Data_Sheet_1_RIOK-1 Is a Suppressor of the p38 MAPK Innate Immune Pathway in Caenorhabditis elegans.DOCX

<p>Innate immunity is the primary defense mechanism against infection in metazoans. However, aberrant upregulation of innate immune-signaling pathways can also be detrimental to the host. The p38 MAPK/PMK-1 innate immune-signaling pathway has been demonstrated to play essential roles in cellular defenses against numerous infections in metazoans, including Caenorhabditis elegans. However, the negative regulators that maintain the homeostasis of this important innate immune pathway remain largely understudied. By screening a focused RNAi library against the kinome of C. elegans, we identified RIOK-1, a human RIO kinase homolog, as a novel suppressor of the p38 MAPK/PMK-1 signal pathway. We demonstrated that the suppression of riok-1 confers resistance to Aeromonas dhakensis infection in C. elegans. Using quantitative real time-PCR and riok-1 reporter worms, we found the expression levels of riok-1 to be significantly upregulated in worms infected with A. dhakensis. Our genetic epistasis analysis suggested that riok-1 acts on the upstream of the p38 MAPK/pmk-1 genetic pathway. Moreover, the suppression of riok-1 enhanced the p38 MAPK signal, suggesting that riok-1 is a negative regulator of this innate pathway in C. elegans. Our epistatic results put riok-1 downstream of skn-1, which encodes a p38 MAPK downstream transcription factor and serves as a feedback loop to the p38 MAPK pathway during an A. dhakensis infection. In conclusion, riok-1 is proposed as a novel innate immune suppressor and as a negative feedback loop model involving p38 MAPK, SKN-1, and RIOK-1 in C. elegans.</p

Table_1_RIOK-1 Is a Suppressor of the p38 MAPK Innate Immune Pathway in Caenorhabditis elegans.XLSX

<p>Innate immunity is the primary defense mechanism against infection in metazoans. However, aberrant upregulation of innate immune-signaling pathways can also be detrimental to the host. The p38 MAPK/PMK-1 innate immune-signaling pathway has been demonstrated to play essential roles in cellular defenses against numerous infections in metazoans, including Caenorhabditis elegans. However, the negative regulators that maintain the homeostasis of this important innate immune pathway remain largely understudied. By screening a focused RNAi library against the kinome of C. elegans, we identified RIOK-1, a human RIO kinase homolog, as a novel suppressor of the p38 MAPK/PMK-1 signal pathway. We demonstrated that the suppression of riok-1 confers resistance to Aeromonas dhakensis infection in C. elegans. Using quantitative real time-PCR and riok-1 reporter worms, we found the expression levels of riok-1 to be significantly upregulated in worms infected with A. dhakensis. Our genetic epistasis analysis suggested that riok-1 acts on the upstream of the p38 MAPK/pmk-1 genetic pathway. Moreover, the suppression of riok-1 enhanced the p38 MAPK signal, suggesting that riok-1 is a negative regulator of this innate pathway in C. elegans. Our epistatic results put riok-1 downstream of skn-1, which encodes a p38 MAPK downstream transcription factor and serves as a feedback loop to the p38 MAPK pathway during an A. dhakensis infection. In conclusion, riok-1 is proposed as a novel innate immune suppressor and as a negative feedback loop model involving p38 MAPK, SKN-1, and RIOK-1 in C. elegans.</p

Kaplan-Meier survival curves for 148 patients with monomicrobial bacteremia caused by <i>Aeromonas veronii</i>, <i>A</i>. <i>dhakensis</i>, <i>A</i>. <i>caviae</i>, and <i>A</i>. <i>hydrophila</i> (Log-rank test, <i>P</i> = 0.02).

<p>Kaplan-Meier survival curves for 148 patients with monomicrobial bacteremia caused by <i>Aeromonas veronii</i>, <i>A</i>. <i>dhakensis</i>, <i>A</i>. <i>caviae</i>, and <i>A</i>. <i>hydrophila</i> (Log-rank test, <i>P</i> = 0.02).</p

Clinical Implications of Species Identification in Monomicrobial <i>Aeromonas</i> Bacteremia

<div><p>Background</p><p>Advances in <i>Aeromonas</i> taxonomy have led to the reclassification of aeromonads. Hereon, we aimed to re-evaluate the characteristics of <i>Aeromonas</i> bacteremia, including those of a novel species, <i>Aeromonas dhakensis</i>.</p><p>Methodology/Principal Findings</p><p>A retrospective study of monomicrobial <i>Aeromonas</i> bacteremia at a medical center in southern Taiwan from 2004–2011 was conducted. Species identification was based on <i>rpoB</i> sequencing. Of bacteremia of 153 eligible patients, <i>A. veronii</i> (50 isolates, 32.7%), <i>A. dhakensis</i> (48, 31.4%), <i>A. caviae</i> (43, 28.1%), and <i>A. hydrophila</i> (10, 6.5%) were the principal causative species. <i>A. dhakensis</i> and <i>A. veronii</i> bacteremia were mainly community-acquired and presented as primary bacteremia, spontaneous bacterial peritonitis, or skin and soft-tissue infection, whereas <i>A. caviae</i> was associated with hospital-onset bacteremia. The distribution of the AmpC β-lactamase and metallo-β-lactamase genes was species-specific: <i>bla</i>_AQU-1, <i>bla</i>_MOX, or <i>bla</i>_CepH was present in <i>A. dhakensis</i>, <i>A. caviae</i>, or <i>A. hydrophila</i>, respectively, and <i>bla</i>_CphA was present in <i>A. veronii</i>, <i>A. dhakensis</i>, and <i>A. hydrophila</i>. The cefotaxime resistance rates of the <i>A. caviae</i>, <i>A. dhakensis</i>, and <i>A. hydrophila</i> isolates were higher than that of <i>A. veronii</i> (39.5%%, 25.0%, and 30% <i>vs.</i> 2%, respectively). <i>A. dhakensis</i> bacteremia was linked to the highest 14-day sepsis-related mortality rate, followed by <i>A. hydrophila</i>, <i>A. veronii</i>, and <i>A. caviae</i> bacteremia (25.5%, 22.2%, 14.0%, and 4.7%, respectively; <i>P</i> = 0.048). Multivariate analysis revealed that <i>A. dhakensis</i> bacteremia, active malignancies, and a Pitt bacteremia score ≥ 4 was an independent mortality risk factor.</p><p>Conclusions/Significance</p><p>Characteristics of <i>Aeromonas</i> bacteremia vary between species. <i>A. dhakensis</i> prevalence and its associated poor outcomes suggest it an important human pathogen.</p></div

Multivariate stepwise logistic regression analysis for mortality (N = 22, 767).

<p>Multivariate stepwise logistic regression analysis for mortality (N = 22, 767).</p

Additional file 1 of Mitochondrial CISD1/Cisd accumulation blocks mitophagy and genetic or pharmacological inhibition rescues neurodegenerative phenotypes in Pink1/parkin models

Additional file 1: Supplementary Figure S1. Characterisation of Cisd in ageing and different tissues. (A) Immunoblot analysis of protein lysates from young (2 days) or old (30 days) whole flies of the indicated genotypes. Samples were homogenised under non-reducing (top panel) and reducing (bottom panel) conditions. Blots were probed for Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. (B) Immunoblot of mitochondrial fractions from WT flies, probed for poly-Ub, Cisd (CISD2, Proteintech, 13318-1-AP) and ATP5a. (C) Immunoblot analysis of protein lysates from 2- and 30-day-old adult heads of the indicated genotypes, probed for pUb, Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. (D) Relative amount of Cisd monomer and dimer quantified from replicate blots shown in C. (E) Immunoblot of protein lysates from whole flies of WT or Cisd null (−/−) or Cisd overexpression (OE) driven by da-GAL4, probed for Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. (F, G) Climbing assay of 2- and 20-day-old WT flies orCisd overexpression driven only in (F) DA neurons (TH-GAL4) or (G) pan-muscle (Mef2-GAL4). (H) Lifespan analysis of flies overexpressing Cisd in muscles as in G versus a WT control genotype. Statistical analysis: (D) unpaired t-test; (F, G) Kruskal-Wallis non-parametric test with Dunn’s post-hoc correction. ** P < 0.01, **** P < 0.0001. Supplementary Figure S2. Quantification of p62, Atg8-II and Cisd protein levels, and Pink1 and parkin mRNA. (A, B) Quantification of immunoblots shown in Fig. 4D. (C) Quantification of Cisd levels from immunoblots shown in Fig. 4I. (D, E) Quantification of Pink1 (D) or parkin (E) transcript levels upon Cisd overexpression (OE) or knockdown (RNAi). Statistical analysis: (A, B, D, E) Welch’s t test, (C) one-way ANOVA with Sidak’s post-hoc correction. * P < 0.05, ** P < 0.01, **** P < 0.0001. Supplementary Figure S3. Characterisation of Cisd loss-of-function on organismal and mitochondrial phenotypes. (A) Immunoblot analysis of protein lysates from whole flies of the indicated genotypes of Cisd loss versus control, probed for Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. (B) Climbing assay of 2- and 20-day-old flies of the indicated genotypes. Knockdown is driven by da-GAL4. (C) Lifespan analysis of Cisd loss as in B. (D) Confocal microscopy analysis of mitochondrial morphology, immunostained for ATP5A mitochondrial marker in flight muscle of the indicated genotypes. Knockdowns are driven by da-GAL4. (E) Confocal analysis of adult neurons of the indicated ages, WT control and Cisd RNAi (KK line) animals co-expressing the mitophagy reporter mito-QC (OMM-localised tandem RFP-GFP) with nSyb-GAL4 to highlight mitolysosomes, shown separately and quantified in F. Statistical analysis: (B) Kruskal-Wallis non-parametric test with Dunn’s post-hoc correction. (F) one-way ANOVA with Sidak’s post-hoc correction. * P < 0.05, **** P < 0.0001. Scale bars = 10 μm. (G) Immunoblot analysis of protein lysates from whole flies of the indicated genotypes probed for pUb and Total Ub. Supplementary Figure S4. Characterisation of Cisdand parkin genetic interaction. (A, B) Climbing assay of tissue-specific Cisd knockdown (KD) in either pan-muscles with Mef2-GAL driver (A) or pan-neurons with nSyb-GAL driver (B) alongside respective controls. (C) Viability assay for genetic interactions between the indicated combinations revealing synthetic lethality of Pink1/parkin mutants and CisdOE with da-GAL driver. (D) Confocal microscopy analysis of mitophagy reporter mito-QC in flight muscle from Cisd knockdown driven by Mef2-GAL4 in WT and parkin mutant backgrounds in 30-day-old animals. (E) Quantification of the number of mitolysosomes shown in D. Data points indicated individual animals analysed. Statistical analysis: one-way ANOVA with Sidak’s post-hoc correction; * P < 0.05, **** P < 0.0001. Scale bars = 10 μm

Characteristics of the 22,777 patients with dengue in Tainan.

<p>Characteristics of the 22,777 patients with dengue in Tainan.</p

Symptoms associated with adverse dengue fever prognoses at the time of reporting in the 2015 dengue outbreak in Taiwan

<div><p>Background</p><p>Tainan experienced the most severe dengue epidemic in Taiwan in 2015. This study investigates the association between the signs and symptoms at the time of reporting with the adverse dengue prognoses.</p><p>Methods</p><p>A descriptive study was conducted using secondary data from the Dengue Disease Reporting System in Tainan, Taiwan, between January 1 and December 31, 2015. A multivariate stepwise logistic regression was used to identify the risk factors for the adverse prognoses: ICU admissions and mortality.</p><p>Results</p><p>There were 22,777 laboratory-confirmed reported cases (mean age 45.6 ± 21.2 years), of which 3.7% were admitted to intensive care units (ICU), and 0.8% were fatal. The most common symptoms were fever (92.8%), myalgia (26.6%), and headache (22.4%). The prevalence of respiratory distress, altered consciousness, shock, bleeding, and thrombocytopenia increased with age. The multivariate analysis indicated that being in 65–89 years old age group [Adjusted Odds Ratio (aOR):4.95], or the 90 years old and above age group (aOR: 9.06), and presenting with shock (aOR: 8.90) and respiratory distress (aOR: 5.31) were significantly associated with the risk of ICU admission. While old age (aOR: 1.11), respiratory distress (aOR: 9.66), altered consciousness (aOR: 7.06), and thrombocytopenia (aOR: 2.55) were significantly associated with the risk of mortality.</p><p>Conclusions</p><p>Dengue patients older than 65 and those with severe and non-specific signs and symptoms at the time of reporting were at a higher risk of ICU admission and mortality. First-line healthcare providers need to be aware of the varied presentations between the different age groups to allow early diagnosis and in-time management, which would prevent ICU admissions and fatalities in dengue patients.</p></div

PRMT-7/PRMT7 activates HLH-30/TFEB to guard plasma membrane integrity compromised by bacterial pore-forming toxins

Bacterial pore-forming toxins (PFTs) that disrupt host plasma membrane integrity (PMI) significantly contribute to the virulence of various pathogens. However, how host cells protect PMI in response to PFT perforation in vivo remains obscure. Previously, we demonstrated that the HLH-30/TFEB-dependent intrinsic cellular defense (INCED) is elicited by PFT to maintain PMI in Caenorhabditis elegans intestinal epithelium. Yet, the molecular mechanism for the full activation of HLH-30/TFEB by PFT remains elusive. Here, we reveal that PRMT-7 (protein arginine methyltransferase-7) is indispensable to the nuclear transactivation of HLH-30 elicited by PFTs. We demonstrate that PRMT-7 participates in the methylation of HLH-30 on its RAG complex binding domain to facilitate its nuclear localization and activation. Moreover, we showed that PRMT7 is evolutionarily conserved to regulate TFEB cellular localization and repair plasma damage caused by PFTs in human intestinal cells. Together, our observations not only unveil a novel PRMT-7/PRMT7-dependent post-translational regulation of HLH-30/TFEB but also shed insight on the evolutionarily conserved mechanism of the INCED against PFT in metazoans.</p

Dielectrophoresis System for Testing Antimicrobial Susceptibility of Gram-Negative Bacteria to β‑Lactam Antibiotics

Gram-negative bacteria (GNBs) are common pathogens causing severe sepsis. Rapid evaluation of drug susceptibility would guide effective antibiotic treatment and promote life-saving. A total of 78 clinical isolates of 13 Gram-negative species collected between April 2013 and November 2013 from two medical centers in Tainan were tested. Bacterial morphology changes in different concentrations of antibiotics were observed under the electric field of a quadruple electrode array using light microscopy. The minimal inhibitory concentrations (MICs) of four antimicrobial agents, namely, cefazolin, ceftazidime, cefepime, and doripenem, were determined by the dielectrophoretic antimicrobial susceptibility testing (dAST) and by the conventional broth dilution testing (BDT). The antibiotics at the concentration of 1× MIC induced obvious morphological changes in susceptible GNBs, including cell elongation, cell swelling, or lysis, at 90 min. In contrast, resistant strains remained unchanged. The MIC results measured by dAST were in good agreement with those of BDT (essential agreement 95.6%). The category agreement rate was 89.2%, and the very major errors rate for dAST was 2.9%. In conclusion, dAST could accurately determine drug susceptibility within 90 min. Comprehensive tests by dAST for more drugs against more GNB species are possible in the future