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Abstract Background The Anaplastic Lymphoma Kinase (ALK) is an orphan receptor tyrosine kinase, which undergoes post-translational N-linked glycosylation. The catalytic domain of ALK was originally identified in the t(2;5) translocation that produces the unglycosylated oncogenic protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL). Recently, both germline and somatic activating missense mutations of ALK have been identified in neuroblastoma (NB), a pediatric cancer arising from neural crest cells. Moreover, we previously reported that ALK expression is significantly upregulated in advanced/metastatic NB. We hypothesized that ALK function may depend on N-linked glycosylation and that disruption of this post-translational modification would impair ALK activation, regardless the presence of either gene mutations or overexpression. Methods We employed tunicamycin to inhibit N-linked glycosylation. The following ALK-positive NB cell lines were used: SH-SY5Y and KELLY (ALK mutation F1174L), UKF-NB3 (ALK mutation R1275Q) and NB1 (ALK amplification). As a control, we used the NB cell lines LA1-5S and NB5 (no ALK expression), and the ALCL cell line SU-DHL1 (NPM-ALK). Results Tunicamycin treatment of ALK-positive NB cells resulted in a hypoglycosylated ALK band and in decreased amounts of mature full size receptor. Concomitantly, we observed a marked reduction of mature ALK phosphorylation. On the contrary, tunicamycin had no effects on NPM-ALK phosphorylation in SU-DHL1 cells. Moreover, phosphorylation levels of ALK downstream effectors (AKT, ERK1/2, STAT3) were clearly impaired only in ALK mutated/amplified NB cell lines, whereas no significant reduction was observed in both ALK-negative and NPM-ALK-positive cell lines. Furthermore, inhibition of N-linked glycosylation considerably impaired cell viability only of ALK mutated/amplified NB cells. Finally, the cleavage of the Poly-ADP-ribose-polymerase (PARP) suggested that apoptotic pathways may be involved in cell death. Conclusions In this study we showed that inhibition of N-linked glycosylation affects ALK phosphorylation and disrupts downstream pro-survival signaling, indicating that inhibition of this post-translational modification may be a promising therapeutic approach. However, as tunicamycin is not a likely candidate for clinical use other approaches to alter N-linked glycosylation need to be explored. Future studies will assess whether the efficacy in inhibiting ALK activity might be enhanced by the combination of ALK specific small molecule and N-linked glycosylation inhibitors.

Background: Uremic pruritus is a quite common condition among patients with chronic kidney disease. Symptom severity and patterns are variable., Aim: To assess how nurses in the field of nephrology perceive the issue of uremic pruritus in dialysis patients and the relevance of this condition., Design: A qualitative survey-based study., Participants: Nurses working in the field of nephrology., Methods: Nurses were administered an online survey with a 10-item survey (5 on socio-demographics, 5 specific to nephrology). Responses were reported in descriptive form and response rates., Results: The study involved 185 nurses working in the field of nephrology, most of which were over the age of 40, and 39% had over 20 years of experience. Most perceived that uremic pruritus has decreased over the years thanks to better dialysis methods and that it is currently independent of the type of dialysis. Fifty-two percent estimate pruritus affects half or more of patients, and 47.6% that it affects only a minority. 75% estimate moderate-to-severe uremic pruritus involves between 1 and 3 cases every 10 patients (40.5% estimating ≤3/10 patients and 34.6% ≤1/10 patients), and 25% it affects at least half of patients. 41% estimate that more than half the patients do not find relief from PU to prevent it from impairing their quality of life., Conclusions: The results highlight a large divergence in the perception of uremic pruritus among nurses. While this evidence the need for greater awareness on uremic pruritus, this also suggests a privileged role for nurses in detecting uremic pruritus occurrence and properly referring patients to nephrologist., (© 2025 European Dialysis and Transplant Nurses Association/European Renal Care Association.)

Sports-related traumatic dental injuries (TDIs) are a significant global concern, particularly in contact sports, where the risk of orofacial injuries is high. Custom-made sports mouthguards (CSMs) are recognized as the most effective means of preventing these injuries, providing both protection and comfort without impairing athletic performance. Despite their proven benefits, there is no globally standardized approach to mouthguard design, fabrication, or usage, primarily due to varying regulations, awareness levels, and cultural attitudes toward sports safety across different countries. This document from the European Association for Sports Dentistry (EA4SD) outlines the latest guidelines for selecting, constructing, clinical use, and maintaining CSMs. It emphasizes the need for mouthguards fabricated from FDA-approved materials, designed to absorb and distribute impact forces effectively, and customized to ensure optimal fit and comfort. The EA4SD also highlights the importance of education for dental professionals and athletes on the benefits of CSMs, advocating for their mandatory use in high-risk sports to reduce the prevalence of TDIs and related complications., (© 2024 The Author(s). Dental Traumatology published by John Wiley & Sons Ltd.)

The regenerative ability of Hydra vulgaris was tested as potential biomarker for the development of a new eco-toxicological index. The test is based on the regeneration rate and the aberration frequency of the columna (body and adhesive foot) after separation from head and tentacles by a bistoury. Particularly, 45 columnae were submerged in the rearing solution (that is Hydra medium) to have control, and 285 in potential contaminated waters to have treatments, collected from 19 sites along 10 rivers in central Italy. ANCOVA and chi-square tests were used to compare values from each site to a laboratory control. Subsequently the values on regeneration rate and aberration frequency were inserted in a double entry matrix, where the match of the two entries in the matrix provides the score of the proposed Teratogenic Risk Index (TRI). Each score corresponded to one of the 5 teratogenic risk classes, to which a risk level was associated: from 1 (no risk) to 5 (very high risk). On the whole, 32% of the studied sites were classified as no teratogenic risk while the remaining showed a variable risk level from low to very high. This study proposed for the first time an early warning system to detect the presence of teratogens in running waters, providing a rapid and cost-effective evaluation method. Therefore, TRI may contribute to initiate adequate measures to manage riverine habitats, and to monitor the running water teratogenic status. Specifically, this index may provide the opportunity to identify the disturbance sources and then to drive the decisions, together with competent authorities, on the catchment and landscape management and on the possible use of waters for urban, agricultural, and industrial activities, since they may show significant effects on the human health.

The ability of mammals to feel noxious stimuli lies in a heterogeneous group of primary somatosensory neurons termed nociceptors, which express specific membrane receptors, such as the Transient Receptor Potential (TRP) family. Here, we show that one of the most important nociceptive-like pathways is conserved in the freshwater coelenterate Hydra vulgaris, the most primitive organism possessing a nervous system. In particular, we found that H. vulgaris expresses TRPM3, a nociceptor calcium channel involved in the detection of noxious heat in mammals. Furthermore, we detected that both heat shock and TRPM3 specific agonist (i.e., pregnenolone sulfate) induce the modulation of the heat shock protein 70 (HSP70) and the nitric oxide synthase (NOS), two genes activated by TRP-mediated heat painful stimuli in mammals. As expected, these effects are inhibited by a TRPM3 antagonist (i.e., mefenamic acid). Interestingly, the TRPM3 agonist and heat shock also induce the expression of nuclear transcription erythroid 2-related factor (Nrf2) and superoxide dismutase (SOD), known markers of oxidative stress; noteworthy gene expression was also inhibited by the TRPM3 antagonist. As a whole, our results demonstrate the presence of conserved molecular oxidative/nociceptive-like pathways at the primordial level of the animal kingdom.

Neuroblastoma is a stroma-poor (SP) aggressive pediatric cancer belonging to neuroblastic tumors, also including ganglioneuroblastoma and ganglioneuroma, two stroma-rich (SR) less aggressive tumors. Our previous gene-expression profiling analysis showed a different CXCL13 mRNA expression between SP and SR tumors. Therefore, we studied 13 SP and 13 SR tumors by reverse transcription quantitative real-time PCR (RT-qPCR) and we found that CXCR5b was more expressed in SP than in SR and CXCL13 was predominantly expressed in SR tumors. Then, we isolated neuroblastic and Schwannian stromal cells by laser capture microdissection and we found that malignant neuroblasts express CXCR5b mRNA, whereas Schwannian stromal cells express CXCL13. Immunohistochemistry confirmed that stroma expresses CXCL13 but not CXCR5. To better understand the role of CXCL13 and CXCR5 in neuroblastic tumors we studied 11 neuroblastoma cell lines and we detected a heterogeneous expression of CXCL13 and CXCR5b. Interestingly, we found that only CXCR5b splice variant was expressed in both tumors and neuroblastoma lines, whereas CXCR5a was never detected. Moreover, we found that neuroblastoma cells expressing CXCR5 receptor migrate toward a source of recombinant CXCL13. Lastly, neuroblastoma cells induced to glial cell differentiation expressed CXCL13 mRNA and protein. The chemokine released in the culture medium was able to stimulate chemotaxis of LA1-5S neuroblastoma cells. Collectively, our data suggest that CXCL13 produced by stromal cells may contribute to the generation of an environment in which the malignant neuroblasts are retained, thus limiting the possible development of metastases in patients with SR tumor., (©2011 AACR.)

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALK(high) 12.8%, ALK(low) 73%, P = 0.0035; cell death: ALK(high) 56.4%, ALK(low) 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs.

We developed an in vivo gene therapy approach to characterize and optimize the anti-angiogenic activity of class I interferons (IFNs), using packaging cell lines producing an amphotropic LXSN-based retrovirus expressing either IFN-alpha1 (alpha1Am12), IFN-beta (betaAm12) murine cDNAs, or the vector alone (neoAm12). Pretreatment of endothelial-like Eahy926 cells in vitro with conditioned media (CM) from alpha1Am12 or betaAm12 cells for 48 hours significantly inhibited their migration and invasion as compared to neoAm12-CM-treated cells. betaAm12-CM also inhibited the formation of capillary-like structures on Matrigel by EAhy926 cells. In vivo, inclusion of the betaAm12 cells strongly inhibited, and alpha1Am12 partially inhibited, the angiogenic response in the Matrigel sponge model in both immune-competent and athymic nude mice. Electron microscopy showed a reduction of host cell infiltration in alpha1Am12- and betaAm12-containing sponges and reduction of invading tubular clefts of host cells as compared to controls. Finally, inoculation of either alpha1Am12 or betaAm12 cells (10%) along with a highly angiogenic Kaposi's sarcoma cell line (90%) resulted in a powerful reduction of tumor growth in nude mice in vivo, as did infection with the interferon-alpha-producing retroviruses. These data suggest that a gene therapy approach using class I interferons can effectively inhibit tumor angiogenesis and growth of vascular tumors.

Metastasis is a sequence of events including proliferation, migration, adhesion, invasion and subsequent metastatic growth of tumour cells in distant organs. We previously showed that highly metastatic variants of murine melanoma cells express higher levels of the basement membrane proteoglycan perlecan than low or non metastatic variants and expression of an antisense perlecan can reduce metastatic potential. In contrast, antisense expression of perlecan in fibrosarcoma cells was reported to enhance tumorigenesis. To better understand the role of perlecan in angiogenesis we have transfected KS-IMM, an immortalized cell line derived from a human Kaposi s sarcoma, with an antisense perlecan construct and investigated the positive/negative role of perlecan in KS. KS-IMM cells were transfected with either empty vector (neo) or the antisense perlecan construct and clones were isolated. Immuno-blot analysis showed a reduction of perlecan levels in two (AP3 and AP4) isolated clones, in Northern blot analysis endogenous perlecan was undetectable in the AP3 and AP4 clones, while it was present in the neo control clones. AP clones had a reduced migration to HGF in Boyden chambers as compared to neo clones. Proliferation in low serum or serum-free conditions was strongly reduced in the AP clones as compared to the neo control cells. The neotransfected cells showed rapid proliferation in low serum supplemented with HGF and VEGF, while antisense transfected clones showed little response. Finally, AP-trasfected KS-IMM cells had significantly reduced migration to VEGF and HGF with respect to controls. In contrast, when the AP transfected cells were injected in nude mice they paradoxically showed enhanced tumor growth as compared to controls. Our preliminary data indicate that perlecan reduction plays a crucial role on Kaposi s sarcoma cell migration and proliferation in vitro. However, in vivo KS-IMM depleted of perlecan had a growth advantage. A possible hypothesis is that perlecan is necessary for growth of KS-IMM cells in vitro, however its down-regulation might promote angiogenesis through increased angiogenic growth factor diffusion, resulting in enhanced tumor growth in vivo.