Controlled Copolymerization of 1‑Octene and (Meth)acrylates via Organotellurium-Mediated Living Radical Polymerization (TERP)

Copolymerization of 1-octene and (meth)­acrylates, such as methyl acrylate, trifluoroethyl acrylate (TFEA), methyl methacrylate, and trifluoroethyl methacrylate, under organotellurium-mediated living radical polymerization (TERP) conditions was investigated. Polymerization under thermal conditions gave copolymers with considerably broad molecular distributions (polydispersity index [PDI] > 1.45), whereas that under photoirradiation greatly increased the PDI control. Structurally well-controlled copolymers with number-average molecular weights (<i>M</i>_n) of 3000–18 000 and low PDIs (1.22–1.45) were obtained. Addition of Brønsted acids, such as 1,3-C₆H₄[C­(CF₃)₂OH]₂ and hexafluoroisopropanol, increased the insertion of 1-octene into the copolymer. The molar fraction of 1-octene (MF_oct) reached ∼0.5 in the copolymerization using TFEA as an acrylate monomer and excess amount of 1-octene in the presence of the acid. The copolymer was used as a macro-chain-transfer agent for the synthesis of block copolymers. This is the first example of the use of this type of copolymer as a macro-chain-transfer agent in the controlled synthesis of block copolymers

Simultaneous Detection of ATP and GTP by Covalently Linked Fluorescent Ribonucleopeptide Sensors

A noncovalent RNA complex embedding an aptamer function and a fluorophore-labeled peptide affords a fluorescent ribonucleopeptide (RNP) framework for constructing fluorescent sensors. By taking an advantage of the noncovalent properties of the RNP complex, the ligand-binding and fluorescence characteristics of the fluorescent RNP can be independently tuned by taking advantage of the nature of the RNA and peptide subunits, respectively. Fluorescent sensors tailored for given measurement conditions, such as a detection wavelength and a detection concentration range for a ligand of interest can be easily identified by screening of fluorescent RNP libraries. The noncovalent configuration of a RNP becomes a disadvantage when the sensor is to be utilized at very low concentrations or when multiple sensors are applied to the same solution. Here, we report a strategy to convert a fluorescent RNP sensor in the noncovalent configuration into a covalently linked stable fluorescent RNP sensor. This covalently linked fluorescent RNP sensor enabled ligand detection at a low sensor concentration, even in cell extracts. Furthermore, application of both ATP and GTP sensors enabled simultaneous detection of ATP and GTP by monitoring each wavelength corresponding to the respective sensor. Importantly, when a fluorescein-modified ATP sensor and a pyrene-modified GTP sensor were co-incubated in the same solution, the ATP sensor responded at 535 nm only to changes in the concentration of ATP, whereas the GTP sensor detected GTP at 390 nm without any effect on the ATP sensor. Finally, simultaneous monitoring by these sensors enabled real-time measurement of adenosine deaminase enzyme reactions

<i>P</i>. <i>acnes</i> in LC3-positive vacuoles.

<p>(A) Confocal microscopic image of LC3-positive vacuoles containing <i>P</i>. <i>acnes</i> found in Raw264.7, MEF, and HeLa cells at 8 h MOI 1000 postinfection. Scale bar: 5 μm. (B) Frequency of cells with LC3-positive vacuoles containing <i>P</i>. <i>acnes</i> at 1, 2, 4, 8, 16, and 24 h MOI 1000 postinfection. Data are representative of at least three independent experiments. Black bars; means + SD.</p

Autophagy Induced by Intracellular Infection of <i>Propionibacterium acnes</i>

<div><p>Background</p><p>Sarcoidosis is caused by Th1-type immune responses to unknown agents, and is linked to the infectious agent <i>Propionibacterium acnes</i>. Many strains of <i>P</i>. <i>acnes</i> isolated from sarcoid lesions cause intracellular infection and autophagy may contribute to the pathogenesis of sarcoidosis. We examined whether <i>P</i>. <i>acnes</i> induces autophagy.</p><p>Methods</p><p>Three cell lines from macrophages (Raw264.7), mesenchymal cells (MEF), and epithelial cells (HeLa) were infected by viable or heat-killed <i>P</i>. <i>acnes</i> (clinical isolate from sarcoid lymph node) at a multiplicity of infection (MOI) of 100 or 1000 for 1 h. Extracellular bacteria were killed by washing and culturing infected cells with antibiotics. Samples were examined by colony assay, electron-microscopy, and fluorescence-microscopy with anti-LC3 and anti-LAMP1 antibodies. Autophagy-deficient (Atg5^-/-) MEF cells were also used.</p><p>Results</p><p>Small and large (≥5 μm in diameter) LC3-positive vacuoles containing few or many <i>P</i>. <i>acnes</i> cells (LC3-positive <i>P</i>. <i>acnes</i>) were frequently found in the three cell lines when infected by viable <i>P</i>. <i>acnes</i> at MOI 1000. LC3-positive large vacuoles were mostly LAMP1-positive. A few small LC3-positive/LAMP1-negative vacuoles were consistently observed in some infected cells for 24 h postinfection. The number of LC3-positive <i>P</i>. <i>acnes</i> was decreased at MOI 100 and completely abolished when heat-killed <i>P</i>. <i>acnes</i> was used. LC3-positive <i>P</i>. <i>acnes</i> was not found in autophagy-deficient Atg5^-/- cells where the rate of infection was 25.3 and 17.6 times greater than that in wild-type Atg5^+/+ cells at 48 h postinfection at MOI 100 and 1000, respectively. Electron-microscopic examination revealed bacterial cells surrounded mostly by a single-membrane including the large vacuoles and sometimes a double or multi-layered membrane, with occasional undigested bacterial cells in ruptured late endosomes or in the cytoplasm.</p><p>Conclusion</p><p>Autophagy was induced by intracellular <i>P</i>. <i>acnes</i> infection and contributed to intracellular bacterial killing as an additional host defense mechanism to endocytosis or phagocytosis.</p></div

Colony assay for intracellular viable <i>P</i>. <i>acnes</i>.

<p>(A) Autophagy-deficiency allowed for survival and persistence of <i>P</i>. <i>acnes</i> within the host cells. Both Atg5^+/+ and Atg5^-/- MEF cells were infected by <i>P</i>. <i>acnes</i> at MOI 100 or 1000. The rate of infection (% intracellular viable <i>P</i>. <i>acnes</i>) was measured by colony assay at 5, 24, and 48 h postinfection. Black bars; means + SD, n = 9. Data were collected from three independent experiments. (B) Raw264.7, MEF, and HeLa cells were infected by <i>P</i>. <i>acnes</i> at MOI 100 or 1000. The rate of infection (% intracellular viable <i>P</i>. <i>acnes</i>) was measured by colony assay at 5, 24, 48, and 72 h postinfection. Black bars; means ± SD, n = 3. *<i>P</i> = 0.0036, **<i>P</i> = 0.036 compared among three cell lines.</p

Large LC3-positive vacuoles containing many <i>P</i>. <i>acnes</i>.

<p>(A) Confocal microscopic image of large (≥5 μm in diameter) LC3-positive vacuoles containing many <i>P</i>. <i>acnes</i> found in Raw264.7, MEF, and HeLa cells at 8 h MOI 1000 postinfection. Scale bar: 5 μm. (B) Frequency of cells with large LC3-positive vacuoles containing <i>P</i>. <i>acnes</i> at 1, 2, 4, 8, 16, and 24 h MOI 1000 postinfection. Data are representative of at least three independent experiments. Black bars; means + SD.</p

Electron microscopic images of intracellular <i>P</i>. <i>acnes</i>.

<p>HeLa cells infected by <i>P</i>. <i>acnes</i> at MOI 1000 were examined at 8 h postinfection. (A) Small (indicated by an arrow) and large (indicated by arrowhead) vacuoles in a single HeLa cell, containing a few and many undigested <i>P</i>. <i>acnes</i> cells, respectively, both surrounded by a single membrane. (B) Two undigested <i>P</i>. <i>acnes</i> cells interfaced with cytosolic components were surrounded by a double-membrane. (C) Three undigested <i>P</i>. <i>acnes</i> cells without cytosolic components were surrounded by a multi-layered membrane. (D) A single undigested <i>P</i>. <i>acnes</i> cell free in cytoplasmic space without a surrounding membrane structure. (E) A late endosome surrounded by a single-membrane with multi-vesicular bodies (arrowheads) containing undigested <i>P</i>. <i>acnes</i> cells. A part of the single-membrane is discontinued (indicated by an arrow) with a small influx of cytosolic components into the endosome. Scale bar: A 5 μm, B-E 500 nm.</p

Trafficking pathways of intracellular <i>P</i>. <i>acnes</i>.

<p>Intracellular <i>P</i>. <i>acnes</i> are digested in both endocytic (E) and autophagic (A) pathways. Autophagy induction is caused by cytosolic <i>P</i>. <i>acnes</i> cells that completely escaped from endosomes (A1) or by damaged endosomes containing <i>P</i>. <i>acnes</i> cells (A2). LC3-associated phagocytosis (L) is especially induced in the presence of infection by a large number of bacteria.</p

90-day mortality by dose category of cumulative steroid dose.

90-day mortality by dose category of cumulative steroid dose.</p

Baseline patient and demographic characteristics.

ObjectiveLong-term steroid use increases the risk of developing Pneumocystis pneumonia (PcP), but there are limited reports on the relation of long-term steroid and PcP mortality.MethodsRetrospective multicenter study to identify risk factors for PcP mortality, including average steroid dose before the first visit for PcP in non-human immunodeficiency virus (HIV)-PcP patients. We generated receiver operating characteristic (ROC) curves for 90-day all-cause mortality and the mean daily steroid dose per unit body weight in the preceding 10 to 90 days in 10-day increments. Patients were dichotomized by 90-day mortality and propensity score-based stabilized inverse probability of treatment weighting (IPTW) adjusted covariates of age, sex, and underlying disease. Multivariate analysis with logistic regression assessed whether long-term corticosteroid use affected outcome.ResultsOf 133 patients with non-HIV-PcP, 37 died within 90 days of initial diagnosis. The area under the ROC curve for 1–40 days was highest, and the optimal cutoff point of median adjunctive corticosteroid dosage was 0.34 mg/kg/day. Past steroid dose, underlying interstitial lung disease and emphysema, lower serum albumin and lower lymphocyte count, higher lactate dehydrogenase, use of therapeutic pentamidine and therapeutic high-dose steroids were all significantly associated with mortality. Underlying autoimmune disease, past immunosuppressant use, and a longer time from onset to start of treatment, were associated lower mortality. Logistic regression analysis after adjusting for age, sex, and underlying disease with IPTW revealed that steroid dose 1–40 days before the first visit for PcP (per 0.1 mg/kg/day increment, odds ratio 1.36 [95% confidence interval = 1.16–1.66], PConclusionA steroid dose before PcP onset was strongly associated with 90-day mortality in non-HIV-PcP patients, emphasizing the importance of appropriate prophylaxis especially in this population.</div