Alkaloid escholidine and its interaction with DNA structures

Berberine, the most known quaternary protoberberine alkaloid (QPA), has been reported to inhibit the SIK3 protein connected with breast cancer. Berberine also appears to reduce the bcl-2 and XIAP expression-proteins responsible for the inhibition of apoptosis. As some problems in the therapy with berberine arose, we studied the DNA binding properties of escholidine, another QPA alkaloid. CD, fluorescence, and NMR examined models of i-motif and G-quadruplex sequences present in the n-myc gene and the c-kit gene. We provide evidence that escholidine does not induce stabilization of the i-motif sequences, while the interaction with G-quadruplex structures appears to be more significant

Cytokine expression by CD163+ monocytes in healthy and Actinobacillus pleuropneumoniae-infected pigs

Distinct monocyte subpopulations have been previously described in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (APP). The CD163+ subpopulation of bone marrow (BM), peripheral blood (PB) and lung monocytes was found to play an important role in the inflammatory process. The inflammation is accompanied by elevation of inflammatory cytokines. The aim of the study was to evaluate the contribution of CD163+ monocytes and macrophages to cytokine production during APP-induced lung inflammation. Cytokine production was assessed by flow cytometry (FC) and quantitative PCR (qPCR) in CD163+ monocytes and by qPCR, immunohistochemistry/fluorescence in lungs and tracheobronchial lymph nodes (TBLN). Despite the systemic inflammatory response after APP infection, BM and PB CD163+ monocytes did not express elevated levels of a wide range of cytokines compared to control pigs. In contrast, significant amounts of IL-1β, IL-6, IL-8 and TNF-α were produced in lung lesions and IL-1β in the TBLN. At the protein level, TNF-α was expressed by both CD163+ monocytes and macrophages in lung lesions, whereas IL-1β, IL-6 and IL-8 expression was found only in CD163+ monocytes; no CD163+ macrophages were found to produce these cytokines. Furthermore, the quantification of CD163+ monocytes expressing the two cytokines IL-1β and IL-8 that were most elevated was performed. In lung lesions, 36.5% IL-1β positive CD163+ monocytes but only 18.3% IL-8 positive CD163+ monocytes were found. In conclusion, PB and BM CD163+ monocytes do not appear to contribute to the elevated cytokine levels in plasma. On the other hand, CD163+ monocytes contribute to inflammatory cytokine expression, especially IL-1β at the site of inflammation during the inflammatory process.Peer reviewe

Study of the interaction of the palmatine alkaloid with hybrid G-quadruplex/duplex and i-motif/duplex DNA structures

There is an increasing interest in the study of guanine or cytosine-rich sequences that may fold into G-quadruplex (G4) or i-motif (iM) structures showing a short hairpin (or stem-loop) stabilized by Watson-Crick base pairs. These hybrid spatial arrangements may be target of ligands that have been shown to interact strongly with B-DNA. In this work, the interaction of the palmatine alkaloid with several sequences forming different G4s, iMs, and hybrid structures has been studied by means of spectroscopic and separation techniques, as well as multivariate data analysis methods. At the experimental conditions used in this work, the results have shown that this ligand strongly stabilizes parallel G4 structures, whereas a weaker interaction was observed with the antiparallel G4 adopted by the thrombin-binding aptamer or iMs. The presence of hairpins within the loops scarcely affects the affinity of this ligand for the hybrid G4/duplex or iM/duplex structures. Fluorescence measurements have provided evidence of a certain interaction with iMs at pH 5.1, despite the absence of thermal stabilization effects

Study of alkaloid berberine and its interaction with the human telomeric i-motif DNA structure

The alkaloid berberine presents many biological activities related to its potential to bind DNA structures, such as duplex or G-quadruplex. Recently, it has been proposed that berberine may interact with i-motif structures formed from the folding of cytosine-rich sequences. In the present work, the interaction of this alkaloid with the i-motif formed by the human telomere cytosine-rich sequence, as well as with several positive and negative controls, has been studied. Molecular fluorescence and circular dichroism spectroscopies, as well as nuclear magnetic resonance spectrometry and competitive dialysis, have been used with this purpose. The results shown here reveal that the interaction of berberine with this i-motif is weak, mostly electrostatics in nature and takes place with bases not involved in C·C+ base pairs. Moreover, this ligand is not selective for i-motif structures, as binds equally to both, folded structure, and unfolded strand, without producing any stabilization of the i-motif. As a conclusion, the development of analytical methods based on the interaction of fluorescent ligands, such as berberine, with i-motif structures should consider the thermodynamic aspects related with the interaction, as well as the selectivity of the proposed ligands with different DNA structures, including unfolded strands

Naturally occurring quaternary benzo[c]phenanthridine alkaloids selectively stabilize G quadruplexes

In this work, the interaction of six natural benzo[c]phenanthridine alkaloids (macarpine, sanguilutine, sanguirubine, chelerythrine, sanguinarine and chelirubine) with parallel and antiparallel G-quadruplex DNA structures was studied. HT22 corresponding to the end of human telomere and the modified promoter oncogenes c-kit21 and Pu22 sequences have been used. Spectroscopically-monitored melting experiments and fluorescence titrations, competitive dialysis and nuclear magnetic resonance spectroscopy were used with this purpose. The results showed that these alkaloids stabilized G-quadruplex structures in terms of increments of Tm values (from 15 to 25 oC) with high selectivity over duplexes and unfolded DNA. The mode of binding was mainly by stacking on the terminal G-tetrads with stoichiometries 1:2 (DNA:ligand). The presence of non-specific electrostatics interaction was also observed. Overall, the results pointed to a strong stabilization of G-quadruplex structures by these alkaloids

Precision immuno-oncology approach for four malignant tumors in siblings with constitutional mismatch repair deficiency syndrome

Abstract Constitutional mismatch repair deficiency (CMMRD) is a rare syndrome characterized by an increased incidence of cancer. It is caused by biallelic germline mutations in one of the four mismatch repair genes (MMR) genes: MLH1, MSH2, MSH6, or PMS2. Accurate diagnosis accompanied by a proper molecular genetic examination plays a crucial role in cancer management and also has implications for other family members. In this report, we share the impact of the diagnosis and challenges during the clinical management of two brothers with CMMRD from a non-consanguineous family harbouring compound heterozygous variants in the PMS2 gene. Both brothers presented with different phenotypic manifestations and cancer spectrum. Treatment involving immune checkpoint inhibitors significantly contributed to prolonged survival in both patients affected by lethal gliomas. The uniform hypermutation also allowed immune-directed treatment using nivolumab for the B-cell lymphoma, thereby limiting the intensive chemotherapy exposure in this young patient who remains at risk for subsequent malignancies