Contact-mediated nucleation of subcooled droplets in melt emulsions: A microfluidic approach

The production of melt emulsions is mainly influenced by the crystallization step, as every single droplet needs to crystallize to obtain a stable product with a long shelf life. However, the crystallization of dispersed droplets requires high subcooling, resulting in a time, energy and cost intensive production processes. Contact-mediated nucleation (CMN) may be used to intensify the nucleation process, enabling crystallization at higher temperatures. It describes the successful inoculation of a subcooled liquid droplet by a crystalline particle. Surfactants are added to emulsions/suspensions for their stabilization against coalescence or aggregation. They cover the interface, lower the specific interfacial energy and form micelles in the continuous phase. It may be assumed that micelles and high concentrations of surfactant monomers in the continuous phase delay or even hinder CMN as the two reaction partners cannot get in touch. Experiments were carried out in a microfluidic chip, allowing for the controlled contact between a single subcooled liquid droplet and a single crystallized droplet. We were able to demonstrate the impact of the surfactant concentration on the CMN. Following an increase in the aqueous micelle concentrations, the time needed to inoculate the liquid droplet increased or CMN was prevented entirely

Inactivation and inducible oncogenic mutation of p53 in gene targeted pigs.

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53(R167H) mutant allele, orthologous to oncogenic human mutant TP53(R175H) and mouse Trp53(R172H), that can be activated by Cre recombination. MSCs carrying the latent TP53(R167H) mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53(R167H) allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53(R167H) mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal

Dual Fluorescent Reporter Pig for Cre Recombination: Transgene Placement at the ROSA26 Locus

We are extending the Cre/loxP site-specific recombination system to pigs, focussing on conditional and tissue-specific expression of oncogenic mutations to model human cancers. Identifying the location, pattern and extent of Cre recombination in vivo is an important aspect of this technology. Here we report pigs with a dual fluorochrome cassette under the control of the strong CAG promoter that switches expression after Cre-recombination, from membrane-targeted tandem dimer Tomato to membrane-targeted green fluorescent protein. The reporter cassette was placed at the porcine ROSA26 locus by conventional gene targeting using primary mesenchymal stem cells, and animals generated by nuclear transfer. Gene targeting efficiency was high, and analysis of foetal organs and primary cells indicated that the reporter is highly expressed and functional. Cre reporter pigs will provide a multipurpose indicator of Cre recombinase activity, an important new tool for the rapidly expanding field of porcine genetic modification

Porcine model elucidates function of p53 isoform in carcinogenesis and reveals novel circTP53 RNA

Recent years have seen an increasing number of genetically engineered pig models of human diseases including cancer. We previously generated pigs with a modified TP53 allele that carries a Cre-removable transcriptional stop signal in intron 1, and an oncogenic mutation TP53R167H (orthologous to human TP53R175H) in exon 5. Pigs with the unrecombined mutant allele (flTP53R167H) develop mainly osteosarcoma but also nephroblastomas and lymphomas. This observation suggested that TP53 gene dysfunction is itself the key initiator of bone tumorigenesis, but raises the question which aspects of the TP53 regulation lead to the development of such a narrow tumour spectrum. Molecular analysis of p53 revealed the presence of two internal TP53 promoters (Pint and P2) equivalent to those found in human. Consequently, both pig and human express TP53 isoforms. Data presented here strongly suggest that P2-driven expression of the mutant R167H-Δ152p53 isoform (equivalent to the human R175H-Δ160p53 isoform) and its circular counterpart circTP53 determine the tumour spectrum and play a critical role in the malignant transformation in flTP53R167H pigs. The detection of Δ152p53 isoform mRNA in serum is indicative of tumorigenesis. Furthermore, we showed a tissue-specific p53-dependent deregulation of the p63 and p73 isoforms in these tumours. This study highlights important species-specific differences in the transcriptional regulation of TP53. Considering the similarities of TP53 regulation between pig and human, these observations provide useful pointers for further investigation into isoform function including the novel circTP53 in both the pig model and human patients.ISSN:0950-9232ISSN:1476-559

Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set

Background: Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency. Results: 109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring. Conclusion: Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models

Genome-wide Maps of Nuclear Lamina Interactions in Single Human Cells

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization.National Institutes of Health (U.S.) (Grant R01 GM114190)National Human Genome Research Institute (U.S.) (Grant R01 HG003143

Nonmicrosurgical reconstruction of the auricle after traumatic amputation due to human bite

BACKGROUND: Traumatic auricular amputation due to human bite is not a common event. Nonetheless, it constitutes a difficult challenge for the reconstructive surgeon. Microsurgery can be performed in some cases, but most microsurgical techniques are complex and their use can only be advocated in specialized centers. Replantation of a severed ear without microsurgery can be a safe alternative as long as a proper technique is selected. METHODS: We present two cases, one of a partial and one of a total traumatic auricular amputation, both caused by human bites, that were successfully managed in our Department. The technique of ear reattachment as a composite graft, with partial burial of the amputated part in the retroauricular region, as first described by Baudet, was followed in both cases. RESULTS AND DISCUSSION: The prementioned technique is described in detail, along with the postoperative management and outcome of the patients. In addition, a brief review of the international literature regarding ear replantation is performed. CONCLUSION: The Baudet technique has been used successfully in two cases of traumatic ear amputation due to human bites. It is a simple technique, without the need for microsurgery, and produces excellent aesthetic results, while preserving all neighboring tissues in case of failure with subsequent need for another operation