Project description:Using an integrated model system for reproducible growth of biofilms, a JPIAMR-funded consortium of researchers* studied the expressed proteome of P. aeruginosa strain MPAO1 under i) planktonic growth, and ii) biofilm formation conditions. The model system included, as a first step, the sequencing and de novo assembly of the complete genome of this opportunistic human pathogen that belongs to the notorious group of Gram-negative ESKAPE pathogens. MPAO1 is also the parental strain for the widely used transposon (Tn) mutant library from the University of Washington. The complete MPAO1 genome sequence turned out to harbor several deletions and insertions compared to the PAO1-UW reference genome including numerous MPAO1-unique genes. As a second step in the model system, a biofilm flow cell based on poly (dimethylsiloxane) (PDMS) was designed to reproducibly study and identify known and novel genes related to biofilm growth and antibiotic resistance (ABR) from the Tn mutant collection. With the complete genome as optimal basis, publicly available TnSeq data were reanalyzed to identify known and novel essential genes. Furthermore, shotgun proteomics data was generated uncovering 1530 (planktonic) and 1728 (biofilm) expressed proteins, respectively, resulting in the identification of 1922 (33.1%) of the 5799 annotated P. aeruginosa MPAO1 proteins. They included proteins known to be differentially expressed during biofilm formation, and proteogenomic evidence for proteins uniquely encoded by MPAO1 as well as novel proteins.
Project description:Informed consent was obtained to collect human mCRPC tissues and generate the patient-derived xenograft tumors as described previously (Labrecque et al., 2019; Nguyen et al., 2017). The study was approved by the University of Washington Human Subjects Division institutional review board (no. 39053). All animal studies were approved by University of Washington IACUC and performed according to NIH guidelines. Molecular characterization of AR+ mCRPC LuCaP PDXs 70CR, 78CR, 81CR, 96CR, 105CR, 136CR and 147CR was previously described (Labrecque et al., 2019; Nguyen et al., 2017). LuCaP PDX 167CR was established from a liver metastasis of 77-year-old Caucasian male who died of abiraterone-, carboplatin- and docetaxel-resistant CRPC. LuCaP 167CR expresses AR, responds to castration and is negative for synaptophysin. PDX cellular morphology recapitulates the original liver metastasis (Supplementary Figure S8A).
Project description:To identify novel genes modulating Candida albicans biofilm formation, a screen of 2451 overexpression strains allowed us to identify 16 genes whose overexpression significantly reduced biofilm formation. Genome-wide expression and binding analyses were conducted upon overexpression of ZCF15 and ZCF26 and wild type planktonic and biofilm cells were performed. A ChIP assays was performed. Briefly, untagged strain (CEC4665) and two replicates each of ZCF15 (CEC5929 and CEC5930) and ZCF26 (CEC5931 and CEC5932) strain were grown in biofilm condition for 18 h and cells were cross-linked with 1% final concentration of formaldehyde for 25 min at 30°C.The DNA was immunoprecipitated with anti-protein A antibodies (Sigma Aldrich Cat. No. P3775). The immunoprecipitated (IP) DNA were used to determine the binding of Zcf15 and Zcf26 across the genome by ChIP-sequencing
Project description:Animals. Male Sprague–Dawley rats (Charles River Laboratories, Wilmington, MA) weighing about 250g were used. The study was approved by the Thomas Jefferson University Institutional Animal Care and Use Committee and was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animals were housed in Thomas Jefferson University's animal care facilities. Animals were anesthetized with isoflurane dissolved in O2 (5% induction; 1% maintenance) and one femoral artery and vein were cannulated (PE-50 tubing) via a small medial incision for measurement of arterial pressure and infusion of drugs, respectively. The cannulae were run subcutaneously to an exit incision between the scapulae. The leg wound was sutured and topical anesthetic (lidocaine) was applied to both skin incisions. Following surgery, after one hour of stable and normal resting blood pressure and heart rate, intravenous infusion was initiated of approximately 1 mL saline, as a control, or phenylephrine (200 µg/mL; 1 mL/hr), to induce hypertension. We followed standard methods in the use of phenylephrine (PE) to elevate blood pressure. PE does not cross the blood brain barrier and the elevated blood pressure it produces has been shown to cause molecular effects in the NTS principally via increased baroreceptor afferent drive by both pharmacological and sinoaortic denervation studies. We titered the PE dose to maintain intermediate levels of elevated blood pressure 25 mmHg above resting blood pressure. Keywords: Hypertension, time series
Project description:This study is aimed to isolate marine actinomycetes from sediments from Andaman and the Gulf of Thailand. All 101 marine actinomycetes were screened for anti-biofilm activity. Streptomyces sp. GKU223 showed significantly inhibited biofilm formation of S. aureus. The evaluation of supernatants of anti-biofilm activity produced by Streptomyces sp. GKU223 has been performed. Since the interaction between marine actinomycetes and biofilm forming bacteria has never been investigated, proteomic analysis has been used to identify whole cell proteins involved in anti–biofilm activity. Understanding the interaction at molecular level will lead to sustainably use for anti-biofilm producing marine actinomycetes in pharmaceutical and medicinal applications in the future.