The zebrafish genome contains two distinct selenocysteine tRNA[Ser]Sec genes

AbstractThe zebrafish is widely used as a model system for studying mammalian developmental genetics and more recently, as a model system for carcinogenesis. Since there is mounting evidence that selenium can prevent cancer in mammals, including humans, we characterized the selenocysteine tRNA[Ser]Sec gene and its product in zebrafish. Two genes for this tRNA were isolated and sequenced and were found to map at different loci within the zebrafish genome. The encoding sequences of both are identical and their flanking sequences are highly homologous for several hundred bases in both directions. The two genes likely arose from gene duplication which is a common phenomenon among many genes in this species. In addition, zebrafish tRNA[Ser]Sec was isolated from the total tRNA population and shown to decode UGA in a ribosomal binding assay

Bloodstream Infections and Clinical Significance of Healthcare-associated Bacteremia: A Multicenter Surveillance Study in Korean Hospitals

Recent changes in healthcare systems have changed the epidemiologic paradigms in many infectious fields including bloodstream infection (BSI). We compared clinical characteristics of community-acquired (CA), hospital-acquired (HA), and healthcare-associated (HCA) BSI. We performed a prospective nationwide multicenter surveillance study from 9 university hospitals in Korea. Total 1,605 blood isolates were collected from 2006 to 2007, and 1,144 isolates were considered true pathogens. HA-BSI accounted for 48.8%, CA-BSI for 33.2%, and HCA-BSI for 18.0%. HA-BSI and HCA-BSI were more likely to have severe comorbidities. Escherichia coli was the most common isolate in CA-BSI (47.1%) and HCA-BSI (27.2%). In contrast, Staphylococcus aureus (15.2%), coagulase-negative Staphylococcus (15.1%) were the common isolates in HA-BSI. The rate of appropriate empiric antimicrobial therapy was the highest in CA-BSI (89.0%) followed by HCA-BSI (76.4%), and HA-BSI (75.0%). The 30-day mortality rate was the highest in HA-BSI (23.0%) followed by HCA-BSI (18.4%), and CA-BSI (10.2%). High Pitt score and inappropriate empirical antibiotic therapy were the independent risk factors for mortality by multivariate analysis. In conclusion, the present data suggest that clinical features, outcome, and microbiologic features of causative pathogens vary by origin of BSI. Especially, HCA-BSI shows unique clinical characteristics, which should be considered a distinct category for more appropriate antibiotic treatment

Down-Regulation of NF-κB Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in Drosophila

The activation of several transcription factors is required for the elimination of infectious pathogens via the innate immune response. The transcription factors NF-κB, AP-1, and STAT play major roles in the synthesis of immune effector molecules during innate immune responses. However, the fact that these immune responses can have cytotoxic effects requires their tight regulation to achieve restricted and transient activation, and mis-regulation of the damping process has pathological consequences. Here we show that AP-1 and STAT are themselves the major inhibitors responsible for damping NF-κB–mediated transcriptional activation during the innate immune response in Drosophila. As the levels of dAP-1 and Stat92E increase due to continuous immune signaling, they play a repressive role by forming a repressosome complex with the Drosophila HMG protein, Dsp1. The dAP-1–, Stat92E-, and Dsp1-containing complexes replace Relish at the promoters of diverse immune effector genes by binding to evolutionarily conserved cis-elements, and they recruit histone deacetylase to inhibit transcription. Reduction by mutation of dAP-1, Stat92E, or Dsp1 results in hyperactivation of Relish target genes and reduces the viability of bacterially infected flies despite more efficient pathogen clearance. These defects are rescued by reducing the Relish copy number, thus confirming that mis-regulation of Relish, not inadequate activation of dAP-1, Stat92E, or Dsp1 target genes, is responsible for the reduced survival of the mutants. We conclude that an inhibitory effect of AP-1 and STAT on NF-κB is required for properly balanced immune responses and appears to be evolutionarily conserved

Promyelocytic leukemia proteins regulate fanconi anemia gene expression

Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.1

HER2 regulates intracellular calcium concentration in SKBR3 cells.

<p>A) PMCA2 levels in control versus HER2KD-SKBR3 cells as assessed by immunoblot. B) Intracellular calcium measurements in HER2KD-SKBR3 cells relative to control. Numbers indicate the mean calcium concentrations estimated by FURA2 measurements. Each bar represents the mean ± SEM of 3 separate experiments. Asterisk denotes statistically significant difference. C) Expression of a NFAT-luciferase indicator construct in control SKBR3 cells, HER2KD-SKBR3 cells and PMCA2KD-SKBR3 cells. Each bar represents the mean ± SEM of 3 separate experiments. Asterisks denote statistically significant differences. D) Con-focal images of immunofluorescence for NFATc1 (red) or DAPI (blue) in control (top row), HER2KD-SKBR3 (middle row) or PMCA2KD-SKBR3 (bottom row) cells. Scale bars represent 10μm. E) Apoptosis as assessed by TUNEL assay in HER2KD-SKBR3 cells relative to controls exposed to differing concentrations of extracellular calcium ± ionomycin. Each bar represents the mean ± SEM of 3 separate experiments. F) Con-focal images of immunofluorescence for NFATc1 (red) or DAPI (blue) in control (top row), HER2KD-SKBR3 (middle row) or PMCA2KD-SKBR3 (bottom row) cells treated with cyclosporine A to block calcineurin activity. Scale bars represent 10μm.</p

Loss of HER2 prevents the formation of membrane protrusion in HER2KD-SKBR3 cells.

<p>A) Examination of relative expression of HER2 by QPCR. Bars represent mean ± SEM for 3 separate experiments. B) Immunoblot analysis from control and HER2KD-SKBR3 cells. C) Time course of pHER2 and pAKT levels in control (black bars) and HER2KD-SKBR3 (gray bars) cells in response to EGF. Cells were serum-starved for 16 hours, treated with growth factors and harvested at times listed on graphs. Each bar represents the mean ± SEM of 3 separate experiments. * represents p<0.05, ** represents p<0.01, *** represents p< 0.001. D) Con-focal images of immunofluorescence for HER2 (green) and phalloidin (actin, red) in control and HER2KD-SKBR3 cells. Insets represent Z-stack images in 2 different orientations and white arrow indicates membrane protrusions. E) Con-focal images of immunofluorescence for HER2 (green) and phospho-HER2 (red) in control and HER2KD-SKBR3 cells. Insets represent Z-stack images in 2 different orientations and white arrow indicates membrane protrusions. F) Con-focal images of immunofluorescence for HER2 (green) and phospho-AKT (red) in control and HER2KD-SKBR3 cells. Insets represent Z-stack images in 2 different orientations and white arrow indicates membrane protrusions. In D-F scale bar represents 10μm. Confocal settings for capturing images of HER2 immunofluorescence were adjusted to detect membrane structure and should not be interpreted as quantitative. G) Scanning (top) and transmission (bottom) electron microscopy of control and HER2KD-SKBR3 cells. Scale bars represent 20μM. H) BrdU incorporation in HER2KD-SKBR3 cells relative to control cells. Asterisk denotes a significant difference with control. (n = 3) I) Viable HER2KD-SKBR3 and control cells as assessed by XTT assay. Asterisk denote significant difference vs. control. (n = 3).</p

PLEKHA8P1 Promotes Tumor Progression and Indicates Poor Prognosis of Liver Cancer

Hepatocellular carcinoma (HCC) records the second-lowest 5-year survival rate despite the avalanche of research into diagnosis and therapy. One of the major obstacles in treatment is chemoresistance to drugs such as 5-fluorouracil (5-FU), making identification and elucidation of chemoresistance regulators highly valuable. As the regulatory landscape grows to encompass non-coding genes such as long non-coding RNAs (lncRNAs), a relatively new class of lncRNA has emerged in the form of pseudogene-derived lncRNAs. Through bioinformatics analyses of the TCGA LIHC dataset, we have systematically identified pseudogenes of prognostic value. Initial experimental validation of selected pseudogene-derived lncRNA (PLEKHA8P1) and its parental gene (PLEKHA8), a well-studied transport protein in Golgi complex recently implicated as an oncogene in both colorectal and liver cancer, indicates that the pseudogene/parental gene pair promotes tumor progression and that their dysregulated expression levels affect 5-FU-induced chemoresistance in human HCC cell line FT3-7. Our study has thus confirmed cancer-related functions of PLEKHA8, and laid the groundwork for identification and validation of oncogenic pseudogene-derived lncRNA that shows potential as a novel therapeutic target in circumventing chemoresistance induced by 5-FU

Activation of HER2 signaling promotes the formation of membrane protrusions.

<p>A) Con-focal images of immunofluorescence for actin (phalloidin, left 2 panels), phsopho-HER2 (middle 2 panels) and phospho-AKT (right 2 panels) in SKBR3 cells in serum-free media. B) Con-focal images of immunofluorescence for actin (phalloidin, left 2 panels), phsopho-HER2 (middle 2 panels) and phospho-AKT (right 2 panels) in SKBR3 cells treated with EGF for 2 hours. C) Con-focal images of immunofluorescence for actin (phalloidin, left 2 panels), phsopho-HER2 (middle 2 panels) and phospho-AKT (right 2 panels) in SKBR3 cells treated with NRG1 for 2 hours. For each pair of panels, the image on the right represents an enlargement of the boxed area from the left panel and the insets represent Z-stack images in 2 different orientations. White arrows point to membrane protrusions. Confocal settings for capturing immunofluorescence images were adjusted to detect membrane structure and should not be interpreted as quantitative. All scale bars represent 10μm.</p