Plant communities of a Central Namib inselberg landscape

Abstract. Three granite inselbergs and six dolerite dykes and their surroundings were investigated in the Central Namib, at the interface between the Namib Desert and Nama Karoo biomes. The main objectives of this study included a phytoso‐ciological interpretation of the described plant communities, explanation of the correlation of the communities with environmental variables and quantification of the relative contribution of different types of variables to structuring plant communities. Nine grassland and shrubland plant communities were recognized, largely organized according to general habitat, elevation, size of inselberg and geology. Soil properties, often thought to play an important role in arid environments, showed no clear patterns in the level of analyses used in this study. Other environmental parameters of importance in arid mountain habitats, such as slope aspect and angle, also played a minor role. The main implications of the study are: 1. Central Namib inselbergs, particularly granite domes, harbour diverse plant communities, often with species from neighbouring higher rainfall areas, and are thus of high conservation value. 2. The poor contribution of environmental variables in this study, which are conventionally used in field studies of plant community – environment relationships, may demand a critical review of additional parameters to be included when analysing plant community – environment relations in arid environments. In particular between‐season variation, phytogeographic aspects and the heterogeneity of microhabitats, often contained within a plant community, need to be taken into account.     

Activation of Type I and III Interferon Response by Mitochondrial and Peroxisomal MAVS and Inhibition by Hepatitis C Virus

Sensing viruses by pattern recognition receptors (PRR) triggers the innate immune system of the host cell and activates immune signaling cascades such as the RIG-I/IRF3 pathway. Mitochondrial antiviral-signaling protein (MAVS, also known as IPS-1, Cardif, and VISA) is the crucial adaptor protein of this pathway localized on mitochondria, peroxisomes and mitochondria-associated membranes of the endoplasmic reticulum. Activation of MAVS leads to the production of type I and type III interferons (IFN) as well as IFN stimulated genes (ISGs). To refine the role of MAVS subcellular localization for the induction of type I and III IFN responses in hepatocytes and its counteraction by the hepatitis C virus (HCV), we generated various functional and genetic knock-out cell systems that were reconstituted to express mitochondrial (mito) or peroxisomal (pex) MAVS, exclusively. Upon infection with diverse RNA viruses we found that cells exclusively expressing pexMAVS mounted sustained expression of type I and III IFNs to levels comparable to cells exclusively expressing mitoMAVS. To determine whether viral counteraction of MAVS is affected by its subcellular localization we employed infection of cells with HCV, a major causative agent of chronic liver disease with a high propensity to establish persistence. This virus efficiently cleaves MAVS via a viral protease residing in its nonstructural protein 3 (NS3) and this strategy is thought to contribute to the high persistence of this virus. We found that both mito- and pexMAVS were efficiently cleaved by NS3 and this cleavage was required to suppress activation of the IFN response. Taken together, our findings indicate comparable activation of the IFN response by pex- and mitoMAVS in hepatocytes and efficient counteraction of both MAVS species by the HCV NS3 protease.     

Repeated H2O2 exposure drives cell cycle progression in an in vitro model of ulcerative colitis

The production of hydrogen peroxide (H₂O₂) drives tumourigenesis in ulcerative colitis (UC). Recently, we showed that H₂O₂ activates DNA damage checkpoints in human colonic epithelial cells (HCEC) through c‐Jun N‐terminal Kinases (JNK) that induces p21^WAF1. Moreover, caspases circumvented the G1/S and intra‐S checkpoints, and cells accumulated in G2/M. The latter observation raised the question of whether repeated H₂O₂ exposures alter JNK activation, thereby promoting a direct passage of cells from G2/M arrest to driven cell cycle progression. Here, we report that increased proliferation of repeatedly H₂O₂‐exposed HCEC cells (C‐cell cultures) was associated with (i) increased phospho‐p46 JNK, (ii) decreased total JNK and phospho‐p54 JNK and (iii) p21^WAF1 down‐regulation. Altered JNK activation and p21^WAF1 down‐regulation were accompanied by defects in maintaining G2/M and mitotic spindle checkpoints through adaptation, as well as by apoptosis resistance following H₂O₂ exposure. This may cause increased proliferation of C‐cell cultures, a defining initiating feature in the inflammation‐carcinoma pathway in UC. We further suggest that dysregulated JNK activation is attributed to a non‐apoptotic function of caspases, causing checkpoint adaptation in C‐cell cultures. Additionally, loss of cell‐contact inhibition and the overcoming of senescence, hallmarks of cancer, contributed to increased proliferation. Furthermore, there was evidence that p54 JNK inactivation is responsible for loss of cell‐contact inhibition. We present a cellular model of UC and suggest a sinusoidal pattern of proliferation, which is triggered by H₂O₂‐induced reactive oxygen species generation, involving an interplay between JNK activation/inactivation, p21^WAF1, c‐Fos, c‐Jun/phospho‐c‐Jun, ATF2/phospho‐ATF2, β‐catenin/TCF4‐signalling, c‐Myc, CDK6 and Cyclin D2, leading to driven cell cycle progression.     

Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.     

Novel High Efficient Coatings for Anti-Microbial Surgical Sutures Using Chlorhexidine in Fatty Acid Slow-Release Carrier Systems

Sutures can cause challenging surgical site infections, due to capillary effects resulting in bacteria permeating wounds. Anti-microbial sutures may avoid these complications by inhibiting bacterial pathogens. Recently, first triclosan-resistances were reported and therefore alternative substances are becoming clinically relevant. As triclosan alternative chlorhexidine, the “gold standard” in oral antiseptics was used. The aim of the study was to optimize novel slow release chlorhexidine coatings based on fatty acids in surgical sutures, to reach a high anti-microbial efficacy and simultaneously high biocompatibility. Sutures were coated with chlorhexidine laurate and chlorhexidine palmitate solutions leading to 11, 22 or 33 µg/cm drug concentration per length. Drug release profiles were determined in aqueous elutions. Antibacterial efficacy against Staphylococcus aureus was assessed in agar diffusion tests. Biocompatibility was evaluated via established cytotoxicity assay (WST-1). A commercially triclosan-containing suture (Vicryl Plus), was used as anti-microbial reference. All coated sutures fulfilled European Pharmacopoeia required tensile strength and proved continuous slow drug release over 96 hours without complete wash out of the coated drug. High anti-microbial efficacy for up to 5 days was observed. Regarding biocompatibility, sutures using 11 µg/cm drug content displayed acceptable cytotoxic levels according to ISO 10993-5. The highest potential for human application were shown by the 11 µg/cm chlorhexidine coated sutures with palmitic acid. These novel coated sutures might be alternatives to already established anti-microbial sutures such as Vicryl Plus in case of triclosan-resistance. Chlorhexidine is already an established oral antiseptic, safety and efficacy should be proven for clinical applications in anti-microbial sutures.     

Pharmacological postconditioning by bolus injection of phosphodiesterase-5 inhibitors vardenafil and sildenafil

Postconditioning enables cardioprotection against ischemia/reperfusion injury either by application of short, repetitive ischemic periods or by pharmacological intervention prior to reperfusion. Pharmacological postconditioning has been described for phosphodiesterase-5 inhibitors when the substances were applied as a permanent infusion. For clinical purposes, application of a bolus is more convenient. In a rat heart in situ model of ischemia reperfusion vardenafil or sildenafil were applied as a bolus prior to reperfusion. Cardioprotective effects were found over a broad dosage range. In accordance with current hypotheses on pharmacological postconditioning signaling, the protective effect was mediated by extracellular signal-regulated kinase and protein kinase C pathway. Interestingly, the extent of protection was independent of the concentration applied for both substances. Full protection comparable to ischemic postconditioning was reached with half-maximal human equivalence dose. In contrast, mean arterial pressure dropped upon bolus application in a dose-dependent manner. Taken together, the current study extends previous findings obtained in a permanent infusion model to bolus application. This is an important step toward clinical application of pharmacological postconditioning with sildenafil and vardenafil, especially because the beneficial effects were proven for concentrations with reduced hemodynamic side effects compared to the dosage applied for erectile dysfunction treatment.     

Comparison between Different Methods for Biomechanical Assessment of Ex Vivo Fracture Callus Stiffness in Small Animal Bone Healing Studies

For ex vivo measurements of fracture callus stiffness in small animals, different test methods, such as torsion or bending tests, are established. Each method provides advantages and disadvantages, and it is still debated which of those is most sensitive to experimental conditions (i.e. specimen alignment, directional dependency, asymmetric behavior). The aim of this study was to experimentally compare six different testing methods regarding their robustness against experimental errors. Therefore, standardized specimens were created by selective laser sintering (SLS), mimicking size, directional behavior, and embedding variations of respective rat long bone specimens. For the latter, five different geometries were created which show shifted or tilted specimen alignments. The mechanical tests included three-point bending, four-point bending, cantilever bending, axial compression, constrained torsion, and unconstrained torsion. All three different bending tests showed the same principal behavior. They were highly dependent on the rotational direction of the maximum fracture callus expansion relative to the loading direction (creating experimental errors of more than 60%), however small angular deviations (<15°) were negligible. Differences in the experimental results between the bending tests originate in their respective location of maximal bending moment induction. Compared to four-point bending, three-point bending is easier to apply on small rat and mouse bones under realistic testing conditions and yields robust measurements, provided low variation of the callus shape among the tested specimens. Axial compressive testing was highly sensitive to embedding variations, and therefore cannot be recommended. Although it is experimentally difficult to realize, unconstrained torsion testing was found to be the most robust method, since it was independent of both rotational alignment and embedding uncertainties. Constrained torsional testing showed small errors (up to 16.8%, compared to corresponding alignment under unconstrained torsion) due to a parallel offset between the specimens’ axis of gravity and the torsional axis of rotation.