Fibroblast Activation Protein Targeted Photodynamic Therapy Selectively Kills Activated Skin Fibroblasts from Systemic Sclerosis Patients and Prevents Tissue Contraction

Systemic sclerosis (SSc) is a rare, severe, auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of this fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. In this study, we explored the use of FAP-tPDT in primary skin fibroblasts from SSc patients, both in 2D and 3D cultures. Method: The FAP targeting antibody 28H1 was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous outgrowth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer construct, washed after 4 h and exposed to λ = 689 nm light. Cell viability was measured using CellTiter Glo^®®. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Results: FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with an irrelevant antibody-photosensitizer construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. Conclusions: Here we have shown, for the first time, the potential of FAP-tPDT for the treatment of fibrosis in SSc skin.     

Cisplatin-Resistant CD44+ Lung Cancer Cells Are Sensitive to Auger Electrons

Cancer stem cells (CSCs) are resistant to conventional therapy and present a major clinical challenge since they are responsible for the relapse of many cancers, including non-small cell lung cancer (NSCLC). Hence, future successful therapy should also eradicate CSCs. Auger electrons have demonstrated promising therapeutic potential and can induce DNA damage while sparing surrounding cells. Here, we sort primary patient-derived NSCLC cells based on their expression of the CSC-marker CD44 and investigate the effects of cisplatin and a thymidine analog (deoxyuridine) labeled with an Auger electron emitter (¹²⁵I). We show that the CD44⁺ populations are more resistant to cisplatin than the CD44⁻ populations. Interestingly, incubation with the thymidine analog 5-[¹²⁵I]iodo-2′-deoxyuridine ([¹²⁵I]I-UdR) induces equal DNA damage, G2/M cell cycle arrest, and apoptosis in the CD44⁻ and CD44⁺ populations. Our results suggest that Auger electron emitters can also eradicate resistant lung cancer CD44⁺ populations.     

Information and computing technology and the gap between school pedagogy and children's culture

This article focuses on the gap between the use of Information and Computing Technology (ICT) in schools and the use made of ICT in children's everyday life outside school. Particular emphasis is placed on the communities of practice that young people create through their use of digital technology. The article is based on data collected over five years in Denmark. Overall, the article raises questions about the future of teaching and learning both in school and out of school.     

Reliability-based calibration of load duration factors for timber structures

The load bearing capacity of timber structures decrease with time depending on the type of load and timber. Based on representative limit states and stochastic models for timber structures, load duration factors are calibrated using probabilistic methods. Load duration effects are estimated on basis of simulation of realizations of wind, snow and imposed loads in accordance with the load models in the Danish structural codes. Three damage accumulation models are considered, namely Gerhards model, Barrett and Foschi’s model and Foschi and Yao’s model. The parameters in these models are fitted by the Maximum Likelihood Method using data relevant for Danish structural timber and the statistical uncertainty is quantified. The reliability is evaluated using representative short- and long-term limit states, and the load duration factor k_mod is estimated using the probabilistic model such that equivalent reliability levels are obtained using short- and long-term design equations. Time variant reliability aspect is considered using a simple, representative limit state with time variant strength and simulation of the whole life time load processes.     

Analysis of histidine phosphorylation using tandem MS and ion-electron reactions

Phosphorylation of proteins is essential in intracellular signal transduction pathways in eukaryotic and prokaryotic cells. Histidine phosphorylation plays an important role in two-component signal transduction in bacteria. In this study, we describe the characterization of a synthetic histidine-phosphorylated peptide with four different mass spectrometric (MS) fragmentation techniques: Collision-induced dissociation (CID), electron capture dissociation, electron-transfer dissociation, and electron detachment dissociation. Furthermore, LC-MS methods were developed to detect histidine-phosphorylated peptides, which are acid-labile, in more complex samples. From these results, we concluded that nonacidic solvent systems or fast LC methods provide the best conditions for separation of histidine-phosphorylated peptides prior to electrospray ionization mass spectrometry analysis. Electron-based fragmentation methods should be used for determination of histidine phosphorylation sites, since CID results in very facile phosphate-related neutral losses. The developed LC-MS/MS methods were successfully applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ, which was in vitro autophosphorylated. Finally, a new method is described for nonretentive solid-phase extraction of histidine-phosphorylated peptides using polymeric Strata-X microcolumns.     

Anaerobic digestion of the organic fraction of municipal solid waste: influence of co-digestion with manure

Anaerobic digestion of the organic fraction of municipal solid waste (OFMSW) was investigated in two thermophilic (55 degrees C) wet digestion treatment systems R1 and R2. Initially OFMSW was co-digested with manure with a successively higher concentration of OFMSW, at a hydraulic retention time (HRT) of 14-18 d and an organic loading rate (OLR) of 3.3-4.0 g-VS/l/d. Adaptation of the co-digestion process to a OFMSW:manure ratio of 50% (VS/VS) was established over a period of 6 weeks. This co-digestion ratio was maintained in reactor R2 while the ratio of OFMSW to manure was slowly increased to 100% in reactor R1 over a period of 8 weeks. Use of recirculated process liquid to adjust the organic loading to R1 was found to have a beneficial stabilization effect. The pH rose to a value of 8 and the reactor showed stable performance with high biogas yield and low VFA levels. The biogas yield from source-sorted OFMSW was 0.63-0.71 l/g-VS both in the co-digestion configuration and in the treatment of 100% OFMSW with process liquid recirculation. This yield is corresponding to 180-220 m3 biogas per ton OFMSW. VS reduction of 69-74% was achieved when treating 100% OFMSW. None of the processes showed signs of inhibition at the free ammonia concentration of 0.45-0.62 g-N/l.     

Novel 3‐Carboxy‐ and 3‐Phosphonopyrazoline Amino Acids as Potent and Selective NMDA Receptor Antagonists: Design,Synthesis, and Pharmacological Characterization

The design and synthesis of new N1‐substituted 3‐carboxy‐ and 3‐phosphonopyrazoline and pyrazole amino acids that target the glutamate binding site of NMDA receptors are described. An analysis of the stereochemical requirements for high‐affinity interaction with these receptors was performed. We identified two highly potent and selective competitive NMDA receptor antagonists, (5S,αR)‐ 1 and (5S,αR)‐ 4 , which exhibit good in vitro neuroprotective activity and in vivo anticonvulsant activity by i.p. administration, suggesting that these molecules may have potential use as therapeutic agents.     

Rhizomucor miehei triglyceride lipase is synthesized as a precursor

ARhizomucor miehie cDNA library constructed inEscherichia coli was screened with synthetic oligonucleotides designed from knowledge of a partial amino acid sequence of the secreted triglyceride lipase (triacyl-glycerol acylhydrolase EC 3.1.1.3) from this fungus. Lipase-specific recombinants were isolated and their insert sequenced. Unlike characterized bacterial and mammalian triglyceride lipases, the fungal enzyme is synthesized as a precursor, including a 70 amino acid residue propeptide between the 24 amino acid residues of the signal peptide and the 269 residues of the mature enzyme. The precursor processing mechanism, which involves cleavage between a methionine and a serine residue, is unknown. By sequence comparison with other lipases, a serine residue involved in substrate binding was identified in the fungal lipase. The sequence around this residue is well-conserved among characterized lipases. Conservation of an intron in an isolated cDNA recombinant and immunoprecipitation of in vitro synthesizedR. miehei translation products indicates that the expression of the lipase gene might involve inefficient mRNA splicing.     

Rhizomucor miehei triglyceride lipase is processed and secreted from transformedAspergillus oryzae

The cDNA encoding the precursor of theRhizomucor miehei triglyceride lipase was inserted in anAspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of theAspergillus oryzae α-amylase gene promoter and theAspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced intoAspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted fromA. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the nativeRhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted fromAspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.     

The performance of the Danish simulation model DAISY in prediction of Nmin at spring

In Denmark the Danish Agricultural Advisory Centre has for some years used the soil content of mineral nitrogen in spring in fertilizer recommendations. Since 1986 these recommendations have been based on soil samples carried out at all intersections of a nationwide 7 km square grid in Denmark. It was hoped that it may be possible to replace soil measurements with values of soil mineral-N calculated with a model. The Danish simulation model DAISY, which among other things simulates changes in the inorganic N content of the soil, was evaluated with respect to the N_min content in the early spring under bare soil and under winter cereal. For both situations the precrop was cereals. The performance of the model was evaluated in farming systems receiving mineral fertilizer and in some instances organic manures. The results were analysed according to type of subsoil: sandy or loamy. Predictions were 11 kg N ha^–1 less than the measured values as a mean and the differences between simulated and measured values were high for fields receiving organic manures. Predictions were less than ± 10 kg N ha^–1 of measured values in 25–58% of cases for the different types of crop cover at the time of soil sampling, type of subsoils and fertilizer strategies, respectively. Predictions were less than ± 20 kg N ha^–1 of measured values in 48–89% of cases for the different situations. The best predictions were obtained for sandy subsoils covered by winter cereal and supplied with mineral fertilizer only. It is concluded that the quality of the data used as input in the model has to be increased and that further developments of parts of the DAISY model are needed before modelling can be a useful tool in N fertilizer recommendations.