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Evaluation of an innovative multi-cancer early detection test: high sensitivity and specificity in differentiating cancer, inflammatory conditions, and healthy individuals
No full textBackground: Cancer is a leading cause of death worldwide, with early detection
crucial for effective treatment. Traditional diagnostic methods, such as imaging
and biopsies, are often limited by invasiveness, cost, and sensitivity. Blood-based
multi-cancer early detection (MCED) tests offer a less invasive and potentially
more comprehensive approach. Recently, a novel screening tool, the Carcimun®
test was reported, detecting conformational changes in plasma proteins through
optical extinction measurements. This study evaluates the Carcimun® test’s
performance, including participants with inflammatory conditions.
Methods: This prospective, single-blinded study included 172 participants: 80
healthy volunteers, 64 cancer patients (various types), and 28 individuals with
inflammatory conditions (fibrosis, sarcoidosis, pneumonia) or benign tumors.
Plasma samples were analyzed using the Carcimun® test. Sensitivity, specificity,
positive predictive value (PPV), and negative predictive value (NPV)were calculated.
Results: Mean extinction values were significantly higher in cancer patients (315.1)
compared to healthy individuals (23.9) and those with inflammatory conditions
(62.7) (p<0.001). The Carcimun® test distinguished these groups with high
accuracy (95.4%), sensitivity (90.6%), and specificity (98.2%). Significant
differences were found between healthy participants and cancer patients
(p<0.001), and between cancer patients and those with inflammation (p<0.001).
Conclusion: The Carcimun® test achieved high accuracy, sensitivity, and
specificity, effectively identifying cancer patients while minimizing false positives
and negatives. By including participants with inflammatory conditions, we
addressed a significant limitation of previous studies, demonstrating the test’s
robustness in real-world clinical scenarios. These findings underscore the potential
of the Carcimun® test as a valuable tool for early cancer detection and screening
Donor-Acceptor-Cyclopropanes: Versatile Starting Materials for the Synthesis of Cyclic Structures
No full textThe aim of this work was the development of ring opening reactions of fused cyclopropanes. The substrate scope of the Rh-catalyzed cyclopropanation was expanded to include cyclic ethers, carbocycles, heterocycles and more. Additionally, the enantioselective cyclopropanation of substituted 2,3-dihydrofurans was developed, using the well-known catalyst Rh₂(R-DOSP)₄ and allowing for ee values of up to 78%. These vinylcyclopropanes are known to undergo the vinylcyclopropane rearrangement to give cyclopentenes. Previously established procedures relied on harsh reaction conditions, whereas the developed procedure allowed for mild and facile rearrangement of a wide variety of vinylcyclopropanes using only 5 mol% of Yb(OTf)₃ at 62 °C, with no chromatography needed in most cases. The method was shown to be versatile, catalyzing the transformation of vinylcyclopropanes fused to cyclic ethers, carbocycles and N-heterocycles, as well as non-cyclic systems in yields of up to 95%. The well-known lactonization was performed on closely related cyclopropanes by both Reiser et al. and Theodorakis et al. on monosubstituted cyclopropanes. The conditions were optimized for the vinylcyclopropanes and the lactones could be obtained in yields of up to 91% as a mixture of 2 diastereomers. An oxygen atom in α-position to the cyclopropane is needed in this case. When the vinyl moiety was substituted with a methyl group, equilibration to the α,β-lactone was achieved with NEt₃. The attempted enantioselective lactonization of the geminal substituted cyclopropanes showed promising beginning results. Enantiomeric excess of up to 16% on one diastereomer could be obtained. Finally, the previously developed photoredox catalyzed reaction to hexahydro[2,3b]furans via an interrupted Nicewicz rearrangement was further developed and investigated. The yield was improved to up to 75%. The mechanistic proposal was supported by various experiments including isolation of intermediates, time-resolved UV-Vis spectroscopy and DFT calculations. The diastereoselectivity is low, and attempts to solve this by using CPAs was unfruitful. Finally, aryl acetate cyclopropanes were subjected to an NIS-mediated rearrangement. This yielded formyl esters in up to 61% yield, which could be further transformed to lactones
Free-energy analysis of the suppression of the amyloid fibril formation induced by ATP
No full textThe formation of amyloid fibrils is strongly connected to neurodegenerative diseases, such as Alzheimer's and Parkinson's, which not only cause detrimental effects to individuals but also pose considerable challenges on society as a whole.
While adenosine triphosphate (ATP) is primarily recognized for its role as the primary energy currency for driving biomolecular reactions, recent experiments have unveiled its ability to inhibit protein aggregation. This finding suggests that ATP might play a pivotal role in preventing the aggregation of amyloid peptides into fibrils, thereby preserving protein solubility within cells.
Aside from the tendency of ATP to adsorb onto amyloid peptides, the underlying mechanism remained largely unknown due to a scarce understanding of the molecular interactions.
In this thesis, a thorough explanation at the molecular scale is provided by addressing multiple aspects of the interactions using all-atom molecular dynamics simulations.
First, the ATP-ATP interactions are investigated, revealing that the commonly employed AMBER force field parameters result in an overestimation of the aggregation of the ATP anions. By scaling the charges of the phosphate moiety by a factor of 0.7, an inconsistency in the treatment of the charges of ionic groups is addressed, resulting in a reduction of the ATP self-aggregation closer to experimental observations.
Following this, the ATP-peptide interactions are systematically analyzed. The analysis demonstrates that ATP interacts favorably with all amino acids, excluding those with negatively charged side chains. However, the repulsion is comparatively weak, indicating that the tendency of ATP to adsorb onto protein and peptide surfaces stems from the ability to adsorb onto almost all amino acids.
Finally, the effect of ATP on the peptide-peptide interactions is studied. It is shown that ATP influences the relative orientation of the peptides to each other.
Furthermore, ATP induces a change in the free energies, resulting in a significant shift in the equilibrium in favor of non-aggregated peptides.
The decomposition of the free energies reveals that the van der Waals interactions between ATP and the peptides are the energetic driving force, despite ATP being highly charged.
The analysis of the energy contributions of the whole system shows that the strong electrostatic interactions of the phosphate moiety are compensated by the water solvent
Prevalence, Predictors and Decompressive Laparotomy in Abdominal Compartment Syndrome in Patients Requiring Extracorporeal Membrane Oxygenation
Full text linkBackground: Critically ill patients requiring extracorporeal membrane oxygenation (ECMO) have several risk factors to suffer from abdominal compartment syndrome (ACS). Little is known about this subgroup. The aim of this study was to investigate the prevalence and associated factors for ACS in patients requiring ECMO to assess the effect of decompressive laparotomy (DL) and the impact on mortality. Methods: This retrospective observational study analyzed adult patients requiring ECMO in four intensive care units at the University Medical Center Regensburg between 01/2010 and 06/2020. Patients with clinically suspected ACS were screened by measuring intra-abdominal pressure (IAP) with the trans-bladder technique. ACS was defined as IAP > 20 mmHg and survival was defined as successful discharge from hospital. Results: The prevalence of ACS in non-ECMO ICU patients was 0.8% (291/36,795) and 2.9% (47/1643) in ECMO patients. In the subgroup of resuscitated ECMO patients, ACS was present in 4.2% (32/766). Procalcitonin was associated with ACS. ECMO patients with ACS receiving DL were significantly more ill compared to those without DL (SOFA score at ICU admission 18 [15; 20], vs. 16 [13; 17], p = 0.048). DL decreased IAP and significantly improved ventilation; vasopressor and lactate stabilized within 24 hours. Survival was comparable between the DL and the non-DL groups (11% [1/9] vs. 14% [1/7], p = 1.000). Conclusions: ECMO patients are at high risk of developing ACS, even more so for resuscitated patients. This and high procalcitonin may be taken into consideration when screening for ACS. Decompressive laparotomy did improve respiratory compliance and stabilized hemodynamic parameters with low rates of complication. Even though patients that received DL were significantly more ill, the mortality rates were not higher
Anoctamin 9 determines Ca2+ signals during activation of T-lymphocytes
Full text linkBackground: Activation of T-cells is initiated by an increase in intracellular Ca2+, which underlies positive and negative regulation. Because the phospholipid scramblase and ion channel ANO9 (TMEM16J) was shown previously to regulated Ca2+ signals in renal epithelial cells, we asked whether ANO9 demonstrates a similar regulation in T-cells.
Methods: We used measurements of the intracellular Ca2+ concentration to
examine the effects of ANO9 on intracellular Ca2+ signaling and demonstrated
expression of ANO9 and its effects on cellular and molecular parameters.
Results: ANO9 was found to be expressed in human lymphocytes, including the
Jurkat T-lymphocyte cell line and mouse lymphocytes. ANO9 has been shown to affect intracellular Ca2+ signals in renal epithelial cells. Here we demonstrate the essential role of ANO9 during initiation of intracellular Ca2+ signals in Jurkat Tcells and isolated mouse lymphocytes. ANO9 is essential for the initial rise in intracellular Ca2+ due to influx of extracellular Ca2+ through store-operated ORAI1 Ca2+ entry channels. ANO9 is indispensable for T-cell function, independent on whether cells are activated by stimulation of the T-cell receptor with CD3-antibody or by PMA/phytohemagglutinin.
Conclusions: Upon activation of T-cells and formation of the immunological
synapse, ANO9 recruits the Ca2+-ATPase (PMCA) to the plasma membrane, which is supported by the scaffolding protein discs large 1 (DLG1). PMCAs maintain low Ca2+ levels near ORAI1 channels thereby suppressing Ca2+-inhibition of ORAI1 and thus retaining store-operated Ca2+ entry (SOCE). It is suggested that ANO9 has a role in interorganelle communication and regulation of cellular protein trafficking, which probably requires its phospholipid scramblase function
Characteristics of self-injurious behaviour and early traumatic experiences: associations with emotional reactivity, depression and aggression in university students
Full text linkBackground
A lifetime history of non-suicidal self-injury (NSSI) is a risk factor for subsequent behavioural and emotional problems, including depression, aggression and heightened emotional reactivity. Traumatic experiences, which are frequently reported by individuals with NSSI, also show predictive links to these mental health problems. However, the exact connections between these areas and their subdomains remain unclear.
Aims
To explore in detail the relationships of specific characteristics of NSSI (e.g. termination in adolescence, duration, frequency, reinforcement mechanisms) and various types of traumatic experience (emotional, physical, sexual) with distinct aspects of emotional reactivity (sensitivity, intensity, persistence), aggression (behavioural, cognitive, affective) and severity of depression in university students.
Method
Via online survey, 150 university students aged 18 to 25 years, who had self-injured at least once, provided information on NSSI, and completed questionnaires including the Childhood Trauma Questionnaire, Patient Health Questionnaire, Emotion Reactivity Scale, and Aggression Questionnaire. Regression analyses were conducted to determine risk factors linked to increased depression scores, aggression and emotional reactivity. The study was pre-registered in the German Clinical Trials Register (DRKS00023731).
Results
Childhood emotional abuse contributed to emotional reactivity, aggression and depressive symptom severity (β = 0.33–0.51). Risk factors for sustained NSSI beyond adolescence included increased automatic positive reinforcement (odds ratio: 2.24).
Conclusions
Childhood emotional abuse significantly contributes to emotional and behavioural problems and needs to be considered in NSSI therapy. NSSI was found to persist into adulthood when used as an emotion regulation strategy
The Dynamics of Non-Belief (with Modesty)
Full text link“Suspension of judgment” is an ambiguous term that may refer either to a doxastic state (“suspended judgment”) or to a doxastic action (“suspending judgment”). Based on a simple non-belief account, this paper presents a formal study of both aspects of suspension. We first introduce the notion of a suspension set (a set of non-beliefs) and determine its logical structure. Then we present the classical AGM operations of belief revision and belief contraction and give characterizations of them that refer to suspension sets rather than belief sets. Finally, we study the suspension operation, a
symmetric cousin of belief contraction, and characterize it both in terms of belief sets and in terms of suspension sets. Belief contraction and belief suspension thereby get reinterpreted as two different forms of the expansion of suspension sets. The project is interesting because in contrast to belief revision and belief contraction, the suspension operation is symmetric with respect to negation: suspending judgment on A is the same as suspending judgment on ¬A. Most of our results are premised on an assumption
of modesty: For every person with consistent beliefs, there is always at least one proposition on which she suspends judgment
Thrombocytopenia During Venovenous Extracorporeal Membrane Oxygenation in Adult Patients With Bacterial, Viral, and COVID-19 Pneumonia
Full text linkContact of blood with artificial surfaces triggers platelet activation. The aim was to compare platelet kinetics after venovenous extracorporeal membrane oxygenation (V-V ECMO) start and after system exchange in different etiologies of acute lung failure. Platelet counts and coagulation parameters were analyzed from adult patients with long and exchange-free (≥8 days) ECMO runs (n = 330) caused by bacterial (n = 142), viral (n = 76), or coronavirus disease 2019 (COVID-19) (n = 112) pneumonia. A subpopulation requiring a system exchange and with long, exchange-free runs of the second oxygenator (≥7 days) (n = 110) was analyzed analogously. Patients with COVID-19 showed the highest platelet levels before ECMO implantation. Independent of the underlying disease and ECMO type, platelet counts decreased significantly within 24 hours and reached a steady state after 5 days. In the subpopulation, at the day of a system exchange, platelet counts were lower compared with ECMO start, but without differences between underlying diseases. Subsequently, platelets remained unchanged in the bacterial pneumonia group, but increased in the COVID-19 and viral pneumonia groups within 2–4 days, whereas D-dimers decreased and fibrinogen levels increased. Thus, overall platelet counts on V-V ECMO show disease-specific initial dynamics followed by an ongoing consumption by the ECMO device, which is not boosted by new artificial surfaces after a system exchange
Immunhistochemische Analyse der GPR43 Expression in intestinalen Biopsien nach allogener Stammzelltransplantation und deren Bedeutung für die Graft-versus-Host-Erkrankung
Full text linkAufgrund der hohen Mortalitätsrate gilt die GvHD nach wie vor als eine der größten Herausforderungen nach ASCT. Neben der Identifikation diverser Biomarker zur Früherkennung und Prognoseeinschätzung einer GvHD beschäftigte sich die Forschung der letzten Jahre vor allem mit der Rolle des Mikrobioms in der Pathogenese der GvHD.
Als einer der wichtigsten Metaboliten des Mikrobioms wurden SCFA zusammen mit ihren Rezeptoren sowohl im Tier- als auch im humanen Modell untersucht. Dabei wurden bisher die Effekte von SCFA auf einzelne Zellgruppen und die Rezeptorexpression der jeweiligen Zellentitäten betrachtet. Bislang gibt es keine in vivo Untersuchung der GPR43 Expression in der humanen Darmwand von Patienten nach ASCT. Ziel dieser Arbeit war es daher, den Zusammenhang zwischen der GPR43 Expression und der GvHD mittels immunhistochemischer Färbemethoden in intestinalen Biopsien von Patienten nach ASCT aufzuzeigen.
Dazu wurden retrospektiv 100 Biopsien von 97 Patienten untersucht, die sich im Zeitraum von 2007 bis 2019 am Uniklinikum Regensburg einer ASCT unterzogen. Es erfolgte eine immunhistochemische Färbung des GPR43 Rezeptors mithilfe zweier unterschiedlicher Antikörper und als Ergänzung die Detektion FoxP3 exprimierender Lymphozyten. Als Kon-trollproben dienten vor der ASCT gewonnene Gewebeproben. Um mögliche Einflussfaktoren für die GPR43 Expression zu identifizieren, erfolgte zusätzlich die Erfassung wichtiger klinischer Daten wie die Ausprägung der GvHD, der Biopsiezeitpunkt, die Dosierung verabreichter Antibiotika und Steroide und 3-IS Werte aus dem Urin.
Beim Vergleich der Färbeergebnisse der beiden Antikörper stellte sich heraus, dass LS-A6599 zur Visualisierung der GPR43 Expression im Stroma am geeignetsten ist, wohingegen die Darstellung der Proteinexpression im Epithel mit LS-A1578 bessere Resultate lieferte. Während die Auswertung im Colon die meisten positiven Stromazellen detektierte, wies das Ileum die geringste Menge auf. Ferner exprimierte das Zottenepithel des Duodenums signifikant mehr GPR43 als jenes im Ileum. Verglichen zu den Kontrollproben nahm die Proteinexpression sowohl im Stroma als auch im Zottenepithel nach ASCT signifikant zu. Innerhalb der postTx Proben stieg die GPR43 Expression im Stroma mit zunehmendem Schweregrad der GvHD an. Demgegenüber veränderte sich die Anzahl GPR43 positiver Zellen im Epithel der Krypten nach ASCT und in Abhängigkeit vom Lerner Grad kaum. Überdies beeinflusste der Zeitpunkt der Biopsie die Expression von GPR43. Diese war im Stroma innerhalb der ersten 30 Tage nach ASCT am höchsten und sank auch danach nicht unter den Ausgangswert vor Transplantation. Einen ähnlichen Trend zeigte das Zottenepithel, während sich die Expression in den Krypten abhängig vom Biopsiezeitpunkt nicht veränderte. Des Weiteren beeinflusste die Gabe von Steroiden und Antibiotika die Menge GPR43 positiver Zellen. Erhielten Patienten mehr als 20 mg Steroide am Tag, stieg die Proteinexpression im Stroma und sank im Epithel der Zotten und oberflächlichen Krypten. Durch die Behandlung mit Breitspektrumantibiotika erhöhte sich die GPR43 Expression sowohl im Stroma als auch im Zottenepithel, während sie in den Krypten abnahm. Die Anzahl FoxP3 positiver Treg korrelierte mit der Anzahl GPR43 positiver Zellen im Stroma. Dieser Zusammenhang verstärkte sich bei Patienten, die nicht mit Breitspektrumantibiotika behandelt wurden oder weniger als 20 mg Steroide erhielten. Eine Korrelation der immunhistochemischen GPR43 Expression mit 3-IS Werten aus dem Urin lag weder im Stroma noch im Epithel der Zotten und Krypten vor. Gleiches galt für den Vergleich der hier gewonnenen Ergebnisse mit den Resultaten aus der PCR-Analyse. In der Überlebenszeitanalyse konnte anhand der GPR43 Expression außerdem keine prognostische Aussage hinsichtlich des Risikos für TRM oder RRM getroffen werden.
Die immunhistochemische Analyse legt nahe, dass die GPR43 Expression in Stroma- und Epithelzellen unterschiedlichen Regulationsmechanismen unterliegt. Um dies zu bestätigen, benötigt es weitere Studien, die sich mit den jeweiligen Signalwegen, den Interaktionen zwischen den Zellentitäten und dem Einfluss von Mikroben und Medikamenten auf die Proteinexpression beschäftigen. Ferner sind entsprechende Untersuchungen notwendig, um zu klären, ob eine Neutropenie für die Hochregulation des FFAR2 im Stroma in der Anfangsphase nach ASCT verantwortlich ist. Überdies gilt es zu erforschen, durch welche genauen Mechanismen Antibiotika und Steroide die GPR43 abhängige Induktion von Treg beeinflussen und ob sich daraus ein therapeutischer Nutzen ergibt. Abschließend sollte der Zusammenhang zwischen BCoAT, einem indirekten Marker für die Anwesenheit Butyrat-produzierender Spezies, und der GPR43 Expression näher analysiert werden. Dadurch kann geklärt werden, ob die Anzahl der Mikroben und die daraus resultierende vermehrte Produktion von SCFA die Expression von GPR43 steigert. Lässt sich dies bestätigen, eröffnet sich mit dem Transfer bestimmter Bakterienstämme eine neue Therapiemöglichkeit, mit der der Verlauf und die Prognose der GvHD günstig beeinflusst werden können. Die deutlichsten Veränderungen hinsichtlich der GPR43 Expression wurden in dieser Arbeit im Stroma festgestellt. Dies deutet darauf hin, dass vor allem die Proteinexpression dieses Kompartiments eine Rolle in der Pathophysiologie der GvHD spielt und den Krankheitsverlauf steuert. Eine Identifikation der einzelnen Stromazellen kann in zukünftigen Arbeiten mithilfe der Multiplex-Chip-Technologie, welche die Koexpression von GPR43 und zellspezifischen Markern visualisiert, erfolgen. Dadurch können weitere Erkenntnisse hinsichtlich der pathophysiologischen Bedeutung des GPR43 Rezeptors für die GvHD gewonnen werden
Testung der RIST-Therapie am in vitro 3D-Modell des Neuroblastoms
Full text linkNeuroblastoma (NB) is the most common extracranial solid tumor in childhood. Despite intensive treatment regimens the outcome for patients with high-risk relapsed or treatment refractory NB remains poor and novel treatment strategies are urgently needed. A new multimodal treatment design based on metronomically combining molecular targeted biologicals (Rapamycin and Dasatinib) with conventional cytostatics (Irinotecan and Temozolomid), called RIST therapy, was evaluated in a prospective randomized phase II clinical trial in relapsed and refractory NB patients (RIST-rNB-2011, NCT01467986) and demonstrated a significant impact on progression-free survival and overall survival in patients with MycN amplification.
Often promising preclinical results show weak in vivo efficacy due to drug testing on conventional two-dimensional (2D) monolayer cell cultures facing the challenge of accurately representing in vivo tumor biology, metabolic gradients and architecture. Aim of this study is to establish a three-dimensional (3D) cell culture model suitable to mimic a wide range of avascular tumor characteristics and include them into testing the RIST protocol to further improve preclinical studies. Therefore we used three oncogene MycN-amplified (MNA) (SK-N-BE(2), Kelly, IMR-32), two MycN-non amplified (MNN) (SK-N-AS, SK-N-SH) and one MycN-inducible (MNI) (Tet21N) NB cell lines. The 3D cell cultures were evaluated by determining the size, number, and viability of spheroids at various time points after seeding increasing cell numbers, applying distinct growth supplements, and providing different types of ultra-low attachment plates. Furthermore, the gene expression of various cancer stem cell (CSC) markers (CXCR4, BMI1, NANOG, ABCG2) was evaluated by reverse transcription and quantitative polymerase chain reaction (RTqPCR) comparing 2D and 3D cell cultures. To assess the RIST protocol on spheroid formation, drug treatment was started directly after cell plating, while spheroid growth was assessed on completely formed spheroids.
Our data demonstrate that the viability of all tested NB spheroids was significantly reduced applying the same half maximal inhibitory concentration (IC50) as applied in 2D cell cultures. In this study, we could verify previously published data of MycN-inducible NB cell line Tet21N demonstrating that expression of high MycN levels being more sensitive to mTOR inhibition by Rapamycin compared to cells expressing lower MycN levels. Further we validated CXCR4 as a CSC marker being higher expressed in all tested NB spheroids compared to 2D monolayer cultures. Interestingly, all tested CSC markers except ABCG2 showed a significant higher upregulation in expression in the MNN cell lines compared to MNA cell lines.
In conclusion, despite the presence of elevated CSC marker expression our data validated the efficacy of the RIST protocol in the in vitro 3D model of NB suggesting that the RIST therapy may also be effective in a more aggressive subset of tumors. Overall, the established 3D cell culture model is a valuable complement for preclinical testing of novel drugs against neuroblastoma even in high-throughput screening (HTS) formats