Studio del trasporto nucleocitoplasmatico in una condizione di assemblaggio difettivo della Lamina Nucleare

La Lamina Nucleare (LN) è una struttura proteica filamentosa situata sul versante nucleoplasmatico della membrana nucleare interna, che fornisce una ''intelaiatura'' per il nucleo della cellula, àncora i Complessi del Poro Nucleare (NPC) e concorre nell'organizzazione dell'eterocromatina periferica. Le mutazioni a carico dei geni delle lamìne (le proteine che costituiscono la Lamina) provocano una serie di malattie che prendono il nome di ''laminopatie''. Tra di esse, la Sindrome di Hutchinson-Gilford (HGPS), nota anche come Progeria, è una grave patologia che causa invecchiamento precoce nei bambini. Nella maggior parte dei casi, l'HGPS deriva da una sostituzione de novo C1824T nel gene della làmina A nucleare (LMNA) che attiva un sito donatore di splicing criptico. L'evento di splincing promosso dalla mutazione provoca una delezione interna di 50 amminoacidi che impedisce il normale processamento della proteina LaA (chiamata, nella sua forma mutante, progerina). Nei nuclei di cellule affette da HGPS appaiono frequentemente erniazioni e vesciche a livello dell'Involucro Nucleare e un ispessimento della LN. Se il difetto genetico e proteico alla base della malattia è stato ampiamente compreso, non è noto, invece, l'effetto che l'accumulo di progerina possa avere sulle proprietà intrinseche del trasporto nucleocitoplasmatico attivo e passivo attraverso gli NPC. Tramite l'impiego della tecnica FRAP (Fluorescenze recovery after photobleaching), è stato valutato se il processo di import nucleare risulta alterato in caso di HGPS, considerando la connessione evidente tra NL e NPC. In particolare, i risultati della FRAP sono interpetati tramite un consolidato modello matematico costruito per lo studio della traslocazione nucleoplasmatica sia attiva che passiva. Gli esperimenti sono stati effettuati su cellule in coltura di tipo U2OS, cotrasfettate transientemente al fine di far esprimere loro la progerina e 1) una proteina di fusione di tipo NLS-EGFP, avente funzione di cargo da importare attivamente nel nucleo e 2) EGFP per lo studio del solo trasporto passivo. Sorprendentemente, i risultati dello studio FRAP hanno dimostrato che non sussiste alcuna alterazione della cinetica del trasporto nucleocitoplasmatico attivo e passivo, suggerendo che l'assemblamento scorretto della Lamina Nucleare nell'HGPS non influenza il trasporto attraverso gli NPC

Probing the role of nuclear-envelope invaginations in the nuclear-entry route of lipofected DNA by multi-channel 3D confocal microscopy.

Nuclear breakdown was found to be the dominant route for DNA entry into the nucleus in actively dividing cells. The possibility that alternative routes contribute to DNA entry into the nucleus, however, cannot be ruled out. Here we address the process of lipofection by monitoring the localization of fluorescently-labelled DNA plasmids at the single-cell level by confocal imaging in living interphase cells. As test formulation we choose the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE) with plasmidic DNA pre-condensed by means of protamine. By exploiting the spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br) we monitor the position of the nuclear envelope (NE), while concomitantly imaging the whole nucleus (by Hoechst) and the DNA (by Cy3 fluorophore) in a multi-channel 3D confocal imaging experiment. Reported results show that DNA clusters are typically associated with the NE membrane in the form of tubular invaginations spanning the nuclear environment, but not completely trapped within the NE invaginations, i.e. the DNA may use these NE regions as entry-points towards the nucleus. These observations pave the way to investigating the molecular details of the postulated processes for a better exploitation of gene-delivery vectors, particularly for applications in non-dividing cells

Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living β-cells

The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living β-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average diffusivity, and anomalous coefficient of ISGs, without the need to extract individual trajectories. Clustering of structural and dynamic quantities in a multidimensional parametric space defines the ISGs’ properties for different conditions. First, we create a reference using INS-1E cells expressing proinsulin fused to a fluorescent protein (FP) under basal culture conditions and validate our analysis by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload. After, we investigate the effect of FP-tagged ISG protein markers on the structural and dynamic properties of the granule. While iMSD analysis produces similar results for most of the lumenal markers, the transmembrane marker phogrin-FP shows a clearly altered result. Phogrin overexpression induces a substantial granule enlargement and higher mobility, together with a partial de-polymerization of the actin cytoskeleton, and reduced cell responsiveness to glucose stimulation. Our data suggest a more careful interpretation of many previous ISG-based reports in living β-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions

Metabolic response of Insulinoma 1E cells to glucose stimulation studied by fluorescence lifetime imaging

A cascade of highly regulated biochemical processes connects glucose stimulation to insulin secretion in specialized cells of mammalian pancreas, the β-cells. Given the importance of this process for systemic glucose homeostasis, noninvasive and fast strategies capable to monitor the response to glucose in living cells are highly desirable. Here, we use the phasor-based approach to Fluorescence Lifetime IMaging (FLIM) microscopy to quantify the ratio between protein-bound and free Nicotinamide adenine dinucleotide (phosphate) species in their reduced form (NAD(P)H), and the Insulinoma cell line INS-1E as a β-like cellular model. Phasor-FLIM analysis shows that the bound/free ratio of NAD(P)H species increases upon pulsed glucose stimulation. Such response is impaired by 48-hours preincubation of cells under hyperglycemic conditions. Phasor-FLIM concomitantly monitors the appearance of long-lifetime species (LLS) as characteristic products of hyperglycemia-induced oxidative stress

Single-cell imaging of α and β cell metabolic response to glucose in living human Langerhans islets

Here we use a combination of two-photon Fluorescence Lifetime Imaging Microscopy (FLIM) of NAD(P)H free/bound ratio in living HIs with post-fixation, immunofluorescence-based, cell-type identification. FLIM allowed to measure variations in the NAD(P)H free/bound ratio induced by glucose; immunofluorescence data allowed to identify single α and β cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 β cells from a cohort of 4 healthy donors, 15 total islets, were measured. Both α and β cells display a wide spectrum of responses, towards either an increase or a decrease in NAD(P)H free/bound ratio. Yet, if single-cell data are averaged according to the respective donor and correlated to donor insulin secretion power, a non-random distribution of metabolic shifts emerges: robust average responses of both α and β cells towards an increase of enzyme-bound NAD(P)H belong to the donor with the lowest insulin-secretion power; by contrast, discordant responses, with α cells shifting towards an increase of free NAD(P)H and β cells towards an increase of enzyme-bound NAD(P)H, correspond to the donor with the highest insulin-secretion power. Overall, data reveal neat anti-correlation of tissue metabolic responses with respect to tissue insulin secretion power

Optimization and validation of a LC-HRMS method for aflatoxins determination in urine samples

Mycotoxins’ exposure by inhalation and/or dermal contact can occur in different branches of industry especially where heavily dusty settings are present and the handling of dusty commodities is performed. This study aims to explore the possible contribution of the occupational exposure to aflatoxins by analysing urine samples for the presence of aflatoxins B1 and M1 and aflatoxin B1-N7-guanine adduct. The study was conducted in 2017 on two groups of volunteers, the workers group, composed by personnel employed in an Italian feed plant (n = 32), and a control group (n = 29), composed by the administrative employees of the same feed plant; a total of 120 urine samples were collected and analysed. A screening method and a quantitative method with high-resolution mass spectrometry determination were developed and fully validated. Limits of detections were 0.8 and 1.5 pg/mLurine for aflatoxin B1 and M1, respectively. No quantitative determination was possible for the adduct aflatoxin B1-N7-guanine. Aflatoxin B1 and its adduct were not detected in the analysed samples, and aflatoxin M1, instead, was found in 14 samples (12%) within the range 1.9–10.5 pg/mLurine. Only one sample showed a value above the limit of quantification (10.5 pg/mLurine). The absence of a statistical difference between the mean values for workers and the control group which were compared suggests that in this specific setting, no professional exposure occurs. Furthermore, considering the very low level of aflatoxin M1 in the collected urine samples, the contribution from the diet to the overall exposure is to be considered negligible

Biomonitoring data for assessing aflatoxins and ochratoxin a exposure by italian feedstuffs workers

Mycotoxins exposure by inhalation and/or dermal contact is possible in different branches of industry especially where heavily dusty settings are present and the handling of dusty commodities is performed. This study aims to explore the validity of the biomonitoring as a tool to investigate the intake of mycotoxins in a population of workers operating in an Italian feed plant. Serum samples were collected for the determination of aflatoxins B1 (AFB1), AFB1-Lysine adduct and ochratoxin A (OTA). A method based on liquid-liquid extraction coupled with high resolution mass spectrometry determination was developed and fully validated. For AFB1, a high number of non-detected samples (90%) was found and no statistical difference was observed comparing workers and control group. None of the analyzed samples showed the presence of AFB1-Lysine adduct. For OTA, the 100% of the analyzed samples was positive with a 33% of the samples showing a concentration higher than the limit of quantification (LOQ), but no statistical difference was highlighted between the average levels of exposed and control groups. In conclusion, the presence of AFB1 and OTA in serum cannot be attributable to occupational exposure

Investigating the mechanism of action of DNA-loaded PEGylated lipid nanoparticles

PEGylated lipid nanoparticles (LNPs) are commonly used to deliver bioactive molecules, but the role of PEGylation in DNA-loaded LNP interactions at the cellular and subcellular levels remains poorly understood. In this study, we investigated the mechanism of action of DNA-loaded PEGylated LNPs using gene reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), and fluorescence confocal microscopy (FCS). We found that PEG has no significant impact on the size or nanostructure of DNA LNPs but reduces their zeta potential and interaction with anionic cell membranes. PEGylation increases the structural stability of LNPs and results in lower DNA unloading. FCS experiments revealed that PEGylated LNPs are internalized intact inside cells and largely shuttled to lysosomes, while unPEGylated LNPs undergo massive destabilization on the plasma membrane. These findings can inform the design, optimization, and validation of DNA-loaded LNPs for gene delivery and vaccine development

Fluorescence lifetime microscopy unveils the supramolecular organization of liposomal Doxorubicin

The supramolecular organization of Doxorubicin (DOX) within the standard Doxoves® liposomal formulation (DOX®) is investigated using visible light and phasor approach to fluorescence lifetime imaging (phasor-FLIM). First, the phasor-FLIM signature of DOX® is resolved into the contribution of three co-existing fluorescent species, each with its characteristic mono-exponential lifetime, namely: crystallized DOX (DOXc, 0.2 ns), free DOX (DOXf, 1.0 ns), and DOX bound to the liposomal membrane (DOXb, 4.5 ns). Then, the exact molar fractions of the three species are determined by combining phasor-FLIM with quantitative absorption/fluorescence spectroscopy on DOXc, DOXf, and DOXb pure standards. The final picture on DOX® comprises most of the drug in the crystallized form (∼98%), with the remaining fractions divided between free (∼1.4%) and membrane-bound drug (∼0.7%). Finally, phasor-FLIM in the presence of a DOX dynamic quencher allows us to suggest that DOXf is both encapsulated and non-encapsulated, and that DOXb is present on both liposome-membrane leaflets. We argue that the present experimental protocol can be applied to the investigation of the supramolecular organization of encapsulated luminescent drugs/molecules all the way from the production phase to their state within living matter

Neurotrophic Activity and Its Modulation by Zinc Ion of a Dimeric Peptide Mimicking the Brain-Derived Neurotrophic Factor N-Terminal Region

Brain-derived neurotrophic factor (BDNF) is a neurotrophin (NT) essential for neuronal development and synaptic plasticity. Dysregulation of BDNF signaling is implicated in different neurological disorders. The direct NT administration as therapeutics has revealed to be challenging. This has prompted the design of peptides mimicking different regions of the BDNF structure. Although loops 2 and 4 have been thoroughly investigated, less is known regarding the BDNF N-terminal region, which is involved in the selective recognition of the TrkB receptor. Herein, a dimeric form of the linear peptide encompassing the 1-12 residues of the BDNF N-terminal (d-bdnf) was synthesized. It demonstrated to act as an agonist promoting specific phosphorylation of TrkB and downstream ERK and AKT effectors. The ability to promote TrkB dimerization was investigated by advanced fluorescence microscopy and molecular dynamics (MD) simulations, finding activation modes shared with BDNF. Furthermore, d-bdnf was able to sustain neurite outgrowth and increase the expression of differentiation (NEFM, LAMC1) and polarization markers (MAP2, MAPT) demonstrating its neurotrophic activity. As TrkB activity is affected by zinc ions in the synaptic cleft, we first verified the ability of d-bdnf to coordinate zinc and then the effect of such complexation on its activity. The d-bdnf neurotrophic activity was reduced by zinc complexation, demonstrating the role of the latter in tuning the activity of the new peptido-mimetic. Taken together our data uncover the neurotrophic properties of a novel BDNF mimetic peptide and pave the way for future studies to understand the pharmacological basis of d-bdnf action and develop novel BDNF-based therapeutic strategies