RAF1-MEK/ERK pathway-dependent ARL4C expression promotes ameloblastoma cell proliferation and osteoclast formation.

Ameloblastoma is an odontogenic neoplasm characterized by slow intraosseous growth with progressive jaw resorption. Recent reports have revealed that ameloblastoma harbours an oncogenic BRAFV600E mutation with mitogen-activated protein kinase (MAPK) pathway activation and described cases of ameloblastoma harbouring a BRAFV600E mutation in which patients were successfully treated with a BRAF inhibitor. Therefore, the MAPK pathway may be involved in the development of ameloblastoma; however, the precise mechanism by which it induces ameloblastoma is unclear. The expression of ADP-ribosylation factor (ARF)-like 4c (ARL4C), induced by a combination of the EGF-MAPK pathway and Wnt/ß-catenin signalling, has been shown to induce epithelial morphogenesis. It was also reported that the overexpression of ARL4C, due to alterations in the EGF/RAS-MAPK pathway and Wnt/ß-catenin signalling, promotes tumourigenesis. However, the roles of ARL4C in ameloblastoma are unknown. We investigated the involvement of ARL4C in the development of ameloblastoma. In immunohistochemical analyses of tissue specimens obtained from 38 ameloblastoma patients, ARL4C was hardly detected in non-tumour regions but tumours frequently showed strong expression of ARL4C, along with the expression of both BRAFV600E and RAF1 (also known as C-RAF). Loss-of-function experiments using inhibitors or siRNAs revealed that ARL4C elevation depended on the RAF1-MEK/ERK pathway in ameloblastoma cells. It was also shown that the RAF1-ARL4C and BRAFV600E-MEK/ERK pathways promoted cell proliferation independently. ARL4C-depleted tumour cells (generated by knockdown or knockout) exhibited decreased proliferation and migration capabilities. Finally, when ameloblastoma cells were co-cultured with mouse bone marrow cells and primary osteoblasts, ameloblastoma cells induced osteoclast formation. ARL4C elevation in ameloblastoma further promoted its formation capabilities through the increased RANKL expression of mouse bone marrow cells and/or primary osteoblasts. These results suggest that the RAF1-MEK/ERK-ARL4C axis, which may function in cooperation with the BRAFV600E-MEK/ERK pathway, promotes ameloblastoma development. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

An accessory abductor digiti minimi as possible risk factor of the median and ulnar nerves compression / Músculo abductor digiti minimi accesorio como posible factor de riesgo de la compresión de los nervios mediano y ulnar

SUMMARY: During routine dissection of a left upper limb of a 68-year-old male human cadaver, an unusual muscle was observed originating from the radius and flexor retinaculum, and continued in the hypothenar region with the muscle belly of the abductor digiti minimi. We checked that it was an accessory abductor digiti minimi (ADM). Its muscular belly was in close relation to the median and ulnar nerves. We review the literature regarding such muscle variations and discuss the potential for compression of the median and ulnar nerves. Although the accessory ADM is usually asymptomatic and only rarely results in nerve compression, it should be taken into account by surgeons when establishing a differential diagnosis in the compression neuropathies of the median and ulnar nerves. An ultrasound scanning can help establish the differential diagnosis.

RESUMEN: Durante la disección de rutina de un miembro superior izquierdo de un cadáver humano masculino de 68 años, se observó un músculo inusual que se originaba en el radio y el retináculo flexor del carpo, y continuuaba en la región hipotenar con el vientre muscular del abductor digiti minimi manus. Verificamos que se trataba del músculo abductor digiti minimi accessorius (ADMA). Su vientre muscular se encontraba en estrecha relación con los nervios mediano y ulnar. Revisamos la literatura sobre variaciones musculares y discutimos la potencial compresión de los nervios mediano y ulnar. Aunque el ADMA suele ser asintomático y rara vez produce compresión nerviosa, los cirujanos deben tenerlo en cuenta al establecer un diagnóstico diferencial en las neuropatías de compresión de los nervios mediano y ulnar. Una ecografía puede ayudar a establecer el diagnóstico diferencial.

Does education about death and dying decrease stress generated in the dissection room? / ¿Disminuye el estrés generado en la sala de disección la formación sobre la muerte y el moribundo?

SUMMARY: Positive effects on reducing students' stress have been reported across numerous university settings when anatomy preparatory seminars have been provided. To date, this type of preparation for coping with cadaver dissection has not been studied in Spanish universities. The aim of this study is to evaluate how first-year Spanish medical students face the dissecting room and whether previous preparation about death and dying reduces the stress generated. We performed an interventional study with students who received preparatory classes before the dissection practices (Experimental Group, EG) and with students who did not (Control Group, CG). Sociodemographic data and a self-assessment on stress symptoms were collected through a questionnaire completed before and after the dissection practices. No differences were found in the self-report of symptoms of stress among students who consider themselves religious or not, or between students who had a family member in the healthcare environment or not. However, in the EG, the students who had ample experience with terminally ill patients or death reported fewer stress symptoms. Unexpectedly, the number of selfreported stress symptoms after the dissection practice was higher in EG students. In conclusion the stress levels of first-year Spanish medical students not only did not improve after receiving preparatory classes about death and dying and discussion groups, but it gets worse. We found a relationship between student stress measured and experience with terminally ill patients or death. Additional studies are needed to identify the most suitable preparation for Spanish medical students.

RESUMEN: Se han informado efectos positivos en la reducción del estrés en los estudiantes de numerosos entornos universitarios cuando se han impartido seminarios preparatorios de anatomía. Hasta la fecha, este tipo de preparación para hacer frente a la disección del cadáver no se ha estudiado en las universidades españolas. El objetivo de este estudio es evaluar cómo los estudiantes de medicina españoles de primer año se enfrentan a la sala de disección y si la preparación previa sobre la muerte y el moribundo reduce el estrés generado. Realizamos un estudio de intervención con estudiantes que recibieron clases preparatorias antes de las prácticas de disección (Grupo Experimental, GE) y con estudiantes que no las recibieron (Grupo de Control, GC). Se recogieron datos sociodemográficos y síntomas de estrés mediante un cuestionario de autoevaluación antes y después de las prácticas de disección. No se encontraron diferencias en los síntomas de estrés valorados, entre los estudiantes que se consideran religiosos y los que no, ni tampoco entre los estudiantes que tenían o no un familiar en el entorno sanitario. Sin embargo, en el GE, en los estudiantes que tenían una amplia experiencia con pacientes con enfermedades terminales o con la muerte se observaron menos síntomas de estrés. Inesperadamente, el número de síntomas de estrés recogidos después de la práctica de disección fue mayor en los estudiantes del GE. En conclusión, los niveles de estrés de los estudiantes españoles de medicina de primer año no solo no mejoraron después de recibir las clases preparatorias sobre la muerte y el moribundo y establecer grupos de discusión, sino que empeoraron. Encontramos una relación entre la medición del estrés en los estudiantes y la experiencia con pacientes con enfermedades terminales o con la muerte. Se necesitan estudios adicionales para identificar la preparación más adecuada para los estudiantes de medicina españoles.

Ameloblastomas Exhibit Stem Cell Potential, Possess Neurotrophic Properties, and Establish Connections with Trigeminal Neurons.

Ameloblastomas are locally invasive and aggressive odontogenic tumors treated via surgical resection, which results in facial deformity and significant morbidity. Few studies have addressed the cellular and molecular events of ameloblastoma onset and progression, thus hampering the development of non-invasive therapeutic approaches. Tumorigenesis is driven by a plethora of factors, among which innervation has been long neglected. Recent findings have shown that innervation directly promotes tumor progression. On this basis, we investigated the molecular characteristics and neurotrophic properties of human ameloblastomas. Our results showed that ameloblastomas express dental epithelial stem cell markers, as well as components of the Notch signaling pathway, indicating persistence of stemness. We demonstrated that ameloblastomas express classical stem cell markers, exhibit stem cell potential, and form spheres. These tumors express also molecules of the Notch signaling pathway, fundamental for stem cells and their fate. Additionally, we showed that ameloblastomas express the neurotrophic factors NGF and BDNF, as well as their receptors TRKA, TRKB, and P75/NGFR, which are responsible for their innervation by trigeminal axons in vivo. In vitro studies using microfluidic devices showed that ameloblastoma cells attract and form connections with these nerves. Innervation of ameloblastomas might play a key role in the onset of this malignancy and might represent a promising target for non-invasive pharmacological interventions.

High frequency of BRAF V600E mutations in ameloblastoma.

Ameloblastoma is a benign but locally infiltrative odontogenic neoplasm. Although ameloblastomas rarely metastasise, recurrences together with radical surgery often result in facial deformity and significant morbidity. Development of non-invasive therapies has been precluded by a lack of understanding of the molecular background of ameloblastoma pathogenesis. When addressing the role of ERBB receptors as potential new targets for ameloblastoma, we discovered significant EGFR over-expression in clinical samples using real-time RT-PCR, but observed variable sensitivity of novel primary ameloblastoma cells to EGFR-targeted drugs in vitro. In the quest for mutations downstream of EGFR that could explain this apparent discrepancy, Sanger sequencing revealed an oncogenic BRAF V600E mutation in the cell line resistant to EGFR inhibition. Further analysis of the clinical samples by Sanger sequencing and BRAF V600E-specific immunohistochemistry demonstrated a high frequency of BRAF V600E mutations (15 of 24 samples, 63%). These data provide novel insight into the poorly understood molecular pathogenesis of ameloblastoma and offer a rationale to test drugs targeting EGFR or mutant BRAF as novel therapies for ameloblastoma.

In vitro studies on odontogenic tumors.

Ameloblastomas are uncommon benign neoplasms of the jaws. They originate from dental epithelial cells, but they are not capable of mineralizing or forming enamel. The study of these tumors is limited to live tissue collected from patients during scheduled surgery. Ameloblastomas grow slowly in vivo and this property is translated to their behavior in vitro. Here, we describe the methods to culture ameloblastomas in organotypic cultures, as well as to isolate stem/progenitor cells from these tumors.

Enamel-free teeth: Tbx1 deletion affects amelogenesis in rodent incisors.

TBX1 is a principal candidate gene for DiGeorge syndrome, a developmental anomaly that affects the heart, thymus, parathyroid, face, and teeth. A mouse model carrying a deletion in a functional region of the Tbx1 gene has been extensively used to study anomalies related to this syndrome. We have used the Tbx1 null mouse to understand the tooth phenotype reported in patients afflicted by DiGeorge syndrome. Because of the early lethality of the Tbx1-/- mice, we used long-term culture techniques that allow the unharmed growth of incisors until their full maturity. All cultured incisors of Tbx1-/- mice were hypoplastic and lacked enamel, while thorough histological examinations demonstrated the complete absence of ameloblasts. The absence of enamel is preceded by a decrease in proliferation of the ameloblast precursor cells and a reduction in amelogenin gene expression. The cervical loop area of the incisor, which contains the niche for the epithelial stem cells, was either severely reduced or completely missing in mutant incisors. In contrast, ectopic expression of Tbx1 was observed in incisors from mice with upregulated Fibroblast Growth Factor signalling and was closely linked to ectopic enamel formation and deposition in these incisors. These results demonstrate that Tbx1 is essential for the maintenance of ameloblast progenitor cells in rodent incisors and that its deletion results in the absence of enamel formation.