Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2

BACKGROUND: Francisella tularensis is a zoonotic intracellular bacterial pathogen that causes tularemia. The subspecies tularensis is highly virulent and is classified as a category A agent of biological warfare because of its low infectious dose by an aerosol route, and its ability to cause severe disease. In macrophages F. tularensis exhibits a rather novel intracellular lifestyle; after invasion it remains in a phagosome for three to six hours before escaping to, and replicating in the cytoplasm. The molecular mechanisms that allow F. tularensis to invade and replicate within a host cell have not been well defined. METHODS: We constructed a stable transposon mutagenesis library of virulent strain Schu S4 using a derivative of the EZ::TN transposon system(®). Approximately 2000 mutants were screened for the inability to invade, and replicate in the hepatic carcinoma cell line HepG2. These mutants were also tested for replication within the J774.1 macrophage-like cell line. RESULTS: Eighteen mutants defective in intracellular replication in HepG2 cells were identified. Eight of these mutants were auxotrophs; seven had mutations in nucleotide biosynthesis pathways. The remaining mutants had insertions in genes that were predicted to encode putative transporters, enzymes involved in protein modification and turnover, and hypothetical proteins. A time course of the intracellular growth of a pyrB mutant revealed that this mutant was only able to grow at low levels within HepG2 cells but grew like wild-type bacteria in J774.1 cells. This pyrB mutant was also attenuated in mice. CONCLUSION: This is the first reported large-scale mutagenesis of a type A strain of F. tularensis and the first identification of mutants specifically defective in intracellular growth in a hepatic cell line. We have identified several genes and pathways that are key for the survival and growth of F. tularensis in a hepatic cell line, and a number of novel intracellular growth-defective mutants that have not been previously characterized in other pathogens. Further characterization of these mutants will help provide a better understanding of the pathogenicity of F. tularensis, and may have practical applications as targets for drugs or attenuated vaccines

Azithromycin effectiveness against intracellular infections of Francisella

<p>Abstract</p> <p>Background</p> <p>Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az) is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B <it>Francisella </it>(<it>F.</it>) <it>tularensis </it>have been reported to be resistant to Az, however our laboratory <it>Francisella </it>strains were found to be sensitive. We hypothesized that different strains/species of <it>Francisella </it>(including Type A) may have different susceptibilities to Az, a widely used and well-tolerated antibiotic.</p> <p>Results</p> <p><it>In vitro </it>susceptibility testing of Az confirmed that <it>F. tularensis subsp. holarctica </it>Live Vaccine Strain (LVS) (Type B) was not sensitive while <it>F. philomiragia, F. novicida</it>, and Type A <it>F. tularensis </it>(NIH B38 and Schu S4 strain) were susceptible. In J774A.1 mouse macrophage cells infected with <it>F. philomiragia, F. novicida</it>, and <it>F. tularensis </it>LVS, 5 μg/ml Az applied extracellularly eliminated intracellular <it>Francisella </it>infections. A concentration of 25 μg/ml Az was required for <it>Francisella-</it>infected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (<it>tolC </it>and <it>ftlC</it>) in <it>F. novicida </it>demonstrated less sensitivity to Az by MIC than the parental strain, but the <it>tolC </it>disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of <it>acrA </it>and <it>acrB </it>mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in <it>F. novicida</it>. In contrast, <it>F. tularensis </it>Schu S4 mutants Δ<it>acrB </it>and Δ<it>acrA </it>were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in <it>F. tularensis </it>Schu S4. <it>F. novicida </it>LPS O-antigen mutants (<it>wbtN, wbtE, wbtQ </it>and <it>wbtA</it>) were found to be less sensitive <it>in vitro </it>to Az compared to the wild-type. Az treatment prolonged the survival of <it>Galleria </it>(<it>G</it>.) <it>mellonella </it>infected with <it>Francisella</it>.</p> <p>Conclusion</p> <p>These studies demonstrate that Type A <it>Francisella </it>strains, as well as <it>F. novicida </it>and <it>F. philomiragia</it>, are sensitive to Az <it>in vitro. Francisella </it>LPS and the RND efflux pump may play a role in Az sensitivity. Az also has antimicrobial activity against intracellular <it>Francisella</it>, suggesting that the intracellular concentration of Az is high enough to be effective against multiple strains/species of <it>Francisella</it>, especially in macrophages. Az treatment prolonged survival an <it>in vivo </it>model of <it>Francisella-</it>infection.</p

In vitro effect of lysophosphatidic acid on proliferation, invasion and migration of human ovarian cancer cells

Purpose: To evaluate the effect of lysophosphatidic acid (LPA) on the proliferation, invasion and migration ability of 3AO, SKOV3 and CAOV3 human ovarian cancer cell lines.Methods: SKOV3, 3AO and CAOV3 cell lines were respectively treated with LPA. Changes in the proliferation rate of these cell lines were observed after LPA treatment. The cell lines that were not treated with LPA served as control group. Boyden chamber was used to assess cell invasion and migration capability. The expression levels of relevant cytokines related to cell migration in the supernatant of CAOV3 cell line were determined using ELISA following LPA stimulation.Results: The cell proliferation rate of human ovarian cancer cell lines was significantly accelerated after in vitro LPA treatment in a concentration-dependent fashion. Boyden chamber assay data indicate that invasion indices in 3AO and CAOV3 cell lines were significantly higher than those in untreated control cell lines (p &lt; 0.05). However, no statistical significance was noted between 3AO and CAOV3 cell lines (p &lt; 0.05). The expression levels of relevant cytokines in the CAOV3 cell line were significantly upregulated after LPA treatment (p &lt; 0.05).Conclusion: LPA intervention in vitro accelerates cell proliferation rate and also significantly upregulates the expression levels of multiple cytokines related to cell migration in human ovarian cancer cell lines, suggesting that LPA plays a significant role in the invasion and migration of SKOV3, 3AO and CAOV3 cell lines.Keywords: Ovarian carcinoma, Tumor infiltration, Lysophosphatidic acid, Cell migration, Cytokine

Proposal of a free-space-to-chip pipeline for transporting single atoms

A free-space-to-chip pipeline is proposed to efficiently transport single atoms from a magneto-optical trap to an on-chip evanescent field trap. Due to the reflection of the dipole laser on the chip surface, the conventional conveyor belt approach can only transport atoms close to the chip surface but with a distance of about one wavelength, which prevents efficient interaction between the atom and the on-chip waveguide devices. Here, based on a two-layer photonic chip architecture, a diffraction beam of the integrated grating with an incident angle of the Brewster angle is utilized to realize free-space-to-chip atom pipeline. Numerical simulation verified that the reflection of the dipole laser is suppressed and that the atoms can be brought to the chip surface with a distance of only 100nm. Therefore, the pipeline allows a smooth transport of atoms from free space to the evanescent field trap of waveguides and promises a reliable atom source for a hybrid photonic-atom chip.Comment: 8 pages, 7 figure

Differential analysis of chromatin accessibility and histone modifications for predicting mouse developmental enhancers

Enhancers are distal cis-regulatory elements that modulate gene expression. They are depleted of nucleosomes and enriched in specific histone modifications; thus, calling DNase-seq and histone mark ChIP-seq peaks can predict enhancers. We evaluated nine peak-calling algorithms for predicting enhancers validated by transgenic mouse assays. DNase and H3K27ac peaks were consistently more predictive than H3K4me1/2/3 and H3K9ac peaks. DFilter and Hotspot2 were the best DNase peak callers, while HOMER, MUSIC, MACS2, DFilter and F-seq were the best H3K27ac peak callers. We observed that the differential DNase or H3K27ac signals between two distant tissues increased the area under the precision-recall curve (PR-AUC) of DNase peaks by 17.5-166.7% and that of H3K27ac peaks by 7.1-22.2%. We further improved this differential signal method using multiple contrast tissues. Evaluated using a blind test, the differential H3K27ac signal method substantially improved PR-AUC from 0.48 to 0.75 for predicting heart enhancers. We further validated our approach using postnatal retina and cerebral cortex enhancers identified by massively parallel reporter assays, and observed improvements for both tissues. In summary, we compared nine peak callers and devised a superior method for predicting tissue-specific mouse developmental enhancers by reranking the called peaks

Effects of lipids with different oxidation levels on protein degradation and biogenic amines formation in Sichuan-style sausages

ABS T R A C T We evaluated the effects of different oxidation levels of lipids on protein degradation and biogenic amines (BAs) formation during Sichuan-style sausages processing. Lipids with varying degrees of oxidation were obtained through storage at different temperatures and added as raw materials of Sichuan-style sausages, followed by the analyses of lipid oxidation, protein degradation, biogenic amine content, and other indicators. During the pro-cessing, with increasing degree of lipid oxidation, the contents of peroxide value (POV), thiobarbituric acid reactive substances (TBARs), protein degradation index (PI), amino acid nitrogen (AAN), free amino acids (FAAs), and BAs increased. Based on the protein electrophoresis results, the higher the oxidation degree of pig backfat, the higher degree of sarcoplasmic protein oxidation, and the greater myofibril protein degradation. Pearson correlation revealed that lipid oxidation, protein degradation, and BAs content correlated significantly (P < 0.05).Peer reviewe