Multifactorial in vivo regulation of the photoreceptor channelrhodopsin-1 abundance.

Oriented movement (phototaxis) is an efficient way to optimize light-driven processes and to avoid photodamage for motile algae. In Chlamydomonas the receptors for phototaxis are the channelrhodopsins ChR1 and ChR2. Both are directly light-gated, plasma membrane-localized cation channels. To optimally adjust its overall light-dependent responses, Chlamydomonas must tightly control the ChRs cellular abundance and integrate their activities into its general photoprotective network. How this is achieved is largely unknown. Here we show that the ChR1 protein level decreases upon illumination in a light-intensity and quality-dependent manner, whereas it is stable in prolonged darkness. Analysis of knockout strains of six major photoreceptors absorbing in the blue-violet range, which is most effective in evoking ChR1 degradation, revealed that only phototropin (PHOT) is involved. Notably, ChR2 degradation was normal in a ΔPHOT strain. Further, our results indicate that a COP1-SPA1 E3 ubiquitin ligase, the transcription factor Hy5 as well as changes in the cellular redox poise and cyclic nucleotide levels are additional components involved in this light acclimation response of Chlamydomonas. Our data highlight the presence of an adaptive framework connecting phototaxis with general photoprotective mechanisms via the use of overlapping signaling components already at the level of the primary photoreceptor.

Deciphering the role of cytoplasmic domain of Channelrhodopsin in modulation of the interactome and SUMOylome of Chlamydomonas reinhardtii.

Translocation of channelrhodopsins (ChRs) is mediated by the intraflagellar transport (IFT) machinery. However, the functional role of the network involving photoreceptors, IFT and other proteins in controlling algal ciliary motility is still not fully delineated. In the current study, we have identified two important motifs at the C-terminus of ChR1, VXPX and LKNE. VXPX is a known ciliary targeting sequence in animals, and LKNE is a well-known SUMOylation motif. To the best of our knowledge, this study gives prima facie insight into the role of SUMOylation in Chlamydomonas. We prove that VMPS of ChR1 is important for interaction with GTPase CrARL11. We show that SUMO motifs are present in the C-terminus of putative ChR1s from green algae. Performing experiments with n-Ethylmaleimide (NEM) and Ubiquitin-like protease 1 (ULP-1), we show that SUMOylation may modulate ChR1 protein in Chlamydomonas. Experiments with 2D08, a known sumoylation blocker, increased the concentration of ChR1 protein. Finally, we show the endogenous SUMOylated proteins (SUMOylome) of C. reinhardtii, identified by using immunoprecipitation followed by nano-LC-MS/MS detection. This report establishes a link between evolutionarily conserved SUMOylation and ciliary machinery for the maintenance and functioning of cilia across the eukaryotes. Our enriched SUMOylome of C. reinhardtii comprehends the proteins related to ciliary development and photo-signaling, along with the orthologue(s) associated to human ciliopathies as SUMO targets.

Gene Editing in Green Alga Chlamydomonas reinhardtii via CRISPR-Cas9 Ribonucleoproteins.

With the establishment of the CRISPR-Cas9 molecular tool as a DNA editing system in 2012, the handling of gene editing experiments was strongly facilitated pushing reverse genetics approaches forward in many organisms. These new gene editing technologies also drastically increased the possibilities for design-driven synthetic biology. Here, we describe a protocol for gene editing in the green algae Chlamydomonas reinhardtii using preassembled CRISPR-Cas9 ribonucleoproteins.The three sections of the protocol guide through a complete gene editing experiment, starting with the experimental design and the choice of suitable CRISPR target sites and how to perform a Cas9 in vitro test digestion. The second part covers the transformation of algal cells with Cas9 RNPs using electroporation. In the last part, the PCR-based screening for mutants and isolation of clones is explained.

Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting.

The use of CRISPR/Cas endonucleases has revolutionized gene editing techniques for research on Chlamydomonas reinhardtii. To better utilize the CRISPR/Cas system, it is essential to develop a more comprehensive understanding of the DNA repair pathways involved in genome editing. In this study, we have analyzed contributions from canonical KU80/KU70-dependent nonhomologous end-joining (cNHEJ) and DNA polymerase theta (POLQ)-mediated end joining on SpCas9-mediated untemplated mutagenesis and homology-directed repair (HDR)/gene inactivation in Chlamydomonas. Using CRISPR/SpCas9 technology, we generated DNA repair-defective mutants ku80, ku70, polQ for gene targeting experiments. Our results show that untemplated repair of SpCas9-induced double strand breaks results in mutation spectra consistent with an involvement of both KU80/KU70 and POLQ. In addition, the inactivation of POLQ was found to negatively affect HDR of the inactivated paromomycin-resistant mut-aphVIII gene when donor single-stranded oligos were used. Nevertheless, mut-aphVIII was still repaired by homologous recombination in these mutants. POLQ inactivation suppressed random integration of transgenes co-transformed with the donor ssDNA. KU80 deficiency did not affect these events but instead was surprisingly found to stimulate HDR/gene inactivation. Our data suggest that in Chlamydomonas, POLQ is the main contributor to CRISPR/Cas-induced HDR and random integration of transgenes, whereas KU80/KU70 potentially plays a secondary role. We expect our results will lead to improvement of genome editing in C. reinhardtii and can be used for future development of algal biotechnology.

Microstructures and Mechanical Properties of Hybrid, Additively Manufactured Ti6Al4V after Thermomechanical Processing.

In the present study, we propose a hybrid manufacturing route to produce high-quality Ti6Al4V parts, combining additive powder laser directed energy deposition (L-DED) for manufacturing of preforms, with subsequent hot forging as a thermomechanical processing (TMP) step. After L-DED, the material was hot formed at two different temperatures (930 °C and 1070 °C) and subsequently heat-treated for stress relief annealing. Tensile tests were performed on small sub-samples, taking into account different sample orientations with respect to the L-DED build direction and resulting in very good tensile strengths and ductility properties, similar or superior to the forged material. The resulting microstructure consists of very fine grained, partially globularized alpha grains, with a mean diameter ~0.8-2.3 µm, within a beta phase matrix, constituting between 2 and 9% of the sample. After forging in the sub-beta transus temperature range, the typical L-DED microstructure was no longer discernible and the anisotropy in tensile properties, common in additive manufacturing (AM), was significantly reduced. However, forging in the super-beta transus temperature range resulted in remaining anisotropies in the mechanical properties as well as an inferior tensile strength and ductility of the material. It was shown, that by combining L-DED with thermomechanical processing in the sub-beta transus temperature range of Ti6Al4V, a suitable microstructure and desirable mechanical properties for many applications can be obtained, with the advantage of reducing the material waste.

Laser beam shaping with circular serrated apertures. I. Spatial filtering.

Propagation of an axially symmetrical beam through an apodizer comprising a circular serrated aperture and a spatial filter is considered on the basis of the parabolic equation solution. In part I, the equation is solved for uniform and Gaussian beams with a flat wavefront, which propagate from the circular serrated aperture to the spatial filter focal plane. By analyzing the field structure in the focal plane, the diameter of the spatial filter pinhole required for recovering the axial symmetry of the field at the spatial filter exit is determined as a function of the serrated aperture and spatial filter parameters.

Laser beam shaping with circular serrated apertures. II. Theory of the beam profile formation.

Propagation of an axially symmetrical beam through an apodizer comprising a circular serrated aperture and a spatial filter is considered on the basis of the parabolic equation solution. In Part II, the equation is solved for uniform and Gaussian beams with a flat wavefront propagating from the spatial filter pinhole to its exit. It is shown that serrations with complex shapes (for instance, cosine or parabolic) have no advantage over a simple triangular shape: practically the same beam profiles can be obtained with triangular serrations of slightly different size.

Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9.

The fast-growing biflagellated single-celled chlorophyte Chlamydomonas reinhardtii is the most widely used alga in basic research. The physiological functions of the 18 sensory photoreceptors are of particular interest with respect to Chlamydomonas development and behavior. Despite the demonstration of gene editing in Chlamydomonas in 1995, the isolation of mutants lacking easily ascertained newly acquired phenotypes remains problematic due to low DNA recombination efficiency. We optimized gene-editing protocols for several Chlamydomonas strains (including wild-type CC-125) using zinc-finger nucleases (ZFNs), genetically encoded CRISPR/associated protein 9 (Cas9) from Staphylococcus aureus and Streptococcus pyogenes, and recombinant Cas9 and developed protocols for rapidly isolating nonselectable gene mutants. Using this technique, we disrupted the photoreceptor genes COP1/2, COP3 (encoding channelrhodopsin 1 [ChR1]), COP4 (encoding ChR2), COP5, PHOT, UVR8, VGCC, MAT3, and aCRY and created the chr1 chr2 and uvr8 phot double mutants. Characterization of the chr1, chr2, and mat3 mutants confirmed the value of photoreceptor mutants for physiological studies. Genes of interest were disrupted in 5 to 15% of preselected clones (∼1 out of 4000 initial cells). Using ZFNs, genes were edited in a reliable, predictable manner via homologous recombination, whereas Cas9 primarily caused gene disruption via the insertion of cotransformed DNA. These methods should be widely applicable to research involving green algae.

Catheter ablation for atrial fibrillation after an unsuccessful surgical ablation and biological prosthetic mitral valve replacement: a pilot study.

BACKGROUND: Patients with mitral valve (MV) disease and atrial fibrillation (AF) undergo simultaneous prosthetic valve replacement and radiofrequency (RF) ablation procedure; however, this combinational procedure restores sinus rhythm (SR) in only 68-82% of the cases. In patients with ineffective surgical ablation, the use of a biological prosthetic valve might not only be a good choice to perform safe catheter ablation procedure in the left atrium (LA), but also provide a way to discontinue administration of oral anticoagulants. The objective of this study was to assess the efficacy of catheter ablation for AF after MV replacement with a biological prosthesis and an ineffective surgical ablation procedure. METHODS: Ten consecutive patients aged 48 ± 7 years were enrolled in this study. All patients had long-persistent AF associated with a rheumatic valve disease, which was treated by MV replacement with a biological prosthesis and a surgical RF ablation procedure. In the late postoperative period, all the patients had recurrent hemodynamically significant AF, which required repeated cardioversions. From 1 year to 3 years after the surgery, catheter ablation was performed, including reisolation of pulmonary veins (PVs) with the ablation of ganglionic plexi or linear lesions on the roof of the LA and mitral isthmus. The efficacy was assessed at 3 months, 6 months, and 12 months after the procedure. RESULTS: Restoration of SR during ablation was achieved in all of the cases. In 6-9 months, all the patients were free of arrhythmia. LA stunning manifested by the absence or decrease of the "A" wave in the transmitral flow and the retrograde wave in the PV flow was observed in nine patients with SR. In five of the patients, LA contractile function was restored in 1-6 months. Prosthetic valve dysfunction was not detected in any of the patients. CONCLUSION: Catheter ablation is an effective method for AF treatment following an ineffective surgical RF ablation procedure and biological prosthetic MV replacement. The use of bioprosthetic MVs allows for performing safe catheter ablation without subsequent prosthetic dysfunction.

Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases.

The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes (i) design of gene-specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3'-phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin-resistant (Pm-R) clones with expressing nucleases. Of these Pm-R clones, 1% also contained a modified COP3 locus. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.

Nuclear gene targeting in Chlamydomonas as exemplified by disruption of the PHOT gene.

Chlamydomonas reinhardtii is the most powerful photosynthetic eukaryotic unicellular model organism. However, its potential is not fully exploitable since as in most green plants specific targeting of nuclear genes is not routinely possible. Recently, we have shown by repair of an introduced truncated model gene that transformation of Chlamydomonas with single stranded DNA greatly suppresses random integration of the DNA in the genome whereas homologous recombination (HR) is left unchanged. However, endogenous genes still could not be targeted. Here we present optimized transformation conditions that further improved HR and suppressed non-homologous DNA integration (NHI). The improved transformation strategy allowed us now to specifically inactivate in two different Chlamydomonas strains the nuclear PHOT gene, which encodes for the blue light photoreceptor phototropin (PHOT). The option to target moderately expressed Chlamydomonas nuclear genes with high efficiency now further improves the utility of this this alga for basic science and biotechnology.

Nuclear-gene targeting by using single-stranded DNA avoids illegitimate DNA integration in Chlamydomonas reinhardtii.

Homologous DNA recombination (HR) allows the deletion (knockout), repair (rescuing), and modification of a selected gene, thereby rendering a functional analysis of the gene product possible. However, targeting of nuclear genes has been an inefficient process in most eukaryotes, including algae, plants, and animals, due to the dominance of integration of the applied DNA into nonhomologous regions of the genome. We have shown for the green alga Chlamydomonas reinhardtii by repairing a previously introduced truncated aminoglycoside 3'-phosphotransferase gene, aphVIII, that single-stranded DNA can recombine with a homologous endogenous DNA region of interest. Nonhomologous DNA integration appeared to be more than 100-fold reduced compared with the use of double-stranded DNA, thus allowing isolation of the homologous recombinants. We propose that this method will be applicable to direct targeting of nuclear C. reinhardtii genes.