The CeCDC-14 phosphatase is required for cytokinesis in the Caenorhabditis elegans embryo

In all eukaryotic organisms, the physical separation of two nascent cells must be coordinated with chromosome segregation and mitotic exit. In Saccharomyces cerevisiae and Schizosaccharomyces pombe this coordination depends on a number of genes that cooperate in intricate regulatory pathways termed mitotic exit network and septum initiation network, respectively. Here we have explored the function of potentially homologous genes in a metazoan organism, Caenorhabditis elegans, using RNA-mediated interference. Of all the genes tested, only depletion of CeCDC-14, the C. elegans homologue of the budding yeast dual-specificity phosphatase Cdc14p (Clp1/Flp1p in fission yeast), caused embryonic lethality. We show that CeCDC-14 is required for cytokinesis but may be dispensable for progression of the early embryonic cell cycles. In response to depletion of CeCDC-14, embryos fail to establish a central spindle, and several proteins normally found at this structure are mislocalized. CeCDC-14 itself localizes to the central spindle in anaphase and to the midbody in telophase. It colocalizes with the mitotic kinesin ZEN-4, and the two proteins depend on each other for correct localization. These findings identify the CDC14 phosphatase as an important regulator of central spindle formation and cytokinesis in a metazoan organism

Characterisation and mapping of a <i>Globodera pallida</i> resistance derived from the wild potato species <i>Solanum spegazzinii</i>

The potato cyst nematodes (PCN) Globodera pallida and Globodera rostochiensis are economically important potato pests in almost all regions where potato is grown. One important management strategy involves deployment through introgression breeding into modern cultivars of new sources of naturally occurring resistance from wild potato species. We describe a new source of resistance to G. pallida from wild potato germplasm. The diploid species Solanum spegazzinii Bitter accession CPC 7195 shows resistance to G. pallida pathotypes Pa1 and Pa2/3. A cross and first backcross of S. spegazzinii with Solanum tuberosum Group Phureja cultivar Mayan Gold were performed, and the level of resistance to G. pallida Pa2/3 was determined in progeny clones. Bulk-segregant analysis (BSA) using generic mapping enrichment sequencing (GenSeq) and genotyping-by-sequencing were performed to identify single-nucleotide polymorphisms (SNPs) that are genetically linked to the resistance, using S. tuberosum Group Phureja clone DM1-3 516 R44 as a reference genome. These SNPs were converted into allele-specific PCR assays, and the resistance was mapped to an interval of roughly 118 kb on chromosome VI. This newly identified resistance, which we call Gpa VIlspg, can be used in future efforts to produce modern cultivars with enhanced and broad-spectrum resistances to the major pests and pathogens of potato

Characterisation and mapping of a <i>Globodera pallida</i> resistance derived from the wild potato species <i>Solanum spegazzinii</i>

The potato cyst nematodes (PCN) Globodera pallida and Globodera rostochiensis are economically important potato pests in almost all regions where potato is grown. One important management strategy involves deployment through introgression breeding into modern cultivars of new sources of naturally occurring resistance from wild potato species. We describe a new source of resistance to G. pallida from wild potato germplasm. The diploid species Solanum spegazzinii Bitter accession CPC 7195 shows resistance to G. pallida pathotypes Pa1 and Pa2/3. A cross and first backcross of S. spegazzinii with Solanum tuberosum Group Phureja cultivar Mayan Gold were performed, and the level of resistance to G. pallida Pa2/3 was determined in progeny clones. Bulk-segregant analysis (BSA) using generic mapping enrichment sequencing (GenSeq) and genotyping-by-sequencing were performed to identify single-nucleotide polymorphisms (SNPs) that are genetically linked to the resistance, using S. tuberosum Group Phureja clone DM1-3 516 R44 as a reference genome. These SNPs were converted into allele-specific PCR assays, and the resistance was mapped to an interval of roughly 118 kb on chromosome VI. This newly identified resistance, which we call Gpa VIlspg, can be used in future efforts to produce modern cultivars with enhanced and broad-spectrum resistances to the major pests and pathogens of potato

Delay of transfer from the intensive care unit: a prospective observational analysis on economic effects of delayed in-house transfer

BACKGROUND: Intensive care unit (ICU) capacity is a scant and precious resource in hospitals. Therefore, an optimal occupancy rate as well as detailed occupation planning is of great importance. Most literature deals with admission to the ICU, while only few discuss discharge from the ICU. Specifically, a delay of transfer from the ICU can cause a shortness of beds, jeopardize urgent patient treatment and lead to a decrease in treatment quality as well as economic downsides. This study examined the incidence, costs and reasons for delayed discharge from the ICU and analyzed the influence of the department the patient was admitted to. METHODS: Over the course of 12 months, the discharges of all 1643 patients of two surgical intensive care units of a large academic medical center were analyzed. Delay in minutes and reasons were recorded and translated into financial figures. A univariate logistic regression model was developed to evaluate the impact of length of stay at the ICU, age, gender, subspecialty and specific ICU on the delay of transfer. In a next step, significant factors of the univariate logistic regression were incorporated into a multivariate regression model. RESULTS: In 326 out of 1312 patients ready for discharge (24.8%), the transfer to the floor was delayed. Time of delay for all patients added up to a total of 265,691 min in 1 year. The application of the internal cost allocation, in which 1 min corresponds to 0.75 Euro cents, led to costs of 199,268 Euros (~$240,000) for the study period. In 91.7% of the cases, the reason for the delay was the lack of an available or appropriate bed on the regular ward. Multivariate regression analysis revealed that the type of department the patient is admitted to poses a significantly influencing factor for delayed discharge from the ICU. CONCLUSION: Delay in discharge from the ICU is a common problem of economic relevance. The main reason is a lack of appropriate floor beds. Patients from certain specific departments are at a higher risk to be discharged with delay. A solution to this problem lies in the focus on the downstream units. A proper use of the scarce resources is to be pursued because of ethical as well as economic reasons in an increasingly aging population

Lysyl hydroxylase 3 localizes to epidermal basement membrane and Is reduced in patients with Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention

Exploring and exploiting the resistance to Globodera pallida in potato

The potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis are economically important potato pests in almost all regions where potato is grown. Studying the composition, distribution and virulence of PCN populations in fields, and finding new sources of naturally occurring resistance in wild potato species is important for the management of these pests. In Scotland, up to three different introductions of G. pallida, determined by mitotyping, were found to be present in fields. To investigate whether cysts in a population show a correlation between mitotype and different virulence levels, “single cyst” lines were generated, mitotyped, and their virulence to different potato cultivars was determined. One mitotype was shown to correlate with G. pallida pathotype Pa3, but overall, the mitotypes are not usable as reliable virulence markers. A phylogenetic analysis was performed to determine the relationships between British G. pallida populations. In addition, single nucleotide polymorphisms (SNPs) in genomic DNA were identified that represent candidate virulence markers. A screen of wild potato germplasm was undertaken to identify new resistance against G. pallida. The diploid species Solanum spegazzinii Bitter accession 7195 shows resistance to G. pallida pathotypes Pa1 and Pa2/3. A cross and first backcross of S. spegazzinii with S. tuberosum group Phureja cultivar Mayan Gold was performed, and the level of resistance to G. pallida Pa2/3 was determined in progeny. Bulked-segregant analysis sing generic mapping enrichment sequencing and genotyping-by-sequencing was performed to identify SNPs that are genetically linked to the resistance, using S. tuberosum group Phureja clone DM1-3 516 R44 as a reference genome. These SNPs were converted into allele specific PCR assays, and the resistance was mapped using graphical genotyping. The resistance was successfully introgressed into a tetraploid potato cultivar by a tetraploid-diploid interploidy cross

A Lack of premature termination codon read-through efficacy of PTC124 (Ataluren) in a diverse array of reporter assays

The drug molecule PTC124 (Ataluren) has been described as a read-through agent, capable of suppressing premature termination codons (PTCs) and restoring functional protein production from genes disrupted by nonsense mutations. Following the discovery of PTC124 there was some controversy regarding its mechanism of action with two reports attributing its activity to an off-target effect on the Firefly luciferase (FLuc) reporter used in the development of the molecule. Despite questions remaining as to its mechanism of action, development of PTC124 continued into the clinic and it is being actively pursued as a potential nonsense mutation therapy. To thoroughly test the ability of PTC124 to read through nonsense mutations, we conducted a detailed assessment comparing the efficacy of PTC124 with the classical aminoglycoside antibiotic read-through agent geneticin (G418) across a diverse range of in vitro reporter assays. We can confirm the off-target FLuc activity of PTC124 but found that, while G418 exhibits varying activity in every read-through assay, there is no evidence of activity for PTC124