Characterization and comparison of differentially expressed genes involved in Chlamydia psittaci persistent infection in vitro and in vivo.

Chlamydia psittaci is an obligate intracellular zoonotic pathogen that can enter a persistence state in host cells. While the exact pathogenesis is not well understood, this persistence state may play an important role in chronic Chlamydia disease. Here, we assess the effects of chlamydial persistence state in vitro and in vivo by transmission electron microscopy (TEM) and cDNA microarray assays. First, IFN-γ-induced C. psittaci persistence in HeLa cells resulted in the upregulation of 68 genes. These genes are involved in protein translation, carbohydrate metabolism, nucleotide metabolism, lipid metabolism and general stress. However, 109 genes were downregulated following persistent C. psittaci infection, many of which are involved in the TCA cycle, expression regulation and transcription, protein secretion, proteolysis and transport, membrane protein, presumed virulence factor, cell division and late expression. To further study differential gene expression of C. psittaci persistence in vivo, we established an experimentally tractable mouse model of C. psittaci persistence. The C. psittaci-infected mice were gavaged with either water or amoxicillin (amox), and the results indicated that the 20 mg/kg amox-exposed C. psittaci were viable but not infectious. Differentially expressed genes (DEGs) screened by cDNA microarray were detected, and interestingly, the results showed upregulation of three genes (euo, ahpC, prmC) and downregulation of five genes (pbp3, sucB_1, oppA_4, pmpH, ligA) in 20 mg/kg amox-exposed C. psittaci, which suggests that antibiotic treatment in vivo can induce chlamydial persistence state and lead to differential gene expression. However, the discrepancy on inducers between the two models requires more research to supplement. The results may help researchers better understand survival advantages during persistent infection and mechanisms influencing C. psittaci pathogenesis or evasion of the adaptive immune response.

pHmScarlet is a pH-sensitive red fluorescent protein to monitor exocytosis docking and fusion steps.

pH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events. Superecliptic pHluorin (SEP), a green pH-sensitive FP, has been widely used for imaging single-vesicle exocytosis. However, the docking step cannot be visualized using this FP, since the fluorescence signal inside vesicles is too low to be observed during docking process. Among the available red pH-sensitive FPs, none is comparable to SEP for practical applications due to unoptimized pH-sensitivity and fluorescence brightness or severe photochromic behavior. In this study, we engineer a bright and photostable red pH-sensitive FP, named pHmScarlet, which compared to other red FPs has higher pH sensitivity and enables the simultaneous detection of vesicle docking and fusion. pHmScarlet can also be combined with SEP for dual-color imaging of two individual secretory events. Furthermore, although the emission wavelength of pHmScarlet is red-shifted compared to that of SEP, its spatial resolution is high enough to show the ring structure of vesicle fusion pores using Hessian structured illumination microscopy (Hessian-SIM).

Fast Super-Resolution Imaging Technique and Immediate Early Nanostructure Capturing by a Photoconvertible Fluorescent Protein.

Low temporal resolution and limited photocontrollable fluorescent protein probes have restricted the widespread application of single-molecule localization microscopy (SMLM). In the current study, we developed a new photoconvertible fluorescent protein (PCFP), pcStar, and quick single molecule-guided Bayesian localization microscopy (Quick-SIMBA). The combination of pcStar and Quick-SIMBA achieved the highest temporal resolution (0.1-0.25 s) with large field-of-view (76 × 9.4 µm2 -76 × 31.4 µm2) among the SMLM methods, which enabled the dynamic movements of the endoplasmic reticulum dense tubular matrix to be resolved. Moreover, pcStar extended the application of SMLM to imaging the immediate early nanostructures in Drosophila embryos and revealed a specific "parallel three-pillar" structure in the neuronal-glial cell junction, helping to elucidate glial cell "locking" and support of neurons during Drosophila embryogenesis.

Helicobacter pylori induces IL-1ß and IL-18 production in human monocytic cell line through activation of NLRP3 inflammasome via ROS signaling pathway.

This study investigated whether Helicobacter pylori could activate the nucleotide-binding oligomerization domain-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome in human macrophages and the involvement of reactive oxygen species (ROS) in inflammasome activation. Phorbol-12-myristate-13-acetate (PMA)-differentiated human acute monocytic leukemia cell line THP-1 was infected with H. pylori. The levels of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 in supernatant were measured by ELISA. Intracellular ROS level was analyzed by flow cytometry. Quantitative real-time PCR and western blot analysis were employed to determine the mRNA and protein expression levels of NLRP3 and caspase-1 in THP-1 cells, respectively. Our results showed that H. pylori infection could induce IL-1ß and IL-18 production in PMA-differentiated THP-1 cells in a dose- and time-dependent manner. Moreover, secretion of IL-1ß and IL-18 in THP-1 cells following H. pylori infection was remarkably reduced by NLRP3-specific small interfering RNA treatment. In addition, the intracellular ROS level was elevated by H. pylori infection, which could be eliminated by the ROS scavenger N-acetylcysteine (NAC). Furthermore, NAC treatment could inhibit NLRP3 inflammasome formation and caspase-1 activation and suppress the release of IL-1ß and IL-18 from H. pylori-infected THP-1 cells. These findings provide novel insights into the innate immune response against H. pylori infection, which could potentially be used for the prevention and treatment of H. pylori-related diseases.

Distribution and location of Daxx in cervical epithelial cells with high risk human papillomavirus positive.

AIMS: To provide the basis for further exploring the effect and its mechanism of Death domain associated protein (Daxx) on the progress of cervical carcinoma induced by human papillomavirus (HPV), the distribution and location of Daxx in cervical carcinoma with high risk HPV(HR-HPV) positive was analyzed. METHODS: The samples of normal cervical epithelial cells, cervical intraepithelial neoplasia grade I (CINI), CINII CINIII and cervical cancers were collected. Immunohistochemistry assay was used to analyze the distributions and locations of Daxx in the cervical tissue. Indirect immunoinfluorescence test was utilized to observe the locations of Daxx in Caski cells with HPV16 positive. RESULTS: Under the light microscopy, the brown signals of Daxx distributed in the nuclei of normal cervical epithelial cells; Daxx mainly distributed in nuclear membrane and there were a small amount of Daxx in the nuclei in CINI. Daxx intensively distributed in the cytoplasm and cell membrane in CINII, CINIII and cervical cancer. Under fluorescent microscopy, the distribution and location of Daxx in Caski cells was similarly to that in cervical cells of CINII, CINIII and cervical cancer. CONCLUSION: In the progress of the cervical cancer, Daxx gradually translocates from nucleus into nuclear membrane, cytoplasm and cell membrane. Daxx locates in the cytoplasm and cell membrane in CINII, CINIII and cervical cancer. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/4671548951113870.