Transcriptional analysis of ESAT-6 cluster 3 in Mycobacterium smegmatis

<p>Abstract</p> <p>Background</p> <p>The ESAT-6 (early secreted antigenic target, 6 kDa) family collects small mycobacterial proteins secreted by <it>Mycobacterium tuberculosis</it>, particularly in the early phase of growth. There are 23 ESAT-6 family members in <it>M. tuberculosis </it>H37Rv. In a previous work, we identified the Zur- dependent regulation of five proteins of the ESAT-6/CFP-10 family (<it>esxG</it>, <it>esxH</it>, <it>esxQ</it>, <it>esxR</it>, and <it>esxS</it>). <it>esxG </it>and <it>esxH </it>are part of ESAT-6 cluster 3, whose expression was already known to be induced by iron starvation.</p> <p>Results</p> <p>In this research, we performed EMSA experiments and transcriptional analysis of ESAT-6 cluster 3 in <it>Mycobacterium smegmatis </it>(<it>msmeg0615</it>-<it>msmeg0625</it>) and <it>M. tuberculosis</it>. In contrast to what we had observed in <it>M. tuberculosis</it>, we found that in <it>M. smegmatis </it>ESAT-6 cluster 3 responds only to iron and not to zinc. In both organisms we identified an internal promoter, a finding which suggests the presence of two transcriptional units and, by consequence, a differential expression of cluster 3 genes. We compared the expression of <it>msmeg0615 </it>and <it>msmeg0620 </it>in different growth and stress conditions by means of relative quantitative PCR. The expression of <it>msmeg0615 </it>and <it>msmeg0620 </it>genes was essentially similar; they appeared to be repressed in most of the tested conditions, with the exception of acid stress (pH 4.2) where <it>msmeg0615 </it>was about 4-fold induced, while <it>msmeg0620 </it>was repressed. Analysis revealed that in acid stress conditions <it>M. tuberculosis rv0282 </it>gene was 3-fold induced too, while <it>rv0287 </it>induction was almost insignificant.</p> <p>Conclusion</p> <p>In contrast with what has been reported for <it>M. tuberculosis</it>, our results suggest that in <it>M. smegmatis </it>only IdeR-dependent regulation is retained, while zinc has no effect on gene expression. The role of cluster 3 in <it>M. tuberculosis </it>virulence is still to be defined; however, iron- and zinc-dependent expression strongly suggests that cluster 3 is highly expressed in the infective process, and that the cluster contributes to the antigenic profile during the course of infection. Moreover, cluster 3 induction in acid stress conditions strengthens the hypothesis that cluster 3 is expressed in the course of infection.</p> <p>In <it>M. smegmatis</it>, the expression of <it>msmeg0615 </it>and <it>msmeg0620 </it>genes is broadly similar in differing growth phases and in stress conditions, with the exception of acid stress (pH 4.2). Differences in expression between cluster 3 genes can be explained by the presence of internal promoters, both in <it>M. smegmatis </it>and <it>M. tuberculosis</it>.</p

La 'Presidenzializzazione' del sistema di governo britannico

2008-2009Nell'ambito di un approccio giuridico-politologico allo studio delle forme di Stato e di governo, il punto fondamentale sarà quello di chiarire il grado di importanza da attribuire alle variabili esogene nella individuazione delle categorie della modellistica costituzionale tradizionale e, soprattutto, comprendere in che rapporto esse si pongono con gli elementi strutturali-formali che definiscono uno specifico modello istituzionale. In altri termini, la necessità di dar vita a una tassonomia il più possibile esaustiva e realistica dei sistemi istituzionali effettivamente operanti induce il giurista ad abbandonare il suo "metodo tradizionale" optando per criteri classificatori "estranei alla sua scienza" o, pur prendendo atto della loro rilevanza, egli continua a considerare fondamentale la distinzione tra elementi costitutivi e situazioni condizionanti? La risposta a tale quesito sarà graduale. Dopo aver fatto ampio riferimento ai diversi criteri utilizzati nella classificazione tradizionale delle forme di governo (rilevandone di volta in volta limiti e meriti) e aver individuato quale (o quali) tra essi sembra conciliare meglio rispetto agli altri l'esigenza di esaustività e accuratezza, nell'intento di valutare la fondatezza della tesi della presidenzializzazione del sistema britannico, si presterà particolare attenzione, nel corso del secondo e terzo capitolo, all'interrelazione che, dall'analisi della realtà istituzionale, emerge tra le variabili strutturali-formali e gli elementi dinamico-funzionali nella definizione del modello costituzionale. Proprio il rapporto tra le due tipologie di variabili sarà il filo conduttore dell'intero lavoro, nel senso che, dopo esserci soffermati su di esso nell'ambito dell'inquadramento generale delle forme di governo, l'analisi proseguirà prendendo in considerazione lo specifico modello parlamentare britannico per arrivare, infine, a considerare il fenomeno della presidenzializzazione della politica e a valutare l'attendibilità della tesi relativa all'esistenza di una British Presidency su cui da tempo costituzionalisti e politologi dibattono. Nel prendere in considerazione la tesi della presidenzializzazione obiettivo primario di questo lavoro sarà quello di valutare l'attendibilità dell'approccio giuridico classico, ovvero, ciò su cui ci si interrogherà prevalentemente sarà l'opportunità o meno di "liberare" l'analisi di un modello istituzionale dalla vincolo dei suoi elementi giuridico-foemali considerando rilevanti a riguardo anche le dinamiche funzionali ad esso collegate. Onde pervenire a una valutazione scientificamente adeguata, i meccanismi tradizionali della British Constitution saranno oggetto di una duplice valutazione. Da un alto, mediante la comparazione diretta con alcuni aspetti della American Constitution, si cercherà di capire cosa rende possibile e, quindi, fonda il ricorso all'"analogia presidenziale", avvicinando il parlamentarismo britannico al modello presidenziale U.S.A. Dall'altro, considerando le vicende istituzionali collegate soprattutto alle "innovative" premierships di M. Thatcher e T. Blair, si cercherà di capire il valore di tali meccanismi nella definizione degli equilibri attuali della forma di governo. In particolare, consapevoli delle molteplici innovazioni intervenute nella gestione e conduzione dell'attività politica si cercherà di capire quale valore attribuire ad esse nella "riqualificazione in senso presidenziale" del Regno Unito.VIII n.s

Nanoparticle-guided brain drug delivery: Expanding the therapeutic approach to neurodegenerative diseases

Neurodegenerative diseases (NDs) represent a heterogeneous group of aging-related disorders featured by progressive impairment of motor and/or cognitive functions, often accompanied by psychiatric disorders. NDs are denoted as ‘protein misfolding’ diseases or proteinopathies, and are classified according to their known genetic mechanisms and/or the main protein involved in disease onset and progression. Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD) are included under this nosographic umbrella, sharing histopathologically salient features, including deposition of insoluble proteins, activation of glial cells, loss of neuronal cells and synaptic connectivity. To date, there are no effective cures or disease-modifying therapies for these NDs. Several compounds have not shown efficacy in clinical trials, since they generally fail to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells that greatly limits the brain internalization of endogenous substances. By engineering materials of a size usually within 1–100 nm, nanotechnology offers an alternative approach for promising and innovative therapeutic solutions in NDs. Nanoparticles can cross the BBB and release active molecules at target sites in the brain, minimizing side effects. This review focuses on the state-of-the-art of nanoengineered delivery systems for brain targeting in the treatment of AD, PD and HD. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

RNA sequencing and de novo assembly of the digestive gland transcriptome in Mytilus galloprovincialis fed with toxinogenic and non-toxic strains of Alexandrium minutum

Background The Mediterranean mussel Mytilus galloprovincialis is marine bivalve with a relevant commercial importance as well as a key sentinel organism for the biomonitoring of environmental pollution. Here we report the RNA sequencing of the mussel digestive gland, performed with the aim: a) to produce a high quality de novo transcriptome assembly, thus improving the genetic and molecular knowledge of this organism b) to provide an initial assessment of the response to paralytic shellfish poisoning (PSP) on a molecular level, in order to identify possible molecular markers of toxin accumulation. Results The comprehensive de novo assembly and annotation of the transcriptome yielded a collection of 12,079 non-redundant consensus sequences with an average length of 958 bp, with a high percentage of full-length transcripts. The whole-transcriptome gene expression study indicated that the accumulation of paralytic toxins produced by the dinoflagellate Alexandrium minutum over a time span of 5 days scarcely affected gene expression, but the results need further validation with a greater number of biological samples and naturally contaminated specimens. Conclusion The digestive gland reference transcriptome we produced significantly improves the data collected from previous sequencing efforts and provides a basic resource for expanding functional genomics investigations in M. galloprovincialis. Although not conclusive, the results of the RNA-seq gene expression analysis support the classification of mussels as bivalves refractory to paralytic shellfish poisoning and point out that the identification molecular biomarkers of PSP in the digestive gland of this organism is problematic

High-density EEG power topography and connectivity during confusional arousal.

Confusional arousal is the milder expression of a family of disorders known as Disorders of Arousal (DOA) from non-REM sleep. These disorders are characterized by recurrent abnormal behaviors that occur in a state of reduced awareness for the external environment. Despite frequent amnesia for the nocturnal events, when actively probed, patients are able to report vivid hallucinatory/dream-like mental imagery. Traditional (low-density) scalp and stereo-electroencephalographic (EEG) recordings previously showed a pathological admixture of slow oscillations typical of NREM sleep and wake-like fast-mixed frequencies during these phenomena. However, our knowledge about the specific neural EEG dynamics over the entire brain is limited. We collected 2 consecutive in-laboratory sleep recordings using high-density (hd)-EEG (256 vertex-referenced geodesic system) coupled with standard video-polysomnography (v-PSG) from a 12-year-old drug-naïve and otherwise healthy child with a long-lasting history of sleepwalking. Source power topography and functional connectivity were computed during 20 selected confusional arousal episodes (from -6 to +18 sec after motor onset), and during baseline slow wave sleep preceding each episode (from - 3 to -2 min before onset). We found a widespread increase in slow wave activity (SWA) theta, alpha, beta, gamma power, associated with a parallel decrease in the sigma range during behavioral episodes compared to baseline sleep. Bilateral Broadman area 7 and right Broadman areas 39 and 40 were relatively spared by the massive increase in SWA power. Functional SWA connectivity analysis revealed a drastic increase in the number and complexity of connections from baseline sleep to full-blown episodes, that mainly involved an increased out-flow from bilateral fronto-medial prefrontal cortex and left temporal lobe to other cortical regions. These effects could be appreciated in the 6 sec window preceding behavioral onset. Overall, our results support the idea that DOA are the expression of peculiar brain states, compatible with a partial re-emergence of consciousness

Mycobacterium tuberculosis Phosphoribosylpyrophosphate Synthetase: Biochemical Features of a Crucial Enzyme for Mycobacterial Cell Wall Biosynthesis

The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase) is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg2+ or Mn2+ and inhibited by Ca2+ and Cu2+ but, differently from other characterized PRPPases, it showed a better affinity for the Mn2+ and Cu2+ ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer

Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington’s disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer

Editorial: Nucleic acid-based aptamers in therapeutics and diagnostics

Aptamers have emerged as versatile and powerful tools in the fields of therapeutics and diagnostics. These molecules are single-stranded nucleic acids—either DNA or RNA—that fold into unique three-dimensional structures, allowing them to selectively bind to target molecules with high affinity and specificity. They can be identified through an iterative selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment).The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.Peer reviewe