Neuropilin1 is a direct downstream target of Nurr1 in the developing brain stem

The orphan nuclear receptor Nurr1 is expressed in the developing and adult central nervous system. Previous studies have shown that Nurr1 is essential for the generation of midbrain dopamine neurons. Furthermore, Nurr1 is critical for respiratory functions associated with the brain stem. Very few Nurr1 regulated genes have been identified and it remains unclear how Nurr1 influences the function and development of neurons. To identify novel Nurr1 target genes we have searched for regulated genes in the dopaminergic MN9D cell line. These experiments identified Neuropilin-1 (Nrp1), a receptor protein involved in axon guidance and angiogenesis, as a novel Nurr1 target gene. Nrp1 expression was rapidly up-regulated by Nurr1 in MN9D cells and in situ hybridization analysis showed that Nrp1 was coexpressed with Nurr1 in the brain stem dorsal motor nucleus. Importantly, Nrp1 expression was down-regulated in this area in Nurr1 null mice. Moreover, two functional Nurr1 binding sites were identified in the Nrp1 promoter and Nurr1 was found to be recruited to these sites in MN9D cells, further supporting that Nrp1 is a direct downstream target of Nurr1. Taken together, our findings suggest that Nurr1 might influence the processes of axon guidance and/or angiogenesis via the regulation of Nrp1 expression.     

Structure-activity relationship study on a novel series of cyclopentane-containing macrocyclic inhibitors of the hepatitis C virus NS3/4A protease leading to the discovery of TMC435350

SAR analysis performed with a limited set of cyclopentane-containing macrocycles led to the identification of N-[17-[2-(4-isopropylthiazole-2-yl)-7-methoxy-8-methylquinolin-4-yloxy]-13-methyl-2,14-dioxo-3,13-diazatricyclo [13.3.0.0^4,6]octadec-7-ene-4-carbonyl](cyclopropyl)sulfonamide (TMC435350, 32c) as a potent inhibitor of HCV NS3/4A protease (K_i = 0.36 nM) and viral replication (replicon EC₅₀ = 7.8 nM). TMC435350 also displayed low in vitro clearance and high permeability, which were confirmed by in vivo pharmacokinetic studies. TMC435350 is currently being evaluated in the clinics.     

Lack of collagen XVIII accelerates cutaneous wound healing, while overexpression of its endostatin domain leads to delayed healing

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII (Col18a1(-/-)). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1(-/-) mice, and the vascularisation rate was accelerated in the Col18a1(-/-) mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half (n=10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal-dermal junction in the presence of an excess of endostatin.     

Perceived needs and satisfaction with care in people with multiple sclerosis: A two-year prospective study

Background  

Considering the costs of multiple sclerosis (MS), it is crucial that the health-related services supplied are in accordance with needs as they are perceived by people with MS (PwMS). Satisfaction with care is related to quality of care and can provide health care providers with the means for improvement. The aim was to explore the perceived needs and satisfaction with care amongst PwMS over a two-year period, also taking sex and disease severity into consideration.     

Integrating the Concept of Resilience into an Ecosystem Approach to Bivalve Aquaculture Management

Bivalve aquaculture has become increasingly important for marine protein production and is an alternative to exploiting natural resources. Its further and sustainable development should follow an ecosystem approach, to maintain both biodiversity and ecosystem functioning. The identification of critical thresholds to development remains difficult. The present work aims at combining the calculation of the system’s ecological carrying capacity (ECC) with the ecosystem view of resilience for a bay system exposed to bivalve (scallop) aquaculture. Using a trophic food-web model, a stepwise further expansion of culture activities was simulated, and the impact on the system was evaluated twofold: First, a recently developed approach to estimating ECC was used, and second, a resilience indicator was calculated, which is based on the distribution of consumption flows within the trophic network (sensu Arreguín-Sanchez in Ecol Model 272: 27–276, 2014). Results suggest that a culture expansion beyond present-day scale would (a) cause a shift in community composition towards a system dominated by secondary consumers, (b) lead to the loss of system compartments, affecting ecosystem functioning, and (c) result in a decrease in resilience, emphasizing the need to regulate aquaculture activities. The applicability and potential of this presented method in the context of an ecosystem-based approach to aquaculture is discussed. This work aims at adding to the ongoing discussion on sustainable bivalve aquaculture and is expected to help guide aquaculture management.     

Light-dependent induction of cFos during subjective day and night in PACAP-containing ganglion cells of the retinohypothalamic tract.

Environmental light stimulation via the retinohypothalamic tract (RHT) is necessary for stable entrainment of circadian rhythms generated in the suprachiasmatic nucleus (SCN). In the current report, the authors characterized the functional activity and phenotype of retinal ganglion cells that give rise to the RHT of the rat. Retinal ganglion cells that give rise to the RHT were identified by transsynaptic passage of an attenuated alpha herpesvirus known to have selective affinity for this pathway. Dual labeling immunocytochemistry demonstrated co-localization of viral antigen and pituitary adenylate cyclase activating polypeptide (PACAP) in retinal ganglion cells. This was confirmed using the anterograde tracer cholera toxin subunit B (ChB). In normal and retinally degenerated monosodium glutamate (MSG)-treated rats, ChB co-localized with PACAP in axons of the retinorecipient zone of the SCN. Light-induced Fos-immunoreactivity (Fos-IR) was apparent in all PACAP-containing retinal ganglion cells and a population of non-PACAP-containing retinal ganglion cells at dawn of normal and MSG-treated animals. Within the next 3 h, Fos disappeared in all non-PACAP-immunoreactive cells but persisted in all PACAP-containing retinal ganglion cells until dusk. When animals were exposed to constant light, Fos-IR was sustained only in the PACAP-immunoreactive (PACAP-IR) retinal ganglion cells. Darkness eliminated Fos-IR in all PACAP-IR retinal ganglion cells, demonstrating that the induction of Fos gene expression was light dependent. When animals were maintained in constant darkness and exposed to light pulses at ZT 14, ZT 19, or ZT 6, Fos-IR was induced in PACAP-IR retinal ganglion cells in a pattern similar to that seen at dawn. Collectively, these data indicate that PACAP is present in ganglion cells that give rise to the RHT and suggest a role for this peptide in the light entrainment of the clock.     

Archaeal diversity and community structure in a Swedish barley field: Specificity of the EK510R/(EURY498) 16S rDNA primer

The aim of this study was to analyze a total euryarchaeal community at DNA and RNA levels in a Swedish barley field with relation to soil depth (0-10 and 20-30 cm layers), soil fraction (bulk soil and rhizosphere) and time (August and November sample collection). Amplification of 16S rRNA gene using the archaeal universal A2F and Euryarchaea specific EK510R/(EURY498) primer pair, combined with denaturing gradient gel electrophoresis (DGGE), revealed distinct differences between rDNA and rRNA DGGE profiles. The soil depth, time, or rhizosphere effects did not significantly influence Archaeal community structure. Surprisingly, sequence analysis of DGGE-derived amplicons revealed the presence of Euryarchaea as well as uncultured soil Crenarchaea affiliated with group 1. In agreement, sequence comparison analyses showed that the majority of uncultured Crenarchaea group 1 had almost 100% sequence complementarity to the 3' end of the EK510R/(EURY498) primer. Therefore, we propose that EK510R/(EURY498R) is a universal archaeal primer rather than a Euryarchaea specific SSUrRNA primer. Hence, considerable care should be taken during application of this primer in studies of euryarchaeal biodiversity in soil environments.     

Afforested fields benefit nutrient‐demanding fungi

Impaired ecosystems are converted back to natural ecosystems or some other target stage by means of restoration and management. Due to their agricultural legacy, afforested fields might be valuable compensatory habitats for rare fungal species that require nutrient‐rich forest soils. Using a large‐scale field experiment in Finland, we studied community composition of macrofungi (agarics and boletes) on former fields, which had been afforested as monocultures 20 years ago using native spruce Picea abies, pine Pinus sylvestris, and birch Betula pendula. We studied the effect of soil quality, tree species, and site on community composition and structure. Many nutrient‐demanding as well as rare fungal species were recorded, particularly from pine and spruce plots. Pine plots supported more nutrient‐demanding fungi than birch plots. There was no relationship between soil pH, bulk density, P, N, or Ca, and species richness of nutrient‐demanding fungi. Fungal community composition was more similar within sites than among sites for all tree species. Among sites, spruce plots had the smallest fungal species turnover, and birch plots largest. Within sites, however, fungal species turnover from plot to plot was similar among tree species. Our results indicate that tree species has a relatively mild influence on species composition of fungi after 20 years of succession. Interestingly, the results show that afforested fields can be valuable complementary habitats for rare, red‐listed, and nutrient‐demanding fungal species. Field afforestation is a potential conservation tool that could be used to complement the poor representation of rare habitat types in highly fragmented protected area networks.     

Immunogenicity and immunosensitivity of ex vivo human carcinomas: interferon γ and tumour necrosis factor α treatment of tumour cells potentiates their interaction with autologous blood lymphocytes

Human carcinoma cells vary appreciably in the expression of MHC class I, class II, ICAM-1 (CD54) and B7 (CD80) molecules. Short-term in vitro exposure of ex vivo carcinoma cells to interferon and tumour necrosis factor elevated/induced the surface expression of MHC class I, class II and ICAM-1, but only rarely of B7. We found that cytokine treatment elevated the cytotoxic susceptibility and the stimulatory potential of ex vivo tumour cells. This was demonstrated (a) by the increased frequency and elevated level of auto-tumour lysis and (b) by induction of DNA synthesis and generation of cytotoxic lymphocytes in autologous mixed lymphocyte/tumour cell culture (MLTC). The MHC class I and ICAM-1 molecules on the tumour cells were required for interaction with the lymphocytes as indicated by the inhibitory effect of specific mAb both in the stimulation and in the cytotoxic tests. While the cytokine-induced increases in MHC and ICAM-1 on the low-expression tumours were probably important for the modification of functional interaction with the autologous lymphocytes, it is likely that alterations in other properties of tumour cells were also induced which contributed to the phenomenon. This was indicated by the results obtained with several tumours, which expressed indigenously high levels of these molecules but activated the autologous lymphocytes only after cytokine treatment. In several experiments the untreated targets that did not activate the lymphocytes were sensitive to the cytotoxicity of the effectors activated in MLTC. The results show that the afferent and efferent arms of the immune response have different requirements for functional interactions between lymphocytes and tumour cells.     

Genetic heterogeneity of the porphobilinogen deaminase gene in Swedish families with acute intermittent porphyria

Summary Acute intermittent porphyria (AIP) is an autosomal dominant metabolic disorder affecting the enzyme porphobilinogen (PBG) deaminase in the heme biosynthetic pathway. The highest prevalence of the disorder has been observed in Scandinavia, especially in northern Sweden (Lappland) where it occurs with a prevalence of 1 in 1500. Biochemical assays of the activity and concentration of PBG deaminase in red blood cells, haplotyping with 4 intragenic restriction fragment length polymorphisms (RFLPs) (MspI, PstI, BstNI, ApaLI) using the polymerase chain reaction (PCR) and screening for known base substitutions by oligonucleotide probes was performed in 28 Swedish AIP families. There was no close relationship between haplotype, biochemical findings (PBG deaminase activity, enzyme-linked immuno-sorbent assay [ELISA], and excess urinary excretion of delta-aminolevulinic acid or PBG), and a specific mutation. Three different haplotypes were identified. The haplotype 2/1/1/2 (MspI/PstI/BstNI/ApaLI; +/-/-/+) was found to be the most frequent among gene carriers (P < 0.001). The disease segregated with the haplotype 2/1/1/2 in the 10 families originating from northern Sweden. All 28 families were screened for three known point mutations. Only one was found to carry one of these mutations. Thus, the genetic background of AIP is heterogeneous in Sweden.