Development of a rapid,small-scale DNA repair assay for use on clinical samples

Double-strand breaks (DSBs) are the most lethal form of DNA damage. They can be repaired by one of two pathways, homologous recombination and non-homologous end joining (NHEJ). A NHEJ assay has previously been reported which measures joining using cell-free extracts and a linearised plasmid as DNA substrate. This assay was designed for 3 × 10⁹ cells grown in vitro and utilised radioactively labelled substrate. We have scaled down the method to use smaller cell numbers in a variety of cell lines. Altering the cellular extraction procedure decreased background DNA contamination. The cleaner preparations allowed us to use SYBR Green I staining to identify joined products, which was as sensitive as ³²P-end-labelled DNA. NHEJ was found in established tumour cell lines from different originating tissues, though actual levels and fidelity of repair differed. This method also allowed end joining to be assessed in clinical specimens (human blood, brain and bladder tumours) within 24 h of receiving samples. The application of this method will allow investigation of the role of DSB DNA repair pathways in human tumours.     

Rapid turnover of extracellular signal-regulated kinase 3 by the ubiquitin-proteasome pathway defines a novel paradigm of mitogen-activated protein kinase regulation during cellular differentiation

Mitogen-activated protein (MAP) kinases are stable enzymes that are mainly regulated by phosphorylation and subcellular targeting. Here we report that extracellular signal-regulated kinase 3 (ERK3), unlike other MAP kinases, is an unstable protein that is constitutively degraded in proliferating cells with a half-life of 30 min. The proteolysis of ERK3 is executed by the proteasome and requires ubiquitination of the protein. Contrary to other protein kinases, the catalytic activity of ERK3 is not responsible for its short half-life. Instead, analysis of ERK1/ERK3 chimeras revealed the presence of two destabilization regions (NDR1 and -2) in the N-terminal lobe of the ERK3 kinase domain that are both necessary and sufficient to target ERK3 and heterologous proteins for proteasomal degradation. To assess the physiological relevance of the rapid turnover of ERK3, we monitored the expression of the kinase in different cellular models of differentiation. We observed that ERK3 markedly accumulates during differentiation of PC12 and C2C12 cells into the neuronal and muscle lineage, respectively. The accumulation of ERK3 during myogenic differentiation is associated with the time-dependent stabilization of the protein. Terminal skeletal muscle differentiation is accompanied by cell cycle withdrawal. Interestingly, we found that expression of stabilized forms of ERK3 causes G(1) arrest in NIH 3T3 cells. We propose that ERK3 biological activity is regulated by its cellular abundance through the control of protein stability.     

A novel 2'-(N-methylpyridinium acetate) prodrug of paclitaxel induces superior antitumor responses in preclinical cancer models

The development of novel strategies for the treatment of malignancies by successful intervention in advanced stage disease is a major challenge in oncology. We tested the hypothesis that this can be achieved by the rational design of taxoid onium salts modified at C-7 and C-2' positions. The characterization of these molecules revealed a dramatically improved water solubility and prodrug behavior in plasma. Specifically, all compounds released parental paclitaxel with half-lives ranging from 0.9 to 180 min. In the absence of plasma, only the 2'-(N-methylpyridinium acetate) derivative of paclitaxel (2'-MPA-paclitaxel) revealed a complete abrogation of paclitaxel specific microtubule assembly disassembly dynamics and a 3 log reduction in cellular binding, indicating that reversible blockage of the C-2' position by methylpyridinium acetate yields a true paclitaxel prodrug. Structure/activity profiles of all compounds in tissue culture revealed cytotoxicity effective at picomolar concentrations with a panel of 16 cancer cell lines in contrast to 4 nonmalignant cell lines. Importantly, the decisive cytotoxic potential observed in vitro for all compounds correlated only with in vivo findings for 2'-MPA-paclitaxel. Specifically, the 2'-MPA-paclitaxel prodrug induced regression of primary tumors in three xenograft models of nonsmall cell lung carcinoma, ovarian carcinoma and prostate cancer, in contrast to ineffective C-7 derivatives and parental paclitaxel. At the same time, a reduced systemic toxicity of 2'-MPA-paclitaxel was observed in contrast to a far more toxic parental paclitaxel. Taken together, these findings demonstrate that the 2'-MPA-paclitaxel prodrug is a promising new candidate for cancer therapy.     

DIFFERENTIAL SUCCESS OF POLLEN DONORS IN A SELF-COMPATIBLE LILY

If pollen donors are equally effective at siring seeds, the presence of equal proportions of pollen from two pollen donors on a stigma will lead to equal proportions of seeds sired by each pollen donor. Variation in germination rates, pollen-tube growth, and embryo viability may cause one donor to sire more seed than another. We looked for differential donor success in the field by simultaneously applying equal amounts of pollen from two pollen donors. We simultaneously applied equal amounts of self and outcross pollen to receptive stigmas and simultaneously applied pollen from two donors at different physical distances from the recipient. Following simultaneous application of self and outcross pollen, significantly more of the seeds were sired by outcross pollen donors. Seed set following simultaneous application of two outcross donors was also nonrandom. Pollen donors from 100 m were more likely to sire seeds when competing with pollen from plants nearby (1 m). To determine whether pollen-tube growth rates were responsible for these patterns of paternity, we varied the timing of deposition of outcross pollen allowing self pollen tubes a head start on the stigma. Outcross pollen was applied 3 or 24 h after self pollen. In spite of this time delay, the majority of the seeds were again sired by outcross pollen. There was no significant difference in the amount of seeds sired by self pollen between the two delay treatments. This result suggests that mechanisms operating after ovule fertilization may contribute to the discordance between the proportions of the pollen present and the proportions of seeds sired.     

Bioinformatics-driven identification and examination of candidate genes for non-alcoholic fatty liver disease

Objective

Candidate genes for non-alcoholic fatty liver disease (NAFLD) identified by a bioinformatics approach were examined for variant associations to quantitative traits of NAFLD-related phenotypes.

Research Design and Methods

By integrating public database text mining, trans-organism protein-protein interaction transferal, and information on liver protein expression a protein-protein interaction network was constructed and from this a smaller isolated interactome was identified. Five genes from this interactome were selected for genetic analysis. Twenty-one tag single-nucleotide polymorphisms (SNPs) which captured all common variation in these genes were genotyped in 10,196 Danes, and analyzed for association with NAFLD-related quantitative traits, type 2 diabetes (T2D), central obesity, and WHO-defined metabolic syndrome (MetS).

Results

273 genes were included in the protein-protein interaction analysis and EHHADH, ECHS1, HADHA, HADHB, and ACADL were selected for further examination. A total of 10 nominal statistical significant associations (P<0.05) to quantitative metabolic traits were identified. Also, the case-control study showed associations between variation in the five genes and T2D, central obesity, and MetS, respectively. Bonferroni adjustments for multiple testing negated all associations.

Conclusions

Using a bioinformatics approach we identified five candidate genes for NAFLD. However, we failed to provide evidence of associations with major effects between SNPs in these five genes and NAFLD-related quantitative traits, T2D, central obesity, and MetS.     

Barriers in the Delivery of Emergency Obstetric and Neonatal Care in Post-Conflict Africa: Qualitative Case Studies of Burundi and Northern Uganda

Objectives

Maternal and neonatal mortality and morbidity rates are particularly grim in conflict, post-conflict and other crisis settings, a situation partly blamed on non-availability and/or poor quality of emergency obstetric and neonatal care (EmONC) services. The aim of this study was to explore the barriers to effective delivery of EmONC services in post-conflict Burundi and Northern Uganda, in order to provide policy makers and other relevant stakeholders context-relevant data on improving the delivery of these lifesaving services.

Methods

This was a qualitative comparative case study that used 42 face-to-face semi-structured in-depth interviews and 4 focus group discussions for data collection. Participants were 32 local health providers and 37 staff of NGOs working in the area of maternal health. Data was analysed using the framework approach.

Results

The availability, quality and distribution of EmONC services were major challenges across the sites. The barriers in the delivery of quality EmONC services were categorised into two major themes; human resources-related challenges, and systemic and institutional failures. While some of the barriers were similar, others were unique to specific sites. The common barriers included shortage of qualified staff; lack of essential installations, supplies and medications; increasing workload, burn-out and turnover; and poor data collection and monitoring systems. Barriers unique to Northern Uganda were demoralised personnel and lack of recognition; poor referral system; inefficient drug supply system; staff absenteeism in rural areas; and poor coordination among key personnel. In Burundi, weak curriculum; poor harmonisation and coordination of training; and inefficient allocation of resources were the unique challenges. To improve the situation across the sites, efforts are ongoing to improve the training and recruitment of more staff; harmonise and strengthen the curriculum and training; increase the number of EmONC facilities; and improve staff supervision, monitoring and support.

Conclusions

Post-conflict health systems face different challenges in the delivery of EmONC services and as such require context-specific interventions to improve the delivery of these services.     

High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1β in osteoarthritic synoviocytes

Introduction  

High mobility group box 1 (HMGB1) is released by necrotic cells or secreted in response to inflammatory stimuli. Extracellular HMGB1 may act as a pro-inflammatory cytokine in rheumatoid arthritis. We have recently reported that HMGB1 is released by osteoarthritic synoviocytes after activation with interleukin-1beta (IL-1β) The present study investigated the role of HMGB1 in synovial inflammation in osteoarthritis (OA).