Neue molekularzytogenetische Strategien für die Analyse seltener Zellen

Aufgrund fehlender Methoden wurde bis zum heutigen Zeitpunkt nur selten eine molekulargenetische Analyse von Einzelzellen durchgeführt. In vielen Bereichen aber, wie beispielsweise den Tumoren, ist sie von entscheidender Bedeutung. Das Auftreten eines genetisch heterogenen Musters in einem Tumor könnte die Suche nach krankheitsauslösenden oder -fördernden Veränderungen erschweren. Auch bei der Untersuchung von seltenen Zellen (so genannte „rare cell events“), z.B. bei einer minimalen residualen Tumorerkrankung sind geeignete Methoden zur Einzelzellanalyse notwendig, da nur wenig Material zur Verfügung steht. Ziel dieser Doktorarbeit war es, zwei molekulargenetische Analysemethoden zu etablieren, weiterzuentwickeln und in einer geeigneten Strategie bei der Untersuchung von disseminierten Tumorzellen bei fünf Mammakarzinomen einzusetzen. Im ersten Teil der Doktorarbeit erfolgte die Auswahl eines geeigneten Markierungssystems für die Identifizierung disseminierter Tumorzellen im Knochenmark. Die Benutzung des pan-Zytokeratin Antikörpers A45 B/B3 direkt konjugiert mit dem Fluoreszenzfarbstoff Cy3 oder FluorX zeigte in verschiedenen Testsystemen die besten Ergebnisse. Sowohl seine Spezifität als auch die Durchführbarkeit einer Fluoreszenz-in-situ-Hybridisierung (FISH) war möglich. Anhand disseminierter Tumorzellen von Nierentumoren wurde die Möglichkeit einer Untersuchung mittels zweier Interphase FISH Ansätze überprüft. Die Hybridisierung der Zentromernahen YAC Sonden konnte auf dem Knochenmarksgewebe bei pan-Zytokeratin positiven Zellen nach Erproben mehrerer Protokolle nicht durchgeführt werden, allerdings war die Hybridisierung von Zentromersonden erfolgreich. Die Optimierung des veröffentlichten Protokolls zur Einzelzell CGH (C. Klein et al. 1999; N. Stöcklein et al.2002) war das zweite Ziel der Arbeit. Diese Optimierung war notwendig, weil sich das ursprünglich publizierte Protokoll als Artefakt anfällig herausstellte. Durch die in dieser Arbeit beschriebenen Protokollveränderungen konnte die für Einzelzell Analysen notwendige Reproduzierbarkeit erreicht werden. Als Testsystem wurde eine molekulargenetisch ausreichend charakterisierte und in ihren Veränderungen stabile Zelllinie gewählt (RCC-26). Die ersten Versuche zur Einzelzellanalyse zeigten Veränderungen in der Zelllinie, die in allen vorangegangenen Analysen mit etablierten Techniken (M-FISH und CGH) nicht gezeigt werden konnten. Eine Optimierung des Protokolls führte zu übereinstimmenden Ergebnissen von Einzelzell CGH, CGH und M-FISH. Anschließend konnte die Reproduzierbarkeit dieser Methode anhand der Zelllinie RCC-26 gezeigt werden. Die Anwendung beider Methoden zur Analyse von seltenen Zellen wurde mit der Untersuchung von fünf Mammakarzinomen dargelegt. Der Vergleich von Primärtumor und kultivierten disseminierten Tumorzellen ins Knochenmark zeigte im Rahmen der untersuchten Fälle gemeinsame molekulargenetische Veränderungen beider Tumorentitäten. Mit der Anwendung dieser neuen Methoden ist es möglich detaillierte Informationen über seltene Zellen zu erhalten und diese in den biologischen Kontext einzuordnen

Psychosocial burden and associated factors among nurses in care homes during the COVID-19 pandemic: findings from a retrospective survey in Germany

Background: Care homes were hit hard by the COVID-19 pandemic. Although high levels of psychosocial burden (i.e., anxiety, depression and stress) during the pandemic have been described for healthcare workers in hospitals, evidence on the psychosocial burden for nurses in care homes during the pandemic is scarce. Methods: A total of 811 nurses participated in a retrospective online survey between November 2020 and February 2021. Information about the COVID-19 situation (i.e., working demands, COVID-19 cases in their facility, and COVID-19-related burden) of nurses in German care homes during the first wave of the pandemic (March 2020 to June 2020) was gathered. The Stress Scale of the Depression Anxiety and Stress Scales (SDASS-21), the Generalized Anxiety Disorder Scale-2 (GAD-2), the Patients-Health-Questionnaire-2 (PHQ-2), and the Copenhagen Psychosocial Questionnaire (COPSOQ) were used to screen for psychosocial burden. Results: Among nurses, 94.2% stated that working demands since the COVID-19 pandemic increased. Further, 59.1% showed clinically relevant levels of either stress, anxiety, and/or depression. Multiple regression analysis showed significant associations between COVID-19-related burden and qualification (p < .01), dissatisfaction with COVID-19 management of care home manager (p < .05), COVID-19-related anxiety (p < .001), and dementia as a focus of care (p < .05). Stress, depression, and anxiety showed associations with COVID-19 related burden at work (p < .01), COVID-19-related anxiety (p < .001), social support (p < .01), and sense of community (p < .05). Stress was also associated with COVID-19 cases among residents (p < .05), and size of care home (p < .05). Conclusion: Short- and long-term strategies (i.e., psychosocial counseling, mandatory team meetings, more highly qualified nurses, additional training) in the work environment of nursing, in crises, but beyond, should be encouraged to reduce the burden on nursing staff in care homes

General practitioner care in nursing homes during the first wave of the COVID-19 pandemic in Germany: a retrospective survey among nursing home managers

Background: Though evidence on the detrimental impact of the COVID-19 pandemic in nursing homes is vast, research focusing on general practitioners' (GP) care during the pandemic in nursing homes is still scarce. Methods: A retrospective online survey among 1,010 nursing home managers in Germany was conducted during the first wave of the COVID-19 pandemic between November 2020 and February 2021. Associations between perceived deficits in GP care (routine and acute visits) and both general and COVID-19-related characteristics of nursing homes were analysed using multiple logistic regression analyses. Results: The majority of nursing home managers reported no deficits in GP care (routine visits, 84.3%; acute visits, 92.9%). Logistic regression analyses revealed that deficits in GP care (routine visits) were significantly associated with visiting restrictions for GPs and nursing home size. Small nursing homes (1-50 residents) were significantly more likely to report deficits in GP care (routine visits) compared to medium (51-100 residents) and large nursing homes (> 100 residents). Further, deficits in GP care (acute visits) were significantly associated with dementia as a focus of care and the burden of insufficient testing for SARS-CoV-2 among residents. Moreover, visiting restrictions for GPs were significantly associated with dementia as the focus of care and the COVID-19 incidence at the federal state level. Finally, COVID-19 cases in nursing homes were significantly associated with size of nursing homes, COVID-19-incidence on the federal state level and the burden of insufficient testing capacities for SARS-CoV-2 among residents. Conclusion: We found structural factors associated with GP care deficits during the pandemic. New concepts for GP care should be implemented in pandemic preparedness plans to ensure high quality, consistent, and reliable GP care as well as effective infection prevention measures in nursing homes

Non-Equilibrium Statistical Mechanics of Classical Lattice ϕ4\phi^4 Field Theory

Classical $\phi^4$ theory in weak and strong thermal gradients is studied on the lattice in (1+1) dimensions. Classical $\phi^4$ theory in weak and strong thermal gradients is studied on the lattice in (1+1) dimensions. The steady state physics of the theory is investigated from first principles and classified into dynamical regimes. We derive the bulk properties associated with thermal transport, and explore in detail the non-equilibrium statistical mechanics of the theory as well as connections to equilibrium and irreversible thermodynamics. Linear response predictions are found to be valid for systems quite far from equilibrium and are seen to eventually break down simultaneously with local equilibrium.Comment: 28 pages, 20 fig

Biogeochemical structure of the Laptev Sea in 2015-2020 associated with the River Lena plume

The discharge of rivers and the subsequent dispersion of their plumes play a pivotal role in the biogeochemical cycling of the Arctic Ocean. Based on the data collected during annual transects conducted in the autumn period (September-October) from 2015-2020, this study explores the effect of River Lena plume dispersion on the seasonal and interannual changes in the hydrophysical and biogeochemical structure of the southeastern Laptev Sea. The temperature-salinity relationship (T-S), Redfield ratio and multiparameter cluster analysis were used to investigate variations in the water mass structure along the transect. The results revealed that the plume’s interannual and seasonal spreading patterns play a crucial role in regulating the local physical, biogeochemical, and biological processes in the southern Laptev Sea. During September-October, the hydrochemical water mass structure along the transects shifted from highly stratified to unstratified as the plume’s mixing intensity increased. Anomalous hydrochemical distributions were observed due to coastal upwelling, which was primarily characterized by high total alkalinity and nitrate levels, and low organic phosphorus, nitrite, and ammonia levels in the seawater. Wind and cold weather conditions drive deep vertical mixing of seawater, causing the resuspension of bottom sediment and the subsequent enrichment of bottom water by nutrients. Multi-parameter cluster analysis is used to describe the details of water mass structures in the highly dynamic southern Laptev Sea, with water mass structures typically undergoing significant changes within two weeks between September and October. The migration and transformation of water masses throughout the seasons are influenced by the volume of river discharge, fall-winter cooling, and atmospheric circulation patterns. Furthermore, the general atmospheric circulation is confirmed to be the primary cause of the interannual variation in the spread of the Lena River plume over the southeast Laptev Sea.publishedVersio

Experimental validation of an extended Jones matrix calculus model to study the 3D structural orientation of the collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography

We report results to verify a theoretical framework to analyze the 3D depth-wise structural organization of collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography. Apparent birefringence data obtained from multi-angle measurements using a time domain polarization-sensitive optical coherence tomography system has been compared with simulated data based on the extended Jones matrix calculus. Experimental data has been shown to agree with the lamellar model previously proposed for the cartilage microstructure based on scanning electron microscopy data. This tool could have potential application in mapping the collagen structural orientation information of cartilage non-invasively during arthroscopy

Metabolomic Profiles in Jamaican Children With and Without Autism Spectrum Disorder

BACKGROUND: Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with a wide range of behavioral and cognitive impairments. While genetic and environmental factors are known to contribute to its etiology, the underlying metabolic perturbations associated with ASD which can potentially connect genetic and environmental factors, remain poorly understood. Therefore, we conducted a metabolomic case-control study and performed a comprehensive analysis to identify significant alterations in metabolite profiles between children with ASD and typically developing (TD) controls. OBJECTIVE: To elucidate potential metabolomic signatures associated with ASD in children and identify specific metabolites that may serve as biomarkers for the disorder. METHODS: We conducted metabolomic profiling on plasma samples from participants in the second phase of Epidemiological Research on Autism in Jamaica (ERAJ-2), which was a 1:1 age (±6 months)-and sex-matched cohort of 200 children with ASD and 200 TD controls (2-8 years old). Using high-throughput liquid chromatography-mass spectrometry techniques, we performed a targeted metabolite analysis, encompassing amino acids, lipids, carbohydrates, and other key metabolic compounds. After quality control and imputation of missing values, we performed univariable and multivariable analysis using normalized metabolites while adjusting for covariates, age, sex, socioeconomic status, and child\u27s parish of birth. RESULTS: Our findings revealed unique metabolic patterns in children with ASD for four metabolites compared to TD controls. Notably, three of these metabolites were fatty acids, including myristoleic acid, eicosatetraenoic acid, and octadecenoic acid. Additionally, the amino acid sarcosine exhibited a significant association with ASD. CONCLUSIONS: These findings highlight the role of metabolites in the etiology of ASD and suggest opportunities for the development of targeted interventions

Metabolomic profiles in Jamaican children with and without autism spectrum disorder

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with a wide range of behavioral and cognitive impairments. While genetic and environmental factors are known to contribute to its etiology, the underlying metabolic perturbations associated with ASD which can potentially connect genetic and environmental factors, remain poorly understood. Therefore, we conducted a metabolomic case-control study and performed a comprehensive analysis to identify significant alterations in metabolite profiles between children with ASD and typically developing (TD) controls. The objective of this study is to elucidate potential metabolomic signatures associated with ASD in children and identify specific metabolites that may serve as biomarkers for the disorder. We conducted metabolomic profiling on plasma samples from participants in the second phase of Epidemiological Research on Autism in Jamaica, a cohort of 200 children with ASD and 200 TD controls (2-8 years old). Using high-throughput liquid chromatography-mass spectrometry techniques, we performed a targeted metabolite analysis, encompassing amino acids, lipids, carbohydrates, and other key metabolic compounds. After quality control and imputation of missing values, we performed univariable and multivariable analysis using normalized metabolites while adjusting for covariates, age, sex, socioeconomic status, and child's parish of birth. Our findings revealed unique metabolic patterns in children with ASD for four metabolites compared to TD controls. Notably, three of these metabolites were fatty acids, including myristoleic acid, eicosatetraenoic acid, and octadecenoic acid. Additionally, the amino acid sarcosine exhibited a significant association with ASD. These findings highlight the role of metabolites in the etiology of ASD and suggest opportunities for the development of targeted interventions

Nitrous Oxide Dynamics in the Siberian Arctic Ocean and Vulnerability to Climate Change

Nitrous oxide (N2O) is a strong greenhouse gas and stratospheric ozone-depleting substance. Around 20% of global emissions stem from the ocean, but current estimates and future projections are uncertain due to poor spatial coverage over large areas and limited understanding of drivers of N2O dynamics. Here, we focus on the extensive and particularly data-lean Arctic Ocean shelves north of Siberia that experience rapid warming and increasing input of land-derived nitrogen with permafrost thaw. We combine water column N2O measurements from two expeditions with on-board incubation of intact sediment cores to assess N2O dynamics and the impact of land-derived nitrogen. Elevated nitrogen concentrations in water column and sediments were observed near large river mouths. Concentrations of N2O were only weakly correlated with dissolved nitrogen and turbidity, reflecting particulate matter from rivers and coastal erosion, and correlations varied between river plumes. Surface water N2O concentrations were on average close to equilibrium with the atmosphere, but varied widely (N2O saturation 38%–180%), indicating strong local N2O sources and sinks. Water column N2O profiles and low sediment-water N2O fluxes do not support strong sedimentary sources or sinks. We suggest that N2O dynamics in the region are influenced by water column N2O consumption under aerobic conditions or in anoxic microsites of particles, and possibly also by water column N2O production. Changes in biogeochemical and physical conditions will likely alter N2O dynamics in the Siberian Arctic Ocean over the coming decades, in addition to reduced N2O solubility in a warmer ocean.publishedVersio

High resolution array-CGH analysis of single cells

Heterogeneity in the genome copy number of tissues is of particular importance in solid tumor biology. Furthermore, many clinical applications such as pre-implantation and non-invasive prenatal diagnosis would benefit from the ability to characterize individual single cells. As the amount of DNA from single cells is so small, several PCR protocols have been developed in an attempt to achieve unbiased amplification. Many of these approaches are suitable for subsequent cytogenetic analyses using conventional methodologies such as comparative genomic hybridization (CGH) to metaphase spreads. However, attempts to harness array-CGH for single-cell analysis to provide improved resolution have been disappointing. Here we describe a strategy that combines single-cell amplification using GenomePlex library technology (GenomePlex(®) Single Cell Whole Genome Amplification Kit, Sigma-Aldrich, UK) and detailed analysis of genomic copy number changes by high-resolution array-CGH. We show that single copy changes as small as 8.3 Mb in single cells are detected reliably with single cells derived from various tumor cell lines as well as patients presenting with trisomy 21 and Prader–Willi syndrome. Our results demonstrate the potential of this technology for studies of tumor biology and for clinical diagnostics