Benzo[c]phenanthridine alkaloids are known for their stabilizing effects on non-canonical DNA structures, particularly G-quadruplexes (G4s). In this study, the interaction of fagaronine, a rare benzo[c]phenanthridine alkaloid, with several DNA structures (including B-DNA, parallel, antiparallel and hybrid G4s) is studied using molecular fluorescence and circular dichroism (CD) spectroscopy. It has been found that fagaronine significantly enhances the stability of all tested G4 conformations. Furthermore, a study by NMR spectroscopy provided valuable information on the mechanism of interaction of the ligand with the parallel G4 structure adopted by Pu22T14T23, a sequence mutated with respect to that found within the promoter region of the c-myc gene. Remarkably, when compared with data reported in the literature, fagaronine appears to exhibit one of the strongest G4 thermal stabilization effects ever recorded for a small ligand.

• Klíčová slova
• Circular dichroism spectroscopy, Docking simulation, Fagaronine, Fluorescence spectroscopy, G-quadruplex, NMR spectroscopy,
• MeSH
• alkaloidy * chemie   MeSH
• cirkulární dichroismus MeSH
• DNA * chemie   MeSH
• fenantridiny * chemie   MeSH
• G-kvadruplexy * účinky léků   MeSH
• magnetická rezonanční spektroskopie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alkaloidy * MeSH
• DNA * MeSH
• fenantridiny * MeSH

BACKGROUND: Human milk harbors diverse bacterial communities that contribute to infant health. Although pumping and storing milk is a common practice, the viable bacterial composition of pumped milk and the impact of storage practice on these bacteria remains under-explored. This metagenomic observational study aimed to characterize viable bacterial communities in freshly pumped human milk and its changes under different storage conditions. METHODS: In 2023, twelve lactating mothers from the CELSPAC: TNG cohort (Czech Republic) provided freshly pumped milk samples. These samples were stored under various conditions (refrigeration for 24 h, 48 h, or freezing for six weeks) and treated with propidium monoazide (PMA) to selectively identify viable cells. The DNA extracted from individual samples was subsequently analyzed using 16S rRNA amplicon sequencing on the Illumina platform. RESULTS: The genera Streptococcus, Staphylococcus, Diaphorobacter, Cutibacterium, and Corynebacterium were the most common viable bacteria in fresh human milk. The median sequencing depth and Shannon index of fresh human milk samples treated with PMA (+ PMA) were significantly lower than in untreated (-PMA) samples (p < 0.05 for all), which was true also for each time point. Also, significant changes in these parameters were observed between fresh human milk samples and their paired frozen samples (p < 0.05), while no differences were found between fresh human milk samples and those refrigerated for up to 48 h (p > 0.05). Of specific genera, only + PMA frozen human milk samples showed a significant decrease in the central log-ratio transformed relative abundances of the genera Diaphorobacter and Cutibacterium (p < 0.05) in comparison to + PMA fresh human milk samples. CONCLUSIONS: The study demonstrated that the bacterial profiles significantly differed between human milk samples treated with PMA, which represent only viable bacteria, and those untreated. While storage at 4 °C for up to 48 h did not significantly alter the overall diversity and composition of viable bacteria in human milk, freezing notably affected both the viability and relative abundances of some bacterial genera.

• Klíčová slova
• 16S rRNA, Human milk, Microbiome, Milk expression, Next-generation sequencing, Propidium monoazide, Pumped milk, Storage, Viable bacteria,
• MeSH
• azidy MeSH
• Bacteria * izolace a purifikace   genetika   klasifikace   MeSH
• chlazení MeSH
• dospělí MeSH
• lidé MeSH
• mateřské mléko * mikrobiologie   MeSH
• mikrobiota * MeSH
• propidium analogy a deriváty   MeSH
• RNA ribozomální 16S MeSH
• skladování potravin * metody   MeSH
• zmrazování MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• pozorovací studie MeSH
• Názvy látek
• azidy MeSH
• propidium monoazide MeSH Prohlížeč
• propidium MeSH
• RNA ribozomální 16S MeSH

This phytochemical study presents the isolation of eight alkaloids from Zephyranthes citrina Baker. The structures of the new alkaloids, zephycitrine (1) and 6-oxonarcissidine (2), were established by analysis of spectroscopic and spectrometric data. Processing the EtOH extract under acid-base conditions yielded the unreported isolation artifacts 3 and 4. This work also provides analytical data for alkaloids not properly described in the literature (5 and 6). The hippeastidine/zephyranine scaffolds in derivatives 3, 4, and 8-10 are also thoroughly discussed. Furthermore, a cytotoxicity screening of 25 Amaryllidaceae alkaloids isolated from Z. citrina was performed. Only the known alkaloids haemanthamine (12), haemanthidine (13), and lycorine (27) showed significant cell growth inhibition.

• MeSH
• alkaloidy amarylkovitých * farmakologie   chemie   izolace a purifikace   MeSH
• Amaryllidaceae * chemie   MeSH
• fenantridiny farmakologie   chemie   MeSH
• fytogenní protinádorové látky farmakologie   chemie   izolace a purifikace   MeSH
• lidé MeSH
• molekulární struktura MeSH
• screeningové testy protinádorových léčiv MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alkaloidy amarylkovitých * MeSH
• fenantridiny MeSH
• fytogenní protinádorové látky MeSH
• hemanthamine MeSH Prohlížeč
• lycorine MeSH Prohlížeč

BACKGROUND: Multidrug resistance is the major obstacle to cancer chemotherapy. Modulation of P-glycoprotein and drug combination approaches have been considered important strategies to overcome drug resistance. PURPOSE: Aiming at generating a small library of Amaryllidaceae-type alkaloids to overcome drug resistance, two major alkaloids, isolated from Pancratium maritimum, lycorine (1), and 2α-10bα-dihydroxy-9-O-demethylhomolycorine (2), were derivatized, giving rise to nineteen derivatives (3 - 21). METHODS: The main chemical transformation of lycorine resulted from the cleavage of ring E of the diacetylated lycorine derivative (3) to obtain compounds that have carbamate and amine functions (5 - 16), while acylation of compound 2 provided derivatives 17 - 21. Compounds 1 - 21 were evaluated for their effects on cytotoxicity, and drug resistance reversal, using resistant human ovarian carcinoma cells (HOC/ADR), overexpressing P-glycoprotein (P-gp/ABCB1), as model. RESULTS: Excluding lycorine (1) (IC50 values of 1.2- 2.5 µM), the compounds were not cytotoxic or showed moderate/weak cytotoxicity. Chemo-sensitization assays were performed by studying the in vitro interaction between the compounds and the anticancer drug doxorubicin. Most of the compounds have shown synergistic interactions with doxorubicin. Compounds 5, 6, 9 - 14, bearing both carbamate and aromatic amine moieties, were found to have the highest sensitization rate, reducing the dose of doxorubicin 5-35 times, highlighting their potential to reverse drug resistance in combination chemotherapy. Selected compounds (4 - 6, 9 - 14, and 21), able of re-sensitizing resistant cancer cells, were further evaluated as P-gp inhibitors. Compound 11, which has a para‑methoxy-N-methylbenzylamine moiety, was the strongest inhibitor. In the ATPase assay, compounds 9-11 and 13 behaved as verapamil, suggesting competitive inhibition of P-gp. At the same time, none of these compounds affected P-gp expression at the mRNA or protein level. CONCLUSIONS: This study provided evidence of the potential of Amaryllidaceae alkaloids as lead candidates for the development of MDR reversal agents.

• Klíčová slova
• Amaryllidaceae-type alkaloid, Homolycorine, Lycorine, Multidrug resistance, P-gycoprotein, Pancratium maritimum, Synergism,
• MeSH
• adenokarcinom * MeSH
• alkaloidy amarylkovitých * farmakologie   MeSH
• alkaloidy * farmakologie   MeSH
• chemorezistence MeSH
• doxorubicin farmakologie   MeSH
• fenantridiny * MeSH
• karbamáty farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• P-glykoprotein metabolismus   MeSH
• P-glykoproteiny metabolismus   MeSH
• protinádorové látky * farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alkaloidy amarylkovitých * MeSH
• alkaloidy * MeSH
• doxorubicin MeSH
• fenantridiny * MeSH
• homolycorine MeSH Prohlížeč
• karbamáty MeSH
• lycorine MeSH Prohlížeč
• P-glykoprotein MeSH
• P-glykoproteiny MeSH
• protinádorové látky * MeSH

1, 4-naphthoquinone, a plant-based quinone derivative, has gained much attention for its effectiveness against several biofilm-linked diseases. The biofilm inhibitory effect of 1, 4-naphthoquinone against Staphylococcus aureus has already been reported in our previous study. We observed that the extracellular DNA (eDNA) could play an important role in holding the structural integrity of the biofilm. Hence, in this study, efforts have been directed to examine the possible interactions between 1, 4-naphthoquinone and DNA. An in silico analysis indicated that 1, 4-naphthoquinone could interact with DNA through intercalation. To validate the same, UV-Vis spectrophotometric analysis was performed in which a hypochromic shift was observed when the said molecule was titrated with calf-thymus DNA (CT-DNA). Thermal denaturation studies revealed a change of 8℃ in the melting temperature (Tm) of CT-DNA when complexed with 1, 4-naphthoquinone. The isothermal calorimetric titration (ITC) assay revealed a spontaneous intercalation between CT-DNA and 1, 4-naphthoquinone with a binding constant of 0.95 ± 0.12 × 108. Furthermore, DNA was run through an agarose gel electrophoresis with a fixed concentration of ethidium bromide and increasing concentrations of 1, 4-naphthoquinone. The result showed that the intensity of ethidium bromide-stained DNA got reduced concomitantly with the gradual increase of 1, 4-naphthoquinone suggesting its intercalating nature. To gain further confidence, the pre-existing biofilm was challenged with ethidium bromide wherein we observed that it could also show biofilm disintegration. Therefore, the results suggested that 1, 4-naphthoquinone could exhibit disintegration of the pre-existing biofilm of Staphylococcus aureus through eDNA intercalation.

• Klíčová slova
• 1, 4-naphthoquinone, Biofilm, Intercalation, Staphylococcus aureus, eDNA,
• MeSH
• biofilmy MeSH
• DNA farmakologie   MeSH
• ethidium farmakologie   MeSH
• lidé MeSH
• naftochinony * farmakologie   MeSH
• stafylokokové infekce * MeSH
• Staphylococcus aureus genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• ethidium MeSH
• naftochinony * MeSH

Defects in cell death signaling pathways are one of the hallmarks of cancer and can lead to resistance to conventional therapy. Natural products are promising compounds that can overcome this resistance. In the present study we studied the effect of six quaternary benzophenanthridine alkaloids (QBAs), sanguinarine, chelerythrine, sanguirubine, chelirubine, sanguilutine, and chelilutine, on Jurkat leukemia cells, WT, and cell death deficient lines derived from them, CASP3/7/6-/- and FADD-/-, and on solid tumor, human malignant melanoma, A375 cells. We demonstrated the ability of QBAs to overcome the resistance of these deficient cells and identified a novel mechanism for their action. Sanguinarine and sanguirubine completely and chelerythrine, sanguilutine, and chelilutine partially overcame the resistance of CASP3/7/6-/- and FADD-/- cells. By detection of cPARP, a marker of apoptosis, and pMLKL, a marker of necroptosis, we proved the ability of QBAs to induce both these cell deaths (bimodal cell death) with apoptosis preceding necroptosis. We identified the new mechanism of the cell death induction by QBAs, the downregulation of the apoptosis inhibitors cIAP1 and cIAP2, i.e., an effect similar to that of Smac mimetics.

• Klíčová slova
• Smac mimetic drug resistance, apoptosis, benzophenanthridine alkaloids, cIAP, cancer, cell death, chelerythrine, sanguinarine,
• MeSH
• alkaloidy * farmakologie   metabolismus   MeSH
• apoptóza * MeSH
• benzofenantridiny farmakologie   MeSH
• kaspasa 3 metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alkaloidy * MeSH
• benzofenantridiny MeSH
• kaspasa 3 MeSH

Propidium iodide (PI) and YO-PRO-1 (YPI) dyes are routinely used to determine sperm viability in many livestock species. It is commonly accepted that these dyes penetrate only sperm cells with damaged plasma membranes. Recently, however, the mechanism of dye uptake unrelated to damaged plasma membranes, but instead related to pannexin channels in dog and stallion sperm cells was demonstrated. This pilot study aimed to evaluate the role of pannexins in the uptake of PI and YPI dyes on Wallachian frozen-thawed ram spermatozoa by flow cytometry using probenecid, a specific inhibitor of pannexin channels. Additionally, the expression of pannexins in Wallachian sperm was evaluated directly (by qRT-PCR). The results demonstrate the active role of pannexin channels in the uptake of PI and YPI dyes on frozen-thawed Wallachian ram sperm. In conclusion, when using the PI or YPI exclusion assay to determine Wallachian frozen-thawed ram sperm viability, the danger of overestimating the number of spermatozoa with the damaged plasma membrane must be considered. The observed breed-specific, and more importantly, individual differences in gene expression as well as in dye uptake indicate the need for further studies.

• Klíčová slova
• PANX1, PANX2, flow cytometry, frozen-thawed spermatozoa, qRT-PCR,
• MeSH
• barvicí látky MeSH
• benzoxazoly MeSH
• chinolinové sloučeniny * MeSH
• jodidy * MeSH
• koně MeSH
• kryoprezervace metody   veterinární   MeSH
• pilotní projekty MeSH
• propidium MeSH
• psi MeSH
• sperma MeSH
• spermie MeSH
• uchování spermatu * veterinární   metody   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• barvicí látky MeSH
• benzoxazoly MeSH
• chinolinové sloučeniny * MeSH
• jodidy * MeSH
• propidium MeSH
• YO-PRO 1 MeSH Prohlížeč

• MeSH
• barvení a značení MeSH
• buněčné jádro * MeSH
• fluorescenční barviva MeSH
• fluorescenční protilátková technika MeSH
• indoly * MeSH
• lidé MeSH
• propidium MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DAPI MeSH Prohlížeč
• fluorescenční barviva MeSH
• indoly * MeSH
• propidium MeSH

Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 µM bis(benzonitrile)dichloropalladium(II) or 30 µM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.

• MeSH
• azidy farmakologie   MeSH
• biotest MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• mikrobiální viabilita MeSH
• Mycobacterium avium subsp. paratuberculosis * genetika   MeSH
• palladium farmakologie   MeSH
• paratuberkulóza * mikrobiologie   MeSH
• propidium farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azidy MeSH
• palladium MeSH
• propidium MeSH

Extracellular pH has been assumed to play little if any role in how bacteria respond to antibiotics and antibiotic resistance development. Here, we show that the intracellular pH of Escherichia coli equilibrates to the environmental pH following treatment with the DNA damaging antibiotic nalidixic acid. We demonstrate that this allows the environmental pH to influence the transcription of various DNA damage response genes and physiological processes such as filamentation. Using purified RecA and a known pH-sensitive mutant variant RecA K250R we show how pH can affect the biochemical activity of a protein central to control of the bacterial DNA damage response system. Finally, two different mutagenesis assays indicate that environmental pH affects antibiotic resistance development. Specifically, at environmental pH's greater than six we find that mutagenesis plays a significant role in producing antibiotic resistant mutants. At pH's less than or equal to 6 the genome appears more stable but extensive filamentation is observed, a phenomenon that has previously been linked to increased survival in the presence of macrophages.

• MeSH
• antibakteriální látky farmakologie   MeSH
• Escherichia coli účinky léků   genetika   účinky záření   MeSH
• koncentrace vodíkových iontů MeSH
• kyselina nalidixová farmakologie   MeSH
• mikrobiální viabilita účinky léků   účinky záření   MeSH
• nestabilita genomu účinky léků   genetika   účinky záření   MeSH
• poškození DNA účinky léků   genetika   účinky záření   MeSH
• propidium farmakologie   MeSH
• průtoková cytometrie MeSH
• retardační test MeSH
• rifampin farmakologie   MeSH
• ultrafialové záření MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• kyselina nalidixová MeSH
• propidium MeSH
• rifampin MeSH