Plating Efficiency Research Articles

Chemopreventive mechanism of polypeptides from Chlamy Farreri (PCF) against UVB-induced malignant transformation of HaCaT cells.

To investigate polypeptide from Chlamy Farreri (PCF)'s protective effect against skin cancer, we used a cellular model of ultraviolet B (UVB)-induced malignant transformation. The human keratinocyte cell line HaCaT was repeatly exposed to UVB (10 mJ/cm(2), 20 times) and malignant transformation was confirmed by Gimesa staining, cell cycle analysis and various assays [anchorage independent growth, matrix metalloproteinase-9 (MMP9) activity, plating efficiency]. The malignant transformation was found to be effectively prevented by PCF pretreatment (2.84mM for 2h prior to each UVB exposure). We investigated the mechanism of PCF-mediated action by determining its effect on DNA methylation status of the tumour suppressor genes [P16 and ras association domain family 1 A (RASSF1A)] in the UVB-transformed cells. Both genes were found to be hypermethylated by chronic UVB exposure. The expression levels of P16, RASSF1A, DNA methyltransferases (DNMTs) and DNA damage inducible protein a (GADD45a) were measured by reverse transcriptase-polymerase chain reaction and western blotting. While chronic UVB exposure was found to suppress the expression of P16 and RASSF1A, it enhanced the expression of DNMT3b. In the early phase of UVB-induced malignant transformation, the GADD45a expression was increased, however, it declined with a continued irradiation of the cells. The UVB-induced DNA hypermethylation of P16 and RASSF1A and subsequent gene silencing was reversed by PCF treatment. The inhibition of DNMTs expression suggested that PCF blocked DNA methylation and thereby the silencing of tumour suppressor genes. Furthermore, the PCF-mediated substantial increase in GADD45a expression indicated that PCF promoted demethylation of tumour suppressor genes via GADD45a induction.

Efficacy of Palladium Solution Concentration on Electroless Fabrication of Dense Metal Ceramic Composite Membranes Coupled with Surfactant and Sonication

This article addresses the effect of varying palladium solution concentration (0.005–0.015 mol/L) on the cost-effective fabrication of dense palladium composite membranes. Laboratory fabricated clay (kaolin) based ceramic disc were used as substrate along with a modified electroless plating technique consisting of a coupled effect of ultrasound and surfactant. Further to increase the efficacy of the process controlled addition of reducing agent was opted for all baths. It was analyzed that the optimal concentration that provided higher plating efficiencies (95.9%), higher transport efficiency (41.3%), higher plating rates (1.5 × 10−4 mol/m2 · s), minimal total plating time (10.5 h), and maximum pore densification (99.7%) corresponds to 0.01 mol/L.

Genetic interactions among homologous recombination mutants in Candida albicans

rad52-ΔΔ and, to a lesser extent, rad51-ΔΔ deletants of Candidaalbicans displayed slow growth and aberrant filamentous morphology whereas rad59-ΔΔ mutants, both by growth rate and morphology resembled wild type. In this study, we have constructed pair-wise double deletants to analyze genetic interactions among these homologous recombination (HR) proteins that affect growth and morphology traits. When grown in liquid YPD medium, double mutant rad51-ΔΔ rad59-ΔΔ exhibited growth rates, cell and colony morphologies, and plating efficiencies that were not significantly different from those observed for rad51-ΔΔ. The same was true for rad52-ΔΔ rad59-ΔΔ compared to rad52-ΔΔ. Slow growth and decreased plating efficiency were caused, at least in part, by a decreased viability, as deduced from FUN1 staining. Flow cytometry and microscopic studies of filamentous mutant populations revealed major changes in cell ploidy, size and morphology, whereas DAPI staining identified complex nuclear rearrangements in yeast and filamentous cells. These phenotypes were not observed in the rad59-ΔΔ mutant populations. Our results show that abolishing Rad51 functions induces the appearance of a subpopulation of aberrant yeast and filamentous forms with increased cell size and ploidy. The size of this complex subpopulation was exacerbated in rad52-ΔΔ mutants. The combination of filamentous cell morphology and viability phenotypes was reflected on the colony morphology of the respective mutants. We conclude that the rad52 mutation is epistatic to rad51 for all the morphological traits analyzed. We discuss these results in the light of the several functions of these recombination genes.

Abstract 1579: Induction of cytotoxicity by soluble chromium (VI) compounds in cultured C3H/10T1/2 Cl 8 mouse embryo cells effects of ascorbate and dehydroascorbate

Abstract Hexavalent chromium [Cr(VI)]-containing compounds are human carcinogens. They cause cancers in the respiratory system when inhaled, and stomach/other internal cancers when ingested. Soluble and insoluble hexavalent chromium [Cr(VI)] compounds induced base substitution, deletion, addition, and frameshift mutations. Cr(VI) compounds also induce DNA-DNA cross links and DNA-protein cross-links in mammalian cells. We examined the ability of the soluble chromium compounds, sodium chromate (Na2CrO4), calcium chromate (CaCrO4) and potassium chromate (K2CrO4) to induce cytotoxicity and morphological transformation in C3H/10T½ Cl 8 (10T1/2) mouse embryo cells. We tested the hypothesis that the intracellular reductants, ascorbate and dehydroascorbate, can reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III) intracellularly, making it a strong cytotoxin in mammalian cells. Ascorbate is present in serum at concentrations in the mM range under physiological conditions in humans, but is only present in the μM range in mouse embryo cells grown in BME cell culture medium plus 10% fetal calf serum. We hypothesized the relatively weak responses for induction of morphological transformation of mammalian cells by Cr(VI) compounds in culture (dose-dependent but weak induction of foci by lead chromate, and no induction of foci by calcium chromate, potassium chromate, and sodium chromate) is due to the small amounts of ascorbate in cultures of mammalian cells that are insufficient to reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III). Therefore, we investigated the cytotoxic effects of ascorbate on 10T½ mouse embryo cells, and the effects of the highest non-cytotoxic concentrations of ascorbate on Cr(VI)-induced cytotoxicity in 10T½ cells, using reduction in plating efficiency as our cytotoxicity assay. Cell survival data showed that ascorbate itself exerted significant cytotoxic effects at concentrations of 0.00625 mM and higher on 10T½ mouse embryo cells. Furthermore, when 10T½ cells were treated with both Cr(VI) and ascorbate, ascorbate played dual roles. It served as a pro-oxidant (enhancer of the cytotoxicity of chromate) at concentrations up to 0.1 mM, and as an anti-oxidant (reducer of the cytotoxicity of chromate) at concentrations of 0.25 mM and higher. This information is important for our ongoing and future experiments, where we designed a protocol to incorporate ascorbate into our assays assessing the cytotoxicity and cell transforming activity of Cr(VI) compounds. We project that at concentrations of 0.1 mM and lower, ascorbate will enhance the cell transforming activity of Cr(VI) compounds. Supported by undergraduate fellowships from the Provost's Office at the University of Southern California (USC) (P. I., JRL), by funding from the M. S. Program in Molecular Microbiology and Immunology to JRL, by discretionary funding to JRL, and by prior grant ES03341 from NIEHS/NIH (P. I., JRL). Citation Format: Sophia A. Shahin, William Liao, Laureen Tran, Qasim A. Akinwumi, Farn-shuan Tseng, Alyssa Mathew-Joseph, Joseph R. Landolph. Induction of cytotoxicity by soluble chromium (VI) compounds in cultured C3H/10T1/2 Cl 8 mouse embryo cells effects of ascorbate and dehydroascorbate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1579. doi:10.1158/1538-7445.AM2014-1579

Abstract 5566: Functional new insights into the regulation of the androgen receptor by PMEPA1/NEDD4-1 in prostate cancer

Abstract Introduction: Androgen receptor (AR) stability has been shown to be regulated by MDM2 E3 ubiquitin ligase. Our data suggest a role for PMEPA1 and NEDD4-1 in the regulation of AR protein in prostate cancer cells. The AR inducible PMEPA1 recruits NEDD4-1, an E3 ubiquitin ligase, to degrade AR. Decreased or absent PMPEA1 was observed in more than 65% of prostate tumors. PMEPA1 depleted transfectant is established in LNCaP cells with a shRNA lentivirus system and shown elevated AR protein levels, AR signaling and increased cell growth. In the current study, we investigated the growth and AR signaling of PMEPA1 depleted prostate cancer cells in the presence of AR inhibitors. Also we investigated the role of PMEPA1 silencing by DNA methylation in the regulation of AR. Methods: PMEPA1 depleted LNCaP cells were treated with AR inhibitors (MDV3100 or bicalutamide) in the presence or absence of the synthetic androgen R1881. Cell growth was monitored by cell counting, BrdU incorporation, colony plating and soft agar assays. Cell cycle was analyzed with propidium iodide staining based flowcytometry. AR, PMEPA1, PSA proteins were assessed by immunoblot assay. Methylation of the PMEPA1 gene was evaluated in LCM-enriched tumor samples by combing precipitation of methylated-DNA and methylation-sensitive restriction enzyme digestion (COMPARE-MS). The transcript levels of PMEPA1 gene was assessed by qRT-PCR. Results: PMEPA1 depleted LNCaP cells are more resistance to growth inhibition by MDV3100 or bicalutamide in both hormone and hormone-depleted conditions, which demonstrated stronger BrdU incorporation abilities, cell plating efficiency and colony formation capacities in soft agar under MDV3100 or bicalutamid treatment compared to controls. The half life of AR was increased, and more cells in S-phase and less apoptotic cells in PMEPA1 depleted LNCaP cells. Methylation of PMEPA1 gene and mRNA levels showed significant correlation in prostate tumors (n=77, P=0.001). The higher frequency of PMEPA1 gene methylation was noticed in Caucasian American patients. Conclusions: PMEPA1 depleted prostate cancer cells develop increased resistance to AR inhibitors through enhanced AR signaling. Silencing of PMEPA1 may contribute to AR activation in prostate cancer. These data highlight that PMEPA1 deficiency may confer resistance to AR inhibitors during prostate cancer progression. This study is supported by the NIH grant (R01CA106653) to SS. Citation Format: Hua Li, Shashwat Sharad, Lakshmi Ravindranath, Michael Haffner, Daniel Heidenberg, Srinivasan Yegnasubramanian, Yongmei Chen, Ahmed Mohamed, Alagarsamy Srinivasan, Gyorgy Petrovics, Shiv Srivastava. Functional new insights into the regulation of the androgen receptor by PMEPA1/NEDD4-1 in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5566. doi:10.1158/1538-7445.AM2014-5566

Abstract 857: Preclinical evaluation: efficacy of DI17E6, a therapeutic antibody against alpha v integrins, compared to cilengitide in sensitizing human cancers to radiation therapy

Abstract Purpose: Integrins are implicated in resistance of solid tumors to radiation therapy, suggesting that their inhibition would enhance therapeutic efficacy. We investigated the efficacy of DI17E6, a therapeutic antibody targeting alpha v (αv) integrins, in enhancing the radiosensitivity of non-small cell lung carcinoma (NSCLC) and head & neck cancer (HNSCC) cells using in vitro and in vivo models, and compared to the previously reported effects of the cyclic peptide cilengitide (inhibits αvβ3 and αvβ5 integrins). Methods: NSCLC lines (A549 and H460) and HNSCC lines (FaDu and HN-5) were examined. Basal level of αv integrin was assessed by viable cell flow-cytometry and by Western blot. The effects of DI17E6 on cell viability were determined by MTS assay, and on cellular radiosensitivity by clonogenic cell survival assay after exposing the cells to γ-radiation with or without DI17E6. In vivo effect of DI17E6 on radioresponse of xenografts was evaluated by tumor growth delay assay. DI17E6 was given i.p.; as a single loading dose of 180 mg/kg followed by weekly doses of 10, 30 or 100 mg/kg given for 6 weeks; fractionated doses of local tumor irradiation was delivered using 137Cs γ-ray source (2 Gy daily for 7 days). When combined, the loading dose of DI17E6 was given either 4 h before or after the first radiation dose. Results: DI17E6 (0.01-10 µg/ml, 24 h) had a minimal effect on the cell viability. However, DI17E6 reduced the plating efficiency of A549, H460 and FaDu with no effect on HN-5 cells. When combined with radiation, DI17E6 enhanced the radiosensitivity of NSCLC cells but not the HNSCC cells. These data are consistent with previously observed effects of cilengitide in vitro. In vivo experiments with A549 xenografts showed that DI17E6 as a single agent had no antitumor activity, but in combination with radiation it suppressed the tumor growth in a schedule- and dose-dependent manner. These data were compared to the previously reported effect of cilengitide, which did not enhance the response of A549 xenografts to radiation therapy. DI17E6 has an extended pharmacokinetics (with a half-life of >7 days) and blocks more αv integrins than cilengitide. The results imply that when αvβ3 and αvβ5 integrins were inhibited by cilengitide either the duration of blockade was insufficient, or that other αv integrins may have provided tumor growth advantage. In vivo studies on other tumor types are underway. Conclusions: Inhibition of αv integrins by DI17E6 in combination with radiation enhanced radiosensitivity of NSCLC cell lines in vitro and in vivo enhanced A549 tumor xenograft response to radiation in mice. These results suggest that DI17E6 may improve the treatment outcome of patients with NSCLC when combined with radiotherapy in a clinical setting. Supported by EMD Serono Merck KGaA, Germany Citation Format: Amit A. Deorukhkar, David P. Molkentine, David R. Valdecanas, Kathryn A. Mason, Uma Raju. Preclinical evaluation: efficacy of DI17E6, a therapeutic antibody against alpha v integrins, compared to cilengitide in sensitizing human cancers to radiation therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 857. doi:10.1158/1538-7445.AM2014-857

Abstract 1952: Notch-1 regulated stemness and EMT in colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer death nationwide. In 2013, nearly 156,000 patients will be diagnosed with CRC. Surgical resection for early stage disease offers the best chance for cure; however, up to 30% of patients will relapse. Current models of CRC progression hold that tumor development is driven by a small subpopulation of cancer initiating (CIC) or cancer stem (CSC) cells that share many properties of stem cells. These cells have the capacity to drive tumor growth, are largely responsible for relapse and have been implicated in metastasis via epithelial to mesenchymal transition (EMT), similar to normal counterparts in organogenesis. Notch-1 is the major receptor for a highly conserved signaling pathway that is well-documented to control stem cell and lineage commitment in various organs, including the colon. The function of Notch signaling in colorectal cancer, however, is not well-defined. Based on preliminary data in which colon tumor samples from CRC patients expressed Notch-1 but surrounding benign tissue did not, we sought to test the hypothesis that Notch-1 signaling in colon cells promotes tumorigenesis by enhancing stem cell and EMT characteristics of tumor cells. The human colon tumor line HCT-116 was transduced with a retroviral construct that expresses constitutively active Notch-1 (ICN1). Hes-1 protein levels were increased in the activated Notch-1 transduced cells, indicating that the construct was functional in activating Notch-signaling. These HCT116/ICN1 cells exhibited higher plating efficiency than the parental cell line. The average colony size of the HCT116/ICN1 cells was about two-fold smaller than the parental cell line (p<0.001). In addition, cells with constitutively active Notch-1 (HCT116/ICN1) had a longer duplication time than parental HCT-116 cells (p<0.03). Consistent with longer duplication time, HCT116/ICN1 cells were two-fold slower in completing wound healing as compared with the parental HCT-116 cell line (p<0.001). Transwell migration assays revealed a two-fold increase in migration of HCT116/ICN1 cells as compared with the parental control (p<0.03). Western blot analysis demonstrated that the HCT116/ICN1 cells with activated Notch-1 expressed highly increased levels of the EMT associated proteins Smad3, CD44 and Slug. This was accompanied by a four-fold decreased expression of E-cadherin. Meanwhile, E-cadherin was not altered in HCT116 cells transduced with a Notch-1 shRNA lentiviral construct that inactivated Notch-1 signaling. Finally, levels of the numb protein, a Notch-1 antagonist, appeared to be significantly reduced (five-fold decrease) by constitutive Notch-1 signaling. Alternatively, inactivation of Notch-1 resulted in a four-fold increased expression of the numb protein. Collectively, our data point to the Notch-1 pathway as being a potential key regulator of EMT in colorectal tumors and as such, may be of therapeutic or prognostic utility. Citation Format: Alex Fender, Makenzie Nutter, Timothy Fitzgerald, Fred Bertrand, George Sigounas. Notch-1 regulated stemness and EMT in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1952. doi:10.1158/1538-7445.AM2014-1952

Isolation, propagation, characterization, cryopreservation, and application of novel cardiovascular endothelial cell line from Channa striatus (Bloch, 1793).

There are only few primary endothelial cell cultures developed from fishes to date, but in this work the development of an endothelial cell line from Channa striatus is described. The vascular explants were plated into fibronectin (5 µg ml(-1)) and anti-CD31 antibody (100 ng ml(-1))-coated flask; after 60 h incubation explants were removed from the flask. The flask contained only endothelial and blood cells. Blood cells were cleared out after subsequent passages. The culture medium used was Leibovitz's L-15 supplemented with 20 % serum and antibiotics. The cultures were incubated at 28 °C in a normal atmosphere incubator. The plating efficiency was high (53.72 %). The endothelial cells were cryopreserved at different passage levels and revived successfully with 75-85 % survival. Polymerase chain reaction amplification of mitochondrial 16S rRNA using primer specific to C. striatus confirmed the origin of C. striatus cardiovascular endothelial (CSCVE) cell line from C. striatus. This cell line was further characterized for chromosome number, transfection, mycoplasma, cell cycle distribution, mitochondrial staining, and phagocytic activity. Cells were analyzed according to morphological appearance and expression of specific endothelial markers by fluorescent staining (von Willebrand Factor, anti-platelet endothelial cell adhesion molecule-1, and anti-Endoglin). The formation of tubules in the Matrigel and endothelial co-cultured with fibroblast like cells was observed. The cytotoxicity of ciprofloxacin on the CSCVE cell line was determined by MTT, AB, and R-123 cytotoxicity end points. Susceptibility of CSCVE cell line to nodavirus was confirmed by cytopathic effect and reverse transcriptase-polymerase chain reaction.

Hypothermic Storage of Human Hepatocytes for Transplantation

Transplantation of human hepatocytes is gaining recognition as a bridge or an alternative to orthotopic liver transplantation for patients with acute liver failure and genetic defects. Since most patients require multiple cell infusions over an extended period of time, we investigated hepatic functions in cells maintained in University of Wisconsin solution at 4°C up to 72 h. Eleven different assessments of hepatic viability and function were investigated both pre- and posthypothermic storage, including plating efficiency, caspase-3/7 activity, ammonia metabolism, and drug-metabolizing capacity of isolated hepatocytes. Long-term function, basal, and induced cytochrome P450 activities were measured after exposure to prototypical inducing agents. Cells from 47 different human liver specimens were analyzed. Viability significantly decreased in cells cold stored in UW solution, while apoptosis level and plating efficiency were not significantly different from fresh cells. Luminescent and fluorescent methods assessed phases I and II activities both pre- and post-24-72 h of cold preservation. A robust induction (up to 200-fold) of phase I enzymes was observed in cultured cells. Phase II and ammonia metabolism remained stable during hypothermic storage, although the inductive effect of culture on each metabolic activity was eventually lost. Using techniques that characterize 11 measurements of hepatic viability and function from plating efficiency, to ammonia metabolism, to phases I and II drug metabolism, it was determined that while viability decreased, the remaining viable cells in cold-stored suspensions retained critical hepatic functions for up to 48 h at levels not significantly different from those observed in freshly isolated cells.

Cryopreservation of microencapsulated human hepatocytes

To establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for therapeutic usage in hepatocyte transplantation and bioartificial liver support systems for the treatment of acute and chronic liver diseases,and for experimental usage as an in vitro model of the liver. Adult hepatocytes from 20 human donors undergoing partial hepatectomy were isolated using a two-step extracoporeal collagenase perfusion technique.Seven preincubation time points (2h,6h,12h,24h,36h,48h and 72h) were selected for optimization.After pre-incubation at 4 degrees C for 12-24h in HepatoZYME-SFM (the optimal condition),hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules,transferred to a complete medium containing 10% dimethyl sulphoxide and immediately placed into an isopropanol progressive freezing container for overnight freezing at -80 degrees C followed by immersion in liquid nitrogen the next day.During the post-thawing culture period,the cells were tested for albumin secretion,urea synthesis,cell cycling,transcription and protein synthesis (measuring mRNA and protein levels),and the morphological structure and pathology,for comparison with the features from before microencapsulated cryopreservation (PMC). The viability and plating efficiency of the hepatocytes isolated using the two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0%,respectively.The pre-incubation times of 12h and 24h (viability:61.4+/-4.8% and 62.0+/-5.6%; plating efficiency:3.2+/-5.8% and 62.6+/-3.6%,respectively) showed significantly higher albumin secretion than all other time points tested (F =40.3,all P less than 0.05).Compared with the immediate cryopreservation (immediately frozen control) hepatocytes,the PMC hepatocytes showed significantly better transcription and protein synthesis and higher albumin secretion and urea levels.The PMC group did not show a significantly different level of albumin production from the directly cultured hepatocytes (culture day 2:ll9.2ng/ml vs.131.36ng/ml,P =0.051; day 3:110ng/ml vs.120.4ng/ml,P=0.063; day 4:98.2ng/ml vs.109.8ng/ml,P more than 0.05).However,over culturing days 2,3 and 4,comparison of the PMC hepatocytes to the immediate cryopreservation hepatoeytes showed the former to have significantly higher secretion of albumin (119.2ng/ml vs.101.2ng/ml,110.0ng/ml vs.87.6ng/ml and 98.2ng/ml vs.73.8ng/ml; all P less than 0.05) and urea level (7.83 mug/ml vs.6.79 mug/ml,6.83 mug/ml vs.5.89 mug/ml and 5.85 mug/ml vs.4.83 mug/ml; all P less than 0.05).The post-thawed PMC hepatoeytes showed preservation of the morphological structure,while the immediate cryopreservation hepatocytes did not. The two-step extracorporeal collagenase perfusion technique after partial hepatectomy is a novel,simple,and reliable method for hepatocyte isolation.Pre-incubation at 4 degrees C for 12-24h before the microencapsulation cryopreservation allows for efficient recovery of functional and morphological integrity after thawing and provides viable hepatoeytes that may be useful for clinical applications in pharmacotoxicology,bioartificial liver therapy and cell therapy in humans.

Statil suppresses cancer cell growth and proliferation by the inhibition of tumor marker AKR1B10.

Aldo-keto reductase 1B10 (AKR1B10) is an oncogenic carbonyl reductase that eliminates α,β-unsaturated carbonyl compounds/lipid peroxides and mediates retinoic acid signaling. Targeted inhibition of AKR1B10 activity is a newly emerging strategy for cancer therapy. This study evaluated the inhibitory activity of a small chemical statil towards AKR1B10 and tested its antiproliferative activity in breast (BT-20) and lung (NCI-H460) cancer cells that express AKR1B10. Experimental results showed that statil inhibited AKR1B10 enzyme activity efficiently, with an IC50 at 0.21±0.06 µmol/l. Exposing BT-20 and NCI-H460 cells to statil and diclofenac, a selective AKR1B10 inhibitor, led to dose-dependent inhibition of cell growth and proliferation and plating efficiency. At higher doses (50 µmol/l or higher), statil induced cell death with apoptotic characteristics, such as DNA fragmentation and Annexin-V staining. Furthermore, statil enhanced the susceptibility of cells to acrolein, an active substrate of AKR1B10. Taken together, these data suggest that statil possesses potent antiproliferative activity by inhibiting AKR1B10 activity.

Mixed effects of long-term frozen storage on cord tissue stem cells

Background aimsCord tissue (CT) storage is promoted as an opportunity to preserve a source of mesenchymal stromal cells (MSCs) for future use. We analyzed maximal MSC yields from fresh and frozen CT including functional capacity after long-term cryopreservation as a means of assessing potential utility. MethodsCT was evaluated immediately upon harvest or frozen and banked for 5 years before analysis. Upon thawing, cell viability and yield were determined, as were growth characteristics and the ability to differentiate into various tissues. After thawing, enzymatic digestion of CT to release MSCs resulted in poor cell recoveries and few viable cells, requiring explant cultures to recover sufficient cell numbers for analysis. Upon expansion of surviving cells, fluorescence-activated cell sorter analysis showed the cells to be MSCs based on phenotype (CD34–, 45–, 44+, 90+, 105+) and function (ability to form adipocytes and osteoblasts). Frozen CT, however, exhibited decreased plating efficiency, increased doubling times but near equivalent maximum cell expansion, compared with fresh CT. ConclusionsPoor cell yields and recoveries, along with slower growth characteristics, make frozen CT a less-than-optimal choice for MSC banking, despite good functional recovery. In addition, because the amount of fresh CT available at birth is limited and total MSC yields are low, even fresh CT-MSC requires extensive in vitro expansion before clinical use, which limits it application.

The Effect of High Charge Rate Cycling on Coulombic Efficiency Measurements

Recent work has shown good correlation between short term measurements of the coulombic efficiency and long term capacity retention during relatively slow cycling (greater than 20 hours per cycle) and high temperature (above room temperature) [1-3] using the High Precision Charger built at Dalhousie University which is able to measure coulombic efficiency to < 0.01% error [4]. Smith et al. [5] studied how the charge rate impacts the coulombic efficiency in commercial cylindrical cells of different positive electrodes and graphite negative electrodes at different temperatures. This work showed that the departure of the coulombic efficiency from the ideal value of 1.00000 was dependant on the time of one cycle and independent of charge rate for slow rates and elevated temperatures. This work could not investigate the dependence of coulombic efficiency on high charge rates due to limitations of the equipment.Smith et al. [5] showed that as the charge rate of Li-ion cells was increased from ~C/100 to ~C/20, the coulombic efficiency (CE) increased such that (1 – CE) = k*t, where k is a constant that increases with temperature and t is the time of one charge-discharge cycle. Extending this idea to higher cycle rates means that at very high rates the coulombic efficiency approaches 1.00000 since there is very little time per cycle for degradation. However, it is well known that lithium plating can occur at high charge rates with graphite negative electrodes [6, 7] and this would lower the coulombic efficiency. It has been shown that the coulombic efficiency of plating and stripping metallic lithium from graphite electrodes is 0.97-0.98 [8] compared to the coulombic efficiency of intercalating and de-intercalating lithium from a graphite electrode which is > 0.995 [9]. Therefore as the charge rate continues to increase past the onset of lithium plating, the coulombic efficiency should begin to depart further from 1.00000 as a larger fraction of the lithium is involved in plating/stripping instead of intercalation/de-intercalation reactions. Figure 1 shows CE versus charge rate results for two temperatures that shows the time dependent degradation regime at low rates on the left (low rates) and the regime governed by lithium plating on the right (high rates).This study will show results of high charge rate cycling on Li[Ni1/3Mn1/3Co1/3]O2 (NMC)/graphite cells at different temperatures to more thoroughly understand the shape of these curves and the impact of lithium plating on precision measurements of coulombic efficiency and endpoint capacity slippage. This behavior needs to be well understood as many applications for Li-ion batteries, including electrified vehicles, desire high charge rates.

Loss of Malignancy in Mouse Melanoma Cells by Long-Term Impact of Interferon-Beta In Vitro is Associated with N- and VE-Cadherins Suppression without Inhibition of Expression of EMT-Associated Twist and Slug Proteins

Aim: To study the effect of long-term impact of mouse interferon-beta (IFN-b) on the behavior of mouse melanoma cells in vitro and in vivo and the expression of epithelial-mesenchymal transition (EMT) associated proteins. Materials and Methods: Studies were performed on mouse B16 melanoma cells as a tumor model (MM-4 cell line). Immunocytochemical and tumor cell biology approaches have been used in this study. Results: Long-time treatmentof MM-4 melanoma cellswith low-dose IFN-b (1/2 of IC50) leads to change their morphology, significant inhibition of cell growth and plating efficiency, suppression of cell migration and anchorage-independent growth in semisolid agar. Moreover, IFN-modification of melanoma cells is accompanied by the significant suppression of their malignancy in vivo: growth of tumor induced by IFN-treated cells has inhibited on 50% and growth of metastases - on 90%. Also, IFN-modification of MM-4 cells affects on the expression of proteins involved in cell cycle regulation and inhibits expression of some molecules of adhesion (N-, VE-cadherins), but not influence on the expression of EMT-associated Twist and Slug proteins and E-cadherin. Conclusions: Long-term impact of mouse IFN-beta in low dose on melanoma cells in vitro changes their phenotype and inhibits their proliferative potential, signs of malignancy in vitro, tumorigenicity and metastatic ability in vivo. Loss of malignancy is associated with inhibition of N- and VE-cadherins expression, but not associated with the change of expression and subcellular localization of E-cadherin and EMT transcription factors Twist and Slug.

Strategies for short-term storage of hepatocytes for repeated clinical infusions.

Hepatocyte transplantation is an upcoming treatment for patients with metabolic liver diseases. Repeated cell infusions over 1-2 days improve clinical outcome. Isolated hepatocytes are usually cold stored in preservation solutions between repeated infusions. However, during cold storage isolated hepatocytes undergo cell death. We investigated if tissue preservation and repeated isolations are better than storage of isolated hepatocytes when cold preserving human hepatocytes. Liver tissue obtained from liver surgery or organ donors was divided into two pieces. Hepatocytes were isolated by collagenase digestion. Hepatocytes were analyzed directly after isolation (fresh) or after storage for 48 h at 4°C in University of Wisconsin solution (UW cells). Liver tissue from the same donor was stored at 4°C in UW and hepatocytes were isolated after 48 h (UW tissue cells). Hepatocyte viability and function was evaluated by trypan blue exclusion, plating efficiency, ammonia metabolism, CYP 1A1/2, 2C9, 3A7, and 3A4 activities, phase II conjugation, and apoptosis evaluation by TUNEL assay and caspase-3/7 activities. Hepatocytes stored in UW showed a significantly lower viability compared to fresh cells or hepatocytes isolated from tissue stored for 48 h (54% vs. 71% vs. 79%). Plating efficiency was significantly decreased for cells stored in UW (40%) compared to fresh and UW tissue cells (63% vs. 55%). No significant differences between UW cells and UW tissue cells could be shown for CYP activities or ammonia metabolism. Hepatocytes stored in UW showed a strong increase in TUNEL-positive cells, whereas TUNEL staining in cold-stored liver tissue and hepatocytes isolated after 48 h was unchanged. This observation was confirmed by increased caspase-3/7 activities in UW cells. Although preservation of isolated hepatocytes in UW maintains function, cold storage of liver tissue and repeated hepatocyte isolations is superior to cold storage of isolated hepatocytes in preserving hepatocyte viability and function.

REVIEW ON ELECTROLESS PLATING Ni–P COATINGS FOR IMPROVING SURFACE PERFORMANCE OF STEEL

Electroless plating has been considered as an effective approach to provide protection and enhancement for metallic materials with many excellent properties in engineering field. This paper begins with a brief introduction of the fundamental aspects underlying the technological principles and conventional process of electroless nickel–phosphorus ( Ni – P ) coatings. Then this paper discusses different electroless nickel plating, including binary plating, ternary composite plating and nickel plating with nanoparticles and rare earth, with the intention of improving the surface performance on steel substrate in recent years in detail. Based on different coating process, the varied features depending on the processing parameters are highlighted. Separately, diverse preparation techniques aiming at improvement of plating efficiency are summarized. Moreover, in view of the outstanding performance, such as corrosion resistance, abrasive resistance and fatigue resistance, this paper critically reviews the behaviors and features of various electroless coatings under different conditions.

Maximizing plating density and efficiency for a negative deposition reaction in a flow battery

Flow batteries utilizing a plating reaction as the negative reaction are limited in energy capacity by the available void area in the cell stack. Large scale energy storage applications necessitate maximizing the plating density within the flow battery. Six porous negative electrode configurations using conductive and non-conductive materials are considered for use in a hybrid flow battery. Plating results using a Cu–Fe sulfate chemistry demonstrated a plating density in a carbon felt electrode (145 mAh cm−2 at 40 mA cm−2). Two layered electrode configurations were able to achieve 150 mAh cm−2 of plating density: carbon felt with non-conductive felt, and carbon felt-Daramic-carbon felt. Both electrode designs were tested with the all-iron chemistry using shallow charge/discharge cycles. The carbon felt with non-conducting felt electrode configuration maintained a voltaic efficiency of 81% over six cycles.

Effect of β-lactam antibiotics on plant regeneration in carrot protoplast cultures.

Protoplasts of three carrot cultivars were isolated from in vitro-grown plantlets by overnight incubation in an enzyme mixture composed of 1% (w/v) cellulase Onozuka R-10 and 0.1% (w/v) pectolyase Y-23. After cell immobilization in modified thin alginate layers, three types of β-lactam antibiotics (cefotaxime, carbenicillin, or timentin) at five different concentrations (100, 200, 300, 400, or 500 mg L−1) were added to the culture medium. In 20-d-old cultures, a different number of cell colonies had formed and varied on average from 27 to 56% in carbenicillin- and cefotaxime-containing media, respectively. Supplementation of the culture media with antibiotics at concentrations higher than 100 mg L−1 resulted in a decrease in plating efficiency in comparison with the controls. However, from all antibiotic treatments, except carbenicillin at concentrations of 400–500 mg L−1, efficient plant regeneration occurred. For this reason, we believe that cefotaxime and timentin in the concentrations analyzed here may be used in complex in vitro procedures or valuable carrot cultures as a prophylactic agent for prevention against occasional contaminations.

Irradiated U937 Cells Trigger Inflammatory Bystander Responses in Human Umbilical Vein Endothelial Cells through the p38 Pathway

Radiation-induced bystander effects are a well-known phenomenon that are observed when treating cancer and other diseases after radiotherapy, and even after occupational exposure to radiation. However, little is known about the crosstalk between irradiated macrophages and endothelial cells that line the circulatory system, which may play a role in the development of atherosclerosis. In the current study, we found that the expression of inducible nitric oxide synthase (iNOS) and the intracellular level of nitric oxide (NO) in gamma-irradiated U937 macrophage cells were significantly increased. When human umbilical vein endothelial cells (HUVECs) were co-cultured with gamma-irradiated U937 cells, additional micronuclei (MN) and apoptosis were induced so that the plating efficiency of the bystander HUVECs decreased and P38 was overexpressed in the bystander HUVECs cells. In addition, the contents of vascular cell adhesion molecule 1 (VCAM-1) and the activities of matrix metalloproteinase-9 (MMP-9) in the culture medium of bystander HUVECs were increased. Furthermore, during cell co-culture the adhesive ability of irradiated U937 cells to the bystander HUVECs increased. When U937 cells were treated with 500 μM S-methylisothiourea sulfate (SMT) (iNOS inhibitor) before irradiation, and HUVECs were treated with 10 μM SB203580 (p38 inhibitor) before cell co-culture or treated with 20 μM c-PTIO (NO scavenger) in the co-culture medium, the bystander micronuclei and the amounts of VCAM-1 and MMP-9 in the medium of bystander HUVECs were diminished, and the ability of irradiated U937 cells adhering to HUVECs was also reduced, while the plating efficiency of bystander HUVECs partially recovered. These results demonstrated that irradiated U937 cells appear to release nitric oxide and thereby further trigger apoptosis and inflammatory responses in the bystander HUVECs through a p38-dependent pathway.

Lithium dendrite and solid electrolyte interphase investigation using OsO4

Osmium tetroxide (OsO4) staining, commonly used to enhance scattering contrast in electron microscopy of biologic tissue and polymer blends, has been adopted for studies of graphite anodes in lithium-ion batteries. OsO4 shows a coordinated reaction with components of the solid electrolyte interphase (SEI) and lithium dendrites, thereby increasing material contrast for scanning electron microscopy investigations. Utilizing the high affinity of lithium metal to react with osmium tetroxide it was possible to localize even small lithium deposits on graphite electrodes. In spite of their reaction with the OsO4 fume, the lithium dendrite morphology remains almost untouched by the staining procedure, offering information on the dendrite growth process. Correlating the quantity of osmium detected with the amount of residual (“dead”) lithium of a discharged electrode, it was possible to obtain a practical measure for lithium plating and stripping efficiencies. EDX mappings allowed for a localization of electrochemically stripped lithium dendrites by their residual stained SEI shells. Cross sections, prepared by focused ion beam (FIB) of cycled graphite electrodes treated with OsO4, revealed important information about deposition and distribution of metallic lithium and the electrolyte reduction layer across the electrode.