MicroRNA-155 Is Required for Mycobacterium bovis BCG-Mediated Apoptosis of Macrophages

Pathogenic rnycobacteria, including Mycobacterium tuberculosis and Mycobacterium bovis, cause significant morbidity and mortality worldwide. However, the vaccine strain Mycobacterium bovis BCG, unlike virulent strains, triggers extensive apoptosis of infected macrophages, a step necessary for the elicitation of robust protective immunity. We here demonstrate that M. bovis BCG triggers Toll-like receptor 2 (TLR2)-dependent microRNA-155 (miR-155) expression, which involves signaling cross talk among phosphatidylinositol 3-kinase (PI3K), protein kinase C delta (PKC delta), and mitogen-activated protein kinases (MAPKs) and recruitment of NF-kappa B and c-ETS to miR-155 promoter. Genetic and signaling perturbations presented the evidence that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-alpha). Enhanced activation of PKA signaling resulted in the generation of PKA C-alpha; phosphorylation of MSK1, cyclic AMP response element binding protein (CREB), and histone H3; and recruitment of phospho-CREB to the apoptotic gene promoters. The miR-155-triggered activation of caspase-3, BAK1, and cytochrome c translocation involved signaling integration of MAPKs and epigenetic or posttranslational modification of histones or CREB. Importantly, M. bovis BCG infection-induced apoptosis was severely compromised in macrophages derived from miR-155 knockout mice. Gain-of-function and loss-of-function studies validated the requirement of miR-155 for M. bovis BCG's ability to trigger apoptosis. Overall, M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, strongly implicating a novel role for miR-155 in orchestrating cellular reprogramming during immune responses to mycobacterial infection.

Novel Levamisole Derivative Induces Extrinsic Pathway of Apoptosis in Cancer Cells and Inhibits Tumor Progression in Mice

Background: Levamisole, an imidazo(2,1-b) thiazole derivative, has been reported to be a potential antitumor agent. In the present study, we have investigated the mechanism of action of one of the recently identified analogues, 4a (2-benzyl-6-(4'-fluorophenyl)-5-thiocyanato-imidazo2,1-b]1,3,4]thi adiazole). Materials and Methods: ROS production and expression of various apoptotic proteins were measured following 4a treatment in leukemia cell lines. Tumor animal models were used to evaluate the effect of 4a in comparison with Levamisole on progression of breast adenocarcinoma and survival. Immunohistochemistry and western blotting studies were performed to understand the mechanism of 4a action both ex vivo and in vivo. Results: We have determined the IC50 value of 4a in many leukemic and breast cancer cell lines and found CEM cells most sensitive (IC50 5 mu M). Results showed that 4a treatment leads to the accumulation of ROS. Western blot analysis showed upregulation of pro-apoptotic proteins t-BID and BAX, upon treatment with 4a. Besides, dose-dependent activation of p53 along with FAS, FAS-L, and cleavage of CASPASE-8 suggest that it induces death receptor mediated apoptotic pathway in CEM cells. More importantly, we observed a reduction in tumor growth and significant increase in survival upon oral administration of 4a (20 mg/kg, six doses) in mice. In comparison, 4a was found to be more potent than its parental analogue Levamisole based on both ex vivo and in vivo studies. Further, immunohistochemistry and western blotting studies indicate that 4a treatment led to abrogation of tumor cell proliferation and activation of apoptosis by the extrinsic pathway even in animal models. Conclusion: Thus, our results suggest that 4a could be used as a potent chemotherapeutic agent.

Extracts of Strawberry Fruits Induce Intrinsic Pathway of Apoptosis in Breast Cancer Cells and Inhibits Tumor Progression in Mice

Background: The consumption of berry fruits, including strawberries, has been suggested to have beneficial effects against oxidative stress mediated diseases. Berries contain multiple phenolic compounds and secondary metabolites that contribute to their biological properties. Methodology/Principal Findings: Current study investigates the anticancer activity of the methanolic extract of strawberry (MESB) fruits in leukaemia (CEM) and breast cancer (T47D) cell lines ex vivo, and its cancer therapeutic and chemopreventive potential in mice models. Results of MTT, trypan blue and LDH assays suggested that MESB can induce cytotoxicity in cancer cells, irrespective of origin, in a concentration-and time-dependent manner. Treatment of mice bearing breast adenocarcinoma with MESB blocked the proliferation of tumor cells in a time-dependent manner and resulted in extended life span. Histological and immunohistochemical studies suggest that MESB treatment affected tumor cell proliferation by activating apoptosis and did not result in any side effects. Finally, we show that MESB can induce intrinsic pathway of apoptosis by activating p73 in breast cancer cells, when tumor suppressor gene p53 is mutated. Conclusions/Significance: The present study reveals that strawberry fruits possess both cancer preventive and therapeutic values and we discuss the mechanism by which it is achieved.

Mapping the apoptosis inducing domain of an immunomodulatory protein: glycodelin A

Glycodelin A (GdA) is a dimeric glycoprotein synthesized by the human endometrium under progesterone regulation. Based on the high sequence similarity with beta-lactoglobulin, it is placed under the lipocalin superfamily. The protein is one of the local immunomodulators present at the feto-maternal interface which affects both the innate as well as the acquired arms of the immune system, thereby bringing about successful establishment and progression of pregnancy. Our previous studies revealed that the domain responsible for the immunosuppressive activity of glycodelin lies on its protein backbone and the glycans modulate the same. This study attempts to further delineate the apoptosis inducing region of GdA. Our results demonstrate that the stretch of amino acid sequence between Met24 to Leu105 is necessary and sufficient to inhibit proliferation of T cells and induce apoptosis in them. Further, within this region the key residues involved in harboring the activity were shown to be present between Asp52 and Ser65.

Analysis of integrated optofluidic lab-on-a-chip sensor based on refractive index and absorbance sensing

The analysis of a fully integrated optofluidic lab-on-a-chip sensor is presented in this paper. This device is comprised of collinear input and output waveguides that are separated by a microfluidic channel. When light is passed through the analyte contained in the fluidic gap, optical power loss occurs owing to absorption of light. Apart from absorption, a mode-mismatch between the input and output waveguides occurs when the light propagates through the fluidic gap. The degree of mode-mismatch and quantum of optical power loss due to absorption of light by the fluid form the basis of our analysis. This sensor can detect changes in refractive index and changes in concentration of species contained in the analyte. The sensitivity to detect minute changes depends on many parameters. The parameters that influence the sensitivity of the sensor are mode spot size, refractive index of the fluid, molar concentration of the species contained in the analyte, width of the fluidic gap, and waveguide geometry. By correlating various parameters, an optimal fluidic gap distance corresponding to a particular mode spot size that achieves the best sensitivity is determined both for refractive index and absorbance-based sensing.

A novel DNA intercalator, 8-methoxy pyrimido4 `,5 `:4,5]thieno (2,3-b)quinoline-4(3H)-one induces apoptosis in cancer cells, inhibits the tumor progression and enhances lifespan in mice with tumor

Polycyclic aromatic molecules such as ellipticine intercalate into double-stranded DNA and interfere with physiological functions. In the present study, we evaluate the chemotherapeutic potential of MPTQ on animal models and its mode of action. In order to test the antitumor activity, monohydrochloride of MPTQ was orally administered in mice bearing tumor. Results showed a significant inhibition of tumor growth compared to that of untreated controls. More importantly, mean lifespan of tumor bearing animals treated with MPTQ was significantly higher as compared to that of untreated tumor bearing mice suggesting that the treatment affected viability of cancerous cells, but not of normal cells. Consistent with this, we find that administration of MPTQ to normal mice did not cause any major side effects as observed upon hematological and serum profiling. We also found that MPTQ induces cytotoxicity in cancer cell lines, by activating apoptosis both by intrinsic and extrinsic pathways. Thus, MPTQ could be used as a potential cancer therapeutic agent.

Intracellular reactive oxidative stress, cell proliferation and apoptosis of Schwann cells on carbon nanofibrous substrates

Despite considerable research to develop carbon based materials for biomedical applications, the toxicity of carbon remains a major concern. In order to address this issue as well as to investigate the cell fate processes of neural cells from the perspective of neural tissue engineering applications, the in vitro cytocompatibility of polyacrylonitrile (PAN) derived continuous carbon nanofibers and PAN derived carbon thin films were investigated both quantitatively and qualitatively using in vitro biochemical assays followed by extensive flow cytometry analysis. The experimental results of Schwann cell fate, i.e. cell proliferation, cell metabolic activity and cell apoptosis on amorphous carbon substrates are discussed in reference to the time dependent evolution of intracellular oxidative stress. Apart from providing evidence that an electrospun carbon nanofibrous substrate can physically guide the cultured Schwann cells, this study suggested that continuous carbon nanofibers and amorphous carbon films are not cytotoxic in vitro and do not significantly induce apoptosis of Schwann cells, but in fact even facilitate their proliferation and growth.

Influence of crack parameters on wavelet energy correlated damage index

This paper illustrates a Wavelet Coefficient based approach using experiments to understand the sensitivity of ultrasonic signals due to parametric variation of a crack configuration in a metal plate. A PZT patch sensor/actuator system integrated to a metal plate with through-thickness crack is used. The proposed approach uses piezoelectric patches, which can be used to both actuate and sense the ultrasonic signals. While this approach leads to more flexibility and reduced cost for larger scalability of the sensor/actuator network, the complexity of the signals increases as compared to what is encountered in conventional ultrasonic NDE problems using selective wave modes. A Damage Index (DI) has been introduced, which is function of wavelet coefficient. Experiments have been carried out for various crack sizes, crack orientations and band-limited tone-burst signal through FIR filter. For a 1 cm long crack interrogated with 20 kHz tone-burst signal, the Damage Index (DI) for the horizontal crack orientation increases by about 70% with respect to that for 135 degrees oriented crack and it increases by about 33% with respect to the vertically oriented crack. The detailed results reported in this paper is a step forward to developing computational schemes for parametric identification of damage using sensor/actuator network and ultrasonic wave.

CXI-benzo-84 reversibly binds to tubulin at colchicine site and induces apoptosis in cancer cells

Here, we have discovered CXI-benzo-84 as a potential anticancer agent from a library of benzimidazole derivatives using cell based screening strategy. CXI-benzo-84 inhibited cell cycle progression in metaphase stage of mitosis and accumulated spindle assembly checkpoint proteins Mad2 and BubR1 on kinetochores, which subsequently activated apoptotic cell death in cancer cells. CXI-benzo-84 depolymerized both interphase and mitotic microtubules, perturbed EB1 binding to microtubules and inhibited the assembly and GTPase activity of tubulin in vitro. CXI-benzo-84 bound to tubulin at a single binding site with a dissociation constant of 1.2 +/- 0.2 mu M. Competition experiments and molecular docking suggested that CXI-benzo-84 binds to tubulin at the colchicine-site. Further, computational analysis provided a significant insight on the binding site of CXI-benzo-84 on tubulin. In addition to its potential use in cancer chemotherapy, CXI-benzo-84 may also be useful to screen colchicine-site agents and to understand the colchicine binding site on tubulin. (C) 2013 Elsevier Inc. All rights reserved.

Propyl-2-(8-(3,4-Difluorobenzyl)-2 `,5 `-Dioxo-8-AzaspiroBicyclo3.2.1] Octane-3,4 `-Imidazolidine]-1 `-yl) Acetate Induces Apoptosis in Human Leukemia Cells through Mitochondrial Pathway following Cell Cycle Arrest

Background: Due to the functional defects in apoptosis signaling molecules or deficient activation of apoptosis pathways, leukemia has become an aggressive disease with poor prognosis. Although the majority of leukemia patients initially respond to chemotherapy, relapse is still the leading cause of death. Hence targeting apoptosis pathway would be a promising strategy for the improved treatment of leukemia. Hydantoin derivatives possess a wide range of important biological and pharmacological properties including anticancer properties. Here we investigated the antileukemic activity and mechanism of action of one of the potent azaspiro hydantoin derivative, (ASHD). Materials and Methods: To investigate the antileukemic efficacy of ASHD, we have used MTT assay, cell cycle analysis by FACS, tritiated thymidine incorporation assay, Annexin V staining, JC1 staining and western blot analysis. Results: Results showed that ASHD was approximately 3-fold more potent than the parent compounds in inducing cytotoxicity. Tritiated thymidine assay in conjunction with cell cycle analysis suggests that ASHD inhibited the growth of leukemic cells. The limited effect of ASHD on cell viability of normal cells indicated that it may be specifically directed to cancer cells. Translocation of phosphatidyl serine, activation of caspase 3, caspase 9, PARP, alteration in the ratio of BCL2/BAD protein expression as well as the loss of mitochondrial membrane potential suggests activation of the intrinsic pathway of apoptosis. Conclusion: These results could facilitate the future development of novel hydantoin derivatives as chemotherapeutic agents for leukemia.

Flow cytometry analysis of human fetal osteoblast fate processes on spark plasma sintered hydroxyapatite-titanium biocomposites

Hydroxyapatite (HA)-based biocomposites have been widely investigated for a multitude of applications and these studies have been largely driven to improve mechanical properties (toughness and strength) without compromising cytocompatibility properties. Apart from routine cell viability/proliferation analysis, limited efforts have been made to quantify the fate processes (cell proliferation, cell cycle, and cell apoptosis) of human fetal osteoblast (hFOB) cells on HA-based composites, in vitro. In this work, the osteoblast cell fate process has been studied on a model hydroxyapatite-titanium (HA-Ti) system using the flow cytometry. In order to retain both HA and Ti, the novel processing technique, that is, spark plasma sintering, was suitably adopted. The cell fate processes of hFOBs, as evaluated using a flow cytometry, revealed statistically insignificant differences among HA-10 wt % Ti and HA and control (tissue culture polystyrene surface) in terms of osteoblast apoptosis, proliferation index as well as division index. For the first time, we provide quantified flow cytometry results to demonstrate that 10 wt % Ti additions to HA do not have any significant influence on the fate processes of human osteoblast-like cells, in vitro.

Inhibition of cancer cell proliferation and apoptosis-inducing activity of fungal taxol and its precursor baccatin III purified from endophytic Fusarium solani

Background: Taxol (generic name paclitaxel), a plant-derived antineoplastic agent, used widely against breast, ovarian and lung cancer, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. The limited supply of the drug has prompted efforts to find alternative sources, such as chemical synthesis, tissue and cell cultures of the Taxus species both of which are expensive and yield low levels. Fermentation processes with microorganisms would be the methods of choice to lower the costs and increase yields. Previously we have reported that F. solani isolated from T. celebica produced taxol and its precursor baccatin III in liquid grown cultures J Biosci 33: 259-67, 2008. This study was performed to evaluate the inhibition of proliferation and induction of apoptosis of cancer cell lines by the fungal taxol and fungal baccatin III of F. solani isolated from T. celebica. Methods: Cell lines such as HeLa, HepG2, Jurkat, Ovcar3 and T47D were cultured individually and treated with fungal taxol, baccatin III with or without caspase inhibitors according to experimental requirements. Their efficacy on apoptotic induction was examined. Results: Both fungal taxol and baccatin III inhibited cell proliferation of a number of cancer cell lines with IC50 ranging from 0.005 to 0.2 mu M for fungal taxol and 2 to 5 mu M for fungal baccatin III. They also induced apoptosis in JR4-Jurkat cells with a possible involvement of anti-apoptotic Bcl2 and loss in mitochondrial membrane potential, and was unaffected by inhibitors of caspase-9,-2 or -3 but was prevented in presence of caspase-10 inhibitor. DNA fragmentation was also observed in cells treated with fungal taxol and baccatin III. Conclusions: The cytotoxic activity exhibited by fungal taxol and baccatin III involves the same mechanism, dependent on caspase-10 and membrane potential loss of mitochondria, with taxol having far greater cytotoxic potential.

Epoch extraction based on integrated linear prediction residual using plosion index

Epoch is defined as the instant of significant excitation within a pitch period of voiced speech. Epoch extraction continues to attract the interest of researchers because of its significance in speech analysis. Existing high performance epoch extraction algorithms require either dynamic programming techniques or a priori information of the average pitch period. An algorithm without such requirements is proposed based on integrated linear prediction residual (ILPR) which resembles the voice source signal. Half wave rectified and negated ILPR (or Hilbert transform of ILPR) is used as the pre-processed signal. A new non-linear temporal measure named the plosion index (PI) has been proposed for detecting `transients' in speech signal. An extension of PI, called the dynamic plosion index (DPI) is applied on pre-processed signal to estimate the epochs. The proposed DPI algorithm is validated using six large databases which provide simultaneous EGG recordings. Creaky and singing voice samples are also analyzed. The algorithm has been tested for its robustness in the presence of additive white and babble noise and on simulated telephone quality speech. The performance of the DPI algorithm is found to be comparable or better than five state-of-the-art techniques for the experiments considered.

Detection of the closure-burst transitions of stops and affricates in continuous speech using the plosion index

Automatic and accurate detection of the closure-burst transition events of stops and affricates serves many applications in speech processing. A temporal measure named the plosion index is proposed to detect such events, which are characterized by an abrupt increase in energy. Using the maxima of the pitch-synchronous normalized cross correlation as an additional temporal feature, a rule-based algorithm is designed that aims at selecting only those events associated with the closure-burst transitions of stops and affricates. The performance of the algorithm, characterized by receiver operating characteristic curves and temporal accuracy, is evaluated using the labeled closure-burst transitions of stops and affricates of the entire TIMIT test and training databases. The robustness of the algorithm is studied with respect to global white and babble noise as well as local noise using the TIMIT test set and on telephone quality speech using the NTIMIT test set. For these experiments, the proposed algorithm, which does not require explicit statistical training and is based on two one-dimensional temporal measures, gives a performance comparable to or better than the state-of-the-art methods. In addition, to test the scalability, the algorithm is applied on the Buckeye conversational speech corpus and databases of two Indian languages. (C) 2014 Acoustical Society of America.