Characterization of Langmuir-Blodgett films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphat idylcholine by FTIR-ATR

Monomolecular films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidylc holine (PPDPC) were transferred from an air/water interface onto a germanium attenuated total reflection crystal by the Langmuir-Blodgett (LB) technique. The assemblies were thereafter investigated by Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. To determine the molecular organization in the deposited layers we monitored the CH2 and C = O stretching and the CH2 bending regions of the infrared spectra of these lipids in detail. Using Fourier self-deconvolution technique, the carbonyl stretching mode was resolved into two models corresponding to the conformational differences in the ester linkages of the phospholipid sn-1 and sn-2 acyl chains. By varying the temperature of the subphase and using different surface pressures, we were able to transfer different conformational states of DPPC onto a germanium ATR crystal. Deposition of DPPC at 40 mN m-1 and at 15 degrees C or at 20 mN m-1 and at 35 degrees C results in LB-assemblies in ordered or disordered states, respectively, as judged by the IR spectra. These structures in LB films correspond to the state of DPPC in liposomes below and above the temperature of the order-disorder phase transition. Irrespective of the surface pressure and subphase temperature used during the deposition, an ordering process was found in DPPC films when the number of the transferred layers was increased from one to five. The pyrene-labelled phosphatidylcholine analogue, PPDPC, behaved differently from DPPC. In the case where one to three layers of PPDPC transferred at 35 mN m-1 and at 20 degrees C only conformational structures resembling those in fully hydrated liposomes above the main transition temperature were observed.     

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.     

Hypoxia-inducible factor and vascular endothelial growth factor in the neuroretina and retinal blood vessels after retinal ischemia

Retinal ischemia arises from circulatory failure. As the retinal blood vessels are key organs in circulatory failure, our aim was to study the retinal vasculature separately from the neuroretina to elucidate the role of hypoxia-inducible factor (HIF) 1α and 1β and vascular endothelial growth factor (VEGF) in retinal ischemia. Retinal ischemia was induced in porcine eyes by applying an intraocular pressure, followed by 12 h of reperfusion. HIF-1α mRNA expression was not affected by ischemia, while immunofluorescence staining was higher after ischemia in the neuroretina. HIF-1β immunoreactivity and mRNA expression were unaffected. VEGF protein levels in the vitreous humor and VEGF staining in the neuroretina were more pronounced in eyes subjected to ischemia than in the sham eyes. VEGF may be activated downstream of HIF-1 and is known to stimulate retinal neovascularization, which causes sight-threatening complications. These results emphasize the need for pharmacological treatment to block the HIF and VEGF signaling pathways in retinal ischemia.     

A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells

Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze–thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10⁹ ± 4.74 × 10⁹ cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5–66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.     

Surface-assisted delivery of fluorescent groups to hGST A1-1 and a lysine mutant

Human glutathione transferase (hGST) A1-1 and a lysine mutant (A216K) can both be rapidly and site-specifically acylated on Y9 and K216, respectively, using a range of thiolesters of glutathione (GS-thiolesters) as modifying reagents. The present investigation was aimed at developing a method with which to deliver a fluorescent acyl group from a solid support under conditions compatible with standard protein purification schemes. A number of fluorescent GS-thiolesters with modified peptide backbones were therefore prepared and tested for reactivity toward hGST A1-1 and the A216K mutant. Substitutions at the alpha-NH2 part of the glutathione backbone were not tolerated by the proteins. However, two fluorescent reagents that carry a biotin moiety at the C-terminal part of glutathione were found through MALDI-MS experiments to react in solution with Y9 of the wild-type protein and one reagent with K216 of A216K. The reaction can take place in the presence of glutathione and even in a crude E. coli lysate of cells expressing A216K. Delivery of the fluorescent group to Y9 or K216 was possible using NeutrAvidin (NA) beads that had been preincubated with biotinylated reagent. Alternatively, excess reagent can be removed by a brief incubation with NA beads. We have thus now developed a system for protein labeling with easy removal of excess and used up low-molecular weight reagent. This strategy can conceivably be utilized in future protein purification and labeling experiments.     

Identification of a novel idiopathic epilepsy locus in Belgian Shepherd dogs

Epilepsy is the most common neurological disorder in dogs, with an incidence ranging from 0.5% to up to 20% in particular breeds. Canine epilepsy can be etiologically defined as idiopathic or symptomatic. Epileptic seizures may be classified as focal with or without secondary generalization, or as primary generalized. Nine genes have been identified for symptomatic (storage diseases) and one for idiopathic epilepsy in different breeds. However, the genetic background of common canine epilepsies remains unknown. We have studied the clinical and genetic background of epilepsy in Belgian Shepherds. We collected 159 cases and 148 controls and confirmed the presence of epilepsy through epilepsy questionnaires and clinical examinations. The MRI was normal while interictal EEG revealed abnormalities and variable foci in the clinically examined affected dogs. A genome-wide association study using Affymetrix 50K SNP arrays in 40 cases and 44 controls mapped the epilepsy locus on CFA37, which was replicated in an independent cohort (81 cases and 88 controls; combined p = 9.70×10⁻¹⁰, OR = 3.3). Fine mapping study defined a ∼1 Mb region including 12 genes of which none are known epilepsy genes or encode ion channels. Exonic sequencing was performed for two candidate genes, KLF7 and ADAM23. No variation was found in KLF7 but a highly-associated non-synonymous variant, G1203A (R387H) was present in the ADAM23 gene (p = 3.7×10⁻⁸, OR = 3.9 for homozygosity). Homozygosity for a two-SNP haplotype within the ADAM23 gene conferred the highest risk for epilepsy (p = 6.28×10⁻¹¹, OR = 7.4). ADAM23 interacts with known epilepsy proteins LGI1 and LGI2. However, our data suggests that the ADAM23 variant is a polymorphism and we have initiated a targeted re-sequencing study across the locus to identify the causative mutation. It would establish the affected breed as a novel therapeutic model, help to develop a DNA test for breeding purposes and introduce a novel candidate gene for human idiopathic epilepsies.     

Electrophilic Affibodies Forming Covalent Bonds to Protein Targets

Antibody affinity limits sensitivity of detection in many areas of biology and medicine. High affinity usually depends on achieving the optimal combination of the natural 20 amino acids in the antibody binding site. Here, we investigate the effect on recognition of protein targets of placing an unnatural electrophile adjacent to the target binding site. We positioned a weak electrophile, acrylamide, near the binding site between an affibody, a non-immunoglobulin binding scaffold, and its protein target. The proximity between cysteine, lysine, or histidine on the target protein drove covalent bond formation to the electrophile on the affibody. Covalent bonds did not form to a non-interacting point mutant of the target, and there was minimal cross-reactivity with serum, cell lysate, or when imaging at the cell surface. Electrophilic affibodies showed more stable protein imaging at the surface of mammalian cells, and the sensitivity of protein detection in an immunoassay improved by two orders of magnitude. Thus electrophilic affibodies combined good specificity with improved detection of protein targets.     

PYY(3-36) reduces food intake and body weight and improves insulin sensitivity in rodent models of diet-induced obesity

The gut hormone peptide YY (PYY) was recently proposed to comprise an endogenous satiety factor. We have studied acute anorectic functions of PYY(3-36) in mice and rats, as well as metabolic effects of chronic PYY(3-36) administration to diet-induced obese (DIO) mice and rats. A single intraperitoneal injection of PYY(3-36) inhibited food intake in mice, but not in rats. We next investigated the effects of increasing doses (100, 300, and 1,000 microg.kg-1.day-1) of PYY(3-36) administered subcutaneously via osmotic minipumps on food intake and body weight in DIO C57BL/6J mice. Whereas only the highest dose (1,000 microg.kg-1.day-1) of PYY(3-36) significantly reduced food intake over the first 3 days, body weight gain was dose dependently reduced, and on day 28 the group treated with 1,000 microg.kg-1.day-1 PYY(3-36) weighed approximately 10% less than the vehicle-treated group. Mesenteric, epididymal, retroperitoneal, and inguinal fat pad weight was dose dependently reduced. Subcutaneous administration of PYY(3-36) (250 and 1,000 microg.kg-1.day-1) for 28 days reduced body weight and improved glycemic control in glucose-intolerant DIO rats. Neither 250 nor 1,000 microg/kg PYY(3-36) elicited a conditioned taste aversion in male rats.     

Cost-effective HLA typing with tagging SNPs predicts celiac disease risk haplotypes in the Finnish, Hungarian, and Italian populations

Human leukocyte antigen (HLA) genes, located on chromosome 6p21.3, have a crucial role in susceptibility to various autoimmune and inflammatory diseases, such as celiac disease and type 1 diabetes. Certain HLA heterodimers, namely DQ2 (encoded by the DQA1*05 and DQB1*02 alleles) and DQ8 (DQA1*03 and DQB1*0302), are necessary for the development of celiac disease. Traditional genotyping of HLA genes is laborious, time-consuming, and expensive. A novel HLA-genotyping method, using six HLA-tagging single-nucleotide polymorphisms (SNPs) and suitable for high-throughput approaches, was described recently. Our aim was to validate this method in the Finnish, Hungarian, and Italian populations. The six previously reported HLA-tagging SNPs were genotyped in patients with celiac disease and in healthy individuals from Finland, Hungary, and two distinct regions of Italy. The potential of this method was evaluated in analyzing how well the tag SNP results correlate with the HLA genotypes previously determined using traditional HLA-typing methods. Using the tagging SNP method, it is possible to determine the celiac disease risk haplotypes accurately in Finnish, Hungarian, and Italian populations, with specificity and sensitivity ranging from 95% to 100%. In addition, it predicts homozygosity and heterozygosity for a risk haplotype, allowing studies on genotypic risk effects. The method is transferable between populations and therefore suited for large-scale research studies and screening of celiac disease among high-risk individuals or at the population level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lotta Koskinen and Jihane Romanos are authors with equal contribution.