Comparative proteomic analysis reveals mechanistic insights into Pseudomonas putida F1 growth on benzoate and citrate

Pseudomonas species are capable to proliferate under diverse environmental conditions and thus have a significant bioremediation potential. To enhance our understanding of their metabolic versatility, this study explores the changes in the proteome and physiology of Pseudomonas putida F1 resulting from its growth on benzoate, a moderate toxic compound that can be catabolized, and citrate, a carbon source that is assimilated through central metabolic pathways. A series of repetitive batch cultivations were performed to ensure a complete adaptation of the bacteria to each of these contrasting carbon sources. After several growth cycles, cell growth stabilized at the maximum level and exhibited a reproducible growth profile. The specific growth rates measured for benzoate (1.01 ± 0.11 h-1) and citrate (1.11 ± 0.12 h-1) were similar, while a higher yield was observed for benzoate (0.6 and 0.3 g cell mass per g of benzoate and citrate, respectively), reflecting the different degrees of carbon reduction in the two substrates. Comparative proteomic analysis revealed an enrichment of several oxygenases/dehydrogenases in benzoate-grown cells, indicative of the higher carbon reduction of benzoate. Moreover, the upregulation of all 14 proteins implicated in benzoate degradation via the catechol ortho-cleavage pathway was observed, while several stress-response proteins were increased to aid cells to cope with benzoate toxicity. Unexpectedly, citrate posed more challenges than benzoate in the maintenance of pH homeostasis, as indicated by the enhancement of the Na+/H+ antiporter and carbonic anhydrase. The study provides important mechanistic insights into Pseudomonas adaptation to varying carbon sources that are of great relevance to bioremediation efforts

The XXL Survey: : XXIX. GMRT 610 MHz continuum observations

Accepted for publication in a forthcoming issue of Astronomy & Astrophysics. Reproduced with permission from Astronomy & Astrophysics. © 2018 ESO.We present the 25 square-degree GMRT-XXL-N 610 MHz radio continuum survey, conducted at 50 cm wavelength with the Giant Metrewave Radio Telescope (GMRT) towards the XXL Northern field (XXL-N). We combined previously published observations of the XMM-Large Scale Structure (XMM-LSS) field, located in the central part of XXL-N, with newly conducted observations towards the remaining XXL-N area, and imaged the combined data-set using the Source Peeling and Atmospheric Modeling (SPAM) pipeline. The final mosaic encompasses a total area of 30:4 square degrees, with rms <150 μJy beam -1 over 60% of the area. The rms achieved in the inner 9.6 square degree area, enclosing the XMM-LSS field, is about 200 μJy beam -1, while that over the outer 12.66 square degree area (which excludes the noisy edges) is about 45 μJy beam -1. The resolution of the final mosaic is 6.5 arcsec. We present a catalogue of 5434 sources detected at ≥7 × rms. We verify, and correct the reliability of, the catalog in terms of astrometry, flux, and false detection rate. Making use of the (to date) deepest radio continuum survey over a relatively large (2 square degree) field, complete at the flux levels probed by the GMRT-XXL-N survey, we also assess the survey's incompleteness as a function of flux density. The radio continuum sensitivity reached over a large field with a wealth of multi-wavelength data available makes the GMRTXXL- N 610 MHz survey an important asset for studying the physical properties, environments and cosmic evolution of radio sources, in particular radio-selected active galactic nuclei (AGN).Peer reviewedFinal Accepted Versio

Channeling macrophage polarization by rocaglates increases macrophage resistance to Mycobacterium tuberculosis

Macrophages contribute to host immunity and tissue homeostasis via alternative activation programs. M1-like macrophages control intracellular bacterial pathogens and tumor progression. In contrast, M2-like macrophages shape reparative microenvironments that can be conducive for pathogen survival or tumor growth. An imbalance of these macrophages phenotypes may perpetuate sites of chronic unresolved inflammation, such as infectious granulomas and solid tumors. We have found that plant-derived and synthetic rocaglates sensitize macrophages to low concentrations of the M1-inducing cytokine IFN-gamma and inhibit their responsiveness to IL-4, a prototypical activator of the M2-like phenotype. Treatment of primary macrophages with rocaglates enhanced phagosome-lysosome fusion and control of intracellular mycobacteria. Thus, rocaglates represent a novel class of immunomodulators that can direct macrophage polarization toward the M1-like phenotype in complex microenvironments associated with hypofunction of type 1 and/or hyperactivation of type 2 immunity, e.g., chronic bacterial infections, allergies, and, possibly, certain tumors.R35 GM118173 - NIGMS NIH HHS; R01 HL126066 - NHLBI NIH HHS; R01 GM120272 - NIGMS NIH HHS; R01 CA218500 - NCI NIH HHS; R01 HL133190 - NHLBI NIH HHS; R33 AI105944 - NIAID NIH HHSPublished versio

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells.

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up-regulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial- and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells.

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up-regulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial- and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production

Host Cell Protein Profiling by Targeted and Untargeted Analysis of Data Independent Acquisition Mass Spectrometry Data with Parallel Reaction Monitoring Verification

Host cell proteins (HCPs) are process-related impurities of biopharmaceuticals that remain at trace levels despite multiple stages of downstream purification. Currently, there is interest in implementing LC-MS in biopharmaceutical HCP profiling alongside conventional ELISA, because individual species can be identified and quantitated. Conventional data dependent LC-MS is hampered by the low concentration of HCP-derived peptides, which are 5–6 orders of magnitude less abundant than the biopharmaceutical-derived peptides. In this paper, we present a novel data independent acquisition (DIA)-MS workflow to identify HCP peptides using automatically combined targeted and untargeted data processing, followed by verification and quantitation using parallel reaction monitoring (PRM). Untargeted data processing with DIA-Umpire provided a means of identifying HCPs not represented in the assay library used for targeted, peptide-centric, data analysis. An IgG1 monoclonal antibody (mAb) purified by Protein A column elution, cation exchange chromatography, and ultrafiltration was analyzed using the workflow with 1D-LC. Five protein standards added at 0.5 to 100 ppm concentrations were detected in the background of the purified mAb, demonstrating sensitivity to low ppm levels. A calibration curve was constructed on the basis of the summed peak areas of the three highest intensity fragment ions from the highest intensity peptide of each protein standard. Sixteen HCPs were identified and quantitated on the basis of the calibration curve over the range of low ppm to over 100 ppm in the purified mAb sample. The developed approach achieves rapid HCP profiling using 1D-LC and specific identification exploiting the high mass accuracy and resolution of the mass spectrometer