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In this study, the effect of inorganic polyphosphate (polyP) on the initial phase of angiogenesis and vascularization was investigated, applying the HUVEC cell tube formation assay. PolyP is a physiological and high energy phosphate polymer which has been proposed to act as a metabolic fuel in the extracellular space with only a comparably low ATP content. The experiments revealed that polyP accelerates tube formation of human umbilical vein endothelial cells (HUVEC), seeded onto a solidified basement membrane extract matrix which contains polyP-metabolizing alkaline phosphatase (ALP) activity. This effect is abolished by co-addition of apyrase, which degrades ATP to AMP and inorganic phosphate. The assumption that ATP, derived from polyP, activates HUVEC cells leading to tube formation was corroborated by experiments showing that addition of polyP to the cells causes a strong rise of ATP level in the culture medium. Finally, we show that at a later stage of cultivation of HUVEC cells, after 3 d, polyP causes a strong enhancement of the expression of the genes encoding for the two major matrix metalloproteinases (MMPs) released by endothelial cells during tube formation, MMP-9 and MMP-2. This stimulatory effect is again abrogated by addition of apyrase together with polyP. From these results, we propose that polyP is involved either directly or indirectly in energy supply, via ALP-mediated transfer of energy-rich phosphate under ATP formation. This ATP is utilized for the activation and oriented migration of endothelial cells and for the matrix organization during the initial phases of tube formation.

Objective In this study we demonstrate that inorganic polyphosphate (polyP) exhibits a dual protective effect on teeth: it elicits a strong antibacterial effect against the cariogenic bacterium Streptococcus mutans and, in form of amorphous calcium polyP microparticles (size of 100–400 nm), it efficiently reseals cracks/fissures in the tooth enamel and dentin. Methods Three different formulations of amorphous polyP microparticles (Ca-polyP, Zn-polyP and Sr-polyP) were prepared. Results Among the different polyP microparticles tested, the Ca-polyP microparticles, as a component of a newly developed formulation of a dentifrice, turned out to be most effective in inhibiting growth of S. mutans . Further studies have shown that it is mainly the soluble polyP, which has a strong antibacterial activity, either given as sodium salt of polyP or formed by partial disintegration of the microparticles via the alkaline phosphatase present in the oropharyngeal cavity. In addition, we demonstrate that the developed toothpaste containing incorporated amorphous polyP microparticles, efficiently reduces dental biofilm formation. Significance From our results we conclude that polyP microparticles, if added to toothpaste in an amorphous state, might be beneficial not only for restoring tooth damages but also because they provide a suitable depot of functionally/antibacterially active soluble polyP.

We describe the fabrication of a new scaffold, an inorganic–organic hybrid biomaterial, consisting of the physiological polymers: the inorganic polymer polyphosphate (polyP), as well as the organic macromolecules collagen and chondroitin sulfate. The polyP polymer is composed of multiple phosphate orthophosphate units linked together by high-energy phosphoanhydride bonds. Chondroitin sulfate has been included due to its hydrogel-forming properties. In the presence of Ca2+ ions, the randomly coiled polyP reorganizes together with collagen and chondroitin sulfate to linear molecules which undergo hardening. This scaffold is deposited as amorphous Ca–polyP nanoparticles (size ≈20–40 nm large) around the collagen fibers. Applying the method of freeze-extraction a stable macroporous 3D hydrogel scaffold with a Young's modulus of ≈0.4 MPa is formed comprising cavities/channels larger than 75 μm. The scaffold exhibits a pronounced capacity for induction of cell proliferation (primary human osteoblasts, phOSB) and differentiation (alkaline phosphatase gene expression). We propose that incorporation of the physiological polymer polyP into the collagen-based scaffold might allow a dynamic opening and resealing and by that facilitates the exchange of nutrients for the growth of cells. This physiological hybrid biomaterial might have the potential to be used in bone and cartilage repair.

In this study, we investigated the effect of the two flavonoids, baicalin (baicalein 7-O-[Formula: see text]- d-glucuronic acid) and its aglycone, baicalein (5,6,7-trihydroxyflavone), after encapsulation into amorphous calcium polyphosphate (Ca-polyP) microparticles on mineralization of primary human osteoblasts (phOSB). Both flavonoids, which come from root extracts of Scutellaria baicalensis Georgi, are used in Traditional Chinese Medicine, and are nontoxic in cells up to a concentration of 3[Formula: see text][Formula: see text]g/ml. The morphogenetically active, energy-rich Ca-polyP particles with a stoichiometric P:Ca ratio of 1:2 are degraded by cellular alkaline phosphatase (ALP) to ortho-phosphate used for bone hydroxyapatite formation. Here we show that the flavone-loaded Ca-polyP microparticles are readily taken up by phOSB, resulting in the accumulation of polyP around the nuclei and the formation of intracellular vesicles containing the ALP. In addition, we demonstrate that baicalin/baicalein causes a rise of the intracellular calcium [Ca[Formula: see text]]i a level which markedly is augmented after encapsulation into Ca-polyP, through activation of the phospholipase C. Moreover, both flavones, either alone or associated with Ca-polyP microparticles, upregulate the expression of the osteoblast calcium efflux channel, the plasma membrane Ca[Formula: see text]-ATPase (PMCA), while the expression of ALP, which promotes bone mineralization, is induced by Ca-polyP and by the flavones only if present in the Ca-polyP-microparticle-associated form. As a result, the extent of bone mineralization is markedly enhanced. Based on the two-armed activating function, new applications of baicalin/baicalein as a component of nutriceuticals for osteoporosis prevention or bone implants can be envisaged.

Actuellement, l’impact des interventions therapeutiques basees sur la pleine conscience commence a etre documente en population pediatrique [1] . Le refus scolaire anxieux est un motif extremement frequent de consultation en pedopsychiatrie, mais aucun consensus n’est etabli concernant sa prise en charge therapeutique. L’objectif de notre etude etait d’etudier l’impact d’un programme de regulation emotionnelle base sur la pleine conscience (START’UP) dans une population clinique d’adolescents avec refus scolaire anxieux. Dix patients presentant une descolarisation totale ou partielle d’origine anxieuse ont participe en groupe au programme START’UP compose de 11 seances hebdomadaires de 2 heures. Au debut (T1) et a la fin (T2) du programme, les patients remplissaient des auto-questionnaires evaluant la depression chez l’adolescent (ADRS version patient), l’anxiete (MASC) et l’evitement experientiel (AFQY). Deux hetero-questionnaires ont egalement ete remplis a T1 et T2 lors d’entretiens semi-structures afin d’evaluer l’anxiete (Echelle d’anxiete de Hamilton) la depression chez l’adolescent (ADRS version clinicien), et l’evolution de la descolarisation. Les resultats indiquent que la symptomatologie depressive a diminue de maniere significative en hetero-questionnaire (p = 0,028) et en auto-questionnaire (p = 0,021). Concernant l’anxiete, une baisse significative est constatee en hetero-questionnaire (p = 0,004) ainsi que pour les composantes « symptomes physiques » (p = 0,014) et « anxiete de separation » (p = 0,033) en auto-questionnaire. Les scores d’evitement baissent egalement significativement (p = 0,017). Une augmentation du temps scolaire est constatee cliniquement. Cette etude montre l’interet de poursuivre les investigations concernant les approches de type regulation emotionnelle basee sur la pleine conscience dans la prise en charge du refus scolaire anxieux chez l’adolescent.

Inorganic polyphosphates (polyP) consist of linear chains of orthophosphate residues, linked by high-energy phosphoanhydride bonds. They are evolutionarily old biopolymers that are present from bacteria to man. No other molecule concentrates as much (bio)chemically usable energy as polyP. However, the function and metabolism of this long-neglected polymer are scarcely known, especially in higher eukaryotes. In recent years, interest in polyP experienced a renaissance, beginning with the discovery of polyP as phosphate source in bone mineralization. Later, two discoveries placed polyP into the focus of regenerative medicine applications. First, polyP shows morphogenetic activity, i.e., induces cell differentiation via gene induction, and, second, acts as an energy storage and donor in the extracellular space. Studies on acidocalcisomes and mitochondria provided first insights into the enzymatic basis of eukaryotic polyP formation. In addition, a concerted action of alkaline phosphatase and adenylate kinase proved crucial for ADP/ATP generation from polyP. PolyP added extracellularly to mammalian cells resulted in a 3-fold increase of ATP. The importance and mechanism of this phosphotransfer reaction for energy-consuming processes in the extracellular matrix are discussed. This review aims to give a critical overview about the formation and function of this unique polymer that is capable of storing (bio)chemically useful energy.

Introduction/Background Endometrial carcinoma (EC) is the most frequent gynecological malignancy in the developed world. Currently there are no valid non-invasive diagnostic or prognostic methods available and therefore, diagnosis and treatment of EC patients is guided by histopathological and surgical findings. The lack of non-invasive diagnostic and prognostic biomarkers of EC is addressed in the current clinical study titled ‘Biomarkers for Diagnosis and Prognosis of Endometrial Carcinoma’ (BioEndoCar). Methodology Prospective observational case - control study. Patient recruitment takes place at three medical centers (University Medical Centre Ljubljana, Slovenia; Maastricht University, The Netherlands; Lublin Medical University, Poland). Plasma samples from women with diagnosed EC and controls will be examined using non-targeted and targeted metabolomics (Helmholtz Zentrum Munchen, Germany) and semi-targeted proteomics approaches (Sciomics GmbH, Heidelberg, Germany). Combined blood metabolome (>850 metabolites), proteome (900 different proteins), clinical and epidemiological data will be analyzed (University of Tartu, Estonia) in order to construct diagnostic/prognostic algorithms for early diagnosis of EC and to identify patients with low/high risk for cancer progression and recurrence. Results BioEndoCar project has defined inclusion/exclusion criteria and a strict standard operating procedure for sample collection, processing and sample storage that is followed by in all three medical centers. Since the beginning of the project (April 1st 2018) we recruited nearly 100 patients in total. Great effort was put into informing the lay and expert public about the importance of the translational studies in EC. We have established an official website (https://bioendocar.eu/) and Facebook page where we post all news concerning the project. Conclusion We expect to find different metabolic and protein profiles in patients with early stages of EC as compared to controls and in patients with poor prognosis with high risk of disease progression and recurrence as compared to those with favorable prognosis. The BioEndoCar project is currently ongoing. Disclosure Nothing to disclose.

Halogenated L- or D-tryptophan obtained by biocatalytic halogenation was incorporated into RGD peptides together with a variety of alkyl or aryl boronic acids. Suzuki-Miyaura cross-coupling either in solution or on-resin results in side chain-to-tail-cyclized RGD peptides, for example, with biaryl moieties, providing a new dimension of structure-activity relationships. An array of RGD peptides differing in macrocycle size, the presence of D-amino acid, N-methylation, or connectivity between the indole moiety and the boronic acid showed that, in particular, connectivity exhibits a major impact on affinities toward integrins, for example, a(v)beta(3). Structure-activity relationship studies yielded peptides with affinities toward a(v)beta(3) in the low nanomolar range, good selectivity, and high plasma stability. Structural characteristics of representative molecules have been investigated by molecular dynamics simulations, which allowed understanding the observed activity differences.

Impaired regeneration of the corneal epithelium, as found in many ocular surface diseases, is a major clinical problem in ophthalmology. We hypothesized that corneal epithelial regeneration can be promoted by the physiological, energy-delivering as well as “morphogenetically active” polymer, inorganic polyphosphate (polyP). Corneal limbal explants (diameter, 4 mm) were cultivated on collagen-coated well plates in the absence or presence of polyP (chain length, ∼40 Pi units; 50 μg ml−1) or human platelet lysate (hp-lysate; 5% v/v). Cell outgrowth and differentiation were analyzed after staining with DRAQ5 (nuclei) and rhodamine phalloidin (cytoskeleton), as well as by environmental scanning electron microscopy (ESEM). Cell growth/viability of hCECs was assessed by XTT assay. The expression of SDF-1 was quantitated by qRT-PCR. Exposure to hp-lysate (also containing polyP) increased cell migration already at day 1. Even stronger was the effect of polyP. This effect was blocked by a mast cell serine protease. The formation of cell multilayers was enhanced by hp-lysate or even more by polyP. ESEM revealed continuous cell junctions and prominent microvilli on the surface of adjacent cells exposed to polyP; those structures were only rarely seen in the controls. The hp-lysate and, more potently, polyP increased the proliferation of hCECs, as well as SDF-1 expression. The findings indicate the potential usefulness of the natural polymer, polyP, for topical treatment of corneal epithelial defects. Future studies are directed to develop suitable formulations of polyP, such as biomimetic polyP nano/microparticles showing an adjustable release kinetics.

BACKGROUND The diagnostic significance of the atopy patch test for the management of dermatitis possibly triggered by aeroallergens is still controversial. However, sufficiently large studies with routinely tested standardized aeroallergen patch test preparations in dermatitis patients are lacking. OBJECTIVE To evaluate the reaction frequency and the reaction profiles of 10 until mid-2015 commercially available, standardized aeroallergen patch test preparations of the 'Stallerpatch' test series (Stallergenes, Antony Cedex, France) in a large multicentre patient cohort. METHODS A retrospective data analysis of patients with suspected aeroallergen-dependent eczematous skin lesions was performed, who were patch tested in 15 Information Network of Departments of Dermatology-associated clinics between 2000 and 2015. Patients were stratified according to their atopic dermatitis (AD) status. RESULTS The study group included 3676 patients (median age 41 years, 34.8% males, 54.5% AD). The most common aeroallergens causing positive patch test reactions were Dermatophagoides pteronyssinus (19.6%), Dermatophagoides farinae (16.9%), birch (6.2%), timothy grass (6.0%), cat dander (5.4%), mugwort (4.9%) and dog dander (4.6%). Reactions to other pollen allergen preparations, that is 5 grasses (3.2%), cocksfoot (2.1%) and plantain (1.6%), were less common. Positive patch test reactions to aeroallergens were consistently more frequent in patients with AD. These patients showed proportionally less dubious, follicular, irritant and weak positive reactions. Independent of AD status, a patient history of past or present allergic rhinitis was associated with an increased chance of a positive aeroallergen patch test reaction to pollen allergens. CONCLUSION The aeroallergen patch test is a useful add-on tool in clinical routine, especially in patients with AD and/or respiratory allergy. A patch test series comprising Dermatophagoides pteronyssinus, Dermatophagoides farinae, birch, timothy grass, cat dander and mugwort seems to be suitable. Controlled studies with specific provocation and elimination procedures are required to further evaluate the diagnostic significance of the proposed screening series.

The preparation and characterization of a porous hybrid cryogel based on the two organic polymers, poly(vinyl alcohol) (PVA) and karaya gum (KG), into which polyphosphate (polyP) nanoparticles have been incorporated, are described. The PVA/KG cryogel is prepared by intermolecular cross‐linking of PVA via freeze‐thawing and Ca2+‐mediated ionic gelation of KG to form stable salt bridges. The incorporation of polyP as amorphous nanoparticles with Ca2+ ions (Ca‐polyP‐NP) is achieved using an in situ approach. The polyP constituent does not significantly affect the viscoelastic properties of the PVA/KG cryogel that are comparable to natural soft tissue. The exposure of the Ca‐polyP‐NP within the cryogel to medium/serum allows the formation of a biologically active polyP coacervate/protein matrix that stimulates the growth of human mesenchymal stem cells in vitro and provides the cells a suitable matrix for infiltration superior to the polyP‐free cryogel. In vivo biocompatibility studies in rats reveal that already two to four weeks after implantation into muscle, the implant regions containing the polyP‐KG/PVA material become replaced by initial granulation tissue, whereas the controls are free of any cells. It is proposed that the polyP‐KG/PVA cryogel has the potential to become a promising implant material for soft tissue engineering/repair.

Adams-Oliver syndrome (AOS) is a rare congenital disease characterized by aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It shows significant genetic heterogeneity and can be transmitted by autosomal dominant or recessive inheritance. Recessive inheritance is associated with mutations of DOCK6 or EOGT; however, only few cases have been published so far. We present two families with EOGT-associated AOS. Due to pseudodominance in one family, the recognition of the recessive inheritance pattern was difficult. We identified two novel AOS-causing mutations (c.404G>A/p.Cys135Tyr and c.311+1G>T). The phenotype in the presented families was dominated by large ACC, whereas TTLD were mostly subtle or even absent and no major malformations occured. Our observations along with the previously published cases indicate that the two types of recessive AOS (EOGT- vs. DOCK6-associated) differ significanty regarding the frequency of neurologic or ocular deficits.

The enigmatic interplay between electronic and magnetic phenomena observed in many early experiments and outlined in Maxwell's equations propelled the development of modern electromagnetism

Hepatitis E virus (HEV) is one major cause of acute clinical hepatitis among humans throughout the world. In industrialized countries an increasing number of autochthonous HEV infections have been identified over the last years triggered by food borne as well as – to a much lower degree – by human to human transmission via blood transfusion. Pigs have been recognised as main reservoir for HEV genotype 3 (HEV-3), and zoonotic transmission to humans through undercooked/raw meat is reported repeatedly. The minimal infectious dose of HEV-3 for pigs is so far unknown.

Simple Summary Bleomycin (BLM) is a medication introduced used to treat various types of cancer, including testicular cancer, ovarian cancer, and Hodgkin’s disease. Its most serious side effect is pulmonary fibrosis and impaired lung function. Using A549 human lung cells it is shown that, in parallel to an increased cell toxicity and DNA damage, BLM causes a marked enlargement of the cell nucleus. This effect is abolished by inorganic polyphosphate (polyP), if this physiological polymer is administered together with BLM. The detoxification of BLM is–most likely–caused by the upregulation of the gene encoding the BLM hydrolase which inactivates BLM in vitro and in vivo. This study contributes also to a rational application in COVID-19 patients since polyP prevents binding of SARS-CoV-2 to host cells. Abstract The anti-cancer antitumor antibiotic bleomycin(s) (BLM) induces athyminic sites in DNA after its activation, a process that results in strand splitting. Here, using A549 human lung cells or BEAS-2B cells lunc cells, we show that the cell toxicity of BLM can be suppressed by addition of inorganic polyphosphate (polyP), a physiological polymer that accumulates and is released from platelets. BLM at a concentration of 20 µg ml−1 causes a decrease in cell viability (by ~70%), accompanied by an increased DNA damage and chromatin expansion (by amazingly 6-fold). Importantly, the BLM-caused effects on cell growth and DNA integrity are substantially suppressed by polyP. In parallel, the enlargement of the nuclei/chromatin in BLM-treated cells (diameter, 20–25 µm) is normalized to ~12 µm after co-incubation of the cells with BLM and polyP. A sequential application of the drugs (BLM for 3 days, followed by an exposure to polyP) does not cause this normalization. During co-incubation of BLM with polyP the gene for the BLM hydrolase is upregulated. It is concluded that by upregulating this enzyme polyP prevents the toxic side effects of BLM. These data might also contribute to an application of BLM in COVID-19 patients, since polyP inhibits binding of SARS-CoV-2 to cellular ACE2.

Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were prepared and loaded with the secondary plant metabolite quercetin or with dexamethasone to study their effects on the respiratory epithelium using human alveolar basal epithelial A549 cells as a model. The results revealed that both compounds embedded into the polyP nanoparticles significantly increased the steady-state-expression of the MUC5AC gene. This mucin species is the major mucus glycoprotein present in the secreted gel-forming mucus. The level of gene expression caused by quercetin or with dexamethasone, if caged into polyP NP, is significantly higher compared to the individual drugs alone. Both quercetin and dexamethasone did not impair the growth-supporting effect of polyP on A549 cells even at concentrations of quercetin which are cytotoxic for the cells. A possible mechanism of the effects of the two drugs together with polyP on mucin expression is proposed based on the scavenging of free oxygen species and the generation of ADP/ATP from the polyP, which is needed for the organization of the protective mucin-based mucus layer.

The mucus layer of the nasopharynx and bronchial epithelium has a barrier function against inhaled pathogens such as the coronavirus SARS-CoV-2. We recently found that inorganic polyphosphate (polyP), a physiological, metabolic energy (ATP)-providing polymer released from blood platelets, blocks the binding of the receptor binding domain (RBD) to the cellular ACE2 receptor in vitro. PolyP is a marine natural product and is abundantly present in marine bacteria. Now, we have approached the in vivo situation by studying the effect of polyP on the human alveolar basal epithelial A549 cells in a mucus-like mucin environment. These cells express mucins as well as the ectoenzymes alkaline phosphatase (ALP) and adenylate kinase (ADK), which are involved in the extracellular production of ATP from polyP. Mucin, integrated into a collagen-based hydrogel, stimulated cell growth and attachment. The addition of polyP to the hydrogel significantly increased cell attachment and also the expression of the membrane-tethered mucin MUC1 and the secreted mucin MUC5AC. The increased synthesis of MUC1 was also confirmed by immunostaining. This morphogenetic effect of polyP was associated with a rise in extracellular ATP level. We conclude that the nontoxic and non-immunogenic polymer polyP could possibly also exert a protective effect against SARS-CoV-2-cell attachment, first, by stimulating the innate antiviral response by strengthening the mucin barrier with its antimicrobial proteins, and second, by inhibiting virus attachment to the cells, as deduced from the reduction in the strength of binding between the viral RBD and the cellular ACE2 receptor.

Graphical abstract The inorganic physiological polymer, polyphosphate, blocks binding of SARS-CoV-2 spike protein to ACE2 receptor at physiological concentrations. This discovery proposes polyphosphate as a new member of the host's antiviral innate immune defense., Inorganic polyphosphate (polyP) is a morphogenetically active and metabolic energy-delivering physiological polymer that is released from blood platelets. Here, we show that polyP efficiently inhibits the binding of the envelope spike (S)-protein of the coronavirus SARS-CoV-2, the causative agent of COVID-19, to its host cell receptor ACE2 (angiotensin-converting enzyme 2). To stabilize polyP against the polyP-degrading alkaline phosphatase, the soluble polymer was encapsulated in silica/polyP nanoparticles. Applying a binding assay, soluble Na-polyP (sizes of 40 Pi and of 3 Pi units) as well as silica-nanoparticle-associated polyP significantly inhibit the interaction of the S-protein with ACE2 at a concentration of 1 µg/mL, close to the level present in blood. This inhibition is attributed to an interaction of polyP with a basic amino acid stretch on the surface of the receptor binding domain of S-protein. PolyP retains its activity in a flushing solution, opening a new strategy for the prevention and treatment of SARS-CoV-2 infection in the oropharyngeal cavity. The data suggest that supplementation of polyP might contribute to a strengthening of the human innate immunity system in compromised, thrombocytopenic COVID-19 patients.

We present a catalogue of 261 new infrared selected members of the 3C129 galaxy cluster. The cluster, located at $z \approx$ 0.02, forms part of the Perseus-Pisces filament and is obscured at optical wavelengths due to its location in the zone of avoidance. We identified these galaxies using the $J-$ and $K-$band imaging data provided by the UKIDSS Galactic Plane Survey within an area with a radius of $1.1^{\circ}$ centred on the X-ray emission of the cluster at $\ell, b \approx 160.52^{\circ}, 0.27^{\circ}$. A total of 26 of the identified galaxy members have known redshifts 24 of which are from our 2016 Westerbork HI survey and two are from optical spectroscopy. An analysis of the galaxy density at the core of the 3C129 cluster shows it to be less dense than the Coma and Norma clusters, but comparable to the galaxy density in the core of the Perseus cluster. From an assessment of the spatial and velocity distributions of the 3C129 cluster galaxies that have redshifts, we derived a velocity of $cz = 5227 \pm 171$ km/s and $\sigma = 1097 \pm 252$ km/s for the main cluster, with a substructure in the cluster outskirts at $cz = 6923 \pm 71$ km/s with $\sigma = 422 \pm 100$ km/s. The presence of this substructure is consistent with previous claims based on the X-ray analysis that the cluster is not yet virialised and may have undergone a recent merger., Comment: 33 pages, 16 figures, includes an appendix, accepted for publication in A&A

Inorganic polyphosphate (polyP) is a naturally occurring polymer that is bioresorbable and anabolically active on bone forming cells in vitro. In order to demonstrate if polyP also shows morphogenetic activity in vivo, animal studies are performed applying the rat calvarial defect model. Poly(D,L-lactide-co-glycolide) (PLGA) microspheres with a narrow size distribution (≈820 μm) are prepared, containing either encapsulated polyP or β-tricalcium phosphate (β-TCP), used as a reference material. Discs are prepared from the microspheres and inserted into 10 mm large defects created in the calvaria of rats. Both the formation of COL-I and the expression of ALP is upregulated, as well as the extent of mineralization within and around the polyP implant. Our data demonstrate that amorphous and resorbable inorganic polyP is also morphogenetically active in vivo.

There is increasing evidence that inorganic calcium-polyphosphates (polyP) are involved in human bone hydroxyapatite (HA) formation. Here we investigated the morphology of the particles, containing calcium phosphate (CaP) with different concentrations of various Na-polyP concentrations, as well as their effects in cell culture. We used both SaOS-2 cells and human mesenchymal stem cells. The polymeric phosphate readily binds calcium ions under formation of insoluble precipitates. We found that addition of low concentrations of polyP (10wt.%, referred to the CaP deposits) results in an increased size of the HA crystals. Surprisingly, at higher polyP concentrations (10wt.%) the formation of crystalline HA is prevented and amorphous polyP/HA hybrid particles with a size of ≈50nm are formed, most likely consisting of polyP molecules linked via Ca(2+) bridges to the surface of the CaP deposits. Further studies revealed that the polyP-CaP particles cause a strong upregulation of the expression of the genes encoding for two marker proteins of bone formation, collagen type I and alkaline phosphatase. Based on their morphogenetic activity the amorphous polyP-CaP particles offer a promising material for the development of bone implants, formed from physiological inorganic precursors/polymers.Hydroxyapatite (HA) is a naturally occurring mineral of vertebrate bone. Natural HA, a bio-ceramic material which is crystalline to different scale, has been used as a biomaterial to fabricate scaffolds for in situ bone regeneration and other tissue engineering purposes. In contrast to natural HA, synthetic apatite is much less effective. In general, while HA is bioactive, its interaction and biocompatibility with existing bone tissue is low. These properties have been attributed to a minimal degradability in the physiological environment. In the present study we introduce a new Ca-phosphate (CaP) fabrication technology, starting from calcium chloride and dibasic ammonium phosphate with the HA characteristic Ca/P molar ratio of 10:6 and report that after addition10% (by weight) of polyphosphate (polyP) amorphous CaP/HA samples were obtained. Those samples elicits strong morphogenetic activity let us to conclude that polyP/HA-based material might be beneficial for application as bone substitute implant.

Inorganic polyphosphate (polyP) is a physiological polymer composed of tens to hundreds of phosphate units linked together via phosphoanhydride bonds. Here we compared the biological activity of polyP (chain length of 40 phosphate units), complexed with Gd(3+) (polyP·Gd), with the one caused by polyP (as calcium salt) and by GdCl3 alone, regarding their potencies to induce hydroxyapatite (HA) formation in SaOS-2 cells in vitro. The three compounds, GdCl3, polyP and polyP·Gd were found to be non-toxic at concentrations up to at least 30μM. Selecting a low, 5μM, concentration it was found that polyP·Gd significantly induced HA formation, as determined by Alizarin Red S staining and by quantitative determinations using that dye. Under those conditions polyP·Gd and to a smaller extent also polyP or GdCl3 (5μM each) caused HA crystal formation arranged in a nest-like pattern. Exposure of cells to polyP·Gd resulted in a strong increase in alkaline phosphatase activity; this enzyme did not cause a distinct degradation of polyP but of polyP·Gd which was extensively hydrolyzed. The morphogenetic activity of gadolinium, in the form of polyP·Gd, is underscored by the finding that this polymer causes a strong upregulation of the genes encoding morphogenetic protein-2 (BMP2) as well as collagen type I. It is concluded that polyP·Gd is not an inert polymer but acts as a morphogenetically active polymer and induces HA formation in vitro.

Stichverletzungen stellen in der rechtsmedizinischen Praxis keine Seltenheit dar. Immer wieder wird in Kasuistiken uber ungewohnliche, bizarre oder zahlreiche Stich- und Schnittverletzungen berichtet, die die Frage aufwerfen: Homizid oder Suizid? Der Fall eines 34 Jahre alten Mannes, der sich mithilfe eines Samuraischwerts und eigens gebauter Stichkonstruktion suizidierte, erinnerte aufgrund der vorliegenden Szenerie an die alte, spirituell angehauchte Form der japanischen Selbsttotung, das „Seppuku“.

Here we describe an artificial cartilage-like material based on a hyaluronic acid-Mg/Ca-polyphosphate paste (HA-aMg/Ca-polyP-p) that is fabricated from a water-soluble Na-salt of energy-rich inorganic polyphosphate (polyP) and soluble hyaluronic acid in the presence of water-insoluble CaCO3. The resulting material, after conversion of Na-polyP into the less soluble Mg/Ca-salt consisting of amorphous Mg/Ca-polyP microparticles, was found to mimic the physiological cartilage tissue and to bind Ca2+ ions present in the synovial fluid. After the Mg2+/Ca2+ exchange and water extrusion, the polyP becomes more stable, but is still susceptible to hydrolytic cleavage by the alkaline phosphatase (ALP). The material shows biomechanical properties, comparable to cartilage. Treatment with CaCl2 resulted in an increase of the hardness (Young's modulus) from 1.27 MPa to 3.23 MPa. In addition, the CaCl2-treated material showed a faster stress increase, almost no micro-crumbling and an extended creep period and elastic/viscoelastic recovery period. Moreover, the material exhibits morphogenetic activity through upregulation of the marker genes in chondrocytes encoding for ALP, collagen 2A1, collagen 3A1 and aggrecan. We propose that Mg-polyP is a promising material for cartilage repair that is able to scavenge, from the synovial fluid, Ca2+ ions implicated in crystal formation in osteoarthritis patients, as well as hyaluronic acid, through Ca2+-bridge formation, and to act as a bonding material for cartilage and bone.

Peptidotriazolamers are hybrid foldamers with features of peptides and triazolamers, containing alternation of amide bonds and 1,4-disubstituted 1H-1,2,3-triazoles with conservation of the amino acid side chains. We report on the synthesis of a new class of peptidomimetics, containing 1,4-disubstituted 1H-1,2,3-triazoles in alternation with amide bonds and the elucidation of their conformational properties in solution. Based on enantiomerically pure propargylamines bearing the stereogenic center in the propargylic position and α-azido esters, building blocks were obtained by copper-catalyzed azide-alkyne cycloaddition. With these building blocks the peptidotriazolamers were readily available by solution phase synthesis. A panel of homo- and heterochiral tetramers, hexamers, and heptamers was synthesized and the heptamer Boc-Ala-Val-Ψ[4Tz]Phe-LeuΨ[4Tz]Phe-LeuΨ[4Tz]Val-OAll as well as an heterochiral and a Gly-containing equivalent were structurally characterized by NMR-based molecular dynamics simulations using a specifically tailored force field to determine their conformational and solvation properties. All three variants adopt a compact folded conformation in DMSO as well as in water. In addition to the heptamers we predicted the conformational behavior of similar longer oligomers i.e., Boc-Ala-(AlaΨ[4Tz]Ala)6-OAll as well as Boc-Ala-(d-AlaΨ[4Tz]Ala)6-OAll and Boc-Ala-(GlyΨ[4Tz]Ala)6-OAll. Our calculations predict a clear secondary structure of the first two molecules in DMSO that collapses in water due to the hydrophobic character of the side chains. The homochiral compound folds into a regular helical structure and the heterochiral one shows a twisted “S”-shape, while the Gly variant exhibits no clear secondary structure.

L’insomnie chronique touche environ 19,8 % de la population et 10 % souffrent d’insomnie severe, survenant a tous les âges de la vie (enfants, adolescents, adultes et sujets âges) et dont la prise en charge variera en fonction de la physiologie du sommeil. L’insomnie represente un enjeu de sante publique majeure, notamment sur le plan cardiovasculaire et psychopathologique. L’insomnie est un facteur de risque de depression, participe aux rechutes et recidives de certaines pathologies psychiatriques, et est impliquee dans l’utilisation abusive des traitements psychotropes et leur prise au long cours. La prise en charge de l’insomnie est cependant un paradoxe en soi : alors que la frequence de l’insomnie est elevee, son cout socioeconomique non negligeable, et les consequences pour la sante importantes, le traitement (les therapies cognitivo-comportementales adaptees a l’insomnie) reconnu efficace de longue date, reste peu accessible. A cela s’ajoute des campagnes virulentes contre les hypnotiques (notamment benzodiazepiniques et apparentes) alors que des prises en charges, comme la sophrologie, la relaxation, l’hypnose, la melatonine, la phytotherapie… sont accueillies avec une certaine bienveillance, en depit d’un niveau de preuve tres limite, voire absent. Le conseil scientifique de la SFRMS a constitue un groupe de travail d’experts en insomnie et psychopathologies associees, comportant des psychiatres, des pedopsychiatres, des pediatres, des medecins generalistes, des pneumologues, des medecins du travail, des geriatres, des psychologues, des chercheurs (homme et animal) afin de proposer un consensus et des recommandations concernant la conduite a tenir devant une insomnie chronique quelles que soient les comorbidites associees, et en particulier des comorbidites psychopathologiques. La presentation de ce consensus sera l’objet de ce symposium propose par la SFRMS : Isabelle Poirot presentera le consensus sur la prise ne charge de l’insomnie en psychiatrie d’adultes et Carmen Schroder en pedopsychiatrie.

Background: Childhood asthma prevalence is significantly greater in urban areas compared with rural/farm environments. Murine studies have shown that TNF-alpha-induced protein 3 (TNFAIP3; A20), an anti-inflammatory regulator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) signaling, mediates environmentally induced asthma protection. Objective: We aimed to determine the role of TNFAIP3 for asthma development in childhood and the immunomodulatory effects of environmental factors. Methods: In a representative selection of 250 of 2168 children from 2 prospective birth cohorts and 2 cross-sectional studies, we analyzed blood cells of healthy and asthmatic children from urban and rural/farm environments from Europe and China. PBMCs were stimulated ex vivo with dust from "asthma-protective'' farms or LPS. NF-kappa B signaling-related gene and protein expression was assessed in PBMCs and multiplex gene expression assays (NanoString Technologies) in isolated dendritic cells of schoolchildren and in cord blood mononuclear cells from newborns. Results: Anti-inflammatory TNFAIP3 gene and protein expression was consistently decreased, whereas proinflammatory Toll-like receptor 4 expression was increased in urban asthmatic patients (P

To complement the 2MASS Redshift Survey (2MRS) and the 2MASS Tully-Fisher survey (2MTF) a search for 21cm HI line emission of 2MASS bright galaxy candidates has been pursued along the dust-obscured plane of the Milky Way with the 100m Nancay Radio Telescope. For our sample selection we adopted an isophotal extinction-corrected K-band magnitude limit of $K_s^o = 11.25$mag, corresponding to the first 2MRS data release and 2MTF, for which the 2MASX completeness level remains fairly constant deep into the Zone of Avoidance (ZoA). About one thousand galaxies without prior redshift measurement accessible from Nancay (Dec > -40\deg) were observed to an rms noise level of ~3 mJy for the velocity range -250 to 10'600 km/s. This resulted in 220 clear and 12 marginal detections of the target sample. Only few detections have redshifts above 8000 km/s due to recurring radio frequency interference (RFI). A further 29 detections and 6 marginals have their origin in non-target galaxies in the telescope beam. The newly detected galaxies are on average considerably more \HI-rich (mostly $10^9 - 10^{10}$M$_\odot$) compared to systematic (blind) HI surveys. The HI detections reveal various new filaments crossing the mostly uncharted northern ZoA (e.g. at $\ell \sim 90\deg, 130\deg, 160\deg$), whilst consolidating galaxy agglomerations in Monoceros and Puppis ($\ell \sim 220\deg, 240\deg$). Considerably new insight has been gained about the extent of the Perseus-Pisces Supercluster through the confirmation of a ridge ($\ell \sim 160\deg$) encompassing the 3C129 cluster that links Perseus-Pisces to Lynx, and the continuation of the second Perseus-Pisces arm ($\ell \sim 90\deg$) across the ZoA., Comment: 19 pages, 11 figures, 7 tables; accepted for publication in MNRAS; supplementary material available at http://www.acgc.uct.ac.za/~kraan/NRT_ZOA/Supplement.pdf

Objective In the present study, we investigated the fusion process between amorphous microparticles of the calcium salt of the physiological polymer comprising orthophosphate units, of inorganic polyphosphate (polyP), and enamel. Methods This polymer was incorporated as an ingredient into toothpaste and the fusion process was studied by electron microscopy and by synchrotron-based X-ray tomography microscopy (SRXTM) techniques. Results The data showed that toothpaste, supplemented with the amorphous Ca-polyP microparticles (aCa-polyP-MP), not only reseals tooth defects on enamel, like carious lesions, and dentin, including exposed dentinal tubules, but also has the potential to induce re-mineralization in the enamel and dentin regions. The formation of a regeneration mineralic zone on the tooth surface induced by aCa-polyP-MP was enhanced upon exposure to artificial saliva, as demonstrated by SRXTM. Energy dispersive X-ray analysis revealed an increase in the calcium/phosphorus atomic ratio of the enamel deposits to values characteristic for the particles during the treatment with polyP applied in the toothpaste, indicating a fusion of the particles with the tooth mineral. Significance Our results suggest that toothpaste enriched with aCa-polyP-MP is a promising biomimetic material for accelerating enamel and dentin restoration.

Inorganic polyphosphate [polyP] has proven to be a promising physiological biopolymer for potential use in regenerative medicine because of its morphogenetic activity and function as an extracellular energy-donating system. Amorphous Ca2+ -polyP nanoparticles [Ca-polyP-NPs] are characterized by a high zeta potential with -34 mV (at pH 7.4). This should contribute to the stability of suspensions of the spherical nanoparticles (radius 94 nm), but make them less biocompatible. The zeta potential decreases to near zero after exposure of the Ca-polyP-NPs to protein/peptide-containing serum or medium plus serum. Electron microscopy analysis reveals that the particles rapidly change into a coacervate phase. Those mats are amorphous, but less stable than the likewise amorphous Ca-polyP-NPs and are morphogenetically active. Mesenchymal stem cells grown onto the polyP coacervate show enhanced growth/proliferation and become embedded in the coacervate. These results suggest that the Ca-polyP coacervate, formed from Ca-polyP-NPs in the presence of protein, can act as an adaptable framework that mimics a niche and provides metabolic energy in bone/cartilage engineering.

The cosmic evolution of galaxies' neutral atomic gas content is a major science driver for the Square Kilometre Array (SKA), as well as for its South African (MeerKAT) and Australian (ASKAP) precursors. Among the HI large survey programs (LSPs) planned for ASKAP and MeerKAT, the deepest and narrowest tier of the "wedding cake" will be defined by the combined L-band+UHF-band Looking At the Distant Universe with the MeerKAT Array (LADUMA) survey, which will probe HI in emission within a single "cosmic vuvuzela" that extends to z = 1.4, when the universe was only a third of its present age. Through a combination of individual and stacked detections (the latter relying on extensive multi-wavelength studies of the survey's target field), LADUMA will study the redshift evolution of the baryonic Tully--Fisher relation and the cosmic HI density, the variation of the HI mass function with redshift and environment, and the connection between HI content and galaxies' stellar properties (mass, age, etc.). The survey will also build a sample of OH megamaser detections that can be used to trace the cosmic merger history. This proceedings contribution provides a brief introduction to the survey, its scientific aims, and its technical implementation, deferring a more complete discussion for a future article after the implications of a recent review of MeerKAT LSP project plans are fully worked out.

The in vitro tube formation assay with human umbilical vein endothelial cells (HUVEC) was applied to identify the extra- and intracellular sources of metabolic energy/ATP required for cell migration during the initial stage of microvascularization. Extracellularly, the physiological energy-rich polymer, inorganic polyphosphate (polyP), applied as biomimetic amorphous calcium polyP microparticles (Ca-polyP-MP), is functioning as a substrate for ATP generation most likely via the combined action of the alkaline phosphatase (ALP) and the adenylate kinase (AK). The linear Ca-polyP-MP with a size of 40 phosphate units, close to the polyP in the acidocalcisomes in the blood platelets, were found to increase endothelial cell tube formation, as well as the intracellular ATP levels. Depletion of extracellular ATP with apyrase suppressed tube formation during the initial incubation period. Inhibition experiments revealed that inhibitors (levamisole and Ap5A) of the enzymes involved in extracellular ATP generation strongly reduce the Ca-polyP-MP-induced tube formation. The stimulatory effect of Ca-polyP-MP was also diminished by the glycolysis inhibitor oxamate and trifluoperazine which blocks endocytosis, as well as by MRS2211, an antagonist of the P2Y13 receptor. Oligomycin, an inhibitor of the mitochondrial F0F1-ATP synthase, displayed no effect at lower concentrations on tube formation. Electron microscopic data revealed that after cellular uptake, the Ca-polyP-MP accumulate close to the cell membrane. We conclude that in HUVEC exposed to polyP, ATP is formed extracellularly via the coupled ALP-AK reaction, and intracellularly during glycolysis. The results suggest an autocrine signaling pathway of ATP with polyP as an extracellular store of metabolic energy for endothelial cell migration during the initial vascularization process.

Neutron diffraction is exquisitely sensitive to the positions of H atoms in protein crystal structures. IMAGINE is a high-intensity, quasi-Laue neutron crystallography beamline developed at the High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory. This state-of-the-art facility for neutron diffraction has enabled detailed structural analysis of macromolecules. IMAGINE is especially suited to resolve individual H atoms in protein structures, enabling neutron protein structures to be determined at or near atomic resolutions from crystals with volumes of less than 1 mm3 and unit-cell edges of less than 150 Å. Beamline features include elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2 mm focal spot at the sample position, and variable short- and long-wavelength cutoff optics that provide automated exchange between multiple wavelength configurations. This review gives an overview of the IMAGINE beamline at the HFIR, presents examples of the scientific questions being addressed at this beamline, and highlights important findings in enzyme chemistry that have been made using the neutron diffraction capabilities offered by IMAGINE.

Inorganic polyphosphate (polyP) is a physiological energy-rich polymer with multiple phosphoric anhydride bonds. In cells such as bone-forming osteoblasts, glycolysis is the main pathway generating metabolic energy in the form of ATP. In the present study, we show that, under hypoxic culture conditions, the growth/viability of osteoblast-like SaOS-2 cells is not impaired. The addition of polyP to those cells, administered as amorphous calcium polyP nanoparticles (aCa-polyP-NP; approximate size 100 nm), significantly increased the proliferation of the cells. In the presence of polyP, the cells produce significant levels of lactate, the end product of anaerobic glycolysis. Under those conditions, an eight-fold increase in the steady-state level of the membrane-associated carbonic anhydrase IX is found, as well as a six-fold induction of the hypoxia-inducible factor 1. Consequently, biomineral formation onto the SaOS-2 cells decreases under low oxygen tension. If the polyP nanoparticles are added to the cells, the degree of mineralization is enhanced. These changes had been measured also in human mesenchymal stem cells. The assumption that the bicarbonate, generated by the carbonic anhydrase in the presence of polyP under low oxygen, is deposited as a constituent of the bioseeds formed during initial hydroxyapatite formation is corroborated by the identification of carbon besides of calcium, oxygen and phosphorus in the initial biomineral deposit onto the cells using the sensitive technology of high-resolution energy dispersive spectrometry mapping. Based on these data, we conclude that polyP is required for the supply of metabolic energy during bone mineral formation under physiological, hypoxic conditions, acting as a 'metabolic fuel' for the cells to grow.

Background Early life farm exposures have been shown to decrease the risk of allergic diseases. Dendritic cells (DCs) may mediate asthma-protective effect of farm exposures as they play an important role in the development of immunity and tolerance. Our aim was to investigate whether the numbers and phenotypes of circulating DCs at age 6 are associated with farming, asthma, and atopy in a selected sample of French and Finnish children from the PASTURE study. Methods We studied 82 farm and 86 nonfarm children with and without asthma. Using flow cytometry, BDCA1+ CD11c+ myeloid DC1s (mDC1), BDCA3+high mDC2s and BDCA2+ plasmacytoid DCs (pDCs) were identified and expressions of CD86, immunoglobulin-like transcript 3 (ILT3) and ILT4 were analyzed. Questionnaires were used to assess prenatal and lifetime patterns of farm exposures and to define asthma. Atopic sensitization was defined by specific IgE measurements. Results The percentage of mDC2 cells was lower in farm children (0.033 ± 0.001) than in nonfarm children (0.042 ± 0.001; P = 0.008). Similar associations were found between mDC2 percentage and prenatal (P = 0.02) and lifetime exposure to farm milk (P = 0.03) and stables (P = 0.003), but these associations were not independent from farming. Asthma was positively associated with ILT4 + mDCs (P = 0.04) and negatively with CD86 + pDCs (P = 0.048) but only in nonfarm children. Conclusions Inverse association between farm exposure and mDC2 percentage suggest that this DC subset may play a role in farm-related immunoregulation.

The systematic assessment of the human immune system bears huge potential to guide rational development of novel immunotherapies and clinical decision making. Multiple assays to monitor the quantity, phenotype, and function of Ag-specific T cells are commonly used to unravel patients’ immune signatures in various disease settings and during therapeutic interventions. When compared with tests measuring soluble analytes, cellular immune assays have a higher variation, which is a major technical factor limiting their broad adoption in clinical immunology. The key solution may arise from continuous control of assay performance using TCR-engineered reference samples. We developed a simple, stable, robust, and scalable technology to generate reference samples that contain defined numbers of functional Ag-specific T cells. First, we show that RNA-engineered lymphocytes, equipped with selected TCRs, can repetitively deliver functional readouts of a controlled size across multiple assay platforms. We further describe a concept for the application of TCR-engineered reference samples to keep assay performance within or across institutions under tight control. Finally, we provide evidence that these novel control reagents can sensitively detect assay variation resulting from typical sources of error, such as low cell quality, loss of reagent stability, suboptimal hardware settings, or inaccurate gating.

Both the quality and quantity of collagen, the major structural component of the skin, decrease in aging skin. We succeeded to encapsulate retinol into amorphous inorganic polyphosphate (polyP) nanoparticles together with calcium ions ("aCa-polyP-NP"), under formation of amorphous Ca-polyP/retinol nanospheres ("retinol/aCa-polyP-NS"). The globular nanospheres are not cytotoxic, show an almost uniform size of ≈ 45 nm and have a retinol content of around 25%. Both components of those nanospheres, retinol and the aCa-polyP-NP, if administered together, caused a strong increase in proliferation of mouse calvaria MC3T3 cells. The expressions of collagen types I, II and III genes, but not the expression of collagen type V gene, were significantly enhanced if retinol is added together with aCa-polyP-NP. This synergistic effect was especially pronounced for the expression of the collagen type III gene. We propose that the synergistic effect of the retinol/aCa-polyP-NS on cell growth and collagen type III expression is induced via two routes, first through cellular uptake of the 45 nm nanospheres by clathrin-mediated endocytosis and second through extracellular disintegration of the nanospheres resulting in the release of retinol which is then taken up into the cells after binding to the retinal binding protein receptor.

Polyphosphate (polyP) is a physiologically occurring polyanion that is synthesized especially in bone-forming osteoblast cells and blood platelets. We used amorphous polyP nanoparticles, complexed with Ca2+, that have a globular size of ∼100 nm. Because polyP comprises inorganic orthophosphate units that are linked together through high-energy phosphoanhydride bonds, we questioned whether the observed morphogenetic effect, elicited by polyP, is correlated with the energy-generating machinery within the cells. We show that exposure of SaOS-2 osteoblast-like cells to polyP results in a strong accumulation of mitochondria and a parallel translocation of the polyP-degrading enzyme alkaline phosphatase to the cell surface. If SaOS-2 cells are activated by the mineralization activation cocktail (comprising β-glycerophosphate, ascorbic acid and dexamethasone) and additionally incubated with polyP, a tenfold intracellular increase of the ATP level occurs. Even more, in those cells, an intensified release of ATP into the extracellular space is also seen. We propose and conclude that polyP acts as metabolic fuel after the hydrolytic cleavage of the phosphoanhydride linkages, which contributes to hydroxyapatite formation on the plasma membranes of osteoblasts.

The initial mineralization centers during human bone formation onto osteoblasts are composed of CaCO3 . Those bioseeds are enzymatically formed via carbonic anhydrase(s) in close association with the cell surface of the osteoblasts. Subsequently, the bicarbonate/carbonate anions are exchanged non-enzymatically by inorganic phosphate [Pi ]. One source for the supply of Pi is polyphosphate [polyP] which is a physiological polymer, formed in the osteoblasts as well as in the platelets. The energy-rich acid anhydride bonds within the polyP chain are cleaved by phosphatase(s); during this reaction free-energy might be released that could be re-used, as metabolic fuel, for the maintenance of the steady-state concentrations of the substrates/products during mineralization. Finally it is outlined that polyP, as a morphogenetically active scaffold, is even suitable for 3D cell printing.

As a further step towards a new generation of bone implant materials, we developed a procedure for biological functionalization of titanium alloy surfaces with inorganic calcium polyphosphate (Ca-polyP). This polymer has been demonstrated to exhibit morphogenetic activity. The coating of titanium oxidized Ti-6Al-4V scaffolds with biologically active amorphous Ca-polyP microparticles is formed by Ca2+ ion bridges to the silane coupling agent APTMS. This surface is durable and stable as an almost homogeneous Ca-polyP layer onto the metal. The homogenously coated Ca-polyP titanium scaffold was found to be biologically active and supported the growth and functional activity of bone cell-related SaOS-2 cells despite the reduced surface roughness, while the non-modified titanium surfaces are biologically inert. Quantitative qRT-PCR experiments revealed that the Ca-polyP coated titanium alloy markedly increased the steady-state levels of transcripts of the two marker enzymes involved in initiation of bone mineral deposition, the carbonic anhydrase IX and the alkaline phosphatase. The innovative coating of titanium alloy with Ca-polyP offers a promising technique for the fabrication of morphogenetically active bone implants with advantageous mechanical and regeneration-inducing properties.

Biomimetic materials have been gaining increasing importance in tissue engineering since they may provide regenerative alternatives to the use of autologous tissues for transplantation. In the present study, we applied for bioprinting of a functionalized three-dimensional template, N,O-carboxymethyl chitosan (N,O-CMC), mimicking the physiological extracellular matrix. This polymer, widely used in tissue engineering, has been provided with functional activity by integration of polyphosphate (polyP), an osteogenically acting natural polymer. The two polymers, N,O-CMC and polyP, are linked together via Ca2+ bridges. This N,O-CMC + polyP material was proven to be printable and durable. The N,O-CMC + polyP printed layers and tissue units retain their properties to induce SaOS-2 bone-like cells to biomineralization. Subsequent in vivo experiments revealed a strong regeneration-inducing activity of the material in the rat calvarial defect model. In turn, N,O-CMC + polyP represents a promising hybrid material useful as a potential custom-designed scaffold for alternative tissue-engineering solutions.

Background Laccases are copper-containing enzymes that catalyze the oxidation of a wide variety of phenolic substrates. Methods We describe the first poriferan laccase from the marine demosponge Suberites domuncula. Results This enzyme comprises three characteristic multicopper oxidase homologous domains. Immunohistological studies revealed that the highest expression of the laccase is in the surface zone of the animals. The expression level of the laccase gene is strongly upregulated after exposure of the animals to the bacterial endotoxin lipopolysaccharide. To allow the binding of the recombinant enzyme to ferromagnetic nanoparticles, a recombinant laccase was prepared which contained in addition to the His-tag, a Glu-tag at the N-terminus of the enzyme. The recombinant laccase was enzymatically active. The apparent Michaelis constant of the enzyme is 114 μM, using syringaldazine as substrate. Exposure of E. coli to the nanoparticles, coated with Glu-tagged laccase, and to the mediator 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in the presence of lignin, as the oxidizable substrate, resulted in an almost complete inhibition of colony formation. Quantitative studies of the effect of the laccase-coated iron oxide nanoparticles were performed using E. coli grown in suspension in reaction tubes within a magnetic nanoparticle separator. Conclusions This newly designed magnetic nanoparticle separator allowed a removal of the nanoparticles after terminating the reaction. Using this system, a strong dose-dependent inhibition of the growth of E. coli by the laccase iron oxide nanoparticles was determined. General significance From our data we conclude that the sponge laccase is involved in the anti-bacterial defense of the sponge organism.