Project description:Burkholderia cenocepacia J2315 was repeatedly and intermittently exposed to tobramycin, ciprofloxacin or meropenem. Bacteria were grown on cryobeads submerged in liquid BHI medium. After 24 hours, the beads were washed and fresh medium with of without antibiotics added. After another 24 hours of incubation, the beads were washed, the bacteria removed from the beads, and used for inoculation of fresh beads. This was repeated to a total of up to ten cycles. Evolved lineages were then DNA-sequenced to screen for genome changes.
Project description:Pseudomonas aeruginosa AA2 was repeatedly and intermittently exposed to tobramycin, ciprofloxacin or meropenem. Bacteria were grown on cryobeads submerged in liquid BHI medium. After 24 hours, the beads were washed and fresh medium with of without antibiotics added. After another 24 hours of incubation, the beads were washed, the bacteria removed from the beads, and used for inoculation of fresh beads. This was repeated to a total of up to ten cycles. Evolved lineages were then DNA-sequenced to screen for genome changes.
Project description:Pseudomonas aeruginosa was repeatedly and intermittently exposed to tobramycin. Bacteria were grown in synthetic cystic fibrosis medium in wells of a 96-well microtiter plate. After 24 hours, more medium with or without tobramycin was added. After another 24 hours of incubation, a subsample of the well content was used to inoculate fresh synthetic cystic fibrosis medium in a 96-well microtiter plate. This was repeated for a total of 15 cycles. Evolved lineages were then DNA-sequenced to screen for genome changes.
Project description:We analyzed a developmental time course of ten different mutants with a combination of single and double mutants in 6 genes. The samples were collected at the following time points following starvation: 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours. Dissimilarities between mutant time courses were measured by calculating both Euclidean distances and Pearson's correlation based distances between the scaled mutant time courses.
Project description:RNA-seq analysis to assess the effect of different drugs (34503, 36009, 34503, 36018, Moxi) on Mycobacterium tuberculosis compared to untreated control cells (DMSO).
Project description:Exponentially growing S.aureus ATCC 25923 was treated with linezolid (LZD) at concentrations of 1/2× MIC, 1× MIC, and 5× MIC, or an equivalent amount of DMSO for 1 hour. The bacteria were pelleted by centrifugation and washed twice with saline. The samples were then re-suspended in bacteria lysis buffer (100 mM NH4HCO3, 8 M urea, 1 mM PMSF, 1× Protease Inhibitor Cocktail) and mechanically lysed using 0.1 mm silica beads in a Precellys tissue homogenizer at 4°C. Subsequently, the samples were crushed with a non-contact ultrasonic cell crusher for 1 hour. Silica beads and bacterial debris were removed by centrifugation at 1200×g and 4°C for 20 minutes. The protein concentration in the supernatant was measured using a Bradford assay kit (Beyotime, China). Next, the samples were reduced with 10 mM DTT for 1 hour, followed by alkylation in the dark with 45 mM iodoacetamide to break disulfide bonds. Each sample underwent trypsin digestion, with 50 μg of total protein digested using 2 μg trypsin overnight at 37°C or 1.5 hours at 56°C, and an additional 1 μg trypsin added twice during digestion. The digested peptides were filtered using Ultra Centrifugal Filters (Millipore, USA) and desalted using MonoSpin C18 (Shimadzu, Japan) following the manufacturer's protocol. The peptide samples were dried using a freeze-dryer and resuspended in 25 μl of water with 0.1% formic acid (vol/vol).Data acquisition was performed using a reverse-phase LC-MS/MS system comprising an Easy nLC1200 system (ThermoFisher, USA) coupled with a Q Exactive Plus Orbitrap MS System (ThermoFisher, USA).
Project description:Vitamin D is the strongest known natural anti-proliferative. A large number of studies in a wide spectrum of cancers, including epidemiological, in vitro and animal models, demonstrate that the active form of Vitamin D has anti-cancer benefits, affecting both progression and metastasis. Alike the role in calcium Vitamin D regulation, its anti-proliferative effect is thought to function through the Vitamin D receptor (VDR), although convincing evidence is lacking. Notwithstanding, separation of the calcemic and the anti-proliferative activity of Vitamin D analogues has been a major obstacle in developing new drugs for the treatment of cancer. The work presented attempts to unveil the molecular mechanism behind the anti-proliferative action of Vitamin D using genomic tools. For that purpose four independently developed Vitamin D sensitive/resistant MCF7 cell line pairs were collected. These unique biological replicates enabled us, both to increase the power of our study and to omit the use of Vitamin D. We deem this omission crucial since in the presence of Vitamin D only downstream genes involved in proliferation and cell cycle would be identified rather than causal resistance genes. The use of a variety of genomic techniques including expression, NMD and oligo CGH arrays reveal in the resistant cell lines the 11q13-14 as a region of DNA copy number loss and an altered expression of EGFR signaling pathway genes. Surprisingly, no genes known from calcium Vitamin D regulation were identified, nor did the VDR silencing by RNAi induce resistance to the sensitive cell lines. Keywords: comparative genomic hybridization, cell type comparision Several MCF7 breast tumor cell lines independently derived from the human breast cancer cell line, both resistant and sensitive to Vitamin D, were used. The pairs of cell lines (parental-resistant) were developed in different laboratories and using different methodologies; while some cell lines acquired resistance by long exposure to the physiological concentration of Vitamin D (100nM), others were induced to resistant by being exposed to increasing amounts of Vitamin D. A total of 4 microarray expression experiments were carried out. In all microarray experiments DNA from the Vitamin D resistant cell line was hybrizided agains DNA from the respsective sensitive/parental cell line.
Project description:Activation of telomerase often endows cancer cells, but rarely normal somatic cells, with immortality. Especially, fetal lung fibroblasts are known to be hardly immortalized by TERT overexpression. We here established an immortal non-transformed lung fibroblast cell line only by TERT transfection, as well as an immortal transformed cell line by transfection of TERT and SV40 early antigens. Comparing the expression profiles of these cell lines with those of mortal cell strains with elongated lifespan after TERT transfection, 51 genes, including 19 upregulated and 32 downregulated, were explored to be the candidates responsible for regulation of cellular proliferation of lung fibroblasts. These included the genes previously reported to be involved in cellular proliferation, transformation, or self-renewal capacity, and those highly expressed in lung tissues obtained from patients with idiopathic pulmonary fibrosis or hypersensitivity pneumonitis. This set of lung fibrobrast cell lines/strains of identical genetic background with different proliferative capacity, mortal and immortal non-transformed fibroblasts may become useful model cells for research on lung fibroblast growth regulation and the candidate genes explored in this study may provide promising biomarkers or molecular targets of pulmonary fibrosis. Keywords: Cell type comparison We analyzed 6 arrays with Affymetrix, 4 arrays with Agilent, and 7 arrays with CodeLink oligonucleotide microarrays for lung fibroblasts with various lifespan with or without transfection of TERT and/or SV40 early antigens.
Project description:Transposon insertion site sequencing (TIS) is a powerful tool that has significantly advanced our knowledge of functional genomics. While providing valuable insights, these applications of TIS focus on (conditional) gene essentiality and neglect possibly interesting but subtle differences in the importance of genes for fitness. Notably, data from TIS experiments can be used for fitness quantification and constructing genetic interaction maps, though this potential is only sporadically exploited. We aimed to develop a method to quantify the fitness of gene disruption mutants using data obtained from the TIS screen SATAY. This dataset was used to determine the reproducibility of the fitness estimates across biological and technical replicates of the same strain of S. cerevisiae. In addition, a mutant bem3∆ strain was utilized to compare the genetic interactions inferred from these fitness estimates with those documented in published databases. The dataset for the wild-type strain was created by transforming strain yWT01 with plasmid pBK549 and picking 4 different colonies from the transformation plate. These 4 biological replicates were then renamed to FD7, FD9, FD11 and FD12.