Video_1_NF-κB-mediated anti-inflammatory effects of an organic light-emitting diode (OLED) device in lipopolysaccharide (LPS)-induced in vitro and in vivo inflammation models.wmv

Inflammation is the body’s physiological response to harmful agents. However, if not regulated properly, inflammation can become pathological. Macrophages are key players in the inflammatory process, and modulate the immune response. Due to the side effects of anti-inflammatory drugs, non-pharmaceutical therapies for inflammatory diseases must be developed. Photobiomodulation is a non-invasive therapeutic approach to treating certain pathological conditions using light energy. Light-emitting diodes (LEDs) are commonly used as light sources for photobiomodulation treatment, but their clinical applications are limited. Organic LEDs (OLEDs) are thin, lightweight and flexible, enabling consistent and even delivery of light energy to target areas; this makes OLED promising components for therapeutic devices. In the present study, we examined the effects of OLED treatment on inflammation in vitro using a lipopolysaccharide (LPS)-induced macrophage RAW264.7 cell model, and in vivo using a pinna skin mouse model. We found that LPS-induced morphological changes and inflammatory cytokine expression were significantly reduced in RAW264.7 cells subjected to OLED treatment compared to the LPS-induced controls. This work provides evidence for the anti-inflammatory effects of OLEDs, demonstrating their potential to be incorporated into medical devices in the future.</p

DataSheet_1_NF-κB-mediated anti-inflammatory effects of an organic light-emitting diode (OLED) device in lipopolysaccharide (LPS)-induced in vitro and in vivo inflammation models.docx

Inflammation is the body’s physiological response to harmful agents. However, if not regulated properly, inflammation can become pathological. Macrophages are key players in the inflammatory process, and modulate the immune response. Due to the side effects of anti-inflammatory drugs, non-pharmaceutical therapies for inflammatory diseases must be developed. Photobiomodulation is a non-invasive therapeutic approach to treating certain pathological conditions using light energy. Light-emitting diodes (LEDs) are commonly used as light sources for photobiomodulation treatment, but their clinical applications are limited. Organic LEDs (OLEDs) are thin, lightweight and flexible, enabling consistent and even delivery of light energy to target areas; this makes OLED promising components for therapeutic devices. In the present study, we examined the effects of OLED treatment on inflammation in vitro using a lipopolysaccharide (LPS)-induced macrophage RAW264.7 cell model, and in vivo using a pinna skin mouse model. We found that LPS-induced morphological changes and inflammatory cytokine expression were significantly reduced in RAW264.7 cells subjected to OLED treatment compared to the LPS-induced controls. This work provides evidence for the anti-inflammatory effects of OLEDs, demonstrating their potential to be incorporated into medical devices in the future.</p

Multiple Reaction Monitoring of Multiple Low-Abundance Transcription Factors in Whole Lung Cancer Cell Lysates

Lung cancer-related transcription factors (TFs) were identified by integrating previously reported genomic, transcriptomic, and proteomic data and were quantified by multiple reaction monitoring (MRM) in various cell lines. All experiments were performed without affinity depletion or subfractionation of cell lysates. Since the target proteins were expected to be present in low abundance, we experimentally optimized MRM transition parameters with chemically synthesized peptides. Quantitation was based on stable isotope-labeled standard peptides (SIS peptides). Out of 288 MRM measurements (36 peptides representing 28 TFs × 8 cell lines), 241 were successfully obtained within a quantitation limit of 15 amol, 221 measurements (91.7%) showed coefficients of variation (CVs) of ≤20%, and 149 (61.8%) showed CVs of ≤10%, quantifying as low as 19.4 amol/μg protein for STAT2 with a CV of 6.3% in an A549 cell. Comparisons between MRM measurements and levels of the corresponding mRNAs revealed linear, nonlinear, or no relationship between protein and mRNA levels, indicating the need for an MRM assay. An integrative analysis of MRM and gene expression profiles from doxorubicin-resistant H69AR and sensitive H69 cells further showed that 14 differentially expressed TFs, such as STAT1 and SMAD4, regulated genes associated with drug resistance and cell differentiation-related processes. Thus, the analytical performance of MRM for the quantitation of low abundance TFs suggests its usefulness for biological application

Multiple Reaction Monitoring of Multiple Low-Abundance Transcription Factors in Whole Lung Cancer Cell Lysates

Lung cancer-related transcription factors (TFs) were identified by integrating previously reported genomic, transcriptomic, and proteomic data and were quantified by multiple reaction monitoring (MRM) in various cell lines. All experiments were performed without affinity depletion or subfractionation of cell lysates. Since the target proteins were expected to be present in low abundance, we experimentally optimized MRM transition parameters with chemically synthesized peptides. Quantitation was based on stable isotope-labeled standard peptides (SIS peptides). Out of 288 MRM measurements (36 peptides representing 28 TFs × 8 cell lines), 241 were successfully obtained within a quantitation limit of 15 amol, 221 measurements (91.7%) showed coefficients of variation (CVs) of ≤20%, and 149 (61.8%) showed CVs of ≤10%, quantifying as low as 19.4 amol/μg protein for STAT2 with a CV of 6.3% in an A549 cell. Comparisons between MRM measurements and levels of the corresponding mRNAs revealed linear, nonlinear, or no relationship between protein and mRNA levels, indicating the need for an MRM assay. An integrative analysis of MRM and gene expression profiles from doxorubicin-resistant H69AR and sensitive H69 cells further showed that 14 differentially expressed TFs, such as STAT1 and SMAD4, regulated genes associated with drug resistance and cell differentiation-related processes. Thus, the analytical performance of MRM for the quantitation of low abundance TFs suggests its usefulness for biological application

Immunofluorescent (IF) detection of MMP-2 isoform expression in HK2 cells: effect of culture in normal (5 mM) vs. high (30 mM) glucose.

<p>Panel I. IF staining for FL-MMP-2 isoform of HK2 cells cultured in normal (5 mM) glucose for 24 and 48 hours (A, C). IF staining for FL-MMP-2 isoform of HK2 cells cultured in high (30 mM) glucose for 24 and 48 hours (B, D). Upper panel x 400; lower panel x 800. Panel II. IF staining for NTT-MMP-2 isoform of HK2 cells cultured in normal (5 mM) glucose for 24 and 48 hours (A, C). IF staining for NTT-MMP-2 isoform cultured in high (30 mM) glucose for 24 and 48 hours (B, D). Upper panel x 400; lower panel x 800. Panels III and IV: Quantitation of IF staining for the FL-MMP-2 isoform (panel III) and NTT-MMP-2 (panel IV) of HK2 cells cultured in normal (5 mM) and high (30 mM) glucose medium for 24 and 48 hours. (N = 5 for all study groups; *p<0.05).</p

Immunohistochemical staining of renal cortices for FL-MMP-2 (panel I) and NTT-MMP-2 (panel II) of normoglycemic and diabetic mice (24 weeks).

<p>Panel I: There is detectable FL-MMP-2 in the renal cortex of control mice (A, B; x 200 and x 400 respectively) and this is increased in renal cortex of mice following 24 weeks of diabetes (C, D; x 200 and x 400 respectively). Panel II: NTT-MMP-2 protein expression is not detectable in the renal cortex of control mice (A, B; x 200 and x 400 respectively) but is greatly increased in the renal cortex of mice with 24 weeks of diabetes (C, D; x 200 and x 400 respectively). Note strong NTT-MMP-2 staining in necrotic tubular epithelial cells in tubular lumen of diabetic kidneys (D, arrow).</p

Quantitative polymerase chain reaction primer sequences.

<p>Quantitative polymerase chain reaction primer sequences.</p

Induction of MMP-2 isoform expression in cultured HK2 cells by high glucose and HHE oxidative stress.

<p>HK2 cells were cultured in either normal glucose concentrations (5 mM) or high glucose concentrations (30 mM) and transcript levels of the FL-MMP-2 and NTT-MMP-2 isoforms determined by qPCR at 2, 24 and 48 hours of incubation. To determine the effect of oxidative stress on MMP-2 isoform expression, HK2 cells were cultured in the presence or absence of HHE (10 μM) for 2, 24 and 48 hours. All control cells were harvest at 2hr because the expression of MMP-2 was not significantly different according to varying time point (data not shown). N = 3 for all study groups. (* p<0.05)</p

Induction of renal expression of FL-MMP-2 and NTT-MMP-2 in diabetic mice.

<p><b>Diabetes mellitus was induced in C57/BL6 male mice by daily injection of streptozotocin (40 mg/kg) for five days.</b> Panel I A: Gelatin zymography of renal cortical extracts from control and diabetic mice (24 weeks). Zones of lysis denote gelatin enzymatic activity for MMP-9 (98 kDa) and MMP-2 (68 kDa). MMP-2 enzymatic activity is increased in the renal cortex of diabetic mice as compared to normoglycemic controls. There is no significant increase in MMP-9 activity in the renal cortex of diabetic mice as compared to normoglycemic controls. Panel I B: The band intensity was quantified by measuring pixel intensity. More MMP-2 enzymatic acitivities were noted in diabetic group as compared to control group. There is no increase in the lytic band corresponding to MMP-9 in the diabetic samples as compared to controls (*: p<0.05, NS: not significant). Panel II A: Quantitation of FL-MMP-2 transcript abundance in renal cortices of normoglycemic (control) and diabetic mice at 12 and 24 weeks. There is a modest, but significant increase in FL-MMP-2 transcript abundance after 24 weeks of diabetes (N = 8;* p<0.05). Panel II B: Quantitation of NTT-MMP-2 transcript abundance in renal cortices of normoglycemic (control) and diabetic mice at 12 and 24 week. NTT-MMP-2 transcript abundance is significantly increased at both 12 and 24 weeks of diabetes (N = 8; *p<0.05).</p

The characteristics of human diabetics and control group.

<p>The characteristics of human diabetics and control group.</p