Novel cellular systems unveil mucosal melanoma initiating cells and a role for PI3K/Akt/mTOR pathway in mucosal melanoma fitness.

BACKGROUND: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. METHODS: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. RESULTS: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. CONCLUSIONS: Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Retrospective Analysis of Patient-Reported Adverse Drug Reactions in an Italian Allergy Unit: ALLERG-RAF Study.

INTRODUCTION: The Italian Medicines Agency indicates that about 5% of hospital admissions are due to adverse drug reactions (ADRs). Several factors are recognized to be associated with an increased risk for ADRs, such as the female gender and polytherapy. The aim of this study was to retrospectively analyze the suspected ADRs reported by patients during the anamnestic interview at the Allergy Unit. PATIENTS AND METHODS: ALLERG-RAF study is a retrospective analysis of the medical records of patients evaluated in the Allergy Unit of ASST Spedali Civili and the University of Brescia from 2000 to 2016. The inclusion criteria were age ≥18 years and medical consultation requested for suspected ADRs. Data relating to the patient's intrinsic characteristics, the drug supposed to be the cause, and the prescribed pharmacological therapy were collected. Pseudonymized data from each patient were collected in an informatics database. RESULTS: From 2000 to 2016, 35,817 accesses to the Allergy Unit were made, and 2,171 unique events related to a suspected ADR were collected in 1,840 patients. More than two-thirds of the reports concerned females (70.4%). Antibiotics were involved in the majority of the self-reported suspected ADRs (48.7%), particularly beta-lactams (61.1%). Anti-inflammatory drugs, mainly NSAIDs, were second in incidence and suspected in 25.2% of reports. As a site of ADR manifestation, most of the reported reactions involve the skin. No clinical sequelae were reported. CONCLUSIONS: Our results underline the importance of patient reporting in pharmacovigilance. Furthermore, gender gap data emphasizes the importance of the gender-specific medicine approach.

Three-dimensional assessment on digital cast of spontaneous upper first molar distorotation after Ni-ti leaf springs expander and rapid maxillary expander: A two-centre randomized controlled trial.

OBJECTIVE: The aim of this randomized controlled trial (RCT) was to evaluate the spontaneous distorotation of upper first permanent molars and the transverse dentoalveolar changes on digital casts in growing patients following maxillary expansion treatment using either the Leaf Expander® or the rapid maxillary expander (RME), both anchored to the deciduous second molar. TRIAL DESIGN AND SETTING: This study was a two-arm, parallel-assignment, RCT with a dual-centre design conducted at two teaching hospitals in Italy. PARTICIPANTS: Inclusion criteria included maxillary transverse deficiency, prepubertal development stage (cervical vertebra maturation stage [CVMS] 1-2) and early mixed dentition with fully erupted upper first permanent molars. Exclusion criteria were systemic diseases or syndromes, CVMS 3-6, agenesis of upper second premolars, unavailability of the second deciduous molar for anchorage and Class III malocclusion. RANDOMIZATION: Patients were randomly assigned to the Leaf Expander® or RME group using a computer-generated randomization list created by a central randomization centre. Randomization was conducted immediately before the start of treatment. INTERVENTION: The intervention involved treatment with either the Leaf Expander® or the RME. Both devices were anchored to the second deciduous molars. Following randomization, patients were further categorized based on the presence of no crossbite, unilateral crossbite or bilateral crossbite. MAIN OUTCOME MEASURE: The primary outcome measure was the distorotation of the upper first molar (U6). Secondary outcomes included measurements of interdental linear dimensions, specifically upper inter-canine width (53-63), upper inter-molar width (MV16-MV26) and upper inter-deciduous second molar width (55-65). BLINDING: The examiner analysing the digital casts was blinded to the treatment groups to prevent detection bias and ensure objective assessment. However, due to the nature of the intervention, blinding was not feasible for the patients and clinicians involved in administering the treatment. RESULTS: A total of 150 patients were enrolled and randomly assigned to two groups: 75 to the Leaf Expander® group and 75 to the RME group. Recruitment started in November 2021 and was completed in November 2022. At the time of analysis, the trial was complete with no ongoing follow-ups. ANOVA tests revealed no significant differences between the three subgroups (no-cross, unilateral-cross and bilateral-cross) within both the Leaf Expander® and RME groups at T0. The Leaf Expander® demonstrated significantly greater distorotation in the unilateral crossbite subgroup compared to the RME (p = .014). In terms of total molar distorotation, the Leaf Expander® appliance showed a significantly greater effect (12.66°) compared with conventional RME (7.83°). Linear regression analysis demonstrated a significant correlation between the extent of expansion and the degree of molar rotation. CONCLUSIONS: Maxillary expansion resulted in significant spontaneous molar distorotation when the appliance was bonded to the second deciduous molars. The Leaf Expander® exhibited significantly greater molar distorotation compared with conventional RME. The degree of molar distorotation was correlated with the extent of expansion obtained on the second deciduous molar. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov (ID: NCT05135962).

Comparison of the skeletal, dentoalveolar, and periodontal changes after Ni-Ti leaf spring expander and rapid maxillary expansion: a three-dimensional CBCT based evaluation.

BACKGROUND: The aim of the present study was twofold:(1) three-dimensionally evaluate the quantitative skeletal and dentoalveolar changes after Ni-Ti leaf spring expander (leaf expander) and rapid maxillary expansion (RME) in mixed dentition patients;(2) analyze the modifications of the buccal alveolar bone plate of the maxillary first permanent molars. METHODS: Patients who underwent CBCT scans before and after maxillary expansion were randomly selected from the records archived at the Department of Biomedical Surgical and Dental Sciences, University of Milan, Italy. Inclusion criteria were the following: no systemic disease or syndromes; maxillary transverse deficiencies (difference between the upper intermolar width and the lower intermolar width of at least 3 mm and/or clinical need based on radiographic evaluation), early mixed dentition with ages between 7 to 10 years old; cervical vertebra maturation stage (CVMS) 1 or 2; no pathologic periodontal status; skeletal class I or II; maxillary expander cemented on the upper second deciduous molars. Exclusion criteria were the following: patients with pubertal or post-pubertal stage of development (CVMS 3-6); late deciduous or late mixed dentition, impossibility to use the second primary molar as anchorage; skeletal class III malocclusion; craniofacial syndromes; patients unable to be followed during the treatment period. Twenty-three patients treated with Leaf Expander, 11 males (mean age 7.8 ± 0.6 years) and 12 females (mean age 8.1 ± 0.8 years), met the inclusion criteria and constituted the case group. Twenty-four (control group) treated with conventional RME, 12 males (mean age 8.4 ± 0.9 years) and 12 females (mean age 8.1 ± 0.7 years). The paired-sample T test was used for intra-group comparison to evaluate the difference between before (T1) and after (T2) maxillary expansion. Independent sample t-test was computed to perform between groups comparison of the skeletal, dentoalveolar, and periodontal changes. RESULTS: The Leaf Expander and RME group showed a significant increase between T1 and T2 for most of the skeletal and dentoalveolar variables. Concerning the skeletal variables only the RME demonstrated a significant increase at the level of the posterior nasal (PNW) and apical base width (PABW) and maxillary mid-alveolar width (MMW). Despite this, when compare with the Leaf Expander, the RME group exhibited a statistically larger width increase for only two skeletal parameters: PNW (p = 0.03) and MMW (p = 0.02). No significant changes at the periodontal level were found in either group. CONCLUSIONS: According to the current research, the authors confirm the effectiveness of the Leaf Expander and RME to produce similar skeletal and dentoalveolar effects in mixed dentition subjects. Moreover, the devices anchored to deciduous teeth did not reduce the thickness and height of the buccal bone at the level of the maxillary permanent first molars in either of the two groups.

Preclinical Evidence of Progesterone as a New Pharmacological Strategy in Human Adrenocortical Carcinoma Cell Lines.

BACKGROUND: Adrenocortical cancer (ACC) is a rare malignancy with a dismal prognosis. The treatment includes mitotane and EDP chemotherapy (etoposide, doxorubicin, and cisplatin). However, new therapeutic approaches for advanced ACC are needed, particularly targeting the metastatic process. Here, we deepen the role of progesterone as a new potential drug for ACC, in line with its antitumoral effect in other cancers. METHODS: NCI-H295R, MUC-1, and TVBF-7 cell lines were used and xenografted in zebrafish embryos. Migration and invasion were studied using transwell assays, and MMP2 activity was studied using zymography. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Progesterone significantly reduced xenograft tumor area and metastases formation in embryos injected with metastatic lines, MUC-1 and TVBF-7. These results were confirmed in vitro, where the reduction of invasion was mediated, at least in part, by the decrease in MMP2 levels. Progesterone exerted a long-lasting effect in metastatic cells. Progesterone caused apoptosis in NCI-H295R and MUC-1, inducing changes in the cell-cycle distribution, while autophagy was predominantly activated in TVBF-7 cells. CONCLUSION: Our results give support to the role of progesterone in ACC. The involvement of its analog (megestrol acetate) in reducing ACC progression in ACC patients undergoing EDP-M therapy is now under investigation in the PESETA phase II clinical study.

A PNU-Based Methodology to Improve the Reliability of Biometric Systems.

Face recognition is an important application of pattern recognition and image analysis in biometric security systems. The COVID-19 outbreak has introduced several issues that can negatively affect the reliability of the facial recognition systems currently available: on the one hand, wearing a face mask/covering has led to growth in failure cases, while on the other, the restrictions on direct contact between people can prevent any biometric data being acquired in controlled environments. To effectively address these issues, we designed a hybrid methodology that improves the reliability of facial recognition systems. A well-known Source Camera Identification (SCI) technique, based on Pixel Non-Uniformity (PNU), was applied to analyze the integrity of the input video stream as well as to detect any tampered/fake frames. To examine the behavior of this methodology in real-life use cases, we implemented a prototype that showed two novel properties compared to the current state-of-the-art of biometric systems: (a) high accuracy even when subjects are wearing a face mask; (b) whenever the input video is produced by deep fake techniques (replacing the face of the main subject) the system can recognize that it has been altered providing more than one alert message. This methodology proved not only to be simultaneously more robust to mask induced occlusions but also even more reliable in preventing forgery attacks on the input video stream.

3D cephalometry on reduced FOV CBCT: skeletal class assessment through AF-BF on Frankfurt plane-validity and reliability through comparison with 2D measurements.

OBJECTIVES: To test the validity of a novel protocol for 3D sagittal jaw discrepancy assessment (skeletal class determination) through comparison with common 2D indexes by the use of reduced FOV (10 × 10) CBCT which shows at least from the Frankfurt plane to the B point vertically, and from the most anterior between A and B point to Po point horizontally. METHODS: A sample of CBCT scans of 109 adult patients (46 females; 63 males; mean age 30 years ± 11.6) equally distributed between I, II and III class was selected. Skeletal class was evaluated with specific software using the distance of A and B point's projection (AF-BF) on FHp (Frankfurt horizontal plane) and compared to 2D common indexes (ANB and Witts appraisal). The validity and reliability of the aforementioned analyses were determined using intra-class correlation coefficients, quadratic weighted Cohen's K and sensitivity. RESULT: A selected range of values of 2.5 ± 2.5 AF-BF showed a solid correlation with the ANB angle (r = 0.846, K = 0.838, p < 0.001) and moderate with Wits appraisal (r = 0.723, K = 0.720, p < 0.001). CONCLUSIONS: AF-BF showed high reliability in skeletal class determination on reduced FOV CBCT without the use of S and N cephalometric landmarks. KEY POINTS: • Reduced FOV CT allows skeletal class determination for orthodontic purposes. • A new 3D-reduced FOV cephalometry is proposed. • AF-BF is a reliable alternative to ANB.

Assessment of condylar volume and ramus height in JIA patients with unilateral and bilateral TMJ involvement: retrospective case-control study.

OBJECTIVES: The objectives of the study are to analyze volumetric differences of condylar volumes in patients with unilateral and bilateral JIA and to compare results with control condylar volumes. MATERIALS AND METHODS: Forty-six CBCT images were analyzed for all patients affected by JIA, 37 females and 9 males (mean age 10.8 ± 4.2) with TMJ involvement (19 unilaterally, mean age 10.9 ± 4.5; 27 bilaterally, mean age 10.7 ± 4.5), and 25 CBCT of subjects without diagnosis of JIA were selected as controls (mean age 10.8 ± 4.2 years). In the case of unilateral JIA, condylar volumes and ramus lengths were compared with healthy condyle and with the compromised one. In the case of bilateral JIA, condyle volume and ramus lengths were compared with healthy one. The Shapiro-Wilk test was used to assess whether the data was normally distributed. Paired t test was applied to compare affected and non-affected condyle in the same patients (P < 0.05). Independent t test was used to evaluate whether the difference between the groups were comparable or significantly different (P < 0.05). RESULTS: For the unilateral JIA group, significant differences comparing affected and non-affected condyles were found. A statistically significant reduction of the volume of the head, neck, and ramus was found in the affected side (P < 0.01). For the bilateral JIA group, statistically significant differences have been found considering the condylar head and neck, the whole condylar volume, and the ramus length compared with the control group (P < 0.05). CONCLUSIONS: Subjects with unilateral JIA have condyles volumetrically smaller than those of the unaffected side and those found in healthy patients. A considerable decrease of the volume of all the anatomical structures considered in the patients with bilateral JIA was found compared with control group. CLINICAL RELEVANCE: The study presents the effects of JIA on different anatomical structures highlighting their dimensional changes, whose sequelae are irreversible if not diagnosed and treated early.

Comparison of Mechanical Behavior of Clear Aligner and Rapid Palatal Expander on Transverse Plane: An In Vitro Study.

(1) Background: This study aims to investigate, within a controlled laboratory environment, the magnitude of the transversal load and the force decay over time produced by clear aligners in comparison to a Rapid Palatal Expander (RPE). (2) Methods: Resin models of a dental maxillary arch, additively manufactured from an intraoral scan, were inserted in a testing machine with uniaxial load cells to measure the force trend over time expressed by RPE and clear aligners. The mechanical load was recorded during a certain timeframe for both appliances. (3) Results: The force expressed by the RPE ranged from 30 to 50 N for each activation, decreasing with a nonlinear pattern over time. The force expressed by the clear aligner ranged from 3 to 5 N, decreasing with a linear pattern over time. In contrast, the force generated by the clear aligner fell within the range of 3 to 5 N, showing a linear reduction in force magnitude over the observed period of time. (4) Conclusions: The RPE exerted a force magnitude approximately ten times greater than that generated by clear aligners. Nevertheless, it is essential to acknowledge that the oral environment can significantly influence these results. These limitations underscore the need for caution when applying these findings to clinical settings.

Cone-Beam Computed Tomography (CBCT) Analysis of Mandibular Condyles' Diameters in Patient with Juvenile Idiopathic Arthritis and Temporomandibular Joint Affection: A Cross-Sectional Investigation.

Objectives: The aim of this study was to evaluate through analysis using CBCT the transverse and axial diameters of the mandibular condyles in subjects affected by juvenile idiopathic arthritis (JIA) and compare them with those of healthy subjects. Methods: The study was conducted on CBCT scans from the digital archive of the Department of Biomedical Surgical and Dental Sciences, University of Milan, including patients with JIA and using healthy subjects as controls. Inclusion criteria: aged between 7 and 25 years old at the time of the CBCT examination; Caucasian ethnicity; diagnosis of JIA according to the International League of Associations for Rheumatology (ILAR) criteria documented in patients' records; TMJ involvement; good quality CBCTs covering our region of interest (ROI), from the glabella to the mandibular inferior border; no previous orthodontic/orthopedic treatment; no history of craniofacial trauma or congenital birth defects involving the craniofacial area. Each CBCT scan underwent examination using 3Diagnosys® software. Since data were normally distributed, parametric tests were used for analysis. The sample was divided into three groups: (1) bilateral JIA subjects, (2) unilateral JIA subjects, and (3) healthy controls. Results: We found a statistically significant reduction (p < 0.0001) in the transverse diameter (TR-Diam) of the affected condyles by an average of 1.7 mm, while the axial diameter (AX-Diam) again showed a slight reduction, on average by 0.1 mm, with a non-statistically significant value. Another comparison was made between the unaffected condyles of patients with unilateral JIA and the healthy condyles of the control group. The unilateral unaffected condyles were found to be slightly smaller than those of healthy patients, but without statistically significant differences. We found that in both JIA males and females, the condylar growth tends to stop earlier than the healthy ones. Conclusions: The transverse diameter was found to be more affected than the axial one, causing typical bone resorption and condylar shape. Moreover, we showed that the pathology, in the case of unilateral JIA, does not compromise only the affected condyles; the corresponding condyle that seems to be healthy is actually partially compromised. In addition, we observed that the growth of affected condyles of JIA subjects tends to stop earlier than the condyles of the healthy controls.

Spontaneous Mandibular Dentoalveolar Changes after Rapid Maxillary Expansion (RME), Slow Maxillary Expansion (SME), and Leaf Expander-A Systematic Review.

BACKGROUND: This systematic review aims to analyze the spontaneous dentoalveolar changes in the mandibular arch after maxillary expansion in growing patients obtained with different expansion protocols: Rapid Maxillary Expansion (RME), Slow Maxillary Expansion (SME), and Leaf Expander. METHODS: The study adhered to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines. Eligibility criteria were established in the PICO format, involving patients who underwent slow, rapid, or leaf maxillary expansion during the mixed or early permanent dentitions. A comprehensive search of electronic databases and manual searches was conducted up to December 2023. The outcome measures included inter-mandibular first permanent molar width, inter-deciduous molar and canine width, arch perimeter, and arch length; both short- and long-term results were considered. The articles that met the inclusion criteria were included in this systematic review and were qualitatively evaluated using a methodological quality scoring system with a 13-point scale. To assess the inter-examiner agreement concerning the article selection and the qualitative assessment of the included studies, Kappa statistics were computed. RESULTS: A total of 1184 articles were identified through electronic and manual searches. After the removal of duplicates and the initial examination of the titles and abstracts, 57 articles were considered for the full text analysis, and according to the eligibility and exclusion criteria, 22 studies were finally selected, composed of 8 randomized controlled trials (RCTs) and 14 retrospective/case-control studies. The qualitative assessment of the included studies showed the following scores: 6 papers have high research quality, 5 have moderate quality, and 11 have low quality. SME demonstrated negligible mandibular changes, with less than 1 mm variation on average (range 0.46-2.00 mm) in the selected parameters and relapses observed in the long term. RME induced more significant increases, particularly in intermolar width greater than 1 mm, which ranged between 0.93 and 3.3 mm, and good stability over the long term. Leaf Expander exhibited promising short-term lower intermolar width increases greater than 1 mm and ranged from 0.5 to 1.69 mm, but long-term stability was not thoroughly evaluated. CONCLUSIONS: SME results in negligible short- and long-term effects, while RME, especially with Haas-type appliances, exhibits significant intermolar width increases that remain stable over the years. Leaf Expander shows short-term lower intermolar width increases, requiring further investigation into long-term stability.

The CDK Inhibitor Dinaciclib Improves Cisplatin Response in Nonseminomatous Testicular Cancer: A Preclinical Study.

BACKGROUND: Most patients with testicular germ cell tumors (GCTs) are treated with cisplatin (CP)-based chemotherapy. However, some of them may develop CP resistance and therefore represent a clinical challenge. Cyclin-dependent kinase 5 (CDK5) is involved in chemotherapy resistance in different types of cancer. Here, we investigated the possible role of CDK5 and other CDKs targeted by dinaciclib in nonseminoma cell models (both CP-sensitive and CP-resistant), evaluating the potential of the CDK inhibitor dinaciclib as a single/combined agent for the treatment of advanced/metastatic testicular cancer (TC). METHODS: The effects of dinaciclib and CP on sensitive and resistant NT2/D1 and NCCIT cell viability and proliferation were evaluated using MTT assays and direct count methods. Flow cytometry cell-cycle analysis was performed. The protein expression was assessed via Western blotting. The in vivo experiments were conducted in zebrafish embryos xenografted with TC cells. RESULTS: Among all the CDKs analyzed, CDK5 protein expression was significantly higher in CP-resistant models. Dinaciclib reduced the cell viability and proliferation in each cell model, inducing changes in cell-cycle distribution. In drug combination experiments, dinaciclib enhances the CP effect both in vitro and in the zebrafish model. CONCLUSIONS: Dinaciclib, when combined with CP, could be useful for improving nonseminoma TC response to CP.

A Database Tool Integrating Genomic and Pharmacologic Data from Adrenocortical Carcinoma Cell Lines, PDX, and Patient Samples.

Adrenocortical carcinoma (ACC) is a rare and highly heterogeneous disease with a notably poor prognosis due to significant challenges in diagnosis and treatment. Emphasizing on the importance of precision medicine, there is an increasing need for comprehensive genomic resources alongside well-developed experimental models to devise personalized therapeutic strategies. We present ACC_CellMinerCDB, a substantive genomic and drug sensitivity database (available at https://discover.nci.nih.gov/acc_cellminercdb) comprising ACC cell lines, patient-derived xenografts, surgical samples, and responses to more than 2,400 drugs examined by the NCI and National Center for Advancing Translational Sciences. This database exposes shared genomic pathways among ACC cell lines and surgical samples, thus authenticating the cell lines as research models. It also allows exploration of pertinent treatment markers such as MDR-1, SOAT1, MGMT, MMR, and SLFN11 and introduces the potential to repurpose agents like temozolomide for ACC therapy. ACC_CellMinerCDB provides the foundation for exploring larger preclinical ACC models. SIGNIFICANCE: ACC_CellMinerCDB, a comprehensive database of cell lines, patient-derived xenografts, surgical samples, and drug responses, reveals shared genomic pathways and treatment-relevant markers in ACC. This resource offers insights into potential therapeutic targets and the opportunity to repurpose existing drugs for ACC therapy.

Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma.

Disruption of processes involved in tissue development and homeostatic self-renewal is increasingly implicated in cancer initiation, progression, and recurrence. The adrenal cortex is a dynamic tissue that undergoes life-long turnover. Here, using genetic fate mapping and murine adrenocortical carcinoma (ACC) models, we have identified a population of adrenocortical stem cells that express delta-like non-canonical Notch ligand 1 (DLK1). These cells are active during development, near dormant postnatally but are re-expressed in ACC. In a study of over 200 human ACC samples, we have shown DLK1 expression is ubiquitous and is an independent prognostic marker of recurrence-free survival. Paradoxically, despite its progenitor role, spatial transcriptomic analysis has identified DLK1 expressing cell populations to have increased steroidogenic potential in human ACC, a finding also observed in four human and one murine ACC cell lines. Finally, the cleavable DLK1 ectodomain is measurable in patients' serum and can discriminate between ACC and other adrenal pathologies with high sensitivity and specificity to aid in diagnosis and follow-up of ACC patients. These data demonstrate a prognostic role for DLK1 in ACC, detail its hierarchical expression in homeostasis and oncogenic transformation and propose a role for its use as a biomarker in this malignancy.

Three-dimensional evaluation of the maxillary sinus in patients with different skeletal classes and cranio-maxillary relationships assessed with cone beam computed tomography.

The objective was to evaluate the relationship between the dimensions of the maxillary sinuses (MSs) and various cephalometric parameters. MS volume (MSV), MS surface (MSS), linear maximum depth (LMD), linear maximum width (LMW), and linear maximum height (LMH) were calculated on CBCT scans of 99 adults. Two sets of two-way (ANOVA) assessed the influence respectively of ANB and SNA angles and of the gender on MS dimensions. Pearson's correlation was calculated between MS dimensions and different cephalometric variables. Reliability and accuracy of the proposed method was tested with intra-operator and inter-operator intraclass correlation coefficient (ICC). Two-way ANOVA showed no statistically significant difference in MSV, MSS and LMH between ANB groups, whilst males were associated with bigger sinuses. LMW showed statistically significant difference in both ANB and gender groups. LMD showed no statistically significant difference. The second Two-way ANOVA showed significantly larger MSV, MSS and LMD in patients with increased or reduced SNA angle but not between genders. LMW and LMH also showed a significant difference between genders. All linear measurements showed a significant interaction of the two factors. The intra-observer and inter-observer ICC scored high for all the tested measurements. MSV and MSS showed a positive correlation with S-N, PNS-A, S-Go, N-Me, N-Ans and the distance between Mx points. LMW had a negative correlation with Ba-S-N angle and N-Me, LMH with Ba-S-N angle, S-Go and Mx r-Mx l and LMD with N-Me and N-ANS. LMW had a positive correlation with Mx r-Mx l, LMH with S-N, S-N^Ans-Pns, N-Me, N-Ans and LMD with S-N, Ba-S-N, PNS-A, S-Go and distance between Mx points. In conclusion, MSV and MSS did not differ between the three skeletal classes, males showed significantly larger MS than in females. Concerning the influence of the cranio-maxillary relationship (SNA) and gender on MS dimension, subjects with a retrusion (SNA < 80°) or protusion (SNA > 84°) of the maxillary alveolar bone had larger MSV, MSS, LMW, LMH and LMD than subjects with a normal cranio-maxillary relationship (SNA 82 ± 2°). A statistically significant high positive correlation was observed between S-N, Pns-A, S-Go, Mx-R/Mx-r and MS dimension. Further studies that evaluate similar outcomes in different races may be able to enrich our knowledge on this topic.

Evaluation of Masticatory Muscles in Adult Patients with Maxillary Hypoplasia Treated with Surgically Assisted Rapid Maxillary Expansion (SARME): A Retrospective Study.

Aim: The aim of the present study was to investigate modifications in electromyographic activity of temporal and masseter muscles before and after surgically assisted rapid maxillary expansion (SARME) in adult subjects. Materials and Methods: Data from 20 patients with unilateral posterior crossbite were selected retrospectively from the Orthodontics Department of the University of Genoa and the Department of Biomedical Surgical and Dental Sciences of the University of Milan, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico Milan. Inclusion criteria were set as skeletal class I; adult patients (age > 18); good general health; patients with a transverse maxillary deficiency with unilateral posterior crossbite and maxillary constriction ≥ 5 mm; Superficial electromyographic (EMG) examinations at T0 and T1. Exclusion criteria were smoking, metabolic bone diseases (e.g., hyperparathyroidism, vitamin C deficiency), chronic use of corticoids before or during treatment, parafunctional habits (e.g., bruxism), and temporomandibular joint dysfunction. The Shapiro−Wilk test was performed to check whether the data were normally distributed. Differences for each variable before and after SARME were analyzed with a paired t-test (p < 0.05). Results: The statistical analysis demonstrated no statistically significant differences between the EMG values taken before and after SARME regarding the standardized electrical activity of the masticatory muscles (masseter and anterior temporalis (p > 0.05)). Conclusions: Considering the specific conditions of this study, it can be concluded that SARME did not alter the EMG activity of the masseter and temporal muscles. The present study has shown that the masticatory musculature evaluated after approximately 8 months of therapy can adapt well to SARME.

The limitations for expression recognition in computer vision introduced by facial masks.

Facial Expression recognition is a computer vision problem that took relevant benefit from the research in deep learning. Recent deep neural networks achieved superior results, demonstrating the feasibility of recognizing the expression of a user from a single picture or a video recording the face dynamics. Research studies reveal that the most discriminating portions of the face surfaces that contribute to the recognition of facial expressions are located on the mouth and the eyes. The restrictions for COVID pandemic reasons have also revealed that state-of-the-art solutions for the analysis of the face can severely fail due to the occlusions of using the facial masks. This study explores to what extend expression recognition can deal with occluded faces in presence of masks. To a fairer comparison, the analysis is performed in different occluded scenarios to effectively assess if the facial masks can really imply a decrease in the recognition accuracy. The experiments performed on two public datasets show that some famous top deep classifiers expose a significant reduction in accuracy in presence of masks up to half of the accuracy achieved in non-occluded conditions. Moreover, a relevant decrease in performance is also reported also in the case of occluded eyes but the overall drop in performance is not as severe as in presence of the facial masks, thus confirming that, like happens for face biometric recognition, occluded faces by facial mask still represent a challenging limitation for computer vision solutions.

Correction: Sigala et al. A Comprehensive Investigation of Steroidogenic Signaling in Classical and New Experimental Cell Models of Adrenocortical Carcinoma. Cells 2022, 11, 1439.

The authors made the following changes to their paper [...].

Feasibility and Activity of Megestrol Acetate in Addition to Etoposide, Doxorubicin, Cisplatin, and Mitotane as First-Line Therapy in Patients with Metastatic/Unresectable Adrenocortical Carcinoma with Low Performance Status.

(1) Background: The standard first-line therapy for advanced adrenocortical carcinoma (ACC) is represented by EDP-M (etoposide, doxorubicin, cisplatin + mitotane). Progestins have shown cytotoxic activity both in vitro and in vivo on ACC; better EDP-M tolerability and efficacy have been hypnotized due to the association with progestins. (2) Methods: The feasibility and tolerability of EDP-M combined with oral megestrol acetate (EDP-MM) were tested in 24 patients (pts) affected by metastatic ACC with a low performance status (PS); the case group was compared with a 48 pts control group according to the propensity score. The secondary objectives were clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS). (3) Results: Thirteen pts (54.2%) in the EDP-MM population experienced progestin-related toxicities; in particular, five pts experienced vaginal bleeding (20.8%); four pts experienced weight gain (16.7%); and thromboembolic events, worsening of hypertension, skin rashes, and hyperglycemia were registered in one patient each (4.2%). This led to the discontinuation of megestrol acetate in four pts (16.7%). EDP-M-related toxicities were similar in both groups. No differences in PFS and OS curves were observed; the CBR was 75.0% and 60.4%, respectively. (4) Conclusions: The association of EDP-M + megestrol acetate in ACC pts with a low PS is feasible and well tolerated; its efficacy appeared to be non-inferior to EDP-M administered to pts with a good PS.

Trabectedin impairs invasiveness and metastasis in adrenocortical carcinoma preclinical models.

The pharmacological approach to adrenocortical carcinoma (ACC) is based on mitotane with/without etoposide, doxorubicin, and cisplatin, according to the disease stage. Considering the limited efficacy and toxicity of this treatment, new strategies are required. Trabectedin is a marine-derivated antitumoral agent that inhibits oncogenic transcription. We have already demonstrated trabectedin cytotoxic activity at sub-nanomolar concentrations in ACC cells. Here, we expanded the investigation of trabectedin effect on ACC preclinical models, evaluating whether trabectedin could affect ACC cells' invasiveness and metastasis formation. NCI-H295R, MUC-1, and TVBF-7 cell lines were used. Cell tumor xenografts in Danio rerio embryos were performed. The tumor mass areas and the number of embryos with metastasis were evaluated. The in vitro invasiveness of cells was evaluated. Effects of trabectedin of MMP2, TIMP1, and TIMP2 were evaluated at gene level qRT-PCR. MMP2 secreted in the cell medium was evaluated by Western blot and by zymography. Xenograft experiments demonstrated that trabectedin significantly reduced the tumor area in each ACC cell model and metastasis formation in embryos injected with metastasis-derived cell lines. Trabectedin treatment reduced the invasiveness of ACC cells across the matrix, which was greater at baseline for the metastatic models. In metastatic cell models, protein analysis demonstrated a reduction of MMP2 secretion and activity in the culture medium after treatment. Our results indicate that trabectedin interferes with invasiveness and metastasis processes, both dramatic features of ACC. Furthermore, these results support those previously published in providing the rationale for a clinical evaluation of the efficacy of trabectedin in ACC patients.