Nonlinear laser scanning microscopy of oral multispecies-biofilms: Fixative induced fluorescence as a fast and economical in vitro screening method

In this letter we report a fast and easy method which could be used for initial screening of multispecies-biofilm development on putative new dental implant materials. Most staining methods require numerous washing steps that can result in detachment of loosely bound biofilms and therefore falsify the results. Thus, we used glutaraldehyde fixation, which induces autofluorescence through bacterial membrane protein cross-linking and concurrently stabilizes the biofilm structure. We analyzed the biofilms with nonlinear laser scanning microscopy and were able to (I) evaluate the multispecies-biofilm growth and (II) distinguish between bacterial species based on different two-photon autofluorescence intensities. © 2016 by De Gruyter

New Tetromycin Derivatives with Anti-Trypanosomal and Protease Inhibitory Activities †

Four new tetromycin derivatives, tetromycins 1–4 and a previously known one, tetromycin B (5) were isolated from Streptomyces axinellae Pol001T cultivated from the Mediterranean sponge Axinella polypoides. Structures were assigned using extensive 1D and 2D NMR spectroscopy as well as HRESIMS analysis. The compounds were tested for antiparasitic activities against Leishmania major and Trypanosoma brucei, and for protease inhibition against several cysteine proteases such as falcipain, rhodesain, cathepsin L, cathepsin B, and viral proteases SARS-CoV Mpro, and PLpro. The compounds showed antiparasitic activities against T. brucei and time-dependent inhibition of cathepsin L-like proteases with Ki values in the low micromolar range

GestaltMatcher Database - A global reference for facial phenotypic variability in rare human diseases

The most important factor that complicates the work of dysmorphologists is the significant phenotypic variability of the human face. Next-Generation Phenotyping (NGP) tools that assist clinicians with recognizing characteristic syndromic patterns are particularly challenged when confronted with patients from populations different from their training data. To that end, we systematically analyzed the impact of genetic ancestry on facial dysmorphism. For that purpose, we established the GestaltMatcher Database (GMDB) as a reference dataset for medical images of patients with rare genetic disorders from around the world. We collected 10,980 frontal facial images - more than a quarter previously unpublished - from 8,346 patients, representing 581 rare disorders. Although the predominant ancestry is still European (67%), data from underrepresented populations have been increased considerably via global collaborations (19% Asian and 7% African). This includes previously unpublished reports for more than 40% of the African patients. The NGP analysis on this diverse dataset revealed characteristic performance differences depending on the composition of training and test sets corresponding to genetic relatedness. For clinical use of NGP, incorporating non-European patients resulted in a profound enhancement of GestaltMatcher performance. The top-5 accuracy rate increased by +11.29%. Importantly, this improvement in delineating the correct disorder from a facial portrait was achieved without decreasing the performance on European patients. By design, GMDB complies with the FAIR principles by rendering the curated medical data findable, accessible, interoperable, and reusable. This means GMDB can also serve as data for training and benchmarking. In summary, our study on facial dysmorphism on a global sample revealed a considerable cross ancestral phenotypic variability confounding NGP that should be counteracted by international efforts for increasing data diversity. GMDB will serve as a vital reference database for clinicians and a transparent training set for advancing NGP technology.</p

Utveckling av testsystem för Interface Test Adapter till Gripen ATE

The work has been carried out as a product development project at Saab AB in Arboga, focusing on construction. Saab in Arboga develops test systems for JAS 39 Gripen, whose purpose is to verify all the contacts. This is done by using the test system, ATE. Before testing can begin, the wiring harness in the ITA must be verified. Currently this is done manually, and due to wiring extent, the risks of incorrect measurements are apparent. In addition to incorrect measuring, the extent of the wiring can also lead to incorrect wiring in production. These errors can result in the test system fails, which means great financial loss for the company. The project's aim was to develop an automated measurement process and produce a prototype, in order to verify that the ITA is correctly connected before the simulation is done. The main issue for the project has been if an automated measurement process can obtain sufficient precision to replace manual measurement. By analyzing the market for automated movement, a number of concepts for the overall measurement process were generated. The concepts were evaluated with decision-matrix method, as well with regard to the complexity of the required software management for each concept. The measurement process that was chosen based on the 3D printer and its underlying mechanics and automation. To realize the concept to a fully working prototype the process of product development was broken down in segments. This reduced the level of complexity, while it contributed to simplify optimization. In order to achieve optimum design every segment consisted of concept generation and concept evaluation. The construction which is the basis for the prototype is an off-the-shelf solution, and based on the parts used in the milling machine x-Carve. The overall structure is based on the milling machine, however modified to the extent that the purpose can be achieved. The choice to base the design on the X-Carve was made against the background that it harmonized well with the overall concept of measuring process, but also well with the concepts generated in the broken down segments, mentioned above. The underlying factor to the use of components which is an off-the-shelf solution to the design is a result of the project vastness and the provided time. The developed prototype lives up to the requirements. Empirical tests indicate that the prototype obtains sufficient precision to perform measurements. The results of the tests carried out shows that manual labor can be replaced, and therefore leads to the conclusion that the prototype proved useful. The prototype has some potential for development, why it is recommended to examine each segment if additional optimizations must be made

Comparison of Corneal Riboflavin Gradients Using Dextran and HPMC Solutions.

PURPOSE To determine the riboflavin concentration gradient in the anterior corneal stroma when using hydroxypropyl methylcellulose (HPMC) or dextran as the carrier agent. METHODS Four different groups of porcine corneas (5 each) were compared regarding the riboflavin concentration in the anterior stroma. Prior to all experiments, stable hydration conditions were established for the corresponding solution. The dextran groups were treated with 0.1% riboflavin in 20% dextran for 10 and 30 minutes and the HPMC groups with 0.1% riboflavin in 1.1% HPMC for 10 and 30 minutes. After imbibition, nonlinear microscopy and consecutive image analysis were used to determine two-photon fluorescence intensities. To determine the riboflavin concentration, corneas were saturated and measured a second time by two-photon microscopy. With this measurement, a proper correction for absorption and scattering could be performed. Ultraviolet-A (UVA) transmission was measured after the application time for each group. RESULTS Riboflavin concentration decreased with increasing depth and increased with longer application times in all groups. Comparing the dextran for 30 minutes and HPMC for 10 minutes groups, a significantly higher stromal riboflavin concentration was found within the most anterior 70 µm in the dextran group for 30 minutes, whereas deeper than 260 µm HPMC-assisted imbibition for 10 minutes yielded higher concentrations. In dextran-treated corneas, values obtained from pachymetry were substantially reduced, whereas HPMC-assisted imbibition led to a decent swelling. UVA transmission values were higher in dextran-assisted imbibition than in HPMC-assisted imbibition. CONCLUSIONS Stromal riboflavin gradients are similar when applied in dextran for 30 minutes and HPMC for 10 minutes. When using HPMC solutions, a shallower cross-linked volume is expected due to a higher corneal hydration. [J Refract Surg. 2016;32(12):798-802.]

Spectral behavior of second harmonic signals from organic and non-organic materials in multiphoton microscopy

Multimodal nonlinear microscopy allows imaging of highly ordered biological tissue due to spectral separation of nonlinear signals. This requires certain knowledge about the spectral distribution of the different nonlinear signals. In contrast to several publications we demonstrate a factor of 1 2 2 relating the full width at half maximum of a gaussian laser pulse spectrum to the corresponding second harmonic pulse spectrum in the spatial domain by using a simple theoretical model. Experiments on monopotassium phosphate crystals (KDP-crystals) and on porcine corneal tissue support our theoretical predictions. Furthermore, no differences in spectral width were found for epi- and trans-detection of the second harmonic signal. Overall, these results may help to build an optimized multiphoton setup for spectral separation of nonlinear signals

Erratum: Spectral behavior of second harmonic signals from organic and non-organic materials in multiphoton microscopy (vol 5, 084903, 2015)

Erratum to: “Spectral behavior of second harmonic signals from organic and non-organic materials in multiphoton microscopy” in: AIP Advances 5, 084903 (2015

Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines

Brandt D, Simunovic M, Busche T, et al. Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines. Viruses. 2021;13(9): 1870.Genomic surveillance of the SARS-CoV-2 pandemic is crucial and mainly achieved by amplicon sequencing protocols. Overlapping tiled-amplicons are generated to establish contiguous SARS-CoV-2 genome sequences, which enable the precise resolution of infection chains and outbreaks. We investigated a SARS-CoV-2 outbreak in a local hospital and used nanopore sequencing with a modified ARTIC protocol employing 1200 bp long amplicons. We detected a long deletion of 168 nucleotides in the ORF8 gene in 76 samples from the hospital outbreak. This deletion is difficult to identify with the classical amplicon sequencing procedures since it removes two amplicon primer-binding sites. We analyzed public SARS-CoV-2 sequences and sequencing read data from ENA and identified the same deletion in over 100 genomes belonging to different lineages of SARS-CoV-2, pointing to a mutation hotspot or to positive selection. In almost all cases, the deletion was not represented in the virus genome sequence after consensus building. Additionally, further database searches point to other deletions in the ORF8 coding region that have never been reported by the standard data analysis pipelines. These findings and the fact that ORF8 is especially prone to deletions, make a clear case for the urgent necessity of public availability of the raw data for this and other large deletions that might change the physiology of the virus towards endemism

Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines

Genomic surveillance of the SARS-CoV-2 pandemic is crucial and mainly achieved by amplicon sequencing protocols. Overlapping tiled-amplicons are generated to establish contiguous SARS-CoV-2 genome sequences, which enable the precise resolution of infection chains and outbreaks. We investigated a SARS-CoV-2 outbreak in a local hospital and used nanopore sequencing with a modified ARTIC protocol employing 1200 bp long amplicons. We detected a long deletion of 168 nucleotides in the ORF8 gene in 76 samples from the hospital outbreak. This deletion is difficult to identify with the classical amplicon sequencing procedures since it removes two amplicon primer-binding sites. We analyzed public SARS-CoV-2 sequences and sequencing read data from ENA and identified the same deletion in over 100 genomes belonging to different lineages of SARS-CoV-2, pointing to a mutation hotspot or to positive selection. In almost all cases, the deletion was not represented in the virus genome sequence after consensus building. Additionally, further database searches point to other deletions in the ORF8 coding region that have never been reported by the standard data analysis pipelines. These findings and the fact that ORF8 is especially prone to deletions, make a clear case for the urgent necessity of public availability of the raw data for this and other large deletions that might change the physiology of the virus towards endemism