Compartmentalized Toxoplasma EB1 bundles spindle microtubules to secure accurate chromosome segregation

Toxoplasma gondii replicates asexually by a unique internal budding process characterized by interwoven closed mitosis and cytokinesis. Although it is known that the centrosome coordinates these processes, the spatiotemporal organization of mitosis remains poorly defined. Here we demonstrate that centrosome positioning around the nucleus may signal spindle assembly: spindle microtubules (MTs) are first assembled when the centrosome moves to the basal side and become extensively acetylated after the duplicated centrosomes reposition to the apical side. We also tracked the spindle MTs using the MT plus end–binding protein TgEB1. Endowed by a C-terminal NLS, TgEB1 resides in the nucleoplasm in interphase and associates with the spindle MTs during mitosis. TgEB1 also associates with the subpellicular MTs at the growing end of daughter buds toward the completion of karyokinesis. Depletion of TgEB1 results in escalated disintegration of kinetochore clustering. Furthermore, we show that TgEB1’s MT association in Toxoplasma and in a heterologous system (Xenopus) is based on the same principles. Finally, overexpression of a high-MT-affinity TgEB1 mutant promotes the formation of overstabilized MT bundles, resulting in avulsion of otherwise tightly clustered kinetochores. Overall we conclude that centrosome position controls spindle activity and that TgEB1 is critical for mitotic integrity.     

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis

Background

Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed.

Results

Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated.

Conclusion

The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1448-x) contains supplementary material, which is available to authorized users.     

Chemical immobilization of adult female Weddell seals with tiletamine and zolazepam: effects of age, condition and stage of lactation

Background

Postweaning diarrhoea (PWD) in pigs is usually the main infectious problem of large-scale farms and is responsible for significant losses worldwide. The disease is caused mainly by enterotoxigenic E. coli (ETEC) and Shiga-toxin producing E. coli (STEC). In this study a total of 101 E. coli isolated from pigs with PWD in Slovakia were characterized using phenotypic and genotypic methods.

Results

These 101 isolates belonged to 40 O:H serotypes. However, 57% of the isolates belonged to only six serotypes (O9:H51, O147:H-, O149:H10, O163:H-, ONT:H-, and ONT:H4), including two new serotypes (O163:H- and ONT:H4) not previously found among porcine ETEC and STEC isolated in other countries. Genes for EAST1, STb, STa, LT and Stx2e toxins were identified in 64%, 46%, 26%, 20%, and 5% of isolates, respectively. PCR showed that 35% of isolates carried genes for F18 colonization factor, and further analyzed by restriction endonuclease revealed that all of them were F18ac. Genes for F4 (K88), F6 (P987), F17, F5 (K99), F41, and intimin (eae gene) adhesins were detected in 19 %, 5%, 3%, 0.9%, 0.9%, and 0.9% of the isolates, respectively. The study of genetic diversity, carried out by PFGE of 46 representative ETEC and STEC isolates, revealed 36 distinct restriction profiles clustered in eight groups. Isolates of the same serotype were placed together in the dendrogram, but high degree of polymorphism among certain serotypes was detected.

Conclusion

Seropathotype O149:H10 LT/STb/EAST1/F4 (14 isolates) was the most commonly detected followed by O163:H- EAST1/F18 (six isolates), and ONT:H4 STa/STb/Stx2e/F18 (five isolates). Interestingly, this study shows that two new serotypes (O163:H- and ONT:H4) have emerged as pig pathogens in Slovakia. Furthermore, our results show that there is a high genetic variation mainly among ETEC of O149:H10 serotype.     

The cell cycle and Toxoplasma gondii cell division: tightly knit or loosely stitched?

The flexibility displayed by apicomplexan parasites to vary their mode of replication has intrigued biologists since their discovery by electron microscopy in the 1960s and 1970s. Starting in the 1990s we began to understand the cell biology of the cytoskeleton elements driving cytokinesis. By contrast, the molecular mechanisms that regulate the various division modes and how they translate into the budding process that uniquely characterizes this parasite family are much less understood. Although growth mechanisms are a neglected area of study, it is an important pathogenic parameter as fast division rounds are associated with fulminant infection whereas slower growth attenuates virulence, as is exploited in some vaccine strains. In this review we summarize a recent body of cell biological experiments that are rapidly leading to an understanding of the events that yield successful mitosis and cytokinesis in Toxoplasma. We place these observations within a cell cycle context with comments on how these events may be regulated by known eukaryotic checkpoints active in fission and budding yeasts as well as mammalian cells. The presence of cell cycle control mechanisms in the Apicomplexa is supported by our findings that identify several cell cycle checkpoints in Toxoplasma. The progress of the cell cycle is ultimately controlled by cyclin-Cdk pair activities, which are present throughout the Apicomplexa. Although many of the known controllers of cyclin-Cdk activity are present, several key controls cannot readily be identified, suggesting that apicomplexan parasites deviate at these points from the higher eukaryotic models. Altogether, new insights in Toxoplasma replication are reciprocally applied to hypothesize how other division modes in the Toxoplasma life cycle and in other Apicomplexa species could be controlled in terms of cell cycle checkpoint regulation.     

HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase

Background

Deficiency of 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) caused by HIBCH mutations is a rare cerebral organic aciduria caused by disturbance of valine catabolism. Multiple mitochondrial respiratory chain (RC) enzyme deficiencies can arise from a number of mechanisms, including defective maintenance or expression of mitochondrial DNA. Impaired biosynthesis of iron-sulphur clusters and lipoic acid can lead to pyruvate dehydrogenase complex (PDHc) deficiency in addition to multiple RC deficiencies, known as the multiple mitochondrial dysfunctions syndrome.

Methods

Two brothers born to distantly related Pakistani parents presenting in early infancy with a progressive neurodegenerative disorder, associated with basal ganglia changes on brain magnetic resonance imaging, were investigated for suspected Leigh-like mitochondrial disease. The index case had deficiencies of multiple RC enzymes and PDHc in skeletal muscle and fibroblasts respectively, but these were normal in his younger brother. The observation of persistently elevated hydroxy-C4-carnitine levels in the younger brother led to suspicion of HIBCH deficiency, which was investigated by biochemical assay in cultured skin fibroblasts and molecular genetic analysis.

Results

Specific spectrophotometric enzyme assay revealed HIBCH activity to be below detectable limits in cultured skin fibroblasts from both brothers. Direct Sanger sequence analysis demonstrated a novel homozygous pathogenic missense mutation c.950G <A; p.Gly317Glu in the HIBCH gene, which segregated with infantile-onset neurodegeneration within the family.

Conclusions

HIBCH deficiency, a disorder of valine catabolism, is a novel cause of the multiple mitochondrial dysfunctions syndrome, and should be considered in the differential diagnosis of patients presenting with multiple RC deficiencies and/or pyruvate dehydrogenase deficiency.

     

Hypercholesterolemia boosts joint destruction in chronic arthritis. An experimental model aggravated by foam macrophage infiltration

Objective

The aim of this study was to determine whether hypercholesterolemia increases articular damage in a rabbit model of chronic arthritis.

Methods

Hypercholesterolemia was induced in 18 rabbits by administrating a high-fat diet (HFD). Fifteen rabbits were fed normal chow as controls. Chronic antigen-induced arthritis (AIA) was induced in half of the HFD and control rabbits, previously immunized, by intra-articular injections of ovalbumin. After sacrifice, lipid and systemic inflammation markers were analyzed in blood serum. Synovium was analyzed by Krenn score, multinucleated cell counting, immunohistochemistry of RAM11 and CD31, and TNF-α and macrophage chemoattractant protein-1 (MCP-1) gene expression. Active bone resorption was assessed by protein expression of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and quantification of cathepsin K, contact surface and the invasive area of pannus into bone.

Results

Rabbits receiving the HFD showed higher total serum cholesterol, HDL, triglycerides and CRP levels than rabbits fed a normal diet. Synovitis score was increased in HFD, and particularly in AIA and AIA + HFD groups. AIA + HFD synovium was characterized by a massive infiltration of RAM11+ cells, higher presence of multinucleated foam cells and bigger vascularization than AIA. Cathepsin K+ osteoclasts and the contact surface of bone resorbing pannus were also increased in rabbits with AIA + HFD compared with AIA alone. Synovial TNF-α and MCP-1 gene expression was increased in AIA and HFD rabbits compared with healthy animals. RANKL protein expression in AIA and AIA + HFD groups was higher compared with either HFD or normal groups.

Conclusions

This experimental model demonstrates that hypercholesterolemia increments joint tissue damage in chronic arthritis, with foam macrophages being key players in this process.     

(Cost)-effectiveness of a multi-component intervention for adults with epilepsy: study protocol of a Dutch randomized controlled trial (ZMILE study)

Background

In patients with epilepsy, poor adherence to anti-epileptic drugs has been shown to be the most important cause of poorly controlled epilepsy. Furthermore, it has been noted that the quality of life among patients with epilepsy can be improved by counseling and treatments aimed at increasing their self-efficacy and concordance, thus stimulating self-management skills. However, there is a need for evidence on the effectiveness of such programs, especially within epilepsy care. Therefore, we have developed a multi-component intervention (MCI) which combines a self-management/education program with e-Health interventions. Accordingly, the overall objective of this study is to assess the (cost)-effectiveness and feasibility of the MCI, aiming to improve self-efficacy and concordance in patients with epilepsy.

Methods

A RCT in two parallel groups will be conducted to compare the MCI with a control condition in epilepsy patients. One hundred eligible epilepsy patients will be recruited and allocated to either the intervention or control group. The intervention group will receive the MCI consisting of a self-management/education program of six meetings, including e-Health interventions, and will be followed for 12 months. The control group will receive care as usual and will be followed for 6 months, after which patients will be offered the possibility of participating in the MCI. The study will consist of three parts: 1) a clinical effectiveness study, 2) a cost-effectiveness study, and 3) process evaluation. The primary outcome will be self-efficacy. Secondary outcomes include adherence, side effects, change in seizure severity & frequency, improved quality of life, proactive coping, and societal costs. Outcome assessments will be done using questionnaires at baseline and after 3, 6, 9, and 12 months (last two applicable only for intervention group).

Discussion

In times of budget constraints, MCI could be a valuable addition to the current healthcare provision for epilepsy, as it is expected that higher concordance and self-efficacy will result in reduced use of healthcare resources and an increased QOL. Accordingly, this study is aimed helping patients to be their own provider of health care, shifting epilepsy management from professionals to self-care by patients equipped with appropriate skills and tools.

Trial registration number

NTR4484.

     

Whole genome profiling of spontaneous and chemically induced mutations in Toxoplasma gondii

Background

Next generation sequencing is helping to overcome limitations in organisms less accessible to classical or reverse genetic methods by facilitating whole genome mutational analysis studies. One traditionally intractable group, the Apicomplexa, contains several important pathogenic protozoan parasites, including the Plasmodium species that cause malaria.Here we apply whole genome analysis methods to the relatively accessible model apicomplexan, Toxoplasma gondii, to optimize forward genetic methods for chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) and ethylmethane sulfonate (EMS) at varying dosages.

Results

By comparing three different lab-strains we show that spontaneously generated mutations reflect genome composition, without nucleotide bias. However, the single nucleotide variations (SNVs) are not distributed randomly over the genome; most of these mutations reside either in non-coding sequence or are silent with respect to protein coding. This is in contrast to the random genomic distribution of mutations induced by chemical mutagenesis. Additionally, we report a genome wide transition vs transversion ratio (ti/tv) of 0.91 for spontaneous mutations in Toxoplasma, with a slightly higher rate of 1.20 and 1.06 for variants induced by ENU and EMS respectively. We also show that in the Toxoplasma system, surprisingly, both ENU and EMS have a proclivity for inducing mutations at A/T base pairs (78.6% and 69.6%, respectively).

Conclusions

The number of SNVs between related laboratory strains is relatively low and managed by purifying selection away from changes to amino acid sequence. From an experimental mutagenesis point of view, both ENU (24.7%) and EMS (29.1%) are more likely to generate variation within exons than would naturally accumulate over time in culture (19.1%), demonstrating the utility of these approaches for yielding proportionally greater changes to the amino acid sequence. These results will not only direct the methods of future chemical mutagenesis in Toxoplasma, but also aid in designing forward genetic approaches in less accessible pathogenic protozoa as well.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-354) contains supplementary material, which is available to authorized users.     

Intradiscal application of rhBMP-7 does not induce regeneration in a canine model of spontaneous intervertebral disc degeneration

IntroductionStrategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain.MethodsCanine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue.ResultsIn vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7.ConclusionsAn intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0625-2) contains supplementary material, which is available to authorized users.