TreEducation: A Visual Education Platform for Teaching Treemap Layout Algorithms.

Treemaps are a powerful tool for representing hierarchical data in a space-efficient manner and are used in various domains, including network security or software development. However, interpreting the topology encoded by nested rectangles can be challenging, particularly compared to tree-structured representations like node-link diagrams or icicle plots. To address this challenge, we introduce TreEducation, a visual education platform designed to improve the visualization literacy skills required for reading treemaps among non-expert users. TreEducation is an online application that combines visualizations, interactions, and gamification elements to facilitate understanding of eight different treemap layout algorithms and enhance students' learning process. We evaluated TreEducation in a classroom setting and a controlled environment. Our results indicate a significant knowledge gain of students training exclusively with TreEducation and the usefulness of competition as a social gamification element included in our competitive quiz.

Corrective osteotomy of the distal radius with palmar locking plate osteosynthesis without bone grafting and without cortical contact.

The aim of this study was to assess bone healing and secondary fracture displacement after corrective osteotomy of the distal radius without any cortical contact using palmar locking plates without bone grafting. Between 2009 and 2021, 11 palmar corrective osteotomies of extra-articular malunited distal radius fractures and palmar plate fixations without the use of bone grafts and without cortical contact, were assessed. All patients showed complete osseous restoration and significant improvement in all radiographic parameters. Except for one patient, there were no secondary dislocations or loss of reduction in the postoperative follow-up. Bone grafts may not be mandatory for bone healing and prevention of secondary fracture displacement after palmar corrective osteotomy without cortical contact and fixation with palmar locking plate.Level of evidence: IV.

Mucor osteomyelitis after traumatic forearm amputation in a 38-year-old patient.

We report the case of a forest ranger who developed a polymicrobial infection with Mucor circinelloides after traumatic forearm amputation. Based on our case report we discuss epidemiology and management of this rare and potentially fatal infection.

Lung macrophages utilize unique cathepsin K-dependent phagosomal machinery to degrade intracellular collagen.

Resident tissue macrophages are organ-specialized phagocytes responsible for the maintenance and protection of tissue homeostasis. It is well established that tissue diversity is reflected by the heterogeneity of resident tissue macrophage origin and phenotype. However, much less is known about tissue-specific phagocytic and proteolytic macrophage functions. Here, using a quantitative proteomics approach, we identify cathepsins as key determinants of phagosome maturation in primary peritoneum-, lung-, and brain-resident macrophages. The data further uncover cathepsin K (CtsK) as a molecular marker for lung phagosomes required for intracellular protein and collagen degradation. Pharmacological blockade of CtsK activity diminished phagosomal proteolysis and collagenolysis in lung-resident macrophages. Furthermore, profibrotic TGF-ß negatively regulated CtsK-mediated phagosomal collagen degradation independently from classical endocytic-proteolytic pathways. In humans, phagosomal CtsK activity was reduced in COPD lung macrophages and non-COPD lung macrophages exposed to cigarette smoke extract. Taken together, this study provides a comprehensive map of how peritoneal, lung, and brain tissue environment shapes phagosomal composition, revealing CtsK as a key molecular determinant of lung phagosomes contributing to phagocytic collagen clearance in lungs.

In vivo Characterization of Endogenous Protein Interactomes in Drosophila Larval Brain, Using a CRISPR/Cas9-based Strategy and BioID-based Proximity Labeling.

Understanding protein-protein interactions (PPIs) and interactome networks is essential to reveal molecular mechanisms mediating various cellular processes. The most common method to study PPIs in vivo is affinity purification combined with mass spectrometry (AP-MS). Although AP-MS is a powerful method, loss of weak and transient interactions is still a major limitation. Proximity labeling (PL) techniques have been developed as alternatives to overcome these limitations. Proximity-dependent biotin identification (BioID) is one such widely used PL method. The first-generation BioID enzyme BirA*, a promiscuous bacterial biotin ligase, has been effectively used in cultured mammalian cells; however, relatively slow enzyme kinetics make it less effective for temporal analysis of protein interactions. In addition, BirA* exhibits reduced activity at temperatures below 37°C, further restricting its use in intact organisms cultured at lower optimal growth temperatures ( e.g., Drosophila melanogaster ). TurboID, miniTurbo, and BirA*-G3 are next generation BirA* variants with improved catalytic activity, allowing investigators to use this powerful tool in model systems such as flies. Here, we describe a detailed experimental workflow to efficiently identify the proximal proteome (proximitome) of a protein of interest (POI) in the Drosophila brain using CRISPR/Cas9-induced homology-directed repair (HDR) strategies to endogenously tag the POI with next generation BioID enzymes.

Multiscale Visualization: A Structured Literature Analysis.

Multiscale visualizations are typically used to analyze multiscale processes and data in various application domains, such as the visual exploration of hierarchical genome structures in molecular biology. However, creating such multiscale visualizations remains challenging due to the plethora of existing work and the expression ambiguity in visualization research. Up to today, there has been little work to compare and categorize multiscale visualizations to understand their design practices. In this article, we present a structured literature analysis to provide an overview of common design practices in multiscale visualization research. We systematically reviewed and categorized 122 published journal or conference articles between 1995 and 2020. We organized the reviewed articles in a taxonomy that reveals common design factors. Researchers and practitioners can use our taxonomy to explore existing work to create new multiscale navigation and visualization techniques. Based on the reviewed articles, we examine research trends and highlight open research challenges.

In vivo Profiling of the Alk Proximitome in the Developing Drosophila Brain.

Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed that Alk signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. In this study, we used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila central nervous system (CNS). To do this, we used CRISPR/Cas9 induced homology-directed repair (HDR) to modify the endogenous Alk locus to produce first and next generation Alk::BioID chimeras. This approach allowed identification of Alk proximitomes under physiological conditions and without overexpression. Our results show that the next generation of BioID proteins (TurboID and miniTurbo) outperform the first generation BirA* fusion in terms of labeling speed and efficiency. LC-MS3-based BioID screening of AlkTurboID and AlkminiTurbo larval brains revealed an extensive neuronal Alk proximitome identifying numerous potential components of Alk signaling complexes. Validation of Alk proximitome candidates further revealed co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx) and Rugose (Rg) with Alk in the CNS and identified the protein-tyrosine-phosphatase Corkscrew (Csw) as a modulator of Alk signaling.

Mass Spectrometry Proteotyping-Based Detection and Identification of Staphylococcus aureus, Escherichia coli, and Candida albicans in Blood.

Bloodstream infections (BSIs), the presence of microorganisms in blood, are potentially serious conditions that can quickly develop into sepsis and life-threatening situations. When assessing proper treatment, rapid diagnosis is the key; besides clinical judgement performed by attending physicians, supporting microbiological tests typically are performed, often requiring microbial isolation and culturing steps, which increases the time required for confirming positive cases of BSI. The additional waiting time forces physicians to prescribe broad-spectrum antibiotics and empirically based treatments, before determining the precise cause of the disease. Thus, alternative and more rapid cultivation-independent methods are needed to improve clinical diagnostics, supporting prompt and accurate treatment and reducing the development of antibiotic resistance. In this study, a culture-independent workflow for pathogen detection and identification in blood samples was developed, using peptide biomarkers and applying bottom-up proteomics analyses, i.e., so-called "proteotyping". To demonstrate the feasibility of detection of blood infectious pathogens, using proteotyping, Escherichia coli and Staphylococcus aureus were included in the study, as the most prominent bacterial causes of bacteremia and sepsis, as well as Candida albicans, one of the most prominent causes of fungemia. Model systems including spiked negative blood samples, as well as positive blood cultures, without further culturing steps, were investigated. Furthermore, an experiment designed to determine the incubation time needed for correct identification of the infectious pathogens in blood cultures was performed. The results for the spiked negative blood samples showed that proteotyping was 100- to 1,000-fold more sensitive, in comparison with the MALDI-TOF MS-based approach. Furthermore, in the analyses of ten positive blood cultures each of E. coli and S. aureus, both the MALDI-TOF MS-based and proteotyping approaches were successful in the identification of E. coli, although only proteotyping could identify S. aureus correctly in all samples. Compared with the MALDI-TOF MS-based approaches, shotgun proteotyping demonstrated higher sensitivity and accuracy, and required significantly shorter incubation time before detection and identification of the correct pathogen could be accomplished.

BioID-Screening Identifies PEAK1 and SHP2 as Components of the ALK Proximitome in Neuroblastoma Cells.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib. LC/MS-MS analysis identified multiple proteins, including PEAK1 and SHP2, which were validated as ALK interactors in NB cells. Further analysis of the ALK-SHP2 interaction confirmed that the ALK-SHP2 interaction as well as SHP2-Y542 phosphorylation was dependent on ALK activation. Use of the SHP2 inhibitors, SHP099 and RMC-4550, resulted in inhibition of cell growth in ALK-driven NB cells. In addition, we noted a strong synergistic effect of combined ALK and SHP2 inhibition that was specific to ALK-driven NB cells, suggesting a potential therapeutic option for ALK-driven NB.

The Value of Immersive Visualization.

In recent years, research on immersive environments has experienced a new wave of interest, and immersive analytics has been established as a new research field. Every year, a vast amount of different techniques, applications, and user studies are published that focus on employing immersive environments for visualizing and analyzing data. Nevertheless, immersive analytics is still a relatively unexplored field that needs more basic research in many aspects and is still viewed with skepticism. Rightly so, because in our opinion, many researchers do not fully exploit the possibilities offered by immersive environments and, on the contrary, sometimes even overestimate the power of immersive visualizations. Although a growing body of papers has demonstrated individual advantages of immersive analytics for specific tasks and problems, the general benefit of using immersive environments for effective analytic tasks remains controversial. In this article, we reflect on when and how immersion may be appropriate for the analysis and present four guiding scenarios. We report on our experiences, discuss the landscape of assessment strategies, and point out the directions where we believe immersive visualizations have the greatest potential.

Effects of liraglutide on the metabolism of triglyceride-rich lipoproteins in type 2 diabetes.

AIM: To elucidate the impact of liraglutide on the kinetics of apolipoprotein (apo)B48- and apoB100-containing triglyceride-rich lipoproteins in subjects with type 2 diabetes (T2D) after a single fat-rich meal. MATERIALS AND METHODS: Subjects with T2D were included in a study to investigate postprandial apoB48 and apoB100 metabolism before and after 16 weeks on l.8 mg/day liraglutide (n = 14) or placebo (n = 4). Stable isotope tracer and compartmental modelling techniques were used to determine the impact of liraglutide on chylomicron and very low-density lipoprotein (VLDL) production and clearance after a single fat-rich meal. RESULTS: Liraglutide reduced apoB48 synthesis in chylomicrons by 60% (p < .0001) and increased the triglyceride/apoB48 ratio (i.e. the size) of chylomicrons (p < .001). Direct clearance of chylomicrons, a quantitatively significant pathway pretreatment, decreased by 90% on liraglutide (p < .001). Liraglutide also reduced VLDL1 -triglyceride secretion (p = .017) in parallel with reduced liver fat. Chylomicron-apoB48 production and particle size were related to insulin sensitivity (p = .015 and p < .001, respectively), but these associations were perturbed by liraglutide. CONCLUSIONS: In a physiologically relevant setting that mirrored regular feeding in subjects with T2D, liraglutide promoted potentially beneficial changes on postprandial apoB48 metabolism. Using our data in an integrated metabolic model, we describe how the action of liraglutide in T2D on chylomicron and VLDL kinetics could lead to decreased generation of remnant lipoproteins.

Revision rate of THA in patients younger than 40 years depends on primary diagnosis - a retrospective analysis with a minimum follow-up of 10 years.

BACKGROUND: Treating osteoarthritis in elderly patients with THA is very successful. However, surgeons hesitate to recommend THA in younger patients. The spectrum of etiologies for end stage hip disease in the younger population is diverse and therefore different courses may be assumed. Our objective was to evaluate THA revision rate within a minimum follow-up period of 10 years in young patients and to analyze the difference between different primary diagnoses. METHODS: We included 144 consecutive hips in 127 patients younger than 40 years, who received a primary THA from 01/1996 to 12/2007. Operative reports, clinical and radiographic documentation were reviewed to determine primary diagnosis, prior hip surgery, component specifications and revision surgery. 111 hips in 97 patients were available for outcome analysis with a minimum follow-up of 10 years. RESULTS: The mean age was 33 years (range 15-40 years) at the time of the index THA, 68 patients were female and 59 were male. Ten years revision rate on the prosthetic components was 13%. The most common primary diagnosis was DDH. DDH was associated with a risk of 17% for requiring a reoperation on the prosthetic components because of mechanical fatigue and therefore, significantly higher than for any other primary diagnosis (p = 0.005). CONCLUSION: THA in young patients is associated with a high revision rate of 13% in 10 years. 17% of patients with DDH required revision surgery for mechanical fatigue within 10 years, which was significantly higher than for any other primary diagnosis (1.2%, OR 16.8).

Effects of Evolocumab on the Postprandial Kinetics of Apo (Apolipoprotein) B100- and B48-Containing Lipoproteins in Subjects With Type 2 Diabetes.

OBJECTIVE: Increased risk of atherosclerotic cardiovascular disease in subjects with type 2 diabetes is linked to elevated levels of triglyceride-rich lipoproteins and their remnants. The metabolic effects of PCSK9 (proprotein convertase subtilisin/kexin 9) inhibitors on this dyslipidemia were investigated using stable-isotope-labeled tracers. Approach and Results: Triglyceride transport and the metabolism of apos (apolipoproteins) B48, B100, C-III, and E after a fat-rich meal were investigated before and on evolocumab treatment in 13 subjects with type 2 diabetes. Kinetic parameters were determined for the following: apoB48 in chylomicrons; triglyceride in VLDL1 (very low-density lipoprotein) and VLDL2; and apoB100 in VLDL1, VLDL2, IDL (intermediate-density lipoprotein), and LDL (low-density lipoprotein). Evolocumab did not alter the kinetics of apoB48 in chylomicrons or apoB100 or triglyceride in VLDL1. In contrast, the fractional catabolic rates of VLDL2-apoB100 and VLDL2-triglyceride were both increased by about 45%, which led to a 28% fall in the VLDL2 plasma level. LDL-apoB100 was markedly reduced by evolocumab, which was linked to metabolic heterogeneity in this fraction. Evolocumab increased clearance of the more rapidly metabolized LDL by 61% and decreased production of the more slowly cleared LDL by 75%. ApoC-III kinetics were not altered by evolocumab, but the apoE fractional catabolic rates increased by 45% and the apoE plasma level fell by 33%. The apoE fractional catabolic rates was associated with the decrease in VLDL2- and IDL-apoB100 concentrations. CONCLUSIONS: Evolocumab had only minor effects on lipoproteins that are involved in triglyceride transport (chylomicrons and VLDL1) but, in contrast, had a profound impact on lipoproteins that carry cholesterol (VLDL2, IDL, LDL). Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02948777.

Different Routes of Protein Folding Contribute to Improved Protein Production in Saccharomyces cerevisiae.

Protein folding is often considered the flux controlling process in protein synthesis and secretion. Here, two previously isolated Saccharomyces cerevisiae strains with increased α-amylase productivity were analyzed in chemostat cultures at different dilution rates using multi-omics data. Based on the analysis, we identified different routes of the protein folding pathway to improve protein production. In the first strain, the increased abundance of proteins working on the folding process, coordinated with upregulated glycogen metabolism and trehalose metabolism, helped increase α-amylase productivity 1.95-fold compared to the level in the original strain in chemostat culture at a dilution rate of 0.2/h. The second strain further strengthened the folding precision to improve protein production. More precise folding helps the cell improve protein production efficiency and reduce the expenditure of energy on the handling of misfolded proteins. As calculated using an enzyme-constrained genome-scale metabolic model, the second strain had an increased productivity of 2.36-fold with lower energy expenditure than that of the original under the same condition. Further study revealed that the regulation of N-glycans played an important role in the folding precision control and that overexpression of the glucosidase Cwh41p can significantly improve protein production, especially for the strains with improved folding capacity but lower folding precision. Our findings elucidated in detail the mechanisms in two strains having improved protein productivity and thereby provided novel insights for industrial recombinant protein production as well as demonstrating how multi-omics analysis can be used for identification of novel strain-engineering targets.IMPORTANCE Protein folding plays an important role in protein maturation and secretion. In recombinant protein production, many studies have focused on the folding pathway to improve productivity. Here, we identified two different routes for improving protein production by yeast. We found that improving folding precision is a better strategy. Dysfunction of this process is also associated with several aberrant protein-associated human diseases. Here, our findings about the role of glucosidase Cwh41p in the precision control system and the characterization of the strain with a more precise folding process could contribute to the development of novel therapeutic strategies.

Absolute yeast mitochondrial proteome quantification reveals trade-off between biosynthesis and energy generation during diauxic shift.

Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.

Teaching Clustering Algorithms With EduClust: Experience Report and Future Directions.

We share our experiences teaching university students about clustering algorithms using EduClust, an online visualization we developed. EduClust supports professors in preparing teaching material and students in visually and interactively exploring cluster steps and the effects of changing clustering parameters. We used EduClust for two years in our computer science lectures on clustering algorithms and share our experience integrating the online application in a data science curriculum. We also point to opportunities for future development.

Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation.

The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery.

Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping.

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

Visual Analytics of Volunteered Geographic Information: Detection and Investigation of Urban Heat Islands.

Urban heat islands are local areas where the temperature is much higher than in the vicinity and are a modern phenomenon that occurs mainly in highly developed areas, such as large cities. This effect has a negative impact on energy management in buildings, and also has a direct impact on human health, especially for elderly people. With the advent of volunteered geographic information from private weather station networks, more high-resolution data are now available within cities to better analyze this effect. However, such datasets are large and have heterogeneous characteristics requiring visual-interactive applications to support further analysis. We use machine learning methods to predict urban heat islands occurrences and utilize temporal and spatio-temporal visualizations to contextualize the emergence of urban heat islands to comprehend the influencing causes and their effects. Subsequently, we demonstrate the analysis capabilities of our application by presenting two use cases.

Absolute quantification of cohesin, CTCF and their regulators in human cells.

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.