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The article presents the results of measurements regarding the impact of the MHT9002HV tracked harvester on surface deformations and changing the physical parameters of the soil of three operational trails. The measurements were made in the terrain with a longitudinal slope of up to 14.9° (33.2%) and a transverse slope of up to 8.8° (17.9%). Spruce deadwood trees in mountain forest habitats were harvested. Static Eijkelkamp Penetrologger 0615SA and dynamic own design penetrometer were used to measure penetration resistance, soil samples were taken to determine bulk density, moisture content, and ground deformations on the trails were measured with a laser profilometer. A statistically significant increase in soil penetration resistance measured with penetrometers occurred for trails in the left rut at a depth of 16–20 cm. The change in the bulk density and moisture content proved statistically insignificant. The maximum ground deformation on the trails reached an average of 5.9 cm. The selection of a machine with low unit pressures (33 kPa), under the given favorable atmospheric conditions (there was a high temperature reaching 35 °C), with low soil moisture, protective organic layer of high thickness, and post-limbing residues, was optimal. The comparison of the results of the compactness measurements made with different penetrometers shows that the values obtained for the static penetrometer 0615SA are lower than those of the dynamic penetrometer of our own design. This is due to the lack of registration of high compactness in the memory of the 0615SA device. In the case of the impact penetrometer measurement, this problem does not occur, however, the presented solution does not allow performing a large number of measurements, and data processing in the case of such a simple solution is tedious. There is a need to develop a new penetrometer useful for determining soil compaction under similar difficult measurement conditions.

The results of epidemiological and pathophysiological studies suggest that type 2 diabetes mellitus (T2DM) may predispose to Alzheimer’s disease (AD). The two conditions present similar glucose levels, insulin resistance, and biochemical etiologies such as inflammation and oxidative stress. The diabetic state also contributes to increased acetylcholinesterase (AChE) activity, which is one of the factors leading to neurodegeneration in AD. The aim of this study was to assess in vitro the effects of metformin, phenformin, and metformin sulfenamide prodrugs on the activity of human AChE and butyrylcholinesterase (BuChE) and establish the type of inhibition. Metformin inhibited 50% of the AChE activity at micromolar concentrations (2.35 μmol/mL, mixed type of inhibition) and seemed to be selective towards AChE since it presented low anti-BuChE activity. The tested metformin prodrugs inhibited cholinesterases (ChE) at nanomolar range and thus were more active than metformin or phenformin. The cyclohexyl sulfenamide prodrug demonstrated the highest activity towards both AChE (IC50 = 890 nmol/mL, noncompetitive inhibition) and BuChE (IC50 = 28 nmol/mL, mixed type inhibition), while the octyl sulfenamide prodrug did not present anti-AChE activity, but exhibited mixed inhibition towards BuChE (IC50 = 184 nmol/mL). Therefore, these two bulkier prodrugs were concluded to be the most selective compounds for BuChE over AChE. In conclusion, it was demonstrated that biguanides present a novel class of inhibitors for AChE and BuChE and encourages further studies of these compounds for developing both selective and nonselective inhibitors of ChEs in the future.

Hyaluronan is an acidic heteropolysaccharide comprising alternating N -acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix 1 . The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis 2 . Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors 3 , 4 . Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body. A cryo-electron microscopy analysis reveals how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore.

Fluorescence molecular imaging plays a vital role in image‐guided surgery. In this context, the urokinase plasminogen activator receptor (uPAR) is an interesting biomarker enabling the detection and delineation of various tumor types due to its elevated expression on both tumor cells and the tumor microenvironment. In this study, anti‐uPAR Nanobodies (Nbs) are generated through llama immunization with human and murine uPAR protein. Extensive in vitro characterization and in vivo testing with radiolabeled variants are conducted to assess their pharmacokinetics and select lead compounds. Subsequently, the selected Nbs are converted into fluorescent agents, and their application for fluorescence‐guided surgery is evaluated in various subcutaneous and orthotopic tumor models. The study yields a panel of high‐affinity anti‐uPAR Nbs, showing specific binding across multiple types of cancer cells in vitro and in vivo. Lead fluorescently‐labeled compounds exhibit high tumor uptake with high contrast at 1 h after intravenous injection across all assessed uPAR‐expressing tumor models, outperforming a non‐targeting control Nb. Additionally, rapid and accurate tumor localization and demarcation are demonstrated in an orthotopic human glioma model. Utilizing these Nbs can potentially enhance the precision of surgical tumor resection and, consequently, improve survival rates in the clinic. This study details the generation and characterization of a panel of high‐affinity anti‐uPAR Nanobodies specific for various cancer types. The selected lead fluorescently‐labeled Nanobody demonstrates rapid and precise tumor localization and demarcation in an orthotopic human glioma model. These outcomes indicate the potential of these Nanobodies for applications in preclinical and clinical settings, providing real‐time guidance during surgical procedures.

Intra-operative fluorescence imaging has demonstrated its ability to improve tumor lesion identification. However, the limited tissue penetration of the fluorescent signals hinders the detection of deep-lying or occult lesions. Integrating fluorescence imaging with SPECT and/or intra-operative gamma-probing synergistically combines the deep tissue penetration of gamma rays for tumor localization with the precision of fluorescence imaging for precise tumor resection. In this study, we detail the use of a genetically encoded multifunctional handle, henceforth referred to as a GEM-handle, for the development of fluorescent/radioactive bimodal single-domain antibody (sdAb)-based tracers. A sdAb that targets the urokinase plasminogen activator receptor (uPAR) was engineered to carry a GEM-handle containing a carboxy-terminal hexahistidine-tag and cysteine-tag. A two-step labeling strategy was optimized and applied to site-specifically label IRDye800CW and 99m Tc to the sdAb. Bimodal labeling of the sdAbs proved straightforward and successful. 99m Tc activity was however restricted to 18.5 MBq per nmol fluorescently-labeled sdAb to prevent radiobleaching of IRDye800CW without impeding SPECT/CT imaging. Subsequently, the in vivo biodistribution and tumor-targeting capacity of the bimodal tracer were evaluated in uPAR-positive tumor-bearing mice using SPECT/CT and fluorescence imaging. The bimodal sdAb showed expected renal background signals due to tracer clearance, along with slightly elevated non-specific liver signals. Four hours post-injection, both SPECT/CT and fluorescent images achieved satisfactory tumor uptake and contrast, with significantly higher values observed for the anti-uPAR bimodal sdAb compared to a control non-targeting sdAb. In conclusion, the GEM-handle is a convenient method for designing and producing bimodal sdAb-based tracers with adequate in vivo characteristics.

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

Intra-operative fluorescence imaging has demonstrated its ability to improve tumor lesion identification. However, the limited tissue penetration of the fluorescent signals hinders the detection of deep-lying or occult lesions. Integrating fluorescence imaging with SPECT and/or intra-operative gamma-probing synergistically combines the deep tissue penetration of gamma rays for tumor localization with the precision of fluorescence imaging for precise tumor resection. In this study, we detail the use of a genetically encoded multifunctional handle, henceforth referred to as a GEM-handle, for the development of fluorescent/radioactive bimodal single-domain antibody (sdAb)-based tracers. A sdAb that targets the urokinase plasminogen activator receptor (uPAR) was engineered to carry a GEM-handle containing a carboxy-terminal hexahistidine-tag and cysteine-tag. A two-step labeling strategy was optimized and applied to site-specifically label IRDye800CW and Tc to the sdAb. Bimodal labeling of the sdAbs proved straightforward and successful. Tc activity was however restricted to 18.5 MBq per nmol fluorescently-labeled sdAb to prevent radiobleaching of IRDye800CW without impeding SPECT/CT imaging. Subsequently, the biodistribution and tumor-targeting capacity of the bimodal tracer were evaluated in uPAR-positive tumor-bearing mice using SPECT/CT and fluorescence imaging. The bimodal sdAb showed expected renal background signals due to tracer clearance, along with slightly elevated non-specific liver signals. Four hours post-injection, both SPECT/CT and fluorescent images achieved satisfactory tumor uptake and contrast, with significantly higher values observed for the anti-uPAR bimodal sdAb compared to a control non-targeting sdAb. In conclusion, the GEM-handle is a convenient method for designing and producing bimodal sdAb-based tracers with adequate characteristics.