High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.     

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca²⁺ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca²⁺ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca²⁺ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca²⁺ indicator and a hydrophilic fluorescent dye/Ca²⁺ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F₀.     

DUF538 Protein Super Family is Predicted to be the Potential Homologue of Bactericidal/Permeability-Increasing Protein in Plant System

DUF538 protein super family includes a number of plant proteins that their role is not yet clear. These proteins have been frequently reported to be expressed in plants under various stressful stimuli such as bacteria and elicitors. In order to further understand about this protein family we utilized bioinformatics tools to analyze its structure in details. As a result, plants DUF538 was predicted to be the partial structural homologue of BPI (bactericidal/permeability increasing) proteins in mammalian innate immune system that provides the first line of defense against different pathogens including bacteria, fungi, viruses and parasites. Moreover, on the base of the experimental data, it was identified that exogenously applied purified fused product of Celosia DUF538 affects the bacterial growth more possibly similar to BPI through the binding to the bacterial membranes. In conclusion, as the first ever time report, we nominated DUF538 protein family as the potential structural and functional homologue of BPI protein in plants, providing a basis to study the novel functions of this protein family in the biological systems in the future.     

Effect of Polymer Crystallinity in P3HT:PCBM Solar Cells on Band Gap Trap States and Apparent Recombination Order

The non‐geminate recombination of charge carriers in polymer‐fullerene solar cells has been modeled in the last few years with a trap‐assisted recombination model, which states that the apparent recombination order depends on the concentration of trapped charges tailing into the band gap. Higher concentrations of trapped charges lead to higher apparent recombination orders. In this work, the mass fraction f of highly crystalline nanofibrillar P3HT to the total P3HT content in P3HT:PCBM solar cells is consistently varied, controlling the temperature of a nanofibers‐P3HT casting dispersion. A systematic study of the apparent recombination order, measured with a transient photovoltage technique, as a function of f is presented. A correlation is shown between the apparent recombination order, the P3HT crystallinity, and the trap concentration in the band gap measured with an admittance spectroscopy technique.     

Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.     

Immobilization rapidly selects for chemoresistant ovarian cancer cells with enhanced ability to enter dormancy

Around 20–30% of ovarian cancer patients exhibit chemoresistance, but there are currently no methods to predict whether a patient will respond to chemotherapy. Here, we discovered that chemoresistant ovarian cancer cells exhibit enhanced survival in a quiescent state upon experiencing the stress of physical confinement. When immobilized in stiff silica gels, most ovarian cancer cells die within days, but surviving cells exhibit hallmarks of single-cell dormancy. Upon extraction from gels, the cells resume proliferation but demonstrate enhanced viability upon reimmobilization, indicating that initial immobilization selects for cells with a higher propensity to enter dormancy. RNA-seq analysis of the extracted cells shows they have signaling responses similar to cells surviving cisplatin treatment, and in comparison to chemoresistant patient cohorts, they share differentially expressed genes that are associated with platinum-resistance pathways. Furthermore, these extracted cells demonstrate greater resistance to cisplatin and paclitaxel, despite being proliferative. In contrast, serum starvation and hypoxia could not effectively select for chemoresistant cells upon removal of the environmental stress. These findings demonstrate that ovarian cancer chemoresistance and the ability to enter dormancy are linked, and immobilization rapidly distinguishes chemoresistant cells. This platform could be suitable for mechanistic studies, drug development, or as a clinical diagnostic tool.     

Molecular genetic diversity within Myrmeleontidae family

Antlions are insects which feed on ants, insect which dig a pit and lies in wait for ants and other insects. Twelve species of Myrmeleontidae family as antlions and many specimens were identified in different locations in Fars province in Iran. To unveil the genetic similarity between these species, their DNA was extracted by modified CTAB method and with the use of seventeen 10-nucleotides primers of random amplified polymorphic DNA (RAPD); the genetic analysis of them was investigated. After PCR, agarose 1.5?% was used for electrophoresis. The obtained electrophoresis bands had base pairs range between 150 and 1,000?bp. The maximum of polymorphic bands belonged to OPH5, N13, and the minimum of polymorphic bands belonged to OPA7 primers. Different genetic similarity indices were found between eight species of antlions. Possibility of use of RAPD marker together with morphological studies for classification and identification of antlions is discussed.