Development and application of cytotoxic T lymphocyte-associated antigen 4 as a protein scaffold for the generation of novel binding ligands

AbstractWe have explored the possibilities of using human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) as a single immunoglobulin fold-based scaffold for the generation of novel binding ligands. To obtain a suitable protein library selection system, the extracellular domain of CTLA-4 was first displayed on the surface of a filamentous phage as a fusion product of the phage coat protein p3. CTLA-4 was shown to be functionally intact by binding to its natural ligands B7-1 (CD80) and B7-2 (CD86) both in vitro and in situ. Secondly, the complementarity determining region 3 (CDR3) loop of the CTLA-4 extracellular domain was evaluated as a permissive site. We replaced the nine amino acid CDR3-like loop of CTLA-4 with the sequence XXX-RGD-XXX (where X represents any amino acid). Using phage display we selected several CTLA-4-based variants capable of binding to human αvβ3 integrin, one of which showed binding to integrins in situ. To explore the construction of bispecific molecules we also evaluated one other potential permissive site diametrically opposite the natural CDR-like loops, which was found to be tolerant of peptide insertion. Our data suggest that CTLA-4 is a suitable human scaffold for engineering single-domain molecules with one or possibly more binding specificities

Reconstructing Asian faunal introductions to eastern Africa from multi-proxy biomolecular and archaeological datasets

Human-mediated biological exchange has had global social and ecological impacts. In subS-aharan Africa, several domestic and commensal animals were introduced from Asia in the pre-modern period; however, the timing and nature of these introductions remain contentious. One model supports introduction to the eastern African coast after the mid-first millennium CE, while another posits introduction dating back to 3000 BCE. These distinct scenarios have implications for understanding the emergence of long-distance maritime connectivity, and the ecological and economic impacts of introduced species. Resolution of this longstanding debate requires new efforts, given the lack of well-dated fauna from high-precision excavations, and ambiguous osteomorphological identifications. We analysed faunal remains from 22 eastern African sites spanning a wide geographic and chronological range, and applied biomolecular techniques to confirm identifications of two Asian taxa: domestic chicken (Gallus gallus) and black rat (Rattus rattus). Our approach included ancient DNA (aDNA) analysis aided by BLAST-based bioinformatics, Zooarchaeology by Mass Spectrometry (ZooMS) collagen fingerprinting, and direct AMS (accelerator mass spectrometry) radiocarbon dating. Our results support a late, mid-first millennium CE introduction of these species. We discuss the implications of our findings for models of biological exchange, and emphasize the applicability of our approach to tropical areas with poor bone preservation

Palaeogenomic analysis of black rat (Rattus rattus) reveals multiple European introductions associated with human economic history

The distribution of the black rat (Rattus rattus) has been heavily influenced by its association with humans. The dispersal history of this non-native commensal rodent across Europe, however, remains poorly understood, and different introductions may have occurred during the Roman and medieval periods. Here, in order to reconstruct the population history of European black rats, we first generate a de novo genome assembly of the black rat. We then sequence 67 ancient and three modern black rat mitogenomes, and 36 ancient and three modern nuclear genomes from archaeological sites spanning the 1st-17th centuries CE in Europe and North Africa. Analyses of our newly reported sequences, together with published mitochondrial DNA sequences, confirm that black rats were introduced into the Mediterranean and Europe from Southwest Asia. Genomic analyses of the ancient rats reveal a population turnover in temperate Europe between the 6th and 10th centuries CE, coincident with an archaeologically attested decline in the black rat population. The near disappearance and re-emergence of black rats in Europe may have been the result of the breakdown of the Roman Empire, the First Plague Pandemic, and/or post-Roman climatic cooling.Peer reviewe

Qualification of an ultrasonic instrument for real-time monitoring of size and concentration of nanoparticles during liquid phase bottom-up synthesis

Both in design and production of nanoparticles and nanocomposites it is of vital importance to have information about their size and concentration. During the formation of nanoparticles, real-time monitoring of particle size and concentration during bottom-up synthesis in liquids allows for a detailed study of nucleation and growth. This provides valuable insights into the formation of nanoparticles that can be used for process optimization and scale up. In the production of nanoparticles, real-time monitoring enables intervention to minimize the number of off-spec batches. In this paper we will qualify an ultrasound nanoparticle sizer (UNPS) as a real-time monitor for the growth of nanoparticles (or sub-micro particles) in the 100 nm-1 μm range. Nanoparticles affect the speed and attenuation of ultrasonic waves in the dispersion. The size of the change depends, amongst other things, on the size and concentration of the nanoparticles. This dependency is used in the UNPS method. The qualification of the UNPS was undertaken in two successful experiments. The first experiment consisted of static measurements on commercially available silica particles, and the second experiment was real-time monitoring of the size and concentration during the growth of silica nanoparticles in Stöber synthesis in a water-alcohol mixture starting from the molecular precursor tetraethyl orthosilicate. The results of the UNPS were verified by measurements of a dynamic light scattering device and a transmission electron microscope

Qualification of an ultrasonic instrument for real-time monitoring of size and concentration of nanoparticles during liquid phase bottom-up synthesis

\u3cp\u3eBoth in design and production of nanoparticles and nanocomposites it is of vital importance to have information about their size and concentration. During the formation of nanoparticles, real-time monitoring of particle size and concentration during bottom-up synthesis in liquids allows for a detailed study of nucleation and growth. This provides valuable insights into the formation of nanoparticles that can be used for process optimization and scale up. In the production of nanoparticles, real-time monitoring enables intervention to minimize the number of off-spec batches. In this paper we will qualify an ultrasound nanoparticle sizer (UNPS) as a real-time monitor for the growth of nanoparticles (or sub-micro particles) in the 100 nm-1 μm range. Nanoparticles affect the speed and attenuation of ultrasonic waves in the dispersion. The size of the change depends, amongst other things, on the size and concentration of the nanoparticles. This dependency is used in the UNPS method. The qualification of the UNPS was undertaken in two successful experiments. The first experiment consisted of static measurements on commercially available silica particles, and the second experiment was real-time monitoring of the size and concentration during the growth of silica nanoparticles in Stöber synthesis in a water-alcohol mixture starting from the molecular precursor tetraethyl orthosilicate. The results of the UNPS were verified by measurements of a dynamic light scattering device and a transmission electron microscope.\u3c/p\u3

Qualification of an ultrasonic instrument for real-time monitoring of size and concentration of nanoparticles during liquid phase bottom-up synthesis

Both in design and production of nanoparticles and nanocomposites it is of vital importance to have information about their size and concentration. During the formation of nanoparticles, real-time monitoring of particle size and concentration during bottom-up synthesis in liquids allows for a detailed study of nucleation and growth. This provides valuable insights into the formation of nanoparticles that can be used for process optimization and scale up. In the production of nanoparticles, real-time monitoring enables intervention to minimize the number of off-spec batches. In this paper we will qualify an ultrasound nanoparticle sizer (UNPS) as a real-time monitor for the growth of nanoparticles (or sub-micro particles) in the 100 nm-1 μm range. Nanoparticles affect the speed and attenuation of ultrasonic waves in the dispersion. The size of the change depends, amongst other things, on the size and concentration of the nanoparticles. This dependency is used in the UNPS method. The qualification of the UNPS was undertaken in two successful experiments. The first experiment consisted of static measurements on commercially available silica particles, and the second experiment was real-time monitoring of the size and concentration during the growth of silica nanoparticles in Stöber synthesis in a water-alcohol mixture starting from the molecular precursor tetraethyl orthosilicate. The results of the UNPS were verified by measurements of a dynamic light scattering device and a transmission electron microscope.ImPhys/Acoustical Wavefield Imagin

Pathological findings in the red fox (Vulpes vulpes), stone marten (Martes foina) and raccoon dog (Nyctereutes procyonoides), with special emphasis on infectious and zoonotic agents in Northern Germany

Anthropogenic landscape changes contributed to the reduction of availability of habitats to wild animals. Hence, the presence of wild terrestrial carnivores in urban and peri-urban sites has increased considerably over the years implying an increased risk of interspecies spillover of infectious diseases and the transmission of zoonoses. The present study provides a detailed characterisation of the health status of the red fox (Vulpes vulpes), stone marten (Martes foina) and raccoon dog (Nyctereutes procyonoides) in their natural rural and peri-urban habitats in Schleswig-Holstein, Germany between November 2013 and January 2016 with focus on zoonoses and infectious diseases that are potentially threatening to other wildlife or domestic animal species. 79 red foxes, 17 stone martens and 10 raccoon dogs were collected from traps or hunts. In order to detect morphological changes and potential infectious diseases, necropsy and pathohistological work-up was performed. Additionally, in selected animals immunohistochemistry (influenza A virus, parvovirus, feline leukemia virus, Borna disease virus, tick-borne encephalitis, canine adenovirus, Neospora caninum, Toxoplasma gondii and Listeria monocytogenes), next-generation sequencing, polymerase chain reaction (fox circovirus) and serum-neutralisation analysis (canine distemper virus) were performed. Furthermore, all animals were screened for fox rabies virus (immunofluorescence), canine distemper virus (immunohistochemistry) and Aujeszky's disease (virus cultivation). The most important findings included encephalitis (n = 16) and pneumonia (n = 20). None of the investigations revealed a specific cause for the observed morphological alterations except for one animal with an elevated serum titer of 1:160 for canine distemper. Animals displayed macroscopically and/or histopathologically detectable infections with parasites, including Taenia sp., Toxocara sp. and Alaria alata. In summary, wildlife predators carry zoonotic parasitic disease and suffer from inflammatory diseases of yet unknown etiology, possibly bearing infectious potential for other animal species and humans. This study highlights the value of monitoring terrestrial wildlife following the "One Health" notion, to estimate the incidence and the possible spread of zoonotic pathogens and to avoid animal to animal spillover as well as transmission to humans

Pathological findings in the red fox (<i>Vulpes vulpes</i>), stone marten (<i>Martes foina</i>) and raccoon dog (<i>Nyctereutes procyonoides</i>), with special emphasis on infectious and zoonotic agents in Northern Germany

<div><p>Anthropogenic landscape changes contributed to the reduction of availability of habitats to wild animals. Hence, the presence of wild terrestrial carnivores in urban and peri-urban sites has increased considerably over the years implying an increased risk of interspecies spillover of infectious diseases and the transmission of zoonoses. The present study provides a detailed characterisation of the health status of the red fox (<i>Vulpes vulpes</i>), stone marten (<i>Martes foina</i>) and raccoon dog (<i>Nyctereutes procyonoides</i>) in their natural rural and peri-urban habitats in Schleswig-Holstein, Germany between November 2013 and January 2016 with focus on zoonoses and infectious diseases that are potentially threatening to other wildlife or domestic animal species. 79 red foxes, 17 stone martens and 10 raccoon dogs were collected from traps or hunts. In order to detect morphological changes and potential infectious diseases, necropsy and pathohistological work-up was performed. Additionally, in selected animals immunohistochemistry (influenza A virus, parvovirus, feline leukemia virus, Borna disease virus, tick-borne encephalitis, canine adenovirus, <i>Neospora caninum</i>, <i>Toxoplasma gondii and Listeria monocytogenes)</i>, next-generation sequencing, polymerase chain reaction (fox circovirus) and serum-neutralisation analysis (canine distemper virus) were performed. Furthermore, all animals were screened for fox rabies virus (immunofluorescence), canine distemper virus (immunohistochemistry) and Aujeszky’s disease (virus cultivation). The most important findings included encephalitis (n = 16) and pneumonia (n = 20). None of the investigations revealed a specific cause for the observed morphological alterations except for one animal with an elevated serum titer of 1:160 for canine distemper. Animals displayed macroscopically and/or histopathologically detectable infections with parasites, including <i>Taenia sp</i>., <i>Toxocara sp</i>. and <i>Alaria alata</i>. In summary, wildlife predators carry zoonotic parasitic disease and suffer from inflammatory diseases of yet unknown etiology, possibly bearing infectious potential for other animal species and humans. This study highlights the value of monitoring terrestrial wildlife following the “One Health” notion, to estimate the incidence and the possible spread of zoonotic pathogens and to avoid animal to animal spillover as well as transmission to humans.</p></div

Photodynamic therapy versus topical imiquimod versus topical fluorouracil for treatment of superficial basal-cell carcinoma: a single blind, non-inferiority, randomised controlled trial

Background Superficial basal-cell carcinoma is most commonly treated with topical non-surgical treatments, such as photodynamic therapy or topical creams. Photodynamic therapy is considered the preferable treatment, although this has not been previously tested in a randomised control trial. We assessed the effectiveness of photodynamic therapy compared with imiquimod or fluorouracil in patients with superficial basal-cell carcinoma. Methods In this single blind, non-inferiority, randomised controlled multicentre trial, we enrolled patients with a histologically proven superficial basal-cell carcinoma at seven hospitals in the Netherlands. Patients were randomly assigned to receive treatment with methylaminolevulinate photodynamic therapy (MAL-PDT; two sessions with an interval of 1 week), imiquimod cream (once daily, five times a week for 6 weeks), or fluorouracil cream (twice daily for 4 weeks). Follow-up was at 3 and 12 months post-treatment. Data were collected by one observer who was blinded to the assigned treatment. The primary outcome was the proportion of patients free of tumour at both 3 and 12 month follow up. A pre-specified non-inferiority margin of 10% was used and modified intention-to-treat analyses were done. This trial is registered as an International Standard Randomised controlled trial (ISRCTN 79701845). Findings 601 patients were randomised: 202 to receive MAL-PDT, 198 to receive imiquimod, and 201 to receive fluorouracil. A year after treatment, 52 of 196 patients treated with MAL-PDT, 31 of 189 treated with imiquimod, and 39 of 198 treated with fluorouracil had tumour residue or recurrence. The proportion of patients tumour-free at both 3 and 12 month follow-up was 72.8% (95% CI 66.8-79.4) for MAL-PDT, 83.4% (78.2-88.9) for imiquimod cream, and 80.1% (74.7-85.9) for fluorouracil cream. The difference between imiquimod and MAL-PDT was 10.6% (95% CI 1.5-19.5; p=0.021) and 7.3% (-1.9 to 16.5; p=0.120) between fluorouracil and MAL-PDT, and between fluorouracil and imiquimod was -3.3% (-11.6 to 5.0; p=0.435. For patients treated with MAL-PDT, moderate to severe pain and burning sensation were reported most often during the actual MAL-PDT session. For other local adverse reactions, local skin redness was most often reported as moderate or severe in all treatment groups. Patients treated with creams more often reported moderate to severe local swelling, erosion, crust formation, and itching of the skin than patients treated with MAL-PDT. In the MAL-PDT group no serious adverse events were reported. One patient treated with imiquimod and two patients treated with fluorouracil developed a local wound infection and needed additional treatment in the outpatient setting. Interpretation Topical fluorouracil was non-inferior and imiquimod was superior to MAL-PDT for treatment of superficial basal-cell carcinoma. On the basis of these findings, imiquimod can be considered the preferred treatment, but all aspects affecting treatment choice should be weighted to select the best treatment for patients