1,721,111 research outputs found
New approaches to unveil the Transcriptional landscape of dopaminergic neurons
Get PDFRecent advances in studying the mammalian transcriptome arised new questions about how genes are organized and what is the function of noncoding RNAs. Furthermore, the discovery of large amounts of polyA- transcripts and antisense transcription proved that a portion of the transcriptome has still to be characterized.
The complex anatomo-functional organization of the brain has prevented a comprehensive analysis of the transcriptional landscape of this tissue. New techniques must be developed to approach neuronal heterogeneity.
In this study we combined Laser Capture Microdissection (LCM) and nanoCAGE, based on Cap Analysis of Gene Expression (CAGE), to describe expressed genes and map their transcription start sites (TSS) in two specific populations, A9 and A10, of mouse mesencephalic dopaminergic cells.
Although sharing common dopaminergic marker genes, these two populations are part of different midbrain anatomical structures, substantia nigra (SN) for A9 and ventral tegmental area (VTA) for A10, project to relatively distinct areas, participate to distinct ascending dopaminergic pathways, exhibit different electrophysiological properties and different susceptibility to neurodegeneration in Parkinson`s disease.
Specific neurons were identified by the expression of Green Fluorescent Protein driven by a celltype specific promoter in transgenic mice. High-quality RNAs were purified from 1000-2500 cells collected by LCM.
We adapted the CAGE technique to analyze limiting amounts of RNAs (nanoCAGE). We took advantage of the cap-switching properties of the reverse transcriptase to specifically tag the 5`end of transcripts with a sequence containing a class III restriction site for EcoP15I. By creating 32bp 5`tags, we considerably improved the TSS mapping rate on the genome. A semi-suppressive PCR strategy was used to prevent primer dimers formation. The use of random priming in the 1st strand synthesis allowed to capture poly(A)- RNAs. 5`tags were sequenced with Illumina-Solexa platform.
Here we show that this new nanoCAGE technology ensures a true high-throughput coverage of the transcriptome of a small number of identified neurons and can be used as an effective mean for gene discovery in the noncoding RNAs, to uncover putative alternative promoters associated to variants of protein coding transcripts and to detect potentially regulatory antisense transcripts. A further experimental validation by 5`RACE (Rapid Amplification of cDNA Ends) and RT-PCR on few candidate genes, have confirmed the existence in vivo of alternative TSS in the case of key regulatory genes involved in specifying and maintaining the dopaminergic phenotype of these neurons such as α-synuclein (Snca), dopamine transporter (Dat), vescicular monoamine transporter 2 (Vmat2), catechol-O-methyltransferase (Comt). Furthermore the differential expression of an antisense transcript overlapping to the polyubiquitin (Ubc) gene was detected as potentially interesting candidate gene accounting for differences in the ubiquitin-proteasome system (UPS) function in the two neuron populations. The potential implications deriving from these newly discovered alternative promoters and transcripts are discussed, considering also the potential consequences for the corresponding protein isoforms
Rischio contagio per la responsabilità sanitaria?
No full textContagion risk for health liability?
The raging crisis from Covid-19 has triggered the alarm
on possible contentious by-products, soon translated into
proposals, not only domestic, to launch a «legal shield» able
to protect health care structures and providers from the
threatening spectrum of rising tides of judicial initiatives.
The ensuing widespread debate focused on the assumption
that the problem is simply that of preventing health professionals from being subject to treacherous litigation; and it
has essentially unfolded along the axis represented by the
question of whether that result postulates the intervention of
the legislator or can be achieved on a hermeneutical level, in
the light of the current legislation, on the assumption that
the system has developed within itself the antibodies capable
to «secure» those who with journalistic emphasis someone has
promoted to the rank of «heroes», when if not «angels». If, however, one overcomes the torpor of a monotonic
analysis and opts for a comprehensive overview, the turning
point of the analysis changes radically. The proposals for a
regulatory shield appear, therefore, incongruous, for different orders of reasons
Il danno da perdita del rapporto parentale: giudice (-legislatore?) in fuga da Milano
No full textUna recente pronunzia della Corte di legittimità ha ritenuto che le tabelle milanesi, accreditate di un carattere quasi normativo per quel che attiene alla quantificazione del danno alla salute, risutano men che soddisfacenti per quel che attiene alla valorizzazione del pregiudizio sofferto dai gongiunti per la perdita o menomazione del proprio caro. Viene perciò suggerito un approccio, ispirato alla metodologia adottata dal Tribunale di Roma, che riduce i margini di discrezionalità e promette maggiore uniformità/preverdibilità
Cefalee croniche e "overuse" di farmaci
No full textLinee Guida Nazionali di riferimento per la prevenzione e la terapia delle cefale
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
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