Modification of Superoxide Dismutase 1 (SOD1) Properties by a GFP Tag – Implications for Research into Amyotrophic Lateral Sclerosis (ALS)

Background

Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whether they behave similarly to their untagged SOD1 counterparts.

Methodology/Principal Findings

Here we compare the physicochemical properties of SOD1 and the effects of GFP-tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showed identical patterns of cytoplasmic expression and of movement within the axon. However, GFP tagging of SOD1 was found to alter some of the intrinsic properties of SOD1, including stability and specific activity. Evaluation of wildtype and mutant SOD1, tagged at either the N- or C-terminus with GFP, in PC12 cells demonstrated that some chimaeric proteins were degraded to the individual proteins, SOD1 and GFP.

Conclusions/Significance

Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag.     

Falling giants and the rise of gene editing: ethics,private interests and the public good

This paper considers the tensions created in genomic research by public and private for-profit ideals. Our intent is to strengthen the public good at a time when doing science is strongly motivated by market possibilities and opportunities. Focusing on the emergence of gene editing, and in particular CRISPR, we consider how commercialisation encourages hype and hope—a sense that only promise and idealism can achieve progress. At this rate, genomic research reinforces structures that promote, above all else, private interests, but that may attenuate conditions for the public good of science. In the first part, we situate genomics using the aphorism that ‘on the shoulders of giants we see farther’; these giants are infrastructures and research cultures rather than individual ‘heroes’ of science. In this respect, private initiatives are not the only pivot for successful discovery, and indeed, fascination in those could impinge upon the fundamental role of public-supported discovery. To redress these circumstances, we define the extent to which progress presupposes research strategies that are for the public good. In the second part, we use a ‘falling giant’ narrative to illustrate the risks of over-indulging for-profit initiatives. We therefore offer a counterpoint to commercialised science, using three identifiable ‘giants’—scientists, publics and cultures—to illustrate how the public good contributes to genomic discovery.     

Autophagy orchestrates adaptive responses to targeted therapy in endometrial cancer

Targeted therapies in endometrial cancer (EC) using kinase inhibitors rarely result in complete tumor remission and are frequently challenged by the appearance of refractory cell clones, eventually resulting in disease relapse. Dissecting adaptive mechanisms is of vital importance to circumvent clinical drug resistance and improve the efficacy of targeted agents in EC. Sorafenib is an FDA-approved multitarget tyrosine and serine/threonine kinase inhibitor currently used to treat hepatocellular carcinoma, advanced renal carcinoma and radioactive iodine-resistant thyroid carcinoma. Unfortunately, sorafenib showed very modest effects in a multi-institutional phase II trial in advanced uterine carcinoma patients. Here, by leveraging RNA-sequencing data from the Cancer Cell Line Encyclopedia and cell survival studies from compound-based high-throughput screenings we have identified the lysosomal pathway as a potential compartment involved in the resistance to sorafenib. By performing additional functional biology studies we have demonstrated that this resistance could be related to macroautophagy/autophagy. Specifically, our results indicate that sorafenib triggers a mechanistic MAPK/JNK-dependent early protective autophagic response in EC cells, providing an adaptive response to therapeutic stress. By generating in vivo subcutaneous EC cell line tumors, lung metastatic assays and primary EC orthoxenografts experiments, we demonstrate that targeting autophagy enhances sorafenib cytotoxicity and suppresses tumor growth and pulmonary metastasis progression. In conclusion, sorafenib induces the activation of a protective autophagic response in EC cells. These results provide insights into the unopposed resistance of advanced EC to sorafenib and highlight a new strategy for therapeutic intervention in recurrent EC.     

A 12 kb multi-allelic copy number variation encompassing a GC gene enhancer is associated with mastitis resistance in dairy cattle

Clinical mastitis (CM) is an inflammatory disease occurring in the mammary glands of lactating cows. CM is under genetic control, and a prominent CM resistance QTL located on chromosome 6 was reported in various dairy cattle breeds. Nevertheless, the biological mechanism underpinning this QTL has been lacking. Herein, we mapped, fine-mapped, and discovered the putative causal variant underlying this CM resistance QTL in the Dutch dairy cattle population. We identified a ~12 kb multi-allelic copy number variant (CNV), that is in perfect linkage disequilibrium with a lead SNP, as a promising candidate variant. By implementing a fine-mapping and through expression QTL mapping, we showed that the group-specific component gene (GC), a gene encoding a vitamin D binding protein, is an excellent candidate causal gene for the QTL. The multiplicated alleles are associated with increased GC expression and low CM resistance. Ample evidence from functional genomics data supports the presence of an enhancer within this CNV, which would exert cis-regulatory effect on GC. We observed that strong positive selection swept the region near the CNV, and haplotypes associated with the multiplicated allele were strongly selected for. Moreover, the multiplicated allele showed pleiotropic effects for increased milk yield and reduced fertility, hinting that a shared underlying biology for these effects may revolve around the vitamin D pathway. These findings together suggest a putative causal variant of a CM resistance QTL, where a cis-regulatory element located within a CNV can alter gene expression and affect multiple economically important traits.     

Vegetation Zonation in a Neotropical Montane Forest: Environment,Disturbance and Ecotones

Gradual changes in vegetation structure and composition are expected to result from continuous environmental change with increasing elevation on mountains. Hence, the occurrence of abrupt or discrete ecotones in vegetation patterns is intriguing and may suggest key controls on community assembly in montane forests. We review tropical montane forest (TMF) zonation patterns focusing on a case study from the Cordillera Central, Hispaniola where a striking discontinuity in forest composition occurs consistently at ～2000 m elevation, with cloud forest below and monodominant pine forest above. We propose that a discontinuity in climatic factors (temperature, humidity) associated with the trade‐wind inversion (TWI) is the primary cause of this and other ecotones in TMFs that occur at a generally consistent elevation. Low humidity, fires and occasional frost above the TWI favor pine over cloud forest species. Fires in the high‐elevation pine forest have repeatedly burned down to the ecotone boundary and extinguished in the cloud forest owing to its low flammability, reinforced by high humidity, cloud immersion and epiphytic bryophyte cover. Small‐scale fire patterns along the ecotone are influenced by topography and where forest structure is impacted by hurricanes and landslides. Analogous patterns are observed worldwide in other TMFs where the TWI is important, high‐elevation fires are frequent, and the flora contains frost‐tolerant species (often of temperate lineage). The response of this and other TMFs to anthropogenic climate change is highly uncertain owing to potentially countervailing effects of different climatic phenomena, including warming temperatures and decreased frost; changes in the TWI, high‐elevation drought or cloudiness; and increased frequency or intensity of hurricanes and El Niño‐Southern Oscillation events.     

Locomotor behaviors in zebrafish (Danio rerio) larvae

Locomotor behaviors were examined in two experiments using zebrafish (Danio rerio) larvae at 4, 5, 6 and 7 days post fertilization (dpf). Larvae were observed in individual wells of a 12-well plate for 1 h a day. In Experiment 1, the same larvae were observed for four consecutive days beginning on post-fertilization day 4; in Experiment 2, different groups of larvae from the same egg collection were observed at 4, 5, 6 and 7 dpf. Automated images collected every 6 s were analyzed for information about larval location, orientation and general activity. In both experiments, 4 dpf larvae rested significantly more, used a smaller area of the well more frequently, and were generally less active than older larvae. All larvae exhibited a preference for facing away from the center of the well and for the edge of the well. However, prolonged exposure to the well influenced overall activity, orientation, and preference for the edge region. The implications of these results for understanding the development of larval behavior and for the design of procedures to measure the effects of experience in zebrafish larvae are discussed.     

Distribution and range expansion of deer in Ireland

• 1 Throughout Europe, the range of many deer species is expanding. We provide current distribution maps for red deer Cervus elaphus, sika Cervus nippon, fallow deer Dama dama and muntjac deer Muntiacus sp. in Ireland, and estimates of range expansion rates for red deer, sika and fallow deer.
• 2 There was a considerable expansion in the ranges of red deer, sika and fallow deer between 1978 and 2008. The compound annual rate of expansion was 7% for red deer, 5% for sika and 3% for fallow deer. The total range increase was 565% for red deer, 353% for sika and 174% for fallow deer. The potential implications of these expansions are discussed.
• 3 There are unknown numbers of red‐sika hybrid deer in some parts of Ireland. Range expansion is likely to lead to further hybridizations with implications for the genetic integrity of deer stocks.
• 4 Sightings of free‐roaming muntjac deer were first recorded in 2007. The distribution of confirmed sightings of single and multiple animals in the eastern region of Ireland suggests multiple releases.
• 5 Deer are already impacting on both the economic and biodiversity values of habitats in Ireland, where, at present, no sustainable deer management policy exists.