Fresh DMEM Research Articles

Sinonasal Stent Coated with Sustained-Release Varnish of Mometasone Furoate Inhibits Pro-Inflammatory Cytokine Release from Macrophages: An In Vitro Study

The aim of the study was to develop a sustained-release varnish (SRV) containing mometasone furoate (MMF) for sinonasal stents (SNS) to reduce mucosa inflammation in the sinonasal cavity. The SNS' segments coated with SRV-MMF or an SRV-placebo were incubated daily in a fresh DMEM at 37 °C for 20 days. The immunosuppressive activity of the collected DMEM supernatants was tested on the ability of mouse RAW 264.7 macrophages to secrete the cytokines' tumor necrosis factor α (TNFα) and interleukin (IL)-10 and IL-6 in response to lipopolysaccharide (LPS). The cytokine levels were determined by respective Enzyme-Linked Immunosorbent Assays (ELISAs). We found that the daily amount of MMF released from the coated SNS was sufficient to significantly inhibit LPS-induced IL-6 and IL-10 secretion from the macrophages up to days 14 and 17, respectively. SRV-MMF had, however, only a mild inhibitory effect on LPS-induced TNFα secretion as compared to the SRV-placebo-coated SNS. In conclusion, the coating of SNS with SRV-MMF provides a sustained delivery of MMF for at least 2 weeks, maintaining a level sufficient for inhibiting pro-inflammatory cytokine release. This technological platform is, therefore, expected to provide anti-inflammatory benefits during the postoperative healing period and may play a significant role in the future treatment of chronic rhinosinusitis.

The inhibition of pancreatic cancer progression by PI3K-activated MSC proteomes

Background and Hypothesis:Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a very poor survival rate. The PI3K pathway has been studied extensively in cancer cells which has led to the creation of PI3K inhibitor drugs. To explore an uncharted and counterintuitive use of the PI3K pathway, PI3K was activated in mesenchymal stem cells (MSCs) and the action of their conditioned medium (CM) on PDAC was examined. We hypothesized that PI3K-activated MSC-derived CM is capable of suppressing tumor growth and migration.
 Project Methods:To generate the treatment conditioned media, we first isolated and grew MSCs in DMEM + 10% serum and added 50 μM of PI3K activator (YS49) and incubated for 24 hours. Afterwards, we exchanged the media with fresh DMEM + 10% serum and incubated for another 24 hours. Next, we hyper-centrifuged the media to extract the conditioned media and used the media to treat PANC-1, PA03C, ASPC-1, PANC10.05, and PANC198 pancreatic cancer cell lines. In vitro viability and migration assays were conducted on these five treated cell lines. The regulatory mechanism was evaluated using Western blotting, immunoprecipitation, and ELISAs.
 Results:PI3K-activated MSC-derived CM reduced the proliferation (N=5) and migration (N=2) of PDAC (p < 0.01). MSC CM was enriched with polyubiquitin C (Ubc), which acted as an extracellular tumor-suppressing protein by interacting with CXCR4. While the anti-tumor efficacy of Ubc differed depending on the CXCR4 expression level of PDAC, MSC CM was commonly effective in the tested cell lines and tissues (N=5).
 Conclusion and potential impact:Collectively, this study demonstrated that MSC CM can be converted into a tumor-suppressing agent by the activation of PI3K, and a Ubc-CXCR4 regulatory axis is at least in part responsible. The use of MSC CM treatment can potentially serve as an alternative or supplemental treatment to current chemotherapeutic agents in treating pancreatic cancer without inducing life-threatening side effects.

Fresh Medium or L-Cystine as an Effective Nrf2 Inducer for Cytoprotection in Cell Culture.

The Nrf2 gene encodes a transcription factor best known for regulating the expression of antioxidant and detoxification genes. A long list of small molecules has been reported to induce Nrf2 protein via Keap1 oxidation or alkylation. Many of these Nrf2 inducers exhibit off-target or toxic effects due to their nature as electrophiles. In searching for non-toxic Nrf2 inducers, we found that a culture medium change to fresh DMEM is capable of inducing Nrf2 protein in HeLa, HEK293, AC16 and MCF7 cells. Testing the components of DMEM led to the discovery of L-Cystine as an effective Nrf2 inducer. L-Cystine induces a dose-dependent increase of Nrf2 protein, from 0.1 to 1.6 mM. RNA-seq analyses and RT-PCR revealed an induction of multiple Nrf2 downstream genes, including NQO1, HMOX1, GCLC, GCLM, SRXN1, TXNRD1, AKR1C and OSGIN1 by 0.8 mM L-Cystine. The induction of Nrf2 protein was dependent on L-Cystine entering cells via the cystine/glutamate antiporter and the presence of Keap1. The half-life of Nrf2 protein increased from 19.4 min to 30.9 min with 0.8 mM L-Cystine treatment. L-Cystine was capable of eliciting cytoprotection by reducing ROS generation and protecting against oxidant- or doxorubicin-induced apoptosis. As an amino acid derivative, L-Cystine is considered a non-toxic Nrf2 inducer that exhibits the potential for protection against oxidative stress and tissue injury.

Cytoplasmic and Nuclear HER4 Expression in HER2 Negative Breast Cancer Cell Lines

HER4 cleavage and its subcellular localisations have been previously proven to mediate anti-HER2 resistance. The description of the HER4 subcellular localisations in HER2 negative breast cancer, on the other hand, is incomplete. The objective of this study was to determine the cytoplasmic and nuclear expression of HER4 in HER2 negative breast cancer cell lines in order to gain a better understanding of the key features of HER4 signalling in the context of anti-HER2 resistance. MCF-7 and MDA-MB-231 cells were cultured for 48 h at 37 °C and 5% CO2 in fresh DMEM medium with 10% foetal bovine serum and 1% penicillin-streptomycin. The western blot analysis for HER4 protein was done on cytoplasmic and nuclear extracts that had been obtained previously using the NE-PER Nuclear and Cytoplasmic Extraction Kit. Cytoplasmic and nuclear HER4 proteins were expressed in a variety of sizes, including 120 kDa, 55 kDa, 50 kDa, and 43 kDa. MCF-7 cells expressed significantly more cytoplasmic HER4120kDa than MDA-MB-231 cells. MCF-7 exhibited a single nuclear HER4 variant with a molecular weight of 50 kDa, whereas MDA-MB-231 expressed two nuclear HER4 variants with molecular weights of 50 kDa and 43 kDa. In comparison to MDA-MB-231 cells, MCF-7 cells exhibit higher level of cytoplasmic HER4120kDa with positive nuclear HER450kDa expression. Due to the presence of ER and PR in MCF-7, it is important to investigate if the interaction of these HER4 variants with ER and PR confers resistance to anti-HER2 treatment on breast cancer. Meanwhile, results from MDA-MB-231 cells indicate that nuclear HER4 contributes to the development of TNBC.

Trophoblast Stem-Cell-Derived Exosomes Improve Doxorubicin-Induced Dilated Cardiomyopathy by Modulating the let-7i/YAP Pathway

Trophoblast stem cells (TSCs) have been confirmed to play a cardioprotective role in heart failure. However, whether TSC-derived exosomes (TSC-exos) can protect against cardiac injury remains unclear. In the present study, TSC-exos were isolated from the supernatant of TSCs using the ultracentrifugation method and characterized by transmission electron microscopy and western blotting. Utilizing the public Gene Expression Omnibus (GEO) database, we found that let-7i and Yes-associated protein 1 (YAP) could participate in the development of heart failure. In vitro, AC16 cardiomyocytes subjected to doxorubicin (DOX) were treated with TSC-exos or let-7i mimic. Flow cytometry showed that TSC-exos and let-7i both decreased cardiomyocyte apoptosis. In vivo, mice that were intraperitoneally injected into DOX received either PBS, TSC-exos, or AAV9-let7iup for let-7i overexpression. Mice receiving TSC-exos and AAV9-let7iup showed improved cardiac function and decreased inflammatory responses, accompanied by downregulated YAP signaling. Mechanistically, TSC-exos could transfer let-7i to cardiomyocytes and silence the YAP signaling pathway. In conclusion, TSC-exos could alleviate DOX-induced cardiac injury via the let-7i/YAP pathway, which sheds new light on the application of TSC-exos as a potential therapeutic tool for heart failure.

Lipophilic tracer Dil and fluorescence labeling of acridine orange used for Leishmania major tracing in the fibroblast cells

BackgroundThis study aims to evaluate the use of fluorescent dye Dil and super vital dye acridine orange (AO) in vitro tracking of labeled L. major in the fibroblast cells. MethodsDil crystal and AO were used to stain L. major in a co-culture of the fibroblasts with the parasite. AO staining solution was added to 1 × 106 parasites. After 10 min, the stained parasites were centrifuged and washed seven times with phosphate buffered saline (PBS). The stained promastigote was incubated with fibroblasts for 6–8 h. The presence of stained parasites with AO in the fibroblast was assessed using a fluorescence microscope. 1 × 106/mL promastigote of L. major was gently suspended and mixed by Dil staining solution with an ultimate concentration of 0.002 μg/mL and it was kept for 20 min at the room temperature. Subsequently, after washing it in PBS for seven times, it was centrifuged at 3000 rpm for 10 min. The supernatant was removed and the precipitate containing stained promastigote was suspended in fresh DMEM F12 with fibroblasts at 37 °C for 6 h. The presence of stained parasites with Dil in fibroblast was assessed using a fluorescence microscope. Fibroblast characterization was undertaken by a real-time polymerase chain reaction (PCR). ResultsAcridine orange staining assisted in detection of the live parasite in the fibroblast cells. Free promastigote looked green before entering into the fibroblasts after 12 h culture. The parasite entered the cytoplasm of fibroblasts at the beginning of the exposure and gradually entered the nucleus of the fibroblast. The fibroblast nucleus was entirely stained green by AO. The L. major appeared green under the fluorescent microscope. Dil staining revealed that the internalized parasites with red/orange color were localized within the cytoplasm after 6-hours and the nucleus of the fibroblasts after 72-hours following culture. Human fibroblasts were positive at the expression of CD10, CD26, matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-3 (MMP-3) and negative for CD106 and integrin alpha 11. ConclusionThe fluorescent dye Dil staining is a safe, easy to use, inexpensive and fast method for labeling of the Leishmania parasite in the fibroblast cells. Acridine orange staining could be useful for tracing the parasites in the fibroblasts too. In this study, both Dil and AO were compared and considered as suitable vital dyes for identifying labeled Leishmania in the fibroblast in vitro, but Dil was superior to AO with its feature does not transfer from the labeled to unlabeled cells.

Assessment of biological functions for C3A cells interacting with adverse environments of liver failure plasma

BackgroundFor its better differentiated hepatocyte phenotype, C3A cell line has been utilized in bioartificial liver system. However, up to now, there are only a few of studies working at the metabolic alternations of C3A cells under the culture conditions with liver failure plasma, which mainly focus on carbohydrate metabolism, total protein synthesis and ureagenesis. In this study, we investigated the effects of acute liver failure plasma on the growth and biological functions of C3A cells, especially on CYP450 enzymes. MethodsC3A cells were treated with fresh DMEM medium containing 10% FBS, fresh DMEM medium containing 10% normal plasma and acute liver failure plasma, respectively. After incubation, the C3A cells were assessed for cell viabilities, lactate dehydrogenase leakage, gene transcription, protein levels, albumin secretion, ammonia metabolism and CYP450 enzyme activities. ResultsCell viabilities decreased 15%, and lactate dehydrogenase leakage had 1.3-fold elevation in acute liver failure plasma group. Gene transcription exhibited up-regulation, down-regulation or stability for different hepatic genes. In contrast, protein expression levels for several CYP450 enzymes kept constant, while the CYP450 enzyme activities decreased or remained stable. Albumin secretion reduced about 48%, and ammonia accumulation increased approximately 41%. ConclusionsC3A cells cultured with acute liver failure plasma showed mild inhibition of cell viabilities, reduction of albumin secretion, and increase of ammonia accumulation. Furthermore, CYP450 enzymes demonstrated various alterations on gene transcription, protein expression and enzyme activities.

Isolation and expansion of OCT4/Sox2 specific cytotoxic T lymphocytes in vitro.

e14578 Background: The main reason for tumor MDR (multidrug resistance ,MDR) is the presence of CSC(cancer stem cells,CSC). In the CSC of lung cancer, OCT4 and Sox2 are closely related to the tumor MDR. Therefore, in the current study, we introduced a method for the generation of OCT4+/Sox2+ CSC specific CTL(cytotoxic T lymphocytes,CTL) through co-culture with CD154+ feeder layer cells. Methods: Lenti/CD154 was constructed. The NIH3T3 cells were incubated with Lenti/CD154 (103 vp per cell) for 24 h at 37 °C and 5% CO2. Then the NIH3T3 cells were subsequently maintained in fresh complete DMEM medium. Lenti/GFP was used as a control. On the 6th day post infection, the infection rate was determined by flow cytometry after incubation with CD154-PE antibody. The feeder layer cells were co-cultured with CD40B cells and the biomarkers of CD40B cells were determined by flow cytometry. The CTL were treated with OCT4 plus Sox2 antigen and then the biomarkers of CTL were examined by flow cytometry and IL-2 and IFNγ were determined by ELISA. Results: The rate of CD154+ NIH3T3 cells was 99.7%, which was higher than that of uninfected-cells (14.8%). After co-culture with CD154+ NIH3T3 cells, the rates of CD40B expressing CD80, CD86 and HLA-A were significantly increased. OCT4 plus Sox2 treatment further increased the CD3, CD4 and CD8 expression and elevated the secretion of IL-2 and IFNγ. After co-culture of CTL with MDR PC9-drived cancer stem cells (CSC) and parental PC9 cells at different ratio, CTL significantly killed the tumor cells compared with control. Conclusions: The generation and expansion of OCT/Sox2 specific CTL is a feasible method for the treatment of lung cancer.

Analysis of clusterin expression changes as a biomarker of osteoarthritis

Purpose: The discovery and validation of arthritis-related biomarkers and establishment of methodology for proteomic studies in osteoarthritis (OA) are needed. Proteomics strategies have identified many proteins that may relate to pathological mechanisms of OA, however targeted approaches are required to validate the roles of these proteins. This study aimed to use mass spectrometry and western blotting to identify peptides from several proteins in the secretome of chondrocytes, cartilage explants and osteochondral biopsies treated with inflammatory cytokines over a 2-week period, to evaluate their potential as biomarkers of OA progression. Methods: Healthy cartilage was obtained from fetlock joints of skeletally mature horses, euthanized for unrelated veterinary reasons. Cartilage explants were isolated using a 6 mm biopsy, with discs placed into wells (3 discs per 1 ml DMEM + 1% Pen/Strep) before incubation for 24 hours (37 °C, 5% CO2). After this equilibration period, the media was removed and replaced with either fresh DMEM + 1% Pen/Strep or DMEM supplemented with 1% Pen/Strep containing TNFα and IL-1β both at 10ng/ml. Explants were culture for 7–14 days with the cytokines replaced every 4th day. For cell based assays chondrocytes were isolated from tissue using 70U pronase for 1hr at 37 °C and overnight digestion at 37°C using a 0.2% collagenase II solution. The cell suspension was filtered and washed before being seeded into culture flasks and cultured until confluence was reached (37°C, 5% CO2). Once cultures were established cells were split into two groups: healthy control (DMEM supplemented with 1% Pen/Strep and 10% foetal calf serum) or stimulated cells (DMEM as above plus TNFα and IL-1β both at 10ng/ml). Chondroyctes were cytokine-stimulated for up to one week. Cells were used in experiments up to the 2nd passage. Results: Mass spectrometry data showed that peptides representative of clusterin were found to decrease following 7 days of inflammatory stimulation. Western blotting of secreted proteins in media of cartilage explants or chondrocyte showed that clusterin expression was reduced following 7 days of cytokine treatment. Catabolic matrix metalloproteinase enzymes MMP1, MMP3 and MMP13, as well the matrix component cartilage oligomeric protein (COMP) were all found to have an increased abundance in the media of the cytokine treated samples. This data was supported by qPCR for clusterin gene expression which showed initially mRNA levels increased 3 day after inflammatory stimulation but expression was lost after 7 days. Western blotting of media from the osteochondral biopsies showed an increase in clusterin expression after 7 days of inflammatory stimulation however clusterin protein expression could not be detected after 14 days of treatment, indicating a delayed response compared to cartilage tissue alone. Conclusions: The equine chondrocytes, cartilage explant and osteochondral biopsy models exhibited highest clusterin secretion in untreated cultures. IL-1β and TNFα treatment caused a reduction in clusterin secretion. Clusterin acts as a chaperone to aid protein refolding in situations of stress and is constitutively secreted by mammalian cells. IL-1β and TNFα appear to interrupt clusterin secretion and therefore the protection it may offer healthy functioning cells. Previous studies have reported variable data, with some studies indicating a decrease in clusterin in OA, while others indicate an increase in clusterin expression. Our results suggest the clusterin increases immediately after inflammatory stimulation but is lost after prolonged exposure. Therefore, levels of secreted clusterin may be a candidate biomarker for OA progression.

Collagen matrix stiffness influencing on fibroblast contraction force

Event Abstract Back to Event Collagen matrix stiffness influencing on fibroblast contraction force Tianrong Jin1*, Richard C. Siow2*, Li Li2 and Kuo-Kang Liu1* 1 University of Warwick, School of Engineering, United Kingdom 2 King’s College London, Cardiovascular Division, Faculty of Life Sciences and Medicine, United Kingdom Introduction: Fibroblast-seeded collagen gel which constructs a 3D bio-artificial extracellular matrix (ECM) has been widely used in wound healing, tissue engineering and regenerative medicine. Previous studies using fibroblast-populated collagen based assay to measure cell contractility were limited to examine the radius changes of gel and hence accurate determination of cell contraction force was difficult to achieve[1],[2]. Up to now, little has been known about how the stiffness of collagen matrix influences on cell contraction force. We have recently developed a novel nano-indentation device to measure the gel elasticity and in combination with a mathematical model, the cell contraction force can be determined accurately based on the measured gel thickness and area[3]. In this study, we apply the new technique to investigate the effect of gel stiffness on cell contraction force in a quantitative manner. Materials and Methods: Human Aortic Adventitial Fibroblast (HAoAF) at Passage 7-10 were used to seed into collagen gel matrix. Type I collagen diluted with sterile water, resulting the final concentration of 1.5, 2.0 and 2.5 mg/ml, was mixed with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 5% FCS. The collagen matrix suspension was transferred into 35 mm Petri dishes (1.2 ml/dish) and incubated for 20 minutes to polymerise. After incubation overnight, the culture medium was replaced with fresh DMEM containing 5% FCS. Some dishes were then stimulated with the agonist histamine (100 µM) to elicit cell contraction. A depth-sensing indentation device (shown as Figure 1) is used to measure thickness and Young’s modulus of the cell-embedded collagen gel. The system has ultimate force and displacement resolutions of 10 nN and 100 nm, respectively. The gel indentation was performed at a controlled speed of 40 µm/sec to generate Force-displacement (F-d) curve. The first 30% of F-d curve was extracted to determine Young’s modulus (E) by fitting with a non-linear strain dependent elasticity model. The cell contraction force (F) can be calculated by the following equation. F = πE(h0+h1)(r0-r1) where h and r with subscript 0 and 1 represent the thickness and radius of disk shaped collagen gel at beginning and end of contraction. Results and Discussion: Figure 2(a) shows overall contraction force of cells decreased as the collagen concentration increases. Meanwhile, the contraction forces were nearly doubled when cells were treated with histamine (100 µM). Figure 2(b) demonstrates a linear decrease in cell contraction force as the gel concentration increases. Figure 2(c) shows the Young’s modulus of gels increases in a higher collagen concentration. The results suggest that the concentration of collagen matrix regulates the fibroblast contraction force in response to the stimulation of agonist Histamine. The findings also show cells response differently to the stiffness change of collagen matrix. As a result, the Young’s modulus of gel matrix should be considered as a key parameter to assess the cell contraction force. Conclusion: In 3D collagen matrix, the cells exhibit different contractility in response to the matrix stiffness change. The finding confirms the mechanical property of collagen matrix should be considered in the cellular contraction events induced by drugs. Overall the study has shown the importance of matrix stiffness in the design of collagen-based biomaterial for clinical applications.

Citotoxicity evaluation of three dental adhesives on vero cells in vitro.

BackgroundTo evaluate, in vitro, the potential cytotoxicity of three different dental adhesives systems (Adper Single Bond 2 -SB, Silorane System Adhesive Bond -SSAB and Single Bond Universal -SBU) on cultivated Vero cells after different contact times.Material and MethodsThe cells were cultured in a concentration of 2 x 105 cells/mL for 24h and grown to sub-confluent monolayers. VERO cells were exposed to 25µl of conditioned extracts obtained from 24h, 48h and 72h immersion of adhesive samples in culture medium (DMEM), immediately after polymerization. Fresh DMEM was used as negative control. Cell metabolism was evaluated by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyl-tetrazolium bromide). The data were analyzed statistically by ANOVA, considering a significance of 5%.ResultsThe values of cell viability ranged from 94.2% at 72h (SBU) to 109.6% at 48h (SB). The mean percentage of viability after exposure to the extracts of SB, SSAB and SBU were 103.2%, 100.63% and 97.43%, respectively. There was no statistically significant difference (p= 0.342) between the experimental and negative control groups.ConclusionsAt all exposure times, all adhesives tested in this study presented no cytotoxicity to Vero cells in vitro. Key words:Biocompatibility, cytotoxicity, dental adhesives, Vero cells.

Arresting a Torsin ATPase Reshapes the Endoplasmic Reticulum

Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures.

Scavenger receptor-BI is a receptor for lipoprotein(a)

Scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that binds a variety of lipoproteins, including high density lipoprotein (HDL) and low density lipoprotein (LDL), but lipoprotein(a) [Lp(a)] has not been investigated as a possible ligand. Stable cell lines (HEK293 and HeLa) expressing human SR-BI were incubated with protein- or lipid-labeled Lp(a) to investigate SR-BI-dependent Lp(a) cell association. SR-BI expression enhanced the association of both (125)I- and Alexa Fluor-labeled protein from Lp(a). By confocal microscopy, SR-BI was also found to promote the internalization of fluorescent lipids (BODIPY-cholesteryl ester (CE)- and DiI-labeled) from Lp(a), and by immunocytochemistry the cellular internalization of apolipoprotein(a) and apolipoprotein B. When dual-labeled ((3)H-cholesteryl ether,(125)I-protein) Lp(a) was added to cells expressing SR-BI, there was a greater relative increase in lipid uptake over protein, indicating that SR-BI mediates selective lipid uptake from Lp(a). Compared with C57BL/6 control mice, transgenic mice overexpressing human SR-BI in liver were found to have increased plasma clearance of (3)H-CE-Lp(a), whereas mouse scavenger receptor class B type I knockout (Sr-b1-KO) mice had decreased plasma clearance (fractional catabolic rate: 0.63 ± 0.08/day, 1.64 ± 0.62/day, and 4.64 ± 0.40/day for Sr-b1-KO, C57BL/6, and human scavenger receptor class B type I transgenic mice, respectively). We conclude that Lp(a) is a novel ligand for SR-BI and that SR-BI mediates selective uptake of Lp(a)-associated lipids.

In vitro transdentinal effect of low-level laser therapy

Low-level laser therapy (LLLT) has been used for the treatment of dentinal hypersensitivity. However, the specific LLL dose and the response mechanisms of these cells to transdentinal irradiation have not yet been demonstrated. Therefore, this study evaluated the transdentinal effects of different LLL doses on stressed odontoblast-like pulp cells MDPC-23 seeded onto the pulpal side of dentin discs obtained from human third molars. The discs were placed in devices simulating in vitro pulp chambers and the whole set was placed in 24-well plates containing plain culture medium (DMEM). After 24 h incubation, the culture medium was replaced by fresh DMEM supplemented with either 5% (simulating a nutritional stress condition) or 10% fetal bovine serum (FBS). The cells were irradiated with doses of 15 and 25 J cm−2 every 24 h, totaling three applications over three consecutive days. The cells in the control groups were removed from the incubator for the same times as used in their respective experimental groups for irradiation, though without activating the laser source (sham irradiation). After 72 h of the last active or sham irradiation, the cells were evaluated with respect to succinic dehydrogenase (SDH) enzyme production (MTT assay), total protein (TP) expression, alkaline phosphatase (ALP) synthesis, reverse transcriptase polymerase chain reaction (RT-PCR) for collagen type 1 (Col-I) and ALP, and morphology (SEM). For both tests, significantly higher values were obtained for the 25 J cm−2 dose. Regarding SDH production, supplementation of the culture medium with 5% FBS provided better results. For TP and ALP expression, the 25 J cm−2 presented higher values, especially for the 5% FBS concentration (Mann–Whitney p < 0.05). Under the tested conditions, near infrared laser irradiation at 25 J cm−2 caused transdentinal biostimulation of odontoblast-like MDPC-23 cells.

Molecular Mechanisms Underlying the Apoptotic Effect of KCNB1 K+ Channel Oxidation

Potassium (K(+)) channels are targets of reactive oxygen species in the aging nervous system. KCNB1 (formerly Kv2.1), a voltage-gated K(+) channel abundantly expressed in the cortex and hippocampus, is oxidized in the brains of aging mice and of the triple transgenic 3xTg-AD mouse model of Alzheimer's disease. KCNB1 oxidation acts to enhance apoptosis in mammalian cell lines, whereas a KCNB1 variant resistant to oxidative modification, C73A-KCNB1, is cytoprotective. Here we investigated the molecular mechanisms through which oxidized KCNB1 channels promote apoptosis. Biochemical evidence showed that oxidized KCNB1 channels, which form oligomers held together by disulfide bridges involving Cys-73, accumulated in the plasma membrane as a result of defective endocytosis. In contrast, C73A-mutant channels, which do not oligomerize, were normally internalized. KCNB1 channels localize in lipid rafts, and their internalization was dynamin 2-dependent. Accordingly, cholesterol supplementation reduced apoptosis promoted by oxidation of KCNB1. In contrast, cholesterol depletion exacerbated apoptotic death in a KCNB1-independent fashion. Inhibition of raft-associating c-Src tyrosine kinase and downstream JNK kinase by pharmacological and molecular means suppressed the pro-apoptotic effect of KCNB1 oxidation. Together, these data suggest that the accumulation of KCNB1 oligomers in the membrane disrupts planar lipid raft integrity and causes apoptosis via activating the c-Src/JNK signaling pathway.

Insights into Ectodomain Shedding and Processing of Protein-tyrosine Pseudokinase 7 (PTK7)

The membrane PTK7 pseudokinase, a component of both the canonical and noncanonical/planar cell polarity Wnt pathways, modulates cell polarity and motility in biological processes as diverse as embryo development and cancer cell invasion. To determine the individual proteolytic events and biological significance of the ectodomain shedding in the PTK7 function, we used highly invasive fibrosarcoma HT1080 cells as a model system. Current evidence suggested a likely link between PTK7 shedding and cell invasion in our HT1080 cell model system. We also demonstrated that in HT1080 cells the cleavage of the PTK7 ectodomain by an ADAM proteinase was coupled with the membrane type-1 matrix metalloproteinase (MT1-MMP) cleavage of the PKP(621)↓LI site in the seventh Ig-like domain of PTK7. Proteolytic cleavages led to the generation of two soluble, N-terminal and two matching C-terminal, cell-associated fragments of PTK7. This proteolysis was a prerequisite for the intramembrane cleavage of the C-terminal fragments of PTK7 by γ-secretase. γ-Secretase cleavage was predominantly followed by the efficient decay of the resulting C-terminal PTK7 fragment via the proteasome. In contrast, in HT1080 cells, which overexpressed the C-terminal PTK7 fragment, the latter readily entered the nucleus. Our data imply that therapeutic inhibition of PTK7 shedding may be used to slow cancer progression.

Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (K(d) = ∼54 nm). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.

Cytotoxicity evaluation of Curcuma zedoaria (Christm.) Roscoe fluid extract used in oral hygiene products

Objective. This in vitro study evaluated the cytotoxic effects of the Curcuma zedoaria (Christm.) Roscoe (popular name: zedoary) fluid extract, as used in preparations for oral hygiene, mostly for anti-septic purposes. Materials and methods. The cell viability and cell growth were assessed by Trypan blue dye exclusion assay using the LMF cell line derived from oral mucosa. Cell viability (short-term assay) was measured 0, 6, 12 and 24 h after contact with the fluid extract. Cell growth (long-term assay) was analyzed in 1, 3, 5 and 7 days. The experimental groups were those testing the fluid extract obtained from the zedoary rhizome and the extractor liquid (ethanol 70° GL) in the concentrations of 0.01–0.0001% v/v. Fresh DMEM were used in the control cultures. Results. Short-term assay—all studied cultures maintained stable cell viability; Long-term assay—there was progressive cell growth in all studied cultures. Conclusion. According to the results, the zedoary fluid extract presents low cytotoxicity and probably can be used in the oral hygiene products.

Auto-activation of c-JUN Gene by Amino Acid Deprivation of Hepatocellular Carcinoma Cells Reveals a Novel c-JUN-mediated Signaling Pathway

Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. This study demonstrates that in hepatocellular carcinoma cells, expression of c-JUN, JUN-B, c-FOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, c-JUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in c-JUN expression was greater in transformed cells compared with nontransformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of c-JUN transcription was ATF4-independent. The increased expression of c-JUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of c-JUN-ATF2-activated heterodimers was increased after AA limitation, and c-JUN or ATF2 knockdown suppressed the induction of c-JUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing c-JUN protein by JNK and subsequent auto-activation of the c-JUN gene by recruitment of c-JUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the c-JUN gene can function as transcriptional amino acid-response elements.

Versican targeting by RNA interference suppresses aggregative growth of dermal papilla cells

Dermal papilla cells (DPCs) are specialized fibroblasts found in the hair follicle papilla, which are associated with the development and cycle regulation of hair follicles (HFs). DPCs exhibit a multilayer aggregative growth character, which is closely related to induction of HF formation. Versican, a large chondroitin sulphate proteoglycan and one of the major components of the extracellular matrix, is involved in the formation of HF. To confirm the relationship between versican and the aggregative growth of DPCs, we first induced and established an aggregative cell model in DPCs in vitro, with cells taken to passage 8. Simultaneously, aggregative passage 2 DPCs and nonaggregative passage 8 DPCs were selected as parallel controls. RNA interference (RNAi) targeted to versican was used in passage 2 DPCs using a lentiviral vector. Reverse transcriptase (RT)-PCR and western blotting were used to assay the expression of versican in DPCs. RNAi targeted to versican efficiently suppressed the aggregative growth of passage 2 DPCs, and the inhibitory effect was significant 3 days after RNAi treatment. The mRNA and protein levels of versican were also downregulated in passage 2 DPCs, and were lower than levels in nonaggregative passage 8 DPCs. Notably, the aggregative growth of nonaggregative passage 8 DPCs was restored after induction in a 1 : 1 v/v mixture of fresh DMEM and medium recycled from a previous passage. Versican is a key gene for the aggregative growth of DPCs, and might be significant in the regeneration of HF.