A regional-based newborn hearing screening program: the Emilia-Romagna model after ten years of legislation.

Background: Hearing loss, occurring in 1-3/1,000 newborns in the well-babies population, is one of the most common congenital diseases, and hearing screening at birth still represents the only means for its early detection. Since 2011 the Emilia Romagna Regional Health Agency has recommended Newborn Hearing Screening for all babies at its birth points and for newborns moving to the region. The aims of this study are to analyze the results of this regional-based Newborn Hearing Screening program and to discuss the impact of the legislative endorsement on the organization. Material and methods: This is an observational retrospective chart study. The recordings of well-babies and babies at Neonatal Intensive Care Units were collected during the period from January 1st 2015 to December 31st 2020. The following data were included: Newborn Hearing Screening coverage, percentage of refer at otoacoustic emissions, prevalence and entity of hearing loss, unilateral/bilateral rate, presence of audiological risk factors. Results: More than 99% of a total of 198,396 newborns underwent the Newborn Hearing Screening test during the period January 1st 2015 to December 31st 2020, with a coverage ranging between 99.6% and 99.9%. Overall, the percentage of confirmed hearing loss cases was about 17-30 % of refer cases, 745 children received a diagnosis of hearing loss (prevalence 3.7/1,000). Considering profound hearing loss cases, these represent 13% of bilateral hearing loss. Conclusion: A regional-based Newborn Hearing Screening program is valuable and cost-effective. In our experience, the centralization of the data system and of the data control is crucial in order to implement its efficiency and effectiveness. Healthcare policies, tracking systems and public awareness are decisive for a successful programme implementation.

Polariton Bose-Einstein condensate from a bound state in the continuum.

Bound states in the continuum (BICs)1-3 are peculiar topological states that, when realized in a planar photonic crystal lattice, are symmetry-protected from radiating in the far field despite lying within the light cone4. These BICs possess an invariant topological charge given by the winding number of the polarization vectors5, similar to vortices in quantum fluids such as superfluid helium and atomic Bose-Einstein condensates. In spite of several reports of optical BICs in patterned dielectric slabs with evidence of lasing, their potential as topologically protected states with theoretically infinite lifetime has not yet been fully exploited. Here we show non-equilibrium Bose-Einstein condensation of polaritons-hybrid light-matter excitations-occurring in a BIC thanks to its peculiar non-radiative nature, which favours polariton accumulation. The combination of the ultralong BIC lifetime and the tight confinement of the waveguide geometry enables the achievement of an extremely low threshold density for condensation, which is reached not in the dispersion minimum but at a saddle point in reciprocal space. By bridging bosonic condensation and symmetry-protected radiation eigenmodes, we reveal ways of imparting topological properties onto macroscopic quantum states with unexplored dispersion features. Such an observation may open a route towards energy-efficient polariton condensation in cost-effective integrated devices, ultimately suited for the development of hybrid light-matter optical circuits.

Mice harboring a Hajdu Cheney Syndrome mutation are sensitized to osteoarthritis.

Osteoarthritis is a joint disease characterized by cartilage degradation, altered gene expression and inflammation. NOTCH1 and NOTCH2 receptors and the JAGGED1 ligand regulate chondrocyte biology; however, the contribution of Notch signaling to osteoarthritis is controversial. Hajdu Cheney Syndrome (HCS) is a rare genetic disorder affecting the skeleton and associated with NOTCH2 mutations that lead to NOTCH2 gain-of-function. A murine model of the disease (Notch2tm1.1Ecan) was used to test whether the HCS mutation increases the susceptibility to osteoarthritis. The knee of three-month-old Notch2tm1.1Ecan male mice and control sex-matched littermates was destabilized by resection of the medial meniscotibial ligament, and changes in the joint analyzed two months thereafter. Expression of Notch target genes was increased in the femoral heads of Notch2tm1.1Ecan mice, documenting Notch signal activation. Periarticular bone and cartilage structures were unaffected in Notch2tm1.1Ecan mutants subjected to sham surgery, indicating that NOTCH2 gain-of-function had no discernible impact on joint structure under basal conditions. However, destabilization of the medial meniscus increased osteophyte volume and thickened subchondral bone in Notch2tm1.1Ecan mice compared to wild type littermates. Moreover, destabilized Notch2tm1.1Ecan mutants exhibited histological signs of moderate to severe cartilage degeneration, demonstrating joint sensitization to the development of osteoarthritis. Chondrocyte cultures from Notch2tm1.1Ecan mutants expressed increased Il6 mRNA levels following exposure to JAGGED1, possibly explaining the susceptibility of Notch2tm1.1Ecan mice to osteoarthritis. In conclusion, Notch2tm1.1Ecan mutants are sensitized to the development of osteoarthritis in destabilized joints and NOTCH2 activation may play a role in the pathogenesis of the disease.

Tackling muscle fibrosis: From molecular mechanisms to next generation engineered models to predict drug delivery.

Muscle fibrosis represents the end stage consequence of different diseases, among which muscular dystrophies, leading to severe impairment of muscle functions. Muscle fibrosis involves the production of several growth factors, cytokines and proteolytic enzymes and is strictly associated to inflammatory processes. Moreover, fibrosis causes profound changes in tissue properties, including increased stiffness and density, lower pH and oxygenation. Up to now, there is no therapeutic approach able to counteract the fibrotic process and treatments directed against muscle pathologies are severely impaired by the harsh conditions of the fibrotic environment. The design of new therapeutics thus need innovative tools mimicking the obstacles posed by the fibrotic environment to their delivery. This review will critically discuss the role of in vivo and 3D in vitro models in this context and the characteristics that an ideal model should possess to help the translation from bench to bedside of new candidate anti-fibrotic agents.

A fourth case of POMT2-related limb girdle muscle dystrophy with mild reduction of α-dystroglycan glycosylation.

BACKGROUND: POMT2 mutations have been identified in Walker-Warburg syndrome or muscle-eye-brain-like, but rarely in limb girdle muscular dystrophy (LGMD). RESULTS: Two POMT2 mutations, one null and one missense, were found in a patient with LGMD and mild mental impairment, no brain or ocular involvement, minor histopathological features, and slight reduction of α-dystroglycan (α-DG) glycosylation and α-DG laminin binding. CONCLUSIONS: Our case, the fourth LGMD POMT2-mutated reported to date, provides further evidence of correlation between level of α-DG glycosylation and phenotype severity.

Interleukin 6 mediates selected effects of Notch in chondrocytes.

OBJECTIVE: Notch receptors determine cell fate by regulating transcription, an event mediated by the Notch intracellular domain (NICD), which is generated by proteolysis brought about by Notch-ligand interactions. Since Notch activation or exposure to interleukin (Il)6 have similar effects in chondrocytes, we explored whether interleukin 6 (Il6) contributes to the mechanisms of Notch action in these cells. METHOD: NICD was overexpressed in primary chondrocytes from Rosa(Notch) mice, where the Rosa26 promoter precedes a loxP-flanked STOP cassette followed by the NICD coding sequence. Cells were infected with adenoviral vectors expressing Cre to induce NICD or green fluorescent protein (GFP) as control. Gene expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Il6 protein concentration in the culture media was determined by enzyme-linked immunosorbent assay (ELISA). To test the mechanisms of Notch action on Il6 expression, cells were transfected with a fragment of the Il6 promoter or control vector pGL3, or transcriptionally arrested with 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole. Il6 was inhibited with a neutralizing antibody, whereas a normal immunoglobulin G (IgG) was used as control. RESULTS: NICD induced Il6 mRNA and protein, and transactivated the Il6 promoter without affecting Il6 mRNA stability. Il6 neutralization had no impact on gene expression under basal conditions, and did not modify the effects of NICD on sex determining region-Y-related high mobility group-box gene (Sox)9, collagen type II α1 (Col2a1) and collagen type X α1 (Col10a1) expression. Conversely, Il6 neutralization opposed aggrecan (Acan) suppression and prevented matrix metalloprotease (Mmp)13 induction by NICD. CONCLUSION: Il6 mediates suppression of Acan and induction of Mmp13 expression by Notch in chondrocytes.

Fibroblasts from the muscles of Duchenne muscular dystrophy patients are resistant to cell detachment apoptosis.

Extracellular matrix (ECM) proteins, including collagen and growth factors, are greatly increased in tissue fibrosis and mainly secreted by fibroblasts. We previously demonstrated that muscle-derived fibroblasts from Duchenne muscular dystrophy (DMD) patients have a profibrotic phenotype, that includes significantly reduced expression of tissue inhibitor of metalloprotease 3 (TIMP-3) compared to control. Since TIMP-3 induces apoptosis in various cell types, we hypothesized increased resistance of DMD fibroblasts to apoptosis. To address this, we evaluated apoptotic nuclei, caspase 3, caspase 3 substrate expression, and migration and adhesion properties of muscle-derived fibroblasts, after applying different apoptosis-inducing treatments. We found that DMD fibroblasts were less susceptible to cell death, more adhesive, and had greater tendency to migrate than control fibroblasts - findings further supported by alterations in FAK and ERK/MAPK expression. Resistance to apoptosis and greater adhesion are likely to contribute to muscle fibrosis so a pharmacological treatment that targets dysregulated pathways involved in cell detachment apoptosis (anoikis) may limit the progressive fibrotic remodeling characteristic of DMD.

[Ectopic breast fibroadenoma. Case report]. / Fibroadenoma in mammella ectopica. Case report.

Among the rare anomalies of the breast development, polythelia is the most common, between 1% and 5% of women and men present supernumerary nipples. Polymastia, usually presenting as ectopic breast tissue without areola-nipple complex, is seen mostly along the milk line, extending from the axilla to the pubic region. Ectopic breast tissue is functionally analogous to mammary gland and it is subjected to the same alterations and diseases, whether benign or malignant, that affect normal breast tissue. We report the case of a 21 years-old female evaluated by the medical staff after founding a solid nodular mass by suspect axillary lymphadenopathy. Differential diagnosis with lymphoma is the major problem in these cases. The mass was removed and the intraoperative histological examination showed fibroadenoma in axillary supernumerary breast. Presence of ectopic breast tissue is a rare condition; development of benign mass or malignant degeneration is possible, but it is very unusual. In case of polymastia diagnosis is simple; in case of isolated nodule, without local inflammation or infection, there are greater difficulties. Ultrasonography is diagnostic in case of breast fibroadenoma, but it might be inadequate in ectopic localizations owing to the shortage of mammary tissue around the mass. Preoperative diagnosis is important to plan an adequate surgical treatment; lumpectomy is indicated in case of benign tissue; in case of malignancy, therapy is based on the standard treatment used for breast cancer (surgery, chemotherapy and radiation therapy).

Can elderly patients with colorectal cancer tolerate planned surgical treatment? A practical approach to a common dilemma.

AIM: Analysing the effectiveness of a surgical procedure is mandatory in every modern health-care system. The aging of the population stresses the need for a good standard of care. This study tests the hypothesis that porthsmouth-physiologic operative severity score for enumeration of morbidity and mortality (P-POSSUM) and colorectal-POSSUM (CR-POSSUM) would be useful clinical auditing tools in colorectal cancer surgery for aged patients. METHOD: One hundred and seventy-seven consecutive patients over 70 years of age underwent emergency or elective surgery from January 2003 to December 2005. Demographic, clinical and surgical information, score systems' prediction, complications and 30-day mortality data were prospectively entered in a comprehensive database. The observed over expected morbidity and mortality rate was calculated. RESULTS: Thirty-day observed mortality was 10.3% (19/177) while P-POSSUM and CR-POSSUM expected mortality were, respectively, 11.21% (P = NS) and 13.08% (P = NS). Overall observed morbidity was 42.7%, P-POSSUM prediction was 59.3% (P = 0.002). Morbidity and mortality data were analysed for specific subgroups of patients (resection and anastomosis/resection and stoma/palliative; emergency/elective). CONCLUSION: P-POSSUM and CR-POSSUM are useful tools to predict mortality in elderly patients. P-POSSUM significantly overestimated the risk of complications. A more accurate tool for preoperative assessment for aged patients is probably needed to predict the post-surgical outcome.

Sarcomatoid anaplastic carcinoma of the small bowel in cardiac transplant bearer.

Sarcomatoid carcinoma is a rare form of primitive carcinoma of the small bowel; it is considered a variant of adenocarcinoma, histologically characterized by a typical biphasic pattern with epithelial- and mesenchymal-like cells. Twenty-one cases have been described in the literature, presenting as small bowel obstructions (twenty cases) or superior vena cava syndrome (one case). The authors report the case of a 56 year-old man on immunosuppressive therapy for a heart transplant, who underwent surgery after a history of repeated episodes of melena, anemization and bowel obstruction. The operation brought to light an intraluminal occlusive mass in the distal ileum, associated with other intraluminal neoplasms of different sizes throughout the small bowel. No evidence of mesenteric adenopathies or hepatic metastases were found. Histological examination and immunohistochemical stain showed an anaplastic sarcomatoid carcinoma. The tumor cells showed strong positivity for cytokeratin and vimentin, and negativity for CD117 and CD34, as well as for all other characteristic markers of mesenchymal tumors. Early diagnosis is usually very difficult, due to the lack of any stereotyped clinical expression and the difficult to study the small bowel. Small-bowel barium follow-through or video capsule endoscopy can be helpful. In most cases, an emergency surgical operation is performed without a clear preoperative diagnosis. The case report is completed by a review of the literature.

Altered extracellular matrix transcript expression and protein modulation in primary Duchenne muscular dystrophy myotubes.

Extent of muscle fibrosis contributes to disease severity in muscular dystrophies. To investigate whether extracellular matrix (ECM) components contribute to the severe fibrosis observed in Duchenne muscular dystrophy (DMD) skeletal muscle, we quantitated several ECM components (transcripts and proteins) in primary DMD and control myotube cultures. We evaluated the fibrogenic transforming growth factor- beta1 (TGF-beta1); the small pleiotropic proteoglycan decorin, involved in collagen fibrillogenesis and TGF-beta1 modulation; metalloproteinases MMP-2 and MMP-9; tissue inhibitors of metalloproteinase (TIMP) 1, 2 and 3; collagens I and VI; and the tissue factor myostatin that inhibits muscle growth. Dystrophic myotube cultures had significantly lower levels of decorin mRNA, as also observed in DMD muscle biopsies, and significantly higher levels of TGF-beta1, myostatin, and collagens I and VI. MMP-2, TIMP-1 and TIMP-2 transcript levels were also significantly increased in DMD, but MMP-9 and TIMP-3 transcripts were unchanged. By zymography, MMP-2 activity was significantly higher in DMD than control. Protein levels were similar in DMD and controls but myostatin protein was significantly increased in DMD. We have found that transcript expression and protein modulation of several ECM components is altered in DMD muscle cells in vitro, indicating that these cells contribute fundamentally to the pathological process, since the inflammation and degeneration characterizing DMD muscle in vivo are presumably absent in culture. Our findings that myostatin-potent inhibitor of satellite cell activation and muscle renewal--is increased, and that decorin-binder and downregulator of TGFbeta1 and myostatin--is decreased, may have implications for DMD therapy to reduce muscle fibrosis.

FMS*Calciumfluor specifically increases mRNA levels and induces signaling via MAPK 42,44 and not FAK in differentiating rat osteoblasts.

The homeopathic compound of resonance FMS*Calciumfluor (FMS*) reportedly promotes osteogenic differentiation of rat pre-osteoblasts in vitro. Here, we show that the continuous exposure of differentiating rat osteogenic cells (ROB) to FMS* modulates the level of expression of mRNAs for 7 of the 8 osteogenic markers tested. Alkaline phosphatase (AP), osteocalcin (OC), metalloproteinases (MMP-2 and -14), procollagenase C (BMP-1), biglycan (BG) and integrin 1 are expressed at higher levels in FMS*-treated osteoblasts than in control cultures. MMP-2 and -14 mRNA are not down-modulated at mineralization. Also, the pattern of expression induced by FMS* for some of these genes (BMP-1, BG and integrin 1) is changed, but collagen type I (Coll I) mRNA levels are not affected by treatment with FMS*. This suggests that FMS* modulates mRNA levels and that this is not generalized, but gene(s) specific. We also report that exposure to FMS* rapidly and transiently induces activation of mitogen-activated protein kinases (MAPKs) 42,44 in populations of early osteoblasts, but not in pre-osteoblasts, with a cell differentiation stage-dependent and pertussis toxin (PTX)-sensitive response. Subsequent to FMS* MAPK signaling activation, an increase in AP and MMP-14 mRNA is detected, which is also inhibited by PTX, suggesting that FMS* activation of MAPK signaling could be an early event required for the induction of these genes. Exposure to FMS* does not cause changes in the activity of p125 (FAK)-mediated signaling.

Expression profile of epidermal differentiation complex genes in normal and anal cancer cells.

Anal cancer originates from a peculiar histological region and provides a useful model for investigating alterations in proliferation and/or differentiation of neoplastic keratinocytes. Epidermal differentiation complex (EDC) genes, which form one of the major gene clusters in the human genome, are involved in the terminal differentiation of epithelial cells and in many instances have been implicated in epithelial tumours. We constructed a DNA macroarray capable of characterising the expression profiles of the entire EDC gene complex in normal mucosa and anal cancer biopsies of seven unrelated patients. Brain tissue and cultured keratinocytes were used as controls. All anal cancer samples showed expression profiles in which none of the EDC genes was silent, as evaluated by phosphor-imager analysis. Variance analysis showed significantly lower expression of SPRR2 with respect to SPRR1 or SPRR3, and significantly higher expression of S100A8 than of other S100A subfamily members. At hierarchical clustering analysis, the four basaloid anal cancer cases conglomerated in the top five positions. The macroarray method used by us provides the first demonstration of the expression profile of the EDC gene family in anal cancer, and is capable of producing significant information on the subgrouping of epithelial tumours such as anal cancer.

Prenatal sonographic diagnosis of axillary cystic lymphangiomas.

Cystic lymphangiomas are congenital malformations of the lymphatic system that occur most commonly in the nuchal region. Nuchal lymphangiomas are often referred to as cystic hygromas. Few cases of axillary cystic lymphangioma have been reported, but the prognosis of this lesion appears to be less grave than that of its nuchal counterpart. We present 2 cases of axillary cystic lymphangiomas that were diagnosed on prenatal sonography. In 1 case, prenatal sonography showed a multilocular, large cystic mass on the fetal left side, in the area of the lower chest and upper abdomen. The mass extended from at least the level of the umbilicus up to the neck but did not involve the neck. In the second case, prenatal sonography showed a multilocular, septate axillary cystic lymphangioma external to the chest with no flow on Doppler sonography. Follow-up sonography revealed normal fetal growth and enlargement of the cystic mass surrounding the left humerus. Both infants were delivered without complications and have been managed conservatively.

Expression analysis and mutational screening of the epithelium-specific ets gene-1 (ESE-1) in patients with squamous anal cancer.

To investigate whether ESE-1 gene abnormalities are involved in alterations of epithelial cell differentiation in squamous anal cancer ESE-1 expression and structure were screened in six patients by reverse transcriptase-polymerase chain reaction (RT-PCR) and automated sequence analysis. The complete cDNA of isoform ESE-1b was always expressed and correctly spliced, with single nucleotide polymorphism being observed in two cases. Presence of ESE-1b point mutations was excluded. Expression of SPRR2A and ENDOA/CK8, two epithelium-specific ESE-1 target genes, were revealed by RT-PCR in all cases. This first report of expression of ESE-1, and of SPRR2A and ENDOA/CK8 (both related to terminal differentiation in different types of epithelia lining) in anal cancer excludes the hypothesis that these genes influenced carcinogenesis in our patients. Despite selecting of patients without clinical evidence of HPV infection, PCR consistently revealed HPV-16 DNA, highlighting the importance of HPV infection in anal cancer.

Changes of synaptotagmin interaction with t-SNARE proteins in vitro after calcium/calmodulin-dependent phosphorylation.

The regulation of multiple phases of the life cycle of synaptic vesicles is carried out by a complex series of protein-protein interactions. According to the SNARE hypothesis the core of these interactions is a heterotrimeric complex formed by syntaxin, SNAP-25, and VAMP-synaptobrevin. Other proteins interacting with the core of the SNARE complex, such as voltage-activated calcium channels and synaptotagmin (a putative calcium sensor), are considered crucial for the calcium dependence of release and also molecular mediators of synaptic plasticity. Here the interaction of synaptotagmin with SNARE proteins was studied in immunoprecipitated native complexes, and the effects of previous phosphorylation-dephosphorylation on this interaction were analyzed. It is surprising that the interaction of synaptotagmin with syntaxin and SNAP-25 in native complexes was not found to be calcium-dependent. However, previous incubation under dephosphorylating conditions decreased the synaptotagmin-syntaxin interaction. Stimulation of Ca2+/calmodulin-dependent protein kinase II, which endogenously phosphorylates synaptotagmin in synaptic vesicles, increased the interaction of syntaxin and SNAP-25 with synaptotagmin (particularly when measured in the presence of calcium), as well as increasing the binding of the kinase itself. These results suggest that calcium decreases synaptotagmin-t-SNARE interactions after dephosphorylation and increases them after phosphorylation. Overall, these results imply a phosphorylation-dephosphorylation balance in regulation of the synaptotagmin-t-SNARE interaction and suggest a role for protein phosphorylation in the modulation of calcium sensitivity in transmitter release.

Genetic dissection of dome formation in a mammary cell line: identification of two genes with opposing action.

In this work, we extend the study of the genes controlling the formation of domes in the rat mammary cell line LA7 under the influence of DMSO. The role of the rat8 gene has already been demonstrated. We have now studied two additional genes. The first, called 133, is the rat ortholog of the human epithelial membrane protein 3 (EMP3), a member of the peripheral myelin protein 22 (PMP22)/EMP/lens-specific membrane protein 20 (MP20) gene family that encodes for tetratransmembrane proteins; it is expressed in the LA7 line in the absence of DMSO but not in its presence. The second gene is the beta subunit of the amiloride-sensitive Na(+) channel. Studies with antisense oligonucleotides show that the formation of domes is under the control of all three genes: the expression of rat8 is required for both their formation and their persistence; the expression of the Na(+) channel beta subunit is required for their formation; and the expression of gene 133 blocks the expression of the Na(+) channel genes, thus preventing formation of the domes. The formation of these structures is also accompanied by the expression of alpha(6)beta(1) integrin, followed by that of E-cadherin and cytokeratin 8. It appears, therefore, that dome formation requires the activity of the Na(+) channel and the rat8-encoded protein and is under the negative control of gene 133. DMSO induces dome formation by blocking this control.

Modifications in brain cAMP- and calcium/calmodulin-dependent protein kinases induced by treatment with S-adenosylmethionine.

Several lines of evidence suggest that the mechanism of action of antidepressant drugs (AD) involves adaptive changes occurring in intraneuronal post-receptor signal transduction cascades. Protein phosphorylation has a key role in signal transduction and was previously found to be a target in the action of AD (5-HT and/or NA reuptake blockers). Several studies showed that cAMP- and type II Ca2+/calmodulin-dependent protein kinases (PKA and CaMKII) are markedly affected by typical AD in two different and complementary cellular districts, respectively microtubules (a somatodendritic compartment) and synaptic vesicles (a presynaptic terminal compartment). In order to investigate whether the effect on protein kinases may be involved in the therapeutic action of drugs it is interesting to compare the effect of atypical AD with that of typical drugs. In this study the effect of the atypical AD S-adenosylmethionine (SAMe) was tested. Repeated (12 days) SAMe treatment induced in cerebrocortical microtubules an increase in the binding of cAMP to the RII PKA regulatory subunit and an increase in the endogenous phosphorylation of microtubule-associated protein 2, an effect resembling that of typical AD. In synaptic terminals the treatment induced an increase in the activity of CaMKII and in the endogenous phosphorylation of vesicular substrates. However, this modification was found in the cerebral cortex rather than in the hippocampus, where typical AD affect CaMKII. In addition the synapsin I level was decreased in the hippocampus and increased in the cerebral cortex, an effect not detected with typical AD.

Spiral intercellular calcium waves in hippocampal slice cultures.

Complex patterns of intercellular calcium signaling occur in the CA1 and CA2 regions of hippocampal slice organotypic cultures from neonatal mice. Spontaneous localized intercellular Ca2+ waves involving 5-15 cells propagate concentrically from multiple foci in the stratum oriens and s. radiatum. In these same regions, extensive Ca2+ waves involving hundreds of cells propagate as curvilinear and spiral wavefronts across broad areas of CA1 and CA2. Ca2+ waves travel at rates of 5-10 mu m/s, are abolished by thapsigargin, and do not require extracellular Ca2+. Staining for astrocytes and neurons indicates that these intercellular waves occur primarily in astrocytes. The frequency and amplitude of Ca2+ waves increase in response to bath application of N-methyl-D-aspartate (NMDA) and decrease in response to removal of extracellular Ca2+ or application of tetrodotoxin. This novel pattern of intercellular Ca2+ signaling is characteristic of the behavior of an excitable medium. Networks of glial cells in the hippocampus may behave as an excitable medium whose spatial and temporal signaling properties are modulated by neuronal activity.

Role of the TFG N-terminus and coiled-coil domain in the transforming activity of the thyroid TRK-T3 oncogene.

The thyroid TRK-T3 oncogene results from the fusion of the tyrosine kinase (TK) domain of NTRK1 (one of the receptors for the Nerve Growth Factor) on chromosome 1 to sequences of a novel gene, TFG, on chromosome 3. The 68 kDa TRK-T3 fusion oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform mouse NIH3T3 cells. The TFG portion of TRK-T3 contains a coiled-coil domain most likely responsible for the constitutive, ligand-independent activation of the receptor tyrosine kinase activity. We have previously shown that TRK-T3 oncoprotein forms, in vivo, complexes of three or four molecules. By mean of different experimental approaches, we show here that TRK-T3 activity depends on oligomers formation. In addition, the analysis of different TRK-T3 mutants indicates that the TFG coiled-coil domain and its N-terminal region are both required for the activation and the fully transforming activity of the TRK-T3 oncoprotein, although, most likely, they play a role in different steps of the transforming process. The deletion of the coiled-coil domain abrogates the oligomers formation leading to a constitutive activation; the deletion of the N-terminal region, although not affecting phosphorylation and complexes formation, abrogates transformation, thus suggesting a role in cellular localization and/or interaction with substrata.