Scalable optical-to-terahertz converter with a prism-coupled plane-parallel lithium niobate plate.

A nonlinear optical converter of femtosecond laser pulses to terahertz radiation, which combines the tilted-pulse-front pumping and prism coupling techniques, is proposed and experimentally tested. In contrast to the conventional tilted-pulse-front scheme with a prism-shaped LiNbO3 crystal, the converter consists of a plane-parallel LiNbO3 plate sandwiched between two dielectric prisms. One prism is used to couple the pump beam into the LiNbO3 plate, another prism couples the generated terahertz radiation out of the plate. The proposed scheme enables scaling to large-diameter LiNbO3 wafers and large-aperture high-energy pump laser beams resulting in generation of strong terahertz fields. In a proof-of-principle experiment with a 1-mm thick, small size (1 × 2 cm2) LiNbO3 plate pumped by a mJ-class laser, the conversion efficiency comparable to that of the conventional scheme (∼0.1%) was demonstrated.

FGFR3-TACC3 fusion proteins act as naturally occurring drivers of tumor resistance by functionally substituting for EGFR/ERK signaling.

The epidermal growth factor receptor (EGFR) is a clinically validated target in head and neck squamous cell carcinoma (HNSCC), where EGFR-blocking antibodies are approved for first-line treatment. However, as with other targeted therapies, intrinsic/acquired resistance mechanisms limit efficacy. In the FaDu HNSCC xenograft model, we show that combined blockade of EGFR and ERBB3 promotes rapid tumor regression, followed by the eventual outgrowth of resistant cells. RNA sequencing revealed that resistant cells express FGFR3-TACC3 fusion proteins, which were validated as drivers of the resistant phenotype by several approaches, including CRISPR-mediated inactivation of FGFR3-TACC3 fusion genes. Interestingly, analysis of signaling in resistant cell lines demonstrated that FGFR3-TACC3 fusion proteins promote resistance by preferentially substituting for EGFR/RAS/ERK signaling rather than ERBB3/PI3K/AKT signaling. Furthermore, although FGFR3-TACC3 fusion proteins promote resistance of additional EGFR-dependent HNSCC and lung cancer cell lines to EGFR blockade, they are unable to compensate for inhibition of PI3K signaling in PIK3CA-mutant HNSCC cell lines. Validation of FGFR3-TACC3 fusion proteins as endogenous drivers of resistance in our screen provides strong evidence that these fusions are capable of substituting for EGFR signaling. Thus, FGFR3-TACC3 fusion proteins may represent a novel mechanism of acquired resistance in EGFR-dependent cancers of multiple cell lineages.

ROLE OF P38 MAP-KINASE IN THE STRESS-INDUCED SENESCENCE PROGRESSION OF HUMAN ENDOMETRIUM-DERIVED MESENCHYMAL STEM CELLS.

Our recent findings clearly demonstrate that human endometrium-derived mesenchymal stem cells (hMESCs) respond to the sublethal oxidative stress by the premature senescence induction via ÀÒÌ/Chk2/p53/ p21/Rb pathway. Furthermore, based on the application of the SB203580 (SB) we suggested p38 MAP-kinase involvement in senescence progression. However, there are several disadvantages concerning this inhibitor: 1) using SB would not be suitable for in vivo experiments due to toxicity issue; 2) the poor kinase selectivity profile of SB complicates interpretation of the obtained data. Here, in order to confirm the implication of p38 in the H2O2-induced senescence of hMESCs, we applied another highly specific inhibitor of p38 ­ BIRB796 (BIRB). In presence of BIRB we revealed cell size decrease, reduction in the levels of reactive oxygen species, partial restoration of proliferation and increase in Rb phosphorylation levels in comparison to H2O2-treated hMESCs. Summarizing the obtained results we can postulate p38 implication in H2O2-induced senescence of hMESCs, and suggest p38 inhibition as a promising approach in prevention of premature senescence.

Reactive Oxygen Species Are Required for Human Mesenchymal Stem Cells to Initiate Proliferation after the Quiescence Exit.

The present study focuses on the involvement of reactive oxygen species (ROS) in the process of mesenchymal stem cells "waking up" and entering the cell cycle after the quiescence. Using human endometrial mesenchymal stem cells (eMSCs), we showed that intracellular basal ROS level is positively correlated with the proliferative status of the cell cultures. Our experiments with the eMSCs synchronized in the G0 phase of the cell cycle revealed a transient increase in the ROS level upon the quiescence exit after stimulation of the cell proliferation. This increase was registered before the eMSC entry to the S-phase of the cell cycle, and elimination of this increase by antioxidants (N-acetyl-L-cysteine, Tempol, and Resveratrol) blocked G1-S-phase transition. Similarly, a cell cycle arrest which resulted from the antioxidant treatment was observed in the experiments with synchronized human mesenchymal stem cells derived from the adipose tissue. Thus, we showed that physiologically relevant level of ROS is required for the initiation of human mesenchymal stem cell proliferation and that low levels of ROS due to the antioxidant treatment can block the stem cell self-renewal.

[RELATIONSHIP BETWEEN p53/p21/Rb AND MAPK SIGNALING PATHWAYS IN HUMAN ENDOMETRIUM-DERIVED STEM CELLS UNDER OXIDATIVE STRESS].

Human endometrium-derived mesenchymal stem cells (hMESC) under the sublethal oxidative stress induced by H2O2 activate both p53/p21/Rb and p38MAPK/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However the mutual relations between p53/p21/Rb and MAPK signaling pathways, including ERK, p38 and JNK remain unexplored as yet. Here, we used the specific inhibitors--pifithrin-α (PFT), U0126, SB203580 and SP600125 to "switch off" one of the proteins in these cascades and to evaluate the functional status alterations of the rest proteins. Suppression each of the MAPK significantly increased the p53 phosphorylation levels, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation compared with p38 and JNK. These results suppose the existence of the reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. Analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as the ERK antagonists: JNK is capable to activate ERK, while p38 may block the ERK activation. Together, these results demonstrate complex links between different signaling cascades in stressed hMESC, implicating ERK, p38 and JNK in regulation of the premature senescence via p53/p21/Rb pathway.

[OXIDATIVE STRESS-PROMOTED RESPONSES IN HUMAN ENDOMETRIAL STEM CELLS AND LUNG EMBRYONIC FIBROBLASTS].

Human mesenchymal stem cells are an attractive cell source for tissue engineering. During transplantation they may be subjected to oxidative stress due to unfavorable cellular microenvironment, which is characterized by increased levels of reactive oxygen species. Recently, we have demonstrated that oxidative stress responses of human mesenchymal stem cells derived from endometrium (hMESCs) depend upon the oxidizer concentration. Besides, the duration of the H2O2-treatment duration. The effects of the high H2O2 doses on hMESCs and human lung embryonic fibroblasts were compared. In both cell types, H2O2-treatment for 60 min was shown to promote the multiphase cell cycle arrest, as well as to the dose-dependent cell death that occurred equally from all phases of cell cycle. However, the cell death dynamics in hMESCs and fibroblasts were different. Interestingly, in both cell types, shortening of H2O2-treatment duration from 60 to 10 min induced growth retardation, G1-phase accumulation and the cell size increase. Together, these findings allow us to suggest an induction of the premature senescence as a result of the short cell exposure to the high H2O2 doses. Thus, regarding both human endometrial stem cells and human embryonic fibroblasts, shortening of oxidative stress duration induced by high H2O2 doses enables to avoid the cell death and to produce the features of the premature senescence.

Intracellular oxidation of hydroethidine: compartmentalization and cytotoxicity of oxidation products.

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli.

[Differences in defense mechanisms against oxidative stress in both human embryonic and endometrium-derived mesenchymal stem cells].

Oxidative stress has been shown to induce either apoptosis or stress-induced premature senescence (SIPS) in different cell types. At present, it is generally accepted that stem cells have high resistance to oxidative stress; however data reported by various authors are controversial. In this study, we investigated stress responses of human embryonic stem cells (hESC) and human mesenchymal stem cells (hMESC) derived from desquamated endometrium to hydrogen peroxide (H2O2). Cell viability was evaluated by MTT assay. LD50 were determined as 300-350, 350-400 and 600-700 µM for hESC, human embryonic fibroblasts and hMESC, respectively. Thus, among the cell lines studied, hMESC demonstrated the most resistance to increased H2O2 concentration. We have found for the first time that sub-lethal doses of H2O2 induce premature senescence phenotype in hMESC, like in HEF, which is characterized by increased expression of cyclin-dependent kinase inhibitor p21(Waf1/Cip1), an irreversible cell cycle arrest, the permanent loss of proliferative potential, cell hypertrophy and SA-ß-Gal staining. While a sub-lethal H2O2 dose (200 µM) promoted in hMESC only SIPS, the higher H2O2 doses induced also apoptosis in the part of the cell population. On the contrary, in hESC, H2O2 regardless of the doses tested (from 50 to 500 µM) triggered apoptosis, that was the only pronounced response of these cells to oxidative damage. The data obtained demonstrate that stem cells of various origins under oxidative stress utilize the different defense mechanisms: hESC rapidly eliminate damaged cells through apoptosis, whereas hMESC may enter SIPS.

[Comparison of human endometrial stem cells and fibroblasts resistance to oxidative stress].

The response of human endometrial stem cells (hESCs) to oxidative stress has been investigated by flow cytometry. Two terminally differentiated cell lines were used for the comparison: human embryonic lung fibroblasts and human dermal fibroblasts. The oxidative stress was designed by hydrogen peroxide (H2O2) action in the wide range of concentrations (50-1500 microM) during 24 h. It has been shown that the H2O2 amount per one cell (pM/cell), but not H2O2 concentration in the growth medium, should be taken into account for the accurate evaluation of H2O2 effect on different cell lines. Therefore, in our experiments LD50 reflects the amount of H2O2 per cell, at which 50% cells survived after 24 h. We have demonstrated that hESCs are more resistant to H2O2 than embryonic lung fibroblasts, but less resistant than dermal fibroblasts.

[Effect of formaldehyde in low concentrations on the proliferation and organization of the cytoskeleton of cultured cells].

3-4% (1.07-1.42 M) formaldehyde is one of the most popular and well-known organs, tissues and cells fixer. In this manuscript we have shown that formaldehyde in concentrations of up to 60 microM (0.0002%) does not have any negative effect on the viability of cell lines A431, HEK293 and primary rat fibroblasts, but it is also increases the proliferative activity of A431. The influence on A431 cells might be explained by the activation of epidermal growth factor receptors as a result of their interaction with formaldehyde.

[Metalloprotease-mediated HB-EGF release regulates EGF receptor transactivation in A431 cells under oxidative stress].

We have shown earlier that H2O2 induces EGF receptor transactivation in different cells overexpressing EGF receptor. Mechanism of H2O2-induced EGF receptor transactivation in A431 human epidermoid carcinoma cells was examined in this work. We have demonstrated autophosphorylation of Tyr1045, 1068, 1148, 1173 as well as phosphorylation of Tyr845 of EGF receptor in response to H2O2, as assessed by autophosphorylation specific antibody. Tyrosine phosphorylation of EGF receptor by H2O2 did not involve receptor autophosphorylation at Tyr992. Blocking functions of metalloproteases by broad-spectrum inhibitor GM6001 suppressed H2O2-induced phosphorylation of EGF receptor, suggesting dependence of the transactivation on metalloproteases activity. To elucidate the possible role of EGF receptor agonists in its activation we used HB-EGF and TGF-alpha neutralizing antibody. H2O2-induced EGF receptor phosphorylation was inhibited by HB-EGF, but not TGF-alpha, neutralizing antibody. Taken together, our data suggest that, in human epidermoid carcinoma A431 cells, H2O2 stimulates EGF receptor transactivation via metalloprotease-dependent HB-EGF release and autophosphorylation.

[Interferon gamma-mediated growth regulation of epithelial cells].

Interferon gamma (IFNgamma) is known to inhibit proliferation of certain transformed cell lines. Recently, we have demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to IFNgamma (Burova et al., 2007) and provided direct evidence for the dependence of IFNgamma-induced EGFR transactivation upon EGFR expression level in epithelial cells (Gonchar et al., 2008). This study examines an antiproliferative effect of IFNgamma on human epithelial cells lines A431 and HeLa which express high levels of EGFR, as well as HEK293, which expresses low levels of EGFR. We characterized the IFNgamma-induced changes in these cells by studying cell growth, the cell cycle and induction of apoptosis. The response to IFNgamma differed in the tested cell lines: cell growth was inhibited in both A431 and HeLa cells, but not in HEK293 cells, as shown by cell counts and MTT. The cell cycle phases analyzed by flow cytometry were disturbed in A431 and HeLa cells in response to IFNgamma. In contrast, IFNgamma treatment did not alter distribution by cell cycle phases in HEK293. Our results indicate that IFNgamma exhibit an antiproliferative effect depending on the increased expression of EGFR in A431 and HeLa cells. Further, it was demonstrated that IFNgamma induced the caspase 3 activation in A431 cells, suggesting an involvement of active caspase 3 in IFNgamma-induced apoptosis.

[The level of EGF receptor expression effects its transactivation by IFN gamma in epithelial cells].

Earlier, we demonstrated transactivation of the epidermal growth factor receptor (EGFR) in response to interferon gamma (IFNgamma) in epidermal carcinoma A431 cells. It was shown that IFNgamma-induced EGFR transactivation is impossible in some cancer epithelial cells. Here, we hypothesize that IFNgamma-dependent EGFR transactivation in these cells correlates with EGFR quantity on the cell surface. To test this suggestion, a line of stably transfected HEK293 cells (HEK293delta99 cells) expressing high level of mutant EGFR lacking 99 C-terminal residues has been established. HEK293delta99 cells demonstrated EGFR transactivation in response to IFNgamma unlike the parent HEK293 cells, in which transactivation lacked. In HEK293delta99 and A431 cells, the time courses of EGFR activation induced by IFNgamma have the same pattern. In HEK293delta99 cells like A431, IFNgamma-induced EGFR transactivation requires EGFR kinase activity and occurs via autophosphorylation mechanism. Taken together, these data provide direct evidence of the dependence of IFNgamma-induced EGFR transactivation upon EGFR expression level in epithelial cells.

Whither... the future for contact dermatitis? A report from the 2007 International Review of Current Problems in Contact Dermatitis.

In our report we summarize presentations made at the International Review of Current Problems in Contact Dermatitis meeting which took place at the St John's Institute of Dermatology, London, on 1 June 2007, and which brought together over 100 dermatologists from the U.K., continental Europe and the U.S.A. During this small and informal meeting, the state-of-the art lectures on various aspects of contact dermatitis were followed by energetic discussions.

[Oxidized glutathione induces activation of the epidermal growth factor receptor and MAP kinases ERK 1,2].

Ligand-independent activation ("transactivation") of the epidermal growth factor receptor (EGFR) was demonstrated upon cell stimulation with cytokines, activators of G-protein-coupled receptors and various stressors. Recently, we showed transactivation of EGFR and activation of transcription factor STAT3, rather than STAT1, induced by glutathione disulfide (GSSG) and glutoxim in epidermoid carcinoma A431 cells (Burova et al., Dokl. Akad. Nauk., 2005, 404: 1-3). Glutoxim (PHARMA-VAM, Moscow) is a pharmacological synthetic analogue of GSSG, whose therapeutic use as an immunomodulator has been permitted. In this study, we investigated dynamics of EGFR activation upon A431 cell stimulation with GSSG and glutoxim. The time course of activation has a sinuous pattern. It has been shown that the intrinsic EGFR tyrosine kinase is responsible for the receptor phosphorylation induced by GSSG and glutoxim. Here, we also demonstrated the activation of ERK 1,2 upon treatment of A431 cells and HER14 cells (HIN 3T3 fibroblasts transfected with full-length EGFR) with these drugs. ERK 1,2 activation was abolished by AG1478, a pharmacological inhibitor of EGFR tyrosine kinase, implicating intrinsic EGFR tyrosine kinase in this process.

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications.

In recent years, recombinant adenoviral and adeno-associated viral (AAV) vectors have been exploited in a number of gene delivery approaches. The use of these vectors in clinical gene transfer has increased the demand for their characterization, production and purification. Although the classical method of adenovirus or AAV purification by density gradient centrifugation is effective on a small scale, chromatographic separation is the most versatile and powerful method for large-scale production of recombinant adenovirus or AAV. This review describes different chromatographic modes for adenovirus or AAV purification and process development, as well as the utility of different purification steps for virus production. Advances in the development of viral vectors for gene therapy, such as the discovery of new AAV serotypes, adenoviral and AAV retargeting and improved production of helper-dependent adenoviral vectors, require further development of efficient purification methods.

[Effect of nocodazole on the activation of transcription factors STAT1 and STAT3 in A431 cells]. / Vliianie nokodazola na aktivatsiiu transkriptsionnykh faktorov STAT1 i STAT3 v kletkakh A431.

The STAT transcription factors (signal transducers and activators of transcription), STAT1 and STAT3, are involved in signal transduction from growth factors and different cytokine receptors. STAT1 and STAT3 activation mechanisms are not sufficiently investigated, but they are known to depend upon both cell type and stimulus for either of them. Recently, we have shown that nocodazole blocked EGF-induced STAT1 transport to the nucleus. Here, we have compared STAT1 and STAT3 activation in response to IFNgamma, IFNalpha and epidermal growth factor (EGF) in A431 cells. We have shown the STAT1 activation by all these agents; unlike, STAT3 was activated by EGF only. STAT1 and STAT3 activation upon EGF is blocked by both nocodazole and Src-kinase family inhibitor. STAT1 activation upon IFNgamma influence is blocked by nocodazole, but does not depend on the activity of Src-family kinases. The increased STAT3 phosphorylation results from a combined action of Src-kinase inhibitor and IFNgamma. IFNalpha-induced activation of STAT1 was not inhibited by either nocodazole or Src-kinase inhibitor. Taken together, the data obtained suggest that the activation of both STAT1 and STAT3 in A431 cells is accomplished by different mechanisms.