Колесный робот автосопровождения

В работе рассмотрен процесс проектирования и сборки мобильного робота автоматического слежения на базе микроконтроллера Arduino Uno. Была подобрана материально-техническая база для реализации, изучены и внедрены алгоритмы ПИД-регулирования и широтно-импульсной модуляции. Была произведена сборка робота и апробация его работы.Arduino based mobile robot of automatic following design and assembling are considered. Components and devices were selected. PID-control and pulse-width modulation algorithms were studied and implemented. Final assembling and testing were provided

Local amplifiers of IL-4Rα-mediated macrophage activation promote repair in lung and liver

The type 2 immune response controls helminth infection and maintains tissue homeostasis but can lead to allergy and fibrosis if not adequately regulated. We have discovered local tissue-specific amplifiers of type 2-mediated macrophage activation. In the lung, surfactant protein A (SP-A) enhanced interleukin-4 (IL-4)-dependent macrophage proliferation and activation, accelerating parasite clearance and reducing pulmonary injury after infection with a lung-migrating helminth. In the peritoneal cavity and liver, C1q enhancement of type 2 macrophage activation was required for liver repair after bacterial infection, but resulted in fibrosis after peritoneal dialysis. IL-4 drives production of these structurally related defense collagens, SP-A and C1q, and the expression of their receptor, myosin 18A. These findings reveal the existence within different tissues of an amplification system needed for local type 2 responses

Surfactant protein-A modulates LPS-induced TLR4 localization and signaling via β-arrestin 2.

The soluble C-type lectin surfactant protein (SP)-A mediates lung immune responses partially via its direct effects on alveolar macrophages (AM), the main resident leukocytes exposed to antigens. SP-A modulates the AM threshold of lipopolysaccharide (LPS) activity towards an anti-inflammatory phenotype both in vitro and in vivo through various mechanisms. LPS responses are tightly regulated via distinct pathways including subcellular TLR4 localization and thus ligand sensing. The cytosolic scaffold and signaling protein β-arrestin 2 acts as negative regulator of LPS-induced TLR4 activation. Here we show that SP-A neither increases TLR4 abundancy nor co-localizes with TLR4 in primary AM. SP-A significantly reduces the LPS-induced co-localization of TLR4 with the early endosome antigen (EEA) 1 by promoting the co-localization of TLR4 with the post-Golgi compartment marker Vti1b in freshly isolated AM from rats and wild-type (WT) mice, but not in β-arrestin 2(-/-) AM. Compared to WT mice pulmonary LPS-induced TNF-α release in β-arrestin 2(-/-) mice is accelerated and enhanced and exogenous SP-A fails to inhibit both lung LPS-induced TNF-α release and TLR4/EEA1 positioning. SP-A, but not LPS, enhances β-arrestin 2 protein expression in a time-dependent manner in primary rat AM. The constitutive expression of β-arrestin 2 in AM from SP-A(-/-) mice is significantly reduced compared to SP-A(+/+) mice and is rescued by SP-A. Prolonged endosome retention of LPS-induced TLR4 in AM from SP-A(-/-) mice is restored by exogenous SP-A, and is antagonized by β-arrestin 2 blocking peptides. LPS induces β-arrestin 2/TLR4 association in primary AM which is further enhanced by SP-A. The data demonstrate that SP-A modulates LPS-induced TLR4 trafficking and signaling in vitro and in vivo engaging β-arrestin 2

SP-A enhances β-arrestin 2 protein expression and LPS-induced β-arrestin 2/TLR4 interaction in primary AM.

<p>A, Western blot of total β-arrestin 2 protein expression in primary rat AM treated with LPS (100 ng/ml, 1 h and 3 h), SP-A (40 µg/ml, 1 h and 3 h) or both (SP-A 1 h plus LPS 1 h or 3 h) as indicated. Equal amounts of whole cell lysates were subjected to SDS-PAGE and immunoblotted for β-arrestin 2 and β-actin. Data of at least four independent experiments were normalized to β-actin, basal β-arrestin 2 expression in untreated cells was set 100%, and calculated data were statistically analyzed by two-way ANOVA with Bonferroni's post test (mean ± SEM). *p<0.05; **p<0.01. B, Immunoprecipitation (IP) of β-arrestin 2 and TLR4 immunoblot (IB) from rat AM lysates treated with LPS (100 ng/ml), SP-A (40 µg/ml), or both as indicated. Data of seven independent experiments were analyzed by one-way Anova with Dunett's posttest (mean ± SEM). *p<0.05; **p<0.01 (versus untreated control).</p

Reduced β-arrestin 2 expression and prolonged TLR4/EEA1 co-localization in SP-A^−/− AM are rescued by SP-A.

<p>A, Western blot of total β-arrestin 2 protein expression in primary AM from WT and SP-A^−/− mice treated with SP-A (40 µg/ml; 30 min). Equal amounts of whole cell lysates were subjected to SDS-PAGE and immunoblotted for β-arrestin 2 and β-actin. Data of four independent experiments were normalized to β-actin, basal β-arrestin 2 expression in WT mice was set 100%, and calculated data were statistically analyzed by one-way ANOVA with Bonferroni's posttest (mean ± SEM). *p<0.05 (WT versus SP-A^−/−); ^#p<0.05 (SP-A-treated versus basal). B, Representative IF for TLR4 localization in primary AM from SP-A^−/− mice treated with LPS (100 ng/ml), SP-A (40 µg/ml), LPS plus SP-A, or pre-treated with cell permeable β-arrestin 2 blocking peptides (15 min, 20 µg/ml) prior to LPS plus SP-A. Images are representative of two independent experiments in which over 80% of the cells had similar staining patterns. Early endosomes were stained with EEA1. Arrows indicate the area of co-localization. Upper panels, DIC image. Lower panels, overlay of single stainings. Scale bars, 10 µm. C, Analysis of TLR4 and EEA1 co-localization after treatment with LPS, SP-A plus LPS, or pretreatment with cell permeable β-arrestin 2 blocking peptides prior to LPS plus SP-A. Values are expressed as percentage ± SEM of two independent experiments with 20 cells per condition. Data were statistically analyzed by one-way ANOVA with Bonferroni's posttest. *p<0.05; ***p<0.001.</p

SP-A inhibits LPS-enhanced TLR4 expression in AM.

<p>A, Western blot of total TLR4 protein expression in primary rat AM treated with LPS (100 ng/ml, 1 h and 3 h), SP-A (40 µg/ml, 1 h and 3 h), or both (SP-A 1 h plus LPS 1 h or 3 h) as indicated. Equal amounts of whole cell lysates were subjected to SDS-PAGE and immunoblotted for TLR4 and β-actin. Data of at least five independent experiments were normalized to β-actin, basal TLR4 expression in untreated cells was set 100%, and calculated data were statistically analyzed by two-way ANOVA with Bonferroni's post test (mean ± SEM). *p<0.05; **p<0.01. B, Representative IF for TLR4 in LPS- and SP-A-treated primary rat AM. TLR4 distribution was monitored in cells treated with LPS (100 ng/ml) or SP-A (40 µg/ml) for the times indicated. Images are representative of at least two independent experiments in which over 80% of the cells had similar staining patterns. Scale bars, 10 µm. Isotype controls are also shown. Upper panel, LPS-treated cells. Lower panel, SP-A-treated cells. C, Representative IF for TLR4 in BAL cells from WT mice after intratracheal administration of 2.5 µg/kg BW of LPS or 5 mg/kg of SP-A plus 2.5 µg/kg of LPS. Images are representative of at least two independent experiments in which over 80% of the cells had similar staining patterns. Scale bars, 10 µm. D, Quantification of pixel intensity from confocal microscopy images stained for TLR4. Values are expressed as percentage change in mean pixel intensity ± SEM of one experiment with at least 20 cells per condition. Untreated control was set to 100% (dotted line) and data were statistically analyzed by one-way ANOVA with Bonferroni's posttest. *p<0.05; **p<0.01, ***p<0.001. E, Representative IF for TLR4 in primary rat AM treated A555-labeled SP-A for 15 min. Images are representative of two independent experiments in which over 80% of the cells had similar staining patterns. Scale bars, 10 µm.</p

SP-A inhibits the LPS-induced co-localization of TLR4/EEA 1 in rat AM.

<p>A and B, Representative IF for TLR4 localization in primary rat AM treated with LPS (100 ng/ml), SP-A (40 µg/ml), or both as indicated. Images are representative of at least three independent experiments in which over 80% of the cells had similar staining patterns. Early endosomes were stained with EEA1. Arrows indicate the area of co-localization. Upper panels, DIC image. Middle panels, TLR4 staining. Lower panels, overlay of single stainings. Scale bars, 10 µm. C, Analysis of TLR4 and EEA1 co-localization. Values are expressed as percentage ± SEM of at least two independent experiments with at least 20 cells per condition. Data were statistically analyzed by one-way ANOVA with Bonferroni's posttest. *p<0.05.</p

SP-A fails to inhibit enhanced LPS responsiveness in β-arrestin 2^−/− mice.

<p>A and B, TNF-α release in BAL fluid obtained from WT (n = 4–5) and β-arrestin 2^−/− mice (n = 3–5) at different times after intratracheal administration of 2.5 µg/kg BW of LPS or 5 mg/kg of SP-A plus 2.5 µg/kg of LPS. The control group received LPS-free PBS. *p<0.05 (LPS- versus PBS-treated WT mice); ^### p<0.001 (LPS-versus PBS-treated β-arrestin 2^−/− mice); **p<0.01 (LPS-treated WT mice versus LPS-treated β-arrestin 2^−/− mice). C, Western blot of cytosolic IκB-α protein expression in BAL cells from WT and β-arrestin 2^−/− mice 4 h after intratracheal administration of LPS or SP-A plus 2.5 µg/kg of LPS. Equal amounts of cytosolic fractions were subjected to SDS-PAGE and immunoblotted for IκB-α and β-actin. Data of three independent experiments were normalized to β-actin, basal IκB-α expression was set 100%, and calculated data were statistically analyzed by one-way ANOVA with Bonferroni's posttest. *p<0.05; **p<0.01. D, Representative IF for TLR4 localization in BAL cells from WT and β-arrestin 2^−/− mice after intratracheal administration of LPS or SP-A plus LPS. Images are representative of at least two independent experiments in which over 80% of the cells had similar staining patterns. Early endosomes were stained with EEA1. Arrows indicate the area of co-localization. Scale bars, 10 µm. E, Analysis of TLR4 and EEA1 co-localization. Values are expressed as percentage ± SEM of two independent experiments with at least 20 cells per condition. Data were statistically analyzed by one-way ANOVA with Bonferroni's posttest. *p<0.05.</p

SP-A fails to inhibit the LPS-induced co-loclization of TLR4/EEA1 in β-arrestin 2^−/− AM.

<p>A + B and D + E, Representative IF for TLR4 localization in primary AM from WT (A and D) and β-arrestin 2^−/− mice (B and E) treated with LPS (100 ng/ml), SP-A (40 µg/ml), or both as indicated. Images are representative of at least three independent experiments in which over 80% of the cells had similar staining patterns. Early endosomes were stained with EEA1 and the post-Golgi compartment was stained with Vti1b. Arrows indicate the area of co-localization. Scale bars, 10 µm. C and F, Analysis of TLR4 and EEA (C) or TLR4 and Vti1b (F) co-localization. Values are expressed in percentage ± SEM of at least three independent experiments with at least 50 cells per condition. Data were statistically analyzed by one-way ANOVA with Bonferroni's posttest. *p<0.05; ***p<0.001.</p