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Transfection of mammalian and human cell lines in medical research both are key technologies in molecular biology and genetic engineering. A vast variety of techniques to facilitate transfection exists including different chemical and nanoparticle-based agents as mediators of nucleic acid uptake, with nanoparticles composed of the lipids DOSPA/DOPE belonging to the established type of agents. We show that inverse-nanoemulsion-derived protein nanohydrogels (NanoTrans-gels), prepared by a simple synthesis protocol, are suited to transfect two model cancer cell lines (MCF7 and A549) with high efficiency. The transfection efficiency was analyzed in comparison to the DOSPA/DOPE-dependent protocols as a reference method. Since nanogel-based transfection outperformed the Lipofectamine-dependent technique in our experiments, we believe that the NanoTrans-gels loaded with plasmid DNA may open new avenues for simple and efficient transfection for humans and probably also other mammalian cell lines and may develop into a general tool for standard transfection procedures in cell biology laboratories. [ABSTRACT FROM AUTHOR]

Infections caused by yeasts of the genus Candida are likely to occur not only in immunocompromised patients but also in healthy individuals, leading to infections of the gastrointestinal tract, urinary tract, and respiratory tract. Due to the rapid increase in the frequency of reported Candidiasis cases in recent years, diagnostic research has become the subject of many studies, and therefore, we developed a polyclonal aptamer library-based fluorometric assay with high specificity and affinity towards Candida spec. to quantify the pathogens in clinical samples with high sensitivity. We recently obtained the specific aptamer library R10, which explicitly recognized Candida and evolved it by mimicking an early skin infection model caused by Candida using the FluCell-SELEX system. In the follow-up study presented here, we demonstrate that the aptamer library R10-based bioassay specifically recognizes invasive clinical Candida isolates, including not only C. albicans but also strains like C. tropcialis, C. krusei, or C. glabrata. The next-generation fluorometric bioassay presented here can reliably and easily detect an early Candida infection and could be used for further clinical research or could even be developed into a full in vitro diagnostic tool. [ABSTRACT FROM AUTHOR]

The establishment of sustainable processes for the production of commodity chemicals is one of today's central challenges for biotechnological industries. The chemo-autotrophic fixation of CO2 and the subsequent production of acetate by acetogenic bacteria via anaerobic gas fermentation represents a promising platform for the ecologically sustainable production of high-value biocommodities via sequential fermentation processes. In this study, the applicability of acetate-containing cell-free spent medium of the gas-fermenting acetogenic bacterium A. woodii WP1 as the feeder strain for growth and the recombinant production of P. aeruginosa PAO1 mono-rhamnolipids in the well-established nonpathogenic producer strain P. putida KT2440 were investigated. Additionally, the potential possibility of a simplified production process without the necessary separation of feeder strain cells was elucidated via the cultivation of P. putida in cell-containing A. woodii culture broth. For these cultures, the content of both strains was investigated by examining the relative quantification of strain-exclusive genes via qPCR. The recombinant production of mono-rhamnolipids was successfully achieved with maximum titers of approximately 360–400 mg/L for both cell-free and cell-containing A. woodii spent medium. The reported processes therefore represent a successful proof of principle for gas fermentation-derived acetate as a potential sustainable carbon source for future recombinant rhamnolipid production processes by P. putida KT2440. [ABSTRACT FROM AUTHOR]

The diminishing portfolio of mankind's available antibiotics urges science to develop novel potent drugs. Here, we present a peptide fitting the typical blueprint of amphipathic and membrane-active antimicrobial peptides, denominated C14R. This 2 kDa peptide consists of 16 amino acid residues, with seven being either hydrophobic, aromatic, or non-polar, and nine being polar or positively charged, strictly separated on opposite sides of the predicted α-helix. The affinity of the peptide C14R to P. aeruginosa membranes and its intrinsic tendency to productively insert into membranes of such composition were analyzed by dynamic simulations. Its biological impact on the viability of two different P. aeruginosa reference strains was demonstrated by determining the minimal inhibitory concentrations (MICs), which were found to be in the range of 10–15 µg/mL. C14R's pore-forming capability was verified in a permeabilization assay based on the peptide-triggered uptake of fluorescent dyes into the bacterial cells. Finally, the peptide was used in radial diffusion assays, which are commonly used for susceptibility testing of antimicrobial peptides in clinical microbiology. In comparison to reference strains, six clinical P. aeruginosa isolates were clearly affected, thereby paving the way for further in-depth analyses of C14R as a promising new AMP drug in the future. [ABSTRACT FROM AUTHOR]

Rikenella microfusus is an essential intestinal probiotic with great potential. The latest research shows that imbalance in the intestinal flora are related to the occurrence of various diseases, such as intestinal diseases, immune diseases, and metabolic diseases. Rikenella may be a target or biomarker for some diseases, providing a new possibility for preventing and treating these diseases by monitoring and optimizing the abundance of Rikenella in the intestine. However, the current monitoring methods have disadvantages, such as long detection times, complicated operations, and high costs, which seriously limit the possibility of clinical application of microbiome-based treatment options. Therefore, the intention of this study was to evolve an enriched aptamer library to be used for specific labeling of R. microfusus, allowing rapid and low-cost detection methods and, ultimately the construction of aptamer-based biosensors. In this study, we used Rikenella as the target bacterium for an in vitro whole Cell-SELEX (Systematic Evolution of Ligands by EXponential Enrichment) to evolve and enrich specific DNA oligonucleotide aptamers. Five other prominent anaerobic gut bacteria were included in this process for counterselection and served as control cells. The aptamer library R.m-R13 was evolved with high specificity and strong affinity (Kd = 9.597 nM after 13 rounds of selection). With this enriched aptamer library, R. microfusus could efficiently be discriminated from the control bacteria in complex mixtures using different analysis techniques, including fluorescence microscopy or fluorometric suspension assays, and even in human stool samples. These preliminary results open new avenues toward the development of aptamer-based microbiome bio-sensing applications for fast and reliable monitoring of R. microfusus. [ABSTRACT FROM AUTHOR]

The concept of Building Information Modelling (BIM) is heavily promoted but rarely used in the Facility Management (FM) industry. Especially for event locations, where FM and Event Management organizations have to collaborate within a short period in a highly flexible manner with various external partners and public authorities. Hence, applying the method of BIM for Event Management is even more difficult compared to regular building operations. As a first attempt to dissolve this issue, the presented article emphasizes on the adoption of agile project management framework for BIM-based Event Management, where the focus lays on the creation of a suitable Building Information Model (BIM Model), at one hand for general FM, on the other hand for event management purposes. The paper shows results of a collaborative research project BIM4Events founded by KiraDIGI between the HTW Berlin, Berlin Airport of Tempelhof and Metropolia UAS Helsinki, as a Scrum-based BIM Model development at the case of a public event, ‘Sommerfest – Tag der offenen Tür’, organized in September 2018.

In more than 30 years of aptamer research, it has become widely accepted that aptamers are fascinating binding molecules for a vast variety of applications. However, the majority of targets have been proteins, although special variants of the so-called SELEX process for the molecular evolution of specific aptamers have also been developed, allowing for the targeting of small molecules as well as larger structures such as cells and even cellular networks of human (tumor) tissues. Although the provocative thesis is widely accepted in the field, that is, in principle, any level of complexity for SELEX targets is possible, the number of studies on whole organs or at least parts of them is limited. To pioneer this thesis, and based on our FluCell-SELEX process, here, we have developed polyclonal aptamer libraries against apices and the elongation/differentiation zones of plant roots as examples of organs. We show that dedicated libraries can specifically label the respective parts of the root, allowing us to distinguish them in fluorescence microscopy. We consider this achievement to be an initial but important evidence for the robustness of this SELEX variant. These libraries may be valuable tools for plant research and a promising starting point for the isolation of more specific individual aptamers directed against root-specific epitopes. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated that changes in the abundance of the intestinal bacterium Blautia producta, a potential probiotic, are closely associated with the development of various diseases such as obesity, diabetes, some neurodegenerative diseases, and certain cancers. However, there is still a lack of an effective method to detect the abundance of B. producta in the gut rapidly. Especially, DNA aptamers are now widely used as biometric components for medical testing due to their unique characteristics, including high chemical stability, low production cost, ease of chemical modification, low immunogenicity, and fast reproducibility. We successfully obtained a high-affinity nucleic acid aptamer library (B.p-R14) after 14 SELEX rounds, which efficiently discriminates B. producta in different analysis techniques including fluorometric suspension assays or fluorescence microscopy from other major gut bacteria in complex mixtures and even in human stool samples. These preliminary findings will be the basis towards aptamer-based biosensing applications for the fast and reliable monitoring of B. producta in the human gut microbiome. [ABSTRACT FROM AUTHOR]

Easy and reliable identification of pathogenic species such as yeasts, emerging as problematic microbes originating from the genus Candida, is a task in the management and treatment of infections, especially in hospitals and other healthcare environments. Aptamers are seizing an already indispensable role in different sensing applications as binding entities with almost arbitrarily tunable specificities and optimizable affinities. Here, we describe a polyclonal SELEX library that not only can specifically recognize and fluorescently label Candida cells, but is also capable to differentiate C. albicans, C. auris and C. parapsilosis cells in flow-cytometry, fluorometric microtiter plate assays and fluorescence microscopy from human cells, exemplified here by human dermal fibroblasts. This offers the opportunity to develop diagnostic tools based on this library. Moreover, these specific and robust affinity molecules could also serve in the future as potent binding entities on biomaterials and as constituents of technical devices and will thus open avenues for the development of cost-effective and easily accessible next generations of electronic biosensors in clinical diagnostics and novel materials for the specific removal of pathogenic cells from human bio-samples. [ABSTRACT FROM AUTHOR]

Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes. [ABSTRACT FROM AUTHOR]

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In this work, we present the results of the first round robin test of different kinds of micro- and nanoscaled samples for total reflection X-ray fluorescence (TXRF) analysis. Therefore preselected, well-characterized samples including an internal standard were provided to the participants of the round robin test. Three different kinds of samples were produced ensuring highly homogeneous mass depositions: first, manually produced μL droplets, representing the most common sample preparation in TXRF. Second, nL droplets pipetted with a nL dispenser, having the potential of being μL (total volume) samples distributed in an optimized manner with respect to reproducibility and homogeneity. Third, multi-elemental sub-monolayers, coated over the entire sample surface, simulating surface contamination and thereby representing ideal samples for the TXRF method. One of the several elements coated as sub-monolayers was selected as an internal standard and quantified with physically traceable XRF. The approach for an accurate and precise round robin activity was to separate the influence of the TXRF instrumental response and internal standard based quantification from any impact related to the sample preparation, in particular spatial inhomogeneity revealed by different X-ray spectrometric techniques. The results of the round robin test are in line with expectations and lie within about 5% deviation for all droplets and about 3% for the layers, showing the strength and reliability of the TXRF method for simultaneous multi-element analysis when decoupled from unfavorable sample preparations. For validation purposes, physically traceable XRF quantification was performed for one selected sample, and the absolute mass deposition of the respective elements was determined. [ABSTRACT FROM AUTHOR]

With the advent of both modern X-ray fluorescence (XRF) methods and improved analytical reliability requirements the demand for suitable reference samples has increased. Especially in nanotechnology with the very low areal mass depositions, quantification becomes considerably more difficult. However, the availability of suited reference samples is drastically lower than the demand. Physical vapor deposition techniques have been enhanced significantly in the last decade driven by the need for extremely precise film parameters in multilayer production. We have applied those techniques for the development of layer-like reference samples with mass depositions in the ng-range and well below for Ca, Cu, Pb, Mo, Pd, Pb, La, Fe and Ni. Numerous other elements would also be possible. Several types of reference samples were fabricated: multi-elemental layer and extremely low (sub-monolayer) samples for various applications in XRF and total-reflection XRF analysis. Those samples were characterized and compared at three different synchrotron radiation beamlines at the BESSY II electron storage ring employing the reference-free XRF approach based on physically calibrated instrumentation. In addition, the homogeneity of the multi-elemental coatings was checked at the P04 beamline at DESY. The measurements demonstrate the high precision achieved in the manufacturing process as well as the versatility of application fields for the presented reference samples. [ABSTRACT FROM AUTHOR]

Efficient soft X-ray spectroscopy in the laboratory is still a challenging task. Here, we report on new toroidal multilayer optics designed and applied with the laser-produced plasma (LPP) source of the Berlin Laboratory for innovative X-ray technologies. The optics are described and characterized, and the application of the updated source to scanning-free grazing emission X-ray fluorescence is demonstrated on thermoelectric gold-doped copper oxide nanofilms. The comparison with synchrotron measurements allows estimating a flux on the sample of approximately 7.5 × 109 photons/s in the 1 keV range on a 100 µm × 100 µm spot, emphasizing the suitability of the updated LPP source for the application in photon hungry experiments. [ABSTRACT FROM AUTHOR]

A microfocus x-ray tube with multiple targets and an electron gun with a focal spot size of 10 μm in diameter has been developed. The electron gun contains a LaB6 cathode and an Einzel lens. The x-ray tube can be operated at 50 W (50 kV, 1 mA) and has three targets, namely, Cr, W, and Rh on the anode that can be selected completely by moving the anode position. A focal spot size of 10 μm in diameter can be achieved at 0.5 mA current. As demonstration of the usability of a multiexcitation x-ray tube, the fluorescence x-rays have been measured using a powder specimen mixed of TiO2, Co, and Zr of the same quantity. The differences of excitation efficiency have clearly appeared according to the change in excitation source. From the results discussed here, it can be expected that the presented x-ray tube will be a powerful tool in microx-ray fluorescence spectrometers and various x-ray instruments. [ABSTRACT FROM AUTHOR]

Abstract: Doubly-curved crystals (DCCs) can be very useful devices to focus and monochromatize radiation from an X-ray tube or a synchrotron, especially for X-ray fluorescence analysis. However, production of a DCC is somewhat difficult. In this paper we will present some basic theory about DCC geometries, different ways of DCC production and methods to test the quality of the DCCs. [Copyright &y& Elsevier]

Antimicrobial peptides (AMPs) are an alternative group for the therapy of infectious diseases, with activity against a wide range of diverse pathogens. However, classical AMPs have significant side effects in human cells due to their unspecific pore formation in biomembranes. Nevertheless, AMPs are promising therapeutics and can be isolated from natural sources, which include sea and freshwater molluscs. The AMPs identified in these organisms show promising antimicrobial activities, as pathogens are mainly fought by innate defence mechanisms. An auspicious candidate among molluscs is the Cuban freshwater snail Pomacea poeyana, from which the peptides Pom-1 and Pom-2 have been isolated and studied. These studies revealed significant antimicrobial activities for both AMPs. Based on the activities determined, Pom-1 was used for further optimization. In order to meet the emerging requirements of improved anti-biofilm activity against naturally occurring Candida species, the six derivatives Pom-1A to F were developed and investigated. Analysis of the derivatives acting on the most abundant naturally occurring Candida yeast Candida albicans (C. albicans) revealed a strong anti-biofilm activity, especially induced by Pom-1 B, C, and D. Furthermore, a moderate decrease in the metabolic activity of planktonic yeast cells was observed. [ABSTRACT FROM AUTHOR]

Single-stranded DNA aptamers as affinity molecules for the rapid, reliable detection of intestinal bacteria are of particular interest to equip health systems with novel robust and cheap diagnostic tools for monitoring the success of supplementation strategies with selected probiotic gut bacteria in the fight against major widespread threats, such as obesity and neurodegenerative diseases. The human gut bacterium Parabacteroides distasonis (P. distasonis) is positively associated with diseases such as obesity, non-alcoholic fatty liver disease and multiple sclerosis with reduced cell counts in these diseases and is thus a promising potential probiotic bacterium for future microbial supplementation. In this paper we report on the evolution of a specific polyclonal aptamer library by the fluorescence based FluCell-SELEX directed against whole cells of P. distasonis that specifically and efficiently binds and labels P. distasonis. The aptamer library showed high binding affinity and was suited to quantitatively discriminate P. distasonis from other prominent gut bacteria also in mixtures. We believe that this library against a promising probiotic bacterium as a prototype may open new routes towards the development of novel biosensors for the easy and efficient quantitative monitoring of microbial abundance in human microbiomes in general. [ABSTRACT FROM AUTHOR]

Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa, we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG4-SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis. [ABSTRACT FROM AUTHOR]

An experimental method for the verification of the individually different energy dependencies of L1-, L2-, and L3- subshell photoionization cross sections is described. The results obtained for Pd and Mo are well in line with theory regarding both energy dependency and absolute values, and confirm the theoretically calculated cross sections by Scofield from the early 1970 s and, partially, more recent data by Trzhaskovskaya, Nefedov, and Yarzhemsky. The data also demonstrate the questionability of quantitative x-ray spectroscopical results based on the widely used fixed jump ratio approximated cross sections with energy independent ratios. The experiments are carried out by employing the radiometrically calibrated instrumentation of the Physikalisch-Technische Bundesanstalt at the electron storage ring BESSY II in Berlin; the obtained fluorescent intensities are thereby calibrated at an absolute level in reference to the International System of Units. Experimentally determined fixed fluorescence line ratios for each subshell are used for a reliable deconvolution of overlapping fluorescence lines. The relevant fundamental parameters of Mo and Pd are also determined experimentally in order to calculate the subshell photoionization cross sections independently of any database. [ABSTRACT FROM AUTHOR]

Grazing-Incidence X-ray fluorescence (GIXRF) analysis, which is closely related to total-reflection XRF, is a very powerful technique for the in-depth analysis of many types of technologically relevant samples, e.g. nanoparticle depositions, shallow dopant profiles, thin layered samples or even well-ordered nanostructures. However, the GIXRF based determination of the depth-dependent information about the sample is usually based on a modeling of the experimental data. This requires profound knowledge of the geometrical parameters of the setup employed, especially the incident beam profile as well as the detector aperture parameters. Together they determine the incident angle dependent so-called effective solid angle of detection which must be known in order to model any experimental data set. In this work, we demonstrate how these instrumental parameters, which are typically not known with sufficient accuracy, can be determined using dedicated experiments with a well-known calibration sample. In addition, this paves the way for a full calibration of the setup, as also information about other parameters, e.g. the incident photon flux is gained. Here, we are using a commercially available instrument for this demonstration but the principle can also be applied for other GIXRF setups. Image 1 • Route for solid angle characterization and partial calibration for TXRF/GIXRF tools • Enables quantitative depth profiling using GIXRF • Principle is transferable to other commercially available or custom build tools [ABSTRACT FROM AUTHOR]