A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii has emerged as a superb model species in plant biology. Although the alga is easily transformable, the low efficiency of transgene expression from the Chlamydomonas nuclear genome has severely hampered functional genomics research. For example, poor transgene expression is held responsible for the lack of sensitive reporter genes to monitor gene expression in vivo, analyze subcellular protein localization or study protein–protein interactions. Here, we have tested the luciferase from the marine copepod Gaussia princeps (G-Luc) for its suitability as a sensitive bioluminescent reporter of gene expression in Chlamydomonas. We show that a Gaussia luciferase gene variant, engineered to match the codon usage in the Chlamydomonas nuclear genome, serves as a highly sensitive reporter of gene expression from both constitutive and inducible algal promoters. Its bioluminescence signal intensity greatly surpasses previously developed reporters for Chlamydomonas nuclear gene expression and reaches values high enough for utilizing the reporter as a tool to monitor responses to environmental stresses in vivo and to conduct high-throughput screenings for signaling mutants in Chlamydomonas

A knowledge translation collaborative to improve the use of therapeutic hypothermia in post-cardiac arrest patients: protocol for a stepped wedge randomized trial

<p>Abstract</p> <p>Background</p> <p>Advances in resuscitation science have dramatically improved survival rates following cardiac arrest. However, about 60% of adults that regain spontaneous circulation die before leaving the hospital. Recently it has been shown that inducing hypothermia in cardiac arrest survivors immediately following their arrival in hospital can dramatically improve both overall survival and neurological outcomes. Despite the strong evidence for its efficacy and the apparent simplicity of this intervention, recent surveys show that therapeutic hypothermia is delivered inconsistently, incompletely, and often with delay.</p> <p>Methods and design</p> <p>This study will evaluate a multi-faceted knowledge translation strategy designed to increase the utilization rate of induced hypothermia in survivors of cardiac arrest across a network of 37 hospitals in Southwestern Ontario, Canada. The study is designed as a stepped wedge randomized trial lasting two years. Individual hospitals will be randomly assigned to four different wedges that will receive the active knowledge translation strategy according to a sequential rollout over a number of time periods. By the end of the study, all hospitals will have received the intervention. The primary aim is to measure the effectiveness of a multifaceted knowledge translation plan involving education, reminders, and audit-feedback for improving the use of induced hypothermia in survivors of cardiac arrest presenting to the emergency department. The primary outcome is the proportion of eligible OHCA patients that are cooled to a body temperature of 32 to 34°C within six hours of arrival in the hospital. Secondary outcomes will include process of care measures and clinical outcomes.</p> <p>Discussion</p> <p>Inducing hypothermia in cardiac arrest survivors immediately following their arrival to hospital has been shown to dramatically improve both overall survival and neurological outcomes. However, this lifesaving treatment is frequently not applied in practice. If this trial is positive, our results will have broad implications by showing that a knowledge translation strategy shared across a collaborative network of hospitals can increase the number of patients that receive this lifesaving intervention in a timely manner.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov Trial Identifier: <a href="http://www.clinicaltrials.gov/ct2/show/NCT00683683">NCT00683683</a></p

Protein expression in experimental malignant glioma varies over time and is altered by radiotherapy treatment

Radiotherapy is one of the mainstays of glioblastoma (GBM) treatment. This study aims to investigate and characterise differences in protein expression patterns in brain tumour tissue following radiotherapy, in order to gain a more detailed understanding of the biological effects. Rat BT4C glioma cells were implanted into the brain of two groups of 12 BDIX-rats. One group received radiotherapy (12 Gy single fraction). Protein expression in normal and tumour brain tissue, collected at four different time points after irradiation, were analysed using surface enhanced laser desorption/ionisation – time of flight – mass spectrometry (SELDI-TOF-MS). Mass spectrometric data were analysed by principal component analysis (PCA) and partial least squares (PLS). Using these multivariate projection methods we detected differences between tumours and normal tissue, radiation treatment-induced changes and temporal effects. 77 peaks whose intensity significantly changed after radiotherapy were discovered. The prompt changes in the protein expression following irradiation might help elucidate biological events induced by radiation. The combination of SELDI-TOF-MS with PCA and PLS seems to be well suited for studying these changes. In a further perspective these findings may prove to be useful in the development of new GBM treatment approaches

Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation

Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus

Imprinted CDKN1C Is a Tumor Suppressor in Rhabdoid Tumor and Activated by Restoration of SMARCB1 and Histone Deacetylase Inhibitors

SMARCB1 is deleted in rhabdoid tumor, an aggressive paediatric malignancy affecting the kidney and CNS. We hypothesized that the oncogenic pathway in rhabdoid tumors involved epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodelling, attributable to loss of SMARCB1, and that this hypothesis if proven could provide a biological rationale for testing epigenetic therapies in this disease. We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter. We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression. The histone deacetylase inhibitor (HDACi), Romidepsin, specifically restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC allelic expression, and induced cell cycle arrest in G401 and STM91-01 rhabdoid tumor cell lines. CDKN1C expression was also shown to be generally absent in clinical specimens of rhabdoid tumor, however CDKN1A and CDKN1B expression persisted. Our observations suggest that maintenance of CDKN1C expression plays a critical role in preventing rhabdoid tumor growth. Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor

Periodontitis and diabetes: a two-way relationship

Periodontitis is a common chronic inflammatory disease characterised by destruction of the supporting structures of the teeth (the periodontal ligament and alveolar bone). It is highly prevalent (severe periodontitis affects 10–15% of adults) and has multiple negative impacts on quality of life. Epidemiological data confirm that diabetes is a major risk factor for periodontitis; susceptibility to periodontitis is increased by approximately threefold in people with diabetes. There is a clear relationship between degree of hyperglycaemia and severity of periodontitis. The mechanisms that underpin the links between these two conditions are not completely understood, but involve aspects of immune functioning, neutrophil activity, and cytokine biology. There is emerging evidence to support the existence of a two-way relationship between diabetes and periodontitis, with diabetes increasing the risk for periodontitis, and periodontal inflammation negatively affecting glycaemic control. Incidences of macroalbuminuria and end-stage renal disease are increased twofold and threefold, respectively, in diabetic individuals who also have severe periodontitis compared to diabetic individuals without severe periodontitis. Furthermore, the risk of cardiorenal mortality (ischaemic heart disease and diabetic nephropathy combined) is three times higher in diabetic people with severe periodontitis than in diabetic people without severe periodontitis. Treatment of periodontitis is associated with HbA1c reductions of approximately 0.4%. Oral and periodontal health should be promoted as integral components of diabetes management

Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains

Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20–100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient β-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events

Childhood obesity, prevalence and prevention

Childhood obesity has reached epidemic levels in developed countries. Twenty five percent of children in the US are overweight and 11% are obese. Overweight and obesity in childhood are known to have significant impact on both physical and psychological health. The mechanism of obesity development is not fully understood and it is believed to be a disorder with multiple causes. Environmental factors, lifestyle preferences, and cultural environment play pivotal roles in the rising prevalence of obesity worldwide. In general, overweight and obesity are assumed to be the results of an increase in caloric and fat intake. On the other hand, there are supporting evidence that excessive sugar intake by soft drink, increased portion size, and steady decline in physical activity have been playing major roles in the rising rates of obesity all around the world. Consequently, both over-consumption of calories and reduced physical activity are involved in childhood obesity. Almost all researchers agree that prevention could be the key strategy for controlling the current epidemic of obesity. Prevention may include primary prevention of overweight or obesity, secondary prevention or prevention of weight regains following weight loss, and avoidance of more weight increase in obese persons unable to lose weight. Until now, most approaches have focused on changing the behaviour of individuals in diet and exercise. It seems, however, that these strategies have had little impact on the growing increase of the obesity epidemic. While about 50% of the adults are overweight and obese in many countries, it is difficult to reduce excessive weight once it becomes established. Children should therefore be considered the priority population for intervention strategies. Prevention may be achieved through a variety of interventions targeting built environment, physical activity, and diet. Some of these potential strategies for intervention in children can be implemented by targeting preschool institutions, schools or after-school care services as natural setting for influencing the diet and physical activity. All in all, there is an urgent need to initiate prevention and treatment of obesity in children

Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana

The potential use of algae in biofuels applications is receiving significant attention. However, none of the current algal model species are competitive production strains. Here we present a draft genome sequence and a genetic transformation method for the marine microalga Nannochloropsis gaditana CCMP526. We show that N. gaditana has highly favourable lipid yields, and is a promising production organism. The genome assembly includes nuclear (~29 Mb) and organellar genomes, and contains 9,052 gene models. We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis. Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed. The availability of a genome sequence and transformation methods will facilitate investigations into N. gaditana lipid biosynthesis and permit genetic engineering strategies to further improve this naturally productive alga