Complementary Effects of Two Growth Factors in Multifunctionalized Silk Nanofibers for Nerve Reconstruction

With the aim of forming bioactive guides for peripheral nerve regeneration, silk fibroin was electrospun to obtain aligned nanofibers. These fibers were functionalized by incorporating Nerve Growth Factor (NGF) and Ciliary NeuroTrophic Factor (CNTF) during electrospinning. PC12 cells grown on the fibers confirmed the bioavailability and bioactivity of the NGF, which was not significantly released from the fibers. Primary neurons from rat dorsal root ganglia (DRGs) were grown on the nanofibers and anchored to the fibers and grew in a directional fashion based on the fiber orientation, and as confirmed by growth cone morphology. These biofunctionalized nanofibers led to a 3-fold increase in neurite length at their contact, which was likely due to the NGF. Glial cell growth, alignment and migration were stimulated by the CNTF in the functionalized nanofibers. Organotypic culture of rat fetal DRGs confirmed the complementary effect of both growth factors in multifunctionalized nanofibers, which allowed glial cell migration, alignment and parallel axonal growth in structures resembling the ‘bands of Bungner’ found in situ. Graftable multi-channel conduits based on biofunctionalized aligned silk nanofibers were developed as an organized 3D scaffold. Our bioactive silk tubes thus represent new options for a biological and biocompatible nerve guidance conduit.     

Covalent immobilization of lipase in organic solvents

Lipase from Rhizopus sp. has been immobilized covalently on tresyl activated silica. Three different coupling media were evaluated: aqueous buffer, n-hexane, and a microemulsion based on n-hexane, aqueous buffer, and the nonionic surfactant triethylene glycol monododecyl ether. In addition, coupling via a very long, hydrophilic spacer arm, polyethylene glycol 1500 (PEG 1500), was compared with attachment to the silica via a short silane bridge only. The enzyme preparations were tested in hydrolysis and transesterification reactions. In the hydrolysis no marked differences in activity were found between the coupling media used. In the transesterification, on the other hand, the choice of immobilization medium had a very large effect on lipase activity, the preparation from microemulsion being the most active one. The use of the hydrophilic spacer had a large effect on activity in the hydrolysis reaction. Whereas direct coupling gave an activity of immobilized lipase of 26-34% of that of free enzyme, depending on the reaction medium, lipase bound via the spacer exhibited 56-67% activity. The latter values are considerably higher than previously reported in the literature for covalently immobilized lipase. The hydrophilic spacer had no effect on enzyme activity in the transesterification, however, a fact which is attributed to the hydrophobic medium of this reaction. The spacer is incompatible with the reaction medium and will, therefore, adsorb on the particles rather than stretch out into the bulk phase. The stability of the bound lipase was extremely good, no loss in activity being observed after a period of three weeks in aqueous solution of 37 degrees C.     

Real-time online monitoring of insect cell proliferation and baculovirus infection using digital differential holographic microscopy and machine learning

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.     

Hdm2 nuclear export, regulated by insulin-like growth factor-I/MAPK/p90Rsk signaling, mediates the transformation of human cells

Insulin-like growth factor (IGF)-I receptor activation leads to enhanced proliferation and cell survival via the MAP kinase and phosphatidylinositol 3-kinase-signaling pathways. Upon stimulation by IGF-I, the Hdm2 oncoprotein is phosphorylated by AKT, leading to its rapid nuclear translocation and subsequent inhibition of p53. We now show that IGF-I stimulation regulates the nuclear export of Hdm2 and p53 via the MAP kinase pathway. Inhibition of p38 MAPK or MEK via pharmacological means or expression of dominant negative proteins inhibited the cytoplasmic accumulation of Hdm2 and increased Hdm2 and p53 protein levels, whereas constitutively active p90Rsk promoted the nuclear export of Hdm2. Expression of constitutively active p90Rsk with E1A, oncogenic H-Ras, and hTERT resulted in the anchorage-independent growth of normal human fibroblasts. Our findings link p90Rsk-mediated modulation of Hdm2 nuclear to cytoplasmic shuttling with the diminished ability of p53 to regulate cell cycle checkpoints that ultimately leads to transformation.     

The cytosolic Arabidopsis thaliana cysteine desulfurase ABA3 delivers sulfur to the sulfurtransferase STR18

The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain–containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins. We report here the biochemical relationships between two cytosolic proteins from Arabidopsis thaliana, a Rhd domain–containing protein, the sulfurtransferase 18 (STR18), and a CD isoform referred to as ABA3, and compare these biochemical features to those of a natural CD–Rhd fusion protein from the bacterium Pseudorhodoferax sp. We observed that the bacterial enzyme is bifunctional exhibiting both CD and STR activities using l-cysteine and thiosulfate as sulfur donors but preferentially using l-cysteine to catalyze transpersulfidation reactions. In vitro activity assays and mass spectrometry analyses revealed that STR18 stimulates the CD activity of ABA3 by reducing the intermediate persulfide on its catalytic cysteine, thereby accelerating the overall transfer reaction. We also show that both proteins interact in planta and form an efficient sulfur relay system, whereby STR18 catalyzes transpersulfidation reactions from ABA3 to the model acceptor protein roGFP2. In conclusion, the ABA3–STR18 couple likely represents an uncharacterized pathway of sulfur trafficking in the cytosol of plant cells, independent of ABA3 function in molybdenum cofactor maturation.     

Synomones in necrophagous larvae of the blow flies Lucilia sericata and Calliphora vomitoria

Chemical signals are widespread in insects, but those resulting in interspecific communication (i.e., synomones) remain understudied. Here, we analysed chemicals left on substrates by two species of blow fly larvae, Lucilia sericata (Meigen) and Calliphora vomitoria (Linneaus) (Diptera: Calliphoridae), which can aggregate together on carrion. Using solid-phase microextraction and dynamic headspace analysis, we identified six compounds common to both species: the decanoic, tetradecanoic, pentadecanoic, hexadecanoic and octadecanoic acids, and the 2-ethylhexyl salicylate. We then tested the behavioural effects of the decanoic and pentadecanoic acids using binary-choice experiments, along with the (Z)-9-tricosene, a pheromone found in many arthropods. The time spent by a larva and its average crawling speed were measured in two sides of an arena, where only one contained a compound at 0.25 or 25 μg/μl. No effect was observed when testing the decanoic acid. The pentadecanoic acid only reduced the speed of C. vomitoria larvae at 25 μg/μl. Finally, L. sericata larvae spent less time in the side containing the (Z)-9-tricosene at 0.25 μg/μl, whereas C. vomitoria spent more time and crawled faster in this side at 25 μg/μl. Although these results did not directly evidence synomones, they suggest that the (Z)-9-tricosene could regulate larval aggregations on carrion.     

Expression of Trisk 51, agrin and nicotinic-acetycholine receptor ε-subunit during muscle development in a novel three-dimensional muscle-neuronal co-culture system

The purpose of our study was to create functional muscle tissue in vitro and to investigate the influence of organotypic neuronal slice cultures from rat spinal cord on the differentiation and function of primary rat myoblasts in a novel three-dimensional culture system. Three-dimensional muscle-neuronal cultures were established by co-cultivating primary rat skeletal muscle cells of newborn rats with organotypic slice cultures of the spinal cord prepared from isogenic rats in a fibrin matrix. These constructs were cultured for up to 4 weeks. Differentiation and fusion of the myoblasts to myofibers was evaluated by analyzing the expression pattern and localization of muscle- and neuron-specific markers. The fibrin matrix provided a suitable environment for three-dimensional myoblast culture. Co-culturing of organotypic spinal cord slices with myoblasts induced the formation of spontaneously contracting multinuclear and parallel-aligned myofibers. Pharmacological tests suggested the formation of neuromuscular junctions. The analysis of neural agrin expression and myogenic desmin, myogenin, MyoD, Trisk 51, and nicotinic-acetycholine receptor (nACh-receptor) -subunit expression revealed the differentiation of the myoblasts to myofibers. The presented novel three-dimensional co-culture system allows the in vitro investigation of myoblast differentiation and neuron-myoblast interaction. Our results suggest the existence of an alternative pathway for the maturation of the nAChR -subunit to the -subunit without neural agrin activity.A.D.B. and J.P.B. contributed equally to this studyThis work was supported by a major grant from the State of Baden-Württemberg within the scope of the Valley TEC (Valley Tissue Engineering Center)     

Spatiotemporal Interactions Among Three Neighboring Groups of Free-Ranging White-Footed Tamarins (Saguinus leucopus) in Colombia

Successful conservation requires an understanding of animal movement patterns and space use. Such data are hard to obtain, however, when difficult terrain, nocturnal habits, or lack of habituation make direct observation impractical. White-footed tamarins (Saguinus leucopus) are small primates endemic to Colombia that are in danger of extinction due to habitat loss, fragmentation, and the illegal pet trade. Here, we report the results of the first study to use radio-tracking to investigate white-footed tamarin ranging behavior. We recorded the movements of three neighboring tamarin groups simultaneously for 3 month using radio-telemetry. Home range sizes (estimated using both minimum convex polygon and fixed kernel contour methods) were substantially larger than reported in previous studies that did not use remote-tracking. Monte Carlo resampling procedures revealed that home range size differed significantly among the three groups but that the mean daily path length did not. As in other tamarin species, the degree of range overlap between neighboring social groups was high, ranging from 27 to 81%. Using a randomization test, we showed that the observed mean distance between groups was significantly lower than expected by chance for two of the three group dyads. This pattern of intergroup “attraction,” in conjunction with substantial range overlap and high population density, implies that the Bellavista Forest, one of the few remaining habitats of Saguinus leucopus, may be saturated, and promoting habitat restoration should be a priority for the conservation of this species.     

New lead elements for histamine H3 receptor ligands in the pyrrolo[2,3-d]pyrimidine class

This work describes the microwave assisted synthesis of twelve novel histamine H₃ receptor ligands. They display pyrrolo[2,3-d]pyrimidine derivatives with rigidized aliphatic amines as warheads. The compounds were screened for H₃R and H₄R binding affinities in radioligand displacement assays and the most potent compounds were evaluated for H₃R binding properties in vitro and in docking studies. The combination of a rigidized H₃R warhead and the pyrrolo[2,3-d]pyrimidine scaffold resulted in selective activity at the H₃ receptor with a pK_i value of 6.90 for the most potent compound. A bipiperidine warhead displayed higher affinity than a piperazine or morpholine motif, while a naphthyl moiety in the arbitrary region increased affinity compared to a phenyl derivative. The compounds can be starting points for novel, simply synthesized histamine H₃ receptor ligands.