Co-culture of human bone marrow mesenchymal stem cells and macrophages attenuates lipopolysaccharide-induced inflammation in human corneal epithelial cells

<p>Dry eye syndrome (DES) is considered as an ocular surface inflammatory disease. Previous studies have shown inflammation plays an important role in the progression and onset of DES. Co-culture of human bone marrow mesenchymal stem cells (HBMSCs) and macrophages showed immunomodulatory effects via regulation of cytokine regulation. Thus, the aim of this study was to investigate the effect of the interaction of these cells on <i>in vitro</i> DES model. The conditioned media (CM) from macrophages, HBMSCs, and HBMSCs + macrophages were treated to human corneal epithelial cells, which showed significant reduction in IL-1α and IL-1β expression levels in HBMSCs + macrophages group. Moreover, the IL-1 Receptor Antagonist (IL-1RA) was highly expressed in the CM from the HBMSCs + macrophages group. Wounded eyes of mice were treated with IL-1RA at 0–100 ng/mL for 16 h, the wound size was reduced. The results of this study might lead to the identification of new therapeutic targets for DES.</p> <p>Conditioned media from co-cultured HBMSCs and macrophages had a synergistic immunomodulatory effect on corneal epithelial cells.</p

Cytotoxicity of Gallium–Indium Liquid Metal in an Aqueous Environment

Eutectic gallium–indium alloy (EGaIn) liquid metal is highly conductive, moldable, and extremely deformable and has attracted significant attention for many applications, ranging from stretchable electronics to drug delivery. Even though EGaIn liquid metal is generally known to have low toxicity, the toxicity of the metal, rather than a salt form of Ga or In, has not been systematically studied yet. In this paper, we investigate the time-dependent concentration of the ions released from EGaIn liquid metal in an aqueous environment and their cytotoxicity to human cells. It is observed that only the Ga ion is dominantly released from EGaIn when no external agitation is applied, whereas the concentration of the In ion drastically increases with sonication. The cytotoxicity study reveals that all human cells tested are viable in the growth media with naturally released EGaIn ions, but the cytotoxicity becomes significant with sonication-induced EGaIn releasates. On the basis of the comparative study with other representative toxic elements, that is, Hg and Cd, it could be concluded that EGaIn is reasonably safe to use in an aqueous environment; however, it should be cautiously handled when any mechanical agitation is applied

Autoactivated MMP3 (actMMP3) induced mitochondrial ROS generation and the Nox1 expression.

<p>N27 cells were transfected with mock control, catalytically inactive mutant MMP3 (MutMMP3), or actMMP3 vector for 24, 48, or 72 h. Mitochondrial ROS levels were determined by the MitoSox staining (A) and MitoSox level (B) was quantified at 24 h after transfected with MutMMP3 and ActMMP3. Cells expressing GFP (green) represent vector-transduced cells. Scale bar  = 10 µm (C) MMP-3 enzyme activity assay on lysates of N27 cells transfected with MutMMP3 and ActMMP3 for 48 or 72 h. (D) Representative photomicrographs of Western blots against Nox1 on lysates of N27 cells transfected with MutMMP3 and ActMMP3 for 48 h. Densitometric analysis of the western blots results normalized against β-actin and expressed as % induction compared with MutMMP3-transfected cells. Results are presented as the mean ± SEM, n = 8. The whole experiment has been repeated three times with similar results. ** p<0.01, *** p<0.001 vs. cells transfected with MutMMP3 vector. (E) Representative photomicrographs of Western blots against Nox1 on lysates of N27 cells treated with 0.25, 0.5, 1, 2 mM of N-acetylcystein (NAC) in the presense or absence of 6-OHDA. Cells were cotreated with 6-OHDA and NAC for 24 h. Densitometric analysis of the western blots results normalized against β-actin and expressed as % induction compared with non-treated control cells. Results are presented as the mean ± SEM, n = 4. The whole experiment has been repeated three times with similar results. * p<0.05, *** p<0.001 vs. non-treated control cells; ## p<0.01, ### p<0.001 vs. 6-OHDA treated cells.</p

MMP3 regulate the expression of Nox1 in 6-OHDA treated N27 cells.

<p>(A) A representative RT-PCR performed on various Nox subunits from RNA preparation of N27 cells treated with 100 µM 6-OHDA. (B) A representative RT-PCR performed on MMP3 and Nox1 from RNA preparation of N27 cells. N27 cells were transfected with two different siRNAs against MMP-3 and Nox1 and their scrambled sequences (scr-siRNA) and subsequently treated with 6-OHDA for 6 h. Densitometric analysis of the RT-PCR results normalized against GAPDH and expressed as % induction compared with respective untreated control. * p<0.05, ** p<0.01, ***p <0.001 vs. control ; ###p<0.001 vs. 6-OHDA control. (C) Representative photographs of western blot for MMP3 and Nox1 on lysate of N27 cells transfected with two different siRNAs against MMP-3 and Nox1 and scr-siRNA and subsequently treated with 6-OHDA for 24 h. Densitometric analysis of the Western blot results normalized against β-actin and expressed as % induction compared with respective untreated control. ** p<0.01 vs. scr-siRNA control ; ##p<0.01 vs. scr-siRNA+6-OHDA control. (D) A representative western blot against Nox1 on lysate of N27 cells pre-treated with 1 µM and 5 µM N-isobutyl-N-(4-methoxyphenylsulfonyl) glycyl hydroxamic acid (NNGH) for 30 min followed by exposure to 100 µM 6-OHDA for 24 h. Densitometric analysis of the western blots results normalized against β-actin and expressed as % expression compared with respective untreated control. ***p<0.001 vs. control ; ##p<0.01vs. 6-OHDA control. Results are presented as the mean ± SEM, n = 8. The whole experiment has been repeated four times with similar results.</p

(A) Ribbon diagrams of PedB (left), EntA-im (middle), and ImB2 (right) shown with secondary structures labeled

<p><b>Copyright information:</b></p><p>Taken from "High resolution crystal structure of PedB: a structural basis for the classification of pediocin-like immunity proteins"</p><p>http://www.biomedcentral.com/1472-6807/7/35</p><p>BMC Structural Biology 2007;7():35-35.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1904221.</p><p></p> The axes of α3 and α4 are indicated by dotted lines. The angle between α3 and α4 is 33° for PedB and 31° for EntA-im. (B) Stereo view of the superimposed structures of PedB (red) and EntA-im (cyan). The flexible C-terminal loop of EntA-im that is thought to be important for its immunity is highlighted by a black ellipse. N and C indicate the N- and C-terminus of PedB, respectively

Increase in Nox1 protein levels in the SN of rats injected with autoactivated MMP3 (actMMP3).

<p>(A) Representative photomicrographs of Nox1 -immunoreactivity in the SN sections. AAV2 particles containing mutMMP3 or actMMP3 were stereotaxically injected into the rat SN. After 1, 2, 3 weeks later, DA neurons in the SN were visualized with Nox1 immunostaining. Scale bar  = 50 µm. (B) Stereologic counts of Nox1-positive neurons in the SN shown as percentage of mutMMP3. ** p<0.01 vs. mutMMP3 injected rats. (C and D) Nox1 knockdown efficiency in the SN was verified by western blot analysis performed at 7 weeks after AAV2 injection. n = 4/group, *** p<0.001 vs. shNC and mutMMP3 injected rats. ### p<0.001 vs. shNC and actMMP3 injected rats. shNC, scramble RNA/AAV2. (E) Representative photomicrographs of TH staining in the rat SN sections. To knockdown Nox1 in the SN, AAV2 particles harboring Nox1 shRNA (shNox1) or T17N Rac1 were stereotaxically injected into the SN. After 4 weeks incubation, AAV2 particles containing mutMMP3 or actMMP3 were stereotaxically injected into the rat SN. Three weeks later, DA neurons in the SN were visualized with TH immunostaining. Scale bar  = 200 µm (F) Stereologic counts of TH-positive neurons in the SN shown as percentage of mutMMP3. Results are presented as the mean ± SEM. n = 6–7. Significance is indicated by * p<0.05 vs. mutMMP3 control; # p<005, ## p<0.01 vs. scr-shRNA and actMMP injected rats. (G) Representative photomicrographs of MDA staining in the rat SN sections. Scale bar  = 30 µm. (H) Stereologic counts of MDA-positive neurons in the SN shown as percentage of mutMMP3. ** p<0.01 vs. mutMMP3 injected rats; ## p<0.01 vs. scr-shRNA and actMMP3 injected rats. mutMMP3, mutMMP3/AAV2 particles in the SN; actMMP3, actMMP3/AAV2 particles in the SN; scr-shRNA, injection of scramble shRNA/AAV2 particles into the SN; shNox1, injection of Nox1 shRNA/AAV2 particles into the SN; T17N Rac1, injection of T17N Rac1/AAV2 particles into the SN; +actMMP3, SN injection of actMMP3/AAV2 particles 4 weeks after Nox1 shRNA/AAV2 or T17N Rac1/AAV2.</p

Nox1 expression abolished MPTP treated MMP3 knockout mice.

<p>(A) MMP3 and Nox1 expression was increased in the rat SN DA neurons after MPTP administration. TH (red), MMP3 (green), and Nox1 (blue) were visualized in the mouse SN at 7 days post-i.p. injection of vehicle or MPTP. MMP3 and Nox1 coexpression in TH⁺ DA neurons is demonstrated as white staining after merging red (TH), green (MMP3) and blue (Nox1) images. Scale bars  = 10 µm. (B) Representative photomicrographs of Western blots against MMP3 on lysates of SN tissue at 7 days post-i.p. injection of MPTP or vehicle. Densitometric analysis of the western blots results normalized against β-actin and expressed as % expression compared with vehicle injected rat. (C) Nox1 expression was increased in the rat SN DA neurons after MPTP administration in wild type mouse (WT) not in MMP3 knockout mouse (MMP3KO). TH (green) and Nox1 (red) were visualized in the SN at 7 days post-i.p. injection of MPTP in WT (middle panels), MMP3 KO (lower panels) or vehicle in WT (upper panels). Nox1 expression in TH⁺ DA neurons is demonstrated as yellow staining after merging green (TH) and red (MMP3) images. Scale bars  = 20 µm. (D) Representative photomicrographs of Western blots against Nox1 and TH on lysates of SN tissue at 7 days post-i.p. injection of MPTP or vehicle in WT or MMP3KO mice. Densitometric analysis of the western blots results normalized against β-actin and expressed as % expression compared with vehicle injected WT rat. Results are presented as the mean ± SEM, n = 5. ** p<0.01, *** p<0.001 vs. saline treated rats, ### p<0.001 vs. MPTP treated rats.</p

Recombinant catalytic domain of MMP3 (rCD MMP3) induced mitochondrial ROS generation in mitochondrial fraction.

<p>(A) Representive photographs of Western blots for tubulin, histone H3, Tim 23 in the total lysates and mitochondrial fractions of the brain tissues. (B) Mitochondrial H₂O₂ levels were determined by the Amplex red assay. H₂O₂ levels after incubation with 1, 3, 10, and 30 µM of rCD MMP3 or 10 and 100 nM of rotenone in mitochondrial fraction for 1 h. *** p<0.001 vs. control, ### p<0.001 vs. 3 µM of rCD MMP3 or 10 nM of Rotenone, <a href="mailto:@@@" target="_blank">@@@</a> p<0.001 vs. 10 µM of rCD MMP3. (C) H₂O₂ levels after incubation with 30 µM of rCD MMP3 in the absence or presence of NNGH (30 µM) for 1 h. *** p<0.01 vs. control, ### p<0.001 vs. rCD MMP3. (D) MMP3 activity was measured in mitochondrial fraction after rCD MMP3 (30 µM) incubation in the absence or presence of NNGH (30 uM) for 1 h. *** p<0.01 vs. control, ## p<0.001 vs. rCD MMP3.</p

MMP3 induced mitochondrial ROS regulate the Nox1 expression in 6-OHDA treated N27 cells.

<p>(A) Mitochondrial ROS generation was measured using the MitoSox staining in N27 dopaminergic cells treated with post 6-OHDA (100 µM) for 1 h. NNGH significantly reduced 6-OHDA-mediated ROS generation. (B) Mitochondrial membrane potential (ΔΨm was measured using the TMRM staining in N27 dopaminergic cells treated with post 6-OHDA (100 µM) for 6 h. NNGH, apocynin, and GKT significantly inhibited decreasing of 6-OHDA-mediated ΔΨm. Apo: apocynin, a Nox inhibitor; GKT: GKT137831, a Nox1 selective inhibitor; AA, antimycin A (100 µM); FCCP, an oxidative phosphorylation uncoupler (100 nM), Results are presented as the mean ± SEM, n = 8. ** p<0.01, ***p<0.001 vs. nontreated control; # p<0.05, ## p<0.01, ### p<0.001 vs. 6-OHDA control. (C to E) ROS level was measured using the MitoSox (C), DHE (D), and DCFDA (E) in N27 cells transiently transfected with rat Nox1 siRNA, MMP3 siRNA or control siRNA for 36 h, followed by 6-OHDA exposure for 1, 3, and 6 h. Results are presented as the mean ± SEM, n = 8. The whole experiment has been repeated four times with similar results. ** p<0.01, ***p<0.001 vs. nontreated control at 0 h; # p<0.05, ## p<0.01, ### p<0.001 vs. 6-OHDA control at 3 or 6 h. <a href="mailto:@@" target="_blank">@@</a> p<0.01 vs. siRNA Nox1 treated with 6-OHDA at 3 h. (E to F) Representative photomicrograph of the MitoSox (E), DHE (F), and DCFDA (F) staining showing ROS levels in N27 cells transiently transfected with rat Nox1 siRNA, MMP3 siRNA or control siRNA for 36 h, followed by 6-OHDA exposure for 3, 6, or 9 h. Scale bar  = 50 µm.</p

(A) Sequence alignment of pediocin PP-1 from (pediocinPP) with pediocin PA-1 from (pediocinPA), EnterocinA, and Carnobacteriocin B2 (CarnobacB2)

<p><b>Copyright information:</b></p><p>Taken from "High resolution crystal structure of PedB: a structural basis for the classification of pediocin-like immunity proteins"</p><p>http://www.biomedcentral.com/1472-6807/7/35</p><p>BMC Structural Biology 2007;7():35-35.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1904221.</p><p></p> The substitution between pediocin PP-1 and pediocin PA-1 is indicated by black box. The color scheme of white on dark grey indicates the consensus residue derived from the occurrence of >70% of a single residue at a given position. (B) Structure-based alignment of PedB from (PedB-PP) with PedB from (PedB-PA), EntA-im, and ImB2. Secondary structure of PedB is presented above the alignment. The additional fifth helix of ImB2 is indicated by black bar. The color scheme is same to (A). The conservative substitution between PedB proteins for pediocin PP-1 and pediocin PA-1 is indicated by black box. (C) Pediocin PP-1 susceptibility of strain harboring gene and control plasmid. The MIC is the concentration of bacteriocin that inhibited growth of the indicator strain by 50%. The immunity activity is presented as the -fold increase in MIC observed for strains expressing PedB variants relative to MICs for strains containing only the control plasmid. The results represent the averaged data from at least three experiments. The psodA indicates the promoter of encoding Mn-containing superoxide dismutase in . (D) Determination of oligomeric state of PedB. Oligomeric state of PedB was analysed by gel filtration chromatography. Predicted molecular mass of PedB (See Methods) is 14,428 Da (= 0.42), indicating that PedB exists as a monomer in solution