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- ItemLaboratory-Developed Tests: Design of a Regulatory Strategy in Compliance with the International State-of-the-Art and the Regulation (EU) 2017/746 (EU IVDR [In Vitro Diagnostic Medical Device Regulation])([New York] : Springer Nature, 2022) Spitzenberger, Folker; Patel, Jaimin; Gebuhr, Inga; Kruttwig, Klaus; Safi, Abdulrahim; Meisel, ChristianPurpose: This study aimed at the development of a regulatory strategy for compliance of laboratory-developed tests (LDTs) with requirements of the Regulation (EU) 2017/746 (“EU-IVDR”) under consideration of international requirements for LDTs as established in major regulatory regions. Furthermore, it was analysed in how far elements of current LDT regulation could qualify for an internationally harmonised concept ensuring quality, safety and performance of LDTs. Methods: A review of regulatory literature including legislation as well as guidance documents was performed. The regulatory strategy was adapted from international guidance concepts used for commercially marketed IVD. It was then applied to the example of a large medical laboratory in the EU. A high-level comparison was conducted to identify gaps and matches between the different international regulatory requirements for LDTs. Results: A four-step strategy for compliance of LDTs with the EU IVDR was implemented in an exemplary medical laboratory. On the basis of an internationally used LDT definition, LDTs constitute nearly 50% of the total IVD devices used in the laboratory. While an ISO 15189-compliant QMS is a major component, it should be accompanied by the application of appropriate processes for risk management, performance evaluation and continuous monitoring of LDTs. At least six criteria represent common characteristics of a potential, internationally convergent concept for the regulation/standardization of LDTs. Conclusions: This study confirms the impact of LDTs for individualized and innovative medical laboratory testing. Prerequisites for LDT use as especially given by the IVDR and missing interpretation in the EU with regard to the scope of LDT definition, the application of standards and the extent of documentation for LDTs currently lead to uncertainties for both laboratories and regulatory bodies responsible for LDT oversight. The characteristics identified as common criteria for ensuring quality, safety and performance of LDTs may be considered as central elements of future international consensus guidance. © 2021, The Author(s).
- ItemThe Superconducting Gravimeter CD-034 at Moxa Observatory: More than 20 Years of Scientific Experience and a Reanimation(Basel : Birkhäuser, 2022) Jahr, T.; Stolz, R.In this work, advanced methods and processing schemes for the analysis of data from a Superconducting Gravimeter (SG) will be introduced and their relevance on acquired data proved. The SG CD-034 was installed on Easter of 1999 in the Geodynamic Observatory Moxa of the Friedrich Schiller University Jena, Germany. Initially, the quality of the recorded data was examined, spectra for the detection of the parasitic modes were calculated and the calibration values for the two sensors were determined. Ever since very high-quality gravity data of this SG and most of the other worldwide SGs were made available through the storage archive of the Global Geodynamics Project (GGP later changed to IGETS, International Geodynamics and Earth Tide Service) for global scientific investigations at that time. SG’s such as the one in Moxa (Germany) still deliver significant scientific value for global gravitational field studies as well as for regional/local studies which will be shortly reviewed. Examples are the detection of polar motion, the influence of continental water loading in general and in particular river basin loads, the gravimetric effect of North Sea storm surges and the study of hydro-gravimetric signals, which could be compared with satellite observations and global hydraulic models. The long-term, low-noise operation of complex SG’s requires some effort on maintenance. In order to evaluate the correct operation of the SG, new data processing steps were introduced to assist in the analysis of the data in case of issues with the instrumentation. For example, in 2012/2013 and 2020/2021 severe interference in the gravimeter electronics in Moxa led to a significant loss of data. In both cases, however, the cause could be determined, and the corresponding electronic components renewed. Since July 2021, the SG in Moxa registers again with high data quality comparable or slightly better than before the incident. Initial tests and tidal analyses confirm the validity of the old calibration factors, and the authors now look forward to the re-established long-term recording with excitement and confidence.
- ItemConsensusPrime—A Bioinformatic Pipeline for Ideal Consensus Primer Design(Basel : MDPI, 2022) Collatz, Maximilian; Braun, Sascha D.; Monecke, Stefan; Ehricht, RalfBackground: High-quality oligonucleotides for molecular amplification and detection procedures of diverse target sequences depend on sequence homology. Processing input sequences and identifying homogeneous regions in alignments can be carried out by hand only if they are small and contain sequences of high similarity. Finding the best regions for large and inhomogeneous alignments needs to be automated. Results: The ConsensusPrime pipeline was developed to sort out redundant and technical interfering data in multiple sequence alignments and detect the most homologous regions from multiple sequences. It automates the prediction of optimal consensus primers for molecular analytical and sequence-based procedures/assays. Conclusion: ConsensusPrime is a fast and easy-to-use pipeline for predicting optimal consensus primers that is executable on local systems without depending on external resources and web services. An implementation in a Docker image ensures platform-independent executability and installability despite the combination of multiple programs. The source code and installation instructions are publicly available on GitHub.
- ItemBackground Reduction in STED-FCS Using a Bivortex Phase Mask(Washington, DC : ACS Publications, 2020) Barbotin, Aurélien; Urbančič, Iztok; Galiani, Silvia; Eggeling, Christian; Booth, MartinFluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics in living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes on the nanoscale in living cells. In two-dimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volume, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern created by a bivortex phase mask reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS-based investigations of 3D diffusion on the subdiffraction scale. Copyright © 2020 American Chemical Society.
- ItemGiant refractometric sensitivity by combining extreme optical Vernier effect and modal interference([London] : Macmillan Publishers Limited, part of Springer Nature, 2020) Gomes, André D.; Kobelke, Jens; Bierlich, Jörg; Dellith, Jan; Rothhardt, Manfred; Bartelt, Hartmut; Frazão, OrlandoThe optical Vernier effect consists of overlapping responses of a sensing and a reference interferometer with slightly shifted interferometric frequencies. The beating modulation thus generated presents high magnified sensitivity and resolution compared to the sensing interferometer, if the two interferometers are slightly out of tune with each other. However, the outcome of such a condition is a large beating modulation, immeasurable by conventional detection systems due to practical limitations of the usable spectral range. We propose a method to surpass this limitation by using a few-mode sensing interferometer instead of a single-mode one. The overlap response of the different modes produces a measurable envelope, whilst preserving an extremely high magnification factor, an order of magnification higher than current state-of-the-art performances. Furthermore, we demonstrate the application of that method in the development of a giant sensitivity fibre refractometer with a sensitivity of around 500 µm/RIU (refractive index unit) and with a magnification factor over 850.
- ItemIMAGE-IN: Interactive web-based multidimensional 3D visualizer for multi-modal microscopy images(San Francisco, California, US : PLOS, 2022) Gupta, Yubraj; Costa, Carlos; Pinho, Eduardo; A. Bastião Silva, Luís; Heintzmann, RainerAdvances in microscopy hardware and storage capabilities lead to increasingly larger multidimensional datasets. The multiple dimensions are commonly associated with space, time, and color channels. Since “seeing is believing”, it is important to have easy access to user-friendly visualization software. Here we present IMAGE-IN, an interactive web-based multidimensional (N-D) viewer designed specifically for confocal laser scanning microscopy (CLSM) and focused ion beam scanning electron microscopy (FIB-SEM) data, with the goal of assisting biologists in their visualization and analysis tasks and promoting digital work-flows. This new visualization platform includes intuitive multidimensional opacity fine-tuning, shading on/off, multiple blending modes for volume viewers, and the ability to handle multichannel volumetric data in volume and surface views. The software accepts a sequence of image files or stacked 3D images as input and offers a variety of viewing options ranging from 3D volume/surface rendering to multiplanar reconstruction approaches. We evaluate the performance by comparing the loading and rendering timings of a heterogeneous dataset of multichannel CLSM and FIB-SEM images on two devices with installed graphic cards, as well as comparing rendered image quality between ClearVolume (the ImageJ open-source desktop viewer), Napari (the Python desktop viewer), Imaris (the closed-source desktop viewer), and our proposed IMAGE-IN web viewer.
- Item1D p–n Junction Electronic and Optoelectronic Devices from Transition Metal Dichalcogenide Lateral Heterostructures Grown by One-Pot Chemical Vapor Deposition Synthesis(Weinheim : Wiley-VCH, 2021) Najafidehaghani, Emad; Gan, Ziyang; George, Antony; Lehnert, Tibor; Ngo, Gia Quyet; Neumann, Christof; Bucher, Tobias; Staude, Isabelle; Kaiser, David; Vogl, Tobias; Hübner, Uwe; Kaiser, Ute; Eilenberger, Falk; Turchanin, AndreyLateral heterostructures of dissimilar monolayer transition metal dichalcogenides provide great opportunities to build 1D in-plane p–n junctions for sub-nanometer thin low-power electronic, optoelectronic, optical, and sensing devices. Electronic and optoelectronic applications of such p–n junction devices fabricated using a scalable one-pot chemical vapor deposition process yielding MoSe2-WSe2 lateral heterostructures are reported here. The growth of the monolayer lateral heterostructures is achieved by in situ controlling the partial pressures of the oxide precursors by a two-step heating protocol. The grown lateral heterostructures are characterized structurally and optically using optical microscopy, Raman spectroscopy/microscopy, and photoluminescence spectroscopy/microscopy. High-resolution transmission electron microscopy further confirms the high-quality 1D boundary between MoSe2 and WSe2 in the lateral heterostructure. p–n junction devices are fabricated from these lateral heterostructures and their applicability as rectifiers, solar cells, self-powered photovoltaic photodetectors, ambipolar transistors, and electroluminescent light emitters are demonstrated. © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH
- ItemSpatial proteomics revealed a CX3CL1-dependent crosstalk between the urothelium and relocated macrophages through IL-6 during an acute bacterial infection in the urinary bladder(New York, NY : Nature Publishing Group, 2020) Bottek, Jenny; Soun, Camille; Lill, Julia K.; Dixit, Akanksha; Thiebes, Stephanie; Beerlage, Anna-Lena; Horstmann, Marius; Urbanek, Annett; Heuer, Heike; Uszkoreit, Julian; Eisenacher, Martin; Bracht, Thilo; Sitek, Barbara; Hoffmann, Franziska; Vijitha, Nirojah; von Eggeling, Ferdinand; Engel, Daniel R.The urothelium of the urinary bladder represents the first line of defense. However, uropathogenic E. coli (UPEC) damage the urothelium and cause acute bacterial infection. Here, we demonstrate the crosstalk between macrophages and the urothelium stimulating macrophage migration into the urothelium. Using spatial proteomics by MALDI-MSI and LC-MS/MS, a novel algorithm revealed the spatial activation and migration of macrophages. Analysis of the spatial proteome unravelled the coexpression of Myo9b and F4/80 in the infected urothelium, indicating that macrophages have entered the urothelium upon infection. Immunofluorescence microscopy additionally indicated that intraurothelial macrophages phagocytosed UPEC and eliminated neutrophils. Further analysis of the spatial proteome by MALDI-MSI showed strong expression of IL-6 in the urothelium and local inhibition of this molecule reduced macrophage migration into the urothelium and aggravated the infection. After IL-6 inhibition, the expression of matrix metalloproteinases and chemokines, such as CX3CL1 was reduced in the urothelium. Accordingly, macrophage migration into the urothelium was diminished in the absence of CX3CL1 signaling in Cx3cr1gfp/gfp mice. Conclusively, this study describes the crosstalk between the infected urothelium and macrophages through IL-6-induced CX3CL1 expression. Such crosstalk facilitates the relocation of macrophages into the urothelium and reduces bacterial burden in the urinary bladder. © 2020, The Author(s).
- ItemSide-view holographic endomicroscopy via a custom-terminated multimode fibre(Washington, DC : Soc., 2021) Silveira, Beatriz M.; Pikálek, Tomáš; Stibůrek, Miroslav; Ondráčková, Petra; Jákl, Petr; Leite, Ivo T.; Čižmár, TomášMicroendoscopes based on optical fibres have recently come to the fore as promising candidates allowing in-vivo observations of otherwise inaccessible biological structures in animal models. Despite being still in its infancy, imaging can now be performed at the tip of a single multimode fibre, by relying on powerful holographic methods for light control. Fibre based endoscopy is commonly performed en face, resulting in possible damage of the specimen owing to the direct contact between the distal end of the probe and target. On this ground, we designed an all-fibre probe with an engineered termination that reduces compression and damage to the tissue under investigation upon probe insertion. The geometry of the termination brings the field of view to a plane parallel to the fibre’s longitudinal direction, conveying the probe with off-axis imaging capabilities. We show that its focusing ability also benefits from a higher numerical aperture, resulting in imaging with increased spatial resolution. The effect of probe insertion was investigated inside a tissue phantom comprising fluorescent particles suspended in agarose gel, and a comparison was established between the novel side-view probe and the standard en face fibre probe. This new concept paves the way to significantly less invasive deep-tissue imaging.
- ItemMolecular characterisation of extended-spectrum ß-lactamase producing Escherichia coli in wild birds and cattle, Ibadan, Nigeria(London : BioMed Central, 2021) Fashae, Kayode; Engelmann, Ines; Monecke, Stefan; Braun, Sascha D.; Ehricht, RalfBackground: Antimicrobial resistance (AMR) is an increasing global health concern reducing options for therapy of infections and also for perioperative prophylaxis. Many Enterobacteriaceae cannot be treated anymore with third generation cephalosporins (3GC) due to the production of certain 3GC hydrolysing enzymes (extended spectrum beta-lactamases, ESBLs). The role of animals as carriers and vectors of multi-resistant bacteria in different geographical regions is poorly understood. Therefore, we investigated the occurrence and molecular characteristics of ESBL-producing Escherichia coli (E. coli) in wild birds and slaughtered cattle in Ibadan, Nigeria. Cattle faecal samples (n = 250) and wild bird pooled faecal samples (cattle egrets, Bubulcus ibis, n = 28; white-faced whistling duck, Dendrocygna viduata, n = 24) were collected and cultured on cefotaxime-eosin methylene blue agar. Antimicrobial susceptibility was determined by agar diffusion assays and all 3GC resistant isolates were genotypically characterised for AMR genes, virulence associated genes (VAGs) and serotypes using DNA microarray-based assays. Results: All 3GC resistant isolates were E. coli: cattle (n = 53), egrets (n = 87) and whistling duck (n = 4); cultured from 32/250 (12.8%), 26/28 (92.9%), 2/24(8.3%), cattle, egrets and whistling duck faecal samples, respectively. blaCTX-M gene family was prevalent; blaCTX-M15 (83.3%) predominated over blaCTX-M9 (11.8%). All were susceptible to carbapenems. The majority of isolates were resistant to at least one of the other tested antimicrobials; multidrug resistance was highest in the isolates recovered from egrets. The isolates harboured diverse repositories of other AMR genes (including strB and sul2), integrons (predominantly class 1) and VAGs. The isolates recovered from egrets harboured more AMR genes; eight were unique to these isolates including tetG, gepA, and floR. The prevalent VAGs included hemL and iss; while 14 (including sepA) were unique to certain animal isolates. E. coli serotypes O9:H9, O9:H30 and O9:H4 predominated. An identical phenotypic microarray profile was detected in three isolates from egrets and cattle, indicative of a clonal relationship amongst these isolates. Conclusion: Wild birds and cattle harbour diverse ESBL-producing E. coli populations with potential of inter-species dissemination and virulence. Recommended guidelines to balance public health and habitat conservation should be implemented with continuous surveillance. © 2021, The Author(s).