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Vascular guidance is critical in developmental vasculogenesis and pathological angiogenesis. Brain tumors are strongly vascularized, and antiangiogenic therapy was anticipated to exhibit a strong anti-tumor effect in this tumor type. However, vascular endothelial growth factor A (VEGFA) specific inhibition had no significant impact in clinical practice of gliomas. More research is needed to understand the failure of this therapeutic approach. EphrinB2 has been found to directly interact with vascular endothelial growth factor receptor 2 (VEGFR2) and regulate its activity. Here we analyzed the expression of ephrinB2 and EphB4 in human glioma, we observed vascular localization of ephrinB2 in physiology and pathology and found a significant survival reduction in patients with elevated ephrinB2 tumor expression. Induced endothelial specific depletion of ephrinB2 in the adult mouse (efnb2i∆EC) had no effect on the quiescent vascular system of the brain. However, we found glioma growth and perfusion altered in efnb2i∆EC animals similar to the effects observed with antiangiogenic therapy. No additional anti-tumor effect was observed in efnb2i∆EC animals treated with antiangiogenic therapy. Our data indicate that ephrinB2 and VEGFR2 converge on the same pathway and intervention with either molecule results in a reduction in angiogenesis.
Background: The aim of this study was to identify pre-operative parameters able to predict length of stay (LoS) based on clinical data and patient-reported outcome measures (PROMs) from a scorecard database in patients with significant aortic stenosis who underwent TAVI (transfemoral aortic valve implantation). Methods: 302 participants (51.7% males, age range 78.2–84.2 years.) were prospectively recruited. After computing the median LoS value (=6 days, range = 5–8 days), we implemented a decision tree algorithm by setting dichotomized values at median LoS as the dependent variable and assessed baseline clinical variables and PROMs (Clinical Frailty Scale (CFS), EuroQol-5 Dimension-5 Levels (EQ-5D) and Kansas City Cardiomyopathy Questionnaire (KCCQ)) as potential predictors. Results: Among clinical parameters, only peripheral arterial disease (p = 0.029, HR = 1.826) and glomerular filtration rate (GFR, cut-off < 33 mL/min/1.73 m2, p = 0.003, HR = 2.252) were predictive of LoS. Additionally, two PROMs (CFS; cut-off = 3, p < 0.001, HR = 1.324 and KCCQ; cut-off = 30, p = 0.003, HR = 2.274) were strong predictors. Further, a risk score for LoS (RS_LoS) was calculated based on these predictors. Patients with RS_LoS = 0 had a median LoS of 5 days; patients RS_LoS ≥ 3 had a median LoS of 8 days. Conclusions: based on the pre-operative values of the above four predictors, a personalized prediction of LoS after TAVI can be achieved.
Evidence-based clinical guidelines generally consider single conditions, and rarely multimorbidity. We developed an evidence-based guideline for a structured care program to manage polypharmacy in multimorbidity by using a realist synthesis to update the German polypharmacy guideline including the following five methods: formal prioritization in focus groups; systematic guideline review of evidence-based multimorbidity/polypharmacy guidelines; evidence search/synthesis and recommendation development; multidisciplinary consent of recommendations; feasibility test of updated guideline. We identified the need for a better description of the target group, decision support, prioritization of medication, consideration of patient preferences and anticholinergic properties, and of healthcare interfaces. We conducted a systematic guideline review of eight guidelines and extracted and synthesized recommendations using the Ariadne principles. We also included 48 systematic reviews. We formulated and agreed upon 34 recommendations for the revised guideline. During the feasibility test, guideline use enabled 57% of GPs to identify problems, leading to medication changes in 49% and self-assessed improvement in 56% of patients. Although 58% of GPs felt that it was too long, 92% recommended it. Polypharmacy should be systematically reviewed at least annually. Patients, family members, and healthcare professionals should monitor and adjust it using prospective process validation, taking into account patient preferences and agreed treatment goals.
Invasive fungal infections are associated with increased mortality in hematological patients. Despite considerable advances in antifungal therapy, the evaluation of suspected treatment failure is a common clinical challenge requiring extensive diagnostic testing to rule out potential causes, such as mixed infections. We present a 64-year-old patient with secondary AML, diabetes mellitus, febrile neutropenia, and sinusitis. While cultures from nasal tissue grew Aspergillus flavus, a microscopic examination of the tissue was suggestive of concomitant mucormycosis. However, fluorescence in situ hybridization (FISH) using specific probes targeting Aspergillus and Mucorales species ruled out mixed infection. This was confirmed by specific qPCR assays amplifying the DNA of Aspergillus, but not of Mucorales. These results provided a rational basis for step-down targeted therapy, i.e., the patient received posaconazole after seven days of calculated dual therapy with liposomal amphotericin B and posaconazole. Despite clinical response to the antifungal therapy, he died due to the progression of the underlying disease within two weeks after diagnosis of fungal infection. Molecular diagnostics applied to tissue blocks may reveal useful information on the etiology of invasive fungal infections, including challenging situations, such as with mixed infections. A thorough understanding of fungal etiology facilitates targeted therapy that may improve therapeutic success while limiting side effects.
Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by the cytochrome P450-(CYP450)-mediated epoxygenation of arachidonic acid. EETs have numerous biological effects on the vascular system, but aspects including their species specificity make their effects on vascular tone controversial. CYP450 enzymes require the 450-reductase (POR) for their activity. We set out to determine the contribution of endothelial CYP450 to murine vascular function using isolated aortic ring preparations from tamoxifen-inducible endothelial cell-specific POR knockout mice (ecPOR−/−). Constrictor responses to phenylephrine were similar between control (CTR) and ecPOR−/− mice. Contrastingly, sensitivity to the thromboxane receptor agonist U46619 and prostaglandin E2 (PGE2) was increased following the deletion of POR. Ex vivo incubation with a non-hydrolyzable EET (14,15-EE-8(Z)-E, EEZE) reversed the increased sensitivity to U46619 to the levels of CTR. EETs had no effect on vascular tone in phenylephrine-preconstricted vessels, but dilated vessels contracted with U46619 or PGE2. As U46619 acts through RhoA-dependent kinase, this system was analyzed. The deletion of POR affected the expression of genes in this pathway and the inhibition of Rho-GTPase with SAR407899 decreased sensitivity to U46619. These data suggest that EET and prostanoid crosstalk at the receptor level and that lack of EET production sensitizes vessels to vasoconstriction via the induction of the Rho kinase system.
Previous studies towards reduced oxygen availability have mostly focused on changes in total mRNA expression, neglecting underlying transcriptional and post-transcriptional events. Therefore, we generated a comprehensive overview of hypoxia-induced changes in total mRNA expression, global de novo transcription, and mRNA stability in monocytic THP-1 cells. Since hypoxic episodes often persist for prolonged periods, we further compared the adaptation to acute and chronic hypoxia. While total mRNA changes correlated well with enhanced transcription during short-term hypoxia, mRNA destabilization gained importance under chronic conditions. Reduced mRNA stability not only added to a compensatory attenuation of immune responses, but also, most notably, to the reduction in nuclear-encoded mRNAs associated with various mitochondrial functions. These changes may prevent the futile production of new mitochondria under conditions where mitochondria cannot exert their full metabolic function and are indeed actively removed by mitophagy. The post-transcriptional mode of regulation might further allow for the rapid recovery of mitochondrial capacities upon reoxygenation. Our results provide a comprehensive resource of functional mRNA expression dynamics and underlying transcriptional and post-transcriptional regulatory principles during the adaptation to hypoxia. Furthermore, we uncover that RNA stability regulation controls mitochondrial functions in the context of hypoxia.
LFA-1 (Lymphocyte function-associated antigen-1) is a heterodimeric integrin (CD11a/CD18) present on the surface of all leukocytes; it is essential for leukocyte recruitment to the site of tissue inflammation, but also for other immunological processes such as T cell activation and formation of the immunological synapse. Absent or dysfunctional expression of LFA-1, caused by mutations in the ITGB2 (integrin subunit beta 2) gene, results in a rare immunodeficiency syndrome known as Leukocyte adhesion deficiency type I (LAD I). Patients suffering from severe LAD I present with recurrent infections of the skin and mucosa, as well as inflammatory symptoms complicating the clinical course of the disease before and after allogeneic hematopoietic stem cell transplantation (alloHSCT); alloHSCT is currently the only established curative treatment option. With this review, we aim to provide an overview of the intrinsic role of inflammation in LAD I.
Activation of the tumor-associated stroma to support tumor growth is a common feature observed in different cancer entities. This principle is exemplified by cancer-associated fibroblasts (CAFs), which are educated by the tumor to shape its development across all stages. CAFs can alter the extracellular matrix (ECM) and secrete a variety of different molecules. In that manner they have the capability to affect activation, survival, proliferation, and migration of other stromal cells and cancer cell themselves. Alteration of the ECM, desmoplasia, is a common feature of breast cancer, indicating a prominent role for CAFs in shaping tumor development in the mammary gland. In this review, we summarize the multiple roles CAFs play in mammary carcinoma. We discuss experimental and clinical strategies to interfere with CAFs function in breast cancer. Moreover, we highlight the issues arising from CAFs heterogeneity and the need for further research to identify CAFs subpopulation(s) that can be targeted to improve breast cancer therapy.
Germany experienced a 6-month second lockdown (November 2020–April 2021) during the COVID-19 pandemic, which included the closure of all physical activity (PA) facilities. The use of online exercise classes (OECs) was promoted by public health and exercise organizations. Using the present cross-sectional online survey, we assess the use of and opinion towards OECs in Germany during the second lockdown. We used contingency tables and the Chi2 test to calculate the frequency of awareness and use of OECs according to PA status, well-being and demographic data, and conducted a binary logistic regression with OEC awareness or use and dichotomized independent predictors. The associations between opinion and activity status, frequency of use, educational attainment, age and body mass index were calculated using Spearman correlations. A total of 993 datasets were analyzed in detail. Of the 785 (79.1%) participants reporting awareness of OECs, 536 tried them, and 262, 188 and 85 used them <1 per week, 1–2 per week and ≥3 per week, respectively. The users were typically active, female participants with poorer mental well-being. The opinions towards OECs varied according to participant characteristics, such as activity status, BMI and age. Overall, regular OEC use was quite limited, and, as such, cannot replace in-person exercise opportunities. Keeping physical activity facilities open and safe must be prioritized in the ongoing pandemic.
Background: Intraoperative blood salvage (IBS) is regarded as an alternative to allogeneic blood transfusion excluding the risks associated with allogeneic blood. Currently, IBS is generally avoided in tumor surgeries due to concern for potential metastasis caused by residual tumor cells in the erythrocyte concentrate.
Methods: The feasibility, efficacy and safety aspects of the new developed Catuvab procedure using the bispecific trifunctional antibody Catumaxomab was investigated in an ex-vivo pilot study in order to remove residual EpCAM positive tumor cells from the autologous erythrocyte concentrates (EC) from various cancer patients, generated by a IBS device.
Results: Tumor cells in intraoperative blood were detected in 10 of 16 patient samples in the range of 69–2.6 × 105 but no residual malignant cells in the final erythrocyte concentrates after Catuvab procedure. IL-6 and IL-8 as pro-inflammatory cytokines released during surgery, were lowered in mean 28-fold and 52-fold during the Catuvab procedure, respectively, whereas Catumaxomab antibody was detected in 8 of 16 of the final EC products at a considerable decreased and uncritical residual amount (37 ng in mean).
Conclusion: The preliminary study results indicate efficacy and feasibility of the new medical device Catuvab allowing potentially the reinfusion of autologous erythrocyte concentrates (EC) produced by IBS device during oncological high blood loss surgery. An open-label, multicenter clinical study on the removal of EpCAM-positive tumor cells from blood collected during tumor surgery using the Catuvab device is initiated to validate these encouraging results.
Hintergrund: Durch COVID-19 kam es weltweit, insbesondere in den ersten Wochen der Pandemie, zu einer Verschiebung und Absage elektiver Operationen in allen chirurgischen Fachdisziplinen. Eine Beschreibung der spezifischen Situation in gefäßchirurgischen Kliniken in Deutschland während dieser Periode ist bislang nicht erfolgt.
Ziel der Arbeit: Zweck der Befragung war die Erfassung der gefäßchirurgischen Leistungserbringung in der Zeit von März 2020 bis Dezember 2020, sowie von logistischen und infrastrukturellen Veränderungen, die sich durch die pandemische Lage ergeben hatten. Hierbei lag der Fokus der Umfrage auf der möglichst realitätsnahen Abbildung der Versorgungssituation anhand der Einschätzung der leitenden Gefäßchirurg*innen.
Material und Methoden: In Zusammenarbeit mit der Deutschen Gesellschaft für Gefäßchirurgie und Gefäßmedizin (DGG) wurde das leitende ärztliche Personal von gefäßchirurgischen Einrichtungen in Deutschland aufgefordert, an der Umfrage teilzunehmen. Die Beantwortung der Fragen erfolgte anonym.
Ergebnisse: Durch COVID-19 und korrespondierende Maßnahmen kam und kommt es zu relevanten Absagen und Verschiebungen von Operationen, Verlust an Kapazitäten und einer gesteigerten Personalbelastung. Es traten im Beobachtungszeitraum verspätete Versorgungen gefäßchirurgischer Krankheitsbilder und ein gehäuftes Auftreten schwererer klinischer Stadien verglichen mit dem entsprechenden Vorjahreszeitraum auf. Betroffen sind alle Versorgungsstufen, größtenteils dauern diese Veränderungen an.
Diskussion: Um der strukturellen Schwächung und den Einschränkungen in der Patientenversorgung zu begegnen, sind klinische Abläufe, Patientenaufklärung und Priorisierung zu optimieren. Neue Konzepte wie z. B. Telemedizin und engmaschigere klinische Kontrolle sind ggf. sinnvoll. Eine erforderliche Infrastruktur für Notfallmanagement (COVID) darf im Alltag nicht die Versorgungsqualität der gefäßchirurgischen Patient*innen negativ beeinflussen.
Neben einer guten Tanztechnik spielt die Körperform von professionellen Tänzer*innen eine entscheidende Rolle. Im klassischen Tanz gilt eine leptosom-grazile Form mit langen Gliedmaßen bei Frauen als ideal. Dementsprechend ist die Prävalenz von Untergewicht hoch. Dabei ist es nicht ausgeschlossen, dass dieses physische Erscheinungsbild mittels Diäten erreicht wird. Auffällig ist eine höhere Prävalenz von Störungen des Essverhaltens und Essstörungen vor allem bei Tänzerinnen im Vergleich zu nicht tanzenden Vergleichsgruppen. Im Zusammenhang mit dem Untergewicht können hormonelle Störungen, wie Amenorrhoe, Osteoporose oder Leistungseinschränkungen auftreten. Daher ist es wichtig, Tänzer*innen regelmäßig zu untersuchen und präventive Maßnahmen zu ergreifen. Ziel der vorliegenden Übersicht ist die Darstellung der Literatur zu diesem Thema.
Background: Orthodontic root resorptions are frequently investigated in small animals, and micro-computed tomography (μCT) enables volumetric comparison. Despite, due to overlapping histograms from dentine and bone, accurate quantification of root resorption is challenging. The present study aims at (i) validating a novel automated approach for tooth segmentation (ATS), (ii) to indicate that matching of contralateral teeth is eligible to assess orthodontic tooth movement (OTM) and root resorption (RR), (iii) and to apply the novel approach in an animal trial performing orthodontic tooth movement.
Methods: The oral apparatus of three female mice were scanned with a μCT. The first molars of each jaw and animal were segmented using ATS (test) and manually (control), and contralateral volumes were compared. Agreement in root volumes and time efficiency were assessed for method validation. In another n = 14 animals, the left first upper molar was protracted for 11 days at 0.5 N, whereas the contralateral molar served as control. Following ATS, OTM and RR were estimated.
Results: ATS was significantly more time efficient compared to the manual approach (81% faster, P < 0.01), accurate (volume differences: − 0.01 ± 0.04 mm3), and contralateral roots had comparable volumes. Protracted molars had significantly lower root volumes (P = 0.03), whereas the amount of OTM failed to reveal linear association with RR (P > 0.05).
Conclusions: Within the limits of the study, it was demonstrated that the combination of ATS and registration of contralateral jaws enables measurements of OTS and associated RR in μCT scans.
The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin β4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies.
Background: Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T cell therapy, has demonstrated efficacy in children and young adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) in two multicenter phase 2 trials (ClinicalTrials.gov, NCT02435849 (ELIANA) and NCT02228096 (ENSIGN)), leading to commercialization of tisagenlecleucel for the treatment of patients up to age 25 years with B-ALL that is refractory or in second or greater relapse.
Methods: A pooled analysis of 137 patients from these trials (ELIANA: n=79; ENSIGN: n=58) was performed to provide a comprehensive safety profile for tisagenlecleucel.
Results: Grade 3/4 tisagenlecleucel-related adverse events (AEs) were reported in 77% of patients. Specific AEs of interest that occurred ≤8 weeks postinfusion included cytokine-release syndrome (CRS; 79% (grade 4: 22%)), infections (42%; grade 3/4: 19%), prolonged (not resolved by day 28) cytopenias (40%; grade 3/4: 34%), neurologic events (36%; grade 3: 10%; no grade 4 events), and tumor lysis syndrome (4%; all grade 3). Treatment for CRS included tocilizumab (40%) and corticosteroids (23%). The frequency of neurologic events increased with CRS severity (p<0.001). Median time to resolution of grade 3/4 cytopenias to grade ≤2 was 2.0 (95% CI 1.87 to 2.23) months for neutropenia, 2.4 (95% CI 1.97 to 3.68) months for lymphopenia, 2.0 (95% CI 1.87 to 2.27) months for leukopenia, 1.9 (95% CI 1.74 to 2.10) months for thrombocytopenia, and 1.0 (95% CI 0.95 to 1.87) month for anemia. All patients who achieved complete remission (CR)/CR with incomplete hematologic recovery experienced B cell aplasia; however, as nearly all responders also received immunoglobulin replacement, few grade 3/4 infections occurred >1 year postinfusion.
Conclusions: This pooled analysis provides a detailed safety profile for tisagenlecleucel during the course of clinical trials, and AE management guidance, with a longer follow-up duration compared with previous reports.
Patients with unilateral hip osteoarthritis show a characteristic gait pattern in which they unload the affected leg and overload the unaffected leg. Information on the gait characteristics of patients with bilateral hip osteoarthritis is very limited. The main purposes of this study were to investigate whether the gait pattern of both legs of patients with bilateral hip osteoarthritis deviates from healthy controls and whether bilateral hip osteoarthritis patients show a more symmetrical joint load compared to unilateral hip osteoarthritis patients. In this prospective study, 26 patients with bilateral hip osteoarthritis, 26 patients with unilateral hip osteoarthritis and 26 healthy controls were included. The three groups were matched for gender, age and walking speed. Patients were scheduled for a unilateral total hip arthroplasty on the more affected/more painful side. All participants underwent a three-dimensional gait analysis. Gait kinematics and gait kinetics of patients and controls were compared using Statistical Parametric Mapping. Corrected for speed, the gait kinematics and kinetics of both legs of patients with bilateral hip osteoarthritis differed from healthy controls. Bilateral patients had symmetrical knee joint loading, in contrast to the asymmetrical knee joint loading in unilateral hip osteoarthritis patients. The ipsilateral leg of the bilateral patients could be included in studies in addition to unilateral hip osteoarthritis patients as no differences were found. Although patients with bilateral hip osteoarthritis show more symmetrical frontal plane knee joint moments, a pathological external knee adduction moment in the second half of stance was present in the ipsilateral leg in patients with unilateral and bilateral hip osteoarthritis. The lateral adjustment of the knee adduction moment may initiate or accelerate progression of degenerative changes in the lateral compartment of the knee.
Um eine gezielte Prophylaxe und Therapie der Urolithiasis zu ermöglichen und um die Rezidivrate zu senken, ist die genaue Kenntnis der Steinzusammensetzung erforderlich. Wegen des hohen Beschaffungspreises sind Röntgendiffraktion und Infrarotspektroskopie nur wenigen Laboratorien vorbehalten, andererseits sollten unspezifisch-chemische Steinanalysenmethoden wegen deren geringeren Sensitivität und Spezifität heute nicht mehr angewendet werden.
In dieser Arbeit wird eine unter dem Mikroskop durchführbare Harnsteinkomponentenanalysenmethode (Harzalith) beschrieben. Es handelt sich um eine mikroskopisch-mikrochemische Harnsteinanalysenmethode. Sie basiert auf der Auswertung mikroskopisch typischer, leicht einprägsamer Farbmuster, die sich je nach Steinzusammensetzung in charakteristischer Weise nach Zugabe des Steinmaterials zum Reagenz innerhalb von Sekunden entwickeln.
Eine Bewertung der mikroskopisch-mikrochemischen Harnsteinkomponentenanalyse gegenüber Röntgendiffraktion, Infrarotspektroskopie und unspezifisch-chemischen Methoden erfolgt anhand von Ergebnissen aus über 10jähriger eigener Anwendungserfahrung.
Folgende Vorteile werden kurz dargestellt:
1. Es werden Steinkomponenten und nicht nur Ionen erfaßt.
2. Es können geringste Steinprobenmengen analysiert werden.
3. Die Ergebnisse der Methode hinsichtlich Richtigkeit, Sensitivität und Spezifität sind in gleicher Größenordnung wie die von Infrarotspektroskopie und Röntgendiffraktion.
4. Die Methode ist einfach zu erlernen, schnell, genau und von leichter Handhabung. Sie ist damit eine echte Alternative gegenüber den anderen apparativ-aufwendigen Harnsteinanalysenverfahren.
Es wird eine neue pH-Indikatormethode zur Harnstoffbestimmung beschrieben. Die Methode zeichnet sich durch gute Präzision und Richtigkeit aus. Der aus 150 Wertepaaren ermittelte Korrelationskoeffizient beträgt mit der DAM-Methode r = 0,9970 und mit der GLDH-Methode r = 0,9971. Die Richtigkeitsversuche charakterisieren die Methode als praxisgerecht. Wegen der schnellen Durchführung ist die Methode auch für Notfall-Laboratorien zu empfehlen.
Der Basismechanismus immunchemischer Reaktionen ist die Antigen-Antikörper-Reaktion. Die nachzuweisenden Substanzen haben in der Bestimmungsreaktion Antigen-Funktion. Die zur Bestimmung der Substanz
im Test eingesetzten Antikörper können polyklonalen oder monoklonalen Ursprungs sein.
Polyklonal gebildete Antikörper werden technisch gewonnen durch die Reinigung der Immunglobulinfraktion immunisierter Tiere. Trotz mehrerer Reinigungsschritte wird vielfach nicht ein spezifischer Antikörper gewonnen, sondern eine heterogene Antikörperfraktion, die neben dem spezifischen Antikörper weitere Antikörper mit unterschiedlicher Affinität Avidität und Spezifität gegen die immunchemisch zu analysierende Substanz enthält.
Die technische Gewinnung monoklonaler Antikörper verläuft im ersten Schritt ähnlich der auf polyklonalem Wege; ein Tier wird immunisiert mit dem immundiagnostisch zu bestimmenden Antigen. Es wird jedoch nachfolgend nicht Blut des immunisierten Tieres zur Antikörpergewinnung entnommen, sondern die MHz. In vitro werden im nächsten Schritt Antigen-sensibilisierte B-Lymphozyten aus der Milz mit Myelomzellen der Maus unter Bildung von Hybridzellen fusioniert. Die Hybridzellen werden selektioniert und diejenigen cloniert, die den gewünschten spezifischen Antikörper bilden. Auf diesem Wege können Antikörper einheitlicher Affinität, Avidität und Spezifität in größeren Mengen gewonnen werden.
Die Vor- und Nachteile poly- und monoklonal gebildeter Antikörper für immunchemische Tests werden dargestellt.
Wir erprobten die Verwendung von Primärgefäßen in einem voll selektiven, patientenorientiert arbeitenden Analysensystem. Unter Routinebedingungen wurden die Veränderungen von 18 Blutbestandteilen (Enzyme, Substrate, Elektrolyte) in Serumproben untersucht, die im Probennahmegefäß nach Zentrifugation längere Zeit über dem Blutkuchen aufbewahrt wurden. Innerhalb von 5 Std. waren nur geringfügige und kaum klinisch relevante Differenzen zu beobachten. Um auch im Einzelfall hämolyse- bzw. diffusionsbedingte Veränderungen von Serumbestandteilen auszuschließen, bemühten wir uns um die Entwicklung eines Ventilfilters, der hinsichtlich seiner Handhabung und Funktionalität Routine-Anforderungen genügt und die Geometrie 'des Primärgefäßes nicht verändert.
Gene therapy has garnered increasing interest over recent decades. Several therapies employing gene transfer mechanisms have been developed, and, of these, adeno-associated virus (AAV) vectors have demonstrated viability for use with in vivo gene therapy. Several AAV-based therapeutics have received regulatory approval in the last few years including those for retinal disease, spinal muscular atrophy or aromatic L-amino acid decarboxylase deficiency. Lately, with the introduction of novel liver-directed AAV vector-based therapeutics for the treatment of haemophilia A and B, gene therapy has attracted significant attention in the hepatology community, with the liver increasingly recognised as a target for gene therapy. However, the introduction of foreign DNA into hepatocytes is associated with a risk of hepatic reactions, with raised ALT (alanine aminotransferase) and AST (aspartate aminotransferase) being – so far – the most commonly reported side effects. The complete mechanisms underlying the ALT flairs remain to be determined and the long-term risks associated with these new treatments is not yet known. The liver community is increasingly being asked to support liver-directed gene therapy to mitigate potential liver associated harm. In this review, we focus on AAV vector-based gene therapy, shedding light on this promising technique and its remarkable success in haemophilia, with a special focus on hepatic complications and their management in daily clinical practice.
Highlights
• High resolution profile of C. pipiens' sugar diet has been obtained using UHPLC-MS.
• Artificial feeding using ornamental plants provides similar sugar profiles as observed in field collected mosquitoes.
• Metabolomic profiling found secondary metabolites and pollutants of anthropogenic use.
Abstract: Culex pipiens (Linnaeus, 1758) mosquitoes search plant sources of sugars to cope with the energetic demand of various physiological processes. The crop as part of the digestive system is devoted to the storage of sugar-based meal obtained from various nectars sources. The profiling of sugars and metabolites in the Culex pipiens’ crop is scarce, and only few studies used Liquid Chromatography – Mass Spectrometry (LC-MS), which provides broad detection for biomonitoring environmental substances and even contaminants in the sugar diet of mosquitoes populations.
Therefore, sugar and metabolite profiling were performed on crops obtained from mosquitoes exposed to plant nectar under laboratory or natural conditions by Ultra High-Performance LC-MS (UHPLC-MS). This method allowed us a precise quantitative and qualitative identification of sugar diet and associated environmental compounds in the crop of the mosquito C. pipiens. Under laboratory condition, mosquitoes were allowed to feed on either glucose solution, commercially-available flowers or field collected flowers. In addition, we collected mosquitoes from the field to compare those crop metabolomes with metabolome patterns occurring after nectar feeding in the lab.
The sugar quantities and quality obtained from the crops of mosquitoes collected in the field were similar to those crops obtained from mosquitoes that fed on commercially-available flowers and from field collected flowers with a limit of detection of 10 μg/L for sucrose, glucose and sucrose. Next to sugar compounds, we identified 2 types of amino acids, 12 natural products, and 9 pesticides.
Next to the diversity of sugar compounds, we could confirm that secondary metabolites and environmental pollutants are typically up taken from floral nectar sources by C. pipiens. The in-depth knowledge on mosquito–plant interactions may inspire the development and further optimization of mosquito trap systems and arboviral surveillance systems.
Quantitative 7gLp(a)- Bestimmungen im Humanserum wurden mit zwei unterschiedlichen immunelektrophoretischenTechniken - der Raketenimmunelektrophorese (RIE) und dem Zonen- Immunelektrophorese- Assay (ZIA) durchge-führt. Ein Vergleich der Ergebnisse beider Methoden zeigte im Bereich von 6 -80 mg l dl eine gute Übereinstimmung(r = 0.9919).Lp(a) -Konzentrationen unter 6 mg /dl können mit der Raketen- Technik nicht mehr quantitativ nachgewiesen werden,da nach Laurell (12) Präzipitathöhen kürzer als 5mm nicht mehr proportional zur Konzentration des Antigens sind.Bei dem ZIA dagegen können Lp(a)-Konzentrationen von 1 -6 mg /dl noch gut und reproduzierbar nachgewiesenwerden.
Highlights
• Microstimulation of visual area V4 improves visual stimulus detection
• Effects of V4 microstimulation extend to the other hemifield
• Microstimulation effects are time dependent and consistent with attention dynamics
Summary
Neuronal activity in visual area V4 is well known to be modulated by selective attention, and there are reports on V4 lesions leading to attentional deficits. However, it remains unclear whether V4 microstimulation can elicit attentional benefits. To test this hypothesis, we performed local microstimulation in area V4 and explored its spatial and time dynamics in two macaque monkeys performing a visual detection task. Microstimulation was delivered via chronically implanted multi-electrode arrays. We found that microstimulation increases average performance by 35% and reduces luminance detection thresholds by −30%. This benefit critically depends on the onset of microstimulation relative to the stimulus, consistent with known dynamics of endogenous attention. These results show that local microstimulation of V4 can improve behavior and highlight the critical role of V4 for attention.
In polarized cells, the multidrug resistance protein MRP2 is localized in the apical plasma membrane, whereas MRP1, another multidrug resistance protein (MRP) family member, is localized in the basolateral membrane. MRP1 and MRP2 are thought to contain an N-terminal region of five transmembrane segments (TMD0) coupled to 2 times six transmembrane segments via an intracellular loop (L0). We previously demonstrated for MRP1 that a mutant lacking TMD0 but still containing L0, called L0ΔMRP1, was functional and routed to the lateral plasma membrane. To investigate the role of the TMD0L0 region of MRP2 in routing to the apical membrane, we generated mutants similar to those made for MRP1. In contrast to L0ΔMRP1, L0ΔMRP2 was associated with an intracellular compartment, most likely endosomes. Co-expression with TMD0, however, resulted in apical localization of L0ΔMRP2 and transport activity. Uptake experiments with vesicles containing L0ΔMRP2 demonstrated that the molecule is able to transport LTC4. An MRP2 mutant without TMD0L0, ΔMRP2, was only core-glycosylated and localized intracellularly. Co-expression of ΔMRP2 with TMD0L0 resulted in an increased protein level of ΔMRP2, full glycosylation of the protein, routing to the apical membrane, and transport activity. Our results suggest that the TMD0 region is required for routing to or stable association with the apical membrane.
The tumor necrosis factor family member Fas ligand (FasL) induces apoptosis in Fas receptor-expressing target cells and is an important cytotoxic effector molecule used by CTL- and NK-cells. In these hematopoietic cells, newly synthesized FasL is stored in specialized secretory lysosomes and only delivered to the cell surface upon activation and target cell recognition. FasL contains an 80-amino acid-long cytoplasmic tail, which includes a proline-rich domain as a bona fide Src homology 3 domain-binding site. This proline-rich domain has been implicated in FasL sorting to secretory lysosomes, and it may also be important for reverse signaling via FasL, which has been described to influence T-cell activation. Here we report the identification of the Src homology 3 domain-containing adaptor protein PSTPIP as a FasL-interacting partner, which binds to the proline-rich domain. PSTPIP co-expression leads to an increased intracellular localization of Fas ligand, thereby regulating extracellular availability and cytotoxic activity of the molecule. In addition, we demonstrate recruitment of the tyrosine phosphatase PTP-PEST by PSTPIP into FasL·PSTPIP·PTP-PEST complexes which may contribute to FasL reverse signaling.
Cyclic AMP analogs containing hydrophobic modification of C(8) at the adenine ring such as 8-(4-chlorophenylthio)-cAMP (8-pCPT-cAMP) and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-methyl-cAMP) can penetrate membranes due to their high lipophilicity and directly activate intracellular cAMP effectors. Therefore, these cAMP analogs have been used in numerous studies, assuming that their effects reflect the consequences of direct activation of cAMP effectors. The present study provides evidence that 8-pCPT-modified cAMP analogs and their corresponding putative hydrolysis products (8-(4-chlorophenylthio)-adenosine (8-pCPT-ado) and 8-(4-chlorophenylthio)-2'-O-methyl-adenosine (8-pCPT-2'-O-methyl-ado)) inhibit the equilibrative nucleoside transporter 1 (ENT1). In PC12 cells, in which nucleoside transport strongly depended on ENT1, 8-pCPT-ado, 8-pCPT-2'-O-methyl-ado, and, to a smaller extent, 8-pCPT-2'-O-methyl-cAMP caused an increase of protein kinase A substrate motif phosphorylation and anti-apoptotic effect by an A(2A) adenosine receptor (A(2A)R)-dependent mechanism. In contrast, the effects of 8-pCPT-cAMP were mainly A(2A)R-independent. In HEK 293 showing little endogenous ENT1-dependent nucleoside transport, transfection of ENT1 conferred A(2A)R-dependent increase in protein kinase A substrate motif phosphorylation. Together, the data of the present study indicate that inhibition of ENT1 and activation of adenosine receptors have to be considered when interpreting the effects of 8-pCPT-substituted cAMP/adenosine analogs.
MutLα is essential for human DNA mismatch repair (MMR). It harbors a latent endonuclease, is responsible for recruitment of process associated proteins and is relevant for strand discrimination. Recently, we demonstrated that the MMR function of MutLα is regulated by phosphorylation of MLH1 at serine (S) 477. In the current study, we focused on S87 located in the ATPase domain of MLH1 and on S446, S456 and S477 located in its linker region. We analysed the phosphorylation-dependent impact of these amino acids on DNA binding, MMR ability and thermal stability of MutLα. We were able to demonstrate that phosphorylation at S87 of MLH1 inhibits DNA binding of MutLα. In addition, we detected that its MMR function seems to be regulated predominantly via phosphorylation of serines in the linker domain, which are also partially involved in the regulation of DNA binding. Furthermore, we found that the thermal stability of MutLα decreased in relation to its phosphorylation status implying that complete phosphorylation might lead to instability and degradation of MLH1. In summary, we showed here, for the first time, a phosphorylation-dependent regulation of DNA binding of MutLα and hypothesized that this might significantly impact its functional regulation during MMR in vivo.
Cadmium-mediated toxicity of cultured proximal tubule (PT) cells is associated with increased production of reactive oxygen species (ROS) and apoptosis. We found that cadmium-dependent apoptosis (Hoechst 33342 and annexin V assays) decreased with prolonged CdCl(2) (10 microM) application (controls: 2.4 +/- 1.6%; 5 h: +5.1 +/- 2.3%, 20 h: +5.7 +/- 2.5%, 48 h: +3.3 +/- 1.0% and 72 h: +2.1 +/- 0.4% above controls), while cell proliferation was not affected. Reduction of apoptosis correlated with a time-dependent up-regulation of the drug efflux pump multidrug resistance P-glycoprotein (mdr1) in cadmium-treated cells ( approximately 4-fold after 72 h), as determined by immunoblotting with the monoclonal antibody C219 and measurement of intracellular accumulation of the fluorescent probe calcein +/- the mdr1 inhibitor PSC833 (0.5 microM). When mdr1 inhibitors (PSC833, cyclosporine A, verapamil) were transiently added to cells with mdr1 up-regulation by pretreatment for 72 h with cadmium, cadmium-induced apoptosis increased significantly and to a percentage similar to that obtained in cells with no mdr1 up-regulation (72-h cadmium: 5.2 +/- 0.9% versus 72-h cadmium + 1-h PSC833: 7.2 +/- 1.4%; p < or = 0.001). Cadmium-induced apoptosis and mdr1 up-regulation depended on ROS, since co-incubation with the ROS scavengers N-acetylcysteine (15 mM) or pyrrolidine dithiocarbamate (0.1 mM) abolished both responses. Moreover, cadmium- and ROS-associated mdr1 up-regulation was linked to activation of the transcription factor NF-kappaB; N-acetylcysteine, pyrrolidine dithiocarbamate, and the IkappaB-alpha kinase inhibitor Bay 11-7082 (20 microM) prevented both, mdr1 overexpression and degradation of the inhibitory NF-kappaB subunit, IkappaB-alpha, induced by cadmium. The data show that 1) cadmium-mediated apoptosis in PT cells is associated with ROS production, 2) ROS increase mdr1 expression by a process involving NF-kappaB activation, and 3) mdr1 overexpression protects PT cells against cadmium-mediated apoptosis. These data suggest that mdr1 up-regulation, at least in part, provides anti-apoptotic protection for PT cells against cadmium-mediated stress.
Requirements for the interaction of mouse Polkappa with ubiquitin and its biological significance
(2008)
Polkappa protein is a eukaryotic member of the DinB/Polkappa branch of the Y-family DNA polymerases, which are involved in the tolerance of DNA damage by replicative bypass. Despite universal conservation through evolution, the precise role(s) of Polkappa in this process has remained unknown. Here we report that mouse Polkappa can physically interact with ubiquitin by yeast two-hybrid screening, glutathione S-transferase pulldown, and immunoprecipitation methods. The association of Polkappa with ubiquitin requires the ubiquitin-binding motifs located at the C terminus of Polkappa. In addition, Polkappa binds with monoubiquitinated proliferating cell nuclear antigen (PCNA) more robustly than with non-ubiquitinated PCNA. The ubiquitin-binding motifs mediate the enhanced association between monoubiquitinated PCNA and Polkappa. The ubiquitin-binding motifs are also required for Polkappa to form nuclear foci after UV radiation. However, the ubiquitin-binding motifs do not affect Polkappa half-life. Finally, we have examined levels of Polkappa expression following the exposure of mouse cells to benzo[a]pyrene-dihydrodiol epoxide or UVB radiation.
The iron-sulfur proteins of the cytochrome bc1 complexes of Schizosaccharomyces pombe and Saccharomyces cerevisiae contain the three amino acid motif RX( downward arrow)(F/L/I)XX(T/S/G)XXXX (downward arrow) that is typical for proteins that are cleaved sequentially in two steps by matrix processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). Despite the presence of this recognition sequence the S. pombe iron-sulfur protein is processed only once during import into mitochondria, whereas the S. cerevisiae protein is processed in two steps. Import of S. pombe iron-sulfur protein in which the putative MIP or MPP recognition sites are eliminated by site-directed mutagenesis and import of iron-sulfur protein into mitochondria from yeast mutants that lack MIP activity indicate that one step processing of the S. pombe iron-sulfur protein is independent of those sites and of MIP activity. Sequencing of the mature protein obtained after import in vitro and of the endogenous iron-sulfur protein isolated from mitochondrial membranes by preparative 2D-electrophoresis shows that MPP recognizes a second site in the presequence and processing occurs between residues 43 and 44. If proline-20 of the S. pombe presequence is changed into a serine, a second cleavage step is induced. Conversely, if serine-24 of the S. cerevisiae presequence is changed to a proline, the first cleavage step that is normally catalyzed by MPP is blocked, causing precursor iron-sulfur protein to accumulate. Together these results indicate that a single amino acid change in the presequence is responsible for one-step processing in S. pombe versus two-step processing in S. cerevisiae.
Respiratory chain complex I contains 8-9 iron-sulfur clusters. In several cases, the assignment of these clusters to subunits and binding motifs is still ambiguous. To test the proposed ligation of the tetranuclear iron-sulfur cluster N5 of respiratory chain complex I, we replaced the conserved histidine 129 in the 75-kDa subunit from Yarrowia lipolytica with alanine. In the mutant strain, reduced amounts of fully assembled but destabilized complex I could be detected. Deamino-NADH: ubiquinone oxidoreductase activity was abolished completely by the mutation. However, EPR spectroscopic analysis of mutant complex I exhibited an unchanged cluster N5 signal, excluding histidine 129 as a cluster N5 ligand.
Stable supercomplexes of bacterial respiratory chain complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) have been isolated as early as 1985 (Berry, E. A., and Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete “respirasome.” Inactivation of the gene for tightly bound cytochrome c552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.
Highlights
• USP32 deubiquitinates the Ragulator complex subunit LAMTOR1 at lysine (K) 20
• LAMTOR1 K20 ubiquitination impairs its binding to the vacuolar H+-ATPase
• USP32 knockout reduces mTORC1 activity and elevates autophagic flux
• Depletion of USP32 in Caenorhabditis elegans inhibits mTOR and induces autophagy
Summary
The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.
Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain–containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show that UBACez binds Lys11-, Lys48-, Lys63-, and Met1-linked ubiquitin chains in vitro, establishing UBACez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBACez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBACez. Our results indicate that UBACez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.
Highlights
• Since there is only a low level of evidence, it is difficult to agree on state-of-the-art standards or to provide recommendations and guidelines.
• The value of combining several monitoring devices for dual or triple guidance must be challenged.
• The principle of fascial plane blocks is suitable to avoid traumatic needle-to-nerve contact. However, local toxicity must be regarded as a possible mechanism for nerve injuries.
• Block procedures might be conducted during sedation or general anesthesia when considering the individual patients' clinical situations and the expertise of the anesthesiologist.
• The quality of ultrasound equipment and education provided by the corresponding anesthesia department is highly relevant
Hypoxia potentiates palmitate-induced pro-inflammatory activation of primary human macrophages
(2015)
Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1β. Although palmitate-induced endoplasmic reticulum stress and nuclear factor κB pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1β gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1β and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation.
Conduct Disorder (CD) is an impairing psychiatric disorder of childhood and adolescence characterized by aggressive and dissocial behavior. Environmental factors such as maternal smoking during pregnancy, socio-economic status, trauma, or early life stress are associated with CD. Although the number of females with CD is rising in Western societies, CD is under-researched in female cohorts. We aimed at exploring the epigenetic signature of females with CD and its relation to psychosocial and environmental risk factors. We performed HpaII sensitive genome-wide methylation sequencing of 49 CD girls and 50 matched typically developing controls and linear regression models to identify differentially methylated CpG loci (tags) and regions. Significant tags and regions were mapped to the respective genes and tested for enrichment in pathways and brain developmental processes. Finally, epigenetic signatures were tested as mediators for CD-associated risk factors. We identified a 12% increased methylation 5’ of the neurite modulator SLITRK5 (FDR = 0.0046) in cases within a glucocorticoid receptor binding site. Functionally, methylation positively correlated with gene expression in lymphoblastoid cell lines. At systems-level, genes (uncorr. P < 0.01) were associated with development of neurons, neurite outgrowth or neuronal developmental processes. At gene expression level, the associated gene-networks are activated perinatally and during early childhood in neocortical regions, thalamus and striatum, and expressed in amygdala and hippocampus. Specifically, the epigenetic signatures of the gene network activated in the thalamus during early childhood correlated with the effect of parental education on CD status possibly mediating its protective effect. The differential methylation patterns identified in females with CD are likely to affect genes that are expressed in brain regions previously indicated in CD. We provide suggestive evidence that protective effects are likely mediated by epigenetic mechanisms impairing specific brain developmental networks and therefore exerting a long-term effect on neural functions in CD. Our results are exploratory and thus, further replication is needed.
Background: The understanding of longitudinal changes in the urinary microbiota of healthy women and its relation to intestinal microbiota is limited.
Methods: From a cohort of 15 premenopausal women without known urogenital disease or current symptoms, we collected catheter urine (CU), vaginal and periurethral swabs, and fecal samples on four visits over six months. Additionally, ten participants provided CU and midstream urine (MU) to assess comparability. Urine was subjected to expanded culture. 16S rRNA gene sequencing was performed on all urine, fecal, and selected vaginal and periurethral samples. Sequence reads were processed (DADA2 pipeline) and analyzed using QIIME 2 and R.
Results: Relative abundances of urinary microbiota were variable over 6–18 months. The degree of intraindividual variability of urinary microbiota was higher than that found in fecal samples. Still, nearly half of the observed beta diversity of all urine samples could be attributed to differences between volunteers (R2 = 0.48, p = 0.001). After stratification by volunteer, time since last sexual intercourse was shown to be a factor significantly contributing to beta diversity (R2 = 0.14, p = 0.001). We observed a close relatedness of urogenital microbial habitats and a clear distinction from intestinal microbiota in the overall betadiversity analysis. Microbiota compositions derived from MU differed only slightly from CU compositions. Within this analysis of low-biomass samples, we identified contaminating sequences potentially stemming from sequencing reagents.
Conclusions: Results from our longitudinal cohort study confirmed the presence of a rather variable individual urinary microbiota in premenopausal women. These findings from catheter urine complement previous observations on temporal dynamics in voided urine. The higher intraindividual variability of urinary microbiota as compared to fecal microbiota will be a challenge for future studies investigating associations with urogenital diseases and aiming at identifying pathogenic microbiota signatures.
The Masquelet technique is used to treat large bone defects; it is a two-stage procedure based on an induced membrane. To improve the induced membrane process, demineralized bone matrix in granular (GDBM) and fibrous form (f-DBM) was tested with and without bone marrow mononuclear cells (BMC) as filling of the membrane against the gold standard filling with syngeneic cancellous bone (SCB). A total of 65 male Sprague–Dawley rats obtained a 5 mm femoral defect. These defects were treated with the induced membrane technique and filled with SCB, GDBM, or f-DBM, with or without BMC. After a healing period of eight weeks, the femurs were harvested and submitted for histological, radiological, and biomechanical analyses. The fracture load in the defect zone was lower compared to SCB in all groups. However, histological analysis showed comparable new bone formation, bone mineral density, and cartilage proportions and vascularization. The results suggest that f-DBM in combination with BMC and the induced membrane technique cannot reproduce the very good results of this material in large, non-membrane coated bone defects, nevertheless it supports the maturation of new bone tissue locally. It can be concluded that BMC should be applied in lower doses and inflammatory cells should be removed from the cell preparation before implantation.
Es wurden 34 polyvalente Immunoglobulinpräparate zur i.m. und i.v. Anwendung verschiedener Hersteller und verschiedener Chargen sowie 9 spezifische Tetanus-Immunglobulinpräparate auf das Vorhandensein von HBsAg-Immunkomplexen untersucht. Möglicherweise vorhandene Immunkomplexe wurden vorher mit der sauren Dissoziationsmethode gespalten. Der anschließende Nachweis von HBsAg erfolgte mit dem von uns modifizierten AUSRIA* II-725-Test der Firma Abbot. Von den polyvalenten Immunglobulinen wurden 22 positiv für HBsAg gefunden. Von den spezifischen Immunglobulinen waren 3 positiv.
Standard values of the upper body posture in healthy adults with special regard to age, sex and BMI
(2023)
In order to classify and analyze the parameters of upper body posture in clinical or physiotherapeutic settings, a baseline in the form of standard values with special regard to age, sex and BMI is required. Thus, subjectively healthy men and women aged 21–60 years were measured in this project. The postural parameters of 800 symptom-free male (n = 397) and female (n = 407) volunteers aged 21–60 years (Ø♀: 39.7 ± 11.6, Ø ♂: 40.7 ± 11.5 y) were studied. The mean height of the men was 1.8 ± 0.07 m, with a mean body weight of 84.8 ± 13.1 kg and an average BMI of 26.0 ± 3.534 kg/m2. In contrast, the mean height of the women was 1.67 ± 0.06 m, with a mean body weight of 66.5 ± 12.7 kg and an average BMI of 23.9 ± 4.6 kg/m2. By means of video rasterstereography, a 3-dimensional scan of the upper back surface was measured when in a habitual standing position. The means or medians, confidence intervals, tolerance ranges, the minimum, 2.5, 25, 50, 75, 97.5 percentiles and the maximum, plus the kurtosis and skewness of the distribution, were calculated for all parameters. Additionally, ANOVA and a factor analyses (sex, BMI, age) were conducted. In both sexes across all age groups, balanced, symmetrical upper body statics were evident. Most strikingly, the females showed greater thoracic kyphosis and lumbar lordosis angles (kyphosis: Ø ♀ 56°, Ø♂ 51°; lordosis: Ø ♀ 49°, Ø♂ 32°) and lumbar bending angles (Ø ♀ 14°, Ø♂ 11°) than the males. The distance between the scapulae was more pronounced in men. These parameters also show an increase with age and BMI, respectively. Pelvic parameters were independent of age and sex. The upper body postures of women and men between the ages of 21 and 60 years were found to be almost symmetrical and axis-conforming with a positive correlation for BMI or age. Consequently, the present body posture parameters allow for comparisons with other studies, as well as for the evaluation of clinical (interim) diagnostics and applications.
We have investigated the role of reactive oxygen species and thiol-oxidizing agents in the induction of cell death and have shown that adenocarcinoma gastric (AGS) cells respond differently to the oxidative challenge according to the signaling pathways activated. In particular, apoptosis in AGS cells is induced via the mitochondrial pathway upon treatment with thiol-oxidizing agents, such as diamide. Apoptosis is associated with persistent oxidative damage, as evidenced by the increase in carbonylated proteins and the expression/activation of DNA damage-sensitive proteins histone H2A.X and DNA-dependent protein kinase. Resistance to hydrogen peroxide is instead associated with Keap1 oxidation and rapid translocation of Nrf2 into the nucleus. Sensitivity to diamide and resistance to hydrogen peroxide are correlated with GSH redox changes, with diamide severely increasing GSSG, and hydrogen peroxide transiently inducing protein-GSH mixed disulfides. We show that p53 is activated in response to diamide treatment by the oxidative induction of the Trx1/p38(MAPK) signaling pathway. Similar results were obtained with another carcinoma cell line, CaCo2, indicating that these findings are not limited to AGS cells. Our data suggest that thiol-oxidizing agents could be exploited as inducers of apoptosis in tumor histotypes resistant to ROS-producing chemotherapeutics.
Thioredoxin 1 and thioredoxin 2 have opposed regulatory functions on hypoxia-inducible factor-1α
(2007)
Hypoxia inducible factor 1 (HIF-1), a key regulator for adaptation to hypoxia, is composed of HIF-1alpha and HIF-1beta. In this study, we present evidence that overexpression of mitochondria-located thioredoxin 2 (Trx2) attenuated hypoxia-evoked HIF-1alpha accumulation, whereas cytosolic thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount. Transactivation of HIF-1 is decreased by overexpression of Trx2 but stimulated by Trx1. Inhibition of proteasomal degradation of HIF-1alpha in Trx2-overexpressing cells did not fully restore HIF-1alpha protein levels, while HIF-1alpha accumulation was enhanced in Trx1-overexpressing cells. Reporter assays showed that cap-dependent translation is increased by Trx1 and decreased by Trx2, whereas HIF-1alpha mRNA levels remained unaltered. These data suggest that thioredoxins affect the synthesis of HIF-1alpha. Trx1 facilitated synthesis of HIF-1alpha by activating Akt, p70S6K, and eIF-4E, known to control cap-dependent translation. In contrast, Trx2 attenuated activities of Akt, p70S6K, and eIF-4E and provoked an increase in mitochondrial reactive oxygen species production. MitoQ, a mitochondria specific antioxidant, reversed HIF-1alpha accumulation as well as Akt activation under hypoxia in Trx2 cells, supporting the notion of translation control mechanisms in affecting HIF-1alpha protein accumulation.
The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of ∼6 μm, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 μm RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.
Resting state fMRI has been employed to identify alterations in functional connectivity within or between brain regions following acute and chronic exposure to Δ9-tetrahydrocannabinol (THC), the psychoactive component in cannabis. Most studies focused a priori on a limited number of local brain areas or circuits, without considering the impact of cannabis on whole-brain network organization. The present study attempted to identify changes in the whole-brain human functional connectome as assessed with ultra-high field (7T) resting state scans of cannabis users (N = 26) during placebo and following vaporization of cannabis. Two distinct data-driven methodologies, i.e. network-based statistics (NBS) and connICA, were used to identify changes in functional connectomes associated with acute cannabis intoxication and history of cannabis use. Both methodologies revealed a broad state of hyperconnectivity within the entire range of major brain networks in chronic cannabis users compared to occasional cannabis users, which might be reflective of an adaptive network reorganization following prolonged cannabis exposure. The connICA methodology also extracted a distinct spatial connectivity pattern of hypoconnectivity involving the dorsal attention, limbic, subcortical and cerebellum networks and of hyperconnectivity between the default mode and ventral attention network, that was associated with the feeling of subjective high during THC intoxication. Whole-brain network approaches identified spatial patterns in functional brain connectomes that distinguished acute from chronic cannabis use, and offer an important utility for probing the interplay between short and long-term alterations in functional brain dynamics when progressing from occasional to chronic use of cannabis.