ABSTRACT: To further gain new insights into how MSMEG_6171 affects bacterial cell envelope properties, we performed global protein expression profiling on the Msm::6171 strains and the WT strains.
Project description:Understanding protein-protein interaction (PPI) network dynamics is key to understanding nominal and perturbed cell states. Here, we develop a new machine learning framework called Tapioca that allows for the study of PPIs in dynamics contexts at a global scale in ex/in vivo conditions. Furthermore, we optimized the thermal denaturation and lysis conditions used in the thermal proximity coaggregation (TPCA) methodology, one of the types of data Tapioca can use to make predictions. Using this optimized protocol and Tapioca, we investigated the temporal dynamics of reactivation from latency of the oncogenic gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV), identifying the host protein NUCKS1 as a factor promoting KSHV genome replication during lytic infection. Integrating this dataset with published TPCA datasets from the alphaherpesvirus herpes simplex virus type-1 (HSV-1) and the betaherpesvirus human cytomegalovirus (HCMV), we determined NUCKS1 to have a proviral role across all three herpesvirus subfamilies.
Project description:A 7-month inhalation study in C57BL/6 mice was conducted to evaluate long-term respiratory toxicity of e-vapor aerosols compared to cigarette smoke and to assess the impact of smoking cessation or switching to an e-vapor product after 3 months of exposure to 3R4F cigarette smoke (CS). In this study, we performed a chronic inhalation (4 h/day, 5 d/week, up to 7 months) study in C57BL/6 mice using a commercial (MarkTen®) e-vapor product and a combustible reference cigarette (3R4F) using a Switching and Cessation study design. A commercial e-vapor product (MarkTen® device [version 2.6.8]; “Test Red”) was supplied by Altria Client Services LLC (Richmond, VA, USA). The Test Red formulation was composed of aerosol formers (propylene glycol [PG] and vegetable glycerol [VG]), ~4% nicotine by weight, and flavors (non-menthol). The 3R4F commercial reference cigarettes were purchased from the University of Kentucky (Lexington, KY). HEPA filtered air at the testing facility (Battelle, West Jefferson, OH) was used as Sham Control. General procedures for animal care and housing met the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) recommendations and requirements stated in the “Guide for Care and Use of Laboratory Animals” [National Research Council (NRC)] and approved by the Institutional Animal Care and Use Committee (IACUC). Female C57BL/6 mice were received from Charles River Kingston (Stone Ridge, NY). Test atmosphere was generated from smoking machines and delivered to the mice through a nose-only exposure system. The modified Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Reference Method 81 regimen (55/30/5: a 55 ± 0.3 mL puff volume, a puff every 30 seconds, a 5-second puff duration) was used to generate e-vapor aerosol for 130 puffs/cartridge. Mainstream smoke from 3R4F cigarette was generated using a modified Health Canada Intense regimen (55/30/2: a 55 ± 0.3 mL puff volume, a puff every 30 seconds, a 2-second puff duration, and a near-square puff profile) for 8 puffs/cigarette. Female C57BL/6 mice (~10 weeks old) were randomly assigned based on body weight to one of five exposure groups: Sham Control, 3R4F CS, Test Red, Switching, and Cessation. Mice were exposed to 3R4F CS (550 µg/L TPM) or e-vapor aerosols (Test Red; 1100 µg/L TPM) via nose-only inhalation up to 4 h/day, 5 d/week for up to 7 months. After the first 3 months of exposure, groups of 3R4F CS mice were subjected to exposures of: (1) Test Red aerosol (“Switching”) or (2) filtered air (“Cessation”), while a group of mice continued to be exposed to 3R4F CS. Here, the protein expression data for lung tissue assessed by iTRAQ®-based quantitative proteomics is reported.
Project description:Osteoarthritis (OA) is a multifactorial pathology which comprises a wide range of distinct phenotypes. The characterization of the different molecular profiles associated to each phenotype can improve the classification of OA for a better personalized medicine. Within the metabolic syndrome phenotype, OA can co-exist with type 2 diabetes (TD2) disease. This study was undertaken to investigate lipidomic and proteomic differences between human OA and OA/TD2 cartilage through a multimodal mass spectrometry approach.
Project description:The objective of this study was to compare the biological and toxicological response of apolipoprotein E-deficient (Apoe-/-) mice to 3R4F mainstream smoke exposure for 2 months in whole-body exposure chambers (WBEC) and nose-only exposure chambers (NOEC). Female ApoE-/- mice were randomized into four groups: two Sham groups, exposed to filtered air, and two 3R4F groups, exposed to CS from the 3R4F reference cigarette (550 µg TPM/L). Half the number of mice in the Sham- and CS-exposed groups were exposed in WBECs and the other half in NOECs. The exposure phase lasted 9 weeks and included 1 week of adaption, during which exposure in both chamber types was escalated in dose and duration to a maximum of 4 h per day. The TPM concentration and exposure duration in WBECs were matched to those in NOECs on the basis of the regimen that the mice tolerated, as determined by in-life findings on acute signs of nicotine toxicity. Fresh air breaks were introduced during the exposure period to maintain carboxyhemoglobin (COHb) concentrations at acceptable levels. More frequent and longer fresh air breaks were required for exposure in the WBEC than in the NOEC because of the greater internal volume—and, consequently, the longer duration—required to clear smoke from the WBEC than from the NOEC. For animals in NOECs, a 30-min fresh air break was introduced after 2 and 3 h of exposure. For animals in WBECs, a 30-min fresh air break was introduced after 1 and 2 h of exposure and a 60-min fresh air break after the third hour of exposure. The general condition and health of the mice following exposure were monitored throughout the study. Full necropsy was performed 16-20 h after the last exposure without prior fasting, in accordance with previously described methods (Vanscheeuwijck et al., 2002). Differences between WBEC and NOEC expsoure were analyzed with regard to aerosol uptake, disease endpoints (adaptive changes in nasal epithelia, changes in lung function and inflammatory parameters, plasma cholesterol/triglyceride levels in lipoprotein fractions, and atherosclerosis plaque development), and systems biology endpoints (changes in the lung proteome and liver, nasal epithelial, and heart transcriptomes). All procedures involving animals were performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and licensed by the Agri-Food & Veterinary Authority of Singapore, with approval from an Institutional Animal Care and Use Committee and in compliance with the National Advisory Committee for Laboratory Animal Research Guidelines on the Care and Use of Animals for Scientific Purposes (NACLAR, 2004). Here, the protein expression data for lung tissue assessed by iTRAQ®-based quantitative proteomics will be described.
Project description:Protein turnover affects protein abundance and phenotypes. Comprehensive investigation of protein turnover dynamics has the potential to provide substantial information about gene expression. Here we report a large-scale protein turnover study in Salmonella Typhimurium during infection by quantitative proteomics. Murine macrophage-like RAW 264.7 cells were infected with SILAC labeled Salmonella. Bacterial cells were extracted after 0, 30, 60, 120 and 240 min. Mass spectrometry analyses yielded information about Salmonella protein turnover dynamics and a software program named Topograph was used for the calculation of protein half lives. The half lives of 311 proteins from intracellular Salmonella were obtained. For bacteria cultured in control medium (DMEM), the half lives for 870 proteins were obtained. The calculated median of protein half lives was 69.13 min and 99.30 min for the infection group and the DMEM group respectively, indicating an elevated protein turnover at the initial stage of infection. Gene ontology analyses revealed that a number of protein functional groups were significantly regulated by infection, including proteins involved in ribosome, periplasmic space, cellular amino acid metabolic process, ion binding and catalytic activity. The half lives of proteins involving in purine metabolism pathway were found to be significantly shortened during infection.
Project description:NUP98 fusion oncoproteins (FOs) are a hallmark of childhood acute myeloid leukemia (AML), and drive leukemogenesis through liquid-liquid phase separation-mediated nuclear condensate formation. However, the composition and consequences of NUP98 FO-associated condensates are incompletely understood. Here we show that histone acetyltransferase (HAT) complex proteins including MOZ associate with NUP98 FOs, and that BRPF1, an epigenetic writer that associates with MOZ is a molecular dependency in NUP98::KDM5A AML. Inactivation of Brpf1 as well as HAT complex member Moz, Hbo1, Brd1 or Meaf6 in Nup98::Kdm5a;Vav-Cre cells impaired fitness of NUP98-rearranged cells. MOZ inhibition decreased global H3K23ac levels, displaced FO from chromatin at the Meis1 locus, and led to myeloid cell differentiation. Additionally, MOZ inhibition decreased leukemic burden in multiple NUP98-rearranged leukemia xenograft models, synergized with Menin inhibitor treatment, and was efficacious in Menin inhibitor-resistant cells. In summary, we show that MOZ is a potentially targetable dependency in NUP98-rearranged AMLs. SIGNIFICANCE STATEMENT MOZ is a member of NUP98 FO condensates with key roles in leukemia phenotypes. MOZ inhibition is effective in multiple preclinical models, including those non-responsive to Menin inhibition. MOZ and Menin inhibition are synergistic in some NUP98-rearranged models, supporting clinical translation to improve outcomes of NUP98 FO-driven leukemias.
Project description:PTEN (Phosphatase and TENsin homolog deleted on chromosome ten) is a major tumor suppressor gene that is frequently mutated or lost under cancerous conditions. PTEN is a dual-specificity phosphatase that negatively regulates the PI3K/AKT/mTOR signaling pathway at the plasma membrane (PM). Its functional regulation and cellular localization are known to be conformationally driven. Access to the PM is phospho-regulated by open and closed PTEN forms. However, clarifying the underlying structural mechanisms is still an open avenue of research. Here, we apply an integrative structural modeling approach, combining coarse-grained and all-atom molecular dynamics with experimental crosslinking mass spectrometry. Conformational exchange between an “eased” form and a “strained” form brings the protein’s phosphatase and C2 domains closer together, blocking the catalytic site, and affecting the loops involved in PM binding. Our full-length PTEN models, AlphaMissense, and RaSP were used to better predict the consequences of PTEN mutations.
Project description:Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co-incubation with Legionella hackeliae and L. pneumophila DeltadotA. One hundred and thirty-one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. The results provide the basis for a better understanding of the complex host-pathogen interactions and for further studies on the Dictyostelium response to Legionella infection. The bacterial strains used in this study were L. pneumophila Philadelphia I JR32, L. pneumophila Philadelphia I JR32 LELA 3118 (dotA3118:Tn903 DLL LacZ) and L. hackeliae (ATCC 35250). The Legionella strains were grown on buffered charcoal yeast extract agar (BCYE) at 37M-BM-0C with 5% CO2 atmosphere for 3 days. The D. discoideum wild-type strain AX2 was grown at 23M-BM-0C in 75 cm2 cell-culture flasks with 10 ml HL5 medium. For infection, Dictyostelium cells were harvested, resuspended in a 1:1 solution of HL5 medium and Soerensen buffer. Fifteen millilitres of a 1M-CM-^W10e6 cells/ml suspension were seeded into a 75 square-cm cell culture flask and the amoebae were inoculated with 10e7 bacteria/ml. Three different pairs of infection were compared: 1. AX2 infected with L. pneumophila JR32 versus uninfected cells; 19 microarrays of seven independent infections; 2. AX2 infected with L. pneumophila JR32 versus AX2 infected with L. pneumophila JR32 delta DotA; 4 microarrays of two independent infections; 3. AX2 infected with L. pneumophila JR32 versus AX2 infected with L. hackeliae; 4 microarrays of two independent infections. 24h post infection the RNA was isolated from 1.5M-CM-^W10e7 Dictyostelium cells and microarray analysis was performed as described (Farbrother et al., 2006).