Expanding the plant genome editing toolbox with recently developed CRISPR–Cas systems

Since its first appearance, CRISPR–Cas9 has been developed extensively as a programmable genome-editing tool, opening a new era in plant genome engineering. However, CRISPR–Cas9 still has some drawbacks, such as limitations of the protospacer-adjacent motif (PAM) sequence, target specificity, and the large size of the cas9 gene. To combat invading bacterial phages and plasmid DNAs, bacteria and archaea have diverse and unexplored CRISPR–Cas systems, which have the potential to be developed as a useful genome editing tools. Recently, discovery and characterization of additional CRISPR–Cas systems have been reported. Among them, several CRISPR–Cas systems have been applied successfully to plant and human genome editing. For example, several groups have achieved genome editing using CRISPR–Cas type I-D and type I-E systems, which had never been applied for genome editing previously. In addition to higher specificity and recognition of different PAM sequences, recently developed CRISPR–Cas systems often provide unique characteristics that differ from well-known Cas proteins such as Cas9 and Cas12a. For example, type I CRISPR–Cas10 induces small indels and bi-directional long-range deletions ranging up to 7.2 kb in tomatoes (Solanum lycopersicum L.). Type IV CRISPR–Cas13 targets RNA, not double-strand DNA, enabling highly specific knockdown of target genes. In this article, we review the development of CRISPR–Cas systems, focusing especially on their application to plant genome engineering. Recent CRISPR–Cas tools are helping expand our plant genome engineering toolbox.

Recently discovered and characterized clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR–Cas) systems allow additional applications to plant genome editing.     

Masters of asymmetry – lessons and perspectives from 50 years of septins

Septins are a unique family of GTPases, which were discovered 50 years ago as essential genes for the asymmetric cell shape and division of budding yeast. Septins assemble into filamentous nonpolar polymers, which associate with distinct membrane macrodomains and subpopulations of actin filaments and microtubules. While structurally a cytoskeleton-like element, septins function predominantly as spatial regulators of protein localization and interactions. Septin scaffolds and barriers have provided a long-standing paradigm for the generation and maintenance of asymmetry in cell membranes. Septins also promote asymmetry by regulating the spatial organization of the actin and microtubule cytoskeleton, and biasing the directionality of membrane traffic. In this 50th anniversary perspective, we highlight how septins have conserved and adapted their roles as effectors of membrane and cytoplasmic asymmetry across fungi and animals. We conclude by outlining principles of septin function as a module of symmetry breaking, which alongside the monomeric small GTPases provides a core mechanism for the biogenesis of molecular asymmetry and cell polarity.

Fifty years ago, Nobel laureate Lee Hartwell published the first three genes from his pioneering screen for mutants that alter the cell division cycle (cdc) of the budding yeast Saccharomyces cerevisiae (Hartwell et al., 1970). Among them, cdc3 and subsequently cdc10, cdc11, and cdc12 were all reported to develop multiple elongated buds, failing to undergo cytokinesis (Hartwell et al., 1970; Hartwell, 1971). Electron microscopy observations indicated that the products of these genes formed a filamentous network at the mother–bud cortex (Byers and Goetsch, 1976). Further characterization and cloning of these genes by John Pringle, who named them septins, marked the birth of a new class of GTP-binding proteins with important functions in the spatial organization of eukaryotic cells (Pringle, 2008).Septins comprise a family of paralogous genes, which arose early in eukaryotic evolution and expanded in fungi and animals, while largely absent from plants (Pan et al., 2007). In mice and humans, 13 septin genes express a diversity of paralogues and isoforms, which are classified under four groups (SEPT2, SEPT6, SEPT7, and SEPT3) and consist of a conserved core GTP-binding domain with variable N- and C-terminal extensions (Figure 1A; Kinoshita, 2003; Mostowy and Cossart, 2012). Opening a new era for the septin field, the x-ray crystal structure of the mammalian SEPT2/6/7 complex revealed that septins dimerize in tandem via their GTP-binding domains by utilizing two different binding interfaces, which alternate every other monomer (Sirajuddin et al., 2007). Through a serial head-to-head and tail-to-tail binding mode, septins assemble linearly into nonpolar oligomers and polymers (Figure 1B). The SEPT2/6/7 structure suggested that the minimal septin unit is a palindromic hexamer, in which SEPT2/6/7 trimers are arranged symmetrically from a central homodimeric SEPT2-SEPT2 interface (Sirajuddin et al., 2007). New evidence, however, shows that SEPT2/6/7 assembles into a hexamer with SEPT7, or SEPT9 in the case of the hetero-octameric SEPT2/6/7/9 complex, forming the central homodimeric contact (McMurray and Thorner, 2019; Mendonca et al., 2019; Soroor et al., 2019). This order is consistent with the arrangement of the budding yeast hetero-octamer (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11) with Cdc10, the yeast septin most homologous to SEPT9, being the central homodimer (Bertin et al., 2008; McMurray and Thorner, 2019). In contrast to the symmetric end-to-end arrangement of their subunits, septin multimers are characterized by a top–bottom asymmetry with their C- and N-terminal extensions extending orthogonally toward opposite sides of the linear axis of multimerization (Figure 1C; John et al., 2007; Sirajuddin et al., 2007; Bertin et al., 2008). Although it is not functionally understood, this asymmetry may underlie an anisotropic mode of interaction with cellular components and protein complexes, which could be further modulated by the identity and combination of septin subunits.Open in a separate windowFIGURE 1:Structure and assembly of septin GTPases. (A) Schematic shows the main domains of septin GTPases, which consist of a highly conserved GTP-binding domain with G1 (GxxxxGK[ST]), G3 (DxxG), and G4 (AKAD) motifs, and a septin unique element. Septins contain a catalytic threonine residue (T*), which corresponds to the Thr of the G2 motif of Ras GTPases and is absent from septin paralogues that lack GTPase activity. The N- and C-terminal extensions of the septin GTP-binding domain vary in sequence and length, and contain proline-rich and coiled-coil domains, respectively. Domains of cytoskeleton- and membrane-binding (amphipathic helices and polybasic sequences) are denoted and differ among septin paralogues. (B, C) The x-ray crystal structure of SEPT2/6/7 (PDB: 2qag; B) shows the NC-NC and G-G interfaces of dimerization, which alternate between the GTP-binding domains of consecutive septin subunits. The C- and N-terminal ends of the G-domains are located at the top and bottom of the dotted vertical lines, respectively, and the guanine nucleotide is depicted in orange. A surface representation of the SEPT2/6/7 heterohexamer (C) shows the symmetric (nonpolar) end-to-end arrangement of septin paralogues from the central homodimeric SEPT7 interface and the asymmetric positioning of the C- and N-terminal ends across the horizontal plane of multimerization. (D) Schematic of the assembly of yeast septin complexes, which is driven by successive events of homo- and heterodimerization that are determined by GTP binding and hydrolysis. (E) Schematic shows the subunit order and identity of mammalian septin hetero-octamers, and the interchangeability of paralogue subunits of the same septin group (Kinoshita rule).Septins are structurally and phylogenetically related to the small GTPases of the Ras superfamily (e.g., Ras, Rho, Rab; Leipe et al., 2002), but most septins hydrolyze and turn over GTP slowly and there are septin paralogues (e.g., SEPT6 group) that lack GTPase activity (Vrabioiu et al., 2004; Sirajuddin et al., 2009; Zent and Wittinghofer, 2014; Abbey et al., 2019). Growing evidence indicates that GTP-binding and hydrolysis stabilize the dimeric interface, determine the order of assembly by selecting for specific dimerization partners, and induce allosteric conformational changes, which could affect the assembly and localization of septin complexes (Figure 1D; Sirajuddin et al., 2009; Zent and Wittinghofer, 2014; Weems and McMurray, 2017; Castro et al., 2020). Based on the activity and structure of their GTPase domains, paralogues of the same group occupy and exchange within the same position of the septin complex—a posit known as the Kinoshita rule (Figure 1E; Kinoshita, 2003). However, high-order septin complexes of homomeric and alternative compositions have also been reported (Mendoza et al., 2002; Mizutani et al., 2013; Sellin et al., 2014; Karasmanis et al., 2018). Higher-order septin structures exhibit slow subunit turnover and thereby persist in place longer than dynamic cytoskeletal polymers (Hu et al., 2008; Hagiwara et al., 2011; Bridges et al., 2014). Hitherto, in the absence of any known septin guanine nucleotide activating and exchange factors (GTPase-activating proteins [GAPs]/ guanine nucleotide exchange factor [GEFs]), septin dynamics and turnover are modulated by posttranslational modifications (Hernandez-Rodriguez and Momany, 2012; Ribet et al., 2017).Septins function broadly as scaffolds and barriers that recruit and exclude proteins, respectively, controlling the localization and interactions of membrane and cytoskeletal proteins (Caudron and Barral, 2009; Mostowy and Cossart, 2012; Spiliotis, 2018). This fundamental property of septins has been adopted by a diversity of molecular mechanisms and cellular processes. In the face of an ever-growing number of biological roles, it is often overlooked that septins function primarily as a module of spatial organization. After 50 years of research, we recount here how septins promote cellular asymmetries in fungi and animals, highlighting the evolutionary adaptation of septins as effectors of asymmetry from fungal cell membranes to the mammalian cytoskeleton.Septin roles in fungal cell asymmetrySeptins perform essential functions in nearly all aspects of the asymmetric growth and division of budding yeast (Figure 2). Prior to budding, a regulatory interplay between septins and the Rho family small GTPase Cdc42 enables yeast cells to polarize growth at a single site. GTP-bound Cdc42 initially recruits septins to a cloudy patch (Gladfelter et al., 2002), where a transient direct interaction between the septin Cdc11 and Cdc24, a GEF for Cdc42, creates a short-term positive feedback loop to reinforce septin recruitment and Cdc42 activation (Chollet et al., 2020). Targeted delivery of Cdc42-containing but septin-free vesicles to the center of the septin patch transforms it into a ring (Gladfelter et al., 2002; Okada et al., 2013). Septin polymerization into circular filaments is thought to render Cdc11 inaccessible to Cdc24 (Chollet et al., 2020). Concomitantly, septins begin to recruit Cdc42 GAPs, which inhibit Cdc42, and they promote the activation of formins, which drive actin cable polymerization (Buttery et al., 2012; Okada et al., 2013). As actin cables direct more vesicles to the center of the septin ring, the nascent bud grows and concomitantly Cdc42 pushes away from septins and localizes to the bud tip, where it promotes further growth (Okada et al., 2013). Hence, septins reinforce the formation of a Cdc42 polar cap during bud emergence. During bud growth, septins pattern the localization of the actin- and formin-binding protein Hof1 along the bud cortex in a manner that aligns actin cables along the mother–bud axis, which is critical for the asymmetric growth of the bud (Garabedian et al., 2020). Septin-mutant cells lose the mother- or bud-specific asymmetric localization of a number of cortical proteins as well as mother–bud asymmetry of actin patch stability and overall cell growth (Barral et al., 2000), though the mechanistic details of how septins control these asymmetries in wild-type cells are unclear.Open in a separate windowFIGURE 2:Septin localization and function in the asymmetric growth and division of the budding yeast S. cerevisiae. The schematic shows in a clockwise order the different stages of the lifecycle of vegetative haploid and diploid, and sporulating S. cerevisiae cells. The localization and organization of septin complexes in vegetative (Cdc11/Shs1-Cdc12-Cdc3-Cdc10) and sporulating (Spr28-Spr3-Cdc3-Cdc10) cells are depicted as single or double rings, an hourglass-shaped collar, and dots. Shaded boxes highlight septin functions in polarized membrane growth and the asymmetric partitioning and inheritance of membrane and cytoplasmic components in different stages of the budding yeast lifecycle.At the mother–bud neck, the septin ring expands into an hourglass collar and splits into two rings before cytokinesis (Figure 2). The hourglass structure provides a scaffold for the assembly of the actomyosin ring (Bi et al., 1998; Schneider et al., 2013) and the asymmetric distribution of many septin interactors, which localize to the mother or bud side of the neck and in between (Gladfelter et al., 2001; McMurray and Thorner, 2009). On the mother side, septins recruit chitin synthase for the generation of the bud scar, whose underlying membrane provides a landmark and spatial memory for the budding site of the next cell cycle (DeMarini et al., 1997; Kozubowski et al., 2005). On the bud side, septins scaffold kinases of the morphogenesis checkpoint, which monitors proper bud growth for entry into mitosis (Barral et al., 1999; Shulewitz et al., 1999; Theesfeld et al., 2003). At the interface of the mother–bud membranes, the double septin ring acts as a barrier to prevent the exocyst complex and membrane remodeling factors from diffusing out of the gap between them, delimiting the site of membrane growth that occurs in late cytokinesis (Dobbelaere and Barral, 2004).Septin barriers appear to be dispensable for cytokinesis (Wloka et al., 2011), but they are essential for mother–daughter asymmetry in organelle content and age. Septins compartmentalize the endoplasmic reticulum (ER) associated with the plasma membrane of the bud neck (Luedeke et al., 2005). The septin ring excludes ribosomes, creating smooth ER at the bud neck (Luedeke et al., 2005). Moreover, as the cortical ER moves into the growing bud, the septin ring protects the bud from inheriting ER membranes with aggregates of misfolded proteins (Clay et al., 2014). Septin-ring–dependent enrichment of sphingolipids in bud neck membranes is required for the diffusion barrier, but it is unknown how septins may control sphingolipid localization (Clay et al., 2014). Septins also act as a sphingolipid-dependent barrier for the diffusion of nuclear pore complexes (NPCs) from the mother to bud ER membranes, which are continuous with the nuclear envelope (NE; Shcheprova et al., 2008). Lack of NPC diffusion correlated with the asymmetric retention of nonchromosomal DNA circles within the mother compartment, which reduces replicative span (Shcheprova et al., 2008). However, the shape of the dividing nucleus and length of mitosis were also proposed to restrict the diffusion of DNA circles, which entered the bud upon artificial tethering to nuclear pores (Gehlen et al., 2011; Khmelinskii et al., 2011). Reconciling these findings, subsequent work showed that in aged cells DNA circles are coupled to only a subset of NPCs by the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, hindering diffusion into the bud beyond the constraints imposed by the nuclear shape (Denoth-Lippuner et al., 2014). Thus, the septin-dependent ER/NE diffusion barrier plays a fundamental role in yeast aging.Polarized budding yeast growth occurs not only intrinsically, taking place adjacent to or opposite the site of the preceding cell division, but also in response to mating pheromones, which override intrinsic signals. Septins encircle polarity factors at the polar cap of the shmoo-shaped tips that grow toward the pheromone gradient, and septin mutants are defective in pheromone tracking and polarized morphogenesis (Kelley et al., 2015). Upon mating, a distinct septin structure at the site of cell fusion limits diffusion of organelles and large cytoplasmic complexes between the mating partners, which promotes the asymmetrical inheritance of mitochondrial DNA by diploid buds (Tartakoff et al., 2013).Septins are similarly required for the polarized membrane growth that occurs during sporulation (Onishi et al., 2010; Heasley and McMurray, 2016). Spore membrane biogenesis is like inside-out budding as new membranes and cell walls form around the four haploid nuclei, which result from the meiosis of a diploid cell. Each membrane emerges from a single point, the pole of a postmeiosis II spindle, and grows outward as a cup before closing at another single point to form a sphere (Figure 2). Septins localize to bar- and horseshoe-shaped structures along the growing membranes. In septin mutants the membranes grow in the wrong directions and often fail to close (Onishi et al., 2010; Heasley and McMurray, 2016). After their formation, spores bud in a prepolarized manner (Joseph-Strauss et al., 2007). Strikingly, a septin spot is the only known polarity marker that demarcates the site of new membrane and wall synthesis. A septin ring of unknown function encircles the site of membrane outgrowth before a new septin ring forms where the spore first buds, completing the S. cerevisiae life cycle (Joseph-Strauss et al., 2007).While budding yeasts have taught us much about septins and asymmetry, we have learned many fundamental lessons from other fungi. In the fission yeast Schizosaccharomyces pombe, septins act as scaffolds and/or diffusion barriers to guide the orderly recruitment of the cytokinetic machinery to the division site, which occurs at the equatorial plane of the cell. Septin mutants undergo cytokinesis, but fail in cell separation due to defects in recruiting the cell wall degrading enzymes (Zheng et al., 2018). In filamentous fungi, multiple distinct septin-based structures are found in a single cell, and function in limiting the polarity sites and branching of hyphae (Khan et al., 2015; Momany and Talbot, 2017). In Ashbya gossypii, septins form rings and lateral bundles, and localize to the membrane curvature of hyphal branch points, which depends on a C-terminal amphipathic helix (Cannon et al., 2019). Notably, septin recognition of micron-scale membrane curvature is a conserved property of septins from fungi to animals (Bridges et al., 2016). The pathogenic rice blast fungus Magnaporthe oryzae undergoes polarized growth to develop a specialized structure called the appressorium, which contacts the plant surface and uses extreme pressure to penetrate the host (Momany and Talbot, 2017). Here, a septin ring acts as a diffusion barrier to retain actin-polymerizing proteins, and scaffolds actin assembly and the localization of the exocyst complex (Dagdas et al., 2012; Gupta et al., 2015). Overall, fungi have provided compelling systems to explore septin assembly and functions in cell asymmetry.Septins promote asymmetry in the plasma membrane of animal cellsStudies of septins in animal cells have revealed a remarkable conservation with their fungal counterparts in promoting cell asymmetry. Septins are required for plasma membrane protrusions and compartments (e.g., uropodia, filopodia, cilia) that underlie the morphogenesis of distinct cell types and tissues and arise from local membrane asymmetries, microdomains of distinct protein enrichment (Caudron and Barral, 2009; Tooley et al., 2009; Hu et al., 2010, 2012; Dolat et al., 2014a). Homozygous deletions of several septin genes in mice cause early embryonic lethality and male sterility, demonstrating how essential septins are for development and physiology (Dolat et al., 2014a).On the plasma membrane of animal cells, septin assembly and localization involves signaling cues and selective binding to microdomains with distinct phospholipid content and curvature (Figure 3). The signaling pathways that instruct the membrane sites of septin assembly in multicellular organisms are not as well studied as in fungi. The WD repeat-containing planar polarity effector and the PAR complex have been implicated in the localization of membrane septins (Cui et al., 2013; Park et al., 2015; Jordan et al., 2016). Septins have intrinsic preferences for select phospholipids including phosphoinositides (e.g, phosphatidylinositol 4,5-bisphosphate) and cardiolipin, a membrane curvature–specific lipid (Zhang et al., 1999; Krokowski et al., 2018). Taken together with a strong affinity for membrane micron-scale curvatures that is mediated by amphipathic helices (Bridges et al., 2016; Cannon et al., 2019), septins become enriched on the inner saddle-like areas of the plasma membrane, outlining the base-neck border of protrusions such as filopodia, lamellipodia, and dendritic spines (Figure 3).Open in a separate windowFIGURE 3:Septins associate with cell membranes and function in plasma membrane asymmetry. Septins associate preferentially with membrane domains of micron-scale curvature with a radius (R) of ∼0.5–1.5 μm or curvature parameter (k) of ∼0.67–2 μm⁻¹. In addition, septins bind preferentially phosphoinositides and cardiolipin, a cone-shaped lipid that localizes in curved membrane domains. Septins promote asymmetry by acting as barriers of lateral diffusion, membrane rigidifiers, and regulators of the organization and dynamics of cortical actin. Septins are essential components of diffusion barriers at the base of primary cilia, dendritic spines, and the midpiece of spermatozoa, where the annulus structure is located. By rigidifying specific subdomains of the plasma membrane, septins delimit areas of membrane activity, which is critical for the position of membrane protrusions in the rear (uropodia) and front (lamellipodia, filopodia) of migrating cells. In gastrulating frog embryos and C. elegans zygotes, septins locally regulate the organization and dynamics of cortical actin, which is critical for the asymmetric contraction of actomyosin that underlies convergent extension and cytokinesis.Septins promote asymmetry by restricting lateral diffusion, enhancing membrane rigidity and spatially regulating membrane–cytoskeleton interactions. Septins are required for the maintenance of diffusion barriers at the base of primary cilia, dendritic spines, and the midpiece of spermatozoa (Hu et al., 2010; Kwitny et al., 2010; Ewers et al., 2014). Diffusion of transmembrane and inner leaflet-anchored proteins across the midbody of dividing cells may also be impeded by septins (Schmidt and Nichols, 2004). It is unclear whether septin filaments directly impede lateral diffusion or whether they are an essential component of a larger actin–spectrin skeleton with barrier properties. Immuno-EM studies indicate that septins are indeed part of the membrane actin skeleton (Hagiwara et al., 2011). Moreover, in the axon initial segment, which functions as a diffusion barrier, septins interact with ankyrin G (Hamdan et al., 2020). Septins have also been proposed to rigidify the plasma membrane, suppressing cortical protrusions and blebs, which are asymmetrically confined to septin-free membrane areas (Gilden and Krummel, 2010). For example, T-cell uropods are delimited by a septin corset-like arrangement, which braces the perinuclear cortex and enables the front–back polarity of migrating T-cells (Gilden et al., 2012).Membrane septins bend actin filaments into circular arrays and scaffold the localization and activation of myosin-II, and thus they can promote asymmetry in membranes by locally regulating the cortical actomyosin (Joo et al., 2007; Mavrakis et al., 2014). In gastrulating frog embryos, septins localize to the vertices of mediolateral cell–cell contacts, where they stabilize actin and maintain the planar polarity of phosphorylated myosin along the anteroposterior contacts (Shindo and Wallingford, 2014). Thereby, actomyosin contraction is spatially restricted along the anteroposterior junctions, which promotes convergent extension by pulling mediolateral contacts closer to one another (Figure 3). In Caenorhabditis elegans embryos, septins polarize to the anterior pole away from the contractile actomyosin ring, inhibiting actin assembly outside the plane of cytokinesis (Jordan et al., 2016). Moreover, C. elegans septins are required for the asymmetric ingression of the cleavage furrow, which progresses directionally with a dorsal-to-ventral orientation (Maddox et al., 2007). Independently of their roles in cytokinesis, cortical septins provide spatial cues for the orientation of the axis of division in Drosophila larval neuroblasts, which is determined by the position of the last-born daughter cell (Loyer and Januschke, 2018). Notably, in neuronal crest cells, which generate dorsal root ganglia neurons after cell division, cortical septins provide a spatial memory for the outgrowth of axons from premitotic sites of membrane protrusions (Boubakar et al., 2017).In sum, septins associate preferentially with micrometer-scale membrane domains of negative Gaussian curvature and distinct phospholipid content. While it is unclear how they impede the lateral diffusion of proteins and lipids, septins promote macroscale asymmetries by enabling molecular partitioning across adjacent membrane compartments. At the nexus, septins also facilitate asymmetries of submicrometer scale by clustering membrane proteins or lipids. For example, septins are required for the organization of PI(4,5)P2 into clusters that surround the calcium release-activated calcium channel ORAI1 at plasma membrane–ER junctions (Sharma et al., 2013). How septins interact with and organize on membrane bilayers, and how they modulate the mobility and distribution of plasma membrane proteins and lipids are key questions. As septins associate with the membranes of various intracellular organelles, it is also unclear how septins function in the generation and/or maintenance of organelle membrane domains and membrane–membrane contacts (Akil et al., 2016; Dolat and Spiliotis, 2016; Pagliuso et al., 2016; Sirianni et al., 2016; Song et al., 2019).Septin roles in cytoskeleton-based mechanisms of cell asymmetryCell asymmetry requires the assembly of local microtubule and actin networks with unique organization and dynamics. Due to their structural polarity and dynamic growth, microtubules and actin filaments can spatially bias membrane traffic and determine the location, shape, and/or directionality of membrane protrusions and adhesions. Hence, microtubules and actin filaments are keys for morphing cells into asymmetric shapes.Septins localize to subsets of microtubules and actin filaments, and promote cell asymmetry by regulating the spatial organization of the cytoskeleton and membrane traffic (Figure 4). In a diversity of mammalian cell types, septins colocalize with microtubules and actin filaments of the perinuclear and peripheral cytoplasm (Spiliotis, 2018). It is not well understood how this region-specific association is established, but it may involve effectors of Rho signaling and depend on the composition (subunit isotypes) and posttranslational modifications of microtubules and actin filaments. Alternatively, cytoskeletal sites of binding might be indirectly determined by septin binding and turnover on proximal membranes. Thus, septin roles in cytoskeleton-based mechanisms of cell asymmetry are not mutually exclusive of their functions in membrane asymmetry, which feeds back locally to the cytoskeleton.Open in a separate windowFIGURE 4:Septins roles in cytoskeleton-based mechanisms of apicobasal, axonodendritic, and front–rear polarity. Microtubule-associated septins guide microtubule organization and membrane traffic along the apicobasal axis of polarizing epithelia. In neuronal dendrites, septin 9 reinforces the polarity of neuronal membrane traffic by impeding the transport of axonal cargo of kinesin-1/KIF5 and enhancing the anterograde movement of dendritic cargo of kinesin-3/KIF1A. In cells undergoing epithelial-to-mesenchymal transition, septins associate with the actin stress fibers of the leading lamellae and promote the asymmetric organization of the actin network and focal adhesion turnover, which is critical for the front–rear polarity of cell migration.In epithelia and neurons, microtubule-associated septins are functionally important for apicobasal and axon–dendrite polarity (Spiliotis et al., 2008; Bowen et al., 2011; Karasmanis et al., 2018). In polarizing epithelia, septins associate with subsets of microtubules, steering microtubule plus end growth and microtubule–­microtubule interactions (Bowen et al., 2011). Hence, septins provide a navigation mechanism for microtubule organization along the developing apicobasal axis of polarity. In addition, microtubule-associated septins are required for Golgi-to-plasma membrane transport of vesicles with apical and basolateral membrane proteins (Spiliotis et al., 2008). Septin depletion abrogates exocytic membrane traffic, disrupting the growth and differentiation of the epithelial cell membrane into apical and basolateral domains (Spiliotis et al., 2008). In fully polarized epithelia, it is unknown whether specific septin paralogues and complexes function exclusively in the apical or basolateral routes of membrane traffic. In hippocampal neurons, however, septin 9 localizes to dendritic microtubules, reinforcing the polarity of membrane traffic by hindering the transport of axonal cargo of kinesin-1/KIF5 and promoting the anterograde movement of dendritic cargo of kinesin-3/KIF1A (Karasmanis et al., 2018). This differential regulation of kinesin-driven transport is critical for axon–dendrite polarity, and in principle may constitute a general mechanism by which septins polarize membrane traffic.Growing evidence indicates that septins function in the mechanisms of mechanotransduction that induce and sustain front–rear polarity in migrating cells (Lam and Calvo, 2019). Cells migrate toward stiffer extracellular matrices and chemoattractants by forming a leading protrusive front and retracting rear. Front–rear polarity is largely supported by an asymmetry in the organization of the actomyosin stress fibers and the turnover of focal adhesions. Septins colocalize with actin stress fibers in a diversity of cell types, and alterations in septin expression impair directional migration. In cells undergoing epithelial-to-mesenchymal transition, septins are enriched in the leading lamella localizing at the interphase of a contractile network of curved actin stress fibers (transverse arc stress fibers) and the radial stress fibers, which are anchored to focal adhesions (Dolat et al., 2014b). Septin depletion collapses the transverse arc actin network with concomitant loss of stability and front-to-rear maturation of focal adhesions. Taken together with loss of directional cell migration, this phenotype underscores a critical role of septins in the front–rear asymmetry of cell migration. In cancer-associated fibroblasts, septins function together with the Cdc42 effector Cdc42EP3 in actin organization in a mechanosensitive manner, responding to stiffer matrices via association with perinuclear actin stress fibers (Calvo et al., 2015). Of note, septins may function in nuclear mechanotransduction, nuclear movement, and/or the regulation of subnuclear focal adhesions (Verdier-Pinard et al., 2017; Lam and Calvo, 2019). In parallel with their functions in the actin cytoskeleton, septins also likely contribute to the front–rear asymmetry of migrating cells by spatially regulating membrane–microtubule interactions (Shindo et al., 2018).Septin association with subsets of actin filaments and microtubules is a salient characteristic that enables local regulation of cytoskeleton-based processes, a key functionality in the generation of cellular asymmetries. Progress in understanding how septins interact with actin and microtubules, and how they mechanistically impact cytoskeletal dynamics has been slow. High-resolution cryoelectron microscopy studies of septins in complex with actin and microtubules are necessary to provide structural insights into how septins modulate cytoskeletal dynamics and interactions with actin-binding and microtubule-associated proteins. Septins have yet to be studied in the context of the basic mechanisms of actin nucleation and polymerization, which can have important implications for actin assembly and symmetry breaking at membrane sites of septin enrichment. Advances in these areas and better understanding of the signaling pathways that determine the cytoskeletal locales of septin function will provide a fuller picture of septins as cytoskeletal agents of cell asymmetry.Principles and unknowns of septin function as a symmetry-breaking moduleThe spatial organization of eukaryotic cells requires microscale asymmetries, which are established and maintained in a region-specific manner, and at the macroscale level promote the polarization of cellular shapes and processes. It is little understood how these asymmetries arise amidst membrane and cytoplasmic fluidity. Part small GTPase-like regulators and part cytoskeleton-like polymers, septins are inherently tailored to break symmetry by facilitating protein localization and interactions that persist in place and time. In summary of past and recent findings, there are four major principles of septin function as a symmetry-breaking module:

1. Septins are spatio-specific and modular. Septins assemble on select intracellular regions (e.g., micron-scale membrane curvatures, perinuclear microtubule bundles, peripheral transverse arc stress fibers). Septin assembly is modular, consisting of alternative combinations of paralogue and isoform subunits, which in turn determine intracellular localization and interactions.
2. Septins form higher-order structures, which turn over slowly and thereby persist spatially and temporally. Thus, septins can serve as imprints or landmarks of previous structures and molecular events, providing a type of spatial memory.
3. Septins scaffold protein localization and interactions. Septin scaffolds induce molecular asymmetries by recruiting and clustering protein interactors, and facilitating the assembly of macromolecular complexes.
4. Septins partition proteins in distinct membrane or cytoskeletal domains. Septins exclude proteins locally by impeding lateral diffusion or binding.

While most evident in membrane septins, these principles also apply to septins that associate with actin and microtubules and the membrane–cytoskeleton interface. On microtubule lattices, where many microtubule-associated proteins (MAPs) undergo diffusion through transient electrostatic interactions, septins selectively inhibit or promote microtubule binding of specific MAPs and kinesin motors (Spiliotis et al., 2008; Karasmanis et al., 2018). Similar modulation is likely to take place on actin filaments, where septins could specify domains of unique actin-binding protein composition promoting the formation of actin networks of distinct architecture and dynamics. A region-specific control of cytoskeletal organization may also occur by membrane-associated septins sequestering actin-­nucleating promoting factors or directly interacting with the dynamic ends of actin filament or microtubules (Dagdas et al., 2012; Hu et al., 2012; Nolke et al., 2016; Nakos et al., 2019). In the cytoplasm, a templated assembly of septins along subpopulations of microtubules and actin filaments may provide a form of cytoskeletal memory as previously proposed for vimentin intermediate filaments (Gan et al., 2016). By outlasting their shorter-lived templates, septins are likely to provide cytoplasmic landmarks for orienting microtubule and actin growth along previously held patterns of spatial organization. In polarized cell types, this region-specific patterning would be critical for the continuous organization of cytoskeletal networks along the axis of cell polarity.Despite much progress over the last two decades, we have merely begun discovering septins as a core mechanism for the spatial organization of cell biology. Many unknowns remain. The signaling inputs that interface with the assembly of mammalian septins are virtually unknown. The spatio-specificity of septin assembly on subnetworks and regions of the cytoskeleton is poorly understood. Septin modularity is little explored, and more work is needed to determine how different combinations of septin paralogues and isoforms bestow alternative localizations and properties. Deciphering this septin code is critical for determining which septin complexes do what, and whether certain septin paralogues can function as homomers independently of the canonical model of heteromeric assembly. In light of GTP hydrolysis favoring septin homodimerization and the recent reordering of the SEPT2/6/7/9 hetero-octamer, it is plausible that paralogues with faster GTPase activities (e.g., SEPT9) and in excess of their cognate partners evade heteromerization into typical complexes. More studies are needed in cell types and disease states, in which certain septin paralogues or isoforms are disproportionally expressed and may take unique or pathogenic functions. Similarly, studies of septins in polarized cell types and stem cell systems, where asymmetry is key for cell fate and renewal, are lacking, and so is our understanding of septin functions in cell regeneration and repair. With many more open questions than answers, the next 50 years of septin research promises important breakthroughs in our basic understanding of cellular asymmetry and potentially the development of septin-based therapies in regenerative medicine and beyond.     

Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene

The CRISPR-Cas9 system uses guide RNAs to direct the Cas9 endonuclease to cleave target sequences. It can, in theory, target essentially any sequence in a genome, but the efficiency of the predicted guide RNAs varies dramatically. If no targeted cells are obtained, it is also difficult to know why the experiment fails. We have developed a transient transfection based method to enrich successfully targeted cells by co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene. Cells are transfected with two guide RNAs that target respectively HPRT and the gene of interest. HPRT targeted cells are selected by resistance to 6-thioguanine (6-TG) and then examined for potential alterations to the gene targeted by the co-transfected guide RNA. Alterations of many genes, such as AAVS1, Exo1 and Trex1, are highly enriched in the 6-TG resistant cells. This method works in both HCT116 cells and U2OS cells and can easily be scaled up to process multiple guide RNAs. When co-targeting fails, it is straightforward to determine whether the target gene is essential or the guide RNA is ineffective. HPRT co-targeting thus provides a simple, efficient and scalable way to enrich gene targeting events and to identify the cause of failure.The CRISPR-Cas9 system is a revolutionary technology for gene targeting in cells (1–3). It consists of two components: a guide RNA and the Cas9 endonuclease that respectively pairs with the target sequence and then cleaves it (4–6). The guide RNA contains 19 nt that in theory can be custom designed to target almost any sequence in a genome (5–8). In practice, the effectiveness of the predicted guide RNAs varies dramatically (8). This problem, when compounded by other commonly encountered problems such as poor efficiency of DNA transfection or low titer of viruses, can make the isolation of successfully targeted cells a really laborious task (9). Low transfection efficiency can be improved by special techniques like nucleofection (6), but they are expensive and not readily available to all labs. A common method to enrich successfully targeted cells is to use drug resistance or green fluorescent protein (GFP) markers to select for cells that have integrated the guide RNA-expressing DNA into chromosomes (7,10). Fluorescence-activated cell sorting (FACS) sorting only enriches cells expressing high levels of GFP but not necessarily Cas9, while integration of foreign DNA, especially viral DNA, into the chromosome is itself an alteration to the genome and might cause oncogenic transformation. In some cases, Cas9 is also integrated into the genome and constitutively expressed (7). Persistent expression of the guide RNA in combination with Cas9 might lead to increasing probabilities of off-target cleavages over long-term culturing (11). In addition, if no targeted cells are obtained, it is difficult to track down the cause of failure. The guide RNA might be ineffective or the correctly targeted cells might have died off and only cells not expressing the guide RNA might have survived.To overcome these shortcomings, we have developed a transient plasmid DNA transfection-based method to enrich successfully targeted cells without the need for DNA integration into chromosomes by co-targeting the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene. This gene encodes a protein that catalyzes the conversion of hypoxanthine to inosinemonophosphate and guanine to guanosine monophosphate in the non-essential purine salvage pathway (12). HPRT⁺ cells are sensitive to 6-thioguanine (6-TG), which can be converted to the nucleotide form by HPRT and incorporated into DNA by DNA polymerase, killing cells by a process involving postreplicative mismatch repair (13–15). The strategy is to transfect cells with two plasmids that express respectively a HPRT guide RNA and a guide RNA for the gene of interest. Cas9 can be expressed from the gene on a separate plasmid, a plasmid carrying the HPRT gRNA or integrated into the chromosome (if such a cell line is already available). If a cell becomes resistant to 6-TG, it would suggest that this cell should also be competent to target the gene of interest as long as the gRNA is effective. Thus if the targeted gene is not altered in the resulting 6-TG resistant cells, it would suggest that the guide RNA is ineffective. On the other hand, if no 6-TG resistant cells can be obtained by co-targeting, it would suggest that the gene of interest might be essential.We have tested this method with guide RNAs for HPRT and the non-essential AAVS1 locus in HCT116 cells, a colorectal cancer cell line with a near diploidic karyotype (16). The results showed a dramatic enrichment of AAVS1 targeting events from below detection without co-targeting to over 80% with co-targeting. Other non-essential genes such as Trex1 and Exo1 were also successfully enriched by HPRT co-targeting. The method also worked in U2OS cells, an osteosarcoma cell line with a complicated karyotype (17). Co-targeting with guide RNAs for DNA topoisomerase 2α (Top2α) gave rise to no 6-TG resistant cells, which is consistent with Top2α being essential for cell proliferation (18). On the other hand, co-targeting with some other guide RNAs gave rise to plenty of 6-TG resistant cells but no alteration to the intended sequences, suggesting that the guide RNAs were ineffective. Together, these results demonstrate that co-targeting the HPRT gene provides a simple and efficient method to enrich successfully targeted cells. It can also be easily used to evaluate the effectiveness of guide RNAs and the essentialness of target genes.     

CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri

Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR–Cas systems, such as the Streptococcus pyogenes CRISPR–Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR–Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR–Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR–Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR–Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR–Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR–Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria.     

Total knee replacement after high tibial osteotomy: time-to-event analysis and predictors

BACKGROUND:An important aim of high tibial osteotomy (HTO) is to prevent or delay the need for total knee replacement (TKR). We sought to estimate the frequency and timing of conversion from HTO to TKR and the factors associated with it.METHODS:We prospectively evaluated patients with osteoarthritis (OA) of the knee who underwent medial opening wedge HTO from 2002 to 2014 and analyzed the cumulative incidence of TKR in July 2019. The presence or absence of TKR on the HTO limb was identified from the orthopedic surgery reports and knee radiographs contained in the electronic medical records for each patient at London Health Sciences Centre. We used cumulative incidence curves to evaluate the primary outcome of time to TKR. We used multivariable Cox proportional hazards analysis to assess potential preoperative predictors including radiographic disease severity, malalignment, correction size, pain, sex, age, body mass index (BMI) and year of surgery.RESULTS:Among 556 patients who underwent 643 HTO procedures, the cumulative incidence of TKR was 5% (95% confidence interval [CI] 3%–7%) at 5 years and 21% (95% CI 17%–26%) at 10 years. With the Cox proportional hazards multivariable model, the following preoperative factors were significantly associated with an increased rate of conversion: radiographic OA severity (adjusted hazard ratio [HR] 1.96, 95% CI 1.12–3.45), pain (adjusted HR 0.85, 95% CI 0.75–0.96)], female sex (adjusted HR 1.67, 95% CI 1.08–2.58), age (adjusted HR 1.50 per 10 yr, 95% CI 1.17–1.93) and BMI (adjusted HR 1.31 per 5 kng/m², 95% CI 1.12–1.53).INTERPRETATION:We found that 79% of knees did not undergo TKR within 10 years after undergoing medial opening wedge HTO. The strongest predictor of conversion to TKR is greater radiographic disease at the time of HTO.

The burden of knee osteoarthritis (OA) on patients and health care systems is substantial and growing.¹ The current treatment strategy that relies largely on total knee replacement (TKR) for end-stage disease may not be sustainable.^2–5 Reduced quality of life and loss of productivity due to knee OA in middle-aged people in the workforce is particularly problematic.^5–8 The global prevalence of knee OA peaks at about 50 years of age.⁹ Worldwide, the estimated years lived with disability is 2.4 million for people younger than 50 years of age, the approximate age of peak prevalence for knee OA.^9,10 Accordingly, the number of middle-aged patients seeking treatment for knee OA, including TKR, is increasing. ¹¹ Joint replacement may not be the most appropriate treatment for these patients.¹² Earlier TKR is associated with prosthesis infection, ¹³ lower patient satisfaction¹⁴ and revision surgery;^15–18 about 25% of all TKRs are considered “likely inappropriate.”¹⁹ Clinicians have identified a clear treatment gap between exhausting nonoperative management and appropriateness for TKR, resulting in years of pain, decreased function, productivity losses and associated costs.^5–9,20,21 It is therefore imperative to identify additional effective treatments for the large group of patients with knee OA.Medial opening wedge high tibial osteotomy (HTO) is a limb realignment surgery intended for patients with medial compartment knee OA who are not suitable candidates for TKR because of less severe disease, younger age and greater physical demands. The purpose of HTO is to correct malalignment, thereby shifting load away from the more involved knee compartment and limit OA progression.^22,23 Substantial shifts in knee loading^24,25 have resulted in clinically important improvements in pain and function after HTO^26,27 and the procedure is cost-effective,^28,29 yet the surgery is uncommon in Canada.³⁰ Unlike the high and increasing rates of other knee surgical procedures including arthroscopy³¹ and TKR,³² rates of HTO remain low.^33,34High tibial osteotomy may help fill the treatment gap between nonsurgical treatments and definitive TKR. At the London Health Sciences Centre in London, Ontario, HTO is performed frequently with a goal of preventing or delaying the need for TKR. Thus, it is appropriate to investigate the duration of benefit of HTO, and the preoperative characteristics associated with it. When quantified as conversion from HTO to TKR, registries using administrative data enable large sample sizes (> 2500 patients) to estimate cumulative incidence of TKR.^30,35,36 However, there can be limitations in using only administrative data, including confirming the correct procedure, limb and diagnosis. Administrative data often lack detailed information assessed preoperatively, such as radiographic features (e.g., disease severity and lower limb alignment) and patient-reported outcome measures. Previously reported predictors of conversion to TKR such as female sex and greater age^30,35–39 may be influenced (perhaps confounded) by other clinical characteristics not typically included in administrative data. Therefore, our objective was to investigate the cumulative incidence of TKR after medial opening wedge HTO and potential predictors using data collected prospectively from a single Canadian centre that focuses on HTO. Specifically, we evaluated the time to conversion from HTO to TKR and investigated the association of HTO preoperative characteristics with subsequent TKR.     

Mitotic spindle: lessons from theoretical modeling

Cell biology is immensely complex. To understand how cells work, we try to find patterns and suggest hypotheses to identify underlying mechanisms. However, it is not always easy to create a coherent picture from a huge amount of experimental data on biological systems, where the main players have multiple interactions or act in redundant pathways. In such situations, when a hypothesis does not lead to a conclusion in a direct way, theoretical modeling is a powerful tool because it allows us to formulate hypotheses in a quantitative manner and understand their consequences. A successful model should not only reproduce the basic features of the system but also provide exciting predictions, motivating new experiments. Much is learned when a model based on generally accepted knowledge cannot explain experiments of interest, as this indicates that the original hypothesis needs to be revised. In this Perspective, we discuss these points using our experiences in combining experiments with theory in the field of mitotic spindle mechanics.

The goal of anyone studying biology is to learn how life works, but for many students the choice of biology is reinforced by a desire to escape mathematics, physics, complex equations, and theoretical work. Yet research in biology often needs theoretical analysis. Theoretical modeling is valuable because it allows us to formulate our hypotheses in a rigorous manner and recognize their implications. Theory in cell biology has been the subject of thought-provoking reviews discussing different types of models as well as why and how to do theoretical modeling (Mogilner et al., 2006; Gunawardena, 2014; Möbius and Laan, 2015; Phillips, 2015; Tyson and Novák, 2015). In this essay, we illustrate the lessons that emerge from the interplay of theory and experiments using examples from spindle mechanics, emphasizing how theory is useful also when it cannot explain experiments and how it becomes especially valuable when it predicts unexpected behavior.The mitotic spindle is a marvelous microtubule-based micromachine that segregates the genome from one cell into two equal parts destined to the future daughter cells (McIntosh, 2016). Spindle microtubules can be divided into three main classes according to their localization and function: kinetochore microtubules that bind the kinetochore, a protein complex at the centromere of each chromosome; overlap microtubules, which extend from the opposite spindle halves and overlap in the middle; and astral microtubules, which grow from the spindle pole toward the cell cortex. Nucleation, dynamics, and forces exerted by spindle microtubules are regulated by hundreds of microtubule-binding and other mitotic proteins, which have multiple mutual interactions. These complex biochemical interactions drive self-organization, a process where order arises from local interactions between initially disordered components, into a molecular machine that can generate large-scale forces to move the chromosomes (Pavin and Tolic´, 2016). Yet, despite the great amount of knowledge about the spindle, this complexity of interactions makes the mechanisms of spindle functioning still largely unclear. Precisely because of the complexity, theoretical modeling is helpful in testing hypotheses and identifying key mechanisms.     

Burden of noninfluenza respiratory viral infections in adults admitted to hospital: analysis of a multiyear Canadian surveillance cohort from 2 centres

BACKGROUND:Data on the outcomes of noninfluenza respiratory virus (NIRV) infections among hospitalized adults are lacking. We aimed to study the burden, severity and outcomes of NIRV infections in this population.METHODS:We analyzed pooled patient data from 2 hospital-based respiratory virus surveillance cohorts in 2 regions of Canada during 3 consecutive seasons (2015/16, 2016/17, 2017/18; n = 2119). We included patients aged ≥ 18 years who developed influenza-like illness or pneumonia and were hospitalized for management. We included patients confirmed positive for ≥ 1 virus by multiplex polymerase chain reaction assays (respiratory syncytial virus [RSV], human rhinovirus/enterovirus (hRV), human coronavirus (hCoV), metapneumovirus, parainfluenza virus, adenovirus, influenza viruses). We compared patient characteristics, clinical severity conventional outcomes (e.g., hospital length-of stay, 30-day mortality) and ordinal outcomes (5 levels: discharged, receiving convalescent care, acute ward or intensive care unit [ICU] care and death) for patients with NIRV infections and those with influenza.RESULTS:Among 2119 adults who were admitted to hospital, 1156 patients (54.6%) had NIRV infections (hRV 14.9%, RSV 12.9%, hCoV 8.2%) and 963 patients (45.4%) had influenza (n = 963). Patients with NIRVs were younger (mean 66.4 [standard deviation 20.4] yr), and more commonly had immunocompromising conditions (30.3%) and delay in diagnosis (median 4.0 [interquartile range (IQR) 2.0–7.0] days). Overall, 14.6% (12.4%–19.5%) of NIRV infections were acquired in hospital. Admission to ICU (18.2%, median 6.0 [IQR 3.0–13.0] d), hospital length-of-stay (median 5.0 [IQR 2.0–10.0] d) and 30-day mortality (8.4%; RSV 9.5%, hRV 6.6%, hCoV 9.2%) and the ordinal outcomes were similar for patients with NIRV infection and those with influenza. Age > 60 years, immunocompromised state and hospital-acquired viral infection were associated with worse outcomes. The estimated median cost per acute care admission was $6000 (IQR $2000–$16 000).INTERPRETATION:The burden of NIRV infection is substantial in adults admitted to hospital and associated outcomes may be as severe as for influenza, suggesting a need to prioritize therapeutics and vaccines for at-risk people.

The global burden of lower respiratory tract infections is substantial, leading to many hospital admissions and deaths, especially among young children and older adults.¹ Respiratory viruses are responsible for almost half of such infections in adults that require in-hospital management; previous studies estimate that 28%–62% are caused by noninfluenza respiratory viruses (NIRVs).^2–4 With some geographical and seasonal variations, respiratory syncytial virus (RSV), human rhinovirus (hRV) and human coronavirus (hCoV) are among the most frequently identified NIRV infections.^1–7 Most infected adults develop mild, self-limiting illnesses, but increasing evidence suggest that NIRVs, either alone or with coinfecting bacteria, can result in severe pneumonia and death.^8,9 For instance, RSV has been shown to cause severe respiratory failure, with fatality rates comparable to or exceeding those observed among adults admitted to hospital with influenza.^10–12 Data on hRV, hCoV and other NIRVs are more limited, owing to the lack of accurate diagnostics and systematic case-finding approaches.^7–9 However, with the increasing availability of multiplex polymerase chain reaction (PCR) assays that can simultaneously detect influenza and NIRVs, these infections are now readily diagnosed as part of a syndromic approach in patients who present with acute respiratory illnesses.^2–5,13,14 The burden, clinical significance and impacts of NIRVs on the health care system remain inadequately characterized.To address this gap, we analyzed the relative frequencies, patient characteristics, location of acquisition (community or hospital), severity and clinical outcomes of patients with NIRV and influenza infections diagnosed by multiplex PCR in a cohort of adults admitted to hospital in 2 large Canadian health care centres during a 3-year surveillance period. The associated health care resource use was also estimated.     

Fifty years of cycling

Fifty years ago, the first isolation of conditional budding yeast mutants that were defective in cell division was reported. Looking back, we now know that the analysis of these mutants revealed the molecular mechanisms and logic of the cell cycle, identified key regulatory enzymes that drive the cell cycle, elucidated structural components that underly essential cell cycle processes, and influenced our thinking about cancer and other diseases. Here, we briefly summarize what was concluded about the coordination of the cell cycle 50 years ago and how that relates to our current understanding of the molecular events that have since been elucidated.

The cell cycle is a process that orders a number of cellular processes to ensure the accurate duplication of the cell. It was hoped that a genetic analysis would reveal how the events were integrated. The inspiration for this was the work of Bob Edgar and Bill Wood on bacteriophage morphogenesis, which revealed the ordered steps by which phage parts were assembled and then put together (Wood and Edgar, 1967). The major questions were how DNA replication and spindle morphogenesis were integrated to achieve accurate chromosome segregation; how cell division was integrated with mitosis to ensure that both daughter cells received a full chromosome complement; and how growth and division were integrated to maintain a constant cell size.Mutants that block cell cycle progression were identified by screening collections of randomly generated temperature sensitive mutants (Hartwell et al., 1970a). Each mutant was screened individually by time-lapse photomicroscopy to identify cell division control (CDC) mutants that caused all cells in the population to arrest at the same point in the cell cycle at the restrictive temperature. The use of budding yeast was critical because the presence and size of the daughter bud provided a simple readout of where cells were in the cell cycle. The first collection of CDC mutants was derived from screening 1500 temperature-sensitive mutants and identified a total of 147 mutants, which fell into 32 complementation groups (Hartwell et al., 1973). An example of one of the first mutants identified is shown in Figure 1. Wild-type cells are found at all stages of the cell cycle at the restrictive temperature (panel A), whereas the CDC mutant cells arrest in late in the cell cycle with large daughter buds (panel B).Open in a separate windowFIGURE 1:An example of one of the first CDC mutants isolated in budding yeast. Wild-type cells and temperature-sensitive mutant cells were grown at the permissive temperature and then shifted to the restrictive temperature, and CDCs were followed by photomicroscopy. (A) Wild-type cells, which are found at all stages of the cell cycle at the restrictive temperature, as indicated by the presence of cells at all stages of the daughter cell budding cycle. (B) A CDC mutant in which all cells have arrested at a cell cycle stage with large daughter buds.The phenotypes of the mutants revealed some preliminary answers to the major questions (Hartwell et al., 1970a, b). Assuming that the primary biochemical defect in a mutant was the process that stopped first, the following conclusions could be drawn. The elongation of the spindle was dependent on prior duplication of the spindle poles and the completion of DNA replication. Cell division and mitosis were coordinated because the formation of the daughter bud was dependent on spindle pole duplication in the previous cycle and cytokinesis was dependent on prior elongation of the spindle. Growth and division were coordinated because the CDC28 (CDK1) function at Start required sufficient growth to initiate all the events of the cell cycle. Cell fusion during mating of haploid cells was coordinated with cell division because mating hormones arrested the cell cycle at the CDC28 step and fusion was restricted to that step in the cell cycle.These observations raised the question of how the dependence of events on one another was controlled. Two models were considered. One, named substrate–product, proposed that a late step was dependent on an early step because the latter was the substrate for the former (e.g., replicated DNA was a substrate for the spindle). The other was regulation, meaning either that signals from completion of an early event induced a late event or that an incomplete early event inhibited a late event. In one example, regulation was evident when a genetic analysis of how damaged DNA arrested nuclear division revealed a signaling pathway (the DNA damage checkpoint) that also accounted for why incomplete DNA replication prevented mitosis (Weinert and Hartwell, 1988).The mechanisms underlying the dependence of cell cycle events upon one another have now been defined in considerable molecular detail. By way of illustration, we will briefly summarize what is known about the dependence of mitosis on replicated chromosomes and the dependence of cell division on mitosis. In some cases, the cdc mutants contributed to this work as a means to identify the relevant genes. However, in many cases, important components were not isolated as cdc mutants. Some of these have since been identified through biochemistry and subsequently shown to have Cdc phenotypes after mutants were created by in vitro mutagenesis. Additional cell cycle components were identified in other genetic screens or isolated using the original cdc mutants as starting points to search for genetic interactors. For example, the cyclins from yeast (which are redundant and nonessential), and even humans, were isolated, in part, as high-copy suppressors of the yeast cdc28 mutant (Hadwiger et al., 1989).One prominent class of cdc mutants affected DNA replication. Mutants targeting two of the three essential replicative polymerases were isolated, as were DNA ligase, a gene required for replication near telomeres, and genes required for the production of deoxyribonucleotides. We now know that these mutations resulted in robust arrest phenotypes because they led to the accumulation of significant amounts of ssDNA, the signal recognized by the replication checkpoint pathway (Zou and Elledge, 2003). This checkpoint signaling pathway both blocks mitosis and feeds back to replication. This feedback to replication helps stalled forks progress and also blocks origins that have not yet fired from doing so. The critical checkpoint phosphorylation events that effect these goals are well understood. Mitotic arrest is largely achieved by phosphorylation and stabilization of Pds1 (Cohen-Fix and Koshland, 1997), an event that blocks sister chromatid separation. Blocking origin firing is mediated by the phosphorylation of two proteins required for origin firing (Lopez-Mosqueda et al., 2010; Zegerman and Diffley, 2010). Finally, the restart of replication forks stalled by either mutational disruption or exogenous agents is promoted by the phosphorylation of several critical targets that increase nucleotide levels and modify the activity of proteins that act at the fork (Ciccia and Elledge, 2010). If measured by viability after fork arrest, this last function is by far the most significant role of this checkpoint pathway, although mitotic arrest and blocking origin firing are also important for preserving genome integrity.While the cdc screen was effective in identifying genes involved in the mechanics of DNA replication, it was less effective in identifying genes that function exclusively to establish origins of replication. An exception to this, CDC6, sheds some light on why this may be. cdc6 mutants brought to a fully nonpermissive temperature do not form replication forks, and thus do not activate the replication checkpoint, although they eventually arrest in mitosis due to the formation of an aberrant spindle that triggers the spindle assembly checkpoint (Piatti et al., 1995; Stern and Murray, 2001). Temperature-sensitive alleles of polymerase or ligases are likely to generate a few nonfunctional replication forks even when the alleles are weak, thus activating the checkpoint and providing a clear Cdc phenotype. Of course, it could not have been foreseen at the time of this screen that mutations in some cell cycle processes might eliminate the very signals for arrest that the screen was designed to identify.Major progress has also been made in understanding the coordination of events needed to ensure successful chromosome segregation to daughter cells during mitosis. The spindle poles duplicate and separate to form a microtubule-based spindle. During DNA replication, the cohesin complex is loaded onto sister chromatids to keep them paired until the metaphase to anaphase transition. At the same time, the kinetochores that mediate attachment of chromosomes to the spindle microtubules assemble on centromeres. These are examples of substrate–product relationships where cohesin and kinetochores will assemble once the chromatin templates are available. Similarly, kinetochores make attachments to the spindle microtubules as soon as they are assembled. The spindle assembly checkpoint, a regulatory pathway, monitors kinetochore–microtubule interactions and halts the metaphase-to-anaphase transition until all chromosomes are properly attached (Hoyt et al., 1991; Li and Murray, 1991). Once the checkpoint is satisfied, cells activate the anaphase-promoting complex to release the linkage between sister chromatids and allow the spindle to elongate and pull chromosomes to opposite poles. As the spindle elongates into the daughter cell, the cell reverses Cdc28 substrate phosphorylations to promote mitotic exit and cytokinesis.How are all of these events coordinated? Surprisingly, although corresponding temperature-sensitive mutants exist for most mitotic genes, the cdc screen did not isolate the structural components of the yeast spindle, spindle pole, or kinetochore, with the exception of one pole mutant, cdc31. In contrast, the screen identified many signaling molecules that regulate the metaphase-to-anaphase transition. The cdc screen identified five subunits of the anaphase-promoting complex, which coordinates this transition by ensuring the degradation of proteins that lead to the removal of cohesion from chromosomes and the down-regulation of Cdc28 activity (King et al., 1995; Sudakin et al., 1995). One of the substrates that must be degraded is Pds1, the same protein that is the target of the DNA checkpoint (Cohen-Fix et al., 1996). Pds1 inhibits the enzyme separase that releases cohesin to ensure the timely separation of sister chromatids (Ciosk et al., 1998; Uhlmann et al., 2000). The targeted degradation of Pds1 and cyclins by a single complex couples spindle elongation and chromosome segregation to Cdc28 inactivation. The spindle assembly checkpoint inhibits the anaphase-promoting complex, reinforcing the coordination between proper spindle attachment to chromosomes and anaphase progression (Hwang et al., 1998; Kim et al., 1998). After chromosome segregation, many Cdc28 substrates must also be dephosphorylated to exit from mitosis. Each of the essential kinases and phosphatases in this control system, called the mitotic exit network, were found in the cdc screen (Shou et al., 1999; Visintin et al., 1999). The mitotic exit network coordinates cytokinesis with the spindle delivering chromosomes to the daughter cell (Bardin et al., 2000; Pereira et al., 2000). Finally, several members of the septin ring that ensures cytokinesis, the last event in the cell cycle, were identified as cdc mutants. The septin mutants continue to bud, replicate DNA, and undergo mitosis in the next cell cycle, showing that completion of all events in the prior cell cycle is not necessarily required for progression. However, looking back, most of the key mitotic events are coordinated by regulatory events that reinforce the dependence of one event on the next, as opposed to the substrate–product relationship. Even in cases where there are clear substrate–product dependencies, such as spindle attachment to kinetochores, the cell has multiple regulatory mechanisms in place to halt the cell cycle until errors are detected and corrected, thus ensuring the proper execution of mitosis.What do the next 50 years hold? The short examples above illustrate the tremendous progress that has been made in understanding the molecular mechanisms that ensure the coordination of cell cycle events. However, there are still major questions about its specificity, accuracy, and complexity, as well as how it is altered in disease. Specialized cell divisions such as meiosis and asymmetric cell division or modified cell cycle states such as quiescence require modifications to the cell cycle. The reconstitution of molecular events has helped to identify the minimal components and regulation required, but this has not accounted for the exquisite precision of these processes in the cell. The complexity of how individual cell cycle events integrate with other cellular processes such as metabolism is still in the early stages. Uncontrolled cell division is the root of cancer, so identifying therapeutic targets that specifically cause cancer vulnerabilities and avoid toxicity to normal cell divisions is still very much needed. In sum, many of the principles gained from cell cycle research have guided our thinking about biological processes; further elucidating the underlying mechanisms of the cell cycle will continue to influence fundamental biology and disease research for decades to come.     

Inflammatory Responses with Left Ventricular Compromise after Induction of Myocardial Infarcts in Sheep (Ovis aries)

Ischemic myocardial disease is a major cause of death among humans worldwide; it results in scarring and pallor of the myocardium and triggers an inflammatory response that contributes to impaired left ventricular function. This response includes and is evidenced by the production of several inflammatory cytokines including TNFα, IL1β, IL4, IFNγ, IL10 and IL6. In the current study, myocardial infarcts were induced in 6 mo old male castrated sheep by ligation of the left circumflex obtuse marginal arteries (OM 1 and 2). MRI was used to measure parameters of left ventricular function that include EDV, ESV, EF, SVI, dp/dt max and dp/dt min at baseline and at 4 wk and 3 mo after infarct induction. We also measured serum concentrations of an array of cytokines. Postmortem histologic findings corroborate the existence of left ventricular myocardial injury and deterioration. Our data show a correlation between serum cytokine concentrations and the development of myocardial damage and left ventricular functional compromise.

Heart failure is a globally significant problem in both humans and lower animals.^3,18 The medical literature is replete with predisposing causes of heart disease,¹³ yet the prevalence of heart failure remained high.^4,5,16 Regardless of the cause of myocardial damage and subsequent left ventricular compromise, the literature indicated that the proinflammatory response that occurs after myocardial infarction is an important contributor to the deterioration of the myocardium^{1,9,12,14,17,18,20,21} Sheep and pigs are excellent translational models of human cardiology because their hearts bear many physiologic and anatomic similarities to the human heart.^4,8,15 The primary use of these models in cardiology is primarily to study myocardial infarction^5,13,16 and to a lesser extent, physiologic processes that develop after myocardial insult.Our study measured some of the major proinflammatory cytokines that contribute to myocardial damage. Most of these cytokines, including: TNFα, IL6, and IFNγ, are important correlates of myocardial ischemia that contribute to a decline in left ventricular myocardial function.^1,9,14 In our study, we detected left ventricular compromise as early as 4 wk after the infarction, while the proinflammatory response was recorded at 48 h after the infarct and peaked at 4 wk. Cardiac functional parameters began to decline early in the study consistent with the proinflammatory response. The cardiac functional parameters continued to decline until 3 mo, which was the termination of the study. These findings may support antiinflammatory intervention as an important adjunct of any therapeutic regimen.     

Symbiotic associations of the deepest recorded photosynthetic scleractinian coral (172 m depth)

The symbiosis between scleractinian corals and photosynthetic algae from the family Symbiodiniaceae underpins the health and productivity of tropical coral reef ecosystems. While this photosymbiotic association has been extensively studied in shallow waters (<30 m depth), we do not know how deeper corals, inhabiting large and vastly underexplored mesophotic coral ecosystems, modulate their symbiotic associations to grow in environments that receive less than 1% of surface irradiance. Here we report on the deepest photosymbiotic scleractinian corals collected to date (172 m depth), and use amplicon sequencing to identify the associated symbiotic communities. The corals, identified as Leptoseris hawaiiensis, were confirmed to host Symbiodiniaceae, predominantly of the genus Cladocopium, a single species of endolithic algae from the genus Ostreobium, and diverse communities of prokaryotes. Our results expand the reported depth range of photosynthetic scleractinian corals (0–172 m depth), and provide new insights on their symbiotic associations at the lower depth extremes of tropical coral reefs.Subject terms: Symbiosis, Microbial ecology

The ecological success of scleractinian corals, the engineers of one of the most productive and diverse ecosystems on Earth, relies on a myriad of symbiotic associations with microorganisms [1]. Among these symbioses, the association between the coral host and unicellular algae from the family Symbiodiniaceae is central to coral health and powers the metabolically expensive process of calcification [2]. The coral host provides limited inorganic nutrients, while Symbiodiniaceae share essential organic compounds derived from their photosynthetic activity [3]. This light-dependent association has mainly been studied in shallow waters (<30 m) because of technical limitations imposed by traditional scientific scuba diving. However, photosynthetic scleractinian corals have been observed in the mesophotic reef slope down to 150–165 m depth [4, 5].As depth increases, the waveband of solar radiation used by most algae for photosynthesis (from 400–700 nm) becomes attenuated in both intensity and width. Even in clear tropical waters, the irradiance levels below 120 m depth can be less than 1% of surface values, and the light spectrum is shifted toward the blue and blue–green wavelengths (~475 nm) (e.g. [4]). These light limitations pose a major constraint for the productivity of benthic organisms that rely on photosynthetic symbionts [6], including reef-building corals (scleractinians). While the scleractinian coral species Leptoseris hawaiiensis has been reported to occur as deep as 153 m in Hawaii and 165 m at Johnston atoll (reviewed in [4]), no live specimens were collected at these extreme depths. The fact that Symbiodiniaceae have been found at much greater depth in association with Antipatharians (396 m) [7], raises the possibility that they might also be present in scleractinian corals deeper than 165 m. Previous studies have genetically confirmed and identified endosymbiotic Symbiodiniaceae in Leptoseris down to 70 m on the Great Barrier Reef [8] and down to 125 m depth in Hawaii [9–11]. A specific host-Symbiodiniaceae association was reported between deep L. hawaiiensis and a Cladocopium from the ancestral C1 radiation [9–11], which represents a diverse group of Symbiodiniaceae commonly found in association with scleractinians on shallow coral reefs [8, 9, 12, 13]. To better understand how scleractinian corals can survive so far away from their presumed light optimum, it is critical to determine if these deep specimens (1) maintain their association with photosynthetic algae and/or (2) if their survival in the deepest mesophotic coral ecosystems requires a shift in their microbial communities, including Symbiodiniaceae and other microorganisms such as endolithic algae and bacteria.Here we report on the observation and collection of the deepest scleractinian corals in association with Symbiodiniaceae and other photosymbionts. Technical divers using closed-circuit rebreathers recovered three L. hawaiiensis colonies from the Gambier archipelago (French Polynesia, Fig. 1A) at 154, 168, and 172 m depth (n = 2 subsamples for each depth; Fig. 1B–D). Irradiance measured at 120 m depth was <2% of that recorded at 6 m depth and irradiance at 172 m was predicted to be <1% (Fig.​(Fig.1E1E and S1). ITS2 sequencing revealed Symbiodiniaceae presence in all three lower mesophotic colonies sampled, with nearly all of the retrieved amplicon sequence variants (ASVs; with most of these representing intragenomic sequence variants) classified as Cladocopium (Fig. 2). The most common ITS2 ASV representative sequence associated with these Leptoseris hosts (S-01, Fig. 2 and S2; 50–57% of total ASVs in each sample) was C1 (GeoSymbio and SymPortal databases; see supplementary methods). This represents one of the most common groups of Symbiodiniaceae, and it has previously been reported in Leptoseris [9, 10, 14], as well as other host species at depths ranging from the surface to 125 m [8, 10, 11, 13–15]. As a complementary approach, ITS2 profiles predicted by SymPortal were used as proxy for Symbiodiniaceae genotypes ([16]; see supplementary methods and data files S1–S4). These predicted ITS2 profiles were largely consistent among replicates but confirmed a different profile for the colony at 172 m depth compared to those at 154 and 168 m depth (Fig. S2). Nonetheless, the Symbiodiniaceae communities shared three ASVs that exactly matched C89 (S-02: 5% at 172 m vs. 17–19% at 154–168 m) and two different C variants (both S-05 and S-07: 7% at 172 m vs. ~2% at 154–168 m) in public databases (Fig. S3; GeoSymbio, SymPortal or Genbank). Of the 26 ASVs identified across all samples, one sequence originated from Durusdinium (S-24 D1 with GeoSymbio and SymPortal databases). This sequence is found in multiple heat-tolerant Durusdinium species including the enigmatic, cosmopolitan [17], host generalist D. trenchii [18]. However, whether or not the Symbiodiniaceae sampled here is D. trenchii or indeed thermally tolerant cannot be confirmed without further genetic and phenotypic data. Low abundance ASVs were observed at all three depths (172 m: 8 ASVs, 154 and 168 m: 10 ASVs, Fig. S3), including nine ASV sequences (Fig. 2) that have not been reported previously in the GeoSymbio [13] and SymPortal (access date: 2020-05-19_07-23-40) [16] databases (Fig. S3). Comparison of the overall Symbiodiniaceae SymPortal predicted ITS2 profiles (Fig. S2) did not confidently identify matches with previously encountered profiles (predominantly from shallow reef environments), indicating that they might be specific to this species and/or mesophotic environment. Given the extreme paucity of light at these depths, we hypothesize that lower mesophotic L. hawaiiensis may use different strategies to photoacclimate. Morphologically, the coral species were characterized by a thin flat skeleton (Fig. 1B–D), which is optimal for light harvesting and reducing skeletal carbonate deposition [19]. Leptoseris hawaiiensis has also been shown to display depth-associated physiological specialization and trophic plasticity (acquiring energy from different food sources) [9], and an unusual light-harvesting system, which enlarges the spectrum of wavelengths for photosynthesis by transforming the short, blue-shifted wavelength with their autofluorescent pigments [19].Open in a separate windowFig. 1Sampling location of the deepest photosymbiotic scleractinian coral recorded to date.A Map of the Gambier archipelago, French Polynesia. Pictures of Leptoseris hawaiiensis collected at 172 m depth in the Gambier archipelago (B) during the in situ sampling (screenshot of video © UTP III), (C) after reaching the surface and (D) after bleaching for taxonomic identification with the green color indicating the presence of endolithic algae. E Variation of the optical index of irradiance (in PAR) along the coral reef depth gradient from 6 to 120 m depth (predictions for 150 and 172 m depths) at Mangareva. For each depth, the three values represent a mean value for 3 days of measurements recorded every 5 min with a PAR logger (DEFI2-L Advantech) at three different time periods of the day (9 h30–10 h00, 12 h30–13 h00 and 15 h30–16 h00).Open in a separate windowFig. 2Microbial communities harbored by the three deep colonies.Composition of the microbial community in Leptoseris hawaiiensis collected at 172, 168, and 154 m. At each depth, two subsamples were analyzed for each colony. The ITS2 marker shows the relative proportion of different Symbiodiniaceae ASVs (with GeoSymbio and SymPortal v.2020-05-19_07-23-40 affiliations). The 16S rDNA marker shows the relative proportions of different ASVs for endolithic algae chloroplast composition and bacteria classes. Asterisk represents sequences with no exact match in the SymPortal database for Symbiodiniaceae.To identify other microorganisms associated with our lower mesophotic scleractinian colonies, we targeted the 16S rRNA gene (V4–V5 region; see supplementary methods). Sequencing data revealed the presence of green algal chloroplast sequences belonging to the genus Ostreobium (Fig. 2). This endolithic alga was abundant in the deep coral colonies as suggested by the marked green color observed below the living tissues (Fig. 1C) and within the skeleton after removing the soft tissues in bleach (Fig. 1D). We identified a single Ostreobium species (ASV ga-01), belonging to clade 2, that was dominant in all the colonies (Fig. 2 and S4), and has been previously reported across the depth gradient in scleractinian corals and octocorals worldwide [20, 21]. The nature of the interaction between corals and Ostreobium has been debated. Evidence supports a mutualistic association under extreme conditions such as coral stress (inducing bleaching) [22] or drastically reduced light exposure [23]. Under the low light conditions of the deep mesophotic fore reef slope, Ostreobium might complement Symbiodiniaceae’s function by providing photosynthates to the host. These endolithic algae are adapted to photosynthesize in near-darkness with increased numbers of light-harvesting xanthophyll pigments that can use shorter wavelengths compared to other green algae and optimize light capture (e.g. [24]).Bacteria associated with the lower mesophotic scleractinian colonies had an observed richness ranging from 106 to 211 ASVs per sample (Fig. S5). These bacteria mainly belonged to the classes Alpha- (19-49%) and Gamma-proteobacteria (8–17%), Bacteroidia (6–20%) and subgroup-6 of Acidobacteria (1–17%) (Fig. 2), which are known to associate with corals [25]. In total, we detected 843 different bacterial ASVs, among which 67–89% were unique to one colony or even unique to one subsample (Fig. S6 and Table S1). Our data suggest that the coral hosts displayed individual microbial signatures with some common ASVs shared between subsamples of the same colony (Fig. S6). However, this result might have been affected by the low-sequencing depth of the microbiome following the removal of the Ostreobium reads. Our results corroborate previous reports describing the high intra-specific variability of coral-associated bacterial communities at different spatial scales (e.g. [25, 26]), which might be driven by biological traits, such as the age [27] or diets of the colonies [28].This study reports a new depth record for scleractinian corals associated with symbiotic algae at 172 m. Similar to conspecifics previously sampled in mesophotic environments between 115 and 125 m depth [10], the deepest L. hawaiiensis reported here associated with symbiotic-microalgae belonging to the highly diverse C1 lineage. The deep colonies were also characterized by the presence and abundance of a single species of endolithic alga from the genus Ostreobium (clade 2). These filamentous green algae adapted to thrive in extreme low light conditions [24] might highly contribute to the survival of L. hawaiiensis at depth through photosynthates translocation [29]. In addition, bacterial communities were diverse, with intraspecific differences in community composition. Our findings provide new insights into the symbioses of scleractinian corals at depth, through the conservation of their associated photosymbiotic algae, raising important questions about the nature and mechanisms involved in the interactions between host and Symbiodiniaceae and/or Ostreobium (e.g. evolutionary theory of symbiosis [30]). Future studies should establish the contribution of photosynthetic symbionts to the energy budget of mesophotic corals. Understanding the biology of ecosystem engineers, such as tropical reef corals, living at the edge of their habitat range is important to determine the plasticity of these organisms and their ability to withstand environmental pressure.     

Training matters! Narrative from a Black scientist

As STEM (Science, Technology, Engineering, and Math) professionals, we are tasked with increasing our understanding of the universe and generating discoveries that advance our society. An essential aspect is the training of the next generation of scientists, including concerted efforts to increase diversity within the scientific field. Despite these efforts, there remains disproportional underrepresentation of Black scientists in STEM. Further, efforts to recruit and hire Black faculty and researchers have been largely unsuccessful, in part due to a lack of minority candidates. Several factors contribute to this including access to opportunities, negative training experiences, lack of effective mentoring, and other more lucrative career options. This is a narrative of a Black male scientist to illustrate some of the issues in retaining Black students in STEM and to highlight the impact of toxic training environments that exists at many institutions. To increase Black participation in STEM careers, we must first acknowledge, then address, the problems that exist within our STEM training environments in hopes to inspire and retain Black students at every level of training.

I write this today as the curtain of systemic racism and oppression has lifted on our nation. I write this today knowing that difficult conversations about race are happening all across America. As a result of tremendous sacrifices and lives lost, there have been demonstrations and rallies internationally demanding change, prompting governments, organizations, and companies to issue statements claiming that Black Lives Matter (Asmelash, 2020). While the rage has sparked the demand for equity in our society, what does this mean for science?My heart is heavy with these discussions as I have reflected on my own journey in science and revisit the toxic environment that often makes up our science culture. The journey has been long and brutal. It has taken me from first realizing that I wanted to become a scientist, to having this dream deferred by racism, to adopting a persona of persistence and resilience, and finally becoming a professor and cell biologist. This trek through science is one that is not traversed by many Black people (Graf et al., 2018).When confronted by the pervasiveness of racism in science, I remember surviving the assault by learning about the resilience story of Carl Brashear (Robbins, 2000). In 1970, Master Chief Petty Officer Brashear became the first African American master diver in the Navy, and he showed unwavering strength and persistence in the face of racism. Brashear faced an onslaught of racism during his training that endangered his life countless times, but he persisted and eventually won the admiration of his fellow divers. Upon reflection, his story has many signs of an abusive hazing relationship. However, at the time, I thought emulating his behaviors of persistence was the answer to success in science. I thought, “All you have to do is not give up.” I focused on what I thought I could control and kept the Japanese proverb, “Fall down seven, stand up eight” above my bench. I worked long hours, made many mistakes, but always got right back up to the bench to try again. I never saw myself as the brightest or smartest, but I would tell myself “I will be the one who does not give up.” When I recall these stories and talk to students about my journey, I would always say I wanted to be like the cockroach. Because, as is commonly known, you can never get rid of the cockroach. What I never realized with this persistence or “grit” mentality was that it never addressed the problems of systemic racism within the culture of science (Das, 2020). This message of persistence is akin to blaming the victim and not dealing with the root problems in science, including the lack of mentoring, implicit bias, and hostile teaching and training environments (Barber et al., 2020; Team, 2020).In her book, We Want to Do More Than Survive, Bettina Love talks about the idea of teaching persistence or “grit” to African American students as the educational equivalent to the Hunger Games, a fictional competition where participants battle to the death until there is only one victor (Love, 2019). Instead of addressing institutional barriers to success for African Americans in science (i.e., dismantling the Hunger Games arena), we prepare them to survive in a toxic environment. We tell African American students at a young age that the system is structured against them and that they have to be twice as good and work twice as hard as white students (Thomas and Wetlaufer, 1997; Cavounidis and Lang, 2015; Danielle, 2015). We heap a tremendous amount of pressure and responsibility on their shoulders without ever addressing the question, why is it like this? We are in effect training them for the Hunger Games. As they enter college as science majors, they are pitted against each other, and the few victors move into science careers.This Hunger Games analogy (Love, 2019) is reflective of my thinking early on in my science career. As a freshman marine biology major, I imagined myself, like Brashear, a soldier during basic training. I was a member of the “people of color” (POC) squad that was given the least amount of resources and the most dangerous duties. As part of the POC squad, we moved forward through our college years. I saw many fellow soldiers drop from science, and there were only a handful of us left when I reached my junior year (Koenig, 2009).Recently, Michael Eisen, Editor-in-Chief of eLife, authored an opinion article entitled “Racism in Science: We need to act now” (Eisen, 2020). In this article, he reflected on the current racial climate in science and examined his role as both a principle investigator (PI) of a research laboratory and an editor of a prestigious journal. Of note, he highlighted the dire lack of African Americans he had worked with over his career, including the number of researchers he trained in his laboratory, senior editors, and even reviewers for the articles sent for publication to eLife. I appreciated his honesty in shedding light on the issue that so many people whisper about in department hallways or during coffee breaks at national conferences. Based on my journey, I truly understand this lack of diversity, as so few of us are victors in the scientific Hunger Games.As we struggle as a nation with the role of policing within our society, I find similarities between aggressive policing in the Black community and training of Black and Brown students (North, 2020). There are strong implicit biases that we hold within our training environment, and Black students usually find themselves very quickly judged (or prejudged) for a perceived lack of commitment, motivation, or focus (Park et al., 2020). They are also stereotyped as lacking in quantitative abilities (especially the ability to do math) (McClain, 2014). Taken together, these biased judgements result in a lack of trust regarding their data (Steele, 1997). In other words, research supervisors may implicitly expect Black students to be untrustworthy. This is extremely problematic because educational research shows that one of the greatest determinants of students’ success is their teachers’ expectations (Boser et al., 2014). Consequently, it is predictable that if research supervisors expect Black students to be untrustworthy, they will fail.As PIs, we must trust our research students because they are extensions of ourselves in the laboratory. Due to our inability to spend significant amounts of time at the bench, we must trust our students to figure it out and get the work done. Inevitably, experimental approaches will fail; however, based on my experiences in science, Black students are often not given the benefit of the doubt. Instead, I have seen mis/distrust of their commitment, values, and abilities that creates the narrative that they are not motivated, do not care about science, and/or are unable to get the work done, resulting in a broken trainer/trainee relationship. I have witnessed too many Black students fall victim to a “one strike” policy. This was true of me in my early training in marine biology, where I was asked to leave after only 6 months of working in a laboratory. The professor suggested that I had a lack of commitment to my project and was told by other lab members that they collected “my” data, thus providing justification to ask me not to continue. However, what the professor did not know (or care to ask about) was that the other lab members deemed me as someone who did not belong. Consequently, without my knowledge, they collected data on my project and sent it to the PI, thereby working to reinforce the narrative of my lack of commitment. This experience significantly hindered my access to research opportunities and blacklisted me from any other marine biology labs at my university because I was labeled as uncommitted to science. This ended my career in marine biology. I lost the Hunger Games.As a graduate student, I found another opportunity in a cell biology laboratory, and I tried to apply lessons learned from my earlier participation in the Games. I overcommitted to lab work, blocking out any activities related to my culture or personal life. Instead, I dedicated myself completely to the lab. Working 12-h days, I found that my research was progressing, but I was burning out and losing any desire toward a research career. In particular, my burnout was connected to the perception that any interest in my culture and community would not be allowed or accepted or would signal a lack of adequate commitment to science. In effect, I was learning that being a scientist meant that I could not be Black. This, coupled with the constant microaggressions that I faced from professors in classes, among my graduate cohort, and my laboratory colleagues, broadcasted the message that I was an intruder in science. Luckily, I received good mentoring and advice on how to succeed in my graduate program, learning that it was not a sprint, but a marathon. I learned how to balance my personal and professional life, and I always kept them separate. Additionally, the mental image of the resilient cockroach helped me repeatedly during my graduate training, from failing my qualifying exams and failed experiments at the bench to rejections of papers and fellowship applications. While all scientists know that being a scientist means accepting significant amounts of failure, I could not help but feel that the failures I experienced were more frequent, more recognized by others, and even expected by some. This culture of expected failure for people of color (i.e., presumed incompetence), combined with implicit biases and microaggressions, can establish significant barriers for entering and staying in STEM training environments (Smith et al., 2007).To overcome barriers to success in STEM, I worked hard to become a professor in cell biology. I believed that as a professor, I could make a difference, change the environment, and contribute to the change that is so desperately needed. However, I have discovered that the current science culture is just as toxic as when I was a student. Yes, there are programs targeting the inclusion of historically underrepresented groups. There are also a growing number of institutions that are adopting inclusive teaching strategies. Further, we are seeing hiring committees require diversity statements from their applicants as well as receiving implicit bias trainings (Wood, 2019). However, there remains nearly a complete lack of Black faculty members at universities and colleges (Jayakumar et al., 2009; Garrison, 2013; Li and Koedel, 2017). This is, in part, because we have not changed the systemic racism that exists within our training environments. In fact, this racism comes from our noninclusive faculty bodies (Hardy, 2020). In essence, we have nearly a complete absence of Black faculty in STEM because so few Black trainees survive the Hunger Games. More troubling, if they survive, they may be found otherwise unacceptable.Changing the system starts with the belief that Black students can be scientists, followed by acting to proactively encourage and support Black students in STEM. As Eisen states, “This is a solvable problem, we have chosen not to solve it” (Eisen, 2020). Recruiting Black students and scientists at every level is a good start, but without changing the scientific environment to be more welcoming and affirming, those recruited to science will continue to be traumatized. In other words, while increasing access to science is required, it is not sufficient. The dominant majority in science also needs to identify and address their own biases to create antiracist environments. This will only happen when scientists from all groups recognize our convergent interests to advance our universal missions, which is to increase our understanding of the world around us and to solve research questions that will benefit our communities. This is best achieved by a diverse and inclusive scientific workforce for greater knowledge, discovery, and innovation.     

Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.     

PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

The multisubunit-pigment-protein complex PSI is an essential component of the electron transport chain in oxygenic photosynthetic organisms. It utilizes solar energy in the form of visible light to transfer electrons from plastocyanin to ferredoxin.PSI consists of a core complex composed of 12 to 14 proteins, which contains the reaction center (RC) and ∼100 chlorophylls (Chls), and a peripheral antenna system, which enlarges the absorption cross section of the core and differs in different organisms (Mazor et al., 2017; Iwai et al., 2018; Pi et al., 2018; Suga et al., 2019; for reviews, see Croce and van Amerongen, 2020; Suga and Shen, 2020). For the antenna system, cyanobacteria use water-soluble phycobilisomes; green algae, mosses, and plants use membrane-embedded light-harvesting complexes (LHCs); and red algae contain both phycobilisomes and LHCs (Busch and Hippler, 2011). In the core complex, PsaA and PsaB, the subunits that bind the RC Chls, are highly conserved, while the small subunits PsaK, PsaL, PsaM, PsaN, and PsaF have undergone substantial changes in their amino acid sequences during the evolution from cyanobacteria to vascular plants (Grotjohann and Fromme, 2013). The appearance of the core subunits PsaH and PsaG and the change of the PSI supramolecular organization from trimer/tetramer to monomer are associated with the evolution of LHCs in green algae and land plants (Busch and Hippler, 2011; Watanabe et al., 2014).A characteristic of the PSI complexes conserved through evolution is the presence of “red” forms, i.e. Chls that are lower in energy than the RC (Croce and van Amerongen, 2013). These forms extend the spectral range of PSI beyond that of PSII and contribute significantly to light harvesting in a dense canopy or algae mat, which is enriched in far-red light (Rivadossi et al., 1999). The red forms slow down the energy migration to the RC by introducing uphill transfer steps, but they have little effect on the PSI quantum efficiency, which remains ∼1 (Gobets et al., 2001; Jennings et al., 2003; Engelmann et al., 2006; Wientjes et al., 2011). In addition to their role in light-harvesting, the red forms were suggested to be important for photoprotection (Carbonera et al., 2005).Two types of LHCs can act as PSI antennae in green algae, mosses, and plants: (1) PSI-specific (e.g. LHCI; Croce et al., 2002; Mozzo et al., 2010), Lhcb9 in Physcomitrella patens (Iwai et al., 2018), and Tidi in Dunaliela salina (Varsano et al., 2006); and (2) promiscuous antennae (i.e. complexes that can serve both PSI and PSII; Kyle et al., 1983; Wientjes et al., 2013a; Drop et al., 2014; Pietrzykowska et al., 2014).PSI-specific antenna proteins vary in type and number between algae, mosses, and plants. For example, the genomes of several green algae contain a larger number of lhca genes than those of vascular plants (Neilson and Durnford, 2010). The PSI-LHCI complex of plants includes only four Lhcas (Lhca1–Lhc4), which are present in all conditions analyzed so far (Ballottari et al., 2007; Wientjes et al., 2009; Mazor et al., 2017), while in algae and mosses, 8 to 10 Lhcas bind to the PSI core (Drop et al., 2011; Iwai et al., 2018; Pinnola et al., 2018; Kubota-Kawai et al., 2019; Suga et al., 2019). Moreover, some PSI-specific antennae are either only expressed, or differently expressed, under certain environmental conditions (Moseley et al., 2002; Varsano et al., 2006; Swingley et al., 2010; Iwai and Yokono, 2017), contributing to the variability of the PSI antenna size in algae and mosses.The colonial green alga Botryococcus braunii (Trebouxiophyceae) is found worldwide throughout different climate zones and has been targeted for the production of hydrocarbons and sugars (Metzger and Largeau, 2005; Eroglu et al., 2011; Tasić et al., 2016). Here, we have purified and characterized PSI from an industrially relevant strain isolated from a mountain lake in Portugal (Gouveia et al., 2017). This B. braunii strain forms colonies, and since the light intensity inside the colony is low, it is expected that PSI in this strain has a large antenna size (van den Berg et al., 2019). We provide evidence that B. braunii PSI differs from that of closely related organisms through the particular organization of its antenna. The structural and functional characterization of B. braunii PSI highlights a large flexibility of PSI and its antennae throughout the green lineage.     

Postpartum mental illness during the COVID-19 pandemic: a population-based,repeated cross-sectional study

BACKGROUND:It is unclear whether the clinical burden of postpartum mental illness has increased during the COVID-19 pandemic. We sought to compare physician visit rates for postpartum mental illness in Ontario, Canada, during the pandemic with rates expected based on prepandemic patterns.METHODS:In this population-based, repeated cross-sectional study using linked health administrative databases in Ontario, Canada, we used negative binomial regression to model expected visit rates per 1000 postpartum people for March–November 2020 based on prepandemic data (January 2016–February 2020). We compared observed visit rates to expected visit rates for each month of the pandemic period, generating absolute rate differences, incidence rate ratios (IRRs) and their 95% confidence intervals (CIs). The primary outcome was a visit to a primary care physician or a psychiatrist for any mental disorder. We stratified analyses by maternal sociodemographic characteristics.RESULTS:In March 2020, the visit rate was 43.5/1000, with a rate difference of 3.11/1000 (95% CI 1.25–4.89) and an IRR of 1.08 (95% CI 1.03–1.13) compared with the expected rate. In April, the rate difference (10.9/1000, 95% CI 9.14–12.6) and IRR (1.30, 95% CI 1.24–1.36) were higher; this level was generally sustained through November 2020. From April–November, we observed elevated visit rates across provider types and for diagnoses of anxiety, depressive and alcohol or substance use disorders. Observed increases from expected visit rates were greater for people 0–90 days postpartum compared with 91–365 days postpartum; increases were small among people living in low-income neighbourhoods. Public health units in the northern areas of the province did not see sustained elevations in visit rates after July; southern health units had elevated rates through to November.INTERPRETATION:Increased visits for mental health conditions among postpartum people during the first 9 months of the COVID-19 pandemic suggest an increased need for effective and accessible mental health care for this population as the pandemic progresses.

Postpartum mental illness affects as many as 1 in 5 mothers,¹ and can result in maternal suffering and diminished functioning.² Related impaired mother–infant interactions are linked to poor social, cognitive and behavioural outcomes in children across their lifespan.³ When mental illness becomes chronic and recurrent, its effects can extend to the entire family and across generations.⁴ With emergence of the novel coronavirus (SARS-CoV-2), the World Health Organization declared a global COVID-19 pandemic on Mar. 11, 2020. Globally, efforts to contain the virus have led to widespread travel restrictions, physical distancing and work limitations, causing broad social and financial disruption that has been associated with substantial mental health effects.^5,6During the COVID-19 pandemic, people have been reporting concerns about postpartum infection,⁷ and difficulty accessing the extended postpartum social support networks and key community programs that protect against mental illness, such as home visits from public health nurses, breastfeeding clinics and support groups, owing to public health measures.⁸ In Canadian surveys, about 50% of pregnant people reported psychological distress in spring 2020,⁹ and alcohol use increased among women, particularly among those with young children.¹⁰ Whether this represents an increased clinical burden of mental illness or need for care is unknown.Using routinely collected health care data from Ontario, Canada, (population of about 14.6 million), we aimed to examine whether rates of maternal visits to physicians for postpartum mental illness from March to November 2020 differed from expected visit rates based on pre-COVID-19 patterns, and to identify variation by provider type, clinical diagnosis, postpartum timing, parity, income, ethnicity and region of residence.     

Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3′-terminal segment of guide RNA

CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR–Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3′-terminal segments. This result implies that the 3′-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3′-terminal stem loops leads to reduced activity during genomic editing.