Retention Assay Research Articles

The effect of the surface properties of poly(methyl methacrylate) on the attachment, adhesion and retention of fungal conidia

Poly(methyl methacrylate) (PMMA) surfaces, (commercial PMMA (PMMAc), spin coated PMMA (PMMAsc) and a 90% methylmethacrylate/10% 3-methacryloxypropyltrimethoxysilane random copolymer (P(MMA-co-gMPS)) were used to determine the effect of surface properties on conidia biofouling. The contact angles of the substrates demonstrated that the PMMAsc and the P(MMA-co-gMPS) polymer (62.8°) were more wettable than the PMMAc surface (71.0°). The PMMAsc had the greatest roughness value (32.0 nm) followed by the PMMAc (3.0 nm), then P(MMA-co-gMPS) (1 nm). Aspergillus niger 1957 conidia were spherical, smooth and hydrophobic (12.1%). Aspergillus niger 1988 conidia were spherical with spikes and hydrophobic (17.1%). Aureobasidium pullulans was elliptical with longitudinal ridges and hydrophilic (79.9%). Following attachment assays, cPMMA attached the greatest numbers of conidia. Following the adhesion and retention assays (washing step included in the protocol), A. niger 1957 and A. niger 1988 were least adhered to the P(MMA-co-gMPS) surface, whilst A. pulluans was least adhered to the PMMAsc surface. This work demonstrated that in the absence of a washing step, only the surface properties influenced the conidia attachment, whilst in the presence of a washing step, both the properties of the surfaces and the conidia affected conidia adhesion and retention. Hence, the methodology used (with or without a washing step) should reflect the environment in which the surface is to be applied.

Zoledronic Acid Inhibits the RhoA-mediated Amoeboid Motility of Prostate Cancer Cells.

The bisphosphonate Zoledronic acid (ZA) is a potent osteoclast inhibitor currently used in the clinic to reduce osteoporosis and cancer-induced osteolysis. Moreover, ZA exerts an anti-tumor effect in several tumors. Despite this evidence, the relevance of ZA in prostate cancer (PCa) is not completely understood. To investigate the effect of ZA administration on the invasive properties of PC3 cells, which are characterised by RhoA-dependent amoeboid motility. The effect of ZA administration on the in vitro invasive properties of PC3 cells was evaluated by cell migration in 3D collagen matrices, immunofluorescence and Boyden assays or transendothelial migration. Lung retention and colonization assays were performed to assess the efficacy of ZA administration in vivo. PC3 cells are characterised by RhoA-dependent amoeboid motility. We now report a clear inhibition of in vitro PC3 cell invasion and RhoA activity upon ZA treatment. Moreover, to confirm a specific role of ZA in the inhibition of amoeboid motility of PC3 cells, we demonstrate that ZA interferes only partially with PC3 cells showing a mesenchymal phenotype due to both treatment with conditioned medium of cancer associated fibroblasts or to the acquisition of chemoresistance. Furthermore, we demonstrate that ZA impairs adhesion to endothelial cells and the trans-endothelial cell migration, two essential properties characterising amoeboid motility and PC3 metastatic dissemination. In vivo experiments prove the ability of ZA to inhibit the metastatic process of PC3 cells as shown by the decrease in lung colonization. This study demonstrates that ZA inhibits Rho-dependent amoeboid motility of PC3 cells, thus suggesting ZA as a potential therapy to impede the metastatic dissemination of PC3 cells.

Tet38 of Staphylococcus aureus Binds to Host Cell Receptor Complex CD36-Toll-Like Receptor 2 and Protects from Teichoic Acid Synthesis Inhibitors Tunicamycin and Congo Red.

Using an affinity column retention assay, we showed that the purified Tet38 membrane transporter of Staphylococcus aureus bound specifically to host cell CD36 and to the complex CD36-Toll-like receptor 2 (TLR-2), but not to TLR-2 alone or TLR-2 and S. aureus lipoteichoic acid (LTA). We tested the effect of LTA on the internalization of S. aureustet38 mutant QT7 versus RN6390 by A549 epithelial cells. Addition of anti-LTA antibody to the bacteria prior to adding to A549 cells reduced internalization of QT7 2-fold compared to that with nonspecific antibody treatment. QT7 internalized 4- to 6-fold less than RN6390 with or without anti-LTA antibody. These data suggested that Tet38 and LTA were independently involved in the invasion process. The wall teichoic acid (WTA) inhibitor tunicamycin had an 8-fold decrease in activity with overexpression of tet38 and a 2-fold increase in activity in QT7 (tet38). Reserpine (an inhibitor of efflux pumps) reduced the effect of tet38 overexpression on tunicamycin resistance 4-fold. In addition, tet38 affected growth in the presence of LTA inhibitor Congo red, with overexpression increasing growth and deletion of tet38 reducing growth. In conclusion, Tet38 contributes to S. aureus invasion of A549 via direct binding to CD36 of the complex CD36-TLR-2, and LTA independently bound to TLR-2. The reduction of tunicamycin resistance in the presence of reserpine and the survival ability of the tet38 overexpressor in the presence of Congo red suggest that Tet38 can also protect the synthesis of LTA and WTA in S. aureus against their inhibitors, possibly functioning as an efflux pump.

Construction and Characterization of a Medium Copy Number Expression Vector Carrying Auto-Inducible dps Promoter to Overproduce a Bacterial Superoxide Dismutase in Escherichia coli.

Medium copy number expression vector and auto-inducible promoter could be a solution for producing recombinant therapeutic proteins in industrial scale regarding plasmid stability, cost, and product quality. This work aimed to construct a medium copy number pBR322-based expression vector carrying auto-inducible promoter, determine its ability to express heterologous gene, and study its segregational stability. Three stationary-phase promoters of Escherichia coli genes (gadA, dps and sbmC) were used to produce a superoxide dismutase from Staphylococcus equorum (rMnSODSeq) coding region from pBR322Δtet (pBR322-mini). Four plasmids were constructed with different promoters, i.e., T7 (pBMsod), gadA (pMCDsod), dps (pCADsod), and sbmC (pCDSsod) using pBR322-mini as backbone. Results showed that rMnSODSeq expression from pBMsod was significantly higher than that from pJExpress414sod (high copy number plasmid). Meanwhile, rMnSODSeq from pCADsod (auto-inducible promoter) was as high as from pBMsod (IPTG-inducible T7 promoter). rMnSODSeq expressed from pCADsod when bacterial cells entered stationary phase appeared as an active protein band of 23.5kDa when analyzed by zymography and SDS-PAGE. pCADsod displayed the highest stability compared with pBMsod and pJEXpress414sod by plasmid retention assay. We demonstrate the use of an auto-inducible dps promoter to express high level of heterologous protein, an SOD of S. equorum, from a stable expression vector with medium copy number.

Regulation of ABCB1 activity by microRNA-200c and microRNA-203a in breast cancer cells: the quest for microRNAs' involvement in cancer drug resistance.

Aim: ABCB1 is a major player in cancer drug resistance. The purpose of this study was to functionally assess the regulation of ABCB1 activity in a doxorubicin-resistant breast cancer cell line by miR-200c and miR-203.Methods: Human breast carcinoma cell lines MCF-7 (Doxorubicin-sensitive and not expressing ABCB1) and KCR (Doxorubicin-resistant and expressing ABCB1) were used to evaluate the expression levels of miR-200c and miR-203 by Real-time quantitative PCR (RT-qPCR). The effects of transient ectopic expression of miRNA-200c and miR-203 on the expression of ABCB1 in KCR and MCF-7 cells was verified by RT-qPCR and Western Blot. The extrusion activity of the ABCB1 pump was analyzed by fluorescence microscopy and flow cytometry through fluorescence substrate retention assays (DiOC2) in the presence and absence of the ABCB1 inhibitor verapamil.Results: RT-qPCR results indicated a 100,000-fold increase in ABCB1 mRNA expression levels in KCR cells compared to MCF-7 cells, and is inversely correlated with the expression of miR-203 and miR-200c. The insertion of miR-200c and miR-203 led to a higher retention of DiOC2 within KCR cells, and slightly reduced the protein levels of ABCB1 in KCR cells, although the high initial expression of ABCB1 masked the reduction in protein levels. The increased intracellular accumulation of the fluorescent due DiOC2 in the presence of the ABCB1 inhibitor verapamil correlated with the inhibition caused by miR-203 and miR-200c in transfected cells.Conclusion: The present study demonstrates that miR-200c and miR-203 exert a negative modulating effect on the activity of ABCB1 associated with doxorubicin resistance.

Cellular Retention Assay to Determine the Interaction Frequency of CD19-directed Chimeric Antigen Receptor (CAR) Engineered Cells against CD19+ Leukemic Cells

Cellular Retention Assay to Determine the Interaction Frequency of CD19-directed Chimeric Antigen Receptor (CAR) Engineered Cells against CD19+ Leukemic Cells

Spatial and temporal variation of three biomarkers in Mytilus edulis

Environmental conditions and xenobiotic exposure can be sources of stress to living organisms. Biological markers are measurable indicator of changes which may happen at any biological level and which can be considered an early warning signal of some biological or environmental state or condition. A structured field study was undertaken to investigate the relationship between three biomarker assays and the spatial and temporal variation of each biomarker in samples of Mytilus edulis. The three biomarkers were the neutral red retention assay, micronucleus assay and comet assay, which indicate damage at different cellular/molecular levels. Three sites in Poole Harbour, an area on the South coast of the UK were sampled on six separate occasions at least three weeks apart. The results for the comet assay showed a significant difference between sites and between sampling dates whereas the results for the other two assays did not show a significant difference for either factor. There was no significant correlation between the results of any pair of the three biomarkers. The results of the micronucleus assay showed a significant correlation with water temperature. This temperature effect, as well as induced repair, may contribute to explain the lack of a strict correspondence between pollution gradients and biomarkers responses.

Introduction of the land snail Cornu aspersum as a bioindicator organism of terrestrial pollution with the use of a suite of biomarkers

The use of organisms in biomonitoring studies allows the determination of the pollution levels, the assessment of the quality of the enviroment and the study of toxic effects of pollutants on the organisms. By studying the responses (biomarkers) of organisms, it is possible to assess the exposure and effects of pollutants. Land gastropods are widely used in relevant studies as they possess several characteristics that make them ideal for use as bioindicators. In the present study we sought to evaluate several biomarkers in the laboratory using snails of the species Cornu aspersum after the exposure to heavy metals (Cd, Hg) and a mixture of polycyclic aromatic hydrocarbons (PAHs) (anthracene, phenanthrene and naphthalene). The biomarkers studied were the following: lysosomic membrane stability using the neutral red retention assay (NRR), the production of reactive oxygen species (ROS) using the compound DCFH-DA and flow cytometry, lipid peroxidation using the TBARs assay that measures the concentration of malondialdehyde (MDA), protein carbonylation (–CO) using ELISA, and single strand breaks of DNA using the comet assay. Our results show significant differences in the responses between the control group and the groups that were exposed to pollutans, enhancing the role of Cornu aspersum as a promising bioindicator organism in terrestrial pollution studies with the use of the biomarkers applied.

Controlling bacteria retention on polymer via replication of laser micro/nano textured metal mould

Polymer materials including paints and objects are widely used in our daily life. Keeping them free from bacteria without the use of any antibacterial agents is of high interest to the public. In this paper, we demonstrate a method for significantly reducing bacteria adhesion and growth on a polymer surfaces by generating a specific surface micro/nano structure. A metallic template was prepared using laser micro/nano surface texturing on Ti6Al4V surfaces. Then the negative surface structure was transferred onto a polydimethylsiloxane (PDMS) polymer using soft lithography. Staphylococcus aureus (S. aureus) retention assays were used to determine the number of retained bacteria on the replicated polymer surfaces. Results showed that on all the replicated plastic surfaces, the number of adhesive bacteria was much reduced (up to a fivefold reduction) compared to that on the non-textured polymer surfaces. This was largely due to the reduced wettability with a water contact angle close to 150° on the replicated polymer surface that contained specific micro/nano structures. This finding could lead to potentially applications for reducing bacteria adhesion or fouling on painted surfaces (e.g. ship hulls) and plastic objects (e.g. phones and computer keyboards) using nanostructure transfer from metal templates by, for example, stamping and hot/cold embossing and injection moulding.

FK506 Attenuates the MRP1-Mediated Chemoresistant Phenotype in Glioblastoma Stem-Like Cells.

Poor response to current treatments for glioblastoma has been attributed to the presence of glioblastoma stem-like cells (GSCs). GSCs are able to expel antitumor drugs to the extracellular medium using the multidrug resistance-associated protein 1 (MRP1) transporter. Tacrolimus (FK506) has been identified as an MRP1 regulator in differentiated glioblastoma (GBM) cells (non-GSCs); however, the effect of FK506 on GSCs is currently unknown. The objective of the following research is to evaluate the effect of FK506 on the MRP1-related chemo-resistant phenotype of GSCs. For this, U87MG and C6 glioma cell lines were used to generate non-GSCs and GSCs. mRNA and MRP1-positive cells were evaluated by RT-qPCR and flow cytometry, respectively. A Carboxyfluorescein Diacetate (CFDA)-retention assay was performed to evaluate the MRP1 activity. Apoptosis and MTT assays were employed to evaluate the cytotoxic effects of FK506 plus Vincristine (MRP1 substrate). GSC-derived subcutaneous tumors were generated to evaluate the in vivo effect of FK506/Vincristine treatment. No differences in transcript levels and positive cells for MRP1 were observed in FK506-treated cells. Lesser cell viability, increased apoptosis, and CFDA-retention in the FK506/Vincristine-treated cells were observed. In vivo, the FK506/Vincristine treatment decreased the tumor size as well as ki67, Glial Fibrillary Acidic Protein (GFAP), and nestin expression. We conclude that FK506 confers a chemo-sensitive phenotype to MRP1-drug substrate in GSCs.

Abstract 3063: Stem cell lineage hierarchy by keratin profiling in normal human prostate epithelial cells and prostate cancer

Abstract Background: Both mechanical and nonmechanical functions of keratins have been implicated in stem cells and cancers. Using a sphere-based label retention assay, we recently isolated and characterized prostate stem cells and cancer stem-like cells from mixed progenitor populations (PMID 28651114), identifying keratin 13 (KRT13) as a specific prostate stem cell marker regulating self-renewal. Herein we utilize detailed keratin profiles to further clarify the human prostate epithelial lineage hierarchy and identify prostate cancer stem-like cells. Methods and Results: Primary prostate epithelial cells were 3D cultured (5 days) to form prostaspheres (PS), followed by PS-based long-term label retention and FACS to separate stem cells from progenitors. In normal prostate tissues from three healthy donors, RNA-seq revealed enrichment of KRT13, 23, 80, 78, 86 and 4 in label-retaining prostate stem cells while KRT6, 17, 14, 5, 8, 18 and P63 were enriched in nonretaining progenitors. We next used Fluidigm C1 captured single-cell RNA-seq and identified three major clusters in the label-retaining stem cell population; Cluster I represents quiescent stem cells (KRT13, 23, 80, 78, 4 enriched) and Clusters II and III contain active stem cells and bipotent progenitors, respectively (KRT16, 17, 6 enriched). GSEA analysis found stem cell and cancer-related pathways enrichment in Cluster I. Three additional clusters were identified in nonretaining progenitor cells, with Cluster IV representing unipotent basal progenitor cells (KRT5, 14, 6, 16 enriched) and Clusters V and VI early- and late-stage unipotent luminal progenitors (KRT8, 18, 10 enriched). Cancer stem-like cells were similarly isolated from three prostate cancer specimens and RNA-seq with MetaCore pathway analysis found enrichment of cytoskeleton remodeling keratin filaments. Interestingly, in addition to normal stem cell keratins (KRT13, 23, 80, 78, 4), other keratins (KRT10, 19, 6, 75, 16, 79, 3, 82) were enriched in cancer stem-like cells. Surprisingly, stem-like cells from patient-matched benign regions revealed a similar keratin profile, suggesting a cancer field effect for stem-like cell populations. Conclusion: Taken together, using gene profiling with an emphasis on keratin patterns, we have delineated the lineage hierarchy of human prostate stem cells originating from the activation of quiescent stem cells to bipotent progenitors that give rise to unipotent basal and luminal progenitor cells. We have identified common keratins enriched in stem cells from normal prostate and cancer/benign tissues as well as keratins unique in stem-like cells from prostate cancer. This clarification of the stem cell lineage hierarchy and keratin profiling of human prostate stem cells and cancer stem-like cells may provide enhanced opportunities for translational studies that target therapeutic-resistant cancer stem-like cells. (CA-172220) Citation Format: Wenyang Hu, Danping Hu, Lishi Xie, Hong Hu, Ye Li, Larisa Nonn, Toshi Shioda, Gail S. Prins. Stem cell lineage hierarchy by keratin profiling in normal human prostate epithelial cells and prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3063.

Design of Nanofiber Coatings for Mitigation of Microbial Adhesion: Modeling and Application to Medical Catheters.

Surface-associated microbial communities, known as biofilms, pose significant challenges in clinical and industrial settings. Micro-/nanoscale substratum surface features have been shown to disrupt firm adhesion of planktonic microbes to surfaces, thereby interfering with the earliest stage of biofilm formation. However, the role of geometry and size of surface features in microbial retention is not completely understood. In this study, we developed a biophysical model that describes the changes in the total free energy (adhesion energy and stretching energy) of an adherent Candida albicans cell on nanofiber-coated surfaces as a function of the geometry (i.e., diameter) and configuration (i.e., interfiber spacing) of the surface features (i.e., nanofibers). We then introduced a new nondimensional parameter, Π, to represent the ratio of cell rigidity to cell-substratum interfacial energy. We show that the total free energy is a strong function of topographical feature size at higher Π and lower spacing values. To confirm our biophysical model predictions, we performed 24 h dynamic retention assays and quantified cell attachment number density on surfaces coated with highly ordered polystyrene nanofibers. We show that the total free energy of a single adherent cell on a patterned surface is a key determinant of microbial retention on that surface. The cell attachment density trend closely correlates with the predictions based on the adherent single-cell total energy. The nanofiber coating design (1.2 μm diameter, 2 μm spacing) that maximized the total energy of the adherent cell resulted in the lowest microbial retention. We further demonstrate the utility of our biophysical model by showing close correlation between the computed single-cell total free energy and biofilm nucleation on fiber-coated urinary and central venous catheters of different materials. This biophysical model could offer a powerful new paradigm in ab initio design of patterned surfaces for controlled biofilm growth for medical applications and beyond.

Picosecond laser treatment production of hierarchical structured stainless steel to reduce bacterial fouling

The design of surfaces that prevent biofouling through their physical structure and chemical properties provides a potential solution to increase their hygienic status. A picosecond laser was used to produce hierarchical textures on stainless steel. The surface topography, chemistry and wettability were characterised. The Sa, and wettability of the surfaces all increased when compared to the control following laser treatment. The Sa, Sq and Spv values ranged between 0.02μm–1.16μm, 0.02μm–1.30μm and 0.82μm–9.84μm respectively whilst the wettability of the surfaces ranged between 99.5°–160°. Following microbial assays, the work demonstrated that on all the surfaces, following attachment, adhesion and retention assays, the number of Escherichia coli on the laser textured surfaces was reduced. One surface was demonstrated to be the best antiadhesive surface, which alongside being superhydrophobic (154.30°) had the greatest Sa and Spv (1.16μm; 6.17μm) values, and the greatest peak (21.63μm) and valley (21.41μm) widths. This study showed that the surface roughness, feature geometry, chemistry and physicochemistry all interplayed to affect bacterial attachment, adhesion and retention Such a modified stainless steel surface may have the ability to reduce specific fouling in an industrial context.

Insights into the toxicity of iron oxides nanoparticles in land snails

The use of manufactured nanoparticles (NPs) is spreading rapidly across technology and medicine fields, posing concerns about their consequence on ecosystems and human health. The present study aims to assess the biological responses triggered by iron oxide NPs (IONPs) and iron oxide NPs incorporated into zeolite (IONPZ) in relation to oxidative stress on the land snail Helix aspersa in order to investigate its use as a biomarker for terrestrial environments. Morphology and structure of both NPs were characterized. Snail food was supplemented with a range of concentrations of IONPs and IONPZ and values of the hemocyte lysosomal membranes' destabilization by 50% were estimated by the neutral red retention (NRRT50) assay. Subsequently, snails were fed with NPs concentrations equal to half of the NRRT50 values, 0.05 mg L−1 for IONPs and 1 mg L−1 for IONPZ, for 1, 5, 10 and 20 days. Both effectors induced oxidative stress in snails' hemocytes compared to untreated animals. The latter was detected by NRRT changes, reactive oxygen species (ROS) production, lipid peroxidation estimation, DNA integrity loss, measurement of protein carbonyl content by an enzyme-linked immunoabsorbent assay (ELISA), determination of ubiquitin conjugates and cleaved caspases conjugates levels. The results showed that the simultaneous use of the parameters tested could constitute possible reliable biomarkers for the evaluation of NPs toxicity. However, more research is required in order to enlighten the disposal and toxic impact of iron oxide NPs on the environment to ensure their safe use in the future.

Metronidazole-Loaded Polyethyleneimine and Chitosan-Based Liquid Crystalline System for Treatment of Staphylococcal Skin Infections.

Staphylococcus aureus is a common gram-positive bacterium of the human skin microbiota. It is also a dangerous pathogen that can cause serious and even lethal skin infections. The topical administration of metronidazole via nanotechnology-based drug delivery systems, such as liquid crystalline systems, can modulate both the drug permeation and activity, decreasing its side effects and increasing the drug potent activity against the gram-positive bacteria. This study aimed at: (1) structurally developing and characterizing a liquid crystalline systems composed of chitosan and polyethyleneimine dispersion as the aqueous phase, oleic acid as the oily phase, and ethoxylated and propoxylated cetyl alcohol as the surfactant (FPC) for metronidazole incorporation (0.5% w/w); (2) evaluating the in vitro release and skin permeation and retention properties of the metronidazole-loaded liquid crystalline systems (FPC-M); (3) investigating the in vitro antibacterial activity of FPC-M against Staphylococcus aureus. Polarised light microscopy indicated that both FPC and FPC-M are hexagonal systems. Rheological, texture, and bioadhesion assays showed that both are elastic and bioadhesive systems. According to the results of the in vitro release, permeation, and retention assays, FPC can modulate metronidazole release and allow metronidazole to stay for a longer time on the skin. The determination of FPC-M activity against Staphylococcus aureus showed that it could target the bacterial cell. In conclusion, the liquid crystalline systems developed in this study can improve the clinical performance of metronidazole in the treatment of staphylococcal skin infections.

Natural membranes of Hevea brasiliensis latex as delivery system for Casearia sylvestris leaf components

Natural latex from Hevea brasiliensis (Wild. ex A.Juss) Müll.Arg., Euphorbiaceae, showed angiogenic action and Casearia sylvestris Sw., Salicaceae, leaf derivatives presented anti-inflammatory and wound healing activities. Therefore, an association of these effects was interesting for wound healing applications. The aims of this study were the development of membranes of natural latex incorporated with C. sylvestris leaf derivatives (ethanolic extract, diterpene concentrated fraction and casearin J), their chemical and physical characterization, and the evaluation of in vitro skin permeation and retention of C. sylvestris bioactive secondary metabolites (diterpenes and phenolic compounds). The membranes were developed mixing hydroethanolic solutions of C. sylvestris derivatives with latex and drying them in a desiccator. They were characterized by infrared spectroscopy, scanning electron microscopy, water vapor permeability and mechanical resistance assays, demonstrating that all membranes were permeable, resistant and homogeneous in surfaces. The permeation and retention assays demonstrated dermal penetration of phenolic compounds for ethanolic extract membrane and of casearin-like clerodane diterpenes for all membranes, indicating that these membranes have great potential for therapeutical application as a topical system for C. sylvestris components releasing.

The impact of expired commercial drugs on non-target marine species: A case study with the use of a battery of biomarkers in hemocytes of mussels

The present study investigated the effects of two expired commercial medicines, like Buscopan Plus and Mesulid, commonly classified as household medical wastes, on hemocytes of mussel Mytilus galloprovincialis. Mussel hemocytes’ lysosomal membrane stability (in terms of neutral red retention assay), superoxide anions (O2·-) and nitric oxides (NO, in terms of nitrites) production, lipid peroxidation (in terms of malondialdehyde/MDA content) and the formation of nuclear abnormalities (using the micronucleus/MN assay) were assessed in hemocytes of mussels treated for 7 days with appropriate amounts of each drug (the concentrations of active substances were considered in each case, due to the absence of data related with the excipients) as well as in hemocytes of post-treated/recovered mussels (7 days post-treatment/recovery period). According to the results, treated mussels showed significantly decreased NRRT values, enhanced O2·-, NO and MDA levels, as well as high frequencies of nuclear abnormalities in both cases. Thοse effects showed a drastic reduction in almost all cases, after the post-treatment/recovery period. Moreover, the “stress on stress” method, commonly performed for estimating mussels’ ability to survive in air, showed significantly reduced LT50 values in challenged mussels, compared to values observed in control mussels. The current findings revealed for the first time that both expired commercial drugs could affect mussels, probably via the formation of active substances bioactivated metabolites, as well as excipients, such as TiO2 and SiO2, at least in case of Buscopan plus. Although further research is needed, the current findings indicate the environmental impact of expired commercial drugs, thus revealing the need for the proper disposal of household medical wastes.

Production of hybrid macro/micro/nano surface structures on Ti6Al4V surfaces by picosecond laser surface texturing and their antifouling characteristics

The development of surfaces which reduce biofouling has attracted much interest in practical applications. Three picosecond laser generated surface topographies (Ti1, Ti2, Ti3) on titanium were produced, treated with fluoroalkylsilane (FAS), then characterised using Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDX), Raman Spectroscopy, Fourier Transform Infra-Red (FTIR) spectroscopy, contact angle measurements and white light interference microscopy. The surfaces had a range of different macro/micro/nano topographies. Ti2 had a unique, surface topography with large blunt conical peaks and was predominantly a rutile surface with closely packed, self-assembled FAS; this was the most hydrophobic sample (water contact angle 160°; ΔGiwi was −135.29mJm−2). Bacterial attachment, adhesion and retention to the surfaces demonstrated that all the laser generated surfaces retained less bacteria than the control surface. This also occurred following the adhesion and retention assays when the bacteria were either not rinsed from the surfaces or were retained in static conditions for one hour. This work demonstrated that picosecond laser generated surfaces may be used to produce antiadhesive surfaces that significantly reduced surface fouling. It was determined that a tri-modally dimensioned surface roughness, with a blunt conical macro-topography, combined with a close-packed fluoroalkyl monolayer was required for an optimised superhydrophobic surface. These surfaces were effective even following surface immersion and static conditions for one hour, and thus may have applications in a number of food or medical industries.

Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome.

Oxidative stress has pervasive effects on cells but how they respond transcriptionally upon the initial insult is incompletely understood. We developed a nuclear walk-on assay that semi-globally quantifies nascent transcripts in promoter-proximal paused RNA polymerase II (Pol II). Using this assay in conjunction with ChIP-Seq, in vitro transcription, and a chromatin retention assay, we show that within a minute, hydrogen peroxide causes accumulation of Pol II near promoters and enhancers that can best be explained by a rapid decrease in termination. Some of the accumulated polymerases slowly move or ‘creep’ downstream. This second effect is correlated with and probably results from loss of NELF association and function. Notably, both effects were independent of DNA damage and ADP-ribosylation. Our results demonstrate the unexpected speed at which a global transcriptional response can occur. The findings provide strong support for the residence time of paused Pol II elongation complexes being much shorter than estimated from previous studies.

Responses of human hepatoma HepG2 cells to silver nanoparticles and polycyclic aromatic hydrocarbons

The nanotechnology has revolutionized the global market with silver nanoparticles (AgNP) occupying a prominent position due to their remarkable anti-bacterial properties. However, there is no data about the adverse and toxic effects of associations of AgNP and ubiquitous compounds, such as polycyclic aromatic hydrocarbons (PAH). In the current study, we investigated the responses of HepG2 cells to realistic concentrations of AgNP (0.09, 0.9, and 9 ng ml−1) and mixture of PAH (30 and 300 ng ml−1), separately and in association. Cell viability and cytotoxicity (neutral red retention and MTT production assays) and proliferation (crystal violet [CV] assay), xenobiotic efflux transporter activity (rhodamine B accumulation assay), ROS levels (dichlorodihydrofluorescein diacetate assay), and lipid peroxidation (pyrenylphosphine-1-diphenyl assay) were analyzed. There was no decreases of cell viability after exposure to AgNP, PAH and most of AgNP + PAH associations, but increases of cell viability/number (CV assay) occurred. Efflux transporter activity was not affected, with exception of one AgNP + PAH associations, ROS levels increased, but lipid peroxidation decreased. Some toxicological interactions occurred, particularly for the highest concentrations of AgNP and PAH, but there is no evidence that these interactions increased the toxicity of AgNP and PAH.