The rapid emergence of antibiotic-resistant microbes worldwide and the urgent need of new antimicrobial agents have stimulated interest in antimicrobial peptides (AMPs) as new therapeutics for treatment of infectious diseases. AMPs are present in all living species and constitute an important part of the innate immune system in multicellular organisms, including humans. AMPs display a remarkably broad spectrum of antimicrobial activity covering both Gram-positive and Gram-negative bacteria, including many antibiotic-resistant strains, as well as fungi, viruses, and protozoa. Further, in contrast to many conventional antibiotics, AMPs rapidly kill bacteria instead of just inhibiting bacterial growth. In addition, AMPs act as modulators of the innate immune system and, importantly, bacteria seem less efficient in developing resistance towards AMPs than towards conventional antibiotics. Together these properties make AMPs highly attractive as a new class of antimicrobials, with clinical potential also extending to diseases where inflammation is part of the pathology. The aim of this thesis was to study novel AMPs with respect to their mechanism of action (MOA), antimicrobial spectrum, propensity to select for resistance, and in vivo efficacy and safety. To achieve this, we used a number of in vitro and in vivo assays, together generating a comprehensive preclinical evaluation of the peptides. The hypothesis was that the AMPs in this thesis have potential to be developed as therapeutic agents for several infectious and inflammatory conditions, including treatment of skin and soft tissue infections and prevention of postsurgical adhesion formation. The results showed that all AMPs tested (i.e. PXL03, PXL150, HLR1r, and five variants of CEN1 HC-Br) had broad antimicrobial spectra in vitro with varying sensitivity to salt and serum. Furthermore, PXL150 caused a rapid permeabilization of bacterial membrane in vitro, indicating that this is at least one part of the MOA of this peptide. Under selection pressure in vitro, bacteria did not develop resistance to the peptides tested, i.e. PXL150 and CEN1 HC. Interestingly, all peptides showed anti-inflammatory activity by inhibiting the secretion of proinflammatory mediators from stimulated human cell lines. In addition, PXL01, PXL150, and HLR1r demonstrated fibrinolytic ability in vitro by suppressing the release of plasminogen activator inhibitor-1 (PAI-1). In ex vivo and in vivo skin/wound infection models, the peptides reduced the number of viable bacteria and yeast cells. Further, PXL01 decreased postsurgical adhesion formation in vivo. Notably, nonclinical safety studies showed that PXL150 was safe and well tolerated. In conclusion, several of the peptides evaluated in this thesis demonstrated a promising preclinical efficacy and safety profile motivating further development as drug candidates for local treatment of infectious and inflammatory conditions.

Antimicrobial peptides (AMPs) have emerged as a new class of drug candidates for the treatment of infectious diseases. Here we describe a novel AMP, HLR1r, which is structurally derived from the human milk protein lactoferrin and demonstrates a broad spectrum microbicidal action in vitro. The minimum concentration of HLR1r needed for killing ≥99% of microorganisms in vitro, was in the range of 3-50 μg/ml for common Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and for the yeast Candida albicans, when assessed in diluted brain-heart infusion medium. We found that HLR1r also possesses anti-inflammatory properties as evidenced by inhibition of tumor necrosis factor alpha (TNF-α) secretion from human monocyte-derived macrophages and by repression of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) secretion from human mesothelial cells, without any cytotoxic effect observed at the concentration range tested (up to 400 μg/ml). HLR1r demonstrated pronounced anti-infectious effect in in vivo experimental models of cutaneous candidiasis in mice and of excision wounds infected with MRSA in rats as well as in an ex vivo model of pig skin infected with S. aureus. In conclusion, HLR1r may constitute a new therapeutic alternative for local treatment of skin infections.

The urgent need to develop novel antimicrobial therapies has stimulated interest in antimicrobial peptides as therapeutic candidates for the treatment of infectious diseases. The aim of this study was to evaluate the anti-infectious effect of the synthetic antimicrobial peptide PXL150, formulated in hydroxypropyl cellulose (HPC) gel, on Pseudomonas aeruginosa in vitro and in an in vivo mouse model of infected burn wounds as well as to assess the in vivo safety profile of PXL150 in rats and rabbits. Minimal microbicidal concentration analysis showed prominent efficacy of PXL150 against P. aeruginosa in vitro, which was further enhanced in formulating the peptide in HPC gel. Application of 1.25, 2.5, 5, 10 and 20 mg/g PXL150 in HPC gel twice daily for four consecutive days significantly reduced bacterial counts in the burn wounds compared with non-treated or placebo-treated controls. Continuous bioluminescence measurements of the bacteria revealed a pronounced anti-infective effect already at the first day post infection by PXL150 in concentrations of ≥2.5 mg/g. In the non-clinical safety studies, PXL150 showed a favourable safety profile following repeated administration systemically and locally in rats and rabbits, respectively. In conclusion, these data support that PXL150 has the potential to be an effective and safe drug candidate for the treatment of infected burn wounds. The findings encourage the progression of PXL150 as a novel topical treatment of microbial infections.

Nanocellulose (bacterial cellulose, BC), such as that produced by Acetobacter xylinum, has shown promising results as a replacement material for small diameter vascular grafts. The surface morphology of the lumen and mechanical properties of such tubes are crucial for their performance. The growth of a BC tube in a vertical fermentation bioreactor using silicone tubing for support and as an oxygen delivery membrane has not been studied in detail previously. Oxygen concentration and the number of bacteria added influence the production of the BC tubes. A dense and smooth luminal surface was formed after 4 days on a 3 mm silicone support. The bacteria were found to be in high concentration close to the silicon support and decreased in number further away. In the region with a high bacteria concentration, dense thin layers of BC were formed since the bacteria moved close together in this region. The presented observations were summarized in a theoretical model of BC tube growth.

The spatial localization of amyloid-β peptide deposits, the major component of senile plaques in Alzheimer's disease (AD), was mapped in transgenic AD mouse brains using time-of-flight secondary ion mass spectrometry (ToF-SIMS), simultaneously with several endogenous molecules that cannot be mapped using conventional immunohistochemistry imaging, including phospholipids, cholesterol and sulfatides. Whereas the endogenous lipids were detected directly, the amyloid-β deposits, which cannot be detected as intact entities with ToF-SIMS because of extensive ion-induced fragmentation, were identified by specific binding of deuterated liposomes to antibodies directed against amyloid-β. Comparative investigation of the amyloid-β deposits using conventional immunohistochemistry and fluorescence microscopy suggests similar sensitivity but a more surface-confined identification due to the shallow penetration depth of the ToF-SIMS signal. The recorded ToF-SIMS images thus display the localization of lipids and amyloid-β in a narrow (∼10 nm) two-dimensional plane at the tissue surface. As compared to a frozen nontreated tissue sample, the liposome preparation protocol generally increased the signal intensity of endogenous lipids, likely caused by matrix effects associated with the removal of salts, but no severe effects on the tissue integrity and the spatial distribution of lipids were observed with ToF-SIMS or scanning electron microscopy (SEM). This method may provide an important extension to conventional tissue imaging techniques to investigate the complex interplay of different kinds of molecules in neurodegenerative diseases, in the same specimen. However, limitations in target accessibility of the liposomes as well as unspecific binding need further consideration.

The pathological mechanisms underlying Alzheimer's disease (AD) are still not understood. The disease pathology is characterized by the accumulation and aggregation of amyloid-β (Aβ) peptides into extracellular plaques, however the factors that promote neurotoxic Aβ aggregation remain elusive. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides and proteins in biological tissues. In the present study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)-based imaging was used to study Aβ deposition in transgenic mouse brain tissue and to elucidate the plaque-associated chemical microenvironment. The imaging experiments were performed in brain sections of transgenic Alzheimer's disease mice carrying the Arctic and Swedish mutation of amyloid-beta precursor protein (tgArcSwe). Multivariate image analysis was used to interrogate the IMS data for identifying pathologically relevant, anatomical features based on their chemical identity. This include cortical and hippocampal Aβ deposits, whose amyloid peptide content was further verified using immunohistochemistry and laser microdissection followed by MALDI MS analysis. Subsequent statistical analysis on spectral data of regions of interest revealed brain region-specific differences in Aβ peptide aggregation. Moreover, other plaque-associated protein species were identified including macrophage migration inhibitory factor suggesting neuroinflammatory processes and glial cell reactivity to be involved in AD pathology. The presented data further highlight the potential of IMS as a powerful approach in neuropathology. Hanrieder et al. described an imaging mass spectrometry based study on comprehensive spatial profiling of C-terminally truncated Aβ species within individual plaques in tgArcSwe mice. Here, brain region-dependent differences in Aβ truncation and other plaque-associated proteins, such as macrophage migration inhibitory factor, were observed. The data shed further light on plaque-associated molecular mechanisms implicated in Alzheimer's pathogenesis.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been proven to successfully image different kinds of molecules, especially a variety of lipids, in biological samples. Proteins, however, are difficult to detect as specific entities with this method due to extensive fragmentation. To circumvent this issue, the authors present in this work a method developed for detection of proteins using antibody-conjugated liposomes, so called immunoliposomes, which are able to bind to the specific protein of interest. In combination with the capability of ToF-SIMS to detect native lipids in tissue samples, this method opens up the opportunity to analyze many different biomolecules, both lipids and proteins, at the same time, with high spatial resolution. The method has been applied to detect and image the distribution of amyloid-β (Aβ), a biologically relevant peptide in Alzheimer’s disease (AD), in transgenic mouse brain tissue. To ensure specific binding, the immunoliposome binding was verified on a model surface using quartz crystal microbalance with dissipation monitoring. The immunoliposome binding was also investigated on tissue sections with fluorescence microscopy, and compared with conventional immunohistochemistry using primary and secondary antibodies, demonstrating specific binding to Aβ. Using ToF-SIMS imaging, several endogenous lipids, such as cholesterol and sulfatides, were also detected in parallel with the immunoliposome-labeled Aβ deposits, which is an advantage compared to fluorescence microscopy. This method can thus potentially provide further information about lipid–protein interactions, which is important to understand the mechanisms of neurodegeneration in AD.

Factors determining the pH-controlled dissolution kinetics of nilotinib formulations with the pH-titrable polymer hydroxypropyl methylcellulose phthalate, obtained by carbon dioxide-mediated precipitation, were mechanistically examined in acid and neutral environment. The matrix effect, modulating the drug dissolution, was characterized with a battery of physicochemical methodologies, including ToF-SIMS for surface composition, SAXS/WAXS and modulated DSC for crystallization characterization, and simultaneous UV-imaging and Raman spectroscopy for monitoring the dissolution process in detail. The hybrid particle formulations investigated consisted of amorphous nilotinib embedded in a polymer matrix in single continuous phase, displaying extended retained amorphicity also under wet conditions. It was demonstrated by Raman and FTIR spectroscopy that the efficient drug dispersion and amorphization in the polymer matrix were mediated by hydrogen bonding between the drug and the phthalate groups on the polymer. Simultaneous Raman and UV-imaging studies of the effect of drug load on the swelling and dissolution of the polymer matrix revealed that high nilotinib load prevented matrix swelling on passage from acid to neutral pH, thereby preventing re-precipitation and re-crystallization of incorporated nilotinib. These findings provide a mechanistic foundation of formulation development of nilotinib and other protein kinase inhibitors, which are now witnessing an intense therapeutic and industrial attention due to the difficulty in formulating these compounds so that efficient oral bioavailability is reached.

The effects of chocolate shell particle size were investigated by means of its influence on rate of oil migration and fat bloom development. The particle size of the non-fat particles in the chocolate, i.e. sugar and cocoa particles was varied between 15, 22 and 40 μm. A novel set of analytical techniques was used and by combining migration results with surface topology results clear differences could be observed between the samples. At 23 °C storage the samples with a particle size of 15 μm showed higher rate of oil migration and further, the earliest development of fat bloom at the surface. This could be observed both macroscopically and microscopically. Thus, it appears as a larger specific surface area of the non-fat particles facilitates migration of filling oil, possibly due to a more heterogeneous and coarser crystal network with higher permeability. Molecular diffusion cannot explain the level of oil migration observed and, thus, convective flow is assumed to be an important contribution in addition to the molecular diffusion.

A challenge for the next generation marine antifouling (AF) paints is to deliver minimum amounts of biocides to the environment. The candidate AF compound medetomidine is here shown to be released at very low concentrations, ie ng ml(-1) day(-1). Moreover, the release rate of medetomidine differs substantially depending on the formulation of the paint, while inhibition of barnacle settlement is independent of release to the ambient water, ie the paint with the lowest release rate was the most effective in impeding barnacle colonisation. This highlights the critical role of chemical interactions between biocide, paint carrier and the solid/aqueous interface for release rate and AF performance. The results are discussed in the light of differential affinity states of the biocide, predicting AF activity in terms of a high surface affinity and preserved bioavailability. This may offer a general framework for the design of low-release paint systems using biocides for protection against biofouling on marine surfaces.

Background: Patterning medical devices at the nanoscale level enables the manipulation of cell behavior and tissue regeneration, with topographic features recognized as playing a significant role inthe osseointegration of implantable devices. Methods: In this study, we assessed the ability of titanium-coated hemisphere-like topographic nanostructures of different sizes (approximately 50, 100, and 200 nm) to influence the morphology, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Results: We found that the proliferation and osteogenicdifferentiation of hMSCs was influenced by the size of the underlying structures, suggesting that size variations in topographic features at the nanoscale level, independently of chemistry, can be exploited to control hMSC behavior in a size-dependent fashion. Conclusion: Our studies demonstrate that colloidal lithography, in combination with coating technologies, can be exploited to investigate the cell response to well defined nanoscale topography and to develop next-generation surfaces that guide tissue regeneration and promote implant integration.

The presence of solid carbonaceous matter in cometary dust was established by the detection of elements such as carbon, hydrogen, oxygen and nitrogen in particles from comet 1P/Halley1, 2. Such matter is generally thought to have originated in the interstellar medium3, but it might have formed in the solar nebula—the cloud of gas and dust that was left over after the Sun formed4. This solid carbonaceous material cannot be observed from Earth, so it has eluded unambiguous characterization5. Many gaseous organic molecules, however, have been observed6, 7, 8, 9; they come mostly from the sublimation of ices at the surface or in the subsurface of cometary nuclei8. These ices could have been formed from material inherited from the interstellar medium that suffered little processing in the solar nebula10. Here we report the in situ detection of solid organic matter in the dust particles emitted by comet 67P/Churyumov–Gerasimenko; the carbon in this organic material is bound in very large macromolecular compounds, analogous to the insoluble organic matter found in the carbonaceous chondrite meteorites11, 12. The organic matter in meteorites might have formed in the interstellar medium and/or the solar nebula, but was almost certainly modified in the meteorites’ parent bodies11. We conclude that the observed cometary carbonaceous solid matter could have the same origin as the meteoritic insoluble organic matter, but suffered less modification before and/or after being incorporated into the comet.

This paper describes strategies to search for, detect, and identify organic material on the surface and subsurface of Mars. The strategies described include those applied by landed missions in the past and those that will be applied in the future. The value and role of ESA's ExoMars rover and of her key science instrument Mars Organic Molecule Analyzer (MOMA) are critically assessed.

Recent studies have revealed that ozone ultraviolet (UVO) illumination of titanium (Ti) implants improves bone-implant anchorage by altering the physico-chemical and immune activating properties of the titanium dioxide (TiO2) layer. In the present rat tibia model, the authors compared the early events of inflammation and bone formation around UVO-treated Ti and complement activating immunoglobin g (IgG)-coated Ti. Machined Ti and machined Ti coated with a physical vapour-deposited Ti layer were used as references. Screw-shaped test and reference implants were implanted into rat tibia and harvested after 1, 7 and 28 days. Messenger RNA expression of implant adhered cells and peri-implant tissue ∼250 μm from the surface were subsequently analysed with regard to IL-1β, TNF-α, osteocalcin, cathepsin K, BMP-2 and PDGF. Separate implants were retrieved after 7 and 28 days for removal torque measurements, and histological staining and histomorphometric analysis of bone area and bone-to-implant contact. While enhanced expression of inflammatory markers, TNF-α and IL-1β, was observed on IgG-coated surfaces throughout the observation time, UVO-treated surfaces indicated a significantly lower early inflammatory response. In the early phases (1 and 7 days), the UVO-treated surfaces displayed a significantly higher expression of osteoblast markers BMP-2 and osteocalcin. In summary, complement activating Ti implants elicited a stronger inflammatory response than UVO-treated Ti, with low complement activation during the first week of healing. In spite of this, the UVO-treated Ti induced only marginally more bone growth outside the implants.

Uterus transplantation (UTx) may be the only possible curative treatment for absolute uterine factor infertility, which affects 1 in every 500 females of fertile age. We recently presented the 6-month results from the first clinical UTx trial, describing nine live-donor procedures. This routine involves complicated surgery and requires potentially harmful immune suppression to prevent rejection. However, tissue engineering applications using biomaterials and stem cells may replace the need for a live donor, and could prevent the required immunosuppressive treatment. To investigate the basic aspects of this, we developed a novel whole-uterus scaffold design for uterus tissue engineering experiments in the rat. Decellularization was achieved by perfusion of detergents and ionic solutions. The remaining matrix and its biochemical and mechanical properties were quantitatively compared from using three different protocols. The constructs were further compared with native uterus tissue composition. Perfusion with Triton X-100/dimethyl sulfoxide/H2O led to a compact, weaker scaffold that showed evidence of a compromised matrix organization. Sodium deoxycholate/dH2O perfusion gave rise to a porous scaffold that structurally and mechanically resembled native uterus better. An innovative combination of two proteomic analyses revealed higher fibronectin and versican content in these porous scaffolds, which may explain the improved scaffold organization. Together with other important protocol-dependent differences, our results can contribute to the development of improved decellularization protocols for assorted organs. Furthermore, our study shows the first available data on decellularized whole uterus, and creates new opportunities for numerous in vitro and in vivo whole-uterus tissue engineering applications.

The COmetary Secondary Ion Mass Analyser instrument on board ESA's Rosetta mission has collected dust particles in the coma of comet 67P/Churyumov-Gerasimenko. During the early-orbit phase of the Rosetta mission, particles and particle agglomerates have been imaged and analyzed in the inner coma at distances between 100 km and 10 km off the cometary nucleus and at more than 3 AU from the Sun. We identified 585 particles of more than 14 μm in size. The particles are collected at low impact speeds and constitute a sample of the dust particles in the inner coma impacting and fragmenting on the targets. The sizes of the particles range from 14 μm up to sub-millimeter sizes and the differential dust flux size distribution is fitted with a power law exponent of -3.1. After impact, the larger particles tend to stick together, spread out or consist of single or a group of clumps, and the flocculent morphology of the fragmented particles is revealed. The elemental composition of the dust particles is heterogeneous and the particles could contain typical silicates like olivine and pyroxenes, as well as iron sulfides. The sodium to iron elemental ratio is enriched with regard to abundances in CI carbonaceous chondrites by a factor from ∼1.5 to ∼15. No clear evidence for organic matter has been identified. The composition and morphology of the collected dust particles appear to be similar to that of interplanetary dust particles.

Doxorubicin is a chemotherapeutic agent indicated for the treatment of a variety of cancer types, including leukaemia, lymphomas, and many solid tumours. The use of doxorubicin is, however, associated with severe cardiotoxicity, often resulting in early discontinuation of the treatment. Importantly, the toxic symptoms can occur several years after the termination of the doxorubicin administration. In this study, the toxic effects of doxorubicin exposure have been investigated in cardiomyocytes derived from human embryonic stem cells (hESC). The cells were exposed to different concentrations of doxorubicin for up to 2 days, followed by a 12 day recovery period. Notably, the cell morphology was altered during drug treatment and the cells showed a reduced contractile ability, most prominent at the highest concentration of doxorubicin at the later time points. A general cytotoxic response measured as Lactate dehydrogenase leakage was observed after 2 days’ exposure compared to the vehicle control, but this response was absent during the recovery period. A similar dose-dependant pattern was observed for the release of cardiac specific troponin T (cTnT) after 1 day and 2 days of treatment with doxorubicin. Global transcriptional profiles in the cells revealed clusters of genes that were differentially expressed during doxorubicin exposure, a pattern that in some cases was sustained even throughout the recovery period, suggesting that these genes could be used as sensitive biomarkers for doxorubicin-induced toxicity in human cardiomyocytes. The results from this study show that cTnT release can be used as a measurement of acute cardiotoxicity due to doxorubicin. However, for the late onset of doxorubicin-induced cardiomyopathy, cTnT release might not be the most optimal biomarker. As an alternative, some of the genes that we identified as differentially expressed after doxorubicin exposure could serve as more relevant biomarkers, and may also help to explain the cellular mechanisms behind the late onset apoptosis associated with doxorubicin-induced cardiomyopathy.

Antimicrobial peptides have recently emerged as a promising new group to be evaluated in the therapeutic intervention of infectious diseases. This study evaluated the anti-infectious effect of the short, synthetic, broad-spectrum antimicrobial peptide PXL150 in a mouse model of staphylococcal surgical site infections. We found that administration of PXL150, formulated in an aqueous solution or in a hydroxypropyl cellulose gel, significantly reduced the bacterial counts in the wound compared with placebo treatment, warranting further investigations of the potential of this peptide as a novel local treatment of microbial infections.

Purpose: Mechanisms governing the cellular interactions with well-defined nanotopography are not well described in vivo. This is partly due to the difficulty in isolating a particular effect of nanotopography from other surface properties. This study employed colloidal lithography for nanofabrication on titanium implants in combination with an in vivo sampling procedure and different analytical techniques. The aim was to elucidate the effect of well-defined nanotopography on the molecular, cellular, and structural events of osseointegration. Materials and methods: Titanium implants were nanopatterned (Nano) with semispherical protrusions using colloidal lithography. Implants, with and without nanotopography, were implanted in rat tibia and retrieved after 3, 6, and 28 days. Retrieved implants were evaluated using quantitative polymerase chain reaction, histology, immunohistochemistry, and energy dispersive X-ray spectroscopy (EDS). Results: Surface characterization showed that the nanotopography was well defined in terms of shape (semispherical), size (79±6 nm), and distribution (31±2 particles/μm2). EDS showed similar levels of titanium, oxygen, and carbon for test and control implants, confirming similar chemistry. The molecular analysis of the retrieved implants revealed that the expression levels of the inflammatory cytokine, TNF-α, and the osteoclastic marker, CatK, were reduced in cells adherent to the Nano implants. This was consistent with the observation of less CD163-positive macrophages in the tissue surrounding the Nano implant. Furthermore, periostin immunostaining was frequently detected around the Nano implant, indicating higher osteogenic activity. This was supported by the EDS analysis of the retrieved implants showing higher content of calcium and phosphate on the Nano implants. Conclusion: The results show that Nano implants elicit less periimplant macrophage infiltration and downregulate the early expression of inflammatory (TNF-α) and osteoclastic (CatK) genes. Immunostaining and elemental analyses show higher osteogenic activity at the Nano implant. It is concluded that an implant with the present range of well-defined nanocues attenuates the inflammatory response while enhancing mineralization during osseointegration.

Additive manufacturing is an emerging manufacturing technology that enables production of patient specific implants, today primarily out of titanium. For optimal functionality and proper integration between the titanium implant and the body tissues surface properties, such as surface oxide thickness is of particular importance, as it is primarily the surface of the material which interacts with the body. Hence, in this study the surface oxidation behavior of titanium parts manufactured by Electron Beam Melting (EBM®) is investigated using the surface sensitive techniques ToF-SIMS and AES. Oxide thicknesses comparable to those found on conventionally machined surfaces are found by both analysis techniques. However, a build height dependency is discovered for different locations of the EBM® manufactured parts due to the presence of trapped moisture in the machine and temperature gradients in the build.

Electron Beam Melting (EBM) has been recognized as a revolutionary technique to produce mass-customized parts to near-net-shape from various metallic materials. The technique produces parts with unique geometries from a powder stock material and uses an electron beam to melt the powder layer-by-layer to fully solid structures. In this study we have investigated the use of two different Ti-6Al-4V powders of different size fractions in the EBM process; a larger 45-100 μm powder, and a smaller 25-45 μm powder. We have also investigated the effects of two build layer thicknesses, 70 μm and 50 μm, respectively. We hypothesize that the smaller powder has the potential to improve surface resolution of parts produced in the EBM process. The EBM as-built parts were investigated regarding surface and bulk chemistry, surface oxide thickness, macro- and microstructure, surface appearance and mechanical properties. We conclude from the results that both powders and both build layer thicknesses are feasible to use in the EBM process. The investigated material properties were not significantly affected by powder size or layer thickness within the studied range of process parameters. However, the surface appearance was found to be different for the samples made with the different powder sizes.

COmetary Secondary Ion Mass Analyzer (COSIMA) is a time-of-flight secondary ion mass spectrometry (TOF-SIMS) instrument on board the Rosetta space mission. COSIMA has been designed to measure the composition of cometary dust particles. It has a mass resolution m/Δm of 1400 at mass 100 u, thus enabling the discrimination of inorganic mass peaks from organic ones in the mass spectra. We have evaluated the identification capabilities of the reference model of COSIMA for inorganic compounds using a suite of terrestrial minerals that are relevant for cometary science. Ground calibration demonstrated that the performances of the flight model were similar to that of the reference model. The list of minerals used in this study was chosen based on the mineralogy of meteorites, interplanetary dust particles and Stardust samples. It contains anhydrous and hydrous ferromagnesian silicates, refractory silicates and oxides (present in meteoritic Ca-Al-rich inclusions), carbonates, and Fe-Ni sulfides. From the analyses of these minerals, we have calculated relative sensitivity factors for a suite of major and minor elements in order to provide a basis for element quantification for the possible identification of major mineral classes present in the cometary particles.

Mid-Mesozoic kalligrammatid lacewings (Neuroptera) entered the fossil record 165 million years ago (Ma) and disappeared 45 Ma later. Extant papilionoid butterflies (Lepidoptera) probably originated 80–70 Ma, long after kalligrammatids became extinct. Although poor preservation of kalligrammatid fossils previously prevented their detailed morphological and ecological characterization, we examine new, well-preserved, kalligrammatid fossils from Middle Jurassic and Early Cretaceous sites in northeastern China to unravel a surprising array of similar morphological and ecological features in these two, unrelated clades. We used polarized light and epifluorescence photography, SEM imaging, energy dispersive spectrometry and time-of-flight secondary ion mass spectrometry to examine kalligrammatid fossils and their environment. We mapped the evolution of specific traits onto a kalligrammatid phylogeny and discovered that these extinct lacewings convergently evolved wing eyespots that possibly contained melanin, and wing scales, elongate tubular proboscides, similar feeding styles, and seed–plant associations, similar to butterflies. Long-proboscid kalligrammatid lacewings lived in ecosystems with gymnosperm–insect relationships and likely accessed bennettitalean pollination drops and pollen. This system later was replaced by mid-Cretaceous angiosperms and their insect pollinators.

The aim of this work was to study the feasibility to use a biomimetic method to prepare biomimetic hydroxyapatite (HA) coatings on CoCr substrates with short soaking times and to characterize the properties of such coatings. A second objective was to investigate if the coatings could be applied to porous CoCr implants manufactured by electron beam melting (EBM). The coating was prepared by immersing the pretreated CoCr substrates and EBM implants into the phosphate-buffered solution with Ca2+ in sealed plastic bottles, kept at 60 °C for 3 days. The formed coating was partially crystalline, slightly calcium deficient and composed of plate-like crystallites forming roundish flowers in the size range of 300-500 nm. Cross-section imaging showed a thickness of 300-500 nm. In addition, dissolution tests in Tris-HCl up to 28 days showed that a substantial amount of the coating had dissolved, however, undergoing only minor morphological changes. A uniform coating was formed within the porous network of the additive manufactured implants having similar thickness and morphology as for the flat samples. In conclusion, the present coating procedure allows coatings to be formed on CoCr and could be used for complex shaped, porous implants made by additive manufacturing.

Colour, derived primarily from melanin and/or carotenoid pigments, is integral to many aspects of behaviour in living vertebrates, including social signalling, sexual display and crypsis. Thus, identifying biochromes in extinct animals can shed light on the acquisition and evolution of these biological traits. Both eumelanin and melanin-containing cellular organelles (melanosomes) are preserved in fossils, but recognizing traces of ancient melanin-based coloration is fraught with interpretative ambiguity, especially when observations are based on morphological evidence alone. Assigning microbodies (or, more often reported, their ‘mouldic impressions’) as melanosome traces without adequately excluding a bacterial origin is also problematic because microbes are pervasive and intimately involved in organismal degradation. Additionally, some forms synthesize melanin. In this review, we survey both vertebrate and microbial melanization, and explore the conflicts influencing assessment of microbodies preserved in association with ancient animal soft tissues.We discuss the types of data used to interpret fossil melanosomes and evaluate whether these are sufficient for definitive diagnosis. Finally, we outline an integrated morphological and geochemical approach for detecting endogenous pigment remains and associated microstructures in multimillion-year-old fossils.

Feathers are amongst the most complex epidermal structures known, and they have a well-documented evolutionary trajectory across non-avian dinosaurs and basal birds. Moreover, melanosome-like microbodies preserved in association with fossil plumage have been used to reconstruct original colour, behaviour and physiology. However, these putative ancient melanosomes might alternatively represent microorganismal residues, a conflicting interpretation compounded by a lack of unambiguous chemical data. We therefore used sensitive molecular imaging, supported by multiple independent analytical tests, to demonstrate that the filamentous epidermal appendages in a new specimen of the Jurassic paravian Anchiornis comprise remnant eumelanosomes and fibril-like microstructures, preserved as endogenous eumelanin and authigenic calcium phosphate. These results provide novel insights into the early evolution of feathers at the sub-cellular level, and unequivocally determine that melanosomes can be preserved in fossil feathers.

Antimicrobial peptides (AMPs), also known as host defense peptides, are short and generally positively charged peptides found in a wide variety of life forms from microorganisms to humans. Most AMPs have the ability to kill microbial pathogens directly, whereas others act indirectly by modulating the host defense systems. Against a background of rapidly increasing resistance development to conventional antibiotics all over the world, efforts to bring AMPs into clinical use are accelerating. Several AMPs are currently being evaluated in clinical trials as novel anti-infectives, but also as new pharmacological agents to modulate the immune response, promote wound healing, and prevent post-surgical adhesions. In this review, we provide an overview of the biological role, classification, and mode of action of AMPs, discuss the opportunities and challenges to develop these peptides for clinical applications, and review the innovative formulation strategies for application of AMPs.

Elucidating cardiac evolution has been frustrated by lack of fossils. One celebrated enigma in cardiac evolution involves the transition from a cardiac outflow tract dominated by a Multi-Valved conus arteriosus in basal actinopterygians, to an outflow tract commanded by the Non- Valved, elastic, bulbus arteriosus in higher actinopterygians. We demonstrate that cardiac preservation is possible in the extinct fish Rhacolepis buccalis from the Brazilian Cretaceous. Using X-Ray synchrotron microtomography, we show that Rhacolepis fossils display hearts with a conus arteriosus containing at least five valve rows. This represents a transitional morphology between the primitive, multivalvar, conal condition and the derived, monovalvar, bulbar state of the outflow tract in modern actinopterygians. Our data rescue a Long-Lost cardiac phenotype (119-113 Ma) and suggest that outflow tract simplification in actinopterygians is compatible with a gradual, rather than a drastic saltation event. Overall, our results demonstrate the feasibility of studying cardiac evolution in fossils.

Peptide AF-16, which includes the active site of Antisecretory Factor protein, has antisecretory and anti-inflammatory properties, making it a potent drug candidate for treatment of secretory and inflammatory diseases such as diarrhea, inflammatory bowel diseases, and intracranial hypertension. Despite remarkable physiological effects and great pharmaceutical need for drug discovery, very little is yet understood about AF-16 mechanism of action. In order to address interaction mechanisms, we investigated the binding of AF-16 to sulfated glycosaminoglycan, heparin, with focus on the effect of pH and ionic strength, and studied the influence of cell-surface proteoglycans on cellular uptake efficiency. Confocal laser scanning microscopy and flow cytometry experiments on wild type and proteoglycan-deficient Chinese hamster ovary cells reveal an endocytotic nature of AF-16 cellular uptake that is, however, less efficient for the cells lacking cell-surface proteoglycans. Isothermal titration calorimetry provides quantitative thermodynamic data and evidence for that the peptide affinity to heparin increases at lower pH and ionic strength. Experimental data, supported by theoretical modeling, of peptide-glycosaminoglycan interaction indicate that it has a large electrostatic contribution, which will be enhanced in diseases accompanied by decreased pH and ionic strength. These observations show that cell-surface proteoglycans are of general and crucial importance for the antisecretory and anti-inflammatory activities of AF-16. 

A facile methodology for the fabrication of functional crosslinked three dimensional (3D) networks has herein been explored via the benign and UV initiated thiol-ene coupling (TEC) chemistry. The careful selection of monomers or polymers and their feed ratio resulted in straightforward design of organic, inorganic and hydrogel networks with readily available alkenes or thiol functional groups. All crosslinked networks were fabricated within 1 second of UV exposure at wavelengths of 320-390 nm and generally exhibited excellent gel fractions around 90%. By introducing off-stoichiometric thiol and ene (OSTE) monomer feed ratios the window of mechanical properties could be manipulated. For the organic triazine system, the Young's modulus was altered from 780 MPa at an equimolar monomer ratio to soft 106 kPa for 2.5 equiv. with excess of thiol compared to enes. Postfunctionalizations with hydrophilic polyethylene glycols or acrylic acid and hydrophobic heneicosafluorododecyl acrylate were explored for the manipulation of functional networks. In this case, the rigid networks with excess of thiols were used as model substrates of which the initial contact angle (CA) of 60°was decreased to 43°by the introduction of acrylic acid and increased to 140°by successful attachment of fluorinated molecules. Finally, amalgamating micropatterning strategy with simple postfunctionalizations of hydrophobic groups resulted in superhydrophobic rigid surfaces with a CA of 173°.

The marine secondary metabolite stryphnusin (1) was isolated from the boreal sponge Stryphnus fortis, collected off the Norwegian coast. Given its resemblance to other natural acetylcholinesterase antagonists, it was evaluated against electric eel acetylcholinesterase and displayed inhibitory activity. A library of twelve synthetic phenethylamine analogs, 2a-7a and 2b-7b, containing tertiary and quaternary amines respectively were synthesized to investigate the individual structural contributions to the activity. Compound 7b was the strongest competitive inhibitor of both acetylcholinesterase and butyrylcholinesterase with IC50 values of 57 and 20 μM, respectively. This inhibitory activity is one order of magnitude higher than the positive control physostigmine, and is comparable with several other marine acetylcholinesterase inhibitors. The physiological effect of compound 7b on muscle function and neuromuscular transmission was studied and revealed a selective mode of action at the investigated concentration. This data is of importance as the interference of therapeutic acetylcholinesterase inhibitors with neuromuscular transmission can be problematic and lead to unwanted side effects. The current findings also provide additional insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.

We investigate theoretically and experimentally the spontaneous imbibition of water based inkjet formulations utilizing paper capillary rise and imbibition of inkjet drops. We approximate the paper structure to a two dimensional anisotropic porous material, and using Darcy's law as a base, we derive dimensionless groups that scale drop imbibition. This derivation is based on a previous dimensional scaling of drop imbibition on thick isotropic porous material. We apply this scaling to a paper substrate by measuring the average drop imbibition rate, and perform paper capillary rise experiments to obtain the average system parameters required for the scaling. The results suggest that this approach is a valuable tool to predict drop imbibition rates on paper. We then continue and perform the same sets of experiments on a different paper with similar structure that is surface treated (surface sized) with CaCl2 salt, an additive that is known to improve print quality. We find that due to rapid aggregation of the colorant ink by the CaCl2, the imbibition rate is slowed down in the capillary rise experiments, i.e., on much larger scales compared to a single inkjet drop. However, the presence of CaCl2 has only minor effect over the average imbibition rates of single drops. Imbibition rates on the CaCl2 surface sized paper did not give adequate scaling as a result of the fact that the aggregation was not included the theoretical assumptions behind the scaling.

Vascular diseases are increasing health problems affecting >25 million individuals in westernized societies. Such patients could benefit fromtransplantation of tissue-engineered vascular grafts using autologous cells. One challenge that has limited this development is the need for cell isolation, and risks associated with ex vivo expanded stem cells. Herewe demonstrate a novel approach to generate transplantable vascular grafts using decellularized allogeneic vascular scaffolds, repopulatedwith peripheralwhole blood (PWB) in vitro in a bioreactor. Circulating, VEGFR-2+/CD45+ and a smaller fraction of VEGFR-2+/CD14+ cells contributed to repopulation of the graft. SEMmicrographs showed flat cells on the luminal surface of the grafts consistentwith endothelial cells. For clinical validation, two autologous PWBtissue-engineered vein conduits were prepared and successfully used for bypass procedures in two pediatric patients. These results provide a proof of principle for the generation of transplantable vascular grafts using a simple autologous blood sample, making it clinically feasible globally.

Barettin, 8,9-dihydrobarettin, bromoconicamin and a novel brominated marine indole were isolated from the boreal sponge Geodia barretti collected off the Norwegian coast. The compounds were evaluated as inhibitors of electric eel acetylcholinesterase. Barettin and 8,9-dihydrobarettin displayed significant inhibition of the enzyme, with inhibition constants (Ki) of 29 and 19 μM respectively towards acetylcholinesterase via a reversible noncompetitive mechanism. These activities are comparable to those of several other natural acetylcholinesterase inhibitors of marine origin. Bromoconicamin was less potent against acetylcholinesterase, and the novel compound was inactive. Based on the inhibitory activity, a library of 22 simplified synthetic analogs was designed and prepared to probe the role of the brominated indole, common to all the isolated compounds. From the structure-activity investigation it was shown that the brominated indole motif is not sufficient to generate a high acetylcholinesterase inhibitory activity, even when combined with natural cationic ligands for the acetylcholinesterase active site. The four natural compounds were also analysed for their butyrylcholinesterase inhibitory activity in addition and shown to display comparable activities. The study illustrates how both barettin and 8,9-dihydrobarettin display additional bioactivities which may help to explain their biological role in the producing organism. The findings also provide new insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.

The efficacy of antifouling coatings designed to minimise the release of biocide, either by embedded (non-covalent) or tethered (covalently bonded) biocides, relies on sufficient bioavailability of the active compound upon contact between the organism and the coating. This investigation is focused on whether coating hardness affects the efficacy of embedded coating systems. Two experimental, non-eroding and waterborne latex paint formulations composed mainly of polystyrene (PS) or polyvinyl versatate (PV) were chosen for their difference in mechanical properties measured in terms of Buchholz indentation resistance. Ivermectin was added to both formulations to a final concentration of 0.1% (w/v) and the steady state release rate was measured according to ISO 15181 at between 34 and 70 ng cm-2 day-1 for both formulations. Field trials conducted over 3 months showed significant differences in anti-barnacle efficacy between the formulations despite their similar release profiles. The softer PV coating showed complete anti-barnacle efficacy, ie no barnacles were detected, while the harder PS coating showed no efficacy against barnacle colonisation during the same time period. The results indicate a new antifouling strategy whereby a route of intoxication is triggered by the organism itself upon interaction with the coating and its embedded biocide. This finding opens new possibilities in controlling macrofouling by low emission antifouling coatings.

In orthopaedic surgery, cobalt chromium (CoCr) based alloys are used extensively for their high strength and wear properties, but with concerns over stress shielding and bone resorption due to the high stiffness of CoCr. The structural stiffness, principally related to the bulk and the elastic modulus of the material, may be lowered by appropriate design modifications, to reduce the stiffness mismatch between metal/alloy implants and the adjacent bone. Here, 3D printed CoCr and Ti6Al4V implants of similar macro-geometry and interconnected open-pore architecture prepared by electron beam melting (EBM) were evaluated following 26 week implantation in adult sheep femora. Despite higher total bone-implant contact for Ti6Al4V (39 ± 4%) than CoCr (27 ± 4%), bone formation patterns were similar, e.g., densification around the implant, and gradual ingrowth into the porous network, with more bone in the outer half (periphery) than the inner half (centre). Raman spectroscopy revealed no major differences in mineral crystallinity, the apatite-to-collagen ratio, or the carbonate-to-phosphate ratio. Energy dispersive X-ray spectroscopy showed similar Ca/P ratio of the interfacial tissue adjacent to both materials. Osteocytes made direct contact with CoCr and Ti6Al4V. While osteocyte density and distribution in the new-formed bone were largely similar for the two alloys, higher osteocyte density was observed at the periphery of the porous network for CoCr, attributable to slower remodelling and a different biomechanical environment. The results demonstrate the possibility to achieve bone ingrowth into open-pore CoCr constructs, and attest to the potential for fabricating customised osseointegrated CoCr implants for load-bearing applications. Statement of Significance Although cobalt chromium (CoCr) based alloys are used extensively in orthopaedic surgery, stress shielding due to the high stiffness of CoCr is of concern. To reduce the stiffness mismatch between CoCr and bone, CoCr and Ti6Al4V implants having an interconnected open-pore architecture were prepared by electron beam melting (EBM). After six months of submerged healing in sheep, both alloys showed similar patterns of bone formation, with densification around the implant and gradual ingrowth into the porous network. The molecular and elemental composition of the interfacial tissue was similar for both alloys. Osteocytes made direct contact with both alloys, with similar overall osteocyte density and distribution. The work attests to the potential for achieving osseointegration of EBM manufactured porous CoCr implants.

The osteocyte network, through the numerous dendritic processes of osteocytes, is responsible for sensing mechanical loading and orchestrates adaptive bone remodelling by communicating with both the osteoclasts and the osteoblasts. The osteocyte network in the vicinity of implant surfaces provides insight into the bone healing process around metallic implants. Here, we investigate whether osteocytes are able to make an intimate contact with topologically modified, but micrometre smooth (Sa < 0.5 µm) implant surfaces, and if sub-micron topography alters the composition of the interfacial tissue. Screw shaped, commercially pure (cp-Ti) titanium implants with (i) machined (Sa = ~0.2 µm), and (ii) two-step acid-etched (HF/HNO3 and H2SO4/HCl; Sa = ~0.5 µm) surfaces were inserted in Sprague Dawley rat tibia and followed for 28 days. Both surfaces showed similar bone area, while the bone-implant contact was 73 % higher for the acid-etched surface. By resin cast etching, osteocytes were observed to maintain a direct intimate contact with the acid-etched surface. Although well mineralised, the interfacial tissue showed lower Ca/P and apatite-to-collagen ratios at the acid-etched surface, while mineral crystallinity and the carbonate-to-phosphate ratios were comparable for both implant surfaces. The interfacial tissue composition may therefore vary with changes in implant surface topography, independently of the amount of bone formed. Implant surfaces that influence bone to have higher amounts of organic matrix without affecting the crystallinity or the carbonate content of the mineral phase presumably result in a more resilient interfacial tissue, better able to resist crack development during functional loading than densely mineralised bone.