Resistance of Bacteria toward 475 nm Blue Light Exposure and the Possible Role of the SOS Response.

The increase in antibiotic resistance represents a major global challenge for our health systems and calls for alternative treatment options, such as antimicrobial light-based therapies. Blue light has shown promising results regarding the inactivation of a variety of microorganisms; however, most often, antimicrobial blue light (aBL) therapy is performed using wavelengths close to the UV range. Here we investigated whether inactivation was possible using blue light with a wavelength of 475 nm. Both Gram-positive and -negative bacterial strains were treated with blue light with fluences of 7.5-45 J/cm2. Interestingly, only some bacterial strains were susceptible to 475 nm blue light, which was associated with the lack of RecA, i.e., a fully functional DNA repair mechanism. We demonstrated that the insertion of the gene recA reduced the susceptibility of otherwise responsive bacterial strains, indicating a protective mechanism conveyed by the bacterial SOS response. However, mitigating this pathway via three known RecA inhibiting molecules (ZnAc, curcumin, and Fe(III)-PcTs) did not result in an increase in bactericidal action. Nonetheless, creating synergistic effects by combining a multitarget therapy, such as aBL, with an RecA targeting treatment could be a promising strategy to overcome the dilemma of antibiotic resistance in the future.

Antimicrobial photodynamic therapy fighting polymicrobial infections - a journey from in vitro to in vivo.

Rapidly evolving multidrug resistance renders conventional antimicrobial strategies increasingly inefficient. This urges the exploration of alternative strategies with a lower potential of resistance development to control microbial infections. A promising option is antimicrobial photodynamic therapy (aPDT), especially in the setting of wound infections. In this study its effectiveness was tested as a treatment option for polymicrobially infected wounds in both in vitro and in vivo models. First, aPDT was applied to wound-relevant Gram-positive and Gram-negative bacteria in planktonic culture as the standard in vitro test system and compared different media to show a possible dependency of the therapy on the surrounding environment. In a second step, aPDT was investigated in an in vitro model mimicking the wound bed conditions using fibrin-coated culture plates. Finally, we tested aPDT in vivo in a polymicrobial infected wound healing model in immunocompromised BALB/c mice. In vitro, it was shown that the bactericidal effectiveness of aPDT was strongly dependent on the surrounding environment of the phototoxic reaction. In vivo, the significant delay in wound healing induced by polymicrobial infection was drastically diminished by a two-times application of aPDT using 100 µM methylene blue (generally regarded as safe for topical application on human skin) and 24 J cm-2 pulsed red LED light. Our experiments suggest that aPDT is capable of significantly improving wound healing also in complicated polymicrobially infected wound situations.

Evaluation of BMP-2 Minicircle DNA for Enhanced Bone Engineering and Regeneration.

BACKGROUND: To date, the significant osteoinductive potential of bone morphogenetic protein 2 (BMP-2) non-viral gene therapy cannot be fully exploited therapeutically. This is mainly due to weak gene delivery and brief expression peaks restricting the therapeutic effect. OBJECTIVE: Our objective was to test the application of minicircle DNA, allowing prolonged expression potential. It offers notable advantages over conventional plasmid DNA. The lack of bacterial sequences and the resulting reduction in size, enables safe usage and improved performance for tissue regeneration. METHODS: We inserted an optimized BMP-2 gene cassette with minicircle plasmid technology. BMP-2 minicircle plasmids were produced in E. coli yielding plasmids lacking bacterial backbone elements. Comparative studies of these BMP-2 minicircles and conventional BMP-2 plasmids were performed in vitro in cell systems, including bone marrow derived stem cells. Tests performed included gene expression profiles and cell differentiation assays. RESULTS: A C2C12 cell line transfected with the BMP-2-Advanced minicircle showed significantly elevated expression of osteocalcin, alkaline phosphatase (ALP) activity, and BMP-2 protein amount when compared to cells transfected with conventional BMP-2-Advanced plasmid. Furthermore, the plasmids show suitability for stem cell approaches by showing significantly higher levels of ALP activity and mineralization when introduced into human bone marrow stem cells (BMSCs). CONCLUSION: We have designed a highly bioactive BMP-2 minicircle plasmid with the potential to fulfil clinical requirements for non-viral gene therapy in the field of bone regeneration.

Spatiotemporal Differences in Gene Expression Between Motor and Sensory Autografts and Their Effect on Femoral Nerve Regeneration in the Rat.

To improve the outcome after autologous nerve grafting in the clinic, it is important to understand the limiting variables such as distinct phenotypes of motor and sensory Schwann cells. This study investigated the properties of phenotypically different autografts in a 6 mm femoral nerve defect model in the rat, where the respective femoral branches distally of the inguinal bifurcation served as homotopic, or heterotopic autografts. Axonal regeneration and target reinnervation was analyzed by gait analysis, electrophysiology, and wet muscle mass analysis. We evaluated regeneration-associated gene expression between 5 days and 10 weeks after repair, in the autografts as well as the proximal, and distal segments of the femoral nerve using qRT-PCR. Furthermore we investigated expression patterns of phenotypically pure ventral and dorsal roots. We identified highly significant differences in gene expression of a variety of regeneration-associated genes along the central - peripheral axis in healthy femoral nerves. Phenotypically mismatched grafting resulted in altered spatiotemporal expression of neurotrophic factor BDNF, GDNF receptor GFRα1, cell adhesion molecules Cadm3, Cadm4, L1CAM, and proliferation associated Ki67. Although significantly higher quadriceps muscle mass following homotopic nerve grafting was measured, we did not observe differences in gait analysis, and electrophysiological parameters between treatment paradigms. Our study provides evidence for phenotypic commitment of autologous nerve grafts after injury and gives a conclusive overview of temporal expression of several important regeneration-associated genes after repair with sensory or motor graft.

Delivery of the improved BMP-2-Advanced plasmid DNA within a gene-activated scaffold accelerates mesenchymal stem cell osteogenesis and critical size defect repair.

Gene-activated scaffolds have been shown to induce controlled, sustained release of functional transgene both in vitro and in vivo. Bone morphogenetic proteins (BMPs) are potent mediators of osteogenesis however we found that the delivery of plasmid BMP-2 (pBMP-2) alone was not sufficient to enhance bone formation. Therefore, the aim of this study was to assess if the use of a series of modified BMP-2 plasmids could enhance the functionality of a pBMP-2 gene-activated scaffold and ultimately improve bone regeneration when implanted into a critical sized bone defect in vivo. A multi-cistronic plasmid encoding both BMP-2 and BMP-7 (BMP-2/7) was employed as was a BMP-2-Advanced plasmid containing a highly truncated intron sequence. With both plasmids, the highly efficient cytomegalovirus (CMV) promoter sequence was used. However, as there have been reports that the elongated factor 1-α promoter is more efficient, particularly in stem cells, a BMP-2-Advanced plasmid containing the EF1α promoter was also tested. Chitosan nanoparticles (CS) were used to deliver each plasmid to MSCs and induced transient up-regulation of BMP-2 protein expression, in turn significantly enhancing MSC-mediated osteogenesis when compared to untreated controls (p < 0.001). When incorporated into a bone mimicking collagen-hydroxyapatite scaffold, the BMP-2-Advanced plasmid, under the control of the CMV promotor, induced MSCs to produce approximately 2500 µg of calcium per scaffold, significantly higher (p < 0.001) than all other groups. Just 4 weeks post-implantation in vivo, this cell-free gene-activated scaffold induced significantly more bone tissue formation compared to a pBMP-2 gene-activated scaffold (p < 0.001) as indicated by microCT and histomorphometry. Immunohistochemistry revealed that the BMP-2-Advanced plasmid accelerated differentiation of osteoprogenitor cells to mature osteoblasts, thus causing rapid healing of the bone defects. This study confirms that optimising the plasmid construct can enhance the functionality of gene-activated scaffolds and translate to accelerated bone formation in a critical sized defect.

Pushing the Right Buttons: Improving Efficacy of Therapeutic DNA Vectors.

Gene therapy represents a potent therapeutical application for regenerative medicine. So far, viral and nonviral approaches suffer from major drawbacks hindering efficient gene therapeutic applicability: the immunogenicity of viral systems on the one hand, and the low gene transfer efficiency of nonviral systems on the other hand. Therefore, there is a high demand for improvements of therapeutical systems at several levels. This review summarizes different DNA vector modifications to enhance biological efficacy and efficiency of therapeutical vectors, aiming for low toxicity, high specificity, and biological efficacy-the cornerstones for successful translation of gene therapy into the clinic. We aim to provide a step-by-step instruction to optimize their vectors to achieve the desired outcome of gene therapy. Our review provides the means to either construct a potent gene therapeutic vector de novo or to specifically address a bottleneck in the chain of events mandatory for therapeutic success. Although most of the introduced techniques can be translated into different areas, this review primarily addresses improvements for applications in transient gene therapy in the field of tissue engineering.

Mitochondria-Targeted Antioxidants SkQ1 and MitoTEMPO Failed to Exert a Long-Term Beneficial Effect in Murine Polymicrobial Sepsis.

Mitochondrial-derived reactive oxygen species have been deemed an important contributor in sepsis pathogenesis. We investigated whether two mitochondria-targeted antioxidants (mtAOX; SkQ1 and MitoTEMPO) improved long-term outcome, lessened inflammation, and improved organ homeostasis in polymicrobial murine sepsis. 3-month-old female CD-1 mice (n = 90) underwent cecal ligation and puncture (CLP) and received SkQ1 (5 nmol/kg), MitoTEMPO (50 nmol/kg), or vehicle 5 times post-CLP. Separately, 52 SkQ1-treated CLP mice were sacrificed at 24 h and 48 h for additional endpoints. Neither MitoTEMPO nor SkQ1 exerted any protracted survival benefit. Conversely, SkQ1 exacerbated 28-day mortality by 29%. CLP induced release of 10 circulating cytokines, increased urea, ALT, and LDH, and decreased glucose but irrespectively of treatment. Similar occurred for CLP-induced lymphopenia/neutrophilia and the NO blood release. At 48 h post-CLP, dying mice had approximately 100-fold more CFUs in the spleen than survivors, but this was not SkQ1 related. At 48 h, macrophage and granulocyte counts increased in the peritoneal lavage but irrespectively of SkQ1. Similarly, hepatic mitophagy was not altered by SkQ1 at 24 h. The absence of survival benefit of mtAOX may be due to the extended treatment and/or a relatively moderate-risk-of-death CLP cohort. Long-term effect of mtAOX in abdominal sepsis appears different to sepsis/inflammation models arising from other body compartments.

SNEVhPrp19/hPso4 Regulates Adipogenesis of Human Adipose Stromal Cells.

Aging is accompanied by loss of subcutaneous adipose tissue. This may be due to reduced differentiation capacity or deficiency in DNA damage repair (DDR) factors. Here we investigated the role of SNEVhPrp19/hPso4, which was implicated in DDR and senescence evasion, in adipogenic differentiation of human adipose stromal cells (hASCs). We showed that SNEV is induced during adipogenesis and localized both in the nucleus and in the cytoplasm. Knockdown of SNEV perturbed adipogenic differentiation and led to accumulation of DNA damage in hASCs upon oxidative stress. In addition, we demonstrated that SNEV is required for fat deposition in Caenorhabditis elegans. Consequently, we tested other DDR factors and found that WRN is also required for adipogenesis in both models. These results demonstrate that SNEV regulates adipogenesis in hASCs and indicate that DDR capacity in general might be a pre-requisite for this process.

A Noninvasive In Vitro Monitoring System Reporting Skeletal Muscle Differentiation.

Monitoring of cell differentiation is a crucial aspect of cell-based therapeutic strategies depending on tissue maturation. In this study, we have developed a noninvasive reporter system to trace murine skeletal muscle differentiation. Either a secreted bioluminescent reporter (Metridia luciferase) or a fluorescent reporter (green fluorescent protein [GFP]) was placed under the control of the truncated muscle creatine kinase (MCK) basal promoter enhanced by variable numbers of upstream MCK E-boxes. The engineered pE3MCK vector, coding a triple tandem of E-Boxes and the truncated MCK promoter, showed twentyfold higher levels of luciferase activation compared with a Cytomegalovirus (CMV) promoter. This newly developed reporter system allowed noninvasive monitoring of myogenic differentiation in a straining bioreactor. Additionally, binding sequences of endogenous microRNAs (miRNAs; seed sequences) that are known to be downregulated in myogenesis were ligated as complementary seed sequences into the reporter vector to reduce nonspecific signal background. The insertion of seed sequences improved the signal-to-noise ratio up to 25% compared with pE3MCK. Due to the highly specific, fast, and convenient expression analysis for cells undergoing myogenic differentiation, this reporter system provides a powerful tool for application in skeletal muscle tissue engineering.

A Luciferase-Based Quick Potency Assay to Predict Chondrogenic Differentiation.

Chondrogenic differentiation of adipose-derived stem cells (ASC) is challenging but highly promising for cartilage repair. Large donor variability of chondrogenic differentiation potential raises the risk for transplantation of cells with reduced efficacy and a low chondrogenic potential. Therefore, quick potency assays are required to control the potency of the isolated cells before cell transplantation. Current in vitro methods to analyze the differentiation capacity are time-consuming, and thus, a novel enhancer and tissue-specific promoter combination was used for the detection of chondrogenic differentiation of ASC in a novel quick potency bioassay. Human primary ASC were cotransfected with the Metridia luciferase-based collagen type II reporter gene pCMVE_ACDCII-MetLuc together with a Renilla control plasmid and analyzed for their chondrogenic potential. On day 3 after chondrogenic induction, the luciferase activity was induced in all tested donors under three-dimensional culture conditions and, in a second approach, also under two-dimensional (2D) culture conditions. With our newly developed quick potency bioassay, we can determine chondrogenic potential already after 3 days of chondrogenic induction and under 2D culture conditions. This will enhance the efficiency of testing cell functionality, which should allow in the future to predict the suitability of cells derived from individual patients for cell therapies in a very short time and at low costs.

In toto differentiation of human amniotic membrane towards the Schwann cell lineage.

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100ß, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100ß was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.