Dynamics of Oxygen-Independent Photocleavage of Blebbistatin as a One-Photon Blue or Two-Photon Near-Infrared Light-Gated Hydroxyl Radical Photocage

Development of versatile, chemically tunable photocages for photoactivated chemotherapy (PACT) represents an excellent opportunity to address the technical drawbacks of conventional photodynamic therapy (PDT) whose oxygen-dependent nature renders it inadequate in certain therapy contexts such as hypoxic tumors. As an alternative to PDT, oxygen free mechanisms to generate cytotoxic reactive oxygen species (ROS) by visible light cleavable photocages are in demand. Here, we report the detailed mechanisms by which the small molecule blebbistatin acts as a one-photon blue light-gated or two-photon near-infrared light-gated photocage to directly release a hydroxyl radical (•OH) in the absence of oxygen. By using femtosecond transient absorption spectroscopy and chemoselective ROS fluorescent probes, we analyze the dynamics and fate of blebbistatin during photolysis under blue light. Water-dependent photochemistry reveals a critical process of water-assisted protonation and excited state intramolecular proton transfer (ESIPT) that drives the formation of short-lived intermediates, which surprisingly culminates in the release of •OH but not superoxide or singlet oxygen from blebbistatin. CASPT2//CASSCF calculations confirm that hydrogen bonding between water and blebbistatin underpins this process. We further determine that blue light enables blebbistatin to induce mitochondria-dependent apoptosis, an attribute conducive to PACT development. Our work demonstrates blebbistatin as a controllable photocage for •OH generation and provides insight into the potential development of novel PACT agents

Additional file 1: of Co-stimulation of LPAR1 and S1PR1/3 increases the transplantation efficacy of human mesenchymal stem cells in drug-induced and alcoholic liver diseases

Figure S1. Optimal concurrent dose selection of LPA and S1P on LPS/H2O2- (a) or ethanol-induced (b) stem cell injury. Figure S2. LPA and/or S1P attenuates ethanol-induced stem cell death. (a) Changes in hADMSC viability. (b) Distribution of hADMSC apoptotic fractions. (c) Changes in hADMSC caspase-3/7 activity. Figure S3. LPA and/or S1P attenuates ethanol-induced stem cell oxidative stress and inflammation. (a) Quantified data of DMPO-stained signals, (b) cellular GSH/GSSG ratio, (c) cell-secreted TNF-α protein level, and (d) cell-secreted IL-6 protein level changes in hADMSC. Figure S4. Stimulation of LPAR1/Gi by LPA or S1PR1/3 by S1P is critical for stem cell protection. (a) Changes in cell viability and caspase-3/7 activity of hADMSCs after ethanol/LPA treatments, with or without AM966 (LPAR1 inhibitor), or PTX (Gi inhibitor) co-treatment. (b) with or without the W146 (S1PR1 inhibitor), JTE013 (S1PR2 inhibitor), or CAY10444 (S1PR3 inhibitor) co-treatment. (c) with or without PTX (Gi inhibitor) co-treatment. (d) with or without G12/13 shRNA co-transfection. Figure S5. The RAS/ERK, PI3K/Akt, and NF-κB/IL-10 pathways are the downstream targets of LPAR1/S1PR1/3-mediated stem cell protection from ethanol-induced damage. (a) Representative images of Western blot results and quantitative data. (b) Changes in cell viability and caspase-3/7 activity of hADMSCs after ethanol and LPA/S1P treatments, with or without the co-administration of salirasib (RAS inhibitor), UO126 (ERK inhibitor), wortmannin (PI3K inhibitor), or MK2206 (Akt inhibitor). (c) Changes in nuclear translocation and activation of NF-κB p65 subunit. (d) (left) Changes in IL-10 secretion ; and (right) cell viability

Supplementary_data_all-in-one - Functional 3D Human Liver Bud Assembled from MSC-Derived Multiple Liver Cell Lineages

<p>Supplementary_data_all-in-one for Functional 3D Human Liver Bud Assembled from MSC-Derived Multiple Liver Cell Lineages by Jing Li, Feiyue Xing, Feng Chen, Liumin He, Kwok-Fai So, Yingxia Liu, and Jia Xiao in Cell Transplantation</p

sj-pdf-1-jcb-10.1177_0271678X231151621 - Supplemental material for Dialysis adequacy and hemoglobin levels predict cerebral atrophy in maintenance-hemodialysis patients

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X231151621 for Dialysis adequacy and hemoglobin levels predict cerebral atrophy in maintenance-hemodialysis patients by Ming-Xuan Cao, Jia Xiao, Hua-Min Qin, Zhi-Hong Wang, Johannes Boltze, , Shu-Xin Liu, Shen Li in Journal of Cerebral Blood Flow & Metabolism</p

Human Umbilical Cord Mesenchymal Stem Cells and Derived Hepatocyte-Like Cells Exhibit Similar Therapeutic Effects on an Acute Liver Failure Mouse Model

<div><p>Mesenchymal stem cells (MSCs) have exhibited therapeutic effects in multiple animal models so that are promising liver substitute for transplantation treatment of end-stage liver diseases. However, it has been shown that over-manipulation of these cells increased their tumorigenic potential, and that reducing the <i>in vitro</i> culture time could minimize the risk. In this study, we used a D-galactosamine plus lipopolysaccharide (Gal/LPS)-induced acute liver failure mouse model, which caused death of about 50% of the mice with necrosis of more than 50% hepatocytes, to compare the therapeutic effects of human umbilical cord MSCs (hUCMSCs) before and after induction of differentiation into hepatocyte (i-Heps). Induction of hUCMSCs to become i-Heps was achieved by treatment of the cells with a group of growth factors within 4 weeks. The resulted i-Heps exhibited a panel of human hepatocyte biomarkers including cytokeratin (hCK-18), α-fetoprotein (hAFP), albumin (hALB), and hepatocyte-specific functions glycogen storage and urea metabolism. We demonstrated that transplantation of both cell types through tail vein injection rescued almost all of the Gal/LPS-intoxicated mice. Although both cell types exhibited similar ability in homing at the mouse livers, the populations of the hUCMSCs-derived cells, as judged by expressing hAFP, hCK-18 and human hepatocyte growth factor (hHGF), were small. These observations let us to conclude that the hUCMSCs was as effective as the i-Heps in treatment of the mouse acute liver failure, and that the therapeutic effects of hUCMSCs were mediated largely via stimulation of host hepatocyte regeneration, and that delivery of the cells through intravenous injection was effective.</p></div

Hepatic metabolism functions of hUCMSCs and i-Hep <i>in vitro</i>.

<p>(A) In the culture free of α-amylase, hUCMSCs showed modest accumulation of glycogen, while i-Hep demonstrated increased amount of glycogen. In the existence of the α-amylase, no glycogen was seen in either cell types. Periodic acid–Schiff staining, 200x. (B) When supplementation of NH₄Cl in the medium, i-Hep produced more than 12-fold of urea than hUCMSCs (<i>p</i><0.05).</p

Quantification and function characterization of transplanted stem cells in the mouse livers.

<p>(A) Quantitative real-time PCR measurement of engrafted human stem cell in the host liver. The ratios between human genome vs host genome in mouse livers of Gal/LPS- group was 12.6-fold of that of Vehicle-hUCMSCs group (<i>p</i><0.05), and a 10.8-fold difference was demonstrated between Gal/LPS- and Vehicle-i-Hep group (<i>p</i><0.05), respectively. Data were expressed as means ± SEM (n = 3–7). Representative images of (B) hCK-18- and (C) hAFP-positive cells in host livers. Immunohistological staining with individual antibodies against human epitopes. 200x. (D) Expression level of selective human genes in mouse liver was quantified by measuring the intensity of hCK-18- and (E) hAFP-positive cells in liver sections. Data were analyzed with ImageJ software and expressed as means ± SEM. Different superscript letters (e.g. a vs. b, or b vs. c) represent a statistically significant difference (<i>p</i><0.05) between two variables.</p

Characterization of hUCMSCs and i-Hep.

<p>(A1) hUCMSCs, bright field, x200; (A2) iHep, bright field, x200. (B) Demonstration of mesodermal marker vimentin in hUCMSCs, x200. (C) Flow cytometry analysis of hUCMSCs at passage 2. The hUCMSCs at passage 2 were subjected to immunocytostaining with individual antibodies before subjected to FASC analysis. More than 90% of the cells were positively stained with stem cell markers CD90-FITC-A, CD73-PEA and CD105-PECA; and extracellular matrix makers CD44-FITCA and CD29-FITCA; but more than 98% of the cells are negative in staining with hematopoietic stem cell markers CD45-FITCA, CD34-PEA, CD19-APC-CY7-A and CD14-FITC-A; almost all the cells were negative in staining of the major histocompatibility complex HLA-PRE-CPA. Results are expressed as percentage of the positive cells (%).</p

Effect of transplantation of MSCs on mouse liver histology.

<p>(A) Survival curve of each group of mice after D-galactosamine/lipopolysaccharide (Gal/LPS) intoxication with or without stem cell administration. Representative images of hematoxylin and eosin (H&E) staining of (B) control, (C, D, E) 1, 3 and 14 days post Gal/LPS treatment, respectively; (F, G, H) 1, 3 and 14 days post Gal/LPS treatment with transplantation of human umbilical cord mesenchymal stem cells (hUCMSCs), respectively; (I, J, K) 1, 3, 14 days post Gal/LPS treatment with induced-differentiation of hepatocytes (i-Hep), respectively; and (L) Double-blind quantification of necrotic area of the hepatic histology of each group. Data were analyzed with ImageJ software and expressed as means ± SEM. Different superscript letters (e.g. a vs. b, or b vs. c) represent a statistically significant difference (<i>p</i><0.05) between two variables. Magnification 200x.</p

Adipogenic and osteogenic differentiation of hUCMSCs.

<p>After 14-day’s induction, adipogenic character of the cells was characterized by formation of intracytoplasmic lipid droplets which became red in color and easily to be identified when stained with Oil Red O. Osteogenic character of hUCMSCs was shown by formation of multiple red bone nodules outside the cells when stained with Alizarin Red. 200x.</p