1,721,674 research outputs found
Assess the utility of the PIP-ON system in M. tb and overexpress or silence several mycobacterial genes
No full textIn this system the gene of interest is under transcriptional control of the Streptomyces Pip protein, the pristinamycin sensitive repressor. The pip gene of S. coelicolor has been cloned downstream of a constitutive mycobacterial promoter (the mutant pfurA102 of M. tb), and introduced in Mycobacterium smegmatis (see pMYS696 in Fig. 1). Furthermore, a Pip-ON system has been constructed, by cloning the S. pristinaespiralis ptr promoter upstream of a reporter gene (lacZ), in the above plasmid (see pMY718 in Fig. 1). These constructs have been introduced in M. smegmatis and beta-galactosidase activity measured: the activity expressed from pMY718 is very low (less than 10 Miller units) and inducible, in a dose dependent manner, by addition of pristinamycin
Mycomancy : Construction of Mycobacterium tuberculosis mutants affected in cell cycle regulation
No full textCeppi batterici ricombinanti per la produzione di nucleoside naturali e analoghi modificati
No full textLa presente invenzione riguarda un vettore ricombinante di espressione plasmidico, basato sul plasmide pUC18, comprendente:
a) Almeno na sequenza genica di un batterio mesofilo codificante per un polipeptide avente attività enzimatica di timidina fosforilasi, e
b) Almeno una sequenza genica che codifica per la resistenza ad almeno un antibiotico, nonché cellule ospite contenenti l’anzidetto vettore, ed usi di cellule ospite
Essentiality testing with inducible promoters
No full textObjectives: Assess the utility of the PIP-ON system in M.tb.
Progress: The PIP-ON system was transferred in M. tb where we could show a very efficient repression of Pip on the ptr promoter. In response to pristinamicin we could see a dose dependent induction of the promoter obtaining a maximum induction of about 100 fold with 150 ng/ml of pristinamicin.
The construction of plasmids where the expression of pknB, glnA1 and fad32 genes and of their antisense RNA is under the ptr promoter control and thus responding to pristinamicin is under way.
An alternative approach using a TET/PIP repressible system was developed. The reporter gene is represented by the same one used for the PIP ON system (lacZ under ptr promoter transcriptional control). In this case the PIP repressor is provided in trans under the control of a TET dependant promoter. In the absence of inducer PIP should not be produced and the ptr promoter should be expressed constitutively. Addition of tetracycline should induce the expression of the PIP repressor structural gene causing the repression of transcription of the ptr promoter. Removal of tetracycline and/or addition of pristinamycin should restore its transcription. The system, introduced in M. smegmatis did not give the expected results probably due to the high strength of the TET-dependant promoter used in these constructs. We decided to place PIP under the control of a weaker promoter. We chose a mutated furA promoter previously shown to express PIP at functional level. The promoter was mutagenized to introduce two tet-operators, one immediately upstream the -35 region and the other between the -35 and the -10, placed upstream the PIP conding region and finally cloned into an integrative vector containing a lacZ gene under ptr- promoter transcriptional control. This plasmid has been sequenced and introduced in a M smegmatis strain expressing the TET repressor from a replicative plasmid and is actually under analysi
Phage therapy against Pseudomonas aeruginosa infections in cystic fibrosis patients
No full textPseudomonas aeruginosa is the most common pathogen found in the lung of cystic fibrosis patients. The use of phage therapy could help in fighting the alarming diffusion of antibiotic multi-resistant strains.
A number of new phages were isolated from sewage samples in Milan, and tested for growth on a panel of P. aeruginosa strains collected in Italian hospitals. Comprehensively, we analyzed 23 new phages on 57 clinical or environmental P. aeruginosa strains. Six phages belonging to different classes, i.e. Myoviridae, Podoviridae and Siphoviridae, as assessed by TEM analysis, were mixed in a cocktail. The host range of the phage cocktail was larger than that of individual phages. Infection in liquid culture of strain PAO1 indicated that the phage cocktail efficiently killed the bacterial cells, although resistant mutants appeared at the end. The ability to infect P. aeruginosa growing in biofilm demonstrated that the phage cocktail was able to reduce the biomass of a preformed biofilm. DNA was extracted from the selected phages and send for whole genome shotgun sequencing using the Illumina MiSeq platform at the CNR IBBE institute in Bari.
This project is financed by Italian Foundation of Cystic Fibrosis (# 17/2015)
Pristinamycin-inducible gene regulation in mycobacteria
No full textIn this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This system is based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and the inducer pristinamycin I. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reporter gene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated that the ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold in Mycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was fully repressed in the absence of the inducer.
Three mycobacterial genes were cloned under pptr –Pip control, both in sense and antisense direction; both proteins and antisense RNAs could be overexpressed, the antisenses causing a partial reduction of the amount of the targeted proteins. This system was used to obtain two M. tuberculosis conditional mutants in the fadD32 and pknB genes: the mutant strains grew only in the presence of the inducer pristinamycin I. Thus it showed to be an effective inducible system in mycobacteria
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
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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