Quadruplex-duplex junction in LTR-III: A molecular insight into the complexes with BMH-21, namitecan and doxorubicin.

Quadruplex-Duplex (Q-D) junctions are unique structural motifs garnering increasing interest as drug targets, due to their frequent occurrence in genomic sequences. The viral HIV LTR-III sequence was chosen as a Q-D junction model to study the affinity of the selected compounds BMH-21, namitecan (ST-1968), and doxorubicin (DOXO), all containing a planar polycyclic aromatic moiety, linked to either one short aminoalkyl or an aminoglycosyl group. A multidisciplinary approach that combines NMR spectroscopy, molecular modelling, circular dichroism (CD) and fluorescence spectroscopy was employed. The studied ligands induced moderate but clear stabilization to the Q-D junction by interacting with the interfacial tetrad. DOXO was found to be the best Q-D junction binder. Interestingly, the removal of the aminoglycosyl group significantly changed the pattern of the interactions, indicating that highly polar substituents have a stronger affinity with the exposed regions of the Q-D junction, particularly at the level of the interfacial tetrad.

Food Plants and Environmental Contamination: An Update.

Food plants are the basis of human nutrition, but, in contaminated places, they can uptake contaminants. Environmental contamination and climate change can modify food quality; generally, they have a negative impact on and imply risks to human health. Heavy metals, like lead, arsenic, cadmium, and chromium, can be present at various environmental levels (soil, water, and atmosphere), and they are widely distributed in the world. Food plants can carry out heavy metal bioaccumulation, a defense pathway for plants, which is different for every plant species. Accumulation is frequent in the roots and the leaves, and heavy metals can be present in fruits and seeds; As and Cd are always present. In addition, other contaminants can bioaccumulate in food plants, including emerging contaminants, like persistent organic pollutants (POPs), pesticides, and microplastics. In food plants, these are present in the roots but also in the leaves and fruits, depending on their chemical structure. The literature published in recent years was examined to understand the distribution of contaminants among food plants. In the literature, old agronomical practices and new integrated technology to clean the water, control the soil, and monitor the crops have been proposed to mitigate contamination and produce high food quality and high food safety.

Exploring the Interaction of New Pyridoquinazoline Derivatives with G-Quadruplex in the c-MYC Promoter Region.

Novel amino-substituted pyridoquinazolinone derivatives have been designed and synthesized as potential c-MYC G-quadruplex (G4) ligands, employing an efficient methodology. All the new compounds exhibited moderate to good antiproliferative activity against the human osteosarcoma U2OS cell line. NMR and docking experiments revealed that the recently synthesized compounds interact with the Pu22 G-quadruplex in the c-MYC promoter region, establishing a 2:1 complex, with each molecule positioned over the tetrads at the 3'- and 5'-ends.

Exploring the stabilizing effect on the i-motif of neighboring structural motifs and drugs.

Cytosine-rich DNA sequences may fold into a structure known as i-motif, with potential in vivo modulation of gene expression. The stability of the i-motif is residual at neutral pH values. To increase it, the addition of neighboring moieties, such as Watson-Crick stabilized loops, tetrads, or non-canonical base pairs have been proposed. Taking a recently described i-motif structure as a model, the relative effect of these structural moieties, as well as several DNA ligands, on the stabilization of the i-motif has been studied. To this end, not only the original sequence but different mutants were considered. Spectroscopic techniques, PAGE, and multivariate data analysis methods have been used to model the folding/unfolding equilibria induced by changes of pH, temperature, and the presence of ligands. The results have shown that the duplex is the moiety that is responsible of the stabilization of the i-motif structure at neutral pH. The T:T base pair, on the contrary, shows little stabilization of the i-motif. From several selected DNA-binding ligands, the G-quadruplex ligand BA41 is shown to interact with the duplex moiety, whereas non-specific interaction and little stabilization has been observed within the i-motif.

Evaluating the potential of hydrochar as a soil amendment.

In this study, hydrochar (HC), a carbon-rich product originated from hydrothermal conversion treatment (HTC), was obtained from wastes of the wine and dairy industries. The effect of mixing secondary char and compost was tested, before and after the aerobic mixing of compost (COM) and HC at increasing doses (from 15 to 75 Mg ha-1 DM), in an effort to lower the HC phytotoxicity due to potential phytotoxic compounds of secondary char. The results indicated that, after the aerobic stabilization, the mix HC/COM was able to double the plant growth in comparison to COM alone. The presence of easily degradable organic compounds probably led to poor stability of HC, increased microbial activity and, consequently, root anoxia when used at high doses. Chemical, spectroscopic and thermal investigation confirmed this hypothesis. In particular, HC shows a high content of dissolved organic matter, characterized by the presence of small molecules, which is negatively correlated with the growth index of lettuce. Furthermore, thermal characterization suggests a higher proportion of less complex and thermally stable molecular compounds in HC in comparison to COM. Therefore, co-composting of HC allows obtaining a useful amendment to support soil organic matter and fertility.

Investigation of the Interaction between Aloe vera Anthraquinone Metabolites and c-Myc and C-Kit G-Quadruplex DNA Structures

G-quadruplexes are nucleotide sequences present in the promoter region of numerous oncogenes, having a key role in the suppression of gene transcription. Recently, the binding of anthraquinones from Aloe vera to G-quadruplex structures has been studied through various physico-chemical techniques. Intrigued by the reported results, we investigated the affinity of aloe emodin, aloe emodin-8-glucoside, and aloin to selected G-quadruplex nucleotide sequences by NMR spectroscopy. The structural determinants for the formation of the ligand/nucleotide complexes were elucidated and a model of the interactions between the tested compounds and C-Kit and c-Myc G-quadruplex DNA structures was built by integrated NMR and molecular modeling studies. Overall, the obtained results confirmed and implemented the previously reported findings, pointing out the complementarity of the different approaches and their contribution to a more detailed overview of the ligand/nucleotide complex formation. Furthermore, the proposed models of interaction could pave the way to the design of new nature-derived compounds endowed with increased G-quadruplex stabilizing activity.

Antifungal Activity and Biochemical Profiling of Exudates from Germinating Maize Nostrano di Storo Local Variety.

Plant pathogens are responsible for important damages to valuable crops causing important economic losses. Agrobiodiversity protection is crucial for the valorization of local varieties that could possess higher resistance to biotic and abiotic stress. At the beginning of germination, seeds are susceptible to pathogens attacks, thus they can release endogenous antimicrobial compounds of different natures in the spermosphere, to contrast proliferation of microorganisms. The work aimed at characterizing the maize of local variety Nostrano di Storo seed exudates secreted during the first phases of germination, to identify compounds active in the defense towards pathogens. Storo seed exudates were proven to inhibit F. verticilloides germination. In order to investigate the cause of the described effect, compositional profiling of the exudates was performed through NMR, lipidomic, and proteomic analyses. This study suggests an important role of microbial endophytic communities in the protection of the seed during the early phases of the germination process and their interplay with fatty acids released by the seeds, rather than a specific antifungal compound. The valorization of agronomically acceptable maize lines with pre-harvest enhanced resistances to pathogens contamination could lead, in the near future, to commercially available varieties potentially requiring more limited chemical protective treatments.

Exploring the Interaction of G-quadruplex Binders with a (3 + 1) Hybrid G-quadruplex Forming Sequence within the PARP1 Gene Promoter Region.

The enzyme PARP1 is an attractive target for cancer therapy, as it is involved in DNA repair processes. Several PARP1 inhibitors have been approved for clinical treatments. However, the rapid outbreak of resistance is seriously threatening the efficacy of these compounds, and alternative strategies are required to selectively regulate PARP1 activity. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter was recently identified. In this study, we explore the interaction of known G-quadruplex binders with the G-quadruplex structure found in the PARP gene promoter region. The results obtained by NMR, CD, and fluorescence titration, also confirmed by molecular modeling studies, demonstrate a variety of different binding modes with small stabilization of the G-quadruplex sequence located at the PARP1 promoter. Surprisingly, only pyridostatin produces a strong stabilization of the G-quadruplex-forming sequence. This evidence makes the identification of a proper (3+1) stabilizing ligand a challenging goal for further investigation.

Properties of Parallel Tetramolecular G-Quadruplex Carrying N-Acetylgalactosamine as Potential Enhancer for Oligonucleotide Delivery to Hepatocytes.

The development of oligonucleotide conjugates for in vivo targeting is one of the most exciting areas for oligonucleotide therapeutics. A major breakthrough in this field was the development of multifunctional GalNAc-oligonucleotides with high affinity to asialoglycoprotein receptors (ASGPR) that directed therapeutic oligonucleotides to hepatocytes. In the present study, we explore the use of G-rich sequences functionalized with one unit of GalNAc at the 3'-end for the formation of tetrameric GalNAc nanostructures upon formation of a parallel G-quadruplex. These compounds are expected to facilitate the synthetic protocols by providing the multifunctionality needed for the binding to ASGPR. To this end, several G-rich oligonucleotides carrying a TGGGGGGT sequence at the 3'-end functionalized with one molecule of N-acetylgalactosamine (GalNAc) were synthesized together with appropriate control sequences. The formation of a self-assembled parallel G-quadruplex was confirmed through various biophysical techniques such as circular dichroism, nuclear magnetic resonance, polyacrylamide electrophoresis and denaturation curves. Binding experiments to ASGPR show that the size and the relative position of the therapeutic cargo are critical for the binding of these nanostructures. The biological properties of the resulting parallel G-quadruplex were evaluated demonstrating the absence of the toxicity in cell lines. The internalization preferences of GalNAc-quadruplexes to hepatic cells were also demonstrated as well as the enhancement of the luciferase inhibition using the luciferase assay in HepG2 cell lines versus HeLa cells. All together, we demonstrate that tetramerization of G-rich oligonucleotide is a novel and simple route to obtain the beneficial effects of multivalent N-acetylgalactosamine functionalization.

Synthesis and Investigation of the G-Quadruplex Binding Properties of Kynurenic Acid Derivatives with a Dihydroimidazoquinoline-3,5-dione Core.

G-quadruplexes are secondary structures originating from nucleic acid regions rich in guanines, which are well known for their involvement in gene transcription and regulation and DNA damage repair. In recent studies from our group, kynurenic acid (KYNA) derivative 1 was synthesized and found to share the structural features typical of G-quadruplex binders. Herein, structural modifications were conducted on this scaffold in order to assist the binding with a G-quadruplex, by introducing charged hydrophilic groups. The antiproliferative activity of the new analogues was evaluated on an IGROV-1 human ovarian cancer cell line, and the most active compound, compound 9, was analyzed with NMR spectrometry in order to investigate its binding mode with DNA. The results indicated that a weak, non-specific interaction was set with duplex nucleotides; on the other hand, titration in the presence of a G-quadruplex from human telomere d(TTAGGGT)4 showed a stable, although not strong, interaction at the 3'-end of the nucleotidic sequence, efficiently assisted by salt bridges between the quaternary nitrogen and the external phosphate groups. Overall, this work can be considered a platform for the development of a new class of potential G-quadruplex stabilizing molecules, confirming the crucial role of a planar system and the ability of charged nitrogen-containing groups to facilitate the binding to G-quadruplex grooves and loops.

In Streptococcus thermophilus, Ammonia from Urea Hydrolysis Paradoxically Boosts Acidification and Reveals a New Regulatory Mechanism of Glycolysis.

Streptococcus thermophilus is widely used in the dairy industry for the manufacturing of fermented milk and cheeses and probiotic formulations. S. thermophilus evolved from closely phylogenetically related pathogenic streptococci through loss-of-function events counterbalanced by the acquisition of relevant traits, such as lactose and urea utilization for the adaptation to the milk environment. In the context of regressive evolution, the urease gene cluster accounts for 0.9% of the total coding sequence belonging to known functional categories. The fate of ammonia and carbon dioxide derived by urea hydrolysis in several biosynthetic pathways have been depicted, and the positive effect of urease activity on S. thermophilus growth fitness and lactic acid fermentation in milk has been already addressed by several authors. However, the mechanistic effect of urea hydrolysis on the energetic metabolisms of S. thermophilus is still unclear. This study aimed to assess the effect of urease activity on the growth and energy metabolism of Streptococcus thermophilus in milk. In milk, 13C-urea was completely hydrolyzed in the first 150 min of S. thermophilus growth, and urea hydrolysis was accompanied by an increase in cell density and a reduction in the generation time. By using energetically discharged cells with gene transcription and translation blocked, we showed that in the presence of fermentable carbon sources, urease activity, specifically the production of ammonia, could dramatically boost glycolysis and, in cascade, homolactic fermentation. Furthermore, we showed that ammonia, specifically ammonium ions, were potent effectors of phosphofructokinase, a key glycolytic enzyme. IMPORTANCE Finding that ammonia-generating enzymes, such as urease, and exogenous ammonia act on phosphofructokinase activity shed new light on the regulatory mechanisms that govern glycolysis. Phosphofructokinase is the key enzyme known to exert a regulatory role on glycolytic flux and, therefore, ammonia as an effector of phosphofructokinase acts, in cascade, modulating the glycolytic pathway. Apart from S. thermophilus, due to the high conservation of glycolytic enzymes in all branches of the tree of life and being aware of the role of ammonia as an effector of phosphofructokinase, we propose to reevaluate the physiological role of the ammonia production pathways in all organisms whose energy metabolism is supported by glycolysis.

Correction: Clua et al. Properties of Parallel Tetramolecular G-Quadruplex Carrying N-Acetylgalactosamine as Potential Enhancer for Oligonucleotide Delivery to Hepatocytes. Molecules 2022, 27, 3944.

In the original article [...].

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.

Grapevine stilbenoids as natural food preservatives: calorimetric and spectroscopic insights into the interaction with model cell membranes.

Food contamination with pathogenic microorganisms, such as Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus and Bacillus cereus, is a common health concern. Natural products, which have been the main source of antimicrobials for centuries, may represent a turning point in alleviating the antibiotic crisis, and plant polyphenolic compounds are considered a promising source for new antibacterial agents. Resveratrol and resveratrol-derived monomers and oligomers (stilbenoids) have been shown to exert a variegated pattern of efficacy as antimicrobials depending on both the polyphenols' structure and the nature of the microorganisms, and the bacterial cell membrane seems to be one of their primary targets.In this scenario and based on the thermodynamic information reported in the literature about cell membranes, this study aimed at the investigation of the direct interaction of selected stilbenoids with a simple but informative model cell membrane. Three complete stilbenoid "monomer/dimer/dehydro-dimer" sets were chosen according to different geometries and substitution patterns. Micro-DSC was performed on 2 : 3 DPPC : DSPC small unilamellar vesicles with incorporated polyphenols at physiological pH and the results were integrated using complementary NMR data. The study highlighted the molecular determinants and mechanisms involved in the stilbenoid-membrane interaction, and the results were well correlated with the microbiological evidence previously assessed.

Investigation of the Complexes Formed between PARP1 Inhibitors and PARP1 G-Quadruplex at the Gene Promoter Region.

DNA repair inhibitors are one of the latest additions to cancer chemotherapy. In general, chemotherapy produces DNA damage but tumoral cells may become resistant if enzymes involved in DNA repair are overexpressed and are able to reverse DNA damage. One of the most successful drugs based on modulating DNA repair are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Several PARP1 inhibitors have been recently developed and approved for clinical treatments. We envisaged that PARP inhibition could be potentiated by simultaneously modulating the expression of PARP 1 and the enzyme activity, by a two-pronged strategy. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter has been recently identified. In this study, we explored the potential binding of clinically approved PARP1 inhibitors to the G-quadruplex structure found at the gene promoter region. The results obtained by NMR, CD, and fluorescence titration confirmed by molecular modeling demonstrated that two out the four PARP1 inhibitors studied are capable of forming defined complexes with the PARP1 G-quadruplex. These results open the possibility of exploring the development of better G-quadruplex binders that, in turn, may also inhibit the enzyme.

Exploring the Interaction of Curaxin CBL0137 with G-Quadruplex DNA Oligomers.

Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor activities in multiple cancers such as glioblastoma, melanoma and others. Although most of the authors suggest that their mechanism of action comes from the activation of p53 and inactivation of NF-kB by targeting FACT, there is evidence supporting the involvement of DNA binding in their antitumor activity. In this work, the DNA binding properties of curaxin CBL0137 with model quadruplex DNA oligomers were studied by 1H NMR, CD, fluorescence and molecular modeling. We provided molecular details of the interaction of curaxin with two G-quadruplex structures, the single repeat of human telomere d(TTAGGGT)4 and the c-myc promoter Pu22 sequence. We also performed 1H and 31P NMR experiments were also performed in order to investigate the interaction with duplex DNA models. Our data support the hypothesis that the interaction of curaxin with G-quadruplex may provide a novel insight into the DNA-binding properties of CBL0137, and it will be helpful for the design of novel selective DNA-targeting curaxin analogues.

Plant-Derived Stilbenoids as DNA-Binding Agents: From Monomers to Dimers.

Stilbenoids are natural compounds endowed with several biological activities, including cardioprotection and cancer prevention. Among them, (±)-trans-δ-viniferin, deriving from trans-resveratrol dimerization, was investigated in its ability to target DNA duplex and G-quadruplex structures by exploiting NMR spectroscopy, circular dichroism, fluorescence spectroscopy and molecular docking. (±)-trans-δ-Viniferin proved to bind both the minor and major grooves of duplexes, whereas it bound the 3'- and 5'-ends of a G-quadruplex by stacking on the outer quartets, accompanied by rearrangement of flanking residues. Specifically, (±)-trans-δ-viniferin demonstrated higher affinity for the investigated DNA targets than its monomeric counterpart. Additionally, the methoxylated derivatives of (±)-trans-δ-viniferin and trans-resveratrol, i. e. (±)-pterostilbene-trans-dihydrodimer and trans-pterostilbene, respectively, were evaluated, revealing similar binding modes, affinities and stoichiometries with the DNA targets as their parent analogues. All tested compounds were cytotoxic at µM concentration on several cancer cell lines, showing DNA damaging activity consistent with their ability to tightly interact with duplex and G-quadruplex structures.

Structural Effects of Incorporation of 2'-Deoxy-2'2'-Difluorodeoxycytidine (Gemcitabine) in A- and B-Form Duplexes.

We report the structural effect of 2'-deoxy-2',2'-difluorocytidine (dFdC) insertions in the DNA strand of a DNA : RNA hybrid duplex and in a self-complementary DNA : DNA duplex. In both cases, the modification slightly destabilizes the duplex and provokes minor local distortions that are more pronounced in the case of the DNA : RNA hybrid. Analysis of the solution structures determined by NMR methods show that dFdC is an adaptable derivative that adopts North type sugar conformation when inserted in pure DNA, or a South sugar conformation in the context of DNA : RNA hybrids. In this latter context, South sugar pucker favors the formation of a 2'F⋅⋅H8 attractive interaction with a neighboring purine, which compensates the destabilizing effect of base pair distortions. These interactions share some features with pseudohydrogen bonds described previously in other nucleic acids structures with fluorine modified sugars.

Thermophilic anaerobic digestion as suitable bioprocess producing organic and chemical renewable fertilizers: A full-scale approach.

This work reports a full-scale study in which organic wastes were transformed by high-solid thermophilic anaerobic digestion (HSAD), into N fertilizers and organic fertilizers, i.e. digestate. The produced fertilizers were characterized over 42 months and their properties were discussed in comparisons with literature data. HSAD coupled with N stripping technology led to ammonia sulphate production having high N concentration (74 ± 2 g kg-1 wet weight), neutral pH (6.8 ± 1.3) and low traces of other elements. Digestate showed both higher carbon (C) content (314 ± 30 g kg-1 on dry matter (DM) and biological stability than green composts, indicating good amendment properties. Digestate was also interesting for its N (77 ± 3.7 g kg-1 dry matter - DM) content, half of it in the ammonia form, and P content (28 ± 4.1 g kg-1 DM) that was 43% readily available as soluble P-orthophosphate. K content was low (6.5 ± 1.3 g kg-1 DM), indicating poor fertilizing ability of digestate for this element. All organic pollutants investigated were much lower than the limits required for agricultural use and levels of some of them were lower than the content revealed for other organic matrices such as agricultural and energy crop digestates and compost. Emerging pollutants (i.e., pharmaceuticals) were tested as markers and they were found to be below the detection limit (<0.01 mg kg-1 DM) indicating very low content. The results obtained showed that HSAD coupled with N stripping allowed transforming sewage sludge into fertilizers and soil improvers exploitable in agriculture.