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TBX1 Transcription Factor: mechanisms of gene regulation

TBX1 Transcription Factor: mechanisms of gene regulation

The gene studied in the thesis work was identified in an effort to isolate the gene/s involved in DiGeorge syndrome (DGS) or 22q11.2 deletion syndrome (22q11.2DS) (Linsday EA et al., 2001; Merscher S et al., 2001). Congenital heart disease (CHD) affects 8/1000 live births. A common genetic cause of CHD is the 22q11.2 deletion, also known as DiGeorge syndrome (DGS) (McDonald-McGinn DM et al., 2015). It has been estimated that a substantial portion of patients with some specific heart defects have 22q11.2 deletions: 52% of those with interrupted aortic arch type B, 34% with truncus arteriosus, 16% with tetralogy of Fallot and 5-10% with Ventricular Septal Defects (VSD). Besides CHD, patients have a number of other phenotypic features, for example cleft palate, development disabilities and schizophrenia. At the genetic level, the deletion could result from aberrant homologous recombination between low copy repeat (LCR) sequences, which flank the deleted regions (Edelman L et al., 1999). The name of this genetic disorders derives from Angelo DiGeorge who, in the late ‘60s, described this syndrome characterized by aberrant development of the thymus and parathyroid. Some of the development anomalies of DGS were also reproduced on chick models of neural crest ablation (Farrel MJ et al., 1999) leading investigators to hypothesize that DGS may derive from abnormal development of neural crest cell-contributed organs. However, more recent studies have implicated other cell lineages, specifically the cardiopharyngeal mesoderm, that will be discussed later. DGS is caused by chromosomal microdeletion of chromosome 22, at q11.2 locus; therefore, the disease is now commonly referred to as 22q11.2 deletion syndrome. Most patients with this syndrome have a large (3Mb) genomic deletion. About 10% of patients have a smaller deletion of about 1.5 Mb. Most genes localized in the region are conserved in the mouse on chromosome 16 (Gong W. et al., 1996; Botta A et al., 1997; Sutherland HF et al., 1998; Puech A et al., 1997; Lund J et al., 1999). This system allowed for the engineering of the first model, named Df1, which carries a deletion that encompasses mouse homologues of 18 genes that are deleted in patients with 1.5 Mb deletion whose phenotype is characterized by heart defects, thymus and parathyroid defects (Linsday EA et al., 1999). In particular, the cardiac defects may be rescued in Df1 mice on chromosome 16 by reciprocal duplication (Dp1) on the homolog, restoring normal dosage of the Df1 region.
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Transcriptional regulation by inositol polyphosphates

Transcriptional regulation by inositol polyphosphates

Studies about the role of IPs as nuclear-signaling molecules show a series of discoveries in the phosphate-responsive signaling pathway (PHO). The PHO pathway is important for chromatin remodeling and it carefully coordinates cellular responses to phosphate starvation. Recent reports identified IP7 as a negative regulator of Pho80-Pho85 CDK complex in a Pho81-dependent manner. Pho-85 is a cyclin-dependent kinase and it is associated with cyclin Pho-80. In the presence of high Pi levels, the complex Ph80-Pho85 is active and makes the phosphorylation of transcription factor Pho4. Phosphorylated Pho4 is exported to the cytoplasm and the gene targets remain turned off. Otherwise, in absence of Pi or in Pi starvation, Pho81, that is a CDK1 inhibitor, prevents Pho80-Pho85 phosphorylation and exportation of Pho4. Pho4 remains in the nucleus and binds activation sequences in the PHO5 promoter. Furthermore, Pho4 recruits the chromatin- remodeling complexes INO80 and SWI/SNF, which help displace four positioned nucleosomes from the PHO5 promoter facilitating PHO5 transcription. Therefore, during Pi starvation, yeast cells increase IP7 levels. The IP7 binds to the Pho80-Pho85 complex and its link is sufficient enough to inactivate the Pho80-Pho85 complex, the Pho4’s phosphorylation and consequently transcriptional activation and mRNA synthesis (19).
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Genetic background dictates pro-angiogenic factor production in preclinical models of colorectal cancer

Genetic background dictates pro-angiogenic factor production in preclinical models of colorectal cancer

Many groups showed that BRAF V600E CRCs express higher levels of EGFR, as compared to BRAF-wt CRCs [75], and that signaling from EGFR could lead to re-accumulation of p-ERK and resistance to EGFR-inhibitors [76]. Based on the observations described here and on literature data, we propose a working model whereby in BRAF-wt contexts dabrafenib causes paradoxical ERK activation (possibly further increased by contextual increased signaling through EGFR family members), thereby causing increased IL-8 transcription, whereas trametinib shuts down ERK activation and IL-8 production (Figure 17, right panel). On the other hand, similar molecular mechanisms are in place in BRAF V600E CRC models in response to trametinib, resulting in ERK inhibition and decrease IL-8 production; however, in response to dabrafenib in BRAF-mut contexts, ERK activation is maintained by sustained signaling through EGFR family members despite BRAF inhibition and the observed decrease in IL-8 production is due to yet unknown mechanisms regulating gene transcription and/or RNA/protein stability (Figure 17, left panel). In that respect, preliminary data from our group show that the activation of EGFR family members (particularly EGFR and HER3) is consistently observed in response to dabrafenib in both BRAF-mut and -wt contexts in CRC models (Bazzichetto C., unpublished observations).
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MtDNA variability and nuclear gene expression in oxidative stress response: Sirtuins and Transcription Factors

MtDNA variability and nuclear gene expression in oxidative stress response: Sirtuins and Transcription Factors

An exhaustive description of all transcription factors that may be involved in mitochondrial signaling pathways is out of the aim of this thesis. In the following, essential information will be given only on the five transcription factors whose corresponding genes have been screened in the present work: Nuclear Factor Erythroid- derived 2-like 1 (NFE2L-1), Nuclear Factor Erythroid-derived 2-like 2 (NFE2L-2), Nuclear Respiratory Factor 1 (NRF1), Mitochondrial Transcription Factor A (TFAM), Nuclear Factor of kappa light polipeptide gene enhancer in B-cells 1 (NFKB1). All of them are conditionally active factors that have a pivotal role in protecting cells from different stress factors thus contributing to maintain cellular homeostasis and survival. The genes NFE2L-1 and NFE2L-2 are component of the Cap”n”collar-basic leucine zipper (bZIP) subfamily, termed also NFE2 family (Moi et al., 1994). These genes encode for transcription factors that are ubiquitously expressed in tissues, but whose expression is different in different tissues (Chan et al., 1996). The binding sequence of the NFE2 family shows high similarity to the antioxidant responsive element consensus sequence. In fact, NFE2L-1 and NFE2L-2 can form heterodimers with the Maf proteins to bind antioxidant responsive element-binding complex which modulate the expression of specific genes in response to ROS or oxidative stress (Wild et al., 1999; Alam et al., 1999). In addition, NFE2L-1 is involved in the regulation of the uncoupling protein-1 (UCP1) to mediate adaptive thermogenesis against cold exposure (Rim and Kozak, 2002).
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Characterization of tomato (Solanum lycopersicum L.) male sterile mutants putatively affected in class B MADS-box transcription factors

Characterization of tomato (Solanum lycopersicum L.) male sterile mutants putatively affected in class B MADS-box transcription factors

58 2003). Another MADS-box gene was characterized, TM4 or TDR4, which is homologous of FRUITFULL (FUL) (Pnueli et al., 1991; Busi et al., 2003; Lozano et al., 2009) and TM8, which is important for anthers, ovary and fruit development (Pnueli et al., 1991; Daminato et al., 2014). The floral organ identity genes, particularly members of class B and C are activated by floral meristem identity genes (Lohmann and Weigel, 2002). In Arabidopsis it was observed that AP3 and PI are activated through the action of these genes in the meristems precursor of petals and stamens, while AG in those precursor of stamens and carpels (Lohmann and Weigel, 2002). LFY promotes directly the expression of floral organ identity genes suppressing the expression of negative regulators for MADS-box genes, such as EMBRYONIC FLOWER 1 (EMF1) protein (Winter et al., 2011). It is known that, for the activation of class B MADS-box genes, LFY requires other factors that act in the same pathway (Jack, 2004). The class B genes are also activated by the UNUSUAL FLORAL ORGAN (UFO) protein (Levin and Meyerowitz, 1995; Wilkinson and Haughn, 1995) and also by the action of AP1 (Weigel and Meyerowitz, 1993). Recently, through the use of new technologies such as microarray analysis and ChIP-Seq, it was understood that the MADS-box factors influenced directly or indirectly the expression of an enormous number of downstream genes (Ito et al., 2004; Wellmer et al., 2004, 2006; Gomez-Mena et al., 2005; Mara and Irish, 2008; Kaufmann et al., 2009, 2010a; Jiao and Meyerowitz, 2010; Wuest et al., 2012). An example of genes that are directly activated by AP3/PI and also AG expression is represented by NOZZLE/SPOROCYTELESS (NZZ/SPL), a transcription factor that is involved in the regulation of sporogenesis (Yang et al., 1999). AP3 and PI suppress the expression of genes that are necessary in carpel and ovule development (Wuest et al., 2012), such as CRABS CLAW (CRC). CRC is involved in carpel development and is expressed early in the third whorl of knock-out mutants for class B MADS-box genes (Bowman and Smyth, 1999). The multitude of downstream genes regulated by class B MADS-box factors encode for proteins involved in cell wall formation (polygalacturonases, pectate lyases and cell wall structural proteins), in stresses response, proteins similar to receptor-like kinases and phosphatases (Zik and Irish, 2003). These genes controlled downstream by MADS- boxes are necessary for different processes and indicate that in floral organ specification are involved a multitude of development pathways (Gomez-Mena et al., 2005; Mara and Irish, 2008; Kaufmann et al., 2010; Wuest et al., 2012).
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The transcription factor Foxm1 controls pro-stemness microRNAs in cerebellar neural stem cells (NSCs)

The transcription factor Foxm1 controls pro-stemness microRNAs in cerebellar neural stem cells (NSCs)

The main goal of this study was to characterize new molecular Hh-related mechanisms responsible for maintaining stemness features in cerebellar NSCs. These results permit the proposal of a model where Hh-Gli signalling controls the self-renewal of murine NSCs through the transcription of a series of microRNAs. As shown in Figure 16, two stemness miRNAs networks are described, via Nanog and via Foxm1, that partially overlap in our system and shed light to the role of Foxm1 in NSCs self- renewal. Hereby identifying the transcription factor Foxm1 as transcriptionally regulated by Hh through Gli 1 and 2 and Nanog. These results expand the list of known downstream effectors in the canonical Hh-Gli signalling pathway, as well as the list of molecules capable of activating Foxm1. Foxm1 is an activating transcription factor that plays a role in several different cellular contexts. In human epidermal stem cells, Foxm1 sustains the balance between self-renewal and terminal differentiation (Gemenetzidis, E. et al., 2010). Foxm1 is known to regulate neuronal precursor induced mitosis (Gemenetzidis, E. et al. 2010), as well as several stem cell-like properties. It is required for proper execution of mitosis and this is highlighted by the fact that Foxm1 knockout is embryonically lethal (Wierstra, I. 2013). It has been reported that Foxm1 upregulates the expression of the neural stemness marker Nestin1 in NSCs from the embryonic cerebral cortices of mouse and is critical for their self-renewal (Wierstra, I. 2013). Mutants conducted with loss-of-function Foxm1 suggest that in the murine cerebellum its main function is the adjustment of the transition in G2 / M phase (Schuller U. et al, 2007; Gage, F.H. 2000). All previous studies did not however provide information regarding Foxm1’s regulation or other functions in cerebellar NSCs.
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Regulation of quorum sensing and virulence in pseudomonas aeruginosa

Regulation of quorum sensing and virulence in pseudomonas aeruginosa

Since rsaL transcription is strongly dependent upon LasR, this factor should not be expressed in a lasR mutant. Hence it is interesting to discuss our results by considering that lasR mutants are frequently isolated from the lungs of CF patients. It is still under debate whether these mutants arise because they are social cheaters gaining a growth advantage by utilizing ‘‘public goods’’ (i.e., virulence factors) produced by neighbour wild type cells, rather than producing their own [45,48,49], or whether they are better adapted than the wild type to the peculiar environment of the CF lung [50,51]. Overall, it is still unclear whether and how the emergence of lasR mutants could contribute to the CF lung decline. However, a recent work showed that lasR mutants were able to produce very high levels of pyocyanin under the slow- growing conditions typical of the chronic infection, while wild type cells did not [13]. Moreover, in co-cultivation experiments, the lasR mutant was able to cooperate with the wild type for pyocyanin production [13]. Pyocyanin overproduction in the lasR mutant is due to the loss of repression normally exerted by RsaL on phenazine biosynthetic genes, because RsaL itself is not expressed as a consequence of lasR mutation [13]. However, mutations in rsaL are not commonly isolated in CF clinical samples, suggesting that the constitutive expression of QS regulated factors caused by this mutation is unfavourable in the CF lung environment, and that a mutation in the rsaL gene can be tolerated only when associated to the lack of expression of the entire LasR regulon.
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The role of Yin and Yang 1 (YY1) transcription factor in acute myeloid leukemia (AML)

The role of Yin and Yang 1 (YY1) transcription factor in acute myeloid leukemia (AML)

Recent studies challenged the classical hierarchy of human hematopoiesis, working out a refined model where multipotent cells, such as HSCs and MPPs, give rise to committed unipotent progenitors, without an intermediate oligopotent CMP and MEP stage (4). However, the hierarchical model is suitable and widely used to investigate the molecular mechanisms that drive HSPCs commitment and differentiation (1). The entire differentiation process is highly regulated by both extrinsic and intrinsic factors, including transcription factors (TFs), signaling pathways, cytokines, niche factors (2) and epigenetic regulators of gene expression (3).
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Novel molecular mechanisms involved in the stimulatory action of zinc in breast cancer

Novel molecular mechanisms involved in the stimulatory action of zinc in breast cancer

Numerous studies have shown that GPER contrib- utes to the progression of certain tumors including breast cancer [45–50]. In addition, clinical studies have indicated that GPER may be a predictor of aggressive cancer behavior as its expression has been associated with negative clinical outcomes in several cancer histotypes [51–55]. The activation of GPER has been shown to trigger EGFR transactivation, subsequent transduction events such as the activation of MAPK and PI3K cascades, gene expression changes, and relevant biological responses such as proliferation, migration, and angiogenesis in diverse cancer cell types and CAFs [56,57]. In this context, it should be mentioned that the metal cadmium may induce cAMP increase, ERK1/2 activation, and proliferation of breast cancer cells in a GPER-dependent manner [58]. Rece- ntly, we also demonstrated that copper activates the HIF-1 a/GPER/VEGF signaling in cancer cells leading to angiogenesis and tumor progression [57]. Further extending these findings, in the present study we have demonstrated that in breast cancer cells exposed to Zn the activation of GPER leads to rapid signaling events such as the phosphorylation of EGFR and IGF-IR, and their downstream effectors ERK and AKT, the up-regulation of c-fos and EGR1, two main GPER target genes largely involved in growth responses. It is worth noting that Zn induced also GPER targets namely metallothioneins MT1X and MT2A, whose overexpression correlates with chemoresistance and poor prognosis in breast tumors [59,60]. Moreover, in line with the known capability of GPER to trigger the transcription of genes associated with cell growth [27], we assessed the potential of Zn to regulate the Figure 9. Schematic representation of the functional cooperation of GPER with IGF-IR and EGFR upon zinc
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Identification and characterization of regulatory networks controlling the expression and activity of the alternative sigma factor pvds in Pseudomonas aeruginosa

Identification and characterization of regulatory networks controlling the expression and activity of the alternative sigma factor pvds in Pseudomonas aeruginosa

reduced T3SS expression and cytotoxicity, and diminished twitching motility. This characteristic phenotype depends on the ability of RsmA to directly inhibit the translation of genes responsible in the biosynthesis of the exopolysaccharide Psl, and to indirectly promote the transcription of the genes involved in the production of the T3SS, type IV pili and flagella (Irie et al., 2010; Brencic and Lory 2009, Burrowes et al., 2006). Moreover transcriptomic analyses of rsmA mutants indicated that RsmA negatively influence the expression of the genes encoding the type VI secretion system and of the pel operon which encodes the enzymes involved in the synthesis of the exopolysaccaride Pel (Brencic and Lory 2009). Transcriptomic analyses also suggested that the Gac system influences the expression of several iron uptake genes, although different studies obtained opposite results (Brencic and Lory 2009; Burrowes et al., 2006). In one study, the deletion of rsmA in the P. aeruginosa reference strain PAO1 caused an increase in the transcription of some iron uptake genes (Burrowes et al., 2006), while in another study, performed on the P. aeruginosa strain PAK, the rsmA deletion resulted in a decrease in iron uptake gene expression (Brencic and Lory 2009). Additional studies are clearly required to clarify the role of the Gac system in the regulation of iron uptake in P. aeruginosa. Irrespective of its effect on iron uptake, the ability of the Gac system to coordinately control the expression of many virulence genes involved in the acute infection and those related to chronic infection makes the Gac system an important factor in P. aeruginosa pathogenicity, as it has been experimentally demonstrated in different models of infection. Indeed, deletion mutants in gacA showed a strongly reduced ability to cause infection in mice, plants, insects and nematodes (Rahme et al., 1995; Tan et al., 1999; Jander et al., 2000, Coleman et al., 2003). Accordingly it has been demonstrated that the deletion of rsmA, hence constitutive activation of Gac system, causes an increase in the ability of P. aeruginosa to persist in the mouse lung (Mulcahy et al., 2007). The enhanced persistence of the P. aeruginosa rsmA mutant could depend on the fact that in this mutant the production of the exopolysaccharides Pel and Psl is not repressed by RsmA, ultimately resulting in increased exopolysaccharide synthesis and biofilm formation compared to the wild type strain. Indeed, in vitro experiments have demonstrated that the deletion of rsmA causes a strong increase in the ability to form biofilms, while the deletion of the two small RNA genes rsmY and rsmZ or of gacA almost completely abolishes biofilm formation (Brencic et al., 2009).
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Regulation of the spermatogonial niche factor GDNF by retinoic acid

Regulation of the spermatogonial niche factor GDNF by retinoic acid

(Cunningham & Duester, 2015). RARs receptors act as a molecular platform to recruit numerous factors that regulate gene transcription. In the classical model, the RA-dependent transcriptional activation requires that the RAR / RXR heterodimer in the absence of retinoic acid binds to the RARE sequence and represses the transcription of the target genes also through epigenetic modifiers. RAR / RXR in the repressive state recruits co- repressors such as NCOR-1 (Kumar et al., 2016), the histone deacetylases HDACs and the repressive chromatin-remodeling complex PRC2 (Polycomb repressive complex 2) (Figure 3 A). This leads to an increase in trimethylation of the lysine 27 of histone H3 (H3K27me3) which induces chromatin condensation and gene silencing. When retinoic acid is present, it binds to RAR and induces a conformational change of the RAR/RXR heterodimer that promotes the release of repressors and the recruitment of factors that activate transcription such as NCOA1 or SRC1, NCOA2 and NCOA3. In turn, the co-activators recruits the HATs histoneacetylases and the Tritorax complex that remove the negative epigenetic modifications (H3K27me3) and add histone activating transcription modifications: histone H4 acetylations and the histone H3 lysine 4 trimethylation. These modifications induce relaxation of the chromatin, allowing access on the promoter of the transcription factor target gene and the RNA polymerase.
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Regulation of cell physiology through the modulation of estrogen receptors activities by natural and synthetic compounds

Regulation of cell physiology through the modulation of estrogen receptors activities by natural and synthetic compounds

ChIP assays were performed essentially as previously described (Barnett et al., 2008). After starvation and ligand treatment, MCF-7 cells were cross-linked using 1% formaldehyde at 37° C for 10 min. Glycine (0.125 M) was than added for 5 min at RT. Cells were next washed twice with PBS and harvested in ice-cold PBS. Cell pellets were first re- suspended in nuclei isolation buffer [50 mM Tris (pH 8.0), 60 mM KCl, 0.5% NP40, protease inhibitor, and 10 mM DTT], centrifuged at 3,000 × g for 5 min, and resuspended in 200 µl lysis buffer [0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris (pH 8.0), protease inhibitor, and 10 mM DTT]. Nuclei were sonicated (Fisher Scientific, Sonic Dismembrator Model 100) three times at 80% maximum power for 5 s and the sonicate was centrifuged at 14,000 × g for 10 min. The supernatant was diluted up to 500 µl with dilution buffer [1% Triton X- 100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris (pH 8), protease inhibitor, and 10 mM DTT] and 1/10 was taken aside as input for qPRC analysis. The samples were than pre-cleared with 50 µl of protein G beads for 1 h rotating at 4° C. Following protein G beads removal, lysates were incubated at 4° C rotating o.n. with 5 µg of anti-ERα antibody (MC-20, Santa Cruz Biotechnology), then pulled down at 4° C for 1 h with 50 µl of protein G beads. After brief centrifugation, precipitates were sequentially washed twice with 1 ml of washing buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), and 150 mM NaCl], once with 1 ml washing buffer II [1% NP-40, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0), 250 mM LiCl], and twice with 1 ml of TE buffer [1 mM EDTA and 10 mM Tris-HCl (pH 8.0)]. Chromatin complexes were centrifuged and than eluted by incubating at R.T. for 30 min the beads with 50 µl 1% SDS e 0,1 M NaHCO 3 . Following
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Study of Enzymatic Mechanisms Using Density Functional Theory and QM/MM Methods

Study of Enzymatic Mechanisms Using Density Functional Theory and QM/MM Methods

In relation to the likelihood of the reactants state, in order to calculate the exact energy of converting the N-MIO mechanism reactants into the Single-Step mechanism reactants, we would need to do some kind of free energy calculation like thermody- namic integration. In the absence of that information, and even without an energy value, it is easy to see that the reactants states of the Single-Step mechanism should be much more probable. The side chain of a Tyrosine has a pKa of 10: it is much more probable to find a protonated tyrosine amino acid than a deprotonated one in standard conditions. This predisposition should only be reversed if in the enzymatic vicinities of the tyrosine there were positive or partially positive amino acids (with a strong dipole, pointing in the opposite direction, for example). In the initial state of the N-MIO mechanism, it is required the active center of TAL to possess two deprotonated tyrosine residues. This is a very improbable event, not only because of the multiplicative relation between the two low probabilities, but also because the presence of a negative charge in the active center makes the presence of another negative charge yet more unlikely. Even if one considers the intermediate 1 structure, with only one deprotonated tyrosine, to be the reactants state (it is possible that the substrate enters the enzyme in the deprotonated amine form), it is very difficult to justify the depro- tonation of Tyr60, since there are no residues in the vicinity to sta- bilize the negative charge. On the other side, the initial state of the Single-Step mechanism seems much more natural. For once, the overall charge of the active center is zero, whereas in the MIO mechanism it was 1. Also, the Single-Step mechanism only requires one deprotonated tyrosine amino acid, Tyr300, and the
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Intragenic modulators of CFTR gene expression involved in cystic fibrosis clinical variability

Intragenic modulators of CFTR gene expression involved in cystic fibrosis clinical variability

of CFTR on different alleles. If no mutations are found (or even if only one mutation is not found) even at third level, the genetic test contributes to a sensible exclusion of diagnosis of CF. On the contrary, in the second case, since it can be difficult to apply all levels of mutational search to each subject to check its carrier status, an appropriate residual genetic risk is usually chosen and the mutation search is performed with a suitable panel of mutations. Following the above considerations, it can be said that the often incomplete genetic characterization of CF and CF-like patients may often be due to technical limitations and constitutes the major obstacle to the understanding of the genotype-phenotype relationship. Although the techniques have progressively evolved, becoming increasingly more sensitive and effective, the situation still remains complex for CF-like forms, characterized by mutational patterns that are different from those of the classical forms of CF and with a definition of the diagnosis often not clear.
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Innate, non-cytolytic CD8+ T cell-mediated suppression of HIV replication by MHC-independent inhibition of virus transcription

Innate, non-cytolytic CD8+ T cell-mediated suppression of HIV replication by MHC-independent inhibition of virus transcription

An incubation at 4 °C overnight, with gentle shaking, was performed by adding 1.5 ml of the diluted mono- and co-culture cell supernatants to each cytokine array. The chemiluminescent signals were captured using the ChemiDoc XRS+ Imaging system (BioRad), and numerical signal densities were processed with ImageJ software. The average signal of the pair of duplicate spots representing each cytokine was determined and average background signal from blank was subtracted from each spot. Values that were below background after blank subtraction were set to 0. Values were then normalized using the average internal positive control signals according to manufacturer’s data analysis instructions. This normalization process allows for a consistent comparison of results across multiple arrays. Comparisons between 5:1 (E:T CD4/CD8) co- cultures and CD4 mono-cultures were carried out using parametric donor-matched pairwise t-test (test of variance) and batch t-test (test of the mean and standard deviation of the set). Normality was assessed using Kolmogorov-Smirnov test, and significance was attributed at p<0.05.
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<<The>> role of intrinsic and extrinsic signals in the regulation of brest cancer stem cell activity

<<The>> role of intrinsic and extrinsic signals in the regulation of brest cancer stem cell activity

As a final step of the current study, we used the R2C tumor xenograft model to examine the effects of DEXA on Leydig tumor growth in vivo. To this aim, we injected R2C cells into the intrascapular region of male nude mice and followed tumor growth after administration of DEXA at 1 and 10 mg/Kg/day. This administration was well tolerated because no change in body weight or in food and water consumption was observed along with no evidence of reduced motor function. In addition, no significant differences in the mean weights or histological features of the major organs (liver, spleen, and kidney) after sacrifice were observed between vehicle-treated mice and those that received treatment, indicating a lack of toxic effects at the dose given. As shown in Figure 4a, DEXA (1 and 10 mg/kg/day) induced a regression in tumor growth. This effect was evident as early as day 12 of treatment, and tumor volumes continued to reduce over control for the duration of experiment. 30 days after injection, tumor weigth as well as tumor size were markedly smaller in animals treated with DEXA respect to vehicle-treated mice (Figure 4b). In agreement with our in vitro findings, we observed in R2C xenograft tumors from mice treated with DEXA a significant decrease of aromatase expression, evaluated by both western blotting and immunohistochemistry analysis (Figure 5a and b). This was concomitant with a reduction in the expression of Ki-67, a well-known marker for cell proliferation, in R2C xenograft tumors from mice treated with DEXA than in tumors from vehicle-treated controls (Figure 5c).
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Neurophysiological mechanisms of quiet vigilance in neurodegenerative diseases: an electroencephalography research program

Neurophysiological mechanisms of quiet vigilance in neurodegenerative diseases: an electroencephalography research program

The diagnosis of the probable DLB was carried out in agreement with the consensus guidelines (McKeith IG et al. 2005; 2017). Twenty-six out of 46 DLB patients performed DaTSCAN to confirm the diagnosis of probable DLB. Concerning the detection of the core and suggestive features of DLB, the Neuropsychiatric inventory (NPI) item-2 investigated the occurrence frequency and the severity of hallucinations (Cummings JL et al., 1994). Frontal Assessment Battery (FAB) (Dubois et al., 2000) and Clinician Assessment of Fluctuations (Walker et al., 2000a; 200b) were included to investigate, respectively, the severity of the frontal dysfunction and the presence and severity of the cognitive fluctuations. Unified Parkinson Disease Rating Scale-III (UPDRS-III) assessed the presence and severity of the extrapyramidal signs (Fahn et al., 1987). The presence and/or absence of rapid eye movement (REM) sleep behavior disorder (RBD) was determined according to minimal International Classification of Sleep Disorders criteria (1992). As this retrospective study was based on data of several clinical units that did not follow a harmonized protocol, the DLBMCI subjects underwent a different battery of clinical scales including the Neuropsychiatric Inventory (NPI), the scale for the assessment of Behavioral and Psychological Symptoms of Dementia (BPSD), the MMSE, the Epworth Sleepiness Scale (ESS) for estimating subjective sleep disturbances, and the Alzheimer’s Disease Cooperative Study for the Activities of Daily Living (ADCS-ADL). Furthermore, DLBMCI subjects underwent different battery of neuropsychological tests to evaluate the status of MCI (Donaghy et al., 2018). This battery included neuropsychological tests assessing the general cognitive performance in the domains of memory, language, executive function/attention, and visuo-construction abilities (some of them received the CERAD-plus battery). The diagnostic criteria for ADD and ADMCI have been described diagnostic criteria of “I and II studies” as weel as inclusion criteria for Nold subjects.
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The evolution of human-dog communication mechanisms during the domestication process

The evolution of human-dog communication mechanisms during the domestication process

modify the behavior of a recipient. Intraspecific aggression is directed toward members of the same species, whereas interspecific aggressiveness is directed toward members of different species. Bouts of aggression may also be elicited following a threatening event not related to another living being. Displays of aggression are linked to the instinct of preservation and are well documented in predation scenarios, often evoking an aggressive defensive response (fear-induced) in the prey species. Aggression may also be aimed at defending a territory for monopolizing resources (e.g., food and mating partners) or achieving and maintaining a higher social status. Thus, from a behavioral ecology perspective, aggression is a tool to achieve a competitive advantage; however, it is a behavior that is energetically expensive, time-consuming and potentially dangerous (Fitzpatrick et al., 1995; Shuster and Wade, 2003). The energy employed in the expression of the aggressive behavior is no longer available for other functions, such as pregnancy and caring for offspring. For males, the cost of the aggressiveness to defend a territory and obtain access to females is balanced by ameliorative reproductive success, whereas females have fewer direct advantages, considering that the energy invested in the expression of aggressive displays is detracted by the functions related to the sex-specific behaviors linked to reproduction (Andersson, 1994). Thus, it is expected that females would express fewer aggressive behaviors than males in several contexts. In many species, including humans, a higher incidence of aggressive behavior has been reported in males and is well documented (Johnson, 1972; Leshner, 1978). However, in some species (e.g., pigs), males and females show the same level of aggressive behaviors (D’Eath and Lawrence, 2004) and in some cases, a sex-reversed trait, with more aggressive females, has also been identified. For example, as an effect of the particular social structure in which females are dominant, female spotted hyenas (Crocuta crocuta) appear to be consistently more aggressive than males (Glickman et al., 1987). Considering that dogs do not belong to a species with a sex-reversed role, more aggressive behavior is expected in males unless the domestication process has affected sex differences related to aggression.
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Transcriptional regulation and dynamics of Arabidopsis nuclear proteome in response to auxin and oligogalacturonides

Transcriptional regulation and dynamics of Arabidopsis nuclear proteome in response to auxin and oligogalacturonides

elongation factor EF-Tu (Kunze et al., 2004). However, there are exceptions; the elicitor effect of NPP1 (necrosis-inducing Phytophthora protein 1) requires an intact protein and overlapping peptide fragments were inactive (Fellbrich et al., 2002), perhaps indicating that it is the activity of this protein that is detected by the plant rather than a specific amino acid sequence. Presumably, there would be a huge selective advantage for mutations within these epitopes that rendered them inactive as elicitors of plant defense systems. However, it would seem that, in many cases, such mutations also have a deleterious effect on the function of these proteins in the pathogen. For example, in Pep-13, a 13 amino acid internal peptide of a 42 kDa transglutaminase enzyme from the cell wall of Phytophthora sojae, substitution of Trp231 to Ala abolished elicitor activity in parsley but with a concurrent 98% reduction in transglutaminase activity (Brunner and et al., 2002). Thus, it appears that plants have evolved receptors that recognize short highly conserved amino acid stretches of certain microbial proteins that cannot easily be altered without loss of the protein function. That said, certain microbes may have evolved the capacity to avoid detection by specific PRRs. For example, Agrobacterium tumefaciens and Ralstonia solanacearum (pathogens) as well as Rhizobium meliloti (symbiont) possess functional flagellins that do not elicit a defence response in Arabidopsis and the N-terminal peptide from Pseudomonas syringae pv. tomato DC3000 (Pst) EF-Tu is not as potent an elicitor in Arabidopsis as those from other bacteria (Kunze et al., 2004; Sun et al., 2006). The evolution of non-eliciting PAMPs is one way in which pathogens can overcome non-host resistance in plants; however, the lack of a single eliciting PAMP has not yet been directly shown to affect the virulence of the pathogen. Some experiments have shown that deletion of a specific PRR in the host affects susceptibility; however, in other studies wild-type plants and plants lacking a PRR were equally susceptible (Sun et al., 2006) (Zipfel et al., 2004). This could be explained by the evolution in plants of recognition systems for multiple PAMPs from the same micro-organism. For example, Arabidopsis recognizes both flagellin and EF-Tu and these PAMPs activate the same signalling and defence responses in a nonsynergistic manner (Zipfel et al., 2006). A recent gene expression profiling study has also demonstrated that the lack of flagellin perception does not dramatically alter PAMP-induced gene expression during infection of Arabidopsis by Pst (Thilmony et al., 2006).
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Gene expression as a digital communication system

Gene expression as a digital communication system

Biologically, the results of gene expression in eukaryotic cells are the proteins that play a fundamental role in a target organ in the body. When this target organ is in the long range of nanocommunication, the proteins are called hormones, and the endocrine system uses them to control the physiological functions in the human body. The transmission media in this long-range nanocommunication system are the blood vessels (which are considered random with a drifting medium), which include arteries, veins and capillaries. Continuously monitoring concentrations of certain parameters in blood capillaries could greatly improve the detection of a potentially critical situation in a patient and could be a fundamental way to administer drugs quickly. Therefore, in Sun et al. [20], a communication system in the blood vessel environments is proposed in which the transmitters are platelets, information is propagated through the flow of blood vessels, and the receivers are the endocrine cells (specifically soluble CD40 ligand sCD40L). At the transmitter end, the platelets secrete and release cytokines (i.e., the information molecules), which are small cell-signalling protein molecules, while at the receiver end, the cytokines are decoded by the sCD40 ligand receptors.
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