1,720,989 research outputs found
Protein clustering in chemically stressed HeLa cells studied by infrared nanospectroscopy
Full text linkPhoto-Thermal Induced Resonance (PTIR) nanospectroscopy, tuned towards amide-I absorption, was used to study the distribution of proteic material in 34 different HeLa cells, of which 18 were chemically stressed by oxidative stress with Na3AsO3. The cell nucleus was found to provide a weaker amide-I signal than the surrounding cytoplasm, while the strongest PTIR signal comes from the perinuclear region. AFM topography shows that the cells exposed to oxidative stress undergo a volume reduction with respect to the control cells, through an accumulation of the proteic material around and above the nucleus. This is confirmed by the PTIR maps of the cytoplasm, where the pixels providing a high amide-I signal were identified with a space resolution of ∼300 × 300 nm. By analyzing their distribution with two different statistical procedures we found that the probability to find protein clusters smaller than 0.6 μm in the cytoplasm of stressed HeLa cells is higher by 35% than in the control cells. These results indicate that it is possible to study proteic clustering within single cells by label-free optical nanospectroscopy
Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors
No full textWe describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings
Live-cell imaging reveals that SMG6 and UPF1 inhibit release of nonsense mRNA from the transcription site
No full textDirect conversion of human pluripotent stem cells into cranial motor neurons using a piggyBac vector
Full text linkHuman pluripotent stem cells (PSCs) are widely used for in vitro disease modeling. One of the challenges in the field is represented by the ability of converting human PSCs into specific disease-relevant cell types. The nervous system is composed of a wide variety of neuronal types with selective vulnerability in neurodegenerative diseases. This is particularly relevant for motor neuron diseases, in which different motor neurons populations show a different susceptibility to degeneration. Here we developed a fast and efficient method to convert human induced Pluripotent Stem Cells into cranial motor neurons of the branchiomotor and visceral motor subtype. These populations represent the motor neuron subgroup that is primarily affected by a severe form of amyotrophic lateral sclerosis with bulbar onset and worst prognosis. This goal was achieved by stable integration of an inducible vector, based on the piggyBac transposon, allowing controlled activation of Ngn2, Isl1 and Phox2a (NIP). The NIP module effectively produced electrophysiologically active cranial motor neurons. Our method can be easily extended to PSCs carrying disease-associated mutations, thus providing a useful tool to shed light on the cellular and molecular bases of selective motor neuron vulnerability in pathological conditions
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Humanized archaeal ferritin as a tool for cell targeted delivery
No full textHuman ferritins have been extensively studied to be used as nanocarriers for diverse applications and could represent a convenient alternative for targeted delivery of anticancer drugs and imaging agents. However, the most relevant limitation to their applications is the need for highly acidic experimental conditions during the initial steps of particle/cargo assembly, a process that could affect both drug stability and the complete reassembly of the ferritin cage. To overcome this issue the unique assembly of Archaeoglobus fulgidus ferritin was genetically engineered by changing a surface exposed loop of 12 amino acids connecting B and C helices to mimic the sequence of the analogous human H-chain ferritin loop. This new chimeric protein was shown to maintain the unique, cation linked, association–dissociation properties of Archaeoglobus fulgidus ferritin occurring at neutral pH values, while exhibiting the typical human H-homopolymer recognition by the transferrin receptor TfR1. The chimeric protein was confirmed to be actively and specifically internalized by HeLa cells, thus representing a unique nanotechnological tool for cell-targeted delivery of possible payloads for diagnostic or therapeutic purposes. Moreover, it was demonstrated that the 12 amino acids’ loop is necessary and sufficient for binding to the transferrin receptor. The three-dimensional structure of the humanized Archaeoglobus ferritin has been obtained both as crystals by X-ray diffraction and in solution by cryo-EM
Esculentin-1a derivatives as new antipseudomonal agents: limited induction of resistance and inhibition of biofilm formation
No full textCationic α-helical antimicrobial peptides (AMPs) hold promise as new therapeutics thanks to their broad spectrum of activity and membrane-perturbing mechanism of action. These features make them interesting compounds compared to conventional antibiotics that easily induce microorganisms to acquire resistance to them. One of these microbes is the opportunist Gram-negative bacterium Pseudomonas aeruginosa. It colonizes abiotic surfaces and tissues growing in a more resistant form, named biofilm. Recently, two derivatives of the AMP esculentin-1a, e.g. Esc(1-21) and its diastereomer Esc(1-21)-1c [Esc-peptides] were characterized for their activity against both planktonic and biofilm forms of P. aeruginosa [1,2]. The ability of these peptides to limit the onset of microbial resistance was evaluated by exposing Pseudomonas strains to repeated treatments with different concentrations of each peptide and the results were compared to conventional antibiotics. Interestingly, while after 15 cycles of drug exposure, the minimal growth inhibitory concentrations (MICs) of ciprofloxacin, aztreonam, colistin and tobramycin were found to be 8 to 128-fold higher than the initial ones, the MICs of Esc-peptides did not change. In addition, while sub-MIC levels of antibiotics stimulated Pseudomonas biofilm formation, the D-amino acid containing Esc(1-21)-1c inhibited its formation. This could be explained by the peptide’ binding to the bacterial signaling nucleotide ppGpp, with consequent reduction in the expression of key genes involved in bacterial virulence. Overall, these results suggest Esc-peptides, particularly Esc(1-21)-1c, as promising candidates for the development of new antimicrobials. [1] Luca V. et al, Cell Mol Life Sci. 2013 Aug;70(15):2773-86; [2] Di Grazia A. et al, Amino Acids. 2015 Dec;47(12):2505-19; Acknowledgments: This work was supported by grants from Sapienza University and from the Italian Cystic Fibrosis Foundation (project FFC 15/2017
FUS mutant human motoneurons display altered transcriptome and microRNA pathways with implications for ALS pathogenesis
Full text linkThe FUS gene has been linked to amyotrophic lateral sclerosis (ALS). FUS is a ubiquitous RNA-binding protein, and the mechanisms leading to selective motoneuron loss downstream of ALS-linked mutations are largely unknown. We report the transcriptome analysis of human purified motoneurons, obtained from FUS wild-type or mutant isogenic induced pluripotent stem cells (iPSCs). Gene ontology analysis of differentially expressed genes identified significant enrichment of pathways previously associated to sporadic ALS and other neurological diseases. Several microRNAs (miRNAs) were also deregulated in FUS mutant motoneurons, including miR-375, involved in motoneuron survival. We report that relevant targets of miR-375, including the neural RNA-binding protein ELAVL4 and apoptotic factors, are aberrantly increased in FUS mutant motoneurons. Characterization of transcriptome changes in the cell type primarily affected by the disease contributes to the definition of the pathogenic mechanisms of FUS-linked ALS
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