-targeted mouse models are extensively used to investigate the role of these enzymes in DNA repair and Ig diversification. However, possible species differences in uracil processing in humans and mice are yet not established. To address this, we analyzed UDG activities and quantities in human and mouse cell lines and in splenic B cells from Ung(+/+) and Ung(-/-) backcrossed mice. Interestingly, human cells displayed ～15-fold higher total uracil excision capacity due to higher levels of UNG. In contrast, SMUG1 activity was ～8-fold higher in mouse cells, constituting ～50% of the total U:G excision activity compared with less than 1% in human cells. In activated B cells, both UNG and SMUG1 activities were at levels comparable with those measured for mouse cell lines. Moreover, SMUG1 activity per cell was not down-regulated after activation. We therefore suggest that SMUG1 may work as a weak backup activity for UNG2 during class switch recombination in Ung(-/-) mice. Our results reveal significant species differences in genomic uracil processing. These findings should be taken into account when mouse models are used in studies of uracil DNA repair and adaptive immunity.