Effect of Technically Relevant X-Ray Doses on the Structure and Function of Alcohol Dehydrogenase and Hen Egg-White Lysozyme

Purpose

The effect of different irradiation doses on the structure and activity of lyophilized powders of Hen Egg-White Lysozyme (HEWL) and alcohol dehydrogenase (ADH) was investigated using these substances as models for robust and sensitive proteins, respectively. Three doses were selected to cover the ranges of radio-sterilization (25kGy), treatment of blood products (25Gy) and annual background radiation dose (approximately 2mGy). The results offer an initial screening of different irradiation doses and support the development of X-ray imaging methods as non-destructive process analytical technology (PAT) tools for detecting the visible particulate matters in such products.

Methods

HEWL and ADH were exposed to X-rays in the solid state. The effect of irradiation was determined directly after irradiation and after storage. Structural changes and degradation were investigated using SAXS, SDS-PAGE and HPLC-MS. Protein functionality was assessed via activity assays.

Results

Lower irradiation doses of 25Gy and 2mGy had no significant impact on the structure and enzyme activity. The dose of 25kGy caused a significant decrease in the enzyme activity and structural changes immediately after irradiation of ADH and after storage of irradiated HEWL at ?20°C.

Conclusion

The results emphasize the importance of careful selection of radiation doses for development of X-ray imaging methods as PAT tools inspection of solid biopharmaceutical products.

     

Association of Environmental Factors with Seasonal Intensity of Erysipelothrix rhusiopathiae Seropositivity among Arctic Caribou

Several caribou (Rangifer tarandus) populations have been declining concurrently with increases in infectious diseases in the Arctic. Erysipelothrix rhusiopathiae, a zoonotic bacterium, was first described in 2015 as a notable cause of illness and death among several Arctic wildlife species. We investigated epidemiologic and environmental factors associated with the seroprevalence of E. rhusiopathiae in the Arctic and found that seropositivity was highest during warmer months, peaking in September, and was highest among adult males. Summer seroprevalence increases tracked with the oestrid index from the previous year, icing and snowing events, and precipitation from the same year but decreased with growing degree days in the same year. Seroprevalence of E. rhusiopathiae varied more during the later years of the study. Our findings provide key insights into the influence of environmental factors on disease prevalence that can be instrumental for anticipating and mitigating diseases associated with climate change among Arctic wildlife and human populations.     

Amyloid beta 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization

Following ectodomain shedding by β-secretase, successive proteolytic cleavages within the transmembrane sequence (TMS) of the amyloid precursor protein (APP) catalyzed by γ-secretase result in the release of amyloid-β (Aβ) peptides of variable length. Aβ peptides with 42 amino acids appear to be the key pathogenic species in Alzheimer’s disease, as they are believed to initiate neuronal degeneration. Sulindac sulfide, which is known as a potent γ-secretase modulator (GSM), selectively reduces Aβ42 production in favor of shorter Aβ species, such as Aβ38. By studying APP–TMS dimerization we previously showed that an attenuated interaction similarly decreased Aβ42 levels and concomitantly increased Aβ38 levels. However, the precise molecular mechanism by which GSMs modulate Aβ production is still unclear. In this study, using a reporter gene-based dimerization assay, we found that APP–TMS dimers are destabilized by sulindac sulfide and related Aβ42-lowering compounds in a concentration-dependent manner. By surface plasmon resonance analysis and NMR spectroscopy, we show that sulindac sulfide and novel sulindac-derived compounds directly bind to the Aβ sequence. Strikingly, the attenuated APP–TMS interaction by GSMs correlated strongly with Aβ42-lowering activity and binding strength to the Aβ sequence. Molecular docking analyses suggest that certain GSMs bind to the GxxxG dimerization motif in the APP–TMS. We conclude that these GSMs decrease Aβ42 levels by modulating APP–TMS interactions. This effect specifically emphasizes the importance of the dimeric APP–TMS as a promising drug target in Alzheimer’s disease.Amyloid-β (Aβ) peptides are produced by a sequential cleavage process involving β- and γ-secretases (1). The most prevalent Aβ species generated during amyloid precursor protein (APP) processing are the intermediate products Aβ40 and Aβ42 when Aβ48 and Aβ49 are converted into smaller Aβ fragments by a successive release of tri- and tetrapeptides upon stepwise γ-secretase cleavages (2). Indeed, γ-secretase generates a range of Aβ peptides with variable C termini and the length of Aβ peptides is critical for the pathogenesis of Alzheimer’s disease because the toxic 42-residue isoform causes neurodegeneration that underlies the decline of cognitive functions (3).Previous attempts to understand γ-secretase cleavage specificity have unraveled that processing of the APP C-terminal fragments (β-CTF) is influenced by the GxxxG dimerization motif of the APP transmembrane sequence (TMS) (4, 5). β-CTF is the only known γ-secretase substrate comprising a GxxxG motif in triplicate, thus making dimerization of the APP–TMS unique (6). Mutational analysis revealed that a disruption of the dimer interface by glycine to alanine mutations at positions 29 and 33 of the Aβ sequence gradually attenuated the TMS-dimerization strength and diminished the formation of Aβ42 in favor of Aβ38, whereas Aβ40 levels remained largely unaffected (5). In addition, a subset of nonsteroidal antiinflammatory drugs (NSAIDs), including indomethacin and sulindac sulfide, effectively modulate the production of Aβ peptides in vitro and in vivo (7). Such compounds, renamed γ-secretase modulators (GSMs), either selectively reduced Aβ42 production with a concomitant increase in Aβ38 levels or vice versa (8).The fact that GSMs were found to be active even in cell-free γ-secretase assays raised the possibility that these compounds might alter the γ-site cleavage by directly modifying the conformation of the γ-secretase complex. In particular, presenilin-1 (PS1) was suggested as a candidate molecule to be affected by an allosteric mechanism (9, 10). Other reports assume that GSMs can interact with cellular membranes and alter biophysical properties of the lipid bilayer (11–13). Evidence was also provided that GSMs target the enzyme’s substrate when GSM photoprobes labeled APP (14). However, recent NMR results have questioned specific APP binding sites of GSMs (15). Thus, the precise molecular mechanism by which GSMs modulate Aβ formation is still unclear and the targets will have to be characterized.In this study, we asked whether a subgroup of GSMs might affect Aβ42 generation by binding to the Aβ sequence and if these GSMs can attenuate APP–TMS dimerization. We show that sulindac sulfide and derived compounds directly bind to Aβ42 peptides. Molecular docking experiments suggest that the groove composed of alternating GxxxG glycine residues forms an ideal contact site in an α-helical APP–TMS conformation. In addition, we have found that APP–TMS dimers are destabilized by sulindac sulfide and related Aβ42-lowering compounds in a concentration-dependent manner, whereas sulindac and its sulfone derivative that lack Aβ42-lowering activity neither bind to the Aβ sequence nor reduce APP–TMS-dimerization strength. Our data strongly indicate that certain GSMs may act through an inhibition of helix–helix interactions of membrane-spanning Aβ segments.     

Bone marrow edema syndrome of the femoral head

Summary BACKGROUND: The purpose of this study was to assess the efficacy of the vasoactive drug iloprost in bone marrow edema syndrome (BMES) and to compare it to the results of a control group treated by core decompression. PATIENTS AND METHODS: 38 hips (36 patients) with BMES in the femoral head were investigated. In group A, 18 hips (17 patients; mean age 49 years) were treated with iloprost, a vasoactive drug that dilates arterioles and venules, reduces capillary permeability and suppresses platelet aggregation. The therapy comprised a series of five infusions with 20 g iloprost over 6 hours on 5 consecutive days. Weight bearing was reduced for up to 3 weeks, depending on the severity of symptoms. In group B, 20 hips (19 patients; mean age, 41 years) underwent surgical core decompression of the femoral head followed by 6 weeks of partial weight bearing. Both groups were evaluated clinically, radiographically and by MRI. RESULTS: In group A, one patient had to discontinue therapy on the first day because of severe headache. In the remaining patients the Harris Hip Score (HHS) improved from a mean of 64.7 points (range, 44–89) before therapy to 97.0 points (83–100) after 3 months. MRI controls showed complete remission in all hips. In group B, the preoperative HHS improved from 53.7 points (31–82) to 95.1 points (39–100) after 3 months. MRI controls showed complete remission of BMES in 14 hips, residual focal bone marrow edema in four hips and a small osteonecrotic area in two hips. In both groups the high level of clinical recovery was maintained at the last examination after a mean follow up of 11 months in group A and 12 months in group B. CONCLUSION: The parenteral application of iloprost can achieve equal or better results in the treatment of bone marrow edema syndrome of the hip compared to core decompression.

---

Knochenmarködemsyndrom des HüftkopfesBehandlung mit dem Prostazyklinanalogon Iloprost im Vergleich zur Hüftkopfbohrung: Eine MRT-kontrollierte Studie

Zusammenfassung HINTERGRUND: Ziel der Studie war, die Wirkung des Prostazyklinanalogons Iloprost auf das Knochenmarködemsyndrom zu untersuchen und mit einer durch Hüftkopfbohrung behandelten Kontrollgruppe zu vergleichen. PATIENTEN UND METHODIK: 38 Hüften (36 Patienten) mit Knochenmarködem wurden untersucht. In Gruppe A wurden 18 Hüften (17 Patienten) mit Iloprost, einem vasoaktivem Wirkstoff, der sowohl auf verschiedene Teile der Gefäßendstrombahn, als auch auf die Thrombozytenaggregation wirkt, behandelt. Den Patienten wurde eine Serie von 5 Infusionen mit 20 g Iloprost über 6 Stunden an 5 aufeinander folgenden Tagen verabreicht. In Gruppe B wurden 20 Hüften (19 Patienten) durch Hüftkopfbohrung, gefolgt von 6 Wochen Teilbelastung behandelt. Beide Gruppen wurden klinisch, nativradiologisch und durch MRT bewertet. ERGEBNISSE: In Gruppe A musste ein Patient die Behandlung aufgrund starker Kopfschmerzen abbrechen. Bei den übrigen Patienten steigerte sich der Harris Hip Score (HHS) von durchschnittlich 64,7 Punkten (44 bis 89) vor der Therapie auf 97,0 Punkte (83 bis 100) nach 3 Monaten. Die MRT-Kontrolle zeigte eine vollständige Remission bei allen Hüften. In Gruppe B konnte der HHS von präoperativ 53,7 Punkten (31 bis 82) auf 95,1 Punkte (39 bis 100) gesteigert werden. Die MRT-Kontrolle zeigte eine vollständige Remission bei 14 Hüften, ein Restödem bei 4 Hüften, sowie ein kleines osteonekrotisches Areal bei 2 Hüften. In beiden Gruppen konnten die guten klinischen Ergebnisse bei der letzten Untersuchung nach durchschnittlich 11 Monaten in Gruppe A und 12 Monaten in Gruppe B bestätigt werden. SCHLUSSFOLGERUNG: Durch die parenterale Verabreichung von Iloprost können beim Knochenmarködemsyndrom der Hüfte gleich gute Ergebnisse erzielt werden, wie durch Hüftkopfbohrung.

     

Pharmacological management of aseptic osteonecrosis in children

Aseptic osteonecrosis (AON) in children can progress during ossification of cartilage in periods of increased growth or excessive physical stain and may occur in various locations in the skeleton. Disturbance of blood supply to the bone has been suggested as the main pathological mechanism involved in AON, which is characterised by the death of bone marrow and trabecular bone. The extent and development of osteonecrosis and the duration of disease until restorative healing, depend on the formation of new blood vessels, the spreading of vessels in the affected bony areas, the absorption of osteonecrotic tissue and the structure of new bone. Conservative and operative treatment options for AON vary according to the location and development of the disease and the age of the patient. The goal of all treatment options currently used today is to achieve relief of physical load in the affected bone and to promote and regulate blood supply. Treatment should be started early in order to minimise the extent of osteonecrosis and allow restorative healing. As the processes of myelopoiesis, myelophthisis and fracture healing become more clear, interest has focused on advances in the utilisation of bioactive factors to salvage bone in children affected by AON. Such methods include the use of osteoinductive agents, such as cytokines and bone morphogenetic proteins, as well as factors that stimulate angiogenesis and regulate blood supply. Currently, the prostacyclin analogue, iloprost (Ilomedin, Schering AG), has been successfully used in a pilot study in children suffering from early stages of AON.     

Bone marrow edema of the forefoot after chevron osteotomy--a rare cause of metatarsalgia: a case report

Treatment options of bone marrow edema syndrome, which is associated with vascular disturbances, are protracted nonoperative treatment or core decompression which still demands several weeks until complete recovery. We obtained excellent results by the use of the vasoactive drug iloprost, a stable prostacyclin analogue, leading to a complete relief of symptoms in cases of bone marrow edema which had initially suggested early avascular necrosis of the second metatarsal head. The bone marrow edema of the second metatarsal bone was thought to be due to altered biomechanics following a distal first metatarsal chevron osteotomy. During the five days of iloprost infusion, the patient reported relief of rest pain. After therapy, the pedobarogram was normalized. The AOFAS forefoot score improved from 44 to 85 points after one month, and to 95 points after three months. At that time, the marrow showed normal signals. Without additional intervention the patient was able to resume normal activities.     

Iloprost for the treatment of bone marrow edema in the hindfoot

The parenteral application of the vasoactive drug, iloprost, might be a viable option for the treatment of BMES of different origins, especially ischemic ones. In edema that is secondary to osteoarthrosis or stress, the effect of therapy with iloprost depends on the grade of the basic disease. The natural course of the disease, as well as the normalization of the signal pattern of the MRI, seem to be accelerated.     

Reflex sympathetic dystrophy in children

This report presents the case of an 8-year-old boy who underwent a second clubfoot operation following early-stage reflex sympathetic dystrophy (RSD). After other conditions had been ruled out, the patient was submitted to physiotherapy supported by antiphlogistic and analgesic drugs as well as a partial immobilisation of the affected extremity. He remained asymptomatic during the following 4 weeks. RSD in children is not a well-recognised entity. This case of early-stage RSD illustrates the need to be aware of this possible complications after operation in the differential diagnosis of local pain and swelling of a limb. Received: 13 January 1998