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Background: Chylothorax is a very rare form of pleural effusion in children, especially after the neonatal period, and predominantly occurs secondary to cardiothoracic surgery. It can lead to significant respiratory distress, immunodeficiency, and malnutrition. Effective treatment strategies are therefore required to reduce morbidity., Case Presentation: A previously healthy two-year old boy was admitted with history of heavy coughing followed by progressive dyspnea. The chest X-ray showed an extensive opacification of the right lung. Ultrasound studies revealed a large pleural effusion of the right hemithorax. Pleural fluid analysis delivered the unusual diagnosis of chylothorax, most likely induced by preceded excessive coughing. After an unsuccessful treatment attempt with a fat-free diet and continuous pleural drainage for two weeks, therapy with octreotide was initiated. This led to complete and permanent resolution of his pleural effusion within 15 days, without any side effects., Conclusions: Severe cough may be a rare cause of chylothorax in young children. Octreotide seems to be an effective and safe treatment of spontaneous or traumatic chylothorax in children. There is, however, a lack of comprehensive studies for chylothorax in children and many issues concerning diagnostic strategies and treatment algorithms remain., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m 7 G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.

A serine/threonine-specific protein kinase B (PKB), also known as Akt, is a key factor in the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway that regulates cell survival, metabolism and proliferation. Akt phosphorylates many downstream specific substrates, which subsequently control the nuclear envelope breakdown (NEBD), centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. In vertebrates, Akt is also an important player during oogenesis and preimplantation development. In the signaling pathways regulating mRNA translation, Akt is involved in the control of mammalian target of rapamycin complex 1 (mTORC1) and thereby regulates the activity of a translational repressor, the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). In this review, we summarize the functions of Akt in mitosis, meiosis and early embryonic development. Additionally, the role of Akt in the regulation of mRNA translation is addressed with respect to the significance of this process during early development.

Data stewardship is a term that is understood in heterogenous ways. In recent organisational developments and efforts to build infrastructures and hire professional staff for research data management in various scientific fields in Europe, data stewardship is understood as mainly aiming at optimising data management in line with the FAIR principles (findability, accessibility, interoperability, reusability) forpurposes of reuse in the interests of the scientific community and the public. In addition, especially in the health and biomedical sciences some understandings of data stewardship mainly focus on the responsibility to respect the informational rights of data subjects. Following on from these different understandings and from recent developments to include ever more stakeholders in data stewardship, we propose a comprehensive understanding of data stewardship. According to this comprehensive understanding, data stewardship includes responsibilities towards all pertinent stakeholders and to equally consider and respect their legitimate rights and interests in order to build and maintain an efficient, trusted and fair data ecosystem. We also point out some of the practical challenges implied in such a comprehensive understanding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Receiving complete and undamaged genetic information is vital for the survival of daughter cells after chromosome segregation. The most critical steps in this process are accurate DNA replication during S phase and a faithful chromosome segregation during anaphase. Any errors in DNA replication or chromosome segregation have dire consequences, since cells arising after division might have either changed or incomplete genetic information. Accurate chromosome segregation during anaphase requires a protein complex called cohesin, which holds together sister chromatids. This complex unifies sister chromatids from their synthesis during S phase, until separation in anaphase. Upon entry into mitosis, the spindle apparatus is assembled, which eventually engages kinetochores of all chromosomes. Additionally, when kinetochores of sister chromatids assume amphitelic attachment to the spindle microtubules, cells are finally ready for the separation of sister chromatids. This is achieved by the enzymatic cleavage of cohesin subunits Scc1 or Rec8 by an enzyme called Separase. After cohesin cleavage, sister chromatids remain attached to the spindle apparatus and their poleward movement on the spindle is initiated. The removal of cohesion between sister chromatids is an irreversible step and therefore it must be synchronized with assembly of the spindle apparatus, since precocious separation of sister chromatids might lead into aneuploidy and tumorigenesis. In this review, we focus on recent discoveries concerning the regulation of Separase activity during the cell cycle.

Background: Data Sharing is widely recognised as crucial for accelerating scientific research and improving its quality. However, data sharing is still not a common practice. Funding agencies tend to facilitate the sharing of research data by both providing incentives and requiring data sharing as part of their policies and conditions for awarding grants. The goal of our article is to answer the following question: What challenges do international funding agencies see when it comes to their own efforts to foster and implement data sharing through their policies?, Methods: We conducted a series of sixteen guideline-based expert interviews with representatives of leading international funding agencies. As contact persons for open science at their respective agencies, they offered their perspectives and experiences concerning their organisations' data sharing policies. We performed a qualitative content analysis of the interviews and categorised the challenges perceived by funding agencies., Results: We identify and illustrate six challenges surrounding data sharing policies as perceived by leading funding agencies: The design of clear policies, monitoring of compliance, sanctions for non-compliance, incentives, support, and limitations for funders' own capabilities. However, our interviews also show how funders approach potential solutions to overcome these challenges, for example by coordinating with other agencies or adjusting grant evaluation metrics to incentivise data sharing., Discussion and Conclusion: Our interviews point to existing flaws in funders' data sharing policies, such as a lack of clarity, a lack of monitoring of funded researchers' data sharing behaviour, and a lack of incentives. A number of agencies could suggest potential solutions but often struggle with the overall complexity of data sharing and the implementation of these measures. Funders cannot solve each challenge by themselves, but they can play an active role and lead joint efforts towards a culture of data sharing., Competing Interests: The authors have declared that no competing interests exist.

Hemodiafiltration (HDF) achieves a more efficient reduction of the uremic toxic load compared to standard high-flux hemodialysis (HF-HD) by virtue of the combined diffusive and convective clearances of a broad spectrum of uremic retention solutes. Clinical trials and registry data suggest that HDF improves patient outcomes. Despite the acknowledged need to improve survival rates of dialysis patients and the survival benefit HDF offers, there is little to no utilization in some countries (such as the US) in prescribing HDF to their patients. In this analysis, we present the healthcare value-based case for HDF (relative to HF-HD) from the patient, provider, and payor perspectives. The improved survival and reduced morbidity observed in studies conducted outside the US, as well as the reduced hospitalization, are attractive for each stakeholder. We also consider the potential barriers to greater utilization of HDF therapies, including unfounded concerns regarding additional costs of HDF, e.g., for the preparation and microbial testing of quality of substitution fluids. Ultrapure fluids are easily attainable and prepared from dialysis fluids using established "online" (OL) technologies. OL-HDF has matured to a level whereby little additional effort is required to safely implement it as all modern machine systems are today equipped with the OL-HDF functionality. Countries already convinced of the advantages of HF-HD are thus well positioned to make the transition to OL-HDF to achieve further clinical and associated economic benefits. Healthcare systems struggling to cope with the increasing demand for HD therapies would therefore, like patients, be beneficiaries in the long term with increased usage of OL-HDF for end stage kidney disease patients., (© 2022 Wiley Periodicals LLC.)

Introduction: Here we report our results of a multi-center, open cohort study ("COVID-Kids-Bavaria") investigating the distribution of acute SARS-CoV-2 infections among children and staff in 99 daycare facilities and 48 elementary schools in Bavaria, Germany., Materials and Methods: Overall, 2,568 children (1,337 school children, 1,231 preschool children) and 1,288 adults (466 teachers, 822 daycare staff) consented to participate in the study and were randomly tested in three consecutive phases (September/October 2020, November/December 2020, March 2021). In total, 7,062 throat swabs were analyzed for SARS-CoV-2 by commercial RT-PCR kits., Results: In phase I, only one daycare worker tested positive. In phase II, SARS-CoV-2 was detected in three daycare workers, two preschool children, and seven school children. In phase III, no sample tested positive. This corresponds to a positive test rate of 0.05% in phase I, 0.4% in phase II and 0% in phase III. Correlation of a positive PCR test result with the local-7-day incidence values showed a strong association of a 7-day-incidence of more than 100/100,000 as compared to <100/100,000 (OR = 10.3 [1.5-438], p < 0.005). After phase III, antibody testing was offered to 713 study participants in elementary schools. A seroprevalence rate of 7.7% (students) and 4.5% (teachers) was determined., Discussion: During the initial waves of the SARS-CoV-2 pandemic, the risk of a positive SARS-CoV-2 result correlated positively with the local 7-day incidence. Hence, the occurrence of SARS-CoV-2 infections were reflected in schools and daycare facilities. An increased risk of SARS-CoV-2 transmission in the setting of daycare and elementary schooling was unlikely., Competing Interests: All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kern, Kuhlmann, Matl, Ege, Maison, Eckert, von Both, Behrends, Anger, Frühwald, Gerstlauer, Woelfle, Neubert, Melter, Liese, Goettler, Sing, Liebl, Hübner, Klein and the COVID Kids Bavaria Consortium.)

Informed decision-making is paramount to the improvement of dialysis therapies and patient outcomes. A cornerstone of delivery of optimal dialysis therapy is to delineate which substances (uraemic retention solutes or 'uraemic toxins') contribute to the condition of uraemia in terms of deleterious biochemical effects they may exert. Thereafter, decisions can be made as to which of the accumulated compounds need to be targeted for removal and by which strategies. For haemodialysis (HD), the non-selectivity of membranes is sometimes considered a limitation. Yet, considering that dozens of substances with potential toxicity need to be eliminated, and targeting removal of individual toxins explicitly is not recommended, current dialysis membranes enable elimination of several molecules of a broad size range within a single therapy session. However, because HD solute removal is based on size-exclusion principles, i.e. the size of the substances to be removed relative to the mean size of the 'pores' of the membrane, only a limited degree of selectivity of removal is possible. Removal of unwanted substances during HD needs to be weighed against the unavoidable loss of substances that are recognized to be necessary for bodily functions and physiology. In striving to improve the efficiency of HD by increasing the porosity of membranes, there is a greater potential for the loss of substances that are of benefit. Based on this elementary trade-off and availability of recent guidance on the relative toxicity of substances retained in uraemia, we propose a new evidence-linked uraemic toxin elimination (ELUTE) approach whereby only those clusters of substances for which there is a sufficient body of evidence linking them to deleterious biological effects need to be targeted for removal. Our approach involves correlating the physical properties of retention solutes (deemed to express toxicity) with key determinants of membranes and separation processes. Our analysis revealed that in attempting to remove the relatively small number of 'larger' substances graded as having only moderate toxicity, uncontrolled (and efficient) removal of several useful compounds would take place simultaneously and may compromise the well-being or outcomes of patients. The bulk of the uraemic toxin load comprises uraemic toxins below <30 000 Da and are adequately removed by standard membranes. Further, removal of a few difficult-to-remove-by-dialysis (protein-bound) compounds that express toxicity cannot be achieved by manipulation of pore size alone. The trade-off between the benefits of effective removal of the bulk of the uraemic toxin load and risks (increased loss of useful substances) associated with targeting the removal of a few larger substances in 'high-efficiency' HD treatment strategies needs to be recognized and better understood. The removability during HD of substances, be they toxic, inert or beneficial, needs be revised to establish the pros and cons of current dialytic elimination strategies.  ., (© The Author(s) 2021. Published by Oxford University Press on behalf of ERA.)

The onset of an early development is, in mammals, characterized by profound changes of multiple aspects of cellular morphology and behavior. These are including, but not limited to, fertilization and the merging of parental genomes with a subsequent transition from the meiotic into the mitotic cycle, followed by global changes of chromatin epigenetic modifications, a gradual decrease in cell size and the initiation of gene expression from the newly formed embryonic genome. Some of these important, and sometimes also dramatic, changes are executed within the period during which the gene transcription is globally silenced or not progressed, and the regulation of most cellular activities, including those mentioned above, relies on controlled translation. It is known that the blastomeres within an early embryo are prone to chromosome segregation errors, which might, when affecting a significant proportion of a cell within the embryo, compromise its further development. In this review, we discuss how the absence of transcription affects the transition from the oocyte to the embryo and what impact global transcriptional silencing might have on the basic cell cycle and chromosome segregation controlling mechanisms.

Chromosome segregation during female meiosis is frequently incorrect with severe consequences including termination of further development or severe disorders, such as Down syndrome. Accurate chromosome segregation requires tight control of a protease called separase, which facilitates the separation of sister chromatids by cohesin cleavage. There are several control mechanisms in place, including the binding of specific protein inhibitor securin, phosphorylation by cyclin-dependent kinase 1 (CDK1), and complex with SGO2 and MAD2 proteins. All these mechanisms restrict the activation of separase for the time when all chromosomes are properly attached to the spindle. In our study, we focused on securin and compared the expression profile of endogenous protein with exogenous securin, which is widely used to study chromosome segregation. We also compared the dynamics of securin proteolysis in meiosis I and meiosis II. Our study revealed that the expression of both endogenous and exogenous securin in oocytes is compartmentalized and that this protein accumulates on the spindle during meiosis I. We believe that this might have a direct impact on the regulation of separase activity in the vicinity of the chromosomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pauerova, Radonova, Horakova, Knott and Anger.)

Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7flox/flox Myh6-Mer-Cre-MerTg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7+/+ Myh6-Mer-Cre-MerTg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKTSer473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL71152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart., Competing Interests: The authors have declared that no competing interests exist.

Aneuploidy is the most frequent single cause leading into the termination of early development in human and animal reproduction. Although the mouse is frequently used as a model organism for studying the aneuploidy, we have only incomplete information about the frequency of numerical chromosomal aberrations throughout development, usually limited to a particular stage or assumed from the occurrence of micronuclei. In our study, we systematically scored aneuploidy in in vivo mouse embryos, from zygotes up to 16-cell stage, using kinetochore counting assay. We show here that the frequency of aneuploidy per blastomere remains relatively similar from zygotes until 8-cell embryos and then increases in 16-cell embryos. Due to the accumulation of blastomeres, aneuploidy per embryo increases gradually during this developmental period. Our data also revealed that the aneuploidy from zygotes and 2-cell embryos does not propagate further into later developmental stages, suggesting that embryos suffering from aneuploidy are eliminated at this stage. Experiments with reconstituted live embryos revealed, that hyperploid blastomeres survive early development, although they exhibit slower cell cycle progression and suffer frequently from DNA fragmentation and cell cycle arrest.

In several species, including Xenopus, mouse and human, two members of cyclin A family were identified. Cyclin A2, which is ubiquitously expressed in dividing cells and plays role in DNA replication, entry into mitosis and spindle assembly, and cyclin A1, whose function is less clear and which is expressed in spermatocytes, leukemia cells and in postmitotic multiciliated cells. Deletion of the gene showed that cyclin A1 is essential for male meiosis, but nonessential for female meiosis. Our results revealed, that the cyclin A1 is not only dispensable in oocytes, we show here that its expression is in fact undesirable in these cells. Our data demonstrate that the APC/C and proteasome in oocytes are unable to target sufficiently cyclin A1 before anaphase, which leads into anaphase arrest and direct inhibition of separase. The cyclin A1-induced cell cycle arrest is oocyte-specific and the presence of cyclin A1 in early embryos has no effect on cell cycle progression or chromosome division. Cyclin A1 is therefore not only an important cell cycle regulator with biased expression in germline, being essential for male and damaging for female meiosis, its persistent expression during anaphase in oocytes shows fundamental differences between APC/C function in oocytes and in early embryos.

Embryonic stem (ES) cells are pluripotent cells widely used in cell therapy and tissue engineering. However, the broader clinical applications of ES cells are limited by their genomic instability and karyotypic abnormalities. Thus, understanding the mechanisms underlying ES cell karyotypic abnormalities is critical to optimizing their clinical use. In this study, we focused on proliferating human and mouse ES cells undergoing multipolar divisions. Specifically, we analyzed the frequency and outcomes of such divisions using a combination of time-lapse microscopy and cell tracking. This revealed that cells resulting from multipolar divisions were not only viable, but they also frequently underwent subsequent cell divisions. Our novel data also showed that in human and mouse ES cells, multipolar spindles allowed more robust escape from chromosome segregation control mechanisms than bipolar spindles. Considering the frequency of multipolar divisions in proliferating ES cells, it is conceivable that cell division errors underlie ES cell karyotypic instability., Competing Interests: The authors report no conflicts of interest in this work.

We evaluated event-based prospective memory (EBPM) in adolescents with Autism, varying the load of the to-be-performed intentions. We included measures of inhibition, working memory and binding. Results showed that increasing the retrospective memory load reduced performance in controls. In Autism, adolescents were impaired in the low load condition with normal performance for the ongoing task, with the reverse pattern in the high load condition. EBPM may be impacted in Autism due to difficulty to process ongoing and EBPM tasks simultaneously possibly because of restricted inhibitory control.

In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.

Cognitive studies generally report impaired autobiographical memory in individuals with autism spectrum disorder (ASD), but mostly using verbal paradigms. In the present study, we therefore investigated the properties of both past and future autobiographical productions using visual cues in 16 boys with ASD and 16 typically developing (TD) participants aged between 10 and 18 years. We focused on sensory properties, emotional properties, and recollection, probing past and future productions for both near and distant time periods. Results showed that the ASD group performed more poorly than controls on free recall for recent periods, but performed like them when provided with visual cues. In addition, the ASD group reported fewer sensory details than controls and exhibited difficulties in the experience of recollection for the most remote events. These data suggest a combination of consolidation and binding deficits. Finally, our findings reveal the relevance of using visual cues to probe autobiographical memory, with possible perspectives for memory rehabilitation.

Early embryonic development is characterized by a plethora of very complex and simultaneously operating processes, which are constantly changing cellular morphology and behaviour. After fertilization, blastomeres of the newly created embryo undergo global epigenetic changes and simultaneously initiate transcription from the zygotic genome and differentiation forming separate cell lineages. Some of these mechanisms were extensively studied during the last several decades and valuable insight was gained into how these processes are regulated at the molecular level. We have, however, a still very limited understanding of how multiple events are coordinated during rapid development of an early mammalian embryo. In this review, we discuss some aspects of early embryonic development in mammals, namely the fidelity of chromosome segregation and occurrence of aneuploidy, as well as the clinical applications of cell cycle monitoring in human embryos.

Optimal culture conditions are essential for successful IVM of mammalian oocytes and for their further development into an embryo. In the present study we used live cell imaging microscopy to assess the effects of suboptimal culture temperature on various aspects of IVM, including duration of meiosis I, dynamics of polar body extrusion, chromosome congression, anaphase-promoting complex/cyclosome (APC/C) activation and aneuploidy. The data showed that even a small deviation from the optimal incubation temperature causes marked changes in the duration and synchronicity of meiosis, APC/C activity and the frequency of chromosome congression and segregation errors. In vitro manipulation and maturation of germ cells is widely used in both human and animal artificial reproduction techniques. Mammalian oocytes are naturally prone to chromosomal segregation errors, which are responsible for severe mental and developmental disorders. The data presented herein demonstrate that exposure of mouse oocytes to suboptimal temperature during manipulation and maturation could further increase the frequency of chromosome segregation defects in these cells.

Chromosome segregation in mammalian oocytes is prone to errors causing aneuploidy with consequences such as precocious termination of development or severe developmental disorders. Aneuploidy also represents a serious problem in procedures utilizing mammalian gametes and early embryos in vitro. In our study, we focused on congression defects during meiosis I and observed whole nondisjoined bivalents in meiosis II as a direct consequence, together with a substantially delayed first polar body extrusion. We also show that the congression defects are accompanied by less stable attachments of the kinetochores. Our results describe a process by which congression defects directly contribute to aneuploidy., (© 2017 S. Karger AG, Basel.)

Proper assembly of the spindle apparatus is crucially important for faithful chromosome segregation during anaphase. Thanks to the effort over the last decades, we have very detailed information about many events leading to spindle assembly and chromosome segregation, however we still do not understand certain aspects, including, for example, spindle length control. When tight regulation of spindle size is lost, chromosome segregation errors emerge. Currently, there are several hypotheses trying to explain the molecular mechanism of spindle length control. The number of kinetochores, activity of molecular rulers, intracellular gradients, cell size, limiting spindle components, and the balance of the spindle forces seem to contribute to spindle size regulation, however some of these mechanisms are likely specific to a particular cell type. In search for a general regulatory mechanism, in our study we focused on the role of cell size and nuclear to cytoplasmic ratio in this process. To this end, we used relatively large cells isolated from 2-cell mouse embryos. Our results showed that the spindle size upper limit is not reached in these cells and suggest that accurate control of spindle length requires balanced ratio between nuclear and cytoplasmic volumes.

A hallmark of oocyte development in mammals is the dependence on the translation and utilization of stored RNA and proteins rather than the de novo transcription of genes in order to sustain meiotic progression and early embryo development. In the absence of transcription, the completion of meiosis and early embryo development in mammals relies significantly on maternally synthesized RNAs. Post-transcriptional control of gene expression at the translational level has emerged as an important cellular function in normal development. Therefore, the regulation of gene expression in oocytes is controlled almost exclusively at the level of mRNA and protein stabilization and protein synthesis. This current review is focused on the recently emerged findings on RNA distribution related to the temporal and spatial translational control of the meiotic progression of the mammalian oocyte.

The fully grown mammalian oocyte is transcriptionally quiescent and utilizes only transcripts synthesized and stored during early development. However, we find that an abundant RNA population is retained in the oocyte nucleus and contains specific mRNAs important for meiotic progression. Here we show that during the first meiotic division, shortly after nuclear envelope breakdown, translational hotspots develop in the chromosomal area and in a region that was previously surrounded the nucleus. These distinct translational hotspots are separated by endoplasmic reticulum and Lamin, and disappear following polar body extrusion. Chromosomal translational hotspots are controlled by the activity of the mTOR-eIF4F pathway. Here we reveal a mechanism that-following the resumption of meiosis-controls the temporal and spatial translation of a specific set of transcripts required for normal spindle assembly, chromosome alignment and segregation.

Mammalian female gametes frequently suffer from numerical chromosomal aberrations, the main cause of miscarriages and severe developmental defects. The underlying mechanisms responsible for the development of aneuploidy in oocytes are still not completely understood and remain a subject of extensive research. From studies focused on prevalence of aneuploidy in mouse oocytes, it has become obvious that reported rates of aneuploidy are strongly dependent on the method used for chromosome counting. In addition, it seems likely that differences between mouse strains could influence the frequency of aneuploidy as well; however, up till now, such a comparison has not been available. Therefore, in our study, we measured the levels of aneuploidy which has resulted from missegregation in meiosis I, in oocytes of three commonly used mouse strains-CD-1, C3H/HeJ, and C57BL/6. Our results revealed that, although the overall chromosomal numerical aberration rates were similar in all three strains, a different number of oocytes in each strain contained prematurely segregated sister chromatids (PSSC). This indicates that a predisposition for this type of chromosome segregation error in oocyte meiosis I is dependent on genetic background.

Purpose: Vibrational spectroscopy enables the label-free characterization of cells and tissue by probing the biochemical composition. Here, we evaluated these techniques to identify glioblastoma stem cells., Materials and Methods: The biochemical fingerprints of glioblastoma cells were established in human cell lines with high and low content of CD133 (cluster of differentiation 133)-positive cells using attenuated total reflection Fourier-transform infrared (ATR FT-IR) on vital cells and FT-IR mapping, which delivers spatially resolved spectroscopic datasets. After data preprocessing, unsupervised cluster analysis was applied. CD133 was addressed with flow cytometry and immunohistochemistry and used as a stemness marker., Results: In all preparations, the algorithm was able to correctly classify the spectra, differentiating CD133-rich and -poor populations. The main spectral differences were found in the region of 1000 cm(- 1) to 1150 cm(- 1) that can be assigned to vibrations of chemical bonds of DNA, RNA, carbohydrates and phospholipids. Interestingly, this spectral region is a key feature to discern glioblastoma from normal brain parenchyma, as FT-IR spectroscopic mapping of experimental brain tumors demonstrated., Conclusions: We were able to show biochemical differences between glioblastoma cell populations with high and low content of cancer stem cells that are presumably related to changes in the RNA/DNA content.

Background: Most dialysis patients require phosphate binders to control hyperphosphatemia. Ferric citrate has been tested in phase 2 trials as a phosphate binder. This trial was designed as a dose-response and efficacy trial., Study Design: Prospective, phase 3, multicenter, open-label, randomized clinical trial., Setting & Participants: 151 participants with hyperphosphatemia on maintenance hemodialysis therapy., Intervention: Fixed dose of ferric citrate taken orally as a phosphate binder for up to 28 days (1, 6, or 8 g/d in 51, 52, and 48 participants, respectively)., Outcomes: Primary outcome is dose-response of ferric citrate on serum phosphorus level; secondary outcomes are safety and tolerability., Measurements: Serum chemistry tests including phosphorus, safety data., Results: 151 participants received at least one dose of ferric citrate. Mean baseline phosphorus levels were 7.3 ± 1.7 (SD) mg/dL in the 1-g/d group, 7.6 ± 1.7 mg/dL in the 6-g/d group, and 7.5 ± 1.6 mg/dL in the 8-g/d group. Phosphorus levels decreased in a dose-dependent manner (mean change at end of treatment, -0.1 ± 1.3 mg/dL in the 1-g/d group, -1.9 ± 1.7 mg/dL in the 6-g/d group, and -2.1 ± 2.0 mg/dL in the 8-g/d group). The mean difference in reduction in phosphorus levels between the 6- and 1-g/d groups was 1.3 mg/dL (95% CI, 0.69 to 1.9; P < 0.001), between the 8- and 1-g/d groups was 1.5 mg/dL (95% CI, 0.86 to 2.1; P < 0.001), and between the 8- and 6-g/d groups was 0.21 mg/dL (95% CI, -0.39 to 0.81; P = 0.5). The most common adverse event was stool discoloration., Limitations: Sample size and duration confirm efficacy, but limit our ability to confirm safety., Conclusions: Ferric citrate is efficacious as a phosphate binder in a dose-dependent manner. A phase 3 trial is ongoing to confirm safety and efficacy., (Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

According to the Dobzhansky-Muller model, hybrid sterility is a consequence of the independent evolution of related taxa resulting in incompatible genomic interactions of their hybrids. The model implies that the incompatibilities evolve randomly, unless a particular gene or nongenic sequence diverges much faster than the rest of the genome. Here we propose that asynapsis of heterospecific chromosomes in meiotic prophase provides a recurrently evolving trigger for the meiotic arrest of interspecific F1 hybrids. We observed extensive asynapsis of chromosomes and disturbance of the sex body in >95% of pachynemas of Mus m. musculus × Mus m. domesticus sterile F1 males. Asynapsis was not preceded by a failure of double-strand break induction, and the rate of meiotic crossing over was not affected in synapsed chromosomes. DNA double-strand break repair was delayed or failed in unsynapsed autosomes, and misexpression of chromosome X and chromosome Y genes was detected in single pachynemas and by genome-wide expression profiling. Oocytes of F1 hybrid females showed the same kind of synaptic problems but with the incidence reduced to half. Most of the oocytes with pachytene asynapsis were eliminated before birth. We propose the heterospecific pairing of homologous chromosomes as a preexisting condition of asynapsis in interspecific hybrids. The asynapsis may represent a universal mechanistic basis of F1 hybrid sterility manifested by pachytene arrest. It is tempting to speculate that a fast-evolving subset of the noncoding genomic sequence important for chromosome pairing and synapsis may be the culprit.

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.

It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.

Background: Primary cutaneous melanoma still constitutes the main cause of skin cancer death in developed countries, and its incidence in recent years has been increasing in a steady, worrisome manner., Objectives: This study evaluated the clinical, epidemiological and demographic aspects of this disease, and correlated them with patient prognosis., Methods: Using epidemiologic and clinical data, we analyzed 84 patients with mild to severe primary cutaneous melanoma treated between 1990 and 2007. Slides containing surgical specimens were analyzed, and new slides were made from archived paraffin sections when necessary., Results: The melanoma incidence was higher in areas of sun exposure, with lesions commonly observed in the trunk for males, and lower limbs for females. In addition to Breslow's thickness and ulceration (p = 0.043 and p < 0.001, respectively), the mitotic rate per mm(2) also correlated with worse patient outcome (p = 0.0007). The sum of ulceration (0 when absent or 1 when present), the Breslow index (1 when <1 mm, 2 when >1 mm and <4 mm, 3 when >4 mm) and the mitotic index (0 when absent or 1 when > or =1 per mm(2)) allowed the establishment of a prognostic score: if the sum was equal to or over three, nearly all (91.7%) patients had systemic disease. The 5-year survival was approximately seventy percent., Conclusion: Because American Join Committee of Cancer Staging will update the classification of malignant tumors (TNM) staging in the near future, and introduce mitosis as a prognostic factor, our results show the importance of such a feature. Additional studies are necessary to confirm the importance of a prognostic score as proposed herein.

Background: The trauma resulting from liposuction damages the subcutaneous cellular tissue and subsequently the lymphatic structures. In turn, this damage can reduce lymphatic drainage, which may or may not appear clinically as some degree of edema., Objective: It is possible to evaluate the function of the lymphatic system using lymphoscintigraphy. We conducted a study to evaluate the lymphatic system using this technique both before and after liposuction of the lower limbs., Methods: The study included seven women ranging from 18 to 42 years of age, all of whom were diagnosed with lipodystrophy solely in the lateral and medial regions of the upper third of the thighs. This study was conducted in three stages: preoperative lymphoscintigraphy, liposuction, and postoperative lymphoscintigraphy three months postoperatively. The Wilcoxon signed-rank test was used for the statistical analysis of the results., Results: A comparison of values for the average time of the radiopharmaceutical acquisition curve and clearance in the areas of lymphoscintigraphic interest both before and three months after liposuction did not reveal significant results., Conclusions: A comparison of preoperative and postoperative lymphoscintigraphic findings showed no significant damage to the lymphatic system associated with liposuction of the lower limbs.

Accurate chromosome segregation during mitosis and meiosis depends on shugoshin proteins that prevent precocious dissociation of cohesin from centromeres. Shugoshins associate with PP2A, which is thought to dephosphorylate cohesin and thereby prevent cleavage by separase during meiosis I. A crystal structure of a complex between a fragment of human Sgo1 and an AB'C PP2A holoenzyme reveals that Sgo1 forms a homodimeric parallel coiled coil that docks simultaneously onto PP2A's C and B' subunits. Sgo1 homodimerization is a prerequisite for PP2A binding. While hSgo1 interacts only with the AB'C holoenzymes, its relative, Sgo2, interacts with all PP2A forms and may thus lead to dephosphorylation of distinct substrates. Mutant shugoshin proteins defective in the binding of PP2A cannot protect centromeric cohesin from separase during meiosis I or support the spindle assembly checkpoint in yeast. Finally, we provide evidence that PP2A's recruitment to chromosomes may be sufficient to protect cohesin from separase in mammalian oocytes.

Proteolytic activity of separase is required for chiasma resolution during meiosis I in mouse oocytes. Rec8, the meiosis-specific alpha-kleisin subunit of cohesin, is a key target of separase in yeast. Is the equivalent protein also a target in mammals? We show here that separase cleaves mouse Rec8 at three positions in vitro but only when the latter is hyper-phosphorylated. Expression of a Rec8 variant (Rec8-N) that cannot be cleaved in vitro at these sites causes sterility in male mice. Their seminiferous tubules lack a normal complement of 2 C secondary spermatocytes and 1 C spermatids and contain instead a high proportion of cells with enlarged nuclei. Chromosome spreads reveal that Rec8-N expression has no effect in primary spermatocytes but produces secondary spermatocytes and spermatids with a 4 C DNA content, suggesting that the first and possibly also the second meiotic division is abolished. Expression of Rec8-N in oocytes causes chromosome segregation to be asynchronous and delays its completion by 2-3 hours during anaphase I, probably due to inefficient proteolysis of Rec8-N by separase. Despite this effect, chromosome segregation must be quite accurate as Rec8-N does not greatly reduce female fertility. Our data is consistent with the notion that Rec8 cleavage is important and probably crucial for the resolution of chiasmata in males and females.

Background: Missegregation of chromosomes during meiosis in human females causes aneuploidy, including trisomy 21, and is thought also to be the major cause of age-related infertility. Most errors are thought to occur at the first meiotic division. The high frequency of errors raises questions as to whether the surveillance mechanism known as the spindle assembly checkpoint (SAC) that controls the anaphase-promoting complex or cyclosome (APC/C) operates effectively in oocytes. Experimental approaches hitherto used to inactivate the SAC in oocytes suffer from a number of drawbacks., Results: Bub1 protein was depleted specifically in oocytes with a Zp3-Cre transgene to delete exons 7 and 8 from a floxed BUB1(F) allele. Loss of Bub1 greatly accelerates resolution of chiasmata and extrusion of polar bodies. It also causes defective biorientation of bivalents, massive chromosome missegregation at meiosis I, and precocious loss of cohesion between sister centromeres. By using a quantitative assay for APC/C-mediated securin destruction, we show that the APC/C is activated in an exponential fashion, with activity peaking 12-13 hr after GVBD, and that this process is advanced by 5 hr in oocytes lacking Bub1. Importantly, premature chiasmata resolution does not occur in Bub1-deficient oocytes also lacking either the APC/C's Apc2 subunit or separase. Finally, we show that Bub1's kinase domain is not required to delay APC/C activation., Conclusions: We conclude that far from being absent or ineffective, the SAC largely determines the timing of APC/C and hence separase activation in oocytes, delaying it for about 5 hr.

Background and Objectives: Management of hyperphosphatemia, a predictor of mortality in chronic kidney disease, is challenging. Nonadherence to dietary phosphate binders, in part, contributes to uncontrolled serum phosphorus levels. This phase IIIb trial assessed the efficacy of increased dosages (3000 to 4500 mg/d) of reformulated lanthanum carbonate (500-, 750-, and 1000-mg tablets) in nonresponders to dosages of up to 3000 mg/d., Design, Setting, Participants, & Measurements: This 8-wk study with a 4-mo open-label extension enrolled 513 patients who were undergoing maintenance hemodialysis. Patients who achieved serum phosphorus control at week 4 with

Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals.

The step-wise assembly of a functional nucleolus, which occurs over the first few cell cycles during preimplantation development, is poorly understood. In this study, we examined the function of the evolutionary conserved nucleolar protein SURF6 in preimplantation mouse embryo development. Immunocytochemical analyses revealed that the localization of SURF6 was similar but not identical to those of fibrillarin and B23/nucleophosmin 1, which are involved in rRNA processing and ribosome biogenesis in mammalian somatic cells. Surf6 mRNA, which is expressed in oocytes and maternally inherited in the zygote, reached a peak level of expression during the 8-cell stage of embryo development, at which time rDNA is highly transcribed. Knock-down of Surf6 mRNA by RNAi led to a decrease in both the mRNA and protein levels, and resulted in developmental arrest at the 8-cell/morula stage, as well as a decrease in the level of 18S rRNA. These results suggest that Surf6 is essential for mouse preimplantation development, presumably by regulating ribosome biogenesis.

In yeast, resolution of chiasmata in meiosis I requires proteolytic cleavage along chromosome arms of cohesin's Rec8 subunit by separase. Since activation of separase by the anaphase-promoting complex (APC/C) is supposedly not required for meiosis I in Xenopus oocytes, it has been suggested that animal cells might resolve chiasmata by a separase-independent mechanism related to the so-called "prophase pathway" that removes cohesin from chromosome arms during mitosis. By expressing Cre recombinase from a zona pellucida promoter, we have deleted a floxed allele of separase specifically in mouse oocytes. This prevents removal of Rec8 from chromosome arms and resolution of chiasmata. It also hinders extrusion of the first polar body (PBE) and causes female sterility. mRNA encoding wild-type but not catalytically inactive separase restores chiasma resolution. Both types of mRNA restore PBE. Proteolytic activity of separase is therefore essential for Rec8's removal from chromosome arms and for chiasma resolution but not for PBE.

Background: Intermittent dosing of calcitriol for secondary hyperparathyroidism (SHPT) has been associated with greater parathyroid hormone (PTH) reduction with fewer calcemic and phosphatemic effects than daily (QD) dosing., Methods: Secondary analyses of three randomized, double-blind, placebo-controlled multicenter studies in stage 3 and 4 chronic kidney disease (CKD) patients with SHPT were performed to compare three times per week (TIW) with QD dosing of paricalcitol. The pharmacokinetics of TIW and QD dosing of paricalcitol capsules were assessed in a separate group of healthy subjects., Results: Pharmacokinetics revealed similar steady state paricalcitol exposure between dosing regimens. In CKD patients, baseline data were similar between the TIW studies (n = 72, paricalcitol; n = 73, placebo) and QD studies (n = 35, paricalcitol; n = 40, placebo). Both dosing regimens resulted in similar efficacy (91%) for the primary end point of two consecutive > or = 30% decreases in intact PTH from baseline, but the QD regimen resulted in a greater percent reduction in intact PTH from baseline. The chances for developing increased serum calcium and phosphorus levels or Ca x P product were similar between paricalcitol and placebo groups for both treatment regimens. Furthermore, no difference in the risk for these elevations was detected between the TIW and QD regimens., Conclusions: QD dosing of paricalcitol capsules is as efficacious as TIW dosing in achieving the primary end point (2 consecutive > or = 30% reductions in PTH) in stage 3 and 4 CKD patients with SHPT. Moreover, the QD regimen had no significant effect on hypercalcemia, hyperphosphatemia or Ca x P product as compared with placebo or intermittent dosing., (Copyright 2006 S. Karger AG, Basel)

A master regulator of DNA replication, CDC6 also functions in the DNA-replication checkpoint by preventing DNA rereplication. Cyclin-dependent kinases (CDKs) regulate the amount and localization of CDC6 throughout the cell cycle; CDC6 phosphorylation after DNA replication initiation leads to its proteolysis in yeast or translocation to the cytoplasm in mammals. Overexpression of CDC6 during the late S phase prevents entry into the M phase by activating CHEK1 kinase that then inactivates CDK1/cyclin B, which is essential for the G2/M-phase transition. We analyzed the role of CDC6 during resumption of meiosis in mouse oocytes, which are arrested in the first meiotic prophase with low CDK1/cyclin B activity; this is similar to somatic cells at the G2/M-phase border. Overexpression of CDC6 in mouse oocytes does not prevent resumption of meiosis. The RNA interference-mediated knockdown of CDC6, however, reveals a new and unexpected function for CDC6; namely, it is essential for spindle formation in mouse oocytes.

A Polo-like kinase 1 (Plk1) appears involved in an autocatalytic loop between CDC25C phosphatase and M phase promoting factor (MPF) in Xenopus oocytes and leads to activation of MPF that is required for germinal vesicle breakdown (GVBD). Although similar evidence for such a role of Plk1 in MPF activation during maturation of mammalian oocytes is absent, changes in Plk1 enzyme activity correlate with MPF activation, Plk1 co-localizes with MPF, and microinjection of antibodies neutralizing Plk1 delays GVBD. In this study, we exploited the prolonged time required for maturation of porcine oocytes to define precisely the timing of Plk1 and MPF activation during maturation. GVBD typically occurs between 24 and 26 hr of culture in vitro and meiotic maturation is completed after 40-44-hr culture. We find that Plk1 is activated before MPF, which is consistent with its role in activating MPF in mammalian oocytes.

Both murine endogenous retrovirus-L (MuERV-L) and intracisternal A particle (IAP), two autonomous long terminal repeat (LTR) retrotransposons, are activated during genome activation in the preimplantation mouse embryo, and both sense and antisense transcripts are detected in 2-cell and 8-cell stage embryos. Because RNA interference (RNAi) functions in the preimplantation mouse embryo, we analyzed the relationship between RNAi and MuERV-L and IAP expression by inhibiting RNAi and measuring relative changes of the levels of these transcripts. We inhibited the initial step in the RNAi pathway by injecting 1-cell embryos with mDicer siRNA or long mDicer dsRNA and analyzed MuERV-L and IAP expression at the 8-cell stage. This approach resulted in the targeted destruction of mDicer mRNA, but not Hdac1 mRNA, inhibited the RNAi pathway, and resulted in a 50% increase in IAP and MuERV-L transcript abundance. These results suggest that RNAi constrains expression of repetitive parasitic sequences in preimplantation embryos, and thereby contributes to preserving genomic integrity at a stage of development when the organism consists of only a few cells.

Enzymes of the Polo-like kinase (Plk) family are active in the pathways controlling mitosis in several species. We have cloned cDNA fragments of the porcine homologues of Plk1, Plk2, and Plk3 employing fetal fibroblasts as source. All three partial cDNAs showed high sequence homology with their mouse and human counterparts and contained the Polo box, a domain characteristic for all Polo kinases. The expression levels of Plk1 mRNA at various points of the cell cycle in synchronized porcine fetal fibroblasts were analyzed by both RT-PCR and the ribonuclease protection assay. Plk1 mRNA was barely detectable in G0 and G1, increased during S phase and peaked after the G2/M transition. A monoclonal antibody was generated against an in vitro expressed porcine Plk1-protein fragment and used to detect changes in Plk1 expression at the protein level. Plk1 protein was first detected by immunoblotting at the beginning of S phase and was highest after the G2/M transition. In summary, the Plk1 expression pattern in the pig is similar to that reported for other species. The absence of Plk1 mRNA and protein appears to be a good marker for G0/G1 and thus for the selection of donor cells for nuclear transfer based somatic cloning., (Copyright 2003 Wiley-Liss, Inc.)

The presence of a form of cyclin A2 with an N-terminal truncation has recently been reported in various murine cell lines and tissues. The truncated cyclin A2 binds to and activates the cyclin-dependent kinase 2 (CDK2). However, CDK2 bound by the truncated cyclin A2 is located in the cytoplasm in contrast to CDK2 bound to full-length cyclin A2, which is in the nucleus. Here, we show that proliferating mouse embryonic stem cells (ES cells) contain very little truncated cyclin A2 but as the cells are induced to differentiate the amount of truncated cyclin A2 increases. The expression pattern of truncated cyclin A2 was the same in p27(Kip1) -/- differentiating ES cells as in the differentiating wild-type cells. We conclude that p27(Kip1) is not necessary for the proteolytic cleavage that gives rise to the truncated form of cyclin A2 in differentiating ES cells and that this post-translational modification is not a function of the cell density but is correlated with differentiation.

Studies in mutant organisms deficient in RNA interference (RNAi) and related post-transcriptional gene silencing implicated a role for a single class of RNA-dependent RNA polymerases (RdRp). Nevertheless, sequence homologs to these RdRps have not been found in coelomate organisms such as Drosophila or mammals. This lack of homologous sequences does not exclude that an RdRp functions in RNAi in these organisms because an RdRp could be acquired by horizontal transfer from an RNA virus. In fact, such a sequence is found in mice (Aquarius) and we observe that it is expressed in mouse oocytes and early embryos, which exhibit RNAi. We report here that cordycepin, an inhibitor of RNA synthesis, does not prevent Mos double-strand RNA (dsRNA) to target endogenous Mos mRNA in mouse oocytes and that targeting a chimeric Mos-EGFP mRNA with dsRNA to EGFP does not reduce the endogenous Mos mRNA, but does target the chimeric mRNA. These results indicate that an RdRp is not involved in dsRNA-mediated mRNA degradation in mammalian oocytes, and possibly in mammals in general, and therefore that only homologous sequences to the dsRNA are targeted for degradation.

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases (cdk), is shown to inhibit germinal vesicle breakdown (GVBD) in pig oocytes. Oocytes treated with 100 microM BL I were arrested in the germinal vesicle (GV)-stage and displayed low activity of cdc2 kinase and MAP kinase. Nevertheless, chromosome condensation occurred and highly condensed bivalents were seen within an intact GV after a 24-hr culture in the presence of BL I. The inhibitory effect of BL I on MAP kinase activation during culture was likely mediated through a cdk-dependent pathway, since MAP kinase activity present in extracts derived from metaphase II eggs was not inhibited by BL I. The block of GVBD could be released by treating oocytes with okadaic acid (OA), an inhibitor of type 1 and 2A phosphatases; 82% of the oocytes treated with the combination of OA/BL I underwent GVBD, and MAP kinase became activated, while cdc2 kinase remained inhibited. These results suggest that both chromosome condensation and GVBD could occur without activation of cdc2 kinase, whereas an increase in MAP kinase activity may be a requisite for GVBD in pig oocytes in conditions when cdc2 kinase activation is blocked by BL I., (Copyright 2002 Wiley-Liss, Inc.)