The Optimal Mutagen Dosage to Induce Point-Mutations in <em>Synechocystis</em> sp. PCC6803 and Its Application to Promote Temperature Tolerance

<div><p>Random mutagenesis is a useful tool to genetically modify organisms for various purposes, such as adaptation to cultivation conditions, the induction of tolerances, or increased yield of valuable substances. This is especially attractive for systems where it is not obvious which genes require modifications. Random mutagenesis has been extensively used to modify crop plants, but even with the renewed interest in microalgae and cyanobacteria for biofuel applications, there is relatively limited current research available on the application of random mutagenesis for these organisms, especially for cyanobacteria. In the presented work we characterized the lethality and rate of non-lethal point mutations for ultraviolet radiation and methyl methanesulphonate on the model cyanobacteria <em>Synechocystis</em> sp. PCC6803. Based on these results an optimal dosage of 10–50 J/m² for UV and either 0.1 or 1 v% for MMS was determined. A <em>Synechocystis</em> wildtype culture was then mutagenized and selected for increased temperature tolerance <em>in vivo</em>. During the second round of mutagenesis the viability of the culture was monitored on a cell by cell level from the treatment of the cells up to the growth at an increased temperature. After four distinct rounds of treatment (two with each mutagen) the temperature tolerance of the strain was effectively raised by about 2°C. Coupled with an appropriate <em>in vivo</em> screening, the described methods should be applicable to induce a variety of desirable characteristics in various strains. Coupling random mutagenesis with high-throughput screening methods would additionally allow to select for important characteristics for biofuel production, which do not yield a higher fitness and can not be selected for <em>in vivo</em>, such as fatty acid concentration. In a combined approach with full genome sequencing random mutagenesis could be used to determine suitable target-genes for more focused methods.</p> </div

Characterization of UV.

<p>Survival rate (green; triangle) and point mutation rate (blue; square) plotted over the administered dosage of UV irradiation. The counted colonies for the survival rate and the point mutation rate were normalized by setting the control to 100%.</p

Characterization of MMS.

<p>Survival rate (green; upward triangle: 5*10∧4 cells plated | downward triangle 5*10∧5 cells plated, counted colonies divided by 10) and point mutation rate (red; square) plotted over the administered dosage of MMS exposure. The counted colonies for the survival rate and the point mutation rate were normalized by setting the control to 100%.</p

Summary of the conditions and results of each round of mutagenesis.

*<p>Note that in the 3rd round of mutagenesis there was a technical problem with the temperation, and cultures were also temperated during the night cycle, leading to many more cells dying than usual. A recovery at lower temperature was used to rescue surviving cells.</p

Analysis of embryonic zebrafish NMJ using 3D ART VeSElecT in comparison to manual analysis using IMOD.

<p>We show in Fig 2 A) the original tomogram of 4dpf zebrafish NMJ, in Fig 2 B) the manual reconstruction is included in the tomogram of A), in B') the 3D reconstruction of the manual annotation (vesicles are colored in light blue) is shown. This is compared to Fig 2 C) which shows the semi-automated vesicle recognition overlaid with the original tomogram, and C') which shows the vesicle pool of the semi-automated annotation as 3D reconstruction (vesicles are in arbitrary colors). In D) boxplots show the results of the comparison of 4dpf and 8dpf zebrafish embryos using manual annotation (left) and semi-automated annotation (right). The box of the box plots shows the mid-50% of data. The line in the box represents the median of all data. Whiskers end at lowest value within 1.5 interquartile range (IQR) of the lower quartile and at the highest value within 1.5 IQR of the upper quartile. Data that is not included in between both whiskers are plotted as outliers with a dot.</p

Measurement of inner and outer synaptic vesicle diameter.

<p>In order to get an approximate value of the discrepancies between vesicle diameters of manual and automated measurement we applied the Fiji measurement tool. Fig 3 (A) shows the inner diameter of a vesicle that was annotated by 3D ART VeSElecT, (B) shows the outer diameter. Fig 3 (C) gives the results of the discrepancy of inner and outer diameter of all measured vesicles shown as a histogram (number of measurements = 80).</p

Workflow of vesicle annotation using 3D ART VeSElecT.

<p>First, the automated registration macro is used, which scales the tomogram via user input of the pixel size and applies various filters in the preprocessing step. Afterwards the foreground is separated, the user semi-automatically selects an area of interest, and the macro applies the watershed algorithm for vesicle segmentation and registration. Second, an optional manual proof-reading step can be applied here, if necessary. Finally, the automatic measurement macro is used to extract results using certain characteristics. All manual steps are colored in yellow, semi-automated steps are in turquoise, automated steps are in blue.</p

Tabular schedule showing required time for semi-automated reconstruction.

<p>Tabular schedule showing required time for semi-automated reconstruction.</p

Preclinical Studies Identify Non-Apoptotic Low-Level Caspase-3 as Therapeutic Target in Pemphigus Vulgaris

<div><p>The majority of pemphigus vulgaris (PV) patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis). The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg) 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG), PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice) as well as PV patients’ biopsies (n=6). A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP) and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the <i>in vitro</i> and <i>in vivo</i> PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other diseases including cancer.</p></div

Protective Vaccination against Infectious Bursal Disease Virus with Whole Recombinant <em>Kluyveromyces lactis</em> Yeast Expressing the Viral VP2 Subunit

<div><p>Here we report on vaccination approaches against infectious bursal disease (IBD) of poultry that were performed with complete yeast of the species <em>Kluyveromyces lactis</em> (<em>K. lactis</em>). Employing a genetic system that enables the rapid production of stably transfected recombinant <em>K. lactis</em>, we generated yeast strains that expressed defined quantities of the virus capsid forming protein VP2 of infectious bursal disease virus (IBDV). Both, subcutaneous as well as oral vaccination regiments with the heat-inactivated but otherwise untreated yeast induced IBDV-neutralizing antibodies in mice and chickens. A full protection against a subsequent IBDV infection was achieved by subcutaneous inoculation of only milligram amounts of yeast per chicken. Oral vaccination also generated protection: while mortality was observed in control animals after virus challenge, none of the vaccinees died and ca. one-tenth were protected as indicated by the absence of lesions in the bursa of Fabricius. Recombinant <em>K. lactis</em> was thus indicated as a potent tool for the induction of a protective immune response by different applications. Subcutaneously applied <em>K. lactis</em> that expresses the IBDV VP2 was shown to function as an efficacious anti-IBD subunit vaccine.</p> </div