Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq

Preliminary research for identification of bacteria useful in microbially induced calcium carbonate precipitation

MICP (Microbially Induced Calcite Precipitation) is a new biological method in soil stabilization. This cheap and eco-friendly technique improves strength parameters of the ground such as shear strength and decreases the permeability of gravelly and sandy soil. There are variety of microorganisms that can be used in calcite precipitation. The most popular method is precipitation of calcium carbonate by bacteria. The main purpose of the article is to present the results from Gram staining of bacteria isolated from construction sites, which is the first step of their identification. Gram’s method allows to find out which morphological groups of bacteria are adapted to conditions present in soil from construction sites and therefore are potentially able to produce calcite. The article describes the methodology of isolation, staining and determination of morphological types of bacteria

Effect of aluminium oxide nanoparticles on the enzymatic activity on microorganisms of activated sludge

The increased production and commercial use of nanoparticles (NPs), combined with a lack of regulation regarding their disposal, may result in the unwanted introduction of NPs to wastewater. Wastewater nutrient removal depends on the metabolisms of activated sludge bacteria and their related key enzymes. Therefore, the aim of this work was to determine the effect of aluminium oxide nanoparticles concentrations on the activated sludge enzymatic activity of microorganisms. Tested nanoparticles inhibition cellular respiration in TTC method in the four highest tested concentrations. Moreover, in most samples observed increase dehydrogenase activity. In this study, nano-Al2O3 also caused a clear stimulation of the activity of hydrolytic enzymes microorganisms of activate sludge. Effects of aluminum oxide (compound in bulk forms) on enzymatic activity were different than in the case of the nano from of Al2O3

Effect of aluminium oxide nanoparticles on the enzymatic activity on microorganisms of activated sludge

The increased production and commercial use of nanoparticles (NPs), combined with a lack of regulation regarding their disposal, may result in the unwanted introduction of NPs to wastewater. Wastewater nutrient removal depends on the metabolisms of activated sludge bacteria and their related key enzymes. Therefore, the aim of this work was to determine the effect of aluminium oxide nanoparticles concentrations on the activated sludge enzymatic activity of microorganisms. Tested nanoparticles inhibition cellular respiration in TTC method in the four highest tested concentrations. Moreover, in most samples observed increase dehydrogenase activity. In this study, nano-Al2O3 also caused a clear stimulation of the activity of hydrolytic enzymes microorganisms of activate sludge. Effects of aluminum oxide (compound in bulk forms) on enzymatic activity were different than in the case of the nano from of Al2O3

Effects of aluminium oxide nanoparticles on bacterial growth

Production and wide application of nanomaterials have led to nanotechnology development but their release to environment and the induction of toxic reactions, affects the natural microbial communities. Therefore, studies on the impact of nanoparticles on microorganisms and environment are required and needed. The aim of this study was to assess the impact of aluminium oxide nanoparticles on the growth of Pseudomonas putida. To compare the harmfulness of different forms of aluminium oxide, the ecotoxicity of its macro-forms was also evaluated in the study. Research showed that the exposure to nanoparticles can negatively influence microorganisms. The EC50-16h determined in this study was 0.5 mg/l, and NOEC equaled 0.19 mg/l. Nano-Al2O3 proved to be more toxic to P. putida than aluminium oxide. This indicates that the nano-form of a given substance demonstrates different properties and may constitute a far greater danger for the environment than the same substance in the large form. According to EU and US EPA criteria, nano-Al2O3 proved to be very toxic and highly toxic, respectively. Changes in bacterial communities caused by nanoparticles may affect the normal biological, chemical and nutrient cycle in the ecosystem and the effect triggered by nanomaterials in relation to other organisms is unpredictable

The effect of zirconium oxide nanoparticles on dehydrogenase and hydrolytic activity of activated sludge microorganisms

The pervasive use of engineered nanoparticles (NPs) in a wide range of fields raises concerns about their potential environmental impacts. The impact of NPs on the activity of enzymes in sewage sludge was examined using model engineered zirconium oxide nanoparticles (nano-ZrO2). Dehydrogenase activity was evaluated with the TTC test and hydrolytic activity with FDA test. In this study an inhibitory or a stimulating effect of NPs on the activity of the enzymes was observed. The levels of inhibition/stimulation of the nano-ZrO2 varied in relation to duration of contact NPs with activated sludge and particle size (nano, bulk)

Effects of aluminium oxide nanoparticles on bacterial growth

Production and wide application of nanomaterials have led to nanotechnology development but their release to environment and the induction of toxic reactions, affects the natural microbial communities. Therefore, studies on the impact of nanoparticles on microorganisms and environment are required and needed. The aim of this study was to assess the impact of aluminium oxide nanoparticles on the growth of Pseudomonas putida. To compare the harmfulness of different forms of aluminium oxide, the ecotoxicity of its macro-forms was also evaluated in the study. Research showed that the exposure to nanoparticles can negatively influence microorganisms. The EC50-16h determined in this study was 0.5 mg/l, and NOEC equaled 0.19 mg/l. Nano-Al2O3 proved to be more toxic to P. putida than aluminium oxide. This indicates that the nano-form of a given substance demonstrates different properties and may constitute a far greater danger for the environment than the same substance in the large form. According to EU and US EPA criteria, nano-Al2O3 proved to be very toxic and highly toxic, respectively. Changes in bacterial communities caused by nanoparticles may affect the normal biological, chemical and nutrient cycle in the ecosystem and the effect triggered by nanomaterials in relation to other organisms is unpredictable

The effect of zirconium oxide nanoparticles on dehydrogenase and hydrolytic activity of activated sludge microorganisms

The pervasive use of engineered nanoparticles (NPs) in a wide range of fields raises concerns about their potential environmental impacts. The impact of NPs on the activity of enzymes in sewage sludge was examined using model engineered zirconium oxide nanoparticles (nano-ZrO2). Dehydrogenase activity was evaluated with the TTC test and hydrolytic activity with FDA test. In this study an inhibitory or a stimulating effect of NPs on the activity of the enzymes was observed. The levels of inhibition/stimulation of the nano-ZrO2 varied in relation to duration of contact NPs with activated sludge and particle size (nano, bulk)

Bioaerosol Emission from Biofilters: Impact of Bed Material Type and Waste Gas Origin

Three semi-technical scale biofilters were applied to treat waste gases at different industrial sites in Poland: a mechanical–biological treatment plant of municipal solid waste, a wastewater treatment plant and a food industry plant. Two types of materials were used as beds in the biofilters: stumpwood chips and pine bark, and stumpwood chips, pine bark and compost from green waste. Both bed materials supported the microbial growth and high numbers (106–108 cfu/g dry mass (DM)) of culturable bacteria, and fungi in beds were observed. There was no correlation between the number of microorganisms (cfu/g DM) and the respiratory activity in the biofilter beds. However, microbial respiration activity corresponded with microbial abundance expressed as microbial equivalents (ME), which was calculated based on adenosine triphosphate (ATP) determination. The biofilters either reduced or increased bioaerosol emissions from industrial plants, depending on the microbial content in the waste gases. A high microbial content in the waste gases made the effect of microbial emission from the biofilter bed negligible. The type of biofilter bed and number of microorganisms in the bed also influenced the final bioaerosol emission, but these factors were relevant for biofilters that treated waste gases with low microbial concentrations

Tourism Competitiveness and Attractiveness of Municipalities in Mazowieckie Province Located Around the Liwiec River

Turystyka należy do tych dziedzin gospodarki, które wykazują najbardziej dynamiczny wzrost. Jako dziedzina interdyscyplinarna umożliwia ona osiąganie zysków nie tylko sektorom, które związane są z wytwarzaniem produktu turystycznego, pojmowanego, jako zakwaterowanie i wyżywienie, ale również tym podmiotom, które oferują towary i usługi, gwarantujące satysfakcję podróżnych. Dolina Liwca na mocy „Dyrektywy Ptasiej” została włączona do systemu Natura, 2000 jako obszar PLB 140002. Obszar ten obejmuje dolinę rzeki Liwiec, od źródeł do jej ujścia do Bugu, z łąkami i zalewowymi pastwiskami utworzonymi na zmeliorowanych bagnach. Niektóre odcinki rzeki mają charakter naturalny, na innych odcinkach jest ona uregulowana, lokalnie w dolinie występują wtórne zabagnienia. Walory przyrodnicze i kulturowe doliny Liwca predestynują ten teren do rozwoju różnych form turystyki. Do niedawna była to głównie turystyka letniskowa, obecnie jest to również agroturystyka. Badaniami objęto gminy, przez które płynie rzeka Liwiec. Do oceny ich konkurencyjności ze względu na intensywność ruchu turystycznego zaproponowano następujące wskaźniki: Baretje’a-Deferta, Schneidera, Charvata, wykorzystania pojemności noclegowej, rozwoju bazy noclegowej, gęstości ruchu turystycznego, gęstości bazy noclegowej. Do analizy przestrzennego zróżnicowania atrakcyjności turystycznej gmin wykorzystano zmienne takie jak: ścieki przemysłowe i komunalne ogółem, ludność korzystająca z oczyszczalni ścieków, odpady komunalne w ciągu roku, biochemiczne zużycie tlenu – BZT5, chemiczne zużycie tlenu – ChZT, zawiesina ogólna, obszary prawnie chronione, pomniki przyrody, tereny zieleni urządzonej, lasy i grunty leśne, trwałe użytki zielone, użytki rolne ogółem. Dane do analizy zaczerpnięto z Bazy Danych Lokalnych Głównego Urzędu Statystycznego za 2012 rok. Porównanie i rangowanie gmin pod względem konkurencyjności i atrakcyjności turystycznej oparto o wielowymiarową analizę porównawczą (WAP). Normalizację zmiennych oparto o metodę unitaryzacji zerowanej. O konkurencyjności terenów gminnych położonych nad rzeką Liwiec decydowały zarówno walory środowiska naturalnego, jak i stan bazy noclegowej. Jednoznaczna ocena atrakcyjności turystycznej jest dosyć skomplikowana i w dużej mierze może być uzależniona od zaproponowanego zbioru zmiennych.Tourism is an example of the economy branches which are displaying the most dynamic growth. As an interdisciplinary field, it enables both sectors connected with developing the tourism product, that is accommodation and catering, and entities which offer goods and services to make profits. Pursuant to the “Birds Directive”, the Liwiec River Valley has become part of Natura 2000 with the code PLB 140002. The area encompasses the Liwiec River valley, from its source to the estuary into the Bug River, including meadows and flood pastures formed from drained bog. Natural and cultural value of the Liwiec River Valley render it suitable for the development of various tourism forms. Until recently, summer tourism was the dominant form but now agritourism has gained popularity, too. The competitiveness of communes located around the Liwiec River was conditioned by both the attractiveness of the natural environment and the quality of accommodation facilities. A precise assessment of tourist competitiveness is quite complicated and to a large degree depends on the set of selected variables which reflect the tourist attractiveness of the communes examined