VenoMS-A Website for the Low Molecular Mass Compounds in Spider Venoms.

Spider venoms are highly complex mixtures. Numerous spider venom metabolites are uniquely found in spider venoms and are of interest concerning their potential use in pharmacology, agriculture, and cosmetics. A nontargeted ultra-high performance high-resolution electrospray tandem mass spectrometry (UHPLC-HR-ESI-MS/MS) approach offers a resource-saving way for the analysis of crude spider venom. However, the identification of known as well as the structure elucidation of unknown low molecular mass spider venom compounds based on their MS/MS spectra is challenging because (1) acylpolyamine toxins are exclusively found in spider and wasp venom, (2) reference MS/MS spectra are missing in established mass spectrometry databases, and (3) trivial names for the various toxin metabolites are used in an inconsistent way in literature. Therefore, we introduce the freely accessible MS website for low molecular mass spider venom metabolites, venoMS, containing structural information, MS/MS spectra, and links to related literature. Currently the database contains the structures of 409 acylpolyamine toxins, 36 free linear polyamines, and 81 additional spider venom metabolites. Implemented into this website is a fragment ion calculator (FRIOC) that allows us to predict fragment ions of linear polyamine derivatives. With three metabolites from the venom of the spider Agelenopsis aperta, it was demonstrated how the new website can support the structural elucidation of acylpolyamines using their MS/MS spectra.

The influence of different artificial extracellular matrix implant coatings on the regeneration of a critical size femur defect in rats.

Resorbable biomaterials based on artificial extracellular matrices (aECM) represent promising scaffolds for the treatment of large bone defects. Here, we investigated various glycosaminoglycan (GAG) derivatives of varying sulfation degree with respect to their influence on in vivo bone healing. The materials used in this study consisted of GAG-coated degradable polycaprolactone-co-lactide (PCL). Critical size femur defects in rats were filled with autologous bone serving as positive control or the respective coated or uncoated PCL scaffolds. After 2 and 12 weeks, progress in the healing process was investigated by analyzing the new bone matrix formation, the collagen content and hydroxyapatite formation by using micro-computed tomography (µCT), biomechanical testing, nuclear magnetic resonance spectroscopy (NMR) and histology. The sulfated GAG coating contributed substantially to bone regeneration, increased collagen synthesis and initiated mineralization of the organic matrix. Most substantial collagen production was detected in scaffolds coated with chondroitin sulfate. Scaffolds coated with hypersulfated hyaluronan induced formation of new bone volume comparable to what was observed in the positive control. GAG differing in the sugar backbone and degree of sulfation modulate the healing process at different times, eventually leading to improved bone healing.

Elucidation of the Structure and Synthesis of Neuroprotective Low Molecular Mass Components of the Parawixia bistriata Spider Venom.

The South American social spider Parawixia bistriata produces a venom containing complex organic compounds with intriguing biological activities. The crude venom leads to paralysis in termites and stimulates l-glutamate uptake and inhibits GABA uptake in rat brain synaptosomes. Glutamate is the major neurotransmitter at the insect neuromuscular junction and at the mammalian central nervous system, suggesting a modulation of the glutamatergic system by the venom. Parawixin1, 2, and 10 (Pwx1, 2 and 10) are HPLC fractions that demonstrate this bioactivity. Pwx1 stimulates l-glutamate uptake through the main transporter in the brain, EAAT2, and is neuroprotective in in vivo glaucoma models. Pxw2 inhibits GABA and glycine uptake in synaptosomes and inhibits seizures and neurodegeneration, and Pwx10 increases l-glutamate uptake in synaptosomes and is neuroprotective and anticonvulsant, shown in in vivo epilepsy models. Herein, we investigated the low molecular mass compounds in this venom and have found over 20 small compounds and 36 unique acylpolyamines with and without amino acid linkers. The active substances in fractions Pwx1 and Pwx2 require further investigation. We elucidated and confirmed the structure of the active acylpolyamine in Pwx10. Both fraction Pwx10 and the synthesized component enhance the activity of transporters EAAT1 and EAAT2, and, importantly, offer in vitro neuroprotection against excitotoxicity in primary cultures. These data suggest that compounds with this mechanism could be developed into therapies for disorders in which l-glutamate excitotoxicity is involved.

Biomaterials in repairing rat femoral defects: In vivo insights from small animal positron emission tomography/computed tomography (PET/CT) studies.

Biomaterials coated with artificial extracellular matrices (aECM) are intended to support the healing of critical size bone defects. This pilot study investigated (i) the feasibility of dual-tracer PET/CT imaging for functional characterization of biomaterial-assisted bone healing in a rat femoral defect model and (ii) the bone healing ability of polycaprolactone-co-lactide (PCL) scaffolds, coated with various aECM consisting of collagen type I (Col) and glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) or polysulfated hyaluronan (sHA3). [18F]FDG and [18F]fluoride PET 4 and 8 weeks after implantation of aECM-coated PCL scaffolds, which provide an in vivo measure of cellular activation and bone mineralization, respectively, combined with CT imaging (in vivo/ex vivo) and histological/immunohistochemical investigations (ex vivo) showed that coating with CS in particular is beneficial for bone healing. The possible involvement of COX-2 and TGase 2, key enzymes of inflammation and ECM remodeling, in these processes offers starting points for targeted adjuvant therapy in the course of various bone healing phases. Our investigations show the feasibility of the selected dual-tracer approach for PET/CT imaging. In principle, this approach can be extended by further PET tracers for the functional characterization of physiological processes such as hypoxia/reperfusion or selected molecular players.

3D Plotted Biphasic Bone Scaffolds for Growth Factor Delivery: Biological Characterization In Vitro and In Vivo.

Bioprinting enables the integration of biological components into scaffolds during fabrication that has the advantage of high loading efficiency and better control of release and/or spatial positioning. In this study, a biphasic scaffold fabricated by extrusion-based 3D multichannel plotting of a calcium phosphate cement (CPC) paste and an alginate/gellan gum (AlgGG) hydrogel paste laden with the angiogenic factor VEGF (vascular endothelial growth factor) is investigated with regard to biological response in vitro and in vivo. Rat mesenchymal stromal cells are able to adhere and grow on both CPC and AlgGG strands, and differentiate toward osteoblasts. A sustained VEGF release is observed, which is able to stimulate endothelial cell proliferation as well as angiogenesis in vitro that indicates maintenance of its biological activity. After implantation into a segmental bone defect in the femur diaphysis of rats, a clear reduction of the defect size by newly formed bone tissue occurs from the distal and proximal ends of the host bone within 12 weeks. The CPC component shows excellent osteoconductivity whereas the local VEGF release from the AlgGG hydrogel gives rise to an enhanced vascularization of the defect region. This work contributes to the development of novel therapeutic concepts for improved bone regeneration which are based on 3D bioprinting.

Design and Fabrication of Complex Scaffolds for Bone Defect Healing: Combined 3D Plotting of a Calcium Phosphate Cement and a Growth Factor-Loaded Hydrogel.

Additive manufacturing enables the fabrication of scaffolds with defined architecture. Versatile printing technologies such as extrusion-based 3D plotting allow in addition the incorporation of biological components increasing the capability to restore functional tissues. We have recently described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of an oil-based CPC paste under mild conditions. In the present study, we have developed a strategy for growth factor loading based on multichannel plotting: a biphasic scaffold design was realised combining CPC with VEGF-laden, highly concentrated hydrogel strands. As hydrogel component, alginate and an alginate-gellan gum blend were evaluated; the blend exhibited a more favourable VEGF release profile and was chosen for biphasic scaffold fabrication. After plotting, two-step post-processing was performed for both, hydrogel crosslinking and CPC setting, which was shown to be compatible with both materials. Finally, a scaffold was designed and fabricated which can be applied for testing in a rat critical size femur defect. Optimization of CPC plotting enabled the fabrication of highly resolved structures with strand diameters of only 200 µm. Micro-computed tomography revealed a precise strand arrangement and an interconnected pore space within the biphasic scaffold even in swollen state of the hydrogel strands.

Chromatographic Separation of the Codonocarpine Type Alkaloids from the Root Bark of Capparis decidua.

Various parts of the caper tree Capparis decidua have found application in traditional medicine. The isolation and structural elucidation of the codonocarpine type alkaloids contained in the root bark, however, is not trivial and has probably led to misinterpretation in the past. This protocol describes the extraction and chromatographic separation of the four major alkaloids of the root bark of Capparis decidua. The delivered samples of cadabicine, codonocarpine, isocodonocarpine and capparidisinine were suitable for their unambiguous structural elucidation by NMR, MS and MS/MS.

Collagen/glycosaminoglycan coatings enhance new bone formation in a critical size bone defect - A pilot study in rats.

Bone regeneration in critical size bone defects still represents an important but unsolved clinical problem. Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) or hyaluronan (HA) are important multifunctional components of the extracellular matrix (ECM) in bone and may stimulate bone healing by recruitment of mesenchymal stromal cells and by supporting their differentiation. Sulfation of GAGs affects their biological activity and thus their interactions with growth factors and/or cells involved in the bone healing process. The aim of this pilot study was to evaluate the osteogenic capacity of chemically high-sulfated chondroitin sulfate (sCS3) and hyaluronan (sHA3) with an average degree of sulfation DS≈3 on bone healing. Titanium-coated polyetheretherketone (Ti-PEEK) plates were coated with collagen type I (col), collagen-based artificial ECMs containing CS or HA and compared to col/sCS3 and col/sHA3 coatings bridging a critical size bone defect in rat femur. After 4weeks the gap size of 5.1mm±0.1mm following surgery was significantly reduced to 1.4mm±0.9mm for col/sHA3 and to 0.9mm±0.7mm for col/CS. The highest amount of newly formed bone was detected for col/CS (79%±30%) and col/sHA3 (36%±20%) compared to uncoated plates (13%±3%) or col-coated plates (18%±16%). Enchondral ossification could be confirmed for col/CS, col/HA, and col/sHA3 by positive staining for Alcian blue and collagen type II. These results suggest that an artificial ECM has osteogenic effects and is able to enhance bone healing in critical situations.

Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles.

Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis.

A new view on the codonocarpine type alkaloids of Capparis decidua.

Several spermidine alkaloids are described in literature as constituents of the root bark of Capparis decidua. Since some of the proposed structures, however, are in conflict with the expected biosynthetic paths, an extract of the root bark of the plant was re-investigated. Four major spermidine alkaloids of the codonocarpine type were identified and their structures elucidated: of the four compounds, isocodonocarpine was described previously for C. decidua and cadabicine was proposed as a possible constituent as well. Codonocarpine was found for the first time in an extract of C. decidua but was previously isolated from a closely related plant. Capparidisinine, finally, is an alkaloid with a structure that has never been described before. The structures of the four alkaloids are substantiated by NMR and MS data, and the four compounds are in logical agreement with biosynthetic considerations: they would arise from α,ω-bis-adducts of spermidine with coumaric and/or ferulic acids, followed by phenol oxidation.

Proteomics and metabolomics for in situ monitoring of wound healing.

Wound healing of soft tissue and bone defects is a complex process in which cellular differentiation and adaption are regulated by internal and external factors, among them are many different proteins. In contrast to insights into the significance of various single proteins based on model systems, the knowledge about the processes at the actual site of wound healing is still limited. This is caused by a general lack of methods that allow sampling of extracellular factors, metabolites, and proteins in situ. Sampling of wound fluids in combination with proteomics and metabolomics is one of the promising approaches to gain comprehensive and time resolved data on effector molecules. Here, we describe an approach to sample metabolites by microdialysis and to extract proteins simultaneously by adsorption. With this approach it is possible (i) to collect, enrich, and purify proteins for a comprehensive proteome analysis; (ii) to detect more than 600 proteins in different defects including more than 100 secreted proteins, of which many proteins have previously been demonstrated to have diagnostic or predictive power for the wound healing state; and (iii) to combine continuous sampling of cytokines and metabolites and discontinuous sampling of larger proteins to gain complementary information of the same defect.

Monitoring of the first stages of bone healing with microdialysis.

BACKGROUND AND PURPOSE: Bone healing is a complex process influenced by growth factors, cytokines, and other mediators. The regulation of this process is not well understood. In this pilot study, we used microdialysis technology in a critical-size bone defect in rat femurs to determine the feasibility of measuring cytokines and growth factors in the first 24 h after injury. METHODS: A 5-mm defect, stabilized by a plate, was created in the femurs of 30 male Wistar rats. The microdialysis probe (with 100 kDa molecular weight cutoff) was inserted into the defect and microdialysates were collected continuously for up to 24 h. Total protein concentration, interleukin-6 (IL-6) concentration, and transforming growth factor-ß1 (TGF-ß1) concentration were assessed under different conditions. RESULTS: Microdialysis allowed continuous and consistent protein collection over 24 h from a critical-size bone defect starting at the time of injury. IL-6 was secreted within the first 3 h after the injury. The highest IL-6 concentration (344 pg/mL) was measured between 12 and 15 h after surgery. Addition of bovine serum albumin to the perfusate resulted in detectable concentrations of TGF-ß1 ranging from 10 to 23 pg/mL. INTERPRETATION: Continuous sampling over 24 h of proteins from a bone defect directly after the injury is feasible and provides the opportunity for a detailed analysis of the initial stages of bone healing.

The pore size of PLGA bone implants determines the de novo formation of bone tissue in tibial head defects in rats.

PURPOSE: The influence of the pore size of biodegradable poly(lactic-co-glycolic acid) scaffolds on bone regeneration was investigated. METHODS: Cylindrical poly(lactic-co-glycolic acid) scaffolds were implanted into a defect in the tibial head of rats. Pore sizes of 100-300, 300-500, and 500-710 µm were tested and compared to untreated defects as control. Two and four weeks after implantation, the specimens were explanted and defect regeneration and de novo extracellular matrix generation were investigated by MRI, quantitative solid-state NMR, and mass spectrometry. RESULTS: The pore size of the scaffolds had a pronounced influence on the quantity of the extracellular matrix synthesized in the graft; most collagen was synthesized within the first 2 weeks of implantation, while the amount of hydroxyapatite increased in the second 2 weeks. After 4 weeks, the scaffolds contained large quantities of newly formed lamellar bone while the control defects were filled by inhomogenous woven bone. Best results were obtained for scaffolds of a pore size of 300-500 µm. CONCLUSION: Our analysis showed that the structure and dynamics of the regenerated extracellular matrix was very similar to that of the native bone, suggesting that biomineralization was significantly enhanced by the choice of the most appropriate implant material.

Surface modification of implants in long bone.

Coatings of orthopedic implants are investigated to improve the osteoinductive and osteoconductive properties of the implant surfaces and thus to enhance periimplant bone formation. By applying coatings that mimic the extracellular matrix a favorable environment for osteoblasts, osteoclasts and their progenitor cells is provided to promote early and strong fixation of implants. It is known that the early bone ongrowth increases primary implant fixation and reduces the risk of implant failure. This review presents an overview of coating titanium and hydroxyapatite implants with components of the extracellular matrix like collagen type I, chondroitin sulfate and RGD peptide in different small and large animal models. The influence of these components on cells, the inflammation process, new bone formation and bone/implant contact is summarized.

Efficient suppression of tissue factor synthesis using antisense oligonucleotides selected by an enhanced strategy for evaluation of structural characteristics.

Selection of optimal antisense constructs is still a problem. Among a huge number of antisense oligonucleotides (AS-ONs) only a small piece show inhibitory efficacy. We want to develop an enhanced strategy for specific selection of effective AS-ONs based on prediction of secondary structure of the target messenger RNA (mRNA) and analysis of thermodynamic properties of the mRNA/AS-ON hybrid. Numerous AS-ONs targeted on human tissue factor (TF) mRNA were investigated to evaluate the relevance of different thermodynamic and structural properties on inhibitory efficacy. Cell viability, TF protein and TF mRNA were determined after transfection of bladder cancer cell line J82. Inhibitory efficacy was related to GC content, target region within the TF mRNA and stability of the mRNA/AS-ON hybrid or affinity of the AS-ON to the target mRNA. We found effective AS-ONs targeted on translated region or 3'-untranslated region of TF RNA. We also detected a great correlation between inhibitory efficacy and GC content as well as stability of the mRNA/AS-ON hybrid.

Tissue factor and tumor: clinical and laboratory aspects.

This review summarizes data demonstrating the role of TF in tumor development, metastasis and angiogenesis. TF is a transmembrane protein that is expressed constitutively in some kinds of extravascular cells and transiently in intravascular cells after stimulation with cytokines and growth factors. Originally TF was considered to have a function in the initiation of coagulation. In the last years it became evident that TF plays a role in physiological and pathological processes outside the hemostasis. Up-regulation of TF expression appears to be characteristic of tumor tissue. In a variety of human tumors it was shown by immunohistochemistry, that TF can be expressed in malignant cells as well as in tumor-infiltrating macrophages or endothelial cells. Such abnormal TF expression contributes to the angiogenic process by a shift in the balance between endogenous proangiogenic and antiangiogenic factors. Observations of a significant correlation between elevated TF expression with increased microvessel density and VEGF expression underline the TF involvement in tumor angiogenesis. Furthermore, TF expression influences also metastasis. The effect of TF on metastasis may result from its angiogenic effect, but also from the production of growth factors or adhesion proteins.

Vascular endothelial growth factor antisense pretreatment of bladder cancer cells significantly enhances the cytotoxicity of mitomycin C, gemcitabine and Cisplatin.

PURPOSE: Due to unsatisfactory success in the treatment of local and systemic bladder cancer and the low response rates to commonly used chemotherapy (CT) alternative and additive approaches must be found. The function of vascular endothelial growth factor (VEGF) in neo-angiogenesis and, therefore, in solid tumors makes it a promising target for a specific antitumor therapy. We investigated the possibility of sensitizing transitional bladder cancer cell lines to CT by pretreatment with VEGF antisense (AS) oligodeoxynucleotides (AS-ODNs). MATERIALS AND METHODS: The human bladder cancer cell lines EJ28 and 5637 were transiently transfected with 3 antiVEGF AS-ODNs, followed by incubation with 3 doses of mitomycin C, gemcitabine or cisplatin CT. WST-1 (a sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay (Roche, Mannheim, Germany) was performed to assess effects on cell viability. Apoptosis was examined by Annexin V staining. In all experiments a nonsense ODN served as a control. RESULTS: Each cell line responded in a dose dependent manner to all CTs. Combined treatment with VEGF AS-ODNs and CT resulted in decreased viability compared with isolated CT. VEGF857 plus CT significantly decreased the viability of the 2 cell lines compared with nonsense ODN plus CT for all 3 CT agents (p <0.007). This detected chemosensitization was based on an AS mediated increase in apoptosis. CONCLUSIONS: One of the 3 AS-ODNs tested (VEGF857) significantly sensitizes human transitional cell carcinoma cells to CT. We suggest VEGF as an additional putative target to enhance the therapeutic benefit of, for example mitomycin C and gemcitabine instillation treatment schedules.

Antisense-mediated VEGF suppression in bladder and breast cancer cells.

Angiogenesis plays a key role in tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is one of the major angiogenic factors. In the study we have evaluated the efficiency of antisense oligodeoxynucleotides (AS-ODN) against VEGF selected from computational prediction of VEGF mRNA structure. Twenty-five different AS-ODN in two different tumor cell lines were investigated. Treatment of cell line EJ28 by VEGF723 resulted in a 83.5% suppression of VEGF protein when compared with control-ODN. Three further AS-ODN reduced VEGF protein more than 45% in comparison to control-ODN. This was caused by an antisense-specific downregulation of the VEGF transcript determined by real-time PCR. Furthermore, antisense-mediated inhibition of VEGF was associated by a reduced cell viability. In MCF-7 cells VEGF protein was inhibited more than 45% by two AS-ODN. In conclusion, we found that computational prediction of potential single strand mRNA motifs is a well suitable method to elect effective AS-ODN.

Inhibition of TF gene expression by antisense oligonucleotides in different cancer cell lines.

Human tissue factor (TF) is the initiator of blood coagulation. Beside this function it is involved in tumor angiogenesis and metastasis. In the study we have evaluated the efficiency of antisense oligonucleotides (AS-ODNs) against TF selected from computational prediction of TF mRNA structure. Fourteen different AS-ODNs were tested in three cell lines of different origin with a high TF content. In cell line MCF-7 expression of TF gene was inhibited up to 50% by the AS-ODN AS-7 in comparison to reference. To investigate the dependence of inhibition efficiency on the AS-ODN position inside a potential target motive we designed further AS-ODNs shifted 2-3 nt among AS-7. One AS-ODN was found as more effective than AS-7. In cell line T508 were obtained moderate effects in inhibition of TF gene expression of 30% by AS-4. In J82 cells TF protein was inhibited up to 68% by two AS-ODNs. In conclusion, we compared inhibition of TF gene expression in different cancer cell lines and found that all effective AS-ODNs were located in the translated region of TF mRNA. Suitability of a target region of an AS-ODN is relatively independent on cell line. In contrast, optimal transfection conditions are dependent on cell line.

Calretinin expression in human normal and neoplastic tissues: a tissue microarray analysis on 5233 tissue samples.

Calretinin is a calcium-binding protein expressed in different normal and neoplastic tissues. Early studies suggested that calretinin is a useful marker to differentiate adenocarcinomas from malignant mesotheliomas of the lung, but subsequent work has shown that calretinin can be expressed in several other tumor types. To systematically investigate the epidemiology of calretinin expression in normal and neoplastic tissues, we used tissue microarrays (TMAs) to analyze the immunohistochemically detectable expression of calretinin in 5233 tissue samples from 128 different tumor categories and 76 different normal tissue types. At least 1 case with weak expression could be found in 74 of 128 (58%) different tumor types and 46 entities (36%) had at least 1 tumor with strong positivity. In normal tissues, a particularly strong expression was found in Leydig cells of the testis, neurons of the brain, theca-lutein and theca interna cells of the ovary, and mesothelium. In tumors, strong calretinin expression was most frequently found in malignant mesotheliomas (6 of 7), Leydig cell tumors of the testis (5 of 5), adenomas of adrenal gland (5 of 9), and adenomatoid tumors (4 of 9). In summary, calretinin is frequently expressed in many different tumor types. Metastases of various different origins must be included in the differential diagnosis of calretinin-positive pleura tumors.