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A Spatial Transcriptomics Browser for Discovering Gene Expression Landscapes across Microscopic Tissue Sections
No full textA crucial feature of life is its spatial organization and compartmentalization on the molecular, cellular, and tissue levels. Spatial transcriptomics (ST) technology has opened a new chapter of the sequencing revolution, emerging rapidly with transformative effects across biology. This technique produces extensive and complex sequencing data, raising the need for computational methods for their comprehensive analysis and interpretation. We developed the ST browser web tool for the interactive discovery of ST images, focusing on different functional aspects such as single gene expression, the expression of functional gene sets, as well as the inspection of the spatial patterns of cell–cell interactions. As a unique feature, our tool applies self-organizing map (SOM) machine learning to the ST data. Our SOM data portrayal method generates individual gene expression landscapes for each spot in the ST image, enabling its downstream analysis with high resolution. The performance of the spatial browser is demonstrated by disentangling the intra-tumoral heterogeneity of melanoma and the microarchitecture of the mouse brain. The integration of machine-learning-based SOM portrayal into an interactive ST analysis environment opens novel perspectives for the comprehensive knowledge mining of the organization and interactions of cellular ecosystems
Added value of FDG-PET for detection of progressive supranuclear palsy
No full textBackground Diagnostic criteria for progressive supranuclear palsy (PSP) include midbrain atrophy in MRI and hypometabolism in [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) as supportive features. Due to limited data regarding their relative and sequential value, there is no recommendation for an algorithm to combine both modalities to increase diagnostic accuracy. This study evaluated the added value of sequential imaging using state-of-the-art methods to analyse the images regarding PSP features.
Methods The retrospective study included 41 PSP patients, 21 with Richardson’s syndrome (PSP-RS), 20 with variant PSP phenotypes (vPSP) and 46 sex- and age-matched healthy controls. A pretrained support vector machine (SVM) for the classification of atrophy profiles from automatic MRI volumetry was used to analyse T1w-MRI (output: MRI-SVM-PSP score). Covariance pattern analysis was applied to compute the expression of a predefined PSP-related pattern in FDG-PET (output: PET-PSPRP expression score).
Results The area under the receiver operating characteristic curve for the detection of PSP did not differ between MRI-SVM-PSP and PET-PSPRP expression score (p≥0.63): about 0.90, 0.95 and 0.85 for detection of all PSP, PSP-RS and vPSP. The MRI-SVM-PSP score achieved about 13% higher specificity and about 15% lower sensitivity than the PET-PSPRP expression score. Decision tree models selected the MRI-SVM-PSP score for the first branching and the PET-PSPRP expression score for a second split of the subgroup with normal MRI-SVM-PSP score, both in the whole sample and when restricted to PSP-RS or vPSP.
Conclusions FDG-PET provides added value for PSP-suspected patients with normal/inconclusive T1w-MRI, regardless of PSP phenotype and the methods to analyse the images for PSP-typical features
Clinical findings and outcome in feline tetanus: a multicentric retrospective study of 27 cases and review of the literature
No full textTetanus is a toxigenic illness caused by the action of Clostridium tetani neurotoxin (TeNT), which results in partial or generalized muscle stiffness in infected mammals and birds. The disease is rarely reported in cats due to their innate resistance to the toxin. This multicentric retrospective study aimed to describe a significant population of cats with a diagnosis of tetanus and report their signalment, clinical and neurological signs, diagnostic findings, treatment, and outcome. A retrospective search through medical records from 11 referral centers in Europe resulted in the identification of 27 cases of feline tetanus from July 2005 to April 2023. These cases were further compared with previously reported cases in the veterinary literature. Young cats were more commonly represented than older cats, with a median age of 4 years. Clinical signs were initially characterized by a lame and/or stiff limb, near the primary injury site, in 17/26 (65%) cats. Signs were focal or multifocal in 21/27 (78%) cats of this study and one typical sign was the inability to flex the most severely affected limbs. Electrodiagnostic studies revealed characteristic changes, such as continuous spontaneous motor unit discharges in both agonist and antagonist muscles. Such studies are particularly useful in focal and multifocal cases and should be performed to further support the diagnosis. The toxin was successfully identified in one case using the mouse bioassay. Treatment included antibiotherapy (metronidazole) in most cases, muscle relaxants, appropriate nursing cares and handling of potential complications. Overall, the outcome appeared to be positive, with only 1/27 (3.7%) cats being euthanized due to financial restrains. 23/25 (92%) cats returned to an independent ambulatory capacity on all limbs within a median delay of 25 days. Mild to moderate long-term sequelae were reported in eight (30%) cats. This multicentric study is the first to bring together such a large number of cats affected with tetanus. Presentation of the disease in cats differs from that observed in humans and dogs, with most cats being locally affected. Compared to previous reports of tetanus, this series of cats had a better outcome overall, especially for cats affected with generalized tetanus
Cytokine and Microbiome Changes in Adolescents with Anorexia Nervosa at Admission, Discharge, and One-Year Follow-Up
No full textAnorexia nervosa (AN) is a severe eating disorder that predominantly affects females and typically manifests during adolescence. There is increasing evidence that serum cytokine levels are altered in individuals with AN. Previous research has largely focused on adult patients, assuming a low-grade pro-inflammatory state. The serum levels of the cytokine tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6 and IL-15, which are pro-inflammatory, were examined in 63 female adolescents with AN and 41 age-matched healthy controls (HC). We included three time points (admission, discharge, and 1-year follow-up) and investigated the clinical data to assess whether the gut microbiota was associated with cytokine alterations. Relative to the HC group, serum levels of IL-1β and IL-6 were significantly lower during the acute phase (admission) of AN. IL-1β expression was normalised to control levels after weight recovery. TNF-α levels were not significantly different between the AN and HC groups. IL-15 levels were significantly elevated in patients with AN at all time points. We found associations between cytokines and bodyweight, illness duration, depressive symptoms, and the microbiome. In contrast to most findings for adults, we observed lower levels of the pro-inflammatory cytokines IL-1β and IL-6 in adolescent patients, whereas the level of IL-15 was consistently increased. Thus, the presence of inflammatory dysregulation suggests a varied rather than uniform pro-inflammatory state
Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment
No full textFor the monitoring of chimeric antigen receptor (CAR) T-cell therapies, antigen-based CAR detection methods are usually applied. However, for each target-antigen, a separate detection system is required. Furthermore, when monitored CAR T-cells in the blood of patients treated with bispecific antibodies or T-cell engagers (bsAbs/BiTEs) recognize the same antigen, these methods produce false-positive results in clinical diagnostics. Anti-CAR-linker monoclonal antibodies (mAbs) targeting the linker sequence between the variable domains of the antigen binding CAR fragment promise a universal and unbiased CAR detection. To test this, we analyzed clinical specimens of all BCMA- and CD19-targeting CAR T-cell products currently approved for clinical use. We found a highly specific and sensitive CAR detection using anti-CAR-linker mAb in blood cells from patients treated with Ide-cel, Tisa-cel, Axi-cel, Brexu-cel, and Liso-cel. For Ide-cel and Tisa-cel, the sensitivity was significantly lower compared to that for antigen-based CAR detection assays. Strikingly, the specificity of anti-CAR linker mAb was not affected by the simultaneous presence of bispecific blinatumomab or teclistamab for Axi-cel, Brexu-cel, Liso-cel, or Ide-cel, respectively. Cilta-cel (containing a monomeric G4S-CAR linker) could not be detected by anti-CAR linker mAb. In conclusion, anti-CAR-linker mAbs are highly specific and useful for CAR T-cell monitoring but are not universally applicable
Räumliche und zeitliche Organisation von subplasmalemmalen Ca2+-Spikes und Fusionsereignissen Insulin-haltiger Vesikel in einem β-Zellmodell
No full textDie basale und Glukose-induzierte Insulinfreisetzung aus β-Zellen ist ein streng regulierter Prozess und wird durch Veränderungen der intrazellulären Ca2+-Konzentration ([Ca2+]i), den Einfluss verschiedener Hormone, medikamentöse Interventionen und viele weitere Faktoren moduliert (Ammälä, Ashcroft et al., 1993; Mayendraraj, Rosenkilde et al., 2022). Durch die Arbeit von Langlhofer et al. wurde klar, dass die [Ca2+]i im Bereich subplasmalemmaler Mikrodomänen in Spikes oder Gruppen von Spikes (Bursts) fluktuiert, die eine Höhe von 2-6 µM erreichen und damit ausreichen könnten, um Ca2+-abhängige Sensorproteine zu aktivieren, die ihrerseits die Freisetzung insulinhaltiger Vesikel triggern können. Diese Spikes treten synchronisiert über die ganze INS-1-Zelle auf. Mithilfe des Exozytosemarkers SynaptopHluorin konnten wir zeigen, dass es prinzipiell Fusionsereignisse gibt, die in Einzelzellen nahezu gleichzeitig, d.h. im Zeitraum zwischen 50-150 ms ablaufen. Mittels Zweifarbkanal LED-TIRF (total internal reflection fluorescence)-Mikroskopie führten wir dann Messungen durch, die simultan die Ca2+-Signale mit der PKCαmCherry2 als Indikator und die durch SynaptopHluorin dargestellten Fusionsereignisse abbilden konnten, um herauszufinden, ob eine Ca2+-getriggerte Exozytose zur Freisetzung von mehr als einem Vesikel und damit zum Ansprechen des „readily releasable pool“ (RRP) in INS-1-Zellen führen kann.
Wir nutzten dabei verschiedene Techniken und pharmakologische Modulatoren, um
Störfaktoren auszuschalten, aber dennoch in einem für die Zellen möglichst physiologischen Umfeld zu arbeiten. Durch die Anwendung von Thapsigargin konnten wir sicherstellen, dass eine Freisetzung aus intrazellulären Ca2+-Speichern nicht zu den Spitzenkonzentrationen der subplasmalemmalen Ca2+-Signale beiträgt, die uns Cal520ff oder die PKCαmCherry2 anzeigen. Um den Einfluss der Ca2+-Mikrodomänen auf die Exozytoseleistung der Zellen zu untersuchen, arbeiteten wir mit verschiedenen Ca2+-Puffersystemen. Wurden die Zellen mit 30 µM EGTA-AM beladen, konnte Cal520ff keine Ca2+-Signale mehr darstellen, einer kompletten Pufferung entsprechend. Das führte zu einer Verminderung der Fusionsrate um 23,3 %. Nach Beladung mit dem schneller puffernden BAPTA-AM in gleicher Konzentration konnte eine noch stärkere und signifikante Verminderung um 44,8 %, unter extrazellulär Ca2+-freien Bedingungen sogar um 57,4 % erreicht werden.
Um eine zeitliche Korrelation zwischen Ca2+-Spikes und Exozytoseereignissen festzustellen, setzten wir zunächst einen empirisch gewählten Zeitrahmen von 500 ms, 100 ms vor und 400 ms nach einem Ca2+-Spike. Etwa 65 % aller Fusionsereignisse fanden außerhalb dieses Intervalls statt und wurden somit als asynchron angesehen. Die übrigen 35 % erschienen als synchronisiert, ein Teil davon könnte aber lediglich zufällig in diesem Zeitfenster liegen, ohne kausal durch das akute Ca2+-Signal getriggert worden zu sein. Wurden diese mittels einer mathematischen Kompensation herausgerechnet, so sank die Fraktion synchronisierter Exozytoseereignisse noch einmal um 22 %. Unter zusätzlicher Stimulation mit 20 µM Forskolin stieg zwar die Rate asynchroner Fusionsereignisse stark an, aber die der synchronisierten Fusionen änderte sich nicht signifikant. Ähnliche Ergebnisse erhielten wir unter Zugabe von 50 µM Tolbutamid.
Um die Kausalität eines Ca2+-Spikes für eine durch ihn ausgelöste Vesikelfusion genauer zu bestimmen, haben wir einen zusätzlichen Modellierungsansatz entwickelt. Dabei gingen wir davon aus, dass im Fall einer kausal getriggerten Fusion ein Ca2+-Signal vorausgehen, nicht aber folgen sollte. Eine Synchronisation würde sich dann als asymmetrische Poisson-Verteilung des zeitlich nächstliegenden Ca2+-Spikes darstellen, welcher der Fusion mit einem Erwartungswert λ vorauseilt. Nicht kausal zu einer synchronisierten Fusion führende Ca2+-Spikes würden sich hingegen zeitlich symmetrisch um das Fusionsereignis verteilen. Ca2+-Signale wurden durch 5 oder 15 mM Glukose ausgelöst und weiterhin mit Forskolin oder Tolbutamid stimuliert. Die zeitlich nächsten Ca2+-Spikes zu einem Fusionsereignis (Δt < 1 s)
wurden in ein Histogramm aufgenommen. Nach Berechnung der Histogramme passten wir die Parameter der Formeln mit Hilfe eines Fehlerminimierungsverfahrens (nicht-lineare Regressionsanalyse) so an, dass sie den Messdaten am besten entsprachen. Viele Fusionsereignisse waren aber nicht von einem Ca2+-Signal im 1-s-Zeitfenster umgeben. Auch wenn sich der Großteil des Histogramms als symmetrisch darstellt, ist vor allem der mittlere Teil leicht asymmetrisch und lässt damit auf kausal durch Ca2+-Spikes getriggerte Events schließen. Nach Stimulation mit Forskolin oder Tolbutamid zeigte sich eine nur leicht erhöhte Synchronisation von 1,6 Events/Minute/Zelle (7,2 %) im Vergleich zu 0,61 Events/Minute/Zelle (5,3 %) vor Stimulation.
Diese Erkenntnis wird zusätzlich davon unterstützt, dass die Fusionsereignisse häufig auch räumlich getrennt von den Ca2+-Mikrodomänen abzulaufen schienen. Fusionsereignisse fanden favorisiert im Zentrum des Zellbodens statt, während die Orte der Ca2+-Mikrodomänen eher in Zellausläufern zu finden waren und damit bis zu 20 µm von Fusionsereignissen entfernt stattfanden („loose coupling“).:I Abkürzungsverzeichnis
1 Einführung
1.1 Hochfrequente Glukose-getriggerte Ca2+-Spikes als Exozytosesignal
1.2 Signalwege der Insulinsekretion
1.2.1 Ca2+-abhängige Insulinsekretion
1.2.2 Ca2+-unabhängige Insulinsekretion
1.3 Fragestellung
1.4 Zur Beantwortung der Fragestellung gewählte Methoden
1.4.1 Ca2+-Indikatoren
1.4.2 Darstellung der Exozytoseereignisse
1.4.3 Zweifarbkanal LED-TIRF-Mikroskopie
2 Publikation
3 Zusammenfassung und Ausblick
4 Literaturverzeichnis
II Darstellung des eigenen wissenschaftlichen Beitrags
III Erklärung über die eigenständige Abfassung der Arbeit
IV Lebenslauf
V Verzeichnis der wissenschaftlichen Veröffentlichungen
VI Danksagun
The Role of Sports Club Participation on Stability of Motor Performance and Body Composition: A Longitudinal Study in Primary School Children
No full textBackground and Objective. Research has shown that children’s motor performance (MP) and body composition (BC) are positively related to health and influence physical activity across the lifespan. As an intervention for improving MP and BC in childhood, engagement in a sports club (SC) is an important key strategy. Therefore, the aim of the study is to examine the stability of MP and BC and their associations with SC participation. Methods. MP and BC of 295 children (161 girls) with a mean age of 8.42 ± 0.36 years were measured annually with the German Motor Test 6-18. Based on self-reports, children were divided into three groups according to consistent (CON), partial (PAR), and nonparticipation (NO) in SC. NO and PAR were then combined into NO-PAR. The stability of MP and BC was determined using Pearson’s correlation coefficient (r). Associations of SC participation, MP, and BC were examined using robust mixed-model ANOVA (mmANOVA) additionally with first grade as covariate (ANCOVA). Results. More girls (39%) than boys (25%) were classified in PAR. The stability of MP (r = .755) and BMI (r = .889) was moderately high. Children in CON (r = .847) showed lower stability in BMI than NO-PAR (r = .923). mmANOVA revealed better overall MP for both sexes in CON except for balance and BMI. Boys in CON showed better performance in flexibility, endurance, and speed than NO-PAR. ANCOVA confirmed unadjusted results for boys. Conclusion. Lower stability of BMI due to CON indicates a greater variance in ranking and thus the chance to adjust weight management in childhood. Girls’ MP benefited less from SC participation, which may be due to their more frequent rates in PAR. Therefore, interventions to increase engagement in SC should focus on a stepwise approach from none to partial and finally to long-term participation
Identification of Lipid Droplet-Associated Genes in Breast Cancer Patients
No full textLipid droplets (LDs) are known to be involved in the invasion and migration of breast
cancer (BC) cells. This study aimed to identify LD-associated genes as prognostic markers in BC
through comprehensive literature research and integration with lipid composition studies in BC cell
lines. The GEPIA platform was used to analyze the differential expression of LD-associated genes in
BC. The lipid composition of cell lines (MCF-10A, MDA-MB 436 and 231) was obtained by extraction
and thin-layer chromatography coupled with mass spectrometry (MS). Additionally, cell lines were
co-cultured with fatty tissue and analyzed by confocal fluorescence microscopy. A total of 143 genes
were identified as LD-associated genes through literature research and were subsequently analyzed
using GEPIA. Among these, three genes were found to be over-expressed and 45 under-expressed
in BC. Notably, FABP7 showed a statistically significant rank for all bioinformatics criteria as a
prognostic factor. Experimental results showed only minor changes from MCF-10A to both MDA-MB
cell lines for apolar lipids (triacylglycerols and cholesteryl esters) compared to phospholipids (PLs).
Microscopic analyses showed that MDA-MB-231 had larger LDs compared to MCF-10A after 10 days
of cultivation. Our bioinformatics analysis identified 26 genes that play important roles in metastatic
transition in BC via LD-related mechanisms, though these findings could be only partially confirmed
by experimental lipid compositional analyses, so far
The Role of Atmospheric Circulation in Favouring Forest Fires in the Extreme Southern Portugal
No full textThe study explores two forest fires in extreme southern Portugal aiming to increase the knowledge of how atmospheric circulation influenced the fire dynamics in each event. The meteorological conditions were simulated by the Meso-NH full-physics non-hydrostatic limited-area research model. The two numerical simulations were performed using a two-way nested domain configuration with horizontal resolutions of 2500 m and 500 m. In both cases, the large-scale atmospheric environment was marked by the Azores Anticyclone west of Portugal which induced northerly winds over the coastal of Mainland Portugal. The analysis of Tavira’s fire (18–21 July 2012, 24,800 ha of total burned area) revealed flow characteristics resembling a low-level jet located below 1 km, with stronger winds during the second day resulting in enhanced fire spread rates. The second case study (Aljezur, 19–21 June 2020; 2302 ha of burned area) highlights a fire occurring under atypical fire weather conditions, namely lower air temperature and higher relative humidity; however, orographic effects produced downslope winds favouring fire propagation. This study provides a better understanding of the fire critical conditions in extreme South Portugal and investigates the atmosphere–orography interactions in the region that played an important role in the development of these two forest fires. Increasing knowledge about large fires in Southern Portugal can support fire management practices and encourage the sustainable development of the region
Multiple barriers for micropollutants in nutrient recovery from centrate – combining membrane bioreactor and electrodialysis
No full textCentrate from digested sludge dewatering holds promise for nutrient recovery, but concerns about organic
and inorganic contaminants must be addressed. This study investigates the effectiveness of a two-stage
system in retaining organic micropollutants, metals, and metalloids during recovery of a multi-nutrient
solution from centrate. In combination, the lab-scale membrane bioreactor (MBR) and electrodialysis (ED)
effectively reduced contaminant loads by >90% for 21 of the monitored 22 organic micropollutants and
for six of nine metals and metalloids. The combined process demonstrated resilience to fluctuations in the
MBR stage, with a temporary 87% decrease in MBR removal efficiency for carbamazepine translating to
only 6% decrease after the ED. Despite this robust performance, individual compounds such as valsartan
acid or benzotriazole were detected at around 10–20 μg L−1 in the recovered nutrient solution. Zn was
present at around 400 μg L−1 with the highest concentrations of monitored metals. Still, all metals ranged
at least one order of magnitude below recommended values for wastewater reuse. Therefore, the risk
associated with contaminant uptake into hydroponically cultivated produce is considered low, given the
high retention in the system and the necessary dilution of the multi-nutrient solution before its application
as fertilizer. This study demonstrates the effective removal of contaminants by the combination of MBR
and ED for nutrient recovery from centrate, achieving a fit-for-purpose quality of the derived multi-nutrient
solution