Empathy at school project: Effects of didactics of emotions® on emotional competence, cortisol secretion and inflammatory profile in primary school children. A controlled longitudinal psychobiological study.

Background: There is mounting evidence of the presence of chronic stress among children during primary school: girls and boys under the age of 15 years often experience anxiety, irritability and sleeping problems with negative consequences on scholastic climate and the spread of bullying and dropping out of school. The promotion of emotion regulation within school environment through innovative didactic methodologies represents a valuable tool for teachers and parents to reduce emotional distress and associated risk behaviours and to promote wellbeing. Aim: Our research aims to explore the psychological and biological consequences of teaching emotional training in an experimental group of Italian Primary School children. Methods: A sample of pupils (81 children aged between 6 and 8) was divided into an experimental group (33 subjects) and a control group (30 subjects). A further advanced group of 18 subjects, who have experienced the method in the previous school year, was also included. The experimental study lasted one school year (from October 2021 to May 2022). The following psychological tests were administered to all groups: TEC (Test of Emotion Comprehension) to measure the children's different emotional abilities and the Projective test (PT) 'A person in the rain', to identify the coping skills of children in a stressful condition. Morning salivary cortisol, IL-6 and TNF-alpha assays were conducted in all three groups. Psychological and biological tests were administered at the beginning of the study and at the end of the study. Results: The MR-Anova model for TEC score showed that there was not a significant group effect [Fgroup = 2.24, p = 0.114]. Pairwise comparisons showed that mean score significantly increased only in the Experimental group (pB < 0.001) and at the end of the project there was a significant difference between Experimental group and Control group (pB = 0.012). The mean score of PT test increased significantly from baseline to the end of the project for the Experimental group (pB < 0.001) and for the Advanced group (pB = 0.004). At the end of the project, there were significant differences between the Experimental group and the Control group (pB = 0.004) and between the Advanced group and the Control group (pB < 0.001). Salivary cortisol analysis revealed a significant effect between subjects [Fgroup = 9.66; p < 0.001] and significant effects within subjects with the main effect of the time [Ftime = 35.41; p < 0.001] and the significant interaction "time x group" [Ftimexgroup = 3.38; p = 0.040]. Pairwise comparisons showed that cortisol levels decreased significantly over time only in the Experimental group (pB < 0.001). Regarding to IL-6 levels, there was not a significant effect between subjects [Fgroups = 0.0481; p = 0.953]. The mean level decreased significantly for each group from baseline to post project (pB < 0.001). With respect to TNF-alpha levels, the mean levels decreased over time for all groups (pB = 0.006 for Experimental group; pB < 0.001 either for the Advanced or Control group). Conclusion: the results documented in the experimental groups who experienced didactics of emotion for at least one school year show a significant increase in children's ability to cope with reality, stress and anxiety, and an improvement of their emotional competence. Meanwhile, a significant reduction in the amount of salivary cortisol was observed in the experimental group at the end of the scholastic year; meantime a stable reduced amount of salivary cortisol in advanced group throughout the project was also observed. These findings show that an intervention through an emotional education program is able to regulate interpersonal skills and the stress axis response.

A role for D-aspartate oxidase in schizophrenia and in schizophrenia-related symptoms induced by phencyclidine in mice.

Increasing evidence points to a role for dysfunctional glutamate N-methyl-D-aspartate receptor (NMDAR) neurotransmission in schizophrenia. D-aspartate is an atypical amino acid that activates NMDARs through binding to the glutamate site on GluN2 subunits. D-aspartate is present in high amounts in the embryonic brain of mammals and rapidly decreases after birth, due to the activity of the enzyme D-aspartate oxidase (DDO). The agonistic activity exerted by D-aspartate on NMDARs and its neurodevelopmental occurrence make this D-amino acid a potential mediator for some of the NMDAR-related alterations observed in schizophrenia. Consistently, substantial reductions of D-aspartate and NMDA were recently observed in the postmortem prefrontal cortex of schizophrenic patients. Here we show that DDO mRNA expression is increased in prefrontal samples of schizophrenic patients, thus suggesting a plausible molecular event responsible for the D-aspartate imbalance previously described. To investigate whether altered D-aspartate levels can modulate schizophrenia-relevant circuits and behaviors, we also measured the psychotomimetic effects produced by the NMDAR antagonist, phencyclidine, in Ddo knockout mice (Ddo(-)(/-)), an animal model characterized by tonically increased D-aspartate levels since perinatal life. We show that Ddo(-/-) mice display a significant reduction in motor hyperactivity and prepulse inhibition deficit induced by phencyclidine, compared with controls. Furthermore, we reveal that increased levels of D-aspartate in Ddo(-/-) animals can significantly inhibit functional circuits activated by phencyclidine, and affect the development of cortico-hippocampal connectivity networks potentially involved in schizophrenia. Collectively, the present results suggest that altered D-aspartate levels can influence neurodevelopmental brain processes relevant to schizophrenia.

Chromatin and DNA methylation dynamics during retinoic acid-induced RET gene transcriptional activation in neuroblastoma cells.

Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci.

HAND1 gene expression is negatively regulated by the High Mobility Group A1 proteins and is drastically reduced in human thyroid carcinomas.

HMGA1 proteins exert their major physiological function during embryonic development and play a critical role in neoplastic transformation. Here, we show that Hand1 gene, which codes for a transcription factor crucial for differentiation of trophoblast giant cells and heart development, is upregulated in hmga1 minus embryonic stem cells. We demonstrate that HMGA1 proteins bind directly to Hand1 promoter both in vitro and in vivo and inhibit Hand1 promoter activity. We have also investigated HAND1 expression in human thyroid carcinoma cell lines and tissues, in which HMGA proteins are overexpressed, with respect to normal thyroid; an inverse correlation between HMGA1 and HAND1 expression was found in all thyroid tumor histotypes. A correlation between HAND1 gene repression and promoter hypermethylation was found in anaplastic carcinomas but not in other thyroid tumor histotypes. Therefore, we can hypothesize that HMGA1 overexpression plays a key role on HAND1 silencing in differentiated thyroid carcinomas and that promoter hypermethylation occurs in later stages of thyroid tumor progression. Finally, the restoration of the HAND1 gene expression reduces the clonogenic ability of two human thyroid carcinoma-derived cell lines, suggesting that HAND1 downregulation may have a role in the process of thyroid carcinogenesis.

DNA methylation in intron 1 of the frataxin gene is related to GAA repeat length and age of onset in Friedreich ataxia patients.

BACKGROUND: The most frequent mutation of Friedreich ataxia (FRDA) is the abnormal expansion of a GAA repeat located within the first intron of FXN gene. It is known that the length of GAA is directly correlated with disease severity. The effect of mutation is a severe reduction of mRNA. Recently, a link among aberrant CpG methylation, chromatin organisation and GAA repeat was proposed. METHODS: In this study, using pyrosequencing technology, we have performed a quantitative analysis of the methylation status of five CpG sites located within the region upstream of GAA repeat, in 67 FRDA patients. RESULTS: We confirm previous observation about differences in the methylation degree between FRDA individuals and controls. We showed a direct correlation between CpG methylation and triplet expansion size. Significant differences were found for each CpG tested (ANOVA p<0.001). These differences were largest for CpG1 and CpG2: 84.45% and 76.80%, respectively, in FRDA patients compared to 19.65% and 23.34% in the controls. Most importantly, we found a strong inverse correlation between CpG2 methylation degree and age of onset (Spearman's rho = -0.550, p<0.001). CONCLUSION: Because epigenetic changes may cause or contribute to gene silencing, our data may have relevance for the therapeutic approach to FRDA. Since the analysis can be performed in peripheral blood leucocytes (PBL), evaluation of the methylation status of specific CpG sites in FRDA patients could be a convenient biomarker.

PATZ1 gene has a critical role in the spermatogenesis and testicular tumours.

PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. To gain insights into its biological role, we generated mice lacking the PATZ1 gene. Male PATZ1(-/-) mice were unfertile, suggesting a crucial role of this gene in spermatogenesis. Consistently, most of adult testes from these mice showed only few spermatocytes, associated with increased apoptosis, and complete absence of spermatids and spermatozoa, with the subsequent loss of tubular structure. The analysis of PATZ1 expression, by northern blot, western blot and immunohistochemistry, revealed its presence in Sertoli cells and, among the germ cells, exclusively in the spermatogonia. Since PATZ1 has been indicated as a potential tumour suppressor gene, we also looked at its expression in tumours deriving from testicular germ cells (TGCTs). Although expression of PATZ1 protein was increased in these tumours, it was delocalized in the cytoplasm, suggesting an impaired function. These results indicate that PATZ1 plays a crucial role in normal male gametogenesis and that its up-regulation and mis-localization could be associated to the development of TGCTs.

Isolation of a SIR-like gene, SIR-T8, that is overexpressed in thyroid carcinoma cell lines and tissues.

We used subtractive library screening to identify the changes that occur in gene expression during thyroid cell neoplastic transformation. Complementary DNA from normal thyroid cells (HTC 2) was subtracted from a complementary DNA library constructed from a human thyroid papillary carcinoma cell line. The library was screened for genes upregulated in human thyroid papillary carcinoma cell line cells, and several cDNA clones were isolated. One of these clones has a sirtuin core and high homology with the human silent information regulator protein family. This clone, designated "SIR-T8", was overexpressed in human thyroid carcinoma cell lines and tissues, but not in adenomas. The human SIR-T8 protein has a molecular weight of 39 kDa and is primarily located in the cytoplasm under the nuclear membrane. The SIR-T8 gene is located on chromosome 17q25-1.

High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth.

The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with different transcriptional factors. The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours. In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait. This analysis led to the isolation of homeodomain-interacting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase. HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells. The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y). We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay. In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory effect on normal thyroid cells. Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle. Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory effect is independent from the interaction with the HMGI(Y) protein.

RNF4 is a growth inhibitor expressed in germ cells but not in human testicular tumors.

The RING-finger protein RNF4 modulates both steroid-receptor-dependent and basal transcription and interacts with a variety of nuclear proteins involved in cell growth control. RNF4 is expressed at very high levels in testis and at much lower levels in several other tissues. We show that in germ cells RNF4 expression is strongly modulated during progression of spermatogonia to spermatids, with a peak in spermatocytes. Analysis of human testicular germ cell tumors shows that RNF4 is not expressed in all tumors analyzed including seminomas, the highly malignant embryonal carcinomas, yolk sac, and mixed germ cell tumors. We also show that the ectopically expressed RNF4 gene inhibits cell proliferation of both somatic and germ cell tumor-derived cells. Mutation of critical cysteine residues in the RING finger domain abolished the RNF4 growth inhibition activity. Our results suggest that the lack of RNF4 expression may play a role in the progression of testicular tumors.

Rat protein tyrosine phosphatase eta physically interacts with the PDZ domains of syntenin.

The tyrosine phosphatase r-PTPeta is able to suppress the malignant phenotype of rat thyroid tumorigenic cell lines. To identify r-PTPeta interacting proteins, a yeast two-hybrid screening was performed and an insert corresponding to the full-length syntenin cDNA was isolated. It encodes a protein containing two PDZ domains that mediates the binding of syntenin to proteins such as syndecan, proTGF-alpha, beta-ephrins and neurofascin. We show that r-PTPeta is able to interact with syntenin also in mammalian cells, and although syntenin is a tyrosine-phosphorylated protein it is not a substrate of r-PTPeta. The integrity of both PDZ domains of syntenin and the carboxy-terminal region of r-PTPeta are required for the interaction between syntenin and r-PTPeta.

The overlap of Inr and TATA elements sets the use of alternative transcriptional start sites in the mouse galectin-1 gene promoter.

In the mouse gene encoding the protein galectin-1, transcription initiation at the +1 site is directed by a TATA box. Here we show that a consensus Inr element (TCCAGTT), which spans residues -34 to -28 and overlaps the TATA box, directs RNA initiation also from a previously uncharacterized site located at position -31. Upstream transcripts are polyadenylated and contribute to more than half of the galectin-1 mRNA population in all tissues analyzed. The promoter architecture is evolutionarily conserved to man, and galectin-1 mRNA size variants accumulate also in human HeLa cells. The 5' end terminus of the transcripts initiated at residue -31 is extremely GC-rich, and may fold into a relative stable hairpin which could influence translation and thus modulate the intracellular levels of galectin-1. The interval -63/+45 contains sufficient information to ensure RNA initiation from both -31 and +1 sites, and a Sp1 site spanning residues -57 to -48 is crucial for promoter functioning. The unusual overlap of core promoter elements suggests that RNA initiation from the -31 and the +1 sites may take place in a sequential manner.

Galectin-1 gene expression and methylation state in human T leukemia cell lines.

Galectin-1 has been demonstrated to be a mediator of T-cell apoptosis acting on activated T-cells and, in a selective manner, on different T leukemia cell lines. Here we show that the sensitivity to galectin-1 is associated with repression of the endogenous galectin-1 gene whereas non-sensitive cells express high levels of galectin-1. Repression of galectin-1 gene in sensitive cells is associated with hyper-methylation of the promoter region. Transient treatment of non-expressing cells with the demethylating agent 5-azacytidine led to irreversible demethylation and subsequent reactivation of galectin-1 gene.

Pivotal role of the RB family proteins in in vitro thyroid cell transformation.

Rat thyroid differentiated cells (PC Cl 3) are an excellent model system with which to study the interaction between differentiation and cell transformation. We previously demonstrated that PC Cl 3 cells expressing the adenovirus E1A gene no longer depend on thyrotropin for growth and do not express thyroid differentiation markers. Here we show that an E1A mutant unable to bind the RB protein failed to transform the PC Cl 3 cells. Conversely, mutations in the E1A p300 interacting region did not affect its transforming ability. The pivotal role of RB family proteins in the thyroid cell transformation is supported by the thyrotropin independence induced by the E7 gene of human papilloma virus type 16, but not by a mutated form in the RB-binding region.

Glial cell line-derived neutrotrophic factor and neurturin can act as paracrine growth factors stimulating DNA synthesis of Ret-expressing spermatogonia.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are related growth factors which exert trophic effects on several neuronal populations and developing kidney. GDNF-family ligands interact with membrane receptors designated GFRalphas which, in turn, mediate stimulation of the Ret receptor tyrosine kinase. Here we show that Ret, GFRalpha-1 (the GDNF receptor), and GFRalpha-2 (the NTN receptor) are expressed by testicular germ cells, while GDNF and NTN are expressed by Sertoli cells. Both GDNF and NTN stimulate DNA synthesis in spermatogonia. Furthermore, Ret, ligands and co-receptors are expressed in germ cell tumors. Thus, GDNF-family ligands may act as paracrine factors in spermatogenesis and this circuit may be active in germ cell tumors.

A novel member of the BTB/POZ family, PATZ, associates with the RNF4 RING finger protein and acts as a transcriptional repressor.

We have identified a novel human gene encoding a 59-kDa POZ-AT hook-zinc finger protein (PATZ) that interacts with RNF4, a mediator of androgen receptor activity, and acts as a transcriptional repressor. PATZ cDNA was isolated through a two-hybrid interaction screening using the RING finger protein RNF4 as a bait. In vitro and in vivo interaction between RNF4 and PATZ was demonstrated by protein-protein affinity chromatography and coimmunoprecipitation experiments. Such interaction occurred through a small region of PATZ containing an AT-hook DNA binding domain. Immunofluorescence staining and confocal microscopy showed that PATZ localizes in distinct punctate nuclear regions and colocalizes with RNF4. Functional analysis was performed by cotransfection assays: PATZ acted as a transcriptional repressor, whereas its partner RNF4 behaved as a transcriptional activator. When both proteins were overexpressed a strong repression of the basal transcription was observed, indicating that the association of PATZ with RNF4 switches activation to repression. In addition, RNF4 was also found to associate with HMGI(Y), a chromatin-modeling factor containing AT-hook domains.

Control of galectin gene expression.

In this review we summarize the available information on the expression of mammalian galectins in normal and transformed cells. From all these studies it is apparent that each cell might express most of galectins; yet, during development or in various differentiation stages or under different physiological or pathological conditions, one or more galectins are preferentially expressed in each cell type. This implies a fine control of gene expression and suggests that such control should be coordinated. Nevertheless, to date very few studies have been performed on the mechanisms responsible for the regulation of galectin genes. We review the current knowledge on galectin promoter function. We believe that this area of galectin research will expand rapidly in the near future.