Shotgun DNA mapping (SDM) is the ability to identify the genomic location of a random DNA fragmen... more Shotgun DNA mapping (SDM) is the ability to identify the genomic location of a random DNA fragment based on its naked DNA unzipping forces compared with simulated unzipping forces of a published genome. We have previously demonstrated proof of principle for shotgun DNA mapping by using plasmid pBR322 unzipping data amongst yeast genome background[cite preprint]. Currently we are validating the technique using unzipping data from yeast genomic DNA. Genomic DNA from yeast (S. cerevisiae) has been digested with restriction endonucleases to produce a library of random fragments, which we used to create a limited library of shotgun clones. Single-molecule unzipping constructs derived from these clones will be unzipping with optical tweezers (OT). In parallel, we have created a library of simulated possible unzipping force profiles, based on the known yeast genome sequence. The OT data and the library will be used in our existing SDM algorithms to identify each shotgun clone, and success rate will be determined via DNA sequencing of the clones. A major application of SDM we are working towards is mapping of nucleosomes and RNA Polymerase II molecules on native chromatin. We will report our progress towards this goal and also discuss other applications of SDM, including splice variant and telomere analysis.
Ability to m a p polymerases and nucleoso m e s on chro m atin is important for understanding the... more Ability to m a p polymerases and nucleoso m e s on chro m atin is important for understanding the impact of chro m atin remo deling on key cellular processes. Current m ethods (such as ChIP and C hIPchip) have produced a w ealth of information that de m o n strates this importance, but key information is elusive in these ense m ble m ethods. W e're pursuing a ne w singlem olecule chro m atin m a p ping m ethod based on unzipping native chro m atin m olecules with optical tweezers. The first step w e are taking towards this ability is shotgun D N A m a p ping (SD M). This is the ability to identify the geno mic location of a rando m D N A frag me nt based on its naked D N A unzipping forces co m p ared with simulated unzipping forces of a published geno m e. W e sho w that [~32] separate experimental unzipping curves for pB R 3 2 2 w ere correctly m atched to their simulated unzipping curves hidden in a background of the [~2700] sequences neighboring XhoI sites in the S. cerevisiae (yeast) geno m e. W e describe this m ethod and characterize its robustness as w ell as discuss future applications. Abbreviations S D M = Shotgun D N A M a p ping; S M = singlem olecule; ChIP = C hro m atin Im m u n o precipitation;
To investigate any gender effect of the beta-1 adrenergic blocker, landiolol, on cardiac performa... more To investigate any gender effect of the beta-1 adrenergic blocker, landiolol, on cardiac performance and energy metabolism in septic rats, and to explore the expression of genes and proteins involved in this process. Randomized animal study. University research laboratory. Male and female Wistar rats. One hour after cecal ligation and puncture, male and female rats were randomly allocated to the following groups: sham male, cecal ligation and puncture male, cecal ligation and puncture + landiolol male, sham female, cecal ligation and puncture female, and cecal ligation and puncture + landiolol female. Cardiac MRI was carried out 18 hours after cecal ligation and puncture to assess in vivo cardiac function. Ex vivo cardiac function measurement and P magnetic resonance spectroscopy were subsequently performed using an isovolumic isolated heart preparation. Finally, we assessed cardiac gene and protein expression. In males, landiolol increased indexed stroke volume by reversing the ind...
Streptococcus suis infection is an emerging zoonosis in Asia. We determined the detailed epidemio... more Streptococcus suis infection is an emerging zoonosis in Asia. We determined the detailed epidemiological, clinical, and microbiological characteristics of S. suis meningitis in adults. We prospectively studied 450 patients with suspected bacterial meningitis. Four hundred thirty-five (96.7%) of the patients participated in a trial to determine the effect of adjunctive dexamethasone treatment. For patients with S. suis infection, bacterial DNA load at hospital admission and during treatment was analyzed in cerebrospinal fluid specimens using quantitative real-time polymerase chain reaction. S. suis strains were characterized using pulsed-field gel electrophoresis and multilocus sequence typing. Putative virulence factors, including extracellular protein factor, suilysin, and muramidase released protein, were detected using polymerase chain reaction and Western blot assay. Predictors of outcome were identified using logistic regression analysis. S. suis was the most common pathogen an...
Shotgun DNA mapping (SDM) is the ability to identify the genomic location of a random DNA fragmen... more Shotgun DNA mapping (SDM) is the ability to identify the genomic location of a random DNA fragment based on its naked DNA unzipping forces compared with simulated unzipping forces of a published genome. We have previously demonstrated proof of principle for shotgun DNA mapping by using plasmid pBR322 unzipping data amongst yeast genome background[cite preprint]. Currently we are validating the technique using unzipping data from yeast genomic DNA. Genomic DNA from yeast (S. cerevisiae) has been digested with restriction endonucleases to produce a library of random fragments, which we used to create a limited library of shotgun clones. Single-molecule unzipping constructs derived from these clones will be unzipping with optical tweezers (OT). In parallel, we have created a library of simulated possible unzipping force profiles, based on the known yeast genome sequence. The OT data and the library will be used in our existing SDM algorithms to identify each shotgun clone, and success rate will be determined via DNA sequencing of the clones. A major application of SDM we are working towards is mapping of nucleosomes and RNA Polymerase II molecules on native chromatin. We will report our progress towards this goal and also discuss other applications of SDM, including splice variant and telomere analysis.
Ability to m a p polymerases and nucleoso m e s on chro m atin is important for understanding the... more Ability to m a p polymerases and nucleoso m e s on chro m atin is important for understanding the impact of chro m atin remo deling on key cellular processes. Current m ethods (such as ChIP and C hIPchip) have produced a w ealth of information that de m o n strates this importance, but key information is elusive in these ense m ble m ethods. W e're pursuing a ne w singlem olecule chro m atin m a p ping m ethod based on unzipping native chro m atin m olecules with optical tweezers. The first step w e are taking towards this ability is shotgun D N A m a p ping (SD M). This is the ability to identify the geno mic location of a rando m D N A frag me nt based on its naked D N A unzipping forces co m p ared with simulated unzipping forces of a published geno m e. W e sho w that [~32] separate experimental unzipping curves for pB R 3 2 2 w ere correctly m atched to their simulated unzipping curves hidden in a background of the [~2700] sequences neighboring XhoI sites in the S. cerevisiae (yeast) geno m e. W e describe this m ethod and characterize its robustness as w ell as discuss future applications. Abbreviations S D M = Shotgun D N A M a p ping; S M = singlem olecule; ChIP = C hro m atin Im m u n o precipitation;
To investigate any gender effect of the beta-1 adrenergic blocker, landiolol, on cardiac performa... more To investigate any gender effect of the beta-1 adrenergic blocker, landiolol, on cardiac performance and energy metabolism in septic rats, and to explore the expression of genes and proteins involved in this process. Randomized animal study. University research laboratory. Male and female Wistar rats. One hour after cecal ligation and puncture, male and female rats were randomly allocated to the following groups: sham male, cecal ligation and puncture male, cecal ligation and puncture + landiolol male, sham female, cecal ligation and puncture female, and cecal ligation and puncture + landiolol female. Cardiac MRI was carried out 18 hours after cecal ligation and puncture to assess in vivo cardiac function. Ex vivo cardiac function measurement and P magnetic resonance spectroscopy were subsequently performed using an isovolumic isolated heart preparation. Finally, we assessed cardiac gene and protein expression. In males, landiolol increased indexed stroke volume by reversing the ind...
Streptococcus suis infection is an emerging zoonosis in Asia. We determined the detailed epidemio... more Streptococcus suis infection is an emerging zoonosis in Asia. We determined the detailed epidemiological, clinical, and microbiological characteristics of S. suis meningitis in adults. We prospectively studied 450 patients with suspected bacterial meningitis. Four hundred thirty-five (96.7%) of the patients participated in a trial to determine the effect of adjunctive dexamethasone treatment. For patients with S. suis infection, bacterial DNA load at hospital admission and during treatment was analyzed in cerebrospinal fluid specimens using quantitative real-time polymerase chain reaction. S. suis strains were characterized using pulsed-field gel electrophoresis and multilocus sequence typing. Putative virulence factors, including extracellular protein factor, suilysin, and muramidase released protein, were detected using polymerase chain reaction and Western blot assay. Predictors of outcome were identified using logistic regression analysis. S. suis was the most common pathogen an...
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