BBF RFC 36: Fusion protein BioBrick assembly standard with optional linker extension

This RFC 36 describes an extension of the original BioBrick assembly standard (BBF RFC 10) and Freiburg assembly standard (BBF RFC 25). The Fusion Assembly strategy described here is fully compatible with RFC 25 (Freiburg) and RFC 10 standard and supports in-frame fusion and controlled potentially infinite extension of linker between protein fusion domains

BBF RFC 37: Fusion protein BioBrick assembly standard with optional linker extension

BBF RFC 37 replaces BBF RFC 36.This RFC 37 describes an extension of the original BioBrick assembly standard (BBF RFC 10) and Freiburg assembly standard (BBF RFC 25). The Fusion Assembly strategy described here is fully compatible with RFC 25 (Freiburg) and RFC 10 standard and supports in-frame fusion and controlled potentially infinite extension of linker between protein fusion domains

Toll-Like Receptor 4 Activation in Cancer Progression and Therapy

Cancer immunotherapy has been the focus of intense research since the late 19th century when Coley observed that bacterial components can contribute to cancer regression by eliciting an antitumor immune response. Successful activation and maturation of tumor-specific immune cells is now known to be mediated by bacterial endotoxin, which activates Toll-like receptor 4 (TLR4). TLR4 is expressed on a variety of immune as well as tumor cells, but its activation can have opposing effects. While TLR4 activation can promote antitumor immunity, it can also result in increased tumor growth and immunosuppression. Nevertheless, TLR4 engagement by endotoxin as well as by endogenous ligands represents notable contribution to the outcome of different cancer treatments, such as radiation or chemotherapy. Further research of the role and mechanisms of TLR4 activation in cancer may provide novel antitumor vaccine adjuvants as well as TLR4 inhibitors that could prevent inflammation-induced carcinogenesis

A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation

Inflammation, which is a highly regulated host response against danger signals, may be harmful if it is excessive and deregulated. Ideally, anti-inflammatory therapy should autonomously commence as soon as possible after the onset of inflammation, should be controllable by a physician, and should not systemically block beneficial immune response in the long term. We describe a genetically encoded anti-inflammatory mammalian cell device based on a modular engineered genetic circuit comprising a sensor, an amplifier, a “thresholder” to restrict activation of a positive-feedback loop, a combination of advanced clinically used biopharmaceutical proteins, and orthogonal regulatory elements that linked modules into the functional device. This genetic circuit was autonomously activated by inflammatory signals, including endogenous cecal ligation and puncture (CLP)-induced inflammation in mice and serum from a systemic juvenile idiopathic arthritis (sIJA) patient, and could be reset externally by a chemical signal. The microencapsulated anti-inflammatory device significantly reduced the pathology in dextran sodium sulfate (DSS)-induced acute murine colitis, demonstrating a synthetic immunological approach for autonomous anti-inflammatory therapy

Effect of Hydrophobic Mutations in the H2-H3 Subdomain of Prion Protein on Stability and Conversion In Vitro and In Vivo

Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes

The CRISPR/Cas9 genome editing system has already proved its efficiency, versatility and simplicity in numerous applications in human, animal, microbe and plant cells. Together with the vast amount of genome and transcriptome databases available, it represents an enormous potential for plant breeding and research. Although most changes produced with CRISPR/Cas9 do not differ from naturally occurring mutations, the use of transgenesis during varietal development can still trigger GMO legislation in countries that rely on process-based regulation. Moreover, stable integration of DNA coding for genome-editing tools into plant genomes can result in insertional mutagenesis, while its prolonged expression can cause mutations in off-target sites. These pitfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro-transcribed or synthesized sgRNA. We therefore aimed to develop a DNA-free protocol for site-directed mutagenesis of three species of the genus Brassica (B. oleracea, B. napus, and B. rapa) with the use of RNPs. We chose cabbage, rapeseed and Chinese cabbage as species representatives and introduced RNPs into their protoplasts with PEG 4000. Four sgRNAs targeting two endogenous genes (the FRI and PDS genes, two sgRNAs per gene) were introduced into all three species. No mutations were detected after transfection of rapeseed protoplasts, while we obtained mutation frequencies of 0.09 to 2.25% and 1.15 to 24.51% in cabbage and Chinese cabbage, respectively. In both species, a positive correlation was displayed between the amount (7.5, 15, 30, and 60 μg) of Cas9 enzyme and sgRNA introduced and mutation frequency. Nucleotide changes (insertions and deletions) were detected 24 h after transfection and did not differ 72 h after transfection. They were species-, gene- and locus-dependent. In summary, we demonstrated the suitability of RNP transfection into B. oleracea and B. rapa protoplasts for high-efficiency indel induction of two endogenous genes. Due to the relatively high mutation frequencies detected (up to 24.51%), this study paves the way for regeneration of precisely mutated Brassica plants without the use of transgenesis

Comparative analysis of selected methods for the assessment of antimicrobial and membrane-permeabilizing activity: a case study for lactoferricin derived peptides

<p>Abstract</p> <p>Background</p> <p>Growing concerns about bacterial resistance to antibiotics have prompted the development of alternative therapies like those based on cationic antimicrobial peptides (APs). These compounds not only are bactericidal by themselves but also enhance the activity of antibiotics. Studies focused on the systematic characterization of APs are hampered by the lack of standard guidelines for testing these compounds. We investigated whether the information provided by methods commonly used for the biological characterization of APs is comparable, as it is often assumed. For this purpose, we determined the bacteriostatic, bactericidal, and permeability-increasing activity of synthetic peptides (n = 57; 9–13 amino acid residues in length) analogous to the lipopolysaccharide-binding region of human lactoferricin by a number of the most frequently used methods and carried out a comparative analysis.</p> <p>Results</p> <p>While the minimum inhibitory concentration determined by an automated turbidimetry-based system (Bioscreen) or by conventional broth microdilution methods did not differ significantly, bactericidal activity measured under static conditions in a low-ionic strength solvent resulted in a vast overestimation of antimicrobial activity. Under these conditions the degree of antagonism between the peptides and the divalent cations differed greatly depending on the bacterial strain tested. In contrast, the bioactivity of peptides was not affected by the type of plasticware (polypropylene vs. polystyrene). Susceptibility testing of APs using cation adjusted Mueller-Hinton was the most stringent screening method, although it may overlook potentially interesting peptides. Permeability assays based on sensitization to hydrophobic antibiotics provided overall information analogous – though not quantitatively comparable- to that of tests based on the uptake of hydrophobic fluorescent probes.</p> <p>Conclusion</p> <p>We demonstrate that subtle changes in methods for testing cationic peptides bring about marked differences in activity. Our results show that careful selection of the test strains for susceptibility testing and for screenings of antibiotic-sensitizing activity is of critical importance. A number of peptides proved to have potent permeability-increasing activity at subinhibitory concentrations and efficiently sensitized <it>Pseudomonas aeruginosa </it>both to hydrophilic and hydrophobic antibiotics.</p

Novel carboxylate-based glycolipids: TLR4 antagonism, MD-2 binding and self-assembly properties

New monosaccharide-based lipid A analogues were rationally designed through MD-2 docking studies. A panel of compounds with two carboxylate groups as phosphates bioisosteres, was synthesized with the same glucosamine-bis-succinyl core linked to different unsaturated and saturated fatty acid chains. The binding of the synthetic compounds to purified, functional recombinant human MD-2 was studied by four independent methods. All compounds bound to MD-2 with similar affinities and inhibited in a concentration-dependent manner the LPS-stimulated TLR4 signaling in human and murine cells, while being inactive as TLR4 agonists when provided alone. A compound of the panel was tested in vivo and was not able to inhibit the production of proinflammatory cytokines in animals. This lack of activity is probably due to strong binding to serum albumin, as suggested by cell experiments in the presence of the serum. The interesting self-assembly property in solution of this type of compounds was investigated by computational methods and microscopy, and formation of large vesicles was observed by cryo-TEM microscopy.TOLLerant project (H2020-MSC-ETN-642157), the Italian Ministry for Foreign Affairs and International Cooperation (MAECI) and Spanish MINECO (CTQ2014-57141-R and CTQ2017-88353-R grants) are acknowledged. Vesna Hodnik of University of Ljubljana for the help with SPR, Sandra Delgado of CIC BioGUNE for the Cryo-TEM images. RJ was partially funded by the research program P4-0176 by the Slovenian Research Agency

Zwiespältige Bilanz für Afrika : die Milleniumsziele der UN haben keine Gleichberechtigung in der Entwicklungszusammenarbeit gebracht

An increasing number of pathologies have been linked to Toll-like receptor 4 (TLR4) activation and signaling, therefore new hit and lead compounds targeting this receptor activation process are urgently needed. We report on the synthesis and biological properties of glycolipids based on glucose and trehalose scaffolds which potently inhibit TLR4 activation and signaling in vitro and in vivo. Structure–activity relationship studies on these compounds indicate that the presence of fatty ester chains in the molecule is a primary prerequisite for biological activity and point to facial amphiphilicity as a preferred architecture for TLR4 antagonism. The cationic glycolipids here presented can be considered as new lead compounds for the development of drugs targeting TLR4 activation and signaling in infectious, inflammatory, and autoimmune diseases. Interestingly, the biological activity of the best drug candidate was retained after adsorption at the surface of colloidal gold nanoparticles, broadening the options for clinical development.Italian, Ministry of University and Research (MIUR), PRIN 2010 - 11Ministerio de Economía y Competitividad AF2013-44021- R, CTQ2010 - 15848Junta de Andalucía FQM 1467Slovenian Research Agency P4-176, J1 - 417