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Post-labelling cleavable substrates for self-labelling protein tags, such as SNAP- and Halo-tags, can be used to study cell surface receptor trafficking events by stripping dyes from non-internalized protein pools. Since the complexity of receptor biology requires the use of multiple and orthogonal approaches to simultaneously probe multiple receptor pools, we report the development of four membrane impermeable probes that covalently bind to either the SNAP- or the Halo-tag in the red to far-red range. These molecules bear a disulfide bond to release the non-internalized probe using the reducing agent sodium 2-mercaptoethane sulfonate (MESNA). As such, our approach allows the simultaneous visualization of multiple internalized cell surface proteins in two colors which we showcase using G protein-coupled receptors. We use this approach to detect internalized group II metabotropic glutamate receptor (mGluRs), homo- and heterodimers, and to reveal unidirectional crosstalk between co-expressed glucagon-like peptide 1 (GLP1R) and glucose-dependent insulinotropic polypeptide receptors (GIPR). In these applications, we translate our method to both high resolution imaging and quantitative, high throughput assays, demonstrating the value of our approach for a wide range of applications., Competing Interests: The authors declare no competing interests., (This journal is © The Royal Society of Chemistry.)

Communication between cells is largely orchestrated by proteins on the cell surface, which allow information transfer across the cell membrane. Super-resolution and single-molecule visualization of these proteins can be achieved by genetically grafting HTP (HaloTag Protein) into the protein of interest followed by brief incubation of cells with a dye-HTL (dye-linked HaloTag Ligand). This approach allows for use of cutting-edge fluorophores optimized for specific optical techniques or a cell-impermeable dye-HTL to selectively label surface proteins without labeling intracellular copies. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Traditional methods to eliminate cell permeability face synthetic bottlenecks and risk altering photophysical properties. Here we report that dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed. This simple, one-step method requires no purification and is compatible with both the original HTL and second-generation HTL.2, the latter offering accelerated labeling. We validate such compounds, termed dye-SHTL ('dye shuttle') conjugates, in live cells via widefield microscopy, demonstrating exclusive membrane staining of extracellular HTP fusion proteins. In transduced primary hippocampal neurons, we label mGluR2, a neuromodulatory G protein-coupled receptor (GPCR), with dyes optimized for stimulated emission by depletion (STED) super-resolution microscopy, allowing unprecedented accuracy in distinguishing surface and receptors from those in internal compartments of the presynaptic terminal, important in neural communication. This approach offers broad utility for surface-specific protein labelling., Competing Interests: COMPETING INTERESTS NL is a member of the scientific advisory board of Trace Neuroscience. MRT and BCS are on patent applications describing HTL.2. All other authors declare no competing interests.

Chemical photoswitches have become a widely used approach for the remote control of biological functions with spatiotemporal precision. Several molecular scaffolds have been implemented to improve photoswitch characteristics, ranging from the nature of the photoswitch itself (e.g. azobenzenes, dithienylethenes, hemithioindigo) to fine-tuning of aromatic units and substituents. Herein, we present deuterated azobenzene photoswitches as a general means of enhancing the performance of photopharmacological molecules. Deuteration can improve azobenzene performance in terms of light sensitivity (higher molar extinction coefficient), photoswitch efficiency (higher photoisomerization quantum yield), and photoswitch kinetics (faster macroscopic rate of photoisomerization) with minimal alteration to the underlying structure of the photopharmacological ligand. We report synthesized deuterated azobenzene-based ligands for the optimized optical control of ion channel and G protein-coupled receptor (GPCR) function in live cells, setting the stage for the straightforward, widespread adoption of this approach., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The metabotropic glutamate receptors (mGluRs) are family C, dimeric G protein-coupled receptors (GPCRs), which play critical roles in synaptic transmission. Despite an increasing appreciation of the molecular diversity of this family, how distinct mGluR subtypes are regulated remains poorly understood. We reveal that different group II/III mGluR subtypes show markedly different beta-arrestin (β-arr) coupling and endocytic trafficking. While mGluR2 is resistant to internalization and mGluR3 shows transient β-arr coupling, which enables endocytosis and recycling, mGluR8 and β-arr form stable complexes, which leads to efficient lysosomal targeting and degradation. Using chimeras and mutagenesis, we pinpoint carboxyl-terminal domain regions that control β-arr coupling and trafficking, including the identification of an mGluR8 splice variant with impaired internalization. We then use a battery of high-resolution fluorescence assays to find that heterodimerization further expands the diversity of mGluR regulation. Together, this work provides insight into the relationship between GPCR/β-arr complex formation and trafficking while revealing diversity and intricacy in the regulation of mGluRs.

Background: Colloid cysts, although benign, may occasionally cause obstructive hydrocephalus and sudden death. Reliable prognostic factors for symptomatic progression have been sought, with heterogenous results., Methods: We conducted a retrospective review of all cases of colloid cysts of the third ventricle managed at our center between 2009 and 2019. Clinical and neuroimaging characteristics were analyzed using logistic regression in relation to symptomatic status and hydrocephalus. The cutoff values for outcome prediction were calculated using the receiver operating characteristic curve analysis., Results: There were 82 patients with colloid cysts, of whom 60 were asymptomatic and 22 symptomatic. None of the asymptomatic patients experienced acute neurologic decline or hydrocephalus during follow-up, whereas half (n = 11) of the symptomatic patients presented with hydrocephalus, 8 of whom had acute hydrocephalus. We found 3 putative candidate risk factors for symptomatic colloid cysts: T1-weighted magnetic resonance imaging hyperintense/mixed signal appearance (P = 0.004), location in risk zone I (P = 0.007), and a volume >236.49 mm 3 (P = 0.007). Cyst diameter and volume/foramen of Monro diameter ratios had a decreasing trend over time among asymptomatic patients, providing new insights into the natural history of the disease., Conclusions: Only a few asymptomatic colloid cysts showed progression requiring surgery, with no acute deterioration or fatal events, whereas the rest remained stable over time, thus supporting a more conservative approach for this group of patients. Higher risk for developing symptomatic colloid cyst was defined by a risk score that included T1-weighted magnetic resonance imaging appearance, risk zone, and colloid cyst volume, aiding the detection of patients at risk of clinical deterioration., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

TGFβs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFβ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways., (© 2023. The Author(s).)

The (in)ability to permeate membranes is a key feature of chemical biology probes that defines their suitability for specific applications. Here we report sulfonated rhodamines that endow xanthene dyes with cellular impermeability for analysis of surface proteins. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to benzylguanine and choloralkane substrates for SNAP-tag and Halo-tag labelling. Sulfonated rhodamine-conjugated fluorophores maintain desirable photophysical properties, such as brightness and photostability. While transfected cells with a nuclear localized SNAP-tag remain unlabelled, extracellular exposed tags can be cleanly visualized. By multiplexing with a permeable rhodamine, we are able to differentiate extra- and intracellular SNAP- and Halo-tags, including those installed on the glucagon-like peptide-1 receptor, a prototypical class B G protein-coupled receptor. Sulfo549 and Sulfo646 also labelled transfected neurons derived from induced pluripotent stem cells (iPSCs), allowing STED nanoscopy of the axonal membrane. Together, this work provides a new avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags. We anticipate that Sulfo549, Sulfo646 and their congeners will be useful for a number of cell biology applications where labelling of intracellular sites interferes with accurate surface protein analysis.

Rhodamine fluorophores are setting benchmarks in fluorescence microscopy. Herein, we report the deuterium (d12) congeners of tetramethyl(silicon)rhodamine, obtained by isotopic labelling of the four methyl groups, show improved photophysical parameters ( i.e. brightness, lifetimes) and reduced chemical bleaching. We explore this finding for SNAP- and Halo-tag labelling in live cells, and highlight enhanced properties in several applications, such as fluorescence activated cell sorting, fluorescence lifetime microscopy, stimulated emission depletion nanoscopy and single-molecule Förster-resonance energy transfer. We finally extend this idea to other dye families and envision deuteration as a generalizable concept to improve existing and to develop new chemical biology probes., Competing Interests: JB has a licensing deal with Celtarys Research for LUXendin distribution., (This journal is © The Royal Society of Chemistry.)

The glucagon-like peptide-1 receptor (GLP1R) is expressed in peripheral tissues and the brain, where it exerts pleiotropic actions on metabolic and inflammatory processes. Detection and visualization of GLP1R remains challenging, partly due to a lack of validated reagents. Previously, we generated LUXendins , antagonistic red and far-red fluorescent probes for specific labeling of GLP1R in live and fixed cells/tissues. We now extend this concept to the green and near-infrared color ranges by synthesizing and testing LUXendin492 , LUXendin551 , LUXendin615 , and LUXendin762 . All four probes brightly and specifically label GLP1R in cells and pancreatic islets. Further, LUXendin551 acts as a chemical beta cell reporter in preclinical rodent models, while LUXendin762 allows noninvasive imaging, highlighting differentially accessible GLP1R populations. We thus expand the color palette of LUXendins to seven different spectra, opening up a range of experiments using wide-field microscopy available in most labs through super-resolution imaging and whole animal imaging. With this, we expect that LUXendins will continue to generate novel and specific insights into GLP1R biology., Competing Interests: The authors declare the following competing financial interest(s): D.J.H. and J.B. have a licensing deal with Celtarys Research for LUXendin distribution., (© 2022 The Authors. Published by American Chemical Society.)

Background: The rise of an infectious disease crisis such as the SARS-CoV‑2 pandemic posed significant challenges for the administrative structures of the public health service, which resulted in varying levels of efficiency in outbreak management as a function of staffing and digital resources. This substantially impeded the integration of innovative pandemic outbreak management tools. Innovative crisis management, such as cluster tracking, risk group testing, georeferencing, or the integration of wastewater surveillance recommended by the EU Commission, was made significantly more difficult., Aim: In this case study in Berchtesgadener Land, we present the integration of an area-wide georeferenced wastewater surveillance system that captured 95% of the entire population since November 2020., Methodology: Sampling occurred twice a week at nine municipal wastewater treatment plants and directly from the main sewer at three locations. Samples were pre-treated by centrifugation and subsequently analyzed by digital droplet polymerase chain reaction (PCR) targeting four specific genes of SARS-CoV‑2., Results: The integration of an area-wide georeferenced wastewater surveillance system was successful. Wastewater occurrences are plotted for each municipality against cumulative infections over seven days per 100,000 inhabitants. Changes in the infection pattern in individual communities are noticeable ten days ahead of the official case numbers with a sensitivity of approximately 20 in 100,000 inhabitants., Discussion: The integration of this innovative approach to provide a comprehensive overview of the situation by employing a digital dashboard and the use of an early warning system via quantitative wastewater surveillance resulted in very efficient, proactive management, which might serve as a blueprint for other municipalities in Germany., (© 2021. The Author(s).)

The Medical Intelligence and Information (MI2) Unit of the German Armed Forces (Bundeswehr) is experienced in crisis support in military missions since several years. It gained additional experiences during the current coronavirus 2019 (COVID-19) pandemic on different levels of the response to crisis and was requested to share the findings and expertise with the overloaded civil public health agencies inside Germany. Since the beginning of the pandemic, the unit is constantly developing new products for crisis communication, knowledge sharing techniques in new databases, dashboards for leadership, and training for laypersons in contact tracing. Hence, trying to innovate in crisis since the first severe acute respiratory syndrome coronavirus (SARS-CoV)-2-disease wave. During the second wave, the unit was requested to evaluate the outbreak management of different national civil public health agencies in southern Germany, and to support the development of dashboards in a comprehensive public health approach as a necessary start toward digitalization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Roßmann, Wegner, Stark, Großmann, Jansen and Frangoulidis.)

Background: The Syria crisis has forced more than 4 million people to leave their homeland. As a result, in 2016, an overwhelming number of refugees reached Germany. In response to this, it was of utmost importance to set up refugee camps and to provide humanitarian aid, but a health surveillance system was also implemented in order to obtain rapid information about emerging diseases., Objective: The present study describes the effects of using digital paper and pen (DPP) technology on the speed, sequence, and behavior of epidemiological documentation in a refugee camp., Methods: DPP technology was used to examine documentation speed, sequence, and behavior. The data log of the digital pens used to fill in the documentation was analyzed, and each pen stroke in a field was recorded using a timestamp. Documentation time was the difference between first and last stroke on the paper, which includes clinical examination and translation., Results: For three months, 495 data sets were recorded. After corrections had been made, 421 data sets were considered valid and subjected to further analysis. The median documentation time was 41:41 min (interquartile range 29:54 min; mean 45:02 min; SD 22:28 min). The documentation of vital signs ended up having the strongest effect on the overall time of documentation. Furthermore, filling in the free-text field clinical findings or therapy or measures required the most time (mean 16:49 min; SD 20:32 min). Analysis of the documentation sequence revealed that the final step of coding the diagnosis was a time-consuming step that took place once the form had been completed., Conclusions: We concluded that medical documentation using DPP technology leads to both an increase in documentation speed and data quality through the compliance of the data recorders who regard the tool to be convenient in everyday routine. Further analysis of more data sets will allow optimization of the documentation form used. Thus, DPP technology is an effective tool for the medical documentation process in refugee camps., (©Kai Kehe, Roland Girgensohn, Walter Swoboda, Dan Bieler, Axel Franke, Matthias Helm, Martin Kulla, Kerstin Luepke, Thomas Morwinsky, Markus Blätzinger, Katalyn Rossmann. Originally published in JMIR Mhealth and Uhealth (http://mhealth.jmir.org), 19.08.2019.)

Dependence of the speed of various screen/film systems on x-ray energy was studied using the nearly monoenergetic x rays emitted by a filtered fluorescent source. The results show that response depends on screen phosphor composition and thickness. Barium and rare earth screens having K absorption energies lower than that of calcium tungstate are relatively more sensitive to x rays in the 40-70-keV region.

Two methods for computing x-ray images of small blood vessels in angiography are presently available, namely, convolution and ray tracing. The convolution method, which is simpler and more powerful than the ray-tracing method, is based on the assumption that blood vessel imaging is isoplantic, whereas ray tracing is considered to provide correct images. In this study, the approximation error (difference between two images, normalized by the maximum value) due to nonisoplanatic imaging was determined by computation of blood vessel images according to both methods. The approximation error for geometric conditions normally encountered in angiography was less than 0.01. It is concluded that an approximation error of this magnitude is negligible and that the convolution method can be applied instead of the ray-tracing method for the computation of images of small blood vessels.

High-resolution radiography may be done using either optical or radiographic magnification. In the former technique, industrial Type M film was used without screens and the image was viewed with 4-10X optical magnification. In the latter technique, RP film was used with Detail screens and 4X geometric magnification together with a microfocus x-ray tube having a nominal focal spot size of 50 mum. The imaging properties of both techniques were evaluated by means of H & D curves, modulation transfer functions, and Wiener spectra. It was found that for thin objects such as the hand, optical magnification provides better bone images than radiographic magnification; whereas for thicker parts such as the knee, radiographic magnification is superior.

A new method of stereoscopic cerebral angiography has been developed which employs 2X radiographic magnification. In order to obtain the same depth perception in the object as with conventional contact stereoscopic angiography, one can made the x-ray exposures at two focal spot positions which are separated by only 1 inch (2.5 cm), whereas the contact technique requires a separation of 4 inches (10 cm). The smaller distance is possible because, with 2X magnification, the transverse detail in the object is magnified by a factor of two, but the longitudinal detail, which is related to the stereo effect, is magnified by a factor of four, due to the longitudinal magnification effect. The small focal spot separation results in advantages such as improved stereoscopic image detail, better image quality, and low radiation exposure to the patient.

From a computer simulation study of blood vessel imaging with uniform, triangular, gaussian, and twin gaussian line spread functions (LSF) corresponding to various focal spot distributions, it is found that vessel images magnified less than 6 times are not strongly dependent upon the focal spot distributions when the size of the focal spot is equal to or smaller than the vessel diameter. When the focal spot is larger than the vessel, image distributions vary appreciably with focal spot distributions, except at low magnification. It is concluded that, in many practical cases, the focal spot distribution does not seriously affect magnified image distributions of blood vessels.

The computer-simulation technique for imaging of vessels in angiography is being recognized as a useful tool for the study of the effect of physical parameters on the vessel image. We have examined the validity of this technique by comparing the computed image distribution directly with experimental results. The imput x-ray pattern of a blood vessel phantom, the LSF of the focal spot, and the LSF of a screen-film system were measured. From the data obtained, the final image distributions of the phantom were computed and compared with the experimentally determined distributions. A high-precision device for the alignment and positioning of the vessel phantom was employed in the experiments. Results indicate that computer simulation can correctly predict the image distribution of the blood vessel phantom.

Modulation transfer functions (MTFs) and test object radiographs were used to study the effect of geometric and recording system unsharpness in mammography with the CGR Senographe x-ray unit. Results show that geometric unsharpness can be a significant factor in the detection of microcalcifications within the breast, depending on the size and shape of the focal spot, the focal spot-to-recording system distance, and the object-to-recording system distance (o.r.d.). A new recording system for mammography, the DuPont Lo-dose system, requires approximately 1/15th the exposure of a direct x-ray film, such as Kodak RP/M, to provide mammograms with comparable photographic density. With the Lo-dose system, geometric unsharpness can be reduced by use of a specially designed long cone with an increased focal spot-to-recording system distance. This cannot be accomplished with direct x-ray films because the x-ray unit is operating at near-maximum output conditions even when short cones are used. Although direct x-ray films have a higher resolution than the Lo-dose system, at certain o.r.d.s total resolution is found to be affected more significantly by geometric unsharpness than by the Lo-dose recording system. In several cases, clinical results show improved detection of microcalcifications at larger o.r.d.s by the Lo-dose system with a long one, combined with a reduction by a factor of 15 in patient exposure.

The measured x-ray spectra from a molybdenum anode Senographe mammography unit and a tungsten anode unit were used to calculate the x-ray energy spectra transmitted through various thicknesses of fat and water (breast-equivalent materials). The dependence of subject contrast and patient exposure on (a) x-ray spectra, (b) attenuation properties of two breast-equivalent materials, and (c) thickness of breast-equivalent material was predicted. Radiographs of resected breast tissue confirmed these predictions and demonstrated the general relative effects of x-ray beam quality on image contrast in mammography.

Ointments for the protection against poisoning of the skin by highly toxic organophosphates or organophosphate insecticides were prepared and examined for their protective effect both on guinea-pig skin in vitro and on guinea-pigs in vivo. The results of these investigations were as follows: 1. The prophylactic application of the ointments yields 20 fold reduction of the permeation rate of the toxic organophosphate compared with unprotected skin. 2. The ointments on a polyethyleneglycol basis show their best protective effect if they contain both a base and an active substance. 3. Maximum possible protection can be achieved by applying a thick layer of ointment which should be mechanically removed (by wiping with a damp cellulose tissue) from the contamination site within 30 min.

A new device has been developed with which the focal spot distribution can be measured accurately. The alignment and localization of the focal spot relative to the device are accomplished by adjustment of three micrometer screws in three orthogonal directions and by comparison of red reference light spots with green fluorescent pinhole images at five locations. The standard deviations for evaluating the reproducibility of the adjustments in the horizontal and vertical directions were 0.2 and 0.5 mm, respectively. Measurements were made of the pinhole images as well as of the line-spread functions (LSFs) and modulation transfer functions (MTFs) for an x-ray tube with focal spots of 1-mm and 50-mum nominal size. The standard deviations for the LSF and MTF of the 1-mm focal spot were 0.017 and 0.010, respectively.

The basic theory of x-ray image formation of blood vessels, which is related to isoplanatism, is discussed. The x-ray intensity distributions of the blood vessel images are derived first for the actual case, which gives "correct" x-ray images obtained by the ray-tracing method. Secondly, as an approximation of the actual case, the image distributions are derived by the convolution method, which corresponds to an isoplanatic case. It is concluded that, under practical conditions, x-ray images of blood vessels are given approximately by a convolution integral of the object distribution, that is, the input x-ray pattern of the vessel exposed with a parallel x-ray beam, with the line spread function of geometric unsharpness. Therefore, this theory provides support for the experimental procedure commonly used in obtaining blood vessel images, and for the validity of applying in angiography the concept of the line spread function and the modulation transfer function of geometric unsharpness.

New concepts which are useful for evaluating image resolution and distortion in magnification radiography are introduced. It is shown theoretically and experimentally that "longitudinal magnification" defined as the ratio of the magnified image size to the conventional image size for objects located in a plane perpendicular to the film, is given approximately by the square of the conventional magnification. Resolution in the radiographically magnified image of such objects is much higher than that obtained by optical magnification. This is confirmed experimentally by radiographs of square-wave test objects. The related image distortion is also discussed.

The purpose of this study was to determine the physical activity of physical therapists during clinical practice. Nineteen physical therapists from three clinical facilities wore portable accelerometers on their waist belts or chest pockets for the duration of their work shifts. The subjects repeated the experiment on the second day. The accelerometer readings (in arbitrary units) were converted to oxygen consumption VO2 and energy expenditure (kcal) values using previously derived regression equations. The PTs worked an average of 7.9 hours during each shift. The total energy expended during the work shift ranged from 933.6 to 1,689.6 kcal. A correlation coefficient of .91 was found between the accelerometer readings (count X hr-1) recorded on day 1 and day 2 of the study. The analysis of variance revealed that the PTs in the three clinical facilities were not significantly different in age, height, and work-shift duration, but were significantly different (p less than .05) in weight, mean accelerometer readings, and estimated VO2 (L X min-1). When the VO2 was expressed in mL X kg-1 X min-1, no significant difference was found between the PTs in the three clinical facilities. The implications and limitations of the findings are discussed.