[Calcaneoplasty with radiofrequency cementing following balloon reduction]. / Kalkaneoplastie mit Radiofrequenzzementierung nach Ballonaufrichtung.

The standard surgical procedure for complex calcaneal fractures is open reduction, internal reduction and internal stable angle plate osteosynthesis via a lateral approach. More recently, options for minimally invasive and percutaneous surgical strategies have been presented [4, 7]. As a possible procedural alternative for a covered, surgical treatment of calcaneal fractures, calcaneoplasty is discussed and applied in this context [5]. In this case series of five complex calcaneal fractures presented here, a balloon catheter was used for percutaneous reduction to restore the alignment of the calcaneus.This was followed by placement of PMMA cement in radiofrequency application and osteosynthesis using percutaneous cannulated screws. This Vicenti technique allows stable reduction and retention with early partial weight bearing with an overall low complication rate [17].

Penetrating injuries in Germany - epidemiology, management and outcome an analysis based on the TraumaRegister DGU®.

BACKGROUND: The management of penetrating wounds is a rare challenge for trauma surgeons in Germany and Central Europe as a result of the low incidence of this type of trauma. In Germany, penetrating injuries are reported to occur in 4-5 % of the severely injured patients who are enrolled in the TraumaRegister DGU® (trauma registry of the German Trauma Society). They include gunshot injuries, knife stab injuries, which are far more common, and penetrating injuries of other origin, for example trauma caused by accidents. The objective of this study was to assess the epidemiology and outcome of penetrating injuries in Germany, with a particular focus on the level of care provided by the treating trauma centre to gain more understanding of this trauma mechanism and to anticipate the necessary steps in the initial treatment. MATERIALS AND METHODS: Since 2009, the TraumaRegister DGU® has been used to assess not only whether a trauma was penetrating but also whether it was caused by gunshot or stabbing. Data were taken from the standard documentation forms that participating German hospitals completed between 2009 and 2018. Excluded were patients with a maximum abbreviated injury scale (MAIS) score of 1 with a view to obtaining a realistic idea of this injury entity, which is rare in Germany. RESULTS: From 2009 to 2018, there were 1123 patients with gunshot wounds, corresponding to a prevalence rate of 0.5 %, and 4333 patients with stab wounds (1.8 %), which were frequently caused by violent crime. The high proportion of intentionally self-inflicted gunshot wounds to the head resulted in a cumulative mortality rate of 41 % for gunshot injuries. Stab wounds were associated with a lower mortality rate (6.8 %). Every fourth to fifth patient with a gunshot or stab wound presented with haemorrhagic shock, which is a problem that is seen during both the prehospital and the inhospital phase of patient management. Of the patients with penetrating injuries, 18.3 % required transfusions. This percentage was more than two times higher than that of the basic group of patients of the TraumaRegister DGU®, which consists of patients with a MAIS ≥ 3 and patients with a MAIS of 2 who died or were treated on the intensive care unit. CONCLUSIONS: In Germany, gunshot and stab wounds have a low incidence and are mostly caused by violent crime or attempted suicides. Depending on the site of injury, they have a high mortality and are often associated with major haemorrhage. As a result of the low incidence of these types of trauma, further data and analyses are required in order to provide the basis for evaluating the long-term quality of the management of patients with stab or gunshot wounds.

Impact of seed-applied pesticides on rhizobial survival and legume nodulation.

AIMS: Compatibility of seed-applied pesticides and rhizobial inoculants is an important consideration for farmers when sowing legumes. Some of the seed-applied pesticides may influence rhizobial growth and nodulation, but there is currently little available information on the potential inhibitory effects. Therefore, common seed fungicidal and insecticidal treatments were assessed to determine adverse impacts on rhizobial inoculants both in vitro, on treated seed, and in the field. METHODS AND RESULTS: Initially, the in vitro toxicity of the seed-applied fungicides Thiram 600, P-Pickel T (PPT), their active ingredients (thiram and thiabendazole) and the insecticide Gaucho to rhizobia was measured with filter discs containing varying concentrations of the pesticides. Pea and chickpea seed was then coated with the same pesticides and inoculated with rhizobia in different inoculant substrates to determine bacterial survival and nodulation. Finally, a field trial using the fungicide PPT and commercial inoculants was conducted. Some seed fungicide treatments were found to be inhibitory to rhizobia and reduce nodulation under monoxenic conditions and in the field. SIGNIFICANCE AND IMPACT OF THE STUDY: These data provide more detailed information on the compatibility of specific rhizobial inoculants with common seed-applied pesticides. This research will provide information on the compatibility of rhizobia and seed-applied pesticides, and assist farmers to select sowing practices which reduce the risk of crop nodulation failures.

Understanding pluripotency--how embryonic stem cells keep their options open.

Embryonic stem (ES) cells have the capacity to proliferate indefinitely in culture while maintaining the ability to differentiate to form any of the cells of the body. This unique combination of functions suggests that these cells could provide a potentially unlimited source of differentiated cells for the treatment of disease and aging. Understanding the molecular processes that underpin these functions in ES cells will allow us to harness their potential and develop strategies that control their differentiation. Combination of controlled differentiation with ground-breaking technologies for the reversal of somatic cells to an ES cell-like state promise the generation of patient-derived pluripotent cell lines for the treatment of disease in the future.

Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development.

Formation and differentiation of a pluripotent cell population is central to mammalian development, and the isolation, identification and manipulation of human pluripotent cells is predicted to be of therapeutic use. Within the early mammalian embryo, two distinct populations of pluripotent cells have been described: the inner cell mass (ICM), which differentiates to form a second pluripotent cell populations, the primitive ectoderm. Indirect evidence suggests the existence of temporally distinct intermediate pluripotent cell populations as primitive ectoderm is formed. We coupled an in vitro model of primitive ectoderm formation (the transition of embryonic stem cells to early primitive ectoderm-like (EPL) cells) with ddPCR-based techniques to identify three novel genes, Psc1, CRTR-1 and PRCE, that were expressed differently during pluripotent cell progression. Detailed mapping of these genes with Oct4, Rex1 and Fgf5 on pregastrulation embryos provided the first molecular evidence for the existence of successive, temporally distinct pluripotent cell populations in the embryo between the ICM and primitive ectoderm. No evidence was found for spatial heterogeneity within the Oct4(+) pool. The transition between populations correlated with morphological or developmental alterations in pluripotent cells in vivo. Genes that are temporally expressed during pluripotent cell progression may provide an opportunity for molecular discrimination of pluripotent cells at different stages of maturation in vivo and an understanding of the cellular origins and properties of pluripotent cell lines isolated from diverse sources. Furthermore, the strong correlation of gene expression demonstrated between EPL cell formation in vitro and primitive ectoderm formation in vivo validates EPL cells as a model for primitive ectoderm, thereby providing a model system for the investigation of pluripotent differentiation and an opportunity for directed differentiation of pluripotent cells to therapeutically useful cell populations.

Lineage specific differentiation of pluripotent cells in vitro: a role for extraembryonic cell types.

The controlled differentiation of pluripotent cells will be a prerequisite for many cell therapies. We have previously reported homogeneous conversion of embryonic stem (ES) cells in vitro to early primitive ectoderm-like (EPL) cells, equivalent to early primitive ectoderm, an obligatory differentiation intermediate between ES cells and somatic cell populations. Early primitive ectoderm-like cells differentiated within aggregates form mesodermal lineages at the expense of ectoderm. In this work we demonstrate that the failure of EPL cells to form ectodermal cell types does not reflect an inherent restriction in developmental potential. Early primitive ectoderm-like cells form ectodermal derivatives such as neurons in response to neural inducers such as retinoic acid, or when differentiated in the environment provided by ES cell embryoid bodies. This could be explained by signals from the extraembryonic cell type visceral endoderm which forms in differentiating ES cell but not EPL cell aggregates. Consistent with this possibility, culture of EPL cell aggregates in the presence of visceral endoderm-like signals did not prevent differentiation of the pluripotent cells, but resulted in suppression of mesoderm formation. These results suggest a role for visceral endoderm in regulation of germ layer specification from pluripotent cells, and can be integrated into a model for cell differentiation in vitro and in vivo.

Mouse ES cells: experimental exploitation of pluripotent differentiation potential.

Pluripotent ES cells can be used to generate a wide variety of cell populations in vitro in a manner resembling embryonic development. Recent advances in controlling ES cell differentiation, combined with the power of genetic and biochemical manipulation, are providing insights into cell biology and the determination of cell fate.

Structure, sequence and function of a marsupial LIF gene: conservation of IL-6 family cytokines.

Leukaemia Inhibitory Factor (LIF) is a multifunctional cytokine with an obligate role in the mouse in embryonic implantation. In this paper we demonstrate the existence of a functional LIF gene in the marsupial Sminthopsis crassicaudata, and the presence of LIF-related sequences in the monotreme Tachyglossus aculeatus (Australian echidna). Isolation of genomic and cDNA clones from S. crassicaudata, indicated that the LIF gene is highly conserved between marsupials and monotremes in terms of sequence and genomic organisation. Critical functional residues within the LIF sequence were also conserved including residues implicated in intracellular LIF activity, and in interaction with the receptor subunits LIFR and gp130. These findings suggest that the structure and biochemical function of the protein is likely to be conserved. Consistent with this, purified recombinant S. crassicaudata LIF interacted functionally with mouse receptor components and was sufficient for maintenance of mouse embryonic stem (ES) cells in the undifferentiated state. Conservation of LIF outside eutherians is intriguing given the markedly divergent reproductive strategies which include, for some marsupial species, embryonic diapause, and in monotremes, the absence of implantation. The availability of marsupial LIF probes provides an opportunity to investigate conservation of expression and function in these mammals.

avrPto enhances growth and necrosis caused by Pseudomonas syringae pv.tomato in tomato lines lacking either Pto or Prf.

AvrPto was introduced into three tomato genotypes with two biotic agents to study its role in compatible interactions. avrPto enhanced the capacity of the Pseudomonas syringae pv. tomato strain T1 to induce necrotic symptoms on tomato plants that lacked either Pto or Prf genes. The enhanced necrosis correlated with a small increase in bacterial growth. In planta expression of avrPto in isolation did not elicit necrosis in the absence of a functional Prf gene.

Reversible programming of pluripotent cell differentiation.

We have undertaken an in vitro differentiation analysis of two related, interconvertible, pluripotent cell populations, ES and early primitive ectoderm-like (EPL) cells, which are most similar in morphology, gene expression, cytokine responsiveness and differentiation potential in vivo to ICM and early primitive ectoderm, respectively. Pluripotent cells were differentiated in vitro as aggregates (embryoid bodies) and the appearance and abundance of cell lineages were assessed by morphology and gene expression. Differentiation in EPL cell embryoid bodies recapitulated normal developmental progression in vivo, but was advanced in comparison to ES cell embryoid bodies, with the rapid establishment of late primitive ectoderm specific gene expression, and subsequent loss of pluripotent cell markers. Nascent mesoderm was formed earlier and more extensively in EPL cell embryoid bodies, and resulted in the appearance of terminally differentiated mesodermal cell types prior to and at higher levels than in ES cell embryoid bodies. Nascent mesoderm in EPL cell embryoid bodies was not specified but could be programmed to alternative fates by the addition of exogenous factors. EPL cells remained competent to form primitive endoderm even though this is not the normal fate of primitive ectoderm in vivo. The establishment of primitive ectoderm-like gene expression and inability to participate in embryogenesis following blastocyst injection is therefore not directly associated with restriction in the ability to form extra-embryonic lineages. However, the EPL cell embryoid body environment did not support differentiation of primitive endoderm to visceral endoderm, indicating the lack of an inductive signal for visceral endoderm formation deduced to originate from the pluripotent cells. Similarly, the inability of EPL cells to form neurons when differentiated as embryoid bodies was attributable to perturbation of the differentiation environment and loss of inductive signals rather than a restricted differentiation potential. Reversion of EPL cells to ES cells was accompanied by restoration of ES cell-like differentiation potential. These results demonstrate the ability of pluripotent cells to adopt developmentally distinct, stable cell states with altered differentiation potentials.

Constitutively active Pto induces a Prf-dependent hypersensitive response in the absence of avrPto.

Resistance in tomato to Pseudomonas syringae pv tomato (avrPto) is conferred by the gene Pto in a gene-for-gene relationship. A hypersensitive disease resistance response (HR) is elicited when Pto and avrPto are expressed experimentally within the same plant cell. The kinase capability of Pto was required for AvrPto-dependent HR induction. Systematic mutagenesis of the activation segment of Pto kinase confirmed the homologous P+1 loop as an AvrPto-binding determinant. Specific amino acid substitutions in this region led to constitutive induction of HR upon expression in the plant cell in the absence of AvrPto. Constitutively active Pto mutants required kinase capability for activity, and were unable to interact with proteins previously shown to bind to wild-type Pto. The constitutive gain-of-function phenotype was dependent on a functional Prf gene, demonstrating activation of the cognate disease resistance pathway and precluding a role for Prf upstream of Pto.

Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors.

The primitive ectoderm of the mouse embryo arises from the inner cell mass between 4.75 and 5.25 days post coitum, around the time of implantation. Positioned at a pivotal time in development, just prior to formation of the three germ layers of the embryo proper, the primitive ectoderm responds directly to the signals generated during gastrulation. We have identified a conditioned medium, MEDII, which caused the homogeneous conversion of ES cells to a morphologically distinct cell population, termed early primitive ectoderm-like (EPL) cells. EPL cells expressed the pluripotent cell markers Oct4, SSEA1 and alkaline phosphatase. However, the formation of EPL cells was accompanied by alterations in Fgf5, Gbx2 and Rex1 expression, a loss in chimaera forming ability, changes in factor responsiveness and modified differentiation capabilities, all consistent with the identification of EPL cells as equivalent to the primitive ectoderm population of the 5.5 to 6.0 days post coitum embryo. EPL cell formation could be reversed in the presence of LIF and withdrawal of MEDII, which suggested that EPL cell formation was not a terminal differentiation event but reflected the ability of pluripotent cells to adopt distinct cell states in response to specific factors. Partial purification of MEDII revealed the presence of two separable biological activities, both of which were required for the induction and maintenance of EPL cells. We show here the first demonstration of uniform differentiation of ES cells in response to biological factors. The formation of primitive ectoderm, both in vivo and in vitro, appears to be an obligatory step in the differentiation of the inner cell mass or ES cells into cell lineages of the embryonic germ layers. EPL cells potentially represent a model for the development of lineage specific differentiation protocols and analysis of gastrulation at a molecular level. An understanding of the active components of MEDII may provide a route for the identification of factors which induce primitive ectoderm formation in vivo.

Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy.

Embryonic stem cells are pluripotent cells derived from the early mouse embryo that can be propagated stably in the undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in an embryonic and adult mouse in vivo, and can be induced to differentiate into many cell types in vitro. Exploitation of ES cell technology for the creation of mice bearing predetermined genetic alterations has received widespread attention because of the sophistication that it brings to the study of gene function in mammals. Analysis of cell differentiation in vitro has also been of value, leading to the identification of novel bioactive factors and the elucidation of cell specification mechanisms. In this paper, we summarise the features of pluripotent cell lines and their applications, foreshadowing the impact that these systems may have on human biology. While the isolation of definitive human pluripotent cell lines has not yet been achieved, potential applications for these cells in the study of human biology, particularly cell specification, can be envisaged. Of particular interest is the possibility that human embryonic stem cells with properties similar to mouse embryonic stem cells might provide a generic system for gene therapy.

Developmental complexity of early mammalian pluripotent cell populations in vivo and in vitro.

Early mammalian embryogenesis is characterised by the coordinated proliferation, differentiation, migration and apoptosis of a pluripotent cell pool that is able to give rise to extraembryonic lineages and all the cell types of the embryo proper. These cells retain pluripotent differentiation capability, defined in this paper as the ability to form all cell types of the embryo and adult, until differentiation into the three embryonic germ layers at gastrulation. Our understanding of pluripotent cell biology and molecular regulation has been hampered by the difficulties associated with experimental manipulation of these cells in vivo. However, a more detailed understanding of pluripotent cell behaviour is emerging from the application of molecular technologies to early mouse embryogenesis. The construction of mouse mutants by gene targeting, mapping of gene expression in vivo, and modelling of cell decisions in vitro are providing insight into the cellular origin, identity and action of key developmental regulators, and the nature of pluripotent cells themselves. In this review we discuss the properties of early embryonic pluripotent cells in vitro and in vivo, focusing on progression from inner cell mass (ICM) cells in the blastocyst to the onset of gastrulation.

Sequence, genomic organization, and expression of the novel homeobox gene Hesx1.

Extensive analyses of homeobox gene expression and function during murine embryogenesis have demonstrated that homeobox gene products are key components in the establishment of pattern formation and regional identity during development. In this paper we report the molecular characterization and expression of a novel murine homeobox sequence, Hesx1, isolated from pluripotent embryonic stem cells. Hesx1 is expressed as two transcripts of 1.0 and 1.2 kilobases which encode an identical 185 amino acid open reading frame. The transcripts differ in the 3'-untranslated region due to the differential utilization of a weak splice donor site located immediately downstream of the translation termination codon. The Hesx1 homeodomain shared 80% identity with the Xenopus homeoprotein XANF-1 and was less than 50% related to other homeodomain sequences. Hesx1 and XANF-1 therefore constitute the founder members of a new homeodomain class. Hesx1 expression was down-regulated during embryonic stem cell differentiation and was detected in tissue-specific RNA samples derived from the embryonic liver, and at lower levels in viscera, amnion, and yolk sac. Expression in adult mice was not detected. These sites of expression are consistent with a role for Hesx1 in the regulation of developmental decisions in the early mouse embryo and during fetal hematopoiesis.

Efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAs in a new plasmid vector.

Full-length cucumber mosaic cucumovirus (CMV) cDNAs were cloned into a new plasmid vector containing a modified plant virus promoter designed to transcribe the inserted sequence from its first nucleotide. cDNA copies of CMV strain Q (Q-CMV) genomic RNAs 1, 2 and 3 cloned into this vector were infectious when inoculated together, producing symptoms indistinguishable from those caused by wildtype Q-CMV infection. The infectivity of the clones could be substantially increased by excision of the viral insert together with the transcriptional promoter and terminator prior to inoculation. A diagnostic but silent mutation was introduced into the RNA 2 cDNA and found to be stably maintained in viral infection, allowing distinction of the recombinant virus from native contaminants. The infectious cDNA clones supported the replication of CMV satellite RNA when co-inoculated with biologically active Q-CMV satellite RNA transcripts. Using the infectious cDNAs described, it was found that a newly-identified overlapping gene (2b) encoded by Q-CMV RNA 2 was not essential for either systemic viral infection of Nicotiana glutinosa or replication of the satellite RNAs.

Soybean dwarf luteovirus contains the third variant genome type in the luteovirus group.

Complementary DNAs covering the entire RNA genome of soybean dwarf luteovirus (SDV) were cloned and sequenced. Computer analysis of the 5861 nucleotide sequence revealed five major open reading frames (ORFs) possessing conservation of sequence and organisation with known luteovirus sequences. Comparative analyses of the genome structure show that SDV shares sequence homology and features of gene organisation with barley yellow dwarf virus (PAV isolate) in the 5' half of the genome, yet is more closely related to potato leafroll virus in its 3' coding regions. In addition, SDV differs from other known luteoviruses in possessing an exceptionally long 3' terminal sequence with no apparent coding capacity. We conclude from these data that the SDV genome represents a third variant genome type in the luteovirus group.

DNA binding of CPF1 is required for optimal centromere function but not for maintaining methionine prototrophy in yeast.

The centromere and promoter factor 1 (CPF1) binds specifically in vitro and in vivo to an octanucleotide (RTCACRTG). This sequence is found in the centromere DNA element I (CDEI) of yeast centromeres and upstream from a number of transcription units including MET25, GAL2 and TRP1. Inactivation of the CPF1 gene results in three phenotypes; slow growth, a partial loss of centromere function and methionine auxotrophy. These phenotypes correlate well with the known binding sites for CPF1 and have led to the suggestion that CPF1 functions as a kinetochore protein at centromeres and as a transcriptional activator at promoters such as MET25. By analysing transcription from the MET25, GAL2, and TRP1 genes in cpf1 strains, we demonstrate that CPF1 plays no direct role in their transcriptional regulation. Further evidence in support of this comes from the analysis of point mutations in the basic region of CPF1 that affect DNA binding. A strain expressing a non-DNA bound form of CPF1 is phenotypically Met+, shows normal growth rate but has sub-optimal centromere function. We conclude that a DNA-bound form of CPF1 is required for the kinetochore function but not for maintaining methionine prototrophy.

CPF1, a yeast protein which functions in centromeres and promoters.

Centromeres and several promoters of Saccharomyces cerevisiae contain a highly conserved octanucleotide, RTCACRTG, called CDEI. Using biochemical, genetic and structural analyses, we show that the same protein binds in vivo to CDEI sites in centromeres and in promoters. This protein, called CPF1 for centromere promoter factor, binds DNA as a dimer. Inactivation of the gene is not lethal but leads to a partial loss of the centromere function and to a Met- phenotype. Changes of the chromatin structure due to inactivation of CPF1 are seen at centromeres and at several CDEI-carrying promoters (e.g. MET25, TRP1, GAL2). However promoter activities are affected in diverse ways making it presently difficult to describe a function for CPF1 in gene expression. The sequence of the cloned gene reveals in the carboxy-terminal part two potential amphipathic helices preceded by a positively charged stretch of amino acids very similar to the helix-loop-helix domains recently identified in factors controlling tissue specific transcription in higher eukaryotes. Carboxy-terminal truncations of CPF1 lacking this domain no longer bind to CDEI. The amino-terminal half of CPF1 carries two clusters of negatively charged amino acid residues. Surprisingly, deletions of these clusters still render cells Met+ and lead only to a marginal decrease in centromere activity.