Identification and characterization of early photoreceptor cis-regulatory elements and their relation to Onecut1

Background: Cone and rod photoreceptors are two of the primary cell types affected in human retinal disease. Potential strategies to combat these diseases are the use of gene therapy to rescue compromised photoreceptors or to generate new functional photoreceptors to replace those lost in the diseased retina. Cis-regulatory elements specific to cones, rods, or both types of photoreceptors are critical components of successful implementation of these two strategies. The purpose of this study was to identify and characterize the cell type specificity and activity of cis-regulatory elements active in developing photoreceptors. Methods: Cis-regulatory elements were introduced into the developing chicken and mouse retina by electroporation. Characterization of reporter activity in relation with cell type markers was determined using confocal microscopy. In addition, two high-throughput flow cytometry assay were developed to assess whether these elements were downstream of Onecut1 in the photoreceptor specification network. Results: The majority of cis-regulatory elements were active in both cone and rod photoreceptors and were largely uninfluenced by a Onecut1 dominant-negative construct. Elements associated with the Thrb, Nr2e3, and Rhodopsin genes showed highly enriched activity in cones or rods, and were affected by interference in Onecut1 signaling. Rhodopsin promoter activity was the most highly influenced by Onecut1 activity and its induction could be modulated by the Maf family transcription factor L-Maf. Nr2e3 elements were observed to have activity in cone photoreceptors and Nr2e3 protein was expressed in developing cone photoreceptors, suggesting a role for this predominant rod gene in cone photoreceptor development. Conclusions: The analysis presented here provides an experimental framework to determine the specificity and strength of photoreceptor elements within specific genetic networks during development. The Onecut1 transcription factor is one such factor that influences the gene regulatory networks specific to cones and rods, but not those that are common to both

The production of fluorescent transgenic trout to study in vitro myogenic cell differentiation

<p>Abstract</p> <p>Background</p> <p>Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (<it>Oncorhynchus mykiss</it>) carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter, and cultivated genetically modified myogenic cells derived from these fish.</p> <p>Results</p> <p>In transgenic trout, green fluorescence appeared in fast muscle fibers as early as the somitogenesis stage and persisted throughout life. Using an <it>in vitro </it>myogenesis system we observed that satellite cells isolated from the myotomal muscle of transgenic trout expressed GFP about 5 days post-plating as they started to fuse. GFP fluorescence persisted subsequently in myosatellite cell-derived myotubes. Using this <it>in vitro </it>myogenesis system, we showed that the rate of muscle cell differentiation was strongly dependent on temperature, one of the most important environmental factors in the muscle growth of poikilotherms.</p> <p>Conclusions</p> <p>We produced MLC2f-gfp transgenic trout that exhibited fluorescence in their fast muscle fibers. The culture of muscle cells extracted from these trout enabled the real-time monitoring of myogenic differentiation. This <it>in vitro </it>myogenesis system could have numerous applications in fish physiology to evaluate the myogenic activity of circulating growth factors, to test interfering RNA and to assess the myogenic potential of fish mesenchymal stem cells. In ecotoxicology, this system could be useful to assess the impact of environmental factors and marine pollutants on fish muscle growth.</p

Automated SNP detection from a large collection of white spruce expressed sequences: contributing factors and approaches for the categorization of SNPs

BACKGROUND: High-throughput genotyping technologies represent a highly efficient way to accelerate genetic mapping and enable association studies. As a first step toward this goal, we aimed to develop a resource of candidate Single Nucleotide Polymorphisms (SNP) in white spruce (Picea glauca [Moench] Voss), a softwood tree of major economic importance. RESULTS: A white spruce SNP resource encompassing 12,264 SNPs was constructed from a set of 6,459 contigs derived from Expressed Sequence Tags (EST) and by using the bayesian-based statistical software PolyBayes. Several parameters influencing the SNP prediction were analysed including the a priori expected polymorphism, the probability score (P(SNP)), and the contig depth and length. SNP detection in 3' and 5' reads from the same clones revealed a level of inconsistency between overlapping sequences as low as 1%. A subset of 245 predicted SNPs were verified through the independent resequencing of genomic DNA of a genotype also used to prepare cDNA libraries. The validation rate reached a maximum of 85% for SNPs predicted with either P(SNP )≥ 0.95 or ≥ 0.99. A total of 9,310 SNPs were detected by using P(SNP )≥ 0.95 as a criterion. The SNPs were distributed among 3,590 contigs encompassing an array of broad functional categories, with an overall frequency of 1 SNP per 700 nucleotide sites. Experimental and statistical approaches were used to evaluate the proportion of paralogous SNPs, with estimates in the range of 8 to 12%. The 3,789 coding SNPs identified through coding region annotation and ORF prediction, were distributed into 39% nonsynonymous and 61% synonymous substitutions. Overall, there were 0.9 SNP per 1,000 nonsynonymous sites and 5.2 SNPs per 1,000 synonymous sites, for a genome-wide nonsynonymous to synonymous substitution rate ratio (Ka/Ks) of 0.17. CONCLUSION: We integrated the SNP data in the ForestTreeDB database along with functional annotations to provide a tool facilitating the choice of candidate genes for mapping purposes or association studies

Dubreuilville (Canada): A Strategic Action Plan for a Rural Community

The town of Dubreuilville, with a population of just under 1,000, is located in Northern Ontario, Canada, approximately one thousand kilometers North-West of Toronto. The region was previously known for lumber production. When the Canadian lumber market collapsed due to the imposition of duties by Washington, the local mill shut down. Large gold deposits were found nearby, and Dubreuilville became a mining town almost overnight. Half of the population had left by the time the mine opened, and part of the workforce commutes from cities located up to six-hour drives away. With the opening of a second gold mine in the area planned for 2023, Dubreuilville had to prepare to attract and retain more workers for the project to succeed and for the town to prosper. Below is the strategic plan that was prepared by Mayor Nantel and Economic Development Officer Melanie Pilon, with the help of a Laurentian University Management professor and three students

Differential analysis of glioblastoma multiforme proteome by a 2D-DIGE approach

<p>Abstract</p> <p>Background</p> <p>Genomics, transcriptomics and proteomics of glioblastoma multiforme (GBM) have recently emerged as possible tools to discover therapeutic targets and biomarkers for new therapies including immunotherapy. It is well known that macroscopically complete surgical excision, radiotherapy and chemotherapy have therapeutic limitations to improve survival in these patients. In this study, we used a differential proteomic-based technique (2D-Difference Gel Electrophoresis) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify proteins that may serve as brain tumor antigens in new therapeutic assays. Five samples of patients presenting a GBM and five samples of microscopically normal brain tissues derived from brain epileptic surgery specimen were labeled and run in 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis) with an internal pool sample on each gel. Five gels were matched and compared with DIA (Difference In-gel Analysis) software. Differential spots were picked, in-gel digested and peptide mass fingerprints were obtained.</p> <p>Results</p> <p>From 51 protein-spots significantly up-regulated in GBM samples, mass spectrometry (MS) identified twenty-two proteins. The differential expression of a selected protein set was first validated by western-blotting, then tested on large cohorts of GBM specimens and non-tumor tissues, using immunohistochemistry and real-time RT-PCR.</p> <p>Conclusions</p> <p>Our results confirmed the importance of previously described proteins in glioma pathology and their potential usefulness as biological markers but also revealed some new interesting targets for future therapies.</p

Better Control of Holder Pasteurization Results in Higher Retention of Human Milk Lactoferrin, IgA, and Lysozyme

Background: Holder pasteurization is commonly used in milk banks. We previously reported that the pattern of temperature and time may be different according to the pasteurizer used.Aim: The aim of our study was to assess the variances in pasteurization using two different devices: a standard pasteurizer (Past STD) and an optimized pasteurizer (Past OPTI).Methods: Immunoglobulin A (IgA), lactoferrin (LF), and lysozyme (LZ) content were assessed before and after pasteurization of 24 donor human milk samples. The impact of the pasteurization device was evaluated by testing 50- to 200-mL samples.Results: Mean temperature and duration of the plateau were 1.5°C lower and 11 min shorter, respectively, with Past OPTI vs. Past STD. The loss of IgA, LF, and LZ was 17.6, 5.6, and 9.8% lower, respectively, with Past OPTI than with Past STD.Conclusions: Accurate control of temperature enabled better preservation of IgA, LF, and LZ in donor milk. Holder pasteurization should be optimized, and new techniques proposed to treat donor milk should be compared with Holder pasteurization performed with a well-controlled device under realistic conditions

Individualized Fortification Influences the Osmolality of Human Milk

Background: Fortification of human milk (HM) increases its osmolality, which is associated with an increased risk of necrotizing enterocolitis. The impact of new fortifiers on osmolality is not well-known, nor are the kinetics regarding the increase in osmolality.Aim: To determine the optimum fortifier composition for HM fortification by measuring the osmolality of fortified HM made with three powder multicomponent fortifiers (MCFs) and a protein fortifier (PF).Methods: The osmolality of HM was assessed at 2 (H2) and 24 (H24) h after fortification to compare the effects of MCF (MCF1–3) and PF used in quantities that ensured that infants' nutrient needs would be met (MCF: 4 g/100 ml HM; PF: 0.5 g or 1 g/100 ml HM). To evaluate the early kinetics associated with the osmolality increase, the osmolality of HM fortified with MCF1 or MCF2 was also measured at 0, 1, 5, 10, 15, 20, 30, 40, 50, 60, 90, and 120 min after fortification.Results: The osmolality increased significantly immediately after fortification, depending on the type of fortification used and the quantity of MCF and PF used, rather than the time elapsed after fortification. The maximum value at H24 was 484 mOsm/kg. The mean increase in osmolality between H2 and H24 was 3.1% (p &lt; 0.01) (range: 0.2–10.8%). Most of the increase (&gt;70%) occurred immediately after fortification.Conclusion: When choosing a fortifier, its effect on HM osmolality should be considered. As most of the increase in osmolality occurred immediately, bedside fortification is not useful to prevent the increase in osmolality, and further research should focus on improving fortifier composition

Effects of caffeine and/or nasal CPAP treatment on laryngeal chemoreflexes in preterm lambs

Current knowledge suggests that laryngeal chemoreflexes (LCR) are involved in the occurrence of certain neonatal apneas/bradycardias, especially in the preterm newborn. While caffeine and/or nasal continuous positive airway pressure (nCPAP) are the most frequent options used for treating apneas in preterm newborns, their effects on LCR-related apneas/bradycardias are virtually unknown. The aim of the present study was to test the hypothesis that caffeine and/or nCPAP decreases LCR-related cardiorespiratory inhibition in a preterm ovine model. Seven preterm lambs were born vaginally on gestational day 133 (normal gestation: 147 days) after intramuscular injections of betamethasone and mifepristone. Five days after birth, a chronic surgical instrumentation was performed to record states of alertness, electrocardiogram, systemic arterial pressure, and electromyographic activity of a laryngeal constrictor muscle, as well as to insert a transcutaneous supraglottal catheter. LCR were induced in quiet sleep under four conditions: 1) control (without caffeine or nCPAP); 2) nCPAP (5 cmH2O, without caffeine); 3) caffeine (10 mg/kg infused intravenously for 30 min, without nCPAP); and 4) nCPAP + caffeine. Our results showed that nCPAP consistently blunted LCR-related cardiorespiratory inhibition vs. control condition, contrary to caffeine whose overall effect was nonsignificant. In addition, nCPAP condition was characterized by a more consistent and rapid arousal after HCl injection. No significant differences were observed between all tested conditions with regard to swallowing and cough. It is concluded that nCPAP should be further assessed for its usefulness in treating neonatal apneas linked to LCR