Modulation in vitro and in vivo of cytotoxicity but not cellular levels of doxorubicin by the calmodulin inhibitor trifluoperazine is dependent on the level of resistance.

PubMed Central

Ganapathi, R.; Schmidt, H.; Grabowski, D.; Melia, M.; Ratliff, N.

1988-01-01

The role of the calmodulin inhibitor trifluoperazine (TFP) in modulating the cellular levels and cytotoxicity in vitro and antitumour effects in vivo of doxorubicin (DOX), was evaluated in progressively DOX-resistant (5- to 40-fold) sublines of B16-BL6 mouse melanoma. In parental-sensitive B16-BL6 cells treated for 3 h, the IC50 of DOX was 0.1 microgram ml-1, and a less than 2-fold enhancement in DOX cell kill in the presence of a noncytotoxic concentration of 5 microM TFP was observed. However, in the DOX-resistant sublines, the IC50 was 0.7 to 5.0 micrograms ml-1 DOX in the absence of 5 microM TFP and 0.3 to 0.7 microgram ml-1 DOX in the presence of 5 microM TFP. The 2- to 7.5-fold decrease in the IC50 of DOX in the presence of 5 microM TFP, was dependent on the level of DOX-resistance in the various sublines. Compared to parental-sensitive cells, a 2-fold decrease in DOX-accumulation was evident only in the 40-fold DOX-resistant subline. Further, maximal enhancement (50%) of cellular DOX accumulation in the presence of 5 microM TFP was observed only in the 40-fold resistant cells treated with 5.0 micrograms ml-1 DOX. Retention of DOX in the 40-fold resistant subline was only 20% lower than similarly treated sensitive cells, and the inclusion of TFP increased DOX retention less than 10-15%. Antitumour studies in mice with experimental pulmonary metastases revealed that although DOX and DOX plus TFP had similar antitumour activity with the parental sensitive B16-BL6 cells, the combination of DOX plus TFP was significantly more effective than DOX alone with the DOX-resistant sublines. No overt toxicity was observed in normal mice treated with doses of TFP, DOX or DOX plus TFP used for in vivo chemotherapy studies. Results from this study suggest that gross cellular DOX levels do not appear to correlate with the magnitude of resistance, and the effects of TFP in modulating DOX resistance is possibly due to mechanisms other than mere alterations in cellular drug accumulation and/or retention. PMID:3179186

Elevated hypothalamic TCPTP in obesity contributes to cellular leptin resistance

PubMed Central

Loh, Kim; Fukushima, Atsushi; Zhang, Xinmei; Galic, Sandra; Briggs, Dana; Enriori, Pablo J.; Simonds, Stephanie; Wiede, Florian; Reichenbach, Alexander; Hauser, Christine; Sims, Natalie A.; Bence, Kendra K.; Zhang, Sheng; Zhang, Zhong-Yin; Kahn, Barbara B.; Neel, Benjamin G.; Andrews, Zane B.; Cowley, Michael A.; Tiganis, Tony

2011-01-01

SUMMARY In obesity, anorectic responses to leptin are diminished, giving rise to the concept of ‘leptin resistance’. Increased expression of protein tyrosine phosphatase 1B (PTP1B) has been associated with the attenuation of leptin signaling and development of cellular leptin resistance. Here we report that hypothalamic levels of the tyrosine phosphatase TCPTP are also elevated in obesity to attenuate the leptin response. We show that mice that lack TCPTP in neuronal cells have enhanced leptin sensitivity and are resistant to high fat diet-induced weight gain and the development of leptin resistance. Also, intracerebroventricular administration of a TCPTP inhibitor enhances leptin signaling and responses in mice. Moreover, the combined deletion of TCPTP and PTP1B in neuronal cells has additive effects in the prevention of diet-induced obesity. Our results identify TCPTP as a critical negative regulator of hypothalamic leptin signaling and causally link elevated TCPTP to the development of cellular leptin resistance in obesity. PMID:22000926

A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

PubMed

Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

2016-11-09

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication. Published by Elsevier Inc.

Paramagnetic fluorinated nanoemulsions for sensitive cellular fluorine-19 magnetic resonance imaging

PubMed Central

Kislukhin, Alexander A.; Xu, Hongyan; Adams, Stephen R.; Narsinh, Kazim H.; Tsien, Roger Y.; Ahrens, Eric T.

2016-01-01

Fluorine-19 magnetic resonance imaging (19F MRI) probes enable quantitative in vivo detection of cell therapies and inflammatory cells. Here, we describe the formulation of perfluorocarbon-based nanoemulsions with improved sensitivity for cellular MRI. Reduction of the 19F spin-lattice relaxation time (T1) enables rapid imaging and an improved signal-to-noise ratio, thereby improving cell detection sensitivity. We synthesized metal-binding β-diketones conjugated to linear perfluoropolyether (PFPE), formulated these fluorinated ligands as aqueous nanoemulsions, and then metalated them with various transition and lanthanide ions in the fluorous phase. Iron(III) tris-β-diketonate ('FETRIS') nanoemulsions with PFPE have low cytotoxicity (<20%) and superior MRI properties. Moreover, the 19F T1 can readily be reduced by an order of magnitude and tuned by stoichiometric modulation of the iron concentration. The resulting 19F MRI detection sensitivity is enhanced by 3-to-5 fold over previously used tracers at 11.7 T, and is predicted to increase by at least 8-fold at clinical field strength of 3 T. PMID:26974409

Sensitivity to methylmercury toxicity is enhanced in oxoguanine glycosylase 1 knockout murine embryonic fibroblasts and is dependent on cellular proliferation capacity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ondovcik, Stephanie L.; Tamblyn, Laura; McPherson, John Peter

2013-07-01

Methylmercury (MeHg) is a persistent environmental contaminant with potent neurotoxic action for which the underlying molecular mechanisms remain to be conclusively delineated. Our objectives herein were twofold: first, to corroborate our previous findings of an increased sensitivity of spontaneously-immortalized oxoguanine glycosylase 1-null (Ogg1{sup −/−}) murine embryonic fibroblasts (MEFs) to MeHg through generation of Simian virus 40 (SV40) large T antigen-immortalized wild-type and Ogg1{sup −/−} MEFs; and second, to determine whether MeHg toxicity is proliferation-dependent. As with the spontaneously-immortalized cells used previously, the SV40 large T antigen-immortalized cells exhibited similar tendencies to undergo MeHg-initiated cell cycle arrest, with increased sensitivity inmore » the Ogg1{sup −/−} MEFs as measured by clonogenic survival and DNA damage. Compared to exponentially growing cells, those seeded at a higher density exhibited compromised proliferation, which proved protective against MeHg-mediated cell cycle arrest and induction of DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), and by its functional confirmation by micronucleus assessment. This enhanced sensitivity of Ogg1{sup −/−} MEFs to MeHg toxicity using discrete SV40 immortalization corroborates our previous studies, and suggests a novel role for OGG1 in minimizing MeHg-initiated DNA lesions that trigger replication-associated DSBs. Furthermore, proliferative capacity may determine MeHg toxicity in vivo and in utero. Accordingly, variations in cellular proliferative capacity and interindividual variability in repair activity may modulate the risk of toxicological consequences following MeHg exposure. - Highlights: • SV40 large T antigen-immortalized Ogg1{sup −/−} cells are more sensitive to MeHg. • Sensitivity to MeHg is dependent on cellular proliferation capacity. • OGG1 maintains genomic integrity following MeHg-initiated DNA damage. • OGG1 may limit MeHg-initiated DNA lesions that trigger replication-associated DSBs. • Variations in proliferation and repair activity may modulate toxicological risk.« less

Perturbations of Amino Acid Metabolism Associated with Glyphosate-Dependent Inhibition of Shikimic Acid Metabolism Affect Cellular Redox Homeostasis and Alter the Abundance of Proteins Involved in Photosynthesis and Photorespiration1[W][OA

PubMed Central

Vivancos, Pedro Diaz; Driscoll, Simon P.; Bulman, Christopher A.; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H.

2011-01-01

The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway. PMID:21757634

Cellular glutathione depletion by diethyl maleate or buthionine sulfoximine: no effect of glutathione depletion on the oxygen enhancement ratio

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, J.B.; Russo, A.; Biaglow, J.E.

1983-11-01

The hypoxic and euoxic radiation response for Chinese hamster lung and A549 human lung carcinoma cells was obtained under conditions where their nonprotein thiols, consisting primarily of glutathione (GSH), were depleted by different mechanisms. The GSH conjugating reagent diethylmaleate (DEM) was compared to DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathionine biosynthesis. Each reagent depleted cellular GSH to less than 5% of control values. A 2-h exposure to 0.5 mM DEM or a 4- or 24-h exposure to BSO at 10 or 1 mM, respectively, depleted cellular GSH to less than 5% of control values. Both agents sensitized cells irradiated under airmore » or hypoxic conditions. When GSH levels are lowered to < 5% by both agents, hypoxic DEM-treated cells exhibited slightly greater x-ray sensitization than hypoxic BSO-treated cells. The aerobic and anoxic sensitization of the cells results in the OER's of 2.8 and 3.0 for the DEM- and BSO-treated cells compared to 2.9 for the V79 control A549. BSO-treated cells showed an OER of 3.3 versus 3 for the control. Our results suggest that GSH depletion by either BSO or DEM sensitizes aerobic cells to radiation but does not appreciably alter the OER.« less

Molecular control of steady-state dendritic cell maturation and immune homeostasis.

PubMed

Hammer, Gianna Elena; Ma, Averil

2013-01-01

Dendritic cells (DCs) are specialized sentinels responsible for coordinating adaptive immunity. This function is dependent upon coupled sensitivity to environmental signs of inflammation and infection to cellular maturation-the programmed alteration of DC phenotype and function to enhance immune cell activation. Although DCs are thus well equipped to respond to pathogens, maturation triggers are not unique to infection. Given that immune cells are exquisitely sensitive to the biological functions of DCs, we now appreciate that multiple layers of suppression are required to restrict the environmental sensitivity, cellular maturation, and even life span of DCs to prevent aberrant immune activation during the steady state. At the same time, steady-state DCs are not quiescent but rather perform key functions that support homeostasis of numerous cell types. Here we review these functions and molecular mechanisms of suppression that control steady-state DC maturation. Corruption of these steady-state operatives has diverse immunological consequences and pinpoints DCs as potent drivers of autoimmune and inflammatory disease.

Towards a high sensitivity small animal PET system based on CZT detectors (Conference Presentation)

NASA Astrophysics Data System (ADS)

Abbaszadeh, Shiva; Levin, Craig

2017-03-01

Small animal positron emission tomography (PET) is a biological imaging technology that allows non-invasive interrogation of internal molecular and cellular processes and mechanisms of disease. New PET molecular probes with high specificity are under development to target, detect, visualize, and quantify subtle molecular and cellular processes associated with cancer, heart disease, and neurological disorders. However, the limited uptake of these targeted probes leads to significant reduction in signal. There is a need to advance the performance of small animal PET system technology to reach its full potential for molecular imaging. Our goal is to assemble a small animal PET system based on CZT detectors and to explore methods to enhance its photon sensitivity. In this work, we reconstruct an image from a phantom using a two-panel subsystem consisting of six CZT crystals in each panel. For image reconstruction, coincidence events with energy between 450 and 570 keV were included. We are developing an algorithm to improve sensitivity of the system by including multiple interaction events.

Poly(styrene-co-maleic acid)-based pH-sensitive liposomes mediate cytosolic delivery of drugs for enhanced cancer chemotherapy.

PubMed

Banerjee, Shubhadeep; Sen, Kacoli; Pal, Tapan K; Guha, Sujoy K

2012-10-15

pH-responsive polymers render liposomes pH-sensitive and facilitate the intracellular release of encapsulated payload by fusing with endovascular membranes under mildly acidic conditions found inside cellular endosomes. The present study reports the use of high-molecular weight poly(styrene-co-maleic acid) (SMA), which exhibits conformational transition from a charged extended structure to an uncharged globule below its pK(1) value, to confer pH-sensitive property to liposomes. The changes in the co-polymer chain conformation resulted in destabilization of the liposomes at mildly acidic pH due to vesicle fusion and/or channel formation within the membrane bilayer, and ultimately led to the release of the encapsulated cargo. The vesicles preserved their pH-sensitivity and stability in serum unlike other polymer-based liposomes and exhibited no hemolytic activity at physiological pH. The lysis of RBCs at endosomal pH due to SMA-based liposome-induced alterations in the bilayer organization leading to spherocyte formation indicated the potential of these vesicles to mediate cytosolic delivery of bio-active molecules through endosome destabilization. The SMA-loaded liposomes exhibiting excellent cytocompatibility, efficiently delivered chemotherapeutic agent 5-Fluorouracil (5-FU) within colon cancer cells HT-29 in comparison to neat liposomes. This caused increased cellular-availability of the drug, which resulted in enhanced apoptosis and highlighted the clinical potential of SMA-based vesicles. Copyright © 2012 Elsevier B.V. All rights reserved.

Enhanced Intracellular Delivery and Tissue Retention of Nanoparticles by Mussel-Inspired Surface Chemistry.

PubMed

Chen, Kai; Xu, Xiaoqiu; Guo, Jiawei; Zhang, Xuelin; Han, Songling; Wang, Ruibing; Li, Xiaohui; Zhang, Jianxiang

2015-11-09

Nanomaterials have been broadly studied for intracellular delivery of diverse compounds for diagnosis or therapy. Currently it remains challenging for discovering new biomolecules that can prominently enhance cellular internalization and tissue retention of nanoparticles (NPs). Herein we report for the first time that a mussel-inspired engineering approach may notably promote cellular uptake and tissue retention of NPs. In this strategy, the catechol moiety is covalently anchored onto biodegradable NPs. Thus, fabricated NPs can be more effectively internalized by sensitive and multidrug resistant tumor cells, as well as some normal cells, resulting in remarkably potentiated in vitro activity when an antitumor drug is packaged. Moreover, the newly engineered NPs afford increased tissue retention post local or oral delivery. This biomimetic approach is promising for creating functional nanomaterials for drug delivery, vaccination, and cell therapy.

Co-delivery of paclitaxel and tetrandrine via iRGD peptide conjugated lipid-polymer hybrid nanoparticles overcome multidrug resistance in cancer cells

PubMed Central

Zhang, Jinming; Wang, Lu; Fai Chan, Hon; Xie, Wei; Chen, Sheng; He, Chengwei; Wang, Yitao; Chen, Meiwan

2017-01-01

One of the promising strategies to overcome tumor multidrug resistance (MDR) is to deliver anticancer drug along with P-glycoprotein (P-gp) inhibitor simultaneously. To enhance the cancer cellular internalization and implement the controlled drug release, herein an iRGD peptide-modified lipid-polymer hybrid nanosystem (LPN) was fabricated to coload paclitaxel (PTX) and tetrandrine (TET) at a precise combination ratio. In this co-delivery system, PTX was covalently conjugated to poly (D,L-lactide-co-glycolide) polymeric core by redox-sensitive disulfide bond, while TET was physically capsulated spontaneously for the aim to suppress P-gp in advance by the earlier released TET in cancer cells. As a result, the PTX+TET/iRGD LPNs with a core-shell structure possessed high drug loading efficiency, stability and redox-sensitive drug release profiles. Owing to the enhanced cellular uptake and P-gp suppression mediated by TET, significantly more PTX accumulated in A2780/PTX cells treated with PTX+TET/iRGD LPNs than either free drugs or non-iRGD modified LPNs. As expected, PTX+TET/iRGD LPNs presented the highest cytotoxicity against A2780/PTX cells and effectively promoted ROS production, enhanced apoptosis and cell cycle arrests particularly. Taken together, the co-delivery system demonstrated great promise as potential treatment for MDR-related tumors based on the synergistic effects of P-gp inhibition, enhanced endocytosis and intracellular sequentially drug release. PMID:28470171

A novel Chk1-binding peptide that enhances genotoxic sensitivity through the cellular redistribution of nuclear Chk1

PubMed Central

Kim, Kwang Seok; Choi, Kyu Jin; Bae, Sangwoo

2016-01-01

Since checkpoint kinase 1 (Chk1) is an essential factor for cell viability following DNA damage, the inhibition of Chk1 has been a major focus of pharmaceutical development to enhance the sensitivity of tumor cells to chemo- and radiotherapy that damage DNA. However, due to the off-target effects of conventional Chk1-targeting strategies and the toxicity of Chk1 inhibitors, alternative strategies are required to target Chk1. To facilitate such efforts, in this study, we identified a specific Chk1-binding 12-mer peptide from the screening of a phage display library and characterized the peptide in terms of cellular cytotoxicity, and in terms of its effect on Chk1 activity and sensitivity to genotoxic agents. This peptide, named N-terminal Chk1-binding peptide (Chk1-NP), bound the kinase domain of Chk1. Simulation of the binding revealed that the very N-terminus of the Chk1 kinase domain is the potential peptide binding site. Of note, the polyarginine-mediated internalization of Chk1-NP redistributed nuclear Chk1 with a prominent decrease in the nucleus in the absence of DNA damage. Treatment with Chk1-NP peptide alone decreased the viability of p53-defective HeLa cells, but not that of p53-functional NCI-H460 cells under normal conditions. The treatment of HeLa or NCI-H460 cells with the peptide significantly enhanced radiation sensitivity following ionizing radiation (IR) with a greater enhancement observed in HeLa cells. Moreover, the IR-induced destabilization of Chk1 was aggravated by treatment with Chk1-NP. Therefore, the decreased nuclear localization and protein levels of Chk1 seem to be responsible for the enhanced cancer cell killing following combined treatment with IR and Chk1-NP. The approach using the specific Chk1-binding peptide may facilitate the mechanistic understanding and potential modulation of Chk1 activities and may provide a novel rationale for the development of specific Chk1-targeting agents. PMID:28025997

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin.

PubMed

Rink, Jonathan S; Yang, Shuo; Cen, Osman; Taxter, Tim; McMahon, Kaylin M; Misener, Sol; Behdad, Amir; Longnecker, Richard; Gordon, Leo I; Thaxton, C Shad

2017-11-06

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

Polarization Sensitive Coherent Anti-Stokes Raman Spectroscopy of DCVJ in Doped Polymer

NASA Astrophysics Data System (ADS)

Ujj, Laszlo

2014-05-01

Coherent Raman Microscopy is an emerging technic and method to image biological samples such as living cells by recording vibrational fingerprints of molecules with high spatial resolution. The race is on to record the entire image during the shortest time possible in order to increase the time resolution of the recorded cellular events. The electronically enhanced polarization sensitive version of Coherent anti-Stokes Raman scattering is one of the method which can shorten the recording time and increase the sharpness of an image by enhancing the signal level of special molecular vibrational modes. In order to show the effectiveness of the method a model system, a highly fluorescence sample, DCVJ in a polymer matrix is investigated. Polarization sensitive resonance CARS spectra are recorded and analyzed. Vibrational signatures are extracted with model independent methods. Details of the measurements and data analysis will be presented. The author gratefully acknowledge the UWF for financial support.

Fluorescence enhancement of quercetin complexes by silver nanoparticles and its analytical application

NASA Astrophysics Data System (ADS)

Liu, Ping; Zhao, Liangliang; Wu, Xia; Huang, Fei; Wang, Minqin; Liu, Xiaodan

2014-03-01

It is found that the plasmon effect of silver nanoparticles (AgNPs) helps to enhance the fluorescence intensity of the quercetin (Qu) and nucleic acids system. Qu exhibited strong fluorescence enhancement when it bound to nucleic acids in the presence of AgNPs. Based on this, a sensitive method for the determination of nucleic acids was developed. The detection limits for the nucleic acids (S/N = 3) were reduced to the ng mL-1 level. The interaction mechanism of the AgNPs-fish sperm DNA (fsDNA)-Qu system was also investigated in this paper. This complex system of Qu and AgNPs was also successfully used for the detection of nucleic acids in agarose gel electrophoresis analysis. Preliminary results indicated that AgNPs also helped to improve sensitivity in the fluorescence image analysis of Qu combined with cellular contents in Arabidopsis thaliana protoplasts.

Salicylate effects on proton gradient dissipation by isolated gastric mucosal surface cells.

PubMed

Olender, E J; Woods, D; Kozol, R; Fromm, D

1986-11-01

The effects of salicylate were examined on Na+/H+ exchange by isolated gastric mucosal surface cells loaded with H+ and resuspended in a buffered medium. Choline salicylate (pH 7.4) increases the dissipation of an intracellular proton gradient which was measured using acridine orange. The exchange of extracellular Na+ with intracellular H+ by surface cells not only remains intact but also is enhanced upon exposure to salicylate. This was confirmed by cellular uptake of 22Na and titration of cellular H+ efflux. Salicylate increases Na+/H+ exchange via a pathway predominantly sensitive to amiloride. However, the data also suggest that salicylate dissipates an intracellular proton gradient by an additional mechanism. The latter is independent of extracellular Na+ and not due to a generalized increase in cellular permeability.

Breast MR segmentation and lesion detection with cellular neural networks and 3D template matching.

PubMed

Ertaş, Gökhan; Gülçür, H Ozcan; Osman, Onur; Uçan, Osman N; Tunaci, Mehtap; Dursun, Memduh

2008-01-01

A novel fully automated system is introduced to facilitate lesion detection in dynamic contrast-enhanced, magnetic resonance mammography (DCE-MRM). The system extracts breast regions from pre-contrast images using a cellular neural network, generates normalized maximum intensity-time ratio (nMITR) maps and performs 3D template matching with three layers of 12x12 cells to detect lesions. A breast is considered to be properly segmented when relative overlap >0.85 and misclassification rate <0.10. Sensitivity, false-positive rate per slice and per lesion are used to assess detection performance. The system was tested with a dataset of 2064 breast MR images (344slicesx6 acquisitions over time) from 19 women containing 39 marked lesions. Ninety-seven percent of the breasts were segmented properly and all the lesions were detected correctly (detection sensitivity=100%), however, there were some false-positive detections (31%/lesion, 10%/slice).

Frontiers of optofluidics in synthetic biology.

PubMed

Tan, Cheemeng; Lo, Shih-Jie; LeDuc, Philip R; Cheng, Chao-Min

2012-10-07

The development of optofluidic-based technology has ushered in a new era of lab-on-a-chip functionality, including miniaturization of biomedical devices, enhanced sensitivity for molecular detection, and multiplexing of optical measurements. While having great potential, optofluidic devices have only begun to be exploited in many biotechnological applications. Here, we highlight the potential of integrating optofluidic devices with synthetic biological systems, which is a field focusing on creating novel cellular systems by engineering synthetic gene and protein networks. First, we review the development of synthetic biology at different length scales, ranging from single-molecule, single-cell, to cellular population. We emphasize light-sensitive synthetic biological systems that would be relevant for the integration with optofluidic devices. Next, we propose several areas for potential applications of optofluidics in synthetic biology. The integration of optofluidics and synthetic biology would have a broad impact on point-of-care diagnostics and biotechnology.

Sulfasalazine, an inhibitor of the cystine-glutamate antiporter, reduces DNA damage repair and enhances radiosensitivity in murine B16F10 melanoma

PubMed Central

Shibata, Yuki; Shimizu, Takuto; Yoshioka, Chie; Maruo, Takuya

2018-01-01

The sodium-independent cystine-glutamate antiporter plays an important role in extracellular cystine uptake. It comprises the transmembrane protein, xCT and its chaperone, CD98. Because glutathione is only weakly cell membrane permeable, cellular uptake of its precursor, cystine, is known to be a key step in glutathione synthesis. Moreover, it has been reported that xCT expression affects the progression of tumors and their resistance to therapy. Sulfasalazine is an inhibitor of xCT that is known to increase cellular oxidative stress, giving it anti-tumor potential. Here, we describe a radio-sensitizing effect of sulfasalazine using a B16F10 melanoma model. Sulfasalazine decreased glutathione concentrations and resistance to H2O2 in B16F10 melanoma cells, but not in mouse embryonic fibroblasts. It synergistically enhanced the cyto-killing effect of X-irradiation in B16F10 cells. It inhibited cellular DNA damage repair and prolonged cell cycle arrest after X-irradiation. Furthermore, in an in vivo transplanted melanoma model, sulfasalazine decreased intratumoral glutathione content, leading to enhanced susceptibility to radiation therapy. These results suggest the possibility of using SAS to augment the treatment of radio-resistant cancers. PMID:29649284

Inhibition of autophagy significantly enhances combination therapy with sorafenib and HDAC inhibitors for human hepatoma cells.

PubMed

Yuan, Hang; Li, Ai-Jun; Ma, Sen-Lin; Cui, Long-Jiu; Wu, Bin; Yin, Lei; Wu, Meng-Chao

2014-05-07

To clarify whether histone deacetylase inhibitors histone deacetylase inhibitors (HDACIs) can sensitize hepatocellular carcinoma (HCC) cells to sorafenib treatment. Bax, Bcl-2, ATG5-ATG12, p21, and p27 protein levels in Hep3B, HepG2, and PLC/PRF/5 cells were examined by Western blot. CCK8 and a fluorometric caspase-3 assay were used to examine cellular viability and apoptosis levels. The effect of Beclin-1 on sensitization of HCC cells to sorafenib was examined by transfecting Beclin-1 siRNA into Hep3B, HepG2, and PLC/PRF/5 cells. Autophagy inhibition enhances the inhibitory effects of vorinostat and sorafenib alone or in combination on HCC cell growth. Vorinostat and sorafenib synergistically induced apoptosis and cell cycle alterations. Western blot data indicated that HDACIs and Beclin-1 knockdown increased the p53 acetylation level. The knockdown of Beclin-1 enhanced the synergistic effect of the combination of vorinostat with sorafenib. HDACIs can sensitize HCC cells to sorafenib treatment by regulating the acetylation level of Beclin-1.

Oncostatin M (OSM) protects against cardiac ischaemia/reperfusion injury in diabetic mice by regulating apoptosis, mitochondrial biogenesis and insulin sensitivity.

PubMed

Sun, Dongdong; Li, Shuang; Wu, Hao; Zhang, Mingming; Zhang, Xiaotian; Wei, Liping; Qin, Xing; Gao, Erhe

2015-06-01

Oncostatin M (OSM) exhibits many unique biological activities by activating Oβ receptor. However, its role in myocardial I/R injury in diabetic mice remains unknown. The involvement of OSM was assessed in diabetic mice which underwent myocardial I/R injury by OSM treatment or genetic deficiency of OSM receptor Oβ. Its mechanism on cardiomyocyte apoptosis, mitochondrial biogenesis and insulin sensitivity were further studied. OSM alleviated cardiac I/R injury by inhibiting cardiomyocyte apoptosis through inhibition of inositol pyrophosphate 7 (IP7) production, thus activating PI3K/Akt/BAD pathway, decreasing Bax expression while up-regulating Bcl-2 expression and decreasing the ratio of Bax to Bcl-2 in db/db mice. OSM enhanced mitochondrial biogenesis and mitochondrial function in db/db mice subjected to cardiac I/R injury. On the contrary, OSM receptor Oβ knockout exacerbated cardiac I/R injury, increased IP7 production, enhanced cardiomyocyte apoptosis, impaired mitochondrial biogenesis, glucose homoeostasis and insulin sensitivity in cardiac I/R injured diabetic mice. Inhibition of IP7 production by TNP (IP6K inhibitor) exerted similar effects of OSM. The mechanism of OSM on cardiac I/R injury in diabetic mice is partly associated with IP7/Akt and adenine mononucleotide protein kinase/PGC-1α pathway. OSM protects against cardiac I/R Injury by regulating apoptosis, insulin sensitivity and mitochondrial biogenesis in diabetic mice through inhibition of IP7 production. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with onemore » GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.« less

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay.

PubMed

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil; Kim, Dong-Myung; Yoo, Tae Hyeon; Kim, Yong-Sung

2015-11-27

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Interference of mobile phones and digitally enhanced cordless telecommunications mobile phones in renal scintigraphy.

PubMed

Stegmayr, Armin; Fessl, Benjamin; Hörtnagl, Richard; Marcadella, Michael; Perkhofer, Susanne

2013-08-01

The aim of the study was to assess the potential negative impact of cellular phones and digitally enhanced cordless telecommunication (DECT) devices on the quality of static and dynamic scintigraphy to avoid repeated testing in infant and teenage patients to protect them from unnecessary radiation exposure. The assessment was conducted by performing phantom measurements under real conditions. A functional renal-phantom acting as a pair of kidneys in dynamic scans was created. Data were collected using the setup of cellular phones and DECT phones placed in different positions in relation to a camera head to test the potential interference of cellular phones and DECT phones with the cameras. Cellular phones reproducibly interfered with the oldest type of gamma camera, which, because of its single-head specification, is the device most often used for renal examinations. Curves indicating the renal function were considerably disrupted; cellular phones as well as DECT phones showed a disturbance concerning static acquisition. Variable electromagnetic tolerance in different types of γ-cameras could be identified. Moreover, a straightforward, low-cost method of testing the susceptibility of equipment to interference caused by cellular phones and DECT phones was generated. Even though some departments use newer models of γ-cameras, which are less susceptible to electromagnetic interference, we recommend testing examination rooms to avoid any interference caused by cellular phones. The potential electromagnetic interference should be taken into account when the purchase of new sensitive medical equipment is being considered, not least because the technology of mobile communication is developing fast, which also means that different standards of wave bands will be issued in the future.

Combinatorial contextualization of peptidic epitopes for enhanced cellular immunity.

PubMed

Ito, Masaki; Hayashi, Kazumi; Adachi, Eru; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka

2014-01-01

Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our "motif-programming" approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freund's adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment.

ATM Expression Predicts Veliparib and Irinotecan Sensitivity in Gastric Cancer by Mediating P53-Independent Regulation of Cell Cycle and Apoptosis.

PubMed

Subhash, Vinod Vijay; Tan, Shi Hui; Yeo, Mei Shi; Yan, Fui Leng; Peethala, Praveen C; Liem, Natalia; Krishnan, Vaidehi; Yong, Wei Peng

2016-12-01

Identification of synthetically lethal cellular targets and synergistic drug combinations is important in cancer chemotherapy as they help to overcome treatment resistance and increase efficacy. The Ataxia Telangiectasia Mutated (ATM) kinase is a nuclear protein that plays a major role in the initiation of DNA repair signaling and cell-cycle check points during DNA damage. Although ATM was shown to be associated with poor prognosis in gastric cancer, its implications as a predictive biomarker for cancer chemotherapy remain unexplored. The present study evaluated ATM-induced synthetic lethality and its role in sensitization of gastric cancer cells to PARP and TOP1 inhibitors, veliparib (ABT-888) and irinotecan (CPT-11), respectively. ATM expression was detected in a panel of gastric cell lines, and the IC 50 against each inhibitors was determined. The combinatorial effect of ABT-888 and CPT-11 in gastric cancer cells was also determined both in vitro and in vivo ATM deficiency was found to be associated with enhanced sensitivity to ABT-888 and CPT-11 monotherapy, hence suggesting a mechanism of synthetic lethality. Cells with high ATM expression showed reduced sensitivity to monotherapy; however, they showed a higher therapeutic effect with ABT-888 and CPT-11 combinatorial therapy. Furthermore, ATM expression was shown to play a major role in cellular homeostasis by regulating cell-cycle progression and apoptosis in a P53-independent manner. The present study highlights the clinical utility of ATM expression as a predictive marker for sensitivity of gastric cancer cells to PARP and TOP1 inhibition and provides a deeper mechanistic insight into ATM-dependent regulation of cellular processes. Mol Cancer Ther; 15(12); 3087-96. ©2016 AACR. ©2016 American Association for Cancer Research.

Polymeric nanocomposites loaded with fluoridated hydroxyapatite Ln3+ (Ln = Eu or Tb)/iron oxide for magnetic targeted cellular imaging

PubMed Central

Pan, Jie; Liu, Wei-Jiao; Hua, Chao; Wang, Li-Li; Wan, Dong; Gong, Jun-Bo

2015-01-01

Objective To fabricate polymeric nanocomposites with excellent photoluminescence, magnetic properties, and stability in aqueous solutions, in order to improve specificity and sensitivity of cellular imaging under a magnetic field. Methods Fluoridated Ln3+-doped HAP (Ln3+-HAP) NPs and iron oxides (IOs) can be encapsulated with biocompatible polymers via a modified solvent exaction/evaporation technique to prepare polymeric nanocomposites with fluoridated Ln3+-HAP/iron oxide. The nanocomposites were characterized for surface morphology, fluorescence spectra, magnetic properties and in vitro cytotoxicity. Magnetic targeted cellular imaging of such nanocomposites was also evaluated with confocal laser scanning microscope using A549 cells with or without magnetic field. Results The fabricated nanocomposites showed good stability and excellent luminescent properties, as well as low in vitro cytotoxicity, indicating that the nanocomposites are suitable for biological applications. Nanocomposites under magnetic field achieved much higher cellular uptake via an energy-dependent pathway than those without magnetic field. Conclusion The nanocomposites fabricated in this study will be a promising tool for magnetic targeted cellular imaging with improved specificity and enhanced selection. PMID:26487962

Murine Hyperglycemic Vasculopathy and Cardiomyopathy: Whole-Genome Gene Expression Analysis Predicts Cellular Targets and Regulatory Networks Influenced by Mannose Binding Lectin

PubMed Central

Zou, Chenhui; La Bonte, Laura R.; Pavlov, Vasile I.; Stahl, Gregory L.

2012-01-01

Hyperglycemia, in the absence of type 1 or 2 diabetes, is an independent risk factor for cardiovascular disease. We have previously demonstrated a central role for mannose binding lectin (MBL)-mediated cardiac dysfunction in acute hyperglycemic mice. In this study, we applied whole-genome microarray data analysis to investigate MBL’s role in systematic gene expression changes. The data predict possible intracellular events taking place in multiple cellular compartments such as enhanced insulin signaling pathway sensitivity, promoted mitochondrial respiratory function, improved cellular energy expenditure and protein quality control, improved cytoskeleton structure, and facilitated intracellular trafficking, all of which may contribute to the organismal health of MBL null mice against acute hyperglycemia. Our data show a tight association between gene expression profile and tissue function which might be a very useful tool in predicting cellular targets and regulatory networks connected with in vivo observations, providing clues for further mechanistic studies. PMID:22375142

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib.

PubMed

Wang, Chen; Jette, Nicholas; Moussienko, Daniel; Bebb, D Gwyn; Lees-Miller, Susan P

2017-04-01

The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Statistical mechanics of tuned cell signalling: sensitive collective response by synthetic biological circuits

NASA Astrophysics Data System (ADS)

Voliotis, M.; Liverpool, T. B.

2017-03-01

Living cells sense and process environmental cues through noisy biochemical mechanisms. This apparatus limits the scope of engineering cells as viable sensors. Here, we highlight a mechanism that enables robust, population-wide responses to external stimulation based on cellular communication, known as quorum sensing. We propose a synthetic circuit consisting of two mutually repressing quorum sensing modules. At low cell densities the system behaves like a genetic toggle switch, while at higher cell densities the behaviour of nearby cells is coupled via diffusible quorum sensing molecules. We show by systematic coarse graining that at large length and timescales that the system can be described using the Ising model of a ferromagnet. Thus, in analogy with magnetic systems, the sensitivity of the population-wide response, or its ‘susceptibility’ to a change in the external signal, is highly enhanced for a narrow range of cell-cell coupling close to a critical value. We expect that our approach will be used to enhance the sensitivity of synthetic bio-sensing networks.

Comparative proteomic analysis of the stolon cold stress response between the C4 perennial grass species Zoysia japonica and Zoysia metrella.

PubMed

Xuan, Jiping; Song, Yufeng; Zhang, Hongxiao; Liu, Jianxiu; Guo, Zhongren; Hua, Yuelou

2013-01-01

Zoysiagrass, the most cold-tolerant grass among the warm-season turfgrasses, is often used as a model species for isolating cellular components related to cold stress. To understand the proteomic responses to cold stress in zoysiagrass stolons, we extracted stolon proteins from Zoysiajaponica, cv. Meyer (cold-tolerant) and Z. metrella, cv. Diamond (cold-sensitive), which were grown with or without cold treatment. Approximately 700 proteins were resolved on 2-DE gels, and 70 protein spots were differentially accumulated. We further observed that 45 of the identified proteins participate in 10 metabolic pathways and cellular processes. A significantly greater number of proteins accumulated in the Meyer than in the Diamond and 15 increased proteins were detected only in the Meyer cultivar under cold stress. Furthermore, we propose a cold stress-responsive protein network composed of several different functional components that exhibits a balance between reactive oxygen species (ROS) production and scavenging, accelerated protein biosynthesis and proteolysis, reduced protein folding, enhanced photosynthesis, abundant energy supply and enhanced biosynthesis of carbohydrates and nucleotides. Generally, the cold-tolerant Meyer cultivar showed a greater ROS scavenging ability, more abundant energy supply and increased photosynthesis and protein synthesis than did the cold-sensitive Diamond cultivar, which may partly explain why Meyer is more cold tolerant.

Metabolic response to MMS-mediated DNA damage in Saccharomyces cerevisiae is dependent on the glucose concentration in the medium.

PubMed

Kitanovic, Ana; Walther, Thomas; Loret, Marie Odile; Holzwarth, Jinda; Kitanovic, Igor; Bonowski, Felix; Van Bui, Ngoc; Francois, Jean Marie; Wölfl, Stefan

2009-06-01

Maintenance and adaptation of energy metabolism could play an important role in the cellular ability to respond to DNA damage. A large number of studies suggest that the sensitivity of cells to oxidants and oxidative stress depends on the activity of cellular metabolism and is dependent on the glucose concentration. In fact, yeast cells that utilize fermentative carbon sources and hence rely mainly on glycolysis for energy appear to be more sensitive to oxidative stress. Here we show that treatment of the yeast Saccharomyces cerevisiae growing on a glucose-rich medium with the DNA alkylating agent methyl methanesulphonate (MMS) triggers a rapid inhibition of respiration and enhances reactive oxygen species (ROS) production, which is accompanied by a strong suppression of glycolysis. Further, diminished activity of pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase upon MMS treatment leads to a diversion of glucose carbon to glycerol, trehalose and glycogen accumulation and an increased flux through the pentose-phosphate pathway. Such conditions finally result in a significant decline in the ATP level and energy charge. These effects are dependent on the glucose concentration in the medium. Our results clearly demonstrate that calorie restriction reduces MMS toxicity through increased respiration and reduced ROS accumulation, enhancing the survival and recovery of cells.

Genetic manipulation of iron biomineralization enhances MR relaxivity in a ferritin-M6A chimeric complex.

PubMed

Radoul, Marina; Lewin, Limor; Cohen, Batya; Oren, Roni; Popov, Stanislav; Davidov, Geula; Vandsburger, Moriel H; Harmelin, Alon; Bitton, Ronit; Greneche, Jean-Marc; Neeman, Michal; Zarivach, Raz

2016-05-23

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene - ferritin-M6A - in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies.

Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells

PubMed Central

2014-01-01

Background Cholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but do not interfere with CD-20 in chemo immunotherapy of chronic lymphocytic leukemia (CLL). Methods MEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients were treated with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations. Results We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients. Conclusion Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment. PMID:25401046

Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells.

PubMed

Benakanakere, Indira; Johnson, Tyler; Sleightholm, Richard; Villeda, Virgilio; Arya, Monika; Bobba, Ravi; Freter, Carl; Huang, Chunfa

2014-01-01

Cholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but do not interfere with CD-20 in chemo immunotherapy of chronic lymphocytic leukemia (CLL). MEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients were treated with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations. We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients. Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment.

Foliar Nutrient Distribution Patterns in Sympatric Maple Species Reflect Contrasting Sensitivity to Excess Manganese.

PubMed

Fernando, Denise R; Marshall, Alan T; Lynch, Jonathan P

2016-01-01

Sugar maple and red maple are closely-related co-occurring tree species significant to the North American forest biome. Plant abiotic stress effects including nutritional imbalance and manganese (Mn) toxicity are well documented within this system, and are implicated in enhanced susceptibility to biotic stresses such as insect attack. Both tree species are known to overaccumulate foliar manganese (Mn) when growing on unbuffered acidified soils, however, sugar maple is Mn-sensitive, while red maple is not. Currently there is no knowledge about the cellular sequestration of Mn and other nutrients in these two species. Here, electron-probe x-ray microanalysis was employed to examine cellular and sub-cellular deposition of excessively accumulated foliar Mn and other mineral nutrients in vivo. For both species, excess foliar Mn was deposited in symplastic cellular compartments. There were striking between-species differences in Mn, magnesium (Mg), sulphur (S) and calcium (Ca) distribution patterns. Unusually, Mn was highly co-localised with Mg in mesophyll cells of red maple only. The known sensitivity of sugar maple to excess Mn is likely linked to Mg deficiency in the leaf mesophyll. There was strong evidence that Mn toxicity in sugar maple is primarily a symplastic process. For each species, leaf-surface damage due to biotic stress including insect herbivory was compared between sites with acidified and non-acidified soils. Although it was greatest overall in red maple, there was no difference in biotic stress damage to red maple leaves between acidified and non-acidified soils. Sugar maple trees on buffered non-acidified soil were less damaged by biotic stress compared to those on unbuffered acidified soil, where they are also affected by Mn toxicity abiotic stress. This study concluded that foliar nutrient distribution in symplastic compartments is a determinant of Mn sensitivity, and that Mn stress hinders plant resistance to biotic stress.

Foliar Nutrient Distribution Patterns in Sympatric Maple Species Reflect Contrasting Sensitivity to Excess Manganese

PubMed Central

Fernando, Denise R.; Marshall, Alan T.; Lynch, Jonathan P.

2016-01-01

Sugar maple and red maple are closely-related co-occurring tree species significant to the North American forest biome. Plant abiotic stress effects including nutritional imbalance and manganese (Mn) toxicity are well documented within this system, and are implicated in enhanced susceptibility to biotic stresses such as insect attack. Both tree species are known to overaccumulate foliar manganese (Mn) when growing on unbuffered acidified soils, however, sugar maple is Mn-sensitive, while red maple is not. Currently there is no knowledge about the cellular sequestration of Mn and other nutrients in these two species. Here, electron-probe x-ray microanalysis was employed to examine cellular and sub-cellular deposition of excessively accumulated foliar Mn and other mineral nutrients in vivo. For both species, excess foliar Mn was deposited in symplastic cellular compartments. There were striking between-species differences in Mn, magnesium (Mg), sulphur (S) and calcium (Ca) distribution patterns. Unusually, Mn was highly co-localised with Mg in mesophyll cells of red maple only. The known sensitivity of sugar maple to excess Mn is likely linked to Mg deficiency in the leaf mesophyll. There was strong evidence that Mn toxicity in sugar maple is primarily a symplastic process. For each species, leaf-surface damage due to biotic stress including insect herbivory was compared between sites with acidified and non-acidified soils. Although it was greatest overall in red maple, there was no difference in biotic stress damage to red maple leaves between acidified and non-acidified soils. Sugar maple trees on buffered non-acidified soil were less damaged by biotic stress compared to those on unbuffered acidified soil, where they are also affected by Mn toxicity abiotic stress. This study concluded that foliar nutrient distribution in symplastic compartments is a determinant of Mn sensitivity, and that Mn stress hinders plant resistance to biotic stress. PMID:27391424

Dynamic-SERS Optophysiology: A Nanosensor for Monitoring Cell Secretion Events.

PubMed

Lussier, Félix; Brulé, Thibault; Vishwakarma, Medhavi; Das, Tamal; Spatz, Joachim P; Masson, Jean-François

2016-06-08

We monitored metabolite secretion near living cells using a plasmonic nanosensor. The nanosensor created from borosilicate nanopipettes analogous to the patch clamp was decorated with Au nanoparticles and served as a surface-enhanced Raman scattering (SERS) substrate with addressable location. With this nanosensor, we acquired SERS locally near Madin-Darby canine kidney (MDCKII) epithelial cells, and we detected multiple metabolites, such as pyruvate, lactate, ATP, and urea simultaneously. These plasmonic nanosensors were capable of monitoring metabolites in the extracellular medium with enough sensitivity to detect an increase in metabolite concentration following the lyses of MDCKII cells with a nonionic surfactant. The plasmonic nanosensors also allowed a relative quantification of a chemical gradient for a metabolite near cells, as demonstrated with a decrease in relative lactate to pyruvate concentration further away from the MDCKII cells. This SERS optophysiology technique for the sensitive and nondestructive monitoring of extracellular metabolites near living cells is broadly applicable to different cellular and tissue models and should therefore provide a powerful tool for cellular studies.

Irigenin sensitizes TRAIL-induced apoptosis via enhancing pro-apoptotic molecules in gastric cancer cells.

PubMed

Xu, Ying; Gao, Cheng-Cheng; Pan, Zhen-Guo; Zhou, Chuan-Wen

2018-02-12

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds promising value for cancer therapy due to its capacity to induce apoptosis in cancer cells. Nevertheless, TRAIL therapy is greatly hampered by its resistance. Irigenin (Iri), isoflavonoids, can be isolated from the rhizome of Belamcanda chinensis, and has been shown anti-cancer properties. In this study, we explored if Iri could enhance TRAIL-regulated apoptosis in TRAIL resistant gastric cancer cells. Iri significantly potentiated TRAIL-triggered cytotoxicity. Iri alone and TRAIL alone showed no effective role in apoptosis induction, whereas combined treatment with Iri and TRAIL markedly induced apoptosis in cancer cells, as evidenced by the up-regulation of cleaved Caspase-8/-9/-3 and PARP. Additionally, the sensitization to TRAIL was along with the enhancement of pro-apoptotic proteins, including FAS-associated protein with death domain (FADD), death receptor 5 (DR5) and Bax. And suppressing FADD, DR5 and Bax by si RNA significantly reduced the apoptosis and enhanced the cell viability induced by the co-application of Iri and TRAIL. Moreover, the sensitization to TRAIL was accompanied by the decrease of Cellular-FLICE inhibitory protein (c-FLIP), Bcl-2 and Survivin. Additionally, Iri could sensitize TRAIL to produce reactive oxygen species (ROS). Pre-treatment of N-acetyl-cysteine (NAC), ROS scavenger, attenuated Iri plus TRAIL-induced apoptosis and improved cell viability. Finally, combination of Iri and TRAIL inhibited tumor growth in the xenograft model. Collectively, our present study gave new insights into the effects of Iri on potentiating TRAIL-sensitivity, and suggested that Iri could be a potential candidate for sensitizer of TRAIL-resistant cancer cell treatment. Copyright © 2018. Published by Elsevier Inc.

Quantification of α-tubulin isotypes by sandwich ELISA with signal amplification through biotinyl-tyramide or immuno-PCR.

PubMed

Dráberová, Eduarda; Stegurová, Lucie; Sulimenko, Vadym; Hájková, Zuzana; Dráber, Petr; Dráber, Pavel

2013-09-30

Microtubules formed by αβ-tubulin dimers represent cellular structures that are indispensable for the maintenance of cell morphology and for cell motility generation. Microtubules in intact cells are in highly regulated equilibrium with cellular pools of soluble tubulin dimers. Sensitive, reproducible and rapid assays are necessary to monitor tubulin changes in cytosolic pools after treatment with anti-mitotic drugs, during the cell cycle or activation and differentiation events. Here we describe new assays for α-tubulin quantification. The assays are based on sandwich ELISA, and the signal is amplified with biotinyl-tyramide or immuno-PCR. Matching monoclonal antibody pair recognizes phylogenetically highly conserved epitopes localized outside the C-terminal isotype-defining region. This makes it possible to detect α-tubulin isotypes in different cell types of various species. Biotinyl-tyramide amplification and immuno-PCR amplification enable detection of tubulin at concentrations 2.5ng/ml and 0.086ng/ml, respectively. Immuno-PCR detection shows enhanced sensitivity and wider dynamic range when compared to ELISA with biotinyl-tyramide detection. Our results on taxol-treated and activated bone marrow-derived mast cells demonstrate, that the assays allow sensitive quantification of tubulin in complex biological fluids. © 2013.

A mechanism of extreme growth and reliable signaling in sexually selected ornaments and weapons.

PubMed

Emlen, Douglas J; Warren, Ian A; Johns, Annika; Dworkin, Ian; Lavine, Laura Corley

2012-08-17

Many male animals wield ornaments or weapons of exaggerated proportions. We propose that increased cellular sensitivity to signaling through the insulin/insulin-like growth factor (IGF) pathway may be responsible for the extreme growth of these structures. We document how rhinoceros beetle horns, a sexually selected weapon, are more sensitive to nutrition and more responsive to perturbation of the insulin/IGF pathway than other body structures. We then illustrate how enhanced sensitivity to insulin/IGF signaling in a growing ornament or weapon would cause heightened condition sensitivity and increased variability in expression among individuals--critical properties of reliable signals of male quality. The possibility that reliable signaling arises as a by-product of the growth mechanism may explain why trait exaggeration has evolved so many different times in the context of sexual selection.

Prospects of third-generation femtosecond laser technology in biological spectromicroscopy

NASA Astrophysics Data System (ADS)

Fattahi, Hanieh; Fattahi, Zohreh; Ghorbani, Asghar

2018-05-01

The next generation of biological imaging modalities will be a movement towards super-resolution, label-free approaches to realize subcellular images in a nonperturbative, non-invasive manner and towards new detection metrologies to reach a higher sensitivity and dynamic range. In this paper, we discuss how the third generation femtosecond laser technology in combination with the already existing concepts in time-resolved spectroscopy could fulfill the requirements of these exciting prospects. The expected enhanced specificity and sensitivity of the envisioned super-resolution microscope could lead us to a better understanding of the inter- and intra-cellular molecular transport and DNA-protein interaction.

Biotin-Pt (IV)-indomethacin hybrid: A targeting anticancer prodrug providing enhanced cancer cellular uptake and reversing cisplatin resistance.

PubMed

Hu, Weiwei; Fang, Lei; Hua, Wuyang; Gou, Shaohua

2017-10-01

A Pt(IV) prodrug (2) composed of cancer-targeting biotin and nonsteroidal anti-inflammatory drug indomethacin in the axial positions of the six-coordinated octahedral geometry derived from cisplatin was developed, which could be highly accumulated in cancer cells more than normal ones and activated by endogenous reducing molecules to release cisplatin and indomethacin moieties simultaneously to inhibit tumor progression synergistically. In vitro assays revealed that 2 exhibited significantly selective inhibition to the tested cancer cell lines and sensitivity to cisplatin resistant cancer cells. Moreover, 2 presented cyclooxygenases inhibition properties to reduce tumor-associated inflammation, reduced the invasiveness of the highly aggressive PC-3 cells, and disrupted capillary-like tube formation in EA.hy926 cells. In all, this study offers a new strategy to enhance sensitivity and reduce toxicity of cisplatin. Copyright © 2017 Elsevier Inc. All rights reserved.

Substance P enhances the activation of AMPK and cellular lipid accumulation in 3T3‑L1 cells in response to high levels of glucose.

PubMed

Dubon, Maria Jose; Byeon, Yeji; Park, Ki-Sook

2015-12-01

The rescue of glucose tolerance and insulin‑sensitivity in peripheral tissues, including adipose tissue, is essential in therapeutic strategies for diabetes. The present study demonstrated that substance P (SP) increases the accumulation of lipids in 3T3‑L1 cells during their differentiation into adipocytes in response to a high concentration of glucose. SP reciprocally regulated the activities of AMP‑activated protein kinase (AMPK) and Akt: SP enhanced the activation of AMPK, although the activity of Akt was downregulated. Notably, SP induced an increase in the expression level of glucose transporter 4 in the 3T3‑L1 adipocytes. Therefore, it is possible that SP leads to an increase in glucose uptake and the accumulation of lipids in adipocytes, and may contribute towards the rescue of insulin‑sensitivity in diabetes.

Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents.

PubMed

Mailloux, Ryan J; Adjeitey, Cyril Nii-Klu; Harper, Mary-Ellen

2010-10-13

Uncoupling protein-2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ∼22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.

Zwitterionic poly(carboxybetaine)-based cationic liposomes for effective delivery of small interfering RNA therapeutics without accelerated blood clearance phenomenon.

PubMed

Li, Yan; Liu, Ruiyuan; Shi, Yuanjie; Zhang, Zhenzhong; Zhang, Xin

2015-01-01

For efficient delivery of small interfering RNA (siRNA) to the target diseased site in vivo, it is important to design suitable vehicles to control the blood circulation of siRNA. It has been shown that surface modification of cationic liposome/siRNA complexes (lipoplexes) with polyethylene glycol (PEG) could enhance the circulation time of lipoplexes. However, the first injection of PEGylated lipoplexes in vivo induces accelerated blood clearance and enhances hepatic accumulation of the following injected PEGylated lipoplexes, which is known as the accelerated blood clearance (ABC) phenomenon. Herein, we developed zwitterionic poly(carboxybetaine) (PCB) modified lipoplexes for the delivery of siRNA therapeutics, which could avoid protein adsorption and enhance the stability of lipoplexes as that for PEG. Quite different from the PEGylation, the PCBylated lipoplexes could avoid ABC phenomenon, which extended the blood circulation time and enhanced the tumor accumulation of lipoplexes in vivo. After accumulation in tumor site, the PCBylation could promote the cellular uptake and endosomal/lysosomal escape of lipoplexes due to its unique chemical structure and pH-sensitive ability. With excellent tumor accumulation, cellular uptake and endosomal/lysosomal escape abilities, the PCBylated lipoplexes significantly inhibited tumor growth and induced tumor cell apoptosis.

Recent advances in intracellular and in vivo ROS sensing: focus on nanoparticle and nanotube applications.

PubMed

Uusitalo, Larissa M; Hempel, Nadine

2012-01-01

Reactive oxygen species (ROS) are increasingly being implicated in the regulation of cellular signaling cascades. Intracellular ROS fluxes are associated with cellular function ranging from proliferation to cell death. Moreover, the importance of subtle, spatio-temporal shifts in ROS during localized cellular signaling events is being realized. Understanding the biochemical nature of the ROS involved will enhance our knowledge of redox-signaling. An ideal intracellular sensor should therefore resolve real-time, localized ROS changes, be highly sensitive to physiologically relevant shifts in ROS and provide specificity towards a particular molecule. For in vivo applications issues such as bioavailability of the probe, tissue penetrance of the signal and signal-to-noise ratio also need to be considered. In the past researchers have heavily relied on the use of ROS-sensitive fluorescent probes and, more recently, genetically engineered ROS sensors. However, there is a great need to improve on current methods to address the above issues. Recently, the field of molecular sensing and imaging has begun to take advantage of the unique physico-chemical properties of nanoparticles and nanotubes. Here we discuss the recent advances in the use of these nanostructures as alternative platforms for ROS sensing, with particular emphasis on intracellular and in vivo ROS detection and quantification.

Altered expression of prohibitin in psoriatic lesions and its cellular implication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Soon Young; Kim, Younghwa; Hwang, Ha Young

2007-08-31

Psoriasis is characterized by excessive proliferation of keratinocytes accompanying acanthosis and incomplete differentiation. Prohibitin was investigated by examining its function of HaCaT as well as psoriasis. Psoriatic involved skin revealed high level of prohibitin in the basal layer. Prohibitin was analyzed by applying RNAi (PHBi) with HaCaT, which demonstrated increased S-phase. PHBi showed enhanced sensitivity to anthralin-mediated cell death due to enhanced loss of mitochondrial membrane potential, suggesting a protective role of prohibitin against apoptosis. Collectively, prohibitin plays a role both in cell cycle regulation and in maintaining mitochondrial integrity, implying its association with pathogenesis of psoriasis.

Engineered peptide-based nanobiomaterials for electrochemical cell chip.

PubMed

Kafi, Md Abdul; Cho, Hyeon-Yeol; Choi, Jeong-Woo

2016-01-01

Biomaterials having cell adhesion ability are considered to be integral part of a cell chip. A number of researches have been carried out to search for a suitable material for effective immobilization of cell on substrate. Engineered ECM materials or their components like collagen, Poly-l-Lysine (PLL), Arg-Gly-Asp (RGD) peptide have been extensively used for mammalian cell adhesion and proliferation with the aim of tissue regeneration or cell based sensing application. This review focuses on the various approaches for two- and three-dimensionally patterned nanostructures of a short peptide i.e. RGD peptide on chip surfaces together with their effects on cell behaviors and electrochemical measurements. Most of the study concluded with positive remarks on the well-oriented engineered RGD peptide over their homogenous thin film. The engineered RGD peptide not only influences cell adhesion, spreading and proliferation but also their periodic nano-arrays directly influence electrochemical measurements of the chips. The electrochemical signals found to be enhanced when RGD peptides were used in well-defined two-dimensional nano-arrays. The topographic alteration of three-dimensional structure of engineered RGD peptide was reported to be suitably contacted with the integrin receptors of cellular membrane which results indicated the enhanced cell-electrode adhesion and efficient electron exchange phenomenon. This enhanced electrochemical signal increases the sensitivity of the chip against the target analytes. Therefore, development of engineered cellular recognizable peptides and its 3D topological design for fabrication of cell chip will provide the synergetic effect on bio-affinity, sensitivity and accuracy for the in situ real-time monitoring of analytes.

Toluidine Blue with a Synergistic Effect in Morphological Assessment of Oral Cytosmears.

PubMed

Ratna Kumari, T V N; Ahmed Mujib, B R

2018-01-01

One percent toluidine blue being the most effective adjunct is often used to detect dysplasia. Not much attention has been given to the effect of toluidine blue in enhancement of cytological smears. The present study assessed the smears before and after application of toluidine blue in smokers against non-smokers using three different stains [Papanicolaou (PAP), Hematoxylin and Eosin (H and E), and Giemsa]. Twenty-five individuals from each group participated in the study. The oral cytosmears were obtained before and after application of toluidine blue and assessed for clumping of squamous cells, cellular and nuclear pleomorphism, micronuclei, binucleation, bacterial colony units, and keratin flakes. In smokers, the maximum enhancement in cytological smears post-toluidine blue application was shown by Giemsa stain than PAP and H and E stains. Among the individual parameters, nuclear pleomorphism exhibited greatest significant difference between smokers and non-smokers. Toluidine blue enhanced the staining characteristics both in terms of sensitivity and specificity and thereby was found to be synergistic in assessment of cytosmears. The cellular alterations noticed in the smears of smokers with clinically normal buccal mucosa can be used as a means of education tool in counselling for smoking cessation.

Label-free surface-enhanced Raman scattering imaging to monitor the metabolism of antitumor drug 6-mercaptopurine in living cells.

PubMed

Han, Guangmei; Liu, Renyong; Han, Ming-Yong; Jiang, Changlong; Wang, Jianping; Du, Shuhu; Liu, Bianhua; Zhang, Zhongping

2014-12-02

The molecular processes of drugs from cellular uptake to intracellular distribution as well as the intracellular interaction with the target molecule are critically important for the development of new antitumor drugs. In this work, we have successfully developed a label-free surface-enhanced Raman scattering (SERS) technique to monitor and visualize the metabolism of antitumor drug 6-mercaptopurine in living cells. It has been clearly demonstrated that Au@Ag NPs exhibit an excellent Raman enhancement effect to both 6-mercaptopurine and its metabolic product 6-mercaptopurine-ribose. Their different ways to absorb at the surface of Au@Ag NPs lead to the obvious spectral difference for distinguishing the antitumor drug and its metabolite by SERS spectra. The Au@Ag NPs can easily pass through cell membranes in a large amount and sensitively respond to the biological conversion of 6-mercaptopurine in tumor cells. The Raman imaging can visualize the real-time distribution of 6-mercaptopurine and its biotransformation with the concentrations in tumor cells. The SERS-based method reported here is simple and efficient for the assessments of drug efficacy and the understanding of the molecular therapeutic mechanism of antitumor drugs at the cellular level.

Toluidine Blue with a Synergistic Effect in Morphological Assessment of Oral Cytosmears

PubMed Central

Ratna Kumari, T V. N.; Ahmed Mujib, B. R.

2018-01-01

Objectives: One percent toluidine blue being the most effective adjunct is often used to detect dysplasia. Not much attention has been given to the effect of toluidine blue in enhancement of cytological smears. The present study assessed the smears before and after application of toluidine blue in smokers against non-smokers using three different stains [Papanicolaou (PAP), Hematoxylin and Eosin (H and E), and Giemsa]. Study Design: Twenty-five individuals from each group participated in the study. The oral cytosmears were obtained before and after application of toluidine blue and assessed for clumping of squamous cells, cellular and nuclear pleomorphism, micronuclei, binucleation, bacterial colony units, and keratin flakes. Results: In smokers, the maximum enhancement in cytological smears post-toluidine blue application was shown by Giemsa stain than PAP and H and E stains. Among the individual parameters, nuclear pleomorphism exhibited greatest significant difference between smokers and non-smokers. Conclusion: Toluidine blue enhanced the staining characteristics both in terms of sensitivity and specificity and thereby was found to be synergistic in assessment of cytosmears. The cellular alterations noticed in the smears of smokers with clinically normal buccal mucosa can be used as a means of education tool in counselling for smoking cessation. PMID:29403163

Comparative Proteomic Analysis of the Stolon Cold Stress Response between the C4 Perennial Grass Species Zoysia japonica and Zoysia metrella

PubMed Central

Xuan, Jiping; Song, Yufeng; Zhang, Hongxiao; Liu, Jianxiu; Guo, Zhongren; Hua, Yuelou

2013-01-01

Zoysiagrass, the most cold-tolerant grass among the warm-season turfgrasses, is often used as a model species for isolating cellular components related to cold stress. To understand the proteomic responses to cold stress in zoysiagrass stolons, we extracted stolon proteins from Zoysia japonica , cv. Meyer (cold-tolerant) and Z . metrella , cv. Diamond (cold-sensitive), which were grown with or without cold treatment. Approximately 700 proteins were resolved on 2-DE gels, and 70 protein spots were differentially accumulated. We further observed that 45 of the identified proteins participate in 10 metabolic pathways and cellular processes. A significantly greater number of proteins accumulated in the Meyer than in the Diamond and 15 increased proteins were detected only in the Meyer cultivar under cold stress. Furthermore, we propose a cold stress-responsive protein network composed of several different functional components that exhibits a balance between reactive oxygen species (ROS) production and scavenging, accelerated protein biosynthesis and proteolysis, reduced protein folding, enhanced photosynthesis, abundant energy supply and enhanced biosynthesis of carbohydrates and nucleotides. Generally, the cold-tolerant Meyer cultivar showed a greater ROS scavenging ability, more abundant energy supply and increased photosynthesis and protein synthesis than did the cold-sensitive Diamond cultivar, which may partly explain why Meyer is more cold tolerant. PMID:24086619

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression.

PubMed

Zhang, Yin; Carr, Theresa; Dimtchev, Alexandre; Zaer, Naghmeh; Dritschilo, Anatoly; Jung, Mira

2007-07-01

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.

Xc- inhibitor sulfasalazine sensitizes colorectal cancer to cisplatin by a GSH-dependent mechanism.

PubMed

Ma, Ming-zhe; Chen, Gang; Wang, Peng; Lu, Wen-hua; Zhu, Chao-feng; Song, Ming; Yang, Jing; Wen, Shijun; Xu, Rui-hua; Hu, Yumin; Huang, Peng

2015-11-01

Sulfasalazine (SSZ) is an anti-inflammatory drug that has been demonstrated to induce apoptosis and tumor regression through inhibition of plasma membrane cystine transporter xc(-). Cysteine is a rate-limiting precursor for intracellular glutathione (GSH) synthesis, which is vital for compound detoxification and maintaining redox balance. Platinum-based chemotherapy is an important regimen used in clinics for various cancers including colorectal cancer (CRC). We hypothesized that targeting xc(-) transporter by SSZ may annihilate cellular detoxification through interruption of GSH synthesis and may enhance the anti-cancer activity of cisplatin (CDDP) by increasing drug transport. In the present study, we revealed that xCT, the active subunit of xc(-), is highly expressed in CRC cell lines and human colorectal carcinoma tissues compared with their normal counterparts. SSZ effectively depleted cellular GSH, leading to significant accumulation of reactive oxygen species and growth inhibition in CRC cells. In contrast, the normal epithelial cells of colon origin were less sensitive to SSZ, showing a moderate ROS elevation. Importantly, SSZ effectively enhanced the intracellular platinum level and cytotoxicity of CDDP in CRC cells. The synergistic effect of SSZ and CDDP was reversed by antioxidant N-acetyl-L-cysteine (NAC). Together, these results suggest that SSZ, a relatively non-toxic drug that targets cystine transporter, may, in combination with CDDP, have effective therapy for colorectal cancer. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells.

PubMed

He, Jinpeng; Feng, Xiu; Hua, Junrui; Wei, Li; Lu, Zhiwei; Wei, Wenjun; Cai, Hui; Wang, Bing; Shi, Wengui; Ding, Nan; Li, He; Zhang, Yanan; Wang, Jufang

2017-10-18

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3'-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.

A superior bright NIR luminescent nanoparticle preparation and indicating calcium signaling detection in cells and small animals.

PubMed

Zhang, Jian; Lakowicz, Joseph R

2018-01-01

Near-field fluorescence (NFF) effects were employed to develop a novel near-infrared (NIR) luminescent nanoparticle (LNP) with superior brightness. The LNP is used as imaging contrast agent for cellular and small animal imaging and furthermore suggested to use for detecting voltage-sensitive calcium in living cells and animals with high sensitivity. NIR Indocyanine green (ICG) dye was conjugated with human serum albumin (HSA) followed by covalently binding to gold nanorod (AuNR). The AuNR displayed dual plasmons from transverse and longitudinal axis, and the longitudinal plasmon was localized at the NIR region which could efficiently couple with the excitation and emission of ICG dye leading to a largely enhanced NFF. The enhancement factor was measured to be about 16-fold using both ensemble and single nanoparticle spectral methods. As an imaging contrast agent, the ICG-HSA-Au complex (abbreviate as ICG-Au) was conjugated on HeLa cells and fluorescence cell images were recorded on a time-resolved confocal microscope. The emission signals of ICG-Au complexes were distinctly resolved as the individual spots that were observed over the cellular backgrounds due to their strong brightness as well as shortened lifetime. The LNPs were also tested to have a low cytotoxicity. The ICG-Au complexes were injected below the skin surface of mouse showing emission spots 5-fold brighter than those from the same amount of free ICG-HSA conjugates. Based on the observations in this research, the excitation and emission of NIR ICG dyes were found to be able to sufficiently couple with the longitudinal plasmon of AuNRs leading to a largely enhanced NFF. Using the LNP with super-brightness as a contrast agent, the ICG-Au complex could be resolved from the background in the cell and small animal imaging. The novel NIR LNP has also a great potential for detection of voltage-gated calcium concentration in the cell and living animal with a high sensitivity.

Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity.

PubMed

Shi, Hao; Munk, Alexander; Nielsen, Thomas S; Daughtry, Morgan R; Larsson, Louise; Li, Shize; Høyer, Kasper F; Geisler, Hannah W; Sulek, Karolina; Kjøbsted, Rasmus; Fisher, Taylor; Andersen, Marianne M; Shen, Zhengxing; Hansen, Ulrik K; England, Eric M; Cheng, Zhiyong; Højlund, Kurt; Wojtaszewski, Jørgen F P; Yang, Xiaoyong; Hulver, Matthew W; Helm, Richard F; Treebak, Jonas T; Gerrard, David E

2018-05-01

Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle. We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO) mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses. We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2). Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression

PubMed Central

Redmond, Catherine J.; Dooley, Katharine E.; Fu, Haiqing; Gillison, Maura L.; Akagi, Keiko; Symer, David E.; Aladjem, Mirit I.

2018-01-01

Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted de novo super-enhancer formation. Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression. PMID:29364907

Hierarchical Nafion enhanced carbon aerogels for sensing applications

NASA Astrophysics Data System (ADS)

Weng, Bo; Ding, Ailing; Liu, Yuqing; Diao, Jianglin; Razal, Joselito; Lau, King Tong; Shepherd, Roderick; Li, Changming; Chen, Jun

2016-02-01

This work describes the fabrication of hierarchical 3D Nafion enhanced carbon aerogels (NECAGs) for sensing applications via a fast freeze drying method. Graphene oxide, multiwalled carbon nanotubes and Nafion were mixed and extruded into liquid nitrogen followed by the removal of ice crystals by freeze drying. The addition of Nafion enhanced the mechanical strength of NECAGs and effective control of the cellular morphology and pore size was achieved. The resultant NECAGs demonstrated high strength, low density, and high specific surface area and can achieve a modulus of 20 kPa, an electrical conductivity of 140 S m-1, and a specific capacity of 136.8 F g-1 after reduction. Therefore, NECAG monoliths performed well as a gas sensor and as a biosensor with high sensitivity and selectivity. The remarkable sensitivity of 8.52 × 103 μA mM-1 cm-2 was obtained in dopamine (DA) detection, which is two orders of magnitude better than the literature reported values using graphene aerogel electrodes made from a porous Ni template. These outstanding properties make the NECAG a promising electrode candidate for a wide range of applications. Further in-depth investigations are being undertaken to probe the structure-property relationship of NECAG monoliths prepared under various conditions.This work describes the fabrication of hierarchical 3D Nafion enhanced carbon aerogels (NECAGs) for sensing applications via a fast freeze drying method. Graphene oxide, multiwalled carbon nanotubes and Nafion were mixed and extruded into liquid nitrogen followed by the removal of ice crystals by freeze drying. The addition of Nafion enhanced the mechanical strength of NECAGs and effective control of the cellular morphology and pore size was achieved. The resultant NECAGs demonstrated high strength, low density, and high specific surface area and can achieve a modulus of 20 kPa, an electrical conductivity of 140 S m-1, and a specific capacity of 136.8 F g-1 after reduction. Therefore, NECAG monoliths performed well as a gas sensor and as a biosensor with high sensitivity and selectivity. The remarkable sensitivity of 8.52 × 103 μA mM-1 cm-2 was obtained in dopamine (DA) detection, which is two orders of magnitude better than the literature reported values using graphene aerogel electrodes made from a porous Ni template. These outstanding properties make the NECAG a promising electrode candidate for a wide range of applications. Further in-depth investigations are being undertaken to probe the structure-property relationship of NECAG monoliths prepared under various conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08631k

Folate-receptor-targeted NIR-sensitive polydopamine nanoparticles for chemo-photothermal cancer therapy

NASA Astrophysics Data System (ADS)

Li, Hao; Jin, Zhen; Cho, Sunghoon; Jeon, Mi Jeong; Du Nguyen, Van; Park, Jong-Oh; Park, Sukho

2017-10-01

We propose the use of folate-receptor-targeted, near-infrared-sensitive polydopamine nanoparticles (NPs) for chemo-photothermal cancer therapy as an enhanced type of drug-delivery system which can be synthesized by in situ polymerization and conjugation with folic acid. The NPs consist of a Fe3O4/Au core, coated polydopamine, conjugated folic acid, and loaded anti-cancer drug (doxorubicin). The proposed multifunctional NPs show many advantages for therapeutic applications such as good biocompatibility and easy bioconjugation. The polydopamine coating of the NPs show a higher photothermal effect and thus more effective cancer killing compared to Fe3O4/Au nanoparticles at the same intensity as near-infrared laser irradiation. In addition, the conjugation of folic acid was shown to enhance cancer cellular uptake efficiency via the folate receptor and thus improve chemotherapeutic efficiency. Through in vitro cancer cell treatment testing, the proposed multifunctional NPs showed advanced photothermal and chemotherapeutic performance. Based on these enhanced anti-cancer properties, we expect that the proposed multifunctional NPs can be used as a drug-delivery system in cancer therapy.

CD8+ T cells produce a dialyzable antigen-specific activator of dendritic cells

PubMed Central

Myles, Ian A.; Zhao, Ming; Nardone, Glenn; Olano, Lisa R.; Reckhow, Jensen D.; Saleem, Danial; Break, Timothy J.; Lionakis, Michail S.; Myers, Timothy G.; Gardina, Paul J.; Kirkpatrick, Charles H.; Holland, Steven M.; Datta, Sandip K.

2017-01-01

Cellular lysates from PPD+ donors have been reported to transfer tuberculin reactivity to naïve recipients, but not diphtheria reactivity, and vice versa. A historically controversial topic, the terms "transfer factor" and "DLE" were used to characterize the reactivity-transferring properties of lysates. Intrigued by these reported phenomena, we found that the cellular extract derived from antigen-specific memory CD8+ T cells induces IL-6 from antigen-matched APCs. This ultimately elicits IL-17 from bystander memory CD8+ T cells. We have identified that dialyzable peptide sequences, S100a9, and the TCR β chain from CD8+ T cells contribute to the molecular nature of this activity. We further show that extracts from antigen-targeted T cells enhance immunity to Staphylococcus aureus and Candida albicans. These effects are sensitive to immunization protocols and extraction methodology in ways that may explain past discrepancies in the reproducibility of passive cellular immunity. PMID:27515950

[Relation between physical activity, weight balance and breast cancer].

PubMed

Maître, Carole

2013-05-01

Many epidemiologic studies, with a good methodology, support the evidence of the positive role of regular physical activity on primary and tertiary prevention of breast cancer on the risk of recurrence and mortality. This relation depends on the level of total energy expenditure by week, which helps balance weight on lifetime, an essential part of benefit. The beneficial effects of physical activity are linked to many interrelated additional mechanisms: in a short-term, contraction of skeletal muscles involves aerobic metabolism which utilizes glucose and amino acids like glutamine, improves insulin sensitivity and lowers plasma insulin; in a long-term, physical activity produces favorable changes in body composition, decreasing body fat and increasing lean mass. That is a key point to reduce the intake of energy substrates stimulating carcinogenesis, to improve insulin sensitivity, to change the ratio of leptin and adiponectin, to enhance cellular immunity and to block cellular pathways of cell proliferation and angiogenesis. Maintaining a healthy weight through regular physical activity well balanced with energy intake is it a goal for prevention of breast cancer. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Cannabidiol induced a contrasting pro-apoptotic effect between freshly isolated and precultured human monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Hsin-Ying; Chang, An-Chi; Wang, Chia-Chi

2010-08-01

It has been documented that cannabidiol (CBD) induced apoptosis in a variety of transformed cells, including lymphocytic and monocytic leukemias. In contrast, a differential sensitivity between normal lymphocytes and monocytes to CBD-mediated apoptosis has been reported. The present study investigated the pro-apoptotic effect of CBD on human peripheral monocytes that were either freshly isolated or precultured for 72 h. CBD markedly enhanced apoptosis of freshly isolated monocytes in a time- and concentration-dependent manner, whereas precultured monocytes were insensitive. By comparison, both cells were sensitive to doxorubicin-induced apoptosis. CBD significantly diminished the cellular thiols and glutathione in freshly isolated monocytes. Themore » apoptosis induced by CBD was abrogated in the presence of N-acetyl-{sub L}-cysteine, a precursor of glutathione. In addition, precultured monocytes contained a significantly greater level of glutathione and heme oxygenase-1 (HO-1) compared to the freshly isolated cells. The HO-1 competitive inhibitor zinc protoporphyrin partially but significantly restored the sensitivity of precultured monocytes to CBD-mediated apoptosis. Collectively, our results demonstrated a contrasting pro-apoptotic effect of CBD between precultured and freshly isolated monocytes, which was closely associated with the cellular level of glutathione and the antioxidative capability of the cells.« less

Lipid composition affects the rate of photosensitized dissipation of cross-membrane diffusion potential on liposomes

PubMed Central

Ytzhak, Shany; Wuskell, Joseph P.; Loew, Leslie M.; Ehrenberg, Benjamin

2010-01-01

Hydrophobic or amphiphilic tetrapyrrole sensitizers are taken up by cells and are usually located in cellular lipid membranes. Singlet oxygen is photogenerated by the sensitizer and it diffuses in the membrane and causes oxidative damage to membrane components. This damage can occur to membrane lipids and to membrane-localized proteins. Depolarization of the Nernst electric potential on cells’ membranes has been observed in cellular photosensitization, but it was not established whether lipid oxidation is a relevant factor leading to abolishing the resting potential of cells’ membranes and to their death. In this work we studied the effect of liposomes’ lipid composition on the kinetics of hematoporphyrin-photosensitized dissipation of K+-diffusion electric potential that was generated across the membranes. We employed an electrochromic voltage-sensitive spectroscopic probe that possesses a high fluorescence signal response to the potential. We found a correlation between the structure and unsaturation of lipids and the leakage of the membrane, following photosensitization. As the extent of non-conjugated unsaturation of the lipids is increased from 1 to 6 double bonds, the kinetics of depolarization become faster. We also found that the kinetics of depolarization is affected by the percentage of the unsaturated lipids in the liposome: as the fraction of the unsaturated lipids increases the leakage trough the membrane is enhanced. When liposomes are composed of a lipid mixture similar to that of natural membranes and photosensitization is being carried out under usual photodynamic therapy (PDT) conditions, photodamage to the lipids is not likely to cause enhanced permeability of ions through the membrane, which would have been a mechanism that leads to cell death. PMID:20536150

Enhancing Docetaxel Delivery to Multidrug-Resistant Cancer Cells with Albumin-Coated Nanocrystals.

PubMed

Gad, Sheryhan F; Park, Joonyoung; Park, Ji Eun; Fetih, Gihan N; Tous, Sozan S; Lee, Wooin; Yeo, Yoon

2018-01-29

Intravenous delivery of poorly water-soluble anticancer drugs such as docetaxel (DTX) is challenging due to the low bioavailability and the toxicity related to solubilizing excipients. Colloidal nanoparticles are used as alternative carriers, but low drug loading capacity and circulation instability limit their clinical translation. To address these challenges, DTX nanocrystals (NCs) were prepared using Pluronic F127 as an intermediate stabilizer and albumin as a functional surface modifier, which were previously found to be effective in producing small and stable NCs. We hypothesize that the albumin-coated DTX NCs (DTX-F-alb) will remain stable in serum-containing medium so as to effectively leverage the enhanced permeability and retention effect. In addition, the surface-bound albumin, in its native form, may contribute to cellular transport of NCs through interactions with albumin-binding proteins such as secreted protein acidic and rich in cysteine (SPARC). DTX-F-alb NCs showed sheet-like structure with an average length, width, and thickness of 284 ± 96, 173 ± 56, and 40 ± 8 nm and remained stable in 50% serum solution at a concentration greater than 10 μg/mL. Cytotoxicity and cellular uptake of DTX-F-alb and unformulated (free) DTX were compared on three cell lines with different levels of SPARC expression and DTX sensitivity. While the uptake of free DTX was highly dependent on DTX sensitivity, DTX-F-alb treatment resulted in relatively consistent cellular levels of DTX. Free DTX was more efficient in entering drug-sensitive B16F10 and SKOV-3 cells than DTX-F-alb, with consistent cytotoxic effects. In contrast, multidrug-resistant NCI/ADR-RES cells took up DTX-F-alb more than free DTX with time and responded better to the former. This difference was reduced by SPARC knockdown. The high SPARC expression level of NCI/ADR-RES cells, the known affinity of albumin for SPARC, and the opposing effect of SPARC knockdown support that DTX-F-alb have exploited the surface-bound albumin-SPARC interaction in entering NCI/ADR-RES cells. Albumin-coated NC system is a promising formulation for the delivery of hydrophobic anticancer drugs to multidrug-resistant tumors.

The German ISS-Experiment Cellular Responses to Radiation in Space (CERASP): The Effects of Single and Combined Space Flight Conditions on Mammalian Cells

NASA Astrophysics Data System (ADS)

Baumstark-Khan, C.; Hellweg, C. E.; Arenz, A.

The combined action of ionizing radiation and microgravity will continue to influence future space missions with special risks for astronauts on the Moon surface or for long duration missions to Mars Previous space flight experiments have reported additive neither sensitization nor protection as well as synergistic increased radiation effect under microgravity interactions of radiation and microgravity in different cell systems Although a direct effect of microgravity on enzymatic mechanisms can be excluded on thermo dynamical reasons modifications of cellular repair can not be excluded as such processes are under the control of cellular signal transduction systems which are controlled by environmental parameters presumably also by gravity DNA repair studies in space on bacteria yeast cells and human fibroblasts which were irradiated before flight gave contradictory results from inhibition of repair by microgravity to enhancement whereas others did not detect any influence of microgravity on repair At the Radiation Biology Department of the German Aerospace Center DLR recombinant bacterial and mammalian cell systems were developed as reporters for cellular signal transduction modulation by genotoxic environmental conditions The space experiment CERASP Cellular Responses to Radiation in Space to be performed at the International Space Station ISS will make use of such reporter cell lines thereby supplying basic information on the cellular response to radiation applied in microgravity One of the biological endpoints will be survival

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

PubMed

Kim, Sun Yee; Park, Jeen-Woo

2003-03-01

Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

Glycyrrhetinic acid-decorated and reduction-sensitive micelles to enhance the bioavailability and anti-hepatocellular carcinoma efficacy of tanshinone IIA.

PubMed

Chen, Fengqian; Zhang, Jinming; He, Yao; Fang, Xiefan; Wang, Yitao; Chen, Meiwan

2016-01-01

It remains a challenge to increase drug tumor-specific accumulation as well as to achieve intracellular-controlled drug release for hepatocellular carcinoma (HCC) chemotherapy. Herein, we developed a dual-functional biodegradable micellar system constituted by glycyrrhetinic acid coupling poly(ethylene glycol)-disulfide linkage-poly(lactic-co-glycolic acid) (GA-PEG-SS-PLGA) to achieve both hepatoma-targeting and redox-responsive intracellular drug release. Tanshinone IIA (TAN IIA), an effective anti-HCC drug, was encapsulated. Notably, it exhibited rapid aggregation and faster drug release in 10 mM dithiothreitol compared with the redox-insensitive control. Furthermore, GA-decorated micelles revealed HCC-specific cellular uptake in human liver cancer HepG2 cells with an energy-dependent manner, in which micropinocytosis and caveolae-mediated endocytosis were demonstrated as the major cellular pathways. The enhanced cytotoxicity and pro-apoptotic effects against HepG2 cells in vitro were observed, mediated by up-regulation of the intracellular ROS level, the increased cell cycle arrest at S phase, enhanced necrocytosis and up-regulation of caspase 3/7, P38 protein expression. In addition, TAN IIA-loaded micelles had a significantly prolonged circulation time, improved bioavailability, and resulted in an increased accumulation of TAN IIA in the liver. With the synergistic effects of HCC-targeting and controlled drug release, TAN IIA-loaded GA-PEG-SS-PLGA micelles significantly inhibited tumor growth and increased survival time in a mouse HCC-xenograft model. Collectively, the GA-PEG-SS-PLGA micelles with HCC-targeting and redox-sensitive characters would provide a novel strategy to deliver TAN IIA effectively for HCC therapy.

Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates.

PubMed

Cordina, Nicole M; Sayyadi, Nima; Parker, Lindsay M; Everest-Dass, Arun; Brown, Louise J; Packer, Nicolle H

2018-03-14

Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.

A Quantitative Study of Oxygen as a Metabolic Regulator

NASA Technical Reports Server (NTRS)

Radhakrishnan, Krishnan; LaManna, Joseph C.; Cabera, Marco E.

2000-01-01

An acute reduction in oxygen delivery to a tissue is associated with metabolic changes aimed at maintaining ATP homeostasis. However, given the complexity of the human bio-energetic system, it is difficult to determine quantitatively how cellular metabolic processes interact to maintain ATP homeostasis during stress (e.g., hypoxia, ischemia, and exercise). In particular, we are interested in determining mechanisms relating cellular oxygen concentration to observed metabolic responses at the cellular, tissue, organ, and whole body levels and in quantifying how changes in tissue oxygen availability affect the pathways of ATP synthesis and the metabolites that control these pathways. In this study; we extend a previously developed mathematical model of human bioenergetics, to provide a physicochemical framework that permits quantitative understanding of oxygen as a metabolic regulator. Specifically, the enhancement - sensitivity analysis - permits studying the effects of variations in tissue oxygenation and parameters controlling cellular respiration on glycolysis, lactate production, and pyruvate oxidation. The analysis can distinguish between parameters that must be determined accurately and those that require less precision, based on their effects on model predictions. This capability may prove to be important in optimizing experimental design, thus reducing use of animals.

pH and redox-responsive mixed micelles for enhanced intracellular drug release.

PubMed

Cai, Mengtan; Zhu, Kun; Qiu, Yongbin; Liu, Xinrong; Chen, Yuanwei; Luo, Xianglin

2014-04-01

In order to prepare pH and redox sensitive micelles, amphiphilic copolymers of poly (epsilon-caprolactone)-b-poly(2-(diethylamino) ethyl methacrylate) (PCL-PDEA) and disulfide-linked poly(ethyl glycol)-poly(epsilon-caprolactone) (mPEG-SS-PCL) were synthesized. The double-sensitive micelles were prepared simply by solvent-evaporating method with the mixed two copolymers. The pH sensitivity of the mixed micelles was confirmed by the change of micelle diameter/diameter distribution measured by dynamic lighting scattering (DLS) and the redox sensitivity of the mixed micelles was testified by the change of micellar morphous observed by scanning electron microscope (SEM). In vitro drug release showed that drug-loaded mixed micelles (mass ratio 5:5) could achieve above 90% of drug release under low pH and reducing condition within 10h. Moreover, the drug-loaded mixed micelles (mass ratio 5:5) showed the largest cellular toxicity compared with other drug-loaded micelles, while blank mixed micelles exhibited no toxicity. These results meant that the mixed micelles composed by the two amphiphilic copolymers can enhance intracellular drug release. It is concluded that the newly developed mixed micelles can serve as a potential drug delivery system for anticancer drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

Cellular imaging by targeted assembly of hot-spot SERS and photoacoustic nanoprobes using split-fluorescent protein scaffolds.

PubMed

Köker, Tuğba; Tang, Nathalie; Tian, Chao; Zhang, Wei; Wang, Xueding; Martel, Richard; Pinaud, Fabien

2018-02-09

The in cellulo assembly of plasmonic nanomaterials into photo-responsive probes is of great interest for many bioimaging and nanophotonic applications but remains challenging with traditional nucleic acid scaffolds-based bottom-up methods. Here, we address this quandary using split-fluorescent protein (FP) fragments as molecular glue and switchable Raman reporters to assemble gold or silver plasmonic nanoparticles (NPs) into photonic clusters directly in live cells. When targeted to diffusing surface biomarkers in cancer cells, the NPs self-assemble into surface-enhanced Raman-scattering (SERS) nanoclusters having hot spots homogenously seeded by the reconstruction of full-length FPs. Within plasmonic hot spots, autocatalytic activation of the FP chromophore and near-field amplification of its Raman fingerprints enable selective and sensitive SERS imaging of targeted cells. This FP-driven assembly of metal colloids also yields enhanced photoacoustic signals, allowing the hybrid FP/NP nanoclusters to serve as contrast agents for multimodal SERS and photoacoustic microscopy with single-cell sensitivity.

Enhanced depigmenting effects of N-glycosylation inhibitors delivered by pH-sensitive liposomes into HM3KO melanoma cells.

PubMed

Park, Ju Young; Choi, Hyunjung; Hwang, Jae Sung; Kim, Junoh; Chang, Ih-Seop

2008-01-01

Delivery activity of pH-sensitive 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE):cholesteryl hemisuccinate (CHEMS) liposomes was assessed as an in vitro intracellular carrier system to increase the bioavailability of depigmentation actives. N-glycosylation inhibitors have a glycosylation-inhibiting effect, which is useful for the skin depigmentation that operates by interfering with the maturation of tyrosinase. However, an N-glycosylation inhibitor does not easily pass through skin or even cellular membranes due to its water-soluble property. Therefore, it should be transported to target cells by an efficient delivery carrier to reduce the glycosylated tyrosinase. Glycosylation-inhibiting and depigmentation effects of N-butyldeoxynojirimycine (NB-DNJ) and 1-deoxynojirimycine (DNJ)-loaded liposomes were evaluated using Western blotting and measurement of synthesized melanin. Interestingly, it was found that the pH-sensitive liposomes increased the glycosylation-inhibiting and thus, pigment-lightening effects of N-glycosylation inhibitors in vitro. In addition, cargo materials loaded in pH-sensitive liposomes were found to be much more efficiently delivered into the cytoplasm, as observed in fluorescent-activated cell sorting (FACS) and confocal laser-scanning microscopic (CLSM) analysis. These results indicate that pH-sensitive DOPE:CHEMS liposomes have a strong potential as a carrier system to promote delivery efficiency and to enhance the biological effects of water-soluble actives for applications in cosmetics, personal care products, and pharmaceutics.

Reversal of in vitro cellular MRP1 and MRP2 mediated vincristine resistance by the flavonoid myricetin.

PubMed

van Zanden, Jelmer J; de Mul, Anika; Wortelboer, Heleen M; Usta, Mustafa; van Bladeren, Peter J; Rietjens, Ivonne M C M; Cnubben, Nicole H P

2005-06-01

In the present study, the effects of myricetin on either MRP1 or MRP2 mediated vincristine resistance in transfected MDCKII cells were examined. The results obtained show that myricetin can inhibit both MRP1 and MRP2 mediated vincristine efflux in a concentration dependent manner. The IC50 values for cellular vincristine transport inhibition by myricetin were 30.5+/-1.7 microM for MRP1 and 24.6+/-1.3 microM for MRP2 containing MDCKII cells. Cell proliferation analysis showed that the MDCKII control cells are very sensitive towards vincristine toxicity with an IC50 value of 1.1+/-0.1 microM. The MDCKII MRP1 and MRP2 cells are less sensitive towards vincristine toxicity with IC50 values of 33.1+/-1.9 and 22.2+/-1.4 microM, respectively. In both the MRP1 and MRP2 cells, exposure to 25 microM myricetin enhances the sensitivity of the cells towards vincristine toxicity to IC50 values of 7.6+/-0.5 and 5.8+/-0.5 microM, respectively. The increase of sensitivity represents a reversal of the resistance towards vincristine as a result of MRP1 and MRP2 inhibition. Thus, the present study demonstrates the ability of the flavonoid myricetin to modulate MRP1 and MRP2 mediated resistance to the anticancer drug vincristine in transfected cells, indicating that flavonoids might be a valuable adjunct to chemotherapy to block MRP mediated resistance.

Specific telomere dysfunction induced by GRN163L increases radiation sensitivity in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gomez-Millan, Jaime; Goldblatt, Erin M.; Gryaznov, Sergei M.

Purpose: Telomerase is expressed in 80-90% of tumor cells, but is absent in most somatic cells. The absence of telomerase activity results in progressive telomere shortening, leading to cellular senescence or death through deoxyribonucleic acid (DNA) damage signals. In addition, a role for telomerase in DNA damage repair has also been suggested. A specific telomerase inhibitor, GRN163L that is complementary to the template region of the telomerase ribonucleic acid component (hTR). We hypothesized that exposure to GRN163L, either through immediate inhibition of telomerase activity or through eventual telomere shortening and dysfunction, may enhance radiation sensitivity. Our goal was to testmore » whether the treatment with GRN163L enhances sensitivity to irradiation (IR) in MDA-MB-231 breast cancer cells. Methods and Materials: The MDA-MB-231 breast cancer cells were treated with or without GRN163L for 2-42 days. Inhibition of telomerase activity and shortening of telomeres were confirmed. Cells were then irradiated and clonogenic assays were performed to show cell survival differences. In vivo studies using MDA-MB-231 xenografts were performed to corroborate the in vitro results. Results: We show that cells with shortened telomeres due to GRN163L enhance the effect on IR reducing survival by an additional 30% (p < 0.01). These results are confirmed in vivo, with a significant decrease in tumor growth in mice exposed to GRN163L. Conclusions: We found that GRN163L is a promising adjuvant treatment in combination with radiation therapy that may improve the therapeutic index by enhancing the radiation sensitivity. These studies prompt further investigation as to whether this combination can be applied to other cancers and the clinic.« less

A full computation-relevant topological dynamics classification of elementary cellular automata.

PubMed

Schüle, Martin; Stoop, Ruedi

2012-12-01

Cellular automata are both computational and dynamical systems. We give a complete classification of the dynamic behaviour of elementary cellular automata (ECA) in terms of fundamental dynamic system notions such as sensitivity and chaoticity. The "complex" ECA emerge to be sensitive, but not chaotic and not eventually weakly periodic. Based on this classification, we conjecture that elementary cellular automata capable of carrying out complex computations, such as needed for Turing-universality, are at the "edge of chaos."

Reversal to cisplatin sensitivity in recurrent human ovarian cancer cells by NCX-4016, a nitro derivative of aspirin

PubMed Central

Bratasz, Anna; Weir, Nathan M.; Parinandi, Narasimham L.; Zweier, Jay L.; Sridhar, Rajagopalan; Ignarro, Louis J.; Kuppusamy, Periannan

2006-01-01

Ovarian cancer is a gynecological malignancy that is commonly treated by cytoreductive surgery followed by cisplatin treatment. However, the cisplatin treatment, although successful initially, is not effective in the treatment of the recurrent disease that invariably surfaces within a few months of the initial treatment. The refractory behavior is attributed to the increased levels of cellular thiols apparently caused by the cisplatin treatment. This observation prompted us to choose a cytotoxic drug whose activity is potentiated by cellular thiols with enhanced specificity toward the thiol-rich cisplatin-resistant cells. We used NCX-4016 [2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester], a derivative of aspirin containing a nitro group that releases nitric oxide in a sustained fashion for several hours in cells and in vivo, and we studied its cytotoxic efficacy against human ovarian cancer cells (HOCCs). Cisplatin-sensitive and cisplatin-resistant (CR) HOCCs were treated with 100 μM NCX-4016 for 6 h, and/or 0.5 μg/ml cisplatin for 1 h and assayed for clonogenecity. NCX-4016 significantly reduced the surviving fractions of cisplatin-sensitive (63 ± 6%) and CR (70 ± 10%) HOCCs. NCX-4016 also caused a 50% reduction in the levels of cellular glutathione in CR HOCCs. Treatment of cells with NCX-4016 followed by cisplatin showed a significantly greater extent of toxicity when compared with treatment of cells with NCX-4016 or cisplatin alone. In conclusion, this study showed that NCX-4016 is a potential inhibitor of the proliferation of CR HOCCs and thus might specifically kill cisplatin-refractory cancer cells in patients with recurrent ovarian cancer. PMID:16497833

The potential role of postsynaptic phospholipase C activity in synaptic facilitation and behavioral sensitization in Aplysia.

PubMed

Fulton, Daniel; Condro, Michael C; Pearce, Kaycey; Glanzman, David L

2008-07-01

Previous findings indicate that synaptic facilitation, a cellular mechanism underlying sensitization of the siphon withdrawal response (SWR) in Aplysia, depends on a cascade of postsynaptic events, including activation of inositol triphosphate (IP3) receptors and release of Ca2+ from postsynaptic intracellular stores. These findings suggest that phospholipase C (PLC), the enzyme that catalyzes IP3 formation, may play an important role in postsynaptic signaling during facilitation and learning in Aplysia. Using the PLC inhibitor U73122, we found that PLC activity is required for synaptic facilitation following a 10-min treatment with 5-HT, as measured at 20 min after 5-HT washout. Prior work has indicated that facilitation at this time is supported primarily by postsynaptic processes. To determine whether postsynaptic PLC activity is involved in 5-HT-mediated facilitatory actions, we examined the effect of U73122 on enhancement of the response of motor neurons isolated in cell culture to glutamate, the sensory neuron transmitter. A 10-min application of 5-HT induced persistent (>40 min) enhancement of glutamate-evoked potentials (Glu-EPs) recorded from isolated motor neurons, and this enhancement was blocked by U73122. Finally, we showed that injecting U73122 into intact animals before behavioral training impaired intermediate-term sensitization, indicating that PLC activity contributes to this form of nonassociative learning.

Variable Delay Multi-Pulse Train for Fast Chemical Exchange Saturation Transfer and Relayed-Nuclear Overhauser Enhancement MRI

PubMed Central

Xu, Jiadi; Yadav, Nirbhay N.; Bar-Shir, Amnon; Jones, Craig K.; Chan, Kannie W. Y.; Zhang, Jiangyang; Walczak, P.; McMahon, Michael T.; van Zijl, Peter C. M.

2013-01-01

Purpose Chemical exchange saturation transfer (CEST) imaging is a new MRI technology allowing the detection of low concentration endogenous cellular proteins and metabolites indirectly through their exchangeable protons. A new technique, variable delay multi-pulse CEST (VDMP-CEST), is proposed to eliminate the need for recording full Z-spectra and performing asymmetry analysis to obtain CEST contrast. Methods The VDMP-CEST scheme involves acquiring images with two (or more) delays between radiofrequency saturation pulses in pulsed CEST, producing a series of CEST images sensitive to the speed of saturation transfer. Subtracting two images or fitting a time series produces CEST and relayed-nuclear Overhauser enhancement CEST maps without effects of direct water saturation and, when using low radiofrequency power, minimal magnetization transfer contrast interference. Results When applied to several model systems (bovine serum albumin, crosslinked bovine serum albumin, l-glutamic acid) and in vivo on healthy rat brain, VDMP-CEST showed sensitivity to slow to intermediate range magnetization transfer processes (rate < 100–150 Hz), such as amide proton transfer and relayed nuclear Overhauser enhancement-CEST. Images for these contrasts could be acquired in short scan times by using a single radiofrequency frequency. Conclusions VDMP-CEST provides an approach to detect CEST effect by sensitizing saturation experiments to slower exchange processes without interference of direct water saturation and without need to acquire Z-spectra and perform asymmetry analysis. PMID:23813483

Bakery flour dust exposure causes non-allergic inflammation and enhances allergic airway inflammation in mice

PubMed Central

Marraccini, Paolo; Brass, David M.; Hollingsworth, John W.; Maruoka, Shuichiro; Garantziotis, Stavros; Schwartz, David A.

2014-01-01

Background Baker’s asthma is one of the most commonly reported occupational lung diseases in countries where fresh bread is baked daily in large quantities, and is characterized by rhinitis, bronchial hyperresponsiveness, and reversible airflow obstruction. Epidemiological studies have identified pre-existing atopy as an important risk factor for developing baker’s asthma, yet the etiology and pathogenesis of baker’s asthma remain poorly understood. Objective We sought to develop a mouse model of baker’s asthma that could be used to characterize the development and progression of baker’s asthma. Methods We were unable to sensitize mice to bakery flour dust or flour dust extract. We assessed total inflammatory cells, cellular differential, total serum IgE and the pro-inflammatory cytokine response to oropharyngeally instilled bakery flour dust or flour dust extract by itself or in the context of OVA sensitization and challenge. Results Both bakery flour dust and flour dust extract consistently elicited a neutrophilic inflammation in a tlr4-independent manner; suggesting that endotoxin is not playing a role in the inflammatory response to flour dust. Moreover, bakery flour dust and dust extract significantly enhance the inflammatory response in OVA sensitized and challenged mice. Conclusions Bakery flour dust and flour dust extract are strongly pro-inflammatory and can cause non-allergic airway inflammation and can enhance allergen-mediated airway inflammation. PMID:18564331

Epigenetic modifications and glucocorticoid sensitivity in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

PubMed

de Vega, Wilfred C; Herrera, Santiago; Vernon, Suzanne D; McGowan, Patrick O

2017-02-23

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating idiopathic disease characterized by unexplained fatigue that fails to resolve with sufficient rest. Diagnosis is based on a list of symptoms and exclusion of other fatigue-related health conditions. Despite a heterogeneous patient population, immune and hypothalamic-pituitary-adrenal (HPA) axis function differences, such as enhanced negative feedback to glucocorticoids, are recurring findings in ME/CFS studies. Epigenetic modifications, such as CpG methylation, are known to regulate long-term phenotypic differences and previous work by our group found DNA methylome differences in ME/CFS, however the relationship between DNA methylome modifications, clinical and functional characteristics associated with ME/CFS has not been examined. We examined the DNA methylome in peripheral blood mononuclear cells (PBMCs) of a larger cohort of female ME/CFS patients using the Illumina HumanMethylation450 BeadChip Array. In parallel to the DNA methylome analysis, we investigated in vitro glucocorticoid sensitivity differences by stimulating PBMCs with phytohaemagglutinin and suppressed growth with dexamethasone. We explored DNA methylation differences using bisulfite pyrosequencing and statistical permutation. Linear regression was implemented to discover epigenomic regions associated with self-reported quality of life and network analysis of gene ontology terms to biologically contextualize results. We detected 12,608 differentially methylated sites between ME/CFS patients and healthy controls predominantly localized to cellular metabolism genes, some of which were also related to self-reported quality of life health scores. Among ME/CFS patients, glucocorticoid sensitivity was associated with differential methylation at 13 loci. Our results indicate DNA methylation modifications in cellular metabolism in ME/CFS despite a heterogeneous patient population, implicating these processes in immune and HPA axis dysfunction in ME/CFS. Modifications to epigenetic loci associated with differences in glucocorticoid sensitivity may be important as biomarkers for future clinical testing. Overall, these findings align with recent ME/CFS work that point towards impairment in cellular energy production in this patient population.

Restoration of the cellular secretory milieu overrides androgen dependence of in vivo generated castration resistant prostate cancer cells overexpressing the androgen receptor.

PubMed

Patki, Mugdha; Huang, Yanfang; Ratnam, Manohar

2016-07-22

It is believed that growth of castration resistant prostate cancer (CRPC) cells is enabled by sensitization to minimal residual post-castrate androgen due to overexpression of the androgen receptor (AR). Evidence is derived from androgen-induced colony formation in the absence of cell-secreted factors or from studies involving forced AR overexpression in hormone-dependent cells. On the other hand, standard cell line models established from CRPC patient tumors (e.g., LNCaP and VCaP) are hormone-dependent and require selection pressure in castrated mice to re-emerge as CRPC cells and the resulting tumors then tend to be insensitive to the androgen antagonist enzalutamide. Therefore, we examined established CRPC model cells produced by castration of mice bearing hormone-dependent cell line xenografts including CRPC cells overexpressing full-length AR (C4-2) or co-expressing wtAR and splice-variant AR-V7 that is incapable of ligand binding (22Rv1). In standard colony formation assays, C4-2 cells were shown to be androgen-dependent and sensitive to enzalutamide whereas 22Rv1 cells were incapable of colony formation under identical conditions. However, both C4-2 and 22Rv1 cells formed colonies in conditioned media derived from the same cells or from HEK293 fibroblasts that were proven to lack androgenic activity. This effect was (i) not enhanced by androgen, (ii) insensitive to enzalutamide, (iii) dependent on AR (in C4-2) and on AR-V7 and wtAR (in 22Rv1) and (iv) sensitive to inhibitors of several signaling pathways, similar to androgen-stimulation. Therefore, during progression to CRPC in vivo, coordinate cellular changes accompanying overexpression of AR may enable cooperation between hormone-independent activity of AR and actions of cellular secretory factors to completely override androgen-dependence and sensitivity to drugs targeting hormonal factors. Copyright © 2016 Elsevier Inc. All rights reserved.

A Pathway for the Control of Anoikis Sensitivity by E-Cadherin and Epithelial-to-Mesenchymal Transition▿‡

PubMed Central

Kumar, Sanjeev; Park, Sun Hee; Cieply, Benjamin; Schupp, Jane; Killiam, Elizabeth; Zhang, Fan; Rimm, David L.; Frisch, Steven M.

2011-01-01

Detachment of epithelial cells from matrix or attachment to an inappropriate matrix engages an apoptotic response known as anoikis, which prevents metastasis. Cellular sensitivity to anoikis is compromised during the oncogenic epithelial-to-mesenchymal transition (EMT), through unknown mechanisms. We report here a pathway through which EMT confers anoikis resistance. NRAGE (neurotrophin receptor-interacting melanoma antigen) interacted with a component of the E-cadherin complex, ankyrin-G, maintaining NRAGE in the cytoplasm. Oncogenic EMT downregulated ankyrin-G, enhancing the nuclear localization of NRAGE. The oncogenic transcriptional repressor protein TBX2 interacted with NRAGE, repressing the tumor suppressor gene p14ARF. P14ARF sensitized cells to anoikis; conversely, the TBX2/NRAGE complex protected cells against anoikis by downregulating this gene. This represents a novel pathway for the regulation of anoikis by EMT and E-cadherin. PMID:21746881

Frequency sensitive mechanism in low-intensity ultrasound enhanced bioeffects

PubMed Central

Chama, Abdoulkadri; Subramanian, Anuradha; Viljoen, Hendrik J.

2017-01-01

This study presents two novel theoretical models to elucidate frequency sensitive nuclear mechanisms in low-intensity ultrasound enhanced bioeffects. In contrast to the typical 1.5 MHz pulsed ultrasound regime, our group previously experimentally confirmed that ultrasound stimulation of anchored chondrocytes at resonant frequency maximized gene expression of load inducible genes which are regulatory markers for cellular response to external stimuli. However, ERK phosphorylation displayed no frequency dependency, suggesting that the biochemical mechanisms involved in enhanced gene expression is downstream of ERK phosphorylation. To elucidate such underlying mechanisms, this study presents a theoretical model of an anchored cell, representing an in vitro chondrocyte, in an ultrasound field. The model results showed that the mechanical energy storage is maximized at the chondrocyte’s resonant frequency and the energy density in the nucleus is almost twice as high as in the cytoplasm. Next, a mechanochemical model was developed to link the mechanical stimulation of ultrasound and the increased mechanical energy density in the nucleus to the downstream targets of the ERK pathway. This study showed for the first time that ultrasound stimulation induces frequency dependent gene expression as a result of altered rates of transcription factors binding to chromatin. PMID:28763448

Delivering anti-cancer drugs with endosomal pH-sensitive anti-cancer liposomes.

PubMed

Moku, Gopikrishna; Gulla, Suresh Kumar; Nimmu, Narendra Varma; Khalid, Sara; Chaudhuri, Arabinda

2016-04-01

Numerous prior studies have been reported on the use of pH-sensitive drug carriers such as micelles, liposomes, peptides, polymers, nanoparticles, etc. that are sensitive to the acidic (pH = ∼6.5) microenvironments of tumor tissues. Such systems have been primarily used in the past as effective drug/gene/microRNA carriers for releasing their anti-cancer payloads selectively to tumor cells/tissues. Herein, we report on the development of new liposomal drug carriers prepared from glutamic acid backbone-based cationic amphiphiles containing both endosomal pH-sensitive histidine as well as cellular uptake & solubility enhancing guanidine moieties in their polar head-group regions. The most efficient one among the four presently described endosomal pH-sensitive liposomal drug carriers not only effectively delivers potent anti-cancer drugs (curcumin & paclitaxel) to mouse tumor, but also significantly contributes to inhibiting mouse tumor growth. The findings in the in vitro mechanistic studies are consistent with apoptosis of tumor cells being mediated through increased cell cycle arrest in the G2/M phase. Findings in the FRET assay and in vitro drug release studies conducted with the liposomes of the most efficient pH-sensitive lipid demonstrated its pH dependent fusogenic and controlled curcumin release properties. Importantly, the presently described liposomal formulation of curcumin & paclitaxel enhanced overall survivability of tumor bearing mice. To the best of our knowledge, the presently described system (curcumin, paclitaxel and liposomal carrier itself) is the first of its kind pH-sensitive liposomal formulation of potent chemotherapeutics in which the liposomal drug itself exhibits significant mouse tumor growth inhibition properties.

Radiosensitization of head/neck sqaumous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rhee, Juong G.; Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD; Li, Daqing

2007-01-01

Purpose: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. Experimental Procedures: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into anmore » adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. Results: Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. Conclusions: These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers.« less

Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies.

PubMed

Friedman, Jay; Morisada, Megan; Sun, Lillian; Moore, Ellen C; Padget, Michelle; Hodge, James W; Schlom, Jeffrey; Gameiro, Sofia R; Allen, Clint T

2018-06-21

Natural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined. Tumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed. Cultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab. Tumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.

Targeting Breast Cancer Recurrence via Hedgehog-Mediated Sensitization of Breast Cancer Stem Cells

DTIC Science & Technology

2011-07-01

identification of Notch3 as a transcriptional target of ΔNp63α and a mediator of cellular quiescence in mammary stem cells. 2. Presentation of a poster...enhanced expression of Notch3 in HC11s and breast cancer cell lines, and ectopic expression of the Notch3 intracellular domain (N3ICD) was sufficient to...signaling or shRNA-mediated suppression of Notch3 were sufficient to bypass quiescence induced by ΔNp63α and other quiescence-inducing stimuli. these

WE-FG-BRA-11: Theranostic Platinum Nanoparticle for Radiation Sensitization in Breast Cancer Radiotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yue, Y; Wagner, S; Medina-Kauwe, L

Purpose: We have developed a novel receptor-targeted theranostic platinum nanoparticle (HER-PtNP) for enhanced radiation sensitization in HER2-positive breast cancer radiation treatment. This study aims to evaluate receptor-targeting specificity, and radiation sensitization of the nanoparticle. Methods: The platinum nanoparticle (PtNP) was synthesized with the diameter of 2nm, and capped with cysteine. The nanoparticle was tagged with a fluorescent dye (cy5) for the fluoresence detection, and conjuated with HER2/3 targeted protein (HerPBK10) for HER2-targeting specificity. We evaluated the theranostic features using in vitro breast cancer cell models: HER2-positive BT-474, and HER2-negative MDA-MB-231. The HER2-targeting specificity was evaluated using immunofluorescence and confocal microscopy.more » For each cell line, three sets of samples, including non-stained control, fluorescence stained PtNP-cy5 treated, and HER-PtNP treated, were imaged by confocal microscopy. Two breast cancer cell lineages were incubated with PtNP and HER-PtNP at 10 µg/mL, and then irradiated with X-rays for 2 Gy dose at 50 kVp. A colonogenic assay was used to determine cellular survival fractions by immediately reseeding 300 cells after irradiation in growth media and allowing colonies to grow for 2 weeks. Results: The results of confocal images show that no apparent nanoparticle cellular uptake was observed in the HER2-(MDA-MB-231) cells with 1% for PtNP-cy5 and 0.5% for HER-PtNP. Similarly no apparent PtNP-cy5 uptake (<1%) for BT474 cells was observed. However, there was significant HER-PtNP uptake (73%) for the HER2+(BT474) cells. The clonogenic assay showed that BT474 cells treated with HER-PtNP had significantly lower survival compared to those treated with PtNP (32% vs 81%, p=0.01). However, no significant radiosensitivity enhancement was observed for MDA-MB-231 cell treated with PtNP and HER-PtNP (89% vs 92%, p=0.78). Conclusion: Our studies suggest that the HER2-targeted platinum nanoparticle has excellent receptor targeting specificity and enhanced radiation sensitization compared to nanoparticle alone, suggesting potential for clinical applications in breast cancer radiotherapy.« less

Targeting DNA repair with PNKP inhibition sensitizes radioresistant prostate cancer cells to high LET radiation

PubMed Central

Srivastava, Pallavi; Sarma, Asitikantha

2018-01-01

High linear energy transfer (LET) radiation or heavy ion such as carbon ion radiation is used as a method for advanced radiotherapy in the treatment of cancer. It has many advantages over the conventional photon based radiotherapy using Co-60 gamma or high energy X-rays from a Linear Accelerator. However, charged particle therapy is very costly. One way to reduce the cost as well as irradiation effects on normal cells is to reduce the dose of radiation by enhancing the radiation sensitivity through the use of a radiomodulator. PNKP (polynucleotide kinase/phosphatase) is an enzyme which plays important role in the non-homologous end joining (NHEJ) DNA repair pathway. It is expected that inhibition of PNKP activity may enhance the efficacy of the charged particle irradiation in the radioresistant prostate cancer cell line PC-3. To test this hypothesis, we investigated cellular radiosensitivity by clonogenic cell survival assay in PC-3 cells.12Carbon ion beam of62 MeVenergy (equivalent 5.16 MeV/nucleon) and with an entrance LET of 287 kev/μm was used for the present study. Apoptotic parameters such as nuclear fragmentation and caspase-3 activity were measured by DAPI staining, nuclear ladder assay and colorimetric caspase-3method. Cell cycle arrest was determined by FACS analysis. Cell death was enhanced when carbon ion irradiation is combined with PNKPi (PNKP inhibitor) to treat cells as compared to that seen for PNKPi untreated cells. A low concentration (10μM) of PNKPi effectively radiosensitized the PC-3 cells in terms of reduction of dose in achieving the same survival fraction. PC-3 cells underwent significant apoptosis and cell cycle arrest too was enhanced at G2/M phase when carbon ion irradiation was combined with PNKPi treatment. Our findings suggest that combined treatment of carbon ion irradiation and PNKP inhibition could enhance cellular radiosensitivity in a radioresistant prostate cancer cell line PC-3. The synergistic effect of PNKPi and carbon ion irradiation could be used as a promising method for carbon-ion therapy in radioresistant cells. PMID:29320576

Systematic analysis of Ca2+ homeostasis in Saccharomyces cerevisiae based on chemical-genetic interaction profiles

PubMed Central

Ghanegolmohammadi, Farzan; Yoshida, Mitsunori; Ohnuki, Shinsuke; Sukegawa, Yuko; Okada, Hiroki; Obara, Keisuke; Kihara, Akio; Suzuki, Kuninori; Kojima, Tetsuya; Yachie, Nozomu; Hirata, Dai; Ohya, Yoshikazu

2017-01-01

We investigated the global landscape of Ca2+ homeostasis in budding yeast based on high-dimensional chemical-genetic interaction profiles. The morphological responses of 62 Ca2+-sensitive (cls) mutants were quantitatively analyzed with the image processing program CalMorph after exposure to a high concentration of Ca2+. After a generalized linear model was applied, an analysis of covariance model was used to detect significant Ca2+–cls interactions. We found that high-dimensional, morphological Ca2+–cls interactions were mixed with positive (86%) and negative (14%) chemical-genetic interactions, whereas one-dimensional fitness Ca2+–cls interactions were all negative in principle. Clustering analysis with the interaction profiles revealed nine distinct gene groups, six of which were functionally associated. In addition, characterization of Ca2+–cls interactions revealed that morphology-based negative interactions are unique signatures of sensitized cellular processes and pathways. Principal component analysis was used to discriminate between suppression and enhancement of the Ca2+-sensitive phenotypes triggered by inactivation of calcineurin, a Ca2+-dependent phosphatase. Finally, similarity of the interaction profiles was used to reveal a connected network among the Ca2+ homeostasis units acting in different cellular compartments. Our analyses of high-dimensional chemical-genetic interaction profiles provide novel insights into the intracellular network of yeast Ca2+ homeostasis. PMID:28566553

IGF-I enhances cellular senescence via the reactive oxygen species-p53 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Handayaningsih, Anastasia-Evi; Takahashi, Michiko; Fukuoka, Hidenori

2012-08-24

Highlights: Black-Right-Pointing-Pointer Cellular senescence plays an important role in tumorigenesis and aging process. Black-Right-Pointing-Pointer We demonstrated IGF-I enhanced cellular senescence in primary confluent cells. Black-Right-Pointing-Pointer IGF-I enhanced cellular senescence in the ROS and p53-dependent manner. Black-Right-Pointing-Pointer These results may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging. -- Abstract: Cellular senescence is characterized by growth arrest, enlarged and flattened cell morphology, the expression of senescence-associated {beta}-galactosidase (SA-{beta}-gal), and by activation of tumor suppressor networks. Insulin-like growth factor-I (IGF-I) plays a critical role in cellular growth, proliferation, tumorigenesis, and regulation of aging. In the presentmore » study, we show that IGF-I enhances cellular senescence in mouse, rat, and human primary cells in the confluent state. IGF-I induced expression of a DNA damage marker, {gamma}H2AX, the increased levels of p53 and p21 proteins, and activated SA-{beta}-gal. In the confluent state, an altered downstream signaling of IGF-I receptor was observed. Treatment with a reactive oxygen species (ROS) scavenger, N-acetylcystein (NAC) significantly suppressed induction of these markers, indicating that ROS are involved in the induction of cellular senescence by IGF-I. In p53-null mouse embryonic fibroblasts, the IGF-I-induced augmentation of SA-{beta}-gal and p21 was inhibited, demonstrating that p53 is required for cellular senescence induced by IGF-I. Thus, these data reveal a novel pathway whereby IGF-I enhances cellular senescence in the ROS and p53-dependent manner and may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging.« less

The A2b adenosine receptor antagonist PSB-603 promotes oxidative phosphorylation and ROS production in colorectal cancer cells via adenosine receptor-independent mechanism.

PubMed

Mølck, Christina; Ryall, James; Failla, Laura M; Coates, Janine L; Pascussi, Jean-Marc; Heath, Joan K; Stewart, Gregory; Hollande, Frédéric

2016-12-01

Adenosine is a multifaceted regulator of tumor progression. It modulates immune cell activity as well as acting directly on tumor cells. The A 2b adenosine receptor (A 2b -AR) is thought to be an important mediator of these effects. In this study we sought to analyze the contribution of the A 2b -AR to the behavior of colorectal cancer cells. The A 2b -AR antagonist PSB-603 changed cellular redox state without affecting cellular viability. Quantification of cellular bioenergetics demonstrated that PSB-603 increased basal oxygen consumption rates, indicative of enhanced mitochondrial oxidative phosphorylation. Unexpectedly, pharmacological and genetic approaches to antagonize AR-related signalling of PSB-603 did not abolish the response, suggesting that it was AR-independent. PSB-603 also induced acute increases in reactive oxygen species, and PSB-603 synergized with chemotherapy treatment to increase colorectal cancer cell death, consistent with the known link between cellular metabolism and chemotherapy response. PSB-603 alters cellular metabolism in colorectal cancer cells and increases their sensitivity to chemotherapy. Although requiring more mechanistic insight into its A 2b -AR-independent activity, our results show that PSB-603 may have clinical value as an anti-colorectal cancer therapeutic. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Fetal Stress and Programming of Hypoxic/Ischemic-Sensitive Phenotype in the Neonatal Brain: Mechanisms and Possible Interventions

PubMed Central

Li, Yong; Gonzalez, Pablo; Zhang, Lubo

2012-01-01

Growing evidence of epidemiological, clinical and experimental studies has clearly shown a close link between adverse in utero environment and the increased risk of neurological, psychological and psychiatric disorders in later life. Fetal stresses, such as hypoxia, malnutrition, and fetal exposure to nicotine, alcohol, cocaine and glucocorticoids may directly or indirectly act at cellular and molecular levels to alter the brain development and result in programming of heightened brain vulnerability to hypoxic-ischemic encephalopathy and the development of neurological diseases in the postnatal life. The underlying mechanisms are not well understood. However, glucocorticoids may play a crucial role in epigenetic programming of neurological disorders of fetal origins. This review summarizes the recent studies about the effects of fetal stress on the abnormal brain development, focusing on the cellular, molecular and epigenetic mechanisms and highlighting the central effects of glucocorticoids on programming of hypoxicischemic-sensitive phenotype in the neonatal brain, which may enhance the understanding of brain pathophysiology resulting from fetal stress and help explore potential targets of timely diagnosis, prevention and intervention in neonatal hypoxic-ischemic encephalopathy and other for brain disorders. PMID:22627492

Detection of angiotensin II binding to single adrenal zona glomerulosa cells by confocal Raman microspectroscopy

NASA Astrophysics Data System (ADS)

McCoy, Michael J.; Habermann, Timothy J.; Hanke, Craig J.; Adar, Fran; Campbell, William B.; Nithipatikom, Kasem

1999-04-01

We developed a confocal Raman microspectroscopic technique to study ligand-receptor bindings in single cells using Raman-labeled ligands and surface-enhanced Raman scattering (SERS). The adrenal zona glomerulosa (ZG) cells were used as a model in this study. ZG cells have a high density of angiotensin II (AII) receptors on the cellular membrane. There are two identified subtypes of AII receptors,namely AT1 and AT2 receptors. AII is a peptidic hormone, which upon binding to its receptors, stimulates the release of aldosterone from ZG cells. The cellular localization of these receptors subtypes was detected in single ZG cells by using immunocomplexation of receptors with specific antibodies and confocal Raman microspectroscopy. In the binding study, we used biotin-labeled AII to bind to its receptors in ZG cells. Then, avidin and Raman-labeled AII. The binding was measure directly on the single ZG cells. The results showed that the binding was displaced with unlabeled AII and specific AII antagonists. This is a rapid and sensitive technique for detection of cellular ligand bindings as well as antagonists screening in drug discovery.

Design, Multicomponent Synthesis, and Anticancer Activity of a Focused Histone Deacetylase (HDAC) Inhibitor Library with Peptoid-Based Cap Groups.

PubMed

Krieger, Viktoria; Hamacher, Alexandra; Gertzen, Christoph G W; Senger, Johanna; Zwinderman, Martijn R H; Marek, Martin; Romier, Christophe; Dekker, Frank J; Kurz, Thomas; Jung, Manfred; Gohlke, Holger; Kassack, Matthias U; Hansen, Finn K

2017-07-13

In this work, we report the multicomponent synthesis of a focused histone deacetylase (HDAC) inhibitor library with peptoid-based cap groups and different zinc-binding groups. All synthesized compounds were tested in a cellular HDAC inhibition assay and an MTT assay for cytotoxicity. On the basis of their noteworthy activity in the cellular HDAC assays, four compounds were further screened for their inhibitory activity against recombinant HDAC1-3, HDAC6, and HDAC8. All four compounds showed potent inhibition of HDAC1-3 as well as significant inhibition of HDAC6 with IC 50 values in the submicromolar concentration range. Compound 4j, the most potent HDAC inhibitor in the cellular HDAC assay, revealed remarkable chemosensitizing properties and enhanced the cisplatin sensitivity of the cisplatin-resistant head-neck cancer cell line Cal27CisR by almost 7-fold. Furthermore, 4j almost completely reversed the cisplatin resistance in Cal27CisR. This effect is related to a synergistic induction of apoptosis as seen in the combination of 4j with cisplatin.

Chromium picolinate enhances skeletal muscle cellular insulin signaling in vivo in obese, insulin-resistant JCR:LA-cp rats.

PubMed

Wang, Zhong Q; Zhang, Xian H; Russell, James C; Hulver, Matthew; Cefalu, William T

2006-02-01

Chromium is one of the few trace minerals for which a specific cellular mechanism of action has not been identified. Recent in vitro studies suggest that chromium supplementation may improve insulin sensitivity by enhancing insulin receptor signaling, but this has not been demonstrated in vivo. We investigated the effect of chromium supplementation on insulin receptor signaling in an insulin-resistant rat model, the JCR:LA-corpulent rat. Male JCR:LA-cp rats (4 mo of age) were randomly assigned to receive chromium picolinate (CrPic) (obese n=6, lean n=5) or vehicle (obese n=5, lean n=5) for 3 mo. The CrPic was provided in the water, and based on calculated water intake, rats randomized to CrPic received 80 microg/(kg.d). At the end of the study, skeletal muscle (vastus lateralis) biopsies were obtained at baseline and at 5, 15, and 30 min postinsulin stimulation to assess insulin signaling. Obese rats treated with CrPic had significantly improved glucose disposal rates and demonstrated a significant increase in insulin-stimulated phosphorylation of insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI)-3 kinase activity in skeletal muscle compared with obese controls. The increase in cellular signaling was not associated with increased protein levels of the IRS proteins, PI-3 kinase or Akt. However, protein tyrosine phosphatase 1B (PTP1B) levels were significantly lower in obese rats administered CrPic than obese controls. When corrected for protein content, PTP1B activity was also significantly lower in obese rats administered CrPic than obese controls. Our data suggest that chromium supplementation of obese, insulin-resistant rats may improve insulin action by enhancing intracellular signaling.

Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNFα and co-treatments

NASA Astrophysics Data System (ADS)

Loo, Lit-Hsin; Bougen-Zhukov, Nicola Michelle; Tan, Wei-Ling Cecilia

2017-03-01

Signaling pathways can generate different cellular responses to the same cytotoxic agents. Current quantitative models for predicting these differential responses are usually based on large numbers of intracellular gene products or signals at different levels of signaling cascades. Here, we report a study to predict cellular sensitivity to tumor necrosis factor alpha (TNFα) using high-throughput cellular imaging and machine-learning methods. We measured and compared 1170 protein phosphorylation events in a panel of human lung cancer cell lines based on different signals, subcellular regions, and time points within one hour of TNFα treatment. We found that two spatiotemporal-specific changes in an intermediate signaling protein, p90 ribosomal S6 kinase (RSK), are sufficient to predict the TNFα sensitivity of these cell lines. Our models could also predict the combined effects of TNFα and other kinase inhibitors, many of which are not known to target RSK directly. Therefore, early spatiotemporal-specific changes in intermediate signals are sufficient to represent the complex cellular responses to these perturbations. Our study provides a general framework for the development of rapid, signaling-based cytotoxicity screens that may be used to predict cellular sensitivity to a cytotoxic agent, or identify co-treatments that may sensitize or desensitize cells to the agent.

Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNFα and co-treatments

PubMed Central

Loo, Lit-Hsin; Bougen-Zhukov, Nicola Michelle; Tan, Wei-Ling Cecilia

2017-01-01

Signaling pathways can generate different cellular responses to the same cytotoxic agents. Current quantitative models for predicting these differential responses are usually based on large numbers of intracellular gene products or signals at different levels of signaling cascades. Here, we report a study to predict cellular sensitivity to tumor necrosis factor alpha (TNFα) using high-throughput cellular imaging and machine-learning methods. We measured and compared 1170 protein phosphorylation events in a panel of human lung cancer cell lines based on different signals, subcellular regions, and time points within one hour of TNFα treatment. We found that two spatiotemporal-specific changes in an intermediate signaling protein, p90 ribosomal S6 kinase (RSK), are sufficient to predict the TNFα sensitivity of these cell lines. Our models could also predict the combined effects of TNFα and other kinase inhibitors, many of which are not known to target RSK directly. Therefore, early spatiotemporal-specific changes in intermediate signals are sufficient to represent the complex cellular responses to these perturbations. Our study provides a general framework for the development of rapid, signaling-based cytotoxicity screens that may be used to predict cellular sensitivity to a cytotoxic agent, or identify co-treatments that may sensitize or desensitize cells to the agent. PMID:28272488

Ca2+ sensitizers: An emerging class of agents for counterbalancing weakness in skeletal muscle diseases?

PubMed

Ochala, Julien

2010-02-01

Ca(2+) ions are key regulators of skeletal muscle contraction. By binding to contractile proteins, they initiate a cascade of molecular events leading to cross-bridge formation and ultimately, muscle shortening and force production. The ability of contractile proteins to respond to Ca(2+) attachment, also known as Ca(2+) sensitivity, is often compromised in acquired and congenital skeletal muscle disorders. It constitutes, undoubtedly, a major physiological cause of weakness for patients. In this review, we discuss recent studies giving strong molecular and cellular evidence that pharmacological modulators of some of the contractile proteins, also termed Ca(2+) sensitizers, are efficient agents to improve Ca(2+) sensitivity and function in diseased skeletal muscle cells. In fact, they compensate for the impaired contractile proteins response to Ca(2+) binding. Currently, such Ca(2+) sensitizing compounds are successfully used for reducing problems in cardiac disorders. Therefore, in the future, under certain conditions, these agents may represent an emerging class of agents to enhance the quality of life of patients suffering from skeletal muscle weakness. Copyright 2009 Elsevier B.V. All rights reserved.

Chronic Ca2+ influx through voltage-dependent Ca2+ channels enhance delayed rectifier K+ currents via activating Src family tyrosine kinase in rat hippocampal neurons.

PubMed

Yang, Yoon-Sil; Jeon, Sang-Chan; Kim, Dong-Kwan; Eun, Su-Yong; Jung, Sung-Cherl

2017-03-01

Excessive influx and the subsequent rapid cytosolic elevation of Ca 2+ in neurons is the major cause to induce hyperexcitability and irreversible cell damage although it is an essential ion for cellular signalings. Therefore, most neurons exhibit several cellular mechanisms to homeostatically regulate cytosolic Ca 2+ level in normal as well as pathological conditions. Delayed rectifier K + channels (I DR channels) play a role to suppress membrane excitability by inducing K + outflow in various conditions, indicating their potential role in preventing pathogenic conditions and cell damage under Ca 2+ -mediated excitotoxic conditions. In the present study, we electrophysiologically evaluated the response of I DR channels to hyperexcitable conditions induced by high Ca 2+ pretreatment (3.6 mM, for 24 hours) in cultured hippocampal neurons. In results, high Ca 2+ -treatment significantly increased the amplitude of I DR without changes of gating kinetics. Nimodipine but not APV blocked Ca 2+ -induced I DR enhancement, confirming that the change of I DR might be targeted by Ca 2+ influx through voltage-dependent Ca 2+ channels (VDCCs) rather than NMDA receptors (NMDARs). The VDCC-mediated I DR enhancement was not affected by either Ca 2+ -induced Ca 2+ release (CICR) or small conductance Ca 2+ -activated K + channels (SK channels). Furthermore, PP2 but not H89 completely abolished I DR enhancement under high Ca 2+ condition, indicating that the activation of Src family tyrosine kinases (SFKs) is required for Ca 2+ -mediated I DR enhancement. Thus, SFKs may be sensitive to excessive Ca 2+ influx through VDCCs and enhance I DR to activate a neuroprotective mechanism against Ca 2+ -mediated hyperexcitability in neurons.

Inhibition of the Nedd8 system sensitizes cells to DNA Inter-strand crosslinking agents

PubMed Central

Kee, Younghoon; Huang, Min; Chang, Sophia; Moreau, Lisa A.; Park, Eunmi; Smith, Peter G.; D’Andrea, Alan D.

2012-01-01

The Fanconi Anemia (FA) pathway is required for repair of DNA interstrand crosslinks (ICLs). FA pathway-deficient cells are hypersensitive to DNA ICL-inducing drugs such as Cisplatin. Conversely, hyperactivation of the FA pathway is a mechanism that may underlie cellular resistance to DNA ICL agents. Modulating FANCD2 monoubiquitination, a key step in the FA pathway, may be an effective therapeutic approach to conferring cellular sensitivity to ICL agents. Here, we show that inhibition of the Nedd8 conjugation system increases cellular sensitivity to DNA ICL-inducing agents. Mechanistically, the Nedd8 inhibition, either by siRNA-mediated knockdown of Nedd8 conjugating enzymes or treatment with a Nedd8 activating enzyme inhibitor MLN4924, suppressed DNA damage-induced FANCD2 monoubiquitination and CHK1 phosphorylation. Our data indicate that inhibition of the FA pathway is largely responsible for the heightened cellular sensitivity to DNA ICLs upon Nedd8 inhibition. These results suggest that a combination of Nedd8 inhibition with ICL-inducing agents may be an effective strategy for sensitizing a subset of drug-resistant cancer cells. PMID:22219386

Tumor-Triggered Geometrical Shape Switch of Chimeric Peptide for Enhanced in Vivo Tumor Internalization and Photodynamic Therapy.

PubMed

Han, Kai; Zhang, Jin; Zhang, Weiyun; Wang, Shibo; Xu, Luming; Zhang, Chi; Zhang, Xianzheng; Han, Heyou

2017-03-28

Geometrical shape of nanoparticles plays an important role in cellular internalization. However, the applicability in tumor selective therapeutics is still scarcely reported. In this article, we designed a tumor extracellular acidity-responsive chimeric peptide with geometrical shape switch for enhanced tumor internalization and photodynamic therapy. This chimeric peptide could self-assemble into spherical nanoparticles at physiological condition. While at tumor extracellular acidic microenvironment, chimeric peptide underwent detachment of acidity-sensitive 2,3-dimethylmaleic anhydride groups. The subsequent recovery of ionic complementarity between chimeric peptides resulted in formation of rod-like nanoparticles. Both in vitro and in vivo studies demonstrated that this acidity-triggered geometrical shape switch endowed chimeric peptide with accelerated internalization in tumor cells, prolonged accumulation in tumor tissue, enhanced photodynamic therapy, and minimal side effects. Our results suggested that fusing tumor microenvironment with geometrical shape switch should be a promising strategy for targeted drug delivery.

Effects of X-shaped reduction-sensitive amphiphilic block copolymer on drug delivery.

PubMed

Xiao, Haijun; Wang, Lu

2015-01-01

To study the effects of X-shaped amphiphilic block copolymers on delivery of docetaxel (DTX) and the reduction-sensitive property on drug release, a novel reduction-sensitive amphiphilic copolymer, (PLGA)2-SS-4-arm-PEG2000 with a Gemini-like X-shape, was successfully synthesized. The formation of nanomicelles was proved with respect to the blue shift of the emission fluorescence as well as the fluorescent intensity increase of coumarin 6-loaded particles. The X-shaped polymers exhibited a smaller critical micelle concentration value and possessed higher micellar stability in comparison with those of linear ones. The size of X-shaped (PLGA)2-SS-4-arm-PEG2000 polymer nanomicelles (XNMs) was much smaller than that of nanomicelles prepared with linear polymers. The reduction sensitivity of polymers was confirmed by the increase of micellar sizes as well as the in vitro drug release profile of DTX-loaded XNMs (DTX/XNMs). Cytotoxicity assays in vitro revealed that the blank XNMs were nontoxic against A2780 cells up to a concentration of 50 µg/mL, displaying good biocompatibility. DTX/XNMs were more toxic against A2780 cells than other formulations in both dose- and time-dependent manners. Cellular uptake assay displayed a higher intracellular drug delivery efficiency of XNMs than that of nanomicelles prepared with linear polymers. Besides, the promotion of tubulin polymerization induced by DTX was visualized by immunofluorescence analysis, and the acceleration of apoptotic process against A2780 cells was also imaged using a fluorescent staining method. Therefore, this X-shaped reduction-sensitive (PLGA)2-SS-4-arm-PEG2000 copolymer could effectively improve the micellar stability and significantly enhance the therapeutic efficacy of DTX by increasing the cellular uptake and selectively accelerating the drug release inside cancer cells.

Chemosensitizing effects of carbon-based nanomaterials in cancer cells: enhanced apoptosis and inhibition of proliferation as underlying mechanisms

NASA Astrophysics Data System (ADS)

Erdmann, Kati; Ringel, Jessica; Hampel, Silke; Rieger, Christiane; Huebner, Doreen; Wirth, Manfred P.; Fuessel, Susanne

2014-10-01

Recent studies have shown that carbon nanomaterials such as carbon nanofibres (CNFs) and multi-walled carbon nanotubes (CNTs) can exert antitumor activities themselves and sensitize cancer cells to conventional chemotherapeutics such as carboplatin and cisplatin. In the present study, the chemosensitizing effect of CNFs and CNTs on cancer cells of urological origin was investigated regarding the underlying mechanisms. Prostate cancer (DU-145, PC-3) and bladder cancer (EJ28) cells were treated with carbon nanomaterials (CNFs, CNTs) and chemotherapeutics (carboplatin, cisplatin) alone as well as in combination for 24 h. Forty-eight (EJ28) or 72 h (DU-145, PC-3) after the end of treatment the effects on cellular proliferation, clonogenic survival, cell death rate and cell cycle distribution were evaluated. Depending on the cell line, simultaneous administration of chemotherapeutics and carbon nanomaterials produced an additional inhibition of cellular proliferation and clonogenic survival of up to 77% and 98%, respectively, compared to the inhibitory effects of the chemotherapeutics alone. These strongly enhanced antiproliferative effects were accompanied by an elevated cell death rate, which was predominantly mediated via apoptosis and not by necrosis. The antitumor effects of combinations with CNTs were less pronounced than those with CNFs. The enhanced effects of the combinatory treatments on cellular function were mostly of additive to partly synergistic nature. Furthermore, cell cycle analysis demonstrated an arrest at the G2/M phase mediated by a monotreatment with chemotherapeutics. Following combinatory treatments, mostly less than or nearly additive increases of cell fractions in the G2/M phase could be observed. In conclusion, the pronounced chemosensitizing effects of CNFs and CNTs were mediated by an enhanced apoptosis and inhibition of proliferation. The combination of carbon-based nanomaterials and conventional chemotherapeutics represents a novel approach in cancer therapy to bypass chemoresistance by minimizing the chemotherapeutic dosing.

pHLIP®-Mediated Delivery of PEGylated Liposomes to Cancer Cells

PubMed Central

Yao, Lan; Daniels, Jennifer; Wijesinghe, Dayanjali; Andreev, Oleg A.; Reshetnyak, Yana K.

2013-01-01

We develop a method for pH-dependent fusion between liposomes and cellular membranes using pHLIP® (pH Low Insertion Peptide), which inserts into lipid bilayer of membrane only at low pH. Previously we establish the molecular mechanism of peptide action and show that pHLIP can target acidic diseased tissue. Here we investigate how coating of PEGylated liposomes with pHLIP might affect liposomal uptake by cells. The presence of pHLIP on the surface of PEGylated-liposomes enhanced membrane fusion and lipid exchange in a pH dependent fashion, leading to increase of cellular uptake and payload release, and inhibition of cell proliferation by liposomes containing ceramide. A novel type of pH-sensitive, “fusogenic” pHLIP-liposomes was developed, which could be used to selectively deliver various diagnostic and therapeutic agents to acidic diseased cells. PMID:23416366

STARD13 promotes hepatocellular carcinoma apoptosis by acting as a ceRNA for Fas.

PubMed

Zhang, Hai; Wang, Fang; Hu, Yahua

2017-02-01

To study the roles of STARD13 in cellular apoptosis of hepatocellular carcinoma (HCC). Quantitative real-time PCR and immunohistochemistry analyses showed that the expression levels of STARD13 and Fas were lower in clinical HCC tissues than in normal tissues and were positively correlated, which is consistent with the results analyzed by The Cancer Genome Atlas (TCGA) data. Patients with higher STARD13 or Fas expression levels had longer overall survival. Additionally, STARD13 3'-UTR enhanced cellular apoptosis and the 3'-UTRs of STARD13 and Fas were predicted to harbor nine similar miRNA binding sites. And STARD13 3'-UTR promoted Fas expression in a 3'-UTR- and miRNA-dependent way and increased the sensitivity of HCC cells to chemotherapy. Importantly, the coding sequence of STARD13 did not increase Fas expression. STARD13 3'-UTR promotes HCC apoptosis through acting as a ceRNA for Fas.

IDH2 knockdown sensitizes tumor cells to emodin cytotoxicity in vitro and in vivo.

PubMed

Ku, Hyeong Jun; Kwon, Oh-Shin; Kang, Boem Sik; Lee, Dong-Seok; Lee, Hyun-Shik; Park, Jeen-Woo

2016-10-01

Although reactive oxygen species (ROS) work as second messengers at sublethal concentrations, higher levels of ROS can kill cancer cells. Since cellular ROS levels are determined by a balance between ROS generation and removal, the combination of ROS generators, and the depletion of reducing substances greatly enhance ROS levels. Emodin (1,3,8-trihydroxy-6-methyl anthraquinone), a natural anthraquinone derivative from the root and rhizome of numerous plants, is a ROS generator that induces apoptosis in cancer cells. The major enzyme to generate mitochondrial NADPH is the mitochondrial isoenzyme of NADP + -dependent isocitrate dehydrogenase (IDH2). In this report, we demonstrate that IDH2 knockdown effectively enhances emodin-induced apoptosis of mouse melanoma B16F10 cells through the regulation of ROS generation. Our findings suggest that suppression of IDH2 activity results in perturbation of the cellular redox balance and, ultimately, exacerbate emodin-induced apoptotic cell death in B16F10 cells. Our results strongly support a therapeutic strategy in the management of cancer that alters the intracellular redox status by the combination of a ROS generator and the suppression of antioxidant enzyme activity.

Probing Novel Roles of the Mitochondrial Uniporter in Ovarian Cancer Cells Using Nanoparticles*♦

PubMed Central

Arvizo, Rochelle R.; Moyano, Daniel F.; Saha, Sounik; Thompson, Michael A.; Bhattacharya, Resham; Rotello, Vincent M.; Prakash, Y. S.; Mukherjee, Priyabrata

2013-01-01

Nanoparticles provide a potent tool for targeting and understanding disease mechanisms. In this regard, cancer cells are surprisingly resistant to the expected toxic effects of positively charged gold nanoparticles (+AuNPs). Our investigations led to the identification of MICU1, regulator of mitochondrial calcium uniporter, as a key molecule conferring cancer cells with resistance to +AuNPs. The increase in cytosolic [Ca2+]cyto in malignant cells induced by +AuNPs is counteracted by MICU1, preventing cell death. Pharmacological or siRNA-mediated inhibition of mitochondrial Ca+2 entry leads to endoplasmic reticulum stress and sensitizes cancer cells to +AuNP-induced cytotoxicity. Silencing MICU1 decreases Bcl-2 expression and increases caspase-3 activity and cytosolic cytochrome c levels, thus initiating the mitochondrial pathway for apoptosis: effects further enhanced by +AuNPs. This study highlights the potential of nanomaterials as a tool to broaden our understanding of cellular processes, establishes MICU1 as a novel regulator of the machinery in cancer cells that prevents apoptosis, and emphasizes the need to synergize nanoparticle design with understanding of mitochondrial machinery for enhancing targeted cellular toxicity. PMID:23615904

An improved, non-functionalized route to plasmonic nanoparticle based cellular probing through osmolyte mediation (Conference Presentation)

NASA Astrophysics Data System (ADS)

Siddhanta, Soumik; Barman, Ishan

2017-02-01

Engineering nanostructured probes for ultra-sensitive detection of specific molecular species, our research seeks to capture the complex changes in cells and tissues that can predict disease progression in an individual. While such nanoparticle-based platforms are rapidly gaining a foothold in cancer diagnostics, one of the most concerning factors is the vulnerability of cells to the interaction with functional nanoparticles thereby raising the specter of systemic toxicity. The nanoparticles end up damaging the cells and disrupting cellular functions thereby impeding their imaging aim. Furthermore, PEGylation, and similar routes, force a tradeoff between desired nanoparticle properties (recognition, uptake, and reduced toxicity) and sensitivity of plasmon-enhanced spectroscopic sensing methods, such as surface-enhanced Raman spectroscopy (SERS) where the proximal presence of noble metal NP and the organic molecule of interest is key. In this work, we report a trehalose-mediated, non-surface functionalized route for cell-nanoparticle interactions that maintains cell viability while allowing selective interaction of the nanoparticle with the cell surface receptors and subsequent internalization. Through careful electron microscopy of nanoparticle-prostate cancer cells interactions, we elucidated that there exists a dynamic equilibrium between "free" cytosolic diffusion of the nanoparticles and endocytosis through vesicle formation - and trehalose tilts the scale in favor of the latter to mask the toxic effects of the nanoparticles. The precise molecular interpretation of this behavior was further probed through SERS, which directly points towards the protein stabilization properties of trehalose mediation during interaction of the nanoparticles with the plasma membrane components.

Hierarchical Targeting Strategy for Enhanced Tumor Tissue Accumulation/Retention and Cellular Internalization.

PubMed

Wang, Sheng; Huang, Peng; Chen, Xiaoyuan

2016-09-01

Targeted delivery of therapeutic agents is an important way to improve the therapeutic index and reduce side effects. To design nanoparticles for targeted delivery, both enhanced tumor tissue accumulation/retention and enhanced cellular internalization should be considered simultaneously. So far, there have been very few nanoparticles with immutable structures that can achieve this goal efficiently. Hierarchical targeting, a novel targeting strategy based on stimuli responsiveness, shows good potential to enhance both tumor tissue accumulation/retention and cellular internalization. Here, the recent design and development of hierarchical targeting nanoplatforms, based on changeable particle sizes, switchable surface charges and activatable surface ligands, will be introduced. In general, the targeting moieties in these nanoplatforms are not activated during blood circulation for efficient tumor tissue accumulation, but re-activated by certain internal or external stimuli in the tumor microenvironment for enhanced cellular internalization. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase.

PubMed

Jo, Seung-Hee; Lee, So-Hyun; Chun, Hang Suk; Lee, Su Min; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

2002-03-29

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury. (c)2002 Elsevier Science (USA).

Cellular Responses to Beta Blocker Exposures in Marine ...

EPA Pesticide Factsheets

β blockers are prescription drugs used for medical treatment of hypertension and arrhythmias. They prevent activation of adenylate cyclase and increases in blood pressure by limiting cAMP production and protein kinase A activation. After being taken therapeutically, β blockers may make their way to coastal habitats via discharge from waste water treatment plants, posing a potential risk to aquatic organisms. The aim of our research is to evaluate cellular biomarkers of β blocker exposure using two drugs, propranolol and metoprolol, in three commercially important marine bivalves -Crassostrea virginica, Mytilus edulis and Mercenaria mercenaria. Bivalves were obtained from Narragansett Bay (Rhode Island, USA) and acclimated in the laboratory. Following acclimation, gills and hepatopancreas tissues were harvested and separately exposed to 0, 1, 10, 100 and 1000 ng/l of each drug for 24 hours. Samples were preserved for cellular biomarker assays. Elevated cellular damage and changes in enzymatic activities were noted at environmentally relevant concentrations, and M. mercenaria was found to be the most sensitive bivalve out of the three species tested. These studies enhance our understanding of the potential impacts of commonly used prescription medication on organisms in coastal ecosystems, and demonstrate that filter feeders such as marine bivalves may serve as good model organisms to examine the effects of water soluble drugs. Evaluating a suite of biomarkers

Cellular, molecular, and epigenetic mechanisms in non-associative conditioning: implications for pain and memory.

PubMed

Rahn, Elizabeth J; Guzman-Karlsson, Mikael C; David Sweatt, J

2013-10-01

Sensitization is a form of non-associative conditioning in which amplification of behavioral responses can occur following presentation of an aversive or noxious stimulus. Understanding the cellular and molecular underpinnings of sensitization has been an overarching theme spanning the field of learning and memory as well as that of pain research. In this review we examine how sensitization, both in the context of learning as well as pain processing, shares evolutionarily conserved behavioral, cellular/synaptic, and epigenetic mechanisms across phyla. First, we characterize the behavioral phenomenon of sensitization both in invertebrates and vertebrates. Particular emphasis is placed on long-term sensitization (LTS) of withdrawal reflexes in Aplysia following aversive stimulation or injury, although additional invertebrate models are also covered. In the context of vertebrates, sensitization of mammalian hyperarousal in a model of post-traumatic stress disorder (PTSD), as well as mammalian models of inflammatory and neuropathic pain is characterized. Second, we investigate the cellular and synaptic mechanisms underlying these behaviors. We focus our discussion on serotonin-mediated long-term facilitation (LTF) and axotomy-mediated long-term hyperexcitability (LTH) in reduced Aplysia systems, as well as mammalian spinal plasticity mechanisms of central sensitization. Third, we explore recent evidence implicating epigenetic mechanisms in learning- and pain-related sensitization. This review illustrates the fundamental and functional overlay of the learning and memory field with the pain field which argues for homologous persistent plasticity mechanisms in response to sensitizing stimuli or injury across phyla. Copyright © 2013 Elsevier Inc. All rights reserved.

RGD-modified pH-sensitive liposomes for docetaxel tumor targeting.

PubMed

Chang, Minglu; Lu, Shanshan; Zhang, Fang; Zuo, Tiantian; Guan, Yuanyuan; Wei, Ting; Shao, Wei; Lin, Guimei

2015-05-01

Phosphatidylethanolamine-based pH-sensitive liposomes of various compositions have been described as efficient systems for delivery of therapeutic molecules into tumor cells. The aim of this work was to develop a drug delivery system based on pH-sensitive liposomes (PLPs) that were modified with arginine-glycine-aspartic acid (RGD) peptide to enhance the effectiveness of docetaxel treatment. Docetaxel/coumarin-6 loaded PLPs were prepared by the thin-film dispersion method and characterized in detail, including by particle size, polydispersity, zeta potential and drug encapsulation efficiency. In vitro studies using MCF-7, HepG2and A549 cells were employed to investigate cytotoxicity and cellular uptake of the drug solution or docetaxel/coumarin-6 loaded PLPs. The accumulation of 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled liposomes in vivo was studied through tumor section imaging of xenograft mouse models of MCF-7 24h after intravenous administration. The particle size of the non-coated or RGD modified PLPs ranged between 146 and 129nm. Drug release in vitro was modestly prolonged and had good pH sensitivity. In the in vitro study, RGD-coated PLPs showed higher cytotoxicity and cellular uptake relative to non-coated ones. The results of the in vivo study showed that RGD-coated PLPs had higher fluorescence, which suggested a more efficient accumulation than normal PLPs in tumors. In conclusion, these results confirmed RGD-modified PLPs as a potential drug delivery system to achieve controlled release and tumor targeting. Copyright © 2015 Elsevier B.V. All rights reserved.

Camptothecin prodrug nanomicelle based on a boronate ester-linked diblock copolymer as the carrier of doxorubicin with enhanced cellular uptake.

PubMed

Gao, Ya; Xiao, Yi; Liu, Shiyuan; Yu, Jiahui

2018-02-01

A novel pH-sensitive polymeric prodrug of camptothecin (CPT) by polymerizing γ-camptothecin-glutamate N-carboxyanhydride (Glu (CPT)-NCA) on boronate ester-linked poly (ethyleneglycol) (PEG) directly via the amine-initiated ring open polymerization (ROP) has been developed. The resulting amphiphilic prodrug (mPEG-BC-PGluCPT) could self-assemble into nanoparticles and encapsulate doxorubicin (Dox) simultaneously in aqueous solution for dual-drug delivery. The formation of polymeric prodrug micelles (mPEG-BC@PGluCPT) was confirmed by the measurements of critical aggregation concentration (CAC), particle size, and morphology observations. The mPEG-BC@PGluCPT micelles were colloidally stable in solutions for two weeks. Polymeric prodrug micelles mPEG-BC@PGluCPT and Dox-loaded micelles mPEG-BC@PGluCPT⋅Dox showed sustained drug release profiles over 48 h. As expected, drug release was accelerated by the decreasement of pH value from 7.4 to 6.0, which demonstrated pH-dependent manner of drug release. Additionally, it was found that cellular uptake of mPEG-BC@PGluCPT⋅Dox micelles on HepG2 cells was higher than that on HL-7702 cells, especially in culture medium at pH 6.0. The enhanced cellular uptake of mPEG-BC@PGluCPT⋅Dox micelles under acidic condition on HepG2 cells resulted in the higher cytotoxicity of mPEG-BC@PGluCPT⋅Dox micelles at acidic pH than that at pH 7.4.

Improvement of the antiproliferative effect of rapamycin on tumor cell lines by poly (monomethylitaconate)-based pH-sensitive, plasma stable liposomes.

PubMed

Ghanbarzadeh, Saeed; Arami, Sanam; Pourmoazzen, Zhaleh; Khorrami, Arash

2014-03-01

pH-responsive polymers produce liposomes with pH-sensitive property which can release their encapsulated drug under mild acidic conditions found inside the cellular endosomes, inflammatory tissues and cancerous cells. The aim of this study was preparing pH-sensitive and plasma stable liposomes in order to enhance the selectivity and antiproliferative effect of Rapamycin. In the present study we used PEG-poly (monomethylitaconate)-CholC6 (PEG-PMMI-CholC6) copolymer and Oleic acid (OA) to induce pH-sensitive property in Rapamycin liposomes. pH-sensitive liposomal formulations bearing copolymer PEG-PMMI-CholC6 and OA were characterized in regard to physicochemical stability, pH-responsiveness and stability in human plasma. The ability of pH-sensitive liposomes in enhancing the cytotoxicity of Rapamycin was evaluated in vitro by using colon cancer cell line (HT-29) and compared with its cytotoxicity on human umbilical vein endothelial cell (HUVEC) line. Both formulations were found to release their contents under mild acidic conditions rapidly. However, unlike OA-based liposomes, the PEG-PMMI-CholC6 bearing liposomes preserved their pH-sensitivity in plasma. Both types of pH-sensitive Rapamycin-loaded liposomes exhibited high physicochemical stability and could deliver antiproliferative agent into HT-29 cells much more efficiently in comparison with conventional liposomes. Conversely, the antiproliferative effect of pH-sensitive liposomes on HUVEC cell line was less than conventional liposomes. This study showed that both OA and PEG-PMMI-CholC6-based vesicles could submit pH-sensitive property, however, only PEG-PMMI-CholC6-based liposomes could preserve pH-sensitive property after incubation in plasma. As a result pH-sensitive PEG-PMMI-CholC6-based liposomal formulation can improve the selectivity, stability and antiproliferative effect of Rapamycin. Copyright © 2014 Elsevier B.V. All rights reserved.

Visualizing individual microtubules by bright field microscopy

NASA Astrophysics Data System (ADS)

Gutiérrez-Medina, Braulio; Block, Steven M.

2010-11-01

Microtubules are slender (˜25 nm diameter), filamentous polymers involved in cellular structure and organization. Individual microtubules have been visualized via fluorescence imaging of dye-labeled tubulin subunits and by video-enhanced, differential interference-contrast microscopy of unlabeled polymers using sensitive CCD cameras. We demonstrate the imaging of unstained microtubules using a microscope with conventional bright field optics in conjunction with a webcam-type camera and a light-emitting diode illuminator. The light scattered by microtubules is image-processed to remove the background, reduce noise, and enhance contrast. The setup is based on a commercial microscope with a minimal set of inexpensive components, suitable for implementation in a student laboratory. We show how this approach can be used in a demonstration motility assay, tracking the gliding motions of microtubules driven by the motor protein kinesin.

Rice Fertilization-Independent Endosperm1 Regulates Seed Size under Heat Stress by Controlling Early Endosperm Development1[W

PubMed Central

Folsom, Jing J.; Begcy, Kevin; Hao, Xiaojuan; Wang, Dong; Walia, Harkamal

2014-01-01

Although heat stress reduces seed size in rice (Oryza sativa), little is known about the molecular mechanisms underlying the observed reduction in seed size and yield. To elucidate the mechanistic basis of heat sensitivity and reduced seed size, we imposed a moderate (34°C) and a high (42°C) heat stress treatment on developing rice seeds during the postfertilization stage. Both stress treatments reduced the final seed size. At a cellular level, the moderate heat stress resulted in precocious endosperm cellularization, whereas severe heat-stressed seeds failed to cellularize. Initiation of endosperm cellularization is a critical developmental transition required for normal seed development, and it is controlled by Polycomb Repressive Complex2 (PRC2) in Arabidopsis (Arabidopsis thaliana). We observed that a member of PRC2 called Fertilization-Independent Endosperm1 (OsFIE1) was sensitive to temperature changes, and its expression was negatively correlated with the duration of the syncytial stage during heat stress. Seeds from plants overexpressing OsFIE1 had reduced seed size and exhibited precocious cellularization. The DNA methylation status and a repressive histone modification of OsFIE1 were observed to be temperature sensitive. Our data suggested that the thermal sensitivity of seed enlargement could partly be caused by altered epigenetic regulation of endosperm development during the transition from the syncytial to the cellularized state. PMID:24590858

Trimethylation enhancement using diazomethane (TrEnDi): rapid on-column quaternization of peptide amino groups via reaction with diazomethane significantly enhances sensitivity in mass spectrometry analyses via a fixed, permanent positive charge.

PubMed

Wasslen, Karl V; Tan, Le Hoa; Manthorpe, Jeffrey M; Smith, Jeffrey C

2014-04-01

Defining cellular processes relies heavily on elucidating the temporal dynamics of proteins. To this end, mass spectrometry (MS) is an extremely valuable tool; different MS-based quantitative proteomics strategies have emerged to map protein dynamics over the course of stimuli. Herein, we disclose our novel MS-based quantitative proteomics strategy with unique analytical characteristics. By passing ethereal diazomethane over peptides on strong cation exchange resin within a microfluidic device, peptides react to contain fixed, permanent positive charges. Modified peptides display improved ionization characteristics and dissociate via tandem mass spectrometry (MS(2)) to form strong a2 fragment ion peaks. Process optimization and determination of reactive functional groups enabled a priori prediction of MS(2) fragmentation patterns for modified peptides. The strategy was tested on digested bovine serum albumin (BSA) and successfully quantified a peptide that was not observable prior to modification. Our method ionizes peptides regardless of proton affinity, thus decreasing ion suppression and permitting predictable multiple reaction monitoring (MRM)-based quantitation with improved sensitivity.

Hydrogen peroxide sensor based on carbon nanowalls grown by plasma-enhanced chemical vapor deposition

NASA Astrophysics Data System (ADS)

Tomatsu, Masakazu; Hiramatsu, Mineo; Foord, John S.; Kondo, Hiroki; Ishikawa, Kenji; Sekine, Makoto; Takeda, Keigo; Hori, Masaru

2017-06-01

Fabrication of an electrochemical sensor for hydrogen peroxide (H2O2) detection was demonstrated. H2O2 is a major messenger molecule in various redox-dependent cellular signaling transductions. Therefore, sensitive detection of H2O2 is greatly important in health inspection and environmental protection. Carbon nanowalls (CNWs) are composed of few-layer graphenes standing almost vertically on a substrate forming a three-dimensional structure. In this work, CNWs were used as a platform for H2O2 sensing, which is based on the large surface area of conducting carbon and surface decoration with platinum (Pt) nanoparticles (NPs). CNWs were grown on carbon fiber paper (CFP) by inductively coupled plasma-enhanced chemical vapor deposition to increase the surface area. Then, the CNW surface was decorated with Pt-NPs by the reduction of H2PtCl6. Cyclic voltammetry results indicate that the Pt-decorated CNW/CFP electrode possesses excellent electrocatalytic activity for the reduction of H2O2. Amperometric responses indicate the high-sensitivity detection capability of the Pt-decorated CNW/CFP electrode for H2O2.

When Pain Hurts: Nociceptive Stimulation Induces a State of Maladaptive Plasticity and Impairs Recovery after Spinal Cord Injury.

PubMed

Grau, James W; Huang, Yung-Jen; Turtle, Joel D; Strain, Misty M; Miranda, Rajesh C; Garraway, Sandra M; Hook, Michelle A

2017-05-15

Spinal cord injury (SCI) is often accompanied by other tissue damage (polytrauma) that provides a source of pain (nociceptive) input. Recent findings are reviewed that show SCI places the caudal tissue in a vulnerable state that exaggerates the effects nociceptive stimuli and promotes the development of nociceptive sensitization. Stimulation that is both unpredictable and uncontrollable induces a form of maladaptive plasticity that enhances nociceptive sensitization and impairs spinally mediated learning. In contrast, relational learning induces a form of adaptive plasticity that counters these adverse effects. SCI sets the stage for nociceptive sensitization by disrupting serotonergic (5HT) fibers that quell overexcitation. The loss of 5HT can enhance neural excitability by reducing membrane-bound K + -Cl - cotransporter 2, a cotransporter that regulates the outward flow of Cl - . This increases the intracellular concentration of Cl - , which reduces the hyperpolarizing (inhibitory) effect of gamma-aminobutyric acid. Uncontrollable noxious stimulation also undermines the recovery of locomotor function, and increases behavioral signs of chronic pain, after a contusion injury. Nociceptive stimulation has a greater effect if experienced soon after SCI. This adverse effect has been linked to a downregulation in brain-derived neurotrophic factor and an upregulation in the cytokine, tumor necrosis factor. Noxious input enhances tissue loss at the site of injury by increasing the extent of hemorrhage and apoptotic/pyroptotic cell death. Intrathecal lidocaine blocks nociception-induced hemorrhage, cellular indices of cell death, and its adverse effect on behavioral recovery. Clinical implications are discussed.

``Sheddable'' PEG-lipid to balance the contradiction of PEGylation between long circulation and poor uptake

NASA Astrophysics Data System (ADS)

Zhao, Caiyan; Deng, Hongzhang; Xu, Jing; Li, Shuyi; Zhong, Lin; Shao, Leihou; Wu, Yan; Liang, Xing-Jie

2016-05-01

PEGylated lipids confer longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. In order to balance the contradiction between the advantages of long circulation and the disadvantages of poor uptake of PEGylated lipids, we prepared a ``sheddable'' PEG-lipid micelle system based on the conjugation of PEG and phosphatidyl ethanolamine (DSPE) with a pH sensitive benzoic imine bond. In a physiological environment, the PEG-protected micelles were not readily taken up by the reticuloendothelial system (RES) and could be successfully delivered to tumor tissue by the EPR effect. In a tumor acidic microenvironment, the PEG chains detached from the surfaces of the micelles while the degree of linker cleavage could not cause a significant particle size change, which facilitated the carrier binding to tumor cells and improved the cellular uptake. Subsequently, the ``sheddable'' PEG-lipid micelles easily internalized into cells and the increased acidity in the lysosomes further promoted drug release. Thus, this ``sheddable'' PEG-lipid nanocarrier could be a good candidate for effective intracellular drug delivery in cancer chemotherapy.PEGylated lipids confer longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. In order to balance the contradiction between the advantages of long circulation and the disadvantages of poor uptake of PEGylated lipids, we prepared a ``sheddable'' PEG-lipid micelle system based on the conjugation of PEG and phosphatidyl ethanolamine (DSPE) with a pH sensitive benzoic imine bond. In a physiological environment, the PEG-protected micelles were not readily taken up by the reticuloendothelial system (RES) and could be successfully delivered to tumor tissue by the EPR effect. In a tumor acidic microenvironment, the PEG chains detached from the surfaces of the micelles while the degree of linker cleavage could not cause a significant particle size change, which facilitated the carrier binding to tumor cells and improved the cellular uptake. Subsequently, the ``sheddable'' PEG-lipid micelles easily internalized into cells and the increased acidity in the lysosomes further promoted drug release. Thus, this ``sheddable'' PEG-lipid nanocarrier could be a good candidate for effective intracellular drug delivery in cancer chemotherapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr02174c

High Tumor Penetration of Paclitaxel Loaded pH Sensitive Cleavable Liposomes by Depletion of Tumor Collagen I in Breast Cancer.

PubMed

Zhang, Li; Wang, Yang; Yang, Yuting; Liu, Yayuan; Ruan, Shaobo; Zhang, Qianyu; Tai, Xiaowei; Chen, Jiantao; Xia, Tai; Qiu, Yue; Gao, Huile; He, Qin

2015-05-13

The network of collagen I in tumors could prevent the penetration of drugs loaded in nanoparticles, and this would lead to impaired antitumor efficacy. In this study, free losartan (an angiotensin inhibitor) was injected before treatment to reduce the level of collagen I, which could facilitate the penetration of nanoparticles. Then the pH-sensitive cleavable liposomes (Cl-Lip) were injected subsequently to exert the antitumor effect. The Cl-Lip was constituted by PEG(5K)-Hydrazone-PE and DSPE-PEG(2K)-R8. When the Cl-Lip reached to the tumor site by the enhanced permeability and retention (EPR) effect, PEG(5K)-Hydrazone-PE was hydrolyzed from the Cl-Lip under the low extra-cellular pH conditions of tumors, then the R8 peptide was exposed, and finally liposomes could be internalized into tumor cells by the mediation of R8 peptide. In vitro experiments showed both the cellular uptake of Cl-Lip by 4T1 cells and cytotoxicity of paclitaxel loaded Cl-Lip (PTX-Cl-Lip) were pH sensitive. In vivo experiments showed the Cl-Lip had a good tumor targeting ability. After depletion of collagen I, Cl-Lip could penetrate into the deep place of tumors, the tumor accumulation of Cl-Lip was further increased by 22.0%, and the oxygen distributed in tumor tissues was also enhanced. The antitumor study indicated free losartan in combination with PTX-Cl-Lip (59.8%) was more effective than injection with PTX-Cl-Lip only (37.8%) in 4T1 tumor bearing mice. All results suggested that depletion of collagen I by losartan dramatically increased the penetration of PTX-Cl-Lip and combination of free losartan and PTX-CL-Lip could lead to better antitumor efficacy of chemical drugs. Thus, the combination strategy might be a promising tactic for better treatment of solid tumors with a high level of collagen I.

Ultrasound-enhanced localized chemotherapy of drug-sensitive and multidrug resistant tumors

NASA Astrophysics Data System (ADS)

Rapoport, Natalya Y.; Gao, Zhonggao; Kamaev, Pavel; Christensen, Douglas A.

2006-05-01

A new modality of targeted tumor chemotherapy is based on the drug encapsulation in polymeric nanoparticles followed by a localized release at the tumor site triggered by focused ultrasound. Effect of 1 MHz and 3 MHz unfocused ultrasound applied locally to the tumor on the Doxorubicin (DOX) biodistribution and tumor growth rates was measured for ovarian carcinoma tumors in nu/nu mice. The bioeffects of ultrasound were investigated on the systemic and cellular levels. Growth rates of A2780 ovarian carcinoma tumors were substantially reduced by combining micellar drug delivery with tumor irradiation. Ultrasound effect was not thermal as manifested by intratumoral temperature measurements during sonication. Biodistribution studies showed that ultrasound did not enhance micelle extravasation. Main mechanisms of the ultrasound-enhanced chemotherapy included (i) passive targeting of drug-loaded micelles to the tumor interstitium; (ii) ultrasound-triggered localized drug release from micelles in the tumor volume; (iii) enhanced micelle and drug diffusion through the tumor interstitium; and (iv) ultrasound-triggered cell membrane damage resulting in the enhanced micelle and drug uptake by tumor cells.

Severe Acneiform Eruption Induced by Cetuximab (Erbitux®)

PubMed Central

Lee, Jung Eun; Lee, Sang Ju; Lee, Hee Jung; Lee, Ju Hee

2008-01-01

Epidermal growth factor has an important role in the regulation of proliferation and differentiation in epidermal keratinocytes, as well as in the survival, angiogenesis and metastasis of cancer cells. Cetuximab is a chimeric monoclonal antibody selective for the epidermal growth factor receptor that induces a broad range of cellular responses that enhance tumor sensitivity to radiotherapy and chemotherapeutic agents. However, it can cause adverse events in the patient including acneiform eruption, asthenia, abdominal pain and nausea/vomiting. We report a case of severe acneiform eruption induced by cetuximab in a 56-year-old man with colorectal cancer and liver metastases. PMID:18972607

Increasing cellular level of phosphatidic acid enhances FGF-1 production in long term-cultured rat astrocytes.

PubMed

Nagayasu, Yuko; Morita, Shin-Ya; Hayashi, Hideki; Miura, Yutaka; Yokoyama, Kazuki; Michikawa, Makoto; Ito, Jin-Ichi

2014-05-14

We found in a previous study that both mRNA expression and release of fibroblast growth factor 1 (FGF-1) are greater in rat astrocytes that are long term-cultured for one month (W/M cells) than in the cells cultured for one week (W/W cells). However, FGF-1 does not enhance phosphorylation of Akt, MEK, and ERK in W/M cells, while it does in W/W cells. In this work we studied the mechanism to cause these differences between W/W and W/M cells in culture. As it is known that long term culture generates oxidative stress, we characterized the stresses which W/M cells undergo in comparison with W/W cells. The levels of superoxide dismutase 1 (SOD1) and mitochondrial Bax were higher in W/M cells than in W/W cells. W/M cells recovered their ability to respond to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK in the presence of antioxidants. Oxidative stress induced by hydrogen peroxide (H2O2) had no effect on mRNA expression of FGF-1 in W/W cells, although H2O2 enhances release of FGF-1 from W/W cells without inducing apoptosis. The influence of cell density was studied on mRNA expression of FGF-1 and cellular response to FGF-1, as an increasing cell density is observed in W/M cells. The increasing cell density enhanced mRNA expression of FGF-1 in W/W cells without suppression of responses to FGF-1. The decrease in cell density lowered the FGF-1 mRNA expression in W/M cells without recovery of the response to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK. These findings suggest that oxidative stress attenuate sensitivity to FGF-1 and higher cell density may enhance FGF-1 expression in W/M cells. In addition, we found that the cellular level of phosphatidic acid (PA) increased in H2O2-treated W/W and W/M cells and decreased by the treatment with antioxidants, and that PA enhances the mRNA expression of FGF-1 in the W/W cells. These findings suggest that the increasing PA production may enhance FGF-1 expression to protect astrocytes against oxidative stress induced by long-term culture. Copyright © 2014 Elsevier B.V. All rights reserved.

In vivo imaging of cancer cell size and cellularity using temporal diffusion spectroscopy.

PubMed

Jiang, Xiaoyu; Li, Hua; Xie, Jingping; McKinley, Eliot T; Zhao, Ping; Gore, John C; Xu, Junzhong

2017-07-01

A temporal diffusion MRI spectroscopy based approach has been developed to quantify cancer cell size and density in vivo. A novel imaging microstructural parameters using limited spectrally edited diffusion (IMPULSED) method selects a specific limited diffusion spectral window for an accurate quantification of cell sizes ranging from 10 to 20 μm in common solid tumors. In practice, it is achieved by a combination of a single long diffusion time pulsed gradient spin echo (PGSE) and three low-frequency oscillating gradient spin echo (OGSE) acquisitions. To validate our approach, hematoxylin and eosin staining and immunostaining of cell membranes, in concert with whole slide imaging, were used to visualize nuclei and cell boundaries, and hence, enabled accurate estimates of cell size and cellularity. Based on a two compartment model (incorporating intra- and extracellular spaces), accurate estimates of cell sizes were obtained in vivo for three types of human colon cancers. The IMPULSED-derived apparent cellularities showed a stronger correlation (r = 0.81; P < 0.0001) with histology-derived cellularities than conventional ADCs (r = -0.69; P < 0.03). The IMPULSED approach samples a specific region of temporal diffusion spectra with enhanced sensitivity to length scales of 10-20 μm, and enables measurements of cell sizes and cellularities in solid tumors in vivo. Magn Reson Med 78:156-164, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Targeting glutamine metabolism in multiple myeloma enhances BIM binding to BCL-2 eliciting synthetic lethality to venetoclax.

PubMed

Bajpai, R; Matulis, S M; Wei, C; Nooka, A K; Von Hollen, H E; Lonial, S; Boise, L H; Shanmugam, M

2016-07-28

Multiple myeloma (MM) is a plasma cell malignancy that is largely incurable due to development of resistance to therapy-elicited cell death. Nutrients are intricately connected to maintenance of cellular viability in part by inhibition of apoptosis. We were interested to determine if examination of metabolic regulation of BCL-2 proteins may provide insight on alternative routes to engage apoptosis. MM cells are reliant on glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. We and others have demonstrated that glucose maintains levels of key resistance-promoting BCL-2 family member, myeloid cell leukemic factor 1 (MCL-1). Cells continuing to survive in the absence of glucose or glutamine were found to maintain expression of MCL-1 but importantly induce pro-apoptotic BIM expression. One potential mechanism for continued survival despite induction of BIM could be due to binding and sequestration of BIM to alternate pro-survival BCL-2 members. Our investigation revealed that cells surviving glutamine withdrawal in particular, enhance expression and binding of BIM to BCL-2, consequently sensitizing these cells to the BH3 mimetic venetoclax. Glutamine deprivation-driven sensitization to venetoclax can be reversed by metabolic supplementation with TCA cycle intermediate α-ketoglutarate. Inhibition of glucose metabolism with the GLUT4 inhibitor ritonavir elicits variable cytotoxicity in MM that is marginally enhanced with venetoclax treatment, however, targeting glutamine metabolism with 6-diazo-5-oxo-l-norleucine uniformly sensitized MM cell lines and relapse/refractory patient samples to venetoclax. Our studies reveal a potent therapeutic strategy of metabolically driven synthetic lethality involving targeting glutamine metabolism for sensitization to venetoclax in MM.

Targeting glutamine metabolism in multiple myeloma enhances BIM binding to BCL-2 eliciting synthetic lethality to venetoclax

PubMed Central

Bajpai, R; Matulis, SM; Wei, C; Nooka, AK; Von Hollen, HE; Lonial, S; Boise, LH; Shanmugam, M

2016-01-01

Multiple myeloma (MM) is a plasma cell malignancy that is largely incurable due to development of resistance to therapy-elicited cell death. Nutrients are intricately connected to maintenance of cellular viability in part by inhibition of apoptosis. We were interested to determine if examination of metabolic regulation of BCL-2 proteins may provide insight on alternative routes to engage apoptosis. MM cells are reliant on glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. We and others have demonstrated that glucose maintains levels of key resistance-promoting BCL-2 family member, myeloid cell leukemic factor 1 (MCL-1). Cells continuing to survive in the absence of glucose or glutamine were found to maintain expression of MCL-1 but importantly induce pro-apoptotic BIM expression. One potential mechanism for continued survival despite induction of BIM could be due to binding and sequestration of BIM to alternate pro-survival BCL-2 members. Our investigation revealed that cells surviving glutamine withdrawal in particular, enhance expression and binding of BIM to BCL-2, consequently sensitizing these cells to the BH3 mimetic venetoclax. Glutamine deprivation-driven sensitization to venetoclax can be reversed by metabolic supplementation with TCA cycle intermediate α-ketoglutarate. Inhibition of glucose metabolism with the GLUT4 inhibitor ritonavir elicits variable cytotoxicity in MM that is marginally enhanced with venetoclax treatment, however, targeting glutamine metabolism with 6-diazo-5-oxo-l-norleucine uniformly sensitized MM cell lines and relapse/refractory patient samples to venetoclax. Our studies reveal a potent therapeutic strategy of metabolically driven synthetic lethality involving targeting glutamine metabolism for sensitization to venetoclax in MM. PMID:26640142

Label-free electrochemical aptasensor constructed by layer-by-layer technology for sensitive and selective detection of cancer cells.

PubMed

Wang, Tianshu; Liu, Jiyang; Gu, Xiaoxiao; Li, Dan; Wang, Jin; Wang, Erkang

2015-07-02

Here, a cytosensor was constructed with ferrocene-appended poly(allylamine hydrochloride) (Fc-PAH) functionalized graphene (Fc-PAH-G), poly(sodium-p-styrenesulfonate) (PSS) and aptamer (AS1411) by layer-by-layer assembly technology. The hybrid nanocomposite Fc-PAH-G not only brings probes on the electrode and also promotes electron transfer between the probes and the substrate electrode. Meanwhile, LBL technology provides more effective probes to enhance amplified signal for improving the sensitivity of the detection. While AS1411 forming G-quardruplex structure and binding cancer cells, the current response of the sensing electrode decreased due to the insulating properties of cellular membrane. Differential pulse voltammetry (DPV) was performed to investigate the electrochemical detection of HeLa cells attributing to its sensitivity of the current signal change. The as-prepared aptasensor showed a high sensitivity and good stability, a widely detection range from 10 to 10(6) cells/mL with a detection limit as low as 10 cells/mL for the detection of cancer cells. Copyright © 2015. Published by Elsevier B.V.

Microglia-Derived Cytokines/Chemokines Are Involved in the Enhancement of LPS-Induced Loss of Nigrostriatal Dopaminergic Neurons in DJ-1 Knockout Mice

PubMed Central

Chien, Chia-Hung; Lee, Ming-Jen; Liou, Houng-Chi; Liou, Horng-Huei; Fu, Wen-Mei

2016-01-01

Mutation of DJ-1 (PARK7) has been linked to the development of early-onset Parkinson’s disease (PD). However, the underlying molecular mechanism is still unclear. This study is aimed to compare the sensitivity of nigrostriatal dopaminergic neurons to lipopolysaccharide (LPS) challenge between DJ-1 knockout (KO) and wild-type (WT) mice, and explore the underlying cellular and molecular mechanisms. Our results found that the basal levels of interferon (IFN)-γ (the hub cytokine) and interferon-inducible T-cell alpha chemoattractant (I-TAC) (a downstream mediator) were elevated in the substantia nigra of DJ-1 KO mice and in microglia cells with DJ-1 deficiency, and the release of cytokine/chemokine was greatly enhanced following LPS administration in the DJ-1 deficient conditions. In addition, direct intranigral LPS challenge caused a greater loss of nigrostriatal dopaminergic neurons and striatal dopamine content in DJ-1 KO mice than in WT mice. Furthermore, the sensitization of microglia cells to LPS challenge to release IFN-γ and I-TAC was via the enhancement of NF-κB signaling, which was antagonized by NF-κB inhibitors. LPS-induced increase in neuronal death in the neuron-glia co-culture was enhanced by DJ-1 deficiency in microglia, which was antagonized by the neutralizing antibodies against IFN-γ or I-TAC. These results indicate that DJ-1 deficiency sensitizes microglia cells to release IFN-γ and I-TAC and causes inflammatory damage to dopaminergic neurons. The interaction between the genetic defect (i.e. DJ-1) and inflammatory factors (e.g. LPS) may contribute to the development of PD. PMID:26982707

γ-radiation induces cellular sensitivity and aberrant methylation in human tumor cell lines.

PubMed

Kumar, Ashok; Rai, Padmalatha S; Upadhya, Raghavendra; Vishwanatha; Prasada, K Shama; Rao, B S Satish; Satyamoorthy, Kapettu

2011-11-01

Ionizing radiation induces cellular damage through both direct and indirect mechanisms, which may include effects from epigenetic changes. The purpose of this study was to determine the effect of ionizing radiation on DNA methylation patterns that may be associated with altered gene expression. Sixteen human tumor cell lines originating from various cancers were initially tested for radiation sensitivity by irradiating them with γ-radiation in vitro and subsequently, radiation sensitive and resistant cell lines were treated with different doses of a demethylating agent, 5-Aza-2'-Deoxycytidine (5-aza-dC) and a chromatin modifier, Trichostatin-A (TSA). Survival of these cell lines was measured using 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium (MTT) and clonogenic assays. The effect of radiation on global DNA methylation was measured using reverse phase high performance liquid chromatography (RP-HPLC). The transcription response of methylated gene promoters, from cyclin-dependent kinase inhibitor 2A (p16(INK4a)) and ataxia telangiectasia mutated (ATM) genes, to radiation was measured using a luciferase reporter assay. γ-radiation resistant (SiHa and MDAMB453) and sensitive (SaOS2 and WM115) tumor cell lines were examined for the relationship between radiation sensitivity and DNA methylation. Treatment of cells with 5-aza-dC and TSA prior to irradiation enhanced DNA strand breaks, G2/M phase arrest, apoptosis and cell death. Exposure to γ-radiation led to global demethylation in a time-dependent manner in tumor cells in relation to resistance and sensitivity to radiation with concomitant activation of p16(INK4a) and ATM gene promoters. These results provide important information on alterations in DNA methylation as one of the determinants of radiation effects, which may be associated with altered gene expression. Our results may help in delineating the mechanisms of radiation resistance in tumor cells, which can influence diagnosis, prognosis and eventually therapy for human cancers.

Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels

PubMed Central

Puissant, Madeleine M.; Mouradian, Gary C.; Liu, Pengyuan; Hodges, Matthew R.

2017-01-01

Ventilation is continuously adjusted by a neural network to maintain blood gases and pH. Acute CO2 and/or pH regulation requires neural feedback from brainstem cells that encode CO2/pH to modulate ventilation, including but not limited to brainstem serotonin (5-HT) neurons. Brainstem 5-HT neurons modulate ventilation and are stimulated by hypercapnic acidosis, the sensitivity of which increases with increasing postnatal age. The proper function of brainstem 5-HT neurons, particularly during post-natal development is critical given that multiple abnormalities in the 5-HT system have been identified in victims of Sudden Infant Death Syndrome. Here, we tested the hypothesis that there are age-dependent increases in expression of pH-sensitive ion channels in brainstem 5-HT neurons, which may underlie their cellular CO2/pH sensitivity. Midline raphe neurons were acutely dissociated from neonatal and mature transgenic SSePet-eGFP rats [which have enhanced green fluorescent protein (eGFP) expression in all 5-HT neurons] and sorted with fluorescence-activated cell sorting (FACS) into 5-HT-enriched and non-5-HT cell pools for subsequent RNA extraction, cDNA library preparation and RNA sequencing. Overlapping differential expression analyses pointed to age-dependent shifts in multiple ion channels, including but not limited to the pH-sensitive potassium ion (K+) channel genes kcnj10 (Kir4.1), kcnj16 (Kir5.1), kcnk1 (TWIK-1), kcnk3 (TASK-1) and kcnk9 (TASK-3). Intracellular contents isolated from single adult eGFP+ 5-HT neurons confirmed gene expression of Kir4.1, Kir5.1 and other K+ channels, but also showed heterogeneity in the expression of multiple genes. 5-HT neuron-enriched cell pools from selected post-natal ages showed increases in Kir4.1, Kir5.1, and TWIK-1, fitting with age-dependent increases in Kir4.1 and Kir5.1 protein expression in raphe tissue samples. Immunofluorescence imaging confirmed Kir5.1 protein was co-localized to brainstem neurons and glia including 5-HT neurons as expected. However, Kir4.1 protein expression was restricted to glia, suggesting that it may not contribute to 5-HT neuron pH sensitivity. Although there are caveats to this approach, the data suggest that pH-sensitive Kir5.1 channels may underlie cellular CO2/pH chemosensitivity in brainstem 5-HT neurons. PMID:28270749

Cellular Mechanisms Underlying Bone-Forming Cell Proliferative Response to Hypergravity

NASA Technical Reports Server (NTRS)

Vercoutere, W.; Parra, M.; DaCosta, M.; Wing, A.; Roden, C.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

2004-01-01

Life on Earth has evolved under the continuous influence of gravity (1-g). As humans explore and develop space, however, we must learn to adapt to an environment with little or no gravity. Studies indicate that lack of weightbearing for vertebrates occurring with immobilization, paralysis, or in a microgravity environment may cause muscle and bone atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) (consisting of molecules such as collagen, fibronectin, and laminin) in mechanosensitive tissues. We test for the presence of gravity-sensitive pathways in bone-forming cells (osteoblasts) using hypergravity applied by a cell culture centrifuge. Stimulation of 50 times gravity (50-g) increased proliferation in primary rat osteoblasts for cells grown on collagen Type I and fibronectin, but not on laminin or uncoated surfaces. Survival was also enhanced during hypergravity stimulation by the presence of ECM. Bromodeoxyuridine incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. Reverse transcription-polymerase chain reaction was used to test for all possible integrins. Our combined results indicate that beta1 and/or beta3 integrin subunits may be involved. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signalling pathways which are sensitive to g-level. Further research to define the mechanisms involved will provide direction so that we may better adapt and counteract bone atrophy caused by the lack of weightbearing.

RADIATION EFFECTS ON IMMUNE MECHANISMS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoner, R.D.; Hale, W.M.

1963-03-01

Experiments were performed on pathogen-free Swiss albino mice to determine the repressive effect of ionizing radiation on immune mechanisms. In animals given sublethal doses of Co/sup 60/ gamma radiation by acute short-term exposure or by chronic long-term exposure at a low dose rate, ability to produce antibody was inhibited or abolished, and natural resistance and active and passive immunity to pneumococcal and Trichinella infections were severely depressed. It appears that the repression resulted from damage to the cellular defensive mechanisms of the host. Active immunity and natural resistance to influenza virus infections were not altered significantly by radiation. Exposure tomore » radiation enhanced the severity of anaphylactic shock markedly in mice previously sensitized to tetanus toxoid and challenged with tetanus toxoid after radiation. Chronic exposure to radiation caused immediate increased sensitivity to fatal anaphylaxis. (auth)« less

A class of parametrically excited calcium oscillation detectors.

PubMed Central

Izu, L T; Spangler, R A

1995-01-01

Intracellular Ca2+ oscillations are often a response to external signals such as hormones. Changes in the external signal can alter the frequency, amplitude, or form of the oscillations suggesting that information is encoded in the pattern of Ca2+ oscillations. How might a cell decode this signal? We show that an excitable system whose kinetic parameters are modulated by the Ca2+ concentration can function as a Ca2+ oscillation detector. Such systems have the following properties: (1) They are more sensitive to an oscillatory than to a steady Ca2+ signal. (2) Their response is largely independent of the signal amplitude. (3) They can extract information from a noisy signal. (4) Unlike other frequency sensitive detectors, they have a flat frequency response. These properties make a Ca(2+)-sensitive excitable system nearly ideal for detecting and decoding Ca2+ oscillations. We suggest that Ca2+ oscillations, in concert with these detectors, can act as cellular timekeepers to coordinate related biochemical reactions and enhance their overall efficiency. PMID:7787048

Role of TRP ion channels in cancer and tumorigenesis.

PubMed

Shapovalov, George; Ritaine, Abigael; Skryma, Roman; Prevarskaya, Natalia

2016-05-01

Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has captured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca(2+) distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca(2+) homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.

Cell tracking with caged xenon: using cryptophanes as MRI reporters upon cellular internalization.

PubMed

Klippel, Stefan; Döpfert, Jörg; Jayapaul, Jabadurai; Kunth, Martin; Rossella, Federica; Schnurr, Matthias; Witte, Christopher; Freund, Christian; Schröder, Leif

2014-01-07

Caged xenon has great potential in overcoming sensitivity limitations for solution-state NMR detection of dilute molecules. However, no application of such a system as a magnetic resonance imaging (MRI) contrast agent has yet been performed with live cells. We demonstrate MRI localization of cells labeled with caged xenon in a packed-bed bioreactor working under perfusion with hyperpolarized-xenon-saturated medium. Xenon hosts enable NMR/MRI experiments with switchable contrast and selectivity for cell-associated versus unbound cages. We present MR images with 10(3) -fold sensitivity enhancement for cell-internalized, dual-mode (fluorescence/MRI) xenon hosts at low micromolar concentrations. Our results illustrate the capability of functionalized xenon to act as a highly sensitive cell tracer for MRI detection even without signal averaging. The method will bridge the challenging gap for translation to in vivo studies for the optimization of targeted biosensors and their multiplexing applications. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Shock enhancement of cellular materials subjected to intensive pulse loading

NASA Astrophysics Data System (ADS)

Zhang, J.; Fan, J.; Wang, Z.; Zhao, L.; Li, Z.

2018-03-01

Cellular materials can dissipate a large amount of energy due to their considerable stress plateau, which contributes to their extensive applications in structural design for crashworthiness. However, in some experiments with specimens subjected to intense impact loads, transmitted stress enhancement has been observed, leading to severe damage to the objects protected. Transmitted stress through two-dimensional Voronoi cellular materials as a protective device is qualitatively studied in this paper. Dimensionless parameters of material properties and loading parameters are defined to give critical conditions for shock enhancement and clarify the correlation between the deformations and stress enhancement. The effect of relative density on this amplifying phenomenon is investigated as well. In addition, local strain fields are calculated by using the optimal local deformation gradient, which gives a clear presentation of deformations and possible local non-uniformity in the crushing process. This research provides valuable insight into the reliability of cellular materials as protective structures.

Inflammatory responses to psychological stress in fatigued breast cancer survivors: relationship to glucocorticoids.

PubMed

Bower, Julienne E; Ganz, Patricia A; Aziz, Najib; Olmstead, Richard; Irwin, Michael R; Cole, Steve W

2007-03-01

Fatigue is a common problem following cancer treatment and our previous studies suggest that a chronic inflammatory process might contribute to cancer-related fatigue. However, immune responses to challenge have not yet been evaluated among individuals with cancer-related fatigue, and it is not known what mechanisms drive increased levels of inflammatory markers in fatigued cancer survivors. We have previously reported that fatigued breast cancer survivors show a blunted cortisol response to an experimental psychological stressor. In this report, we focus on inflammatory responses to this stressor and their relationship to circulating glucocorticoids and cellular sensitivity to glucocorticoid inhibition. Relative to non-fatigued control survivors, participants experiencing persistent fatigue showed significantly greater increases in LPS-stimulated production of IL-1beta and IL-6 following the stressor (Group x Time interaction: p<.05). Fatigued participants did not show any difference in cellular sensitivity to cortisol inhibition of cytokine production, but they did show significantly less salivary cortisol increase in the aftermath of the stressor. Moreover, blunted cortisol responses were associated with significantly increased production of IL-6 in response to LPS stimulation (p<.05). These data provide further evidence of enhanced inflammatory processes in fatigued breast cancer survivors and suggest that these processes may stem in part from decreased glucocorticoid response to stress.

Insulin Resistance in Alzheimer Disease: p53 and MicroRNAs as Important Players.

PubMed

Gasiorowski, Kazimierz; Brokos, Barbara; Leszek, Jerzy; Tarasov, Vadim V; Ashraf, Ghulam Md; Aliev, Gjumrakch

2017-01-01

Glucose homeostasis is crucial for neuronal survival, synaptic plasticity, and is indispensable for learning and memory. Reduced sensitivity of cells to insulin and impaired insulin signaling in brain neurons participate in the pathogenesis of Alzheimer disease (AD). The tumor suppressor protein p53 coordinates with multiple cellular pathways in response to DNA damage and cellular stresses. However, prolonged stress conditions unveil deleterious effects of p53-evoked insulin resistance in neurons; enhancement of transcription of pro-oxidant factors, accumulation of toxic metabolites (e.g. ceramide and products of advanced glycation) and ROS-modified cellular components, together with the activation of proapoptotic genes, could finally induce a suicide death program of autophagy/apoptosis in neurons. Recent studies reveal the impact of p53 on expression and processing of several microRNAs (miRs) under DNA damage-inducing conditions. Additionally, the role of miRs in promotion of insulin resistance and type 2 diabetes mellitus has been well documented. Detailed recognition of the role of p53/miRs crosstalk in driving insulin resistance in AD brains could improve the disease diagnostics and aid future therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Graphene-enhanced imaging of microRNA with enzyme-free signal amplification of catalyzed hairpin assembly in living cells.

PubMed

Liu, Haiyun; Tian, Tian; Ji, Dandan; Ren, Na; Ge, Shenguang; Yan, Mei; Yu, Jinghua

2016-11-15

In situ imaging of miRNA in living cells could help us to monitor the miRNA expression in real time and obtain accurate information for studying miRNA related bioprocesses and disease. Given the low-level expression of miRNA, amplification strategies for intracellular miRNA are imperative. Here, we propose an amplification strategy with a non-destructive enzyme-free manner in living cells using catalyzed hairpin assembly (CHA) based on graphene oxide (GO) for cellular miRNA imaging. The enzyme-free CHA exhibits stringent recognition and excellent signal amplification of miRNA in the living cells. GO is a good candidate as a fluorescence quencher and cellular carrier. Taking the advantages of the CHA and GO, we can monitor the miRNA at low level in living cells with a simple, sensitive and real-time manner. Finally, imaging of miRNAs in the different expression cells is realized. The novel method could supply an effective tool to visualize intracellular low-level miRNAs and help us to further understand the role of miRNAs in cellular processes. Copyright © 2016 Elsevier B.V. All rights reserved.

Thoracic organ transplantation: laboratory methods.

PubMed

Patel, Jignesh K; Kobashigawa, Jon A

2013-01-01

Although great progress has been achieved in thoracic organ transplantation through the development of effective immunosuppression, there is still significant risk of rejection during the early post-transplant period, creating a need for routine monitoring for both acute antibody and cellular mediated rejection. The currently available multiplexed, microbead assays utilizing solubilized HLA antigens afford the capability of sensitive detection and identification of HLA and non-HLA specific antibodies. These assays are being used to assess the relative strength of donor specific antibodies; to permit performance of virtual crossmatches which can reduce the waiting time to transplantation; to monitor antibody levels during desensitization; and for heart transplants to monitor antibodies post-transplant. For cell mediated immune responses, the recent development of gene expression profiling has allowed noninvasive monitoring of heart transplant recipients yielding predictive values for acute cellular rejection. T cell immune monitoring in heart and lung transplant recipients has allowed individual tailoring of immunosuppression, particularly to minimize risk of infection. While the current antibody and cellular laboratory techniques have enhanced the ability to manage thoracic organ transplant recipients, future developments from improved understanding of microchimerism and graft tolerance may allow more refined allograft monitoring techniques.

Platelike WO3 sensitized with CdS quantum dots heterostructures for photoelectrochemical dynamic sensing of H2O2 based on enzymatic etching.

PubMed

Wang, Yanhu; Gao, Chaomin; Ge, Shenguang; Yu, Jinghua; Yan, Mei

2016-11-15

A platelike tungsten trioxide (WO3) sensitized with CdS quantum dots (QDs) heterojunction is developed for solar-driven, real-time, and selective photoelectrochemical (PEC) sensing of H2O2 in the living cells. The structure is synthesized by hydrothermally growing platelike WO3 on fluorine doped tin oxide (FTO) and subsequently sensitized with CdS QDs. The as-prepared WO3-CdS QDs heterojunction achieve significant photocurrent enhancement, which is remarkably beneficial for light absorption and charge carrier separation. Based on the enzymatic etching of CdS QDs enables the activation of quenching the charge transfer efficiency, thus leading to sensitive PEC recording of H2O2 level in buffer and cellular environments. The results indicated that the proposed method will pave the way for the development of excellent PEC sensing platform with the quantum dot sensitization. This study could also provide a new train of thought on designing of self-operating photoanode in PEC sensing, promoting the application of semiconductor nanomaterials in photoelectrochemistry. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell biological effects of hyperthermia alone or combined with radiation or drugs: a short introduction to newcomers in the field.

PubMed

Kampinga, Harm H

2006-05-01

Hyperthermia results in protein unfolding that, if not properly chaperoned by Heat Shock Proteins (HSP), can lead to irreversible and toxic protein aggregates. Elevating HSP prior to heating makes cells thermotolerant. Hyperthermia also can enhance the sensitivity of cells to radiation and drugs. This sensitization to drugs or radiation is not directly related to altered HSP expression. However, altering HSP expression before heat and radiation or drug treatment will affect the extent of thermal sensitization because the HSP will attenuate the heat-induced protein damage that is responsible for radiation- or drug-sensitization. For thermal radiosensitization, nuclear protein damage is considered to be responsible for hyperthermic effects on DNA repair, in particular base excision repair. Hyperthermic drug sensitization can be seen for a number of anti-cancer drugs, especially of alkylating agents. Synergy between heat and drugs may arise from multiple events such as heat damage to ABC transporters (drug accumulation), intra-cellular drug detoxification pathways and repair of drug-induced DNA adducts. This may be why cells with acquired drug resistance (often multi-factorial) can be made responsive to drugs again by combining the drug treatment with heat.

The cochlear CRF signaling systems and their mechanisms of action in modulating cochlear sensitivity and protection against trauma

PubMed Central

Graham, Christine E.; Basappa, Johnvesly; Turcan, Sevin; Vetter, Douglas E.

2011-01-01

A key requirement for encoding the auditory environment is the ability to dynamically alter cochlear sensitivity. However, merely attaining a steady state of maximal sensitivity is not a viable solution since the sensory cells and ganglion cells of the cochlea are prone to damage following exposure to loud sound. Most often, such damage is via initial metabolic insult that can lead to cellular death. Thus, establishing the highest sensitivity must be balanced with protection against cellular metabolic damage that can lead to loss of hair cells and ganglion cells, resulting in loss of frequency representation. While feedback mechanisms are known to exist in the cochlea that alter sensitivity, they respond only after stimulus encoding, allowing potentially damaging sounds to impact the inner ear at times coincident with increased sensitivity. Thus, questions remain concerning the endogenous signaling systems involved in dynamic modulation of cochlear sensitivity and protection against metabolic stress. Understanding endogenous signaling systems involved in cochlear protection may lead to new strategies and therapies for prevention of cochlear damage and consequent hearing loss. We have recently discovered a novel cochlear signaling system that is molecularly equivalent to the classic hypothalamic-pituitary-adrenal (HPA) axis. This cochlear HPA-equivalent system functions to balance auditory sensitivity and susceptibility to noise-induced hearing loss, and also protects against cellular metabolic insults resulting from exposures to ototoxic drugs. We review the anatomy, physiology, and cellular signaling of this system, and compare it to similar signaling in other organs/tissues of the body. PMID:21909974

Elastomeric Cellular Structure Enhanced by Compressible Liquid Filler

NASA Astrophysics Data System (ADS)

Sun, Yueting; Xu, Xiaoqing; Xu, Chengliang; Qiao, Yu; Li, Yibing

2016-05-01

Elastomeric cellular structures provide a promising solution for energy absorption. Their flexible and resilient nature is particularly relevant to protection of human bodies. Herein we develop an elastomeric cellular structure filled with nanoporous material functionalized (NMF) liquid. Due to the nanoscale infiltration in NMF liquid and its interaction with cell walls, the cellular structure has a much enhanced mechanical performance, in terms of loading capacity and energy absorption density. Moreover, it is validated that the structure is highly compressible and self-restoring. Its hyper-viscoelastic characteristics are elucidated.

Double NIR laser stimulation and enhancing the thermal sensitivity of Er3+/Tm3+/Nd3+ doped multilayer core-shell nanoparticles.

PubMed

Ba, Zhaojing; Hu, Min; Zhao, Yiming; Wang, Yiqing; Wang, Jing; Zhang, Zhenxi

2018-08-31

Non-contact thermal sensors are important devices to study cellular processes and monitor temperature in vivo. Herein, a novel highly sensitive nanothermometer based on NaYF 4 :Yb,Er@ NaYF 4 @NaYF 4 :Yb,Tm@ NaYF 4 :Nd (denoted as Er@Y@Tm@Nd) was prepared by a facile solvothermal method. When excited by the near-infrared (NIR) light of 808 and 980 nm, the as-prepared Er@Y@Tm@Nd nanoparticles could emit both blue and green light, respectively, since the lanthanide cations responsible for these emissions are gathered inside this nanostructure. The green and blue light intensity ratio exhibits obvious temperature dependence in the range of the physiological temperature. Additionally, the fluorescence intensity of Er 3+ and Tm 3+ are also greatly enhanced due to the multilayer structure that implies avoiding the Er 3+ and Tm 3+ energy cross-relaxation by introduction of a NaYF 4 wall between them. The as-prepared core-shell-shell-shell structure with Er 3+ and Tm 3+ in different layers improves dozens of times of the thermal sensitivity based on the non-thermal coupling levels of the probe: the maximum values for the sensitivity are 2.95% K -1 (I Er-521 /I Tm-450 ) and 6.30% K -1 (I Tm-474 /I Er-541 ) when excited by 980 and 808 nm laser sources, respectively. These values are well above those previously reported (<0.7% K -1 ), indicating that the prepared nanostructures are temperature sensors with excellent thermal sensitivity and sensitive to NIR wavelength excitation that makes them highly preferred for thermal detection.

Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, X.; Li, L.; Zhang, L.

Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidativemore » stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H{sub 2}O{sub 2} generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H{sub 2}O{sub 2} generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H{sub 2}O{sub 2} accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.« less

Targeting the Oxidative Stress Response System of Fungi with Redox-Potent Chemosensitizing Agents

PubMed Central

Kim, Jong H.; Chan, Kathleen L.; Faria, Natália C. G.; Martins, M. de L.; Campbell, Bruce C.

2012-01-01

The cellular antioxidant system is a target in the antifungal action of amphotericin B (AMB) and itraconazole (ITZ), in filamentous fungi. The sakAΔ mutant of Aspergillus fumigatus, a mitogen-activated protein kinase (MAPK) gene deletion mutant in the antioxidant system, was found to be more sensitive to AMB or ITZ than other A. fumigatus strains, a wild type and a mpkCΔ mutant (a MAPK gene deletion mutant in the polyalcohol sugar utilization system). Complete fungal kill (≥99.9%) by ITZ or AMB was also achieved by much lower dosages for the sakAΔ mutant than for the other strains. It appears msnA, an Aspergillus ortholog to Saccharomyces cerevisiae MSN2 (encoding a stress-responsive C2H2-type zinc-finger regulator) and sakA and/or mpkC (upstream MAPKs) are in the same stress response network under tert-butyl hydroperoxide (t-BuOOH)-, hydrogen peroxide (H2O2)- or AMB-triggered toxicity. Of note is that ITZ-sensitive yeast pathogens were also sensitive to t-BuOOH, showing a connection between ITZ sensitivity and antioxidant capacity of fungi. Enhanced antifungal activity of AMB or ITZ was achieved when these drugs were co-applied with redox-potent natural compounds, 2,3-dihydroxybenzaldehyde, thymol or salicylaldehyde, as chemosensitizing agents. We concluded that redox-potent compounds, which target the antioxidant system in fungi, possess a chemosensitizing capacity to enhance efficacy of conventional drugs. PMID:22438852

Mechanical sensitivity of Piezo1 ion channels can be tuned by cellular membrane tension

PubMed Central

Lewis, Amanda H; Grandl, Jörg

2015-01-01

Piezo1 ion channels mediate the conversion of mechanical forces into electrical signals and are critical for responsiveness to touch in metazoans. The apparent mechanical sensitivity of Piezo1 varies substantially across cellular environments, stimulating methods and protocols, raising the fundamental questions of what precise physical stimulus activates the channel and how its stimulus sensitivity is regulated. Here, we measured Piezo1 currents evoked by membrane stretch in three patch configurations, while simultaneously visualizing and measuring membrane geometry. Building on this approach, we developed protocols to minimize resting membrane curvature and tension prior to probing Piezo1 activity. We find that Piezo1 responds to lateral membrane tension with exquisite sensitivity as compared to other mechanically activated channels and that resting tension can drive channel inactivation, thereby tuning overall mechanical sensitivity of Piezo1. Our results explain how Piezo1 can function efficiently and with adaptable sensitivity as a sensor of mechanical stimulation in diverse cellular contexts. DOI: http://dx.doi.org/10.7554/eLife.12088.001 PMID:26646186

Multimodal imaging of lymph nodes and tumors using glycol-chitosan-coated gold nanoparticles (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sun, In-Cheol; Dumani, Diego S.; Emelianov, Stanislav Y.

2017-03-01

A key step in staging cancer is the diagnosis of metastasis that spreads through lymphatic system. For this reason, researchers develop various methods of sentinel lymph node mapping that often use a radioactive tracer. This study introduces a safe, cost-effective, high-resolution, high-sensitivity, and real-time method of visualizing the sentinel lymph node: ultrasound-guided photoacoustic (US/PA) imaging augmented by a contrast agent. In this work, we use clearable gold nanoparticles covered by a biocompatible polymer (glycol chitosan) to enhance cellular uptake by macrophages abundant in lymph nodes. We incubate macrophages with glycol-chitosan-coated gold nanoparticles (0.05 mg Au/ml), and then fix them with paraformaldehyde solution for an analysis of in vitro dark-field microscopy and cell phantom. The analysis shows enhanced cellular uptake of nanoparticles by macrophages and strong photoacoustic signal from labeled cells in tissue-mimicking cell phantoms consisting gelatin solution (6 %) with silica gel (25 μm, 0.3%) and fixed macrophages. The in-vivo US/PA imaging of cervical lymph nodes in healthy mice (nu/nu, female, 5 weeks) indicates a strong photoacoustic signal from a lymph node 10 minutes post-injection (2.5 mg Au/ml, 80 μl). The signal intensity and the nanoparticle-labeled volume of tissue within the lymph node continues to increase until 4 h post-injection. Histological analysis further confirms the accumulation of gold nanoparticles within the lymph nodes. This work suggests the feasibility of molecular/cellular US/PA imaging with biocompatible gold nanoparticles as a photoacoustic contrast agent in the diagnosis of lymph-node-related diseases.

Bioluminescence enhancement through an added washing protocol enabling a greater sensitivity to carbofuran toxicity.

PubMed

Jia, Kun; Eltzov, Evgeni; Marks, Robert S; Ionescu, Rodica E

2013-10-01

The effects of carbofuran toxicity on a genetically modified bacterial strain E. coli DPD2794 were enhanced using a new bioluminescent protocol which consisted of three consecutive steps: incubation, washing and luminescence reading. Specifically, in the first step, several concentrations of carbofuran aqueous solutions were incubated with different bacterial suspensions at recorded optical densities for different lengths of time. Thereafter, the resulting bacterial/toxicant mixtures were centrifuged and the aged cellular supernatant replaced with fresh medium. In the final step, the carbofuran- induced bioluminescence to the exposed E. coli DPD2794 bacteria was shown to provide a faster and higher intensity when recorded at a higher temperature at30°C which is not usually used in the literature. It was found that the incubation time and the replacement of aged cellular medium were essential factors to distinguish different concentrations of carbofuran in the bioluminescent assays. From our results, the optimum incubation time for a "light ON" bioluminescence detection of the effect of carbofuran was 6h. Thanks to the replacement of the aged cellular medium, a group of additional peaks starting around 30min were observed and we used the corresponding areas under the curve (AUC) at different contents of carbofuran to produce the calibration curve. Based on the new protocol, a carbofuran concentration of 0.5pg/mL can be easily determined in a microtiter plate bioluminescent assay, while a non-wash protocol provides an unexplainable order of curve evolutionswhich does not allow the user to determine the concentration. Copyright © 2013 Elsevier Inc. All rights reserved.

Comprehensive Evaluation of the Contribution of X Chromosome Genes to Platinum Sensitivity

PubMed Central

Gamazon, Eric R.; Im, Hae Kyung; O’Donnell, Peter H.; Ziliak, Dana; Stark, Amy L.; Cox, Nancy J.; Dolan, M. Eileen; Huang, Rong Stephanie

2011-01-01

Utilizing a genome-wide gene expression dataset generated from Affymetrix GeneChip® Human Exon 1.0ST array, we comprehensively surveyed the role of 322 X chromosome gene expression traits on cellular sensitivity to cisplatin and carboplatin. We identified 31 and 17 X chromosome genes whose expression levels are significantly correlated (after multiple testing correction) with sensitivity to carboplatin and cisplatin, respectively, in the combined HapMap CEU and YRI populations (false discovery rate, FDR<0.05). Of those, 14 overlap for both cisplatin and carboplatin. Employing an independent gene expression quantification method, the Illumina Sentrix Human-6 Expression BeadChip, measured on the same HapMap cell lines, we found that 4 and 2 of these genes are significantly associated with carboplatin and cisplatin sensitivity respectively in both analyses. Two genes, CTPS2 and DLG3, were identified by both genome-wide gene expression analyses as correlated with cellular sensitivity to both platinating agents. The expression of DLG3 gene was also found to correlate with cellular sensitivity to platinating agents in NCI60 cancer cell lines. In addition, we evaluated the role of X chromosome gene expression to the observed differences in sensitivity to the platinums between CEU and YRI derived cell lines. Of the 34 distinct genes significantly correlated with either carboplatin or cisplatin sensitivity, 14 are differentially expressed (defined as p<0.05) between CEU and YRI. Thus, sex chromosome genes play a role in cellular sensitivity to platinating agents and differences in the expression level of these genes are an important source of variation that should be included in comprehensive pharmacogenomic studies. PMID:21252287

Citrate- and Succinate-Modified Carbonate Apatite Nanoparticles with Loaded Doxorubicin Exhibit Potent Anticancer Activity against Breast Cancer Cells

PubMed Central

Mehbuba Hossain, Sultana; Chowdhury, Ezharul Hoque

2018-01-01

Biodegradable inorganic apatite-based particle complex is popular for its pH-sensitivity at the endosomal acidic environment to facilitate drug release following cellular uptake. Despite being a powerful anticancer drug, doxorubicin shows severe off-target effects and therefore would need a carrier for the highest effectiveness. We aimed to chemically modify carbonate apatite (CA) with Krebs cycle intermediates, such as citrate and succinate in order to control the growth of the resultant particles to more efficiently carry and transport the anticancer drug into the cancer cells. Citrate- or succinate-modified CA particles were synthesized with different concentrations of sodium citrate or sodium succinate, respectively, in the absence or presence of doxorubicin. The drug loading efficiency of the particles and their cellular uptake were observed by quantifying fluorescence intensity. The average diameter and surface charge of the particles were determined using Zetasizer. Cell viability was assessed by MTT assay. Citrate-modified carbonate apatite (CMCA) exhibited the highest (31.38%) binding affinity for doxorubicin and promoted rapid cellular uptake of the drug, leading to the half-maximal inhibitory concentration 1000 times less than that of the free drug in MCF-7 cells. Hence, CMCA nanoparticles with greater surface area enhance cytotoxicity in different breast cancer cells by enabling higher loading and more efficient cellular uptake of the drug. PMID:29534497

A Novel in Vitro Analog Expressing Learning-Induced Cellular Correlates in Distinct Neural Circuits

ERIC Educational Resources Information Center

Weisz, Harris A.; Wainwright, Marcy L.; Mozzachiodi, Riccardo

2017-01-01

When presented with noxious stimuli, "Aplysia" exhibits concurrent sensitization of defensive responses, such as the tail-induced siphon withdrawal reflex (TSWR) and suppression of feeding. At the cellular level, sensitization of the TSWR is accompanied by an increase in the excitability of the tail sensory neurons (TSNs) that elicit the…

Enhanced cellular uptake of maleimide-modified liposomes via thiol-mediated transport

PubMed Central

Li, Tianshu; Takeoka, Shinji

2014-01-01

With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) – 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG5000-DSPE]/maleimide [M]-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%–45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport. PMID:24940060

Enhanced cellular uptake of maleimide-modified liposomes via thiol-mediated transport.

PubMed

Li, Tianshu; Takeoka, Shinji

2014-01-01

With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) - 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG₅₀₀₀-DSPE]/maleimide [M]-PEG₅₀₀₀-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG₅₀₀₀-DSPE/PEG₅₀₀₀-Glu2C₁₈ at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%-45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport.

Multiple-channel detection of cellular activities by ion-sensitive transistors

NASA Astrophysics Data System (ADS)

Machida, Satoru; Shimada, Hideto; Motoyama, Yumi

2018-04-01

An ion-sensitive field-effect transistor to record cellular activities was demonstrated. This field-effect transistor (bio transistor) includes cultured cells on the gate insulator instead of gate electrode. The bio transistor converts a change in potential underneath the cells into variation of the drain current when ion channels open. The bio transistor has high detection sensitivity to even minute variations in potential utilizing a subthreshold swing region. To open ion channels, a reagent solution (acetylcholine) was added to a human-originating cell cultured on the bio transistor. The drain current was successfully decreased with the addition of acetylcholine. Moreover, we attempted to detect the opening of ion channels using a multiple-channel measurement circuit containing several bio transistors. As a consequence, the drain current distinctly decreased only after the addition of acetylcholine. We confirmed that this measurement system including bio transistors enables to observation of cellular activities sensitively and simultaneously.

Transferrin-conjugated magnetic dextran-spermine nanoparticles for targeted drug transport across blood-brain barrier.

PubMed

Ghadiri, Maryam; Vasheghani-Farahani, Ebrahim; Atyabi, Fatemeh; Kobarfard, Farzad; Mohamadyar-Toupkanlou, Farzaneh; Hosseinkhani, Hossein

2017-10-01

Application of many vital hydrophilic medicines have been restricted by blood-brain barrier (BBB) for treatment of brain diseases. In this study, a targeted drug delivery system based on dextran-spermine biopolymer was developed for drug transport across BBB. Drug loaded magnetic dextran-spermine nanoparticles (DS-NPs) were prepared via ionic gelation followed by transferrin (Tf) conjugation as targeting moiety. The characteristics of Tf conjugated nanoparticles (TDS-NPs) were analyzed by different methods and their cytotoxicity effects on U87MG cells were tested. The superparamagnetic characteristic of TDS-NPs was verified by vibration simple magnetometer. Capecitabine loaded TDS-NPs exhibited pH-sensitive release behavior with enhanced cytotoxicity against U87MG cells, compared to DS-NPs and free capecitabine. Prussian-blue staining and TEM-imaging showed the significant cellular uptake of TDS-NPs. Furthermore, a remarkable increase of Fe concentrations in brain was observed following their biodistribution and histological studies in vivo, after 1 and 7 days of post-injection. Enhanced drug transport across BBB and pH-triggered cellular uptake of TDS-NPs indicated that these theranostic nanocarriers are promising candidate for the brain malignance treatment. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2851-2864, 2017. © 2017 Wiley Periodicals, Inc.

Effects of Cetuximab and Erlotinib on the behaviour of cancer stem cells in head and neck squamous cell carcinoma.

PubMed

Setúbal Destro Rodrigues, Maria Fernanda; Gammon, Luke; Rahman, Muhammad M; Biddle, Adrian; Nunes, Fabio Daumas; Mackenzie, Ian C

2018-03-02

The therapeutic responses of many solid tumours to chemo- and radio-therapies are far from fully effective but therapies targeting malignancy-related cellular changes show promise for further control. In head and neck squamous cell carcinoma, the epidermal growth factor receptor (EGFR) is commonly overexpressed and investigation of agents that block this receptor indicate a limited response when used alone but an ability to enhance the actions of other drugs. The hierarchical stem cell patterns present in tumours generate cellular heterogeneity and this is further complicated by cancer stem cells (CSC) shifting between epithelial (Epi-CSC) and mesenchymal (EMT-CSC) states. To clarify how such heterogeneity influences responses to EGFR blocking, we examined the effects of Cetuximab and Erlotinib on the cell sub-populations in HNSCC cell lines. These agents reduced cell proliferation for all subpopulations but induced little cell death. They did however induce large shifts of cells between the EMT-CSC, Epi-CSC and differentiating cell compartments. Loss of EMT-CSCs reduced cell motility and is expected to reduce invasion and metastasis. EGFR blocking also induced shifts of Epi-CSCs into the differentiating cell compartment which typically has greater sensitivity to chemo/radiation, an effect expected to enhance the overall response of tumour cell populations to adjunctive therapies.

Effects of Cetuximab and Erlotinib on the behaviour of cancer stem cells in head and neck squamous cell carcinoma

PubMed Central

Setúbal Destro Rodrigues, Maria Fernanda; Gammon, Luke; Rahman, Muhammad M.; Biddle, Adrian; Nunes, Fabio Daumas; Mackenzie, Ian C.

2018-01-01

The therapeutic responses of many solid tumours to chemo- and radio-therapies are far from fully effective but therapies targeting malignancy-related cellular changes show promise for further control. In head and neck squamous cell carcinoma, the epidermal growth factor receptor (EGFR) is commonly overexpressed and investigation of agents that block this receptor indicate a limited response when used alone but an ability to enhance the actions of other drugs. The hierarchical stem cell patterns present in tumours generate cellular heterogeneity and this is further complicated by cancer stem cells (CSC) shifting between epithelial (Epi-CSC) and mesenchymal (EMT-CSC) states. To clarify how such heterogeneity influences responses to EGFR blocking, we examined the effects of Cetuximab and Erlotinib on the cell sub-populations in HNSCC cell lines. These agents reduced cell proliferation for all subpopulations but induced little cell death. They did however induce large shifts of cells between the EMT-CSC, Epi-CSC and differentiating cell compartments. Loss of EMT-CSCs reduced cell motility and is expected to reduce invasion and metastasis. EGFR blocking also induced shifts of Epi-CSCs into the differentiating cell compartment which typically has greater sensitivity to chemo/radiation, an effect expected to enhance the overall response of tumour cell populations to adjunctive therapies. PMID:29568372

Simvastatin-induced up-regulation of gap junctions composed of connexin 43 sensitize Leydig tumor cells to etoposide: an involvement of PKC pathway.

PubMed

Wang, Lingzhi; Fu, Yanni; Peng, Jianxin; Wu, Dengpan; Yu, Meiling; Xu, Chengfang; Wang, Qin; Tao, Liang

2013-10-04

Some of lipophilic statins have been reported to enhance toxicities induced by antineoplastic agents but the underling mechanism is unclear. The authors investigated the involvement of Cx43-mediated gap junction intercellular communication (GJIC) in the effect of simvastatin on the cellular toxicity induced by etoposide in this study. The results showed that a major component of the cytotoxicity of therapeutic levels of etoposide is mediated by gap junctions composed of connexin 43(Cx43) and simvastatin at the dosage which does not induce cytotoxicity enhances etoposide toxicity by increasing gap junction coupling. The augmentative effect of simvastatin on GJIC was related to the inhibition of PKC-mediated Cx43 phosphorylation at ser368 and subsequent enhancement of Cx43 membrane location induced by the agent. The present study suggests the possibility that upregulation of gap junctions may be utilized to increase the efficacy of anticancer chemotherapies. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos

PubMed Central

2013-01-01

Background In mammals, ABCB1 constitutes a cellular “first line of defense” against a wide array of chemicals and drugs conferring cellular multidrug or multixenobiotic resistance (MDR/MXR). We tested the hypothesis that an ABCB1 ortholog serves as protection for the sensitive developmental processes in zebrafish embryos against adverse compounds dissolved in the water. Results Indication for ABCB1-type efflux counteracting the accumulation of chemicals in zebrafish embryos comes from experiments with fluorescent and toxic transporter substrates and inhibitors. With inhibitors present, levels of fluorescent dyes in embryo tissue and sensitivity of embryos to toxic substrates were generally elevated. We verified two predicted sequences from zebrafish, previously annotated as abcb1, by cloning; our synteny analyses, however, identified them as abcb4 and abcb5, respectively. The abcb1 gene is absent in the zebrafish genome and we explored whether instead Abcb4 and/or Abcb5 show toxicant defense properties. Quantitative real-time polymerase chain reaction (qPCR) analyses showed the presence of transcripts of both genes throughout the first 48 hours of zebrafish development. Similar to transporter inhibitors, morpholino knock-down of Abcb4 increased accumulation of fluorescent substrates in embryo tissue and sensitivity of embryos toward toxic compounds. In contrast, morpholino knock-down of Abcb5 did not exert this effect. ATPase assays with recombinant protein obtained with the baculovirus expression system confirmed that dye and toxic compounds act as substrates of zebrafish Abcb4 and inhibitors block its function. The compounds tested comprised model substrates of human ABCB1, namely the fluorescent dyes rhodamine B and calcein-am and the toxic compounds vinblastine, vincristine and doxorubicin; cyclosporin A, PSC833, MK571 and verapamil were applied as inhibitors. Additionally, tests were performed with ecotoxicologically relevant compounds: phenanthrene (a polycyclic aromatic hydrocarbon) and galaxolide and tonalide (two polycyclic musks). Conclusions We show that zebrafish Abcb4 is a cellular toxicant transporter and provides protection of embryos against toxic chemicals dissolved in the water. Zebrafish Abcb4 thus is functionally similar to mammalian ABCB1, but differs from mammalian ABCB4, which is not involved in cellular resistance to chemicals but specifically transports phospholipids in the liver. Our data have important implications: Abcb4 could affect bioavailability - and thus toxicologic and pharmacologic potency - of chemicals to zebrafish embryos and inhibition of Abcb4 therefore causes chemosensitization, that is, enhanced sensitivity of embryos to toxicants. These aspects should be considered in (eco)toxicologic and pharmacologic chemical screens with the zebrafish embryo, a major vertebrate model. PMID:23773777

AMP-activated protein kinase and type 2 diabetes.

PubMed

Musi, Nicolas

2006-01-01

AMP-activated protein kinase (AMPK) is an enzyme that works as a fuel gauge, being activated in situations of high-energy phosphate depletion. Upon activation, AMPK functions to restore cellular ATP by modifying diverse metabolic pathways. AMPK is activated robustly by skeletal muscle contraction and myocardial ischemia, and may be involved in the stimulation of glucose transport and fatty acid oxidation produced by these stimuli. In liver, activation of AMPK results in enhanced fatty acid oxidation and in decreased production of glucose, cholesterol, and triglycerides. Recent studies have shown that AMPK is the cellular mediator for many of the metabolic effects of drugs such as metformin and thiazolidinediones, as well as the insulin sensitizing adipocytokines leptin and adiponectin. These data, along with evidence from studies showing that chemical activation of AMPK in vivo with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) improves blood glucose concentrations and lipid profiles, make this enzyme an attractive pharmacological target for the treatment of type 2 diabetes and other metabolic disorders.

Electric-field driven assembly of live bacterial cell microarrays for rapid phenotypic assessment and cell viability testing.

PubMed

Goel, Meenal; Verma, Abhishek; Gupta, Shalini

2018-07-15

Microarray technology to isolate living cells using external fields is a facile way to do phenotypic analysis at the cellular level. We have used alternating current dielectrophoresis (AC-DEP) to drive the assembly of live pathogenic Salmonella typhi (S.typhi) and Escherichia coli (E.coli) bacteria into miniaturized single cell microarrays. The effects of voltage and frequency were optimized to identify the conditions for maximum cell capture which gave an entrapment efficiency of 90% in 60 min. The chip was used for calibration-free estimation of cellular loads in binary mixtures and further applied for rapid and enhanced testing of cell viability in the presence of drug via impedance spectroscopy. Our results using a model antimicrobial sushi peptide showed that the cell viability could be tested down to 5 μg/mL drug concentration under an hour, thus establishing the utility of our system for ultrafast and sensitive detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Stressed to death: implication of lymphocyte apoptosis for psychoneuroimmunology

NASA Technical Reports Server (NTRS)

Shi, Yufang; Devadas, Satish; Greeneltch, Kristy M.; Yin, Deling; Allan Mufson, R.; Zhou, Jian-nian

2003-01-01

Psychological and physical stressors best exemplify the intercommunication of the immune and the nervous systems. It has been shown that stress significantly impacts leukocyte cellularity and immune responses and alters susceptibility to various diseases. While acute stress has been shown to enhance immune responses, chronic stress often leads to immunosuppression. Among many criteria examined upon exposure to chronic stress, the reduction in lymphocyte mitogenic response and lymphocyte cellularity are commonly assessed. We have reported that chronic restraint stress could induce lymphocyte reduction, an effect dependent on endogenous opioids. Interestingly, the effect of endogenous opioids was found to be exerted through increasing the expression of a cell death receptor, Fas, and an increased sensitivity of lymphocytes to apoptosis. Stress-induced lymphocyte reduction was not affected by adrenalectomy. In this review, based on available literature and our recent data, we will discuss the role of the hypothalamic-pituitary-adrenal axis and endogenous opioids and examine the mechanisms by which chronic stress modulates lymphocyte apoptosis.

Effects of Macrophage Depletion on Sleep in Mice

PubMed Central

Ames, Conner; Boland, Erin; Szentirmai, Éva

2016-01-01

The reciprocal interaction between the immune system and sleep regulation has been widely acknowledged but the cellular mechanisms that underpin this interaction are not completely understood. In the present study, we investigated the role of macrophages in sleep loss- and cold exposure-induced sleep and body temperature responses. Macrophage apoptosis was induced in mice by systemic injection of clodronate-containing liposomes (CCL). We report that CCL treatment induced an immediate and transient increase in non-rapid-eye movement sleep (NREMS) and fever accompanied by decrease in rapid-eye movement sleep, motor activity and NREMS delta power. Chronically macrophage-depleted mice had attenuated NREMS rebound after sleep deprivation compared to normal mice. Cold-induced increase in wakefulness and decrease in NREMS, rapid-eye movement sleep and body temperature were significantly enhanced in macrophage-depleted mice indicating increased cold sensitivity. These findings provide further evidence for the reciprocal interaction among the immune system, sleep and metabolism, and identify macrophages as one of the key cellular elements in this interplay. PMID:27442442

Effect of MPS1 Inhibition on Genotoxic Stress Responses in Murine Tumour Cells.

PubMed

Suzuki, Motofumi; Yamamori, Tohru; Yasui, Hironobu; Inanami, Osamu

2016-06-01

The monopolar spindle 1 (MPS1) is a serine/threonine kinase that plays an important role in spindle assembly checkpoint signaling. To determine the possible relationship between MPS1 inhibition and genotoxic stress responses, herein we examined whether MPS1 inhibition influences cellular susceptibility towards two genotoxic treatments, etoposide and ionizing radiation (IR). Two murine tumour cell lines, SCCVII and EMT6, were used. The effect of genotoxic treatments with or without two novel MPS1 inhibitors, NMS-P715 and AZ3146, on cellular survival, cell-cycle distribution, centrosome status and mitotic catastrophe (MC) was evaluated. MPS1 inhibition sensitized murine tumour cells to etoposide but not to IR. In addition, MPS1 inhibition altered cell-cycle progression and exacerbated centrosome abnormalities, resulting in enhanced MC induced by etoposide but not by IR. MPS1 inhibition promotes the etoposide-induced aberrant mitosis and, consequently, the induction of tumour cell death. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Specific Uptake of Lipid-Antibody-Functionalized LbL Microcarriers by Cells.

PubMed

Göse, Martin; Scheffler, Kira; Reibetanz, Uta

2016-11-14

The modular construction of Layer-by-Layer biopolymer microcarriers facilitates a highly specific design of drug delivery systems. A supported lipid bilayer (SLB) contributes to biocompatibility and protection of sensitive active agents. The addition of a lipid anchor equipped with PEG (shielding from opsonins) and biotin (attachment of exchangeable outer functional molecules) enhances the microcarrier functionality even more. However, a homogeneously assembled supported lipid bilayer is a prerequisite for a specific binding of functional components. Our investigations show that a tightly packed SLB improves the efficiency of functional components attached to the microcarrier's surface, as illustrated with specific antibodies in cellular application. Only a low quantity of antibodies is needed to obtain improved cellular uptake rates independent from cell type as compared to an antibody-functionalized loosely packed lipid bilayer or directly assembled antibody onto the multilayer. A fast disassembly of the lipid bilayer within endolysosomes exposing the underlying drug delivering multilayer structure demonstrates the suitability of LbL-microcarriers as a multifunctional drug delivery system.

A fuel-limited isothermal DNA machine for the sensitive detection of cellular deoxyribonucleoside triphosphates.

PubMed

Dong, Jiantong; Wu, Tongbo; Xiao, Yu; Xu, Lei; Fang, Simin; Zhao, Meiping

2016-09-29

A fuel-limited isothermal DNA machine has been built for the sensitive fluorescence detection of cellular deoxyribonucleoside triphosphates (dNTPs) at the fmol level, which greatly reduces the required sample cell number. Upon the input of the limiting target dNTP, the machine runs automatically at 37 °C without the need for higher temperature.

Bicarbonate alters cellular responses in respiration assays.

PubMed

Krycer, James R; Fisher-Wellman, Kelsey H; Fazakerley, Daniel J; Muoio, Deborah M; James, David E

2017-08-05

Metabolic assay buffers often omit bicarbonate, which is susceptible to alkalinisation in an open environment. Here, we assessed the effect of including bicarbonate in respirometry experiments. By supplementing HEPES-buffered media with low concentrations of bicarbonate, we found increased respiration in adipocytes and hepatocytes, but not myotubes. This was observed across multiple respirometry platforms and was independent of effects on enhanced insulin sensitivity, pH drift, or mitochondrial function. Permeabilised cell experiments suggest that bicarbonate increases substrate availability, likely by acting as a cofactor for carboxylase enzymes. This emphasises the importance of buffer choice in experimental biology. Copyright © 2017 Elsevier Inc. All rights reserved.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice.

PubMed

Feeks, James A; Hunter, Jennifer J

2017-05-01

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina.

Promising New Photon Detection Concepts for High-Resolution Clinical and Preclinical PET

PubMed Central

Levin, Craig S.

2013-01-01

The ability of PET to visualize and quantify regions of low concentration of PET tracer representing subtle cellular and molecular signatures of disease depends on relatively complex biochemical, biologic, and physiologic factors that are challenging to control, as well as on instrumentation performance parameters that are, in principle, still possible to improve on. Thus, advances to the latter can somewhat offset barriers of the former. PET system performance parameters such as spatial resolution, contrast resolution, and photon sensitivity contribute significantly to PET’s ability to visualize and quantify lower concentrations of signal in the presence of background. In this report we present some technology innovations under investigation toward improving these PET system performance parameters. We focus particularly on a promising advance known as 3-dimensional position-sensitive detectors, which are detectors capable of distinguishing and measuring the position, energy, and arrival time of individual interactions of multi-interaction photon events in 3 dimensions. If successful, these new strategies enable enhancements such as the detection of fewer diseased cells in tissue or the ability to characterize lower-abundance molecular targets within cells. Translating these advanced capabilities to the clinic might allow expansion of PET’s roles in disease management, perhaps to earlier stages of disease. In preclinical research, such enhancements enable more sensitive and accurate studies of disease biology in living subjects. PMID:22302960

NIR-driven Smart Theranostic Nanomedicine for On-demand Drug Release and Synergistic Antitumour Therapy.

PubMed

Zhao, Pengfei; Zheng, Mingbin; Luo, Zhenyu; Gong, Ping; Gao, Guanhui; Sheng, Zonghai; Zheng, Cuifang; Ma, Yifan; Cai, Lintao

2015-09-24

Smart nanoparticles (NPs) that respond to external and internal stimulations have been developing to achieve optimal drug release in tumour. However, applying these smart NPs to attain high antitumour performance is hampered by limited drug carriers and inefficient spatiotemporal control. Here we report a noninvasive NIR-driven, temperature-sensitive DI-TSL (DOX/ICG-loaded temperature sensitive liposomes) co-encapsulating doxorubicin (DOX) and indocyanine green (ICG). This theranostic system applies thermo-responsive lipid to controllably release drug, utilizes the fluorescence (FL) of DOX/ICG to real-time trace the distribution of NPs, and employs DOX/ICG to treat cancer by chemo/photothermal therapy. DI-TSL exhibits uniform size distribution, excellent FL/size stability, enhanced response to NIR-laser, and 3 times increased drug release through laser irradiation. After endocytosis by MCF-7 breast adenocarcinoma cells, DI-TSL in cellular endosomes can cause hyperthermia through laser irradiation, then endosomes are disrupted and DI-TSL 'opens' to release DOX simultaneously for increased cytotoxicity. Furthermore, DI-TSL shows laser-controlled release of DOX in tumour, enhanced ICG and DOX retention by 7 times and 4 times compared with free drugs. Thermo-sensitive DI-TSL manifests high efficiency to promote cell apoptosis, and completely eradicate tumour without side-effect. DI-TSL may provide a smart strategy to release drugs on demand for combinatorial cancer therapy.

NIR-driven Smart Theranostic Nanomedicine for On-demand Drug Release and Synergistic Antitumour Therapy

NASA Astrophysics Data System (ADS)

Zhao, Pengfei; Zheng, Mingbin; Luo, Zhenyu; Gong, Ping; Gao, Guanhui; Sheng, Zonghai; Zheng, Cuifang; Ma, Yifan; Cai, Lintao

2015-09-01

Smart nanoparticles (NPs) that respond to external and internal stimulations have been developing to achieve optimal drug release in tumour. However, applying these smart NPs to attain high antitumour performance is hampered by limited drug carriers and inefficient spatiotemporal control. Here we report a noninvasive NIR-driven, temperature-sensitive DI-TSL (DOX/ICG-loaded temperature sensitive liposomes) co-encapsulating doxorubicin (DOX) and indocyanine green (ICG). This theranostic system applies thermo-responsive lipid to controllably release drug, utilizes the fluorescence (FL) of DOX/ICG to real-time trace the distribution of NPs, and employs DOX/ICG to treat cancer by chemo/photothermal therapy. DI-TSL exhibits uniform size distribution, excellent FL/size stability, enhanced response to NIR-laser, and 3 times increased drug release through laser irradiation. After endocytosis by MCF-7 breast adenocarcinoma cells, DI-TSL in cellular endosomes can cause hyperthermia through laser irradiation, then endosomes are disrupted and DI-TSL ‘opens’ to release DOX simultaneously for increased cytotoxicity. Furthermore, DI-TSL shows laser-controlled release of DOX in tumour, enhanced ICG and DOX retention by 7 times and 4 times compared with free drugs. Thermo-sensitive DI-TSL manifests high efficiency to promote cell apoptosis, and completely eradicate tumour without side-effect. DI-TSL may provide a smart strategy to release drugs on demand for combinatorial cancer therapy.

Novel thermal-sensitive hydrogel enhances both humoral and cell-mediated immune responses by intranasal vaccine delivery.

PubMed

Wu, Youbin; Wu, Shipo; Hou, Lihua; Wei, Wei; Zhou, Meng; Su, Zhiguo; Wu, Jie; Chen, Wei; Ma, Guanghui

2012-08-01

A novel thermal sensitive hydrogel was formulated with N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) and α, β-glycerophosphate (α, β-GP). A serial of hydrogels containing different amount of GP and HTCC with diverse quarternize degree (QD, 41%, 59%, 79.5%, and 99%) were prepared and characterized by rheological method. The hydrogel was subsequently evaluated for intranasal vaccine delivery with adenovirus based Zaire Ebola virus glycoprotein antigen (Ad-GPZ). Results showed that moderate quarternized HTCC (60% and 79.5%) hydrogel/antigen formulations induced highest IgG, IgG1, and IgG2a antibody titers in serum, as well as mucosal IgA responses in lung wash, which may attributed to the prolonged antigen residence time due to the thermal-sensitivity of this hydrogel. Furthermore, CD8(+) splenocytes for IFN-γ positive cell assay and the release profile of Th1/Th2 type cytokines (IFN-γ, IL-2, IL-10, and IL-4) showed that hydrogel/Ad-GPZ generated an overwhelmingly enhanced Th1 biased cellular immune response. In addition, this hydrogel displayed low toxicity to nasal tissue and epithelial cells even by frequently intranasal dosing of hydrogel. All these results strongly supported this hydrogel as a safe and effective delivery system for nasal immunization. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans.

PubMed

Knowlton, Wendy M; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D; Jin, Yishi

2017-01-01

The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo , we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1 . Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7 , and isp-1 , and the putative oxidoreductase rad-8 . In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1 . Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular adaptations used by neurons under conditions of injury.

A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans

PubMed Central

Knowlton, Wendy M.; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D.; Jin, Yishi

2017-01-01

The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo, we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1. Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7, and isp-1, and the putative oxidoreductase rad-8. In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1. Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular adaptations used by neurons under conditions of injury. PMID:28539870

Theranostic pH-sensitive nanoparticles for highly efficient targeted delivery of doxorubicin for breast tumor treatment.

PubMed

Pan, Changqie; Liu, Yuqing; Zhou, Minyu; Wang, Wensheng; Shi, Min; Xing, Malcolm; Liao, Wangjun

2018-01-01

A multifunctional theranostic nanoplatform integrated with environmental responses has been developed rapidly over the past few years as a novel treatment strategy for several solid tumors. We synthesized pH-sensitive poly(β-thiopropionate) nanoparticles with a supermagnetic core and folic acid (FA) conjugation (FA-doxorubicin-iron oxide nanoparticles [FA-DOX@ IONPs]) to deliver an antineoplastic drug, DOX, for the treatment of folate receptor (FR)-overexpressed breast cancer. In addition to an imaging function, the nanoparticles can release their payloads in response to an environment of pH 5, such as the acidic environment found in tumors. After chemical ( 1 H nuclear magnetic resonance) and physical (morphology and super-magnetic) characterization, FA-DOX@IONPs were shown to demonstrate pH-dependent drug release profiles. Western blotting analysis revealed the expression of FRs in three breast cancer cell lines, MCF-7, BT549, and MD-MBA-231. The cell counting kit-8 assay and transmission electron microscopy showed that FA-DOX@IONPs had the strongest cytotoxicity against breast cancer cells, compared with free DOX and non-FR targeted nanoparticles (DOX@IONPs), and caused cellular apoptosis. The FA-DOX@IONP-mediated cellular uptake and intracellular internalization were clarified by fluorescence microscopy. FA-DOX@IONPs plus magnetic field treatment suppressed in vivo tumor growth in mice to a greater extent than either treatment alone; furthermore, the nanoparticles exerted no toxicity against healthy organs. Magnetic resonance imaging was successfully applied to monitor the nanoparticle accumulation. Our results suggest that theranostic pH-sensitive nanoparticles with dual targeting could enhance the available therapies for cancer.

Proteomic analysis of the response of α-ketoglutarate-producer Yarrowia lipolytica WSH-Z06 to environmental pH stimuli.

PubMed

Guo, Hongwei; Wan, Hui; Chen, Hongwen; Fang, Fang; Liu, Song; Zhou, Jingwen

2016-10-01

During bioproduction of short-chain carboxylates, a shift in pH is a common strategy for enhancing the biosynthesis of target products. Based on two-dimensional gel electrophoresis, comparative proteomics analysis of general and mitochondrial protein samples was used to investigate the cellular responses to environmental pH stimuli in the α-ketoglutarate overproducer Yarrowia lipolytica WSH-Z06. The lower environmental pH stimuli tensioned intracellular acidification and increased the level of reactive oxygen species (ROS). A total of 54 differentially expressed protein spots were detected, and 11 main cellular processes were identified to be involved in the cellular response to environmental pH stimuli. Slight decrease in cytoplasmic pH enhanced the cellular acidogenicity by elevating expression level of key enzymes in tricarboxylic acid cycle (TCA cycle). Enhanced energy biosynthesis, ROS elimination, and membrane potential homeostasis processes were also employed as cellular defense strategies to compete with environmental pH stimuli. Owing to its antioxidant role of α-ketoglutarate, metabolic flux shifted to α-ketoglutarate under lower pH by Y. lipolytica in response to acidic pH stimuli. The identified differentially expressed proteins provide clues for understanding the mechanisms of the cellular responses and for enhancing short-chain carboxylate production through metabolic engineering or process optimization strategies in combination with manipulation of environmental conditions.

Curcumin loaded pH-sensitive nanoparticles for the treatment of colon cancer.

PubMed

Prajakta, Dandekar; Ratnesh, Jain; Chandan, Kumar; Suresh, Subramanian; Grace, Samuel; Meera, Venkatesh; Vandana, Patravale

2009-10-01

The investigation was aimed at designing pH-sensitive, polymeric nanoparticles of curcumin, a natural anti-cancer agent, for the treatment of colon cancer. The objective was to enhance the bioavailability of curcumin, simultaneously reducing the required dose through selective targeting to colon. Eudragit S100 was chosen to aid targeting since the polymer dissolves at colonic pH to result in selective colonic release of the entrapped drug. Solvent emulsion-evaporation technique was employed to formulate the nanoparticles. Various process parameters were optimized and the optimized formulation was evaluated for particle size distribution and encapsulation efficiency before subjecting to freeze-drying. The freeze dried product was characterized for particle size, drug content, DSC studies, particle morphology. Anti-cancer potential of the formulation was demonstrated by MTT assay in HT-29 cell line. Nanometric, homogeneous, spherical particles were obtained with encapsulation efficiency of 72%. Freeze-dried nanoparticles exhibited a negative surface charge, drug content of > 99% and presence of drug in amorphous form which may result in possible enhanced absorption. MTT assay demonstrated almost double inhibition of the cancerous cells by nanoparticles, as compared to curcumin alone, at the concentrations tested. Enhanced action may be attributed to size influenced improved cellular uptake, and may result in reduction of overall dose requirement. Results indicate the potential for in vivo studies to establish the clinical application of the formulation.

Impaired osteogenic differentiation and enhanced cellular receptor of advanced glycation end products sensitivity in patients with type 2 diabetes.

PubMed

Phimphilai, Mattabhorn; Pothacharoen, Peraphan; Kongtawelert, Prachya; Chattipakorn, Nipon

2017-11-01

Preclinical studies have demonstrated impaired osteoblast differentiation in type 2 diabetes (T2DM), which is related to skeletal accumulation of advanced glycation end products (AGEs). However, the role of AGE in osteoblast differentiation in patients with T2DM is unclear. This cross-sectional study was performed to investigate osteoblast differentiation and its association with serum pentosidine and soluble receptor of AGEs (sRAGE). Twenty-seven patients with T2DM and 15 age-matched controls were included to measure sRAGE and osteogenic differentiation in mononuclear cells derived from peripheral blood. The mononuclear cells isolated from patients with T2DM showed a significantly lower rate of osteogenic differentiation (7.4% vs 86.7%, p < 0.0001) with a lower level of ALPL, COL1A1, and BGLAP expression than those of controls by 11-, 44-, and 15-fold respectively, together with nonvisualized mineralization by alizarin red S staining. The levels of pentosidine and sRAGE were comparable in both groups. AGER expression was significantly higher in the T2DM group. BAX expression was also significantly higher in the T2DM group, and showed a strong correlation with AGER expression (r = 0.86, p < 0.0001). Fasting plasma glucose (FPG) level, AGER expression, and BAX expression showed a strong correlation with osteogenic differentiation defects on univariate analysis. However, only FPG showed a correlation with this defect in a multivariate analysis. In conclusion, patients with T2DM showed impairment of osteoblast differentiation, and FPG was an independent risk factor for this impairment. Moreover, T2DM showed a higher cellular sensitivity for activation of receptor of AGEs and higher cellular apoptosis, which may contribute to the defect in osteoblast differentiation.

A highly sensitive nanoscale pH-sensor using Au nanoparticles linked by a multifunctional Raman-active reporter molecule

NASA Astrophysics Data System (ADS)

Lawson, Latevi S.; Chan, James W.; Huser, Thomas

2014-06-01

Chemical sensing on the nanoscale has been breaking new ground since the discovery of surface enhanced Raman scattering (SERS). For nanoparticles, controlled particle aggregation is necessary to achieve the largest SERS enhancements. Therefore, aggregating agents such as salts or linker molecules are used in conjunction with chemically sensitive reporters in order to develop robust environmentally sensitive SERS probes. While salt-induced colloidal nanosphere aggregates have produced robust SERS signals, their variability in aggregate size contributes significantly to poor SERS signal reproducibility, which can complicate their use in in vitro cellular studies. Such systems often also lack reproducibility in spectral measurements between different nanoparticle clusters. Preaggregation of colloids via linkers followed by surface functionalization with reporter molecules results in the linker occupying valuable SERS hotspot volume which could otherwise be utilized by additional reporter molecules. Ideally, both functionalities should be obtained from a single molecule. Here, we report the use of 3,5-dimercaptobenzoic acid, a single multifunctional molecule that creates SERS hotspots via the controlled aggregation of nanoparticles, and also reports pH values. We show that 3,5-dimercaptobenzoic acid bound to Au nanospheres results in an excellent pH nanoprobe, producing very robust, and highly reproducible SERS signals that can report pH across the entire physiological range with excellent pH resolution. To demonstrate the efficacy of our novel pH reporters, these probes were also used to image both the particle and pH distribution in the cytoplasm of human induced pluripotent stem cells (hiPSCs).Chemical sensing on the nanoscale has been breaking new ground since the discovery of surface enhanced Raman scattering (SERS). For nanoparticles, controlled particle aggregation is necessary to achieve the largest SERS enhancements. Therefore, aggregating agents such as salts or linker molecules are used in conjunction with chemically sensitive reporters in order to develop robust environmentally sensitive SERS probes. While salt-induced colloidal nanosphere aggregates have produced robust SERS signals, their variability in aggregate size contributes significantly to poor SERS signal reproducibility, which can complicate their use in in vitro cellular studies. Such systems often also lack reproducibility in spectral measurements between different nanoparticle clusters. Preaggregation of colloids via linkers followed by surface functionalization with reporter molecules results in the linker occupying valuable SERS hotspot volume which could otherwise be utilized by additional reporter molecules. Ideally, both functionalities should be obtained from a single molecule. Here, we report the use of 3,5-dimercaptobenzoic acid, a single multifunctional molecule that creates SERS hotspots via the controlled aggregation of nanoparticles, and also reports pH values. We show that 3,5-dimercaptobenzoic acid bound to Au nanospheres results in an excellent pH nanoprobe, producing very robust, and highly reproducible SERS signals that can report pH across the entire physiological range with excellent pH resolution. To demonstrate the efficacy of our novel pH reporters, these probes were also used to image both the particle and pH distribution in the cytoplasm of human induced pluripotent stem cells (hiPSCs). Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr06277e

Hypersensitivity to acid is associated with impaired esophageal mucosal integrity in patients with gastroesophageal reflux disease with and without esophagitis.

PubMed

Weijenborg, Pim W; Smout, André J P M; Verseijden, Caroline; van Veen, Henk A; Verheij, Joanne; de Jonge, Wouter J; Bredenoord, Albert J

2014-08-01

Increased esophageal sensitivity and impaired mucosal integrity have both been described in patients with gastroesophageal reflux disease, but the relationship between hypersensitivity and mucosal integrity is unclear. The aim of the present study was to investigate acid sensitivity in patients with erosive and nonerosive reflux disease and control subjects to determine the relation with functional esophageal mucosal integrity changes as well as to investigate cellular mechanisms of impaired mucosal integrity in these patients. In this prospective experimental study, 12 patients with nonerosive reflux disease, 12 patients with esophagitis grade A or B, and 11 healthy control subjects underwent an acid perfusion test and upper endoscopy. Mucosal integrity was measured during endoscopy by electrical tissue impedance spectroscopy and biopsy specimens were analyzed in Ussing chambers for transepithelial electrical resistance, transepithelial permeability and gene expression of tight junction proteins and filaggrin. Patients with nonerosive reflux disease and esophagitis were more sensitive to acid perfusion compared with control subjects, having a shorter time to perception of heartburn and higher perceived intensity of heartburn. In reflux patients, enhanced acid sensitivity was associated with impairment of in vivo and vitro esophageal mucosal integrity. Mucosal integrity was significantly impaired in patients with esophagitis, displaying higher transepithelial permeability and lower extracellular impedance. Although no significant differences in the expression of tight junction proteins were found in biopsies among patient groups, mucosal integrity parameters in reflux patients correlated negatively with the expression of filaggrin. In conclusion, sensitivity to acid is enhanced in patients with gastroesophageal reflux disease, irrespective of the presence of erosions, and is associated with impaired esophageal mucosal integrity. Mucosal integrity of the esophagus is associated with the expression of filaggrin. Copyright © 2014 the American Physiological Society.

SERS imaging of cell-surface biomolecules metabolically labeled with bioorthogonal Raman reporters.

PubMed

Xiao, Ming; Lin, Liang; Li, Zefan; Liu, Jie; Hong, Senlian; Li, Yaya; Zheng, Meiling; Duan, Xuanming; Chen, Xing

2014-08-01

Live imaging of biomolecules with high specificity and sensitivity as well as minimal perturbation is essential for studying cellular processes. Here, we report the development of a bioorthogonal surface-enhanced Raman scattering (SERS) imaging approach that exploits small Raman reporters for visualizing cell-surface biomolecules. The cells were cultured and imaged by SERS microscopy on arrays of Raman-enhancing nanoparticles coated on silicon wafers or glass slides. The Raman reporters including azides, alkynes, and carbondeuterium bonds are small in size and spectroscopically bioorthogonal (background-free). We demonstrated that various cell-surface biomolecules including proteins, glycans, and lipids were metabolically incorporated with the corresponding precursors bearing a Raman reporter and visualized by SERS microscopy. The coupling of SERS microscopy with bioorthogonal Raman reporters expands the capabilities of live-cell microscopy beyond the modalities of fluorescence and label-free imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparation of pH-sensitive zwitterionic nano micelles and drug controlled release for enhancing cellular uptake.

PubMed

Wu, Luyan; Ni, Caihua; Zhang, Liping; Shi, Gang

2016-01-01

Zwitterionic copolymers have exhibited high resistance to nonspecific protein adsorption and have wide applications in drug delivery systems. Herein, a pH-responsive poly(Lysine-alt-N,N'-bis(acryloyl) diaminohexane) was synthesized through the Michael addition polymerization between N, N'-bis(acryloyl) diaminohexane and lysine. Subsequently, nano micelles (NMs) were formed by self-assembly of the copolymer in an aqueous solution. The NMs showed a slightly negative charge in blood environment, but a positively charged surface in extracellular pH of tumor. This feature could be used to enhance permeability and retention effect, and reinforce tumor cell uptake. Vitro release studies revealed that the release of DOX from the DOX-loaded NMs was evidently faster at pH 5.0 than at pH 7.4. MTT assays revealed that NMs were nontoxic. Thus, these smart NMs were feasible candidates and could be potentially used in cancer chemotherapy.

Surface-Enhanced Raman Scattering Surface Selection Rules for the Proteomic Liquid Biopsy in Real Samples: Efficient Detection of the Oncoprotein c-MYC.

PubMed

Pazos, Elena; Garcia-Algar, Manuel; Penas, Cristina; Nazarenus, Moritz; Torruella, Arnau; Pazos-Perez, Nicolas; Guerrini, Luca; Vázquez, M Eugenio; Garcia-Rico, Eduardo; Mascareñas, José L; Alvarez-Puebla, Ramon A

2016-11-02

Blood-based biomarkers (liquid biopsy) offer extremely valuable tools for the noninvasive diagnosis and monitoring of tumors. The protein c-MYC, a transcription factor that has been shown to be deregulated in up to 70% of human cancers, can be used as a robust proteomic signature for cancer. Herein, we developed a rapid, highly specific, and sensitive surface-enhanced Raman scattering (SERS) assay for the quantification of c-MYC in real blood samples. The sensing scheme relies on the use of specifically designed hybrid plasmonic materials and their bioderivatization with a selective peptidic receptor modified with a SERS transducer. Peptide/c-MYC recognition events translate into measurable alterations of the SERS spectra associated with a molecular reorientation of the transducer, in agreement with the surface selection rules. The efficiency of the sensor is demonstrated in cellular lines, healthy donors and a cancer patient.

Modulated Raman Spectroscopy for Enhanced Cancer Diagnosis at the Cellular Level

PubMed Central

De Luca, Anna Chiara; Dholakia, Kishan; Mazilu, Michael

2015-01-01

Raman spectroscopy is emerging as a promising and novel biophotonics tool for non-invasive, real-time diagnosis of tissue and cell abnormalities. However, the presence of a strong fluorescence background is a key issue that can detract from the use of Raman spectroscopy in routine clinical care. The review summarizes the state-of-the-art methods to remove the fluorescence background and explores recent achievements to address this issue obtained with modulated Raman spectroscopy. This innovative approach can be used to extract the Raman spectral component from the fluorescence background and improve the quality of the Raman signal. We describe the potential of modulated Raman spectroscopy as a rapid, inexpensive and accurate clinical tool to detect the presence of bladder cancer cells. Finally, in a broader context, we show how this approach can greatly enhance the sensitivity of integrated Raman spectroscopy and microfluidic systems, opening new prospects for portable higher throughput Raman cell sorting. PMID:26110401

Exogenous calcium improves viability of biocontrol yeasts under heat stress by reducing ROS accumulation and oxidative damage of cellular protein.

PubMed

An, Bang; Li, Boqiang; Qin, Guozheng; Tian, Shiping

2012-08-01

In this article, we investigated the effect of exogenous calcium on improving viability of Debaryomyces hansenii and Pichia membranaefaciens under heat stress, and evaluated the role of calcium in reducing oxidant damage of proteins in the yeast cells. The results indicated that high concentration of exogenous calcium in culture medium was beneficial for enhancing the tolerance of the biocontrol yeasts to heat stress. The possible mechanism of calcium improving the viability of yeasts was attributed to enhancement of antioxidant enzyme activities, decrease in ROS accumulation and reduction of oxidative damage of intracellular protein in yeast cells under heat stress. D. hansenii is more sensitive to calcium as compared to P. membranaefaciens. Our results suggest that application of exogenous calcium combined with biocontrol yeasts is a practical approach for the control of postharvest disease in fruit.

Classifying elementary cellular automata using compressibility, diversity and sensitivity measures

NASA Astrophysics Data System (ADS)

Ninagawa, Shigeru; Adamatzky, Andrew

2014-10-01

An elementary cellular automaton (ECA) is a one-dimensional, synchronous, binary automaton, where each cell update depends on its own state and states of its two closest neighbors. We attempt to uncover correlations between the following measures of ECA behavior: compressibility, sensitivity and diversity. The compressibility of ECA configurations is calculated using the Lempel-Ziv (LZ) compression algorithm LZ78. The sensitivity of ECA rules to initial conditions and perturbations is evaluated using Derrida coefficients. The generative morphological diversity shows how many different neighborhood states are produced from a single nonquiescent cell. We found no significant correlation between sensitivity and compressibility. There is a substantial correlation between generative diversity and compressibility. Using sensitivity, compressibility and diversity, we uncover and characterize novel groupings of rules.

Differential retention of alpha-vitamin E is correlated with its transporter gene expression and growth inhibition efficacy in prostate cancer cells.

PubMed

Ni, Jing; Pang, See-Too; Yeh, Shuyuan

2007-04-01

Epidemiological studies showed Vit E has protective effects against prostate cancer (PCa). Interestingly, different prostate cancer cells have different sensitivity to alpha-Vit E or VES treatment. The goal of this study is to determine whether cellular Vit E bioavailability and its transport proteins are important contributing factors. alpha-Vit E and its ester form, VES, were used to treat prostate cancer LNCaP, PC3, and DU145 cells, and their growth rates were determined by MTT assay. Cellular levels of Vit E were quantified using HPLC as the index of bioavailability. The expression levels of Vit E transport proteins were determined by real-time PCR. Among these PCa cells, only LNCaP cells were sensitive to 20 microM alpha-Vit E treatment, while both LNCaP and PC3 cells were sensitive to 20 microM VES treatment. Coordinately, cellular levels of alpha-Vit E and VES positively correlated to their inhibitory effects. Further study found expression levels of Vit E transport proteins, including tocopherol associated protein (TAP), scavenger receptor class B type I (SR-BI), alpha-tocopherol transfer protein (TTP), and ATP binding cassette transporter A1 (ABCA1), were different in various PCa cells, which may contribute to cellular Vit E bioavailability. This notion is further supported by the findings that overexpression or knockdown of TTP could coordinately alter cellular alpha-Vit E levels in PCa cells. Antiproliferative efficacy of alpha-Vit E is correlated with its cellular bioavailability in PCa cells. Modulating the expression of the efflux or influx transporters could sensitize the growth inhibition efficacy of Vit E in prostate cancer cells.

Redox Regulation of Mitochondrial Function

PubMed Central

Handy, Diane E.

2012-01-01

Abstract Redox-dependent processes influence most cellular functions, such as differentiation, proliferation, and apoptosis. Mitochondria are at the center of these processes, as mitochondria both generate reactive oxygen species (ROS) that drive redox-sensitive events and respond to ROS-mediated changes in the cellular redox state. In this review, we examine the regulation of cellular ROS, their modes of production and removal, and the redox-sensitive targets that are modified by their flux. In particular, we focus on the actions of redox-sensitive targets that alter mitochondrial function and the role of these redox modifications on metabolism, mitochondrial biogenesis, receptor-mediated signaling, and apoptotic pathways. We also consider the role of mitochondria in modulating these pathways, and discuss how redox-dependent events may contribute to pathobiology by altering mitochondrial function. Antioxid. Redox Signal. 16, 1323–1367. PMID:22146081

Rocaglamide overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance in hepatocellular carcinoma cells by attenuating the inhibition of caspase-8 through cellular FLICE-like-inhibitory protein downregulation.

PubMed

Luan, Zhou; He, Ying; He, Fan; Chen, Zhishui

2015-01-01

The enhancement of apoptosis is a therapeutic strategy used in the treatment of cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent. However, hepatocellular carcinoma (HCC) cells exhibit marked resistance to the induction of cell death by TRAIL. The present study investigated whether rocaglamide, a naturally occurring product isolated from the genus Aglaia, is able to sensitize resistant HCC cells to TRAIL-mediated apoptosis. Two HCC cell lines, HepG2 and Huh-7, were treated with rocaglamide and/or TRAIL and the induction of apoptosis and effects on the TRAIL signaling pathway were investigated. The in vivo efficacy of rocaglamide was determined in TRAIL-resistant Huh-7-derived tumor xenografts. Rocaglamide significantly sensitized the TRAIL-resistant HCC cells to apoptosis by TRAIL, which resulted from the rocaglamide-mediated downregulation of cellular FLICE-like inhibitory protein and subsequent caspase-8 activation. Furthermore, rocaglamide markedly inhibited tumor growth from Huh-7 cells propagated in severe combined immunodeficient mice, suggesting that chemosentization also occurred in vivo. These data suggest that rocaglamide acted synergistically with TRAIL against the TRAIL-resistant HCC cells. Thus, it is concluded that rocaglamide as an adjuvant to TRAIL-based therapy may present a promising therapeutic approach for the treatment of HCC.

Cyclic AMP differentiates two separate but interacting pathways of phosphoinositide hydrolysis in the DDT1-MF2 smooth muscle cell line.

PubMed

Schachter, J B; Wolfe, B B

1992-03-01

The activation of adenosine A1 receptors in DDT1-MF2 smooth muscle cells resulted in both the inhibition of agonist-stimulated cAMP accumulation and the potentiation of norepinephrine-stimulated phosphoinositide hydrolysis. Pharmacological analysis indicated the involvement of an A1 adenosine receptor subtype in both of these responses. In the absence of norepinephrine, the activation of the adenosine receptor did not directly stimulate phosphoinositide hydrolysis. The adenosine receptor-mediated augmentation of norepinephrine-stimulated phosphoinositide hydrolysis was pertussis toxin sensitive and was selectively antagonized by agents that mimicked cAMP (8-bromo-cAMP) or raised cellular cAMP levels (forskolin). This initially suggested that cAMP might partially regulate the magnitude of the phospholipase C response to norepinephrine and that adenosine agonists might enhance the phospholipase C response by reducing cAMP levels. However, neither the reduction of cellular cAMP levels by other agents nor the inhibition of cAMP-dependent protein kinase was sufficient to replicate the action of adenosine receptor activation on phosphoinositide hydrolysis. Thus, in the presence of norepinephrine, adenosine receptor agonists appear to stimulate phosphoinositide hydrolysis via a pathway that is separate from, but dependent upon, that of norepinephrine. This second pathway can be distinguished from that which is stimulated by norepinephrine on the basis of its sensitivity to inhibition by both cAMP and pertussis toxin.

Ultrasound-guided photoacoustic imaging of lymph nodes with biocompatible gold nanoparticles as a novel contrast agent (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sun, In-Cheol; Dumani, Diego; Emelianov, Stanislav Y.

2017-02-01

A key step in staging cancer is the diagnosis of metastasis that spreads through lymphatic system. For this reason, researchers develop various methods of sentinel lymph node mapping that often use a radioactive tracer. This study introduces a safe, cost-effective, high-resolution, high-sensitivity, and real-time method of visualizing the sentinel lymph node: ultrasound-guided photoacoustic (US/PA) imaging augmented by a contrast agent. In this work, we use clearable gold nanoparticles covered by a biocompatible polymer (glycol chitosan) to enhance cellular uptake by macrophages abundant in lymph nodes. We incubate macrophages with glycol-chitosan-coated gold nanoparticles (0.05 mg Au/ml), and then fix them with paraformaldehyde solution for an analysis of in vitro dark-field microscopy and cell phantom. The analysis shows enhanced cellular uptake of nanoparticles by macrophages and strong photoacoustic signal from labeled cells in tissue-mimicking cell phantoms consisting gelatin solution (6 %) with silica gel (25 μm, 0.3%) and fixed macrophages (13 X 105 cells). The in-vivo US/PA imaging of cervical lymph nodes in healthy mice (nu/nu, female, 5 weeks) indicates a strong photoacoustic signal from a lymph node 10 minutes post-injection (2.5 mg Au/ml, 80 μl). The signal intensity and the nanoparticle-labeled volume of tissue within the lymph node continues to increase until 4 h post-injection. Histological analysis further confirms the accumulation of gold nanoparticles within the lymph nodes. This work suggests the feasibility of molecular/cellular US/PA imaging with biocompatible gold nanoparticles as a photoacoustic contrast agent in the diagnosis of lymph-node-related diseases.

Aldolase B knockdown prevents high glucose-induced methylglyoxal overproduction and cellular dysfunction in endothelial cells.

PubMed

Liu, Jianghai; Mak, Timothy Chun-Ping; Banigesh, Ali; Desai, Kaushik; Wang, Rui; Wu, Lingyun

2012-01-01

We used cultured endothelial cells as a model to examine whether up-regulation of aldolase B and enhanced methylglyoxal (MG) formation play an important role in high glucose-induced overproduction of advanced glycosylation endproducts (AGEs), oxidative stress and cellular dysfunction. High glucose (25 mM) incubation up-regulated mRNA levels of aldose reductase (an enzyme converting glucose to fructose) and aldolase B (a key enzyme that catalyzes MG formation from fructose) and enhanced MG formation in human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA. hy926 cells. High glucose-increased MG production in EA. hy926 cells was completely prevented by siRNA knockdown of aldolase B, but unaffected by siRNA knockdown of aldolase A, an enzyme responsible for MG formation during glycolysis. In addition, inhibition of cytochrome P450 2E1 or semicarbazide-sensitive amine oxidase which produces MG during the metabolism of lipid and proteins, respectively, did not alter MG production. Both high glucose (25 mM) and MG (30, 100 µM) increased the formation of N(ε)-carboxyethyl-lysine (CEL, a MG-induced AGE), oxidative stress (determined by the generation of oxidized DCF, H(2)O(2), protein carbonyls and 8-oxo-dG), O-GlcNAc modification (product of the hexosamine pathway), membrane protein kinase C activity and nuclear translocation of NF-κB in EA. hy926 cells. However, the above metabolic and signaling alterations induced by high glucose were completely prevented by knockdown of aldolase B and partially by application of aminoguanidine (a MG scavenger) or alagebrium (an AGEs breaker). In conclusion, efficient inhibition of aldolase B can prevent high glucose-induced overproduction of MG and related cellular dysfunction in endothelial cells.

Sensitization of human carcinoma cells to alkylating agents by small interfering RNA suppression of 3-alkyladenine-DNA glycosylase.

PubMed

Paik, Johanna; Duncan, Tod; Lindahl, Tomas; Sedgwick, Barbara

2005-11-15

One of the major cytotoxic lesions generated by alkylating agents is DNA 3-alkyladenine, which can be excised by 3-alkyladenine DNA glycosylase (AAG). Inhibition of AAG may therefore result in increased cellular sensitivity to chemotherapeutic alkylating agents. To investigate this possibility, we have examined the role of AAG in protecting human tumor cells against such agents. Plasmids that express small interfering RNAs targeted to two different regions of AAG mRNA were transfected into HeLa cervical carcinoma cells and A2780-SCA ovarian carcinoma cells. Stable derivatives of both cell types with low AAG protein levels were sensitized to alkylating agents. Two HeLa cell lines with AAG protein levels reduced by at least 80% to 90% displayed a 5- to 10-fold increase in sensitivity to methyl methanesulfonate, N-methyl-N-nitrosourea, and the chemotherapeutic drugs temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea. These cells showed no increase in sensitivity to UV light or ionizing radiation. After treatment with methyl methanesulfonate, AAG knockdown HeLa cells were delayed in S phase but accumulated in G2-M. Our data support the hypothesis that ablation of AAG activity in human tumor cells may provide a useful strategy to enhance the efficacy of current chemotherapeutic regimens that include alkylating agents.

Cytomegalovirus: pathophysiological mechanisms of the cytomegalovirus-induced cellular responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nokta, M.A.

1986-01-01

Cytomegalovirus (CMV) infection of fibroblasts of human origin is associated with a cascade of morphologic cellular responses which in other systems have been associated with regulation of intracellular free (IF) (Ca/sup + +/). In the present study, the relationship of specific ion fluxes (Ca/sup + +/, Na/sup +/) to the development of cytomegalovirus (CMV)-induced morphologic cellular responses was investigated. An influx of Ca/sup + +/ was observed by the first hour after CMV infection (PI), and total calcium sequestered by infected cells was enhanced by 5 hr Pl. A gradual rise in intracellular free (IF) (Ca/sup + +/) was observedmore » that continued through 48 hour postinfection (hr Pl). The IF (Ca/sup + +/) response to CMV infection was shown to be multiplicity dependent, require viable virus, and occur under conditions consistent with the expression of immediate early CMV genes. Development and progression of cytomegaly was found to be independent of CMV DNA synthesis and appeared to be dependent on the IF (Ca/sup + +/) response. Ca/sup + +/ influx blockers (e.g. verapamil) and cyclic nucleotide modulators (e.g. papaverine) inhibited both Ca/sup + +/ responses and cytomegaly. Quabain-sensitive /sup 86/Rb uptake and sequestering of Ca/sup + +/ increased in parallel with development of cytomegaly. There may be a relationship between Ca/sup + +/ influx, IF (Ca/sup + +/), activation of the Na/sup +//H/sup +/ exchanger, induction of Na/sup +/, Cl/sup -/, HCO/sub 3/ cotransport, Na/sup +/ entry, Na/sup +//K/sup +/ ATPase activity and development of CMV-induced morphologic cellular responses including cytomegaly.« less

Treatment of a Solid Tumor Using Engineered Drug-Resistant Immunocompetent Cells and Cytotoxic Chemotherapy

PubMed Central

Dasgupta, Anindya; Shields, Jordan E.

2012-01-01

Abstract Multimodal therapy approaches, such as combining chemotherapy agents with cellular immunotherapy, suffers from potential drug-mediated toxicity to immune effector cells. Overcoming such toxic effects of anticancer cellular products is a potential critical barrier to the development of combined therapeutic approaches. We are evaluating an anticancer strategy that focuses on overcoming such a barrier by genetically engineering drug-resistant variants of immunocompetent cells, thereby allowing for the coadministration of cellular therapy with cytotoxic chemotherapy, a method we refer to as drug-resistant immunotherapy (DRI). The strategy relies on the use of cDNA sequences that confer drug resistance and recombinant lentiviral vectors to transfer nucleic acid sequences into immunocompetent cells. In the present study, we evaluated a DRI-based strategy that incorporates the immunocompetent cell line NK-92, which has intrinsic antitumor properties, genetically engineered to be resistant to both temozolomide and trimetrexate. These immune effector cells efficiently lysed neuroblastoma cell lines, which we show are also sensitive to both chemotherapy agents. The antitumor efficacy of the DRI strategy was demonstrated in vivo, whereby neuroblastoma-bearing NOD/SCID/γ-chain knockout (NSG) mice treated with dual drug-resistant NK-92 cell therapy followed by dual cytotoxic chemotherapy showed tumor regression and significantly enhanced survival compared with animals receiving either nonengineered cell-based therapy and chemotherapy, immunotherapy alone, or chemotherapy alone. These data show there is a benefit to using drug-resistant cellular therapy when combined with cytotoxic chemotherapy approaches. PMID:22397715

A role for SIRT1 in cell growth and chemoresistance in prostate cancer PC3 and DU145 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kojima, Keitaro; Department of Longevity and Aging Research, Gifu International Institute of Biotechnology, 1-1 Naka-fudogaoka, Kakamigahara, Gifu 504-0838; Ohhashi, Riyako

2008-08-29

SIRT1, which belongs to the family of type III histone deacetylase, is implicated in diverse cellular processes. We have determined the expression levels of SIRT1 in human prostate cancer cell lines and have examined the roles of SIRT1 in cell growth and chemoresistance. SIRT1 expression was markedly up-regulated in androgen-refractory PC3 and DU145 cells compared with androgen-sensitive LNCaP cells and its expression level was correlated with cell growth in PC3 cells. Treatment with a SIRT1 inhibitor, sirtinol, inhibited cell growth and increased sensitivity to camptothecin and cisplatin. Silencing of SIRT1 expression by siRNA also suppressed cell proliferation and reduced camptothecinmore » resistance in PC3 cells, mimicking the chemosensitizing effect caused by sirtinol. Also in DU145 cells, sirtinol treatment enhanced sensitivity to camptothecin and cisplatin. These results suggest that up-regulation of SIRT1 expression may play an important role in promoting cell growth and chemoresistance in androgen-refractory PC3 and DU145 cells.« less

Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2.

PubMed

Lee, Jung-Seok; Kim, Seul Ki; Jung, Byung-Joo; Choi, Seong-Bok; Choi, Eun-Young; Kim, Chang-Sung

2018-04-01

This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs) expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2) treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia) and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis), and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Hierarchically Self-Assembled Supramolecular Host-Guest Delivery System for Drug Resistant Cancer Therapy.

PubMed

Cheng, Hongwei; Fan, Xiaoshan; Wang, Xiaoyuan; Ye, Enyi; Loh, Xian Jun; Li, Zibiao; Wu, Yun-Long

2018-06-11

In this report, a new star-like copolymer β-CD- g-(PNIPAAm- b-POEGA) x , consisting of a β-CD core, grafted with temperature-responsive poly( N-isopropylacrylamide) (PNIPAAm) and biocompatible poly(oligo(ethylene glycol) acrylate) (POEGA) in a block copolymer of the arms, was used to deliver chemotherapeutics to drug resistant cancer cells and tumors. The first step of the self-assembly process involves the encapsulation of chemotherapeutics through host-guest inclusion complexation between the β-cyclodextrin cavity and the anticancer drug. Next, the chain interaction of the PNIPAAm segment at elevated temperature drives the drug-loaded β-CD- g-(PNIPAAm- b-POEGA) x /PTX inclusion complex to hierarchically self-assemble into nanosized supramolecular assemblies at 37 °C, whereas the presence of poly(ethylene glycol) (PEG) chains in the distal end of the star-like copolymer arms impart enhanced stability to the self-assembled structure. More interestingly, this supramolecular host-guest nanocomplex promoted the enhanced cellular uptake of chemotherapeutics in MDR-1 up-regulated drug resistant cancer cells and exhibited high therapeutic efficacy for suppressing drug resistant tumor growth in an in vivo mouse model, due to the increased stability, improvement in aqueous solubility, enhanced cellular uptake, and partial membrane pump impairment by taking the advantage of PEGylation and supramolecular complex between this star-like copolymer and chemotherapeutics. This work signifies that temperature-sensitive PEGylated supramolecular nanocarriers with good biocompatibility are effective in combating MDR-1 mediated drug resistance in both in vitro and in vivo models, which is of significant importance for the advanced drug delivery platform designed to combat drug resistant cancer.

The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system.

PubMed

Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

2014-01-01

ATP-sensitive potassium channels (K(ATP)) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated K(ATP) channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system. © 2013 The British Pharmacological Society.

Mathematical Modeling and Experimental Validation of Nanoemulsion-Based Drug Transport across Cellular Barriers.

PubMed

Kadakia, Ekta; Shah, Lipa; Amiji, Mansoor M

2017-07-01

Nanoemulsions have shown potential in delivering drug across epithelial and endothelial cell barriers, which express efflux transporters. However, their transport mechanisms are not entirely understood. Our goal was to investigate the cellular permeability of nanoemulsion-encapsulated drugs and apply mathematical modeling to elucidate transport mechanisms and sensitive nanoemulsion attributes. Transport studies were performed in Caco-2 cells, using fish oil nanoemulsions and a model substrate, rhodamine-123. Permeability data was modeled using a semi-mechanistic approach, capturing the following cellular processes: endocytotic uptake of the nanoemulsion, release of rhodamine-123 from the nanoemulsion, efflux and passive permeability of rhodamine-123 in aqueous solution. Nanoemulsions not only improved the permeability of rhodamine-123, but were also less sensitive to efflux transporters. The model captured bidirectional permeability results and identified sensitive processes, such as the release of the nanoemulsion-encapsulated drug and cellular uptake of the nanoemulsion. Mathematical description of cellular processes, improved our understanding of transport mechanisms, such as nanoemulsions don't inhibit efflux to improve drug permeability. Instead, their endocytotic uptake, results in higher intracellular drug concentrations, thereby increasing the concentration gradient and transcellular permeability across biological barriers. Modeling results indicated optimizing nanoemulsion attributes like the droplet size and intracellular drug release rate, may further improve drug permeability.

Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex*

PubMed Central

Ranoa, Diana Rose E.; Kelley, Stacy L.; Tapping, Richard I.

2013-01-01

Bacterial lipoproteins are the most potent microbial agonists for the Toll-like receptor 2 (TLR2) subfamily, and this pattern recognition event induces cellular activation, leading to host immune responses. Triacylated bacterial lipoproteins coordinately bind TLR1 and TLR2, resulting in a stable ternary complex that drives intracellular signaling. The sensitivity of TLR-expressing cells to lipoproteins is greatly enhanced by two lipid-binding serum proteins known as lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14); however, the physical mechanism that underlies this increased sensitivity is not known. To address this, we measured the ability of LBP and sCD14 to drive ternary complex formation between soluble extracellular domains of TLR1 and TLR2 and a synthetic triacylated lipopeptide agonist. Importantly, addition of substoichiometric amounts of either LBP or sCD14 significantly enhanced formation of a TLR1·TLR2 lipopeptide ternary complex as measured by size exclusion chromatography. However, neither LBP nor sCD14 was physically associated with the final ternary complex. Similar results were obtained using outer surface protein A (OspA), a naturally occurring triacylated lipoprotein agonist from Borrelia burgdorferi. Activation studies revealed that either LBP or sCD14 sensitized TLR-expressing cells to nanogram levels of either the synthetic lipopeptide or OspA lipoprotein agonist. Together, our results show that either LBP or sCD14 can drive ternary complex formation and TLR activation by acting as mobile carriers of triacylated lipopeptides or lipoproteins. PMID:23430250

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis

PubMed Central

Phuc, Le Thi Minh; Taniguchi, Akiyoshi

2017-01-01

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF–EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications. PMID:28629179

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis.

PubMed

Phuc, Le Thi Minh; Taniguchi, Akiyoshi

2017-06-19

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF-EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications.

Chronic Intermittent Ethanol Inhalation Increases Ethanol Self-administration in both C57BL/6J and DBA/2J Mice

PubMed Central

McCool, Brian A.; Chappell, Ann M.

2015-01-01

Inbred mouse strains provide significant opportunities to understand the genetic mechanisms controlling ethanol-directed behaviors and neurobiology. They have been specifically employed to understand cellular mechanisms contributing to ethanol consumption, acute intoxication, and sensitivities to chronic effects. However, limited ethanol consumption by some strains has restricted our understanding of clinically relevant endpoints such as dependence-related ethanol intake. Previous work with a novel tastant-substitution procedure using monosodium glutamate (MSG or umami flavor) has shown that the procedure greatly enhances ethanol consumption by mouse strains that express limited drinking phenotypes using other methods. In the current study, we employ this MSG-substitution procedure to examine how ethanol dependence, induced with passive vapor inhalation, modifies ethanol drinking in C57BL/6J and DBA/2J mice. These strains represent ‘high’ and ‘low’ drinking phenotypes, respectively. We found that the MSG substitution greatly facilitates ethanol drinking in both strains, and likewise, ethanol dependence increased ethanol consumption regardless of strain. However, DBA/2J mice exhibited greater sensitivity dependence-enhanced drinking, as represented by consumption behaviors directed at lower ethanol concentrations and relative to baseline intake levels. DBA/2J mice also exhibited significant withdrawal-associated anxiety-like behavior while C57BL/6J mice did not. These findings suggest that the MSG-substitution procedure can be employed to examine dependence-enhanced ethanol consumption across a range of drinking phenotypes, and that C57BL/6J and DBA/2J mice may represent unique neurobehavioral pathways for developing dependence-enhanced ethanol consumption. PMID:25659650

Selective inhibition of histone deacetylase 6 (HDAC6) induces DNA damage and sensitizes transformed cells to anticancer agents.

PubMed

Namdar, Mandana; Perez, Gisela; Ngo, Lang; Marks, Paul A

2010-11-16

Histone deacetylase 6 (HDAC6) is structurally and functionally unique among the 11 human zinc-dependent histone deacetylases. Here we show that chemical inhibition with the HDAC6-selective inhibitor tubacin significantly enhances cell death induced by the topoisomerase II inhibitors etoposide and doxorubicin and the pan-HDAC inhibitor SAHA (vorinostat) in transformed cells (LNCaP, MCF-7), an effect not observed in normal cells (human foreskin fibroblast cells). The inactive analogue of tubacin, nil-tubacin, does not sensitize transformed cells to these anticancer agents. Further, we show that down-regulation of HDAC6 expression by shRNA in LNCaP cells enhances cell death induced by etoposide, doxorubicin, and SAHA. Tubacin in combination with SAHA or etoposide is more potent than either drug alone in activating the intrinsic apoptotic pathway in transformed cells, as evidenced by an increase in PARP cleavage and partial inhibition of this effect by the pan-caspase inhibitor Z-VAD-fmk. HDAC6 inhibition with tubacin induces the accumulation of γH2AX, an early marker of DNA double-strand breaks. Tubacin enhances DNA damage induced by etoposide or SAHA as indicated by increased accumulation of γH2AX and activation of the checkpoint kinase Chk2. Tubacin induces the expression of DDIT3 (CHOP/GADD153), a transcription factor up-regulated in response to cellular stress. DDIT3 induction is further increased when tubacin is combined with SAHA. These findings point to mechanisms by which HDAC6-selective inhibition can enhance the efficacy of certain anti-cancer agents in transformed cells.

Cell-specific radiosensitization by gold nanoparticles at megavoltage radiation energies.

PubMed

Jain, Suneil; Coulter, Jonathan A; Hounsell, Alan R; Butterworth, Karl T; McMahon, Stephen J; Hyland, Wendy B; Muir, Mark F; Dickson, Glenn R; Prise, Kevin M; Currell, Fred J; O'Sullivan, Joe M; Hirst, David G

2011-02-01

Gold nanoparticles (GNPs) have been shown to cause sensitization with kilovoltage (kV) radiation. Differences in the absorption coefficient between gold and soft tissue, as a function of photon energy, predict that maximum enhancement should occur in the kilovoltage (kV) range, with almost no enhancement at megavoltage (MV) energies. Recent studies have shown that GNPs are not biologically inert, causing oxidative stress and even cell death, suggesting a possible biological mechanism for sensitization. The purpose of this study was to assess GNP radiosensitization at clinically relevant MV X-ray energies. Cellular uptake, intracellular localization, and cytotoxicity of GNPs were assessed in normal L132, prostate cancer DU145, and breast cancer MDA-MB-231 cells. Radiosensitization was measured by clonogenic survival at kV and MV photon energies and MV electron energies. Intracellular DNA double-strand break (DSB) induction and DNA repair were determined and GNP chemosensitization was assessed using the radiomimetic agent bleomycin. GNP uptake occurred in all cell lines and was greatest in MDA-MB-231 cells with nanoparticles accumulating in cytoplasmic lysosomes. In MDA-MB-231 cells, radiation sensitizer enhancement ratios (SERs) of 1.41, 1.29, and 1.16 were achieved using 160 kVp, 6 MV, and 15 MV X-ray energies, respectively. No significant effect was observed in L132 or DU145 cells at kV or MV energies (SER 0.97-1.08). GNP exposure did not increase radiation-induced DSB formation or inhibit DNA repair; however, GNP chemosensitization was observed in MDA-MB-231 cells treated with bleomycin (SER 1.38). We have demonstrated radiosensitization in MDA-MB-231 cells at MV X-ray energies. The sensitization was cell-specific with comparable effects at kV and MV energies, no increase in DSB formation, and GNP chemopotentiation with bleomycin, suggesting a possible biological mechanism of radiosensitization. Copyright © 2011 Elsevier Inc. All rights reserved.

Multifunctional pH-sensitive superparamagnetic iron-oxide nanocomposites for targeted drug delivery and MR imaging.

PubMed

Zhu, Lijuan; Wang, Dali; Wei, Xuan; Zhu, Xinyuan; Li, Jianqi; Tu, Chunlai; Su, Yue; Wu, Jieli; Zhu, Bangshang; Yan, Deyue

2013-08-10

A multifunctional pH-sensitive superparamagnetic iron-oxide (SPIO) nanocomposite system was developed for simultaneous tumor magnetic resonance imaging (MRI) and therapy. Small-size SPIO nanoparticles were chemically bonded with antitumor drug doxorubicin (DOX) and biocompatible poly(ethylene glycol) (PEG) through pH-sensitive acylhydrazone linkages, resulting in the formation of SPIO nanocomposites with magnetic targeting and pH-sensitive properties. These DOX-conjugated SPIO nanocomposites exhibited not only good stability in aqueous solution but also high saturation magnetizations. Under an acidic environment, the DOX was quickly released from the SPIO nanocomposites due to the cleavage of pH-sensitive acylhydrazone linkages. With the help of magnetic field, the DOX-conjugated SPIO nanocomposites showed high cellular uptake, indicating their magnetic targeting property. Comparing to free DOX, the DOX-conjugated SPIO nanocomposites showed better antitumor effect under magnetic field. At the same time, the relaxivity value of these SPIO nanocomposites was higher than 146s(-1)mM(-1) Fe, leading to ~4 times enhancement compared to that of free SPIO nanoparticles. As a negative contrast agent, these SPIO nanocomposites illustrated high resolution in MRI diagnosis of tumor-bearing mice. All of these results confirm that these pH-sensitive SPIO nanocomposites are promising hybrid materials for synergistic MRI diagnosis and tumor therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

Vancomycin incorporated chitosan/gelatin coatings coupled with TiO2-SrHAP surface modified cp-titanium for osteomyelitis treatment.

PubMed

D, Nancy; N, Rajendran

2018-04-15

Commercially pure Titanium (Cp-Ti) was electrophoretically modified using double layer coatings consisting of TiO 2 -SrHAP as the first layer (TH) followed by vancomycin incorporated Chitosan/Gelatin as the second layer (THV). The nano crystalline phase of coated Strontium incorporated hydroxyapatite (Sr-HAP) confirmed through X-ray diffraction studies (XRD). The polyelectrolyte complex formation between chitosan and gelatin, the stability of the drug, the bonding between chitosan and Sr-HAP were confirmed through infra-red spectroscopic studies (IR). The average roughness (R a ) value calculated from atomic force microscopy (AFM) corroborates with the water contact angle data, which clearly confirms the tuning property of the surface in relation to the surface energy and roughness of the coated samples. The total amount of vancomycin encapsulated was calculated to be 11.5 μg. Antibacterial activity was found against both Staphylococcus aureus strains methicillin resistant Staphylococcus aureus (MRSA) and methicillin sensitive Staphylococcus aureus (MRSA) for a drug concentration of 2.74 μg released after 12 h of immersion. The in-vitro cell culture studies showed enhanced cellular activity for THV samples. Thus, THV samples have a dual action at the surface, by resisting the bacterial adhesion and enhancing cellular interaction at the bio-interface, making it a promising candidate to treat osteomyelitis infection. Copyright © 2018. Published by Elsevier B.V.

Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

NASA Astrophysics Data System (ADS)

Marquet, P.; Rothenfusser, K.; Rappaz, B.; Depeursinge, C.; Jourdain, P.; Magistretti, P. J.

2016-03-01

Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining1

PubMed Central

Behbehani, Gregory K.; Thom, Colin; Zunder, Eli R.; Finck, Rachel; Gaudilliere, Brice; Fragiadakis, Gabriela K.; Fantl, Wendy J.; Nolan, Garry P.

2015-01-01

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression. PMID:25274027

Upregulation of Nicotinic Acetylcholine Receptor alph4+beta2 through a Ligand-Independent PI3Kbeta Mechanism That Is Enhanced by TNFalpha and the Jak2/p38Mapk Pathways.

PubMed

Rogers, Scott W; Gahring, Lorise C

2015-01-01

High affinity nicotine-binding sites in the mammalian brain are neuronal nicotinic acetylcholine receptors (nAChR) assembled from at least alpha4 and beta2 subunits into pentameric ion channels. When exposed to ligands such as nicotine, these receptors respond by undergoing upregulation, a correlate of nicotine addiction. Upregulation can be measured using HEK293 (293) cells that stably express alpha4 and beta2 subunits using quantification of [3H]epibatidine ([3H]Eb) binding to measure mature receptors. Treatment of these cells with choline also produces upregulation through a hemicholinium3 (HC3)-sensitive (choline kinase) and an HC3-insensitive pathway which are both independent of the mechanism used by nicotine for upregulation. In both cases, upregulation is significantly enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) which signals through its receptor Tnfr1 to activate p38Mapk. Here we report that the inhibition of class1 phosphoinositide 3-kinases isoform PI3Kbeta using the selective antagonist PI828 is alone sufficient to produce upregulation and enhance both nicotine and choline HC3-sensitive mediated upregulation. Further, these processes are impacted upon by an AG-490 sensitive Jak2-associated pathway. Both PI3Kbeta (negative) and Jak2 (positive) modulation of upregulation converge through p38Mapk and both overlap with TNFalpha enhancement of this process. Upregulation through the PI3Kbeta pathway did not require Akt. Collectively these findings support upregulation of endogenous alpha4beta2 as a balance among cellular signaling networks that are highly responsive to multiple environmental, inflammatory and metabolic agents. The findings also suggest how illness and metabolic stress could alter the expression of this important nicotinic receptor and novel avenues to intercede in modifying its expression.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice

PubMed Central

Feeks, James A.; Hunter, Jennifer J.

2017-01-01

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina. PMID:28663886

Telmisartan enhances mitochondrial activity and alters cellular functions in human coronary artery endothelial cells via AMP-activated protein kinase pathway.

PubMed

Kurokawa, Hirofumi; Sugiyama, Seigo; Nozaki, Toshimitsu; Sugamura, Koichi; Toyama, Kensuke; Matsubara, Junichi; Fujisue, Koichiro; Ohba, Keisuke; Maeda, Hirofumi; Konishi, Masaaki; Akiyama, Eiichi; Sumida, Hitoshi; Izumiya, Yasuhiro; Yasuda, Osamu; Kim-Mitsuyama, Shokei; Ogawa, Hisao

2015-04-01

Mitochondrial dysfunction plays an important role in cellular senescence and impaired function of vascular endothelium, resulted in cardiovascular diseases. Telmisartan is a unique angiotensin II type I receptor blocker that has been shown to prevent cardiovascular events in high risk patients. AMP-activated protein kinase (AMPK) plays a critical role in mitochondrial biogenesis and endothelial function. This study assessed whether telmisartan enhances mitochondrial function and alters cellular functions via AMPK in human coronary artery endothelial cells (HCAECs). In cultured HCAECs, telmisartan significantly enhanced mitochondrial activity assessed by mitochondrial reductase activity and intracellular ATP production and increased the expression of mitochondria related genes. Telmisartan prevented cellular senescence and exhibited the anti-apoptotic and pro-angiogenic properties. The expression of genes related anti-oxidant and pro-angiogenic properties were increased by telmisartan. Telmisartan increased endothelial NO synthase and AMPK phosphorylation. Peroxisome proliferator-activated receptor gamma signaling was not involved in telmisartan-induced improvement of mitochondrial function. All of these effects were abolished by inhibition of AMPK. Telmisartan enhanced mitochondrial activity and exhibited anti-senescence effects and improving endothelial function through AMPK in HCAECs. Telmisartan could provide beneficial effects on vascular diseases via enhancement of mitochondrial activity and modulating endothelial function through AMPK activation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Fluorescence Molecular Tomography: Principles and Potential for Pharmaceutical Research

PubMed Central

Stuker, Florian; Ripoll, Jorge; Rudin, Markus

2011-01-01

Fluorescence microscopic imaging is widely used in biomedical research to study molecular and cellular processes in cell culture or tissue samples. This is motivated by the high inherent sensitivity of fluorescence techniques, the spatial resolution that compares favorably with cellular dimensions, the stability of the fluorescent labels used and the sophisticated labeling strategies that have been developed for selectively labeling target molecules. More recently, two and three-dimensional optical imaging methods have also been applied to monitor biological processes in intact biological organisms such as animals or even humans. These whole body optical imaging approaches have to cope with the fact that biological tissue is a highly scattering and absorbing medium. As a consequence, light propagation in tissue is well described by a diffusion approximation and accurate reconstruction of spatial information is demanding. While in vivo optical imaging is a highly sensitive method, the signal is strongly surface weighted, i.e., the signal detected from the same light source will become weaker the deeper it is embedded in tissue, and strongly depends on the optical properties of the surrounding tissue. Derivation of quantitative information, therefore, requires tomographic techniques such as fluorescence molecular tomography (FMT), which maps the three-dimensional distribution of a fluorescent probe or protein concentration. The combination of FMT with a structural imaging method such as X-ray computed tomography (CT) or Magnetic Resonance Imaging (MRI) will allow mapping molecular information on a high definition anatomical reference and enable the use of prior information on tissue's optical properties to enhance both resolution and sensitivity. Today many of the fluorescent assays originally developed for studies in cellular systems have been successfully translated for experimental studies in animals. The opportunity of monitoring molecular processes non-invasively in the intact organism is highly attractive from a diagnostic point of view but even more so for the drug developer, who can use the techniques for proof-of-mechanism and proof-of-efficacy studies. This review shall elucidate the current status and potential of fluorescence tomography including recent advances in multimodality imaging approaches for preclinical and clinical drug development. PMID:24310495

Multidrug resistance reversal and apoptosis induction in human colon cancer cells by some flavonoids present in citrus plants.

PubMed

Wesołowska, Olga; Wiśniewski, Jerzy; Sroda-Pomianek, Kamila; Bielawska-Pohl, Aleksandra; Paprocka, Maria; Duś, Danuta; Duarte, Noélia; Ferreira, Maria-José U; Michalak, Krystyna

2012-11-26

Multidrug resistance (MDR) of cancer cells constitutes one of the main reasons for chemotherapy failure. The search for nontoxic modulators that reduce MDR is a task of great importance. An ability to enhance apoptosis of resistant cells would also be beneficial. In the present study, the MDR reversal and apoptosis-inducing potency of three flavonoids produced by Citrus plants, namely, naringenin (1a), aromadendrin (2), and tangeretin (3), and the methylated naringenin derivatives (1b, 1c), have been studied in sensitive (LoVo) and multidrug-resistant (LoVo/Dx) human colon adenocarcinoma cells. Cytotoxicity of methoxylated flavonoids was higher as compared to hydroxylated analogues. Only 3 turned out to inhibit P-glycoprotein, as demonstrated by a rhodamine 123 accumulation assay. It also increased doxorubicin accumulation in LoVo/Dx cells and enabled doxorubicin to enter cellular nuclei. In addition, 3 was found to be an effective MDR modulator in resistant cells by sensitizing them to doxorubicin. Tangeretin-induced caspase-3 activation and elevated surface phosphatidylserine exposure demonstrated its apoptosis-inducing activity in LoVo/Dx cells, while the other flavonoids evaluated were not active. Additionally, 3 was more toxic to resistant rather than to sensitive cancer cells. Its apoptosis-inducing activity was also higher in LoVo/Dx than in LoVo cells. It was concluded that the activity of 3 against multidrug-resistant cancer cells may be enhanced by its apoptosis-inducing activity.

Role of androgen-mediated enhancement of erythropoiesis in the increased body iron stores of patients with polycystic ovary syndrome.

PubMed

Escobar-Morreale, Héctor F; Luque-Ramírez, Manuel

2011-04-01

To determine whether androgen excess contributes to the increased body iron stores of polycystic ovary syndrome (PCOS) by stimulating erythropoietic activity, by measuring serum soluble transferrin receptor (sTfR) concentrations and its ratio to ferritin levels in patients with PCOS, as surrogate markers of erythropoietic activity and of the appropriateness of cellular iron demands for the total body iron contents, respectively. Case-control study. Academic hospital. One hundred-four patients with PCOS and 100 controls without androgen excess. Blood sampling and oral glucose tolerance test. Serum sTfR and ferritin concentrations, as well as indexes of androgen excess, inflammation, obesity, and insulin and glucose metabolism. Serum ferritin levels increased in women presenting with PCOS, obesity, and/or abnormal glucose tolerance, but these disorders did not influence sTfR concentrations. The sTfR/ferritin ratio decreased with obesity and abnormal glucose tolerance, and its logarithm correlated inversely with body mass index, free T, and C-reactive protein levels and directly with the insulin sensitivity and disposition indexes. A stepwise multiple regression analysis indicated that the changes in the insulin sensitivity index explained 7% of the variability of the logarithm of sTfR/ferritin ratio. Increased serum ferritin levels in patients with PCOS are associated with a reduction in insulin sensitivity but do not result from a putative enhancement of erythropoiesis by androgen excess. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Redox-sensitive micelles composed of disulfide-linked Pluronic-linoleic acid for enhanced anticancer efficiency of brusatol

PubMed Central

Chan, Hon Fai; Lin, Zhixiu; Wang, Yitao

2018-01-01

Brusatol (Bru) exhibits promising anticancer effects, with both proliferation inhibition and chemoresistance amelioration activity. However, the poor solubility and insufficient intracellular delivery of Bru greatly restrict its application. Herein, to simultaneously utilize the advantages of Pluronics as drug carriers and tumor microenvironment-responsive drug release profiles, a flexible amphiphilic copolymer with a polymer skeleton, that is, Pluronic® F68 grafting with linoleic acid moieties by redox-reducible disulfide bonds (F68-SS-LA), was synthesized. After characterization by 1H-nuclear magnetic resonance and Fourier transform infrared spectroscopy, the redox-sensitive F68-SS-LA micelles were self-assembled in a much lower critical micelle concentration than that of the unmodified F68 copolymer. Bru was loaded in micelles (Bru/SS-M) with high loading efficiency, narrow size distribution, and excellent storage stability. The redox-sensitive Bru/SS-M exhibited rapid particle dissociation and drug release in response to a redox environment. Based on the enhanced cellular internalization, Bru/SS-M achieved higher cytotoxicity in both Bel-7402 and MCF-7 cells compared with free Bru and nonreducible micelles. The improved anticancer effect was attributed to the remarkably decreased mitochondrial membrane potential and increased reactive oxygen species level as well as apoptotic rate. These results demonstrated that F68-SS-LA micelles possess great potential as an efficient delivery vehicle for Bru to promote its anticancer efficiency via an oxidation pathway. PMID:29491708

A pH-sensitive micelle composed of heparin, phospholipids, and histidine as the carrier of photosensitizers: Application to enhance photodynamic therapy of cancer.

PubMed

Debele, Tilahun Ayane; Mekuria, Shewaye Lakew; Tsai, Hsieh-Chih

2017-05-01

In this study, we describe the synthesis of a stable, pH-sensitive micelle composed of heparin, 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine, and l-histidine (HDH) through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) chemistry. 1 H-Nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) analyses confirmed the formation of HDH copolymers and dynamic light scattering (DLS) measurements indicated a particle size of 111.57±12.36nm and zeta potential of -59.8±5.2mV for the nanoparticles. The drug-loading and encapsulation efficiency of the micelles were 14.52±1.2% and 65.47±1.87%, respectively. Drug release studies showed approximately 91% zinc phthalocyanine (ZnPc) release from micelles in acidic conditions (pH 5.0) in comparison with 63% in physiological conditions (pH 7.4) after 96h of incubation. Singlet oxygen ( 1 O 2 ) detection revealed that the micelles prevented ZnPc aggregation and enhanced 1 O 2 generation. Cellular uptake of ZnPc-loaded micelles (ZnPc-HDH) was observed using confocal microscopy. Phototoxicity experiments in HeLa cells showed that ZnPc-loaded micelles had higher toxicity than that shown by the same concentration of free ZnPc. Hence, pH-sensitive HDH micelles are a promising carrier for hydrophobic ZnPc and improving PDT efficacy. Copyright © 2017 Elsevier B.V. All rights reserved.

Dynamic Nuclear Polarization NMR in Human Cells Using Fluorescent Polarizing Agents.

PubMed

Albert, Brice J; Gao, Chukun; Sesti, Erika L; Saliba, Edward P; Alaniva, Nicholas; Scott, Faith J; Sigurdsson, Snorri Th; Barnes, Alexander B

2018-06-20

Solid-state nuclear magnetic resonance (NMR) enables atomic resolution characterization of molecular structure and dynamics within complex heterogeneous samples, but it is typically insensitive. Dynamic nuclear polarization (DNP) increases NMR signal intensity by orders of magnitude and can be performed in combination with magic angle spinning (MAS) for sensitive, high-resolution spectroscopy. Here we report MAS DNP experiments, for the first time, within intact human cells with >40-fold DNP enhancement and a sample temperature below 6 K. In addition to cryogenic MAS results below 6 K, we also show in-cell DNP enhancements of 57-fold at 90 K. In-cell DNP is demonstrated using biradicals and sterically-shielded monoradicals as polarizing agents. A novel trimodal polarizing agent is introduced for DNP, which contains a nitroxide biradical, a targeting peptide for cell penetration, and a fluorophore for subcellular localization with confocal microscopy. The fluorescent polarizing agent provides in-cell DNP enhancements of 63-fold at a concentration of 2.7 mM. These experiments pave the way for structural characterization of biomolecules in an endogenous cellular context.

Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine-needle aspirate samples.

PubMed

Dyhdalo, Kathryn; Macnamara, Stephen; Brainard, Jennifer; Underwood, Dawn; Tubbs, Raymond; Yang, Bin

2014-02-01

BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples. This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 10+ cells on a ThinPrep slide: 1+ (1-5 groups), 2+ (6-10 groups), 3+ (11-20 groups), and 4+ (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis. Approximately 49% of thyroid FNA samples had low cellularity (1-2+). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 1+ to 4+ cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 10+ cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells. This study demonstrates that by using 6 groups of 10+ follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens. Cancer (Cancer Cytopathol) 2014;122:114-22 © 2013 American Cancer Society. © 2013 American Cancer Society.

DEEP: a general computational framework for predicting enhancers

PubMed Central

Kleftogiannis, Dimitrios; Kalnis, Panos; Bajic, Vladimir B.

2015-01-01

Transcription regulation in multicellular eukaryotes is orchestrated by a number of DNA functional elements located at gene regulatory regions. Some regulatory regions (e.g. enhancers) are located far away from the gene they affect. Identification of distal regulatory elements is a challenge for the bioinformatics research. Although existing methodologies increased the number of computationally predicted enhancers, performance inconsistency of computational models across different cell-lines, class imbalance within the learning sets and ad hoc rules for selecting enhancer candidates for supervised learning, are some key questions that require further examination. In this study we developed DEEP, a novel ensemble prediction framework. DEEP integrates three components with diverse characteristics that streamline the analysis of enhancer's properties in a great variety of cellular conditions. In our method we train many individual classification models that we combine to classify DNA regions as enhancers or non-enhancers. DEEP uses features derived from histone modification marks or attributes coming from sequence characteristics. Experimental results indicate that DEEP performs better than four state-of-the-art methods on the ENCODE data. We report the first computational enhancer prediction results on FANTOM5 data where DEEP achieves 90.2% accuracy and 90% geometric mean (GM) of specificity and sensitivity across 36 different tissues. We further present results derived using in vivo-derived enhancer data from VISTA database. DEEP-VISTA, when tested on an independent test set, achieved GM of 80.1% and accuracy of 89.64%. DEEP framework is publicly available at http://cbrc.kaust.edu.sa/deep/. PMID:25378307

SU-F-19A-08: Optimal Time Release Schedule of In-Situ Drug Release During Permanent Prostate Brachytherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cormack, R; Ngwa, W; Makrigiorgos, G

Purpose: Permanent prostate brachytherapy spacers can be used to deliver sustained doses of radiosentitizing drug directly to the target, in order to enhance the radiation effect. Implantable nanoplatforms for chemo-radiation therapy (INCeRTs) have a maximum drug capacity and can be engineered to control the drug release schedule. The optimal schedule for sensitization during continuous low dose rate irradiation is unknown. This work studies the optimal release schedule of drug for both traditional sensitizers, and those that work by suppressing DNA repair processes. Methods: Six brachytherapy treatment plans were used to model the anatomy, implant geometry and calculate the spatial distributionmore » of radiation dose and drug concentrations for a range of drug diffusion parameters. Three state partial differential equations (cells healthy, damaged or dead) modeled the effect of continuous radiation (radiosensitivities α,β) and cellular repair (time tr) on a cell population. Radiosensitization was modeled as concentration dependent change in α,β or tr which with variable duration under the constraint of fixed total drug release. Average cell kill was used to measure effectiveness. Sensitization by means of both enhanced damage and reduced repair were studied. Results: Optimal release duration is dependent on the concentration of radiosensitizer compared to the saturation concentration (csat) above which additional sensitization does not occur. Long duration drug release when enhancing α or β maximizes cell death when drug concentrations are generally over csat. Short term release is optimal for concentrations below saturation. Sensitization by suppressing repair has a similar though less distinct trend that is more affected by the radiation dose distribution. Conclusion: Models of sustained local radiosensitization show potential to increase the effectiveness of radiation in permanent prostate brachytherapy. INCeRTs with high drug capacity produce the greatest benefit with drug release over weeks. If in-vivo drug concentrations are not able to approach saturation concentration, durations of days is optimal. DOD 1R21CA16977501; A. David Mazzone Awards Program 2012PD164.« less

Spectral domain phase microscopy: a new tool for measuring cellular dynamics and cytoplasmic flow

NASA Astrophysics Data System (ADS)

McDowell, Emily J.; Choma, Michael A.; Ellerbee, Audrey K.; Izatt, Joseph A.

2005-03-01

Broadband interferometry is an attractive technique for the detection of cellular motions because it provides depth-resolved interferometric phase information via coherence gating. Here a phase sensitive technique called spectral domain phase microscopy (SDPM) is presented. SDPM is a functional extension of spectral domain optical coherence tomography that allows for the detection of cellular motions and dynamics with nanometer-scale sensitivity. This sensitivity is made possible by the inherent phase stability of spectral domain OCT combined with common-path interferometry. The theory that underlies this technique is presented, the sensitivity of the technique is demonstrated by the measurement of the thermal expansion coefficient of borosilicate glass, and the response of an Amoeba proteus to puncture of its cell membrane is measured. We also exploit the phase stability of SDPM to perform Doppler flow imaging of cytoplasmic streaming in A. proteus. We show reversal of cytoplasmic flow in response to stimuli, and we show that the cytoplasmic flow is laminar (i.e. parabolic) in nature. We are currently investigating the use of SDPM in a variety of different cell types.

Progress and challenges in bioinformatics approaches for enhancer identification

PubMed Central

Kleftogiannis, Dimitrios; Kalnis, Panos

2016-01-01

Enhancers are cis-acting DNA elements that play critical roles in distal regulation of gene expression. Identifying enhancers is an important step for understanding distinct gene expression programs that may reflect normal and pathogenic cellular conditions. Experimental identification of enhancers is constrained by the set of conditions used in the experiment. This requires multiple experiments to identify enhancers, as they can be active under specific cellular conditions but not in different cell types/tissues or cellular states. This has opened prospects for computational prediction methods that can be used for high-throughput identification of putative enhancers to complement experimental approaches. Potential functions and properties of predicted enhancers have been catalogued and summarized in several enhancer-oriented databases. Because the current methods for the computational prediction of enhancers produce significantly different enhancer predictions, it will be beneficial for the research community to have an overview of the strategies and solutions developed in this field. In this review, we focus on the identification and analysis of enhancers by bioinformatics approaches. First, we describe a general framework for computational identification of enhancers, present relevant data types and discuss possible computational solutions. Next, we cover over 30 existing computational enhancer identification methods that were developed since 2000. Our review highlights advantages, limitations and potentials, while suggesting pragmatic guidelines for development of more efficient computational enhancer prediction methods. Finally, we discuss challenges and open problems of this topic, which require further consideration. PMID:26634919

AMP-activated Protein Kinase (AMPK): Does This Master Regulator of Cellular Energy State Distinguish Insulin Sensitive from Insulin Resistant Obesity?

PubMed Central

Valentine, Rudy J.; Ruderman, Neil B.

2014-01-01

Although a correlation exists between obesity and insulin resistance, roughly 25 % of obese individuals are insulin sensitive. AMP-activated protein kinase (AMPK) is a cellular energy sensor that among its many actions, integrates diverse physiological signals to restore energy balance. In addition, in many situations it also increases insulin sensitivity. In this context, AMPK activity is decreased in very obese individuals undergoing bariatric surgery who are insulin resistant compared to equally obese patients who are insulin sensitive. In this review, we will both explore what distinguishes these individuals, and evaluate the evidence that diminished AMPK is associated with insulin resistance and metabolic syndrome-associated disorders in other circumstances. PMID:24891985

A Unique Fungal Two-Component System Regulates Stress Responses, Drug Sensitivity, Sexual Development, and Virulence of Cryptococcus neoformans

PubMed Central

Bahn, Yong-Sun; Kojima, Kaihei; Cox, Gary M.

2006-01-01

The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal “two-component” system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component–like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen. PMID:16672377

Cytosolic NADP(+)-dependent isocitrate dehydrogenase regulates cadmium-induced apoptosis.

PubMed

Shin, Seoung Woo; Kil, In Sup; Park, Jeen-Woo

2010-04-01

Cadmium ions have a high affinity for thiol groups. Therefore, they may disturb many cellular functions. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme to supply NADPH, a major source of reducing equivalents to the cytosol. Cadmium decreased the activity of IDPc both as a purified enzyme and in cultured cells. In the present study, we demonstrate that the knockdown of IDPc expression in HEK293 cells greatly enhances apoptosis induced by cadmium. Transfection of HEK293 cells with an IDPc small interfering RNA significantly decreased the activity of IDPc and enhanced cellular susceptibility to cadmium-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation and condensation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. Taken together, our results suggest that suppressing the expression of IDPc enhances cadmium-induced apoptosis of HEK293 cells by increasing disruption of the cellular redox status. Copyright 2009 Elsevier Inc. All rights reserved.

Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells

PubMed Central

Brandt, Benjamin; Munemasa, Shintaro; Wang, Cun; Nguyen, Desiree; Yong, Taiming; Yang, Paul G; Poretsky, Elly; Belknap, Thomas F; Waadt, Rainer; Alemán, Fernando; Schroeder, Julian I

2015-01-01

A central question is how specificity in cellular responses to the eukaryotic second messenger Ca2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca2+-dependent and Ca2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca2+-signaling on a cellular, genetic, and biochemical level. DOI: http://dx.doi.org/10.7554/eLife.03599.001 PMID:26192964

The emerging role of redox-sensitive Nrf2-Keap1 pathway in diabetes.

PubMed

Bhakkiyalakshmi, Elango; Sireesh, Dornadula; Rajaguru, Palanisamy; Paulmurugan, Ramasamy; Ramkumar, Kunka Mohanram

2015-01-01

The pathogenic processes involving in the development of diabetes range from autoimmune destruction of pancreatic β-cells with consequent insulin deficiency to abnormalities that result in resistance to insulin action. The major contributing factor for excessive β-cell death includes oxidative stress-mediated mitochondrial damage, which creates an imbalance in redox homeostasis. Yet, β-cells have evolved adaptive mechanisms to endure a wide range of stress conditions to safeguard its potential functions. These include 'Nrf2/Keap1' pathway, a key cellular defense mechanism, to combat oxidative stress by regulating phase II detoxifying and antioxidant genes. During diabetes, redox imbalance provokes defective Nrf2-dependent signaling and compromise antioxidant capacity of the pancreas which turnout β-cells to become highly vulnerable against various insults. Hence, identification of small molecule activators of Nrf2/Keap1 pathway remains significant to enhance cellular defense to overcome the burden of oxidative stress related disturbances. This review summarizes the molecular mechanism behind Nrf2 activation and the impact of Nrf2 activators in diabetes and its complications. Copyright © 2014 Elsevier Ltd. All rights reserved.

Correction: Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells

DOE PAGES

Brandt, Benjamin; Munemasa, Shintaro; Wang, Cun; ...

2015-07-20

One central question is how specificity in cellular responses to the eukaryotic second messenger Ca 2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca 2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca 2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca 2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruplemore » mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca 2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca 2+-dependent and Ca 2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca 2+-signaling on a cellular, genetic, and biochemical level.« less

Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging.

PubMed

Ben-Zvi, Anat; Miller, Elizabeth A; Morimoto, Richard I

2009-09-01

Protein damage contributes prominently to cellular aging. To address whether this occurs at a specific period during aging or accumulates gradually, we monitored the biochemical, cellular, and physiological properties of folding sensors expressed in different tissues of C. elegans. We observed the age-dependent misfolding and loss of function of diverse proteins harboring temperature-sensitive missense mutations in all somatic tissues at the permissive condition. This widespread failure in proteostasis occurs rapidly at an early stage of adulthood, and coincides with a severely reduced activation of the cytoprotective heat shock response and the unfolded protein response. Enhancing stress responsive factors HSF-1 or DAF-16 suppresses misfolding of these metastable folding sensors and restores the ability of the cell to maintain a functional proteome. This suggests that a compromise in the regulation of proteostatic stress responses occurs early in adulthood and tips the balance between the load of damaged proteins and the proteostasis machinery. We propose that the collapse of proteostasis represents an early molecular event of aging that amplifies protein damage in age-associated diseases of protein conformation.

Diselenolane-mediated cellular uptake.

PubMed

Chuard, Nicolas; Poblador-Bahamonde, Amalia I; Zong, Lili; Bartolami, Eline; Hildebrandt, Jana; Weigand, Wolfgang; Sakai, Naomi; Matile, Stefan

2018-02-21

The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides.

Two-Photon Fluorescence Spectroscopy and Imaging of 4-Dimethylaminonaphthalimide-Peptide and Protein Conjugates

PubMed Central

McLean, Alan M.; Socher, Elke; Varnavski, Oleg; Clark, Travis B.

2014-01-01

We report detailed photophysical studies on the two-photon fluorescence processes of the solvatochromic fluorophore 4-DMN as a conjugate of the important calmodulin (CaM) and the associated CaM-binding peptide M13. Strong two-photon fluorescence enhancement has been observed which is associated with calcium binding. It is found that the two-photon absorption cross-section is strongly dependent on the local environment surrounding the 4-DMN fluorophore in the CaM conjugates, providing sensitivity between sites of fluorophore attachment. Utilizing time-resolved measurements, the emission dynamics of 4-DMN under various environmental (solvent) conditions are analyzed. In addition, anisotropy measurements reveal that the 4-DMN-S38C-CaM system has restricted rotation in the calcium-bound calmodulin. To establish the utility for cellular imaging, two-photon fluorescence microscopy studies were also carried out with the 4-DMN-modified M13 peptide in cells. Together, these studies provide strong evidence that 4-DMN is a useful probe in two-photon imaging, with advantageous properties for cellular experiments. PMID:24245815

Correction: Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells

DOE PAGES

Brandt, Benjamin; Munemasa, Shintaro; Wang, Cun; ...

2015-07-29

A central question is how specificity in cellular responses to the eukaryotic second messenger Ca 2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca 2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca 2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca 2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruplemore » mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca 2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca 2+-dependent and Ca 2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca 2+-signaling on a cellular, genetic, and biochemical level.« less

Complexing Methylene Blue with Phosphorus Dendrimers to Increase Photodynamic Activity.

PubMed

Dabrzalska, Monika; Janaszewska, Anna; Zablocka, Maria; Mignani, Serge; Majoral, Jean Pierre; Klajnert-Maculewicz, Barbara

2017-02-23

The efficiency of photodynamic therapy is limited mainly due to low selectivity, unfavorable biodistribution of photosensitizers, and long-lasting skin sensitivity to light. However, drug delivery systems based on nanoparticles may overcome the limitations mentioned above. Among others, dendrimers are particularly attractive as carriers, because of their globular architecture and high loading capacity. The goal of the study was to check whether an anionic phosphorus dendrimer is suitable as a carrier of a photosensitizer-methylene blue (MB). As a biological model, basal cell carcinoma cell lines were used. We checked the influence of the MB complexation on its singlet oxygen production ability using a commercial fluorescence probe. Next, cellular uptake, phototoxicity, reactive oxygen species (ROS) generation, and cell death were investigated. The MB-anionic dendrimer complex (MB-1an) was found to generate less singlet oxygen; however, the complex showed higher cellular uptake and phototoxicity against basal cell carcinoma cell lines, which was accompanied with enhanced ROS production. Owing to the obtained results, we conclude that the photodynamic activity of MB complexed with an anionic dendrimer is higher than free MB against basal cell carcinoma cell lines.

High resolution surface plasmon microscopy for cell imaging

NASA Astrophysics Data System (ADS)

Argoul, F.; Monier, K.; Roland, T.; Elezgaray, J.; Berguiga, L.

2010-04-01

We introduce a new non-labeling high resolution microscopy method for cellular imaging. This method called SSPM (Scanning Surface Plasmon Microscopy) pushes down the resolution limit of surface plasmon resonance imaging (SPRi) to sub-micronic scales. High resolution SPRi is obtained by the surface plasmon lauching with a high numerical aperture objective lens. The advantages of SPPM compared to other high resolution SPRi's rely on three aspects; (i) the interferometric detection of the back reflected light after plasmon excitation, (ii) the twodimensional scanning of the sample for image reconstruction, (iii) the radial polarization of light, enhancing both resolution and sensitivity. This microscope can afford a lateral resolution of - 150 nm in liquid environment and - 200 nm in air. We present in this paper images of IMR90 fibroblasts obtained with SSPM in dried environment. Internal compartments such as nucleus, nucleolus, mitochondria, cellular and nuclear membrane can be recognized without labelling. We propose an interpretation of the ability of SSPM to reveal high index contrast zones by a local decomposition of the V (Z) function describing the response of the SSPM.

Detection and signal amplification in zebrafish RNA FISH.

PubMed

Hauptmann, Giselbert; Lauter, Gilbert; Söll, Iris

2016-04-01

In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts. Copyright © 2016 Elsevier Inc. All rights reserved.

Relaxometry model of strong dipolar perturbers for balanced-SSFP: application to quantification of SPIO loaded cells.

PubMed

Lebel, R Marc; Menon, Ravi S; Bowen, Chris V

2006-03-01

Magnetic resonance microscopy using magnetically labeled cells is an emerging discipline offering the potential for non-destructive studies targeting numerous cellular events in medical research. The present work develops a technique to quantify superparamagnetic iron-oxide (SPIO) loaded cells using fully balanced steady state free precession (b-SSFP) imaging. An analytic model based on phase cancellation was derived for a single particle and extended to predict mono-exponential decay versus echo time in the presence of multiple randomly distributed particles. Numerical models verified phase incoherence as the dominant contrast mechanism and evaluated the model using a full range of tissue decay rates, repetition times, and flip angles. Numerical simulations indicated a relaxation rate enhancement (DeltaR(2b)=0.412 gamma . LMD) proportional to LMD, the local magnetic dose (the additional sample magnetization due to the SPIO particles), a quantity related to the concentration of contrast agent. A phantom model of SPIO loaded cells showed excellent agreement with simulations, demonstrated comparable sensitivity to gradient echo DeltaR(*) (2) enhancements, and 14 times the sensitivity of spin echo DeltaR(2) measurements. We believe this model can be used to facilitate the generation of quantitative maps of targeted cell populations. Magn Reson Med, 2006. (c) 2006 Wiley-Liss, Inc.

β-elemene sensitizes hepatocellular carcinoma cells to oxaliplatin by preventing oxaliplatin-induced degradation of copper transporter 1

PubMed Central

Li, Xiaoqiang; Lin, Zhenhai; Zhang, Bo; Guo, Lei; Liu, Shuang; Li, Hui; Zhang, Jubo; Ye, Qinghai

2016-01-01

β-elemene, a Curcuma wenyujin plant extract, has been used widely as a tumor adjuvant therapeutic agent. However, how to obtain optimum therapeutic effects by combining this compound with other agents remain unclear. In this study, we found that β-elemene, which alone had little effect on hepatocellular carcinoma (HCC) cell proliferation, exerted a synergistic anti-proliferative effect in HCC cells when dosed in combination with oxaliplatin, which increased the amounts of platinum accumulation and platinum-DNA adduct significantly and augmented the oxaliplatin-induced apoptosis. Western blot and laser scanning confocal microscopy studies indicated that β-elemene enhanced the sensitivity of HCC cells to oxaliplatin by upregulating copper transporter 1 (CTR1), a major controller of intracellular platinum accumulation. In an orthotopic transplantation HCC model in nude mice, HCC tumor growth was inhibited significantly by oxaliplatin combined with β-elemene, as compared with oxaliplatin alone. Notably, CTR1 protein expression in xenograft HCC was upregulated in mice who received β-elemene treatment. Taken together, our findings show that β-elemene can block the reduction of CTR1 resulting from oxaliplatin treatment, and therefore has a synergistic anti-HCC effect with oxaliplatin by enhancing cellular uptake of oxaliplatin. The synergistic effects of β-elemene and oxaliplatin deserve further evaluation in clinical settings. PMID:26867799

Suppression of telomere-binding protein TPP1 resulted in telomere dysfunction and enhanced radiation sensitivity in telomerase-negative osteosarcoma cell line.

PubMed

Qiang, Weiguang; Wu, Qinqin; Zhou, Fuxiang; Xie, Conghua; Wu, Changping; Zhou, Yunfeng

2014-03-07

Mammalian telomeres are protected by the shelterin complex that contains the six core proteins POT1, TPP1, TIN2, TRF1, TRF2 and RAP1. TPP1, formerly known as TINT1, PTOP, and PIP1, is a key factor that regulates telomerase recruitment and activity. In addition to this, TPP1 is required to mediate the shelterin assembly and stabilize telomere. Previous work has found that TPP1 expression was elevated in radioresistant cells and that overexpression of TPP1 led to radioresistance and telomere lengthening in telomerase-positive cells. However, the exact effects and mechanism of TPP1 on radiosensitivity are yet to be precisely defined in the ALT cells. Here we report on the phenotypes of the conditional deletion of TPP1 from the human osteosarcoma U2OS cells using ALT pathway to extend the telomeres.TPP1 deletion resulted in telomere shortening, increased apoptosis and radiation sensitivity enhancement. Together, our findings show that TPP1 plays a vital role in telomere maintenance and protection and establish an intimate relationship between TPP1, telomere and cellular response to ionizing radiation, but likely has the specific mechanism yet to be defined. Copyright © 2014 Elsevier Inc. All rights reserved.

Smart pH-responsive upconversion nanoparticles for enhanced tumor cellular internalization and near-infrared light-triggered photodynamic therapy.

PubMed

Wang, Sheng; Zhang, Lei; Dong, Chunhong; Su, Lin; Wang, Hanjie; Chang, Jin

2015-01-01

A smart pH-responsive photodynamic therapy system based on upconversion nanoparticle loaded PEG coated polymeric lipid vesicles (RB-UPPLVs) was designed and prepared. These RB-UPPLVs which are promising agents for deep cancer photodynamic therapy applications can achieve enhanced tumor cellular internalization and near-infrared light-triggered photodynamic therapy.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Rice Shaker Potassium Channel OsKAT1 Confers Tolerance to Salinity Stress on Yeast and Rice Cells1[OA

PubMed Central

Obata, Toshihiro; Kitamoto, Hiroko K.; Nakamura, Atsuko; Fukuda, Atsunori; Tanaka, Yoshiyuki

2007-01-01

We screened a rice (Oryza sativa L. ‘Nipponbare’) full-length cDNA expression library through functional complementation in yeast (Saccharomyces cerevisiae) to find novel cation transporters involved in salt tolerance. We found that expression of a cDNA clone, encoding the rice homolog of Shaker family K+ channel KAT1 (OsKAT1), suppressed the salt-sensitive phenotype of yeast strain G19 (Δena1–4), which lacks a major component of Na+ efflux. It also suppressed a K+-transport-defective phenotype of yeast strain CY162 (Δtrk1Δtrk2), suggesting the enhancement of K+ uptake by OsKAT1. By the expression of OsKAT1, the K+ contents of salt-stressed G19 cells increased during the exponential growth phase. At the linear phase, however, OsKAT1-expressing G19 cells accumulated less Na+ than nonexpressing cells, but almost the same K+. The cellular Na+ to K+ ratio of OsKAT1-expressing G19 cells remained lower than nonexpressing cells under saline conditions. Rice cells overexpressing OsKAT1 also showed enhanced salt tolerance and increased cellular K+ content. These functions of OsKAT1 are likely to be common among Shaker K+ channels because OsAKT1 and Arabidopsis (Arabidopsis thaliana) KAT1 were able to complement the salt-sensitive phenotype of G19 as well as OsKAT1. The expression of OsKAT1 was restricted to internodes and rachides of wild-type rice, whereas other Shaker family genes were expressed in various organs. These results suggest that OsKAT1 is involved in salt tolerance of rice in cooperation with other K+ channels by participating in maintenance of cytosolic cation homeostasis during salt stress and thus protects cells from Na+. PMID:17586689

SBML-SAT: a systems biology markup language (SBML) based sensitivity analysis tool

PubMed Central

Zi, Zhike; Zheng, Yanan; Rundell, Ann E; Klipp, Edda

2008-01-01

Background It has long been recognized that sensitivity analysis plays a key role in modeling and analyzing cellular and biochemical processes. Systems biology markup language (SBML) has become a well-known platform for coding and sharing mathematical models of such processes. However, current SBML compatible software tools are limited in their ability to perform global sensitivity analyses of these models. Results This work introduces a freely downloadable, software package, SBML-SAT, which implements algorithms for simulation, steady state analysis, robustness analysis and local and global sensitivity analysis for SBML models. This software tool extends current capabilities through its execution of global sensitivity analyses using multi-parametric sensitivity analysis, partial rank correlation coefficient, SOBOL's method, and weighted average of local sensitivity analyses in addition to its ability to handle systems with discontinuous events and intuitive graphical user interface. Conclusion SBML-SAT provides the community of systems biologists a new tool for the analysis of their SBML models of biochemical and cellular processes. PMID:18706080

SBML-SAT: a systems biology markup language (SBML) based sensitivity analysis tool.

PubMed

Zi, Zhike; Zheng, Yanan; Rundell, Ann E; Klipp, Edda

2008-08-15

It has long been recognized that sensitivity analysis plays a key role in modeling and analyzing cellular and biochemical processes. Systems biology markup language (SBML) has become a well-known platform for coding and sharing mathematical models of such processes. However, current SBML compatible software tools are limited in their ability to perform global sensitivity analyses of these models. This work introduces a freely downloadable, software package, SBML-SAT, which implements algorithms for simulation, steady state analysis, robustness analysis and local and global sensitivity analysis for SBML models. This software tool extends current capabilities through its execution of global sensitivity analyses using multi-parametric sensitivity analysis, partial rank correlation coefficient, SOBOL's method, and weighted average of local sensitivity analyses in addition to its ability to handle systems with discontinuous events and intuitive graphical user interface. SBML-SAT provides the community of systems biologists a new tool for the analysis of their SBML models of biochemical and cellular processes.

Complete spinal cord injury (SCI) transforms how brain derived neurotrophic factor (BDNF) affects nociceptive sensitization.

PubMed

Huang, Yung-Jen; Lee, Kuan H; Grau, James W

2017-02-01

Noxious stimulation can induce a lasting increase in neural excitability within the spinal cord (central sensitization) that can promote pain and disrupt adaptive function (maladaptive plasticity). Brain-derived neurotrophic factor (BDNF) is known to regulate the development of plasticity and has been shown to impact the development of spinally-mediated central sensitization. The latter effect has been linked to an alteration in GABA-dependent inhibition. Prior studies have shown that, in spinally transected rats, exposure to regular (fixed spaced) stimulation can counter the development of maladaptive plasticity and have linked this effect to an up-regulation of BDNF. Here it is shown that application of the irritant capsaicin to one hind paw induces enhanced mechanical reactivity (EMR) after spinal cord injury (SCI) and that the induction of this effect is blocked by pretreatment with fixed spaced shock. This protective effect was eliminated if rats were pretreated with the BDNF sequestering antibody TrkB-IgG. Intrathecal (i.t.) application of BDNF prevented, but did not reverse, capsaicin-induced EMR. BDNF also attenuated cellular indices (ERK and pERK expression) of central sensitization after SCI. In uninjured rats, i.t. BDNF enhanced, rather than attenuated, capsaicin-induced EMR and ERK/pERK expression. These opposing effects were related to a transformation in GABA function. In uninjured rats, BDNF reduced membrane-bound KCC2 and the inhibitory effect of the GABA A agonist muscimol. After SCI, BDNF increased KCC2 expression, which would help restore GABAergic inhibition. The results suggest that SCI transforms how BDNF affects GABA function and imply that the clinical usefulness of BDNF will depend upon the extent of fiber sparing. Copyright © 2016 Elsevier Inc. All rights reserved.

The significance of transferrin receptors in oncology: the development of functional nano-based drug delivery systems.

PubMed

Tortorella, Stephanie; Karagiannis, Tom C

2014-01-01

Anticancer therapeutic research aims to improve clinical management of the disease through the development of strategies that involve currently-relevant treatment options and targeted delivery. Tumour-specific and -targeted delivery of compounds to the site of malignancy allows for enhanced cellular uptake, increased therapeutic benefit with high intratumoural drug concentrations, and decreased systemic exposure. Due to the upregulation of transferrin receptor expression in a wide variety of cancers, its function and its highly efficient recycling pathway, strategies involving the selective targeting of the receptor are well documented. Direct conjugation and immunotoxin studies using the transferrin peptide or anti-transferrin receptor antibodies as the targeting moiety have established the capacity to enhance cellular uptake, cross the blood brain barrier, limit systemic toxicity and reverse multi-drug resistance. Limitations in direct conjugation, including the difficulty in linking an adequate amount of therapeutic compound to the ligand or antibody have identified the requirement to develop novel delivery methods. The application of nanoparticulate theory in the development of functional drug delivery systems has proven to be most promising, with the ability to selectively modify size-dependent properties and surface chemistry. The transferrin modification on a range of nanoparticle formulations enhances selective cellular uptake through transferrin-mediated processes, and increases therapeutic benefit through the ability to encapsulate high concentrations of relevant drug to the tumour site. Although ineffective in crossing the blood brain barrier in its free form, chemotherapeutic compounds including doxorubicin, may be loaded into transferrin-conjugated nanocarriers and impart cytotoxic effects in glioma cells in vitro and in vivo. Additionally, transferrin-targeted nanoparticles may be used in selective diagnostic applications with enhanced selectivity and sensitivity. Four transferrin-modified nano-based drug delivery systems are currently in early phases of human clinical trials. Despite the collective promise, inconsistencies in some studies have exposed some limitations in current formulations and the difficulty in translating preliminary studies into clinically-relevant therapeutic options. The main objective of this review is to investigate the development of transferrin targeted nano-based drug delivery systems in order to establish the use of transferrin as a cancer-targeted moiety, and to ultimately evaluate the progression of cancer therapeutic strategies for future research.

Radiation Sensitization in Cancer Therapy.

ERIC Educational Resources Information Center

Greenstock, Clive L.

1981-01-01

Discusses various aspects of radiation damage to biological material, including free radical mechanisms, radiation sensitization and protection, tumor hypoxia, mechanism of hypoxic cell radiosensitization, redox model for radiation modification, sensitizer probes of cellular radiation targets, pulse radiolysis studies of free radical kinetics,…

Differential cellular responses in healthy mice and in mice with established airway inflammation when exposed to hematite nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gustafsson, Åsa, E-mail: asa.gustafsson@foi.se; Dept of Public Health and Clinical Medicine, Umeå University; Bergström, Ulrika

The aim of this study was to investigate the inflammatory and immunological responses in airways and lung-draining lymph nodes (LDLNs), following lung exposure to iron oxide (hematite) nanoparticles (NPs). The responses to the hematite NPs were evaluated in both healthy non-sensitized mice, and in sensitized mice with an established allergic airway disease. The mice were exposed intratracheally to either hematite NPs or to vehicle (PBS) and the cellular responses were evaluated on days 1, 2, and 7, post-exposure. Exposure to hematite NPs increased the numbers of neutrophils, eosinophils, and lymphocytes in the airways of non-sensitized mice on days 1 andmore » 2 post-exposure; at these time points the number of lymphocytes was also elevated in the LDLNs. In contrast, exposing sensitized mice to hematite NPs induced a rapid and unspecific cellular reduction in the alveolar space on day 1 post-exposure; a similar decrease of lymphocytes was also observed in the LDLN. The results indicate that cells in the airways and in the LDLN of individuals with established airway inflammation undergo cell death when exposed to hematite NPs. A possible explanation for this toxic response is the extensive generation of reactive oxygen species (ROS) in the pro-oxidative environment of inflamed airways. This study demonstrates how sensitized and non-sensitized mice respond differently to hematite NP exposure, and it highlights the importance of including individuals with respiratory disorders when evaluating health effects of inhaled nanomaterials. - Highlights: • Hematite NPs induce differential responses in airways of healthy and allergic mice. • Hematite induced an airway inflammation in healthy mice. • Hematite induced cellular reduction in the alveolus and lymph nodes of allergic mice. • Cell death is possible due to extensive pro-oxidative environment in allergic mice. • It is important to include sensitive individuals when valuing health effects of NPs.« less

The enhancement of antiproliferative and proapoptotic activity of HDAC inhibitors by curcumin is mediated by Hsp90 inhibition.

PubMed

Giommarelli, Chiara; Zuco, Valentina; Favini, Enrica; Pisano, Claudio; Dal Piaz, Fabrizio; De Tommasi, Nunziatina; Zunino, Franco

2010-03-01

Curcumin, a natural polyphenol, has been described to exhibit effects on signaling pathways, leading to induction of apoptosis. In this study, we observed that curcumin inhibited Hsp90 activity causing depletion of client proteins implicated in survival pathways. Based on this observation, this study was designed to investigate the cellular effects of curcumin combination with the pan-HDAC inhibitors, vorinostat and panobinostat, which induce hyperacetylation of Hsp90, resulting in inhibition of its chaperone function. The results showed that, at subtoxic concentrations, curcumin markedly sensitized tumor cells to vorinostat- and panobinostat-induced growth inhibition and apoptosis. The sensitization was associated with persistent depletion of Hsp90 client proteins (EGFR, Raf-1, Akt, and survivin). In conclusion, our findings document a novel mechanism of action of curcumin and support the therapeutic potential of curcumin/HDAC inhibitors combination, because the synergistic interaction was observed at pharmacologically achievable concentrations, which were ineffective when each drug was used alone.

Polyamines and Their Metabolites as Diagnostic Markers of Human Diseases

PubMed Central

Park, Myung Hee; Igarashi, Kazuei

2013-01-01

Polyamines, putrescine, spermidine and spermine, are ubiquitous in living cells and are essential for eukaryotic cell growth. These polycations interact with negatively charged molecules such as DNA, RNA, acidic proteins and phospholipids and modulate various cellular functions including macromolecular synthesis. Dysregulation of the polyamine pathway leads to pathological conditions including cancer, inflammation, stroke, renal failure and diabetes. Increase in polyamines and polyamine synthesis enzymes is often associated with tumor growth, and urinary and plasma contents of polyamines and their metabolites have been investigated as diagnostic markers for cancers. Of these, diacetylated derivatives of spermidine and spermine are elevated in the urine of cancer patients and present potential markers for early detection. Enhanced catabolism of cellular polyamines by polyamine oxidases (PAO), spermine oxidase (SMO) or acetylpolyamine oxidase (AcPAO), increases cellular oxidative stress and generates hydrogen peroxide and a reactive toxic metabolite, acrolein, which covalently incorporates into lysine residues of cellular proteins. Levels of protein-conjuagated acrolein (PC-Acro) and polyamine oxidizing enzymes were increased in the locus of brain infarction and in plasma in a mouse model of stroke and also in the plasma of stroke patients. When the combined measurements of PC-Acro, interleukin 6 (IL-6), and C-reactive protein (CRP) were evaluated, even silent brain infarction (SBI) was detected with high sensitivity and specificity. Considering that there are no reliable biochemical markers for early stage of stroke, PC-Acro and PAOs present promising markers. Thus the polyamine metabolites in plasma or urine provide useful tools in early diagnosis of cancer and stroke. PMID:24009852

Molecular imaging and sensing using plasmonic nanoparticles

NASA Astrophysics Data System (ADS)

Crow, Matthew James

Noble metal nanoparticles exhibit unique optical properties that are beneficial to a variety of applications, including molecular imaging. The large scattering cross sections of nanoparticles provide high contrast necessary for biomarkers. Unlike alternative contrast agents, nanoparticles provide refractive index sensitivity revealing information regarding the local cellular environment. Altering the shape and composition of the nanoparticle shifts the peak resonant wavelength of scattered light, allowing for implementation of multiple spectrally distinct tags. In this project, nanoparticles that scatter in different spectral windows are functionalized with various antibodies recognizing extra-cellular receptors integral to cancer progression. A hyperspectral imaging system is developed, allowing for visualization and spectral characterization of cells labeled with these conjugates. Various molecular imaging and microspectroscopy applications of plasmonic nanoparticles are then investigated. First, anti-EGFR gold nanospheres are shown to quantitatively measure receptor expression with similar performance to fluorescence assays. Second, anti-EGFR gold nanorods and novel anti-IGF-1R silver nanospheres are implemented to indicate local cellular refractive indices. Third, because biosensing capabilities of nanoparticle tags may be limited by plasmonic coupling, polarization mapping is investigated as a method to discern these effects. Fourth, plasmonic coupling is tested to monitor HER-2 dimerization. Experiments reveal the interparticle conformation of proximal HER-2 bound labels, required for plasmonic coupling-enhanced dielectric sensing. Fifth, all three functionalized plasmonic tags are implemented simultaneously to indicate clinically relevant cell immunophenotype information and changes in the cellular dielectric environment. Finally, flow cytometry experiments are conducted utilizing the anti-EGFR nanorod tag to demonstrate profiling of receptor expression distribution and potential increased multiplexing capability.

The inhibitory effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and its family members on the activity of cellular microRNAs.

PubMed

Zhang, Hui

2010-01-01

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or APOBEC3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. However, the cellular function of APOBEC3G remains to be further clarified. It has been reported that APOBEC3s can restrict the mobility of endogenous retroviruses and LTR-retrotransposons, suggesting that they can maintain stability in host genomes. However, APOBEC3G is normally cytoplasmic. Further studies have demonstrated that it is associated with an RNase-sensitive high molecular mass (HMM) and located in processing bodies (P-bodies) of replicating T-cells, indicating that the major cellular function of APOBEC3G seems to be related to P-body-related RNA processing and metabolism. As the function of P-body is closely related to miRNA activity, APOBEC3G could affect the miRNA function. Recent studies have demonstrated that APOBEC3G and its family members counteract miRNA-mediated repression of protein translation. Further, APOBEC3G enhances the association of miRNA-targeted mRNA with polysomes, and facilitates the dissociation of miRNA-targeted mRNA from P-bodies. As such, APOBEC3G regulate the activity of cellular miRNAs. Whether this function is related to its potent antiviral activity remains to be further determined.

Codelivery of doxorubicin and triptolide with reduction-sensitive lipid–polymer hybrid nanoparticles for in vitro and in vivo synergistic cancer treatment

PubMed Central

Wu, Bo; Lu, Shu-Ting; Zhang, Liu-Jie; Zhuo, Ren-Xi; Xu, Hai-Bo; Huang, Shi-Wen

2017-01-01

Codelivery is a promising strategy to overcome the limitations of single chemotherapeutic agents in cancer treatment. Despite progress, codelivery of two or more different functional drugs to increase anticancer efficiency still remains a challenge. Here, reduction-sensitive lipid–polymer hybrid nanoparticles (LPNPs) drug delivery system composed of monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16), soybean lecithin, and poly(D,L-lactide-co-glycolide) (PLGA) was used for codelivery of doxorubicin (DOX) and a Chinese herb extract triptolide (TPL). Hydrophobic DOX and TPL could be successfully loaded in LPNPs by self-assembly. More importantly, drug release and cellular uptake experiments demonstrated that the two drugs were reduction sensitive, released simultaneously from LPNPs, and taken up effectively by the tumor cells. DOX/TPL-coloaded LPNPs (DOX/TPL-LPNPs) exhibited a high level of synergistic activation with low combination index (CI) in vitro and in vivo. Moreover, the highest synergistic therapeutic effect was achieved at the ratio of 1:0.2 DOX/TPL. Further experiments showed that TPL enhanced the uptake of DOX by human oral cavity squamous cell carcinoma cells (KB cells). Overall, DOX/TPL-coencapsulated reduction-sensitive nanoparticles will be a promising strategy for cancer treatment. PMID:28331310

Codelivery of doxorubicin and triptolide with reduction-sensitive lipid-polymer hybrid nanoparticles for in vitro and in vivo synergistic cancer treatment.

PubMed

Wu, Bo; Lu, Shu-Ting; Zhang, Liu-Jie; Zhuo, Ren-Xi; Xu, Hai-Bo; Huang, Shi-Wen

2017-01-01

Codelivery is a promising strategy to overcome the limitations of single chemotherapeutic agents in cancer treatment. Despite progress, codelivery of two or more different functional drugs to increase anticancer efficiency still remains a challenge. Here, reduction-sensitive lipid-polymer hybrid nanoparticles (LPNPs) drug delivery system composed of monomethoxy-poly(ethylene glycol)- S - S -hexadecyl (mPEG- S - S -C 16 ), soybean lecithin, and poly(D,L-lactide-co-glycolide) (PLGA) was used for codelivery of doxorubicin (DOX) and a Chinese herb extract triptolide (TPL). Hydrophobic DOX and TPL could be successfully loaded in LPNPs by self-assembly. More importantly, drug release and cellular uptake experiments demonstrated that the two drugs were reduction sensitive, released simultaneously from LPNPs, and taken up effectively by the tumor cells. DOX/TPL-coloaded LPNPs (DOX/TPL-LPNPs) exhibited a high level of synergistic activation with low combination index (CI) in vitro and in vivo. Moreover, the highest synergistic therapeutic effect was achieved at the ratio of 1:0.2 DOX/TPL. Further experiments showed that TPL enhanced the uptake of DOX by human oral cavity squamous cell carcinoma cells (KB cells). Overall, DOX/TPL-coencapsulated reduction-sensitive nanoparticles will be a promising strategy for cancer treatment.

A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins[S

PubMed Central

Gaebler, Anne; Penno, Anke; Kuerschner, Lars; Thiele, Christoph

2016-01-01

The demand to study the cellular localization of specific lipids has led to recent advances in lipid probes and microscopy. Alkyne lipids bear a small, noninterfering tag and can be detected upon click reaction with an azide-coupled reporter. Fluorescent alkyne lipid imaging crucially depends on appropriate azide reporters and labeling protocols that allow for an efficient click reaction and therefore a sensitive detection. We synthesized several azide reporters with different spacer components and tested their suitability for alkyne lipid imaging in fixed cells. The implementation of a copper-chelating picolyl moiety into fluorescent or biotin-based azide reagents strongly increased the sensitivity of the imaging routine. We demonstrate the applicability and evaluate the performance of this approach using different lipid classes and experimental setups. As azide picolyl reporters allow for reduced copper catalyst concentrations, they also enable coimaging of alkyne lipids with multiple fluorescent proteins including enhanced green fluorescent protein. Alternatively, and as we also show, microscopy of alkyne lipids can be combined with protein detection by immunocytochemistry. In summary, we present a robust, sensitive, and highly versatile protocol for the labeling of alkyne lipids with azide-coupled reporters for fluorescence microscopy that can be combined with different protein detection and imaging techniques. PMID:27565170

A Wireless Communications Laboratory on Cellular Network Planning

ERIC Educational Resources Information Center

Dawy, Z.; Husseini, A.; Yaacoub, E.; Al-Kanj, L.

2010-01-01

The field of radio network planning and optimization (RNPO) is central for wireless cellular network design, deployment, and enhancement. Wireless cellular operators invest huge sums of capital on deploying, launching, and maintaining their networks in order to ensure competitive performance and high user satisfaction. This work presents a lab…

Enhanced suppression of tumor growth by concomitant treatment of human lung cancer cells with suberoylanilide hydroxamic acid and arsenic trioxide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chien, Chia-Wen; Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan; Yao, Ju-Hsien

2011-11-15

The efficacy of arsenic trioxide (ATO) against acute promyelocytic leukemia (APL) and relapsed APL has been well documented. ATO may cause DNA damage by generating reactive oxygen intermediates. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, modulates gene and protein expression via histone-dependent or -independent pathways that may result in chromatin decondensation, cell cycle arrest, differentiation, and apoptosis. We investigated whether ATO and SAHA act synergistically to enhance the death of cancer cells. Our current findings showed that combined treatment with ATO and SAHA resulted in enhanced suppression of non-small-cell lung carcinoma in vitro in H1299 cells and in vivomore » in a xenograft mouse model. Flow cytometric analysis of annexin V+ cells showed that apoptotic cell death was significantly enhanced after combined treatment with ATO and SAHA. At the doses used, ATO did not interfere with cell cycle progression, but SAHA induced p21 expression and led to G1 arrest. A Comet assay demonstrated that ATO, but not SAHA, induced DNA strand breaks in H1299 cells; however, co-treatment with SAHA significantly increased ATO-induced DNA damage. Moreover, SAHA enhanced acetylation of histone H3 and sensitized genomic DNA to DNase I digestion. Our results suggest that SAHA may cause chromatin relaxation and increase cellular susceptibility to ATO-induced DNA damage. Combined administration of SAHA and ATO may be an effective approach to the treatment of lung cancer. -- Highlights: Black-Right-Pointing-Pointer ATO and SAHA are therapeutic agents with different action modes. Black-Right-Pointing-Pointer Combination of ATO and SAHA synergistically inhibits tumor cell growth. Black-Right-Pointing-Pointer SAHA loosens chromatin structure resulting in increased sensitivity to DNase I. Black-Right-Pointing-Pointer ATO-induced DNA damage and apoptosis are enhanced by co-treatment with SAHA.« less

Computational adaptive optics for broadband optical interferometric tomography of biological tissue

NASA Astrophysics Data System (ADS)

Boppart, Stephen A.

2015-03-01

High-resolution real-time tomography of biological tissues is important for many areas of biological investigations and medical applications. Cellular level optical tomography, however, has been challenging because of the compromise between transverse imaging resolution and depth-of-field, the system and sample aberrations that may be present, and the low imaging sensitivity deep in scattering tissues. The use of computed optical imaging techniques has the potential to address several of these long-standing limitations and challenges. Two related techniques are interferometric synthetic aperture microscopy (ISAM) and computational adaptive optics (CAO). Through three-dimensional Fourierdomain resampling, in combination with high-speed OCT, ISAM can be used to achieve high-resolution in vivo tomography with enhanced depth sensitivity over a depth-of-field extended by more than an order-of-magnitude, in realtime. Subsequently, aberration correction with CAO can be performed in a tomogram, rather than to the optical beam of a broadband optical interferometry system. Based on principles of Fourier optics, aberration correction with CAO is performed on a virtual pupil using Zernike polynomials, offering the potential to augment or even replace the more complicated and expensive adaptive optics hardware with algorithms implemented on a standard desktop computer. Interferometric tomographic reconstructions are characterized with tissue phantoms containing sub-resolution scattering particles, and in both ex vivo and in vivo biological tissue. This review will collectively establish the foundation for high-speed volumetric cellular-level optical interferometric tomography in living tissues.

Chemical imaging analysis of the brain with X-ray methods

NASA Astrophysics Data System (ADS)

Collingwood, Joanna F.; Adams, Freddy

2017-04-01

Cells employ various metal and metalloid ions to augment the structure and the function of proteins and to assist with vital biological processes. In the brain they mediate biochemical processes, and disrupted metabolism of metals may be a contributing factor in neurodegenerative disorders. In this tutorial review we will discuss the particular role of X-ray methods for elemental imaging analysis of accumulated metal species and metal-containing compounds in biological materials, in the context of post-mortem brain tissue. X-rays have the advantage that they have a short wavelength and can penetrate through a thick biological sample. Many of the X-ray microscopy techniques that provide the greatest sensitivity and specificity for trace metal concentrations in biological materials are emerging at synchrotron X-ray facilities. Here, the extremely high flux available across a wide range of soft and hard X-rays, combined with state-of-the-art focusing techniques and ultra-sensitive detectors, makes it viable to undertake direct imaging of a number of elements in brain tissue. The different methods for synchrotron imaging of metals in brain tissues at regional, cellular, and sub-cellular spatial resolution are discussed. Methods covered include X-ray fluorescence for elemental imaging, X-ray absorption spectrometry for speciation imaging, X-ray diffraction for structural imaging, phase contrast for enhanced contrast imaging and scanning transmission X-ray microscopy for spectromicroscopy. Two- and three-dimensional (confocal and tomographic) imaging methods are considered as well as the correlation of X-ray microscopy with other imaging tools.

Acute spinal cord injury (SCI) transforms how GABA affects nociceptive sensitization.

PubMed

Huang, Yung-Jen; Lee, Kuan H; Murphy, Lauren; Garraway, Sandra M; Grau, James W

2016-11-01

Noxious input can sensitize pain (nociceptive) circuits within the spinal cord, inducing a lasting increase in spinal cord neural excitability (central sensitization) that is thought to contribute to chronic pain. The development of spinally-mediated central sensitization is regulated by descending fibers and GABAergic interneurons. The current study provides evidence that spinal cord injury (SCI) transforms how GABA affects nociceptive transmission within the spinal cord, recapitulating an earlier developmental state wherein GABA has an excitatory effect. In spinally transected rats, noxious electrical stimulation and inflammation induce enhanced mechanical reactivity (EMR), a behavioral index of nociceptive sensitization. Pretreatment with the GABA A receptor antagonist bicuculline blocked these effects. Peripheral application of an irritant (capsaicin) also induced EMR. Both the induction and maintenance of this effect were blocked by bicuculline. Cellular indices of central sensitization [c-fos expression and ERK phosphorylation (pERK)] were also attenuated. In intact (sham operated) rats, bicuculline had the opposite effect. Pretreatment with a GABA agonist (muscimol) attenuated nociceptive sensitization in intact, but not spinally injured, rats. The effect of SCI on GABA function was linked to a reduction in the Cl - transporter, KCC2, leading to a reduction in intracellular Cl - that would attenuate GABA-mediated inhibition. Pharmacologically blocking the KCC2 channel (with i.t. DIOA) in intact rats mimicked the effect of SCI. Conversely, a pharmacological treatment (bumetanide) that should increase intracellular Cl - levels blocked the effect of SCI. The results suggest that GABAergic neurons drive, rather than inhibit, the development of nociceptive sensitization after spinal injury. Copyright © 2016 Elsevier Inc. All rights reserved.

Beneficial effects of intermittent fasting and caloric restriction on the cardiovascular and cerebrovascular systems.

PubMed

Mattson, Mark P; Wan, Ruiqian

2005-03-01

Intermittent fasting (IF; reduced meal frequency) and caloric restriction (CR) extend lifespan and increase resistance to age-related diseases in rodents and monkeys and improve the health of overweight humans. Both IF and CR enhance cardiovascular and brain functions and improve several risk factors for coronary artery disease and stroke including a reduction in blood pressure and increased insulin sensitivity. Cardiovascular stress adaptation is improved and heart rate variability is increased in rodents maintained on an IF or a CR diet. Moreover, rodents maintained on an IF regimen exhibit increased resistance of heart and brain cells to ischemic injury in experimental models of myocardial infarction and stroke. The beneficial effects of IF and CR result from at least two mechanisms--reduced oxidative damage and increased cellular stress resistance. Recent findings suggest that some of the beneficial effects of IF on both the cardiovascular system and the brain are mediated by brain-derived neurotrophic factor signaling in the brain. Interestingly, cellular and molecular effects of IF and CR on the cardiovascular system and the brain are similar to those of regular physical exercise, suggesting shared mechanisms. A better understanding of the cellular and molecular mechanisms by which IF and CR affect the blood vessels and heart and brain cells will likely lead to novel preventative and therapeutic strategies for extending health span.

Concise review: Nanoparticles and cellular carriers-allies in cancer imaging and cellular gene therapy?

PubMed Central

Tang, Catherine; Russell, Pamela J; Martiniello-Wilks, Rosetta; J Rasko, John E; Khatri, Aparajita

2010-01-01

Ineffective treatment and poor patient management continue to plague the arena of clinical oncology. The crucial issues include inadequate treatment efficacy due to ineffective targeting of cancer deposits, systemic toxicities, suboptimal cancer detection and disease monitoring. This has led to the quest for clinically relevant, innovative multifaceted solutions such as development of targeted and traceable therapies. Mesenchymal stem cells (MSCs) have the intrinsic ability to “home” to growing tumors and are hypoimmunogenic. Therefore, these can be used as (a) “Trojan Horses” to deliver gene therapy directly into the tumors and (b) carriers of nanoparticles to allow cell tracking and simultaneous cancer detection. The camouflage of MSC carriers can potentially tackle the issues of safety, vector, and/or transgene immunogenicity as well as nanoparticle clearance and toxicity. The versatility of the nanotechnology platform could allow cellular tracking using single or multimodal imaging modalities. Toward that end, noninvasive magnetic resonance imaging (MRI) is fast becoming a clinical favorite, though there is scope for improvement in its accuracy and sensitivity. In that, use of superparamagnetic iron-oxide nanoparticles (SPION) as MRI contrast enhancers may be the best option for tracking therapeutic MSC. The prospects and consequences of synergistic approaches using MSC carriers, gene therapy, and SPION in developing cancer diagnostics and therapeutics are discussed. STEM CELLS 2010; 28:1686–1702. PMID:20629172

A Biosensor of S100A4 Metastasis Factor Activation: Inhibitor Screening and Cellular Activation Dynamics†

PubMed Central

Garrett, Sarah C.; Hodgson, Louis; Rybin, Andrew; Toutchkine, Alexei; Hahn, Klaus M.; Lawrence, David S.; Bresnick, Anne R.

2011-01-01

S100A4, a member of the S100 family of Ca2+-binding proteins, displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 has a direct role in metastatic progression, likely due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. We developed a fluorescent biosensor (Mero-S100A4) that reports on the Ca2+-bound, activated form of S100A4. Direct attachment of a novel solvatochromatic reporter dye to S100A4 results in a sensor that, upon activation, undergoes a 3-fold enhancement in fluorescence, thus providing a sensitive assay for use in vitro and in vivo. In cells, localized activation of S100A4 at the cell periphery is observed during random migration and following stimulation with lysophosphatidic acid, a known activator of cell motility and proliferation. Additionally, a screen against a library of FDA-approved drugs with the biosensor identified an array of phenothiazines as inhibitors of myosin-II associated S100A4 function. These data demonstrate the utility of the new biosensor both for drug discovery and for probing the cellular dynamics controlled by the S100A4 metastasis factor. PMID:18154362

Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis

PubMed Central

Dragovic, Rebecca A.; Gardiner, Christopher; Brooks, Alexandra S.; Tannetta, Dionne S.; Ferguson, David J.P.; Hole, Patrick; Carr, Bob; Redman, Christopher W.G.; Harris, Adrian L.; Dobson, Peter J.; Harrison, Paul; Sargent, Ian L.

2011-01-01

Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ∼50 nm and is far more sensitive than conventional flow cytometry (lower limit ∼300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. From the Clinical Editor The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry. PMID:21601655

RNA from Trained Aplysia Can Induce an Epigenetic Engram for Long-Term Sensitization in Untrained Aplysia.

PubMed

Bédécarrats, Alexis; Chen, Shanping; Pearce, Kaycey; Cai, Diancai; Glanzman, David L

2018-01-01

The precise nature of the engram, the physical substrate of memory, remains uncertain. Here, it is reported that RNA extracted from the central nervous system of Aplysia given long-term sensitization (LTS) training induced sensitization when injected into untrained animals; furthermore, the RNA-induced sensitization, like training-induced sensitization, required DNA methylation. In cellular experiments, treatment with RNA extracted from trained animals was found to increase excitability in sensory neurons, but not in motor neurons, dissociated from naïve animals. Thus, the behavioral, and a subset of the cellular, modifications characteristic of a form of nonassociative long-term memory (LTM) in Aplysia can be transferred by RNA. These results indicate that RNA is sufficient to generate an engram for LTS in Aplysia and are consistent with the hypothesis that RNA-induced epigenetic changes underlie memory storage in Aplysia .

RNA from Trained Aplysia Can Induce an Epigenetic Engram for Long-Term Sensitization in Untrained Aplysia

PubMed Central

Chen, Shanping; Pearce, Kaycey; Cai, Diancai

2018-01-01

The precise nature of the engram, the physical substrate of memory, remains uncertain. Here, it is reported that RNA extracted from the central nervous system of Aplysia given long-term sensitization (LTS) training induced sensitization when injected into untrained animals; furthermore, the RNA-induced sensitization, like training-induced sensitization, required DNA methylation. In cellular experiments, treatment with RNA extracted from trained animals was found to increase excitability in sensory neurons, but not in motor neurons, dissociated from naïve animals. Thus, the behavioral, and a subset of the cellular, modifications characteristic of a form of nonassociative long-term memory (LTM) in Aplysia can be transferred by RNA. These results indicate that RNA is sufficient to generate an engram for LTS in Aplysia and are consistent with the hypothesis that RNA-induced epigenetic changes underlie memory storage in Aplysia. PMID:29789810

Chronic intermittent ethanol inhalation increases ethanol self-administration in both C57BL/6J and DBA/2J mice.

PubMed

McCool, Brian A; Chappell, Ann M

2015-03-01

Inbred mouse strains provide significant opportunities to understand the genetic mechanisms controlling ethanol-directed behaviors and neurobiology. They have been specifically employed to understand cellular mechanisms contributing to ethanol consumption, acute intoxication, and sensitivities to chronic effects. However, limited ethanol consumption by some strains has restricted our understanding of clinically relevant endpoints such as dependence-related ethanol intake. Previous work with a novel tastant-substitution procedure using monosodium glutamate (MSG or umami flavor) has shown that the procedure greatly enhances ethanol consumption by mouse strains that express limited drinking phenotypes using other methods. In the current study, we employ this MSG-substitution procedure to examine how ethanol dependence, induced with passive vapor inhalation, modifies ethanol drinking in C57BL/6J and DBA/2J mice. These strains represent 'high' and 'low' drinking phenotypes, respectively. We found that the MSG substitution greatly facilitates ethanol drinking in both strains, and likewise, ethanol dependence increased ethanol consumption regardless of strain. However, DBA/2J mice exhibited greater sensitivity dependence-enhanced drinking, as represented by consumption behaviors directed at lower ethanol concentrations and relative to baseline intake levels. DBA/2J mice also exhibited significant withdrawal-associated anxiety-like behavior while C57BL/6J mice did not. These findings suggest that the MSG-substitution procedure can be employed to examine dependence-enhanced ethanol consumption across a range of drinking phenotypes, and that C57BL/6J and DBA/2J mice may represent unique neurobehavioral pathways for developing dependence-enhanced ethanol consumption. Copyright © 2015 Elsevier Inc. All rights reserved.

Pushing the sensitivity envelope of lanthanide-based magnetic resonance imaging (MRI) contrast agents for molecular imaging applications.

PubMed

Aime, Silvio; Castelli, Daniela Delli; Crich, Simonetta Geninatti; Gianolio, Eliana; Terreno, Enzo

2009-07-21

Contrast in magnetic resonance imaging (MRI) arises from changes in the intensity of the proton signal of water between voxels (essentially, the 3D counterpart of pixels). Differences in intervoxel intensity can be significantly enhanced with chemicals that alter the nuclear magnetic resonance (NMR) intensity of the imaged spins; this alteration can occur by various mechanisms. Paramagnetic lanthanide(III) complexes are used in two major classes of MRI contrast agent: the well-established class of Gd-based agents and the emerging class of chemical exchange saturation transfer (CEST) agents. A Gd-based complex increases water signal by enhancing the longitudinal relaxation rate of water protons, whereas CEST agents decrease water signal as a consequence of the transfer of saturated magnetization from the exchangeable protons of the agent. In this Account, we survey recent progress in both areas, focusing on how MRI is becoming a more competitive choice among the various molecular imaging methods. Compared with other imaging modalities, MRI is set apart by its superb anatomical resolution; however, its success in molecular imaging suffers because of its intrinsic insensitivity. A relatively high concentration of molecular agents (0.01-0.1 mM) is necessary to produce a local alteration in the water signal intensity. Unfortunately, the most desirable molecules for visualization in molecular imaging are present at much lower concentrations, in the nano- or picomolar range. Therefore, augmenting the sensitivity of MRI agents is key to the development of MR-based molecular imaging applications. In principle, this task can be tackled either by increasing the sensitivity of the reporting units, through the optimization of their structural and dynamic properties, or by setting up proper amplification strategies that allow the accumulation of a huge number of imaging reporters at the site of interest. For Gd-based agents, high sensitivities can be attained by exploiting a range of nanosized carriers (micelles, liposomes, microemulsions, and the like, as well as biological structures such as apoferritin and lipoproteins) properly loaded with Gd-based chelates. Furthermore, the sensitivity of Gd-based agents can be markedly affected either by their interactions with biological structures or by their cellular localization. For CEST agents, a huge sensitivity enhancement has been obtained by using the water molecules contained in the inner cavity of liposomes as the exchangeable source of protons for magnetization transfer. Several "tricks" (for example, the use of multimeric lanthanide(III) shift reagents, changes in the shape of the liposome container, and so forth) have been devised to improve the chemical shift separation between the intraliposomal water and the "bulk" water resonances. Overall, excellent sensitivity enhancements have been obtained for both classes of agents, enabling their use in MR molecular imaging applications.

Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

PubMed Central

McClellan, Michael J.; Wood, C. David; Ojeniyi, Opeoluwa; Cooper, Tim J.; Kanhere, Aditi; Arvey, Aaron; Webb, Helen M.; Palermo, Richard D.; Harth-Hertle, Marie L.; Kempkes, Bettina; Jenner, Richard G.; West, Michelle J.

2013-01-01

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors. PMID:24068937

Relationship between interpersonal sensitivity and leukocyte telomere length.

PubMed

Suzuki, Akihito; Matsumoto, Yoshihiko; Enokido, Masanori; Shirata, Toshinori; Goto, Kaoru; Otani, Koichi

2017-10-10

Telomeres are repetitive DNA sequences located at the ends of chromosomes, and telomere length represents a biological marker for cellular aging. Interpersonal sensitivity, excessive sensitivity to the behavior and feelings of others, is one of the vulnerable factors to depression. In the present study, we examined the effect of interpersonal sensitivity on telomere length in healthy subjects. The subjects were 159 unrelated healthy Japanese volunteers. Mean age ± SD (range) of the subjects was 42.3 ± 7.8 (30-61) years. Interpersonal sensitivity was assessed by the Japanese version of the Interpersonal Sensitivity Measure (IPSM). Leukocyte telomere length was determined by a quantitative real-time PCR method. Higher scores of the total IPSM were significantly (β = -0.163, p = 0.038) related to shorter telomere length. In the sub-scale analysis, higher scores of timidity were significantly (β = -0.220, p = 0.044) associated with shorter telomere length. The present study suggests that subjects with higher interpersonal sensitivity have shorter leukocyte telomere length, implying that interpersonal sensitivity has an impact on cellular aging.

Polyethyleneimine Coating Enhances the Cellular Uptake of Mesoporous Silica Nanoparticles and Allows Safe Delivery of siRNA and DNA Constructs

PubMed Central

Xia, Tian; Kovochich, Michael; Liong, Monty; Meng, Huan; Kabehie, Sanaz; Zink, Jeffrey I.; Nel, Andre E.

2014-01-01

Surface-functionalized mesoporous silica nanoparticles (MSNP) can be used as an efficient and safe carrier for bioactive molecules. In order to make the MSNP a more efficient delivery system, we modified the surface of the particles by a functional group that enhances cellular uptake and allows nucleic acid delivery in addition to traditional drug delivery. Non-covalent attachment of polyethyleneimine (PEI) polymers to the surface not only increases MSNP cellular uptake, but also generates a cationic surface to which DNA and siRNA constructs could be attached. While efficient for intracellular delivery of these nucleic acids, the 25 KD PEI polymer unfortunately changes the safety profile of the MSNP that is otherwise very safe. By experimenting with several different polymer molecular weights, it was possible to retain high cellular uptake and transfection efficiency while reducing or even eliminating cationic MSNP cytotoxicity. The particles coated with the 10 KD PEI polymer was particularly efficient for transducing HEPA-1 cells with a siRNA construct that was capable of knocking down GFP expression. Similarly, transfection of a GFP plasmid induced effective expression of the fluorescent protein in > 70% cells in the population. These outcomes were quantitatively assessed by confocal microscopy and flow cytometry. We also demonstrated that the enhanced cellular uptake of the non-toxic cationic MSNP enhance the delivery of the hydrophobic anticancer drug, paclitaxel, to pancreatic cancer cells. In summary, we demonstrate that by a careful selection of PEI size, it is possible to construct cationic MSNP that are capable of nucleotide and enhanced drug delivery with minimal or no cytotoxicity. This novel use of a cationic MSNP extends its therapeutic use potential. PMID:19739605

Enhancement of the thermal transport in a culture medium with Au nanoparticles

NASA Astrophysics Data System (ADS)

Jiménez-Pérez, J. L.; Fuentes, R. Gutierrez; Alvarado, E. Maldonado; Ramón-Gallegos, E.; Cruz-Orea, A.; Tánori-Cordova, J.; Mendoza-Alvarez, J. G.

2008-11-01

In this work, it is reported the gold nanoparticles synthesis, their characterization, and their application to the enhancement of the thermal transport in a cellular culture medium. The Au nanoparticles (NPs), with average size of 10 nm, contained into a culture medium (DMEM (1)/F12(1)) (CM) increased considerably the heat transfer in the medium. Thermal lens spectrometry (TLS) was used to measure the thermal diffusivity of the nanofluids. The characteristic time constant of the transient thermal lens was obtained by fitting the theoretical expression, for transient thermal lens, to the experimental data. Our results show that the thermal diffusivity of the culture medium is highly sensitive to the Au nanoparticle concentration and size. The ability to modify the thermal properties to nanometer scale becomes very important in medical applications as in the case of cancer treatment by using photodynamic therapy (PDT). A complementary study with UV-vis and TEM techniques was performed to characterize the Au nanoparticles.

T-cell Receptor Specificity Maintained by Altered Thermodynamics*

PubMed Central

Madura, Florian; Rizkallah, Pierre J.; Miles, Kim M.; Holland, Christopher J.; Bulek, Anna M.; Fuller, Anna; Schauenburg, Andrea J. A.; Miles, John J.; Liddy, Nathaniel; Sami, Malkit; Li, Yi; Hossain, Moushumi; Baker, Brian M.; Jakobsen, Bent K.; Sewell, Andrew K.; Cole, David K.

2013-01-01

The T-cell receptor (TCR) recognizes peptides bound to major histocompatibility molecules (MHC) and allows T-cells to interrogate the cellular proteome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterized by weak affinity (KD = 100 nm to 270 μm). We used phage-display to produce a melanoma-specific TCR (α24β17) with a 30,000-fold enhanced binding affinity (KD = 0.6 nm) to aid our exploration of the molecular mechanisms utilized to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, α24β17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition. PMID:23698002

Identification of osteosarcoma-related specific proteins in serum samples using surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry.

PubMed

Gu, Jianli; Li, Jitian; Huang, Manyu; Zhang, Zhiyong; Li, Dongsheng; Song, Guoying; Ding, Xingpo; Li, Wuyin

2014-01-01

Osteosarcoma (OS) is the most common malignant bone tumor. To identify OS-related specific proteins for early diagnosis of OS, a novel approach, surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF-MS) to serum samples from 25 OS patients, 16 osteochondroma, and 26 age-matched normal human volunteers as controls, was performed. Two proteins showed a significantly different expression in OS serum samples from control groups. Proteomic profiles and external leave-one-out cross-validation analysis showed that the correct rate of allocation, the sensitivity, and the specificity of diagnosis were 100%. These two proteins were further identified by searching the EPO-KB database, and one of the proteins identified as Serine rich region profile is involved in various cellular signaling cascades and tumor genesis. The presence of these two proteins in OS patients but absence from premalignant and normal human controls implied that they can be potential biomarkers for early diagnosis of OS.

Nicotine-mediated suppression of the retinoic acid metabolizing enzyme CYP26A1 limits the oncogenic potential of breast cancer.

PubMed

Osanai, Makoto; Lee, Gang-Hong

2011-06-01

Tobacco smoke influences cancer development in tissues that are not directly exposed, and epidemiological studies have indicated that smoking women might experience decreased risk of breast cancer as a result of antiestrogenic effects. However, it remains to be clarified whether nicotine, one of the major addictive and best-investigated constituents of tobacco smoke, has any effect on breast cancer. Our recent work demonstrated that the retinoic acid metabolizing enzyme CYP26A1 enhances oncogenic and cell survival properties of breast carcinoma cells, implying a role as an oncogene. Here, we present evidence that nicotine significantly suppresses constitutive expression of CYP26A1, and that cells treated with nicotine exhibit enhanced sensitivity to apoptosis. In addition, nicotine may inhibit anchorage independent growth, cellular invasiveness and motility. These data show that nicotine can limit CYP26A1-mediated oncogenic characteristics, and suggest mechanisms by which nicotine might inhibit breast cancer development. © 2011 Japanese Cancer Association.

Applications of conducting polymers and their issues in biomedical engineering

PubMed Central

Ravichandran, Rajeswari; Sundarrajan, Subramanian; Venugopal, Jayarama Reddy; Mukherjee, Shayanti; Ramakrishna, Seeram

2010-01-01

Conducting polymers (CPs) have attracted much interest as suitable matrices of biomolecules and have been used to enhance the stability, speed and sensitivity of various biomedical devices. Moreover, CPs are inexpensive, easy to synthesize and versatile because their properties can be readily modulated by (i) surface functionalization techniques and (ii) the use of a wide range of molecules that can be entrapped or used as dopants. This paper discusses the various surface modifications of the CP that can be employed in order to impart physico-chemical and biological guidance cues that promote cell adhesion/proliferation at the polymer–tissue interface. This ability of the CP to induce various cellular mechanisms widens its applications in medical fields and bioengineering. PMID:20610422

Depletion of intracellular GSH and NPSH by buthionine sulfoximine and diethyl maleate: factors that influence enhancement of aerobic radiation response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varnes, M.E.; Biaglow, J.E.; Roizin-Towle, L.

1984-08-01

Many investigators have observed aerobic sensitization of V79, CHO and A549 (human lung carcinoma) cells upon depletion of GSH using buthionine sulfoximine (BSO). Recently the authors discovered that this aerobic sensitization can be reversed if WR-2721 or N-acetylcysteine is added to the cells just prior to irradiation. Reversal requires that the exogenous thiols be present during the time of irradiation. One possible explanation was that these thiols entered the cells and either increased the pool of cellular nonprotein thiols or reversed the thiol-depleted state by stimulation of GSH synthesis. Cells treated with BSO do not readily regenerate intracellular GSH becausemore » this agent irreversibly inhibits ..gamma..-glutamyl synthetase. They found that addition of WR-2721 or N-acetylcysteine to BSO-treated cells did not affect the rate of regeneration of intracellular GSH. Thus, reversal of the aerobic sensitization of A549 cells by BSO cannot be explained on the basis of intracellular thiol levels alone, or by rapid reversal of BSO inhibition. In addition, diethylmaleate (DEM)-treated cells are considerably different from BSO-treated cells with respect to the ability to regenerate GSH.« less

Three-dimensional WS2 nanosheet networks for H2O2 produced for cell signaling

NASA Astrophysics Data System (ADS)

Tang, Jing; Quan, Yingzhou; Zhang, Yueyu; Jiang, Min; Al-Enizi, Abdullah M.; Kong, Biao; An, Tiance; Wang, Wenshuo; Xia, Limin; Gong, Xingao; Zheng, Gengfeng

2016-03-01

Hydrogen peroxide (H2O2) is an important molecular messenger for cellular signal transduction. The capability of direct probing of H2O2 in complex biological systems can offer potential for elucidating its manifold roles in living systems. Here we report the fabrication of three-dimensional (3D) WS2 nanosheet networks with flower-like morphologies on a variety of conducting substrates. The semiconducting WS2 nanosheets with largely exposed edge sites on flexible carbon fibers enable abundant catalytically active sites, excellent charge transfer, and high permeability to chemicals and biomaterials. Thus, the 3D WS2-based nano-bio-interface exhibits a wide detection range, high sensitivity and rapid response time for H2O2, and is capable of visualizing endogenous H2O2 produced in living RAW 264.7 macrophage cells and neurons. First-principles calculations further demonstrate that the enhanced sensitivity of probing H2O2 is attributed to the efficient and spontaneous H2O2 adsorption on WS2 nanosheet edge sites. The combined features of 3D WS2 nanosheet networks suggest attractive new opportunities for exploring the physiological roles of reactive oxygen species like H2O2 in living systems.Hydrogen peroxide (H2O2) is an important molecular messenger for cellular signal transduction. The capability of direct probing of H2O2 in complex biological systems can offer potential for elucidating its manifold roles in living systems. Here we report the fabrication of three-dimensional (3D) WS2 nanosheet networks with flower-like morphologies on a variety of conducting substrates. The semiconducting WS2 nanosheets with largely exposed edge sites on flexible carbon fibers enable abundant catalytically active sites, excellent charge transfer, and high permeability to chemicals and biomaterials. Thus, the 3D WS2-based nano-bio-interface exhibits a wide detection range, high sensitivity and rapid response time for H2O2, and is capable of visualizing endogenous H2O2 produced in living RAW 264.7 macrophage cells and neurons. First-principles calculations further demonstrate that the enhanced sensitivity of probing H2O2 is attributed to the efficient and spontaneous H2O2 adsorption on WS2 nanosheet edge sites. The combined features of 3D WS2 nanosheet networks suggest attractive new opportunities for exploring the physiological roles of reactive oxygen species like H2O2 in living systems. Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c5nr09236a

Long noncoding RNA MALAT1 enhances the docetaxel resistance of prostate cancer cells via miR-145-5p-mediated regulation of AKAP12.

PubMed

Xue, Dong; Lu, Hao; Xu, Han-Yan; Zhou, Cui-Xing; He, Xiao-Zhou

2018-06-01

Our present work was aimed to study on the regulatory role of MALAT1/miR-145-5p/AKAP12 axis on docetaxel (DTX) sensitivity of prostate cancer (PCa) cells. The microarray data (GSE33455) to identify differentially expressed lncRNAs and mRNAs in DTX-resistant PCa cell lines (DU-145-DTX and PC-3-DTX) was retrieved from the Gene Expression Omnibus (GEO) database. QRT-PCR analysis was performed to measure MALAT1 expression in DTX-sensitive and DTX-resistant tissues/cells. The human DTX-resistant cell lines DU145-PTX and PC3-DTX were established as in vitro cell models, and the expression of MALAT1, miR-145-5p and AKAP12 was manipulated in DTX-sensitive and DTX-resistant cells. Cell viability was examined using MTT assay and colony formation methods. Cell apoptosis was assessed by TUNEL staining. Cell migration and invasion was determined by scratch test (wound healing) and Transwell assay, respectively. Dual-luciferase assay was applied to analyse the target relationship between lncRNA MALAT1 and miR-145-5p, as well as between miR-145-5p and AKAP12. Tumour xenograft study was undertaken to confirm the correlation of MALAT1/miR-145-5p/AKAP12 axis and DTX sensitivity of PCa cells in vivo. In this study, we firstly notified that the MALAT1 expression levels were up-regulated in clinical DTX-resistant PCa samples. Overexpressed MALAT1 promoted cell proliferation, migration and invasion but decreased cell apoptosis rate of PCa cells in spite of DTX treatment. We identified miR-145-5p as a target of MALAT1. MiR-145-5p overexpression in PC3-DTX led to inhibited cell proliferation, migration and invasion as well as reduced chemoresistance to DTX, which was attenuated by MALAT1. Moreover, we determined that AKAP12 was a target of miR-145-5p, which significantly induced chemoresistance of PCa cells to DTX. Besides, it was proved that MALAT1 promoted tumour cell proliferation and enhanced DTX-chemoresistance in vivo. There was an lncRNA MALAT1/miR-145-5p/AKAP12 axis involved in DTX resistance of PCa cells and provided a new thought for PCa therapy. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis.

PubMed

Nakase, Ikuhiko; Noguchi, Kosuke; Fujii, Ikuo; Futaki, Shiroh

2016-10-17

Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained.

A genome-wide RNAi screen identifies novel targets of neratinib sensitivity leading to neratinib and paclitaxel combination drug treatments.

PubMed

Seyhan, Attila A; Varadarajan, Usha; Choe, Sung; Liu, Yan; McGraw, John; Woods, Matthew; Murray, Stuart; Eckert, Amy; Liu, Wei; Ryan, Terence E

2011-06-01

ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.

In vitro characterization of pH-sensitive azithromycin-loaded methoxy poly (ethylene glycol)-block-poly (aspartic acid-graft-imidazole) micelles.

PubMed

Teng, Fangfang; Deng, Peizong; Song, Zhimei; Zhou, Feilong; Feng, Runliang; Liu, Na

2017-06-15

In order to improve azithromycin's antibacterial activity in acidic medium, monomethoxy poly (ethylene glycol)-block-poly (aspartic acid-graft-imidazole) copolymer was synthesized through allylation, free radical addition, ring-opening polymerization and amidation reactions with methoxy poly (ethylene glycol) as raw material. Drug loading capacity and encapsulation efficiency of azithromycin-loaded micelles prepared via thin film hydration method were 11.58±0.86% and 96.06±1.93%, respectively. The drug-loaded micelles showed pH-dependent property in the respects of particle size, zeta potential at the range of pH 5.5-7.8. It could control drug in vitro release and demonstrate higher release rate at pH 6.0 than that at pH 7.4. In vitro antibacterial experiment indicated that the activity of azithromycin-loaded micelles against S. aureus was superior to free azithromycin in medium at both pH 6.0 and pH 7.4. Using fluorescein as substitute with pH-dependent fluorescence decrease property, laser confocal fluorescence microscopy analysis confirmed that cellular uptake of micelles was improved due to protonation of copolymer's imidazole groups at pH 6.0. The enhanced cellular uptake and release of drug caused its activity enhancement in acidic medium when compared with free drug. The micellar drug delivery system should be potential application in the field of bacterial infection treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Oxidative stress and protein aggregation during biological aging.

PubMed

Squier, T C

2001-09-01

Biological aging is a fundamental process that represents the major risk factor with respect to the development of cancer, neurodegenerative, and cardiovascular diseases in vertebrates. It is, therefore, evident that the molecular mechanisms of aging are fundamental to understand many disease processes. In this regard, the oxidation and nitration of intracellular proteins and the formation of protein aggregates have been suggested to underlie the loss of cellular function and the reduced ability of senescent animals to withstand physiological stresses. Since oxidatively modified proteins are thermodynamically unstable and assume partially unfolded tertiary structures that readily form aggregates, it is likely that oxidized proteins are intermediates in the formation of amyloid fibrils. It is, therefore, of interest to identify oxidatively sensitive protein targets that may play a protective role through their ability to down-regulate energy metabolism and the consequent generation of reactive oxygen species (ROS). In this respect, the maintenance of cellular calcium gradients represents a major energetic expense, which links alterations in intracellular calcium levels to ATP utilization and the associated generation of ROS through respiratory control mechanisms. The selective oxidation or nitration of the calcium regulatory proteins calmodulin and Ca-ATPase that occurs in vivo during aging and under conditions of oxidative stress may represent an adaptive response to oxidative stress that functions to down-regulate energy metabolism and the associated generation of ROS. Since these calcium regulatory proteins are also preferentially oxidized or nitrated under in vitro conditions, these results suggest an enhanced sensitivity of these critical calcium regulatory proteins, which modulate signal transduction processes and intracellular energy metabolism, to conditions of oxidative stress. Thus, the selective oxidation of critical signal transduction proteins probably represents a regulatory mechanism that functions to minimize the generation of ROS through respiratory control mechanisms. The reduction of the rate of ROS generation, in turn, will promote cellular survival under conditions of oxidative stress, when reactive oxygen and nitrogen species overwhelm cellular antioxidant defense systems, by minimizing the non-selective oxidation of a range of biomolecules. Since protein aggregation occurs if protein repair and degradative systems are unable to act upon oxidized proteins and restore cellular function, the reduction of the oxidative load on the cell by the down-regulation of the electron transport chain functions to minimize protein aggregation. Thus, ROS function as signaling molecules that fine-tune cellular metabolism through the selective oxidation or nitration of calcium regulatory proteins in order to minimize wide-spread oxidative damage and protein aggregation. Oxidative damage to cellular proteins, the loss of calcium homeostasis and protein aggregation contribute to the formation of amyloid deposits that accumulate during biological aging. Critical to understand the relationship between these processes and biological aging is the identification of oxidatively sensitive proteins that modulate energy utilization and the associated generation of ROS. In this latter respect, oxidative modifications to the calcium regulatory proteins calmodulin (CaM) and the sarco/endoplasmic reticulum Ca-ATPase (SERCA) function to down-regulate ATP utilization and the associated generation of ROS associated with replenishing intracellular ATP through oxidative phosphorylation. Reductions in the rate of ROS generation, in turn, will minimize protein oxidation and facilitate intracellular repair and degradative systems that function to eliminate damaged and partially unfolded proteins. Since the rates of protein repair or degradation compete with the rate of protein aggregation, the modulation of intracellular calcium concentrations and energy metabolism through the selective oxidation or nitration of critical signal transduction proteins (i.e. CaM or SERCA) is thought to maintain cellular function by minimizing protein aggregation and amyloid formation. Age-dependent increases in the rate of ROS generation or declines in cellular repair or degradation mechanisms will increase the oxidative load on the cell, resulting in corresponding increases in the concentrations of oxidized proteins and the associated formation of amyloid.

Growth and cellular ion content of a salt-sensitive symbiotic system Azolla pinnata-Anabaena azollae under NaCl stress.

PubMed

Rai, Vandna; Sharma, Naveen Kumar; Rai, Ashwani K

2006-09-01

Salinity, at a concentration of 10 mM NaCl affected the growth of Azolla pinnata-Anabaena azollae association and became lethal at 40 mM. Plants exposed up to 30 mM NaCl exhibited longer roots than the control, especially during the beginning of incubation. Average root number in plants exposed to 10 and 20 mM NaCl remained almost the same as in control. A further rise in NaCl concentration to 30 mM reduced the root number, and roots shed off at 40 mM NaCl. Presence of NaCl in the nutrient solution increased the cellular Na+ of the intact association exhibiting differential accumulation by individual partners, while it reduced the cellular Ca2+ level. However, cellular K+ content did not show significant change. Cellular Na+ based on fresh weight of respective individual partners (host tissues and cyanobiont) remained higher in the host tissues than the cyanobiont, while reverse was true for K+ and Ca2+ contents. The contribution of A. azollae in the total cellular ion content of the association was a little because of meagre contribution of the cyanobiont mass (19-21%). High salt sensitivity of Azolla-Anabaena complex is due to an inability of the association to maintain low Na+ and high Ca2+ cellular level.

Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation.

PubMed

Yoshitani, Kazuhiro; Kido, Akira; Honoki, Kanya; Akahane, Manabu; Fujii, Hiromasa; Tanaka, Yasuhito

2011-07-15

Bisphosphonates (BPs) are agents used for treating disorders of excessive bone resorption. In addition, due to their cell-killing activity, BPs were potent candidates for adjuvant cancer therapy. On the other hand, low-concentrations of BPs have been reported to increase cellular viability in several types of tumor cells. Therefore, we focused on the effect of BPs on cellular aggressiveness of malignant bone tumors at low concentrations. MTS assay was performed using osteosarcoma cell lines MG63 and HOS, fibrosarcoma cell line HT1080, and prostate cancer cell line PC3. All the cell lines showed toxicity at high concentrations. On the other hand, at lower concentrations, the cellular viabilities of HOS and MG63 were rather higher than those of untreated controls. Since this tendency was most evident, HOS was used for further assays, including cellular motility, bone resorption activity, and cathepsin K activity. The low-concentration of alendronate enhanced cellular viability and motility, which correlated with the expression of connexin 43 at the mRNA and protein levels. Interestingly, oleamide, a potent connexin 43 inhibitor, had an inhibitory effect on the enhanced proliferation. Our data suggest that alendronate may enhance the proliferation of osteoblastic cell line through connexin 43 activation. Copyright © 2011 Elsevier GmbH. All rights reserved.

A continuum mathematical model of endothelial layer maintenance and senescence

PubMed Central

Wang, Ying; Aguda, Baltazar D; Friedman, Avner

2007-01-01

Background The monolayer of endothelial cells (ECs) lining the inner wall of blood vessels deteriorates as a person ages due to a complex interplay of a variety of causes including cell death arising from shear stress of blood flow and cellular oxidative stress, cellular senescence, and decreased rate of replacement of dead ECs by progenitor stem cells. Results A continuum mathematical model is developed to describe the dynamics of large EC populations of the endothelium using a system of differential equations for the number densities of cells of different generations starting from endothelial progenitors to senescent cells, as well as the densities of dead cells and the holes created upon clearing dead cells. Aging of cells is manifested in three ways, namely, losing the ability to divide when the Hayflick limit of 50 generations is reached, decreasing replication rate parameters and increasing death rate parameters as cells divide; due to the dependence of these rate parameters on cell generation, the model predicts a narrow distribution of cell densities peaking at a particular cell generation. As the chronological age of a person advances, the peak of the distribution – corresponding to the age of the endothelium – moves towards senescence correspondingly. However, computer simulations also demonstrate that sustained and enhanced stem cell homing can halt the aging process of the endothelium by maintaining a stationary cell density distribution that peaks well before the Hayflick limit. The healing rates of damaged endothelia for young, middle-aged, and old persons are compared and are found to be particularly sensitive to the stem cell homing parameter. Conclusion The proposed model describes the aging of the endothelium as being driven by cellular senescence, with a rate that does not necessarily correspond to the chronological aging of a person. It is shown that the age of the endothelium depends sensitively on the homing rates of EC progenitor cells. PMID:17692115

A continuum mathematical model of endothelial layer maintenance and senescence.

PubMed

Wang, Ying; Aguda, Baltazar D; Friedman, Avner

2007-08-10

The monolayer of endothelial cells (ECs) lining the inner wall of blood vessels deteriorates as a person ages due to a complex interplay of a variety of causes including cell death arising from shear stress of blood flow and cellular oxidative stress, cellular senescence, and decreased rate of replacement of dead ECs by progenitor stem cells. A continuum mathematical model is developed to describe the dynamics of large EC populations of the endothelium using a system of differential equations for the number densities of cells of different generations starting from endothelial progenitors to senescent cells, as well as the densities of dead cells and the holes created upon clearing dead cells. Aging of cells is manifested in three ways, namely, losing the ability to divide when the Hayflick limit of 50 generations is reached, decreasing replication rate parameters and increasing death rate parameters as cells divide; due to the dependence of these rate parameters on cell generation, the model predicts a narrow distribution of cell densities peaking at a particular cell generation. As the chronological age of a person advances, the peak of the distribution - corresponding to the age of the endothelium - moves towards senescence correspondingly. However, computer simulations also demonstrate that sustained and enhanced stem cell homing can halt the aging process of the endothelium by maintaining a stationary cell density distribution that peaks well before the Hayflick limit. The healing rates of damaged endothelia for young, middle-aged, and old persons are compared and are found to be particularly sensitive to the stem cell homing parameter. The proposed model describes the aging of the endothelium as being driven by cellular senescence, with a rate that does not necessarily correspond to the chronological aging of a person. It is shown that the age of the endothelium depends sensitively on the homing rates of EC progenitor cells.

Stimulation of the human immunodeficiency virus type 1 enhancer by the human T-cell leukemia virus type I tax gene product involves the action of inducible cellular proteins.

PubMed

Böhnlein, E; Siekevitz, M; Ballard, D W; Lowenthal, J W; Rimsky, L; Bogérd, H; Hoffman, J; Wano, Y; Franza, B R; Greene, W C

1989-04-01

The human immunodeficiency virus type 1 (HIV-1) preferentially infects CD4+ T lymphocytes and may exist as a latent provirus within these cells for extended periods. The transition to a productive retroviral infection results in T-cell death and clinically may lead to the acquired immune deficiency syndrome. Accelerated production of infectious HIV-1 virions appears to be closely linked to a heightened state of T-cell activation. The transactivator (Tax) protein of the type I human T-cell leukemia virus (HTLV-I) can produce such an activated T-cell phenotype and augments activity of the HIV-1 long terminal repeat. One Tax-responsive region within the HIV-1 long terminal repeat has been mapped to a locus composed of two 10-base-pair direct repeats sharing homology with the binding site for the eucaryotic transcription factor NF-kappaB (GGGACTTTCC). Tax-expressing Jurkat T cells contain one or more inducible cellular proteins that specifically associate with the HIV-1 enhancer at these binding sites. Microscale DNA affinity precipitation assays identified a Tax-inducible 86-kilodalton protein, HIVEN86A, as one of these HIV-1 enhancer-binding factors. The interaction of HIVEN86A, and presumably other cellular proteins, with the HIV-1 enhancer appears functionally important as oligonucleotides corresponding to this enhancer were sufficient to impart Tax inducibility to an unresponsive heterologous promoter. These findings suggest that the Tax-inducible cellular protein HIVEN86A plays an important role in the transcriptional activation of the HIV-1 enhancer. These specific protein-DNA interactions may also be important for the transition of HIV-1 from a latent to a productive mode of infection. Furthermore, these findings highlight an intriguing biological interplay between HTLV-1 and HIV-1 through a cellular transcriptional pathway that is normally involved in T-cell activation and growth.

A liquid chromatography/tandem mass spectrometry assay for the analysis of atomoxetine in human plasma and in vitro cellular samples

PubMed Central

Appel, David I.; Brinda, Bryan; Markowitz, John S.; Newcorn, Jeffrey H.; Zhu, Hao-Jie

2012-01-01

A simple, rapid and sensitive method for quantification of atomoxetine by liquid chromatography- tandem mass spectrometry (LC-MS/MS) was developed. This assay represents the first LC-MS/MS quantification method for atomoxetine utilizing electrospray ionization. Deuterated atomoxetine (d3-atomoxetine) was adopted as the internal standard. Direct protein precipitation was utilized for sample preparation. This method was validated for both human plasma and in vitro cellular samples. The lower limit of quantification was 3 ng/ml and 10 nM for human plasma and cellular samples, respectively. The calibration curves were linear within the ranges of 3 ng/ml to 900 ng/ml and 10 nM to 10 μM for human plasma and cellular samples, respectively (r2 > 0.999). The intra- and inter-day assay accuracy and precision were evaluated using quality control samples at 3 different concentrations in both human plasma and cellular lysate. Sample run stability, assay selectivity, matrix effect, and recovery were also successfully demonstrated. The present assay is superior to previously published LC-MS and LC-MS/MS methods in terms of sensitivity or the simplicity of sample preparation. This assay is applicable to the analysis of atomoxetine in both human plasma and in vitro cellular samples. PMID:22275222

Similar cisplatin sensitivity of HPV-positive and -negative HNSCC cell lines

PubMed Central

Kriegs, Malte; Gatzemeier, Fruzsina; Krüger, Katharina; Möckelmann, Nikolaus; Fritz, Gerhard; Petersen, Cordula; Knecht, Rainald; Rothkamm, Kai; Rieckmann, Thorsten

2016-01-01

Patients with HPV-positive head and neck squamous cell carcinoma (HNSCC) show better survival rates than those with HPV-negative HNSCC. While an enhanced radiosensitivity of HPV-positive tumors is clearly evident from single modality treatment, cisplatin is never administered as monotherapy and therefore its contribution to the enhanced cure rates of HPV-positive HNSCC is not known. Both cisplatin and radiotherapy can cause severe irreversible side effects and therefore various clinical studies are currently testing deintensified regimes for patients with HPV-positive HNSCC. One strategy is to omit cisplatin-based chemotherapy or replace it by less toxic treatments but the risk assessment of these approaches remains difficult. In this study we have compared the cytotoxic effects of cisplatin in a panel of HPV-positive and -negative HNSCC cell lines alone and when combined with radiation. While cisplatin-treated HPV-positive strains showed a slightly stronger inhibition of proliferation, there was no difference regarding colony formation. Cellular responses to the drug, namely cell cycle distribution, apoptosis and γH2AX-induction did not differ between the two entities but assessment of cisplatin-DNA-adducts suggests differences regarding the mechanisms that determine cisplatin sensitivity. Combining cisplatin with radiation, we generally observed an additive but only in a minority of strains from both entities a clear synergistic effect on colony formation. In summary, HPV-positive and -negative HNSCC cells were equally sensitive to cisplatin. Therefore replacing cisplatin may be feasible but the substituting agent should be of similar efficacy in order not to jeopardize the high cure rates for HPV-positive HNSCC. PMID:27127883

A highly sensitive nanoscale pH-sensor using Au nanoparticles linked by a multifunctional Raman-active reporter molecule.

PubMed

Lawson, Latevi S; Chan, James W; Huser, Thomas

2014-07-21

Chemical sensing on the nanoscale has been breaking new ground since the discovery of surface enhanced Raman scattering (SERS). For nanoparticles, controlled particle aggregation is necessary to achieve the largest SERS enhancements. Therefore, aggregating agents such as salts or linker molecules are used in conjunction with chemically sensitive reporters in order to develop robust environmentally sensitive SERS probes. While salt-induced colloidal nanosphere aggregates have produced robust SERS signals, their variability in aggregate size contributes significantly to poor SERS signal reproducibility, which can complicate their use in in vitro cellular studies. Such systems often also lack reproducibility in spectral measurements between different nanoparticle clusters. Preaggregation of colloids via linkers followed by surface functionalization with reporter molecules results in the linker occupying valuable SERS hotspot volume which could otherwise be utilized by additional reporter molecules. Ideally, both functionalities should be obtained from a single molecule. Here, we report the use of 3,5-dimercaptobenzoic acid, a single multifunctional molecule that creates SERS hotspots via the controlled aggregation of nanoparticles, and also reports pH values. We show that 3,5-dimercaptobenzoic acid bound to Au nanospheres results in an excellent pH nanoprobe, producing very robust, and highly reproducible SERS signals that can report pH across the entire physiological range with excellent pH resolution. To demonstrate the efficacy of our novel pH reporters, these probes were also used to image both the particle and pH distribution in the cytoplasm of human induced pluripotent stem cells (hiPSCs).

Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses

PubMed Central

Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L.; Merritt, Robert; Hackett, Neil R.; Rovelink, Peter W.; Bruder, Joseph T.; Wickham, Thomas J.; Kovesdi, Imi; Crystal, Ronald G.

2004-01-01

Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing β-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the β-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing β-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to β-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-β-galactosidase antibody levels following vector administration. However, cellular responses to β-galactosidase were significantly enhanced, with the frequency of CD4+ as well as the CD8+ β-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing β-galactosidase: BALB/c mice implanted with the CT26 syngeneic β-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif to the Ad fiber knob increases the infectibility of DC and leads to enhanced cellular immune responses to the Ad-transferred transgene, suggesting that the RGD capsid modification may be useful in developing Ad-based vaccines. PMID:14963160

Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice.

PubMed

Siveen, K S; Kuttan, Girija

2012-01-01

Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn's neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.

Simultaneous PML-IRIS after discontinuation of natalizumab in a patient with MS

PubMed Central

Gheuens, S.; Smith, D.R.; Wang, X.; Alsop, D.C.; Lenkinski, R.E.

2012-01-01

Objective: Progressive multifocal leukoencephalopathy (PML) is a severe complication of natalizumab therapy in patients with multiple sclerosis (MS), which is often accompanied by an immune reconstitution inflammatory syndrome (IRIS) after removal of the drug. We describe a patient with MS who presented with simultaneous PML-IRIS 2 months after stopping natalizumab for other reasons. Case Report and Results: The patient had widespread PML and severe IRIS. He received corticosteroids and displayed a vigorous JC virus–specific cellular immune response. Elevated myoinositol and lipid/creatine peaks measured in PML lesions by proton magnetic resonance spectroscopy (1H-MRS) corresponded to episodes of contrast enhancement on MRI scans and persisted after the enhancement subsided. He demonstrated steady clinical improvement, but developed marked residual atrophy in areas affected by PML and inflammation, as well as seizures. Conclusions: New enhancing white matter lesions, occurring after discontinuation of natalizumab, can be the manifestation of PML-IRIS rather than an MS exacerbation. Elevated myoinositol and lipid/creatine peaks appear to be more sensitive markers of inflammation in PML lesions than contrast enhancement. 1H-MRS may become useful as a biomarker for PML-IRIS by helping clinicians determine the need for corticosteroid administration and anticipate continuing clinical recovery. PMID:22517104

Enhancement of radiosensitivity of melanoma cells by pegylated gold nanoparticles under irradiation of megavoltage electrons.

PubMed

Mousavi, Mehdi; Nedaei, Hassan Ali; Khoei, Samideh; Eynali, Samira; Khoshgard, Karim; Robatjazi, Mostafa; Iraji Rad, Rasoul

2017-02-01

Gold nanoparticles (GNP) have significant potential as radiosensitizer agents due to their distinctive properties. Several studies have shown that the surface modification of nanoparticles with methyl polyethylene glycol (mPEG) can increase their biocompatibility. However, the present study investigated the radiosensitization effects of mPEG-coated GNP (mPEG-GNP) in B16F10 murine melanoma cells under irradiation of 6 MeV Electron beam. The synthesized GNP were characterized by UV-Visible spectroscopy, dynamic light scattering, transmission electron microscopy, and zeta potential. Enhancement of radiosensitization was evaluated by the clonogenic assay at different radiation doses of megavoltage electron beams. It was observed that mPEG-GNP with a hydrodynamic size of approximately 50 nm are almost spherical and cellular uptake occurred at all concentrations. Both proliferation efficiency and survival fraction decreased with increasing mPEG-GNP concentration. Furthermore, significant GNP sensitization occurred with a maximum dose enhancement factor of 1.22 at a concentration of 30 μM. Pegylated-GNP are taken up by B16F10 cancer cells and cause radiosensitization in the presence of 6 MeV electrons. The radiosensitization effects of GNP may probably be due to biological processes. Therefore, the underlying biological mechanisms beyond the physical dose enhancement need to be further clarified.

Germline missense pathogenic variants in the BRCA1 BRCT domain, p.Gly1706Glu and p.Ala1708Glu, increase cellular sensitivity to PARP inhibitor olaparib by a dominant negative effect

PubMed Central

Vaclová, Tereza; Woods, Nicholas T.; Megías, Diego; Gomez-Lopez, Sergio; Setién, Fernando; García Bueno, José María; Macías, José Antonio; Barroso, Alicia; Urioste, Miguel; Esteller, Manel; Monteiro, Alvaro N.A.; Benítez, Javier; Osorio, Ana

2016-01-01

Abstract BRCA1-deficient cells show defects in DNA repair and rely on other members of the DNA repair machinery, which makes them sensitive to PARP inhibitors (PARPi). Although carrying a germline pathogenic variant in BRCA1/2 is the best determinant of response to PARPi, a significant percentage of the patients do not show sensitivity and/or display increased toxicity to the agent. Considering previously suggested mutation-specific BRCA1 haploinsufficiency, we aimed to investigate whether there are any differences in cellular response to PARPi olaparib depending on the BRCA1 mutation type. Lymphoblastoid cell lines derived from carriers of missense pathogenic variants in the BRCA1 BRCT domain (c.5117G > A, p.Gly1706Glu and c.5123C > A, p.Ala1708Glu) showed higher sensitivity to olaparib than cells with truncating variants or wild types (WT). Response to olaparib depended on a basal PARP enzymatic activity, but did not correlate with PARP1 expression. Interestingly, cellular sensitivity to the agent was associated with the level of BRCA1 recruitment into γH2AX foci, being the lowest in cells with missense variants. Since these variants lead to partially stable protein mutants, we propose a model in which the mutant protein acts in a dominant negative manner on the WT BRCA1, impairing the recruitment of BRCA1 into DNA damage sites and, consequently, increasing cellular sensitivity to PARPi. Taken together, our results indicate that carriers of different BRCA1 mutations could benefit from olaparib in a distinct way and show different toxicities to the agent, which could be especially relevant for a potential future use of PARPi as prophylactic agents in BRCA1 mutation carriers. PMID:27742776

Whole-cell patch clamp recording of voltage-sensitive Ca²+ channel currents: heterologous expression systems and dissociated brain neurons.

PubMed

Hainsworth, Atticus H; Randall, Andrew D; Stefani, Alessandro

2005-01-01

Voltage-sensitive Ca(2+) channels (VSCC) play a central role in an extensive array of physiological processes. Their importance in cellular function arises from their ability both to sense membrane voltage and to conduct Ca(2+) ions, two facets that couple membrane excitability to a key intracellular second messenger. Through this relationship, activation of VSCCs is tightly coupled to the gamut of cellular functions dependent on intracellular Ca(2+), including muscle contraction, energy metabolism, gene expression, and exocytotic/endocytotic cycling.

Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging.

PubMed

Lee, Sangyeop; Chon, Hyangah; Lee, Jiyoung; Ko, Juhui; Chung, Bong Hyun; Lim, Dong Woo; Choo, Jaebum

2014-01-15

We report a surface-enhanced Raman scattering (SERS)-based cellular imaging technique to detect and quantify breast cancer phenotypic markers expressed on cell surfaces. This technique involves the synthesis of SERS nano tags consisting of silica-encapsulated hollow gold nanospheres (SEHGNs) conjugated with specific antibodies. Hollow gold nanospheres (HGNs) enhance SERS signal intensity of individual particles by localizing surface electromagnetic fields through pinholes in the hollow particle structures. This capacity to enhance imaging at the level of single molecules permits the use of HGNs to detect specific biological markers expressed in living cancer cells. In addition, silica encapsulation greatly enhances the stability of nanoparticles. Here we applied a SERS-based imaging technique using SEHGNs in the multiplex imaging of three breast cancer cell phenotypes. Expression of epidermal growth factor (EGF), ErbB2, and insulin-like growth factor-1 (IGF-1) receptors were assessed in the MDA-MB-468, KPL4 and SK-BR-3 human breast cancer cell lines. SERS imaging technology described here can be used to test the phenotype of a cancer cell and quantify proteins expressed on the cell surface simultaneously. Based on results, this technique may enable an earlier diagnosis of breast cancer than is currently possible and offer guidance in treatment. © 2013 Elsevier B.V. All rights reserved.

Tumor-Cut: segmentation of brain tumors on contrast enhanced MR images for radiosurgery applications.

PubMed

Hamamci, Andac; Kucuk, Nadir; Karaman, Kutlay; Engin, Kayihan; Unal, Gozde

2012-03-01

In this paper, we present a fast and robust practical tool for segmentation of solid tumors with minimal user interaction to assist clinicians and researchers in radiosurgery planning and assessment of the response to the therapy. Particularly, a cellular automata (CA) based seeded tumor segmentation method on contrast enhanced T1 weighted magnetic resonance (MR) images, which standardizes the volume of interest (VOI) and seed selection, is proposed. First, we establish the connection of the CA-based segmentation to the graph-theoretic methods to show that the iterative CA framework solves the shortest path problem. In that regard, we modify the state transition function of the CA to calculate the exact shortest path solution. Furthermore, a sensitivity parameter is introduced to adapt to the heterogeneous tumor segmentation problem, and an implicit level set surface is evolved on a tumor probability map constructed from CA states to impose spatial smoothness. Sufficient information to initialize the algorithm is gathered from the user simply by a line drawn on the maximum diameter of the tumor, in line with the clinical practice. Furthermore, an algorithm based on CA is presented to differentiate necrotic and enhancing tumor tissue content, which gains importance for a detailed assessment of radiation therapy response. Validation studies on both clinical and synthetic brain tumor datasets demonstrate 80%-90% overlap performance of the proposed algorithm with an emphasis on less sensitivity to seed initialization, robustness with respect to different and heterogeneous tumor types, and its efficiency in terms of computation time.

Protons sensitize epithelial cells to mesenchymal transition.

PubMed

Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M; Pluth, Janice M; Cucinotta, Francis A

2012-01-01

Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1.

Metabolic regulation and maximal reaction optimization in the central metabolism of a yeast cell

NASA Astrophysics Data System (ADS)

Kasbawati, Gunawan, A. Y.; Hertadi, R.; Sidarto, K. A.

2015-03-01

Regulation of fluxes in a metabolic system aims to enhance the production rates of biotechnologically important compounds. Regulation is held via modification the cellular activities of a metabolic system. In this study, we present a metabolic analysis of ethanol fermentation process of a yeast cell in terms of continuous culture scheme. The metabolic regulation is based on the kinetic formulation in combination with metabolic control analysis to indicate the key enzymes which can be modified to enhance ethanol production. The model is used to calculate the intracellular fluxes in the central metabolism of the yeast cell. Optimal control is then applied to the kinetic model to find the optimal regulation for the fermentation system. The sensitivity results show that there are external and internal control parameters which are adjusted in enhancing ethanol production. As an external control parameter, glucose supply should be chosen in appropriate way such that the optimal ethanol production can be achieved. For the internal control parameter, we find three enzymes as regulation targets namely acetaldehyde dehydrogenase, pyruvate decarboxylase, and alcohol dehydrogenase which reside in the acetaldehyde branch. Among the three enzymes, however, only acetaldehyde dehydrogenase has a significant effect to obtain optimal ethanol production efficiently.

Pigment epithelium-derived factor stimulates skeletal muscle glycolytic activity through NADPH oxidase-dependent reactive oxygen species production.

PubMed

Carnagarin, Revathy; Carlessi, Rodrigo; Newsholme, Philip; Dharmarajan, Arun M; Dass, Crispin R

2016-09-01

Pigment epithelium-derived factor is a multifunctional serpin implicated in insulin resistance in metabolic disorders. Recent evidence suggests that exposure of peripheral tissues such as skeletal muscle to PEDF has profound metabolic consequences with predisposition towards chronic conditions such as obesity, type 2 diabetes, metabolic syndrome and polycystic ovarian syndrome. Chronic inflammation shifts muscle metabolism towards increased glycolysis and decreased oxidative metabolism. In the present study, we demonstrate a novel effect of PEDF on cellular metabolism in mouse cell line (C2C12) and human primary skeletal muscle cells. PEDF addition to skeletal muscle cells induced enhanced phospholipase A2 activity. This was accompanied with increased production of reactive oxygen species in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner that triggered a shift towards a more glycolytic phenotype. Extracellular flux analysis and glucose consumption assays demonstrated that PEDF treatment resulted in enhanced glycolysis but did not change mitochondrial respiration. Our results demonstrate that skeletal muscle cells express a PEDF-inducible oxidant generating system that enhances glycolysis but is sensitive to antioxidants and NADPH oxidase inhibition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Electromagnetic field therapy delays cellular senescence and death by enhancement of the heat shock response.

PubMed

Perez, Felipe P; Zhou, Ximing; Morisaki, Jorge; Jurivich, Donald

2008-04-01

Hormesis may result when mild repetitive stress increases cellular defense against diverse injuries. This process may also extend in vitro cellular proliferative life span as well as delay and reverse some of the age-dependent changes in both replicative and non-replicative cells. This study evaluated the potential hormetic effect of non-thermal repetitive electromagnetic field shock (REMFS) and its impact on cellular aging and mortality in primary human T lymphocytes and fibroblast cell lines. Unlike previous reports employing electromagnetic radiation, this study used a long wave length, low energy, and non-thermal REMFS (50MHz/0.5W) for various therapeutic regimens. The primary outcomes examined were age-dependent morphological changes in cells over time, cellular death prevention, and stimulation of the heat shock response. REMFS achieved several biological effects that modified the aging process. REMFS extended the total number of population doublings of mouse fibroblasts and contributed to youthful morphology of cells near their replicative lifespan. REMFS also enhanced cellular defenses of human T cells as reflected in lower cell mortality when compared to non-treated T cells. To determine the mechanism of REMFS-induced effects, analysis of the cellular heat shock response revealed Hsp90 release from the heat shock transcription factor (HSF1). Furthermore, REMFS increased HSF1 phosphorylation, enhanced HSF1-DNA binding, and improved Hsp70 expression relative to non-REMFS-treated cells. These results show that non-thermal REMFS activates an anti-aging hormetic effect as well as reduces cell mortality during lethal stress. Because the REMFS configuration employed in this study can potentially be applied to whole body therapy, prospects for translating these data into clinical interventions for Alzheimer's disease and other degenerative conditions with aging are discussed.

The diverse roles of glutathione-associated cell resistance against hypericin photodynamic therapy.

PubMed

Theodossiou, Theodossis A; Olsen, Cathrine E; Jonsson, Marte; Kubin, Andreas; Hothersall, John S; Berg, Kristian

2017-08-01

The diverse responses of different cancers to treatments such as photodynamic therapy of cancer (PDT) have fueled a growing need for reliable predictive markers for treatment outcome. In the present work we have studied the differential response of two phenotypically and genotypically different breast adenocarcinoma cell lines, MCF7 and MDA-MB-231, to hypericin PDT (HYP-PDT). MDA-MB-231 cells were 70% more sensitive to HYP PDT than MCF7 cells at LD 50 . MCF7 were found to express a substantially higher level of glutathione peroxidase (GPX4) than MDA-MB-231, while MDA-MB-231 differentially expressed glutathione-S-transferase (GSTP1), mainly used for xenobiotic detoxification. Eighty % reduction of intracellular glutathione (GSH) by buthionine sulfoximine (BSO), largely enhanced the sensitivity of the GSTP1 expressing MDA-MB-231 cells to HYP-PDT, but not in MCF7 cells. Further inhibition of the GSH reduction however by carmustine (BCNU) resulted in an enhanced sensitivity of MCF7 to HYP-PDT. HYP loading studies suggested that HYP can be a substrate of GSTP for GSH conjugation as BSO enhanced the cellular HYP accumulation by 20% in MDA-MB-231 cells, but not in MCF7 cells. Studies in solutions showed that L-cysteine can bind the GSTP substrate CDNB in the absence of GSTP. This means that the GSTP-lacking MCF7 may use L-cysteine for xenobiotic detoxification, especially during GSH synthesis inhibition, which leads to L-cysteine build-up. This was confirmed by the lowered accumulation of HYP in both cell lines in the presence of BSO and the L-cysteine source NAC. NAC reduced the sensitivity of MCF7, but not MDA-MB-231, cells to HYP PDT which is in accordance with the antioxidant effects of L-cysteine and its potential as a GSTP substrate. As a conclusion we have herein shown that the different GSH based cell defense mechanisms can be utilized as predictive markers for the outcome of PDT and as a guide for selecting optimal combination strategies. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Blockade of the ERK pathway enhances the therapeutic efficacy of the histone deacetylase inhibitor MS-275 in human tumor xenograft models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, Toshiaki; Ozaki, Kei-ichi; Fujio, Kohsuke

2013-04-19

Highlights: •Blockade of the ERK pathway enhances the anticancer efficacy of HDAC inhibitors. •MEK inhibitors sensitize human tumor xenografts to HDAC inhibitor cytotoxicity. •Such the enhanced efficacy is achieved by a transient blockade of the ERK pathway. •This drug combination provides a promising therapeutic strategy for cancer patients. -- Abstract: The ERK pathway is up-regulated in various human cancers and represents a prime target for mechanism-based approaches to cancer treatment. Specific blockade of the ERK pathway alone induces mostly cytostatic rather than pro-apoptotic effects, however, resulting in a limited therapeutic efficacy of the ERK kinase (MEK) inhibitors. We previously showedmore » that MEK inhibitors markedly enhance the ability of histone deacetylase (HDAC) inhibitors to induce apoptosis in tumor cells with constitutive ERK pathway activation in vitro. To evaluate the therapeutic efficacy of such drug combinations, we administered the MEK inhibitor PD184352 or AZD6244 together with the HDAC inhibitor MS-275 in nude mice harboring HT-29 or H1650 xenografts. Co-administration of the MEK inhibitor markedly sensitized the human xenografts to MS-275 cytotoxicity. A dose of MS-275 that alone showed only moderate cytotoxicity thus suppressed the growth of tumor xenografts almost completely as well as induced a marked reduction in tumor cellularity when administered with PD184352 or AZD6244. The combination of the two types of inhibitor also induced marked oxidative stress, which appeared to result in DNA damage and massive cell death, specifically in the tumor xenografts. The enhanced therapeutic efficacy of the drug combination was achieved by a relatively transient blockade of the ERK pathway. Administration of both MEK and HDAC inhibitors represents a promising chemotherapeutic strategy with improved safety for cancer patients.« less

Folic acid-targeted disulfide-based cross-linking micelle for enhanced drug encapsulation stability and site-specific drug delivery against tumors

PubMed Central

Zhang, Yumin; Zhou, Junhui; Yang, Cuihong; Wang, Weiwei; Chu, Liping; Huang, Fan; Liu, Qiang; Deng, Liandong; Kong, Deling; Liu, Jianfeng; Liu, Jinjian

2016-01-01

Although the shortcomings of small molecular antitumor drugs were efficiently improved by being entrapped into nanosized vehicles, premature drug release and insufficient tumor targeting demand innovative approaches that boost the stability and tumor responsiveness of drug-loaded nanocarriers. Here, we show the use of the core cross-linking method to generate a micelle with enhanced drug encapsulation ability and sensitivity of drug release in tumor. This kind of micelle could increase curcumin (Cur) delivery to HeLa cells in vitro and improve tumor accumulation in vivo. We designed and synthesized the core cross-linked micelle (CCM) with polyethylene glycol and folic acid-polyethylene glycol as the hydrophilic units, pyridyldisulfide as the cross-linkable and hydrophobic unit, and disulfide bond as the cross-linker. CCM showed spherical shape with a diameter of 91.2 nm by the characterization of dynamic light scattering and transmission electron microscope. Attributed to the core cross-linking, drug-loaded CCM displayed higher Nile Red or Cur-encapsulated stability and better sensitivity to glutathione than noncross-linked micelle (NCM). Cellular uptake and in vitro antitumor studies proved the enhanced endocytosis and better cytotoxicity of CCM-Cur against HeLa cells, which had a high level of glutathione. Meanwhile, the folate receptor-mediated drug delivery (FA-CCM-Cur) further enhanced the endocytosis and cytotoxicity. Ex vivo imaging studies showed that CCM-Cur and FA-CCM-Cur possessed higher tumor accumulation until 24 hours after injection. Concretely, FA-CCM-Cur exhibited the highest tumor accumulation with 1.7-fold of noncross-linked micelle Cur and 2.8-fold of free Cur. By combining cross-linking of the core with active tumor targeting of FA, we demonstrated a new and effective way to design nanocarriers for enhanced drug encapsulation, smart tumor responsiveness, and elevated tumor accumulation. PMID:27051287

Prediction of redox-sensitive cysteines using sequential distance and other sequence-based features.

PubMed

Sun, Ming-An; Zhang, Qing; Wang, Yejun; Ge, Wei; Guo, Dianjing

2016-08-24

Reactive oxygen species can modify the structure and function of proteins and may also act as important signaling molecules in various cellular processes. Cysteine thiol groups of proteins are particularly susceptible to oxidation. Meanwhile, their reversible oxidation is of critical roles for redox regulation and signaling. Recently, several computational tools have been developed for predicting redox-sensitive cysteines; however, those methods either only focus on catalytic redox-sensitive cysteines in thiol oxidoreductases, or heavily depend on protein structural data, thus cannot be widely used. In this study, we analyzed various sequence-based features potentially related to cysteine redox-sensitivity, and identified three types of features for efficient computational prediction of redox-sensitive cysteines. These features are: sequential distance to the nearby cysteines, PSSM profile and predicted secondary structure of flanking residues. After further feature selection using SVM-RFE, we developed Redox-Sensitive Cysteine Predictor (RSCP), a SVM based classifier for redox-sensitive cysteine prediction using primary sequence only. Using 10-fold cross-validation on RSC758 dataset, the accuracy, sensitivity, specificity, MCC and AUC were estimated as 0.679, 0.602, 0.756, 0.362 and 0.727, respectively. When evaluated using 10-fold cross-validation with BALOSCTdb dataset which has structure information, the model achieved performance comparable to current structure-based method. Further validation using an independent dataset indicates it is robust and of relatively better accuracy for predicting redox-sensitive cysteines from non-enzyme proteins. In this study, we developed a sequence-based classifier for predicting redox-sensitive cysteines. The major advantage of this method is that it does not rely on protein structure data, which ensures more extensive application compared to other current implementations. Accurate prediction of redox-sensitive cysteines not only enhances our understanding about the redox sensitivity of cysteine, it may also complement the proteomics approach and facilitate further experimental investigation of important redox-sensitive cysteines.

Ultra-sensitive high performance liquid chromatography-laser-induced fluorescence based proteomics for clinical applications.

PubMed

Patil, Ajeetkumar; Bhat, Sujatha; Pai, Keerthilatha M; Rai, Lavanya; Kartha, V B; Chidangil, Santhosh

2015-09-08

An ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique has been developed by our group at Manipal, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from volunteers (normal, and different pre-malignant/malignant conditions) were recorded using this set-up. The protein profiles were analyzed using principal component analysis (PCA) to achieve objective detection and classification of malignant, premalignant and healthy conditions with high sensitivity and specificity. The HPLC-LIF protein profiling combined with PCA, as a routine method for screening, diagnosis, and staging of cervical cancer and oral cancer, is discussed in this paper. In recent years, proteomics techniques have advanced tremendously in life sciences and medical sciences for the detection and identification of proteins in body fluids, tissue homogenates and cellular samples to understand biochemical mechanisms leading to different diseases. Some of the methods include techniques like high performance liquid chromatography, 2D-gel electrophoresis, MALDI-TOF-MS, SELDI-TOF-MS, CE-MS and LC-MS techniques. We have developed an ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from healthy and volunteers with different malignant conditions were recorded by using this set-up. The protein profile data were analyzed using principal component analysis (PCA) for objective classification and detection of malignant, premalignant and healthy conditions. The method is extremely sensitive to detect proteins with limit of detection of the order of femto-moles. The HPLC-LIF combined with PCA as a potential proteomic method for the diagnosis of oral cancer and cervical cancer has been discussed in this paper. This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

Cytosolic NADP(+)-dependent isocitrate dehydrogenase status modulates oxidative damage to cells.

PubMed

Lee, Su Min; Koh, Ho-Jin; Park, Dong-Chan; Song, Byoung J; Huh, Tae-Lin; Park, Jeen-Woo

2002-06-01

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose 6-phosphate dehydrogenase (G6PD), malic enzyme, and the cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc). Little information is available about the role of IDPc in antioxidant defense. In this study we investigated the role of IDPc against cytotoxicity induced by oxidative stress by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 3-4-fold higher and 35% lower, respectively, than that in the parental cells carrying the vector alone. Although the activities of other antioxidant enzymes, such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and G6PD, were comparable in all transformed cells, the ratio of GSSG to total glutathione was significantly higher in the cells expressing the lower level of IDPc. This finding indicates that IDPc is essential for the efficient glutathione recycling. Upon transient exposure to increasing concentrations of H(2)O(2) or menadione, an intracellular source of free radicals and reactive oxygen species, the cells with low levels of IDPc became more sensitive to oxidative damage by H(2)O(2) or menadione. Lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against oxidative stress, compared to the control cells. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role in cellular defense against oxidative stress.

Optimization of a Biomimetic Apatite Nanoparticle Delivery System for Non-viral Gene Transfection---a Simulated Body Fluid Approach

NASA Astrophysics Data System (ADS)

Das, Debobrato

Current methods for gene delivery utilize nanocarriers such as liposomes and viral vectors that may produce in vivo toxicity, immunogenicity, or mutagenesis. Moreover, these common high-cost systems have a low efficacy of gene-vehicle transport across the cell plasma membrane followed by inadequate release and weak intracellular stability of the genetic sequence. Thus, this study aims to maximize gene transfection while minimizing cytotoxicity by utilizing supersaturated blood-plasma ions derived from simulated body fluids (SBF). With favorable electrostatic interactions to create biocompatible calcium-phosphate nanoparticles (NPs) derived from biomimetic apatite (BA), results suggest that the SBF system, though naturally sensitive to reaction conditions, after optimization can serve as a tunable and versatile platform for the delivery of various types of nucleic acids. From a systematic exploration of the effects of nucleation pH, incubation temperature, and time on transfection efficiency, the study proposes distinct characteristic trends in SBF BA-NP morphology, cellular uptake, cell viability, and gene modulation. Specifically, with aggressive nucleation and growth of BA-NPs in solution (observed via scanning electron microscopy), the ensuing microenvironment imposes a more toxic cellular interaction (indicated by alamarBlue and BCA assays), limiting particle uptake (fluorescence experiments) and subsequent gene knockdown (quantitative loss of function assays). Controlled precipitation of BA-NPs function to increase particle accessibility by surrounding cells, and subsequently enhance uptake and transfection efficiency. By closely examining such trends, an optimal fabrication condition of pH 6.5-37C can be observed where particle growth is more tamed and less chaotic, providing improved, favorable cellular interactions that increase cell uptake and consequently maximize gene transfection, without compromising cellular viability.

Osteoporosis and alzheimer pathology: Role of cellular stress response and hormetic redox signaling in aging and bone remodeling

PubMed Central

Cornelius, Carolin; Koverech, Guido; Crupi, Rosalia; Di Paola, Rosanna; Koverech, Angela; Lodato, Francesca; Scuto, Maria; Salinaro, Angela T.; Cuzzocrea, Salvatore; Calabrese, Edward J.; Calabrese, Vittorio

2014-01-01

Alzheimer’s disease (AD) and osteoporosis are multifactorial progressive degenerative disorders. Increasing evidence shows that osteoporosis and hip fracture are common complication observed in AD patients, although the mechanisms underlying this association remain poorly understood. Reactive oxygen species (ROS) are emerging as intracellular redox signaling molecules involved in the regulation of bone metabolism, including receptor activator of nuclear factor-κB ligand-dependent osteoclast differentiation, but they also have cytotoxic effects that include lipoperoxidation and oxidative damage to proteins and DNA. ROS generation, which is implicated in the regulation of cellular stress response mechanisms, is an integrated, highly regulated, process under control of redox sensitive genes coding for redox proteins called vitagenes. Vitagenes, encoding for proteins such as heat shock proteins (Hsps) Hsp32, Hsp70, the thioredoxin, and the sirtuin protein, represent a systems controlling a complex network of intracellular signaling pathways relevant to life span and involved in the preservation of cellular homeostasis under stress conditions. Consistently, nutritional anti-oxidants have demonstrated their neuroprotective potential through a hormetic-dependent activation of vitagenes. The biological relevance of dose–response affects those strategies pointing to the optimal dosing to patients in the treatment of numerous diseases. Thus, the heat shock response has become an important hormetic target for novel cytoprotective strategies focusing on the pharmacological development of compounds capable of modulating stress response mechanisms. Here we discuss possible signaling mechanisms involved in the activation of vitagenes which, relevant to bone remodeling and through enhancement of cellular stress resistance provide a rationale to limit the deleterious consequences associated to homeostasis disruption with consequent impact on the aging process. PMID:24959146

Smart Nanoparticles Undergo Phase Transition for Enhanced Cellular Uptake and Subsequent Intracellular Drug Release in a Tumor Microenvironment.

PubMed

Ye, Guihua; Jiang, Yajun; Yang, Xiaoying; Hu, Hongxiang; Wang, Beibei; Sun, Lu; Yang, Victor C; Sun, Duxin; Gao, Wei

2018-01-10

Inefficient cellular uptake and intracellular drug release at the tumor site are two major obstacles limiting the antitumor efficacy of nanoparticle delivery systems. To overcome both problems, we designed a smart nanoparticle that undergoes phase transition in a tumor microenvironment (TME). The smart nanoparticle is generated using a lipid-polypetide hybrid nanoparticle, which comprises a PEGylated lipid monolayer shell and a pH-sensitive hydrophobic poly-l-histidine core and is loaded with the antitumor drug doxorubicin (DOX). The smart nanoparticle undergoes a two-step phase transition at two different pH values in the TME: (i) At the TME (pH e : 7.0-6.5), the smart nanoparticle swells, and its surface potential turns from negative to neutral, facilitating the cellular uptake; (ii) After internalization, at the acid endolysosome (pH endo : 6.5-4.5), the smart nanoparticle dissociates and induces endolysosome escape to release DOX into the cytoplasm. In addition, a tumor-penetrating peptide iNRG was modified on the surface of the smart nanoparticle as a tumor target moiety. The in vitro studies demonstrated that the iNGR-modified smart nanoparticles promoted cellular uptake in the acidic environment (pH 6.8). The in vivo studies showed that the iNGR-modified smart nanoparticles exerted more potent antitumor efficacy against late-stage aggressive breast carcinoma than free DOX. These data suggest that the smart nanoparticles may serve as a promising delivery system for sequential uptake and intracellular drug release of antitumor agents. The easy preparation of these smart nanoparticles may also have advantages in the future manufacture for clinical trials and clinical use.

RhoA-Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate Topography Dependent.

PubMed

Ogino, Yoichiro; Liang, Ruiwei; Mendonça, Daniela B S; Mendonça, Gustavo; Nagasawa, Masako; Koyano, Kiyoshi; Cooper, Lyndon F

2016-03-01

Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho-family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M), and nanotopography (N) to evaluate the effect of surface topography on RhoA-mediated functions (cell spreading, adhesion, migration, and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S < M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness dependent (S < M, N). RhoA activity and -mediated functions are influenced by surface topography. Smooth surface adherent cells appear highly sensitive to RhoA function, while nano-scale topography adherent cell may utilize alternative cellular signaling pathway(s) to influence adherent cellular functions regardless of RhoA activity. © 2015 Wiley Periodicals, Inc.

Cellular immune responses to platelet factor 4 and heparin complexes in patients with heparin-induced thrombocytopenia.

PubMed

Nazy, Ishac; Clare, Rumi; Staibano, Phillip; Warkentin, Theodore E; Larche, Mark; Moore, Jane C; Smith, James W; Whitlock, Richard P; Kelton, John G; Arnold, Donald M

2018-05-03

Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin characterized by thrombocytopenia and thrombotic complications. HIT is caused by pathogenic antibodies that bind to complexes of platelet factor 4 and heparin (PF4/heparin) leading to platelet activation and inducing a hypercoagulable state. Previous studies have shown immunity to PF4/heparin occurs early in life even before heparin exposure; however, the immunogenesis of HIT is not well characterized. The aim of this study was to investigate cellular proliferation in response to PF4/heparin complexes in patients with HIT. Peripheral blood mononuclear cells (PBMCs) from healthy controls (n = 30), postoperative cardiac surgery patients who underwent cardiopulmonary bypass (CPB, n = 17), and patients with confirmed HIT (n = 41) were cultured with PF4 and PF4/heparin. Cellular proliferation was assessed by 3 H-thymidine uptake and 5-ethynyl-2'-deoxyuridine (EdU) detection. PBMCs proliferated in the presence of PF4 and was enhanced by the addition of heparin in all study groups. CPB and HIT patients exhibited significantly higher proliferative responses compared to healthy controls. PBMC proliferation was antigen-specific, depended on the presence of platelets, and only CD14 + cells were identified as proliferating cells. Culture supernatants were tested for the levels of regulatory cytokines and both CPB and HIT patients produced significantly lower levels of IL-10 and TGF-β1 compared to healthy controls. These findings further demonstrate that cellular immune sensitization to PF4/heparin occurs before heparin exposure and suggests that immune dysregulation can contribute to the immunogenesis of HIT. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Hyperglycemia induced damage to mitochondrial respiration in renal mesangial and tubular cells: Implications for diabetic nephropathy.

PubMed

Czajka, Anna; Malik, Afshan N

2016-12-01

Damage to renal tubular and mesangial cells is central to the development of diabetic nephropathy (DN), a complication of diabetes which can lead to renal failure. Mitochondria are the site of cellular respiration and produce energy in the form of ATP via oxidative phosphorylation, and mitochondrial dysfunction has been implicated in DN. Since the kidney is an organ with high bioenergetic needs, we postulated that hyperglycemia causes damage to renal mitochondria resulting in bioenergetic deficit. The bioenergetic profiles and the effect of hyperglycemia on cellular respiration of human primary mesangial (HMCs) and proximal tubular cells (HK-2) were compared in normoglycemic and hyperglycemic conditions using the seahorse bio-analyzer. In normoglycemia, HK-2 had significantly lower basal, ATP-linked and maximal respiration rates, and lower reserve capacity compared to HMCs. Hyperglycemia caused a down-regulation of all respiratory parameters within 4 days in HK-2 but not in HMCs. After 8 days of hyperglycemia, down-regulation of respiratory parameters persisted in tubular cells with compensatory up-regulated glycolysis. HMCs had reduced maximal respiration and reserve capacity at 8 days, and by 12 days had compromised mitochondrial respiration despite which they did not enhance glycolysis. These data suggest that diabetes is likely to lead to a cellular deficit in ATP production in both cell types, although with different sensitivities, and this mechanism could significantly contribute to the cellular damage seen in the diabetic kidney. Prevention of diabetes induced damage to renal mitochondrial respiration may be a novel therapeutic approach for the prevention/treatment of DN. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Genomic basis for coral resilience to climate change.

PubMed

Barshis, Daniel J; Ladner, Jason T; Oliver, Thomas A; Seneca, François O; Traylor-Knowles, Nikki; Palumbi, Stephen R

2013-01-22

Recent advances in DNA-sequencing technologies now allow for in-depth characterization of the genomic stress responses of many organisms beyond model taxa. They are especially appropriate for organisms such as reef-building corals, for which dramatic declines in abundance are expected to worsen as anthropogenic climate change intensifies. Different corals differ substantially in physiological resilience to environmental stress, but the molecular mechanisms behind enhanced coral resilience remain unclear. Here, we compare transcriptome-wide gene expression (via RNA-Seq using Illumina sequencing) among conspecific thermally sensitive and thermally resilient corals to identify the molecular pathways contributing to coral resilience. Under simulated bleaching stress, sensitive and resilient corals change expression of hundreds of genes, but the resilient corals had higher expression under control conditions across 60 of these genes. These "frontloaded" transcripts were less up-regulated in resilient corals during heat stress and included thermal tolerance genes such as heat shock proteins and antioxidant enzymes, as well as a broad array of genes involved in apoptosis regulation, tumor suppression, innate immune response, and cell adhesion. We propose that constitutive frontloading enables an individual to maintain physiological resilience during frequently encountered environmental stress, an idea that has strong parallels in model systems such as yeast. Our study provides broad insight into the fundamental cellular processes responsible for enhanced stress tolerances that may enable some organisms to better persist into the future in an era of global climate change.

Spirulina platensis Improves Mitochondrial Function Impaired by Elevated Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells (ASCs) and Intestinal Epithelial Cells (IECs), and Enhances Insulin Sensitivity in Equine Metabolic Syndrome (EMS) Horses.

PubMed

Nawrocka, Daria; Kornicka, Katarzyna; Śmieszek, Agnieszka; Marycz, Krzysztof

2017-08-03

Equine Metabolic Syndrome (EMS) is a steadily growing life-threatening endocrine disorder linked to insulin resistance, oxidative stress, and systemic inflammation. Inflammatory microenvironment of adipose tissue constitutes the direct tissue milieu for various cell populations, including adipose-derived mesenchymal stromal cells (ASCs), widely considered as a potential therapeutic cell source in the course of the treatment of metabolic disorders. Moreover, elevated oxidative stress induces inflammation in intestinal epithelial cells (IECs)-the first-line cells exposed to dietary compounds. In the conducted research, we showed that in vitro application of Spirulina platensis contributes to the restoration of ASCs' and IECs' morphology and function through the reduction of cellular oxidative stress and inflammation. Enhanced viability, suppressed senescence, and improved proliferation of ASCs and IECs isolated from metabolic syndrome-affected individuals were evident following exposition to Spirulina. A protective effect of the investigated extract against mitochondrial dysfunction and degeneration was also observed. Moreover, our data demonstrate that Spirulina extract effectively suppressed LPS-induced inflammatory responses in macrophages. In vivo studies showed that horses fed with a diet based on Spirulina platensis supplementation lost weight and their insulin sensitivity improved. Thus, our results indicate the engagement of Spirulina platensis nourishing as an interesting alternative approach for supporting the conventional treatment of equine metabolic syndrome.

Nitroxyl Improves Cellular Heart Function by Directly Enhancing Cardiac Sarcoplasmic Reticulum Ca2+ Cycling

PubMed Central

Tocchetti, Carlo G.; Wang, Wang; Froehlich, Jeffrey P.; Huke, Sabine; Aon, Miguel A.; Wilson, Gerald M.; Benedetto, Giulietta Di; O’Rourke, Brian; Gao, Wei Dong; Wink, David A.; Toscano, John P.; Zaccolo, Manuela; Bers, Donald M.; Valdivia, Hector H.; Cheng, Heping; Kass, David A.; Paolocci, Nazareno

2009-01-01

Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca2+. A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca2+ pump and the ryanodine receptor 2, leading to increased Ca2+ uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca2+-release channels and accelerates Ca2+ reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca2+ transport. Contraction improves with no net rise in diastolic calcium. These changes are not induced by NO, are fully reversible by addition of reducing agents (redox sensitive), and independent of both cAMP/protein kinase A and cGMP/protein kinase G signaling. Rather, the data support HNO/thiolate interactions that enhance the activity of intracellular Ca2+ cycling proteins. These findings suggest HNO donors are attractive candidates for the pharmacological treatment of heart failure. PMID:17138943

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance

PubMed Central

Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L.; Abbas, Tarek; Larner, James M.

2017-01-01

Lung cancer resists radiation therapy, making it one of the deadliest forms of cancer. Here we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor p21 protein is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene (CDKN1A) in CHIP knockdown cells. Conversely, over-expression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone heat shock protein 70 (HSP70). These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity; thus, suggesting a novel strategy for the treatment of lung cancer. Implications The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. PMID:28232384

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance.

PubMed

Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L; Abbas, Tarek; Larner, James M

2017-06-01

Lung cancer resists radiotherapy, making it one of the deadliest forms of cancer. Here, we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically, ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor, p21 protein, is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene ( CDKN1A) in CHIP knockdown cells. Conversely, overexpression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone HSP70. These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity, thus suggesting a novel strategy for the treatment of lung cancer. Implications: The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. Mol Cancer Res; 15(6); 651-9. ©2017 AACR . ©2017 American Association for Cancer Research.

Prenatal Exposure to the Environmental Obesogen Tributyltin Predisposes Multipotent Stem Cells to Become Adipocytes

PubMed Central

Kirchner, Séverine; Kieu, Tiffany; Chow, Connie; Casey, Stephanie; Blumberg, Bruce

2010-01-01

The environmental obesogen hypothesis proposes that pre- and postnatal exposure to environmental chemicals contributes to adipogenesis and the development of obesity. Tributyltin (TBT) is an agonist of both retinoid X receptor (RXR) and peroxisome proliferator-activated receptor γ (PPARγ). Activation of these receptors can elevate adipose mass in adult mice exposed to the chemical in utero. Here we show that TBT sensitizes human and mouse multipotent stromal stem cells derived from white adipose tissue [adipose-derived stromal stem cells (ADSCs)] to undergo adipogenesis. In vitro exposure to TBT, or the PPARγ activator rosiglitazone increases adipogenesis, cellular lipid content, and expression of adipogenic genes. The adipogenic effects of TBT and rosiglitazone were blocked by the addition of PPARγ antagonists, suggesting that activation of PPARγ mediates the effect of both compounds on adipogenesis. ADSCs from mice exposed to TBT in utero showed increased adipogenic capacity and reduced osteogenic capacity with enhanced lipid accumulation in response to adipogenic induction. ADSCs retrieved from animals exposed to TBT in utero showed increased expression of PPARγ target genes such as the early adipogenic differentiation gene marker fatty acid-binding protein 4 and hypomethylation of the promoter/enhancer region of the fatty acid-binding protein 4 locus. Hence, TBT alters the stem cell compartment by sensitizing multipotent stromal stem cells to differentiate into adipocytes, an effect that could likely increase adipose mass over time. PMID:20160124

Prenatal exposure to the environmental obesogen tributyltin predisposes multipotent stem cells to become adipocytes.

PubMed

Kirchner, Séverine; Kieu, Tiffany; Chow, Connie; Casey, Stephanie; Blumberg, Bruce

2010-03-01

The environmental obesogen hypothesis proposes that pre- and postnatal exposure to environmental chemicals contributes to adipogenesis and the development of obesity. Tributyltin (TBT) is an agonist of both retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARgamma). Activation of these receptors can elevate adipose mass in adult mice exposed to the chemical in utero. Here we show that TBT sensitizes human and mouse multipotent stromal stem cells derived from white adipose tissue [adipose-derived stromal stem cells (ADSCs)] to undergo adipogenesis. In vitro exposure to TBT, or the PPARgamma activator rosiglitazone increases adipogenesis, cellular lipid content, and expression of adipogenic genes. The adipogenic effects of TBT and rosiglitazone were blocked by the addition of PPARgamma antagonists, suggesting that activation of PPARgamma mediates the effect of both compounds on adipogenesis. ADSCs from mice exposed to TBT in utero showed increased adipogenic capacity and reduced osteogenic capacity with enhanced lipid accumulation in response to adipogenic induction. ADSCs retrieved from animals exposed to TBT in utero showed increased expression of PPARgamma target genes such as the early adipogenic differentiation gene marker fatty acid-binding protein 4 and hypomethylation of the promoter/enhancer region of the fatty acid-binding protein 4 locus. Hence, TBT alters the stem cell compartment by sensitizing multipotent stromal stem cells to differentiate into adipocytes, an effect that could likely increase adipose mass over time.

pH-Triggered Echogenicity and Contents Release from Liposomes

PubMed Central

2015-01-01

Liposomes are representative lipid nanoparticles widely used for delivering anticancer drugs, DNA fragments, or siRNA to cancer cells. Upon targeting, various internal and external triggers have been used to increase the rate for contents release from the liposomes. Among the internal triggers, decreased pH within the cellular lysosomes has been successfully used to enhance the rate for releasing contents. However, imparting pH sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified at a reduced pH. Herein, we report an alternative strategy to render liposomes pH sensitive by encapsulating a precursor which generates gas bubbles in situ in response to acidic pH. The disturbance created by the escaping gas bubbles leads to the rapid release of the encapsulated contents from the liposomes. Atomic force microscopic studies indicate that the liposomal structure is destroyed at a reduced pH. The gas bubbles also render the liposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeted doxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface. In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents released from these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantially reduced viability for the pancreatic cancer cells (14%). PMID:25271780

pH-triggered echogenicity and contents release from liposomes.

PubMed

Nahire, Rahul; Hossain, Rayat; Patel, Rupa; Paul, Shirshendu; Meghnani, Varsha; Ambre, Avinash H; Gange, Kara N; Katti, Kalpana S; Leclerc, Estelle; Srivastava, D K; Sarkar, Kausik; Mallik, Sanku

2014-11-03

Liposomes are representative lipid nanoparticles widely used for delivering anticancer drugs, DNA fragments, or siRNA to cancer cells. Upon targeting, various internal and external triggers have been used to increase the rate for contents release from the liposomes. Among the internal triggers, decreased pH within the cellular lysosomes has been successfully used to enhance the rate for releasing contents. However, imparting pH sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified at a reduced pH. Herein, we report an alternative strategy to render liposomes pH sensitive by encapsulating a precursor which generates gas bubbles in situ in response to acidic pH. The disturbance created by the escaping gas bubbles leads to the rapid release of the encapsulated contents from the liposomes. Atomic force microscopic studies indicate that the liposomal structure is destroyed at a reduced pH. The gas bubbles also render the liposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeted doxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface. In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents released from these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantially reduced viability for the pancreatic cancer cells (14%).

Spirulina platensis Improves Mitochondrial Function Impaired by Elevated Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells (ASCs) and Intestinal Epithelial Cells (IECs), and Enhances Insulin Sensitivity in Equine Metabolic Syndrome (EMS) Horses

PubMed Central

Nawrocka, Daria; Kornicka, Katarzyna; Śmieszek, Agnieszka

2017-01-01

Equine Metabolic Syndrome (EMS) is a steadily growing life-threatening endocrine disorder linked to insulin resistance, oxidative stress, and systemic inflammation. Inflammatory microenvironment of adipose tissue constitutes the direct tissue milieu for various cell populations, including adipose-derived mesenchymal stromal cells (ASCs), widely considered as a potential therapeutic cell source in the course of the treatment of metabolic disorders. Moreover, elevated oxidative stress induces inflammation in intestinal epithelial cells (IECs)—the first-line cells exposed to dietary compounds. In the conducted research, we showed that in vitro application of Spirulina platensis contributes to the restoration of ASCs’ and IECs’ morphology and function through the reduction of cellular oxidative stress and inflammation. Enhanced viability, suppressed senescence, and improved proliferation of ASCs and IECs isolated from metabolic syndrome-affected individuals were evident following exposition to Spirulina. A protective effect of the investigated extract against mitochondrial dysfunction and degeneration was also observed. Moreover, our data demonstrate that Spirulina extract effectively suppressed LPS-induced inflammatory responses in macrophages. In vivo studies showed that horses fed with a diet based on Spirulina platensis supplementation lost weight and their insulin sensitivity improved. Thus, our results indicate the engagement of Spirulina platensis nourishing as an interesting alternative approach for supporting the conventional treatment of equine metabolic syndrome. PMID:28771165

Diffusion and cellular uptake of drugs in live cells studied with surface-enhanced Raman scattering probes

NASA Astrophysics Data System (ADS)

Bálint, Štefan; Rao, Satish; Sánchez, Mónica Marro; Huntošová, Veronika; Miškovský, Pavol; Petrov, Dmitri

2010-03-01

An understanding of the mechanisms of drug diffusion and uptake through cellular membranes is critical for elucidating drug action and in the development of effective drug delivery systems. We study these processes for emodin, a potential anticancer drug, in live cancer cells using surface-enhanced Raman scattering. Micrometer-sized silica beads covered by nanosized silver colloids are passively embedded into the cell and used as sensors of the drug. We demonstrate that the technique offers distinct advantages: the possibility to study the kinetics of drug diffusion through the cellular membrane toward specific cell organelles, the detection of lower drug concentrations compared to fluorescence techniques, and less damage imparted on the cell.

Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy

PubMed Central

Lara-Guerra, Humberto; Kawashima, Hiroyuki; Sakai, Ryo; Jayachandran, Gitanjali; Majidi, Mourad; Mehran, Reza; Wang, Jing; Bekele, B. Nebiyou; Baladandayuthapani, Veerabhadran; Yoo, Suk-Young; Wang, Ying; Ying, Jun; Meng, Feng; Ji, Lin; Roth, Jack A.

2015-01-01

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR. PMID:26053020

IDH1 R132H Mutation Enhances Cell Migration by Activating AKT-mTOR Signaling Pathway, but Sensitizes Cells to 5-FU Treatment as NADPH and GSH Are Reduced.

PubMed

Zhu, Huixia; Zhang, Ye; Chen, Jianfeng; Qiu, Jiangdong; Huang, Keting; Wu, Mindan; Xia, Chunlin

2017-01-01

Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue.

PubMed

Bertsch, M; Mayburd, A L; Kassner, R J

2003-02-15

Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c('). Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.

Determination of Cancer Cell-Based pH-Sensitive Fluorescent Carbon Nanoparticles of Cross-Linked Polydopamine by Fluorescence Sensing of Alkaline Phosphatase Activity on Coated Surfaces and Aqueous Solution.

PubMed

Kang, Eun Bi; Choi, Cheong A; Mazrad, Zihnil Adha Islamy; Kim, Sung Han; In, Insik; Park, Sung Young

2017-12-19

The tumor-specific sensitive fluorescence sensing of cellular alkaline phosphatase (ALP) activity on the basis of host-guest specific and pH sensitivity was conducted on coated surfaces and aqueous states. Cross-linked fluorescent nanoparticles (C-FNP) consisting of β-cyclodextrin (β-CD)/boronic acid (BA) and fluorescent hyaluronic acid [FNP(HA)] were conjugated to fluorescent polydopamine [FNP(pDA)]. To determine the quenching effect of this system, hydrolysis of 4-nitrophenyl phosphate (NPP) to 4-nitrophenol (NP) was performed in the cavity of β-CD in the presence of ALP activated photoinduced electron transfer (PET) between NP and C-FNP. At an ALP level of 30-1000 U/L, NP caused off-emission of C-FNP because of their specific host-guest recognition. Fluorescence can be recovered under pH shock due to cleavage of the diol bond between β-CD and BA, resulting in release of NP from the fluorescent system. Sensitivity of the assays was assessed by confocal imaging not only in aqueous states, but also for the first time on coated surfaces in MDAMB-231 and MDCK cells. This novel system demonstrated high sensitivity to ALP through generation of good electron donor/acceptor pair during the PET process. Therefore, this fluorescence sensor system can be used to enhance ALP monitoring and cancer diagnosis on both coated surfaces and in aqueous states in clinical settings.

Inducing cell death in vitro in cancer cells by targeted delivery of cytochrome c via a transferrin conjugate

PubMed Central

Delgado, Yamixa; Sharma, Rohit Kumar; Sharma, Shweta; Guzmán, Solimar Liz Ponce De León; Tinoco, Arthur D.; Griebenow, Kai

2018-01-01

One of the major drawbacks of many of the currently used cancer drugs are off-target effects. Targeted delivery is one method to minimize such unwanted and detrimental events. To actively target lung cancer cells, we have developed a conjugate of the apoptosis inducing protein cytochrome c with transferrin because the transferrin receptor is overexpressed by many rapidly dividing cancer cells. Cytochrome c and transferrin were cross-linked with a redox sensitive disulfide bond for the intra-cellular release of the protein upon endocytosis by the transferrin receptor. Confocal results demonstrated the cellular uptake of the cytochrome c-transferrin conjugate by transferrin receptor overexpressing A549 lung cancer cells. Localization studies further validated that this conjugate escaped the endosome. Additionally, an in vitro assay showed that the conjugate could induce apoptosis by activating caspase-3. The neo-conjugate not only maintained an IC50 value similar to the well known drug cisplatin (50 μM) in A549 cancer cells but also was nontoxic to the normal lung (MRC5) cells. Our neo-conjugate holds promise for future development to target cancers with enhanced transferrin receptor expression. PMID:29649293

Downregulation of TFAM inhibits the tumorigenesis of non-small cell lung cancer by activating ROS-mediated JNK/p38MAPK signaling and reducing cellular bioenergetics

PubMed Central

Shangguan, Fugeng; Lin, Xiaoming; Chen, Fuhong; Xu, Shan; Zhang, Ya; Chen, Zilei; Huang, Kate; Wang, Rongrong; Wang, Lu; Song, Xiaoxiao; Liu, Yongzhang; Lu, Bin

2016-01-01

Mitochondrial transcription factor A (TFAM) is essential for the replication, transcription and maintenance of mitochondrial DNA (mtDNA). The role of TFAM in non-small cell lung cancer (NSCLC) remains largely unknown. Herein, we report that downregulation of TFAM in NSCLC cells resulted in cell cycle arrest at G1 phase and significantly blocked NSCLC cell growth and migration through the activation of reactive oxygen species (ROS)-induced c-Jun amino-terminal kinase(JNK)/p38 MAPK signaling and decreased cellular bioenergetics. We further found that TFAM downregulation in NSCLC cells led to increased apoptotic cell death and enhanced the sensitivity of NSCLC cells to cisplatin. Tissue microarray (TMA) data showed that elevated expression of TFAM was related to the histological grade and TNM stage of NSCLC patients. We also demonstrated that TFAM is an independent prognostic factor for overall survival of NSCLC patients. Taken together, our findings suggest that TFAM could serve as a potential diagnostic biomarker and molecular target for the treatment of NSCLC, as well as for prediction of the effectiveness of chemotherapy. PMID:26820294

Unveiling molecular events in the brain by noninvasive imaging.

PubMed

Klohs, Jan; Rudin, Markus

2011-10-01

Neuroimaging allows researchers and clinicians to noninvasively assess structure and function of the brain. With the advances of imaging modalities such as magnetic resonance, nuclear, and optical imaging; the design of target-specific probes; and/or the introduction of reporter gene assays, these technologies are now capable of visualizing cellular and molecular processes in vivo. Undoubtedly, the system biological character of molecular neuroimaging, which allows for the study of molecular events in the intact organism, will enhance our understanding of physiology and pathophysiology of the brain and improve our ability to diagnose and treat diseases more specifically. Technical/scientific challenges to be faced are the development of highly sensitive imaging modalities, the design of specific imaging probe molecules capable of penetrating the CNS and reporting on endogenous cellular and molecular processes, and the development of tools for extracting quantitative, biologically relevant information from imaging data. Today, molecular neuroimaging is still an experimental approach with limited clinical impact; this is expected to change within the next decade. This article provides an overview of molecular neuroimaging approaches with a focus on rodent studies documenting the exploratory state of the field. Concepts are illustrated by discussing applications related to the pathophysiology of Alzheimer's disease.

pH-Responsive Triblock Copolymeric Micelles Decorated with a Cell-Penetrating Peptide Provide Efficient Doxorubicin Delivery

NASA Astrophysics Data System (ADS)

Ng, Khen Eng; Amin, Mohd Cairul Iqbal Mohd; Katas, Haliza; Amjad, Muhammad Wahab; Butt, Adeel Masood; Kesharwani, Prashant; Iyer, Arun K.

2016-12-01

This study developed novel triblock pH-responsive polymeric micelles (PMs) using cholic acid-polyethyleneimine-poly- l-arginine (CA-PEI-pArg) copolymers. PEI provided pH sensitivity, while the hydrophilic cell-penetrating pArg peptide promoted cellular PM internalization. The copolymers self-assembled into PMs in aqueous solution at above the critical micelle concentration (2.98 × 10-7 M) and encapsulated doxorubicin in the core region, with a 34.2% ( w/ w) entrapment efficiency. PMs showed pH-dependent swelling, increasing in size by almost sevenfold from pH 7.4 to 5.0. Doxorubicin release was pH-dependent, with about 65% released at pH 5.0, and 32% at pH 7.4. Cellular uptake, assessed by confocal microscopy and flow cytometry, was enhanced by using doxorubicin-loaded CA-PEI-pArg PMs, as compared to free doxorubicin and DOX-loaded CA-PEI PMs. Moreover, 24-h incubation of these PMs with a human breast cancer cell line produced greater cytotoxicity than free doxorubicin. These results indicate that pH-responsive CA-PEI-pArg micelles could provide a versatile delivery system for targeted cancer therapy using hydrophobic drugs.

Tracking transcriptional activities with high-content epifluorescent imaging

NASA Astrophysics Data System (ADS)

Hua, Jianping; Sima, Chao; Cypert, Milana; Gooden, Gerald C.; Shack, Sonsoles; Alla, Lalitamba; Smith, Edward A.; Trent, Jeffrey M.; Dougherty, Edward R.; Bittner, Michael L.

2012-04-01

High-content cell imaging based on fluorescent protein reporters has recently been used to track the transcriptional activities of multiple genes under different external stimuli for extended periods. This technology enhances our ability to discover treatment-induced regulatory mechanisms, temporally order their onsets and recognize their relationships. To fully realize these possibilities and explore their potential in biological and pharmaceutical applications, we introduce a new data processing procedure to extract information about the dynamics of cell processes based on this technology. The proposed procedure contains two parts: (1) image processing, where the fluorescent images are processed to identify individual cells and allow their transcriptional activity levels to be quantified; and (2) data representation, where the extracted time course data are summarized and represented in a way that facilitates efficient evaluation. Experiments show that the proposed procedure achieves fast and robust image segmentation with sufficient accuracy. The extracted cellular dynamics are highly reproducible and sensitive enough to detect subtle activity differences and identify mechanisms responding to selected perturbations. This method should be able to help biologists identify the alterations of cellular mechanisms that allow drug candidates to change cell behavior and thereby improve the efficiency of drug discovery and treatment design.

Protein and cell micropatterning and its integration with micro/nanoparticles assembly.

PubMed

Yap, F L; Zhang, Y

2007-01-15

Micropatterning of proteins and cells has become very popular over the past decade due to its importance in the development of biosensors, microarrays, tissue engineering and cellular studies. This article reviews the techniques developed for protein and cell micropatterning and its biomedical applications. The prospect of integrating micro and nanoparticles with protein and cell micropatterning is discussed. The micro/nanoparticles are assembled into patterns and form the substrate for proteins and cell attachment. The assembled particles create a micro or nanotopography, depending on the size of the particles employed. The nonplanar structure can increase the surface area for biomolecules attachment and therefore enhance the sensitivity for detection in biosensors. Furthermore, a nanostructured substrate can influence the conformation and functionality of protein attached to it, while cellular response in terms of morphology, adhesion, proliferation, differentiation, etc. can be affected by a surface expressing micro or nanoscale structures. Proteins and cells tend to lose their normal functions upon attachment to substrate. By recognizing the types of topography that are favourable for preserving proteins and cell behaviour, and integrating it with micropattering will lead to the development of functional protein and cell patterns.

Enhancing the Biological Relevance of Secretome-Based Proteomics by Linking Tumor Cell Proliferation and Protein Secretion.

PubMed

Gregori, Josep; Méndez, Olga; Katsila, Theodora; Pujals, Mireia; Salvans, Cándida; Villarreal, Laura; Arribas, Joaquin; Tabernero, Josep; Sánchez, Alex; Villanueva, Josep

2014-07-15

Secretome profiling has become a methodology of choice for the identification of tumor biomarkers. We hypothesized that due to the dynamic nature of secretomes cellular perturbations could affect their composition but also change the global amount of protein secreted per cell. We confirmed our hypothesis by measuring the levels of secreted proteins taking into account the amount of proteome produced per cell. Then, we established a correlation between cell proliferation and protein secretion that explained the observed changes in global protein secretion. Next, we implemented a normalization correcting the statistical results of secretome studies by the global protein secretion of cells into a generalized linear model (GLM). The application of the normalization to two biological perturbations on tumor cells resulted in drastic changes in the list of statistically significant proteins. Furthermore, we found that known epithelial-to-mesenchymal transition (EMT) effectors were only statistically significant when the normalization was applied. Therefore, the normalization proposed here increases the sensitivity of statistical tests by increasing the number of true-positives. From an oncology perspective, the correlation between protein secretion and cellular proliferation suggests that slow-growing tumors could have high-protein secretion rates and consequently contribute strongly to tumor paracrine signaling.

Adaptive optics and the eye (super resolution OCT).

PubMed

Miller, D T; Kocaoglu, O P; Wang, Q; Lee, S

2011-03-01

The combination of adaptive optics (AO) and optical coherence tomography (OCT) was first reported 8 years ago and has undergone tremendous technological advances since then. The technical benefits of adding AO to OCT (increased lateral resolution, smaller speckle, and enhanced sensitivity) increase the imaging capability of OCT in ways that make it well suited for three-dimensional (3D) cellular imaging in the retina. Today, AO-OCT systems provide ultrahigh 3D resolution (3 × 3 × 3 μm³) and ultrahigh speed (up to an order of magnitude faster than commercial OCT). AO-OCT systems have been used to capture volume images of retinal structures, previously only visible with histology, and are being used for studying clinical conditions. Here, we present representative examples of cellular structures that can be visualized with AO-OCT. We overview three studies from our laboratory that used ultrahigh-resolution AO-OCT to measure the cross-sectional profiles of individual bundles in the retinal nerve fiber layer; the diameters of foveal capillaries that define the terminal rim of the foveal avascular zone; and the spacing and length of individual cone photoreceptor outer segments as close as 0.5° from the fovea center.

Molecular Thermometry

PubMed Central

McCabe, Kevin M.; Hernandez, Mark

2010-01-01

Conventional temperature measurements rely on material responses to heat, which can be detected visually. When Galileo developed an air expansion based device to detect temperature changes, Santorio, a contemporary physician, added a scale to create the first thermometer. With this instrument, patients’ temperatures could be measured, recorded and related to changing health conditions. Today, advances in materials science and bioengineering provide new ways to report temperature at the molecular level in real time. In this review the scientific foundations and history of thermometry underpin a discussion of the discoveries emerging from the field of molecular thermometry. Intracellular nanogels and heat sensing biomolecules have been shown to accurately report temperature changes at the nano-scale. Various systems will soon provide the ability to accurately measure temperature changes at the tissue, cellular, and even sub-cellular level, allowing for detection and monitoring of very small changes in local temperature. In the clinic this will lead to enhanced detection of tumors and localized infection, and accurate and precise monitoring of hyperthermia based therapies. Some nanomaterial systems have even demonstrated a theranostic capacity for heat-sensitive, local delivery of chemotherapeutics. Just as early thermometry moved into the clinic, so too will these molecular thermometers. PMID:20139796

Redox control of protein-DNA interactions: from molecular mechanisms to significance in signal transduction, gene expression, and DNA replication.

PubMed

Shlomai, Joseph

2010-11-01

Protein-DNA interactions play a key role in the regulation of major cellular metabolic pathways, including gene expression, genome replication, and genomic stability. They are mediated through the interactions of regulatory proteins with their specific DNA-binding sites at promoters, enhancers, and replication origins in the genome. Redox signaling regulates these protein-DNA interactions using reactive oxygen species and reactive nitrogen species that interact with cysteine residues at target proteins and their regulators. This review describes the redox-mediated regulation of several master regulators of gene expression that control the induction and suppression of hundreds of genes in the genome, regulating multiple metabolic pathways, which are involved in cell growth, development, differentiation, and survival, as well as in the function of the immune system and cellular response to intracellular and extracellular stimuli. It also discusses the role of redox signaling in protein-DNA interactions that regulate DNA replication. Specificity of redox regulation is discussed, as well as the mechanisms providing several levels of redox-mediated regulation, from direct control of DNA-binding domains through the indirect control, mediated by release of negative regulators, regulation of redox-sensitive protein kinases, intracellular trafficking, and chromatin remodeling.

Genetically Encoded Catalytic Hairpin Assembly for Sensitive RNA Imaging in Live Cells.

PubMed

Mudiyanselage, Aruni P K K Karunanayake; Yu, Qikun; Leon-Duque, Mark A; Zhao, Bin; Wu, Rigumula; You, Mingxu

2018-06-26

DNA and RNA nanotechnology has been used for the development of dynamic molecular devices. In particular, programmable enzyme-free nucleic acid circuits, such as catalytic hairpin assembly, have been demonstrated as useful tools for bioanalysis and to scale up system complexity to an extent beyond current cellular genetic circuits. However, the intracellular functions of most synthetic nucleic acid circuits have been hindered by challenges in the biological delivery and degradation. On the other hand, genetically encoded and transcribed RNA circuits emerge as alternative powerful tools for long-term embedded cellular analysis and regulation. Herein, we reported a genetically encoded RNA-based catalytic hairpin assembly circuit for sensitive RNA imaging inside living cells. The split version of Broccoli, a fluorogenic RNA aptamer, was used as the reporter. One target RNA can catalytically trigger the fluorescence from tens-to-hundreds of Broccoli. As a result, target RNAs can be sensitively detected. We have further engineered our circuit to allow easy programming to image various target RNA sequences. This design principle opens the arena for developing a large variety of genetically encoded RNA circuits for cellular applications.

Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides.

PubMed

Meade, Bryan R; Dowdy, Steven F

2008-03-01

The major limitation in utilizing information rich macromolecules for basic science and therapeutic applications is the inability of these large molecules to readily diffuse across the cellular membrane. While this restriction represents an efficient defense system against cellular penetration of unwanted foreign molecules and thus a crucial component of cell survival, overcoming this cellular characteristic for the intracellular delivery of macromolecules has been the focus of a large number of research groups worldwide. Recently, with the discovery of RNA interference, many of these groups have redirected their attention and have applied previously characterized cell delivery methodologies to synthetic short interfering RNA duplexes (siRNA). Protein transduction domain and cell penetrating peptides have been shown to enhance the delivery of multiple types of macromolecular cargo including peptides, proteins and antisense oligonucleotides and are now being utilized to enhance the cellular uptake of siRNA molecules. The dense cationic charge of these peptides that is critical for interaction with cell membrane components prior to internalization has also been shown to readily package siRNA molecules into stable nanoparticles that are capable of traversing the cell membrane. This review discusses the recent advances in noncovalent packaging of siRNA molecules with cationic peptides and the potential for the resulting complexes to successfully induce RNA interference within both in vitro and in vivo settings.

Enhanced splicing correction effect by an oligo-aspartic acid-PNA conjugate and cationic carrier complexes.

PubMed

Bae, Yun Mi; Kim, Myung Hee; Yu, Gwang Sig; Um, Bong Ho; Park, Hee Kyung; Lee, Hyun-il; Lee, Kang Taek; Suh, Yung Doug; Choi, Joon Sig

2014-02-10

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA. They recognize specific cellular nucleic acid sequences and form stable complexes with complementary DNA or RNA. Here, we designed an oligo-aspartic acid-PNA conjugate and showed its enhanced delivery into cells with high gene correction efficiency using conventional cationic carriers, such as polyethylenimine (PEI) and Lipofectamine 2000. The negatively charged oligo-aspartic acid-PNA (Asp(n)-PNA) formed complexes with PEI and Lipofectamine, and the resulting Asp(n)-PNA/PEI and Asp(n)-PNA/Lipofectamine complexes were introduced into cells. We observed significantly enhanced cellular uptake of Asp(n)-PNA by cationic carriers and detected an active splicing correction effect even at nanomolar concentrations. We found that the splicing correction efficiency of the complex depended on the kind of the cationic carriers and on the number of repeating aspartic acid units. By enhancing the cellular uptake efficiency of PNAs, these results may provide a novel platform technology of PNAs as bioactive substances for their biological and therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Strong enhancement of dispersion forces from microwave radiation

NASA Astrophysics Data System (ADS)

Sernelius, B. E.

2002-11-01

We have studied non-thermal effects of microwave radiation on the forces between objects. This is the first step in a study of possible effects of microwave radiation from cellular phones on biological tissue. We have used a simplified model for human blood cells in blood. We find for the normal radiation level of cellular phones an enhancement of the attractive force with ten orders of magnitude as compared to the corresponding effect at thermal radiation.

Adiponectin improves insulin sensitivity via activation of autophagic flux.

PubMed

Ahlstrom, Penny; Rai, Esther; Chakma, Suharto; Cho, Hee Ho; Rengasamy, Palanivel; Sweeney, Gary

2017-11-01

Skeletal muscle insulin resistance is known to play an important role in the pathogenesis of diabetes, and one potential causative cellular mechanism is endoplasmic reticulum (ER) stress. Adiponectin mediates anti-diabetic effects via direct metabolic actions and by improving insulin sensitivity, and we recently demonstrated an important role in stimulation of autophagy by adiponectin. However, there is limited knowledge on crosstalk between autophagy and ER stress in skeletal muscle and in particular how they are regulated by adiponectin. Here, we utilized the model of high insulin/glucose (HIHG)-induced insulin resistance, determined by measuring Akt phosphorylation (T308 and S473) and glucose uptake in L6 skeletal muscle cells. HIHG reduced autophagic flux measured by LC3 and p62 Western blotting and tandem fluorescent RFP/GFP-LC3 immunofluorescence (IF). HIHG also induced ER stress assessed by thioflavin T/KDEL IF, pIRE1, pPERK, peIF2α and ATF6 Western blotting and induction of a GRP78-mCherry reporter. Induction of autophagy by adiponectin or rapamycin attenuated HIHG-induced ER stress and improved insulin sensitivity. The functional significance of enhanced autophagy was validated by demonstrating a lack of improved insulin sensitivity in response to adiponectin in autophagy-deficient cells generated by overexpression of dominant negative mutant of Atg5. In summary, adiponectin-induced autophagy in skeletal muscle cells alleviated HIHG-induced ER stress and insulin resistance. © 2017 Society for Endocrinology.

A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins.

PubMed

Gaebler, Anne; Penno, Anke; Kuerschner, Lars; Thiele, Christoph

2016-10-01

The demand to study the cellular localization of specific lipids has led to recent advances in lipid probes and microscopy. Alkyne lipids bear a small, noninterfering tag and can be detected upon click reaction with an azide-coupled reporter. Fluorescent alkyne lipid imaging crucially depends on appropriate azide reporters and labeling protocols that allow for an efficient click reaction and therefore a sensitive detection. We synthesized several azide reporters with different spacer components and tested their suitability for alkyne lipid imaging in fixed cells. The implementation of a copper-chelating picolyl moiety into fluorescent or biotin-based azide reagents strongly increased the sensitivity of the imaging routine. We demonstrate the applicability and evaluate the performance of this approach using different lipid classes and experimental setups. As azide picolyl reporters allow for reduced copper catalyst concentrations, they also enable coimaging of alkyne lipids with multiple fluorescent proteins including enhanced green fluorescent protein. Alternatively, and as we also show, microscopy of alkyne lipids can be combined with protein detection by immunocytochemistry. In summary, we present a robust, sensitive, and highly versatile protocol for the labeling of alkyne lipids with azide-coupled reporters for fluorescence microscopy that can be combined with different protein detection and imaging techniques. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Imaging deep skeletal muscle structure using a high-sensitivity ultrathin side-viewing optical coherence tomography needle probe

PubMed Central

Yang, Xiaojie; Lorenser, Dirk; McLaughlin, Robert A.; Kirk, Rodney W.; Edmond, Matthew; Simpson, M. Cather; Grounds, Miranda D.; Sampson, David D.

2013-01-01

We have developed an extremely miniaturized optical coherence tomography (OCT) needle probe (outer diameter 310 µm) with high sensitivity (108 dB) to enable minimally invasive imaging of cellular structure deep within skeletal muscle. Three-dimensional volumetric images were acquired from ex vivo mouse tissue, examining both healthy and pathological dystrophic muscle. Individual myofibers were visualized as striations in the images. Degradation of cellular structure in necrotic regions was seen as a loss of these striations. Tendon and connective tissue were also visualized. The observed structures were validated against co-registered hematoxylin and eosin (H&E) histology sections. These images of internal cellular structure of skeletal muscle acquired with an OCT needle probe demonstrate the potential of this technique to visualize structure at the microscopic level deep in biological tissue in situ. PMID:24466482

[Development of a Novel Liposomal DDS by Manipulating Pharmacokinetics and Intracellular Trafficking for Drug Therapy and Nucleic Acid Medicine].

PubMed

Hatakeyama, Hiroto

2018-01-01

　Nucleic acid therapy is expected to be a next generation medicine. We recently developed a multifunctional envelope-type nano device (MEND) for use as a novel delivery system. The modification of polyethylene glycol (PEG), i.e., PEGylation, is useful for achieving the delivery of MENDs to tumors via an enhanced permeability and retention (EPR) effect. However, PEGylation strongly inhibits the cellular uptake and endosomal escape of MEND, which results in significant loss of action, and therefore lost effectiveness, of the cargo therapeutic. For successful nucleic acid delivery in cancer treatment, the crucial problem associated with the use of PEG, known as the "PEG dilemma", must be solved. In this review, we describe the development and application of MEND in overcoming the PEG dilemma based on manipulating both the pharmacokinetics and intracellular trafficking of cellular uptake and endosomal release using a cleavable PEG lipid, a pH-sensitive fusogenic peptide, and a pH-sensitive cationic lipid. We also developed dual-ligand liposomes with a controlled diameter of around 300 nm, then modified these with a specific ligand and a cell penetrating peptide designed to target the neovasculature of tumors. Dual-ligand liposomes could induce an anti-tumor effect in drug resistant tumors by delivering drugs to tumor blood vessels, rather than to the cancer cells themselves. Here, we review our recent efforts to develop a novel liposomal drug delivery system (DDS) by manipulating pharmacokinetics and intracellular trafficking for drug therapy and nucleic acid medicine.

Adaptive Cellular Stress Pathways as Therapeutic Targets of Dietary Phytochemicals: Focus on the Nervous System

PubMed Central

Jo, Dong-Gyu; Park, Daeui; Chung, Hae Young

2014-01-01

During the past 5 decades, it has been widely promulgated that the chemicals in plants that are good for health act as direct scavengers of free radicals. Here we review evidence that favors a different hypothesis for the health benefits of plant consumption, namely, that some phytochemicals exert disease-preventive and therapeutic actions by engaging one or more adaptive cellular response pathways in cells. The evolutionary basis for the latter mechanism is grounded in the fact that plants produce natural antifeedant/noxious chemicals that discourage insects and other organisms from eating them. However, in the amounts typically consumed by humans, the phytochemicals activate one or more conserved adaptive cellular stress response pathways and thereby enhance the ability of cells to resist injury and disease. Examplesof such pathways include those involving the transcription factors nuclear factor erythroid 2-related factor 2, nuclear factor-κB, hypoxia-inducible factor 1α, peroxisome proliferator-activated receptor γ, and forkhead box subgroup O, as well as the production and action of trophic factors and hormones. Translational research to develop interventions that target these pathways may lead to new classes of therapeutic agents that act by stimulating adaptive stress response pathways to bolster endogenous defenses against tissue injury and disease. Because neurons are particularly sensitive to potentially noxious phytochemicals, we focus on the nervous system but also include findings from other cell types in which actions of phytochemicals on specific signal transduction pathways have been more thoroughly studied. PMID:24958636

Detection of gastrointestinal cancer by elastic scattering and absorption spectroscopies with the Los Alamos Optical Biopsy System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mourant, J.R.; Boyer, J.; Johnson, T.M.

1995-03-01

The Los Alamos National Laboratory has continued the development of the Optical Biopsy System (OBS) for noninvasive, real-time in situ diagnosis of tissue pathologies. In proceedings of earlier SPIE conferences we reported on clinical measurements in the bladder, and we report here on recent results of clinical tests in the gastrointestinal tract. With the OBS, tissue pathologies are detected/diagnosed using spectral measurements of the elastic optical transport properties (scattering and absorption) of the tissue over a wide range of wavelengths. The use of elastic scattering as the key to optical tissue diagnostics in the OBS is based on the factmore » that many tissue pathologies, including a majority of cancer forms, exhibit significant architectural changes at the cellular and sub-cellular level. Since the cellular components that cause elastic scattering have dimensions typically on the order of visible to near-IR wavelengths, the elastic (Mie) scattering properties will be wavelength dependent. Thus, morphology and size changes can be expected to cause significant changes m an optical signature that is derived from the wavelength-dependence of elastic scattering. Additionally, the optical geometry of the OBS beneficially enhances its sensitivity for measuring absorption bands. The OBS employs a small fiber-optic probe that is amenable to use with any endoscope or catheter, or to direct surface examination, as well as interstitial needle insertion. Data acquistion/display time is <1 second.« less

Surface decoration by Spirulina polysaccharide enhances the cellular uptake and anticancer efficacy of selenium nanoparticles.

PubMed

Yang, Fang; Tang, Quanming; Zhong, Xueyun; Bai, Yan; Chen, Tianfeng; Zhang, Yibo; Li, Yinghua; Zheng, Wenjie

2012-01-01

A simple and solution-phase method for functionalization of selenium nanoparticles (SeNPs) with Spirulina polysaccharides (SPS) has been developed in the present study. The cellular uptake and anticancer activity of SPS-SeNPs were also evaluated. Monodisperse and homogeneous spherical SPS-SeNPs with diameters ranging from 20 nm to 50 nm were achieved under optimized conditions, which were stable in the solution phase for at least 3 months. SPS surface decoration significantly enhanced the cellular uptake and cytotoxicity of SeNPs toward several human cancer cell lines. A375 human melanoma cells were found extremely susceptible to SPS-SeNPs with half maximal (50%) inhibitory concentration value of 7.94 μM. Investigation of the underlying mechanisms revealed that SPS-SeNPs inhibited cancer cell growth through induction of apoptosis, as evidenced by an increase in sub-G(1) cell population, deoxyribonucleic acid fragmentation, chromatin condensation, and phosphatidylserine translocation. Results suggest that the strategy to use SPS as a surface decorator could be an effective way to enhance the cellular uptake and anticancer efficacy of nanomaterials. SPS-SeNPs may be a potential candidate for further evaluation as a chemopreventive and chemotherapeutic agent against human cancers.

Surface decoration by Spirulina polysaccharide enhances the cellular uptake and anticancer efficacy of selenium nanoparticles

PubMed Central

Yang, Fang; Tang, Quanming; Zhong, Xueyun; Bai, Yan; Chen, Tianfeng; Zhang, Yibo; Li, Yinghua; Zheng, Wenjie

2012-01-01

A simple and solution-phase method for functionalization of selenium nanoparticles (SeNPs) with Spirulina polysaccharides (SPS) has been developed in the present study. The cellular uptake and anticancer activity of SPS-SeNPs were also evaluated. Monodisperse and homogeneous spherical SPS-SeNPs with diameters ranging from 20 nm to 50 nm were achieved under optimized conditions, which were stable in the solution phase for at least 3 months. SPS surface decoration significantly enhanced the cellular uptake and cytotoxicity of SeNPs toward several human cancer cell lines. A375 human melanoma cells were found extremely susceptible to SPS-SeNPs with half maximal (50%) inhibitory concentration value of 7.94 μM. Investigation of the underlying mechanisms revealed that SPS-SeNPs inhibited cancer cell growth through induction of apoptosis, as evidenced by an increase in sub-G1 cell population, deoxyribonucleic acid fragmentation, chromatin condensation, and phosphatidylserine translocation. Results suggest that the strategy to use SPS as a surface decorator could be an effective way to enhance the cellular uptake and anticancer efficacy of nanomaterials. SPS-SeNPs may be a potential candidate for further evaluation as a chemopreventive and chemotherapeutic agent against human cancers. PMID:22359460

The aminoglycoside antibiotic kanamycin damages DNA bases in Escherichia coli: caffeine potentiates the DNA-damaging effects of kanamycin while suppressing cell killing by ciprofloxacin in Escherichia coli and Bacillus anthracis.

PubMed

Kang, Tina Manzhu; Yuan, Jessica; Nguyen, Angelyn; Becket, Elinne; Yang, Hanjing; Miller, Jeffrey H

2012-06-01

The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase gene enhances ethanol-induced toxicity in HepG2 cells.

PubMed

Yang, Eun Sun; Lee, Su-Min; Park, Jeen-Woo

2010-07-01

It has been shown that acute and chronic alcohol administrations increase the production of reactive oxygen species, lower cellular antioxidant levels and enhance oxidative stress in many tissues. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme by supplying NADPH to the cytosol. Upon exposure to ethanol, IDPc was susceptible to the loss of its enzyme activity in HepG2 cells. Transfection of HepG2 cells with an IDPc small interfering RNA noticeably downregulated IDPc and enhanced the cells' vulnerability to ethanol-induced cytotoxicity. Our results suggest that suppressing the expression of IDPc enhances ethanol-induced toxicity in HepG2 cells by further disruption of the cellular redox status.

Differential cellular responses in healthy mice and in mice with established airway inflammation when exposed to hematite nanoparticles.

PubMed

Gustafsson, Åsa; Bergström, Ulrika; Ågren, Lina; Österlund, Lars; Sandström, Thomas; Bucht, Anders

2015-10-01

The aim of this study was to investigate the inflammatory and immunological responses in airways and lung-draining lymph nodes (LDLNs), following lung exposure to iron oxide (hematite) nanoparticles (NPs). The responses to the hematite NPs were evaluated in both healthy non-sensitized mice, and in sensitized mice with an established allergic airway disease. The mice were exposed intratracheally to either hematite NPs or to vehicle (PBS) and the cellular responses were evaluated on days 1, 2, and 7, post-exposure. Exposure to hematite NPs increased the numbers of neutrophils, eosinophils, and lymphocytes in the airways of non-sensitized mice on days 1 and 2 post-exposure; at these time points the number of lymphocytes was also elevated in the LDLNs. In contrast, exposing sensitized mice to hematite NPs induced a rapid and unspecific cellular reduction in the alveolar space on day 1 post-exposure; a similar decrease of lymphocytes was also observed in the LDLN. The results indicate that cells in the airways and in the LDLN of individuals with established airway inflammation undergo cell death when exposed to hematite NPs. A possible explanation for this toxic response is the extensive generation of reactive oxygen species (ROS) in the pro-oxidative environment of inflamed airways. This study demonstrates how sensitized and non-sensitized mice respond differently to hematite NP exposure, and it highlights the importance of including individuals with respiratory disorders when evaluating health effects of inhaled nanomaterials. Copyright © 2015 Elsevier Inc. All rights reserved.

Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laszlo, A.; Radke, K.; Chin, S.

1981-10-01

We have investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the synthesis and modification of polypeptides in normal avian cells and cells infected by wild-type and temperature-sensitive Rous sarcoma virus (RSV). Using two-dimensional gel electrophoresis, we have detected alterations in both the abundance of cellular polypeptides and in their phosphorylation that seem unique to TPA treatment. However, the state of phosphorylation of the major putative substrate for the action of the src gene-associated protein kinase, the 34- to 36-kilodalton protein, was not altered. Moreover, examination of the phosphorylated amino acid content of total cellular phosphoproteins revealed that the response tomore » TPA was not associated with detectable increases in their phosphotyrosine content. These results make it unlikely that TPA acts by the activation of the phosphorylating activity of the cellular proto-src gene or by the activation of other cellular phosphotyrosine-specific kinases. We have shown previously that temperature-sensitive RSV-infected cells at nonpermissive temperature demonstrate an increased sensitivity to TPA treatment (Bissell, M.J., Hatie, C. and Calfin, M. (1979) Proc. Natl. Acad. Sci. USA 76, 348-352). Our present results indicate that this is not due to reactivation of the phosphorylating activity of the defective src gene product or to its leakiness, and they lend support to the notion of multistep viral carcinogenesis.« less

Bio indices for 2,4-D sensitivity between two plant species: Azolla pinnata R.Br. and Vernonia cinerea L. with their cellular responses.

PubMed

De, Arnab Kumar; Dey, Narottam; Adak, Malay Kumar

2016-07-01

In the present experiment a pteridophytic species Azolla and an angiospermic species Vernonia were evaluated on the basis of cellular reactivity for herbicidal action through ongoing concentrations. Initially, both the species recorded a significant activity of IAA-oxidase as mark of IAA metabolism with herbicidal sensitivity. Still, Vernonia species were more affected on 2,4-D mediated auxin catabolism. The loss of auxin concentrations on the tissues by 2,4-D reaction was also reflected on growth parameters including relative growth rate and chlorophyll biosynthesis. In a dose dependent manner Vernonia plants were more affected with loss of chlorophyll content and decline in relative growth rate. On the other hand, both those parameters were adjusted significantly with 2,4-D accumulation in Azolla . The stability of cellular metabolism was documented by significant down regulation of protein and lipid peroxidation with concomitant moderation to superoxide and hydrogen peroxide accumulation. The later two were more vulnerable to damage in the Vernonia plant with profuse accumulation of protein and lipid peroxidation products. Similarly, tissue specific reaction to superoxide and hydrogen peroxide accumulation were distinctly demarcated in two species significantly. As a whole, the cellular responses and metabolite distribution to 2,4-D sensitization are the features to describe bio-indices for aquatic fern species Azolla with comparison to angiospermic species Vernonia .

Degradation of Redox-Sensitive Proteins including Peroxiredoxins and DJ-1 is Promoted by Oxidation-induced Conformational Changes and Ubiquitination

NASA Astrophysics Data System (ADS)

Song, In-Kang; Lee, Jae-Jin; Cho, Jin-Hwan; Jeong, Jihye; Shin, Dong-Hae; Lee, Kong-Joo

2016-10-01

Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1.

Vacuolar amino acid transporter Avt5p is responsible for lithium uptake in Schizosaccharomyces pombe.

PubMed

Iwaki, Tomoko; Sekito, Takayuki; Kakinuma, Yoshimi

2010-01-01

The fission yeast Schizosaccharomyces pombe was sensitive to salinity; cell growth was stopped by 0.5 M NaCl and by 10 mM LiCl. The avt5+ gene encodes a vacuolar transporter with a broad specificity for amino acids. We found that the avt5Delta mutant became highly tolerant of Li+ and Na+ in growth. Concanamycin A-sensitive Li+ uptake as well as cellular Li+ content was lower in the avt5 mutant, suggesting a role of Avt5p in cellular uptake of toxic Li+.

Elongin B-mediated epigenetic alteration of viral chromatin correlates with efficient human cytomegalovirus gene expression and replication.

PubMed

Hwang, Jiwon; Saffert, Ryan T; Kalejta, Robert F

2011-01-01

Elongins B and C are members of complexes that increase the efficiency of transcriptional elongation by RNA polymerase II (RNAPII) and enhance the monoubiquitination of histone H2B, an epigenetic mark of actively transcribed genes. Here we show that, in addition to its role in facilitating transcription of the cellular genome, elongin B also enhances gene expression from the double-stranded DNA genome of human cytomegalovirus (HCMV), a pathogenic herpesvirus. Reducing the level of elongin B by small interfering RNA- or short hairpin RNA-mediated knockdown decreased viral mRNA expression, viral protein accumulation, viral DNA replication, and infectious virion production. Chromatin immunoprecipitation analysis indicated viral genome occupancy of the elongating form of RNAPII, and monoubiquitinated histone H2B was reduced in elongin B-deficient cells. These data suggest that, in addition to the previously documented epigenetic regulation of transcriptional initiation, HCMV also subverts cellular elongin B-mediated epigenetic mechanisms for enhancing transcriptional elongation to enhance viral gene expression and virus replication. The genetic and epigenetic control of transcription initiation at both cellular and viral promoters is well documented. Recently, the epigenetic modification of histone H2B monoubiquitination throughout the bodies of cellular genes has been shown to enhance the elongation of RNA polymerase II-initiated transcripts. Mechanisms that might control the elongation of viral transcripts are less well studied. Here we show that, as with cellular genes, elongin B-mediated monoubiquitination of histone H2B also facilitates the transcriptional elongation of human cytomegalovirus genes. This and perhaps other epigenetic markings of actively transcribed regions may help in identifying viral genes expressed during in vitro latency or during natural infections of humans. Furthermore, this work identifies a novel, tractable model system to further study the regulation of transcriptional elongation in living cells.

The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts.

PubMed

Yhee, Ji Young; Yoon, Hong Yeol; Kim, Hyunjoon; Jeon, Sangmin; Hergert, Polla; Im, Jintaek; Panyam, Jayanth; Kim, Kwangmeyung; Nho, Richard Seonghun

2017-01-01

Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6-78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts.

The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts

PubMed Central

Yhee, Ji Young; Yoon, Hong Yeol; Kim, Hyunjoon; Jeon, Sangmin; Hergert, Polla; Im, Jintaek; Panyam, Jayanth; Kim, Kwangmeyung; Nho, Richard Seonghun

2017-01-01

Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6–78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts. PMID:28860768

Osthole enhances TRAIL-mediated apoptosis through downregulation of c-FLIP expression in renal carcinoma Caki cells.

PubMed

Min, Kyoung-Jin; Han, Min Ae; Kim, Shin; Park, Jong-Wook; Kwon, Taeg Kyu

2017-04-01

Osthole, an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson, has been shown to induce various beneficial biochemical effects such as anti-inflammatory and antitumor. In the present study, we examined whether osthole could sensitize TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human renal carcinoma Caki cells. We found that osthole and TRAIL alone, had no effect on apoptosis, but combined treatment with osthole and TRAIL markedly induced apoptosis in Caki (renal carcinoma), U251MG (glioma) and MDA-MB-231 (breast carcinoma) cells. In contrast, combined treatment with osthole and TRAIL did not induce apoptosis in normal human skin fibroblast cells. Osthole induced downregulation of cellular FLICE-like inhibitory protein (c-FLIP) expression, and overexpression of c-FLIP markedly blocked apoptosis induced by the combined treatment with osthole and TRAIL. In addition, osthole markedly reduced mitochondrial membrane potential levels, and increased cytosolic cytochrome c release in combined treatment with osthole and TRAIL. Therefore, these data suggest that osthole may be an efficient TRAIL sensitizer.

Behavioural and physiological limits to vision in mammals

PubMed Central

Field, Greg D.

2017-01-01

Human vision is exquisitely sensitive—a dark-adapted observer is capable of reliably detecting the absorption of a few quanta of light. Such sensitivity requires that the sensory receptors of the retina, rod photoreceptors, generate a reliable signal when single photons are absorbed. In addition, the retina must be able to extract this information and relay it to higher visual centres under conditions where very few rods signal single-photon responses while the majority generate only noise. Critical to signal transmission are mechanistic optimizations within rods and their dedicated retinal circuits that enhance the discriminability of single-photon responses by mitigating photoreceptor and synaptic noise. We describe behavioural experiments over the past century that have led to the appreciation of high sensitivity near absolute visual threshold. We further consider mechanisms within rod photoreceptors and dedicated rod circuits that act to extract single-photon responses from cellular noise. We highlight how these studies have shaped our understanding of brain function and point out several unresolved questions in the processing of light near the visual threshold. This article is part of the themed issue ‘Vision in dim light’. PMID:28193817

Bile resistance in Lactococcus lactis strains varies with cellular fatty acid composition: analysis by using different growth media.

PubMed

Kimoto-Nira, Hiromi; Kobayashi, Miho; Nomura, Masaru; Sasaki, Keisuke; Suzuki, Chise

2009-05-31

Bile resistance is one of the basic characteristics of probiotic bacteria. The aim of this study was to investigate the characteristics of bile resistance in lactococci by studying the relationship between bile resistance and cellular fatty acid composition in lactococcci grown on different media. We determined the bile resistance of 14 strains in lactose-free M17 medium supplemented with either glucose only (GM17) or lactose only (LM17). Gas chromatographic analyses of free lipids extracted from the tested strains were used for determining their fatty acid composition. A correlation analysis of all strains grown in both media revealed significant positive correlations between bile resistance and relative contents of hexadecanoic acid and octadecenoic acid, and negative correlations between bile resistance and relative contents of hexadecenoic acid and C-19 cyclopropane fatty acid. It is also a fact that the fatty acids associated with bile resistance depended on species, strain, and/or growth medium. In L. lactis subsp. cremoris strains grown in GM17 medium, the bile-resistant strains had significantly more octadecenoic acid than the bile-sensitive strains. In LM17 medium, bile-resistant strains had significantly more octadecenoic acid and significantly less C-19 cyclopropane fatty acid than the bile-sensitive strains. In L. lactis subsp. lactis strains, bile resistances of some of the tested strains were altered by growth medium. Some strains were resistant to bile in GM17 medium but sensitive to bile in LM17 medium. Some strains were resistant in both media tested. The strains grown in GM17 medium had significantly more hexadecanoic acid and octadecenoic acid, and significantly less tetradecanoic acid, octadecadienoic acid and C-19 cyclopropane fatty acid than the strains grown in LM17 medium. In conclusion, the fatty acid compositions of the bile-resistant lactococci differed from those of the bile-sensitive ones. More importantly, our data suggest that altering their fatty acid composition (i.e. increased hexadecanoic acid and octadecenoic acid and decreased hexadecenoic acid and C-19 cyclopropane fatty acid) by changing growth conditions may be a useful way to enhance their bile resistance in lactococci.

Targeting Homology-Directed Recombinational Repair (HDR) of Chromosomal Breaks to Sensitize Prostate Cancer Cells to Poly (ADP-Ribose) Polymerase (PARP) Inhibition

DTIC Science & Technology

2012-08-01

Investigator 15 UAB X1219: Molecular determinants of cellular susceptibility to PARP inhibition in an ex- vivo model of human cholangiocarcinoma Role...cellular susceptibility to PARP inhibition in an ex-vivo model of human cholangiocarcinoma Role: Co-Prinicipal Investigator Career Development

A cellular automata model of land cover change to integrate urban growth with open space conservation

EPA Science Inventory

The preservation of riparian zones and other environmentally sensitive areas has long been recognized as one of the most cost-effective methods of managing stormwater and providing a broad range of ecosystem services. In this research, a cellular automata (CA)—Markov chain model ...

Do antioxidant supplements interfere with skeletal muscle adaptation to exercise training?

PubMed Central

Ristow, Michael

2016-01-01

Abstract A popular belief is that reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced during exercise by the mitochondria and other subcellular compartments ubiquitously cause skeletal muscle damage, fatigue and impair recovery. However, the importance of ROS and RNS as signals in the cellular adaptation process to stress is now evident. In an effort to combat the perceived deleterious effects of ROS and RNS it has become common practice for active individuals to ingest supplements with antioxidant properties, but interfering with ROS/RNS signalling in skeletal muscle during acute exercise may blunt favourable adaptation. There is building evidence that antioxidant supplementation can attenuate endurance training‐induced and ROS/RNS‐mediated enhancements in antioxidant capacity, mitochondrial biogenesis, cellular defence mechanisms and insulin sensitivity. However, this is not a universal finding, potentially indicating that there is redundancy in the mechanisms controlling skeletal muscle adaptation to exercise, meaning that in some circumstances the negative impact of antioxidants on acute exercise response can be overcome by training. Antioxidant supplementation has been more consistently reported to have deleterious effects on the response to overload stress and high‐intensity training, suggesting that remodelling of skeletal muscle following resistance and high‐intensity exercise is more dependent on ROS/RNS signalling. Importantly there is no convincing evidence to suggest that antioxidant supplementation enhances exercise‐training adaptions. Overall, ROS/RNS are likely to exhibit a non‐linear (hormetic) pattern on exercise adaptations, where physiological doses are beneficial and high exposure (which would seldom be achieved during normal exercise training) may be detrimental. PMID:26638792

AtNUDT7, a Negative Regulator of Basal Immunity in Arabidopsis, Modulates Two Distinct Defense Response Pathways and Is Involved in Maintaining Redox Homeostasis1[C][OA

PubMed Central

Ge, Xiaochun; Li, Guo-Jing; Wang, Sheng-Bing; Zhu, Huifen; Zhu, Tong; Wang, Xun; Xia, Yiji

2007-01-01

Plants have evolved complicated regulatory systems to control immune responses. Both positive and negative signaling pathways interplay to coordinate development of a resistance response with the appropriate amplitude and duration. AtNUDT7, a Nudix domain-containing protein in Arabidopsis (Arabidopsis thaliana) that hydrolyzes nucleotide derivatives, was found to be a negative regulator of the basal defense response, and its loss-of-function mutation results in enhanced resistance to infection by Pseudomonas syringae. The nudt7 mutation does not cause a strong constitutive disease resistance phenotype, but it leads to a heightened defense response, including accelerated activation of defense-related genes that can be triggered by pathogenic and nonpathogenic microorganisms. The nudt7 mutation enhances two distinct defense response pathways: one independent of and the other dependent on NPR1 and salicylic acid accumulation. In vitro enzymatic assays revealed that ADP-ribose and NADH are preferred substrates of NUDT7, and the hydrolysis activity of NUDT7 is essential for its biological function and is sensitive to inhibition by Ca2+. Further analyses indicate that ADP-ribose is not likely the physiological substrate of NUDT7. However, the nudt7 mutation leads to perturbation of cellular redox homeostasis and a higher level of NADH in pathogen-challenged leaves. The study suggests that the alteration in cellular antioxidant status caused by the nudt7 mutation primes the cells for the amplified defense response and NUDT7 functions to modulate the defense response to prevent excessive stimulation. PMID:17660350

Antibiotics induce redox-related physiological alterations as part of their lethality

PubMed Central

Dwyer, Daniel J.; Belenky, Peter A.; Yang, Jason H.; MacDonald, I. Cody; Martell, Jeffrey D.; Takahashi, Noriko; Chan, Clement T. Y.; Lobritz, Michael A.; Braff, Dana; Schwarz, Eric G.; Ye, Jonathan D.; Pati, Mekhala; Vercruysse, Maarten; Ralifo, Paul S.; Allison, Kyle R.; Khalil, Ahmad S.; Ting, Alice Y.; Walker, Graham C.; Collins, James J.

2014-01-01

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. PMID:24803433

Sonochemically synthesized biocompatible zirconium phosphate nanoparticles for pH sensitive drug delivery application.

PubMed

Kalita, Himani; Prashanth Kumar, B N; Konar, Suraj; Tantubay, Sangeeta; Kr Mahto, Madhusudan; Mandal, Mahitosh; Pathak, Amita

2016-03-01

The present work reports the synthesis of biocompatible zirconium phosphate (ZP) nanoparticles as nanocarrier for drug delivery application. The ZP nanoparticles were synthesized via a simple sonochemical method in the presence of cetyltrimethylammonium bromide and their efficacy for the delivery of drugs has been tested through various in-vitro experiments. The particle size and BET surface area of the nanoparticles were found to be ~48 nm and 206.51 m(2)/g respectively. The conventional MTT assay and cellular localization studies of the particles, performed on MDA-MB-231 cell lines, demonstrate their excellent biocompatibility and cellular internalization behavior. The loading of curcumin, an antitumor drug, onto the ZP nanoparticles shows the rapid drug uptake ability of the particles, while the drug release study, performed at two different pH values (at 7.4 and 5) depicts pH sensitive release-profile. The MTT assay and cellular localization studies revealed higher cellular inhibition and better bioavailability of the nanoformulated curcumin compared to free curcumin. Copyright © 2015 Elsevier B.V. All rights reserved.

Chemosensitizing tumor cells by targeting the Fanconi anemia pathway with an adenovirus overexpressing dominant-negative FANCA.

PubMed

Ferrer, Miriam; de Winter, Johan P; Mastenbroek, D C Jeroen; Curiel, David T; Gerritsen, Winald R; Giaccone, Giuseppe; Kruyt, Frank A E

2004-08-01

Fanconi anemia (FA) is a rare genetic disorder characterized by bone-marrow failure and cellular hypersensitivity to crosslinking agents, including cisplatin. Here, we studied the use of the FA pathway as a possible target for cancer gene therapy with the aim to sensitize tumor cells for cisplatin by interfering with the FA pathway. As proof-of-principle, FA and non-FA lymphoblast-derived tumors were grown subcutaneously in scid mice and treated with two different concentrations of cisplatin. As predicted, the antitumor response was considerably improved in FA tumors. An adenoviral vector encoding a dominant-negative form of FANCA, FANCA600DN, was generated that interfered with endogenous FANCA-FANCG interaction resulting in the disruption of the FA pathway as illustrated by disturbed FANCD2 monoubiquitination. A panel of cell lines, including non-small-cell lung cancer cells, could be sensitized approximately two- to three-fold for cisplatin after Ad.CMV.FANCA600DN infection that may increase upon enhanced infection efficiency. In conclusion, targeting the FA pathway may provide a novel strategy for the sensitization of solid tumors for cisplatin and, in addition, provides a tool for examining the role of the FA pathway in determining chemoresistance in different tumor types.

Activation of the CRABPII/RAR pathway by curcumin induces retinoic acid mediated apoptosis in retinoic acid resistant breast cancer cells

PubMed Central

Thulasiraman, Padmamalini; Garriga, Galen; Danthuluri, Veena; McAndrews, Daniel J.; Mohiuddin, Imran Q.

2017-01-01

Due to the anti-proliferative and anti-apoptotic effects of retinoic acid (RA), this hormone has emerged as a target for several diseases, including cancer. However, development of retinoid resistance is a critical issue and efforts to understand the retinoid signaling pathway may identify useful biomarkers for future clinical trials. Apoptotic responses of RA are exhibited through the cellular RA-binding protein II (CRABPII)/retinoic acid receptor (RAR) signaling cascade. Delivery of RA to RAR by CRABPII enhances the transcriptional activity of genes involved in cell death and cell cycle arrest. The purpose of this study was to investigate the role of curcumin in sensitizing RA-resistant triple-negative breast cancer (TNBC) cells to RA-mediated apoptosis. We provide evidence that curcumin upregulates the expression of CRABPII, RARβ and RARγ in two different TNBC cell lines. Co-treatment of the cells with curcumin and RA results in increased apoptosis as demonstrated by elevated cleavage of poly(ADP-ribose) polymerase and cleaved caspase-9. Additionally, silencing CRABPII reverses curcumin sensitization of TNBC cells to the apoptotic inducing effects of RA. These findings provide mechanistic insights into sensitizing TNBC cells to RA-mediated cell death by curcumin-induced upregulation of the CRABPII/RAR pathway. PMID:28350049

Polarity-defective mutants of Aspergillus nidulans.

PubMed

Osherov, N; Mathew, J; May, G S

2000-12-01

We have identified two polarity-defective (pod) mutants in Aspergillus nidulans from a collection of heat-sensitive lethal mutants. At restrictive temperature, these mutants are capable of nuclear division but are unable to establish polar hyphal growth. We cloned the two pod genes by complementation of their heat-sensitive lethal phenotypes. The libraries used to clone the pod genes are under the control of the bidirectional niaD and niiA promoters. Complementation of the pod mutants is dependent on growth on inducing medium. We show that rescue of the heat-sensitive phenotype on inducing media is independent of the orientation of the gene relative to the niaD or niiA promoters, demonstrating that the intergenic region between the niaD and the niiA genes functions as an orientation-independent enhancer and repressor that is capable of functioning over long distances. The products of the podG and the podH genes were identified as homologues of the alpha subunit of yeast mitochondrial phenylalanyl--tRNA synthetase and transcription factor IIF interacting component of the CTD phosphatase. Neither of these gene products would have been predicted to produce a pod mutant phenotype based on studies of cellular polarity mutants in other organisms. The implications of these results are discussed. Copyright 2000 Academic Press.

T2* mapping and delayed gadolinium-enhanced magnetic resonance imaging in cartilage (dGEMRIC) of humeral articular cartilage--a histologically controlled study.

PubMed

Bittersohl, Bernd; Kircher, Jörn; Miese, Falk R; Dekkers, Christin; Habermeyer, Peter; Fröbel, Julia; Antoch, Gerald; Krauspe, Rüdiger; Zilkens, Christoph

2015-10-01

Cartilage biochemical imaging modalities that include the magnetic resonance imaging (MRI) techniques of T2* mapping (sensitive to water content and collagen fiber network) and delayed gadolinium-enhanced MRI of cartilage (dGEMRIC, sensitive to the glycosaminoglycan content) can be effective instruments for early diagnosis and reliable follow-up of cartilage damage. The purpose of this study was to provide T2* mapping and dGEMRIC values in various histologic grades of cartilage degeneration in humeral articular cartilage. A histologically controlled in vitro study was conducted that included human humeral head cartilage specimens with various histologic grades of cartilage degeneration. High-resolution, 3-dimensional (3D) T2* mapping and dGEMRIC were performed that enabled the correlation of MRI and histology data. Cartilage degeneration was graded according to the Mankin score, which evaluates surface morphology, cellularity, toluidine blue staining, and tidemark integrity. SPSS software was used for statistical analyses. Both MRI mapping values decreased significantly (P < .001) with increasing cartilage degeneration. Spearman rank analysis revealed a significant correlation (correlation coefficients ranging from -0.315 to 0.784; P < .001) between the various histologic parameters and the T2* and T1Gd mapping values. This study demonstrates the feasibility of 3D T2* and dGEMRIC to identify various histologic grades of cartilage damage of humeral articular cartilage. With regard to the advantages of these mapping techniques with high image resolution and the ability to accomplish a 3D biochemically sensitive imaging, we consider that these imaging techniques can make a positive contribution to the currently evolving science and practice of cartilage biochemical imaging. Copyright © 2015 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction

PubMed Central

Poerschke, Robyn L.; Moos, Philip J.

2010-01-01

Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate (p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction. PMID:20920480

Tumor-targeted pH/redox dual-sensitive unimolecular nanoparticles for efficient siRNA delivery.

PubMed

Chen, Guojun; Wang, Yuyuan; Xie, Ruosen; Gong, Shaoqin

2017-08-10

A unique pH/redox dual-sensitive cationic unimolecular nanoparticle (NP) enabling excellent endosomal/lysosomal escape and efficient siRNA decomplexation inside the target cells was developed for tumor-targeted delivery of siRNA. siRNA was complexed into the cationic core of the unimolecular NP through electrostatic interactions. The cationic core used for complexing siRNA contained reducible disulfide bonds that underwent intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH) inside the cells, thereby facilitating the decomplexation of siRNA from the unimolecular NPs. The cationic polymers were conjugated onto the hyperbranched core (H40) via a pH-sensitive bond, which further facilitated the decomplexation of siRNA from the NPs. In vitro studies on the siRNA release behaviors showed that dual stimuli (pH=5.3, 10mM GSH) induced the quickest release of siRNA from the NPs. In addition, the imidazole groups attached to the cationic polymer segments enhanced the endosomal/lysosomal escape of NPs via the proton sponge effect. Intracellular tracking studies revealed that siRNA delivered by unimolecular NPs was efficiently released to the cytosol. Moreover, the GE11 peptide, an anti-EGFR peptide, enhanced the cellular uptake of NPs in MDA-MB-468, an EFGR-overexpressing triple negative breast cancer (TNBC) cell line. The GE11-conjugated, GFP-siRNA-complexed NPs exhibited excellent GFP gene silencing efficiency in GFP-MDA-MB-468 TNBC cells without any significant cytotoxicity. Therefore, these studies suggest that this smart unimolecular NP could be a promising nanoplatform for targeted siRNA delivery to EFGR-overexpressing cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Hepatitis B virus enhances cisplatin-induced hepatotoxicity via a mechanism involving suppression of glucose-regulated protein of 78 Kda.

PubMed

Zhang, Xiaoxue; Zhang, Rui; Yang, HuiOu; Xiang, Qian; Jiang, Qing; He, Qi; Zhang, Ting; Chen, Chen; Zhu, Huifen; Wang, Qiang; Ning, Qin; Li, Yiwu; Lei, Ping; Shen, Guanxin

2016-07-25

Cisplatin is a classical platinum-based chemotherapeutic drug used in the treatment of many cancer types, including hepatocellular carcinoma (HCC). The application of cisplatin is significantly limited by its toxicity, which may be affected by various biological factors. Persistence of Hepatitis B virus (HBV) infection leads to HCC development and may be associated with higher incidence of severe hepatitis during chemotherapy. However, whether HBV alters the susceptibility of hepatocytes to cisplatin remains poorly understood. Here, we demonstrate that HBV transfection enhanced cisplatin-induced hepatotoxicity via a mechanism involving suppression of glucose-regulated protein of 78 KDa (Grp78), a major stress-induced chaperone that localizes to the endoplasmic reticulum. Silencing Grp78 gene increased the susceptibility of HepG2 to cisplatin by activating caspase-3. Grp78 expression was down-regulated by HBV infection both in vitro and in liver tissues of patients. We compared the cisplatin sensitivity of hepatoma cells either expressing (HepG2.2.15 cells) or not expressing the entire Hepatitis B Virus genome (HepG2). HepG2.2.15 cells showed increased sensitivity to cisplatin and a higher apoptosis rate. Overexpression of Grp78 counteracted the increase of sensitivity of HepG2.215 cells to cisplatin. Furthermore, we found that HBV disrupted Grp78 synthesis in response to cisplatin stimulation, which may trigger severe and prolonged endoplasmic reticulum (ER) stress that can induce cellular apoptosis. Our findings provide new information into the effect of HBV in the modulation of Grp78 expression, and, consequently on cisplatin-induced hepatotoxicity during viral infection. Copyright © 2016. Published by Elsevier Ireland Ltd.

On Pulsating and Cellular Forms of Hydrodynamic Instability in Liquid-Propellant Combustion

NASA Technical Reports Server (NTRS)

Margolis, Stephen B.; Sacksteder, Kurt (Technical Monitor)

1998-01-01

An extended Landau-Levich model of liquid-propellant combustion, one that allows for a local dependence of the burning rate on the (gas) pressure at the liquid-gas interface, exhibits not only the classical hydrodynamic cellular instability attributed to Landau but also a pulsating hydrodynamic instability associated with sufficiently negative pressure sensitivities. Exploiting the realistic limit of small values of the gas-to-liquid density ratio p, analytical formulas for both neutral stability boundaries may be obtained by expanding all quantities in appropriate powers of p in each of three distinguished wave-number regimes. In particular, composite analytical expressions are derived for the neutral stability boundaries A(sub p)(k), where A, is the pressure sensitivity of the burning rate and k is the wave number of the disturbance. For the cellular boundary, the results demonstrate explicitly the stabilizing effect of gravity on long-wave disturbances, the stabilizing effect of viscosity (both liquid and gas) and surface tension on short-wave perturbations, and the instability associated with intermediate wave numbers for negative values of A(sub p), which is characteristic of many hydroxylammonium nitrate-based liquid propellants over certain pressure ranges. In contrast, the pulsating hydrodynamic stability boundary is insensitive to gravitational and surface-tension effects but is more sensitive to the effects of liquid viscosity because, for typical nonzero values of the latter, the pulsating boundary decreases to larger negative values of A(sub p) as k increases through O(l) values. Thus, liquid-propellant combustion is predicted to be stable (that is, steady and planar) only for a range of negative pressure sensitivities that lie below the cellular boundary that exists for sufficiently small negative values of A(sub p) and above the pulsating boundary that exists for larger negative values of this parameter.

Measuring spatial and temporal Ca2+ signals in Arabidopsis plants.

PubMed

Zhu, Xiaohong; Taylor, Aaron; Zhang, Shenyu; Zhang, Dayong; Feng, Ying; Liang, Gaimei; Zhu, Jian-Kang

2014-09-02

Developmental and environmental cues induce Ca(2+) fluctuations in plant cells. Stimulus-specific spatial-temporal Ca(2+) patterns are sensed by cellular Ca(2+) binding proteins that initiate Ca(2+) signaling cascades. However, we still know little about how stimulus specific Ca(2+) signals are generated. The specificity of a Ca(2+) signal may be attributed to the sophisticated regulation of the activities of Ca(2+) channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca(2+) signals at both the tissue and cellular levels. Genetically encoded Ca(2+) indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca(2+) signals. Here we describe instructions for the use of two Ca(2+) detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca(2+) imaging and case12 based live cell confocal fluorescence Ca(2+) imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca(2+) signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca(2+) signals at a high resolution.

The correlation between apparent diffusion coefficient and tumor cellularity in patients: a meta-analysis.

PubMed

Chen, Lihua; Liu, Min; Bao, Jing; Xia, Yunbao; Zhang, Jiuquan; Zhang, Lin; Huang, Xuequan; Wang, Jian

2013-01-01

To perform a meta-analysis exploring the correlation between the apparent diffusion coefficient (ADC) and tumor cellularity in patients. We searched medical and scientific literature databases for studies discussing the correlation between the ADC and tumor cellularity in patients. Only studies that were published in English or Chinese prior to November 2012 were considered for inclusion. Summary correlation coefficient (r) values were extracted from each study, and 95% confidence intervals (CIs) were calculated. Sensitivity and subgroup analyses were performed to investigate potential heterogeneity. Of 189 studies, 28 were included in the meta-analysis, comprising 729 patients. The pooled r for all studies was -0.57 (95% CI: -0.62, -0.52), indicating notable heterogeneity (P<0.001). After the sensitivity analysis, two studies were excluded, and the pooled r was -0.61 (95% CI: -0.66, -0.56) and was not significantly heterogeneous (P = 0.127). Regarding tumor type subgroup analysis, there were sufficient data to support a strong negative correlation between the ADC and cellularity for brain tumors. There was no notable evidence of publication bias. There is a strong negative correlation between the ADC and tumor cellularity in patients, particularly in the brain. However, larger, prospective studies are warranted to validate these findings in other cancer types.

Enhancement of myocardial regeneration through genetic engineering of cardiac progenitor cells expressing Pim-1 kinase.

PubMed

Fischer, Kimberlee M; Cottage, Christopher T; Wu, Weitao; Din, Shabana; Gude, Natalie A; Avitabile, Daniele; Quijada, Pearl; Collins, Brett L; Fransioli, Jenna; Sussman, Mark A

2009-11-24

Despite numerous studies demonstrating the efficacy of cellular adoptive transfer for therapeutic myocardial regeneration, problems remain for donated cells with regard to survival, persistence, engraftment, and long-term benefits. This study redresses these concerns by enhancing the regenerative potential of adoptively transferred cardiac progenitor cells (CPCs) via genetic engineering to overexpress Pim-1, a cardioprotective kinase that enhances cell survival and proliferation. Intramyocardial injections of CPCs overexpressing Pim-1 were given to infarcted female mice. Animals were monitored over 4, 12, and 32 weeks to assess cardiac function and engraftment of Pim-1 CPCs with echocardiography, in vivo hemodynamics, and confocal imagery. CPCs overexpressing Pim-1 showed increased proliferation and expression of markers consistent with cardiogenic lineage commitment after dexamethasone exposure in vitro. Animals that received CPCs overexpressing Pim-1 also produced greater levels of cellular engraftment, persistence, and functional improvement relative to control CPCs up to 32 weeks after delivery. Salutary effects include reduction of infarct size, greater number of c-kit(+) cells, and increased vasculature in the damaged region. Myocardial repair is significantly enhanced by genetic engineering of CPCs with Pim-1 kinase. Ex vivo gene delivery to enhance cellular survival, proliferation, and regeneration may overcome current limitations of stem cell-based therapeutic approaches.

The space experiment CERASP: Definition of a space-suited radiation source and growth conditions for human cells

NASA Astrophysics Data System (ADS)

Hellweg, Christine E.; Baumstark-Khan, Christa; Spitta, Luis; Thelen, Melanie; Arenz, Andrea; Franz, Markus; Schulze-Varnholt, Dirk; Berger, Thomas; Reitz, Günther

The combined action of ionizing radiation and microgravity will continue to influence future space missions, with special risks for astronauts on the Moon surface or for long duration missions to Mars. It has been estimated that on a 3-year mission to Mars about 3% of the bodies' cell nuclei would have been hit by one iron ion with the consequence that nuclear DNA will be heavily damaged. There is increasing evidence that basic cellular functions are sensitive not only to radiation but also to microgravity. DNA repair studies in space on bacteria, yeast cells and human fibroblasts, which were irradiated before, flight, gave contradictory results: from inhibition of repair by microgravity to enhancement, whereas others did not detect any influence of microgravity on repair. The space experiment CERASP (CEllular Responses to RAdiation in SPace) to be performed at the International Space Station (ISS) is aimed to supply basic information on the cellular response in microgravity to radiation applied during flight. It makes use of a recombinant human cell line as reporter for cellular signal transduction modulation by genotoxic environmental conditions. The main biological endpoints under investigation will be gene activation based on enhanced green fluorescent protein (EGFP, originally isolated from the bioluminescent jellyfish Aequorea victoria) expression controlled by a DNA damage-dependent promoter element which reflects the activity of the nuclear factor kappa B (NF- κB) pathway. The NF- κB family of proteins plays a major role in the inflammatory and immune response, cell proliferation and differentiation, anti-apoptosis and tumorgenesis. For radiation exposure during space flight a radiation source has been constructed as damage accumulation by cosmic radiation will certainly be insufficient for analysis. The space experiment specific hardware consists of a specially designed radiation source made up of the β-emitter promethium-147, combined with a miniaturized culture vessel and a seeding apparatus. With this prototype hardware, the requirements of CERASP can be fulfilled with cells growing on the polytetrafluoroethylene foil. The radiation source can be enveloped with additional titanium foils for safety issues. The results from the preparatory experimental phase clearly show that the Pm-147 radiation source meets the requirements for the space experiment CERASP.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hofstetter, Markus; Howgate, John; Schmid, Martin

Highlights: Black-Right-Pointing-Pointer Gallium nitride based sensors show promising characteristics to monitor cellular parameters. Black-Right-Pointing-Pointer Cell growth experiments reveal excellent biocompatibiltiy of the host GaN material. Black-Right-Pointing-Pointer We present a biofunctionality assay using ionizing radiation. Black-Right-Pointing-Pointer DNA repair is utilized to evaluate material induced alterations in the cellular behavior. Black-Right-Pointing-Pointer GaN shows no bio-functional influence on the cellular environment. -- Abstract: There is an increasing interest in the integration of hybrid bio-semiconductor systems for the non-invasive evaluation of physiological parameters. High quality gallium nitride and its alloys show promising characteristics to monitor cellular parameters. Nevertheless, such applications not only request appropriatemore » sensing capabilities but also the biocompatibility and especially the biofunctionality of materials. Here we show extensive biocompatibility studies of gallium nitride and, for the first time, a biofunctionality assay using ionizing radiation. Analytical sensor devices are used in medical settings, as well as for cell- and tissue engineering. Within these fields, semiconductor devices have increasingly been applied for online biosensing on a cellular and tissue level. Integration of advanced materials such as gallium nitride into these systems has the potential to increase the range of applicability for a multitude of test devices and greatly enhance sensitivity and functionality. However, for such applications it is necessary to optimize cell-surface interactions and to verify the biocompatibility of the semiconductor. In this work, we present studies of mouse fibroblast cell activity grown on gallium nitride surfaces after applying external noxa. Cell-semiconductor hybrids were irradiated with X-rays at air kerma doses up to 250 mGy and the DNA repair dynamics, cell proliferation, and cell growth dynamics of adherent cells were compared to control samples. The impact of ionizing radiation on DNA, along with the associated cellular repair mechanisms, is well characterized and serves as a reference tool for evaluation of substrate effects. The results indicate that gallium nitride does not require specific surface treatments to ensure biocompatibility and suggest that cell signaling is not affected by micro-environmental alterations arising from gallium nitride-cell interactions. The observation that gallium nitride provides no bio-functional influence on the cellular environment confirms that this material is well suited for future biosensing applications without the need for additional chemical surface modification.« less

DNA Tetrahedron Delivery Enhances Doxorubicin-Induced Apoptosis of HT-29 Colon Cancer Cells

NASA Astrophysics Data System (ADS)

Zhang, Guiyu; Zhang, Zhiyong; Yang, Junen

2017-08-01

As a nano-sized drug carrier with the advantage of modifiability and proper biocompatibility, DNA tetrahedron (DNA tetra) delivery is hopeful to enhance the inhibitory efficiency of nontargeted anticancer drugs. In this investigation, doxorubicin (Dox) was assembled to a folic acid-modified DNA tetra via click chemistry to prepare a targeted antitumor agent. Cellular uptake efficiency was measured via fluorescent imaging. Cytotoxicity, inhibition efficiency, and corresponding mechanism on colon cancer cell line HT-29 were evaluated by MTT assay, cell proliferation curve, western blot, and flow cytometry. No cytotoxicity was induced by DNA tetra, but the cellular uptake ratio increased obviously resulting from the DNA tetra-facilitated penetration through cellular membrane. Accordingly, folic acid-DNA tetra-Dox markedly increased the antitumor efficiency with increased apoptosis levels. In details, 100 μM was the effective concentration and a 6-h incubation period was needed for apoptosis induction. In conclusion, nano-sized DNA tetrahedron was a safe and effective delivery system for Dox and correspondingly enhanced the anticancer efficiency.

Evaluation of a toxicogenomic approach to the local lymph node assay (LLNA).

PubMed

Boverhof, Darrell R; Gollapudi, B Bhaskar; Hotchkiss, Jon A; Osterloh-Quiroz, Mandy; Woolhiser, Michael R

2009-02-01

Genomic technologies have the potential to enhance and complement existing toxicology endpoints; however, assessment of these approaches requires a systematic evaluation including a robust experimental design with genomic endpoints anchored to traditional toxicology endpoints. The present study was conducted to assess the sensitivity of genomic responses when compared with the traditional local lymph node assay (LLNA) endpoint of lymph node cell proliferation and to evaluate the responses for their ability to provide insights into mode of action. Female BALB/c mice were treated with the sensitizer trimellitic anhydride (TMA), following the standard LLNA dosing regimen, at doses of 0.1, 1, or 10% and traditional tritiated thymidine ((3)HTdR) incorporation and gene expression responses were monitored in the auricular lymph nodes. Additional mice dosed with either vehicle or 10% TMA and sacrificed on day 4 or 10, were also included to examine temporal effects on gene expression. Analysis of (3)HTdR incorporation revealed TMA-induced stimulation indices of 2.8, 22.9, and 61.0 relative to vehicle with an EC(3) of 0.11%. Examination of the dose-response gene expression responses identified 9, 833, and 2122 differentially expressed genes relative to vehicle for the 0.1, 1, and 10% TMA dose groups, respectively. Calculation of EC(3) values for differentially expressed genes did not identify a response that was more sensitive than the (3)HTdR value, although a number of genes displayed comparable sensitivity. Examination of temporal responses revealed 1760, 1870, and 953 differentially expressed genes at the 4-, 6-, and 10-day time points respectively. Functional analysis revealed many responses displayed dose- and time-specific induction patterns within the functional categories of cellular proliferation and immune response, including numerous immunoglobin genes which were highly induced at the day 10 time point. Overall, these experiments have systematically illustrated the potential utility of genomic endpoints to enhance the LLNA and support further exploration of this approach through examination of a more diverse array of chemicals.

The fate of chemoresistance in triple negative breast cancer (TNBC)

PubMed Central

O’Reilly, Elma A.; Gubbins, Luke; Sharma, Shiva; Tully, Riona; Guang, Matthew Ho Zhing; Weiner-Gorzel, Karolina; McCaffrey, John; Harrison, Michele; Furlong, Fiona; Kell, Malcolm; McCann, Amanda

2015-01-01

Background Treatment options for women presenting with triple negative breast cancer (TNBC) are limited due to the lack of a therapeutic target and as a result, are managed with standard chemotherapy such as paclitaxel (Taxol®). Following chemotherapy, the ideal tumour response is apoptotic cell death. Post-chemotherapy, cells can maintain viability by undergoing viable cellular responses such as cellular senescence, generating secretomes which can directly enhance the malignant phenotype. Scope of Review How tumour cells retain viability in response to chemotherapeutic engagement is discussed. In addition we discuss the implications of this retained tumour cell viability in the context of the development of recurrent and metastatic TNBC disease. Current adjuvant and neo-adjuvant treatments available and the novel potential therapies that are being researched are also reviewed. Major conclusions Cellular senescence and cytoprotective autophagy are potential mechanisms of chemoresistance in TNBC. These two non-apoptotic outcomes in response to chemotherapy are inextricably linked and are neglected outcomes of investigation in the chemotherapeutic arena. Cellular fate assessments may therefore have the potential to predict TNBC patient outcome. General Significance Focusing on the fact that cancer cells can bypass the desired cellular apoptotic response to chemotherapy through cellular senescence and cytoprotective autophagy will highlight the importance of targeting non-apoptotic survival pathways to enhance chemotherapeutic efficacy. PMID:26676166

Dopamine neurons modulate pheromone responses in Drosophila courtship learning.

PubMed

Keleman, Krystyna; Vrontou, Eleftheria; Krüttner, Sebastian; Yu, Jai Y; Kurtovic-Kozaric, Amina; Dickson, Barry J

2012-09-06

Learning through trial-and-error interactions allows animals to adapt innate behavioural ‘rules of thumb’ to the local environment, improving their prospects for survival and reproduction. Naive Drosophila melanogaster males, for example, court both virgin and mated females, but learn through experience to selectively suppress futile courtship towards females that have already mated. Here we show that courtship learning reflects an enhanced response to the male pheromone cis-vaccenyl acetate (cVA), which is deposited on females during mating and thus distinguishes mated females from virgins. Dissociation experiments suggest a simple learning rule in which unsuccessful courtship enhances sensitivity to cVA. The learning experience can be mimicked by artificial activation of dopaminergic neurons, and we identify a specific class of dopaminergic neuron that is critical for courtship learning. These neurons provide input to the mushroom body (MB) γ lobe, and the DopR1 dopamine receptor is required in MBγ neurons for both natural and artificial courtship learning. Our work thus reveals critical behavioural, cellular and molecular components of the learning rule by which Drosophila adjusts its innate mating strategy according to experience.

A novel type of cellular senescence that can be enhanced in mouse models and human tumor xenografts to suppress prostate tumorigenesis

PubMed Central

Alimonti, Andrea; Nardella, Caterina; Chen, Zhenbang; Clohessy, John G.; Carracedo, Arkaitz; Trotman, Lloyd C.; Cheng, Ke; Varmeh, Shohreh; Kozma, Sara C.; Thomas, George; Rosivatz, Erika; Woscholski, Rudiger; Cognetti, Francesco; Scher, Howard I.; Pandolfi, Pier Paolo

2010-01-01

Irreversible cell growth arrest, a process termed cellular senescence, is emerging as an intrinsic tumor suppressive mechanism. Oncogene-induced senescence is thought to be invariably preceded by hyperproliferation, aberrant replication, and activation of a DNA damage checkpoint response (DDR), rendering therapeutic enhancement of this process unsuitable for cancer treatment. We previously demonstrated in a mouse model of prostate cancer that inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (Pten) elicits a senescence response that opposes tumorigenesis. Here, we show that Pten-loss–induced cellular senescence (PICS) represents a senescence response that is distinct from oncogene-induced senescence and can be targeted for cancer therapy. Using mouse embryonic fibroblasts, we determined that PICS occurs rapidly after Pten inactivation, in the absence of cellular proliferation and DDR. Further, we found that PICS is associated with enhanced p53 translation. Consistent with these data, we showed that in mice p53-stabilizing drugs potentiated PICS and its tumor suppressive potential. Importantly, we demonstrated that pharmacological inhibition of PTEN drives senescence and inhibits tumorigenesis in vivo in a human xenograft model of prostate cancer. Taken together, our data identify a type of cellular senescence that can be triggered in nonproliferating cells in the absence of DNA damage, which we believe will be useful for developing a “pro-senescence” approach for cancer prevention and therapy. PMID:20197621

Enhancing radiotherapy with cyclooxygenase-2 enzyme inhibitors: a rational advance?

PubMed

Choy, Hak; Milas, Luka

2003-10-01

Results of preclinical studies suggesting that the efficacy of molecular therapies is enhanced when they are combined with radiation have generated a surge of clinical trials combining these modalities. We reviewed the literature to identify the rationale and experimental foundation supporting the use of cyclooxygenase-2 (COX-2) inhibitors with standard radiotherapy regimens in current clinical trials. Radiation affects the ability of cells to divide and proliferate and induces the expression of genes involved in signaling pathways that promote cell survival or trigger cell death. Future advances in radiotherapy will hinge on understanding mechanisms by which radiation-induced transcription of genes governs cell death and survival, the selective control of this process, and the optimal approaches to combining this knowledge with existing therapeutic modalities. COX-2 is expressed in all stages of cancer, and in several cancers its overexpression is associated with poor prognosis. Evidence from clinical and preclinical studies indicates that COX-2-derived prostaglandins participate in carcinogenesis, inflammation, immune response suppression, apoptosis inhibition, angiogenesis, and tumor cell invasion and metastasis. Clinical trial results have demonstrated that selective inhibition of COX-2 can alter the development and the progression of cancer. In animal models, selective inhibition of COX-2 activity is associated with the enhanced radiation sensitivity of tumors without appreciably increasing the effects of radiation on normal tissue, and preclinical evidence suggests that the principal mechanism of radiation potentiation through selective COX-2 inhibition is the direct increase in cellular radiation sensitivity and the direct inhibition of tumor neovascularization. Results of current early-phase studies of non-small-cell lung, esophageal, cervical, and brain cancers will determine whether therapies that combine COX-2 inhibitors and radiation will enter randomized clinical trials.

Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis.

PubMed

Rachlin, Kenneth; Moore, Dan H; Yount, Garret

2013-11-01

The development of nontoxic agents that can selectively enhance the cytotoxicity of chemotherapy is an important aim in oncology. This study evaluates the ability of infrasound exposure to sensitize glioblastoma cells to cisplatin-induced apoptosis. The infrasound was delivered using a device designed to replicate the unique infrasound emissions measured during external Qigong treatments. Human glioblastoma cell lines harboring wild-type p53 (U87) or mutant p53 (U251, SF210, and SF188) were treated in culture with cisplatin, infrasound emissions, or the combination of the 2 agents. Induction of apoptosis was quantified after 24 hours by flow cytometry following annexin V/propidium iodide staining. Infrasound emissions alone, delivered at moderate levels (~10 mPa) with dynamic frequency content (7-13 Hz), did not induce apoptosis, yet combining infrasound with cisplatin augmented the induction of apoptosis by cisplatin in all the 4 cell lines (P < .05). Increased cellular uptake of the fluorophore calcein associated with infrasound exposure was quantified by fluorescence microscopy as well as flow cytometry, demonstrating increased cell membrane permeability. The 4 cell lines differed in the degree to which infrasound exposure increased calcein uptake, and these differences were predictive of the extent to which infrasound enhanced cisplatin-induced apoptosis. When exposed to specific frequencies, membrane permeabilization also appeared to be differentially responsive for each cell line, suggesting the potential for selective targeting of tissue types using isolated infrasonic frequencies. Additionally, the pressure amplitudes used in this study were several orders of magnitude less than those used in similar studies involving ultrasound and shock waves. The results of this study provide support for using infrasound to enhance the chemotherapeutic effects of cisplatin in a clinical setting.

Protons Sensitize Epithelial Cells to Mesenchymal Transition

PubMed Central

Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M.; Pluth, Janice M.; Cucinotta, Francis A.

2012-01-01

Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1. PMID:22844446

Caffeine depression of spontaneous activity in rabbit sino-atrial node cells.

PubMed

Satoh, H

1993-05-01

1. Effects of caffeine on the action potentials and the membrane currents in spontaneously beating rabbit sino-atrial (SA) node cells were examined using a two-microelectrode technique. 2. Cumulative administrations of caffeine (1-10 mM) caused a negative chronotropic effect in a concentration-dependent manner, which was not modified by atropine (0.1 microM). At 10 mM, caffeine increased the amplitude and prolonged the duration of action potentials significantly; the other parameters were unaffected. 3. In 3 of 16 preparations, caffeine (5 mM) elicited arrhythmia. At high Ca2+ (8.1 mM), caffeine (5 mM) increased the incidence of arrhythmia. 4. Caffeine (0.5-10 mM) enhanced the slow inward current, but at 10 mM decreased the enhanced peak current by 5 mM. The hyperpolarization-activated inward current was also enhanced by caffeine, but 10 mM caffeine decreased the current peak as compared with that at 5 mM. In addition, caffeine inhibited the delayed rectifying outward current in a concentration-dependent manner, accompanied by a depressed activation curve without any shift in the half-maximum activation voltage. 5. Caffeine elevated the cytoplasmic Ca2+ level in the SA node cells loaded with Ca(2+)-sensitive fluorescent dye (fura-2). 6. These results suggest that caffeine enhances and/or inhibits the ionic currents and elicits arrhythmia due to the induction of cellular calcium overload.

Imaging enhancement of malignancy by cyclophosphamide: surprising chemotherapy opposite effects

NASA Astrophysics Data System (ADS)

Yamauchi, Kensuke; Yang, Meng; Hayashi, Katsuhiro; Jiang, Ping; Xu, Mingxu; Yamamoto, Norio; Tsuchiya, Hiroyuki; Tomita, Katsuro; Moossa, A. R.; Bouvet, Michael; Hoffman, Robert M.

2008-02-01

Although side effects of cancer chemotherapy are well known, "opposite effects" of chemotherapy which enhance the malignancy of the treated cancer are not well understood. We have observed a number of steps of malignancy that are enhanced by chemotherapy pre-treatment of mice before transplantation of human tumor cells. The induction of intravascular proliferation, extravasation, and colony formation by cancer cells, critical steps of metastasis was enhanced by pretreatment of host mice with the commonly-used chemotherapy drug cyclophosphamide. Cyclophosphamide appears to interfere with a host process that inhibits intravascular proliferation, extravasation, and extravascular colony formation by at least some tumor cells. Cyclophosphamide does not directly affect the cancer cells since cyclophosphamide has been cleared by the time the cancer cells were injected. Without cyclophosphamide pretreatment, human colon cancer cells died quickly after injection in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the cancer cells occurred within 6 hours. The number of apoptotic cells rapidly increased within the portal vein within 12 hours of injection. However, when the host mice were pretreated with cyclophosphamide, the cancer cells survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the cancer cells. This review describes an important unexpected "opposite effects" of chemotherapy that enhances critical steps in malignancy rather than inhibiting them, suggesting that certain current approaches to cancer chemotherapy should be modified.

Nuclear delivery of a therapeutic peptide by long circulating pH-sensitive liposomes: benefits over classical vesicles.

PubMed

Ducat, E; Deprez, J; Gillet, A; Noël, A; Evrard, B; Peulen, O; Piel, G

2011-11-28

The purpose of this study is to propose a suitable vector combining increased circulation lifetime and intracellular delivery capacities for a therapeutic peptide. Long circulating classical liposomes [SPC:CHOL:PEG-750-DSPE (47:47:6 molar% ratio)] or pH-sensitive stealth liposomes [DOPE:CHEMS:CHOL:PEG(750)-DSPE (43:21:30:6 molar% ratio)] were used to deliver a therapeutic peptide to its nuclear site of action. The benefit of using stealth pH-sensitive liposomes was investigated and formulations were compared to classical liposomes in terms of size, shape, charge, encapsulation efficiency, stability and, most importantly, in terms of cellular uptake. Confocal microscopy and flow cytometry were used to evaluate the intracellular fate of liposomes themselves and of their hydrophilic encapsulated material. Cellular uptake of peptide-loaded liposomes was also investigated in three cell lines: Hs578t human epithelial cells from breast carcinoma, MDA-MB-231 human breast carcinoma cells and WI-26 human diploid lung fibroblast cells. The difference between formulations in terms of peptide delivery from the endosome to the cytoplasm and even to the nucleus was investigated as a function of time. Characterization studies showed that both formulations possess acceptable size, shape and encapsulation efficiency but cellular uptake studies showed the important benefit of the pH-sensitive formulation over the classical one, in spite of liposome PEGylation. Indeed, stealth pH-sensitive liposomes were able to deliver hydrophilic materials strongly to the cytoplasm. Most importantly, when encapsulated in pH-sensitive stealth liposomes, the peptide was able to reach the nucleus of tumorigenic and non tumorigenic breast cancer cells. Copyright © 2011 Elsevier B.V. All rights reserved.

Feedback, Mass Conservation and Reaction Kinetics Impact the Robustness of Cellular Oscillations

PubMed Central

Baum, Katharina; Kofahl, Bente; Steuer, Ralf; Wolf, Jana

2016-01-01

Oscillations occur in a wide variety of cellular processes, for example in calcium and p53 signaling responses, in metabolic pathways or within gene-regulatory networks, e.g. the circadian system. Since it is of central importance to understand the influence of perturbations on the dynamics of these systems a number of experimental and theoretical studies have examined their robustness. The period of circadian oscillations has been found to be very robust and to provide reliable timing. For intracellular calcium oscillations the period has been shown to be very sensitive and to allow for frequency-encoded signaling. We here apply a comprehensive computational approach to study the robustness of period and amplitude of oscillatory systems. We employ different prototype oscillator models and a large number of parameter sets obtained by random sampling. This framework is used to examine the effect of three design principles on the sensitivities towards perturbations of the kinetic parameters. We find that a prototype oscillator with negative feedback has lower period sensitivities than a prototype oscillator relying on positive feedback, but on average higher amplitude sensitivities. For both oscillator types, the use of Michaelis-Menten instead of mass action kinetics in all degradation and conversion reactions leads to an increase in period as well as amplitude sensitivities. We observe moderate changes in sensitivities if replacing mass conversion reactions by purely regulatory reactions. These insights are validated for a set of established models of various cellular rhythms. Overall, our work highlights the importance of reaction kinetics and feedback type for the variability of period and amplitude and therefore for the establishment of predictive models. PMID:28027301

Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

NASA Astrophysics Data System (ADS)

Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

2006-01-01

Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions.

Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

PubMed Central

Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

2008-01-01

Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions. PMID:19081771

Luminescent single-walled carbon nanotube-sensitized europium nanoprobes for cellular imaging

PubMed Central

Avti, Pramod K; Sitharaman, Balaji

2012-01-01

Lanthanoid-based optical probes with excitation wavelengths in the ultra-violet (UV) range (300–325 nm) have been widely developed as imaging probes. Efficient cellular imaging requires that lanthanoid optical probes be excited at visible wavelengths, to avoid UV damage to cells. The efficacy of europium-catalyzed single-walled carbon nanotubes (Eu-SWCNTs), as visible nanoprobes for cellular imaging, is reported in this study. Confocal fluorescence microscopy images of breast cancer cells (SK-BR-3 and MCF-7) and normal cells (NIH 3T3), treated with Eu-SWCNT at 0.2 μg/mL concentration, showed bright red luminescence after excitation at 365 nm and 458 nm wavelengths. Cell viability analysis showed no cytotoxic effects after the incubation of cells with Eu-SWCNTs at this concentration. Eu-SWCNT uptake is via the endocytosis mechanism. Labeling efficiency, defined as the percentage of incubated cells that uptake Eu-SWCNT, was 95%–100% for all cell types. The average cellular uptake concentration was 6.68 ng Eu per cell. Intracellular localization was further corroborated by transmission electron microscopy and Raman microscopy. The results indicate that Eu-SWCNT shows potential as a novel cellular imaging probe, wherein SWCNT sensitizes Eu3+ ions to allow excitation at visible wavelengths, and stable time-resolved red emission. The ability to functionalize biomolecules on the exterior surface of Eu-SWCNT makes it an excellent candidate for targeted cellular imaging. PMID:22619533

Bovine Foamy Virus Transactivator BTas Interacts with Cellular RelB To Enhance Viral Transcription▿

PubMed Central

Wang, Jian; Tan, Juan; Guo, Hongyan; Zhang, Qicheng; Jia, Rui; Xu, Xuan; Geng, Yunqi; Qiao, Wentao

2010-01-01

Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor κB (NF-κB) pathway through the action of its transactivator BTas to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question, we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced long terminal repeat [LTR] transactivation) and that this process requires both the localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. The knockdown of the cellular endogenous RelB protein using small interfering RNA (siRNA) significantly attenuated BTas-induced LTR transcription. The results of chromatin immunoprecipitation (ChIP) analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages the RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection upregulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive-feedback circuit in which BFV utilizes the host's NF-κB pathway through the RelB protein for efficient viral transcription. PMID:20844054

Bovine foamy virus transactivator BTas interacts with cellular RelB to enhance viral transcription.

PubMed

Wang, Jian; Tan, Juan; Guo, Hongyan; Zhang, Qicheng; Jia, Rui; Xu, Xuan; Geng, Yunqi; Qiao, Wentao

2010-11-01

Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor κB (NF-κB) pathway through the action of its transactivator BTas to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question, we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced long terminal repeat [LTR] transactivation) and that this process requires both the localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. The knockdown of the cellular endogenous RelB protein using small interfering RNA (siRNA) significantly attenuated BTas-induced LTR transcription. The results of chromatin immunoprecipitation (ChIP) analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages the RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection upregulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive-feedback circuit in which BFV utilizes the host's NF-κB pathway through the RelB protein for efficient viral transcription.

Influence of polyunsaturated fatty acid supplementation and membrane fluidity on ozone and nitrogen dioxide sensitivity of rat alveolar macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rietjens, I.M.; van Tilburg, C.A.; Coenen, T.M.

1987-01-01

The phospholipid polyunsaturated fatty acid (PUFA) content and the membrane fluidity of rat alveolar macrophages were modified dose-dependently and in different ways. This was done to study the importance of both membrane characteristics for the cellular sensitivity toward ozone and nitrogen dioxide. Cells preincubated with arachidonic acid (20:4) complexed to bovine serum albumin (BSA) demonstrated an increased in vitro sensitivity versus ozone and nitrogen dioxide. The phenomenon was only observed at the highest 20:4 concentrations tested, whereas the membrane fluidity of the 20:4-treated cells already showed a maximum increase at lower preincubation concentrations. Hence it could be concluded that themore » increased ozone and nitrogen dioxide sensitivity of PUFA-enriched cells is not caused by their increased membrane fluidity, resulting in an increased accessibility of sensitive cellular fatty acid moieties or amino acid residues. This conclusion receives further support from other observations. These results strongly support the involvement of lipid oxidation in the mechanism(s) of toxic action of both ozone and nitrogen dioxide in an intact cell system.« less

On the topological sensitivity of cellular automata

NASA Astrophysics Data System (ADS)

Baetens, Jan M.; De Baets, Bernard

2011-06-01

Ever since the conceptualization of cellular automata (CA), much attention has been paid to the dynamical properties of these discrete dynamical systems, and, more in particular, to their sensitivity to the initial condition from which they are evolved. Yet, the sensitivity of CA to the topology upon which they are based has received only minor attention, such that a clear insight in this dependence is still lacking and, furthermore, a quantification of this so-called topological sensitivity has not yet been proposed. The lack of attention for this issue is rather surprising since CA are spatially explicit, which means that their dynamics is directly affected by their topology. To overcome these shortcomings, we propose topological Lyapunov exponents that measure the divergence of two close trajectories in phase space originating from a topological perturbation, and we relate them to a measure grasping the sensitivity of CA to their topology that relies on the concept of topological derivatives, which is introduced in this paper. The validity of the proposed methodology is illustrated for the 256 elementary CA and for a family of two-state irregular totalistic CA.

Target-specific cellular uptake of PLGA nanoparticles coated with poly(L-lysine)-poly(ethylene glycol)-folate conjugate.

PubMed

Kim, Sun Hwa; Jeong, Ji Hoon; Chun, Ki Woo; Park, Tae Gwan

2005-09-13

Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with anionic surface charge were surface coated with cationic di-block copolymer, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL) conjugate, for enhancing their site-specific intracellular delivery against folate receptor overexpressing cancer cells. The PLGA nanoparticles coated with the conjugate were characterized in terms of size, surface charge, and change in surface composition by XPS. By employing the flow cytometry method and confocal image analysis, the extent of cellular uptake was comparatively evaluated under various conditions. PLL-PEG-FOL coated PLGA nanoparticles demonstrated far greater extent of cellular uptake to KB cells, suggesting that they were mainly taken up by folate receptor-mediated endocytosis. The enhanced cellular uptake was also observed even in the presence of serum proteins, possibly due to the densely seeded PEG chains. The PLL-PEG-FOL coated PLGA nanoparticles could be potentially applied for cancer cell targeted delivery of various therapeutic agents.

[Enhanced ε-poly-L-lysine production by improving cellular activity during fermentation].

PubMed

Liu, Shengrong; Wu, Qingping; Zhang, Jumei; Yang, Xiaojuan; Cai, Shuzhen

2015-06-04

To assess the effect of cellular activity on ε-poly-1-lysine (ε-PL) biosynthesis and thereby to rationally improve the production, we studied the cellular activity, ε-PL formation and other parameters cross flask fermentation by Streptomyces ahygroscopicus. Laser scanning confocal microscopy and a colorimetric method were used to determine cellular activity using BacLight Live/Dead and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) as viable stains. To enhance the activity of the cells in the ε-PL production period, yeast extract was added. During ε-PL submerged fermentation in flasks, most cells were active in the growth period (0 - 16 h); cells had metabolic activity in the growth and earlier ε-PL production periods between 0 and 30 h fermentation. Almost no activity was detected after 48 h fermentation when no ε-PL was produced. The improved fermentation achieved 2. 24 g/L ε-PL from 1.04 g/L. Biosynthesis of ε-PL can be boosted by up-regulating cell activity in its production phase.

Enhanced transfection by antioxidative polymeric gene carrier that reduces polyplex-mediated cellular oxidative stress.

PubMed

Lee, Min Sang; Kim, Nak Won; Lee, Kyuri; Kim, Hongtae; Jeong, Ji Hoon

2013-06-01

To test the hypothesis in which polyplex-induced oxidative stress may affect overall transfection efficiency, an antioxidative transfection system minimizing cellular oxidative stress was designed for enhanced transfection. An amphiphilic copolymer (PEI-PLGA) was synthesized and used as a micelle-type gene carrier containing hydrophobic antioxidant, α-tocopherol. Cellular oxidative stress and the change of mitochondrial membrane potential after transfection was measured by using a fluorescent probe (H₂DCFDA) and lipophilic cationic probe (JC-1), respectively. Transfection efficiency was determined by measuring a reporter gene (luciferase) expression level. The initial transfection study with conventional PEI/plasmid DNA polyplex showed significant generation of reactive oxygen species (ROS). The PEI-PLGA copolymer successfully carried out the simultaneous delivery of α-tocopherol and plasmid DNA (PEI-PLGA/Toco/pDNA polyplex) into cells, resulting in a significant reduction in cellular ROS generation after transfection and helped to maintain the mitochondrial membrane potential (ΔΨ). In addition, the transfection efficiency was dramatically increased using the antioxidative transfection system. This work showed that oxidative stress would be one of the important factors that should be considered in designing non-viral gene carriers and suggested a possible way to reduce the carrier-mediated oxidative stress, which consequently leads to enhanced transfection.

Knockdown of cytosolic NADP(+) -dependent isocitrate dehydrogenase enhances MPP(+) -induced oxidative injury in PC12 cells.

PubMed

Yang, Eun Sun; Park, Jeen-Woo

2011-05-01

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridium ion (MPP(+)) have been shown to induce Parkinson's disease-like symptoms as well as neurotoxicity in humans and animal species. Recently, we reported that maintenance of redox balance and cellular defense against oxidative damage are primary functions of the novel antioxidant enzyme cytosolic NADP(+) -dependent isocitrate dehydrogenase (IDPc). In this study, we examined the role of IDPc in cellular defense against MPP(+) -induced oxidative injury using PC12 cells transfected with IDPc small interfering RNA (siRNA). Our results demonstrate that MPP(+) -mediated disruption of cellular redox status, oxidative damage to cells, and apoptotic cell death were significantly enhanced by knockdown of IDPc.

MAPK/JNK1 activation protects cells against cadmium-induced autophagic cell death via differential regulation of catalase and heme oxygenase-1 in oral cancer cells.

PubMed

So, Keum-Young; Kim, Sang-Hun; Jung, Ki-Tae; Lee, Hyun-Young; Oh, Seon-Hee

2017-10-01

Antioxidant enzymes are related to oral diseases. We investigated the roles of heme oxygenase-1 (HO-1) and catalase in cadmium (Cd)-induced oxidative stress and the underlying molecular mechanism in oral cancer cells. Exposing YD8 cells to Cd reduced the expression levels of catalase and superoxide dismutase 1/2 and induced the expression of HO-1 as well as autophagy and apoptosis, which were reversed by N-acetyl-l-cysteine (NAC). Cd-exposed YD10B cells exhibited milder effects than YD8 cells, indicating that Cd sensitivity is associated with antioxidant enzymes and autophagy. Autophagy inhibition via pharmacologic and genetic modulations enhanced Cd-induced HO-1 expression, caspase-3 cleavage, and the production of reactive oxygen species (ROS). Ho-1 knockdown increased autophagy and apoptosis. Hemin treatment partially suppressed Cd-induced ROS production and apoptosis, but enhanced autophagy and CHOP expression, indicating that autophagy induction is associated with cellular stress. Catalase inhibition by pharmacological and genetic modulations increased Cd-induced ROS production, autophagy, and apoptosis, but suppressed HO-1, indicating that catalase is required for HO-1 induction. p38 inhibition upregulated Cd-induced phospho-JNK and catalase, but suppressed HO-1, autophagy, apoptosis. JNK suppression exhibited contrary results, enhancing the expression of phospho-p38. Co-suppression of p38 and JNK1 failed to upregulate catalase and procaspase-3, which were upregulated by JNK1 overexpression. Overall, the balance between the responses of p38 and JNK activation to Cd appears to have an important role in maintaining cellular homeostasis via the regulation of antioxidant enzymes and autophagy induction. In addition, the upregulation of catalase by JNK1 activation can play a critical role in cell protection against Cd-induced oxidative stress. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineering cellular fibers for musculoskeletal soft tissues using directed self-assembly.

PubMed

Schiele, Nathan R; Koppes, Ryan A; Chrisey, Douglas B; Corr, David T

2013-05-01

Engineering strategies guided by developmental biology may enhance and accelerate in vitro tissue formation for tissue engineering and regenerative medicine applications. In this study, we looked toward embryonic tendon development as a model system to guide our soft tissue engineering approach. To direct cellular self-assembly, we utilized laser micromachined, differentially adherent growth channels lined with fibronectin. The micromachined growth channels directed human dermal fibroblast cells to form single cellular fibers, without the need for a provisional three-dimensional extracellular matrix or scaffold to establish a fiber structure. Therefore, the resulting tissue structure and mechanical characteristics were determined solely by the cells. Due to the self-assembly nature of this approach, the growing fibers exhibit some key aspects of embryonic tendon development, such as high cellularity, the rapid formation (within 24 h) of a highly organized and aligned cellular structure, and the expression of cadherin-11 (indicating direct cell-to-cell adhesions). To provide a dynamic mechanical environment, we have also developed and characterized a method to apply precise cyclic tensile strain to the cellular fibers as they develop. After an initial period of cellular fiber formation (24 h postseeding), cyclic strain was applied for 48 h, in 8-h intervals, with tensile strain increasing from 0.7% to 1.0%, and at a frequency of 0.5 Hz. Dynamic loading dramatically increased cellular fiber mechanical properties with a nearly twofold increase in both the linear region stiffness and maximum load at failure, thereby demonstrating a mechanism for enhancing cellular fiber formation and mechanical properties. Tissue engineering strategies, designed to capture key aspects of embryonic development, may provide unique insight into accelerated maturation of engineered replacement tissue, and offer significant advances for regenerative medicine applications in tendon, ligament, and other fibrous soft tissues.

Proteomic identification of early salicylate- and flg22-responsive redox-sensitive proteins in Arabidopsis

PubMed Central

Liu, Pei; Zhang, Huoming; Yu, Boying; Xiong, Liming; Xia, Yiji

2015-01-01

Accumulation of reactive oxygen species (ROS) is one of the early defense responses against pathogen infection in plants. The mechanism about the initial and direct regulation of the defense signaling pathway by ROS remains elusive. Perturbation of cellular redox homeostasis by ROS is believed to alter functions of redox-sensitive proteins through their oxidative modifications. Here we report an OxiTRAQ-based proteomic study in identifying proteins whose cysteines underwent oxidative modifications in Arabidopsis cells during the early response to salicylate or flg22, two defense pathway elicitors that are known to disturb cellular redox homeostasis. Among the salicylate- and/or flg22-responsive redox-sensitive proteins are those involved in transcriptional regulation, chromatin remodeling, RNA processing, post-translational modifications, and nucleocytoplasmic shuttling. The identification of the salicylate-/flg22-responsive redox-sensitive proteins provides a foundation from which further study can be conducted toward understanding biological significance of their oxidative modifications during the plant defense response. PMID:25720653

Selenium Nanoparticles Induce the Chemo-Sensitivity of Fluorouracil Nanoparticles in Breast and Colon Cancer Cells.

PubMed

Abd-Rabou, Ahmed A; Shalby, Aziza B; Ahmed, Hanaa H

2018-05-11

Drug resistance is a major challenge of breast and colon cancer therapies leading to treatment failure. The main objective of the current study is to investigate whether selenium nanoparticles (nano-Se) can induce the chemo-sensitivity of 5-fluorouracil (FU)-encapsulated poly (D, L-lactide-co-glycolide) nanoparticles (nano-FU) in breast and colon cancer cell lines. Nano-Se and nano-FU were synthesized and characterized, then applied individually or in combination upon MCF7, MDA-MB-231, HCT 116, and Caco-2 cancerous cell lines. Cytotoxicity, cellular glucose uptake, and apoptosis, as well as malondialdehyde (MDA), nitric oxide (NO), and zinc (Zn) levels, were investigated upon the different treatments. We have resulted that nano-FU induced cell death in MCF7 and Caco-2 more effectively than MDA-MB-231 and HCT 116 cell lines. Moreover, nano-FU plus nano-Se potentiate MCF7 and Caco-2 chemo-sensitivity were higher than MDA-MB-231 and HCT 116 cancerous cell lines. It is relevant to note that Se and FU nano-formulations inhibited cancer cell bioenergetics via glucose uptake slight blockage. Furthermore, nano-FU increased the levels of NO and MDA in media over cancer cells, while their combinations with nano-Se rebalance the redox status with Zn increment. We noticed that MCF7 cell line is sensitive, while MDA-MB-231 cell line is resistant to Se and nano-Se. This novel approach could be of great potential to enhance the chemo-sensitivity in breast and colon cancer cells.

3D printed high performance strain sensors for high temperature applications

NASA Astrophysics Data System (ADS)

Rahman, Md Taibur; Moser, Russell; Zbib, Hussein M.; Ramana, C. V.; Panat, Rahul

2018-01-01

Realization of high temperature physical measurement sensors, which are needed in many of the current and emerging technologies, is challenging due to the degradation of their electrical stability by drift currents, material oxidation, thermal strain, and creep. In this paper, for the first time, we demonstrate that 3D printed sensors show a metamaterial-like behavior, resulting in superior performance such as high sensitivity, low thermal strain, and enhanced thermal stability. The sensors were fabricated using silver (Ag) nanoparticles (NPs), using an advanced Aerosol Jet based additive printing method followed by thermal sintering. The sensors were tested under cyclic strain up to a temperature of 500 °C and showed a gauge factor of 3.15 ± 0.086, which is about 57% higher than that of those available commercially. The sensor thermal strain was also an order of magnitude lower than that of commercial gages for operation up to a temperature of 500 °C. An analytical model was developed to account for the enhanced performance of such printed sensors based on enhanced lateral contraction of the NP films due to the porosity, a behavior akin to cellular metamaterials. The results demonstrate the potential of 3D printing technology as a pathway to realize highly stable and high-performance sensors for high temperature applications.

Long-lasting reductions of ethanol drinking, enhanced ethanol-induced sedation, and decreased c-fos expression in the Edinger-Westphal nucleus in Wistar rats exposed to the organophosphate chlorpyrifos.

PubMed

Carvajal, Francisca; López-Grancha, Matilde; Navarro, Montserrat; Sánchez-Amate, Maria del Carmen; Cubero, Inmaculada

2007-04-01

Intermittent or continuous exposure to a wide variety of chemically unrelated environmental pollutants might result in the development of multiple chemical intolerance and increased sensitivity to drugs of abuse. Interestingly, clinical evidence suggests that exposure to organophosphates might be linked to increased ethanol sensitivity and reduced voluntary consumption of ethanol-containing beverages in humans. The growing body of clinical and experimental evidence emerging in this new scientific field that bridges environmental health sciences, toxicology, and drug research calls for well-controlled studies aimed to analyze the nature of the neurobiological interactions of drugs and pollutants. Present study specifically evaluated neurobiological and behavioral responses to ethanol in Wistar rats that were previously exposed to the pesticide organophosphate chlorpyrifos (CPF). In agreement with clinical data, animals pretreated with a single injection of CPF showed long-lasting ethanol avoidance that was not secondary to altered gustatory processing or enhancement of the aversive properties of ethanol. Furthermore, CPF pretreatment increased ethanol-induced sedation without altering blood ethanol levels. An immunocytochemical assay revealed reduced c-fos expression in the Edinger-Westphal nucleus following CPF treatment, a critical brain area that has been implicated in ethanol intake and sedation. We hypothesize that CPF might modulate cellular mechanisms (decreased intracellular cAMP signaling, alpha-7-nicotinic receptors, and/or cerebral acetylcholinesterase inhibition) in neuronal pathways critically involved in neurobiological responses to ethanol.

Interfacial Engineering of Hierarchical Transition Metal Oxide Heterostructures for Highly Sensitive Sensing of Hydrogen Peroxide.

PubMed

Zhang, Wen; Fan, Guozheng; Yi, Huan; Jia, Gan; Li, Zhaosheng; Yuan, Chunwei; Bai, Yunfei; Fu, Degang

2018-05-01

Hydrogen peroxide (H 2 O 2 ) is a major messenger molecule in cellular signal transduction. Direct detection of H 2 O 2 in complex environments provides the capability to illuminate its various biological functions. With this in mind, a novel electrochemical approach is here proposed by integrating a series of CoO nanostructures on CuO backbone at electrode interfaces. High-resolution transmission electron microscopy (HRTEM), X-ray diffraction, and X-ray photoelectron spectroscopy demonstrate successful formation of core-shell CuO-CoO hetero-nanostructures. Theoretical calculations further confirm energy-favorable adsorption of H 2 O 2 on surface sites of CuO-CoO heterostructures. Contributing to the efficient electron transfer path and enhanced capture of H 2 O 2 in the unique leaf-like CuO-CoO hierarchical 3D interface, an optimal biosensor-based CuO-CoO-2.5 h electrode exhibits an ultrahigh sensitivity (6349 µA m m -1 cm -2 ), excellent selectivity, and a wide detection range for H 2 O 2 , and is capable of monitoring endogenous H 2 O 2 derived from human lung carcinoma cells A549. The synergistic effects for enhanced H 2 O 2 adsorption in integrated CuO-CoO nanostructures and performance of the sensor suggest a potential for exploring pathological and physiological roles of reactive oxygen species like H 2 O 2 in biological systems. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

PubMed

Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

2016-03-04

Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Sequential treatment with aurora B inhibitors enhances cisplatin-mediated apoptosis via c-Myc.

PubMed

Ma, Yaxi; Cao, Handi; Lou, Siyue; Shao, Xuejing; Lv, Wen; Qi, Xiaotian; Liu, Yujia; Ying, Meidan; He, Qiaojun; Yang, Xiaochun

2015-04-01

Platinum compound such as cisplatin is the first-line chemotherapy of choice in most patients with ovarian carcinoma. However, patients with inherent or acquired cisplatin resistance often experience relapse. Therefore, novel therapies are urgently required to treat drug-resistant ovarian carcinoma. Here, we showed that compared to the non-functional traditional simultaneous treatment, sequential combination of Aurora B inhibitors followed by cisplatin synergistically enhanced apoptotic response in cisplatin-resistant OVCAR-8 cells. This effect was accompanied by the induction of polyploidy in a c-Myc-dependent manner, as c-Myc knockdown reduced the efficacy of the combination by suppressing the expression of Aurora B and impairing cellular response to Aurora B inhibitor, as indicated by the decreased polyploidy and hyperphosphorylation of histone H1. In c-Myc-deficient SKOV3 cells, c-Myc overexpression restored Aurora B expression, induced polyploidy after inhibition of Aurora B, and sensitized cells to this combination therapy. Thus, our report reveals for the first time that sequential treatment of Aurora B inhibitors and cisplatin is essential to inhibit ovarian carcinoma by inducing polyploidy and downregulating c-Myc and that c-Myc is identified as a predictive biomarker to select cells responsive to chemotherapeutical combinations targeting Aurora B. Collectively, these studies provide novel approaches to overcoming cisplatin chemotherapy resistance in ovarian cancer. Pretreatment of Aurora B inhibitors augment apoptotic effects of cisplatin. The synergy of Aurora B inhibitor with cisplatin is dependent on c-Myc expression. c-Myc-dependent induction of polyploidy sensitizes cells to cisplatin.

Aurora kinase B inhibition reduces the proliferation of metastatic melanoma cells and enhances the response to chemotherapy.

PubMed

Porcelli, Letizia; Guida, Gabriella; Quatrale, Anna E; Cocco, Tiziana; Sidella, Letizia; Maida, Immacolata; Iacobazzi, Rosa M; Ferretta, Anna; Stolfa, Diana A; Strippoli, Sabino; Guida, Stefania; Tommasi, Stefania; Guida, Michele; Azzariti, Amalia

2015-01-27

The poor response to chemotherapy and the brief response to vemurafenib in metastatic melanoma patients, make the identification of new therapeutic approaches an urgent need. Interestingly the increased expression and activity of the Aurora kinase B during melanoma progression suggests it as a promising therapeutic target. The efficacy of the Aurora B kinase inhibitor barasertib-HQPA was evaluated in BRAF mutated cells, sensitive and made resistant to vemurafenib after chronic exposure to the drug, and in BRAF wild type cells. The drug effectiveness has been evaluated as cell growth inhibition, cell cycle progression and cell migration. In addition, cellular effectors of drug resistance and response were investigated. The characterization of the effectors responsible for the resistance to vemurafenib evidenced the increased expression of MITF or the activation of Erk1/2 and p-38 kinases in the newly established cell lines with a phenotype resistant to vemurafenib. The sensitivity of cells to barasertib-HQPA was irrespective of BRAF mutational status. Barasertib-HQPA induced the mitotic catastrophe, ultimately causing apoptosis and necrosis of cells, inhibited cell migration and strongly affected the glycolytic metabolism of cells inducing the release of lactate. In association i) with vemurafenib the gain in effectiveness was found only in BRAF(V600K) cells while ii) with nab-paclitaxel, the combination was more effective than each drug alone in all cells. These findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment.

NanoLuc: A Small Luciferase is Brightening up the Field of Bioluminescence

PubMed Central

Cai, Weibo

2016-01-01

The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a non-ideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a non-biased manner, allowing the audience to adopt their own views of this novel system. PMID:27045664

Suppression of telomere-binding protein TPP1 resulted in telomere dysfunction and enhanced radiation sensitivity in telomerase-negative osteosarcoma cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiang, Weiguang; Department of Oncology, The Third Affiliated Hospital, Soochow University, Changzhou; Wu, Qinqin

Highlights: • Down-regulation of TPP1 shortened telomere length in telomerase-negative cells. • Down-regulation of TPP1 induced cell apoptosis in telomerase-negative cells. • Down-regulation of TPP1 increased radiosensitivity in telomerase-negative cells. - Abstract: Mammalian telomeres are protected by the shelterin complex that contains the six core proteins POT1, TPP1, TIN2, TRF1, TRF2 and RAP1. TPP1, formerly known as TINT1, PTOP, and PIP1, is a key factor that regulates telomerase recruitment and activity. In addition to this, TPP1 is required to mediate the shelterin assembly and stabilize telomere. Previous work has found that TPP1 expression was elevated in radioresistant cells and thatmore » overexpression of TPP1 led to radioresistance and telomere lengthening in telomerase-positive cells. However, the exact effects and mechanism of TPP1 on radiosensitivity are yet to be precisely defined in the ALT cells. Here we report on the phenotypes of the conditional deletion of TPP1 from the human osteosarcoma U2OS cells using ALT pathway to extend the telomeres.TPP1 deletion resulted in telomere shortening, increased apoptosis and radiation sensitivity enhancement. Together, our findings show that TPP1 plays a vital role in telomere maintenance and protection and establish an intimate relationship between TPP1, telomere and cellular response to ionizing radiation, but likely has the specific mechanism yet to be defined.« less

Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer

NASA Astrophysics Data System (ADS)

Kos, Petra; Lächelt, Ulrich; Herrmann, Annika; Mickler, Frauke Martina; Döblinger, Markus; He, Dongsheng; Krhač Levačić, Ana; Morys, Stephan; Bräuchle, Christoph; Wagner, Ernst

2015-03-01

Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06556e

Pulsating Hydrodynamic Instability in a Dynamic Model of Liquid-Propellant Combustion

NASA Technical Reports Server (NTRS)

Margolis, Stephen B.; Sacksteder, Kurt (Technical Monitor)

1999-01-01

Hydrodynamic (Landau) instability in combustion is typically associated with the onset of wrinkling of a flame surface, corresponding to the formation of steady cellular structures as the stability threshold is crossed. In the context of liquid-propellant combustion, such instability has recently been shown to occur for critical values of the pressure sensitivity of the burning rate and the disturbance wavenumber, significantly generalizing previous classical results for this problem that assumed a constant normal burning rate. Additionally, however, a pulsating form of hydrodynamic instability has been shown to occur as well, corresponding to the onset of temporal oscillations in the location of the liquid/gas interface. In the present work, we consider the realistic influence of a nonzero temperature sensitivity in the local burning rate on both types of stability thresholds. It is found that for sufficiently small values of this parameter, there exists a stable range of pressure sensitivities for steady, planar burning such that the classical cellular form of hydrodynamic instability and the more recent pulsating form of hydrodynamic instability can each occur as the corresponding stability threshold is crossed. For larger thermal sensitivities, however, the pulsating stability boundary evolves into a C-shaped curve in the disturbance-wavenumber/ pressure-sensitivity plane, indicating loss of stability to pulsating perturbations for all sufficiently large disturbance wavelengths. It is thus concluded, based on characteristic parameter values, that an equally likely form of hydrodynamic instability in liquid-propellant combustion is of a nonsteady, long-wave nature, distinct from the steady, cellular form originally predicted by Landau.

Pulsating Hydrodynamic Instability and Thermal Coupling in an Extended Landau/Levich Model of Liquid-Propellant Combustion. 1; Inviscid Analysis

NASA Technical Reports Server (NTRS)

Margolis, Stephen B.; Sacksteder, Kurt (Technical Monitor)

1999-01-01

Hydrodynamic (Landau) instability in combustion is typically associated with the onset of wrinkling of a flame surface, corresponding to the formation of steady cellular structures as the stability threshold is crossed. In the context of liquid-propellant combustion, such instability has recently been shown to occur for critical values of the pressure sensitivity of the burning rate and the disturbance wavenumber, significantly generalizing previous classical results for this problem that assumed a constant normal burning rate. Additionally, however, a pulsating form of hydrodynamic instability has been shown to occur as well, corresponding to the onset of temporal oscillations in the location of the liquid/gas interface. In the present work, we consider the realistic influence of a non-zero temperature sensitivity in the local burning rate on both types of stability thresholds. It is found that for sufficiently small values of this parameter, there exists a stable range of pressure sensitivities for steady, planar burning such that the classical cellular form of hydrodynamic instability and the more recent pulsating form of hydrodynamic instability can each occur as the corresponding stability threshold is crossed. For larger thermal sensitivities, however, the pulsating stability boundary evolves into a C-shaped curve in the (disturbance-wavenumber, pressure-sensitivity) plane, indicating loss of stability to pulsating perturbations for all sufficiently large disturbance wavelengths. It is thus concluded, based on characteristic parameter values, that an equally likely form of hydrodynamic instability in liquid-propellant combustion is of a non-steady, long-wave nature, distinct from the steady, cellular form originally predicted by Landau.

Initiation of Autophagy by Photodynamic Therapy

PubMed Central

Kessel, David; Oleinick, Nancy L.

2010-01-01

Photodynamic therapy (PDT) involves the irradiation of photosensitized cells with light. Depending on localization of the photosensitizing agent, the process can induce photodamage to the endoplasmic reticulum (ER), mitochondria, plasma membrane, and/or lysosomes. When ER or mitochondria are targeted, antiapoptotic proteins of the Bcl-2 family are especially sensitive to photodamage. Both apoptosis and autophagy can occur after PDT, autophagy being associated with enhanced survival at low levels of photodamage to some cells. Autophagy can become a cell-death pathway if apoptosis is inhibited or when cells attempt to recycle damaged constituents beyond their capacity for recovery. While techniques associated with characterization of autophagy are generally applicable, PDT introduces additional factors related to unknown sites of photodamage that may alter autophagic pathways. This chapter discusses issues that may arise in assessing autophagy after cellular photodamage. PMID:19216899

A multifunctional nanocarrier based on nanogated mesoporous silica for enhanced tumor-specific uptake and intracellular delivery.

PubMed

Gao, Yaohua; Yang, Cuihong; Liu, Xue; Ma, Rujiang; Kong, Deling; Shi, Linqi

2012-02-01

A multifunctional drug delivery system based on MCM-41-type mesoporous silica nanoparticles is described that behaves as if nanogates were covalently attached to the outlets of the mesopores through a highly acid-sensitive benzoic-imine linker. Tumor-specific uptake and intracellular delivery results from the pH-dependent progressive hydrolysis of the benzoic-imine linkage that starts at tumor extracellular pH = 6.8 and increases with decreasing pH. The cleavage of the benzoic-imine bond leads to the removal of the polypseudorotaxane caps and subsequent release of the payload drugs at tumor sites. At the same time, the carrier surface becomes positively charged, which further facilitates cellular uptake of the nanocarriers, thus offering a tremendous potential for targeted tumor therapy. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

IL-9-producing cells in the development of IgE-mediated food allergy.

PubMed

Shik, Dana; Tomar, Sunil; Lee, Jee-Boong; Chen, Chun-Yu; Smith, Andrew; Wang, Yui-Hsi

2017-01-01

Food allergy is a harmful immune reaction driven by uncontrolled type 2 immune responses. Considerable evidence demonstrates the key roles of mast cells, IgE, and TH2 cytokines in mediating food allergy. However, this evidence provides limited insight into why only some, rather than all, food allergic individuals are prone to develop life-threatening anaphylaxis. Clinical observations suggest that patients sensitized to food through the skin early in life may later develop severe food allergies. Aberrant epidermal thymic stromal lymphopoietin and interleukin (IL) 33 production and genetic predisposition can initiate an allergic immune response mediated by dendritic cells and CD4 + TH2 cells in inflamed skin. After allergic sensitization, intestinal IL-25 and food ingestion enhance concerted interactions between type 2 innate lymphoid cells (ILC2s) and CD4 + TH2 cells, which perpetuate allergic reactions from the skin to the gut. IL-4 and cross-linking of antigen/IgE/FcεR complexes induce emigrated mast cell progenitors to develop into the multi-functional IL-9-producing mucosal mast cells, which produce prodigious amounts of IL-9 and mast cell mediators to drive intestinal mastocytosis in an autocrine loop. ILC2s and TH9 cells may also serve as alternative cellular sources of IL-9 to augment the amplification of intestinal mastocytosis, which is the key cellular checkpoint in developing systemic anaphylaxis. These findings provide a plausible view of how food allergy develops and progresses in a stepwise manner and that atopic signals, dietary allergen ingestion, and inflammatory cues are fundamental in promoting life-threatening anaphylaxis. This information will aid in improving diagnosis and developing more effective therapies for food allergy-triggered anaphylaxis.

IL-9–producing cells in the development of IgE-mediated food allergy

PubMed Central

Shik, Dana; Tomar, Sunil; Lee, Jee-Boong; Chen, Chun-Yu; Smith, Andrew; Wang, Yui-Hsi

2016-01-01

Food allergy is a harmful immune reaction driven by uncontrolled type-2 immune responses. Considerable evidence demonstrates the key roles of mast cells, IgE, and TH2 cytokines in mediating food allergy. However, this evidence provides limited insight into why only some, rather than all, food allergic individuals are prone to develop life-threatening anaphylaxis. Clinical observations suggest that patients sensitized to food through the skin early in life may later develop severe food allergies. Aberrant epidermal thymic stromal lymphopoietin and interleukin (IL) 33 production and genetic predisposition can initiate an allergic immune response mediated by dendritic cells and CD4+TH2 cells in inflamed skin. After allergic sensitization, intestinal IL-25 and food ingestion enhance concerted interactions between type-2 innate lymphoid cells (ILC2s) and CD4+TH2 cells, which perpetuate allergic reactions from skin to the gut. IL-4 and crosslinking of antigen/IgE/FcεR complexes induce emigrated mast cell progenitors to develop into the multi-functional IL-9–producing mucosal mast cells, which produce prodigious amounts of IL-9 and mast cell mediators to drive intestinal mastocytosis in an autocrine loop. ILC2s and TH9 cells may also serve as alternative cellular sources of IL-9 to augment the amplification of intestinal mastocytosis, which is the key cellular checkpoint in developing systemic anaphylaxis. These findings provide a plausible view of how food allergy develops and progresses in a stepwise manner and that atopic signals, dietary allergen ingestion, and inflammatory cues are fundamental in promoting life-threatening anaphylaxis. This information will aid in improving diagnosis and developing more effective therapies for food allergy–triggered anaphylaxis. PMID:27909880

Anthelminthic drug niclosamide sensitizes the responsiveness of cervical cancer cells to paclitaxel via oxidative stress-mediated mTOR inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Liping; Wang, Li; Shen, Haibin

Drug repurposing represents an alternative therapeutic strategy to cancer treatment. The potent anti-cancer activities of a FDA-approved anthelminthic drug niclosamide have been demonstrated in various cancers. However, whether niclosamide is active against cervical cancer is unknown. In this study, we investigated the effects of niclosamide alone and its combination with paclitaxel in cervical cancer in vitro and in vivo. We found that niclosamide significantly inhibited proliferation and induced apoptosis of a panel of cervical cancer cell lines, regardless of their cellular origin and genetic pattern. Niclosamide also inhibited tumor growth in cervical cancer xenograft mouse model. Importantly, niclosamide significantly enhanced the responsivenessmore » of cervical cancer cell to paclitaxel. We further found that niclosamide induced mitochondrial dysfunctions via inhibiting mitochondrial respiration, complex I activity and ATP generation, which led to oxidative stress. ROS scavenge agent N-acetyl-L-cysteine (NAC) completely reversed the effects of niclosamide in increasing cellular ROS, inhibiting proliferation and inducing apoptosis, suggesting that oxidative stress induction is the mechanism of action of niclosamide in cervical cancer cells. In addition, niclosamide significantly inhibited mammalian target of rapamycin (mTOR) signaling pathway in cervical cancer cells and its inhibitory effect on mTOR is modulated by oxidative stress. Our work suggests that niclosamide is a useful addition to the treatment armamentarium for cervical cancer and induction of oxidative stress may be a potential therapeutic strategy in cervical cancer. - Highlights: • Niclosamide is active against cervical cancer cells in vitro and in vivo. • Niclosamide sensitizes cervical cancer cell response to paclitaxel. • Niclosamide induces mitochondrial dysfunction and oxidative damage. • Niclosamide inhibits mTOR signaling in an oxidative stress-dependent manner.« less

Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase.

PubMed

Sinha, Devanjan; Srivastava, Shubhi; D'Silva, Patrick

2016-08-12

Mitochondrial J-proteins play a critical role in governing Hsp70 activity and, hence, are essential for organellar protein translocation and folding. In contrast to yeast, which has a single J-protein Pam18, humans involve two J-proteins, DnaJC15 and DnaJC19, associated with contrasting cellular phenotype, to transport proteins into the mitochondria. Mutation in DnaJC19 results in dilated cardiomyopathy and ataxia syndrome, whereas expression of DnaJC15 regulates the response of cancer cells to chemotherapy. In the present study we have comparatively assessed the biochemical properties of the J-protein paralogs in relation to their association with the import channel. Both DnaJC15 and DnaJC19 formed two distinct subcomplexes with Magmas at the import channel. Knockdown analysis suggested an essential role for Magmas and DnaJC19 in organellar protein translocation and mitochondria biogenesis, whereas DnaJC15 had dispensable supportive function. The J-proteins were found to have equal affinity for Magmas and could stimulate mitochondrial Hsp70 ATPase activity by equivalent levels. Interestingly, we observed that DnaJC15 exhibits bifunctional properties. At the translocation channel, it involves conserved interactions and mechanism to translocate the precursors into mitochondria. In addition to protein transport, DnaJC15 also showed a dual role in yeast where its expression elicited enhanced sensitivity of cells to cisplatin that required the presence of a functional J-domain. The amount of DnaJC15 expressed in the cell was directly proportional to the sensitivity of cells. Our analysis indicates that the differential cellular phenotype displayed by human mitochondrial J-proteins is independent of their activity and association with Magmas at the translocation channel. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Up-regulation of aldolase A and methylglyoxal production in adipocytes.

PubMed

Liu, Jianghai; Desai, Kaushik; Wang, Rui; Wu, Lingyun

2013-04-01

We previously reported that up-regulation of aldolase B, a key enzyme in fructose metabolism, was mainly responsible for vascular methylglyoxal (MG) overproduction under different pathological conditions. Here we investigated whether aldolase A, an enzyme of the glycolytic pathway, also caused MG overproduction in insulin-sensitive adipocytes. The relative contributions of different metabolic pathways or enzymes to MG generation were evaluated in cultured 3T3-L1 adipocytes. Glucose (25 mM) had no effect on aldolase A gene expression, but insulin (100 nM) up-regulated aldolase A mRNA and protein levels in the absence or presence of 25 mM glucose in adipocytes. Treatment with insulin increased levels of basal or glucose (25 mM)-induced MG and glucose 6-phosphate. However, insulin, glucose (25 mM) or their combination had no effect on cellular levels of sorbitol and fructose, but down-regulated gene expression of aldolase B to a similar extent, when compared with the control group. Incubation of 3T3-L1 adipocytes with fructose, acetone, acetol, threonine or glycine (25 mM), with or without insulin did not alter cellular MG levels. The elevated MG levels induced by insulin, glucose (25 mM) or their combination in adipocytes was completely reduced by siRNA knock down of aldolase A or application of 2-deoxy-D-glucose (a non-specific inhibitor of glucose uptake and glycolysis), but not by knock down of aldolase B. Insulin enhanced MG overproduction in insulin-sensitive adipocytes by up-regulating aldolase A, a mechanism that could be involved in the development of insulin resistance and obesity. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Differential sensitivities of cellular XPA and PARP-1 to arsenite inhibition and zinc rescue.

PubMed

Ding, Xiaofeng; Zhou, Xixi; Cooper, Karen L; Huestis, Juliana; Hudson, Laurie G; Liu, Ke Jian

2017-09-15

Arsenite directly binds to the zinc finger domains of the DNA repair protein poly (ADP ribose) polymerase (PARP)-1, and inhibits PARP-1 activity in the base excision repair (BER) pathway. PARP inhibition by arsenite enhances ultraviolet radiation (UVR)-induced DNA damage in keratinocytes, and the increase in DNA damage is reduced by zinc supplementation. However, little is known about the effects of arsenite and zinc on the zinc finger nucleotide excision repair (NER) protein xeroderma pigmentosum group A (XPA). In this study, we investigated the difference in response to arsenite exposure between XPA and PARP-1, and the differential effectiveness of zinc supplementation in restoring protein DNA binding and DNA damage repair. Arsenite targeted both XPA and PARP-1 in human keratinocytes, resulting in zinc loss from each protein and a pronounced decrease in XPA and PARP-1 binding to chromatin as demonstrated by Chip-on-Western assays. Zinc effectively restored DNA binding of PARP-1 and XPA to chromatin when zinc concentrations were equal to those of arsenite. In contrast, zinc was more effective in rescuing arsenite-augmented direct UVR-induced DNA damage than oxidative DNA damage. Taken together, our findings indicate that arsenite interferes with PARP-1 and XPA binding to chromatin, and that zinc supplementation fully restores DNA binding activity to both proteins in the cellular context. Interestingly, rescue of arsenite-inhibited DNA damage repair by supplemental zinc was more sensitive for DNA damage repaired by the XPA-associated NER pathway than for the PARP-1-dependent BER pathway. This study expands our understanding of arsenite's role in DNA repair inhibition and co-carcinogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of alpha 1A- and alpha 1B-adrenoceptor coupling to inositol phosphate formation in rat kidney.

PubMed

Büscher, R; Erdbrügger, W; Philipp, T; Brodde, O E; Michel, M C

1994-12-01

We have compared the coupling mechanisms of rat renal alpha 1A- and alpha 1B-like adrenoceptors to inositol phosphate formation. The experiments were performed in parallel in native renal tissue preparations and in those where alpha 1B-adrenoceptors had been inactivated by treatment with 10 mumol/l chloroethylclonidine for 30 min at 37 degrees C; renal slices were used in most experiments but isolated renal cells were also used in some cases. The Ca2+ chelating agent, EGTA (5 mmol/l), reduced noradrenaline-stimulated inositol phosphate formation in native but enhanced it in chloroethylclonidine-treated renal slices. The inhibitory effect of EGTA was not mimicked by 100 nmol/l nifedipine. Inactivation of 87% of cellular Gi by 16-20 h treatment with 500 ng/ml pertussis toxin did not significantly affect noradrenaline-stimulated inositol phosphate formation in isolated renal cells but abolished the inhibitory effect of chloroethylclonidine. The adenylate cyclase activator, forskolin (20 mumol/l), inhibited noradrenaline-stimulated inositol phosphate formation in native and chloroethylclonidine-treated slices, and the inhibitory effects of chloroethylclonidine treatment and forskolin were additive. We conclude that in rat kidney inositol phosphate formation via alpha 1B-like adrenoceptors may involve the influx of extracellular Ca2+ and a pertussis toxin-sensitive G-protein but is insensitive to inhibition by forskolin. In contrast alpha 1A-like adrenoceptor-mediated inositol phosphate formation does not require the presence of extracellular Ca2+ or of Gi and is sensitive to inhibition by forskolin. In comparison to published data from other model systems we further conclude that the signaling mechanisms of alpha 1-adrenoceptor subtypes may depend on their cellular environment.

Cancerous inhibitor of protein phosphatase 2A determines bortezomib-induced apoptosis in leukemia cells

PubMed Central

Liu, Chun-Yu; Shiau, Chung-Wai; Kuo, Hsin-Yu; Huang, Hsiang-Po; Chen, Ming-Huang; Tzeng, Cheng-Hwai; Chen, Kuen-Feng

2013-01-01

The multiple cellular targets affected by proteasome inhibition implicate a potential role for bortezomib, a first-in-class proteasome inhibitor, in enhancing antitumor activities in hematologic malignancies. Here, we examined the antitumor activity and drug targets of bortezomib in leukemia cells. Human leukemia cell lines were used for in vitro studies. Drug efficacy was evaluated by apoptosis assays and associated molecular events assessed by Western Blot. Gene silencing was performed by small interference RNA. Drug was tested in vivo in xenograft models of human leukemia cell lines and in primary leukemia cells. Clinical samples were assessed by immunohistochemical staining. Bortezomib differentially induced apoptosis in leukemia cells that was independent of its proteasome inhibition. Cancerous inhibitor of protein phosphatase 2A, a cellular inhibitor of protein phosphatase 2A, mediated the apoptotic effect of bortezomib. Bortezomib increased protein phosphatase 2A activity in sensitive leukemia cells (HL-60 and KG-1), but not in resistant cells (MOLT-3 and K562). Bortezomib’s downregulation of cancerous inhibitor of protein phosphatase 2A and phospho-Akt correlated with its drug sensitivity. Furthermore, cancerous inhibitor of protein phosphatase 2A negatively regulated protein phosphatase 2A activity. Ectopic expression of CIP2A up-regulated phospho-Akt and protected HL-60 cells from bortezomib-induced apoptosis, whereas silencing CIP2A overcame the resistance to bortezomib-induced apoptosis in MOLT3 and K562 cells. Importantly, bortezomib exerted in vivo antitumor activity in HL-60 xenografted tumors and induced cell death in some primary leukemic cells. Cancerous inhibitor of protein phosphatase 2A was expressed in leukemic blasts from bone marrow samples. Cancerous inhibitor of protein phosphatase 2A plays a major role in mediating bortezomib-induced apoptosis in leukemia cells. PMID:22983581

A Reduction in Age-Enhanced Gluconeogenesis Extends Lifespan

PubMed Central

Hachinohe, Mayumi; Yamane, Midori; Akazawa, Daiki; Ohsawa, Kazuhiro; Ohno, Mayumi; Terashita, Yuzu; Masumoto, Hiroshi

2013-01-01

The regulation of energy metabolism, such as calorie restriction (CR), is a major determinant of cellular longevity. Although augmented gluconeogenesis is known to occur in aged yeast cells, the role of enhanced gluconeogenesis in aged cells remains undefined. Here, we show that age-enhanced gluconeogenesis is suppressed by the deletion of the tdh2 gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein that is involved in both glycolysis and gluconeogenesis in yeast cells. The deletion of TDH2 restores the chronological lifespan of cells with deletions of both the HST3 and HST4 genes, which encode yeast sirtuins, and represses the activation of gluconeogenesis. Furthermore, the tdh2 gene deletion can extend the replicative lifespan in a CR pathway-dependent manner. These findings demonstrate that the repression of enhanced gluconeogenesis effectively extends the cellular lifespan. PMID:23342062

A reduction in age-enhanced gluconeogenesis extends lifespan.

PubMed

Hachinohe, Mayumi; Yamane, Midori; Akazawa, Daiki; Ohsawa, Kazuhiro; Ohno, Mayumi; Terashita, Yuzu; Masumoto, Hiroshi

2013-01-01

The regulation of energy metabolism, such as calorie restriction (CR), is a major determinant of cellular longevity. Although augmented gluconeogenesis is known to occur in aged yeast cells, the role of enhanced gluconeogenesis in aged cells remains undefined. Here, we show that age-enhanced gluconeogenesis is suppressed by the deletion of the tdh2 gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein that is involved in both glycolysis and gluconeogenesis in yeast cells. The deletion of TDH2 restores the chronological lifespan of cells with deletions of both the HST3 and HST4 genes, which encode yeast sirtuins, and represses the activation of gluconeogenesis. Furthermore, the tdh2 gene deletion can extend the replicative lifespan in a CR pathway-dependent manner. These findings demonstrate that the repression of enhanced gluconeogenesis effectively extends the cellular lifespan.

Investigating cellular network heterogeneity and modularity in cancer: a network entropy and unbalanced motif approach.

PubMed

Cheng, Feixiong; Liu, Chuang; Shen, Bairong; Zhao, Zhongming

2016-08-26

Cancer is increasingly recognized as a cellular system phenomenon that is attributed to the accumulation of genetic or epigenetic alterations leading to the perturbation of the molecular network architecture. Elucidation of network properties that can characterize tumor initiation and progression, or pinpoint the molecular targets related to the drug sensitivity or resistance, is therefore of critical importance for providing systems-level insights into tumorigenesis and clinical outcome in the molecularly targeted cancer therapy. In this study, we developed a network-based framework to quantitatively examine cellular network heterogeneity and modularity in cancer. Specifically, we constructed gene co-expressed protein interaction networks derived from large-scale RNA-Seq data across 8 cancer types generated in The Cancer Genome Atlas (TCGA) project. We performed gene network entropy and balanced versus unbalanced motif analysis to investigate cellular network heterogeneity and modularity in tumor versus normal tissues, different stages of progression, and drug resistant versus sensitive cancer cell lines. We found that tumorigenesis could be characterized by a significant increase of gene network entropy in all of the 8 cancer types. The ratio of the balanced motifs in normal tissues is higher than that of tumors, while the ratio of unbalanced motifs in tumors is higher than that of normal tissues in all of the 8 cancer types. Furthermore, we showed that network entropy could be used to characterize tumor progression and anticancer drug responses. For example, we found that kinase inhibitor resistant cancer cell lines had higher entropy compared to that of sensitive cell lines using the integrative analysis of microarray gene expression and drug pharmacological data collected from the Genomics of Drug Sensitivity in Cancer database. In addition, we provided potential network-level evidence that smoking might increase cancer cellular network heterogeneity and further contribute to tyrosine kinase inhibitor (e.g., gefitinib) resistance. In summary, we demonstrated that network properties such as network entropy and unbalanced motifs associated with tumor initiation, progression, and anticancer drug responses, suggesting new potential network-based prognostic and predictive measure in cancer.

E2F mediates enhanced alternative polyadenylation in proliferation.

PubMed

Elkon, Ran; Drost, Jarno; van Haaften, Gijs; Jenal, Mathias; Schrier, Mariette; Oude Vrielink, Joachim A F; Agami, Reuven

2012-07-02

The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation.

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

PubMed Central

2012-01-01

Background The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib. PMID:23234355

Regulating the Efficacy of Inhibition Through Trafficking of γ-Aminobutyric Acid Type A Receptors.

PubMed

Vien, Thuy N; Moss, Stephen J; Davies, Paul A

2016-11-01

Trafficking of anesthetic-sensitive receptors within the plasma membrane, or from one cellular component to another, occurs continuously. Changes in receptor trafficking have implications in altering anesthetic sensitivity. γ-Aminobutyric acid type A receptors (GABAARs) are anion-permeable ion channels and are the major class of receptor in the adult mammalian central nervous system that mediates inhibition. GABAergic signaling allows for precise synchronized firing of action potentials within brain circuits that is critical for cognition, behavior, and consciousness. This precision depends upon tightly controlled trafficking of GABAARs into the membrane. General anesthetics bind to and allosterically enhance GABAARs by prolonging the open state of the receptor and thereby altering neuronal and brain circuit activity. Subunit composition and GABAAR localization strongly influence anesthetic end points; therefore, changes in GABAAR trafficking could have significant consequences to anesthetic sensitivity. GABAARs are not static membrane structures but are in a constant state of flux between extrasynaptic and synaptic locations and are continually endocytosed and recycled from and to the membrane. Neuronal activity, posttranslational modifications, and some naturally occurring and synthetic compounds can influence the expression and trafficking of GABAARs. In this article, we review GABAARs, their trafficking, and how phosphorylation of GABAAR subunits can influence the surface expression and function of the receptor. Ultimately, alterations of GABAAR trafficking could modify anesthetic end points, both unintentionally through pathologic processes but potentially as a therapeutic target to adjust anesthetic-sensitive GABAARs.

Long duration mild temperature hyperthermia and brachytherapy.

PubMed

Armour, E P; Raaphorst, G P

2004-03-01

Combining long duration mild temperature hyperthermia (LDMH) and low dose-rate (LDR) brachytherapy to enhance therapeutic killing of cancer cells was proposed many years ago. The cellular and tumour research that supports this hypothesis is presented in this review. Research describing LDMH interaction with pulsed brachytherapy and high dose-rate brachytherapy using clinically relevant parameters are compared with LDMH/LDR brachytherapy. The mechanism by which LDMH sensitizes LDR has been established as the inhibition of sublethal damage repair. The molecular mechanisms have been shown to involve DNA repair enzymes, but the exact nature of these processes is still under investigation. The relative differences between LDMH interactions with human and rodent cells are presented to help in the understanding of possible roles of LDMH in clinical application. The role of LDMH in modifying tumour blood flow and its possible role in LDR sensitization of tumours is also presented. The positive aspects of LDMH-brachytherapy for clinical application are sixfold; (1) the thermal goals (temperature, time and volume) are achievable with currently available technology, (2) the hyperthermia by itself has no detectable toxic effects, (3) thermotolerance appears to play a minor if any role in radiation sensitization, (4) TER of around 2 can be expected, (5) hypoxic fraction may be decreased due to blood flow modification and (6) simultaneous chemotherapy may also be sensitized. Combined LDMH and brachytherapy is a cancer therapy that has established biological rationale and sufficient technical and clinical advancements to be appropriately applied. This modality is ripe for clinical testing.

Creation, characterization and utilization of Cryptococcus neoformans mutants sensitive to micafungin.

PubMed

Toh-E, Akio; Ohkusu, Misako; Shimizu, Kiminori; Yamaguchi, Masashi; Ishiwada, Naruhiko; Watanabe, Akira; Kamei, Katsuhiko

2017-12-01

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding β-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of β-1, 3-glucan synthesis in C. neoformans; the inhibition of β-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized β-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.

Evaluation of the BH3-only Protein Puma as a Direct Bak Activator*

PubMed Central

Dai, Haiming; Pang, Yuan-Ping; Ramirez-Alvarado, Marina; Kaufmann, Scott H.

2014-01-01

Interactions among Bcl-2 family proteins play critical roles in cellular life and death decisions. Previous studies have established the BH3-only proteins Bim, tBid, and Noxa as “direct activators” that are able to directly initiate the oligomerization and activation of Bak and/or Bax. Earlier studies of Puma have yielded equivocal results, with some concluding that it also acts as a direct activator and other studies suggesting that it acts solely as a sensitizer BH3-only protein. In the present study we examined the interaction of Puma BH3 domain or full-length protein with Bak by surface plasmon resonance, assessed Bak oligomerization status by cross-linking followed by immunoblotting, evaluated the ability of the Puma BH3 domain to induce Bak-mediated permeabilization of liposomes and mitochondria, and determined the effect of wild type and mutant Puma on cell viability in a variety of cellular contexts. Results of this analysis demonstrate high affinity (KD = 26 ± 5 nm) binding of the Puma BH3 domain to purified Bak ex vivo, leading to Bak homo-oligomerization and membrane permeabilization. Mutations in Puma that inhibit (L141E/M144E/L148E) or enhance (M144I/A145G) Puma BH3 binding to Bak also produce corresponding alterations in Bak oligomerization, Bak-mediated membrane permeabilization and, in a cellular context, Bak-mediated killing. Collectively, these results provide strong evidence that Puma, like Bim, Noxa, and tBid, is able to act as a direct Bak activator. PMID:24265320

Mesoporous silica nanoparticles loading doxorubicin reverse multidrug resistance: performance and mechanism

NASA Astrophysics Data System (ADS)

Shen, Jianan; He, Qianjun; Gao, Yu; Shi, Jianlin; Li, Yaping

2011-10-01

Multidrug resistance (MDR) is one of the major obstacles for successful chemotherapy in cancer. One of the effective approaches to overcome MDR is to use nanoparticle-mediated drug delivery to increase drug accumulation in drug resistant cancer cells. In this work, we first report that the performance and mechanism of an inorganic engineered delivery system based on mesoporous silica nanoparticles (MSNs) loading doxorubicin (DMNs) to overcome the MDR of MCF-7/ADR (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The experimental results showed that DMNs could enhance the cellular uptake of doxorubicin (DOX) and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells. The IC50 of DMNs against MCF-7/ADR cells was 8-fold lower than that of free DOX. However, an improved effect of DOX in DMNs against MCF-7 cells (a DOX-sensitive cancer cell line) was not found. The increased cellular uptake and nuclear accumulation of DOX delivered by DMNs in MCF-7/ADR cells was confirmed by confocal laser scanning microscopy, and could result from the down-regulation of P-gp and bypassing the efflux action by MSNs themselves. The cellular uptake mechanism of DMNs indicated that the macropinocytosis was one of the pathways for the uptake of DMNs by MCF-7/ADR cells. The in vivo biodistribution showed that DMNs induced a higher accumulation of DOX in drug resistant tumors than free DOX. These results suggested that MSNs could be an effective delivery system to overcome multidrug resistance.

Novel Single-Cell Analysis Platform Based on a Solid-State Zinc-Coadsorbed Carbon Quantum Dots Electrochemiluminescence Probe for the Evaluation of CD44 Expression on Breast Cancer Cells.

PubMed

Qiu, Youyi; Zhou, Bin; Yang, Xiaojuan; Long, Dongping; Hao, Yan; Yang, Peihui

2017-05-24

A novel single-cell analysis platform was fabricated using solid-state zinc-coadsorbed carbon quantum dot (ZnCQDs) nanocomposites as an electrochemiluminescence (ECL) probe for the detection of breast cancer cells and evaluation of the CD44 expression level. Solid-state ZnCQDs nanocomposite probes were constructed through the attachment of ZnCQDs to gold nanoparticles and then the loading of magnetic beads to amplify the ECL signal, exhibiting a remarkable 120-fold enhancement of the ECL intensity. Hyaluronic acid (HA)-functionalized solid-state probes were used to label a single breast cancer cell by the specific recognition of HA with CD44 on the cell surface, revealing more stable, sensitive, and effective tagging in comparison with the water-soluble CQDs. This strategy exhibited a good analytical performance for the analysis of MDA-MB-231 and MCF-7 single cells with linear range from 1 to 18 and from 1 to 12 cells, respectively. Furthermore, this single-cell analysis platform was used for evaluation of the CD44 expression level of these two cell lines, in which the MDA-MB-231 cells revealed a 2.8-5.2-fold higher CD44 expression level. A total of 20 single cells were analyzed individually, and the distributions of the ECL intensity revealed larger variations, indicating the high cellular heterogeneity of the CD44 expression level on the same cell line. The as-proposed single-cell analysis platform might provide a novel protocol to effectively study the individual cellular function and cellular heterogeneity.

Altered Antioxidant System Stimulates Dielectric Barrier Discharge Plasma-Induced Cell Death for Solid Tumor Cell Treatment

PubMed Central

Park, Daehoon; Choi, Eun H.

2014-01-01

This study reports the experimental findings and plasma delivery approach developed at the Plasma Bioscience Research Center, Korea for the assessment of antitumor activity of dielectric barrier discharge (DBD) for cancer treatment. Detailed investigation of biological effects occurring after atmospheric pressure non-thermal (APNT) plasma application during in vitro experiments revealed the role of reactive oxygen species (ROS) in modulation of the antioxidant defense system, cellular metabolic activity, and apoptosis induction in cancer cells. To understand basic cellular mechanisms, we investigated the effects of APNT DBD plasma on antioxidant defense against oxidative stress in various malignant cells as well as normal cells. T98G glioblastoma, SNU80 thyroid carcinoma, KB oral carcinoma and a non-malignant HEK293 embryonic human cell lines were treated with APNT DBD plasma and cellular effects due to reactive oxygen species were observed. Plasma significantly decreased the metabolic viability and clonogenicity of T98G, SNU80, KB and HEK293 cell lines. Enhanced ROS in the cells led to death via alteration of total antioxidant activity, and NADP+/NADPH and GSH/GSSG ratios 24 hours (h) post plasma treatment. This effect was confirmed by annexin V-FITC and propidium iodide staining. These consequences suggested that the failure of antioxidant defense machinery, with compromised redox status, might have led to sensitization of the malignant cells. These findings suggest a promising approach for solid tumor therapy by delivering a lethal dose of APNT plasma to tumor cells while sparing normal healthy tissues. PMID:25068311

Re-evaluation of thin layer chromatography as an alternative method for the quantification of prostaglandins from rat Kupffer cells.

PubMed

Pestel, Sabine; Jungermann, Kurt; Schieferdecker, Henrike L

2005-01-01

In contrast to conventionally used immunoassays, thin layer chromatography (TLC)--by prelabeling of cells with radioactive arachidonic acid (AA)--allows to differentiate between cellularly built and added prostanoids and thus to investigate feedback effects of prostanoids on their own release. PGD2, TXB2 and PGE2 released from zymosan-stimulated Kupffer cells were separated with distinct RF-values, corresponding to those of the pure substances. Quantification of PGD2 and PGE2 gave comparable results with TLC and immunoassays, but measurement in the presence of added prostanoids was only possible with TLC. Moreover TLC was superior to immunoassays in having a longer linear range while being comparably sensitive. Cellularly built TXB2 in its radioactively labeled form was not detectable by TLC. Inhibition of TXB2 release by externally added AA or technical artifacts were excluded, suggesting that the cellular AA-pools used for prostaglandin and thromboxane synthesis differ in their accessibility for added AA. Thus, TLC is a simple, sensitive and precise method for the quantification of cellularly built prostaglandins but not of thromboxane even in the presence of added prostanoids.

Mitochondrial correlates of signaling processes involved with the cellular response to eimeria infection in broiler chickens

USDA-ARS?s Scientific Manuscript database

Host cellular responses to coccidiosis infection are consistent with elements of apoptosis, autophagy, and necrosis. These processes are enhanced in the cell through cell-directed signaling or repressed through parasite-derived inhibitors of these processes favoring the survival of the parasite. Acr...

Hapten-specific lymphocyte transformation in humans sensitized with NDMA or DNCB.

PubMed Central

SoebergB; Andersen, V

1976-01-01

The primary immune response to a contact sensitizing dose of para-N-dimethylnitrosaniline (NDMA) and dinitrochlorobenzene (DNCB) was obtained in humans and measured in vitro by increased thymidine incorporation into sensitized lymphocytes. No cross-reaction was found between these two haptens, and it is thus possible on two separate occasions to quantify and follow the primary cellular immune response in man. PMID:963911

Parameterizing the Logistic Model of Tumor Growth by DW-MRI and DCE-MRI Data to Predict Treatment Response and Changes in Breast Cancer Cellularity during Neoadjuvant Chemotherapy1

PubMed Central

Atuegwu, Nkiruka C; Arlinghaus, Lori R; Li, Xia; Chakravarthy, A Bapsi; Abramson, Vandana G; Sanders, Melinda E; Yankeelov, Thomas E

2013-01-01

Diffusion-weighted and dynamic contrast-enhanced magnetic resonance imaging (MRI) data of 28 patients were obtained pretreatment, after one cycle, and after completion of all cycles of neoadjuvant chemotherapy (NAC). For each patient at each time point, the tumor cell number was estimated using the apparent diffusion coefficient and the extravascular extracellular (ve) and plasma volume (vp) fractions. The proliferation/death rate was obtained using the number of tumor cells from the first two time points in conjunction with the logistic model of tumor growth, which was then used to predict tumor cellularity at the conclusion of NAC. The Pearson correlation coefficient between the predicted and the experimental number of tumor cells measured at the end of NAC was 0.81 (P = .0043). The proliferation rate estimated after the first cycle of therapy was able to separate patients who went on to achieve pathologic complete response from those who did not (P = .021) with a sensitivity and specificity of 82.4% and 72.7%, respectively. These data provide preliminary results indicating that incorporating readily available quantitative MRI data into a simple model of tumor growth can lead to potentially clinically relevant information for predicting an individual patient's response to NAC. PMID:23730404

Alternating Magnetic Field Controlled, Multifunctional Nano-Reservoirs: Intracellular Uptake and Improved Biocompatibility

NASA Astrophysics Data System (ADS)

Ghosh, Santaneel; Ghoshmitra, Somesree; Cai, Tong; Diercks, David R.; Mills, Nathaniel C.; Hynds, Dianna L.

2010-01-01

Biocompatible magnetic nanoparticles hold great therapeutic potential, but conventional particles can be toxic. Here, we report the synthesis and alternating magnetic field dependent actuation of a remotely controllable, multifunctional nano-scale system and its marked biocompatibility with mammalian cells. Monodisperse, magnetic nanospheres based on thermo-sensitive polymer network poly(ethylene glycol) ethyl ether methacrylate- co-poly(ethylene glycol) methyl ether methacrylate were synthesized using free radical polymerization. Synthesized nanospheres have oscillating magnetic field induced thermo-reversible behavior; exhibiting desirable characteristics comparable to the widely used poly- N-isopropylacrylamide-based systems in shrinkage plus a broader volumetric transition range. Remote heating and model drug release were characterized for different field strengths. Nanospheres containing nanoparticles up to an iron concentration of 6 mM were readily taken up by neuron-like PC12 pheochromocytoma cells and had reduced toxicity compared to other surface modified magnetic nanocarriers. Furthermore, nanosphere exposure did not inhibit the extension of cellular processes (neurite outgrowth) even at high iron concentrations (6 mM), indicating minimal negative effects in cellular systems. Excellent intracellular uptake and enhanced biocompatibility coupled with the lack of deleterious effects on neurite outgrowth and prior Food and Drug Administration (FDA) approval of PEG-based carriers suggest increased therapeutic potential of this system for manipulating axon regeneration following nervous system injury.

Electrostatics Control Actin Filament Nucleation and Elongation Kinetics*

PubMed Central

Crevenna, Alvaro H.; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L.; Lamb, Don C.; Wedlich-Söldner, Roland

2013-01-01

The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment. PMID:23486468

Acid-degradable lactobionic acid-modified soy protein nanogels crosslinked by ortho ester linkage for efficient antitumor in vivo.

PubMed

Cheng, Xu; Qin, Jiejie; Wang, Xin; Zha, Qian; Yao, Weijing; Fu, Shengxiang; Tang, Rupei

2018-05-03

It remains a crucial challenge to achieve efficient cellular uptake and intracellular drug release in tumor cells for the nanoscale drug delivery systems. Herein, acid-degradable nanogels were prepared by cross-linking methacrylated soy protein with an acid-labile ortho ester cross-linker (NG1), and then modified with lactobionic acid (LA) to give tumor-targeted nanogels (NG2). Both NG1 and NG2 displayed excellent stability in neutral environment, while showed pH-triggered degradation behaviors under mildly acidic conditions resulting from the breakage of ortho ester bonds. Doxorubicin (DOX) was successfully loaded into nanogels, which exhibited an accelerated release at low pH. In vitro cell studies demonstrated that LA-modified nanogels could effectively improve cellular internalization, show higher cytotoxicity and apoptosis toward asialoglycoprotein receptor (ASGPR) over-expressed HepG2 cells. In vivo antitumor experimentproved that LA modification could significantly enhance the tumor-targeting ability of nanogels and increase DOX concentration in tumor site, leading to better therapeutic efficacy. Histological analysis further demonstrated that soy protein-based nanogels did not cause any damage to normal organs. Overall, these pH-sensitive and tumor-targeting soy protein-based nanogels can be potential drug carriers for efficient tumor treatment. Copyright © 2018. Published by Elsevier B.V.

Diselenolane-mediated cellular uptake† †Electronic supplementary information (ESI) available: Detailed procedures and results for all reported experiments. See DOI: 10.1039/c7sc05151d

PubMed Central

Chuard, Nicolas; Poblador-Bahamonde, Amalia I.; Zong, Lili; Bartolami, Eline; Hildebrandt, Jana; Weigand, Wolfgang; Sakai, Naomi

2018-01-01

The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides. PMID:29675232

Distinct Functional Domains of Ubc9 Dictate Cell Survival and Resistance to Genotoxic Stress

PubMed Central

van Waardenburg, Robert C. A. M.; Duda, David M.; Lancaster, Cynthia S.; Schulman, Brenda A.; Bjornsti, Mary-Ann

2006-01-01

Covalent modification with SUMO alters protein function, intracellular localization, or protein-protein interactions. Target recognition is determined, in part, by the SUMO E2 enzyme, Ubc9, while Siz/Pias E3 ligases may facilitate select interactions by acting as substrate adaptors. A yeast conditional Ubc9P123L mutant was viable at 36°C yet exhibited enhanced sensitivity to DNA damage. To define functional domains in Ubc9 that dictate cellular responses to genotoxic stress versus those necessary for cell viability, a 1.75-Å structure of yeast Ubc9 that demonstrated considerable conservation of backbone architecture with human Ubc9 was solved. Nevertheless, differences in side chain geometry/charge guided the design of human/yeast chimeras, where swapping domains implicated in (i) binding residues within substrates that flank canonical SUMOylation sites, (ii) interactions with the RanBP2 E3 ligase, and (iii) binding of the heterodimeric E1 and SUMO had distinct effects on cell growth and resistance to DNA-damaging agents. Our findings establish a functional interaction between N-terminal and substrate-binding domains of Ubc9 and distinguish the activities of E3 ligases Siz1 and Siz2 in regulating cellular responses to genotoxic stress. PMID:16782883

Chlorpyrifos induces oxidative stress in oligodendrocyte progenitor cells.

PubMed

Saulsbury, Marilyn D; Heyliger, Simone O; Wang, Kaiyu; Johnson, Deadre J

2009-05-02

There are increasing concerns regarding the relative safety of chlorpyrifos (CPF) to various facets of the environment. Although published works suggest that CPF is relatively safe in adult animals, recent evidence indicates that juveniles, both animals and humans, may be more sensitive to CPF toxicity than adults. In young animals, CPF is neurotoxic and mechanistically interferes with cellular replication and cellular differentiation, which culminates in the alteration of synaptic neurotransmission in neurons. However, the effects of CPF on glial cells are not fully elucidated. Here we report that chlorpyrifos is toxic to oligodendrocyte progenitors. In addition, CPF produced dose-dependent increases in 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA) and dihydroethidium (DHE) fluorescence intensities relative to the vehicle control. Moreover, CPF toxicity is associated with nuclear condensation and elevation of caspase 3/7 activity and Heme oxygenase-1 mRNA expression. Pan-caspase inhibitor QVDOPh and cholinergic receptor antagonists' atropine and mecamylamine failed to protect oligodendrocyte progenitors from CPF-induced injury. Finally, glutathione (GSH) depletion enhanced CPF-induced toxicity whereas nitric oxide synthetase inhibitor L-NAME partially protected progenitors and the non-specific antioxidant vitamin E (alpha-tocopherol) completely spared cells from injury. Collectively, this data suggests that CPF induced toxicity is independent of cholinergic stimulation and is most likely caused by the induction of oxidative stress.

mTOR inhibitors sensitize thyroid cancer cells to cytotoxic effect of vemurafenib.

PubMed

Hanly, Elyse K; Bednarczyk, Robert B; Tuli, Neha Y; Moscatello, Augustine L; Halicka, H Dorota; Li, Jiangwei; Geliebter, Jan; Darzynkiewicz, Zbigniew; Tiwari, Raj K

2015-11-24

Treatment options for advanced metastatic thyroid cancer patients are limited. Vemurafenib, a BRAFV600E inhibitor, has shown promise in clinical trials although cellular resistance occurs. Combination therapy that includes BRAFV600E inhibition and avoids resistance is a clinical need. We used an in vitro model to examine combination treatment with vemurafenib and mammalian target of rapamycin (mTOR) inhibitors, metformin and rapamycin. Cellular viability and apoptosis were analyzed in thyroid cell lines by trypan blue exclusion and TUNEL assays. Combination of vemurafenib and metformin decreased cell viability and increased apoptosis in both BCPAP papillary thyroid cancer cells and 8505c anaplastic thyroid cancer cells. This combination was also found to be active in vemurafenib-resistant BCPAP cells. Changes in expression of signaling molecules such as decreased mTOR expression in BCPAP and enhanced inhibition of phospho-MAPK in resistant BCPAP and 8505c were observed. The second combination of vemurafenib and rapamycin amplified cell death in BCPAP cells. We conclude that combination of BRAFV600E and mTOR inhibition forms the basis of a treatment regimen that should be further investigated in in vivo model systems. Metformin or rapamycin adjuvant treatment may provide clinical benefits with minimal side effects to BRAFV600E-positive advanced thyroid cancer patients treated with vemurafenib.

mTOR inhibitors sensitize thyroid cancer cells to cytotoxic effect of vemurafenib

PubMed Central

Hanly, Elyse K.; Bednarczyk, Robert B.; Tuli, Neha Y.; Moscatello, Augustine L.; Halicka, H. Dorota; Li, Jiangwei; Geliebter, Jan; Darzynkiewicz, Zbigniew; Tiwari, Raj K.

2015-01-01

Treatment options for advanced metastatic thyroid cancer patients are limited. Vemurafenib, a BRAFV600E inhibitor, has shown promise in clinical trials although cellular resistance occurs. Combination therapy that includes BRAFV600E inhibition and avoids resistance is a clinical need. We used an in vitro model to examine combination treatment with vemurafenib and mammalian target of rapamycin (mTOR) inhibitors, metformin and rapamycin. Cellular viability and apoptosis were analyzed in thyroid cell lines by trypan blue exclusion and TUNEL assays. Combination of vemurafenib and metformin decreased cell viability and increased apoptosis in both BCPAP papillary thyroid cancer cells and 8505c anaplastic thyroid cancer cells. This combination was also found to be active in vemurafenib-resistant BCPAP cells. Changes in expression of signaling molecules such as decreased mTOR expression in BCPAP and enhanced inhibition of phospho-MAPK in resistant BCPAP and 8505c were observed. The second combination of vemurafenib and rapamycin amplified cell death in BCPAP cells. We conclude that combination of BRAFV600E and mTOR inhibition forms the basis of a treatment regimen that should be further investigated in in vivo model systems. Metformin or rapamycin adjuvant treatment may provide clinical benefits with minimal side effects to BRAFV600E-positive advanced thyroid cancer patients treated with vemurafenib. PMID:26284586

Sensitivity enhanced strain and temperature measurements based on FBG and frequency chirp magnification.

PubMed

Du, Jiangbing; He, Zuyuan

2013-11-04

In this work, highly sensitive measurements of strain and temperature have been demonstrated using a fiber Bragg grating (FBG) sensor with significantly enhance sensitivity by all-optical signal processing. The sensitivity enhancement is achieved by degenerated Four Wave Mixing (FWM) for frequency chirp magnification (FCM), which can be used for magnifying the wavelength drift of the FBG sensor induced by strain and temperature change. Highly sensitive measurements of static strain and temperature have been experimentally demonstrated with strain sensitivity of 5.36 pm/με and temperature sensitivity of 54.09 pm/°C. The sensitivity has been enhanced by a factor of five based on a 4-order FWM in a highly nonlinear fiber (HNLF).

Novel mucus-penetrating liposomes as a potential oral drug delivery system: preparation, in vitro characterization, and enhanced cellular uptake

PubMed Central

Li, Xiuying; Chen, Dan; Le, Chaoyi; Zhu, Chunliu; Gan, Yong; Hovgaard, Lars; Yang, Mingshi

2011-01-01

Background The aim of this study was to investigate the intestinal mucus-penetrating properties and intestinal cellular uptake of two types of liposomes modified by Pluronic F127 (PF127). Methods The two types of liposomes, ie, PF127-inlaid liposomes and PF127-adsorbed liposomes, were prepared by a thin-film hydration method followed by extrusion, in which coumarin 6 was loaded as a fluorescence marker. A modified Franz diffusion cell mounted with the intestinal mucus of rats was used to study the diffusion characteristics of the two types of PF127 liposomes. Cell uptake studies were conducted in Caco-2 cells and analyzed using confocal laser scanning microcopy as well as flow cytometry. Results The diffusion efficiency of the two types of PF127-modified liposomes through intestinal rat mucus was 5–7-fold higher than that of unmodified liposomes. Compared with unmodified liposomes, PF127-inlaid liposomes showed significantly higher cellular uptake of courmarin 6. PF127-adsorbed liposomes showed a lower cellular uptake. Moreover, and interestingly, the two types of PF127-modified liposomes showed different cellular uptake mechanisms in Caco-2 cells. Conclusion PF127-inlaid liposomes with improved intestinal mucus-penetrating ability and enhanced cellular uptake might be a potential carrier candidate for oral drug delivery. PMID:22163166

Transcription Factors MYOCD, SRF, Mesp1 and SMARCD3 Enhance the Cardio-Inducing Effect of GATA4, TBX5, and MEF2C during Direct Cellular Reprogramming

PubMed Central

Christoforou, Nicolas; Chellappan, Malathi; Adler, Andrew F.; Kirkton, Robert D.; Wu, Tianyi; Addis, Russell C.; Bursac, Nenad; Leong, Kam W.

2013-01-01

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca2+ transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies. PMID:23704920

Journey of Mesenchymal Stem Cells for Homing: Strategies to Enhance Efficacy and Safety of Stem Cell Therapy

PubMed Central

Kang, Sung Keun; Shin, Il Seob; Ko, Myung Soon; Jo, Jung Youn; Ra, Jeong Chan

2012-01-01

Human mesenchymal stem cells (MSCs) communicate with other cells in the human body and appear to “home” to areas of injury in response to signals of cellular damage, known as homing signals. This review of the state of current research on homing of MSCs suggests that favorable cellular conditions and the in vivo environment facilitate and are required for the migration of MSCs to the site of insult or injury in vivo. We review the current understanding of MSC migration and discuss strategies for enhancing both the environmental and cellular conditions that give rise to effective homing of MSCs. This may allow MSCs to quickly find and migrate to injured tissues, where they may best exert clinical benefits resulting from improved homing and the presence of increased numbers of MSCs. PMID:22754575

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells

NASA Astrophysics Data System (ADS)

Asalla, Suman; Girada, Shravan Babu; Kuna, Ramya S.; Chowdhury, Debabrata; Kandagatla, Bhaskar; Oruganti, Srinivas; Bhadra, Utpal; Bhadra, Manika Pal; Kalivendi, Shasi Vardhan; Rao, Swetha Pavani; Row, Anupama; Ibrahim, A.; Ghosh, Partha Pratim; Mitra, Prasenjit

2016-06-01

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation.

ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease.

PubMed

Nakano, Masaki; Imamura, Hiromi; Sasaoka, Norio; Yamamoto, Masamichi; Uemura, Norihito; Shudo, Toshiyuki; Fuchigami, Tomohiro; Takahashi, Ryosuke; Kakizuka, Akira

2017-08-01

Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances), which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors) are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist) against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor) were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Dusp5 negatively regulates IL-33-mediated eosinophil survival and function

PubMed Central

Holmes, Derek A; Yeh, Jung-Hua; Yan, Donghong; Xu, Min; Chan, Andrew C

2015-01-01

Mitogen-activated protein kinase (MAPK) activation controls diverse cellular functions including cellular survival, proliferation, and apoptosis. Tuning of MAPK activation is counter-regulated by a family of dual-specificity phosphatases (DUSPs). IL-33 is a recently described cytokine that initiates Th2 immune responses through binding to a heterodimeric IL-33Rα (ST2L)/IL-1α accessory protein (IL-1RAcP) receptor that coordinates activation of ERK and NF-κB pathways. We demonstrate here that DUSP5 is expressed in eosinophils, is upregulated following IL-33 stimulation and regulates IL-33 signaling. Dusp5−/− mice have prolonged eosinophil survival and enhanced eosinophil effector functions following infection with the helminth Nippostrongylus brasiliensis. IL-33-activated Dusp5−/− eosinophils exhibit increased cellular ERK1/2 activation and BCL-XL expression that results in enhanced eosinophil survival. In addition, Dusp5−/− eosinophils demonstrate enhanced IL-33-mediated activation and effector functions. Together, these data support a role for DUSP5 as a novel negative regulator of IL-33-dependent eosinophil function and survival. PMID:25398911

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells

PubMed Central

Asalla, Suman; Girada, Shravan Babu; Kuna, Ramya S.; Chowdhury, Debabrata; Kandagatla, Bhaskar; Oruganti, Srinivas; Bhadra, Utpal; Bhadra, Manika Pal; Kalivendi, Shasi Vardhan; Rao, Swetha Pavani; Row, Anupama; Ibrahim, A; Ghosh, Partha Pratim; Mitra, Prasenjit

2016-01-01

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation. PMID:27282931

Enhanced cellular uptake of size-separated lipophilic silicon nanoparticles

NASA Astrophysics Data System (ADS)

Kusi-Appiah, Aubrey E.; Mastronardi, Melanie L.; Qian, Chenxi; Chen, Kenneth K.; Ghazanfari, Lida; Prommapan, Plengchart; Kübel, Christian; Ozin, Geoffrey A.; Lenhert, Steven

2017-03-01

Specific size, shape and surface chemistry influence the biological activity of nanoparticles. In the case of lipophilic nanoparticles, which are widely used in consumer products, there is evidence that particle size and formulation influences skin permeability and that lipophilic particles smaller than 6 nm can embed in lipid bilayers. Since most nanoparticle synthetic procedures result in mixtures of different particles, post-synthetic purification promises to provide insights into nanostructure-function relationships. Here we used size-selective precipitation to separate lipophilic allyl-benzyl-capped silicon nanoparticles into monodisperse fractions within the range of 1 nm to 5 nm. We measured liposomal encapsulation and cellular uptake of the monodisperse particles and found them to have generally low cytotoxicities in Hela cells. However, specific fractions showed reproducibly higher cytotoxicity than other fractions as well as the unseparated ensemble. Measurements indicate that the cytotoxicity mechanism involves oxidative stress and the differential cytotoxicity is due to enhanced cellular uptake by specific fractions. The results indicate that specific particles, with enhanced suitability for incorporation into lipophilic regions of liposomes and subsequent in vitro delivery to cells, are enriched in certain fractions.

Co-Encapsulating the Fusogenic Peptide INF7 and Molecular Imaging Probes in Liposomes Increases Intracellular Signal and Probe Retention

PubMed Central

Martin, Erik W.; Li, Changqing; Lu, Wuyuan; Kao, Joseph P. Y.

2015-01-01

Liposomes are promising vehicles to deliver diagnostic and therapeutic agents to cells in vivo. After uptake into cells by endocytosis, liposomes are degraded in the endolysosomal system. Consequently, the encapsulated cargo molecules frequently remain sequestered in endosomal compartments; this limits their usefulness in many applications (e.g. gene delivery). To overcome this, various fusogenic peptides have been developed to facilitate delivery of liposomally-encapsulated molecules into the cytosol. One such peptide is the pH-sensitive influenza-derived peptide INF7. Liposomal delivery of imaging agents is an attractive approach for enabling cell imaging and cell tracking in vivo, but can be hampered by inadequate intracellular accumulation and retention of probes caused by exocytosis (and possible degradation) of endosome-entrapped probes. Such signal loss could be minimized by facilitating escape of probe molecules from endolysosomal compartments into the cytosol. We investigated the ability of co-encapsulated INF7 to release liposomally-delivered rhodamine fluorophores into the cytosol after endosomal acidification/maturation. We co-encapsulated INF7 and fluorescent rhodamine derivatives having vastly different transport properties to show that after endocytosis by CV1 cells, the INF7 peptide is activated by acidic endosomal pH and facilitates efficient release of the fluorescent tracers into the cytosol. Furthermore, we show that INF7-facilitated escape from endosomes markedly enhanced retention of tracers that cannot be actively extruded from the cytosol. Minimizing loss of intracellular probes improves cellular imaging by increasing the signal-to-noise ratio of images and lengthening the time window that imaging can be performed. In particular, this will enhance in vivo electron paramagnetic resonance imaging, an emergent magnetic resonance imaging modality requires exogenous paramagnetic imaging agents and is highly promising for cellular and molecular imaging. PMID:25816348

BEMER Electromagnetic Field Therapy Reduces Cancer Cell Radioresistance by Enhanced ROS Formation and Induced DNA Damage.

PubMed

Storch, Katja; Dickreuter, Ellen; Artati, Anna; Adamski, Jerzy; Cordes, Nils

2016-01-01

Each year more than 450,000 Germans are expected to be diagnosed with cancer subsequently receiving standard multimodal therapies including surgery, chemotherapy and radiotherapy. On top, molecular-targeted agents are increasingly administered. Owing to intrinsic and acquired resistance to these therapeutic approaches, both the better molecular understanding of tumor biology and the consideration of alternative and complementary therapeutic support are warranted and open up broader and novel possibilities for therapy personalization. Particularly the latter is underpinned by the increasing utilization of non-invasive complementary and alternative medicine by the population. One investigated approach is the application of low-dose electromagnetic fields (EMF) to modulate cellular processes. A particular system is the BEMER therapy as a Physical Vascular Therapy for which a normalization of the microcirculation has been demonstrated by a low-frequency, pulsed EMF pattern. Open remains whether this EMF pattern impacts on cancer cell survival upon treatment with radiotherapy, chemotherapy and the molecular-targeted agent Cetuximab inhibiting the epidermal growth factor receptor. Using more physiological, three-dimensional, matrix-based cell culture models and cancer cell lines originating from lung, head and neck, colorectal and pancreas, we show significant changes in distinct intermediates of the glycolysis and tricarboxylic acid cycle pathways and enhanced cancer cell radiosensitization associated with increased DNA double strand break numbers and higher levels of reactive oxygen species upon BEMER treatment relative to controls. Intriguingly, exposure of cells to the BEMER EMF pattern failed to result in sensitization to chemotherapy and Cetuximab. Further studies are necessary to better understand the mechanisms underlying the cellular alterations induced by the BEMER EMF pattern and to clarify the application areas for human disease.

BEMER Electromagnetic Field Therapy Reduces Cancer Cell Radioresistance by Enhanced ROS Formation and Induced DNA Damage

PubMed Central

Artati, Anna; Adamski, Jerzy

2016-01-01

Each year more than 450,000 Germans are expected to be diagnosed with cancer subsequently receiving standard multimodal therapies including surgery, chemotherapy and radiotherapy. On top, molecular-targeted agents are increasingly administered. Owing to intrinsic and acquired resistance to these therapeutic approaches, both the better molecular understanding of tumor biology and the consideration of alternative and complementary therapeutic support are warranted and open up broader and novel possibilities for therapy personalization. Particularly the latter is underpinned by the increasing utilization of non-invasive complementary and alternative medicine by the population. One investigated approach is the application of low-dose electromagnetic fields (EMF) to modulate cellular processes. A particular system is the BEMER therapy as a Physical Vascular Therapy for which a normalization of the microcirculation has been demonstrated by a low-frequency, pulsed EMF pattern. Open remains whether this EMF pattern impacts on cancer cell survival upon treatment with radiotherapy, chemotherapy and the molecular-targeted agent Cetuximab inhibiting the epidermal growth factor receptor. Using more physiological, three-dimensional, matrix-based cell culture models and cancer cell lines originating from lung, head and neck, colorectal and pancreas, we show significant changes in distinct intermediates of the glycolysis and tricarboxylic acid cycle pathways and enhanced cancer cell radiosensitization associated with increased DNA double strand break numbers and higher levels of reactive oxygen species upon BEMER treatment relative to controls. Intriguingly, exposure of cells to the BEMER EMF pattern failed to result in sensitization to chemotherapy and Cetuximab. Further studies are necessary to better understand the mechanisms underlying the cellular alterations induced by the BEMER EMF pattern and to clarify the application areas for human disease. PMID:27959944

Lipid-based nanoparticles for contrast-enhanced MRI and molecular imaging.

PubMed

Mulder, Willem J M; Strijkers, Gustav J; van Tilborg, Geralda A F; Griffioen, Arjan W; Nicolay, Klaas

2006-02-01

In the field of MR imaging and especially in the emerging field of cellular and molecular MR imaging, flexible strategies to synthesize contrast agents that can be manipulated in terms of size and composition and that can be easily conjugated with targeting ligands are required. Furthermore, the relaxivity of the contrast agents, especially for molecular imaging applications, should be very high to deal with the low sensitivity of MRI. Lipid-based nanoparticles, such as liposomes or micelles, have been used extensively in recent decades as drug carrier vehicles. A relatively new and promising application of lipidic nanoparticles is their use as multimodal MR contrast agents. Lipids are amphiphilic molecules with both a hydrophobic and a hydrophilic part, which spontaneously assemble into aggregates in an aqueous environment. In these aggregates, the amphiphiles are arranged such that the hydrophobic parts cluster together and the hydrophilic parts face the water. In the low concentration regime, a wide variety of structures can be formed, ranging from spherical micelles to disks or liposomes. Furthermore, a monolayer of lipids can serve as a shell to enclose a hydrophobic core. Hydrophobic iron oxide particles, quantum dots or perfluorocarbon emulsions can be solubilized using this approach. MR-detectable and fluorescent amphiphilic molecules can easily be incorporated in lipidic nanoparticles. Furthermore, targeting ligands can be conjugated to lipidic particles by incorporating lipids with a functional moiety to allow a specific interaction with molecular markers and to achieve accumulation of the particles at disease sites. In this review, an overview of different lipidic nanoparticles for use in MRI is given, with the main emphasis on Gd-based contrast agents. The mechanisms of particle formation, conjugation strategies and applications in the field of contrast-enhanced, cellular and molecular MRI are discussed. 2006 John Wiley & Sons, Ltd.

Inhibition of HeLa cell growth by doxorubicin-loaded and tuftsin-conjugated arginate-PEG microparticles.

PubMed

Hu, Tianmu; Qahtan, Anwar Saeed Ahmed; Lei, Lei; Lei, Zhixin; Zhao, Dapeng; Nie, Hemin

2018-03-01

In order to improve the release pattern of chemotherapy drug and reduce the possibility of drug resistance, poly(ethylene glycol amine) (PEG)-modified alginate microparticles (ALG-PEG MPs) were developed then two different mechanisms were employed to load doxorubicin (Dox): 1) forming Dox/ALG-PEG complex by electrostatic attractions between unsaturated functional groups in Dox and ALG-PEG; 2) forming Dox-ALG-PEG complex through EDC-reaction between the amino and carboxyl groups in Dox and ALG, respectively. Additionally, tuftsin (TFT), a natural immunomodulation peptide, was conjugated to MPs in order to enhance the efficiency of cellular uptake. It was found that the Dox-ALG-PEG-TFT MPs exhibited a significantly slower release of Dox than Dox/ALG-PEG-TFT MPs in neutral medium, suggesting the role of covalent bonding in prolonging Dox retention. Besides, the release of Dox from these MPs was pH-sensitive, and the release rate was observably increased at pH 6.5 compared to the case at pH 7.4. Compared with Dox/ALG-PEG MPs and Dox-ALG-PEG MPs, their counterparts further conjugated with TFT more efficiently inhibited the growth of HeLa cells over a period of 48 h, implying the effectiveness of TFT in enhancing cellular uptake of MPs. Over a period of 48 h, Dox-ALG-PEG-TFT MPs inhibited the growth of HeLa cells less efficiently than Dox/ALG-PEG-TFT MPs but the difference was not significant ( p  > 0.05). In consideration of the prolonged and sustained release of Dox, Dox-ALG-PEG-TFT MPs possess the advantages for long-term treatment.