Spatiotemporal localization of proteins in microorganisms via photoactivated localization microscopy

Photoactivated localization microscopy (PALM) is a single molecule fluorescence microscopy technique (SMLM) that relies on the controlled activation and imaging of photo-activatable/convertible fluorescent proteins to determine their position with nanometer scale precision. The analysis of SMLM data is composed of two sequential aspects: the generation of a super-resolution table/image and the subsequent analysis. In recent years, several data analysis packages dedicated to the generation of super-resolved images have been developed. These packages have been extensively characterized and compared in a community-wide effort, therefore allowing researchers to identify optimal solutions for their experiments and providing software developers with a gold standard. On the contrary, the development of data analysis packages dedicated to the study of protein coordinates has been lagging behind, and no comprehensive approach has been developed to date. Here, I present a combination of Fiji and R based scripts for the characterization, filtering and quality assurance of SMLM derived localizations. Furthermore, I demonstrate that specific conventional image analysis techniques can be applied, both quantitatively and qualitatively, to super resolution images. I then apply these analysis tools exemplarily on the characterization of the spatio-temporal localization of a novel DNA repair system in Corynebacterium glutamicum, termed Dip (DNA damage induced protein) C. Finally, I combine the multiple data analysis packages that I developed and/or adapted for the study of specific biological scenarios into a single cohesive pipeline, therefore providing a generalized and comprehensive approach toward the coordinate based analysis of the spatio-temporal localization of proteins in PALM and, in general, in SMLM. Each of the data analysis packages that comprise the pipeline is here presented together with the biological scenario that prompted its development. These include the study of magnetosome formation in Magnetospirillum gryphiswaldense, the study of the chromosome segregation machinery in C. glutamicum and the study of flagellar organization in Trypanosoma brucei.Die photoaktivierte Lokalisationsmikroskopie (PALM) ist eine Einzelmolekül-Fluoreszenzmikroskopie Technik (SMLM), die auf der kontrollierten Aktivierung und Aufnahme von photoaktivierbaren / konvertierbaren fluoreszierenden Proteinen beruht, um ihre Position mit einer Präzision im Nanometerbereich zu bestimmen. Die Analyse von SMLM-Daten besteht aus zwei aufeinander folgenden Aspekten: der Erzeugung einer Tabelle / eines hochauflösenden Bildes und der anschließenden Analyse. In den letzten Jahren wurden mehrere Datenanalysepakete entwickelt, die sich der Berechnung der hochaufgelösten Bilder widmen. Diese Pakete wurden in gemeinschaftsweiten Anstrengungen umfassend charakterisiert und verglichen, sodass Forscher eine optimale Lösung für eigene Experimente wählen können, während Softwareentwicklern einen Goldstandard zur Hand haben. Gegensätzlich wurde jedoch die Entwicklung von Datenanalysepaketen zur spezifischen Untersuchung von Proteinkoordinaten vernachlässigt, so dass in diesem Bereich keine umfassenden Instrumente existieren. In dieser Arbeit präsentiere ich eine Kombination aus Fiji- und R basierten Skripten zur Charakterisierung, Filterung und Qualitätssicherung von SMLM Proteinkoordinaten. Darüber hinaus zeige ich, dass bestimmte konventionelle Bildanalysetechniken sowohl quantitativ als auch qualitativ auf „Superresolution“ Bilder angewandt werden können. Im Folgenden verwende Ich diese Analysewerkzeuge dann beispielhaft zur Charakterisierung der räumlich-zeitlichen Lokalisierung eines neuartigen DNA-Reparatursystems in Corynebacterium glutamicum, welches ich DipC (DNA-Schaden-induziertes Protein) genannt habe. Schließlich kombiniere ich die genannten Datenanalysepakete, die ich für die Untersuchung spezifischer biologischer Szenarien entwickelt und / oder angepasst habe, zu einer einzigen zusammenhängenden Arbeitsroutine. Diese bietet einen allgemeinen und umfassenden Ansatz für die koordinatenbasierte Analyse der räumlich-zeitlichen Lokalisierung von Proteinen aus PALM- und im Allgemeinen aus SMLM-Experimenten. Jedes der Datenanalysepakete, die in beschriebener Routine enthalten sind, wird hier zusammen mit dem biologischen Szenario vorgestellt, das zu ihrer Entwicklung geführt hat. Dazu gehören die Untersuchung der Magnetosomenbildung in Magnetospirillum gryphiswaldense, die Untersuchung der Chromosomensegregationsmaschinerie in C. glutamicum und die Untersuchung der Flagellenorganisation in Trypanosoma brucei

Relationship between human oral lichen planus and oral squamous cell carcinoma at a genomic level: a datamining study

The leader gene approach is a data mining method based on the systematic search for genes involved in a specific process and their ranking according to the number of interconnections with the other genes identified. The genes with the strongest interconnections are termed leader genes, since they may be supposed to play an important role in the process. The potential of malignant progression of OLP to oral squamous cell carcinoma (OSCC) is still not completely clear. In this study, the leader gene approach is applied to investigate the association between OLP and OSCC at a molecular level. Results were integrated with those obtained in an experimental analysis (see paper 1 of this series). Genes involved in OLP and OSCC were identified by systematic queries to dedicated databases. Interconnections among identified genes were calculated and given a confidence value using STRING database. Leader genes were identified by clustering genes according to their interconnections. This theoretical analysis shows that OLP and OSCC share two leader genes: TP53 and CDKN1A, involved in the PI3K signalling events mediated by AKT pathway. This finding and those obtained in the experimental analysis suggest the possible involvement of some key genes/proteins LCK, PIK3CA, BIRC5, TP53 and CDKN1A in the malignant progression from OLP to OSCC. Moreover, these findings support the role of some molecular pathways, namely IL2 signalling events mediated by PI3K, PI3K signalling events mediated by AKT, and, possibly, Aurora A signalling in the association between OLP and OSCC

Chromosome organization by a conserved condensin-ParB system in the actinobacterium Corynebacterium glutamicum

Higher-order chromosome folding and segregation are tightly regulated in all domains of life. In bacteria, details on nucleoid organization regulatory mechanisms and function remain poorly characterized, especially in non-model species. Here, we investigate the role of DNA-partitioning protein ParB and SMC condensin complexes in the actinobacterium Corynebacterium glutamicum. Chromosome conformation capture reveals SMC-mediated long-range interactions around ten centromere-like parS sites clustered at the replication origin (oriC). At least one oriC-proximal parS site is necessary for reliable chromosome segregation. We use chromatin immunoprecipitation and photoactivated single-molecule localization microscopy to show the formation of distinct, parS-dependent ParB-nucleoprotein subclusters. We further show that SMC/ScpAB complexes, loaded via ParB at parS sites, mediate chromosomal inter-arm contacts (as previously shown in Bacillus subtilis). However, the MukBEF-like SMC complex MksBEFG does not contribute to chromosomal DNA-folding;instead, this complex is involved in plasmid maintenance and interacts with the polar oriC-tethering factor DivIVA. Our results complement current models of ParB-SMC/ScpAB crosstalk and show that some condensin complexes evolved functions that are apparently uncoupled from chromosome folding

Quaderno della Banca d'Italia - Serie: Questioni di Economia e Finanza. - Titolo: Strumenti negoziali per la soluzione delle crisi d\u2019impresa: il concordato preventivo ISSN 1972-6627 (stampa) ISSN 1972-6643 (online)

Questo studio \ue8 stato condotto dagli autori con la collaborazione nella raccolta dei dati del CESPEC ossia del Centro Studi Procedure Esecutive e Concorsuali dei magistrati specializzati in materia concorsuale e della Banca d'Italia al fine di comprendere, sulla base di una robusta analisi empirica: se le procedure di concordato preventivo rappresentino o meno un efficace strumento per la soluzione delle crisi di impresa, quali siano le principali finalit\ue0 economico aziendali che sono perseguite dalle imprese mediante questo strumento e quali siano le percentuali di successo conseguite. La ricerca \ue8 stata considerata di notevole impatto in quanto ha contribuito a creare una base informativa utile a supportare riflessioni e conseguentemente anche proposte di modifica e integrazione nel redigendo codice della crisi e dell'insolvenza. Il lavoro fornisce, infatti, un ampio insieme di nuove evidenze empiriche sull\u2019impiego e sul funzionamento delle procedure di concordato preventivo basate su un dataset, appositamente assemblato, che costituisce la pi\uf9 ricca base informativa al momento disponibile. I risultati principali possono essere sintetizzati come segue: i concordati preventivi sono impiegati prevalentemente con finalit\ue0 liquidatoria (circa il 70 per cento dei casi). La percentuale di concordati per i quali quanto previsto nel piano \ue8 effettivamente realizzato \ue8 pari a meno di un quarto delle procedure presentate. Il recupero dei crediti assistiti da garanzia avviene in misura pressoch\ue9 integrale in tutte le tipologie di concordato, mentre per i crediti non garantiti i tassi di recupero variano sensibilmente, ma sono sistematicamente superiori rispetto alle aspettative: dal 18 per cento per i concordati liquidatori al 37 per cento per quelli in continuit\ue0 diretta (23 per cento per quelli in continuit\ue0 indiretta). Analisi di regressione mostrano che migliori performance dei concordati preventivi sono associate a un minore grado di \u201ccronicit\ue0 della crisi\u201d al momento dell\u2019apertura del concordato, misurata con il tempo trascorso dalle prime difficolt\ue0 persistenti che l\u2019impresa incontra nell\u2019adempiere agli obblighi nei confronti dei creditori bancari e l\u2019avvio della procedura. Ne segue l'importanza di anticipare il pi\uf9 possibile il momento da un lato di rilevazione della crisi (early warning), dall'altro e proprio a tale fine la rilevanza strategica degli assetti organizzativi, amministrativi e contabili adeguati, dall'altro ancora l'opportunit\ue0 che siano implementati interventi tempestivi ed utili per il turnaround. La prof.ssa Riva per il Piemonte Orientale e il prof. Danovi per l'Universit\ue0 di Bergamo, autori del presente lavoro, sono stati individualmente auditi dalla Commissione Giustizia 2 del Senato il 21.11.2018 per proporre modifiche al Codice della Crisi e dell'Insolvenza (Atto del Governo 53). Sono stati inoltre invitati a fare parte della "Commissione di studi del Consiglio Nazionale dei Dottori Commercialisti ed Esperti Contabili Codice della Crisi e dell'Insolvenza" e in seno a tale organismo nuovamente auditi nel luglio 2020 alla Camera dei Deputati seconda Commissione Giustizia (Atto del Governo 175). Alla luce dei risultati ottenuti, inoltre, gli autori Riva e Danovi sono stati coinvolti attivamente nella ricerca finanziata dalla UE (European Commission JUST/2014/JCOO/AG/CIVI/7627) "Best practices in european restructuring. Contractualised distress resolution in the shadow of the law" (www.codire.eu

Strumenti negoziali per la soluzione delle crisi d\u2019impresa: il concordato preventivo

Questo studio fornisce un ampio insieme dinuove evidenze empiriche sull\u2019impiego e sul funzionamento delle procedure di concordato preventivo basate su un dataset, appositamente assemblato, che costituisce lapi\uf9 ricca base informativa al momento disponibile. I concordati sono impiegati prevalentemente con finalit\ue0 liquidatoria (circa il 70 per cento dei casi). La percentuale di concordati per i quali quanto previsto nel piano \ue8 effettivamente realizzato \ue8 pari a meno di un quarto. Il recupero dei crediti assistiti da garanzia avviene in misura pressoch\ue9 integrale in tutte le tipologie di concordato, mentre per i crediti non garantiti i tassi di recupero variano sensibilmente: dal 18 per cento per i concordati liquidatori al 37 per cento per quelli in continuit\ue0 diretta (23 per cento per quelli in continuit\ue0 indiretta). Analisi di regressione mostrano che migliori performance dei concordati preventivi sono associate a un minore grado di \u201ccronicit\ue0 della crisi\u201d al momento dell\u2019apertura del concordato, misurata con il tempo trascorso dalle prime difficolt\ue0 persistenti che l\u2019impresa incontra nell\u2019adempiere agli obblighi nei confronti dei creditori bancari e l\u2019avvio della procedura

Comparison of the functional properties of trimeric and monomeric CaiT of Escherichia coli

Secondary transporters exist as monomers, dimers or higher state oligomers. The significance of the oligomeric state is only partially understood. Here, the significance of the trimeric state of the L-carnitine/gamma-butyrobetaine antiporter CaiT of Escherichia coli was investigated. Amino acids important for trimer stability were identified and experimentally verified. Among others, CaiT-D288A and -D288R proved to be mostly monomeric in detergent solution and after reconstitution into proteoliposomes, as shown by blue native gel electrophoresis, gel filtration, and determination of intermolecular distances. CaiT-D288A was fully functional with kinetic parameters similar to the trimeric wild-type. Significant differences in amount and stability in the cell membrane between monomeric and trimeric CaiT were not observed. Contrary to trimeric CaiT, addition of substrate had no or only a minor effect on the tryptophan fluorescence of monomeric CaiT. The results suggest that physical contacts between protomers are important for the substrate-induced changes in protein fluorescence and the underlying conformational alterations

Interleukin (IL)-22 receptor 1 is over-expressed in primary Sjogren's syndrome and Sjögren-associated non-Hodgkin lymphomas and is regulated by IL-18.

The aim of this study was to elucidate more clearly the role of interleukin (IL)-18 in modulating the IL-22 pathway in primary Sjögren's syndrome (pSS) patients and in pSS-associated lymphomas. Minor salivary glands (MSGs) from patients with pSS and non-specific chronic sialoadenitis (nSCS), parotid glands biopsies from non-Hodgkin lymphomas (NHL) developed in pSS patients, were evaluated for IL-18, IL-22, IL-22 receptor 1 (IL-22R1), IL-22 binding protein (IL-22BP) and signal transducer and activator of transcription-3 (STAT-3) expression. MSGs IL-22R1-expressing cells were characterized by confocal microscopy and flow cytometry in pSS, nSCS and healthy controls . The effect of recombinant IL-18 and IL-22 on peripheral blood mononuclear cells (PBMCs) from pSS and nSCS was studied by flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR). MSGs of pSS and NHL were characterized by an imbalance between IL-22 and IL-22BP protein expression, with IL-18 and IL-22BP being expressed in a mutually exclusive manner and IL-18 and IL-22R1 being correlated directly. Aberrant expression of IL-22R1, induced by IL-18, was observed only among tissue and circulating myeloid cells of pSS patients and macrophages of NHL tissues of pSS patients, but not nSCS. IL-22R1 expression on PBMC of pSS was functional, as its stimulation with recombinant IL-22 significantly up-regulated the expression of STAT-3, IL-17 and IL-22. An IL-18-dependent aberrant expression of IL-22R1 on cells of haematopoietic origin seems to be a specific immunological signature of patients with pSS and pSS-associated lymphomas

S-Matrix Unitarity, Impact Parameter Profiles, Gluon Saturation and High-Energy Scattering

A model combining perturbative and non-perturbative QCD is developed to compute high-energy reactions of hadrons and photons and to investigate saturation effects that manifest the S-matrix unitarity. Following a functional integral approach, the S-matrix factorizes into light-cone wave functions and the universal amplitude for the scattering of two color-dipoles which are represented by Wegner-Wilson loops. In the framework of the non-perturbative stochastic vacuum model of QCD supplemented by perturbative gluon exchange, the loop-loop correlation is calculated and related to lattice QCD investigations. With a universal energy dependence motivated by the two-pomeron (soft + hard) picture that respects the unitarity condition in impact parameter space, a unified description of pp, pip, Kp, gamma* p, and gamma gamma reactions is achieved in good agreement with experimental data for cross sections, slope parameters, and structure functions. Impact parameter profiles for pp and longitudinal gamma* p reactions and the gluon distribution of the proton xG(x,Q^2,b) are calculated and found to saturate in accordance with S-matrix unitarity. The c.m. energies and Bjorken x at which saturation sets in are determined.Comment: 65 pages with 13 figures, Introduction, Sec. 3, and Conclusion extende

The Antituberculosis Drug Ethambutol Selectively Blocks Apical Growth in CMN Group Bacteria

Members of the genus Mycobacterium are the most prevalent cause of infectious diseases. Mycobacteria have a complex cell envelope containing a peptidoglycan layer and an additional arabinogalactan polymer to which a mycolic acid bilayer is linked;this complex, multilayered cell wall composition (mAGP) is conserved among all CMN group bacteria. The arabinogalactan and mycolic acid synthesis pathways constitute effective drug targets for tuberculosis treatment. Ethambutol (EMB), a classical antituberculosis drug, inhibits the synthesis of the arabinose polymer. Although EMB acts bacteriostatically, its underlying molecular mechanism remains unclear. Here, we used Corynebacterium glutamicum and Mycobacterium phlei as model organisms to study the effects of EMB at the single-cell level. Our results demonstrate that EMB specifically blocks apical cell wall synthesis, but not cell division, explaining the bacteriostatic effect of EMB. Furthermore, the data suggest that members of the family Corynebacterineae have two dedicated machineries for cell elongation (elongasome) and cytokinesis (divisome). IMPORTANCE Antibiotic treatment of bacterial pathogens has contributed enormously to the increase in human health. Despite the apparent importance of antibiotic treatment of bacterial infections, surprisingly little is known about the molecular functions of antibiotic actions in the bacterial cell. Here, we analyzed the molecular effects of ethambutol, a first-line antibiotic against infections caused by members of the genus Mycobacterium. We find that this drug selectively blocks apical cell growth but still allows for effective cytokinesis. As a consequence, cells survive ethambutol treatment and adopt a pneumococcal cell growth mode with cell wall synthesis only at the site of cell division. However, combined treatment of ethambutol and beta-lactam antibiotics acts synergistically and effectively stops cell proliferation