Low-mass GEM detector with radial zigzag readout strips for forward tracking at the EIC

We present design and construction of a large low-mass Triple-GEM detector prototype for forward tracking at a future Electron-Ion Collider. In this environment, multiple scattering of forward and backward tracks must be minimized so that electron tracks can be cleanly matched to calorimeter clusters and so that hadron tracks can efficiently seed RICH ring reconstruction for particle identification. Consequently, the material budget for the forward tracking detectors is critical. The construction of the detector builds on the mechanical foil stretching and assembly technique pioneered by CMS for the muon endcap GEM upgrade. As an innovation, this detector implements drift and readout electrodes on thin large foils instead of on PCBs. These foils get stretched mechanically together with three GEM foils in a single stack. This reduces the radiation length of the total detector material in the active area by a factor seven from over 4% to below 0.6%. It also aims at improving the uniformity of drift and induction gap sizes across the detector and consequently signal response uniformity. Thin outer frames custom-made from carbon-fiber composite material take up the tension from the stretched foil stack and provide detector rigidity while keeping the detector mass low. The gas volume is closed with thin aluminized polyimide foils. The trapezoidal detector covers an azimuthal angle of 30.1 degrees and a radius from 8 cm to 90 cm. It is read out with radial zigzag strips with pitches of 1.37 mrad at the outer radius and 4.14 mrad at the inner radius that reduce the number of required electronics channels and associated cost while maintaining good spatial resolution. All front-end readout electronics is located away from the active area at the outer radius of the trapezoid.Comment: 4 pages, 7 figures, submitted to conference record of 2017 IEEE Nuclear Science Symposium, Atlanta, G

NADH Dehydrogenases in Pseudomonas aeruginosa Growth and Virulence

Pseudomonas aeruginosa is an opportunistic human pathogen with a complex respiratory chain. The bacterium is predicted to express three NADH:ubiquinone oxidoreductases (NDH-1, NDH-2 and Nqr). We created deletions strains of the predicted NADH:ubiquinone oxidoreductases alone, and in combination to determine the respective roles of the NADH dehydrogenases in growth and virulence. NDH-1 and NDH-2 were largely redundant under aerobic conditions. Aerobic NADH dehydrogenase enzymatic activity assay was lost with deletion of both NDH-1 and NDH-2. Under anaerobic conditions, NDH-1 was required for robust growth, and overexpression of NDH-2 rescued the NDH-1 anaerobic growth defect in rich media. There was not compensatory upregulation of NDH-2 under anaerobic conditions in NDH-1 deletion strains. To test which genes were required for in vivo virulence, we used both an insect and plant disease model. In the Galleria mellonella model, neither deletion of NDH-1 nor NDH-2 led to a change in median lethal dose, although death occurred more slowly in the NDH-1 deletion infections. In a lettuce model of virulence, loss of NDH-1 caused a decrease in recovered viable bacteria and a decrease in visual tissue damage. The compound deletion of NDH-1/NDH-2 causes a severe growth defect, both under aerobic and anaerobic conditions, and was avirulent in a lettuce model. Together, these results demonstrate the redundancy of the P. aeruginosa respiratory chain at the NADH dehydrogenase level in aerobic growth and virulence

Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

Clostridium perfringens enterotoxin (CPE) binds to claudin receptors, e.g., claudin-4, and then forms a pore that triggers cell death. Pure cultures of host cells that do not express claudin receptors, e.g., fibroblasts, are unaffected by pathophysiologically relevant CPE concentrations in vitro. However, both CPE-insensitive and CPE-sensitive host cells are present in vivo. Therefore, this study tested whether CPE treatment might affect fibroblasts when cocultured with CPE-sensitive claudin-4 fibroblast transfectants or Caco-2 cells. Under these conditions, immunofluorescence microscopy detected increased death of fibroblasts. This cytotoxic effect involved release of a toxic factor from the dying CPE-sensitive cells, since it could be reproduced using culture supernatants from CPE-treated sensitive cells. Supernatants from CPE-treated sensitive cells, particularly Caco-2 cells, were found to contain high levels of membrane vesicles, often containing a CPE species. However, most cytotoxic activity remained in those supernatants even after membrane vesicle depletion, and CPE was not detected in fibroblasts treated with supernatants from CPE-treated sensitive cells. Instead, characterization studies suggest that a major cytotoxic factor present in supernatants from CPE-treated sensitive cells may be a 10- to 30-kDa host serine protease or require the action of that host serine protease. Induction of caspase-3-mediated apoptosis was found to be important for triggering release of the cytotoxic factor(s) from CPE-treated sensitive host cells. Furthermore, the cytotoxic factor(s) in these supernatants was shown to induce a caspase-3-mediated killing of fibroblasts. This bystander killing effect due to release of cytotoxic factors from CPE-treated sensitive cells could contribute to CPE-mediated disease

Mixed Communities of Mucoid and Nonmucoid Pseudomonas aeruginosa Exhibit Enhanced Resistance to Host Antimicrobials

Pseudomonas aeruginosa causes chronic pulmonary infections in patients with cystic fibrosis (CF). P. aeruginosa mucoid conversion, defined by overproduction of the exopolysaccharide alginate, correlates with accelerated decline in CF patient lung function. Recalcitrance of the mucoid phenotype to clearance by antibiotics and the immune response is well documented. However, despite advantages conferred by mucoidy, mucoid variants often revert to a nonmucoid phenotype both in vitro and in vivo. Mixed populations of mucoid isolates and nonmucoid revertants are recovered from CF lungs, suggesting a selective benefit for coexistence of these variants. In this study, cocultures of mucoid and nonmucoid variants exhibited enhanced resistance to two host antimicrobials: LL-37, a cationic antimicrobial peptide, and hydrogen peroxide (H2O2). Alginate production by mucoid isolates protected nonmucoid variants in consortia from LL-37, as addition of alginate exogenously to nonmucoid variants abrogated LL-37 killing. Conversely, nonmucoid revertants shielded mucoid variants from H2O2 stress via catalase (KatA) production, which was transcriptionally repressed by AlgT and AlgR, central regulators of alginate biosynthesis. Furthermore, extracellular release of KatA by nonmucoid revertants was dependent on lys, encoding an endolysin implicated in autolysis and extracellular DNA (eDNA) release. Overall, these data provide a rationale to study interactions of P. aeruginosa mucoid and nonmucoid variants as contributors to evasion of innate immunity and persistence within the CF lung

Bacterial Outer Membrane Vesicles and Immune Modulation of the Host

This article reviews the role of outer membrane vesicles (OMVs) in mediating the interaction between Gram-negative bacteria and their human hosts. OMVs are produced by a diverse range of Gram-negative bacteria during infection and play a critical role in facilitating host–pathogen interactions without requiring direct cell-to-cell contact. This article describes the mechanisms by which OMVs are formed and subsequently interact with host cells, leading to the transport of microbial protein virulence factors and short interfering RNAs (sRNA) to their host targets, exerting their immunomodulatory effects by targeting specific host signaling pathways. Specifically, this review highlights mechanisms by which OMVs facilitate chronic infection through epigenetic modification of the host immune response. Finally, this review identifies critical knowledge gaps in the field and offers potential avenues for future OMV research, specifically regarding rigor and reproducibility in OMV isolation and characterization methods

Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection.

Clinical studies report that viral infections promote acute or chronic bacterial infections at multiple host sites. These viral-bacterial co-infections are widely linked to more severe clinical outcomes. In experimental models in vitro and in vivo, virus-induced interferon responses can augment host susceptibility to secondary bacterial infection. Here, we used a cell-based screen to assess 389 interferon-stimulated genes (ISGs) for their ability to induce chronic Pseudomonas aeruginosa infection. We identified and validated five ISGs that were sufficient to promote bacterial infection. Furthermore, we dissected the mechanism of action of hexokinase 2 (HK2), a gene involved in the induction of aerobic glycolysis, commonly known as the Warburg effect. We report that HK2 upregulation mediates the induction of Warburg effect and secretion of L-lactate, which enhances chronic P. aeruginosa infection. These findings elucidate how the antiviral immune response renders the host susceptible to secondary bacterial infection, revealing potential strategies for viral-bacterial co-infection treatment

Immunotransplantation preferentially expands T-effector cells over T-regulatory cells and cures large lymphoma tumors

Ex vivo–expanded tumor-infiltrating lymphocytes infused into lymphodepleted recipients has clear antitumor efficacy. More practical sources of such antitumor lymphocytes would broaden the application of this approach. Previously, we described an in situ vaccination combining chemotherapy with intratumoral injection of CpG-enriched oligonucleotides, which induced T-cell immunity against established lymphoma. An ongoing clinical trial of this maneuver has demonstrated clinical responses in lymphoma patients. Here, we use this vaccine maneuver to generate immune cells for transfer into irradiated, syngeneic recipients. Transferred tumor-specific T-effector (Teff) cells preferentially expanded, increasing the Teff/T-regulatory (Treg) ratio in these “immunotransplantation” recipients and curing large and metastatic tumors. Donor T cells were necessary for tumor protection, and CD8 T-cell immune responses were enhanced by posttransplantation booster vaccination. Hematopoietic stem cell transplantation is a standard therapy for lymphoma. Therefore, in situ tumor vaccination followed by immunotransplantation of harvested tumor-specific T cells could be directly tested in clinical trials to treat otherwise resistant malignancies

Pseudomonas aeruginosa utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium.

Ferroptosis is a death program executed via selective oxidation of arachidonic acid-phosphatidylethanolamines (AA-PE) by 15-lipoxygenases. In mammalian cells and tissues, ferroptosis has been pathogenically associated with brain, kidney, and liver injury/diseases. We discovered that a prokaryotic bacterium, Pseudomonas aeruginosa, that does not contain AA-PE can express lipoxygenase (pLoxA), oxidize host AA-PE to 15-hydroperoxy-AA-PE (15-HOO-AA-PE), and trigger ferroptosis in human bronchial epithelial cells. Induction of ferroptosis by clinical P. aeruginosa isolates from patients with persistent lower respiratory tract infections was dependent on the level and enzymatic activity of pLoxA. Redox phospholipidomics revealed elevated levels of oxidized AA-PE in airway tissues from patients with cystic fibrosis (CF) but not with emphysema or CF without P. aeruginosa. We believe that the evolutionarily conserved mechanism of pLoxA-driven ferroptosis may represent a potential therapeutic target against P. aeruginosa-associated diseases such as CF and persistent lower respiratory tract infections