Dye-Sensitized and Localized Surface Plasmon Resonance Enhanced Visible-Light Photoelectrochemical Biosensors for Highly Sensitive Analysis of Protein Kinase Activity

A novel visible-light photoelectrochemical (PEC) biosensor based on localized surface plasmon resonance (LSPR) enhancement and dye sensitization was fabricated for highly sensitive analysis of protein kinase activity with ultralow background. In this strategy, DNA conjugated gold nanoparticles (DNA@AuNPs) were assembled on the phosphorylated kemptide modified TiO₂/ITO electrode through the chelation between Zr⁴⁺ ions and phosphate groups, then followed by the intercalation of [Ru­(bpy)₃]²⁺ into DNA grooves. The adsorbed [Ru­(bpy)₃]²⁺ can harvest visible light to produce excited electrons that inject into the TiO₂ conduction band to form photocurrent under visible light irradiation. In addition, the photocurrent efficiency was further improved by the LSPR of AuNPs under the irradiation of visible light. Moreover, because of the excellent conductivity and large surface area of AuNPs that facilitate electron-transfer and accommodate large number of [Ru­(bpy)₃]²⁺, the photocurrent was significantly amplified, affording an extremely sensitive PEC analysis of kinase activity with ultralow background signals. The detection limit of as-proposed PEC biosensor was 0.005 U mL^–1 (<i>S</i>/<i>N</i> = 3). The biosensor also showed excellent performances for quantitative kinase inhibitor screening and PKA activities detection in MCF-7 cell lysates under forskolin and ellagic acid stimulation. The developed dye-sensitization and LSPR enhancement visible-light PEC biosensor shows great potential in protein kinases-related clinical diagnosis and drug discovery

TSC1 is required for naive CD8⁺ T cell homeostasis in adoptive transfer mouse models.

<p>Sorted naïve CD8⁺T cells were adoptively transferred into syngeneic Rag1^−/− or irradiated recipients. The levels of transferred naïve CD8⁺T cells in spleen and pLNs of recipients were determined 7 days after transfer. (<b>A</b>) Schematic representation of adoptive transfer of either WT or Tsc1 KO CD45.2⁺ naïve CD8⁺ T cells into Rag^−/− host. (<b>B</b>) One representative staining of CD8⁺T cells in pLN of Rag1^−/− mice after transfer of sorted naïve CD8⁺T cells. (<b>C</b>) Tsc1 KO CD45.2⁺ naïve CD8⁺ T cells showed homeostatic defect in spleen and pLNs. Statistic analysis of percentage and numbers of transferred CD8⁺T cells in spleen as well as pLN. (<b>D</b>) Schematic representation of adoptive transfer of mixed(1∶1) population of WT CD45.1⁺ and Tsc1 KO CD45.2⁺ naïve CD8⁺T cells into Rag^−/− host. (<b>E</b>) One representative staining of CD45.1 and CD45.2 on gated CD8⁺T cells in pLNs of Rag1^−/− mice 7 days after transfer of sorted naïve CD8⁺T cells. (<b>F</b>) The ratio between WT CD45.1⁺CD8⁺ and Tsc1 KO CD45.2⁺CD8⁺ T cells as well as the frequency of CD45.1⁺ or CD45.2⁺CD8⁺ T cells in spleen and pLNs were summarized. (<b>G</b>) Schematic representation of adoptive transfer of either WT or Tsc1 KO CD45.2⁺ naïve CD8⁺ T cells into sublethally irradiated(4 Gy) CD45.1⁺ host. (<b>H</b>) One representative staining of CD45.1 and CD8 for pLN cells of recipients 7 days after transfer of sorted naïve CD8⁺T cells. (<b>I</b>) Lower percentage and cell number of Tsc1 KO CD45.2⁺ naïve CD8⁺ T cells in spleen and pLNs. *p<0.05, **p<0.01, and ***p<0.001 compared with WT group. Data were shown as Mean±SD (3–5 mice each group). One representative of two or three independent experiments with identical results was shown.</p

Generation of the T cell-specific Tsc1 KO mice.

<p>(<b>A</b>) Western blot analysis showed TSC1 was expressed in thymus, spleen as well as pLNs of WT B6 mice, brain serves as the positive control. (<b>B</b>) Tsc1 and Tsc2 expression level in splenic CD8⁺T cells was higher than that in B cells and CD4⁺T cells as detected by Real-time PCR analysis. *p<0.05; **p<0.01; ***p<0.001 compared with the indicated groups. Date were shown as Mean±SD (N = 6). (<b>C</b>) Schematic representation of deletion of Tsc1 exons 17 and 18 by Lck-Cre-mediated recombination in T cells. (<b>D</b>) Genotyping of wild type (Tsc1^+/+, WT), heterozygous(Lck-cre⁺Tsc1^loxp/+, HE) and knockout (Lck-cre⁺Tsc1^loxp/loxp, KO) mice with tail genomic DNA. (<b>E</b>) Detection of Tsc1 deletion in purified CD8⁺T cells, CD4⁺T cells and non-T cells from spleen of Tsc1 KO mice. Tsc1 was specifically deleted in T cells. (<b>F</b>) Western blot analysis showed efficient deletion of TSC1 and TSC2 protein expression in thymocytes. Phosphorylated S6 level was significantly increased in Tsc1 KO mice.</p

Decreased Akt-FoxO1/FoxO3a phosphorylation of Tsc1 KO naïve CD8⁺T cells in response to IL-7.

<p>The expression of CD122, CD127 and CD95 on naïve CD8⁺T cells was determined by gating CD8⁺CD44^low cells. Comparable surface CD122, CD127 (<b>A</b>) and CD95 (<b>B</b>) expression in Tsc1 KO naïve CD8⁺ T cells. The dash-dot line represents staining with an isotype control antibody. The histograms (gray) represent WT whereas the open histograms with solid line show Tsc1 KO staining pattern. Cells are gated with CD8⁺CD44^low population. Representative data are shown from one of two separate experiments, with three mice in each group. (<b>C</b>) Western blot analysis of mTORC2-Akt-FoxO1/FoxO3a-Bim axis in naïve CD8⁺ T cells after stimulation with IL-7. Sorted naïve CD8⁺T cells were cultured with IL-7 in the presence of Rapa or not for 24 hrs. The freshly isolated WT or Tsc1 KO naïve CD8⁺T cells were used as a control. One representative is shown from two or three separate experiments.</p

Naïve CD8⁺ but not CD4⁺ T cells were significantly decreased in the periphery of Tsc1 KO mice.

<p>The peripheral T cell subsets of WT and Tsc1 KO mice were detected using FCM 4–5 wks after birth. <b>A.</b> Total thymic cell number and thymocyte subset profile of Tsc1 KO mice were identical as WT littermates. <b>B.</b> Total cell number of spleen, pLNs and mLNs of WT and Tsc1 KO mice. <b>C.</b> The percentage of CD8⁺T but not CD4⁺T cells decreased dramatically in all peripheral lymphoid organs of Tsc1 KO mice. <b>D.</b> The FACS profile analysis of CD62L, CD45RB and CD44 on pLN CD4⁺ T cells was evaluated. The frequency of naïve CD62L^hi, CD45RB^hi and CD44^low CD4⁺T cells in spleen, pLN and mLN of Tsc1 KO mice was summarized. <b>E.</b> The FACS profile analysis of CD62L, CD45RB and CD44 on the gated pLN CD8⁺ T cells was evaluated. The frequency of naïve CD62L^hi, CD45RB^hi and CD44^low CD8⁺T cells in spleen, pLN and mLN of Tsc1 KO mice was summarized. *p<0.05; **p<0.01; ***p<0.001. Data were shown as Mean±SD (N = 6).</p

TSC1 regulates naïve CD8⁺ T cell survival to IL-7 or IL-15 in a rapamycin-insensitive manner.

<p>Sorted WT and Tsc1 KO mouse naïve CD8⁺CD44^low T cells were cultured in medium alone or supplemented with IL-15 or IL-7(in the presence or absence of Rapa) for 24 hrs or the below indicated time points. (<b>A</b>) Significantly decreased live cell number of Tsc1 KO naïve CD8⁺T cells than WT naïve CD8⁺T cells in the presence of IL-15 or IL-7 for 24 hours. Live cells were determined by trypan blue exclusion assay. (<b>B</b>) Tsc1 KO naïve CD8⁺ T cells showed severe atrophy in the presence of IL-7 for 24 hrs. (<b>C</b>) One representative of PI staining in gated CD8⁺T cells after culture with or without IL-7 and IL-15. (<b>D</b>) Percentage of cell death was measured by PI staining and statistically analyzed. The above data were one representative of three separate experiments, with three wells in each group. (<b>E</b>) Representative staining of CD45.2 and CD8 for pLN cells of CD45.1⁺ host in the presence or absence of Rapa 7 days after transfer. Either sorted WT or Tsc1 KO CD45.2⁺ naïve CD8⁺ T cells were adoptively transferred into sublethally irradiated(4 Gy) CD45.1⁺ host and the donor cells were determined by CD8 and CD45.2 staining. (<b>F</b>) Significantly decreased percentage and cell number of Tsc1 KO CD45.2⁺ naïve CD8⁺ T cells in spleen of sublethally irradiated (4 Gy) CD45.1⁺ host in the presence or absence of Rapa. (<b>G</b>) Significantly decreased percentage and cell number of Tsc1 KO CD45.2⁺ naïve CD8⁺ T cells in pLNs of sublethally irradiated (4 Gy) CD45.1⁺ host in the presence or absence of Rapa. Data were shown as Mean±SD (3 mice each group). *p<0.05; **p<0.01; ***p<0.001 compared with the indicated groups.</p

DataSheet1_Doxorubicin resistance in breast cancer is mediated via the activation of FABP5/PPARγ and CaMKII signaling pathway.DOCX

Breast cancer is the most prevalent malignancy among women. Doxorubicin (Dox) resistance was one of the major obstacles to improving the clinical outcome of breast cancer patients. The purpose of this study was to investigate the relationship between the FABP signaling pathway and Dox resistance in breast cancer. The resistance property of MCF-7/ADR cells was evaluated employing CCK-8, Western blot (WB), and confocal microscopy techniques. The glycolipid metabolic properties of MCF-7 and MCF-7/ADR cells were identified using transmission electron microscopy, PAS, and Oil Red O staining. FABP5 and CaMKII expression levels were assessed through GEO and WB approaches. The intracellular calcium level was determined by flow cytometry. Clinical breast cancer patient’s tumor tissues were evaluated by immunohistochemistry to determine FABP5 and p-CaMKII protein expression. In the presence or absence of FABP5 siRNA or the FABP5-specific inhibitor SBFI-26, Dox resistance was investigated utilizing CCK-8, WB, and colony formation methods, and intracellular calcium level was examined. The binding ability of Dox was explored by molecular docking analysis. The results indicated that the MCF-7/ADR cells we employed were Dox-resistant MCF-7 cells. FABP5 expression was considerably elevated in MCF-7/ADR cells compared to parent MCF-7 cells. FABP5 and p-CaMKII expression were increased in resistant patients than in sensitive individuals. Inhibition of the protein expression of FABP5 by siRNA or inhibitor increased Dox sensitivity in MCF-7/ADR cells and lowered intracellular calcium, PPARγ, and autophagy. Molecular docking results showed that FABP5 binds more powerfully to Dox than the known drug resistance-associated protein P-GP. In summary, the PPARγ and CaMKII axis mediated by FABP5 plays a crucial role in breast cancer chemoresistance. FABP5 is a potentially targetable protein and therapeutic biomarker for the treatment of Dox resistance in breast cancer.</p