Search

Abstract Acinetobacter baumannii is a pathogenic and multidrug-resistant Gram-negative bacterium that causes severe nosocomial infections. To better understand the mechanism of pathogenesis, we compare the proteomes of uninfected and infected human cells, revealing that transcription factor FOS is the host protein most strongly induced by A. baumannii infection. Pharmacological inhibition of FOS reduces the cytotoxicity of A. baumannii in cell-based models, and similar results are also observed in a mouse infection model. A. baumannii outer membrane vesicles (OMVs) are shown to activate the aryl hydrocarbon receptor (AHR) of host cells by inducing the host enzyme tryptophan-2,3-dioxygenase (TDO), producing the ligand kynurenine, which binds AHR. Following ligand binding, AHR is a direct transcriptional activator of the FOS gene. We propose that A. baumannii infection impacts the host tryptophan metabolism and promotes AHR- and FOS-mediated cytotoxicity of infected cells.

During the COVID-19 pandemic, the number of cases continued to rise. As a result, there was a growing demand for alternative control methods to traditional buttons or touch screens. However, most current gesture recognition technologies rely on machine vision methods. However, this method can lead to suboptimal recognition results, especially in situations where the camera is operating in low-light conditions or encounters complex backgrounds. This study introduces an innovative gesture recognition system for large movements that uses a combination of millimeter wave radar and a thermal imager, where the multi-color conversion algorithm is used to improve palm recognition on the thermal imager together with deep learning approaches to improve its accuracy. While the user performs gestures, the mmWave radar captures point cloud information, which is then analyzed through neural network model inference. It also integrates thermal imaging and palm recognition to effectively track and monitor hand movements on the screen. The results suggest that this combined method significantly improves accuracy, reaching a rate of over 80%.

Legionella pneumophila causes a severe pneumonia known as Legionnaires’ disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1’), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host–pathogen interactions.

Group A streptococcus (GAS) is a versatile pathogen that causes a wide spectrum of diseases in humans. Invading host cells is a known strategy for GAS to avoid antibiotic killing and immune recognition. However, the underlying mechanisms of GAS resistance to intracellular killing need to be explored. Endothelial HMEC-1 cells were infected with GAS, methicillin-resistant Staphylococcus aureus (MRSA) and Salmonella Typhimurium under nicotinamide (NAM)-supplemented conditions. The intracellular NAD+ level and cell viability were respectively measured by NAD+ quantification kit and protease-based cytotoxicity assay. Moreover, the intracellular bacteria were analyzed by colony-forming assay, transmission electron microscopy, and confocal microscopy. We found that supplementation with exogenous nicotinamide during infection significantly inhibited the growth of intracellular GAS in endothelial cells. Moreover, the NAD+ content and NAD+/NADH ratio of GAS-infected endothelial cells were dramatically increased, whereas the cell cytotoxicity was decreased by exogenous nicotinamide treatment. After knockdown of the autophagy-related ATG9A, the intracellular bacterial load was increased in nicotinamide-treated endothelial cells. The results of Western blot and transmission electron microscopy also revealed that cells treated with nicotinamide can increase autophagy-associated LC3 conversion and double-membrane formation during GAS infection. Confocal microscopy images further showed that more GAS-containing vacuoles were colocalized with lysosome under nicotinamide-supplemented conditions than without nicotinamide treatment. In contrast to GAS, supplementation with exogenous nicotinamide did not effectively inhibit the growth of MRSA or S. Typhimurium in endothelial cells. These results indicate that intracellular NAD+ homeostasis is crucial for controlling intracellular GAS infection in endothelial cells. In addition, nicotinamide may be a potential new therapeutic agent to overcome persistent infections of GAS.

ABSTRACT Group A streptococcus (GAS) is an important human pathogen which can cause fatal diseases after invasion into the bloodstream. Although antibiotics and immune surveillance are the main defenses against GAS infection, GAS utilizes internalization into cells as a major immune evasion strategy. Our previous findings revealed that light chain 3 (LC3)-associated single membrane GAS-containing vacuoles in endothelial cells are compromised for bacterial clearance due to insufficient acidification after fusion with lysosomes. However, the characteristics and the activation mechanisms of these LC3-positive compartments are still largely unknown. In the present study, we demonstrated that the LC3-positive GAS is surrounded by single membrane and colocalizes with NADPH oxidase 2 (NOX2) complex but without ULK1, which are characteristics of LC3-associated phagocytosis (LAP). Inhibition of NOX2 or reactive oxygen species (ROS) significantly reduces GAS multiplication and enhances autolysosome acidification in endothelial cells through converting LAP to conventional xenophagy, which is revealed by enhancement of ULK1 recruitment, attenuation of p70s6k phosphorylation, and formation of the isolation membrane. We also clarify that the inactivation of mTORC1, which is the initiation signal of autophagy, is inhibited by NOX2- and ROS-activated phosphatidylinositol 3-kinase (PI3K)/AKT and MEK/extracellular signal-regulated kinase (ERK) pathways. In addition, streptolysin O (SLO) of GAS is identified as a crucial inducer of ROS for β1 integrin-mediated LAP induction. After downregulation of β1 integrin, GAS multiplication is reduced, accompanied with LAP inhibition and xenophagy induction. These results demonstrate that GAS infection preferentially induces ineffective LAP to evade xenophagic killing in endothelial cells through the SLO/β1 integrin/NOX2/ROS pathway. IMPORTANCE Our previous reports showed that the LC3-associated GAS-containing single membrane vacuoles are inefficient for bacterial clearance in endothelial cells, which may result in bacteremia. However, the characteristics and the induction mechanisms of these LC3-positive vacuoles are still largely unknown. Here we provide the first evidence that these LC3-positive GAS-containing single membrane compartments appear to be LAPosomes, which are induced by NOX2 and ROS. Through NOX2- and ROS-mediated signaling, GAS preferentially induces LAP and inhibits bacteriostatic xenophagy in endothelial cells. We also provide the first demonstration that β1 integrin acts as the receptor for LAP induction through GAS-produced SLO stimulation in endothelial cells. Our findings reveal the underlying mechanisms of LAP induction and autophagy evasion for GAS multiplication in endothelial cells.

Abstract Annexin A2 (ANXA2), a phospholipid-binding protein, has multiple biological functions depending on its cellular localization. We previously demonstrated that IFN-γ-triggered ANXA2 secretion is associated with exosomal release. Here, we show that IFN-γ-induced autophagy is essential for the extracellular secretion of ANXA2 in lung epithelial cells. We observed colocalization of ANXA2-containing autophagosomes with multivesicular bodies (MVBs) after IFN-γ stimulation, followed by exosomal release. IFN-γ-induced exophagic release of ANXA2 could not be observed in ATG5-silenced or mutant RAB11-expressing cells. Furthermore, knockdown of RAB8A and RAB27A, but not RAB27B, reduced IFN-γ-triggered ANXA2 secretion. Surface translocation of ANXA2 enhanced efferocytosis by epithelial cells, and inhibition of different exophagic steps, including autophagosome formation, fusion of autophagosomes with MVBs, and fusion of amphisomes with plasma membrane, reduced ANXA2-mediated efferocytosis. Our data reveal a novel route of IFN-γ-induced exophagy of ANXA2.

Abstract Activated microglial cells are present in dengue virus (DENV)-infected brains; however, the possible effects of DENV on microglia remain unclear. Here, we demonstrated DENV caused infection, including viral entry, RNA replication, viral protein expression, and virus release, in the murine microglial cell line BV2. DENV infection caused an increase in the formation of the multipolar phenotype in vitro and in vivo without affecting cell growth and cytotoxicity. DENV infection considerably increased cell motility and disrupting either actin filaments or clathrin retarded such effect. Increase in cell migration was only occurred by DENV infection following a clathrin-regulated endocytosis of DENV entry. Ultraviolet-inactivated DENV did not affect cell migration, and pharmacologically blocking toll-like receptor (TLR) 3 and TLR3-related signaling pathways reduced the DENV-induced increase in cell migration. These results demonstrate an advanced effect of DENV infection on microglial migration via a mechanism involving viral entry, RNA release, and TLR3 signal activation.

ABSTRACT Group A streptococcus (GAS) is an important human pathogen that causes a wide variety of cutaneous and systemic infections. Although originally thought to be an extracellular bacterium, numerous studies have demonstrated that GAS can trigger internalization into nonimmune cells to escape from immune surveillance or antibiotic-mediated killing. Epithelial cells possess a defense mechanism involving autophagy-mediated targeting and killing of GAS within lysosome-fused autophagosomes. In endothelial cells, in contrast, we previously showed that autophagy is not sufficient for GAS killing. In the present study, we showed higher galectin-3 (Gal-3) expression and lower Gal-8 expression in endothelial cells than in epithelial cells. The recruitment of Gal-3 to GAS is higher and the recruitment of Gal-8 to GAS is lower in endothelial cells than in epithelial cells. We further showed that Gal-3 promotes GAS replication and diminishes the recruitment of Gal-8 and ubiquitin, the latter of which is a critical protein for autophagy sequestration. After knockdown of Gal-3 in endothelial cells, the colocalization of Gal-8, parkin, and ubiquitin-decorated GAS is significantly increased, as is the interaction of Gal-8 and parkin, an E3 ligase. Furthermore, inhibition of Gal-8 in epithelial cells attenuates recruitment of parkin; both Gal-8 and parkin contribute to ubiquitin recruitment and GAS elimination. Animal studies confirmed that Gal-3-knockout mice develop less-severe skin damage and that GAS replication can be detected only in the air pouch and not in organs and endothelial cells. These results demonstrate that Gal-3 inhibits ubiquitin recruitment by blocking Gal-8 and parkin recruitment, resulting in GAS replication in endothelial cells. IMPORTANCE In epithelial cells, GAS can be efficiently killed within the lysosome-fused autophaosome compartment. However, we previously showed that, in spite of LC-3 recruitment, the autophagic machinery is not sufficient for GAS killing in endothelial cells. In this report, we provide the first evidence that Gal-3, highly expressed in endothelial cells, blocks the tagging of ubiquitin to GAS by inhibiting recruitment of Gal-8 and parkin, leading to an enhancement of GAS replication. We also provide the first demonstration that Gal-8 can interact with parkin, the critical E3 ligase, for resistance to intracellular bacteria by facilitating the decoration of bacteria with ubiquitin chains. Our findings reveal that differential levels of Gal-3 and Gal-8 expression and recruitment to GAS between epithelial cells and endothelial cells may contribute to the different outcomes of GAS elimination or survival and growth of GAS in these two types of cells.

Group A Streptococcus (GAS) is deleterious pathogenic bacteria whose interaction with blood vessels leads to life-threatening bacteremia. Although xenophagy, a special form of autophagy, eliminates invading GAS in epithelial cells, we found that GAS could survive and multiply in endothelial cells. Endothelial cells were competent in starvation-induced autophagy, but failed to form double-membrane structures surrounding GAS, an essential step in xenophagy. This deficiency stemmed from reduced recruitment of ubiquitin and several core autophagy proteins in endothelial cells, as demonstrated by the fact that it could be rescued by exogenous coating of GAS with ubiquitin. The defect was associated with reduced NO-mediated ubiquitin signaling. Therefore, we propose that the lack of efficient clearance of GAS in endothelial cells is caused by their intrinsic inability to target GAS with ubiquitin to promote autophagosome biogenesis for xenophagy.

Recent findings suggest that Notch-1 signaling contributes to neuronal death in ischemic stroke, but the underlying mechanisms are unknown. Hypoxia inducible factor-1α (HIF-1α), a global regulator of cellular responses to hypoxia, can interact with Notch and modulate its signaling during hypoxic stress. Here we show that Notch signaling interacts with the HIF-1α pathway in the process of ischemic neuronal death. We found that a chemical inhibitor of the Notch-activating enzyme, γ-secretase, and a HIF-1α inhibitor, protect cultured cortical neurons against ischemic stress, and combined inhibition of Notch-1 and HIF-1α further decreased neuronal death. HIF-1α and Notch intracellular domain (NICD) are co-expressed in the neuronal nucleus, and co-immunoprecipitated in cultured neurons and in brain tissue from mice subjected to focal ischemic stroke. Overexpression of NICD and HIF-1α in cultured human neural cells enhanced cell death under ischemia-like conditions, and a HIF-1α inhibitor rescued the cells. RNA interference-mediated depletion of endogenous NICD and HIF-1α also decreased cell death under ischemia-like conditions. Finally, mice treated with inhibitors of γ-secretase and HIF-1α exhibited improved outcome after focal ischemic stroke, with combined treatment being superior to individual treatments. Additional findings suggest that the NICD and HIF-1α collaborate to engage pro-inflammatory and apoptotic signaling pathways in stroke.

The infection of bacterial organisms generally causes cell death to facilitate microbial invasion and immune escape, both of which are involved in the pathogenesis of infectious diseases. In addition to the intercellular infectious processes, pathogen-produced/secreted enterotoxins (mostly exotoxins) are the major weapons that kill host cells and cause diseases by inducing different types of cell death, particularly apoptosis and necrosis. Blocking these enterotoxins with synthetic drugs and vaccines is important for treating patients with infectious diseases. Studies of enterotoxin-induced apoptotic and necrotic mechanisms have helped us to create efficient strategies to use against these well-characterized cytopathic toxins. In this article, we review the induction of the different types of cell death from various bacterial enterotoxins, such as staphylococcal enterotoxin B, staphylococcal alpha-toxin, Panton-Valentine leukocidin, alpha-hemolysin of Escherichia coli, Shiga toxins, cytotoxic necrotizing factor 1, heat-labile enterotoxins, and the cholera toxin, Vibrio cholerae. In addition, necrosis caused by pore-forming toxins, apoptotic signaling through cross-talk pathways involving mitochondrial damage, endoplasmic reticulum stress, and lysosomal injury is discussed.

Infection with dengue virus (DENV) causes an increase in proinflammatory responses, such as nitric oxide (NO) generation and TNF-α expression; however, the molecular mechanism underlying this inflammatory activation remains undefined, although the activation of the transcription factor NF-κB is generally involved. In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation. NF-κB is known to be activated by DENV infection. Pharmacologically inhibiting NF-κB activation abolishes iNOS/NO biosynthesis and TNF-α production. With inhibition, the potential role of NF-κB in oxidative signaling regulation was prevented during DENV infection. Heat-inactivated DENV failed to cause the identified inflammatory responses. Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production. In contrast to TLR3, viral protein NS2B3 also independently contributed to NF-κB activation to regulate TNF-α production. These results show the distinct pathways for NF-κB activation caused by DENV infection individually for the regulation of iNOS/NO and TNF-α expression.

BACKGROUND: Anesthetic propofol has immunomodulatory effects, particularly in the area of anti-inflammation. Bacterial endotoxin lipopolysaccharide (LPS) induces inflammation through toll-like receptor (TLR) 4 signaling. We investigated the molecular actions of propofol against LPS/TLR4-induced inflammatory activation in murine RAW264.7 macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Non-cytotoxic levels of propofol reduced LPS-induced inducible nitric oxide synthase (iNOS) and NO as determined by western blotting and the Griess reaction, respectively. Propofol also reduced the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 as detected by enzyme-linked immunosorbent assays. Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKβ (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced. Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages. An in vivo study using C57BL/6 mice also demonstrated the anti-inflammatory properties against LPS in peritoneal macrophages. CONCLUSIONS/SIGNIFICANCE: These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKβ/NF-κB signaling pathways.

Inactivation of glycogen synthase kinase (GSK)-3 has been implicated in cancer progression. Previously, we showed an abundance of inactive GSK-3 in the human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr-Abl tyrosine kinase. In Bcr-Abl signaling, the role of GSK-3 is not well defined. Here, we report that enforced expression of constitutively active GSK-3 reduced proliferation and increased Bcr-Abl inhibition-induced apoptosis by nearly 1-fold. Bcr-Abl inhibition activated GSK-3 and GSK-3-dependent apoptosis. Inactivation of GSK-3 by Bcr-Abl activity is, therefore, confirmed. To reactivate GSK-3, we used glucosylceramide synthase (GCS) inhibitor PDMP to accumulate endogenous ceramide, a tumor-suppressor sphingolipid and a potent GSK-3 activator. We found that either PDMP or silence of GCS increased Bcr-Abl inhibition-induced GSK-3 activation and apoptosis. Furthermore, PDMP sensitized the most clinical problematic drug-resistant CML T315I mutant to Bcr-Abl inhibitor GNF-2-, imatinib-, or nilotinib-induced apoptosis by >5-fold. Combining PDMP and GNF-2 eliminated transplanted-CML-T315I-mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF-2-induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr-Abl restricted and correlated to increased intracellular ceramide levels and acted through GSK-3-mediated apoptosis. This study suggests a feasible novel anti-CML strategy by accumulating endogenous ceramide to reactivate GSK-3 and abrogate drug resistance. [ABSTRACT FROM AUTHOR]

Electromagnetic fields produced in relativistic heavy-ion collisions are affected by the asymmetry of the projectile-target combination as well as the different initial configurations of the nucleus. In this study, the results of the electric and magnetic fields produced for different combinations of ions, namely 12C+197Au, 24Mg+197Au, 64Cu+197Au, and 197Au+197Au at √sNN=200 GeV, are demonstrated with a multiphase transport model. The configuration of the distribution of nucleons of 12C is initialized by a Woods-Saxon spherical structure, a three-α-clustering triangular structure or a three-α-clustering chain structure. It was observed that the electric and magnetic fields display different behavioral patterns for asymmetric combinations of the projectile and target nuclei as well as for different initial configurations of the carbon nucleus. The major features of the process are discussed. [ABSTRACT FROM AUTHOR]

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5-/-)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5-/- or Atg7-/- MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT 1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5-/- MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. [ABSTRACT FROM AUTHOR]