Complement C3 and C5 Deficiency Affects Fracture Healing

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3^-/- and C5^-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3^-/-: -25%, p=0.02; C5^-/-: -20% p=0.052) and newly formed bone (C3^-/-: -38%, p=0.01; C5^-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3^-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.     

Triploid Atlantic salmon Salmo salar have a higher dietary phosphorus requirement for bone mineralization during early development

The effect of a dietary phosphorus regime in freshwater on vertebra bone mineralization was assessed in diploid and triploid Atlantic salmon, Salmo salar. Fish were fed either a low phosphorus (LP) diet containing 10.5 g kg⁻¹ total phosphorus or a normal phosphorus (NP) diet containing 17.4 g kg⁻¹ total phosphorus from ∼3 to ∼65 g (day 126) in body weight. Two further groups were fed the NP diet from ∼3 g in body weight, but were then switched to the LP diet after 38 (∼10 g in body weight) or 77 (∼30 g in body weight) days. Growth, vertebral ash content (% ash) and radiologically detectable vertebra pathologies were assessed. Triploids were initially smaller than diploids, and again on day 77, but there was no ploidy effect on days 38 or 126. Vertebral ash content increased with increasing body size and those fish fed the NP diet had higher vertebral ash content than those groups fed the LP diet during the intervening time period, but this diet effect became less apparent as fish grew, with all groups having relatively equal vertebral ash content at termination. In general, triploids had lower vertebral ash content than diploids on day 38 and this was most evident in the group fed the LP diet. On day 77, those triploids fed the LP diet during the intervening time period had lower vertebral ash content than diploids. At termination on day 126, the triploids had the same vertebral ash content as diploids, irrespective of diet. There was a ploidy × diet interaction on vertebral deformities, with triploids having higher prevalences of fish with ≥1 deformed vertebra in all dietary groups except continuous NP. In conclusion, between days 0 and 77 (3–30 g body size), triploids required more dietary phosphorus than diploids in order to maintain similar vertebral ash content. A possible link between phosphorus feeding history and phosphorus demand is also discussed.     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.     

Transcription profiling during the cell cycle shows that a subset of Polycomb-targeted genes is upregulated during DNA replication

Genome-wide gene expression analyses of the human somatic cell cycle have indicated that the set of cycling genes differ between primary and cancer cells. By identifying genes that have cell cycle dependent expression in HaCaT human keratinocytes and comparing these with previously identified cell cycle genes, we have identified three distinct groups of cell cycle genes. First, housekeeping genes enriched for known cell cycle functions; second, cell type-specific genes enriched for HaCaT-specific functions; and third, Polycomb-regulated genes. These Polycomb-regulated genes are specifically upregulated during DNA replication, and consistent with being epigenetically silenced in other cell cycle phases, these genes have lower expression than other cell cycle genes. We also find similar patterns in foreskin fibroblasts, indicating that replication-dependent expression of Polycomb-silenced genes is a prevalent but unrecognized regulatory mechanism.     

Dissecting direct and indirect genetic effects on chronic obstructive pulmonary disease (COPD) susceptibility

Cigarette smoking is the major environmental risk factor for chronic obstructive pulmonary disease (COPD). Genome-wide association studies have provided compelling associations for three loci with COPD. In this study, we aimed to estimate direct, i.e., independent from smoking, and indirect effects of those loci on COPD development using mediation analysis. We included a total of 3,424 COPD cases and 1,872 unaffected controls with data on two smoking-related phenotypes: lifetime average smoking intensity and cumulative exposure to tobacco smoke (pack years). Our analysis revealed that effects of two linked variants (rs1051730 and rs8034191) in the AGPHD1/CHRNA3 cluster on COPD development are significantly, yet not entirely, mediated by the smoking-related phenotypes. Approximately 30 % of the total effect of variants in the AGPHD1/CHRNA3 cluster on COPD development was mediated by pack years. Simultaneous analysis of modestly (r ² = 0.21) linked markers in CHRNA3 and IREB2 revealed that an even larger (~42 %) proportion of the total effect of the CHRNA3 locus on COPD was mediated by pack years after adjustment for an IREB2 single nucleotide polymorphism. This study confirms the existence of direct effects of the AGPHD1/CHRNA3, IREB2, FAM13A and HHIP loci on COPD development. While the association of the AGPHD1/CHRNA3 locus with COPD is significantly mediated by smoking-related phenotypes, IREB2 appears to affect COPD independently of smoking.     

Surface active adrenoceptor compounds prevent the settlement of cyprid larvae of Balanus improvisus

The barnacle Balanus improvisus is the major fouling macroorganism in Swedish waters and it colonizes most man‐made surfaces submerged in the sea. New or impending legislation restricts the use of traditional, hazardous antifouling coatings based on heavy metals, mainly copper and tin. This calls for the development of new non‐toxic methods that prevent barnacle settlement. In this work several adrenoceptor compounds are shown to be very efficient in preventing the settlement of cyprid larvae of B. improvisus. The settlement rate of laboratory‐reared cyprids was studied in hydrophilised polystyrene dishes containing adrenoceptor antagonists and agonists dissolved in seawater. Two of these drugs, medetomidine and clonidine, repeatedly inhibited settlement at concentrations between 1 nM and 10 nM. In the vertebrate adrenoceptor classification system, which separates pharmacological substances according to their receptor affinity, both of these substances are classified as α₂ adrenoceptor agonists. An inhibiting effect on presyn‐aptic receptors is suggested, but the localization of the receptor effect requires futher studies. Experiments also revealed that the inhibiting effect of medetomidine was reversible. Cyprids incubated with medetomidine for 20 h attached and metamorphosed into juvenile barnacles after washing and transferrence to seawater. The antagonizing compound atipamezole reversed the effect of medetomidine. This observation supports the assumption that this substance acts at the receptor level. Studies of the surface affinity of medetomidine revealed a strong tendency to accumulate in solid/ liquid phase boundaries. This ability makes it particularly attractive as a candidate for the development of a slow‐release carrier in marine coatings. Panels coated with medetomidine in an acrylate polymer and exposed in the field reduced the recruitment of B. improvisus by 96% after 4 weeks and by 70% after 8 weeks.     

Oxygen-depleted surfaces: a new antifouling technology

A novel, non-toxic strategy to combat marine biofouling is presented. The technology is paint with additions of up to 43% of industrial protein. Through microbial degradation of the protein component, an oxygen-depleted layer rapidly forms in a 0.2 mm layer close to the paint surface. With the present paint formulations, a stable, O₂-depleted layer can persist for 16 weeks. Barnacle larvae (cyprids) did not settle on panels where oxygen saturation was <20%, and cyprids were killed when exposed to O₂-free water for more than 1 h. It is also shown that the O₂-depleted layer will rapidly reform (within 15 min) after exposure to turbulent flow. Field exposure of panels for 16 weeks showed that paint with protein reduced fouling by barnacles and bryozoans by 80% and close to 100%, respectively. The results suggest that this novel technology may be developed into a non-toxic alternative to copper-based antifouling paints, especially for pleasure boats in sensitive environments. There is clearly potential for further development of the paint formulation, and a full-scale test on a boat-hull suggested that service-life under realistic operations needs to be improved.