Surface Plasmon Resonance kinetic analysis of the interaction between G-quadruplex nucleic acids and an anti-G-quadruplex monoclonal antibody

Background G-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules. We here aimed at improving the orientation of an anti-G4 antibody on the SPR sensor chip to optimize detection of binding antigens. Methods SPR was employed to characterize the anti-G4 antibody interaction with G4 and non-G4 oligonucleotides. Dextran-functionalized sensor chips were used both in covalent coupling and capturing procedures. Results The use of two leading molecule for orienting the antibody of interest allowed to improve its activity from completely non-functional to 65% active. The specificity of the anti-G4 antobody for G4 structures could thus be assessed with high sensitivity and reliability. Conclusions Optimization of the immobilization protocol for SPR biosensing, allowed us to determine the anti-G4 antibody affinity and specificity for G4 antigens with higher sensitivity with respect to other in vitro assays such as ELISA. Anti-G4 antibody specificity is a fundamental assumption for the future utilization of this kind of antibodies for monitoring G4s directly in cells. General significance The heterogeneous orientation of amine-coupling immobilized ligands is a general problem that often leads to partial or complete inactivation of the molecules. Here we describe a new strategy for improving ligand orientation: driving it from two sides. This principle can be virtually applied to every molecule that loses its activity or is poorly immobilized after standard coupling to the SPR chip surface

Applying biocultural research protocols in ecology: Insider and outsider experiences from Australia

Collaborations between Indigenous and non-Indigenous scientific researchers are increasingly mandated by global to local conservation policy and research ethics guidelines. Breakdowns occur due to misunderstandings around expected protocols of engagement and cooperation, which are compounded by lack of broader awareness of differences in cultural values, priorities and knowledge systems. Using first-hand experiences, we outline eight key protocols and guidelines that researchers should consider when undertaking research with Indigenous peoples, or on Indigenous Country, through exploration of biocultural protocols and guidelines within Australian and Indigenous customary laws. We use the onion as a metaphor to highlight the layers of protocols and guidelines that researchers can peel back to guide their research from international to local scales, with ethics around the research question at the core. This paper draws on the perspectives and experiences of an Indigenous researcher (as ‘insider’/‘outsider’) and non-Indigenous researcher (‘outsider’), working on a cross-cultural and multidisciplinary investigation of past Aboriginal dispersal of rainforest trees on the Australian east coast. This paper is part of the special issue ‘Indigenous and cross-cultural ecology - perspectives from Australia’ published in Ecological Management & Restoration

daily and seasonal variability of co2 saturation and evasion in a free flowing and in a dammed river reach

The daily and seasonal evolution of O2 and CO2 saturation, water-atmosphere fluxes and budgets were measured in two fluvial reaches of the Mincio River (Italy). The northern reach is free flowing and is dominated by macrophytes while the southern reach is dammed, hypertrophic and phytoplankton dominated. We hypothesized short term regulation of gas saturation and fluxes by primary producers and the reversal of CO2 off-gassing in the southern reach. Results indicated that both reaches were always CO2 supersaturated. Higher CO2 evasion rates in the northern compared to the southern reach depended on reaeration coefficient, in turn depending on water velocity. In the northern reach dissolved inorganic carbon (DIC) production was one order of magnitude higher than oxygen consumption, likely due to a combination of anoxic heterotrophic activity in the hyporheic zone and carbonate dissolution. The activity of macrophytes influenced CO2 saturation on short time scales. A net summer abatement of DIC occurred in the southern reach, probably due to fixation by phytoplankton, which attenuated supersaturation but not reversed CO2 efflux. This study demonstrates how in small rivers CO2 evasion can undergo rapid and significant changes due to eutrophication, altered hydrology and shift in primary producer communities

From Monoamine Oxidase Inhibition to Antiproliferative Activity: New Biological Perspectives for Polyamine Analogs

: Monoamine oxidases (MAOs) are well-known pharmacological targets in neurological and neurodegenerative diseases. However, recent studies have revealed a new role for MAOs in certain types of cancer such as glioblastoma and prostate cancer, in which they have been found overexpressed. This finding is opening new frontiers for MAO inhibitors as potential antiproliferative agents. In light of our previous studies demonstrating how a polyamine scaffold can act as MAO inhibitor, our aim was to search for novel analogs with greater inhibitory potency for human MAOs and possibly with antiproliferative activity. A small in-house library of polyamine analogs (2-7) was selected to investigate the effect of constrained linkers between the inner amine functions of a polyamine backbone on the inhibitory potency. Compounds 4 and 5, characterized by a dianiline (4) or dianilide (5) moiety, emerged as the most potent, reversible, and mainly competitive MAO inhibitors (Ki < 1 μM). Additionally, they exhibited a high antiproliferative activity in the LN-229 human glioblastoma cell line (GI50 < 1 μM). The scaffold of compound 5 could represent a potential starting point for future development of anticancer agents endowed with MAO inhibitory activity

Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3 epsilon

RSL3, a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating GPx4. Here we report the purification of the protein indispensable for GPx4 inactivation by RSL3. MS analysis reveals 14-3-3 isoforms as candidates and recombinant human 14-3-3epsilon confirms the identification. The function of 14-3-3\uf065 is redox-regulated. Moreover, overexpression and silencing of the gene coding for 14-3-3\uf065 consistently control the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox-regulated adaptor protein, operating in cell signalling, further contributes to frame it within redox-regulated pathways of cell survival and death and opens new therapeutic perspectives

Insight into the mechanism of ferroptosis inhibition by ferrostatin-1

Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and prevented by GPx4. Ferrostatin-1 (fer-1) inhibits ferroptosis much more efficiently than phenolic antioxidants. Previous studies on the antioxidant efficiency of fer-1 adopted kinetic tests where a diazo compound generates the hydroperoxyl radical scavenged by the antioxidant. However, this reaction, accounting for a chain breaking effect, is only minimally useful for the description of the inhibition of ferrous iron and lipid hydroperoxide dependent peroxidation. Scavenging lipid hydroperoxyl radicals, indeed, generates lipid hydroperoxides from which ferrous iron initiates a new peroxidative chain reaction. We show that when fer-1 inhibits peroxidation, initiated by iron and traces of lipid hydroperoxides in liposomes, the pattern of oxidized species produced from traces of pre-existing hydroperoxides is practically identical to that observed following exhaustive peroxidation in the absence of the antioxidant. This supported the notion that the anti-ferroptotic activity of fer-1 is actually due to the scavenging of initiating alkoxyl radicals produced, together with other rearrangement products, by ferrous iron from lipid hydroperoxides. Notably, fer-1 is not consumed while inhibiting iron dependent lipid peroxidation. The emerging concept is that it is ferrous iron itself that reduces fer-1 radical. This was supported by electroanalytical evidence that fer-1 forms a complex with iron and further confirmed in cells by fluorescence of calcein, indicating a decrease of labile iron in the presence of fer-1. The notion of such as pseudo-catalytic cycle of the ferrostatin-iron complex was also investigated by means of quantum mechanics calculations, which confirmed the reduction of an alkoxyl radical model by fer-1 and the reduction of fer-1 radical by ferrous iron. In summary, GPx4 and fer-1 in the presence of ferrous iron, produces, by distinct mechanism, the most relevant anti-ferroptotic effect, i.e the disappearance of initiating lipid hydroperoxides

Genomic Screening to Identify Food Trees Potentially Dispersed by Precolonial Indigenous Peoples

Over millennia, Indigenous peoples have dispersed the propagules of non-crop plants through trade, seasonal migration or attending ceremonies; and potentially increased the geographic range or abundance of many food species around the world. Genomic data can be used to reconstruct these histories. However, it can be difficult to disentangle anthropogenic from non-anthropogenic dispersal in long-lived non-crop species. We developed a genomic workflow that can be used to screen out species that show patterns consistent with faunal dispersal or long-term isolation, and identify species that carry dispersal signals of putative human influence. We used genotyping-by-sequencing (DArTseq) and whole-plastid sequencing (SKIMseq) to identify nuclear and chloroplast Single Nucleotide Polymorphisms in east Australian rainforest trees (4 families, 7 genera, 15 species) with large (&gt;30 mm) or small (&lt;30 mm) edible fruit, either with or without a known history of use by Indigenous peoples. We employed standard population genetic analyses to test for four signals of dispersal using a limited and opportunistically acquired sample scheme. We expected different patterns for species that fall into one of three broadly described dispersal histories: (1) ongoing faunal dispersal, (2) post-megafauna isolation and (3) post-megafauna isolation followed by dispersal of putative human influence. We identified five large-fruited species that displayed strong population structure combined with signals of dispersal. We propose coalescent methods to investigate whether these genomic signals can be attributed to post-megafauna isolation and dispersal by Indigenous peoples

Determinazione simultanea della capacit\ue0\ua0 e dell'efficienza nell'inibizione della perossidazione lipidica del plasma e delle sue componenti.

L\u2019attivit\ue0 antiossidante del plasma \ue8 un importante parametro che deve essere considerato per valutare lo stress ossidativo di un individuo, soprattutto in relazione alle patologie di tipo cardiovascolare. Una delle principali cause di queste patologie \ue8 attribuibile al metabolismo lipidico ed in particolare alla perossidazione lipidica dei grassi insaturi presenti nelle lipoproteine. A questo proposito, si \ue8 messo a punto un metodo alternativo per valutare la capacit\ue0 e l\u2019efficienza degli antiossidanti endogeni e esogeni presenti nel plasma nel bloccare i radicali perossidici. La PRTC (Peroxyl Radical Trapping Capacity) \ue8 un parametro stechiometrico che viene espresso come moli di radicali perossidici bloccati da 1 litro di plasma, mentre la PRTE (Peroxyl Radical Trapping Efficiency) \ue8 un parametro cinetico che viene espresso come l\u2019inverso dell\u2019IC50, o volume del sistema in cui 1 litro di plasma dimezza la concentrazione dei radicali perossidici. Una rigorosa trattazione delle equazioni cinetiche che descrivono il processo di perossidazione lipidica e l\u2019analisi dettagliata dei tracciati ossigrafici, ottenuti misurando l\u2019inibizione del consumo di ossigeno in un sistema micellare in cui viene innescata l\u2019ossidazione radicalica, ha fornito importanti informazioni sull\u2019azione del plasma e delle sue componenti a basso e ad alto peso molecolare. Se si analizza il profilo ossigrafico ottenuto dall\u2019inibizione della perossidazione lipidica da parte del plasma in toto, si individuano chiaramente due fasi di inibizione. La prima fase \ue8 caratterizzata da una cinetica di inibizione di ordine zero, con 100% di efficienza nel bloccare i radicali perossidici, la seconda fase \ue8 caratterizzata invece da una cinetica di primo ordine. Considerando il contributo dei singoli antiossidanti presenti nel plasma e i valori di capacit\ue0 ed efficienza delle singole fasi si evidenzia un meccanismo di sinergia che si manifesta mediante il trasferimento di attivit\ue0 antiossidante da parte delle componenti a basso peso molecolare, caratterizzate da una bassa efficienza, alle frazioni ad alto peso molecolare. Questo processo \ue8 dovuto principalmente all\u2019efficiente riciclo del tocoferolo presente nelle lipoproteine, da parte del radicale ascorbile che si forma dalla reazione dell\u2019acido ascorbico con i radicali perossidici. Il metodo messo a punto permette di individuare, da una singola misura e in maniera rigorosa, la capacit\ue0 e l\u2019efficienza di inibizione della perossidazione lipidica del plasma in toto e delle sue frazion

SNP dataset for georeferenced samples of Tristaniopsis collina, T. laurina

Single line SNP markers (0,1,2) for 238 samples of Tristaniopsis collina and 301 T. laurina samples collected in the field. SNP calling performed by Diversity Arrays Technology, Canberra. See website for details (https://www.diversityarrays.com/). Geolocation data for samples in separate files