Neural Crest Research Articles

A Snapshot of Early Transcriptional Changes Accompanying the Pro-Neural Phenotype Switch by NGN2, ASCL1, SOX2, and MSI1 in Human Fibroblasts: An RNA-Seq Study

Direct pro-neural reprogramming is a conversion of differentiated somatic cells to neural cells without an intermediate pluripotency stage. It is usually achieved via ectopic expression (EE) of certain transcription factors (TFs) or other reprogramming factors (RFs). Determining the transcriptional changes (TCs) caused by particular RFs in a given cell line enables an informed approach to reprogramming initiation. Here, we characterized TCs in the human fibroblast cell line LF1 on the 5th day after EE of the single well-known pro-neural RFs NGN2, ASCL1, SOX2, and MSI1. As assessed by expression analysis of the bona fide neuronal markers nestin and beta-III tubulin, all four RFs initiated pro-neuronal phenotype conversion; analysis by RNA-seq revealed striking differences in the resulting TCs, although some pathways were overlapping. ASCL1 and SOX2 were not sufficient to induce significant pro-neural phenotype switches using our EE system. NGN2 induced TCs indicative of cell phenotype changes towards neural crest cells, neural stem cells, mature neurons, as well as radial glia, astrocytes, and oligodendrocyte precursors and their mature forms. MSI1 mainly induced a switch towards early stem-like cells, such as radial glia.

Abstract C002: CKLF attracts CCR4-expressing CD4+ cells to foster immune repression and tumor aggressiveness in MYCN-driven neuroblastoma

Abstract Neuroblastoma, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. Here we combined analyses of human neuroblastoma with live tracking and functional studies of the tumor microenvironment (TME) in zebrafish. We identified chemokine-like factor (CKLF) as a key driver of MYCN-mediated immunosuppression and neuroblastoma aggression. We showed that human MYCN-activated neuroblastoma upregulates and secretes CKLF, which is a reliable predictor of poor patient survival. Analysis of primary patient samples demonstrates a strong positive association between MYCN and CKLF expression in neuroblastoma cells together with the enrichment of FOXP3+ T cells in the TME. Taking advantage of in vivo zebrafish models of MYCN-driven neuroblastoma that resemble human high-risk disease, we demonstrated that cklf overexpression in zebrafish neural crest cells induces an immunosuppressive TME while promoting rapid tumor onset and progression. We also demonstrated that as early as the premalignant stage, tumor cells secrete CKLF to attract CCR4-expressing Cd4+ cells to induce immunosuppression and tumor aggression. Conversely, genetic depletion of cklf in tumor cells reduces the recruitment of Cd4+ cells to the TME while increasing cytotoxic Cd8+ and natural killer cells infiltration, inhibiting neuroblastoma development in zebrafish. Our work provides the first example that MYCN can activate CKLF to allure CD4+ immune cells to the TME and incite immunosuppression, positioning CKLF as a potential novel immunotherapeutic target for the treatment of MYCN-driven high-risk neuroblastoma. Citation Format: Xiaodan Qin, Hui Feng, Andrew Lam, Xu Zhang, Satyaki Sengupta, Bryan Iorgulescu, Sanjukta Das, Zhenwei Zhou, Tao Zuo, Grace Meara, Madison Rager, Alexander Floru, Hongru Ni, Chinyere Kemet, Divya Veerapaneni, Daniel Kashy, Liang Lin, Kenneth Lloyd, Lauren Kwok, Kaylee Smith, Raghavendar Nagaraju, Rob Meijers, Craig Ceol, Ching-Ti Liu, Sanda Alexandrescu, Catherine J. Wu, Derin Keskin, Rani George. CKLF attracts CCR4-expressing CD4+ cells to foster immune repression and tumor aggressiveness in MYCN-driven neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C002.

Neural Crest-Derived Mesenchymal Cells Support Thymic Reconstitution After Lethal Irradiation.

Reconstitution of the thymus is essential for assessing thymic function following injury. However, the currently employed cytoreductive regimes unvaryingly affect the thymic microenvironment, thereby impeding the recovery of T lymphopoiesis. The thymic stroma is composed of epithelial and mesenchymal cells. Thymic mesenchymal cells originate from the Neural crest (NC) and mesoderm and contribute to thymus organogenesis, yet their role in thymic regeneration is unclear. In this study, using transgenic mice expressing NC-specific Cre and Cre-driven DT receptors, we investigated the role of NC-derived mesenchymal cells in thymic regeneration following total body irradiation. We revealed that NC-derived mesenchymal cells have reduced susceptibility to irradiation and induce the upregulation of hematopoietic factors that promote thymus regeneration after irradiation. Additionally, using adult thymic organ culture and renal capsule transplantation, depletion of NC-derived mesenchymal cells resulted in a reduction of DN1-like early T-cell progenitors (ETP) and impaired thymic regeneration. Furthermore, among the numerous factors upregulated by NC-derived mesenchymal cells, Periostin and Flt3L were markedly increased after irradiation and promoted abundance of DN1-like ETPs during thymic reconstitution. Collectively, these findings highlight the importance of NC-derived mesenchymal cells in thymic regeneration.

The Ednrb–Aim2–AKT axis regulates neural crest-derived melanoblast proliferation during early development

ABSTRACT Ednrb is specifically required to develop neural crest (NC) stem cell-derived lineages. However, it is still unknown why Ednrb signaling is only needed for the early development of melanoblasts in the skin and eye. We show that Ednrb is required for the proliferation of melanoblasts during early mouse development. To understand the mechanism of melanoblast proliferation, we found that the gene absent in melanoma 2 (Aim2) is upregulated in Ednrb-deficient NC cells by RNA-sequencing analysis. Consequently, the knockdown or knockout of Aim2 partially rescued the proliferation of Ednrb-deficient melanoblasts. Conversely, the overexpression of Aim2 in melanoblasts suppressed their proliferation. We further show that Ednrb signaling could act through the microRNA miR-196b to block the suppression of melanoblast proliferation by Aim2 in primary NC cell cultures. These results reveal the Ednrb–Aim2–AKT axis in regulating melanocyte development and suggest that Ednrb signaling functions as a negative regulator of Aim2, which inhibits the proliferation of melanoblasts in early development. These findings uncover a previously unreported role for Aim2 outside the inflammasome, showing that it is a significant regulator controlling NC stem cell-derived lineage development.

Therapeutic potential of melatonin-pretreated human dental pulp stem cells (hDPSCs) in an animal model of spinal cord injury.

Dental pulp stem cells (DPSCs) show potential for treating neurodegenerative and traumatic diseases due to their neural crest origin. Melatonin (MT), an endogenous neurohormone with well-documented anti-inflammatory and antioxidant properties, has shown promising results with MSCs in terms of engraftment, proliferation, and neuronal differentiation in animal SCI models. However, the effects of melatonin preconditioning on human dental pulp stem cells (hDPSCs) for SCI treatment remain unclear. This study investigates the impact of melatonin preconditioning on hDPSCs engraftment, neural differentiation, and neurological function in rats with SCI. Forty-two male Sprague-Dawley rats were divided into six groups: Control, Sham, Model, Vehicle, Lesion Treatment A(SCI + hDPSCs), and Lesion Treatment B(SCI + MT-hDPSCs). After obtaining hDPSCs, stem cells were evaluated using flow cytometry. Cell viability was assessed using the MTT assay. SCI was induced in the Model, Vehicle, Lesion Treatment A, and Lesion Treatment B groups. The Lesion Treatment A and B groups received hDPSCs and hDPSCs pretreated with melatonin, respectively, 1 week after SCI, while the Vehicle group received only an intravenous injection of DMEM to simulate treatment. The other groups were used for behavioral testing. Immunohistochemistry (IHC) was employed to assess hDPSCs engraftment and differentiation at the SCI site. Motor function across the six groups was evaluated using the Basso, Beattie, and Bresnahan (BBB) score. Histological studies and cell counts confirmed hDPSCs implantation at the injury site, with a significantly higher presence in the MT-hDPSCs compared to hDPSCs(p < 0.01). IHC revealed that hDPSCs and MT-hDPSCs differentiated into neurons and astrocytes, with greater differentiation observed in the MT-hDPSCs compared to the hDPSCs (p < 0.01 and p < 0.05, respectively). Functional improvement was noted in both SCI + hDPSCs and SCI + MT-hDPSCs groups compared to SCI and Vehicle groups from Week 4 onward (p < 0.001). Significant differences were also observed between the SCI + hDPSCs and SCI + MT-hDPSCs groups starting from Week 7 (p < 0.01). Preconditioning hDPSCs with melatonin enhances engraftment, neuronal differentiation, and greater performance improvement compared to hDPSCs alone in the SCI animal model.

Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchymal cells.

Bone Morphogenetic Protein (BMP) signaling is essential for craniofacial development, though little is known about the mechanisms that govern BMP secretion. We show that depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchyme. We show endogenous transient changes in intracellular calcium occur in cranial neural crest cells, the cells from which embryonic palate mesenchyme derives. Waves of transient changes in intracellular calcium suggest that these cells are electrically coupled and may temporally coordinate BMP release. These transient changes in intracellular calcium persist in palate mesenchyme cells from embryonic day 9.5 to 13.5 mice. Disruption of a potassium channel called Kcnj2 significantly decreases the amplitude of calcium transients and the ability of cells to secrete BMP. Kcnj2 knockout mice have cleft palate and reduced BMP signaling. Our data suggest that temporal control of developmental cues is regulated by ion channels, depolarization, and intracellular calcium for mammalian craniofacial morphogenesis.

METTL3/MYCN cooperation drives neural crest differentiation and provides therapeutic vulnerability in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial childhood cancer, caused by the improper differentiation of developing trunk neural crest cells (tNCC) in the sympathetic nervous system. The N6-methyladenosine (m6A) epitranscriptomic modification controls post-transcriptional gene expression but the mechanism by which the m6A methyltransferase complex METTL3/METTL14/WTAP is recruited to specific loci remains to be fully characterized. We explored whether the m6A epitranscriptome could fine-tune gene regulation in migrating/differentiating tNCC. We demonstrate that the m6A modification regulates the expression of HOX genes in tNCC, thereby contributing to their timely differentiation into sympathetic neurons. Furthermore, we show that posterior HOX genes are m6A modified in MYCN-amplified NB with reduced expression. In addition, we provide evidence that sustained overexpression of the MYCN oncogene in tNCC drives METTL3 recruitment to a specific subset of genes including posterior HOX genes creating an undifferentiated state. Moreover, METTL3 depletion/inhibition induces DNA damage and differentiation of MYCN overexpressing cells and increases vulnerability to chemotherapeutic drugs in MYCN-amplified patient-derived xenografts (PDX) in vivo, suggesting METTL3 inhibition could be a potential therapeutic approach for NB.

CNSC-53. SHH ACTIVATION IN DEDIFFERENTIATION DURING TUMOR PROGRESSION IN MPNST

Abstract BACKGROUND Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with little progress on outcomes and treatment strategies. Previously, unsupervised analyses of methylome and transcriptome profiles of 108 peripheral nerve sheath tumors uncovered two subgroups of MPNSTs that predict progression-free survival, MPNST-G1 (characterized by SHH-pathway activation) and MPNST-G2 (characterized by WNT/ß-catenin/CCND1-pathway activation). Further, single nuclear RNA sequencing revealed that MPNST-G1 and MPNST-G2 cells resemble neural crest-like and Schwann cell precursor-like cells, respectively. PURPOSE &amp; HYPOTHESIS To examine whether the activation of the SHH pathway in MPNST-G1 cell lines induces overexpression of factors known to play canonical roles in early neural crest cell specification (TWIST1, SNAI2, PAX3, PAX6, SOX9, OTX2) and to investigate the role of Src activation on SHH pathway-induced dedifferentiation. We hypothesize that MPNST-G1 cells will exhibit SHH pathway activation, synergizing with Src to induce the overexpression of early neural crest cell (ENCC) transcription factors. METHODS SHH pathway activation, expression of ENCC transcription factors, and effects of inhibitors were analyzed in MPNST-G1 cells using RT-PCR, western blotting, Alamar Blue assay, Trypan Blue assay, and Soft Agar Transformation assay. RESULTS Compared to MPNST-G2 cells, MPNST-G1 cells displayed SHH-pathway activation, SMO-dependent activation of Src, and elevated expression of the ENCC transcription factors. Sonidegib (SMO inhibitor) and Dasatinib (Src inhibitor) were able to revert dedifferentiation. CONCLUSIONS The SHH-pathway cooperates with Src to promote expression of important transcription factors in dedifferentiation. This finding provides insights into the transformation process of MPNST and novel therapeutic options.

Enhanced Gαq Signaling in TSC2-deficient Cells Is Required for Their Neoplastic Behavior.

Inherited or sporadic loss of the TSC2 gene can lead to pulmonary lymphangioleiomyomatosis (LAM), a rare cystic lung disease caused by protease-secreting interstitial tumor nodules. The nodules arise by metastasis of cells that exhibit features of neural crest and smooth muscle lineage ('LAM cells'). Their aberrant growth is attributed to increased activity of 'mechanistic target of rapamycin complex 1' (mTORC1), an anabolic protein kinase that is normally suppressed by the TSC1-TSC2 protein complex. The mTORC1 inhibitor rapamycin slows the progression of LAM, but fails to eradicate disease, indicating a role for mTORC1-independent mechanisms in LAM pathogenesis. Our previous studies revealed G-protein coupled urotensin-II receptor (UT) signaling as a candidate mechanism, but how it promotes oncogenic signaling in TSC2-deficient cells remained unknown. Using a human pluripotent stem cell-derived in vitro model of LAM, we now show hyperactivation of UT, which was required for their enhanced migration and pro-neoplastic signaling in a rapamycin-insensitive mechanism that required heterotrimeric Gαq/11 (Gαq). Bioluminescence resonance energy transfer assays in HEK 293T cells lacking TSC2 demonstrated selective and enhanced activation of Gαq and its RhoA-associated effectors compared to wild-type control cells. By immunoprecipitation, recombinant UT was physically associated with Gαq and TSC2. The augmented Gαq signaling in TSC2-deleted cells was independent of mTOR activity, and associated with increased endosomal targeting of p63RhoGEF, a known RhoA-activating effector of Gαq. These studies identify potential mTORC1-independent pro-neoplastic mechanisms that can be targeted for prevention or eradication of pulmonary and extrapulmonary LAM tumors.

Neuroblastoma plasticity during metastatic progression stems from the dynamics of an early sympathetic transcriptomic trajectory.

Despite their indisputable importance in neuroblastoma (NB) pathology, knowledge of the bases of NB plasticity and heterogeneity remains incomplete. They may be rooted in developmental trajectories of their lineage of origin, the sympatho-adrenal neural crest. We find that implanting human NB cells in the neural crest of the avian embryo allows recapitulating the metastatic sequence until bone marrow involvement. Using deep single cell RNA sequencing, we characterize transcriptome states of NB cells and their dynamics over time and space, and compare them to those of fetal sympatho-adrenal tissues and patient tumors and bone marrow samples. Here we report remarkable transcriptomic proximities restricted to an early sympathetic neuroblast branch that co-exist with phenotypical adaptations over disease progression and recapitulate intratumor and interpatient heterogeneity. Combining avian and patient datasets, we identify a list of genes upregulated during bone marrow involvement and associated with growth dependency, validating the relevance of our multimodal approach.

Establishment of Periodontal Ligament Stem Cell-like Cells Derived from Feeder-Free Cultured Induced Pluripotent Stem Cells.

The periodontal ligament (PDL) is a fibrous connective tissue that connects the cementum of the root to the alveolar bone. PDL stem cells (PDLSCs) contained in the PDL can differentiate into cementoblasts, osteoblasts, and PDL fibroblasts, with essential roles in periodontal tissue regeneration. Therefore, PDLSCs are expected to be useful in periodontal tissue regeneration therapy. In a previous study, we differentiated induced pluripotent stem cells (iPSCs) into PDLSC-like cells (iPDLSCs), which expressed PDL-related markers and mesenchymal stem cell (MSC) markers; they also exhibited high proliferation and multipotency. However, the iPSCs used in this differentiation method were cultured on mouse embryonic fibroblasts; thus, they constituted on-feeder iPSCs (OF-iPSCs). Considering the risk of contamination with feeder cell-derived components, iPDLSCs differentiated from OF-iPSCs (ie, OF-iPDLSCs) are unsuitable for clinical applications. In this study, we aimed to obtain PDLSC-like cells from feeder-free iPSCs (FF-iPSCs) using OF-iPDLSC differentiation method. First, we differentiated FF-iPSCs into neural crest cell-like cells (FF-iNCCs) and confirmed that FF-iNCCs expressed NCC markers (eg, Nestin and p75NTR). Then, we cultured FF-iNCCs on human primary PDL cell-derived extracellular matrix for 2 weeks; the resulting cells were named FF-iPDLSCs. FF-iPDLSCs exhibited higher expression of PDL-related and MSC markers compared with OF-iPDLSCs. FF-iPDLSCs also demonstrated proliferation and multipotency in vitro. Finally, we analyzed the ability of FF-iPDLSCs to form periodontal tissue invivo upon subcutaneous transplantation with β-tricalcium phosphate scaffolds into dorsal tissues of immunodeficient mice. Eight weeks after transplantation, FF-iPDLSCs had formed osteocalcin-positive bone/cementum-like tissues and collagen 1-positive PDL-like fibers. These results suggested that we successfully obtained PDLSC-like cells from FF-iPSCs. Our findings will contribute to the development of novel periodontal regeneration therapies.

The bone Gla protein osteocalcin is expressed in cranial neural crest cells.

Osteocalcin is a small protein abundant in the bone extracellular-matrix, that serves as a marker for mature osteoblasts. To become activated, osteocalcin undergoes a specific post-translational carboxylation. Osteocalcin is expressed at advanced stages of embryogenesis and after birth, when bone formation takes place. Neural crest cells (NCCs) are a unique cell population that evolves during early stages of development. While initially NCCs populate the dorsal neural-tube, later they undergo epithelial-to-mesenchymal-transition and migrate throughout the embryo in highly-regulated manner. NCCs give rise to multiple cell types including neurons and glia of the peripheral nervous system, chromaffin cells and skin melanocytes. Remarkably, in the head region, NCCs give rise to cartilage and bone. Here we report that osteocalcin is detected in cranial NCCs. Analysis of chick embryos at stages of cranial NCC migration revealed that osteocalcin mRNA and protein is expressed in pre-migratory and migratory NCCs in-vivo and ex-vivo. Addition of warfarin, an inhibitor of osteocalcin carboxylation, onto neural-tube explants, reduced the amount of NCC migration. These results provide the first evidence of osteocalcin presence in cranial NCCs, much before they give rise to craniofacial skeleton, and propose its possible involvement in the regulation of NCC migration.

Modeling of Skeletal Development and Diseases Using Human Pluripotent Stem Cells.

Human skeletal elements are formed from distinct origins at distinct positions of the embryo. For example, the neural crest produces the facial bones, the paraxial mesoderm produces the axial skeleton, and the lateral plate mesoderm produces the appendicular skeleton. During skeletal development, different combinations of signaling pathways are coordinated from distinct origins during the sequential developmental stages. Models for human skeletal development have been established using human pluripotent stem cells (hPSCs) and by exploiting our understanding of skeletal development. Stepwise protocols for generating skeletal cells from different origins have been designed to mimic developmental trails. Recently, organoid methods have allowed the multicellular organization of skeletal cell types to recapitulate complicated skeletal development and metabolism. Similarly, several genetic diseases of the skeleton have been modeled using patient-derived induced pluripotent stem cells and genome-editing technologies. Model-based drug screening is a powerful tool for identifying drug candidates. This review briefly summarizes our current understanding of the embryonic development of skeletal tissues and introduces the current state-of-the-art hPSC methods for recapitulating skeletal development, metabolism, and diseases. We also discuss the current limitations and future perspectives for applications of the hPSC-based modeling system in precision medicine in this research field.

Alcohol induces p53-mediated apoptosis in neural crest by stimulating an AMPK-mediated suppression of TORC1, S6K, and ribosomal biogenesis

Prenatal alcohol exposure is a leading cause of permanent neurodevelopmental disability and can feature distinctive craniofacial deficits that partly originate from the apoptotic deletion of craniofacial progenitors, a stem cell lineage called the neural crest (NC). We recently demonstrated that alcohol causes nucleolar stress in NC through its suppression of ribosome biogenesis (RBG) and this suppression is causative in their p53/MDM2-mediated apoptosis. Here, we show that this nucleolar stress originates from alcohol’s activation of AMPK, which suppresses TORC1 and the p70/S6K-mediated stimulation of RBG. Alcohol-exposed cells of the pluripotent, primary cranial NC line O9-1 were evaluated with respect to their S6K, TORC1, and AMPK activity. The functional impact of these signals with respect to RBG, p53, and apoptosis were assessed using gain-of-function constructs and small molecule mediators. Alcohol rapidly (<2hr) increased pAMPK, pTSC2, and pRaptor, and reduced both total and pS6K in NC cells. These changes persisted for at least 12hr to 18hr following alcohol exposure. Attenuation of these signals via gain- or loss-of-function approaches that targeted AMPK, S6K, or TORC1 prevented alcohol’s suppression of rRNA synthesis and the induction of p53-stimulated apoptosis. We conclude that alcohol induces ribosome dysbiogenesis and activates their p53/MDM2-mediated apoptosis via its activation of pAMPK, which in turn activates TSC2 and Raptor to suppress the TORC1/S6K-mediated promotion of ribosome biogenesis. This represents a novel mechanism underlying alcohol’s neurotoxicity and is consistent with findings that TORC1/S6K networks are critical for cranial NC survival.

Robotic-Assisted Adrenalectomy of a Giant Dopamine-Only Secreting Pure Adrenal Ganglioneuroma: A Case Report and Operative Video

Introduction: Ganglioneuromas (GNs) are rare, differentiated tumors that originate from neural crest cells and can occasionally develop in the adrenal medulla. Adrenal GNs (AGNs) are typically hormonally silent and asymptomatic. Hormone-secreting pure AGNs in adults are uncommon; however, dopamine-secreting pure AGNs are extremely rare, with only a few cases reported in the literature. Giant AGNs are usually managed with open surgical intervention. Case Presentation: We present a case of a giant dopamine-secreting pure AGN in a 19-year-old woman who complained of mild left flank pain with no other symptoms. On physical examination, a mass was felt in left upper abdomen. MRI showed a left retroperitoneal mass extending from the left suprarenal region inferiorly, anterior to and compressing the left kidney, measuring 9 × 7.5 × 14 cm. Hormonal investigations ruled out Cushing syndrome, and catecholamine studies were negative for metanephrines, normetanephrines, adrenaline, and noradrenaline but were positive for dopamine levels more than 3 times the upper normal level. The patient underwent robotic-assisted adrenalectomy with minimal morbidity. Conclusion: Robotic-assisted adrenalectomy is a feasible and effective treatment option for rare dopamine-secreting pure AGNs. Careful patient selection and thorough preoperative preparation are essential to minimize risks and ensure favorable outcomes.

Pharmacological Inhibition of the Spliceosome SF3b Complex by Pladienolide-B Elicits Craniofacial Developmental Defects in Mouse and Zebrafish.

Mutations in genes encoding spliceosome components result in craniofacial structural defects in humans, referred to as spliceosomopathies. The SF3b complex is a crucial unit of the spliceosome, but model organisms generated through genetic modification of the complex do not perfectly mimic the phenotype of spliceosomopathies. Since the phenotypes are suggested to be determined by the extent of spliceosome dysfunction, an alternative experimental system that can seamlessly control SF3b function is needed. To establish another experimental system for model organisms elucidating relationship between spliceosome function and human diseases, we administered Pladienolide-B (PB), a SF3b complex inhibitor, to mouse and zebrafish embryos and assessed resulting phenotypes. PB-treated mouse embryos exhibited neural tube defect and exencephaly, accompanied by apoptosis and reduced cell proliferation in the neural tube, but normal structure in the midface and jaw. PB administration to heterozygous knockout mice of Sf3b4, a gene coding for a SF3b component, influenced the formation of cranial neural crest cells (CNCCs). Despite challenges in continuous PB administration and a high death rate in mice, PB was stably administered to zebrafish embryos, resulting in prolonged survival. Brain, cranial nerve, retina, midface, and jaw development were affected, mimicking spliceosomopathy phenotypes. Additionally, alterations in cell proliferation, cell death, and migration of CNCCs were detected. We demonstrated that zebrafish treated with PB exhibited phenotypes similar to those observed in human spliceosomopathies. This experimental system may serve as a valuable research tool for understanding spliceosome function and human diseases.

Polytetrafluoroethylene (PTFE) Microplastics Affect Angiogenesis and Central Nervous System (CNS) Development of Duck Embryo

Prolonged exposure to teratogens is known to cause neural tube defects (NTDs), a severe malformation of the central nervous system (CNS) that significantly contributes to global infant mortality. In recent years, exposure to nanoplastics (NPs) has been linked to faulty neural crest closure and altered neurulation by altering cellular adhesion molecules and accumulation of plastic particles in the neural tube leading to NTDs. However, research on the influence of various types of microplastics (MPs) on malformations of the CNS are still limited. In this study, we investigated whether MPs of polytetrafluoroethylene (PTFE)—a type of plastic commonly used as non-stick coatings of cooking utensils can affect angiogenesis and CNS development using ducks as model organisms. PTFE MPs were administered on Day 3 of duck embryo development at varying concentrations (0.01 mg/ml, 0.1 mg/ml, 1 mg/ml, and 5 mg/ml), and angiogenesis was evaluated using a chorioallantoic membrane (CAM) assay. Gross morphology and histology of the spinal column and brain were analyzed on Days 8 and 18, respectively. FTIR confirmed PTFE's structure, while SEM and DLS analyses showed particle sizes between 300 nm and 5 μm, classifying them as MPs. High concentrations (5 mg/ml) of PTFE MPs treated on duck embryos resulted in a 35% mortality rate and reduced vascular density, suggesting anti-angiogenic effects. Brain and spinal abnormalities, such as encephalomalacia and spinal cord discontinuities were observed in the PTFE-treated embryos. Based on these results, PTFE is an anti-angiogenic and teratogenic agent affecting the development of duck embryos.

Point-of-Care Sensor Using Modified Nickel-doped Zinc Oxide Nanoparticle Carbon Paste Electrode for Homovanillic Acid Cancer Biomarker Detection in Urine

The prevalence of cancer worldwide has prompted efforts to develop and produce a range of electrochemical biosensors for cancer diagnosis. Efficient cancer diagnosis can be enhanced by the sensitive detection of biomarkers, which can also lower the cost of medical diagnostics. Neuroblastoma is an embryonic cancer arising from neural crest stem cells and is considered the most common malignancy in infants and the extracranial solid tumor in children. In this paper, we describe the construction of a nanoparticle-modified electrochemical sensor for detecting and quantifying homovanillic acid (HVA), a biomarker for neuroblastoma. The electrooxidation of HVA was studied at a carbon paste electrode (CPE) modified with nickel-doped zinc oxide nanoparticles (Ni-ZnO NPs). The use of these nanoparticles enhanced electrochemical sensitivity and the electrocatalytic activity. The differential pulse voltammetric response of HVA was found to be linear in the concentration range of (3.96 × 10−6 to 3.83 × 10−5 M) with a lower detection limit of 1.01 × 10−6 M. The electrode demonstrated good stability in the HVA determination process, with a minor decrease in response after 10 weeks. The proposed sensor was successfully applied to determine HVA in a urine sample with a good detection result and a worthwhile biological impact.

Leveraging Neural Crest-Derived Tumors to Identify NF1 Cancer Stem Cell Signatures.

Neurofibromatosis type 1 (NF1) is a genetic disorder that predisposes individuals to develop benign and malignant tumors of the nerve sheath. Understanding the signatures of cancer stem cells (CSCs) for NF1-associated tumors may facilitate the early detection of tumor progression. Background: Neural crest cells, the cell of origin of NF1-associated tumors, can initiate multiple tumor types, including melanoma, neuroblastoma, and schwannoma. CSCs within these tumors have been reported; however, identifying and targeting CSC populations remains a challenge. Results: This study aims to leverage existing studies on neural crest-derived CSCs to explore markers pertinent to NF1 tumorigenesis. By focusing on the molecular and cellular dynamics within these tumors, we summarize CSC signatures in tumor maintenance, progression, and treatment resistance. Conclusion: A review of these signatures in the context of NF1 will provide insights into NF1 tumor biology and pave the way for developing targeted therapies and improving treatment outcomes for NF1 patients.

Primary Adrenal Neuroblastoma in an Adult: A Rare Case Report

ABSTRACT Neuroblastoma (NB), an embryonal malignancy originating from neural crest cells, is predominantly a pediatric disease, making adult cases exceedingly rare. This case report documents the presentation and diagnostic process of a 23-year-old female diagnosed with adrenal NB, a condition scarcely encountered in adults. The patient presented with abdominal pain and a palpable mass. Imaging through computed tomography scan revealed a large necrotic mass in the left adrenal region. Ultrasound-guided fine-needle aspiration cytology (FNAC) showed round cells with scant cytoplasm and characteristic nuclear features, suggesting a round cell tumor, potentially NB. Surgical excision confirmed the diagnosis of poorly differentiated NB with low mitotic-karyorrhectic index. Adult NB, with an incidence of 1 in 10 million per year, has a notably worse prognosis compared to pediatric cases. The literature lacks consensus on optimal treatment protocols for adult patients, though a multimodal approach including surgery, chemotherapy, and possibly radioactive iodine therapy is often employed. This case underscores the utility of FNAC in preliminary diagnosis and highlights the clinical challenges posed by adult NB, emphasizing the need for more comprehensive studies to develop standardized treatment guidelines.