Functionalization of graphenic surfaces by oxygen plasma toward enhanced wettability and cell adhesion: experiments corroborated by molecular modelling.

Graphenic materials attract huge attention because of their outstanding properties, and have a wide range of applications as, i.e., components of biomaterials. Due to their hydrophobic nature, however, the surfaces need to be functionalized to improve wettability and biocompatibility. In this study, we investigate the functionalization of graphenic surfaces by oxygen plasma treatment, introducing surface functional groups in a controlled way. The AFM images and LDI-MS results clearly show that the graphenic surface exposed to plasma is decorated with -OH groups, whereas the surface topography remains intact. The measured water contact angle decreases significantly after oxygen plasma treatment from 99° to ca. 5°, making the surface hydrophilic. It is also reflected in the surface free energy values which increase from 48.18 mJ m-2 to 74.53 mJ m-2 when the number of surface oxygen groups reaches 4 -OH/84 Å2. The DFT (VASP) molecular models of unmodified and oxygen-functionalized graphenic surfaces were constructed and used for molecular interpretation of water-graphenic surface interactions. The computational models were validated by comparison of the theoretically determined water contact angle (based on the Young-Dupré equation) to the experimentally determined values. Additionally, the VASPsol (implicit water environment) results were calibrated against the explicit water models that can be used in further research. Finally, the biological role of functional groups on the graphenic surface was examined in terms of cell adhesion with the use of mouse fibroblast cell line (NIH/3T3). The obtained results illustrate the correlation between surface oxygen groups, wettability, and biocompatibility providing the guidelines for the molecular level-driven design of carbon materials for various applications.

Role of integrons in the proliferation of multiple drug resistance in selected bacteria occurring in poultry production.

1. The increase in microbial resistance, and in particular multiple drug resistance (MDR), is an increasing threat to public health. The uncontrolled use of antibiotics and antibacterial chemotherapeutics in the poultry industry, especially in concentrations too low to cause inhibition, and the occurrence of residues in feed and in the environment play a significant role in the development of resistance among zoonotic food-borne microorganisms.2. Determining the presence and transmission methods of resistance in bacteria is crucial for tracking and preventing antibiotic resistance. Horizontal transfer of genetic elements responsible for drug resistance is considered to be the main mechanism for the spread of antibiotic resistance.3. Of the many well-known genetic elements responsible for horizontal gene transfer, integrons are among the most important factors contributing to multiple drug resistance. The mechanism of bacterial drug resistance acquisition through integrons is one of the essential elements of MDR prevention in animal production.

Stage-specific embryonic antigen-4 as a novel marker of ductal cells of human eccrine sweat glands.

BACKGROUND: Different populations of unipotent or multipotent stem cells have been identified in human epidermis and its appendages. It is well documented that these cells maintain tissue homeostasis and actively participate in epidermal regeneration after injury. However, there is no evidence of the presence of pluripotent stem cells in human epidermis. OBJECTIVES: In this study we investigated whether cells positive for embryonic stem cell marker stage-specific embryonic antigen-4 (SSEA-4) are present in adult human epidermis and, if so, whether they are pluripotent and correspond to the population of primitive stem cells. METHODS: The expressions of SSEA-4 and pluripotency transcription factors were analysed using flow cytometry. By means of immunohistochemical staining, we studied the exact localization of these cells in sections of human skin. RESULTS: We show that a population of SSEA-4+ cells is present in human epidermis. In contrast to the commonly accepted belief, the expression of SSEA-4 is not connected with the pluripotent character of isolated cells. We found that these SSEA-4+ cells are localized in the ducts of eccrine sweat glands. CONCLUSIONS: Our results indicate that SSEA-4 is a novel marker identifying the ductal cells of human sweat glands. The surface character of the antigen provides for a simple method of isolating this cell population and suggests applications of SSEA-4 for future cell therapy research.

Mycophenolic acid affects basic functions of human keratinocytes in the IMPDH-dependent manner.

Clinical studies suggest that the immunosuppressant MPA is associated with impaired wound healing. It is believed that the main cause of impairment is the inhibition of inflammatory response. However, it is unknown whether MPA may directly affect epidermal cells. The aim of our study was to examine the direct influence of mycophenolic acid, the selective blocker of de novo purine synthesis, on human epidermal keratinocyte morphology, proliferation, motile activity, and differentiation in in vitro culture. The number of keratinocytes cultured in the presence of MPA was counted and cell motility was measured by a time-lapse computer-aided method. Cell morphology was determined by flow and image cytometry methods. Real-time RT-PCR analysis was employed to investigate the expression of markers of differentiation. We showed that MPA induces irreversible inhibition of cell proliferation, causes cell enlargement and impairs cell locomotion in a time-dependent manner. The level of expression of differentiation markers was significantly reduced by MPA treatment. All these effects were reversed by the addition of guanine. Our results indicated that MPA impairs basic functions of human skin keratinocytes via intracellular guanosine nucleotide depletion, which may be directly reflected in wound healing problems in patients treated with this immunosuppressant.

Gene expression and protein distribution of leptin and its receptor in bovine oocytes and preattachment embryos produced in vitro.

Many investigations point out the important role of leptin during the preimplantation development. Transcripts for the leptin gene (LEP) and its receptor (LEPR) have been identified in several tissues related to reproduction (e.g. ovaries, testis and oviduct) in both human and mouse. This work shows for the first time the expression and distribution patterns of LEP and LEPR in bovine oocytes and in vitro-produced embryos. Gene expression was analysed by reverse transcription PCR and real-time PCR, and the proteins were localised by immunostaining. This study included immature and mature oocytes, zygotes, two-, four-, eight- to 16-cell embryos, morulae and blastocysts and the LEP transcript was identified throughout all stages of bovine preimplantation development. However, mRNA for the LEPR gene was detected at all stages, excluding four-cell embryos. Expression of both LEP and LEPR genes was reduced at the eight- to 16-cell stage. This in addition to the absence of LEPR mRNA in four-blastomere embryos may suggest that maternally derived transcripts degenerate towards the eight- to 16-cell stage coinciding with embryonic genome activation at eight- to 16-cell stage and subsequent appearance of embryonic mRNA. Immunofluorescent staining demonstrated that LEP and LEPR proteins form a spherical rim beneath the oolemma. After maturation, however, the proteins became evenly distributed within the cytoplasm. In two- to eight-cell embryos, fluorescence was observed in the apical surface of the blastomeres, and from 10- to 16-cell stage in the apical region of outer blastomeres. This pattern persisted to the blastocyst stage, leading to LEP and LEPR distribution within trophoblast cells, but not in the inner cell mass. These results support previous findings on polar distribution of proteins within mammalian oocytes and embryos, as well as suggests leptin's potential role during early mammalian development and implantation.

Polymorphism of the porcine leptin gene promoter and analysis of its association with gene expression and fatness traits.

We describe for the first time a 245 bp fragment of the porcine leptin gene promoter in the proximity of the transcription start site. Altogether, 720 pigs were screened with the PCR-SSCP technique for polymorphism in this region. Four SNPs, segregating as two haplotypes, have been identified, one of them (C113G) in the putative consensus site for the AP-2 transcription factor. This polymorphism was evenly distributed in the Duroc breed (n=21) and was absent in the Polish Landrace (n=248) and Pietrain breed (n=12). In the Polish Large White (n=191) and synthetic line 990 (n=243), allele G occurred with a very low frequency. The investigation was performed to test if the C113G SNP affects leptin mRNA level in subcutaneous fat and leptin protein concentration in serum. Additionally, the effect of this polymorphism on fatness traits was statistically analyzed. Although there was a trend toward decreased expression in GG animals, the differences were not significant between genotypes. We also found no evidence for an association of the LEP promoter genotype with the analyzed fatness traits.

Short communication: effect of leptin gene polymorphisms on breeding value for milk production traits.

New molecular techniques focused on genome analysis open new possibilities for complex evaluation of economically important traits in farm animals. Milk production traits are typical quantitative characteristics controlled by a number of genes. Mutations in their sequences may alter animal performance as well as their breeding values. In this study, we investigated the effect of 3 restriction fragment length polymorphisms (RFLP): HphI, Kpn2I, and Sau3AI in the leptin gene, on bull breeding values for milk, fat, and protein yield, and fat and protein content. One hundred seventeen Polish Black and White AI bulls were genotyped. Pedigree analysis indicated a relatively close relationship between the bulls. Statistical analysis indicated that the HphI polymorphism has a significant effect on milk and protein yield. Animals with the TT genotype had approximately 2x higher estimated breeding values for milk and protein yields. No effect was found for the other 2 polymorphisms.

Directional movement of rat prostate cancer cells in direct-current electric field: involvement of voltagegated Na+ channel activity.

A two-part hypothesis has been tested, which proposes that (1) prostate cancer cells are galvanotactic (i.e. respond to an electric field by moving directionally) and (2) voltage-gated Na+ channel activity, which was shown previously to be expressed specifically by strongly metastatic cells, controls galvanotaxis. Two well-defined rat ('Dunning') cell lines, originally derived from the same prostate tumour but differing markedly in their metastatic ability, were used. Cells were exposed to exogenous direct-current electric fields of physiological strength (0.1-4.0 V cm(-1)), their reactions were recorded by light microscopy and analysed by a quantitative tracking method. Voltage-gated Na+ channel activity was modulated pharmacologically using a range of concentrations of a specific channel blocker (tetrodotoxin) or an opener (veratridine). The results showed that the highly metastatic MAT-LyLu cells responded to the application of the electric field strongly by migrating towards the cathode. By contrast, the weakly metastatic AT-2 cells gave no such response. Tetrodotoxin suppressed the galvanotactic response of the MAT-LyLu cells whereas veratridine enhanced it. Both compounds had little effect on the AT-2 cells. These results are consistent with functional voltage-gated Na+ channel expression occurring specifically in highly metastatic cells. This is also the first demonstration of control of galvanotaxis, in any cell type, by voltage-gated Na+ channel activity. The possible underlying mechanisms and the in vivo relevance of these findings are discussed.

Contact-activated migration of melanoma B16 and sarcoma XC cells.

During migration, tumour cells interact with neighbouring neoplastic and normal host cells, and such interaction may influence their motile activity. We investigated the effect of homotypic collisions on the motile activity of two tumour cell lines, mouse melanoma B16 and rat sarcoma XC, and nontransformed human skin fibroblasts. It was found that the tumour cells show only limited motile activity when moving as single cells without contact with neighbours. At a higher density of the culture (and also at a greater number of cell to cell contacts) the activation of motility of investigated tumour cells was observed. On the other hand, the normal human skin fibroblasts showed a typical reaction of density-dependent inhibition of motility. The motile activity of tumour cells was not affected by conditioned media and was visibly dependent on a direct physical contact among colliding cells. The activation of cell movement was observed about 40-50 min after the initial contact between tumour cells. Contact-activated migration of neoplastic cells was inhibited by 50 microM verapamil (a selective voltage-gated calcium channel inhibitor) and 10 microM gadolinium chloride (a nonspecific blocker of mechanosensitive ion channels) but not by pertussis toxin. The observation that homotypic collisions among tumour cells strongly increase their motile activity suggests that contact-activated migration may play a significant role in tumour invasion and metastasis.

Homotypic cell-cell contacts stimulate the motile activity of rat prostate cancer cells.

OBJECTIVE: To characterize the effect of homotypic cell-to-cell collisions upon the motile activities of two rat prostatic cancer cell lines of markedly different metastatic potential. MATERIALS AND METHODS: The movements of strongly and weakly metastatic MAT-LyLu and AT-2 cells, respectively, were recorded under an inverted microscope at 37 degrees C. The motile activities of the cells at various cell densities were characterized quantitatively by computer-aided tracking methods and image analysis. The following variables were assessed: speed of movement, final displacement, coefficient of movement efficiency, diffusion constant and positive flow. RESULTS: MAT-LyLu and AT-2 cells showed only limited motile activity in sparse cultures where there was little contact amongst the cells. However, under these and all other subsequent conditions tested, the motile activity of the MAT-LyLu cells was higher than the AT-2 cells. As the density of the cultured cells was increased (leading to more cell-to-cell contacts) there was a significant increase in motility. This effect was more pronounced for the AT-2 than for the MAT-LyLu cells, resulting in visible acceleration of movement by direct physical contact among the colliding cells. The motile activities of the tumour cells was only slightly affected by conditioned media. CONCLUSION: Homotypic collisions between migrating prostatic cancer cells can strongly stimulate their motility. The effect of increased contact is greater on the weakly metastatic cells, such that at high cell density, the difference in the motilities of weak and strong metastatic cells is greatly reduced.

Contact-mediated acceleration of migration of melanoma B16 cells depends on extracellular calcium ions.

The escape of malignant cells from primary tumour and their active migration to the surrounding tissues are among the most important steps in the metastatic process. During migration, tumour cells interact with neighbouring neoplastic and normal cells and such interactions may affect their motile activity. We investigated the effect of extracellular calcium ions on migration of mouse melanoma B16 cells stimulated by homotypic cell-to-cell contacts. It was found that the decreasing of extracellular Ca2+ influx into B16 cells by lowering Ca2+ concentration in culture medium, or by the application of 0.5 mM La3+ (non-selective inorganic Ca2+ channels blocker), reduced the contact-mediated acceleration of migration of melanoma cells but only slightly affected the basal motile activity of non-stimulated single, separated cells moving without contacts with neighbouring ones in sparse culture. Since it was suggested that contact-mediated acceleration of migration of melanoma B16 cells may be controlled by mechanosensitive and/or voltage-gated ion channels, the presented data support the concept that these channels may affect cell migration by regulation of extracellular Ca2+ influx into stimulated cell.

Immediate and long-term galvanotactic responses of Amoeba proteus to dc electric fields.

The long-term and immediate galvanotactic responses of Amoeba proteus to the direct current electric fields (dcEFs) were studied with the methods of computer-aided image analysis. It was found that in contrast to earlier reports, amoebae continued locomotion towards cathode (the negative pole) for hours and the increase in the field strength in the range 300-600 mV/mm caused the straightening of cell trajectories accompanied by the decreased frequency of the lateral pseudopods formation and lesser change in the speed of cell movement. In the cell regions pointing to the anode, the formation of new pseudopodia was prevented and the higher cEFs strength the more extended were the regions in which formation of new pseudopods was inhibited. Replacement of calcium with magnesium in the extracellular medium reduced the galvanotactic cell responses. Research on the localisation and kinetics of the primary cell responses to the dcEF or to change in its direction revealed that the primary cell responses occurred at the anode oriented cell regions. The cell response to the field reversal appeared to be localised and to take place in less than 1 sec. First the retraction and withdrawal of the anode-directed pseudopodium was observed whereas the uroid (cell tail) moved for 10-40 sec in the original direction before it begun to react to the field reversal. The exposure of amoebae to the dcEFs sensitised them to the reversion in the field direction and induced an acceleration of cell responses. The results presented are difficult to reconcile with the attempt to explain the cell galvanotaxis as a consequence of the membrane protein lateral electrophoresis or electroosmosis. It is suggested that the lateral electrophoresis of ions and the modification of ionic conditions at the vicinity of ion channels may be involved in the induction of fast responses of cells to external dcEFs.

Morphometric analysis of pancreatic carcinoma by computer-assisted image analysis.

This report describes the results of applying the interactive image analysis system for the measurement of some cytological parameters corresponding to features of adenocarcinoma of the pancreas. The present experiments were carried out by means of the digital cell image analysis of haematoxilyn and eosin stained archival standard glass slides of cancer bearing and healthy patients. Four different parameters describing the morphology of nuclei and nucleoli were selected to quantitate the differences between control and malignant tissues: area, perimeter, elongation, and extension. The parameters that showed the greatest differences between cancerous and normal pancreas were: area and elongation in the case of nuclei as well as area and perimeter for nucleoli. However, the results of this study suggest that none of the four analysed parameters can be selected alone to discriminate neoplastic from normal cells, but could be used all together in diagnosis of pancreatic cancer.

Morphometric analysis of LPS-stimulated human monocytes by computer-assisted image analysis.

This report describes the results of applying the computer-assisted image analysis system for the measurement of some cytological parameters of LPS-stimulated and nonstimulated human monocytes. The experiments were carried out by means of the digital cell image analysis of haematoxilyn stained monocytes. Five different parameters describing the morphology of monocytes and their nuclei were selected to quantitate the differences between control and activated cells area, perimeter, elongation, dispersion, and extension of images of cell projections. The results suggest that all of the analysed parameters can be used to discriminate stimulated from nonstimulated monocytes which permits detailed monitoring of the changes in cell morphology during monocyte activation.

Contact of sarcoma cells with aligned fibroblasts accelerates their displacement: computer-assisted analysis of tumour cell locomotion in co-culture.

The shape and locomotion of rat sarcoma XC cells on glass, polystyrene, and confluent monolayer cultures of aligned human skin fibroblasts were studied with quantitative, computer-assisted methods. The cell shape depended upon the substratum; the sarcoma cells seeded on fibroblasts assumed polarized shapes. The tumour cells emigrating from aggregates and in sparse cultures showed random locomotion when plated on glass or on the polystyrene surface of tissue culture dishes in isotropic conditions. However, when sarcoma cell aggregates were plated onto underlying aligned fibroblasts, the sarcoma cells showed contact guidance, migrating along the long axes of fibroblasts. Simultaneously, suppression of migration normal to the axis of fibroblasts orientation was observed. The sarcoma cells displaced a few times faster on aligned fibroblasts than under isotropic conditions in control cultures. This fast displacement was found to result from the less frequent cell turnings and straightening of cell trajectories (i.e., from klinokinesis), and not from an acceleration of cell movement and the longer cell tracks (i.e., not from orthokinesis). The presented results support the suggestion of Abercrombie (M. Abercrombie. 1979. Nature (London). 281: 259-262.) that tumour cells may be guided by the underlying normal cells when invading surrounding tissues and forming metastases.

Isolation, spreading, locomotion on various substrata, and the effect of hypotonicity on locomotion of fish keratinocytes.

The paper describes improved methods for the isolation of fish skin keratinocytes, which spread and locomote 15 min after trypsinization, in the absence of extracellular matrix proteins. The random locomotion of these keratinocytes under isotropic conditions on glass, plastic (polystyrene), and glass covered with poly-L-lysine or collagen IV was studied with computer-aided methods. Several methods for quantitative description of random cell locomotion were compared. The values of some parameters commonly computed showed non-Gaussian distribution. A comparison of keratinocyte locomotion under isotonic and hypotonic conditions revealed that the hypotonic conditions increased cell displacement (net migration) owing to the klinokinetic and not the orthokinetic effect.

Chemotaxis of Amoeba proteus in the developing pH gradient within a pocket-like chamber studied with the computer assisted method.

A new "U" shaped, pocket-like chamber was used to observe the chemotactic responses of individual cells. This method permits monitoring of both the development of the concentration gradient of a tested substance and cell locomotion. We investigated the chemotactic responses of Amoeba proteus and observed that the amoebae moved in positively and negatively developing [H+] gradients towards the solution of lower pH in a pH range 5.75-7.75. The chemotactic response of amoebae to [H+] gradients required the presence of extracellular calcium ions. It was blocked and random locomotion was restored by the replacement of calcium with magnesium in the cell medium. Time-lapse video recording and data processing were accomplished with computer-assisted methods. This made it possible to compare selected methods of data presentation and analysis for cells locomoting in isotropic and anisotropic conditions. The cell trajectories were determined and displayed in circular diagrams, lengths of cell tracks and final cell displacements were estimated and a few parameters characterizing cell locomotion were computed.

Activation of macrophage-like cells by multiple grooved substrata. Topographical control of cell behaviour.

We studied the influence of substrata topography on the behaviour of murine P388D1 macrophage cell line. Cells were plated on plain fused silica substrata or substrata with microfabricated grooves of varying depth and width. Cell spread area, elongation, orientation and F-actin content were measured on plain substratum and 6 sets of gratings. The speed and persistence of cell movement were also studied. We found that patterned substrata substantially activated cell spreading and elongation and significantly increased the persistence and speed of cell movement, shallow grooves being more effective than deep ones. The contact of cells with micropatterned substrata significantly increased the F-actin content in cells. The sensitivity of LPS (lipopolisaccharide) stimulated and unstimulated macrophages to topographical cues was also compared.

Anchorage-dependent growth factor(s) produced by rat sarcoma (XC) cells.

The autocrine growth factor(s) was isolated from serumfree conditioned medium of rat sarcoma (XC) cells. Autocrine activity was enriched by ultrafiltration using Amicon YM 10 membrane, extraction with 1 M acetic acid and partially purified (650-fold) by chromatography on Bio-Gel P-100 and P-60. The final recovery of the autocrine factor(s) was 4 micrograms from 1800 ml of the conditioned medium (a yield of 6%). The factor(s) with molecular weight 6-10 kDa was heat and acid stable but inactivated by trypsin and dithiothreitol. It stimulated anchorage-dependent (but not anchorage-independent) growth of XC cells as well as untransformed, established lines of rat (NRK) and mouse (3T3) cells. The results obtained may suggest that autocrine factor(s) produced by XC cells can be one of EGF-like or/and insulin-like growth factors.

3H-thymidine incorporation method versus colony forming assay in the determination of anchorage-independent cell growth of rat sarcoma (XC) and Morris hepatoma 7777 (MH) cells.

The modification of 3H-thymidine incorporation method of Tanigawa was used to the estimation of anchorage-independent growth of virally and chemically transformed rat cells. The relationship between colony forming assay and 3H-TdR incorporation test was determined, depends on the composition of culture medium and the period of incubation of rat sarcoma (XC) cells with thymidine. The influence of exogenous mitogens (RFG, TGF beta 1 and insulin) and autocrine factor (at different step of purification) on the growth of Morris hepatoma 7777 (MH) cells was estimated by both methods. Regression analysis comparing the results of colony counting and thymidine incorporation revealed good correlation between the two methods. The modification can be used the determination of growth stimulating or growth inhibiting activity and in multistep purification procedure of autocrine growth factors.