In a highly efficient and reproducible process, bovine serum albumin (BSA) nanogels are prepared from inverse nanoemulsions. The concept of independent nanoreactors of the individual droplets in the ...nanoemulsions allows high protein concentrations of up to 0.6% in the inverse total system. The BSA gel networks are generated by the 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride coupling strategy widely used in protein chemistry. In a robust work-up protocol, the hydrophobic continuous phase of the inverse emulsion is stepwise replaced by water without compromising the colloidal stability and non-toxicity of the nanogel particles. Further, the simple process allows the loading of the nanogels with various cargos like a dye (Dy-495), a drug (ibuprofen), another protein FMN-binding fluorescent protein (EcFbFP), and oligonucleotides plasmid DNA for enhanced GFP expression in mammalian cells (pEGFP c3) and a synthetic anti-Pseudomonas aeruginosa aptamer library. These charged nanoobjects work efficiently as carriers for staining and transfection of cells. This is exemplarily shown for a phalloidin dye and a plasmid DNA as cargo with adenocarcinomic human alveolar basal epithelial cells (A549), a cell revertant of the SV-40 cancer rat cell line SV-52 (Rev2), and human breast carcinoma cells (MDA-MB-231), respectively.
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.
Ph
acute lymphoblastic leukemia (ALL) is characterized by the expression of an oncogenic fusion kinase termed BCR-ABL1. Here, we show that interleukin 7 receptor (IL7R) interacts with the chemokine ...receptor CXCR4 to recruit BCR-ABL1 and JAK kinases in close proximity. Treatment with BCR-ABL1 kinase inhibitors results in elevated expression of IL7R which enables the survival of transformed cells when IL7 was added together with the kinase inhibitors. Importantly, treatment with anti-IL7R antibodies prevents leukemia development in xenotransplantation models using patient-derived Ph
ALL cells. Our results suggest that the association between IL7R and CXCR4 serves as molecular platform for BCR-ABL1-induced transformation and development of Ph
ALL. Targeting this platform with anti-IL7R antibody eliminates Ph
ALL cells including those with resistance to commonly used ABL1 kinase inhibitors. Thus, anti-IL7R antibodies may provide alternative treatment options for ALL in general and may suppress incurable drug-resistant leukemia forms.
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.
A TXRF round robin test using preselected micro- and nanoscaled samples has been performed and shows excellent results, demonstrating the strength and reliability of the TXRF method when decoupled from unfavorable sample preparations.
Infections caused by yeasts of the genus
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
spec. to quantify the pathogens in clinical samples with high sensitivity. We recently obtained the specific aptamer library R10, which explicitly recognized
and evolved it by mimicking an early skin infection model caused by
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
isolates, including not only
but also strains like
, or
The next-generation fluorometric bioassay presented here can reliably and easily detect an early
infection and could be used for further clinical research or could even be developed into a full in vitro diagnostic tool.
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.
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.
Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide.
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
, 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 PEG
-SPDP. Specific and quantitative binding of
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.
Building Information Modeling (BIM) is today one of the most promising approaches to achieve digitalization in the construction industry over the complete building life cycle. Meanwhile most of the ...projects in research, industry or public authorities focus on the planning or construction phase, the practical usage of BIM in operational phase within Facility Management (FM) is still rare. Despite of the well agreed benefits of using BIM in the operations to improve maintenance planning and control, ensure speed up of FM hand over from construction or improve start-up management of building operations to name three of them, serious obstacles for practical use of BIM in FM must be considered. Just to mention two of them, today accepted standards helping FM organization to setup their BIM requirements according to different BIM uses are missing or As-Built information of existing buildings to setup digital building model needed for BIM are not available. The lack of exiting (3D) digital building models in FM will not change quickly; far more the 90 % of the buildings to be operated and maintained in FM business are existing buildings. The acquisition of As-Built information may be simplified by digital surveying methods (e.g. 3D Laser Scanning, Aerial photo capturing by Surveying Drone), but there is still an enormous amount of effort for parametric modelling needed to setup digital building models for FM, which cannot be achieved by typical FM organizations today. This paper extents first results of the research project BIM-FM presented on INservFM in 2017 comparing different digital surveying technologies used in field tests and to optimize workflow to make digital building models of existing buildings available for FM purposes. In order to give FM organization access to BIM data over a long period of building operation, new concepts of data management based on semantic web technologies to link BIM data to existing alphanumeric information located in Computer Aided Facility Management (CAFM) systems be discussed.
An experimental method for the verification of the individually different energy dependencies of L(1)-, L(2)-, and L(3)- 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.
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