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The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine.Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application.The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed.
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Glioblastoma, a deadly brain tumor, shows limited response to standard therapies like temozolomide (TMZ). Recent findings from the REGOMA trial underscore a significant survival improvement offered by Regorafenib (REGO) in recurrent glioblastoma. Our study aimed to propose a 3D ex vivo drug response precision medicine approach to investigate recurrent glioblastoma sensitivity to REGO and elucidate the underlying molecular mechanisms involved in tumor resistance or responsiveness to treatment. Three-dimensional glioblastoma organoids (GB-EXPs) obtained from 18 patients' resected recurrent glioblastoma tumors were treated with TMZ and REGO. Drug responses were evaluated using NAD(P)H FLIM, stratifying tumors as responders (Resp) or non-responders (NRs). Whole-exome sequencing was performed on 16 tissue samples, and whole-transcriptome analysis on 13 GB-EXPs treated and untreated. We found 35% (n = 9) and 77% (n = 20) of tumors responded to TMZ and REGO, respectively, with no instances of TMZ-Resp being REGO-NRs. Exome analysis revealed a unique mutational profile in REGO-Resp tumors compared to NR tumors. Transcriptome analysis identified distinct expression patterns in Resp and NR tumors, impacting Rho GTPase and NOTCH signaling, known to be involved in drug response. In conclusion, recurrent glioblastoma tumors were more responsive to REGO compared to TMZ treatment. Importantly, our approach enables a comprehensive longitudinal exploration of the molecular changes induced by treatment, unveiling promising biomarkers indicative of drug response.
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Glioblastoma , Compostos de Fenilureia , Piridinas , Humanos , Antineoplásicos Alquilantes/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Recidiva Local de Neoplasia/patologia , Temozolomida/farmacologiaRESUMO
Oxygen is essential for plant growth and development. Hypoxia occurs in plants due to limited oxygen availability following adverse environmental conditions as well in hypoxic niches in otherwise normoxic environments. However, the existence and functional integration of spatiotemporal oxygen dynamics with plant development remains unknown. In animal systems dynamic fluctuations in oxygen availability are known as cyclic hypoxia. In this study, we demonstrate that cyclic fluctuations in internal oxygen levels occur in young emerging leaves of Arabidopsis plants. Cyclic hypoxia in plants is based on a mechanism requiring the ETHYLENE RESPONSE FACTORS type VII (ERFVII) that are central components of the oxygen-sensing machinery in plants. The ERFVII-dependent mechanism allows precise adjustment of leaf growth in response to carbon status and oxygen availability within plant cells. This study thus establishes a functional connection between internal spatiotemporal oxygen dynamics and developmental processes of plants.
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Proteínas de Arabidopsis , Arabidopsis , Etilenos , Oxigênio/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Hipóxia , Folhas de Planta/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
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.
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Lipídeos , Nanopartículas , Lipídeos/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Nanopartículas/química , DNA , Polietilenoglicóis/química , RNA Interferente PequenoRESUMO
Lipid nanoparticles (LNPs) are currently having an increasing impact on nanomedicines as delivery agents, among others, of RNA molecules (e.g., short interfering RNA for the treatment of hereditary diseases or messenger RNA for the development of COVID-19 vaccines). Despite this, the delivery of plasmid DNA (pDNA) by LNPs in preclinical studies is still unsatisfactory, mainly due to the lack of systematic structural and functional studies on DNA-loaded LNPs. To tackle this issue, we developed, characterized, and tested a library of 16 multicomponent DNA-loaded LNPs which were prepared by microfluidics and differed in lipid composition, surface functionalization, and manufacturing factors. 8 out of 16 formulations exhibited proper size and zeta potential and passed to the validation step, that is, the simultaneous quantification of transfection efficiency and cell viability in human embryonic kidney cells (HEK-293). The most efficient formulation (LNP15) was then successfully validated both in vitro, in an immortalized adult keratinocyte cell line (HaCaT) and in an epidermoid cervical cancer cell line (CaSki), and in vivo as a nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice. Finally, by a combination of confocal microscopy, transmission electron microscopy and synchrotron small-angle X-ray scattering, we were able to show that the superior efficiency of LNP15 can be linked to its disordered nanostructure consisting of small-size unoriented layers of pDNA sandwiched between closely apposed lipid membranes that undergo massive destabilization upon interaction with cellular lipids. Our results provide new insights into the structure-activity relationship of pDNA-loaded LNPs and pave the way to the clinical translation of this gene delivery technology.
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COVID-19 , Nanopartículas , Animais , Camundongos , Humanos , Vacinas contra COVID-19 , Células HEK293 , Lipídeos/química , Camundongos Endogâmicos C57BL , DNA/química , Nanopartículas/química , RNA Interferente PequenoRESUMO
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.
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Fator Neurotrófico Derivado do Encéfalo , Zinco , Zinco/farmacologiaRESUMO
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 ß cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 ß cells from a cohort of 4 healthy donors, 15 total islets, were measured. Both α and ß 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 ß 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 ß 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.
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Glucose , Ilhotas Pancreáticas , Humanos , Glucose/metabolismo , NAD/metabolismo , NADP/metabolismo , Ilhotas Pancreáticas/metabolismo , Insulina/metabolismoRESUMO
Background: Glioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) treatment, and radiotherapy remain the primary therapeutic options for GB, and no new therapies have been introduced in recent years. This therapeutic standstill is primarily due to preclinical approaches that do not fully respect the complexity of GB cell biology and fail to test efficiently anti-cancer treatments. Therefore, better treatment screening approaches are needed. In this study, we have developed a novel functional precision medicine approach to test the response to anticancer treatments in organoids derived from the resected tumors of glioblastoma patients. Methods: GB organoids were grown for a short period of time to prevent any genetic and morphological evolution and divergence from the tumor of origin. We chose metabolic imaging by NAD(P)H fluorescence lifetime imaging microscopy (FLIM) to predict early and non-invasively ex-vivo anti-cancer treatment responses of GB organoids. TMZ was used as the benchmark drug to validate the approach. Whole-transcriptome and whole-exome analyses were performed to characterize tumor cases stratification. Results: Our functional precision medicine approach was completed within one week after surgery and two groups of TMZ Responder and Non-Responder tumors were identified. FLIM-based metabolic tumor stratification was well reflected at the molecular level, confirming the validity of our approach, highlighting also new target genes associated with TMZ treatment and identifying a new 17-gene molecular signature associated with survival. The number of MGMT gene promoter methylated tumors was higher in the responsive group, as expected, however, some non-methylated tumor cases turned out to be nevertheless responsive to TMZ, suggesting that our procedure could be synergistic with the classical MGMT methylation biomarker. Conclusions: For the first time, FLIM-based metabolic imaging was used on live glioblastoma organoids. Unlike other approaches, ex-vivo patient-tailored drug response is performed at an early stage of tumor culturing with no animal involvement and with minimal tampering with the original tumor cytoarchitecture. This functional precision medicine approach can be exploited in a range of clinical and laboratory settings to improve the clinical management of GB patients and implemented on other cancers as well.
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DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field to enhance DNA penetration into cells, has been the method of choice to produce acceptable levels of gene transfer in vivo. However, this method may cause cell damage or rupture, non-specific targeting, and even degradation of pDNA. Skin irritation, muscle contractions, pain, alterations in skin structure, and irreversible cell damage have been frequently reported. To overcome these limitations, in this work, we use a microfluidic platform to generate DNA-loaded lipid nanoparticles (LNPs) which are then characterized by a combination of dynamic light scattering (DLS), synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Despite the clinical successes obtained by LNPs for mRNA and siRNA delivery, little is known about LNPs encapsulating bulkier DNA molecules, the clinical application of which remains challenging. For in vitro screening, LNPs were administered to human embryonic kidney 293 (HEK-293) and Chinese hamster ovary (CHO) cell lines and ranked for their transfection efficiency (TE) and cytotoxicity. The LNP formulation exhibiting the highest TE and the lowest cytotoxicity was then tested for the delivery of the DNA vaccine pVAX-hECTM targeting the human neoantigen HER2, an oncoprotein overexpressed in several cancer types. Using fluorescence-activated cell sorting (FACS), immunofluorescence assays and fluorescence confocal microscopy (FCS), we proved that pVAX-hECTM-loaded LNPs produce massive expression of the HER2 antigen on the cell membrane of HEK-293 cells. Our results provide new insights into the structure-activity relationship of DNA-loaded LNPs and pave the way for the access of this gene delivery technology to preclinical studies.
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Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more detail, Raster Image Correlation Spectroscopy (RICS) performed at fast timescales (i.e., microseconds) reveals the fast motion of fluorescently labeled molecules within two exemplary dynamic subcellular nanostructures of biomedical interest, the lysosome and the insulin secretory granule (ISG). The measurement of molecular diffusion is then used to extract information on the average properties of subcellular nanostructures, such as macromolecular crowding or molecular aggregation. Concerning the lysosome, fast RICS on a fluorescent tracer allowed us to quantitatively assess the increase in organelle viscosity in the pathological condition of Krabbe disease. In the case of ISGs, fast RICS on two ISG-specific secreting peptides unveiled their differential aggregation propensity depending on intragranular concentration. Finally, a combination of fast RICS and feedback-based 3D orbital tracking was used to subtract the slow movement of subcellular nanostructures from the fast diffusion of molecules contained within them and independently validate the results. Results presented here not only demonstrate the acquired ability to address the dynamic behavior of molecules in moving, nanoscopic reference systems, but prove the relevance of this approach to advance our knowledge on cell function at the subcellular scale.
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Nanoestruturas , Transporte Biológico , Difusão , Movimento (Física) , Espectrometria de Fluorescência/métodosRESUMO
New technologies with the capacity to tune immune system activity are highly desired in clinical practice and disease management. Here we demonstrate that nanoparticles with a protein corona enriched with gelsolin (GSN), an abundant plasma protein that acts as a modulator of immune responses, are avidly captured by human monocytic THP-1 cells in vitro and by leukocyte subpopulations derived from healthy donors ex vivo. In human monocytes, GSN modulates the production of tumor necrosis factor alpha (TNF-α) in an inverse dose-dependent manner. Overall, our results suggest that artificial coronas can be exploited to finely tune the immune response, opening new approaches for the prevention and treatment of diseases.
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Coroa de Proteína , Humanos , Imunidade , Imunomodulação , Monócitos/metabolismo , Coroa de Proteína/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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 (â¼98%), with the remaining fractions divided between free (â¼1.4%) and membrane-bound drug (â¼0.7%). 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.
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Doxorrubicina , Polietilenoglicóis , Doxorrubicina/análogos & derivados , Doxorrubicina/química , Lipossomos , Microscopia de Fluorescência/métodosRESUMO
The availability of a reliable molecular assay in species recognition in forensic cases is of paramount importance when visual inspection or morphological methods are not exhaustive, especially from challenging samples. Here, two different caseworks involving bone samples founded during medico-legal outdoor investigations are presented. In order to exclude the human nature of the specimens and to determine the exact species they belong to, we proceeded with the molecular approach trying to generate sequences from the classical mtDNA markers cyt b and COI. However, they both gave critical results. For this reason, a short amplicon of ~ 150 bp of the 12S rRNA gene was used as an alternative.This short fragment was sufficient to identify the biological origin of the bone specimens with a high degree of certainty leading to the exclusion of their human nature. This work highlights the utility of the 12S rRNA and underlines the importance of deepen the choice of alternative shorter markers with respect to the classical ones, in order to achieve species identification even from challenging and degraded material in forensic criminal and wildlife caseworks.
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DNA Mitocondrial , RNA Ribossômico , Primers do DNA/genética , DNA Mitocondrial/genética , Humanos , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico/genéticaRESUMO
ß-cells convert glucose (input) resulting in the controlled release of insulin (output), which in turn has the role to maintain glucose homeostasis. ß-cell function is regulated by a complex interplay between the metabolic processing of the input, its transformation into second-messenger signals, and final mobilization of insulin-containing granules towards secretion of the output. Failure at any level in this process marks ß-cell dysfunction in diabetes, thus making ß-cells obvious potential targets for therapeutic purposes. Addressing quantitatively ß-cell (dys)function at the molecular level in living samples requires probing simultaneously the spatial and temporal dimensions at the proper resolution. To this aim, an increasing amount of research efforts are exploiting the potentiality of biophysical techniques. In particular, using excitation light in the visible/infrared range, a number of optical-microscopy-based approaches have been tailored to the study of ß-cell-(dys)function at the molecular level, either in label-free mode (i.e., exploiting intrinsic autofluorescence of cells) or by the use of organic/genetically-encoded fluorescent probes. Here, relevant examples from the literature are reviewed and discussed. Based on this, new potential lines of development in the field are drawn.
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Corantes Fluorescentes/química , Glucose/metabolismo , Células Secretoras de Insulina/patologia , Microscopia de Fluorescência/métodos , Animais , Homeostase , Humanos , Células Secretoras de Insulina/metabolismoRESUMO
Plants seem to take up exogenous RNA that was artificially designed to target specific genes, followed by activation of the RNA interference (RNAi) machinery. It is, however, not known whether plants use RNAs themselves as signalling molecules in plant-to-plant communication, other than evidence that an exchange of small RNAs occurs between parasitic plants and their hosts. Exogenous RNAs from the environment, if taken up by some living organisms, can indeed induce RNAi. This phenomenon has been observed in nematodes and insects, and host Arabidopsis cells secrete exosome-like extracellular vesicles to deliver plant small RNAs into Botrytis cinerea. Here we show that micro-RNAs (miRNAs) produced by plants act as signalling molecules affecting gene expression in other, nearby plants. Exogenous miRNAs, such as miR156 and miR399, trigger RNAi via a mechanism requiring both AGO1 and RDR6. This emphasizes that the production of secondary small interfering RNAs is required. This evidence highlights the existence of a mechanism in which miRNAs represent signalling molecules that enable communication between plants.
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Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Interferência de RNA , RNA de Plantas/genética , Arabidopsis/metabolismoRESUMO
Glucagon-like peptide-1 receptor (GLP-1R) agonists are being used for the treatment of type 2 diabetes (T2D) and may have beneficial effects on the pancreatic ß-cells. Here, we evaluated the effects of GLP-1R agonism on insulin secretory granule (ISG) dynamics in primary ß-cells isolated from human islets exposed to palmitate-induced lipotoxic stress. Islets cells were exposed for 48 h to 0.5 mM palmitate (hereafter, 'Palm') with or without the addition of a GLP-1 agonist, namely 10 nM exendin-4 (hereafter, 'Ex-4'). Dissociated cells were first transfected with syncollin-EGFP in order to fluorescently mark the ISGs. Then, by applying a recently established spatiotemporal correlation spectroscopy technique, the average structural (i.e., size) and dynamic (i.e., the local diffusivity and mode of motion) properties of ISGs are extracted from a calculated imaging-derived Mean Square Displacement (iMSD) trace. Besides defining the structural/dynamic fingerprint of ISGs in human cells for the first time, iMSD analysis allowed to probe fingerprint variations under selected conditions: namely, it was shown that Palm affects ISGs dynamics in response to acute glucose stimulation by abolishing the ISGs mobilization typically imparted by glucose and, concomitantly, by reducing the extent of ISGs active/directed intracellular movement. By contrast, co-treatment with Ex-4 normalizes ISG dynamics, i.e., re-establish ISG mobilization and ability to perform active transport in response to glucose stimulation. These observations were correlated with standard glucose-stimulated insulin secretion (GSIS), which resulted in being reduced in cells exposed to Palm but preserved in cells concomitantly exposed to 10 nM Ex-4. Our data support the idea that GLP-1R agonism may exert its beneficial effect on human ß-cells under metabolic stress by maintaining ISGs' proper intracellular dynamics.
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BACKGROUND: Phoenician and Punic expansions have been protagonists of intense trade networks and settlements in the Mediterranean Sea. AIMS: The maternal genetic variability of ancient Punic samples from the Sardinian necropolis of Tharros was analysed, with the aim to explore genetic interactions and signatures of past population events. SUBJECTS AND METHODS: The mtDNA HVS-I and coding region SNPs were analysed in 14 Punic samples and 74 modern individuals from Cabras and Belvì (for which the HVS-II region was also analysed). The results were compared with 5,590 modern Euro-Mediterranean sequences and 127 ancient samples. RESULTS: While contemporary groups fall within the genetic variability of other modern Sardinians, our Punic samples reveal proximity to present-day North-African and Iberian populations. Furthermore, Cabras and Belvì cluster mainly with pre-Phoenician groups, while samples from Tharros project with other Punic Sardinian individuals. CONCLUSION: This study provides the first preliminary insights into the population dynamics of the Punic site of Tharros. While the number of currently available samples does not allow definitive investigation of the connection with indigenous Sardinian groups, our results seem to confirm internal migratory phenomena in the central-western Mediterranean and female participation in the Punic mobility.
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DNA Antigo/análise , DNA Mitocondrial/análise , Variação Genética , Migração Humana , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Itália , Masculino , Dinâmica Populacional , TunísiaRESUMO
Imaging-derived mean square displacement (iMSD) is used to address the structural and dynamic properties of subcellular nanostructures, such as vesicles involved in the endo/exocytotic trafficking of solutes and biomolecules. iMSD relies on standard time-lapse imaging, is compatible with any optical setup, and does not need to dwell on single objects to extract trajectories. From each iMSD trace, a unique triplet of average structural and dynamic parameters (i.e., size, local diffusivity, anomalous coefficient) is calculated and combined to build the "iMSD signature" of the nanostructure under study. The potency of this approach is proved here with the exemplary case of macropinosomes. These vesicles evolve in time, changing their average size, number, and dynamic properties passing from early to late stages of intracellular trafficking. As a control, insulin secretory granules (ISGs) are used as a reference for subcellular structures that live in a stationary state in which the average structural and dynamic properties of the whole population of objects are invariant in time. The iMSD analysis highlights these peculiar features quantitatively and paves the way to similar applications at the sub-cellular level, both in the physiological and pathological states.
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Endossomos , Nanoestruturas , Processamento de Imagem Assistida por Computador , Vesículas Secretórias , Análise EspectralRESUMO
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 3ß-[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.
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Lipossomos , Membrana Nuclear , DNA , Microscopia Confocal , TransfecçãoRESUMO
OBJECTIVES: Genetic drift and admixture are driving forces in human evolution, but their concerted impact to population evolution in historical times and at a micro-geographic scale is poorly assessed. In this study we test a demographic model encompassing both admixture and drift to the case of social-cultural isolates such as the so-called "Commons." MATERIALS AND METHODS: Commons are peculiar institutions of medieval origins whose key feature is the tight relationship between population and territory, mediated by the collective property of shared resources. Here, we analyze the Y-chromosomal genetic structure of four Commons (for a total of 366 samples) from the Central and Eastern Padana plain in Northern Italy. RESULTS: Our results reveal that all these groups exhibit patterns of significant diversity reduction, peripheral/outlier position within the Italian/European genetic space and high frequency of Common-specific haplogroups. By explicitly testing different drift-admixture models, we show that a drift-only model is more probable for Central Padana Commons, while additional admixture (~20%) from external population around the same time of their foundation cannot be excluded for the Eastern ones. DISCUSSION: Building on these results, we suggest central Middle Ages as the most probable age of foundation for three of the considered Commons, the remaining one pointing to late antiquity. We conclude that an admixture-drift model is particularly useful for interpreting the genetic structure and recent demographic history of small-scale populations in which social-cultural features play a significant role.