ALBERT

Description: Background: Burkholderia pseudomallei is an emerging pathogen that causes melioidosis, a serious and potentially fatal disease which requires prolonged antibiotics to prevent relapse. However, diagnosis of melioidosis can be difficult, especially in culture-negative cases. While metabolomics represents an uprising tool for studying infectious diseases, there were no reports on its applications to B. pseudomallei. To search for potential specific biomarkers, we compared the metabolomics profiles of culture supernatants of B. pseudomallei (15 strains), B. thailandensis (3 strains), B. cepacia complex (14 strains), P. aeruginosa (4 strains) and E. coli (3 strains), using ultra-high performance liquid chromatography-electrospray ionization-quadruple time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS). Multi- and univariate analyses were used to identify specific metabolites in B. pseudomallei. Results: Principal component and partial-least squares discrimination analysis readily distinguished the metabolomes between B. pseudomallei and other bacterial species. Using multi-variate and univariate analysis, eight metabolites with significantly higher levels in B. pseudomallei were identified. Three of the eight metabolites were identified by MS/MS, while five metabolites were unidentified against database matching, suggesting that they may be potentially novel compounds. One metabolite, m/z 144.048, was identified as 4-methyl-5-thiazoleethanol, a degradation product of thiamine (vitamin B1), with molecular formula C6H9NOS by database searches and confirmed by MS/MS using commercially available authentic chemical standard. Two metabolites, m/z 512.282 and m/z 542.2921, were identified as tetrapeptides, Ile-His-Lys-Asp with molecular formula C22H37N7O7 and Pro-Arg-Arg-Asn with molecular formula C21H39N11O6, respectively. To investigate the high levels of 4-methyl-5-thiazoleethanol in B. pseudomallei, we compared the thiamine degradation pathways encoded in genomes of B. pseudomallei and B. thailandensis. While both B. pseudomallei and B. thailandensis possess thiaminase I which catalyzes degradation of thiamine to 4-methyl-5-thiazoleethanol, thiM, which encodes hydroxyethylthiazole kinase responsible for degradation of 4-methyl-5-thiazoleethanol, is present and expressed in B. thailandensis as detected by PCR/RT-PCR, but absent or not expressed in all B. pseudomallei strains. This suggests that the high 4-methyl-5-thiazoleethanol level in B. pseudomallei is likely due to the absence of hydroxyethylthiazole kinase and hence reduced downstream degradation. Conclusion: Eight novel biomarkers, including 4-methyl-5-thiazoleethanol and two tetrapeptides, were identified in the culture supernatant of B. pseudomallei.

Description: Background: The explanted, developing rodent retina provides an efficient and accessible preparation for use in gene transfer and pharmacological experimentation. Many of the features of normal development are retained in the explanted retina, including retinal progenitor cell proliferation, heterochronic cell production, interkinetic nuclear migration, and connectivity. To date, live imaging in the developing retina has been reported in non-mammalian and mammalian whole-mount samples. An integrated approach to rodent retinal culture/transfection, live imaging, cell tracking, and analysis in structurally intact explants greatly improves our ability to assess the kinetics of cell production. Results: In this report, we describe the assembly and maintenance of an in vitro, CO2-independent, live mouse retinal preparation that is accessible by both upright and inverted, 2-photon or confocal microscopes. The optics of this preparation permit high-quality and multi-channel imaging of retinal cells expressing fluorescent reporters for up to 48h. Tracking of interkinetic nuclear migration within individual cells, and changes in retinal progenitor cell morphology are described. Follow-up, hierarchical cluster screening revealed that several different dependent variable measures can be used to identify and group movement kinetics in experimental and control samples. Conclusions: Collectively, these methods provide a robust approach to assay multiple features of rodent retinal development using live imaging.

Description: Background: The traditional problems of performing skeletal muscle cell cultures derived from mammalian or avian species are limited myotube differentiation, and transient myotube persistence which greatly restricts the ability of myotubes to undergo phenotypic maturation. We report here on a major technical breakthrough in the establishment of a simple and effective method of extended porcine myotube cultures (beyond 50 days) in two-dimension (2D) that recapitulates key features of postnatal fibre types. Results: Primary porcine muscle satellite cells (myoblasts) were isolated from the longissimus dorsi of 4 to 6 weeks old pigs for 2D cultures to optimise myotube formation, improve surface adherence and characterise myotube maturation. Over 95 % of isolated cells were myoblasts as evidenced by the expression of Pax3 and Pax7. Our relatively simple approach, based on modifications of existing surface coating reagents (Maxgel), and of proliferation and differentiation (Ultroser G) media, typically achieved by 5 days of differentiation fusion index of around 80 % manifested in an abundance of discrete myosin heavy chain (MyHC) slow and fast myotubes. There was little deterioration in myotube viability over 50 days, and the efficiency of myotube formation was maintained over seven myoblast passages. Regular spontaneous contractions of myotubes were frequently observed throughout culture. Myotubes in extended cultures were able to undergo phenotypic adaptation in response to different culture media, including the adoption of a dominant postnatal phenotype of fast-glycolytic MyHC 2x and 2b expression by about day 20 of differentiation. Furthermore, fast-glycolytic myotubes coincided with enhanced expression of the putative porcine long intergenic non-coding RNA (linc-MYH), which has recently been shown to be a key coordinator of MyHC 2b expression in vivo. Conclusions: Our revised culture protocol allows the efficient differentiation and fusion of porcine myoblasts into myotubes and their prolonged adherence to the culture surface. Furthermore, we are able to recapitulate in 2D the maturation process of myotubes to resemble postnatal fibre types which represent a major technical advance in opening access to the in vitro study of coordinated postnatal muscle gene expression.

Description: Pseudomonas syringae pv. tabaci (Pst), which is the pathogen responsible for tobacco wildfire disease, has received considerable attention in recent years. The objective of this study ...

Description: Background: Current methods of isolation of muscle satellite cells from different animal species are highlyvariable making inter-species comparisons problematic. This variation mainly stems from theuse of different proteolytic enzymes to release the satellite cells from the muscle tissue(sometimes a single enzyme is used but often a combination of enzymes is preferred) and thedifferent extracellular matrix proteins used to coat culture ware. In addition, isolation ofsatellite cells is frequently laborious and requires pre-plating of the cell preparation onuncoated flasks or Percoll centrifugation to remove contaminating fibroblasts. Themethodology employed to isolate and culture satellite cells in vitro can critically determinethe fusion of myoblasts into multi-nucleated myotubes. These terminally differentiatedmyotubes resemble mature myofibres in the muscle tissue in vivo, therefore optimal fusion isa keystone of in vitro muscle culture. Hence, a simple method of muscle satellite cellisolation and culture of different vertebrate species that can result in a high fusion rate ishighly desirable. Results: We demonstrate here a relatively simple and rapid method of isolating highly enrichedmuscle satellite cells from different avian and mammalian species without the need of Percollcentrifugation or pre-plating. In brief, muscle tissue was mechanically dissociated, digestedwith a single enzyme (pronase), triturated with a 10-ml pipette, filtered and directly platedonto collagen coated flasks. Following this method and after optimization of the cell cultureconditions, excellent fusion rates were achieved in the duck, chicken, horse and cow (withmore than 50% cell fusion), and to a lesser extent pig, pointing to pronase as a highly suitableenzyme to release satellite cells from muscle tissue. Conclusions: Our simplified method presents a quick and simple alternative to isolating highly enrichedmuscle satellite cell cultures which can subsequently rapidly differentiate into well developedprimary myotubes. The use of the same isolation protocol allows better inter-speciescomparisons of muscle satellite cells. Of all the farm animal species investigated, harvestedchicken muscle cells showed the highest percentage of muscle satellite cells, and equinemuscle cells presented the highest fusion index, an impressive [almost equal to] 77%. Porcine cells displayedthe lowest amount of satellite cells but still achieved a modest fusion rate of [almost equal to] 41%.

Description: Background: Neutral lipid storage is enhanced by nitrogen deprivation (ND) in numbers of green microalgal species. However, little is known about the metabolic pathways whose transcription levels are most significantly altered following ND in green microalgae, especially the nonmodel species. Results: To start gaining knowledge on this, we performed transcriptome profiling of the nonmodel green microalga Botryosphaerella sudeticus cells in response to ND. Transcriptome of B. sudeticus is de novo assembled based on millions of HiSEQ short sequence reads using CLC Genomics Workbench software. The resulting non-redundant ESTs are annotated based on the best hits generated from the BLASTX homology comparison against the "best" proteins in the model microalgae Chlamydomonas reinhardtii and Chlorella variabilis. By using a pathway-based approach according to KEGG databases, we show that ESTs encoding ribosomal proteins and photosynthetic functions are the most abundantly expressed ESTs in the rapidly growing B. sudeticus cells. We find that ESTs encoding photosynthetic function but not the ribosomal proteins are most drastically downregulated upon ND. Notably, ESTs encoding lipid metabolic pathways are not significantly upregulated. Further analyses indicate that chlorophyll content is markedly decreased by 3-fold and total lipid content is only slightly increased by 50%, consistent with the transcriptional profiling. On the other hand, carbon content and photosynthesis efficiency are only marginally decreased by 7% and 20%, respectively, indicating that photosynthesis is only slightly reduced upon drastic downregulation of photosynthetic ESTs and chlorophyll content upon ND. In addition, TAG content is found to be greatly increased by 50-fold, though total lipid content is only slightly increased by 1.5-fold. Conclusions: Taken together, our results suggest that light-harvesting proteins and chlorophylls are in excess in B. sudeticus. Degradation of excess photosynthesis proteins is most likely a mechanism for recycling of nitrogen-rich molecules to synthesize new proteins for preparation of gametogenesis and zygospore formation in adaptation and survival upon ND. Furthermore, our analyses indicate that TAG accumulation is largely attributed to the modification of other pre-existing lipid molecules, rather than de novo synthesis. We propose that this is likely an evolutionarily conserved mechanism in many green microalgae species.

Description: Background: Myeloproliferative neoplasms (MPNs) are a group of haematological malignancies that can be characterised by a somatic mutation (JAK2V617F). This mutation causes the bone marrow to produce excessive blood cells and is found in polycythaemia vera (~95%), essential thrombocythaemia and primary myelofibrosis (both ~50%). It is considered as a major genetic factor contributing to the development of these MPNs. No genetic association study of MPN in the Hong Kong population has so far been reported. Here, we investigated the relationship between germline JAK2 polymorphisms and MPNs in Hong Kong Chinese to find causal variants that contribute to MPN development. We analysed 19 tag single nucleotide polymorphisms (SNPs) within the JAK2 locus in 172 MPN patients and 470 healthy controls. Three of these 19 SNPs defined the reported JAK2 46/1 haplotype: rs10974944, rs12343867 and rs12340895. Allele and haplotype frequencies were compared between patients and controls by logistic regression adjusted for sex and age. Permutation test was used to correct for multiple comparisons. With significant findings from the 19 SNPs, we then examined 76 additional SNPs across the 148.7-kb region of JAK2 via imputation with the SNP data from the 1000 Genomes Project. Results: In single-marker analysis, 15 SNPs showed association with JAK2V617F-positive MPNs (n?=?128), and 8 of these were novel MPN-associated SNPs not previously reported. Exhaustive variable-sized sliding-window haplotype analysis identified 184 haplotypes showing significant differences (P?

Description: Background: The population aged 85 + - the "oldest old" - is now the fastest growing age segment in Canada. Although existing research demonstrates high health services utilization and medication burden in this population, little clinically derived evidence is available to guide care. This is a descriptive study in a primary care context seeking to describe the most common health conditions and medications used in the "oldest old". Methods: We conducted a retrospective chart review of all family practice patients aged 85+ (N = 564; 209 males, 355 females) at Sunnybrook Health Sciences Centre in Toronto, Canada. Electronic medical records were reviewed for all current chronic conditions and medication prescriptions, and then stratified by sex and age subgroup (85-89, 90-94, 95+) for descriptive analysis. Results: On average, patients experienced 6.4 concurrent chronic conditions and took 6.8 medications. Most conditions were related to cardiovascular (79%) and bone health (65%). Hypertension (65%) was the most common condition. Bone-related conditions (e.g. osteoarthritis, osteoporosis) and hypothyroidism predominantly affected women, while coronary artery disease and type 2 diabetes were more prevalent in men. The top two prescribed medications were atorvastatin (33%) and aspirin 81 mg (33%). Males were more likely to be prescribed lipid-lowering medications, while females were more likely to receive osteoporosis therapy. Patients received less lipid-lowering therapy with increasing age. Conclusions: Multimorbidity and polypharmacy are highly prevalent in patients in the 85+ age group. The most common clinical conditions are related to cardiovascular and bone health, and the most commonly prescribed medications are directed towards risk factors for these illnesses. In the absence of data to guide clinical decision-making, this study provides a first look at the common health concerns and medication profiles in this population and reveals trends that give rise to reflections on how clinical care for these patients can be improved.

Description: Background: Tissue regeneration in the lungs is gaining increasing interest as a potential influenza management strategy. In this study, we explored the role of microRNAs, short non-coding RNAs involved in post-transcriptional regulation, during pulmonary regeneration after influenza infection. Results: We profiled miRNA and mRNA expression levels following lung injury and tissue regeneration using a murine influenza pneumonia model. BALB/c mice were infected with a sub-lethal dose of influenza A/PR/8(H1N1) virus, and their lungs were harvested at 7 and 15 days post-infection to evaluate the expression of ~300 miRNAs along with ~36,000 genes using microarrays. A global network was constructed between differentially expressed miRNAs and their potential target genes with particular focus on the pulmonary repair and regeneration processes to elucidate the regulatory role of miRNAs in the lung repair pathways. The miRNA arrays revealed a global down-regulation of miRNAs. TargetScan analyses also revealed specific miRNAs highly involved in targeting relevant gene functions in repair such as miR-290 and miR-505 at 7 dpi; and let-7, miR-21 and miR-30 at 15 dpi. Conclusion: The significantly differentially regulated miRNAs are implicated in the activation or suppression of cellular proliferation and stem cell maintenance, which are required during the repair of the damaged lungs. These findings provide opportunities in the development of novel repair strategies in influenza-induced pulmonary injury.

Description: Background: Ralstonia solanacearum, the causal agent of bacterial wilt, is a genetically diverse bacterial plant pathogen present in tropical and subtropical regions of the world that infects more than 200 plant species, including economically important solanaceous crops. Most strains of R. solanacearum are only pathogenic at temperatures between 25 to 30[degree sign]C with strains that can cause disease below 20[degree sign]C considered a threat to agriculture in temperate areas. Identifying key molecular factors that distinguish strains virulent at cold temperatures from ones that are not is needed to develop effective management tools for this pathogen. We compared protein profiles of two strains virulent at low temperature and two strains not virulent at low temperature when incubated in the rhizosphere of tomato seedlings at 30 and 18[degree sign]C using quantitative 2D DIGE gel methods. Spot intensities were quantified and compared, and differentially expressed proteins were sequenced and identified by mass spectrometry (MS/MS). Results: Four hundred and eighteen (418) differentially expressed protein spots sequenced produced 101 unique proteins. The identified proteins were classified in the Gene Ontology biological processes categories of metabolism, cell processes, stress response, transport, secretion, motility, and virulence. Identified virulence factors included catalase (KatE), exoglucanase A (ChbA), drug efflux pump, and twitching motility porin (PilQ). Other proteins identified included two components of a putative type VI secretion system. We confirmed differential expression of 13 candidate genes using real time PCR techniques. Global regulators HrpB and HrpG also had temperature dependent expression when quantified by real time PCR. Conclusions: The putative involvement of the identified proteins in virulence at low temperature is discussed. The discovery of a functional type VI secretion system provides a new potential virulence mechanism to explore. The global regulators HrpG and HrpB, and the protein expression profiles identified suggest that virulence at low temperatures can be partially explained by differences in regulation of virulence factors present in all the strains.