Nanomechanical and topographical imaging of living cells by Atomic Force Microscopy with colloidal probes

Atomic Force Microscopy (AFM) has a great potential as a tool to characterize mechanical and morphological properties of living cells; these properties have been shown to correlate with cells' fate and patho-physiological state in view of the development of novel early-diagnostic strategies. Although several reports have described experimental and technical approaches for the characterization of cell elasticity by means of AFM, a robust and commonly accepted methodology is still lacking. Here we show that micrometric spherical probes (also known as colloidal probes) are well suited for performing a combined topographic and mechanical analysis of living cells, with spatial resolution suitable for a complete and accurate mapping of cell morphological and elastic properties, and superior reliability and accuracy in the mechanical measurements with respect to conventional and widely used sharp AFM tips. We address a number of issues concerning the nanomechanical analysis, including the applicability of contact mechanical models and the impact of a constrained contact geometry on the measured elastic modulus (the finite-thickness effect). We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in order to demonstrate the importance of the correction of the finite-thickness effect and the change in cell elasticity induced by the action of a cytoskeleton-targeting drug.Comment: 51 pages, 12 figures, 3 table

dna based molecular detection of toxic phytoplankton in water samples different tools to get reliable information

The harmful phytoplankton holds sanitary, ecological and economic implications towards the human health, the coastal environments, and aquaculture facilities due to the consequences of recurrent harmful algal blooms (HABs). The adverse effects of HABs include toxin production, fish gill clogging, oxygen depletion and unpleasant water quality. The HABs are phenomena increasing worldwide for several reasons: eutrophication and/or unusual climatological conditions, the increased utilization of coastal waters for aquaculture, the movement of resting cysts caused by human activities (e.g. ships' ballast waters or the translocation of shellfish stocks), and overfishing (1). Nowadays, it is essential to have rapid, sensitive and reliable methods to be applied in monitoring programs of marine coastal ecosystems for accurately and specifically detecting HAB species. Such methods can allow investigating the HAB species distribution and dispersion mechanism, and facilitating the prevention or mitigation of the harmful effects on human health, marine ecosystem and economic related activities. In routine monitoring programs, the detection and quantification of harmful species are based on morphological recognition through microscope analyses, which are time consuming and require considerable taxonomic expertise. In fact, the presence of morphologically similar species co-existing in the marine environments (e.g. Pseudo-nitzschia spp.) or different morphotypes of the same species can sometimes affect the monitoring reliability. Moreover, in some cases the fixation by Lugol or formalin can cause a morphological cellular distortion. Nevertheless, the fixation processes are crucial to preserve samples during the time intercurring between sample collection and laboratory analysis. Due to these limitations, many molecular methods for HAB species monitoring have been developed in the last years. Because of the instability of RNA (particularly mRNA) and proteins, most detection tools used for phytoplankton rely on detecting DNA. Target DNA sequences commonly used for developing specific primers and probes are rRNA genes, which are phylogenetically informative and tandemly repeated in high copy number. Molecular approaches to species identification and quantification may be broadly categorized as "whole cell" or "lysed cell" methods. In the "whole cell" methods the cells remain intact throughout sampling and processing (e.g. FISH); in the "lysed cell" methods, cells are disrupted and the resulting cell homogenate is analyzed, typically with subsequent calibration of the fluorescence or colorimetric signal back to cell number (e.g. sandwich hybridization, microarray hybridization, real-time qPCR). Advantages of the "lysed cell" approaches for bloom monitoring include amenability to automation and the possibility to analyze larger environmental sample volumes, thereby avoiding errors inherent in the small sample size used for whole cell methods. Disadvantages include the fact that it is not possible to visually inspect samples (e.g. to verify that the positive results are not due to crossreactions with non-target organisms), and that all sources of target sequences can contribute to the signal (including senescent/dead cells or cells obscured in food vacuoles or fecal pellets)

ddit4 gene expression is switched on by a new hdac4 function in ataxia telangiectasia

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Identification of the main ubiquitination site in human erythroid α-spectrin

AbstractErythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid α-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of α-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in α-spectrin and may be present in several other proteins

Identification of ä-Spectrin Domains Susceptible to Ubiquitination

Previously, we demonstrated that alpha-spectrin is a substrate for the ubiquitin system and that this conjugation is a dynamic process (Corsi, D., Galluzzi, L., Crinelli, R., and Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935). In this study, we mapped the sites of ubiquitination on erythrocyte alpha-spectrin. A peptide map of digested alpha-spectrin, previously submitted to in vitro 125I-ubiquitin conjugation, revealed the presence of four distinct labeled bands with Mr 40,000, 36,000, 29,000, and 25,500. Western blotting experiments using antibodies against each alpha-spectrin domain revealed that only IgG anti-alphaIII domain recognized the 125I-labeled ubiquitin peptide of 29 kDa, whereas the IgG anti-alphaV domain recognized the Mr 40,000 125I-ubiquitin-labeled peptide. The other two labeled bands of Mr 36,000 and Mr 25,500 were identified as tetra and tri multiubiquitin chains. Ubiquitination of the alphaIII and alphaV domains was further confirmed by anti-alpha-spectrin domain immunoaffinity chromatography. Endoprotease Lys C-digested spectrin conjugated previously to 125I-ubiquitin was incubated with antibodies against each trypsin-resistant domain of alpha-spectrin. Gamma counting of the radiolabeled antigen-antibody complexes purified by protein A chromatography showed labeling in the IgG anti-alphaIII and anti-alphaV complexes alone. Domain alphaIII is not associated with any known function, whereas domain alphaV contains the nucleation site for the association of the alpha and beta chains. Ubiquitination of the latter domain suggests a role for ubiquitin in the modulation of the stability, deformability, and viscoelastic properties of the erythrocyte membrane

In Vitro Reduced Susceptibility to Pentavalent Antimonials of a Leishmania infantum Isolate from a Human Cutaneous Leishmaniasis Case in Central Italy

Cutaneous leishmaniasis (CL) caused by Leishmania (Leishmania) infantum is endemic in the Mediterranean basin. Here we report an autochthonous case of CL in a patient living in central Italy with an unsatisfactory response to treatment with intralesional Meglumine Antimoniate and in vitro demonstration of reduced susceptibility to SbIII. Parasitological diagnosis was first achieved by histopathology on tissue biopsy and the patient was treated with a local infiltration of Meglumine Antimoniate. Since the clinical response at 12 weeks from the treatment’s onset was deemed unsatisfactory, two further skin biopsies were taken for histopathological examination, DNA extraction and parasite isolation. L. (L.) infantum was identified by molecular typing. The low susceptibility to Meglumine Antimoniate was confirmed in vitro: the promastigotes from the patient strain showed significantly lower susceptibility to SbIII (the active trivalent form of antimonial) compared to the reference strain MHOM/TN/80/IPT1. The patient underwent a new treatment course with intravenous liposomal Amphotericin B, reaching complete healing of the lesion. Additional studies are needed to confirm the epidemiological and clinical relevance of reduced susceptibility to SbIII of human L. (L.) infantum isolate in Italy

The host micro-RNA cfa-miR-346 is induced in canine leishmaniasis

none8noBackground Leishmaniases are a group of anthropo-zoonotic parasitic diseases caused by a protozoan of the Leishmania genus, affecting both humans and other vertebrates, including dogs. L. infantum is responsible for the visceral and occasionally cutaneous form of the disease in humans and canine leishmaniasis. Previously, we have shown that L. infantum induces a mild but significant increase in endoplasmic reticulum (ER) stress expression markers to promote parasites survival in human and murine infected macrophages. Moreover, we demonstrated that the miRNA hsa-miR-346, induced by the UPR-activated transcription factor sXBP1, was significantly upregulated in human macrophages infected with different L. infantum strains. However, the ER stress response in infected dogs, which represent an important reservoir for Leishmania parasite, was described once recently, whereas the miR-346 expression was not reported before. Therefore, this study aimed to investigate these pathways in the canine macrophage-like cell line DH82 infected by Leishmania spp. and to evaluate the presence of cfa-miR-346 in plasma of non-infected and infected dogs. The DH82 cells were infected with L. infantum and L. braziliensis parasites and the expression of cfa-mir-346 and several ER stress markers was evaluated by quantitative PCR (qPCR) at different time points. Furthermore, the cfa-miR-346 was monitored in plasma collected from non-infected dogs (n = 11) and dogs naturally infected by L. infantum (n = 18). Results The results in DH82 cells showed that cfa-mir-346 was induced at both 24 h and 48 h post-infection with all Leishmania strains but not with tunicamycin, accounting for a mechanism of induction independent from sXBP1, unlike what was previously observed in human cell lines. Moreover, the cfa-miR-346 expression analysis on plasma revealed a significant increase in infected dogs compared to non-infected dogs. Conclusions Here for the first time, we report the upregulation of cfa-miR-346 induced by Leishmania infection in canine macrophage-like cells and plasma samples of naturally infected dogs. According to our results, the cfa-miR-346 appears to be linked to infection, and understanding its role and identifying its target genes could contribute to elucidate the mechanisms underlying the host–pathogen interaction in leishmaniasis.Buffi, Gloria; Diotallevi, Aurora; Ceccarelli, Marcello; Bruno, Federica; Castelli, Germano; Vitale, Fabrizio; Magnani, Mauro; Galluzzi, LucaBuffi, Gloria; Diotallevi, Aurora; Ceccarelli, Marcello; Bruno, Federica; Castelli, Germano; Vitale, Fabrizio; Magnani, Mauro; Galluzzi, Luc

A Modular Lunar Hotel

The aim of this paper is to propose an innovative modular lunar hotel/outpost that can be assembled using the load capacity of future rockets Space X is at present developing and presumably will be opera- tional by 2025. In particular, the design is based on the Space X' Starship, that will have the capability to land large and heavy payloads on the Moon. The lunar building is essentially made of four cylindrical modules assembled around one central distribution and service hub. These four modules, intended for housing, have a geodesic dome with large windows to observe the lunar environment surrounding the outpost. The entry point to the base is in the lower part of the central module, which is the only part of the building touching the ground and rests on four adjustable legs. The central module will be used for vertical connections and services as well as for hydroponic laboratories and greenhouses in which to grow the food the settlers will eat. The whole structure will be about 15m high and will be protected from cosmic radiation by a magnetic eld generated by a number of electric cables laid on a spherical structure made of in atable high pressure tubes. The modules can be made of light material since the protection form radiation is supplied by the magnetic eld, and need only a thermal insulating layer, which can be fairly light. The whole structure can thus be carried from Earth without the need of manufacturing it on site. As an added advantage, large windows can be present, mainly in the a top domes/observatories, which will be the characteristic elements of the installation. The cylindrical modules have a diameter of 6m, suitable to be transported in the cargo hold of the Starship. To reach an height of 15m, they are made in sections and then assembled on site. The modules will be lowered from the hold of the Starship by means of the crane with which each spaceship is equipped. Before starting the assembly of the modules, self-propelled cranes and vehicles will be carried to the Moon so that the construction site of the hotel/outpost can be relatively distant from the landing area. These construction machines will then remain available for other construction projects on the Moon. A total of about 10 launches are expected to be required to carry to the Moon all parts needed to build the facility

Modular Lunar Hotel

The aim of this paper is to propose an innovative modular lunar hotel or outpost that can be assembled using the load capacity of future rockets Space X is at present developing and presumably will be opera- tional by 2025. In particular, the design is based on the Space X Starship, that will have the capability to land large and heavy payloads on the Moon. The lunar building is essentially made of four cylindrical modules assembled around one central distribution and service hub. These four modules, intended for housing, have a geodesic dome with large windows to observe the lunar environment surrounding the outpost. The entry point to the base is in the lower part of the central module, which is the only part of the building touching the ground and rests on four adjustable legs. The central module will be used for vertical connections and services as well as for hydroponic laboratories and greenhouses in which to grow the food the settlers will eat. The whole structure will be about 15m high and will be protected from cosmic radiation by a magnetic eld generated by a number of electric cables laid on a spherical structure made of in a table high pressure tubes. The modules can be made of light materials since the protection from radiation is supplied by the magnetic eld, and need only a thermal insulating layer, which can be fairly light. The whole structure can thus be carried from Earth without the need of manufacturing it on site. As an added advantage, large windows can be present, mainly in the a top domes/observatories, which will be the characteristic elements of the installation. The cylindrical modules have a diameter of 6m, suitable to be transported in the cargo hold of the Starship. To reach an height of 15m, they are made in sections and then assembled on site. The modules will be lowered from the hold of the Starship by means of the crane with which each spaceship is equipped. Before starting the assembly of the modules, self-propelled cranes and vehicles will be carried to the Moon so that the construction site of the hotel/outpost can be relatively distant from the landing area. These construction machines will then remain available for other construction projects on the Moon. A total of about 10 launches are expected to be required to carry to the Moon all parts needed to build the facility

Extracellular embryo genomic DNA and its potential for genotyping applications

none8noBackground: Preimplantation genetic diagnosis (PGD) currently relies on biopsy of one or few embryo cells. Our aim was to evaluate the embryo extracellular matrices (spent medium and blastocoele fluid) as source of DNA for embryo genotyping. Results/methodology: We first evaluated the amplifiability and the amount of genomic DNA in spent embryo culture media from day 3 (n = 32) and day 5/6 (n = 54). Secondly, we evaluated the possibility to genotype the MTHFR polymorphism C677T from media at day 5/6 (n = 8) and blastocoele fluids (n = 9) by direct sequencing. The C677T polymorphism detection rate was 62.5 and 44.4% in medium and fluid, respectively. Conclusion: A noninvasive approach for embryo genotyping was possible, but still with limitations due to low detection rate and possible allele dropout.openGalluzzi, Luca; Palini, Simone; Destefani, Silvia; Andreoni, Francesca; Primiterra, Mariangela; Diotallevi, Aurora; Bulletti, Carlo; Magnani, MauroGalluzzi, Luca; Palini, Simone; Destefani, Silvia; Andreoni, Francesca; Primiterra, Mariangela; Diotallevi, Aurora; Bulletti, Carlo; Magnani, Maur