Entwicklung und Anwendung neuer Verfahren zur Untersuchung der Beteiligung von Cathepsin E und D am MHC II-Stoffwechsel

The present work aims to contribute to the understanding of differences in biochemical characteristics of cathepsin E and D and their possible functional role in MHC II pathway. More specifically, the project intended to develop and characterize new tools for studying these proteases that are very similar in their enzymatic characteristics. For specific determination of cathepsin E and D activity an assay combining a new monospecific CatE antibody and substrate Mca-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 [where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl] is developed. This substrate is digested by both proteinases and therefore can be used to detect the total aspartic proteinase activity (TAPA) in biological samples. After depletion of CatE by immunoprecipitation, the remaining activity is due to CatD, and the decrease of activity can be assigned to CatE. This assay distinguishes between the activities of enzymatically similar proteinases CatE and CatD and therefore can be used in studies aimed at understanding the involvement of these enzymes in antigen processing and presentation. Moreover, a detailed substrate profiling of cathepsin E and D was performed. It was found that presence of basic residues i.e. R, K and H especially at position P3 is preferred by cathepsin E. Unfortunately we were unable to identify exclusive substrate for CatE or D. However, the detailed specificity profiling of cathepsin E and D led to the identification of preferable residues at different positions that can help in designing of specific substrates or selective inhibitors for cathepsin E or D. Additionally, new cell permeable aspartic protease inhibitors were synthesized by conjugating pepstatin A with well-known cell-penetrating peptides (CPPs). To achieve this, the most frequently used CPPs, namely pAntp(43-58) (penetratin), Tat(49-60), and 9-mer of L-arginine (R9) were synthesized followed by coupling pepstatin A to the peptides. The enzyme inhibition properties of these bioconjugates and their cellular uptake into MCF7 (human breast cancer cell line), Boleths (EBV-transformed B cell line) and dendritic cells (DC) was studied. It was found that the bioconjugate PepA-penetratin (PepA-P) was the most efficient cell-permeable aspartic protease inhibitor in comparison to PepA. Additionally, we found that PepA-P efficiently inhibited the tetanus toxoid C-fragment processing in peripheral blood mononuclear cells (PBMC), primary DC and in primary B cells. This inhibition of tetanus toxoid C-fragment processing by PepA-P clearly implicates the role of aspartic proteinases in antigen processing.Mit der vorliegenden Arbeit soll ein Beitrag zum besseren Verständnis der Unterschiede von Cathepsin E und D in ihren biochemischen Charakteristika sowie ihrer möglichen Rolle im MHC-II-Stoffwechsel geleistet werden. Hierfür wurden neue Verfahren zur Untersuchung dieser in ihren enzymatischen Eigenschaften sehr ähnlichen Proteasen entwickelt und charakterisiert. Zur spezifischen Bestimmung der Aktivität von Cathepsin E und D wurde ein neuer Test entwickelt, der auf neuen monospezifischen CatE-Antikörpern und dem Substrat Mca-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 (Mca: (7-Methoxycoumarin-4-yl)acetyl; Dnp: Dinitrophenyl) basiert. Das Substrat wird von beiden Proteinasen gespalten und kann deshalb zur Messung der Gesamtaktivität der Aspartatproteasen (TAPA) in biologischen Proben benutzt werden. Nach Abtrennung von CatE durch Immunpräzipitation ist CatD für die Restaktivität verantwortlich, während die Aktivitätsabnahme CatE zugeschrieben werden kann. Dieser Test unterscheidet zwischen den Enzymaktivitäten der enzymatisch ähnlichen Proteinasen CatE und CatD und kann deshalb zur Untersuchung der Beteiligung dieser Enzyme an der Antigenprozessierung und –präsentation benutzt werden. Weiterhin wurde ein detailliertes Substratprofil von Cathepsin E und D erstellt. Dabei zeigte sich, dass Substrate mit basischen Resten wie R, K und H, vor allem an Position P3, von Cathepsin E besser gespalten werden als von Cathepsin D. Leider gelang es nicht, ein ausschließlich von CatE oder CatD spaltbares Substrat zu finden. Durch die detaillierte Untersuchung der Spezifitäten der Cathepsine E und D konnten jedoch an verschiedenen Positionen Aminosäurereste identifiziert werden, die jeweils bevorzugt von einem der beiden Enzyme gespalten werden und somit helfen könnten, spezifische Substrate oder selektive Inhibitoren für Cathepsin E und D zu entwickeln. Zusätzlich wurden durch Kombination von Pepstatin A mit bekannten zellpenetrierenden Peptiden (CPPs) neue zellpermeable Aspartatproteaseinhibitoren synthetisiert. Hierfür wurden die meistbenutzten CPPs pAntp(43-58) (Penetratin), Tat(49-60) und ein Nonamer von L-Arginin (R9) synthetisiert und Pepstatin A daran gekoppelt. Bei der Untersuchung der Inhibitionseigenschaften dieser Biokonjugate sowie ihrer Aufnahme in MCF7 (humane Brustkrebs-Zelllinie), Boleth (EBV-transformierte B-Zelllinie) und Dendritische Zellen (DC) erwies sich das Biokonjugat Pepstatin-A-Penetratin (PepA-P) als der im Vergleich zu Pepstatin A effizienteste zellpermeable Aspartatproteaseinhibitor. Außerdem konnten wir zeigen, dass PepA-P die Prozessierung des Tetanustoxin-C-Fragments in Mononukleären Zellen des peripheren Bluts (PBMC), primären Dendritischen Zellen und primären B-Zellen effizient inhibiert. Diese Hemmung der C Fragment-Prozessierung durch PepA-P impliziert deutlich eine Rolle der Aspartatproteasen bei der Antigenprozessierung

Can contracted out health facilities improve access, equity, and quality of maternal and newborn health services? evidence from Pakistan.

BACKGROUND: The case of contracting out government health services to non-governmental organizations (NGOs) has been weak for maternal, newborn, and child health (MNCH) services, with documented gains being mainly in curative services. We present an in-depth assessment of the comparative advantages of contracting out on MNCH access, quality, and equity, using a case study from Pakistan. METHODS: An end-line, cross-sectional assessment was conducted of government facilities contracted out to a large national NGO and government-managed centres serving as controls, in two remote rural districts of Pakistan. Contracting out was specific for augmenting MNCH services but without contractual performance incentives. A household survey, a health facility survey, and focus group discussions with client and spouses were used for assessment. RESULTS: Contracted out facilities had a significantly higher utilization as compared to control facilities for antenatal care, delivery, postnatal care, emergency obstetric care, and neonatal illness. Contracted facilities had comparatively better quality of MNCH services but not in all aspects. Better household practices were also seen in the district where contracting involved administrative control over outreach programs. Contracting was also faced with certain drawbacks. Facility utilization was inequitably higher amongst more educated and affluent clients. Contracted out catchments had higher out-of-pocket expenses on MNCH services, driven by steeper transport costs and user charges for additional diagnostics. Contracting out did not influence higher MNCH service coverage rates across the catchment. Physical distances, inadequate transport, and low demand for facility-based care in non-emergency settings were key client-reported barriers. CONCLUSION: Contracting out MNCH services at government health facilities can improve facility utilization and bring some improvement in quality of services. However, contracting out of health facilities is insufficient to increase service access across the catchment in remote rural contexts and requires accompanying measures for demand enhancement, transportation access, and targeting of the more disadvantaged clientele

Lipid metabolism in cancer cells under metabolic stress

Cancer cells are often exposed to a metabolically challenging environment with scarce availability of oxygen and nutrients. This metabolic stress leads to changes in the balance between the endogenous synthesis and exogenous uptake of fatty acids, which are needed by cells for membrane biogenesis, energy production and protein modification. Alterations in lipid metabolism and, consequently, lipid composition have important therapeutic implications, as they affect the survival, membrane dynamics and therapy response of cancer cells. In this article, we provide an overview of recent insights into the regulation of lipid metabolism in cancer cells under metabolic stress and discuss how this metabolic adaptation helps cancer cells thrive in a harsh tumour microenvironment.status: publishe

Service quality in contracted facilities.

PURPOSE: The purpose of this paper is to explore the readiness of contracted and non-contracted first-level healthcare facilities in Pakistan to deliver quality maternal and neonatal health (MNH) care. A balanced scorecard (BSC) was used as the assessment framework. DESIGN/METHODOLOGY/APPROACH: Using a cross-sectional study design, two rural health centers (RHCs) contracted out to Aga Khan Health Service, Pakistan were compared with four government managed RHCs. A BSC was designed to assess RHC readiness to deliver good quality MNH care. In total 20 indicators were developed, representing five BSC domains: health facility functionality, service provision, staff capacity, staff and patient satisfaction. Validated data collection tools were used to collect information. Pearson χ2, Fisher\u27s Exact and the Mann-Whitney tests were applied as appropriate to detect significant service quality differences among the two facilities. FINDINGS: Contracted facilities were generally found to be better than non-contracted facilities in all five BSC domains. Patients\u27 inclination for facility-based delivery at contracted facilities was, however, significantly higher than non-contracted facilities (80 percent contracted vs 43 percent non-contracted, p=0.006). PRACTICAL IMPLICATIONS: The study shows that contracting out initiatives have the potential to improve MNH care. ORIGINALITY/VALUE: This is the first study to compare MNH service delivery quality across contracted and non-contracted facilities using BSC as the assessment framework

Correction to: Anthropometric and metabolic indices in assessment of type and severity of dyslipidemia

Abstract After the publication of this work [1] an error was noticed in one of the formulas

Population-level median cycle threshold (Ct) values for asymptomatic COVID-19 cases can predict the trajectory of future cases.

BackgroundRecent studies indicate that the population-level SARS-CoV-2 cycle threshold (Ct) values can inform the trajectory of the pandemic. The presented study investigates the potential of Ct values in predicting the future of COVID-19 cases. We also determined whether the presence of symptoms could change the correlation between Ct values and future cases.MethodsWe examined the individuals (n = 8660) that consulted different sample collection points of a private diagnostic center in Pakistan for COVID-19 testing between June 2020 and December 2021. The medical assistant collected clinical and demographic information. The nasopharyngeal swab specimens were taken from the study participants and real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect SARS-CoV-2 in these samples.ResultsWe observed that median Ct values display significant temporal variations, which show an inverse relationship with future cases. The monthly overall median Ct values negatively correlated with the number of cases occurring one month after specimen collection (r = -0.588, p ConclusionsDecreasing population-level median Ct values for asymptomatic COVID-19 cases appear to be a leading indicator for predicting future COVID-19 cases