Analysis of cell cycle stage, replicated DNA, and chromatin-associated proteins using high-throughput flow cytometry.

Flow cytometry is a versatile tool used for cell sorting, DNA content imaging, and determining various cellular characteristics. With the possibility of high-throughput analyses, it combines convenient labelling techniques to serve rapid, quantitative, and qualitative workflows. The ease of sample preparation and the broad range of applications render flow cytometry a preferred approach for many scientific questions. Yet, we lack practical adaptations to fully harness the quantitative and high-throughput capabilities of most cytometers for many organisms. Here, we present simple and advanced protocols for the analysis of total DNA content, de novo DNA synthesis, and protein association to chromatin in budding yeast and human cells. Upon optimization of experimental conditions and choice of fluorescent dyes, up to four parameters can be measured simultaneously and quantitatively for each cell of a population in a multi-well plate format. Reducing sample numbers, plastic waste, costs per well, and hands-on time without compromising signal quality or single-cell accuracy are the main advantages of the presented protocols. In proof-of-principle experiments, we show that DNA content increase in S-phase correlates with de novo DNA synthesis and can be predicted by the presence of the replicative helicase MCM2-7 on genomic DNA.

MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing.

Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome.

Multi-center bonds as resonance hybrids: A real space perspective.

The concept of distinct bonds within molecules has proven to be successful in rationalizing chemical reactivity. However, bonds are not a well-defined physical concept, but rather vague entities, described by different and often contradicting models. With probability density analysis, which can-in principle-be applied to any wave function, bonds are recovered as spin-coupled positions within most likely electron arrangements in coordinate space. While the wave functions of many systems are dominated by a single electron arrangement that is built from two-center two-electron bonds, some systems require several different arrangements to be well described. In this work, a range of these multi-center bonded molecules are classified and investigated with probability density analysis. The results are compared with valence bond theory calculations and data from collision-induced dissociation experiments.

Episodic future thinking improves children's prospective memory performance in a complex task setting with real life task demands.

Research on children's prospective memory (PM) shows an increase of performance across childhood and provides first evidence that encoding strategies such as episodic future thinking (EFT; i.e., engaging in a vivid prospection of oneself performing future tasks) may improve performance. The present study aimed at testing whether the beneficial effects of EFT extend from typical lab-based tasks to more complex tasks with real life demands. Further, it was tested whether children's ability to project themselves into different perspectives (i.e., self-projection) moderates the effects of EFT encoding on PM. Overall, 56 children (mean age: M = 10.73 years) were included in this study who were randomly assigned to either an EFT or control condition. Children participated in a 'sightseeing tour' (ongoing activity) inside the lab with various socially relevant and neutral PM tasks embedded. Results showed significantly higher PM performance in the EFT compared to the control group. There was no difference between neutral and social PM tasks and no interaction between type of PM tasks with encoding condition. Further, self-projection did not moderate the effects of EFT encoding on PM. Results suggest that EFT is an effective strategy to improve children's everyday PM. These beneficial effects seem to occur independent from children's general ability to change perspectives and for different types of PM tasks.

From structure to mechanism-understanding initiation of DNA replication.

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability.

Falling for the dark side of transcription: Nab2 fosters RNA polymerase III transcription.

RNA polymerase III (RNAPIII) synthesizes diverse, small, non-coding RNAs with many important roles in the cellular metabolism. One of the open questions of RNAPIII transcription is whether and how additional factors are involved. Recently, Nab2 was identified as the first messenger ribonucleoprotein particle (mRNP) biogenesis factor with a function in RNAPIII transcription.

The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription.

RNA polymerase III (RNAPIII) synthesizes most small RNAs, the most prominent being tRNAs. Although the basic mechanism of RNAPIII transcription is well understood, recent evidence suggests that additional proteins play a role in RNAPIII transcription. Here, we discovered by a genome-wide approach that Nab2, a poly(A)-binding protein important for correct poly(A) tail length and nuclear mRNA export, is present at all RNAPIII transcribed genes. The occupancy of Nab2 at RNAPIII transcribed genes is dependent on transcription. Using a novel temperature-sensitive allele of NAB2, nab2-34, we show that Nab2 is required for the occupancy of RNAPIII and TFIIIB at target genes. Furthermore, Nab2 interacts with RNAPIII, TFIIIB, and RNAPIII transcripts. Importantly, impairment of Nab2 function causes an RNAPIII transcription defect in vivo and in vitro. Taken together, we establish Nab2, an important mRNA biogenesis factor, as a novel player required for RNAPIII transcription by stabilizing TFIIIB and RNAPIII at promoters.

LINT, a novel dL(3)mbt-containing complex, represses malignant brain tumour signature genes.

Mutations in the l(3)mbt tumour suppressor result in overproliferation of Drosophila larval brains. Recently, the derepression of different gene classes in l(3)mbt mutants was shown to be causal for transformation. However, the molecular mechanisms of dL(3)mbt-mediated gene repression are not understood. Here, we identify LINT, the major dL(3)mbt complex of Drosophila. LINT has three core subunits-dL(3)mbt, dCoREST, and dLint-1-and is expressed in cell lines, embryos, and larval brain. Using genome-wide ChIP-Seq analysis, we show that dLint-1 binds close to the TSS of tumour-relevant target genes. Depletion of the LINT core subunits results in derepression of these genes. By contrast, histone deacetylase, histone methylase, and histone demethylase activities are not required to maintain repression. Our results support a direct role of LINT in the repression of brain tumour-relevant target genes by restricting promoter access.

Human is what is born of a human: personhood, rationality, and an European convention.

In the course of its preparation, the 1997 convention on human rights and biomedicine adopted by the Council of Europe instigated a widespread debate. This article examines one of the core issues: the notion of the human being as depicted in the convention. It is argued that according to the convention, this being may exist in three different legal categories, namely 'human life', 'embryo', and 'personhood', each furnished with an inherent set of somewhat different rights, yet none of them clearly defined, thus leaving it to domestic law to regulate at what point a human being belongs to which category. While this approach is understandable from a political point of view, it creates a vicious circle, since law thereby has to define its own foundation and, in the case of the convention, to protect a being that it cannot define. It appears that this form of life is seen rather as a given entity, taking precedence over the interests of society and science, and its dignity and identity forming criteria for the subsequent systems of culture, simply because this life is human and nothing else. Thus, the convention approaches a natural law position.

[Standardization of the Kent Infant Development Scale: implications for primary care pediatricians]. / Normalización de la Escala de Desarrollo Infantil de Kent. Implicaciones para la práctica pediátrica ambulatoria.

OBJECTIVE: The purpose of this study was the standardization of an infant assessment protocol based on behavioral observations of Spanish parents. The Kent Infant Development (KIDS) scale was translated into Spanish and named "Escala de Desarrollo Infantil de Kent" (EDIK). PATIENTS AND METHODS: The EDIK normative data were collected from the parents of 662 healthy infants (ages 1 to 15 months) in pediatric clinics. Infants born more than 2 weeks premature or who had serious physical or neurological illness were not included. RESULTS: EDIK raw scores of Spanish infants were converted to developmental ages by comparing them with the number of behaviors for each age group in the normative sample. We obtained the mean score and standard deviation for the full scale and different domains (cognitive, motor, social, language, and self-help). CONCLUSIONS: This study shows that EDIK is sensitive to differences in ages and a good instrument that allows one to make a classification between normal infants or those at risk. It should prove useful in developmental pediatric practice.

Success rates following intracytoplasmic sperm injection are improved by using ZIFT vs IVF for embryo transfer.

PURPOSE: The purpose of this study was to analyze whether the mode of embryo transfer (ZIFT vs IVF) affected the outcome in intracytoplasmic sperm injection (ICSI) cycles. METHODS AND RESULTS: Eighty-two ICSI cycles (42 ZIFT and 40 IVF) were analyzed. Several variables, including patient age and weight, numbers of mature eggs collected, injected, and fertilized, fertilization rate, number of fertilized eggs obtained per cycle, numbers of zygotes/embryos transferred, clinical pregnancy rate, and implantation rate, were compared. Mean patient age and weight were identical. The mean number of mature eggs collected and injected and fertilization rate were significantly higher in the ZIFT group, however, the mean numbers of zygotes/embryos transferred were identical. The clinical pregnancy and implantation rates in ZIFT cycles (52.3 and 23.2% respectively) were significantly higher than in IVF cycles (17.5 and 9.7%). CONCLUSIONS: These data suggest that ZIFT is the more appropriate method for transfer of ICSI-derived embryos.

[A new method for evaluating psychomotor development based on information from parents. The Spanish version of the Kent Infant Development Scale]. / Nuevo método de evaluación del desarrollo psicomotor basado en la información de los padres. Versión española de la Kent Infant Development Scale.

OBJECTIVE: The purpose of this dissertation research was to design, standardize and validate the Spanish version of the Kent Infant Development Scale (KIDS). PATIENTS AND METHODS: This questionnaire is based on information obtained from the parents. It was translated into Spanish and named "Escala de Desarrollo Infantil de Kent" (EDIK). The EDIK normative data were collected from the parents of 662 healthy infants (ages 1 to 15 months) in pediatric clinics in Catalonia (Spain). RESULTS: Test-retest reliability (r = 0.99; p < 0.001), interjudge reliability (r = 0.98; p < 0.001) and internal consistency (Cronbach alpha = 0.9947) were determined. An "r' of 0.96 was obtained when EDIK scores were compared to their estimated developmental ages obtained from the Denver Developmental Scale. The correlation of the infants' chronological age and their EDIK was 0.96 (p < 0.001). CONCLUSIONS: The high reliability and validity correlation coefficients demonstrate the sound psychometric properties of the EDIK. It appears to be a useful and acceptable instrument in measuring the developmental status of infants by using the reports of their parents.

Association of oviduct-specific glycoproteins with human and baboon (Papio anubis) ovarian oocytes and enhancement of human sperm binding to human hemizonae following in vitro incubation.

The objectives of this study were 1) to determine whether or not human and baboon oviduct-specific glycoproteins (human OGP, baboon OGP) would associate with ovarian oocytes during in vitro incubation in a manner similar to that detected in vivo for oviductal oocytes and 2) to determine whether the association of OGP with ovarian oocytes influenced sperm binding. In vitro association of OGP with ovarian oocytes was assessed by indirect immunofluorescence assay using a polyclonal antibody prepared against human or baboon OGP. Human and baboon ovarian oocytes incubated in culture media containing OGP showed association of OGP with the zona pellucida (ZP) as detected by bright fluorescence. A similar pattern of fluorescence was observed in baboon oviductal oocytes (positive control). No fluorescence of the ZP was detected from ovarian oocytes incubated with culture medium alone. The pattern of fluorescence for ovarian oocytes incubated with OGP and serum albumin, the major oviductal fluid protein, was similar to that for oocytes incubated with OGP alone. A modified hemizona assay was used to assess whether association of human OGP with human ovarian oocytes influenced sperm binding. The number of sperm bound to hemizonae in the presence of human OGP was significantly greater (p < 0.01) than the number bound to hemizonae in the control culture medium. Addition of antibodies specific for human OGP to the incubation medium 1 h prior to addition of gametes blocked the enhancement of sperm binding seen in the presence of human OGP alone. Finally, human hemizona assays conducted in the presence of baboon OGP resulted in a significant decrease (p < 0.05) in the number of sperm bound per zona compared with that in culture medium alone despite high homology between human and baboon OGP. These results 1) suggest that human OGP associates with ovulated oocytes in vivo; 2) support the hypothesis that association of OGP with the ZP may play a role in fertilization, possibly through enhancing the binding of sperm to the ZP within the oviduct; and 3) suggest that a homologous system (i.e., gametes and oviductal glycoprotein from the same species) is necessary for study of the function of oviductal glycoproteins.

In vitro incubation of golden (Syrian) hamster ovarian oocytes and human sperm with a human oviduct specific glycoprotein.

The objective of this study was to determine if human oviduct specific glycoprotein (huOGP) would associate with hamster ovarian oocytes and human sperm during in vitro incubation. The huOGP used in these studies was partially purified from human hydrosalpinx fluid. Hamster ovarian oocytes and human sperm samples were incubated in culture medium with and without huOGP. Association of huOGP was assessed by indirect immunofluorescence assay using a polyclonal antibody prepared against huOGP. Intense fluorescence of the zona pellucida, and bright but uneven fluorescence of the perivitelline space, were observed in hamster ovarian oocytes following incubation in the presence of huOGP. A similar but more uniform pattern of fluorescence was observed when hamster oviductal oocytes (positive controls) were incubated in culture medium alone. Fluorescence was absent when oocytes were assayed with preimmune serum. The association of huOGP with the zona pellucida and perivitelline space appeared to be specific since thyroglobulin, a large molecular weight glycoprotein, and human serum albumin, the major protein in oviduct fluid, did not associate with the hamster oocytes nor inhibit huOGP association when included in the culture medium. Fluorescence was absent when human sperm incubated with huOGP were assayed with antiserum to huOGP. However, human sperm fluoresced when incubated with a uterine glycoprotein, CUPED, which had previously been shown to bind to cat sperm during in vitro incubation. Sperm also fluoresced brightly when human sperm antibody was used as a positive control. Solubilization of sperm membrane proteins postincubation and analysis of these proteins by 1-D SDS-PAGE followed by immunoblotting also failed to show an association of huOGP with human sperm. Electron microscopy of sperm both pre- and postsolubilization confirmed that the sperm membranes were removed by this process. In conclusion, the association of huOGP with hamster oocytes in vitro suggests that huOGP may associate with human oocytes in vivo, whereas that may not be true for human sperm in vivo. The association of huOGP with oocytes may serve to facilitate the process of fertilization and early embryonic development within the oviduct.

Autosomal dominant congenital cataract. Interocular phenotypic variability.

PURPOSE: While intrafamilial morphologic heterogeneity of autosomal dominant congenital cataracts has been well established, interocular variation in individual patients of described pedigrees is small. The authors describe a seven-generation family with 48 of 138 individuals known to be affected with autosomal dominant congenital cataracts of the pulverulent type. Affected patients exhibit a seemingly random expression of either unilateral or bilateral lens opacities. METHODS: Ophthalmic and medical histories were obtained, complete ophthalmologic examinations were performed, blood samples were drawn, and transformed lymphoblastoid lines were established on 53 patients. Eighty-five members of the family were unable to be examined. RESULTS: Twenty-eight of the 53 individuals examined had congenital cataracts. Of these patients, 19 eyes (8 right eyes and 11 left eyes) were unilateral and 9 were bilateral. The clinically unaffected eye in patients with unilateral cataracts showed no evidence of lenticular opacity under detailed slit-lamp examination. Severity of the cataracts included a subtle unilateral zonular cataract with 20/20 visual acuity, bilateral inner fetal nuclear pulverulent opacities with 20/16 visual acuity in both eyes, and dense unilateral and bilateral nuclear cataracts requiring early surgical removal. Incorporating the historic data on patients who were not examined, we found 48 affected members (28 unilateral, 17 bilateral, 3 obligate carriers who were not examined). CONCLUSIONS: Hereditary cataracts typically are symmetric in affected individuals. The authors describe a large pedigree with the apparently random expression of an autosomal dominant gene as either unilateral or bilateral cataract. To their knowledge, this is the first such family described in the literature. Currently, work is under way to determine if the causative gene is linked to previously defined cataract loci on chromosomes 1, 2, or 16.