Antimicrobial activity of carbon-based fillers

Diplomová práce se zabývá vlivem uhlíkatého plniva na životaschopnost a produkci extracelulárních látek vybrané bakterie Bacillus subtilis (CCM 1999) a kvasinky Yarrowia lipolytica (CCY 29-26-52). Antimikrobiální aktivita těchto částic, přítomných v kultivačním mediu, byla sledována pomocí následujících parametrů: růst daného mikroorganismu, produkce extracelulárních proteinů a v poslední řadě byla monitorována produkce extracelulárních polymerních substancí, které mají úzkou souvislost s tvorbou biofilmu. Suspenze materiálů (0,135 mg/mL) byly připraveny ve dvou rozdílných kultivačních mediích; tzn. živné medium s obsahem glukózy pro Bacillus subtilis a bazální medium s přídavkem Tweenu 80 pro Yarrowia lipolytica, a media byla inokulována příslušným typem mikroorganismu. Experimenty probíhaly po dobu 6 dnů při rychlosti třepání 160 rpm a teplotě 30 °C pro Bacillus subtilis a 28 °C pro Yarrowia lipolytica. Testovány byly celkem tři typy uhlíkatého nanomateriálu, získané z Katedry anorganické chemie, Vysoké školy chemicko-technologické v Praze. Tyto materiály specifikované jako materiál “A”, “B” a “C” se navzájem lišily velikostí částic a stupněm oxidace. Na základě skríningových studií byla vybrána koncentrace testovaného materiálu 0,135 mg/mL a rychlost třepání 160 rpm. Metodou měření optické hustoty vzorku při 600 nm byly sestaveny a porovnány růstové křivky obou mikroorganismů v přítomnosti testovaných nanočástic po dobu 5 dní. Tímto způsobem bylo zjištěno, že přítomnost nanočástic v mediu nemá velký vliv na růst zkoumaného mikroorganismu. Tato metoda, je však pouze orientační, protože se nevyhneme chybě díky přítomnosti mrtvých buněk. Dále byla testována produkce celkových a extracelulárních proteinů daným mikroorganismem v přítomnosti testovaných nanočástic. Nebyla však pozorována výrazná odchylka hodnot od hodnot kontrolního vzorku, který neobsahoval testovaný materiál. Na základě metod počítání kolonií (Bacillus subtilis) a buněk (Yarrowia lipolytica) byly určeny ztráty životaschopnosti mikroorganismu ve 3 časech (6, 48 a 144 hodin); v kratším časovém intervalu byl růst spíše podporován. Dále byla monitorována produkce extracelulárních polymerních substancí (EPS), tedy proteinů, redukujících substancí a polysacharidů. Tyto látky byly vylučovány daným mikroorganismem do prostředí v průběhu 24 hodin. Bacillus subtilis produkoval EPS ve větší míře než Yarrowia lipolytica. Předpokládáme, že produkce EPS by mohla souviset s tvorbou biofilmu, který chrání buňky před toxicitou nanočástic.The aim of this diploma thesis is focused on the impact of carbon-based fillers on viability and extracellular substances production by bacterium Bacillus subtilis (CCM 1999) and yeast Yarrowia lipolytica (CCY 29-26-52). Antimicrobial activity of these particles, present in cultivation nutrient medium was examined using following parameters: growth of mentioned microorganisms, production of extracellular proteins and finally extracellular polymeric substances production, which is strongly connected with biofilm formation. Nanomaterials suspension (0.135 mg/mL) was prepared in two different cultivation media i.e. nutrient medium supplemented with glucose for Bacillus subtilis and basal medium with the addition of 2% (vol.) Tween 80 for Yarrowia lipolytica and media were inoculated by appropriate type of microorganism. Experiments were performed for 6 days under shaking rate at 160 rpm and at temperature of 30 °C for Bacillus subtilis and 28 °C for Yarrowia lipolytica. Three types of carbon nanomaterials obtained from Department of Inorganic Chemistry, Institute of Chemical Technology, Prague were examined. These materials specified as material “A”, “B” and “C” are mutually different by the size of its particles and the degree of oxidation. Based on the screening studies the tested material concentration of 0.135 mg/mL and shaking rate of 160 rpm were chosen. According to the optical density measurement at 600 nm, the growth curves of both microorganisms in the presence of tested nanoparticles during 5 days period were compared. It was find out, that the presence of nanoparticles don’t have a significant influence on tested microorganisms growth, by this technique. However, this method is just wider point of view, due to mistakes caused by presence of dead cells. Further, production of total cells proteins and extracellular proteins by microorganisms in presence of tested nanoparticles was examined. There was not observed any significant deviation from control samples values, where the tested materials were absent. Based on colony counting method (used for Bacillus subtilis) and cells counting in Bürker counting chamber (used for Yarrowia lipolytica), loss of microorganism viability was determined in 3 cultivation periods (6, 48 and 144 hours); there was observed a support of growth of microorganisms rather in shorter incubation period. Thereafter the extracellular polymeric substances (EPS) production that means proteins, reducing substances and polysaccharides was monitored. These substances were secreted into the medium by mentioned microorganisms during 24 hours of incubation. Bacillus subtilis cells produce much more EPS than Yarrowia lipolytica cells. We suppose that the EPS production could be closely associated with production of biofilm, which protects cells against nanoparticles toxicity.

How toxic are gold nanoparticles? The state-of-the-art.

With the growing interest in biotechnological applications of gold nanoparticles and their effects exerted on the body, the possible toxicity is becoming an increasingly important issue. Numerous investigations carried out, in the last few years, under different experimental conditions, following different protocols, have produced in part conflicting results which have leaded to different views about the effective gold nanoparticle safety in human applications. This work is intended to provide an overview on the most recent experimental results in order to summarize the current state-of-the-art. However, rather than to present a comprehensive review of the available literature in this field, that, among other things, is really huge, we have selected some representative examples of both in vivo and in vitro investigations, with the aim of offering a scenario from which clearly emerges the need of an urgent and impelling standardization of the experimental protocols. To date, despite the great potential, the safety of gold nanoparticles is highly controversial and important concerns have been raised with the need to be properly addressed. Factors such as shape, size, surface charge, surface coating and surface functionalization are expected to influence interactions with biological systems at different extents, with different outcomes, as far as gold nanoparticle potentiality in biomedical applications is concerned. Moreover, despite the continuous attempt to establish a correlation between structure and interactions with biological systems, we are still far from assessing the toxicological profile of gold nanoparticles in an unquestionable manner. This review is intended to provide a contribution in this direction, offering some suggestions in order to reach the systematization of data over the most relevant physico-chemical parameters, which govern and control toxicity, at different cellular and organismal levels

Multi-scale cellular engineering: From molecules to organ-on-a-chip.

Recent technological advances in cellular and molecular engineering have provided new insights into biology and enabled the design, manufacturing, and manipulation of complex living systems. Here, we summarize the state of advances at the molecular, cellular, and multi-cellular levels using experimental and computational tools. The areas of focus include intrinsically disordered proteins, synthetic proteins, spatiotemporally dynamic extracellular matrices, organ-on-a-chip approaches, and computational modeling, which all have tremendous potential for advancing fundamental and translational science. Perspectives on the current limitations and future directions are also described, with the goal of stimulating interest to overcome these hurdles using multi-disciplinary approaches

Representation of probabilistic scientific knowledge

This article is available through the Brunel Open Access Publishing Fund. Copyright © 2013 Soldatova et al; licensee BioMed Central Ltd.The theory of probability is widely used in biomedical research for data analysis and modelling. In previous work the probabilities of the research hypotheses have been recorded as experimental metadata. The ontology HELO is designed to support probabilistic reasoning, and provides semantic descriptors for reporting on research that involves operations with probabilities. HELO explicitly links research statements such as hypotheses, models, laws, conclusions, etc. to the associated probabilities of these statements being true. HELO enables the explicit semantic representation and accurate recording of probabilities in hypotheses, as well as the inference methods used to generate and update those hypotheses. We demonstrate the utility of HELO on three worked examples: changes in the probability of the hypothesis that sirtuins regulate human life span; changes in the probability of hypotheses about gene functions in the S. cerevisiae aromatic amino acid pathway; and the use of active learning in drug design (quantitative structure activity relation learning), where a strategy for the selection of compounds with the highest probability of improving on the best known compound was used. HELO is open source and available at https://github.com/larisa-soldatova/HELO.This work was partially supported by grant BB/F008228/1 from the UK Biotechnology & Biological Sciences Research Council, from the European Commission under the FP7 Collaborative Programme, UNICELLSYS, KU Leuven GOA/08/008 and ERC Starting Grant 240186

Graphenic materials for biomedical applications

Graphene-based nanomaterials have been intensively studied for their properties, modifications, and application potential. Biomedical applications are one of the main directions of research in this field. This review summarizes the research results which were obtained in the last two years (2017-2019), especially those related to drug/gene/protein delivery systems and materials with antimicrobial properties. Due to the large number of studies in the area of carbon nanomaterials, attention here is focused only on 2D structures, i.e. graphene, graphene oxide, and reduced graphene oxide.Web of Science912art. no. 175

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring.

The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of "lab-on-a-chip" methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research

Chemical regulators of epithelial plasticity reveal a nuclear receptor pathway controlling myofibroblast differentiation

Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition. In addition to TGF-β receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-β-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-β-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology