Algorithms for phylogenetic footprinting.

Phylogenetic footprinting is a technique that identifies regulatory elements by finding unusually well conserved regions in a set of orthologous noncoding DNA sequences from multiple species. We introduce a new motif-finding problem, the Substring Parsimony Problem, which is a formalization of the ideas behind phylogenetic footprinting, and we present an exact dynamic programming algorithm to solve it. We then present a number of algorithmic optimizations that allow our program to run quickly on most biologically interesting datasets. We show how to handle data sets in which only an unknown subset of the sequences contains the regulatory element. Finally, we describe how to empirically assess the statistical significance of the motifs found. Each technique is implemented and successfully identifies a number of known binding sites, as well as several highly conserved but uncharacterized regions. The program is available at http://bio.cs.washington.edu/software.html.     

Organization of neuropeptide tyrosine-like immunoreactive system in the brain of the Antarctic fish, Trematomus bernacchii

Antarctic notothenioids have developed unique freezing-resistance adaptations, including brain diversification, to survive in the subzero waters of the Southern Ocean surrounding Antarctica. In this study we have investigated the anatomical distribution of neuropeptide tyrosine (NPY)-like immunoreactive elements in the brain of the Antarctic fish Trematomus bernacchii, by using an antiserum raised against porcine NPY. Perikarya exhibiting NPY-like immunoreactivity were observed in distinct regions of the brain. The most rostral group of immunoreactive perikarya was found in the telencephalon, within the entopeduncular nucleus. In the diencephalon, three groups of NPY-like immunoreactive perikarya were found in the hypothalamus. Two groups of positive cell bodies were found in distinct populations of the preoptic nucleus, whereas the other group was found in the nucleus of the lateral recess. More caudally, NPY immunoreactivity was detected in large neurons located in the subependymal layers of the dorsal tegmentum of the mesencephalon, medially to the torus semicircularis. NPY-like immunoreactive nerve fibres were more widely distributed throughout the telencephalon to the rhombencephalon. High densities of nerve fibres and terminals were observed in several regions of the telencephalon, olfactory bulbs, hypothalamus, tectum of the mesencephalon and in the ventral tegmentum of the rhombencephalon. The distribution of NPY-like immunoreactive structures suggests that, in Trematomus, this peptide may be involved in the control of several brain functions, including olfactory activity, feeding behaviour, and somatosensory and visual information. In comparison with other neuropeptides previously described in the brain of Antarctic fish, NPY is more widely distributed. Our data also indicate the existence of differences in the brain distribution of NPY between Trematomus and other teleosts. In contrast with previous results reported in other fish, Trematomus contains positive fibres in the olfactory bulbs and immunoreactive perikarya in the nucleus of the lateral recess, whereas NPY-immunopositive cell bodies are absent in the thalamus and rhombencephalon, and no NPY immunoreactivity is present in the pituitary. These differences could be related to the Antarctic ecological diversity of notothenioids living at subzero temperatures.     

SARS‐CoV‐2 Alpha,Beta, and Delta variants display enhanced Spike‐mediated syncytia formation

Severe COVID‐19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS‐CoV‐2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco‐2, Calu‐3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon‐induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS‐CoV‐2 emerging variants display enhanced syncytia formation.     

Expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a bipartite microtubule organisation centre coordinating mitosis with axoneme assembly

Transmission of malaria-causing parasites to mosquitoes relies on the production of gametocyte stages and their development into gametes. These stages display various microtubule cytoskeletons and the architecture of the corresponding microtubule organisation centres (MTOC) remains elusive. Combining ultrastructure expansion microscopy (U-ExM) with bulk proteome labelling, we first reconstructed in 3D the subpellicular microtubule network which confers cell rigidity to Plasmodium falciparum gametocytes. Upon activation, as the microgametocyte undergoes three rounds of endomitosis, it also assembles axonemes to form eight flagellated microgametes. U-ExM combined with Pan-ExM further revealed the molecular architecture of the bipartite MTOC coordinating mitosis with axoneme formation. This MTOC spans the nuclear membrane linking cytoplasmic basal bodies to intranuclear bodies by proteinaceous filaments. In P. berghei, the eight basal bodies are concomitantly de novo assembled in a SAS6- and SAS4-dependent manner from a deuterosome-like structure, where centrin, γ-tubulin, SAS4 and SAS6 form distinct subdomains. Basal bodies display a fusion of the proximal and central cores where centrin and SAS6 are surrounded by a SAS4-toroid in the lumen of the microtubule wall. Sequential nucleation of axonemes and mitotic spindles is associated with a dynamic movement of γ-tubulin from the basal bodies to the intranuclear bodies. This dynamic architecture relies on two non-canonical regulators, the calcium-dependent protein kinase 4 and the serine/arginine-protein kinase 1. Altogether, these results provide insights into the molecular organisation of a bipartite MTOC that may reflect a functional transition of a basal body to coordinate axoneme assembly with mitosis.     

Receptor-Mediated Transcytosis of Cyclophilin B Through the Blood—Brain Barrier

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein mainly located in intracellular vesicles and secreted in biological fluids. In previous works, we demonstrated that CyPB interacts with T lymphocytes and enhances in vitro cellular incorporation and activity of CsA. In addition to its immunosuppressive activity, CsA is able to promote regeneration of damaged peripheral nerves. However, the crossing of the drug from plasma to neural tissue is restricted by the relative impermeability of the blood-brain barrier. To know whether CyPB might also participate in the delivery of CsA into the brain, we have analyzed the interactions of CyPB with brain capillary endothelial cells. First, we demonstrated that CyPB binds to two types of binding sites present at the surface of capillary endothelial cells from various species of tissues. The first type of binding sites (K(D) = 300 nM; number of sites = 3 x 10(6)) is related to interactions with negatively charged compounds such as proteoglycans. The second type of binding sites, approximately 50,000 per cell, exhibits a higher affinity for CyPB (K(D) = 15 nM) and is involved in an endocytosis process, indicating it might correspond to a functional receptor. Finally, the use of an in vitro model of blood-brain barrier allowed us to demonstrate that CyPB is transcytosed by a receptor-mediated pathway (flux = 16.5 fmol/cm2/h). In these conditions, CyPB did not significantly modify the passage of CsA, indicating that it is unlikely to provide a pathway for CsA brain delivery.     

European common frog (Rana temporaria) recolonized Switzerland from multiple glacial refugia in northern Italy via trans‐ and circum‐Alpine routes

The high mountain ranges of Western Europe had a profound effect on the biotic recolonization of Europe from glacial refugia. The Alps present a particularly interesting case because they form an absolute barrier to dispersal for most taxa, obstructing recolonization from multiple refugia in northern Italy. Here, we investigate the effect of the European Alps on the phylogeographic history of the European common frog Rana temporaria. Based on partial cytochrome b and COXI sequences from Switzerland, we find two mitochondrial lineages roughly north and south of the Alpine ridge, with contact zones between them in eastern and western Switzerland. The northern haplogroup falls within the previously identified Western European haplogroup, while the southern haplogroup is unique to Switzerland. We find that the lineages diverged ~110 kya, at approximately the onset of the last glacial glaciation; this indicates that they are from different glacial refugia. Phylogenetic analyses suggest that the northern and southern haplogroups colonized Switzerland via trans‐ and circum‐Alpine routes from at least two separate refugia in northern Italy. Our results illustrate how a complex recolonization history of the central European Alps can arise from the semi‐permeable barrier created by high mountains.     

Variable Selection and the Coexistence of Multiple mimetic forms of the Butterfly Heliconius numata

Polymorphism in aposematic animals and coexistence of multiple mimicry rings within a habitat are not predicted by classical Müllerian mimicry. The butterfly Heliconius numata Cramer (Lepidoptera: Nymphalidae; Heliconiinae) is both polymorphic and aposematic. The polymorphism is due to variation at a single locus (or `supergene') which determines colour patterns involved in Müllerian mimicry. We sampled 11 sites in a small area (approx. 60×30km) of North-eastern Peru for H. numata and its co-mimics in the genus Melinaea and Athyrtis (Ithomiinae), and examined the role of temporal and spatial heterogeneity in the maintenance of polymorphism. Colour-patterns of Melinaea communities, which constitute the likely `mimetic environment' for H. numata, are differentiated on a more local scale than morphs of H. numata, but the latter do show a strong and significant response to local selection for colour-pattern. In contrast, analysis of enzyme polymorphism in H. numata across the region revealed no spatial structure, which is consistent with a high mobility of this species. Differences in spatial variability in the two taxa may have caused H. numata to become polymorphic, while temporal variability, not significant in this study, probably has a lesser effect. The mimetic polymorphism is therefore explained by means of multiple selection-migration clines at a single locus, a similar process to that which explains narrow hybrid zones between geographic races of other Heliconius butterflies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75

The stable insertion of the retroviral genome into the host chromosomes requires the association between integration complexes and cellular chromatin via the interaction between retroviral integrase and the nucleosomal target DNA. This final association may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To investigate this and better understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase, we used a combination of biochemical and chromosome-binding assays. Our study revealed that retroviral integrase has an intrinsic ability to bind and recognize specific chromatin regions in metaphase even in the absence of its cofactor. Furthermore, this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromosome regions. We also better determined the chromatin features recognized by each partner alone or within the functional intasome, as well as the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. Our data support a new chromatin-binding function of integrase acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate. This work also provides additional information about the behavior of retroviral integration complexes in metaphase chromatin and the mechanism of action of LEDGF/p75 in this specific context.