Topographically and Chemically Enhanced Textile Polycaprolactone Scaffolds for Tendon and Ligament Tissue Engineering.

The use of tissue engineering to address the shortcomings of current procedures for tendons and ligaments is promising, but it requires a suitable scaffold that meets various mechanical, degradation-related, scalability-related, and biological requirements. Macroporous textile scaffolds made from appropriate fiber material have the potential to fulfill the first three requirements. This study aimed to investigate the biocompatibility, sterilizability, and functionalizability of a multilayer braided scaffold. These macroporous scaffolds with dimensions similar to those of the human anterior cruciate ligament consist of fibers with appropriate tensile strength and degradation behavior melt-spun from Polycaprolactone (PCL). Two different cross-sectional geometries resulting in significantly different specific surface areas and morphologies were used at the fiber level, and a Chitosan-graft-PCL (CS-g-PCL) surface modification was applied to the melt-spun substrates for the first time. All scaffolds elicited a positive cell response, and the CS-g-PCL modification provided a platform for incorporating functionalization agents such as drug delivery systems for growth factors, which were successfully released in therapeutically effective quantities. The fiber geometry was found to be a variable that could be manipulated to control the amount released. Therefore, scaled, surface-modified textile scaffolds are a versatile technology that can successfully address the complex requirements of tissue engineering for ligaments and tendons, as well as other structures.

Selenium- and chitosan-modified biochars reduce methylmercury contents in rice seeds with recruiting Bacillus to inhibit methylmercury production.

Biochar could reshape microbial communities, thereby altering methylmercury (MeHg) concentrations in rice rhizosphere and seeds. However, it remains unclear whether and how biochar amendment perturbs microbe-mediated MeHg production in mercury (Hg) contaminated paddy soil. Here, we used pinecone-derived biochar and its six modified biochars to reveal the disturbance. Results showed that selenium- and chitosan-modified biochar significantly reduced MeHg concentrations in the rhizosphere by 85.83% and 63.90%, thereby decreasing MeHg contents in seeds by 86.37% and 75.50%. The two modified bicohars increased the abundance of putative Hg-resistant microorganisms Bacillus, the dominant microbe in rhizosphere. These reductions about MeHg could be facilitated by biochar sensitive microbes such as Oxalobacteraceae and Subgroup_7. Pinecone-derived biochar increased MeHg concentration in rhizosphere but unimpacted MeHg content in seeds was observed. This biochar decreased the abundance in Bacillus but enhanced in putative Hg methylator Desulfovibrio. The increasing MeHg concentration in rhizosphere could be improved by biochar sensitive microbes such as Saccharimonadales and Clostridia. Network analysis showed that Saccharimonadales and Clostridia were the most prominent keystone taxa in rhizosphere, and the three biochars manipulated abundances of the microbes related to MeHg production in rhizosphere by those biochar sensitive microbes. Therefore, selenium- and chitosan-modified biochar could reduce soil MeHg production by these microorganisms, and is helpful in controlling MeHg contamination in rice.

Moonlighting Arabidopsis molybdate transporter 2 family and GSH-complex formation facilitate molybdenum homeostasis.

Molybdenum (Mo) as essential micronutrient for plants, acts as active component of molybdenum cofactor (Moco). Core metabolic processes like nitrate assimilation or abscisic-acid biosynthesis rely on Moco-dependent enzymes. Although a family of molybdate transport proteins (MOT1) is known to date in Arabidopsis, molybdate homeostasis remained unclear. Here we report a second family of molybdate transporters (MOT2) playing key roles in molybdate distribution and usage. KO phenotype-analyses, cellular and organ-specific localization, and connection to Moco-biosynthesis enzymes via protein-protein interaction suggest involvement in cellular import of molybdate in leaves and reproductive organs. Furthermore, we detected a glutathione-molybdate complex, which reveals how vacuolar storage is maintained. A putative Golgi S-adenosyl-methionine transport function was reported recently for the MOT2-family. Here, we propose a moonlighting function, since clear evidence of molybdate transport was found in a yeast-system. Our characterization of the MOT2-family and the detection of a glutathione-molybdate complex unveil the plant-wide way of molybdate.

Bacterial assemblages imply methylmercury production at the rice-soil system.

The plant microbiota can affect plant health and fitness by promoting methylmercury (MeHg) production in paddy soil. Although most well-known mercury (Hg) methylators are observed in the soil, it remains unclear how rice rhizosphere assemblages alter MeHg production. Here, we used network analyses of microbial diversity to identify bulk soil (BS), rhizosphere (RS) and root bacterial networks during rice development at Hg gradients. Hg gradients greatly impacted the niche-sharing of taxa significantly relating to MeHg/THg, while plant development had little effect. In RS networks, Hg gradients increased the proportion of MeHg-related nodes in total nodes from 37.88% to 45.76%, but plant development enhanced from 48.59% to 50.41%. The module hub and connector in RS networks included taxa positively (Nitrososphaeracea, Vicinamibacteraceae and Oxalobacteraceae) and negatively (Gracilibacteraceae) correlating with MeHg/THg at the blooming stage. In BS networks, Deinococcaceae and Paludibacteraceae were positively related to MeHg/THg, and constituted the connector at the reviving stage and the module hub at the blooming stage. Soil with an Hg concentration of 30 mg kg-1 increased the complexity and connectivity of root microbial networks, although microbial community structure in roots was less affected by Hg gradients and plant development. As most frequent connector in root microbial networks, Desulfovibrionaceae did not significantly correlate with MeHg/THg, but was likely to play an important role in the response to Hg stress.

Significance of nitrogen-fixing actinorhizal symbioses for restoration of depleted, degraded, and contaminated soil.

Atmospheric nitrogen (N2)-fixing legume trees are frequently used for the restoration of depleted, degraded, and contaminated soils. However, biological N2 fixation (BNF) can also be performed by so-called actinorhizal plants. Actinorhizal plants include a high diversity of woody species and therefore can be applied in a broad spectrum of environments. In contrast to N2-fixing legumes, the potential of actinorhizal plants for soil restoration remains largely unexplored. In this Opinion, we propose related basic research requirements for the characterization of environmental stress responses that determine the restoration potential of actinorhizal plants for depleted, degraded, and contaminated soils. We identify advantages and unexplored processes of actinorhizal plants and describe a mainly uncharted avenue of future research for this important group of plant species.

Physiological Importance of Molybdate Transporter Family 1 in Feeding the Molybdenum Cofactor Biosynthesis Pathway in Arabidopsis thaliana.

Molybdate uptake and molybdenum cofactor (Moco) biosynthesis were investigated in detail in the last few decades. The present study critically reviews our present knowledge about eukaryotic molybdate transporters (MOT) and focuses on the model plant Arabidopsis thaliana, complementing it with new experiments, filling missing gaps, and clarifying contradictory results in the literature. Two molybdate transporters, MOT1.1 and MOT1.2, are known in Arabidopsis, but their importance for sufficient molybdate supply to Moco biosynthesis remains unclear. For a better understanding of their physiological functions in molybdate homeostasis, we studied the impact of mot1.1 and mot1.2 knock-out mutants, including a double knock-out on molybdate uptake and Moco-dependent enzyme activity, MOT localisation, and protein-protein interactions. The outcome illustrates different physiological roles for Moco biosynthesis: MOT1.1 is plasma membrane located and its function lies in the efficient absorption of molybdate from soil and its distribution throughout the plant. However, MOT1.1 is not involved in leaf cell imports of molybdate and has no interaction with proteins of the Moco biosynthesis complex. In contrast, the tonoplast-localised transporter MOT1.2 exports molybdate stored in the vacuole and makes it available for re-localisation during senescence. It also supplies the Moco biosynthesis complex with molybdate by direct interaction with molybdenum insertase Cnx1 for controlled and safe sequestering.

Split-HaloTag imaging assay for sophisticated microscopy of protein-protein interactions in planta.

An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. Split-GFP-based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization. Because of their simple protocols, they have become some of the most frequently used methods. However, standard fluorescent proteins present several drawbacks for sophisticated microscopy. With the HaloTag system, these drawbacks can be overcome, as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we have established the Split-HaloTag imaging assay in plants, which is based on the reconstitution of a functional HaloTag protein upon protein-protein interaction and the subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein-protein interaction at cellular structures: the anchoring of the molybdenum cofactor biosynthesis complex to filamentous actin. In addition, a specific interaction was visualized in a more distinctive manner with subdiffractional polarization microscopy, Airyscan, and structured illumination microscopy to provide examples of sophisticated imaging. Split-GFP and Split-HaloTag can complement one another, as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interactions using specific microscopy techniques, such as 3D imaging, single-molecule tracking, and super-resolution microscopy.

Impact of wildfires on SO2 detoxification mechanisms in leaves of oak and beech trees.

Frequency and intensity of wildfire occurrences are dramatically increasing worldwide due to global climate change, having a devastating effect on the entire ecosystem including plants. Moreover, distribution of fire-smoke can influence the natural environment over very long distances, i.e. hundreds of kilometres. Dry plant matter contains 0.1-0.9% (w/w) sulphur, which is mainly released during combustion into the atmosphere as sulphur dioxide (SO2) resulting in local concentrations of up to 3000 nL L-1. SO2 is a highly hazardous gas, which enters plants mostly via the stomata. Toxic sulphite is formed inside the leaves due to conversion of SO2. Plants as sessile organisms cannot escape from threats, why they evolved an impressive diversity of molecular defence mechanisms. In the present study, two recent wildfires in Germany were evaluated to analyse the effect of SO2 released into the atmosphere on deciduous trees: the Meppen peat fire in 2018 and the forest fire close to Luebtheen in 2019. Collected leaf material from beech (Fagus sylvatica) and oak (Quercus robur) was examined with respect to detoxification of sulphur surplus due to the exposure to elevated SO2. An induced stress reaction in both species was indicated by a 1.5-fold increase in oxidized glutathione. In beech leaves, the enzymatic activities of the sulphite detoxification enzymes sulphite oxidase and apoplastic peroxidases were increased 5-fold and a trend of sulphate accumulation was observed. In contrast, oaks did not regulate these enzymes during smoke exposure, however, the constitutive activity is 10-fold and 3-fold higher than in beech. These results show for the first time sulphite detoxification strategies of trees in situ after natural smoke exposure. Beech and oak trees survived short-term SO2 fumigation due to exclusion of toxic gases and different oxidative detoxification strategies. Beeches use efficient upregulation of oxidative sulphite detoxification enzymes, while oaks hold a constitutively high enzyme-pool available.

Cytosolic aromatic aldehyde dehydrogenase provides benzoic acid for xanthone biosynthesis in Hypericum.

Benzoic acid is a building block of a multitude of well-known plant natural products, such as paclitaxel and cocaine. Its simple chemical structure contrasts with its complex biosynthesis. Hypericum species are rich in polyprenylated benzoic acid-derived xanthones, which have received attention due to their biological impact on human health. The upstream biosynthetic sequence leading to xanthones is still incomplete. To supply benzoic acid for xanthone biosynthesis, Hypericum calycinum cell cultures use the CoA-dependent non-ß-oxidative pathway, which starts with peroxisomal cinnamate CoA-ligase (HcCNL). Here, we use the xanthone-producing cell cultures to identify the transcript for benzaldehyde dehydrogenase (HcBD), a pivotal player in the non-ß-oxidative pathways. In addition to benzaldehyde, the enzyme efficiently catalyzes the oxidation of trans-cinnamaldehyde in vitro. The enzymatic activity is strictly dependent on the presence of NAD+ as co-factor. HcBD is localized to the cytosol upon ectopic expression of reporter fusion constructs. HcBD oxidizes benzaldehyde, which moves across the peroxisome membrane, to form benzoic acid. Increases in the HcCNL and HcBD transcript levels precede the elicitor-induced xanthone accumulation. The current work addresses a crucial step in the yet incompletely understood CoA-dependent non-ß-oxidative route of benzoic acid biosynthesis. Addressing this step may offer a new biotechnological tool to enhance product formation in biofactories.

Varying the sustained release of BMP-2 from chitosan nanogel-functionalized polycaprolactone fiber mats by different polycaprolactone surface modifications.

Polycaprolactone (PCL) fiber mats with different surface modifications were functionalized with a chitosan nanogel coating to attach the growth factor human bone morphogenetic protein 2 (BMP-2). Three different hydrophilic surface modifications were compared with regard to the binding and in vitro release of BMP-2. The type of surface modification and the specific surface area derived from the fiber thickness had an important influence on the degree of protein loading. Coating the PCL fibers with polydopamine resulted in the binding of the largest BMP-2 quantity per surface area. However, most of the binding was irreversible over the investigated period of time, causing a low release in vitro. PCL fiber mats with a chitosan-graft-PCL coating and an additional alginate layer, as well as PCL fiber mats with an air plasma surface modification boundless BMP-2, but the immobilized protein could almost completely be released. With polydopamine and plasma modifications as well as with unmodified PCL, high amounts of BMP-2 could also be attached directly to the surface. Integration of BMP-2 into the chitosan nanogel functionalization considerably increased binding on all hydrophilized surfaces and resulted in a sustained release with an initial burst release of BMP-2 without detectable loss of bioactivity in vitro.

Nitrate and ammonium differ in their impact on δ13C of plant metabolites and respired CO2 from tobacco leaves.

The carbon isotopic composition (δ13C) of foliage is often used as proxy for plant performance. However, the effect of N O 3 - vs. N H 4 + supply on δ13C of leaf metabolites and respired CO2 is largely unknown. We supplied tobacco plants with a gradient of N O 3 - to N H 4 + concentration ratios and determined gas exchange variables, concentrations and δ13C of tricarboxylic acid (TCA) cycle intermediates, δ13C of dark-respired CO2, and activities of key enzymes nitrate reductase, malic enzyme and phosphoenolpyruvate carboxylase. Net assimilation rate, dry biomass and concentrations of organic acids and starch decreased along the gradient. In contrast, respiration rates, concentrations of intercellular CO2, soluble sugars and amino acids increased. As N O 3 - decreased, activities of all measured enzymes decreased. δ13C of CO2 and organic acids closely co-varied and were more positive under N O 3 - supply, suggesting organic acids as potential substrates for respiration. Together with estimates of intra-molecular 13C enrichment in malate, we conclude that a change in the anaplerotic reaction of the TCA cycle possibly contributes to 13C enrichment in organic acids and respired CO2 under N O 3 - supply. Thus, the effect of N O 3 - vs. N H 4 + on δ13C is highly relevant, particularly if δ13C of leaf metabolites or respiration is used as proxy for plant performance.

A promiscuous coenzyme A ligase provides benzoyl-coenzyme A for xanthone biosynthesis in Hypericum.

Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-ß-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4-coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

Physiological significance of pedospheric nitric oxide for root growth, development and organismic interactions.

Nitric oxide (NO) is essential for plant growth and development, as well as interactions with abiotic and biotic environments. Its importance for multiple functions in plants means that tight regulation of NO concentrations is required. This is of particular significance in roots, where NO signalling is involved in processes, such as root growth, lateral root formation, nutrient acquisition, heavy metal homeostasis, symbiotic nitrogen fixation and root-mycorrhizal fungi interactions. The NO signal can also be produced in high levels by microbial processes in the rhizosphere, further impacting root processes. To explore these interesting interactions, in the present review, we firstly summarize current knowledge of physiological processes of NO production and consumption in roots and, thereafter, of processes involved in NO homeostasis in root cells with particular emphasis on root growth, development, nutrient acquisition, environmental stresses and organismic interactions.

The First Step of Neurospora crassa Molybdenum Cofactor Biosynthesis: Regulatory Aspects under N-Derepressing and Nitrate-Inducing Conditions.

Molybdenum cofactor (Moco) is the active site prosthetic group found in all Moco dependent enzymes, except for nitrogenase. Mo-enzymes are crucial for viability throughout all kingdoms of life as they catalyze a diverse set of two electron transfer reactions. The highly conserved Moco biosynthesis pathway consists of four different steps in which guanosine triphosphate is converted into cyclic pyranopterin monophosphate, molybdopterin (MPT), and subsequently adenylated MPT and Moco. Although the enzymes and mechanisms involved in these steps are well characterized, the regulation of eukaryotic Moco biosynthesis is not. Within this work, we described the regulation of Moco biosynthesis in the filamentous fungus Neurospora crassa, which revealed the first step of the multi-step pathway to be under transcriptional control. We found, that upon the induction of high cellular Moco demand a single transcript variant of the nit-7 gene is increasingly formed pointing towards, that essentially the encoded enzyme NIT7-A is the key player for Moco biosynthesis activity in Neurospora.

Pilot study on the uptake and modification of harmaline in acceptor plants: An innovative approach to visualize the interspecific transfer of natural products.

Substances which have been leached out from decomposing plant parts or exuded from vital plants (donor plants), are taken up by acceptor plants and subsequently modified. This phenomenon was likewise established for harmala alkaloids. Employing hydroponically grown barley seedlings, it becomes evident that harmaline and harmine are taken up by the roots of the acceptor plants. Furthermore, based on HPLC and GC-MS analyses, it was demonstrated that these alkaloids also are present in Setaria viridis plants, which grew in the direct vicinity of the alkaloid containing Peganum harmala plants. Since harmaline exhibits a bright green fluorescence, this alkaloid was employed to visualize the uptake into the acceptor plants by feeding it to roots of barley seedlings. In the further course, the imported harmaline was converted in the leaves to yield harmine, which exhibits a dark blue fluorescence. This conversion was also verified by HPLC and GC-MS analyses. Based on the massive differences in the fluorescence properties, both processes, uptake and modification in the acceptor plants, could be monitored by macroscopical studies as well as by confocal laser scanning microscopical analyses. As result, for the first time, the phenomenon of "Horizontal Natural Product Transfer" is visualized vividly.

Screening for scFv-fragments that are stable and active in the cytosol.

Intrabodies are antibodies that are not secreted but bind to their antigens inside the cell producing them. Intrabodies targeting antigens in the endoplasmatic reticulum were successfully used in vitro and in vivo. However, many target antigens interesting for research or therapy are located in the reducing environment of the cytosol, where correct folding and formation of disulfide bonds cannot be ensured. The majority of different scFv fragments, when expressed in the cytosol of the cell, do not fold correctly, are not stable or cannot bind their antigen. Such scFv antibodies are therefore not suited as intrabodies.In this study, we evaluated fast and simple screening methods to identify scFv fragments that are stable and functional in the cytosol. We analyzed various phage display derived human scFv antibodies recognizing extracellular signal-regulated kinase 2 (Erk2) for stability and antigen binding under reducing and non-reducing conditions. Further, we developed an assay allowing to measure the interaction of the scFv intrabodies with their antigen in the cytosol of in living cells, by using a Split-Luciferase (Split-Luc) assay. ScFv fragments showing antigen binding in the cytosol could successfully be identified.

The genuine localization of indole alkaloids in Vinca minor and Catharanthus roseus.

Based on the occurrence of indole alkaloids in so-called "chloroform leaf surface extracts", it was previously deduced that these alkaloids are present in the cuticle at the leaf surface of Catharanthus roseus and Vinca minor. As no symplastic markers were found in these extracts this deduction seemed to be sound. However, since chloroform is known to destroy biomembranes very rapidly, these data have to be judged with scepticism. We reanalyzed the alleged apoplastic localization of indole alkaloids by employing slightly acidic aqueous surface extracts and comparing the corresponding alkaloid patterns with those of aqueous total leaf extracts. Whereas in the "chloroform leaf surface extracts" all alkaloids are present in the same manner as in the total leaf extracts, no alkaloids occur in the aqueous leaf surface extracts. These results clearly show that chloroform had rapidly destroyed cell integrity, and the related extracts also contain the alkaloids genuinely accumulated within the protoplasm. The related decompartmentation was verified by the massively enhanced concentration of amino acids in aqueous surface extracts of chloroform treated leaves. Furthermore, the chloroform-induced cell disintegration was vividly visualized by confocal laser scanning microscopical analyses, which clearly displayed a strong decrease in the chlorophyll fluorescence in chloroform treated leaves. These findings unequivocally display that the indole alkaloids are not located in the apoplastic space, but exclusively are present symplastically within the cells of V. minor and C. roseus leaves. Accordingly, we have to presume that also other leaf surface extracts employing organic solvents have to be re-investigated.

Cinnamate-CoA ligase is involved in biosynthesis of benzoate-derived biphenyl phytoalexin in Malus × domestica 'Golden Delicious' cell cultures.

Apple (Malus sp.) and other genera belonging to the sub-tribe Malinae of the Rosaceae family produce unique benzoic acid-derived biphenyl phytoalexins. Cell cultures of Malus domestica cv. 'Golden Delicious' accumulate two biphenyl phytoalexins, aucuparin and noraucuparin, in response to the addition of a Venturia inaequalis elicitor (VIE). In this study, we isolated and expressed a cinnamate-CoA ligase (CNL)-encoding sequence from VIE-treated cell cultures of cv. 'Golden Delicious' (M. domestica CNL; MdCNL). MdCNL catalyses the conversion of cinnamic acid into cinnamoyl-CoA, which is subsequently converted to biphenyls. MdCNL failed to accept benzoic acid as a substrate. When scab-resistant (cv. 'Shireen') and moderately scab-susceptible (cv. 'Golden Delicious') apple cultivars were challenged with the V. inaequalis scab fungus, an increase in MdCNL transcript levels was observed in internodal regions. The increase in MdCNL transcript levels could conceivably correlate with the pattern of accumulation of biphenyls. The C-terminal signal in the MdCNL protein directed its N-terminal reporter fusion to peroxisomes in Nicotiana benthamiana leaves. Thus, this report records the cloning and characterisation of a cinnamoyl-CoA-forming enzyme from apple via a series of in vivo and in vitro studies. Defining the key step of phytoalexin formation in apple provides a biotechnological tool for engineering elite cultivars with improved resistance.

Sequential regiospecific gem-diprenylation of tetrahydroxyxanthone by prenyltransferases from Hypericum sp.

Polyprenylated acylphloroglucinol derivatives, such as xanthones, are natural plant products with interesting pharmacological properties. They are difficult to synthesize chemically. Biotechnological production is desirable but it requires an understanding of the biosynthetic pathways. cDNAs encoding membrane-bound aromatic prenyltransferase (aPT) enzymes from Hypericum sampsonii seedlings (HsPT8px and HsPTpat) and Hypericum calycinum cell cultures (HcPT8px and HcPTpat) were cloned and expressed in Saccharomyces cerevisiae and Nicotiana benthamiana, respectively. Microsomes and chloroplasts were used for functional analysis. The enzymes catalyzed the prenylation of 1,3,6,7-tetrahydroxyxanthone (1367THX) and/or 1,3,6,7-tetrahydroxy-8-prenylxanthone (8PX) and discriminated nine additionally tested acylphloroglucinol derivatives. The transient expression of the two aPT genes preceded the accumulation of the products in elicitor-treated H. calycinum cell cultures. C-terminal yellow fluorescent protein fusions of the two enzymes were localized to the envelope of chloroplasts in N. benthamiana leaves. Based on the kinetic properties of HsPT8px and HsPTpat, the enzymes catalyze sequential rather than parallel addition of two prenyl groups to the carbon atom 8 of 1367THX, yielding gem-diprenylated patulone under loss of aromaticity of the gem-dialkylated ring. Coexpression in yeast significantly increased product formation. The patulone biosynthetic pathway involves multiple subcellular compartments. The aPTs studied here and related enzymes may be promising tools for plant/microbe metabolic pathway engineering.

Layer-by-layer deposition of chitosan nanoparticles as drug-release coatings for PCL nanofibers.

Nanogels were prepared by ionotropic gelation of chitosan (CS) with tripolyphosphate (TPP). The use of such nanogels to prepare coatings by layer-by-layer deposition (LbL) was studied. The nanogels were characterized in terms of particle size, zeta-potential and stability. Nanogel suspensions were used to build polyelectrolyte multilayers on silicon wafers and on PCL fiber mats by LbL-deposition. Three different polysaccharides were used as polyanions, namely chondroitin sulfate, alginate and hyaluronic acid. The ellipsometric thickness was demonstrated to depend significantly on the type of polyanion. XPS analysis with depth profiling further substantiated the differences in the chemical composition of the films with the different polyanions. Furthermore, XPS data clearly indicated a strong penetration of the polyanions into the CS-TPP layer, resulting in a complete exchange and release of the TPP ions. The LbL-deposition also was studied with PCL fiber mats, which were modified with a chitosan-PCL-graft polymer and alginate. The possibility to create graded coatings on the fiber mats was shown employing fluorescently labelled CS-TPP nanoparticles. The potential of the coatings as drug delivery system for therapeutic proteins was exemplified with the release of Transforming Growth Factor ß3 (TGF-ß3). The CS-TPP nanogels were shown to encapsulate and release therapeutic proteins. In combination with the layer-by-layer deposition they will allow the creation of PCL fiber mat implants having with drug gradients for applications at tissue transitions.