Dev Biol

Blastomeres of the pre-implantation mouse embryo form trophectoderm and inner cell mass via a process that requires the transcription factors Tead4, Cdx2, Oct4 and Nanog. In mouse morulae cloned by somatic cell nuclear transfer, we observed that the trophectoderm transcription factor Cdx2 is expressed very differently at the protein level compared to time- and stage-matched fertilized counterparts. Protein levels of Cdx2 in cloned embryos appear 'erratic,' i.e. are widely distributed, when plotted as histograms. In contrast to Cdx2, protein levels of the upstream factor Tead4 and of inner cell mass transcription factors Oct4 and Nanog are similar in cloned and fertilized embryos. These observations suggest that trophectoderm formation is initiated but not maintained correctly in cloned mouse morulae, which is consistent with cloned blastocysts' limited implantation and post-implantation success. Because a cell's ability to differentiate is greatly enhanced if it is surrounded by more cells differentiating the same way, a concept designated community effect by Gurdon, we reasoned that the insufficient cell numbers often observed in cloned embryos might lead to premature Cdx2 expression and differentiation of blastomeres into trophectoderm. Therefore, we created larger cloned embryos by aggregating them at the 4-cell stage. Homologous aggregation stimulates expression of multiple signaling pathways' components and results in cloned embryos with levels of Cdx2 similar to fertilized embryos. Most of the resultant morulae and blastocysts consist of cells of all three founders, indicating that aggregation increases stability of all of the individual components. We conclude that the induction of pluripotency in cloned embryos is more efficient than previously assumed, and we propose that a minimum cell number is necessary to stabilize pluripotency and inhibit premature expression of Cdx2 in cloned mouse embryos

Emergence of associative learning in a neuromorphic inference network

Objective. In the theoretical framework of predictive coding and active inference, the brain can be viewed as instantiating a rich generative model of the world that predicts incoming sensory data while continuously updating its parameters via minimization of prediction errors. While this theory has been successfully applied to cognitive processes-by modelling the activity of functional neural networks at a mesoscopic scale-the validity of the approach when modelling neurons as an ensemble of inferring agents, in a biologically plausible architecture, remained to be explored.Approach.We modelled a simplified cerebellar circuit with individual neurons acting as Bayesian agents to simulate the classical delayed eyeblink conditioning protocol. Neurons and synapses adjusted their activity to minimize their prediction error, which was used as the network cost function. This cerebellar network was then implemented in hardware by replicating digital neuronal elements via a low-power microcontroller.Main results. Persistent changes of synaptic strength-that mirrored neurophysiological observations-emerged via local (neurocentric) prediction error minimization, leading to the expression of associative learning. The same paradigm was effectively emulated in low-power hardware showing remarkably efficient performance compared to conventional neuromorphic architectures.Significance. These findings show that: (a) an ensemble of free energy minimizing neurons-organized in a biological plausible architecture-can recapitulate functional self-organization observed in nature, such as associative plasticity, and (b) a neuromorphic network of inference units can learn unsupervised tasks without embedding predefined learning rules in the circuit, thus providing a potential avenue to a novel form of brain-inspired artificial intelligence

Gas chromatography-mass spectrometry analysis of irradiated fluoxetine aqueous samples

The last decade witnessed the drastic increase in the use of antidepressant drugs, being fluoxetine the most prescribed worldwide. Conventional wastewater treatment is inefficient in removing fluoxetine and its accumulation in water bodies and water living organism is inevitable. Among several methods for contaminant removal from wastewater, electron beam irradiation is an efficient and green technology. This work presents the characterization of aqueous fluoxetine samples before and after irradiation. Gas chromatography coupled to mass spectrometry was used to identify the original compound and its irradiation products. Results indicate a drastic reduction in fluoxetine presence after the irradiation process. Radiolysis pathways were proposed based on mass fragments identification

Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development

Conditional Tek Promoter-Driven Deletion of Arginyltransferase in the Germ Line Causes Defects in Gametogenesis and Early Embryonic Lethality in Mice

Posttranslational protein arginylation mediated by Ate1 is essential for cardiovascular development, actin cytoskeleton functioning, and cell migration. Ate1 plays a role in the regulation of cytoskeleton and is essential for cardiovascular development and angiogenesis—capillary remodeling driven by in-tissue migration of endothelial cells. To address the role of Ate1 in cytoskeleton-dependent processes and endothelial cell function during development, we produced a conditional mouse knockout with Ate1 deletion driven by Tek endothelial receptor tyrosine kinase promoter expressed in the endothelium and in the germ line. Contrary to expectations, Tek-Ate1 mice were viable and had no visible angiogenesis-related phenotypes; however, these mice showed reproductive defects, with high rates of embryonic lethality in the second generation, at stages much earlier than the complete Ate1 knockout strain. While some of the early lethality originated from the subpopulation of embryos with homozygous Tek-Cre transgene—a problem that has not previously been reported for this commercial mouse strain—a distinct subpopulation of embryos had lethality at early post-implantation stages that could be explained only by a previously unknown defect in gametogenesis originating from Tek-driven Ate1 deletion in premeiotic germs cells. These results demonstrate a novel role of Ate1 in germ cell development

A multidisciplinary approach to estimating wolf population size for long-term conservation

The wolf (Canis lupus) is among the most controversial of wildlife species. Abundance estimates are required to inform public debate and policy decisions, but obtaining them at biologically relevant scales is challenging. We developed a system for comprehensive population estimation across the Italian alpine region (100,000 km2), involving 1513 trained operators representing 160 institutions. This extensive network allowed for coordinated genetic sample collection and landscape-level spatial capture–recapture analyses that transcended administrative boundaries to produce the first estimates of key parameters for wolf population status assessment. Wolf abundance was estimated at 952 individuals (95% credible interval 816–1120) and 135 reproductive units (i.e., packs) (95% credible interval 112–165). We also estimated that mature individuals accounted for 33–45% of the entire population. The monitoring effort was spatially estimated thereby overcoming an important limitation of citizen science data. This is an important approach for promoting wolf–human coexistence based on wolf abundance monitoring and an endorsement of large-scale harmonized conservation practices

Phosphorylated Nucleolin Interacts with Translationally Controlled Tumor Protein during Mitosis and with Oct4 during Interphase in ES Cells

BACKGROUND: Reprogramming of somatic cells for derivation of either embryonic stem (ES) cells, by somatic cell nuclear transfer (SCNT), or ES-like cells, by induced pluripotent stem (iPS) cell procedure, provides potential routes toward non-immunogenic cell replacement therapies. Nucleolar proteins serve as markers for activation of embryonic genes, whose expression is crucial for successful reprogramming. Although Nucleolin (Ncl) is one of the most abundant nucleolar proteins, its interaction partners in ES cells have remained unidentified. METHODOLOGY: Here we explored novel Ncl-interacting proteins using in situ proximity ligation assay (PLA), colocalization and immunoprecipitation (IP) in ES cells. PRINCIPAL FINDINGS: We found that phosphorylated Ncl (Ncl-P) interacted with translationally controlled tumor protein (Tpt1) in murine ES cells. The Ncl-P/Tpt1 complex peaked during mitosis and was reduced upon retinoic acid induced differentiation, signifying a role in cell proliferation. In addition, we showed that Ncl-P interacted with the transcription factor Oct4 during interphase in human as well as murine ES cells, indicating of a role in transcription. The Ncl-P/Oct4 complex peaked during early stages of spontaneous human ES cell differentiation and may thus be involved in the initial differentiation event(s) of mammalian development. CONCLUSIONS: Here we described two novel protein-protein interactions in ES cells, which give us further insight into the complex network of interacting proteins in pluripotent cells

Mouse Cofactor of BRCA1 (Cobra1) Is Required for Early Embryogenesis

Negative elongation factor (NELF) is a four-subunit protein complex conserved from Drosophila to humans. In vitro biochemical and tissue culture-based studies have demonstrated an important role of NELF in controlling RNA polymerase II (Pol II) pausing in transcription. However, the physiological significance of NELF function is not clear due to the lack of any genetic systems for studying NELF.Here we show that disruption of the mouse B subunit of NELF (NELF-B), also known as cofactor of BRCA1 (Cobra1), causes inner cell mass (ICM) deficiency and embryonic lethality at the time of implantation. Consistent with the phenotype of the Cobra1 knockout (KO) embryos, knockdown of Cobra1 in mouse embryonic stem cells (ESCs) reduces the efficiency of colony formation and increases spontaneous differentiation. Cobra1-depleted ESCs maintain normal levels of Oct4, Nanog, and Sox2, master regulators of pluripotency in ESCs. However, knockdown of Cobra1 leads to precocious expression of developmental regulators including lymphoid enhancer-binding factor 1 (Lef1). Chromatin immunoprecipitation (ChIP) indicates that Cobra1 binds to the Lef1 promoter and modulates the abundance of promoter-bound RNA polymerase.Cobra1 is essential for early embryogenesis. Our findings also indicate that Cobra1 helps maintain the undifferentiated state of mESCs by preventing unscheduled expression of developmental genes

Restricted Expression of Epstein-Barr Virus Latent Genes in Murine B Cells Derived from Embryonic Stem Cells

Background: Several human malignancies are associated with Epstein-Barr virus (EBV) and more than 95 % of the adult human population carries this virus lifelong. EBV efficiently infects human B cells and persists in this cellular compartment latently. EBV-infected B cells become activated and growth transformed, express a characteristic set of viral latent genes, and acquire the status of proliferating lymphoblastoid cell lines in vitro. Because EBV infects only primate cells, it has not been possible to establish a model of infection in immunocompetent rodents. Such a model would be most desirable in order to study EBV’s pathogenesis and latency in a suitable and amenable host. Methodology/Principal Findings: We stably introduced recombinant EBV genomes into mouse embryonic stem cells and induced their differentiation to B cells in vitro to develop the desired model. In vitro differentiated murine B cells maintained the EBV genomes but expression of viral genes was restricted to the latent membrane proteins (LMPs). In contrast to human B cells, EBV’s nuclear antigens (EBNAs) were not expressed detectably and growth transformed murine B cells did not arise in vitro. Aberrant splicing and premature termination of EBNA mRNAs most likely prevented the expression of EBNA genes required for B-cell transformation. Conclusions/Significance: Our findings indicate that fundamental differences in gene regulation between mouse and ma