Quality assurance in immunoassay performance – carbamazepine immunoassay format evaluation and application on surface and waste water

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Carbamazepine (CBZ) is one of the most frequently detected pharmaceuticals in water samples. For the determination of this anthropogenic marker, various immunoassay formats were tested and evaluated in order to identify the most suitable one. For these direct competitive assays, the analyte was labelled with the enzyme horseradish peroxidase (HRP) or alkaline phosphatase (AP), and seven substrates with specific detection properties were used. The quality criteria for the standard curves were fulfilled by all HRP assays and the chemiluminescence AP format. Furthermore, intra- and inter-plate coefficients of variation as a measure of the achievable precision were determined for the samples. The application of the AP assays to surface water was unfeasible due to CBZ concentrations below the quantifiable concentration range. Surface as well as waste water samples could be analyzed with the HRP assays. Here, the HRP assay employing the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine yielded the best results

Chip-based human liver-intestine and liver-skin co-culture : A first step toward systemic repeated dose substance testing in vitro

Systemic repeated dose safety assessment and systemic efficacy evaluation of substances are currently carried out on laboratory animals and in humans due to the lack of predictive alternatives. Relevant international regulations, such as OECD and ICH guidelines, demand long-term testing and oral, dermal, inhalation, and systemic exposure routes for such evaluations. So-called “human-on-a-chip” concepts are aiming to replace respective animals and humans in substance evaluation with miniaturized functional human organisms. The major technical hurdle toward success in this field is the life-like combination of human barrier organ models, such as intestine, lung or skin, with parenchymal organ equivalents, such as liver, at the smallest biologically acceptable scale. Here, we report on a reproducible homeostatic long-term co-culture of human liver equivalents with either a reconstructed human intestinal barrier model or a human skin biopsy applying a microphysiological system. We used a multi-organ chip (MOC) platform, which provides pulsatile fluid flow within physiological ranges at low media-to-tissue ratios. The MOC supports submerse cultivation of an intact intestinal barrier model and an air–liquid interface for the skin model during their co-culture with the liver equivalents respectively at 1/100.000 the scale of their human counterparts in vivo. To increase the degree of organismal emulation, microfluidic channels of the liver–skin co-culture could be successfully covered with human endothelial cells, thus mimicking human vasculature, for the first time. Finally, exposure routes emulating oral and systemic administration in humans have been qualified by applying a repeated dose administration of a model substance – troglitazone – to the chip-based co-cultures.BMBF/0315569/GO-Bio 3: Multi-Organ-Bioreaktoren für die prädiktive Substanztestung im Chipforma

A bibliographic assessment using the degrees of publication method: Medicinal plants from the rural greater Mpigi region (Uganda)

In ethnopharmacological research, many field assessment tools exist. Yet, these miss that critical point of how to really determine which species merit the costly lab studies, e.g., evaluation of traditional use via pharmacological assays and isolation of bioactive secondary metabolites. This gap can be filled with the introduction of a new tool for literature assessment: the Degrees of Publication (DoPs). In this study, its application is illustrated through an extensive bibliographic assessment of 16 medicinal plant species that were recently identified in the Greater Mpigi region of Uganda as being frequently used by local traditional healers in the treatment of medical disorders (namely, Albizia coriaria, Cassine buchananii, Combretum molle, Erythrina abyssinica, Ficus saussureana, Harungana madagascariensis, Leucas calostachys, Microgramma lycopodioides, Morella kandtiana, Plectranthus hadiensis, Securidaca longipedunculata, Sesamum calycinum subsp. angustifolium, Solanum aculeastrum, Toddalia asiatica, Warburgia ugandensis, and Zanthoxylum chalybeum). These species are suspected to be understudied, and a thorough bibliographic assessment has not been previously performed. Thus, the objectives of our study were to undertake a comparative assessment of the degree to which each of these plant species has been studied in the past, including evaluation of the quality of the journals where results were published in. The determination of the DoPs enabled successful assessment of the degrees to which each individual plant species has been studied so far, while also taking into account the methodological “research chain of ethnopharmacology” from ethnobotanical studies (“traditional use”) to pharmacological assays (“bioactivity”) and finally to pharmacognostic research (“structure elucidation”). The significance of a research paper was assessed by determining whether its journal and publishing house were members of the Committee on Publication Ethics (COPE). In total, 634 peer-reviewed publications were reviewed covering the period of 1960–2019, 53.3% of which were published in journals and by publishing houses affiliated with COPE (338 publications). The literature assessment resulted in the identification of understudied plants among the selected species. The majority of plants reviewed have not been sufficiently studied; six species were classified as being highly understudied and three more as being understudied: C. buchananii, F. saussureana, L. calostachys, M. lycopodioides, M. kandtiana, and S. calycinum subsp. angustifolium and A. coriaria, P. hadiensis, and S. aculeastrum, respectively. The newly introduced DoPs are a useful tool for the selection of traditionally used species for future laboratory studies, especially for pharmacological bioassays, isolation procedures, and drug discovery strategies.BMBF, 13FH026IX5, IngenieurNachwuchs 2015: Schutz der Gesundheit durch Einsatz biologischer Fungizide in der Landwirtschaft - Anwendung von Trihydroxy-octadecensäuren (TriOH) als natürliche Pflanzenschutzmittel zur Sicherung einer gesunden Ernährung (OxiLiFungi

Transferring ethnopharmacological results back to traditional healers in rural indigenous communities – The Ugandan greater Mpigi region example

In ethnopharmacology, scientists often survey indigenous communities to identify and collect natural remedies such as medicinal plants that are yet to be investigated pharmacologically in a laboratory setting. The Nagoya Protocol provided international agreements on financial benefit sharing. However, what has yet only been poorly defined in these agreements are the non-financial benefits for local intellectual property right owners, such as traditional healers who originally provided the respective ethnomedicinal information. Unfortunately, ethnopharmacologists still rarely return to local communities. In this video article, the authors present a method for transferring results back to traditional healers in rural indigenous communities, taking the authors’ previous studies among 39 traditional healers in Uganda as an example. The authors’ approach is based on a two-day workshop, and the results are presented as original footage in the video article. The authors’ work demonstrated a successful method for ensuring bidirectional benefit and communication while fostering future scientific and community-work collaborations. The authors believe it is the moral duty of ethnopharmacologists to contribute to knowledge transfer and feedback once a study is completed. The workshop method, as an example for science outreach, might also be regarded as a valuable contribution to research on education theory and science communication

Antiinflammatory Medicinal Plants from the Ugandan Greater Mpigi Region Act as Potent Inhibitors in the COX-2/PGH2 Pathway

Our study investigates 16 medicinal plants via assessment of inhibition of proinflammatory enzymes such as cyclooxygenases (COX). The plants are used by traditional healers in the Greater Mpigi region in Uganda to treat inflammation and related disorders. We present results of diverse in vitro experiments performed with 76 different plant extracts, namely, (1) selective COX-2 and COX-1 inhibitor screening; (2) 15-LOX inhibition screening; (3) antibacterial resazurin assay against multidrug-resistant Staphylococcus aureus, Listeria innocua, Listeria monocytogenes, and Escherichia coli K12; (4) DPPH assay for antioxidant activity; and (5) determination of the total phenolic content (TPC). Results showed a high correlation between traditional use and pharmacological activity, e.g., extracts of 15 out of the 16 plant species displayed significant selective COX-2 inhibition activity in the PGH2 pathway. The most active COX-2 inhibitors (IC50 < 20 µg/mL) were nine extracts from Leucas calostachys, Solanum aculeastrum, Sesamum calycinum subsp. angustifolium, Plectranthus hadiensis, Morella kandtiana, Zanthoxylum chalybeum, and Warburgia ugandensis. There was no counteractivity between COX-2 and 15-LOX inhibition in these nine extracts. The ethyl acetate extract of Leucas calostachys showed the lowest IC50 value with 0.66 µg/mL (COX-2), as well as the most promising selectivity ratio with 0.1 (COX-2/COX-1). The TPCs and the EC50 values for DPPH radical scavenging activity showed no correlation with COX-2 inhibitory activity. This led to the assumption that the mechanisms of action are most likely not based on scavenging of reactive oxygen species and antioxidant activities. The diethyl ether extract of Harungana madagascariensis stem bark displayed the highest growth inhibition activity against S. aureus (MIC value: 13 µg/mL), L. innocua (MIC value: 40 µg/mL), and L. monocytogenes (MIC value: 150 µg/mL). This study provides further evidence for the therapeutic use of the previously identified plants used medicinally in the Greater Mpigi region.BMBF, 13FH066PX5, FHprofUnt 2015: Modifikation von Proteinen bei der technischen Prozessierung von Melasse und Vinasse: Einfluss auf die Produktion von Bioethanol und BiogasDFG, 414051096, Open Access Publizieren 2020 - 2021 / Hochschule NeubrandenburgBMBF, 13FH026IX5, IngenieurNachwuchs 2015: Schutz der Gesundheit durch Einsatz biologischer Fungizide in der Landwirtschaft - Anwendung von Trihydroxy-octadecensäuren (TriOH) als natürliche Pflanzenschutzmittel zur Sicherung einer gesunden Ernährung (OxiLiFungi

Combined Application of Tacrolimus with Cyproconazole, Hymexazol and Novel {2-(3-R-1<i>H</i>-1,2,4-triazol-5-yl)phenyl}amines as Antifungals: <i>In Vitro</i> Growth Inhibition and <i>In Silico</i> Molecular Docking Analysis to Fungal Chitin Deacetylase

Agents with antifungal activity play a vital role as therapeutics in health care, as do fungicides in agriculture. Effectiveness, toxicological profile, and eco-friendliness are among the properties used to select suitable substances. Furthermore, a steady supply of new agents with different modes of action is required to counter the well-known potential of human and phyto-pathogenic fungi to develop resistance against established antifungals. Here, we use an in vitro growth assay to investigate the activity of the calcineurin inhibitor tacrolimus in combination with the commercial fungicides cyproconazole and hymexazol, as well as with two earlier reported novel {2-(3-R-1H-1,2,4-triazol-5-yl)phenyl}amines, against the fungi Aspergillus niger, Colletotrichum higginsianum, Fusarium oxysporum and the oomycete Phytophthora infestans, which are notoriously harmful in agriculture. When tacrolimus was added in a concentration range from 0.25 to 25 mg/L to the tested antifungals (at a fixed concentration of 25 or 50 mg/L), the inhibitory activities were distinctly enhanced. Molecular docking calculations revealed triazole derivative 5, (2-(3-adamantan-1-yl)-1H-1,2,4-triazol-5-yl)-4-chloroaniline), as a potent inhibitor of chitin deacetylases (CDA) of Aspergillus nidulans and A. niger (AnCDA and AngCDA, respectively), which was stronger than the previously reported polyoxorin D, J075-4187, and chitotriose. The results are discussed in the context of potential synergism and molecular mode of action

Targeting ESKAPE pathogens with anti-infective medicinal plants from the Greater Mpigi region in Uganda

Antibiotic resistance poses one of the greatest threats to global health today; conventional drug therapies are becoming increasingly inefficacious and limited. We identified 16 medicinal plant species used by traditional healers for the treatment of infectious and inflammatory diseases in the Greater Mpigi region of Uganda. Extracts were evaluated for their ability to inhibit growth of clinical isolates of multidrug-resistant ESKAPE pathogens. Extracts were also screened for quorum quenching activity against S. aureus, including direct protein output assessment (δ-toxin), and cytotoxicity against human keratinocytes (HaCaT). Putative matches of compounds were elucidated via LC–FTMS for the best-performing extracts. These were extracts of Zanthoxylum chalybeum (Staphylococcus aureus: MIC: 16 μg/mL; Enterococcus faecium: MIC: 32 μg/mL) and Harungana madagascariensis (S. aureus: MIC: 32 μg/mL; E. faecium: MIC: 32 μg/mL) stem bark. Extracts of Solanum aculeastrum root bark and Sesamum calycinum subsp. angustifolium leaves exhibited strong quorum sensing inhibition activity against all S. aureus accessory gene regulator (agr) alleles in absence of growth inhibition (IC50 values: 1–64 μg/mL). The study provided scientific evidence for the potential therapeutic efficacy of these medicinal plants in the Greater Mpigi region used for infections and wounds, with 13 out of 16 species tested being validated with in vitro studies.BMBF, 13FH026IX5, IngenieurNachwuchs 2015: Schutz der Gesundheit durch Einsatz biologischer Fungizide in der Landwirtschaft - Anwendung von Trihydroxy-octadecensäuren (TriOH) als natürliche Pflanzenschutzmittel zur Sicherung einer gesunden Ernährung (OxiLiFungi)DFG, 414051096, Open Access Publizieren 2020 - 2021 / Hochschule Neubrandenbur

Pharmacological Assessment of the Antiprotozoal Activity, Cytotoxicity and Genotoxicity of Medicinal Plants Used in the Treatment of Malaria in the Greater Mpigi Region in Uganda

We investigated the potential antimalarial and toxicological effects of 16 medicinal plants frequently used by traditional healers to treat malaria, fever, and related disorders in the Greater Mpigi region in Uganda. Species studied were Albizia coriaria, Cassine buchananii, Combretum molle, Erythrina abyssinica, Ficus saussureana, Harungana madagascariensis, Leucas calostachys, Microgramma lycopodioides, Morella kandtiana, Plectranthus hadiensis, Securidaca longipedunculata, Sesamum calycinum subsp. angustifolium, Solanum aculeastrum, Toddalia asiatica, Warburgia ugandensis, and Zanthoxylum chalybeum. In addition, the traditional healers indicated that P. hadiensis is used as a ritual plant to boost fertility and prepare young women and teenagers for motherhood in some Ugandan communities where a high incidence of rapidly growing large breast masses in young female patients was observed (not necessarily breast cancer). We present results from various in vitro experiments performed with 56 different plant extracts, namely, 1) an initial assessment of the 16 species regarding their traditional use in the treatment of malaria by identifying promising plant extract candidates using a heme biocrystallization inhibition library screen; 2) follow-up investigations of antiprotozoal effects of the most bioactive crude extracts against chloroquine-resistant P. falciparum K1; 3) a cytotoxicity counterscreen against human MRC-5SV2 lung fibroblasts; 4) a genotoxicity evaluation of the extract library without and with metabolic bioactivation with human S9 liver fraction; and 5) an assessment of the mutagenicity of the ritual plant P. hadiensis. A total of seven extracts from five plant species were selected for antiplasmodial follow-up investigations based on their hemozoin formation inhibition activity in the heme biocrystallization assay. Among other extracts, an ethyl acetate extract of L. calostachys leaves exhibited antiplasmodial activity against P. falciparum K1 (IC50 value: 5.7 µg/ml), which was further characterized with a selectivity index of 2.6 (CC50 value: 14.7 µg/ml). The experiments for assessment of potential procarcinogenic properties of plant extracts via evaluation of in vitro mutagenicity and genotoxicity indicated that few extracts cause mutations. The species T. asiatica showed the most significant genotoxic effects on both bacterial test strains (without metabolic bioactivation at a concentration of 500 µg/plate). However, none of the mutagenic extracts from the experiments without metabolic bioactivation retained their genotoxic activity after metabolic bioactivation of the plant extract library through pre-incubation with human S9 liver fraction. While this study did not show that P. hadiensis has genotoxic properties, it did provide early stage support for the therapeutic use of the medicinal plants from the Greater Mpigi region.BMBF, 13FH026IX5, IngenieurNachwuchs 2015: Schutz der Gesundheit durch Einsatz biologischer Fungizide in der Landwirtschaft - Anwendung von Trihydroxy-octadecensäuren (TriOH) als natürliche Pflanzenschutzmittel zur Sicherung einer gesunden Ernährung (OxiLiFungi)BMBF, 13FH066PX5, FHprofUnt 2015: Modifikation von Proteinen bei der technischen Prozessierung von Melasse und Vinasse: Einfluss auf die Produktion von Bioethanol und BiogasDFG, 414051096, Open Access Publizieren 2020 - 2021 / Hochschule Neubrandenbur

Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine.

DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine