4D printing of smart polymer nanocomposites: integrating graphene and acrylate based shape memory polymers

The ever-increasing demand for materials to have superior properties and satisfy functions in the field of soft robotics and beyond has resulted in the advent of the new field of four-dimensional (4D) printing. The ability of these materials to respond to various stimuli inspires novel applications and opens several research possibilities. In this work, we report on the 4D printing of one such Shape Memory Polymer (SMP) tBA-co-DEGDA (tert-Butyl Acrylate with diethylene glycol diacrylate). The novelty lies in establishing the relationship between the various characteristic properties (tensile stress, surface roughness, recovery time, strain fixity, and glass transition temperature) concerning the fact that the print parameters of the laser pulse frequency and print speed are governed in the micro-stereolithography (Micro SLA) method. It is found that the sample printed with a speed of 90 mm/s and 110 pulses/s possessed the best batch of properties, with shape fixity percentages of about 86.3% and recovery times as low as 6.95 s. The samples built using the optimal parameters are further subjected to the addition of graphene nanoparticles, which further enhances all the mechanical and surface properties. It has been observed that the addition of 0.3 wt.% of graphene nanoparticles provides the best results

Synovial IL-9 facilitates neutrophil survival, function and differentiation of Th17 cells in rheumatoid arthritis

Abstract Background Role of Th9 cells and interleukin-9 (IL-9) in human autoimmune diseases such as psoriasis and ulcerative colitis has been explored only very recently. However, their involvement in human rheumatoid arthritis (RA) is not conclusive. Pathogenesis of RA is complex and involves various T cell subsets and neutrophils. Here, we aimed at understanding the impact of IL-9 on infiltrating immune cells and their eventual role in synovial inflammation in RA. Methods In vitro stimulation of T cells was performed by engagement of anti-CD3 and anti-CD28 monoclonal antibodies. Flow cytometry was employed for measuring intracellular cytokine, RORγt in T cells, evaluating apoptosis of neutrophils. ELISA was used for measuring soluble cytokine, Western blot analysis and confocal microscopy were used for STAT3 phosphorylation and nuclear translocation. Results We demonstrated synovial enrichment of Th9 cells and their positive correlation with disease activity (DAS28-ESR) in RA. Synovial IL-9 prolonged the survival of neutrophils, increased their matrix metalloprotienase-9 production and facilitated Th17 cell differentiation evidenced by induction of transcription factor RORγt and STAT3 phosphorylation. IL-9 also augmented the function of IFN-γ + and TNF-α + synovial T cells. Conclusions We provide evidences for critical role of IL-9 in disease pathogenesis and propose that targeting IL-9 may be an effective strategy to ameliorate synovial inflammation in RA. Inhibiting IL-9 may have wider impact on the production of pathogenic cytokines involved in autoimmune diseases including RA and may offer better control over the disease

Involvement of ROS in Chlorogenic Acid-Induced Apoptosis of Bcr-Abl+ CML Cells

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl+ chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl+ cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chlinduced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells fromCML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl+ cells

Synergistic Apoptosis of CML Cells by Buthionine Sulfoximine and Hydroxychavicol Correlates with Activation of AIF and GSH-ROS-JNK-ERK-iNOS Pathway

<div><p>Background</p><p>Hydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of the study is to optimize the oxidative stress –induced chronic myeloid leukemic (CML) cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO) with HCH and studying the underlying mechanism.</p><p>Materials and Methods</p><p>Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4), non-leukemic (A549, MIA-PaCa2, PC-3, HepG2) cancer cell lines and normal cell lines (NIH3T3, Vero) was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM) after staining with annexinV-FITC/propidium iodide (PI), detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP)-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF) by confocal microscopy. Intracellular reduced glutathione (GSH) was measured by colorimetric assay using GSH assay kit. 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) were used as probes to measure intracellular increase in ROS and nitric oxide (NO) levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action.</p><p>Results</p><p>Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF)-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the expression of inducible nitric oxide synthase (iNOS). iNOS- mediated production of NO was identified as an effector molecule causing apoptosis of CML cells.</p><p>Conclusion/Significance</p><p>BSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.</p></div

Combination of BSO and HCH induces NO production in CML cells that causes apoptosis.

<p>(<b>A</b>) K562 cells were treated as indicated for different time periods and then intracellular nitric oxide (NO) was measured by flow cytometry after staining with DAF-FM. Data represent mean ± SD of three experiments. ** p<0.01 compared to treatment with either BSO or HCH alone. *** p<0.001 compared to treatment with either BSO or HCH alone. (<b>B</b>) Representative histograms of DAF-FM staining in K562 cells and hPBMC after treatment as indicated for 24 h. (<b>C</b>) K562 cells were pretreated with 100 µM cPTIO for 2 h followed by incubation with combination of 100 µM BSO and 10 µM HCH for 24 h. Cells were then subjected to annexin V/PI binding assay by flow cytometry (left panel). Histograms show measurement of NO in the presence or absence of cPTIO after indicated treatment (right panel). Representative of two similar experiments. (<b>D</b>) K562 cells were treated as indicated for 24 hours after pretreatment with 100 µM cPTIO for 2 h. The whole cell lysates were then immunoblotted with indicated antibodies. (<b>E</b>) K562 cells were pre-incubated with indicated concentrations of GME followed by treatment with BSO and HCH as indicated for 24 h. Analysis of intracellular NO was done by flow cytometry after staining with DAF-FM. Histograms are representative of two similar experiments.</p

Apoptosis induced by combination of BSO and HCH is mediated by JNK dependent ERK1/2 activation.

<p>(<b>A</b>) K562 cells were treated as indicated for different time periods and cell lysates were subjected to western blot analysis with indicated antibodies. (<b>B</b>) K562 cells were pretreated with JNK inhibitor SP600125 (20 µM), p38 inhibitor SB203580 (20 µM), ERK inhibitor PD98059 (40 µM) for 1 h before treatment with BSO (100 µM) and HCH (10 µM) in combination. After 36 h of incubation, cells were subjected to Annexin V/PI binding assay by flow cytometry. Data represent mean ± SD of three experiments. *** p<0.001 compared to treatment in absence of MAPK inhibitors. (<b>C</b>) Knockdown of JNK by specific JNK1 siRNA attenuates apoptosis. JNK1 protein level was shown after knockdown (upper panel). Annexin V/PI binding assay by flow cytometry in K562 cells after transfection with indicated siRNAs (lower panel). Dot plots are representative of two similar experiments. (<b>D</b>) Knockdown of ERK by specific ERK2 siRNA attenuates apoptosis. ERK2 protein level was shown after knockdown (upper panel). Annexin V/PI binding assay by flow cytometry after knocking down ERK2 (lower panel). Dot plots are representative of two similar experiments (<b>E</b>) K562 cells were transfected with indicated siRNAs for 48 h and then treated with BSO plus HCH for 18 h. Protein expression and phosphorylation of JNK and ERK1/2 were analysed by western blot on whole cell lysates. (<b>F</b>) K562 cells were pre-incubated with or without 2 mM GME for 1 h and further incubated with BSO (100 µM) and HCH (10 µM) in combination for 18 h. The level of each protein and phosphorylation status was analyzed as indicated by western blot. (<b>G</b>) K562 cells were transfected with indicated siRNAs for 48 h and then treated with BSO plus HCH for 24 h. Analysis of intracellular NO was done by flow cytometry after staining with DAF-FM. Histograms are representative of two similar experiments.</p

Nitric oxide is produced by inducible nitric oxide synthase (iNOS); iNOS expression is dependent on ERK 1/2 and JNK activation.

<p>(<b>A</b>) K562 cells were treated as indicated for varying time periods and the whole cell lysates were subjected to western blot analysis with indicated antibodies. (<b>B</b>) After transfection of K562 cells with indicated siRNAs followed by indicated treatments for 24 h, cells were stained with DAF-FM for measurement of NO. Representative of two similar histograms. (<b>C</b>) K562 cells were transfected with control siRNA or indicated NOS siRNAs for 48 h and then treated with combination of BSO (100 µM) and HCH (10 µM) for 24 h. Whole cell lysates were subjected to western blot for the expression of indicated proteins to confirm knockdown of representative NOS isoforms. (<b>D</b>) K562 cells were subjected to annexin V/PI binding assay by flow cytometry after 36 h. Representative of two similar histograms. (<b>E</b>) K562 cells were transfected with JNK1 or ERK2 siRNA. Cells were then treated with combination of BSO (100 µM) and HCH (10 µM) for 18 h. Cell lysates were then immunoblotted with anti-iNOS antibody. (<b>F</b>) iNOS siRNA transfected K562 cells were treated with combination of BSO (100 µM) and HCH (10µM) for 18 h and subjected to immunoblot analysis with indicated antibodies. (<b>G</b>) K562 cells were pre-incubated with GME for 1 h before treatment with combination of BSO and HCH for 18 h and cell lysates were then immunoblotted with anti-iNOS antibody. (<b>H</b>) GSH measurement was done in iNOS transfected K562 cells after combined treatment with BSO and HCH for 18 h. Data represent mean ± SD of three experiments. *** p<0.001 compared to vehicle control.</p

BSO potentiates HCH- induced apoptosis by depleting intracellular GSH.

<p>(<b>A</b>) K562 cells were treated as indicated for measurement of intracellular GSH as described in Materials and Methods. Data represent mean ± SD of three experiments. ** p<0.01 and *** p<0.001compared to treatment with BSO alone. (<b>B</b>) K562 cells and hPBMC were treated as indicated for 24 h and intracellular GSH was measured. Data represent mean ± SD of three experiments. # p<0.01 compared to vehicle control; ** p<0.01 compared to BSO alone. (<b>C)</b> K562 cells were treated as indicated for different time points and intracellular H₂O₂ were measured by flow cytometry after DCF-DA staining. Data represent mean ± SD of three experiments. ** p<0.01 compared to treatment with BSO or HCH alone. (<b>D</b>) K562 cells were preincubated with varying concentrations of glutathione monoethyle ester (GME) for 1 h and further incubated with BSO (100 µM) and HCH (10 µM) as indicated for 36 h. Percent apoptotic cells were calculated by flow cytometry after annexinV-PI staining. Data represent mean of three experiments. ** p<0.01 compared to treatment in absence of GME. *** p<0.001 compared to treatment in absence of GME. (<b>E</b>) K562 cells were pre-incubated with 2 mM GME for 1 h and further incubated with BSO (100 µM) and HCH (10 µM) in combination for 36 h. Mitochondrial membrane potential was assessed in a flow cytometer after staining with JC-1 dye. Dot plots and histograms are representative of two similar experiments.</p

Combination of BSO and HCH induces AIF translocation in caspase-independent manner.

<p>K562 cells were treated as indicated and processed for confocal microscopy as stated in Materials and Methods. Scale bar, 10 µm.</p

Additional file 1: Table S1. of Synovial IL-9 facilitates neutrophil survival, function and differentiation of Th17 cells in rheumatoid arthritis

Demographic and clinical characteristics of RA patients (n = 28). (DOCX 12 kb