Candida auris undergoes adhesin-dependent and -independent cellular aggregation.

Candida auris is a fungal pathogen of humans responsible for nosocomial infections with high mortality rates. High levels of resistance to antifungal drugs and environmental persistence mean these infections are difficult to treat and eradicate from a healthcare setting. Understanding the life cycle and the genetics of this fungus underpinning clinically relevant traits, such as antifungal resistance and virulence, is of the utmost importance to develop novel treatments and therapies. Epidemiological and genomic studies have identified five geographical clades (I-V), which display phenotypic and genomic differences. Aggregation of cells, a phenotype primarily of clade III strains, has been linked to reduced virulence in some infection models. The aggregation phenotype has thus been associated with conferring an advantage for (skin) colonisation rather than for systemic infection. However, strains with different clade affiliations were compared to infer the effects of different morphologies on virulence. This makes it difficult to distinguish morphology-dependent causes from clade-specific or even strain-specific genetic factors. Here, we identify two different types of aggregation: one induced by antifungal treatment which is a result of a cell separation defect; and a second which is controlled by growth conditions and only occurs in strains with the ability to aggregate. The latter aggregation type depends on an ALS-family adhesin which is differentially expressed during aggregation in an aggregative C. auris strain. Finally, we demonstrate that macrophages cannot clear aggregates, suggesting that aggregation might after all provide a benefit during systemic infection and could facilitate long-term persistence in the host.

Gene conversion: a non-Mendelian process integral to meiotic recombination.

Meiosis is undoubtedly the mechanism that underpins Mendelian genetics. Meiosis is a specialised, reductional cell division which generates haploid gametes (reproductive cells) carrying a single chromosome complement from diploid progenitor cells harbouring two chromosome sets. Through this process, the hereditary material is shuffled and distributed into haploid gametes such that upon fertilisation, when two haploid gametes fuse, diploidy is restored in the zygote. During meiosis the transient physical connection of two homologous chromosomes (one originally inherited from each parent) each consisting of two sister chromatids and their subsequent segregation into four meiotic products (gametes), is what enables genetic marker assortment forming the core of Mendelian laws. The initiating events of meiotic recombination are DNA double-strand breaks (DSBs) which need to be repaired in a certain way to enable the homologous chromosomes to find each other. This is achieved by DSB ends searching for homologous repair templates and invading them. Ultimately, the repair of meiotic DSBs by homologous recombination physically connects homologous chromosomes through crossovers. These physical connections provided by crossovers enable faithful chromosome segregation. That being said, the DSB repair mechanism integral to meiotic recombination also produces genetic transmission distortions which manifest as postmeiotic segregation events and gene conversions. These processes are non-reciprocal genetic exchanges and thus non-Mendelian.

Intragenic meiotic recombination in Schizosaccharomyces pombe is sensitive to environmental temperature changes.

Changes in environmental temperature influence cellular processes and their dynamics, and thus affect the life cycle of organisms that are unable to control their cell/body temperature. Meiotic recombination is the cellular process essential for producing healthy haploid gametes by providing physical links (chiasmata) between homologous chromosomes to guide their accurate segregation. Additionally, meiotic recombination-initiated by programmed DNA double-strand breaks (DSBs)-can generate genetic diversity and, therefore, is a driving force of evolution. Environmental temperature influencing meiotic recombination outcome thus may be a crucial determinant of reproductive success and genetic diversity. Indeed, meiotic recombination frequency in fungi, plants and invertebrates changes with temperature. In most organisms, these temperature-induced changes in meiotic recombination seem to be mediated through the meiosis-specific chromosome axis organization, the synaptonemal complex in particular. The fission yeast Schizosaccharomyces pombe does not possess a synaptonemal complex. Thus, we tested how environmental temperature modulates meiotic recombination frequency in the absence of a fully-fledged synaptonemal complex. We show that intragenic recombination (gene conversion) positively correlates with temperature within a certain range, especially at meiotic recombination hotspots. In contrast, crossover recombination, which manifests itself as chiasmata, is less affected. Based on our observations, we suggest that, in addition to changes in DSB frequency, DSB processing could be another temperature-sensitive step causing temperature-induced recombination rate alterations.

Immediate visualization of recombination events and chromosome segregation defects in fission yeast meiosis.

Schizosaccharomyces pombe, also known as fission yeast, is an established model for studying chromosome biological processes. Over the years, research employing fission yeast has made important contributions to our knowledge about chromosome segregation during meiosis, as well as meiotic recombination and its regulation. Quantification of meiotic recombination frequency is not a straightforward undertaking, either requiring viable progeny for a genetic plating assay, or relying on laborious Southern blot analysis of recombination intermediates. Neither of these methods lends itself to high-throughput screens to identify novel meiotic factors. Here, we establish visual assays novel to Sz. pombe for characterizing chromosome segregation and meiotic recombination phenotypes. Genes expressing red, yellow, and/or cyan fluorophores from spore-autonomous promoters have been integrated into the fission yeast genomes, either close to the centromere of chromosome 1 to monitor chromosome segregation, or on the arm of chromosome 3 to form a genetic interval at which recombination frequency can be determined. The visual recombination assay allows straightforward and immediate assessment of the genetic outcome of a single meiosis by epi-fluorescence microscopy without requiring tetrad dissection. We also demonstrate that the recombination frequency analysis can be automatized by utilizing imaging flow cytometry to enable high-throughput screens. These assays have several advantages over traditional methods for analyzing meiotic phenotypes.

Surface grafted agents with various molecular lengths and photochemically active benzophenone moieties.

Homologues of benzophenone silane, a covalently graftable, photochemically active surface functionalizing agent, are investigated as surface functionalization agents for both small particles and planar substrates. In these homologues, a chlorosilane functional group and a photochemically active benzophenone oxo moiety are separated with an aliphatic spacer of varying length. The species obtained are first investigated by surface grafting on substrates (Si wafers, glass plates, and indium tin oxide coated glass plates). Si wafer samples are investigated with ellipsometry clearly indicating monolayer formation. The monolayer thickness can be controlled by the size of the aliphatic spacer group and also by the doping concentration of the solution used in the spin-casting step. The functionalized surfaces are further investigated by measuring the contact angle of a suitable organic fluid, a nematic liquid crystal. Photo exposure of these samples results in a drastically varied contact angle: The surface grafted species are still photochemically active and photo exposure leads to the addition of a nearby organic molecule (from the liquid crystalline phase) to each activated surface agent molecule. The synthesized species are then investigated as (covalently binding) surfactants in the wet planetary ball milling process aimed to fabricate solid-liquid dispersions (of Fe doped lithium niobate particles). It was found that the use of species with higher molecular length results in dispersions of small particles, functionalized with photochemically active surface agents. Indeed, they show better performance than conventional surfactants.

The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability.

Bidirectionally moving DNA replication forks merge at termination sites composed of accidental or programmed DNA-protein barriers. If merging fails, then regions of unreplicated DNA can result in the breakage of DNA during mitosis, which in turn can give rise to genome instability. Despite its importance, little is known about the mechanisms that promote the final stages of fork merging in eukaryotes. Here we show that the Pif1 family DNA helicase Pfh1 plays a dual role in promoting replication fork termination. First, it facilitates replication past DNA-protein barriers, and second, it promotes the merging of replication forks. A failure of these processes in Pfh1-deficient cells results in aberrant chromosome segregation and heightened genome instability.

Rapid and extensive karyotype diversification in haploid clinical Candida auris isolates.

Candida auris is a newly emerged pathogenic microbe, having been identified as a medically relevant fungus as recently as 2009. It is one of the most drug-resistant yeast species known to date and its emergence and population structure are unusual. Because of its recent emergence, we are largely ignorant about fundamental aspects of its general biology, life cycle, and population dynamics. Here, we report the karyotype variability of 26 C. auris strains representing the four main clades. We demonstrate that all strains are haploid and have a highly plastic karyotype containing five to seven chromosomes, which can undergo marked alterations within a short time frame when the fungus is put under genotoxic, heat, or osmotic stress. No simple correlation was found between karyotype pattern, drug resistance, and clade affiliation indicating that karyotype heterogeneity is rapidly evolving. As with other Candida species, these marked karyotype differences between isolates are likely to have an important impact on pathogenic traits of C. auris.

Fast copolymer network liquid crystals for tunable birefringence colors.

A fast-switching, tunable color filter was found in a copolymer network liquid crystal (LC), which was in situ generated in a conventional LC test cell with parallel aligned glass plates and investigated with polarized light. Polarization filters were used to convert the tunable optical phase retardance of the test cells to birefringence colors as is always possible in a LC test cell with carefully adjusted cell gap and effective birefringence. The cell gap of the samples could be adjusted to a value of 9 µm, which is not easily possible in a polymer LC composite without creating defects. In these samples, the typical pastel colors seen frequently in birefringent samples could be avoided. The transmittance spectra were recorded and converted to CIE 1931 color coordinates, which showed that the colors seen had a reasonable distance to the white point. The electro-optic switching times of the samples were investigated: Fast responses of t on +t off <5 ms were found, which is an impressive speed for tunable birefringence colors in LCs and LC composites. Upon increasing addressing voltages, a blueshift of the peak seen in the transmittance spectra was observed. The samples consisted of copolymer network LC, generated from a reactive mixture with mesogenic monomer and nonmesogenic comonomer. The tunable color was seen selectively in samples with dodecyl acrylate as comonomer. The experiments show how even a straightforward electro-optic experiment still can result in unexpended findings, which may expand the use of LC composites in nondisplay applications. The polymer morphology in samples with a larger cell gap was investigated with scanning electron microscopy, and interdefect distances of ≈40 µm were found. The appearance of defects in test cells with a cell gap of 9 µm could be avoided because the cell gap was much smaller than the measured interdefect distances in test cells with a larger cell gap.

DNA double-strand break formation and repair in Tetrahymena meiosis.

The molecular details of meiotic recombination have been determined for a small number of model organisms. From these studies, a general picture has emerged that shows that most, if not all, recombination is initiated by a DNA double-strand break (DSB) that is repaired in a recombinogenic process using a homologous DNA strand as a template. However, the details of recombination vary between organisms, and it is unknown which variant is representative of evolutionarily primordial meiosis or most prevalent among eukaryotes. To answer these questions and to obtain a better understanding of the range of recombination processes among eukaryotes, it is important to study a variety of different organisms. Here, the ciliate Tetrahymena thermophila is introduced as a versatile meiotic model system, which has the additional bonus of having the largest phylogenetic distance to all of the eukaryotes studied to date. Studying this organism can contribute to our understanding of the conservation and diversification of meiotic recombination processes.

Genetic interactions between the chromosome axis-associated protein Hop1 and homologous recombination determinants in Schizosaccharomyces pombe.

Hop1 is a component of the meiosis-specific chromosome axis and belongs to the evolutionarily conserved family of HORMA domain proteins. Hop1 and its orthologs in higher eukaryotes are a major factor in promoting double-strand DNA break formation and inter-homolog recombination. In budding yeast and mammals, they are also involved in a meiotic checkpoint kinase cascade monitoring the completion of double-strand DNA break repair. We used the fission yeast, Schizosaccharomyces pombe, which lacks a canonical synaptonemal complex to test whether Hop1 has a role beyond supporting the generation of double-strand DNA breaks and facilitating inter-homolog recombination events. We determined how mutants of homologous recombination factors genetically interact with hop1, studied the role(s) of the HORMA domain of Hop1, and characterized a bio-informatically predicted interactor of Hop1, Aho1 (SPAC688.03c). Our observations indicate that in fission yeast, Hop1 does require its HORMA domain to support wild-type levels of meiotic recombination and localization to meiotic chromatin. Furthermore, we show that hop1∆ only weakly interacts genetically with mutants of homologous recombination factors, and in fission yeast likely has no major role beyond break formation and promoting inter-homolog events. We speculate that after the evolutionary loss of the synaptonemal complex, Hop1 likely has become less important for modulating recombination outcome during meiosis in fission yeast, and that this led to a concurrent rewiring of genetic pathways controlling meiotic recombination.

Modulation of meiotic homologous recombination by DNA helicases.

DNA helicases are ATP-driven motor proteins which translocate along DNA capable of dismantling DNA-DNA interactions and/or removing proteins bound to DNA. These biochemical capabilities make DNA helicases main regulators of crucial DNA metabolic processes, including DNA replication, DNA repair, and genetic recombination. This budding topic will focus on reviewing the function of DNA helicases important for homologous recombination during meiosis, and discuss recent advances in how these modulators of meiotic recombination are themselves regulated. The emphasis is placed on work in the two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, which has vastly expanded our understanding of meiotic homologous recombination, a process whose correct execution is instrumental for healthy gamete formation, and thus functioning sexual reproduction. Copyright © 2016 John Wiley & Sons, Ltd.

Laser-induced erasable patterns in a N* liquid crystal on an iron doped lithium niobate surface.

A chiral nematic (N*) liquid crystal (LC) was hybridized with a z-cut iron doped lithium niobate (Fe:LN) substrate and exposed with a focused continuous wave diode laser beam. The N* LC layer was confined with a cover glass to provide a homogeneous LC layer thickness. Two distinct kinds of test cells were investigated, one with an uncoated glass covering slip and one with an indium tin oxide (ITO) coated cover glass. Photo generated electric fields (generated in the Fe:LN) resulted in a localized defect formation and textural transitions in the N* LC. Due to field confinement, the field induced responses were more localized in samples with ITO coated cover glasses. By scanning the laser beam on programmed trajectories, formation of persistent patterns could be achieved in the N* LC layer. Polarized optical microscopy of the exposed samples revealed that these patterns consisted of adjacent circular Frank-Pryce defects. Exposure with a slightly defocused laser beam could be applied selectively to erase these patterns. Thus, a promising method is reported to generate reconfigurable patterns, photonic motives, and touch sensitive devices in a hybridized N* LC with micron accuracy.

The FANCM ortholog Fml1 promotes recombination at stalled replication forks and limits crossing over during DNA double-strand break repair.

The Fanconi anemia (FA) core complex promotes the tolerance/repair of DNA damage at stalled replication forks by catalyzing the monoubiquitination of FANCD2 and FANCI. Intriguingly, the core complex component FANCM also catalyzes branch migration of model Holliday junctions and replication forks in vitro. Here we have characterized the ortholog of FANCM in fission yeast Fml1 in order to understand the physiological significance of this activity. We show that Fml1 has at least two roles in homologous recombination-it promotes Rad51-dependent gene conversion at stalled/blocked replication forks and limits crossing over during mitotic double-strand break repair. In vitro Fml1 catalyzes both replication fork reversal and D loop disruption, indicating possible mechanisms by which it can fulfill its pro- and antirecombinogenic roles.

Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination.

During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.

New cassettes for single-step drug resistance and prototrophic marker switching in fission yeast.

Construction of multiply mutated strains for genetic interaction analysis and of strains carrying different epitope tags at multiple open reading frames for testing protein localization, abundance and protein-protein interactions is hampered by the availability of a sufficient number of different selectable markers. Moreover, strains with single gene deletions or tags often already exist in strain collections; for historical reasons these will mostly carry the ura4(+) gene or the G418-resistance kanMX as marker. Because it is rather cumbersome to produce multiply deleted or tagged strains using the same marker, or to completely reconstruct a particular strain with a different marker, single-step exchange protocols of markers are a time-saving alternative. In recent years, dominant drug resistance markers (DDRMs) against clonNAT, hygromycin B and bleomycin have been adapted and successfully used in Schizosaccharomyces pombe. The corresponding DDRM cassettes, natMX, hphMX and bleMX, carry the TEF promotor and terminator sequences from Ashbya gossypii as kanMX; this provides flanking homologies to enable single-step marker swapping by homologous gene targeting. To expand this very useful toolset for single-step marker exchange, I constructed MX cassettes containing the nutritional markers arg3(+), his3(+), leu1(+) and ura4(+). Furthermore, a set of constructs was created to enable single-step exchange of ura4(+) to kanMX6, natMX4 and hphMX4. The functionality of the cassettes is demonstrated by successful single-step marker swapping at several loci. These constructs allow straightforward and rapid remarking of existing ura4(+) - and MX-deleted and -tagged strains.

Polarization-independent phase modulation using a blue-phase liquid crystal over silicon device.

Liquid crystal over silicon (LCoS) spatial light modulator technology has become dominant in industries such as pico-projection, which require high-quality reflective microdisplays for intensity modulation of light. They are, however, restricted from being used in wider optical applications, such as computer-generated holography, adaptive optics, and optical correlation, due to their phase modulation ability. The main drawback of these devices is that their modulation is based on simple planar or twisted nematic liquid crystals, which are inherently slow mechanisms due to their viscoelastic properties. Their use is also limited due to fact that the phase modulation is dependent on the state of polarization of the illumination. In this paper, we demonstrate that a polymer-stabilized blue-phase liquid crystal can offer both phase modulation and high speed switching in a silicon backplane device which is independent of the input polarization state. The LCoS device shows continuous phase modulation of light with a submillisecond switching time and insensitivity to the input light polarization direction. This type of phase modulation opens up a whole new class of applications for LCoS technology.

Effects of Composition and Polymerization Conditions on the Electro-Optic Performance of Liquid Crystal-Polymer Composites Doped with Ferroelectric Nanoparticles.

The presence of a polymer network and/or the addition of ferroelectric nanoparticles to a nematic liquid crystal are found to lower transition temperatures and birefringence, which indicates reduced orientational order. In addition, the electro-optic switching voltage is considerably increased when a polymer network is formed by in situ polymerization in the nematic state. However, the resulting polymer network liquid crystal switches at similar voltages as the neat liquid crystal when polymerization is performed at an elevated temperature in the isotropic state. When nanoparticle dispersions are polymerized at an applied DC voltage, the transition temperatures and switching voltages are reduced, yet they are larger than those observed for polymer network liquid crystals without nanoparticles polymerized in the isotropic phase.