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By translocating effectors inside the eukaryotic host cell, bacteria can modulate host cellular processes in their favor. Legionella species, which includes the pneumonia-causing Legionella pneumophila, encode a widely diverse set of effectors with only a small subset that is conserved across this genus. Here, we demonstrate that one of these conserved effector families, represented by L. pneumophila VipF (Lpg0103), is a tandem Gcn5-related N-acetyltransferase interacting with the K subunit of human eukaryotic initiation factor 3 complex. VipF catalyzes the acetylation of lysine residues on the C-terminal tail of the K subunit, resulting in the suppression of eukaryotic translation initiation factor 3-mediated protein translation in vitro . These new data provide the first insight into the molecular function of this pathogenic factor family common across Legionellae .

This study develops a hydraulic circuit to drive a conveying system dealing with heavy and delicate loads. Various safety circuits have been added in order to ensure stable working at high pressure and precise controlling. Here we have shown the calculation procedure based on an arbitrarily selected load. Also the circuit design and calculations of various components used is depicted along with the system simulation. The results show that the system is stable and efficient enough to transmit heavy loads by functioning of the circuit. By this information, one can be able to design their own hydrostatic circuits for various heavy loading conditions.

Interferon (IFN) and inflammation are the key early defence mechanisms that combat pathogen infection. The cytosolic DNA sensor cGAS activates immune signaling via the stimulator of interferon genes (STING) protein. Emerging evidence suggests crosstalk between innate immune DNA and RNA sensing, implicating a role of STING protein in RNA virus infection. This study characterizes STING in the context of Japanese encephalitis virus (JEV), an RNA virus of the flaviviridae family. We observe that activation of type I IFN through MAVS is essential for cGAS and STING activation. Knockdown, null mutant and inhibitor studies confirm that STING restricts JEV replication independently of IFNβ signaling and autophagy. Transcriptomic analysis of STING (Goldenticket) bone-marrow derived macrophages (BMDMs) shows enhanced IFN response, but reduced activation of inflammatory cytokines and chemokines. Phosphorylated STING is recruited to the virus replication complex (RC), marked by the non-structural protein NS1, subsequently triggering the assembly of the NLRP3 inflammasome on the RC. STING proton channel activity is essential for NLRP3 inflammasome activation, IL-1β production, and activation of pyroptotic cell death markers. STING mice, show higher viremia, earlier disease onset, reduced survival, and decreased brain inflammation. These findings establish STING as a key regulator of JEV-induced inflammation and antiviral defence.

Gold/chitosan nanocomposites were synthesized and evaluated as a therapeutic agent for the photothermal therapy. Gold nanoparticles (Au NPs) with controllable optical absorption in the near infrared (NIR) region were prepared by the reaction of chloroauric acid and sodium thiosulfate. To apply these particles to cancer therapy, the bare Au NPs were coated with chitosan (CS), O-carboxymethyl chitosan (CMCS), and a blend of CS and CMCS for utilizations in physiologic conditions. The surface properties, optical stability, and photothermal ablation efficiency on hepatocellular carcinoma cells (HepG2) and human dermal fibroblast cells (HDF) demonstrate that these gold nanocomposites have great potential as a therapeutic agent in in vitro tests. The CS-coated nanocomposites show the highest efficiency for the photo-ablation on the HepG2 cells, and the CS and CMCS blended coated particles show the best discrimination between the cancer cell and normal cells. The well-controlled NIR absorption and the biocompatible surface of these nanocomposites allow low-power NIR laser activation and low-dosage particle injection for the cancer cell treatment.

The selective exposure of cancerous tissue to systemically delivered chemotherapeutic agents remains a major challenge facing cancer therapy. To address this question, a near infrared responsive oligonucleotide-coated (AS1411, hairpin, or both) gold nanoplate loaded with doxorubicin is demonstrated to be nontoxic to cells without triggered release, while being acutely toxic to cells after 5 minutes of laser exposure to trigger DOX release. Conjugation of oligonucleotides to the nanoplates is confirmed by an average increase in hydrodynamic diameter of 30.6 nm, an average blue shift of the plasmon resonance peak by 36 nm, and an average −10 mV shift in zeta potential of the particles. DOX loading through intercalation into the hairpin DNA structure is confirmed through fluorescence measurements. For both GNP-Hairpin and GNP-Hairpin-AS1411, ~60% of loaded DOX is released after the first 5 minutes of laser exposure ( λ = 817 nm), with complete release after two more 5-minute exposures. Preliminary proof of concept is demonstrated in vitro using A549 and MDA-MB-231 cell lines as models for breast and lung cancer, respectively. Exposure of cells to untriggered DOX-loaded conjugate with no laser exposure results in little to no toxicity, while laser-triggered release of DOX causes significant cell death.

Interferon (IFN) and inflammation are the key early defence mechanisms that combat pathogen infection. The cytosolic DNA sensor cGAS activates immune signaling via the stimulator of interferon genes (STING) protein. Emerging evidence suggests crosstalk between innate immune DNA and RNA sensing, implicating a role of STING protein in RNA virus infection. This study characterizes STING in the context of Japanese encephalitis virus (JEV), an RNA virus of the flaviviridae family. We observe that activation of type I IFN through MAVS is essential for cGAS and STING activation. Knockdown, null mutant and inhibitor studies confirmed that STING restricts JEV replication independently of IFNβ signaling and autophagy. Transcriptomic analysis of STINGgt/gt bone-marrow derived macrophages (BMDMs) showed enhanced IFN response, but reduced activation of inflammatory cytokines and chemokines. Phosphorylated STING was recruited on the virus replication complex (RC), marked by the non-structural protein NS1, subsequently triggering the assembly of the NLRP3 inflammasome on the RC. STING proton channel activity was essential for NLRP3 inflammasome activation, IL-1β production, and activation of pyroptotic cell death markers. Stinggt/gt mice, showed higher viremia, earlier disease onset, reduced survival, and decreased brain inflammation. These findings establish STING as a key regulator of JEV-induced inflammation and antiviral defence.

5G non-public networks (NPNs) play a key role in enabling critical Industrial Internet of Things (IoT) applications in various vertical industries. Among other features, 5G NPNs enable novel operation models, where the roles and responsibilities for setting up and operating the network can be distributed among several stakeholders, i.e., among the public mobile network operators (MNOs), the industrial party who uses the 5G NPN services and 3rd parties. This results in many theoretically feasible operation models for 5G NPN, each with its own advantages and disadvantages. We investigate the resulting operation models and identify a set of nine prime models taking into account today's practical considerations. Additionally, we define a framework to qualitatively analyze the operation models and use it to evaluate and compare the identified operation models.

Polymyxins are last-resort antimicrobial peptides administered clinically against multi-drug resistant bacteria, including Gram-negative ESKAPE pathogens. However, an increasing number of pathogens employ a defense strategy involving a relay of enzymes encoded by the pmrE(ugd) loci and the arnBCDTEF operon. As a result, an Ara-4N headgroup is added to the lipid-A component of outer membrane (OM) lipopolysaccharides (LPS) rendering polymyxins ineffective. Here, we report the cryo-EM structures of glycosyltransferase ArnC from Salmonella typhimurium resolved in both apo and UDP-bound forms at resolutions 2.75 Å and 3.8 Å, respectively. The structure of the ArnC protomer comprises of three distinct regions: an N-terminal glycosyltransferase domain, transmembrane region, and the interface helices (IHs). ArnC forms a stable tetramer with C2 symmetry through interactions in the C-terminal region, which is expected to protrude into the cytosol, where the β8 strand inserts into the adjacent protomer. ArnC protomers have two distinct types of interfaces involving multiple hydrogen bonds and salt bridges. The binding of UDP induces conformational changes that stabilizes structurally labile A-loop, spanning residues 201 to 213, and part of the putative catalytic pocket formed by IH1 and IH2. The comparative analysis of ArnC structures with homologs GtrB and DPMS suggests the key residues involved in ArnC catalytic activity.

Legionella are freshwater Gram-negative bacteria that in their normal environment infect protozoa. However, this adaptation also allows Legionella to infect human alveolar macrophages and cause pneumonia. Central to Legionella pathogenesis are more than 330 secreted effectors, of which there are 9 core effectors that are conserved in all pathogenic species. Despite their importance, the biochemical function of several core effectors remains unclear. To address this, we have taken a structural approach to characterize the core effector of unknown function LceB, or Lpg1356, from Legionella pneumophila. Here we solve an X-ray crystal structure of LceB using an AlphaFold model for molecular replacement. The experimental structure shows that LceB adopts a Sel1-like repeat fold as predicted. However, the crystal structure captured multiple conformations of LceB all of which differed from the AlphaFold model. Comparison of the predicted model and the experimental models suggests that LceB is highly flexible in solution. Additionally, molecular analysis of LceB using its close structural homologues reveals sequence and structural motifs of known biochemical function. Specifically, LceB harbors a repeated KAAEQG motif that both stabilizes the Sel1-like repeat fold and is known to participate in protein-protein interactions with eukaryotic host proteins. We also observe that LceB forms several higher-order oligomers in solution. Overall, our results have revealed that LceB has conformational flexibility, self-associates, and contains a molecular surface for binding a target host-cell protein. Additionally, our data provides structural insights into the Sel1-like repeat family of proteins that remain poorly studied.

Legionella pneumophila utilizes the Dot/Icm type IVB secretion system to deliver hundreds of effector proteins inside eukaryotic cells to ensure intracellular replication. Our understanding of the molecular functions of this largest pathogenic arsenal known to the bacterial world remains incomplete. By leveraging advancements in 3D protein structure prediction, we provide a comprehensive structural analysis of 368 L. pneumophila effectors, representing a global atlas of predicted functional domains summarized in a database (https://pathogens3d.org/legionella-pneumophila). Our analysis identified 157 types of diverse functional domains in 287 effectors, including 159 effectors with no prior functional annotations. Furthermore, we identified 35 unique domains in 30 effector models that have no similarity with experimentally structurally characterized proteins, thus, hinting at novel functionalities. Using this analysis, we demonstrate the activity of thirteen domains, including three unique folds, predicted in L. pneumophila effectors to cause growth defects in the Saccharomyces cerevisiae model system. This illustrates an emerging strategy of exploring synergies between predictions and targeted experimental approaches in elucidating novel effector activities involved in infection.