HLA-B27 Modulates Intracellular Growth of Salmonella Pathogenicity Island 2 Mutants and Production of Cytokines in Infected Monocytic U937 Cells

BACKGROUND: Salmonella enterica serovar Enteritidis PT4 KS8822/88 replicates rapidly in HLA-B27-transfected human monocytic U937 cells. In this process, Salmonella pathogenicity island 2 (SPI-2) genes play a crucial role. Our previous study indicated that 118 Salmonella genes, including 8 SPI-2 genes were affected by HLA-B27 antigen during Salmonella infection of U937 cells. METHODS/PRINCIPAL FINDINGS: To further investigate Salmonella replication in HLA-B27-positive U937 monocytic cells, two SPI-2 genes, ssaS and sscA up-regulated most during Salmonella infection of HLA-B27-transfected U937 cells, were mutated by using one-step gene disruption method. Intracellular survival and replication of the mutants in the U937 cells was compared to that of the wild type strain. Surprisingly, the two mutated strains replicated significantly more than the wild type bacteria in HLA-B27-transfected cells. Secretion of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) was significantly induced during the infection of HLA-B27-transfected U937 cells with the mutants. The results indicated that the certain SPI-2 genes in wild type bacteria suppress Salmonella intracellular growth and production of cytokines in infected HLA-B27-transfected cells. HLA-B27-associated modulation of Salmonella SPI-2 genes and cytokine production may have importance in the persistent infection of the bacteria and the pathogenesis of reactive arthritis. CONCLUSIONS: The study provides evidence that certain virulence factors of pathogens can reduce the intracellular growth in the host cells. We suggest that the limiting intracellular growth might be a strategy for persistence of bacteria in host cells, keeping a balance between pathogenic growth and pathogenesis

Implicit Identity Leakage: The Stumbling Block to Improving Deepfake Detection Generalization

In this paper, we analyse the generalization ability of binary classifiers for the task of deepfake detection. We find that the stumbling block to their generalization is caused by the unexpected learned identity representation on images. Termed as the Implicit Identity Leakage, this phenomenon has been qualitatively and quantitatively verified among various DNNs. Furthermore, based on such understanding, we propose a simple yet effective method named the ID-unaware Deepfake Detection Model to reduce the influence of this phenomenon. Extensive experimental results demonstrate that our method outperforms the state-of-the-art in both in-dataset and cross-dataset evaluation. The code is available at https://github.com/megvii-research/CADDM.Comment: Accepted by CVPR 202

Ferromagnetic and insulating behavior in both half magnetic levitation and non-levitation LK-99 like samples

Finding materials exhibiting superconductivity at room temperature has long been one of the ultimate goals in physics and material science. Recently, room-temperature superconducting properties have been claimed in a copper substituted lead phosphate apatite (Pb$_{10-x}$Cu$_x$(PO$_4$)$_6$O, or called LK-99) [1-3]. Using a similar approach, we have prepared LK-99 like samples and confirmed the half-levitation behaviors in some small specimens under the influence of a magnet at room temperature. To examine the magnetic properties of our samples, we have performed systematic magnetization measurements on the as-grown LK-99-like samples, including the half-levitated and non-levitated samples. The magnetization measurements show the coexistence of soft-ferromagnetic and diamagnetic signals in both half-levitated and non-levitated samples. The electrical transport measurements on the as-grown LK-99-like samples including both half-levitated and non-levitated samples show an insulating behavior characterized by the increasing resistivity with the decreasing temperature

Experimental Therapy of Ovarian Cancer with Synthetic Makaluvamine Analog: In Vitro and In Vivo Anticancer Activity and Molecular Mechanisms of Action

The present study was designed to determine the biological effects of novel marine alkaloid analog 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ) on human ovarian cancer cells for its anti-tumor potential and the underlying mechanisms as a novel chemotherapeutic agent. Human ovarian cancer cells (A2780 and OVCAR-3), and Immortalized non-tumorigenic human Ovarian Surface Epithelial cells (IOSE-144), were exposed to FBA-TPQ for initial cytotoxicity evaluation (via MTS assay kit, Promega). The detailed in-vitro (cell level) and in-vivo (animal model) studies on the antitumor effects and possible underlying mechanisms of action of the compounds were then performed. FBA-TPQ exerted potent cytotoxicity against human ovarian cancer A2780 and OVCAR-3 cells as an effective inhibitor of cell growth and proliferation, while exerting lesser effects on non-tumorigenic IOSE-144 cells. Further study in the more sensitive OVCAR-3 cell line showed that it could potently induce cell apoptosis (Annexin V-FITC assay), G2/M cell cycle arrest (PI staining analysis) and also dose-dependently inhibit OVCAR-3 xenograft tumors' growth on female athymic nude mice (BALB/c, nu/nu). Mechanistic studies (both in vitro and in vivo) revealed that FBA-TPQ might exert its activity through Reactive Oxygen Species (ROS)-associated activation of the death receptor, p53-MDM2, and PI3K-Akt pathways in OVCAR-3 cells, which is in accordance with in vitro microarray (Human genome microarrays, Agilent) data analysis (GEO accession number: GSE25317). In conclusion, FBA-TPQ exhibits significant anticancer activity against ovarian cancer cells, with minimal toxicity to non-tumorigenic human IOSE-144 cells, indicating that it may be a potential therapeutic agent for ovarian cancer

A single amino acid substitution confers enhanced methylation activity of mammalian Dnmt3b on chromatin DNA

Dnmt3a and Dnmt3b are paralogous enzymes responsible for de novo DNA methylation but with distinguished biological functions. In mice, disruption of Dnmt3b but not Dnmt3a causes global DNA hypomethylation, especially in repetitive sequences, which comprise the large majority of methylated DNA in the genome. By measuring DNA methylation activity of Dnmt3a and Dnmt3b homologues from five species, we found that mammalian Dnmt3b possessed significantly higher methylation activity on chromatin DNA than Dnmt3a and non-mammalian Dnmt3b. Sequence comparison and mutagenesis experiments identified a single amino acid substitution (I662N) in mammalian Dnmt3b as being crucial for its high chromatin DNA methylation activity. Further mechanistic studies demonstrated this substitution markedly enhanced the binding of Dnmt3b to nucleosomes and hence increased the chromatin DNA methylation activity. Moreover, this substitution was crucial for Dnmt3b to efficiently methylate repetitive sequences, which increased dramatically in mammalian genomes. Consistent with our observation that Dnmt3b evolved more rapidly than Dnmt3a during the emergence of mammals, these results demonstrated that the I662N substitution in mammalian Dnmt3b conferred enhanced chromatin DNA methylation activity and contributed to functional adaptation in the epigenetic system

A Novel CAN Tree Coordinate Routing in Content-Addressable Network

In this paper, we propose a novel approach to improve coordination routing while minimizing the maintenance overhead during nodes churn. It bases on “CAN Tree Routing for Content- Addressable Network” 1 which is a solution for peer-to-peer routing. We concentrated on coordinate routing in this paper. The key idea of our approach is a recursion process to calculate target zone code and search in CAN tree 1. Because the hops are via long links in CAN, it enhances routing flexibility and robustness against failures. Nodes automatically adapt routing table to cope with network change. The routing complexity is , which is much better than a uniform greedy routing, while each node maintains two long links in average

The corresponding DNA PCR product in mutated and wild type genes.

<p>The PCR product from three mutated isolates <i>ssaS</i>::Km (lanes 1–3) and the wild type gene <i>ssaS</i> (lanes 4 and 5 (A), and one mutated isolate <i>sscA</i>::Km (lane 3) and wild type gene <i>sscA</i> (lanes 4 and 5) (B) are demonstrated. The genomic DNAs isolated from the mutant and wild type bacteria were used as templates, respectively, and the flanking primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034093#pone-0034093-t001" target="_blank">Table 1</a>) used in the PCR reaction.</p

Intracellular growth of wild type and mutant strains in U937 cells.

<p>The growth of wild type, the <i>ssaS</i>::Km and <i>sscA</i>::Km mutants were compared in HLA-B27-transfected (A) or HLA-A2-transfected U937 cells (B). The comparison for the mutant <i>ssaS</i>::Km growth in HLA-B27 and HLA-A2 cells (C) and for the mutant <i>sscA</i>::Km growth in HLA-B27 and HLA-A2 cells (D) was shown. Values represented the mean and standard deviation of at least three independent experiments with duplicate samples. *, ** and *** indicate <i>P</i><0.05, <0.01 and <0.001, respectively, when the mutant-infected cells compared to the wild type (WT)-infected cells (A and B) or the mutant-infected HLA-B27 cells to HLA-A2 cells (C and D). Data were compared using Student's paired 2-tailed <i>t</i>-test.</p

Uptake of <i>S.</i> Enteritidis wild type and mutant strains into the transfected U937 cells.

<p>Wild type, <i>ssaS</i>::Km or <i>sscA</i>::Km mutant were used to infect HLA-B27-transfected cells and HLA-A2-transfected cells. The numbers of the bacteria were counted at 1 hour post-infection. The data represented the mean and standard deviation of at least three independent experiments with duplicate samples. No difference was found in the uptake of mutants and wild type bacteria in HLA-B27 cells, and no difference was found in the uptake of mutants and wild type bacteria between HLA-B27 and HLA-A2 cells.</p

Microarray Analysis of Response of <it>Salmonella </it>during Infection of HLA-B27- Transfected Human Macrophage-Like U937 Cells

Abstract Background Human leukocyte antigen (HLA)-B27 is strongly associated with the development of reactive arthritis (ReA) in humans after salmonellosis. Human monocytic U937 cells transfected with HLA-B27 are less able to eliminate intracellular Salmonella enterica serovar Enteritidis than those transfected with control HLA antigens (e.g. HLA-A2). To investigate further the mechanisms by which HLA-B27-transfected cells allow increased replication of these bacteria, a DNA-based microarray was used for comparative genomic analysis of S. Enteritidis grown in HLA-B27- or HLA-A2-transfected cells. The microarray consisted of 5080 oligonucleotides from different serovars of Salmonella including S. Enteritidis PT4-specific genes. Bacterial RNA was isolated from the infected HLA-B27- or HLA-A2-transfected cells, reverse-transcribed to cDNA, and hybridized with the oligonucleotides on the microarrays. Some microarray results were confirmed by RT-PCR. Results When gene expression was compared between Salmonella grown in HLA-B27 cells and in HLA-A2 cells, 118 of the 4610 S. Enteritidis-related genes differed in expression at 8 h after infection, but no significant difference was detectable at 2 h after infection. These differentially expressed genes are mainly involved in Salmonella virulence, DNA replication, energy conversion and metabolism, and uptake and metabolism of nutrient substances, etc. The difference suggests HLA-B27-dependent modulation of Salmonella gene expression, resulting in increased Salmonella replication in HLA-B27-positive cells. Among the up-regulated genes were those located in Salmonella pathogenicity island (SPI)-2, which play a central role in intracellular survival and replication of Salmonella. Conclusions This is the first report to show the regulation of Salmonella gene expression by HLA-B27 during infection of host cells. This regulation probably leads to increased Salmonella survival and replication in HLA-B27-positive cells. SPI-2 genes seem to contribute significantly to the increased replication.</p