Induction of Plasminogen Activator in Cultured Cells by Macrocyclic Plant Diterpene Esters and Other Agents Related to Tumor Promotion

In vitro systems that are responsive to tumor-promoting agents may facilitate the identification of such agents and the analysis of their mode of action. We have previously reported that the potent tumor promoter phorbol-12-myristate-13-acetate induces the synthesis of the enzyme plasminogen activator in cultured chick embryo fibroblasts. We have, therefore, tested various compounds for their ability to induce plasminogen activator in chicken embryo fibroblasts. Among these, phorbol esters and other macrocyclic diterpene esters isolated from species of the families Euphorbiaceae and Thymelaeaceae were potent inducers of plasminogen activator. These compounds maximally induced enzyme to the same levels, although they differed in their relative molar potencies. Structural requirements for in vitro activity paralleled the requirements for activity in vivo. These results indicate that induction of plasminogen activator is a useful marker for the biologically active macrocyclic diterpene esters. On the other hand, tumor-promoting agents such as anthralin, cantharidin, Tween 60, and tobacco leaf extract failed to induce plasminogen activator

Measuring shared variants in cohorts of discordant siblings with applications to autism

We develop a method of analysis [affected to discordant sibling pairs (A2DS)] that tests if shared variants contribute to a disorder. Using a standard measure of genetic relation, test individuals are compared with a cohort of discordant sibling pairs (CDS) to derive a comparative similarity score. We ask if a test individual is more similar to an unrelated affected than to the unrelated unaffected sibling from the CDS and then, sum over such individuals and pairs. Statistical significance is judged by randomly permuting the affected status in the CDS. In the analysis of published genotype data from the Simons Simplex Collection (SSC) and the Autism Genetic Resource Exchange (AGRE) cohorts of children with autism spectrum disorder (ASD), we find strong statistical significance that the affected are more similar to the affected than to the unaffected of the CDS (P value approximately 0.00001). Fathers in multiplex families have marginally greater similarity (P value = 0.02) to unrelated affected individuals. These results do not depend on ethnic matching or gender

Damaging de novo mutations diminish motor skills in children on the autism spectrum

In individuals with autism spectrum disorder (ASD), de novo mutations have previously been shown to be significantly correlated with lower IQ but not with the core characteristics of ASD: deficits in social communication and interaction and restricted interests and repetitive patterns of behavior. We extend these findings by demonstrating in the Simons Simplex Collection that damaging de novo mutations in ASD individuals are also significantly and convincingly correlated with measures of impaired motor skills. This correlation is not explained by a correlation between IQ and motor skills. We find that IQ and motor skills are distinctly associated with damaging mutations and, in particular, that motor skills are a more sensitive indicator of mutational severity than is IQ, as judged by mutational type and target gene. We use this finding to propose a combined classification of phenotypic severity: mild (little impairment of either), moderate (impairment mainly to motor skills), and severe (impairment of both IQ and motor skills)

Low load for disruptive mutations in autism genes and their biased transmission

We previously computed that genes with de novo (DN) likely gene-disruptive (LGD) mutations in children with autism spectrum disorders (ASD) have high vulnerability: disruptive mutations in many of these genes, the vulnerable autism genes, will have a high likelihood of resulting in ASD. Because individuals with ASD have lower fecundity, such mutations in autism genes would be under strong negative selection pressure. An immediate prediction is that these genes will have a lower LGD load than typical genes in the human gene pool. We confirm this hypothesis in an explicit test by measuring the load of disruptive mutations in whole-exome sequence databases from two cohorts. We use information about mutational load to show that lower and higher intelligence quotients (IQ) affected individuals can be distinguished by the mutational load in their respective gene targets, as well as to help prioritize gene targets by their likelihood of being autism genes. Moreover, we demonstrate that transmission of rare disruptions in genes with a lower LGD load occurs more often to affected offspring; we show transmission originates most often from the mother, and transmission of such variants is seen more often in offspring with lower IQ. A surprising proportion of transmission of these rare events comes from genes expressed in the embryonic brain that show sharply reduced expression shortly after birth

SMASH, a fragmentation and sequencing method for genomic copy number analysis

Copy number variants (CNVs) underlie a significant amount of genetic diversity and disease. CNVs can be detected by a number of means, including chromosomal microarray analysis (CMA) and whole-genome sequencing (WGS), but these approaches suffer from either limited resolution (CMA) or are highly expensive for routine screening (both CMA and WGS). As an alternative, we have developed a next-generation sequencing-based method for CNV analysis termed SMASH, for short multiply aggregated sequence homologies. SMASH utilizes random fragmentation of input genomic DNA to create chimeric sequence reads, from which multiple mappable tags can be parsed using maximal almost-unique matches (MAMs). The SMASH tags are then binned and segmented, generating a profile of genomic copy number at the desired resolution. Because fewer reads are necessary relative to WGS to give accurate CNV data, SMASH libraries can be highly multiplexed, allowing large numbers of individuals to be analyzed at low cost. Increased genomic resolution can be achieved by sequencing to higher depth

Regenerative endodontics: a true paradigm shift or a bandwagon about to be derailed?

Aims: Regenerative endodontic techniques (RETs) have been hailed as a paradigm shift for the management of traumatised non-vital immature permanent anterior teeth. In this article the aim was to critically appraise the literature with regards to the outcome of regenerative endodontics on root development. Methods: Critical review of the literature where regenerative endodontic techniques have been used in the management of immature non-vital teeth with continuation of root development as the main outcome reported. Results: Most studies published were in the form of case reports and series with very few randomised controlled trials with a high risk of bias. Continuation of root development following the use of RET has been shown to be unpredictable at best with lower success in those teeth losing vitality as a result of dental trauma. Conclusions: Despite the high success of regenerative endodontics in terms of periodontal healing including resolution of clinical and radiographic signs and symptoms of infection, continuation of root development remains an unpredictable outcome. The use of a blood clot as a scaffold in regenerative endodontics should be reviewed carefully as that might offer an environment for repair rather than regeneration. In addition, preservation of structures, such as Hertwig’s epithelial root sheath, may have an important bearing on the success of this approach and should be further investigated

The Splicing Factor Proline-Glutamine Rich (SFPQ/PSF) Is Involved in Influenza Virus Transcription

The influenza A virus RNA polymerase is a heterotrimeric complex responsible for viral genome transcription and replication in the nucleus of infected cells. We recently carried out a proteomic analysis of purified polymerase expressed in human cells and identified a number of polymerase-associated cellular proteins. Here we characterise the role of one such host factors, SFPQ/PSF, during virus infection. Down-regulation of SFPQ/PSF by silencing with two independent siRNAs reduced the virus yield by 2–5 log in low-multiplicity infections, while the replication of unrelated viruses as VSV or Adenovirus was almost unaffected. As the SFPQ/PSF protein is frequently associated to NonO/p54, we tested the potential implication of the latter in influenza virus replication. However, down-regulation of NonO/p54 by silencing with two independent siRNAs did not affect virus yields. Down-regulation of SFPQ/PSF by siRNA silencing led to a reduction and delay of influenza virus gene expression. Immunofluorescence analyses showed a good correlation between SFPQ/PSF and NP levels in infected cells. Analysis of virus RNA accumulation in silenced cells showed that production of mRNA, cRNA and vRNA is reduced by more than 5-fold but splicing is not affected. Likewise, the accumulation of viral mRNA in cicloheximide-treated cells was reduced by 3-fold. In contrast, down-regulation of SFPQ/PSF in a recombinant virus replicon system indicated that, while the accumulation of viral mRNA is reduced by 5-fold, vRNA levels are slightly increased. In vitro transcription of recombinant RNPs generated in SFPQ/PSF-silenced cells indicated a 4–5-fold reduction in polyadenylation but no alteration in cap snatching. These results indicate that SFPQ/PSF is a host factor essential for influenza virus transcription that increases the efficiency of viral mRNA polyadenylation and open the possibility to develop new antivirals targeting the accumulation of primary transcripts, a very early step during infection

Mapping and characterization of structural variation in 17,795 human genomes

A key goal of whole-genome sequencing for studies of human genetics is to interrogate all forms of variation, including single-nucleotide variants, small insertion or deletion (indel) variants and structural variants. However, tools and resources for the study of structural variants have lagged behind those for smaller variants. Here we used a scalable pipeline1 to map and characterize structural variants in 17,795 deeply sequenced human genomes. We publicly release site-frequency data to create the largest, to our knowledge, whole-genome-sequencing-based structural variant resource so far. On average, individuals carry 2.9 rare structural variants that alter coding regions; these variants affect the dosage or structure of 4.2 genes and account for 4.0–11.2% of rare high-impact coding alleles. Using a computational model, we estimate that structural variants account for 17.2% of rare alleles genome-wide, with predicted deleterious effects that are equivalent to loss-of-function coding alleles; approximately 90% of such structural variants are noncoding deletions (mean 19.1 per genome). We report 158,991 ultra-rare structural variants and show that 2% of individuals carry ultra-rare megabase-scale structural variants, nearly half of which are balanced or complex rearrangements. Finally, we infer the dosage sensitivity of genes and noncoding elements, and reveal trends that relate to element class and conservation. This work will help to guide the analysis and interpretation of structural variants in the era of whole-genome sequencing

Preparative Method for Obtaining Enucleated Mammalian-Cells

Mouse L cells can be enucleated in suspension by centrifugation in discontinuous Ficoll density gradients while in the presence of Cytochalasin B. Greater than 50% of the cytoplasts thus obtained attach to glass or plastic and undergo morphologic recovery within 2–4 hours of replating. Protein synthesis in cytoplasts undergoes a biphasic decay from an initial rate of approximately 50% of control nucleated cells. This method can yield up to 5 × 108 cytoplasts with consistently low levels of contamination by nucleated cells (less than 0.2%), and is well suited for obtaining quantitative amounts of cytoplasts or karyoplasts for physiologic or biochemical studies

Tumor Promotor Induces Plasminogen Activator

INFECTION of chick embryo fibroblasts with Rous sarcoma virus (RSV) induces a cell-specified plasminogen activator 1. Induction occurs with transforming viruses but not with lytic viruses or with oncornaviruses which are not themselves transforming 2. Similarly, many mammalian cell lines and embryo cultures transformed with either viruses or chemical carcinogens may be high producers of plasminogen activator in contrast to their untransformed counterparts 3–6. A correlation has been demonstrated between production of plasminogen activator and various features of the transformed phenotype, such as cell locomotion, morphology and loss of anchorage-dependent growth 4,7. Several established cell lines which are not highly tumorigenic or transformed by the usual criteria are, however, active producers of plasminogen activator; there are examples also of transformed or tumorigenic cell lines which do not produce significant levels of plasminogen activator 3,8–10