Bioinformatics tools for marine biotechnology: A practical tutorial with a metagenomic approach

Background: Bioinformatics has pervaded all fields of biology and has become an indispensable tool for almost all research projects. Although teaching bioinformatics has been incorporated in all traditional life science curricula, practical hands-on experiences in tight combination with wet-lab experiments are needed to motivate students. Results: We present a tutorial that starts from a practical problem: finding novel enzymes from marine environments. First, we introduce the idea of metagenomics, a recent approach that extends biotechnology to non-culturable microbes. We presuppose that a probe for the screening of metagenomic cosmid library is needed. The students start from the chemical structure of the substrate that should be acted on by the novel enzyme and end with the sequence of the probe. To attain their goal, they discover databases such as BRENDA and programs such as BLAST and Clustal Omega. Students' answers to a satisfaction questionnaire show that a multistep tutorial integrated into a research wet-lab project is preferable to conventional lectures illustrating bioinformatics tools. Conclusion: Experimental biologists can better operate basic bioinformatics if a problem-solving approach is chosen

Congenital disorders of glycosylation: narration of a story through its patents

Congenital disorders of glycosylation are a group of more than 160 rare genetic defects in protein and lipid glycosylation. Since the first clinical report in 1980 of PMM2-CDG, the most common CDG worldwide, research made great strides, but nearly all of them are still missing a cure. CDG diagnosis has been at a rapid pace since the introduction of whole-exome/whole-genome sequencing as a diagnostic tool. Here, we retrace the history of CDG by analyzing all the patents associated with the topic. To this end, we explored the Espacenet database, extracted a list of patents, and then divided them into three major groups: (1) Drugs/therapeutic approaches for CDG, (2) Drug delivery tools for CDG, (3) Diagnostic tools for CDG. Despite the enormous scientific progress experienced in the last 30 years, diagnostic tools, drugs, and biomarkers are still urgently needed

Terremoto del 29 dicembre 2013 nel Matese (MW = 5.0). Indagine speditiva degli effetti nell’area epicentrale e analisi preliminare della sequenza sismica.

Il 29 dicembre 2013 un terremoto di magnitudo Mw=5.0 (profondità 10.5 km) è avvenuto nell'area dei Monti del Matese alle ore 18:08:43 ora locale

Pharmacological chaperones: A therapeutic approach for diseases caused by destabilizing missense mutations

The term “pharmacological chaperone” was introduced 20 years ago. Since then the approach with this type of drug has been proposed for several diseases, lysosomal storage disorders representing the most popular targets. The hallmark of a pharmacological chaperone is its ability to bind a protein specifically and stabilize it. This property can be beneficial for curing diseases that are associated with protein mutants that are intrinsically active but unstable. The total activity of the affected proteins in the cell is lower than normal because they are cleared by the quality control system. Although most pharmacological chaperones are reversible competitive inhibitors or antagonists of their target proteins, the inhibitory activity is neither required nor desirable. This issue is well documented by specific examples among which those concerning Fabry disease. Direct specific binding is not the only mechanism by which small molecules can rescue mutant proteins in the cell. These drugs and the properly defined pharmacological chaperones can work together with different and possibly synergistic modes of action to revert a disease phenotype caused by an unstable protein

Interseismic Active Deformation in the central-southern Apennine

The GPS results are of utmost relevance for the study of the complex plate boundary geodynamics. The lithosphere strain partitioning is part of the seismic cycle. We present the first GPS kinematic pattern obtained during the interseismic phase by a dense episodic GPS network, the Southern Apennine Geodetic Network - SAGNet (Sepe et al., 2009), in the time span 2002-2013. This network is located across the transition zone between central and southern Apennine, including Meta-Mainarde-Venafro and AltoMolise-Sannio-Matese mounts. This region is characterized by seismogenic fault systems responsible, in the past, for several destructive earthquakes of intensity I ≥ IX MCS and, in more recent years, characterised mainly by some moderate magnitude seismic sequences (max magnitude Mw 5.0, December 29 2013) and single small events (Ml < 2.5).SAGNet GPS data were processed by BERNESE sw v.5.0 and the resulting velocities were least-squares combined with the permanent stations velocity field and with the velocity solution of Giuliani et al. 2009. The combined GPS velocity field, shows a perpendicular maximum extension with respect to the Apennine chain of about 2.0 mm/y.The Matese area was hit on December 29, 2013 by a Mw=5.0 (Convertito et al., 2016) earthquake. It was followed by an intense seismic activity until the beginning of February 2014. After the mainshock a GPS survey was carried out on the SAGnet stations. We collected data from 2013, 30 December to 2014, 4 April. The time series of 17 stations are affect by an offsets on the linear drift. The map of horizontal coseismic displacements (Figure 3) shows a sub-radial displacement shape with respect to the epicentre. Larger displacements are observed in correspondence of NE portion of the Matese massif. Considering the Matese Lake Fault as the probable source of the mainshock (dip 65°, strike 116, rake 270 – MLF, Ferranti et al, 2015), we found that the Okada modelling does not fit the observed displacements and only a small fraction of displacements are resolved with a simple slip.Figure 4 resembles the results of previous studies compared with our GPS analysis. We considered seismological analyses, tomographic models, degassing of CO2 data and conceptual model of processes recognized in South Apennine (L. Bisio, et al., 2004; Chiarabba and Chiodini, 2013; Improta et al., 2014; Ventura et al., 2007, R. Di Stefano and M.G. Ciaccio, 2014; Ferranti et al., 2015; Convertito et al., 2016;). The GPS results indicate that the relative motion between Eurasia and Adria plates is responsible of the active deformation in the Apennines. The most important outcomes of this study are: (i) During the interseismic phase the differential motion between Adriatic and Tyrrhenian domains seems to be accommodated in a narrow belt bordering the westward flank of the Sannio Mts, showing a 2 mm/y extension. (ii) The maximum extension does not follow the topographic high of the chain but is shifted toward the eastern outer belt. (iii) No significant GPS deformation is highlighted in correspondence of major and known fault systems where the GPS velocities appear almost steady. We propose that the observed coseismic displacements are only marginally explained by a slip on the MLF fault. The vertical directivity and depth distribution of the seismic sequence (Convertito et al., 2016), the vertical and horizontal heterogeneity of lower crust and upper mantle (Bisio et al., 2004; Di Stefano and Ciaccio, 2014), the high flux of CO2 degassing (Ventura et al., 2007, Chiarabba e Chiodini, 2013 ), the probable presence of pressurized CO2 bodies fed by fluids uprising from the mantle wedge (Improta et al.,2014 ), suggest instead that the seismic sequence could be caused by sub-vertical cracks that originate at the Moho interface and reach the bottom of the seismogenic layer (10km depth).DPCUnpublishedSan Francisco (USA)2T. Tettonica attivaope

Transcriptional regulation of the urokinase receptor (u-PAR) - A central molecule of invasion and metastasis

The phenomenon of tumor-associated proteolysis has been acknowledged as a decisive step in the progression of cancer. This short review focuses on the urokinase receptor (u-PAR), a central molecule involved in tumor-associated invasion and metastasis, and summarizes the transcriptional regulation of u-PAR. The urokinase receptor (u-PAR) is a heavily glycosylated cell surface protein and binds the serine protease urokinase specifically and with high affinity. It consists of three similar cysteine-rich repeats and is anchored to the cell membrane via a GPI-anchor. The u-PAR gene comprises 7 exons and is located on chromosome 19q13. Transcriptional activation of the u-PAR promoter region can be induced by binding of transcription factors (Sp1, AP-1, AP-2, NF-kappaB). One current study gives an example for transcriptional downregulation of u-PAR through a PEA3/ets transcriptional silencing element. Knowledge of the molecular regulation of this molecule in tumor cells could be very important for diagnosis and therapy in the near future

Proteostasis regulators modulate proteasomal activity and gene expression to attenuate multiple phenotypes in Fabry disease

The lysosomal storage disorder Fabry disease is characterized by a deficiency of the lysosomal enzyme \u3b1-Galactosidase A. The observation that missense variants in the encoding GLA gene often lead to structural destabilization, endoplasmic reticulum retention and proteasomal degradation of the misfolded, but otherwise catalytically functional enzyme has resulted in the exploration of alternative therapeutic approaches. In this context, we have investigated proteostasis regulators (PRs) for their potential to increase cellular enzyme activity, and to reduce the disease-specific accumulation of the biomarker globotriaosylsphingosine in patient-derived cell culture. The PRs also acted synergistically with the clinically approved 1-deoxygalactonojirimycine, demonstrating the potential of combination treatment in a therapeutic application. Extensive characterization of the effective PRs revealed inhibition of the proteasome and elevation of GLA gene expression as paramount effects. Further analysis of transcriptional patterns of the PRs exposed a variety of genes involved in proteostasis as potential modulators. We propose that addressing proteostasis is an effective approach to discover new therapeutic targets for diseases involving folding and trafficking-deficient protein mutants

The topology of plasminogen binding and activation on the surface of human breast cancer cells

The urokinase-dependent activation of plasminogen by breast cancer cells plays an important role in metastasis. We have previously shown that the metastatic breast cancer cell line MDA-MB-231 over-expresses urokinase and binds and efficiently activates plasminogen at the cell surface compared to non-metastatic cells. The aim of this study was to further characterise plasminogen binding and determine the topology of cell surface-bound plasminogen in terms of its potential for activation. The lysine-dependent binding of plasminogen at 4°C to MDA-MB-231 cells was stable and resulted in an activation-susceptible conformation of plasminogen. Topologically, a fraction of bound plasminogen was co-localised with urokinase on the surfaces of MDA-MB-231 cells where it could be activated to plasmin. At 37°C plasmin was rapidly lost from the cell surface. Apart from actin, other candidate plasminogen receptors were either not expressed or did not co-localise with plasminogen at the cell surface. Thus, based on co-localisation with urokinase, plasminogen binding is partitioned into two functional pools on the surface of MDA-MB-231 cells. In conclusion, these results shed further light on the functional organisation of the plasminogen activation cascade on the surface of a metastatic cancer cell. © 2001 Cancer Research Campaignhttp://www.bjcancer.co

Clathrin and LRP-1-Independent Constitutive Endocytosis and Recycling of uPAR

Background: The urokinase receptor (uPAR/CD87) is highly expressed in malignant tumours. uPAR, as a GPI anchored protein, is preferentially located at the cell surface, where it interacts with its ligands urokinase (uPA) and the extracellular matrix protein vitronectin, thus promoting plasmin generation, cell-matrix interactions and intracellular signalling events. Interaction with a complex formed by uPA and its inhibitor PAI-1 induces cell surface down regulation and recycling of the receptor via the clathrin-coated pathway, a process dependent on the association to LRP-1. Methodology/Principal Findings: In this study, we have found that along with the ligand-induced down-regulation, uPAR also internalizes and recycles constitutively through a second pathway that is independent of LRP-1 and clathrin but shares some properties with macropinocytosis. The ligand-independent route is amiloride-sensitive, does not require uPAR partitioning into lipid rafts, is independent of the activity of small GTPases RhoA, Rac1 and Cdc42, and does not require PI3K activity. Constitutively endocytosed uPAR is found in EEA1 positive early/recycling endosomes but does not reach lysosomes in the absence of ligands. Electron microscopy analysis reveals the presence of uPAR in ruffling domains at the cell surface, in macropinosome-like vesicles and in endosomal compartments. Conclusions/Significance: These results indicate that, in addition to the ligand-induced endocytosis of uPAR, efficient surface expression and membrane trafficking might also be driven by an uncommon macropinocytic mechanism couple

“I luoghi di Mercalli”: a travelling exhibition as a tool for scientists to dialogue with the public on volcanoes and earthquakes

On March 19, 1914 Giuseppe Mercalli, a seismologist and volcanologist, well-known around the world for the Intensity scale of earthquakes bearing his name, died tragically. A hundred years after, the Istituto Nazionale di Geofisica e Vulcanologia (INGV) has promoted a variety of activities and cultural events that will take place under the Patronage of the President of the Italian Republic within a year, the so called “Anno Mercalliano” (the Year of Mercalli). The opening ceremony took place in Naples, Italy, on March 19, 2014, in the Convitto Nazionale Vittorio Emanuele II. A scientific conference was held with the participation of experts from INGV and the university of Milano – Bicocca, and presentations of students. On that day the exhibition entitled “I luoghi di Mercalli” (Mercalli's places) was also inaugurated, at the presence of local authorities. The exhibition, organized by INGV, was realized in collaboration with the high school Vittorio Emanuele II, where Mercalli has been teaching for 19 years, and the Università degli Studi Suor Orsola Benincasa, where he was professor of natural sciences. A biographical and geographical description of the places where Mercalli operated introduces the exhibition, which is organized in sections: - Mercalli educator (he taught at high schools in Reggio Calabria and Naples); - Mercalli volcanologist (Mercalli studied Vesuvius volcanic activity for more than twenty years, he was a scientific witness of the Vesuvius 1906 eruption, and of the eruptions occurred at Vulcano (1888-90) and Stromboli (1891) islands. - Mercalli seismologist (Mercalli Intensity scale definition, based on his experience as witness of catastrophic earthquakes, such as Casamicciola in 1883 and Messina in 1908). Another section deals with the Vesuvius Observatory, directed by Mercalli between 1911 and 1914, and the description of the three active volcanoes of the Campania region (Vesuvius, Campi Flegrei and Ischia island), which have been the subject of studies by the well-known scientist. The exhibition is enriched by documents, manuscripts, photos and field notebooks of Mercalli. It is not intended to be only a celebratory exhibition; rather it is designed as a tool for dissemination of scientific culture and to raise awareness about seismic and volcanic hazards. In the exhibition path a continuous thread between the figure of Mercalli as a researcher and the role of an Earth Science researcher today is highlighted, pointing to the development of scientific knowledge in the past century. The goal is to improve the capability of learning from the disasters occurred in the past to implement preventive actions to safely deal with future events. The exhibition is travelling and will be provided on request to institutions and schools.PublishedMilano, Italia1V. Storia e struttura dei sistemi vulcaniciope