Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and in Xenopus extracts. In the present report we analyze in vivo the involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. Unfertilized Sphaerechinus granularis eggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studying in vivo the onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained in Xenopus egg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with this in vitro system in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21(Cip1) does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensable in vivo for the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast to Xenopus egg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.

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We show that a splice variant–derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation.

Several distributed storage solutions that do not rely on a central server have been proposed over the last few years. Most of them are deployed on public networks on the internet. However, these solutions often do not provide a mechanism for access rights to enable the users to control who can access a specific file or piece of data. In this article, we propose Mutida (from the Latin word "Aditum" meaning "access"), a protocol that allows the owner of a file to delegate access rights to another user. This access right can then be delegated to a computing node to process the piece of data. The mechanism relies on the encryption of the data, public key/value pair storage to register the access control list and on a function executed locally by the nodes to compute the decryption key. After presenting the mechanism, its advantages and limitations, we show that the proposed mechanism has similar functionalities to Wave, an authorization framework with transitive delegation. However, Wave does not require fully trusted nodes. We implement our approach in a Java software program and evaluate it on the Grid'5000 testbed. We compare our approach to an approach based on a protocol relying on Shamir key reconstruction, which provides similar features.is that there are no servers that we can rely on to be in charge of the protocol for rights management. Additionally, anonymity is a key point since peers are communicating directly and additional layers need to be in place to guarantee it. [19]. In this paper, we propose Mutida (from the Latin word "Aditum" meaning "access" and written from right to left), a protocol that focuses on enabling users to manage access rights in a network of the second category.In this paper, we consider the files stored on the Interplanetary File System [11] (IPFS), which is a storage solution that relies on a BitTorrent-like [35] protocol to exchange the files between the nodes and on a Kademlia [39] Distributed Hash Table (DHT) to locate the different pieces of data in the network. The essential characteristic of IPFS is that files are immutable and cannot be modified once they are written.Using a DHT forwards all location requests through different nodes. Therefore, any connected node is involved in the routing of requests and can determine the identifiers of popular files [26]. Additionally, since the storage solution does not manage any permission and because IPFS does not provide any encryption mechanism to protect user privacy [47], every user can access all the files stored in it if the content identifier (CID) is known.The consequence of this is that any node in the IPFS network can observe the DHT requests and access the corresponding files. This illustrates and justifies the need to manage access permissions in such a network. With Mutida, all users will still be able to find the files in the network, but only the permitted users will be able to decrypt the content.Because of the use of Merkle trees [40] and the use of a root hash as a file identifier, users of IPFS do not need to tru...