Collaborative Research Stories: Whakawhanaungatanga

Collaborative Research Stories: Whakawhanaungatanga. Russell Bishop. Palmerston North, New Zealand: Dunmore Press, 1996. 273 pp.     

Hsp 12 is a LEA-like protein in Saccharomyces cerevisiae

LEA group I, II and III antibodies all recognised soluble proteins present in an extract of yeast (Saccharomyces cerevisiae). The smaller protein of the two recognised by the group I antibody displayed identical migration on SDS-PAGE to the pea seed LEA group I protein against which the antibody was raised. However, the antibody failed to recognise the predominant protein present after heating the extract at 80 °C for 10 min. This predominant protein, which also displayed identical migration on SDS-PAGE, was purified from the supernatant of the extract heated at 80 °C for 10 min. Peptide sequencing after CNBr cleavage identified the isolated protein as the heat shock protein HSP 12. Despite a previous report that HSP 12 is a heat shock protein, HSP 12 was found to increase in yeast grown at 37 °C compared with growth at 30 °C . However, increased amounts of HSP 12 were present in yeast after entry into stationary phase; this was enhanced by growth in the osmolytes NaCl and mannitol.     

Drug Repurposing: A Systematic Approach to Evaluate Candidate Oral Neuroprotective Interventions for Secondary Progressive Multiple Sclerosis

Objective

To develop and implement an evidence based framework to select, from drugs already licenced, candidate oral neuroprotective drugs to be tested in secondary progressive multiple sclerosis.

Design

Systematic review of clinical studies of oral putative neuroprotective therapies in MS and four other neurodegenerative diseases with shared pathological features, followed by systematic review and meta-analyses of the in vivo experimental data for those interventions. We presented summary data to an international multi-disciplinary committee, which assessed each drug in turn using pre-specified criteria including consideration of mechanism of action.

Results

We identified a short list of fifty-two candidate interventions. After review of all clinical and pre-clinical evidence we identified ibudilast, riluzole, amiloride, pirfenidone, fluoxetine, oxcarbazepine, and the polyunsaturated fatty-acid class (Linoleic Acid, Lipoic acid; Omega-3 fatty acid, Max EPA oil) as lead candidates for clinical evaluation.

Conclusions

We demonstrate a standardised and systematic approach to candidate identification for drug rescue and repurposing trials that can be applied widely to neurodegenerative disorders.     

Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.     

Multi-Parametric MRI at 14T for Muscular Dystrophy Mice Treated with AAV Vector-Mediated Gene Therapy

The objective of this study was to investigate the efficacy of using quantitative magnetic resonance imaging (MRI) as a non-invasive tool for the monitoring of gene therapy for muscular dystrophy. The clinical investigations for this family of diseases often involve surgical biopsy which limits the amount of information that can be obtained due to the invasive nature of the procedure. Thus, other non-invasive tools may provide more opportunities for disease assessment and treatment responses. In order to explore this, dystrophic mdx^4cv mice were systemically treated with a recombinant adeno-associated viral (AAV) vector containing a codon-optimized micro-dystrophin gene. Multi-parametric MRI of T2, magnetization transfer, and diffusion effects alongside 3-D volume measurements were then utilized to monitor disease/treatment progression. Mice were imaged at 10 weeks of age for pre-treatment, then again post-treatment at 8, 16, and 24 week time points. The efficacy of treatment was assessed by physiological assays for improvements in function and quantification of expression. Tissues from the hindlimbs were collected for histological analysis after the final time point for comparison with MRI results. We found that introduction of the micro-dystrophin gene restored some aspects of normal muscle histology and pathology such as decreased necrosis and resistance to contraction-induced injury. T2 relaxation values showed percentage decreases across all muscle types measured (tibialis anterior, gastrocnemius, and soleus) when treated groups were compared to untreated groups. Additionally, the differences between groups were statistically significant for the tibialis anterior as well. The diffusion measurements showed a wider range of percentage changes and less statistical significance while the magnetization transfer effect measurements showed minimal change. MR images displayed hyper-intense regions of muscle that correlated with muscle pathology in histological sections. T2 relaxation, alongside diffusion and magnetization transfer effects provides useful data towards the goal of non-invasively monitoring the treatment of muscular dystrophy.     

Arginase inhibition restores endothelial function in diet-induced obesity

Arginase may play a major role in the regulation of vascular function in various cardiovascular disorders by impairing nitric oxide (NO) production. In the current study, we investigated whether supplementation of the arginase inhibitor N^ω-hydroxy-nor-l-arginine (nor-NOHA) could restore endothelial function in an animal model of diet-induced obesity. Arginase 1 expression was significantly lower in the aorta of C57BL/6J mice fed a high-fat diet (HFD) supplemented with nor-NOHA (40 mg kg^-1/day) than in mice fed HFD without nor-NOHA. Arginase inhibition led to considerable increases in eNOS expression and NO levels and significant decreases in the levels of circulating ICAM-1. These findings were further confirmed by the results of siRNA-mediated knockdown of Arg in human umbilical vein endothelial cells. In conclusion, arginase inhibition can help restore dysregulated endothelial function by increasing the eNOS-dependent NO production in the endothelium, indicating that arginase could be a therapeutic target for correcting obesity-induced vascular endothelial dysfunction.     

Distribution,diversity and drivers of blood-borne parasite co-infections in Alaskan bird populations

Avian species are commonly infected by multiple parasites, however few studies have investigated the environmental determinants of the prevalence of co-infection over a large scale. Here we believe that we report the first, detailed ecological study of the prevalence, diversity and co-infections of four avian blood-borne parasite genera: Plasmodium spp., Haemoproteus spp., Leucocytozoon spp. and Trypanosoma spp. We collected blood samples from 47 resident and migratory bird species across a latitudinal gradient in Alaska. From the patterns observed at collection sites, random forest models were used to provide evidence of associations between bioclimatic conditions and the prevalence of parasite co-infection distribution. Molecular screening revealed a higher prevalence of haematozoa (53%) in Alaska than previously reported. Leucocytozoons had the highest diversity, prevalence and prevalence of co-infection. Leucocytozoon prevalence (35%) positively correlated with Trypanosoma prevalence (11%), negatively correlated with Haemoproteus prevalence (14%) and had no correlation with Plasmodium prevalence (7%). We found temperature, precipitation and tree cover to be the primary environmental drivers that show a relationship with the prevalence of co-infection. The results provide insight into the impacts of bioclimatic drivers on parasite ecology and intra-host interactions, and have implications for the study of infectious diseases in rapidly changing environments.     

A Highlights from MBoC Selection: Mobility,Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae of Ashbya gossypii

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 μm/min), rotations (up to 180° in 30 s), and long-range nuclear bypassing (up to 9 μm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis–oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.