A sensitive LC-MS/MS method for isomer separation and quantitative determination of 51 pyrrolizidine alkaloids and two tropane alkaloids in cow's milk

1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials;however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid-liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 mu g/L and of 0.009 to 0.123 mu g/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSDr) values below 15% at concentration levels of 0.05 and 0.50 mu g/L and below 8% at a concentration level of 3.00 mu g/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected

A Randomized Trial of Nighttime Physician Staffing in an Intensive Care Unit

Background Increasing numbers of intensive care units (ICUs) are adopting the practice of nighttime intensivist staffing despite the lack of experimental evidence of its effectiveness. Methods We conducted a 1-year randomized trial in an academic medical ICU of the effects of nighttime staffing with in-hospital intensivists (intervention) as compared with nighttime coverage by daytime intensivists who were available for consultation by telephone (control). We randomly assigned blocks of 7 consecutive nights to the intervention or the control strategy. The primary outcome was patients’ length of stay in the ICU. Secondary outcomes were patients’ length of stay in the hospital, ICU and in-hospital mortality, discharge disposition, and rates of readmission to the ICU. For length-of-stay outcomes, we performed time-to-event analyses, with data censored at the time of a patient’s death or transfer to another ICU. Results A total of 1598 patients were included in the analyses. The median Acute Physiology and Chronic Health Evaluation (APACHE) III score (in which scores range from 0 to 299, with higher scores indicating more severe illness) was 67 (interquartile range, 47 to 91), the median length of stay in the ICU was 52.7 hours (interquartile range, 29.0 to 113.4), and mortality in the ICU was 18%. Patients who were admitted on intervention days were exposed to nighttime intensivists on more nights than were patients admitted on control days (median, 100% of nights [interquartile range, 67 to 100] vs. median, 0% [interquartile range, 0 to 33]; P\u3c0.001). Nonetheless, intensivist staffing on the night of admission did not have a significant effect on the length of stay in the ICU (rate ratio for the time to ICU discharge, 0.98; 95% confidence interval [CI], 0.88 to 1.09; P=0.72), ICU mortality (relative risk, 1.07; 95% CI, 0.90 to 1.28), or any other end point. Analyses restricted to patients who were admitted at night showed similar results, as did sensitivity analyses that used different definitions of exposure and outcome. Conclusions In an academic medical ICU in the United States, nighttime in-hospital intensivist staffing did not improve patient outcomes. (Funded by University of Pennsylvania Health System and others; ClinicalTrials.gov number, NCT01434823.

The NFκB/STAT3/PD-L1 network and its impact on the tumor aggressiveness of TERT promoter wild-type versus mutated Glioblastoma

Glioblastoma multiforme (GBM) zeichnet sich durch eine Prognose von nur 15 Monaten aus und ist demnach der bösartigste und außerdem der häufigste Gehirntumor in Erwachsenen. Reakivierung des telomerase reverse transcriptase (TERT) Gens geht oft mit Reaktivierung der Telomerase sowie erhöhter Tumoraggressivität einher. 80% der GBM liegen sogenannte aktivierende TERT Promoter Mutationen zugrunde. Neben ihrer Funktion in der Chromosomenverlängerung, hat Telomerase noch viele anderen Funktionen. Unter anderem spielt TERT in der Aktivierung verschiedener Signalwege, wie beispielsweise die nuclear factor kappa B (NFκB) Kaskade, eine Rolle. Ist die NFκB Achse wiederum aktiviert, stimuliert diese schließlich die signal transducer and activator of transcription 3 (STAT3) Kaskade. Des Weiteren exprimieren 88% aller GBM Fälle den Immunregulator programmed death ligand 1 (PD L1). Davon ausgehend beschäftigt sich diese Studie mit den neu definierten Funktionen von TERT und deren Zusammenhang mit der vermutlich erhöhten Tumoraggressivität in TERT Promoter mutierten (MUT) GBM. Neun GBM Zelllinien wurden auf etwaige Unterschiede bezüglich TERT Expressionslevels in Bezug auf ihren TERT Promoter Status analysiert. Außerdem wurden die Expressionsmuster der entsprechenden NFκB, STAT3 und PD L1 Signalwege getestet. Dementsprechend wurde QNZ oder Bay 11 082 als Inhibitor des NFκB Signalweges eingesetzt. Der STAT3 Signalachse wurde jedoch mit WP1066 oder STATTIC inhibiert. Schlussendlich wurden die inhibitorischen Effekte der Chemotherapeutika getestet. mRNA und protein Expression wurde durch Western Blot und quantitative real time PCR getestet und die TERT Promoter und Telomerase Aktivität wurde durch Luciferase Reporter Assays und TRAP Assays analysiert. Unsere Resultate wurden abschließend zusätzlich immunohistochemisch überprüft. GBM Zelllininen mit einem MUT TERT Promoter exprimierten signifikant mehr TERT mRNA und Protein. Die PD L1 Expression war jedoch besonders in den Wildtyp (WT) Modellen erhöht. Die MUT Tumoren hatten außerdem ein auffallend hohes Toleranzlevel gegenüber den NFκB und STAT3 Inhibitoren. Überraschenderweise führte Blockade der NFκB oder der STAT3 Kaskade außerdem zur gleichzeitigen Inhibierung des jeweilig anderen Signalweges. Der TERT Promoter Status hat großen Einfluss auf die Inhibition des NFκB und STAT3 Signalachsen. Besonders eine Blockade beider Signalwege könnte zukünftige Therapiemöglichkeit darstellen.Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults, characterized by a poor prognosis of about 15 months. Several genomic alterations including reactivation of telomerase reverse transcriptase (TERT) are responsible for telomerase reactivation GBM aggressiveness. In this respect, activating point mutations within the TERT promoter are detected in approximately 80% of GBM samples. Recent studies demonstrated that telomerase is not only restricted to chromosomal elongation, but may also have a broad variety of non-canonical functions. Accordingly, TERT promotes several signaling pathways including the nuclear factor (NFκB) axis, which in turn promotes activation of the signal transducer and activator of transcription 3 (STAT3) cascade. Moreover, 88% of all GBM cases are characterized by overexpression of the immune-regulator programmed death ligand 1 (PD-L1). Based on this knowledge, the present thesis focused on the association between non-canonical functions of TERT and suspected higher tumor aggressiveness of TERT promoter mutated (MUT) GBM. This study analyzed differences in endogenous TERT expression levels of nine GBM cell culture models according to their TERT promoter mutation status. Furthermore, the expression profiles of the NFκB, STAT3 as well as PD-L1 signaling cascades were tested. Accordingly, the NFκB pathway was inhibited using QNZ or Bay-11-082, while STAT3 signaling was blocked with WP1066 or STATTIC. The inhibitory potential of these drugs towards their targets and connected pathways as well as towards telomerase reactivation was tested. mRNA and protein expression was analyzed by Western blots and quantitative real-time PCR, respectively. TERT promoter and telomerase activity was investigated performing luciferase reporter assays and TRAP assays, respectively. Finally, levels of PD-L1 and phosphorylated STAT3 were additionally evaluated in patient-derived tissue samples by immunohistochemistry analyses. TERT promoter MUT cell lines were characterized by significantly higher TERT mRNA and protein levels. On the contrary, PD-L1 expression levels were distinctly increased in the wild-type (WT) as compared to MUT GBM cells. Furthermore, the results revealed a considerably more pronounced sensitivity towards NFκB and STAT3 pathway inhibitors in GBM harboring TERT promoter mutations than in WT cell lines. Surprisingly, blockade of either NFκB or STAT3 pathway resulted in concomitant inhibition of the respective other pathway as well. The inhibitory potential on the NFκB as well as on the STAT3 pathway is highly dependent on the TERT promoter mutation status in GBM. Inhibition of both pathways might represent promising therapeutic targets

Lysosomal Sequestration Impairs the Activity of the Preclinical FGFR Inhibitor PD173074

Knowledge of intracellular pharmacokinetics of anticancer agents is imperative for understanding drug efficacy as well as intrinsic and acquired cellular resistance mechanisms. However, the factors driving subcellular drug distribution are complex and poorly understood. Here, we describe for the first time the intrinsic fluorescence properties of the fibroblast growth factor receptor inhibitor PD1703074 as well as utilization of this physicochemical feature to investigate intracellular accumulation and compartmentalization of this compound in human lung cancer cells. Cell-free PD173074 fluorescence, intracellular accumulation and distribution were investigated using analytical chemistry and molecular biology approaches. Analyses on a subcellular scale revealed selective drug accumulation in lysosomes. Coincubation with inhibitors of lysosomal acidification strongly enhanced PD173074-mediated fibroblast growth factor receptor (FGFR) inhibition and cytotoxicity. In conclusion, intrinsic fluorescence enables analysis of molecular factors influencing intracellular pharmacokinetics of PD173074. Lysosome-alkalinizing agents might represent candidates for rational combination treatment, preventing cancer cell-intrinsic PD173074 resistance based on lysosomal trapping

Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes

Coproheme decarboxylases (ChdC) catalyze the hydrogen peroxide-mediated conversion of coproheme to heme <i>b</i>. This work compares the structure and function of wild-type (WT) coproheme decarboxylase from Listeria monocytogenes and its M149A, Q187A, and M149A/Q187A mutants. The UV–vis, resonance Raman, and electron paramagnetic resonance spectroscopies clearly show that the ferric form of the WT protein is a pentacoordinate quantum mechanically mixed-spin state, which is very unusual in biological systems. Exchange of the Met149 residue to Ala dramatically alters the heme coordination, which becomes a 6-coordinate low spin species with the amide nitrogen atom of the Q187 residue bound to the heme iron. The interaction between M149 and propionyl 2 is found to play an important role in keeping the Q187 residue correctly positioned for closure of the distal cavity. This is confirmed by the observation that in the M149A variant two CO conformers are present corresponding to open (A₀) and closed (A₁) conformations. The CO of the latter species, the only conformer observed in the WT protein, is H-bonded to Q187. In the absence of the Q187 residue or in the adducts of all the heme <i>b</i> forms of ChdC investigated herein (containing vinyls in positions 2 and 4), only the A₀ conformer has been found. Moreover, M149 is shown to be involved in the formation of a covalent bond with a vinyl substituent of heme <i>b</i> at excess of hydrogen peroxide

Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer

Abstract Background Studying the intracellular distribution of pharmacological agents, including anticancer compounds, is of central importance in biomedical research. It constitutes a prerequisite for a better understanding of the molecular mechanisms underlying drug action and resistance development. Hyperactivated fibroblast growth factor receptors (FGFRs) constitute a promising therapy target in several types of malignancies including lung cancer. The clinically approved small-molecule FGFR inhibitor nintedanib exerts strong cytotoxicity in FGFR-driven lung cancer cells. However, subcellular pharmacokinetics of this compound and its impact on therapeutic efficacy remain obscure. Methods 3-dimensional fluorescence spectroscopy was conducted to asses cell-free nintedanib fluorescence properties. MTT assay was used to determine the impact of the lysosome-targeting agents bafilomycin A1 and chloroquine combined with nintedanib on lung cancer cell viability. Flow cytometry and live cell as well as confocal microscopy were performed to analyze uptake kinetics as well as subcellular distribution of nintedanib. Western blot was conducted to investigate protein expression. Cryosections of subcutaneous tumor allografts were generated to detect intratumoral nintedanib in mice after oral drug administration. Results Here, we report for the first time drug-intrinsic fluorescence properties of nintedanib in living and fixed cancer cells as well as in cryosections derived from allograft tumors of orally treated mice. Using this feature in conjunction with flow cytometry and confocal microscopy allowed to determine cellular drug accumulation levels, impact of the ABCB1 efflux pump and to uncover nintedanib trapping into lysosomes. Lysosomal sequestration - resulting in an organelle-specific and pH-dependent nintedanib fluorescence - was identified as an intrinsic resistance mechanism in FGFR-driven lung cancer cells. Accordingly, combination of nintedanib with agents compromising lysosomal acidification (bafilomycin A1, chloroquine) exerted distinctly synergistic growth inhibitory effects. Conclusion Our findings provide a powerful tool to dissect molecular factors impacting organismal and intracellular pharmacokinetics of nintedanib. Regarding clinical application, prevention of lysosomal trapping via lysosome-alkalization might represent a promising strategy to circumvent cancer cell-intrinsic nintedanib resistance

Pyrrolizidine alkaloids and tropane alkaloids in milk samples from individual dairy farms of the German federal states of Bavaria and Schleswig-Holstein

1,2-Dehydro-pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO) and tropane alkaloids (TA), are toxic plant metabolites. If plant material, containing these toxins, is present in the feed of dairy cows these toxins can be transferred into milk. Here, milk was sampled directly from dairy farms in the German federal states of Bavaria and Schleswig-Holstein in 2020–2022 in order to investigate a possible contamination of milk at the production stage. In total, 228 milk samples were analysed for 54 PA/PANO and two TA by a sensitive LC–ESI-MS/MS method. In addition, a subset of milk samples (n = 85) was independently analysed for TA by a cooperating laboratory for verification. PA/PANO were found in 26 samples (11%) with a low median sum content of the contaminated samples of 0.024 µg/L. The highest level of contamination was 5.6 µg/L. Senecionine-, lycopsamine- and heliotrine-type PA/PANO were detected. In four samples (1.8%), atropine was determined up to 0.066 µg/L. The toxin levels in the milk samples hardly contributed to the total daily exposure. These data are first-time results on contamination rates and levels occurring in milk from individual dairy farms, based on a large sample number.</p