Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum

Research into diatom biology has now entered the post-genomics era, following the recent completion of the Thalassiosira pseudonana and Phaeodactylum tricornutum whole genome sequences and the establishment of Expressed Sequence Tag (EST) databases. The thorough exploitation of these resources will require the development of molecular tools to analyze and modulate the function of diatom genes in vivo. Towards this objective, we report here the identification of several reference genes that can be used as internal standards for gene expression studies by quantitative real-time PCR (qRT-PCR) in P. tricornutum cells grown over a diel cycle. In addition, we describe a series of diatom expression vectors based on Invitrogen Gateway technology for high-throughput protein tagging and overexpression studies in P. tricornutum. We demonstrate the utility of the diatom Destination vectors for determining the subcellular localization of a protein of interest and for immunodetection. The availability of these new resources significantly enriches the molecular toolbox for P. tricornutum and provides the diatom research community with well defined high-throughput methods for the analysis of diatom genes and proteins in vivo.     

Towards the molecular understanding of glycogen elongation by amylosucrase

Amylosucrase from Neisseria polysaccharea (AS) is a transglucosidase from the glycoside-hydrolase family 13 that catalyzes the synthesis of an amylose-like polymer from sucrose, without any primer. Its affinity towards glycogen is particularly noteworthy since glycogen is the best D-glucosyl unit acceptor and the most efficient activator (98-fold k(cat) increase) known for this enzyme. Glycogen-enzyme interactions were modeled starting from the crystallographic AS: maltoheptaose complex, where two key oligosaccharide binding sites, OB1 and OB2, were identified. Two maltoheptaose molecules were connected by an alpha-1,6 branch by molecular modeling to mimic a glycogen branching. Among the various docking positions obtained, four models were chosen based on geometry and energy criteria. Robotics calculations enabled us to describe a back and forth motion of a hairpin loop of the AS specific B'-domain, a movement that assists the elongation of glycogen branches. Modeling data combined with site-directed mutagenesis experiments revealed that the OB2 surface site provides an anchoring platform at the enzyme surface to capture the polymer and direct the branches towards the OB1 acceptor site for elongation. On the basis of the data obtained, a semiprocessive glycogen elongation mechanism can be proposed.     

Learn from the Best

What is more inspiring than a discussion with the leading scientists in your field? As a student or a young researcher, you have likely been influenced by mentors guiding you in your career and leading you to your current position. Any discussion with or advice from an expert is certainly very helpful for young people. But how often do we have the opportunity to meet experts? Do we make the most out of these situations? Meetings organized for young scientists are a great opportunity not only for the attendees: they are an opportunity for experts to meet bright students and learn from them in return. In this article, we introduce several successful events organized by Regional Student Groups all around the world, bridging the gap between experts and young scientists. We highlight how rewarding it is for all participants: young researchers, experts, and organizers. We then discuss the various benefits and emphasize the importance of organizing and attending such meetings. As a young researcher, seeking mentorship and additional skills training is a crucial step in career development. Keep in mind that one day, you may be an inspiring mentor, too.     

Crossing Borders for Science

Exchanging ideas with like-minded, enthusiastic people interested in the same topic is crucial for the advancement of a scientist''s career. Several Regional Student Groups (RSGs) of the International Society for Computational Biology (ISCB) Student Council have cooperated in the last six years to organize scientific workshops and conferences. With motivated students, it is possible to create a memorable event for fellow scientists; in doing so, the organizers gain valuable experiences. While collaborating across borders and time zones can be difficult, feedback from event organizers was always positive. When limited resources are juxtaposed with great ideas and a network of contacts, the outcome is always an amazing experience, despite organizers being separated geographically across different countries.     

The miR‐379/miR‐410 cluster at the imprinted Dlk1‐Dio3 domain controls neonatal metabolic adaptation

In mammals, birth entails complex metabolic adjustments essential for neonatal survival. Using a mouse knockout model, we identify crucial biological roles for the miR‐379/miR‐410 cluster within the imprinted Dlk1‐Dio3 region during this metabolic transition. The miR‐379/miR‐410 locus, also named C14MC in humans, is the largest known placental mammal‐specific miRNA cluster, whose 39 miRNA genes are expressed only from the maternal allele. We found that heterozygote pups with a maternal—but not paternal—deletion of the miRNA cluster display partially penetrant neonatal lethality with defects in the maintenance of energy homeostasis. This maladaptive metabolic response is caused, at least in part, by profound changes in the activation of the neonatal hepatic gene expression program, pointing to as yet unidentified regulatory pathways that govern this crucial metabolic transition in the newborn's liver. Not only does our study highlight the physiological importance of miRNA genes that recently evolved in placental mammal lineages but it also unveils additional layers of RNA‐mediated gene regulation at the Dlk1‐Dio3 domain that impose parent‐of‐origin effects on metabolic control at birth and have likely contributed to mammal evolution.     

C5orf42 is the major gene responsible for OFD syndrome type VI

Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the “Joubert syndrome related disorders”. Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies.     

Cryptosporidium parvum-induced ileo-caecal adenocarcinoma and Wnt signaling in a mouse model

Cryptosporidium species are apicomplexan protozoans that are found worldwide. These parasites constitute a large risk to human and animal health. They cause self-limited diarrhea in immunocompetent hosts and a life-threatening disease in immunocompromised hosts. Interestingly, Cryptosporidium parvum has been related to digestive carcinogenesis in humans. Consistent with a potential tumorigenic role of this parasite, in an original reproducible animal model of chronic cryptosporidiosis based on dexamethasone-treated or untreated adult SCID mice, we formerly reported that C. parvum (strains of animal and human origin) is able to induce digestive adenocarcinoma even in infections induced with very low inoculum. The aim of this study was to further characterize this animal model and to explore metabolic pathways potentially involved in the development of C. parvum-induced ileo-caecal oncogenesis. We searched for alterations in genes or proteins commonly involved in cell cycle, differentiation or cell migration, such as β-catenin, Apc, E-cadherin, Kras and p53. After infection of animals with C. parvum we demonstrated immunohistochemical abnormal localization of Wnt signaling pathway components and p53. Mutations in the selected loci of studied genes were not found after high-throughput sequencing. Furthermore, alterations in the ultrastructure of adherens junctions of the ileo-caecal neoplastic epithelia of C. parvum-infected mice were recorded using transmission electron microscopy. In conclusion, we found for the first time that the Wnt signaling pathway, and particularly the cytoskeleton network, seems to be pivotal for the development of the C. parvum-induced neoplastic process and cell migration of transformed cells. Furthermore, this model is a valuable tool in understanding the host-pathogen interactions associated with the intricate infection process of this parasite, which is able to modulate host cytoskeleton activities and several host-cell biological processes and remains a significant cause of infection worldwide.KEY WORDS: SCID mouse model, Cryptosporidiosis, Wnt pathway, Cytoskeleton, Digestive cancer     

Adaptation of the Wine Bacterium Oenococcus oeni to Ethanol Stress: Role of the Small Heat Shock Protein Lo18 in Membrane Integrity

Malolactic fermentation in wine is often carried out by Oenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in O. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from O. oeni grown under optimal growth conditions (here, control liposomes), one was prepared from O. oeni grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from Bacillus subtilis or Lactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from O. oeni suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of O. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.