Does the anatomy around the pterygomaxillary suture contribute to the risk of bad fractures in Le Fort I osteotomy?

Le Fort I (LF1) osteotomy, a common orthognathic procedure for the maxilla aimed at achieving maxillary mobility by separating the pterygomaxillary suture, poses a risk of bad fracture that may lead to complications and inadequate mobility. Our study analyzed two- and three-dimensional computed tomography images to identify the anatomical factors associated with bad fractures due to an LF1 osteotomy. Point 'a' is where the lateral pterygomaxillary suture on the axial image aligns with the zygomatic alveolar line near the line used for an LF1 osteotomy, with the base line connecting the bilateral 'a' points.Two risk factors were identified on the pterygoid side: (i) when the distance from point 'a' to the intersection of the base line and the medial pterygoid plate was <6.0 mm; and (ii) when the distance from the piriform aperture margin to the base line was <44.78 mm. Six risk factors were identified on the maxillary side, including the distance between the most anterior and most lateral points of the internal surface of the maxillary sinus being <31.9 mm. Our analyses revealed that fractures that occur during pterygomaxillary suture separation in an LF1 osteotomy are influenced by anatomical factors of the maxilla and pterygoid process, which form the pterygomaxillary suture.

Terrein Exhibits Anti-tumor Activity by Suppressing Angiogenin Expression in Malignant Melanoma Cells.

BACKGROUND/AIM: Malignant melanoma is a tumor with a poor prognosis that can metastasize distally at an early stage. Terrein, a metabolite produced by Aspergillus terreus, suppresses the expression of angiogenin, an angiogenic factor. However, the pharmacological effects of natural terrein have not been elucidated, because only a small amount of terrein can be extracted from large fungal cultures. In this study, we investigated the antineoplastic effects of terrein on human malignant melanoma cells and its underlying mechanisms. MATERIALS AND METHODS: Human malignant melanoma cell lines were cultured in the presence of terrein and analyzed. Angiogenin production was evaluated using ELISA. Ribosome biosynthesis was evaluated using silver staining of the nucleolar organizer region. Intracellular signaling pathways were analyzed using western blotting. Malignant melanoma cells were transplanted subcutaneously into the backs of nude mice. The tumors were removed at 5 weeks and analyzed histopathologically. RESULTS: Terrein inhibited angiogenin expression, proliferation, migration, invasion, and ribosome biosynthesis in malignant melanoma cells. Terrein was shown to inhibit tumor growth and angiogenesis in animal models. CONCLUSION: This study demonstrated that terrein has anti-tumor effects against malignant melanoma. Furthermore, chemically synthesized non-natural terrein can be mass-produced and serve as a novel potential anti-tumor drug candidate.

Lingual nerve revisited-A comprehensive review Part II: Surgery and radiology.

The lingual nerve (LN) is a branch of the mandibular division of the fifth cranial nerve, the trigeminal nerve, arising in the infratemporal fossa. It provides sensory fibers to the mucous membranes of the floor of the mouth, the lingual gingiva, and the anterior two-thirds of the tongue. Although the LN should rarely be encountered during routine and basic oral surgical procedures in daily dental practice, its anatomical location occasionally poses the risk of iatrogenic injury. The purpose of this section is to consider this potential LN injury risk and to educate readers about the anatomy of this nerve and how to treat it.

Risk factors for postoperative facial nerve injury in retromandibular-approach surgery: A retrospective study including CT measurements of maxillofacial bone structure.

The purpose of this retrospective study was to identify risks of postoperative facial nerve injury (FNI) in mandibular condylar fractures. A total of 59 consecutive cases of condyle fracture or plate removal with a retromandibular transparotid approach (RMTA) were divided into FNI and non-FNI groups that were evaluated for associations with age, sex, laterality, fracture type, height, weight, body mass index (BMI), and maxillofacial bone height and width diameters on computed tomography (CT). FNI occurred in 11 of 59 patients (18.64%), all of them female (p = 0.0011). Other statistically significant factors on univariate analysis for FNI included a short height (156.95 ± 8.16 cm vs. 164.29 ± 9.89 cm, p = 0.04), low weight (46.08 ± 8.03 kg vs. 58.94 ± 11.79 kg, p = 0.003), low BMI (18.64 ± 2.63 kg/m2 21.68 ± 3.02 kg/m2, p = 0.007), short condylion-anterior fracture distance (19.34 ± 3.15 mm vs. 22.26 ± 3.96 mm, p = 0.04) and short condylion-posterior fracture distance (20.12 ± 3.98 mm vs. 25.45 ± 5.02 mm, p = 0.009). Our retrospective study suggested that FNI with RMTA surgery occurs particularly in female patients and may occur more frequently in patients who are short, lean or have high condyle fractures.

Double-faced CX3CL1 enhances lymphangiogenesis-dependent metastasis in an aggressive subclone of oral squamous cell carcinoma.

Because cancer cells have a genetically unstable nature, they give rise to genetically different variant subclones inside a single tumor. Understanding cancer heterogeneity and subclone characteristics is crucial for developing more efficacious therapies. Oral squamous cell carcinoma (OSCC) is characterized by high heterogeneity and plasticity. On the other hand, CX3C motif ligand 1 (CX3CL1) is a double-faced chemokine with anti- and pro-tumor functions. Our study reported that CX3CL1 functioned differently in tumors with different cancer phenotypes, both in vivo and in vitro. Mouse OSCC 1 (MOC1) and MOC2 cells responded similarly to CX3CL1 in vitro. However, in vivo, CX3CL1 increased keratinization in indolent MOC1 cancer, while CX3CL1 promoted cervical lymphatic metastasis in aggressive MOC2 cancer. These outcomes were due to double-faced CX3CL1 effects on different immune microenvironments indolent and aggressive cancer created. Furthermore, we established that CX3CL1 promoted cancer metastasis via the lymphatic pathway by stimulating lymphangiogenesis and transendothelial migration of lymph-circulating tumor cells. CX3CL1 enrichment in lymphatic metastasis tissues was observed in aggressive murine and human cell lines. OSCC patient samples with CX3CL1 enrichment exhibited a strong correlation with lower overall survival rates and higher recurrence and distant metastasis rates. In conclusion, CX3CL1 is a pivotal factor that stimulates the metastasis of aggressive cancer subclones within the heterogeneous tumors to metastasize, and our study demonstrates the prognostic value of CX3CL1 enrichment in long-term monitoring in OSCC.

Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system.

BACKGROUND: Cisplatin (CDDP) plays a central role in chemotherapy for head and neck squamous cell carcinoma (HNSCC), but drug resistance in HNSCC chemotherapy remains a problem, and the mechanism of CDDP resistance is unclear. We investigated CDDP-resistance mechanisms mediated by extracellular vesicles (EVs) and ATPase copper transporting beta (ATP7B) in HNSCC. METHODS: We established CDDP-resistant sublines of HNSCC cells and verified their ATP7B expression. We used an EV secretion inhibitor (GW4869) and ATP7B short hairpin (sh)RNA transfection to examine the correlation between EV secretion and ATP7B expression. RESULTS: The CDDP-resistant HNSCC sublines showed decreased CDDP sensitivity and increased ATP7B expression. GW4869 suppressed ATP7B expression, and ATP7B shRNA transfection suppressed EV secretion. The suppressions of EV secretion and ATP7B expression both enhanced CDDP's cell-killing effect. CONCLUSIONS: EVs were involved in the ATP7B-mediated mechanism underlying CDDP resistance. Further clarification of the EV-induced CDDP-resistance mechanism may lead to novel therapeutic strategies for HNSCC.

Mylohyoid Muscle: Current Understanding for Clinical Management-Part I: Anatomy and Embryology.

The mylohyoid is one of the suprahyoid muscles, along with the geniohyoid, digastric, and stylohyoid muscles. It lies between the anterior belly of the digastric muscle inferiorly and the geniohyoid superiorly. In Part I, the anatomy and embryology of the mylohyoid muscle will be reviewed in preparation for the clinical discussion in Part II.

Mylohyoid Muscle: Current Understanding for Clinical Management Part II: Clinical Anatomy, Radiology, and Surgical/Clinical Relevance.

The mylohyoid is one of the suprahyoid muscles along with the geniohyoid, digastric, and stylohyoid muscles that lies between the anterior belly of the digastric muscle inferiorly and the geniohyoid superiorly. In Part II, the radiology and clinical/surgical importance of the mylohyoid muscle will be discussed.

Rab11 suppresses head and neck carcinoma by regulating EGFR and EpCAM exosome secretion.

OBJECTIVES: Rab11(Rab11a and Rab11b) localizes primarily along recycling endosomes in cells and is involved in various intracellular trafficking processes, including membrane receptor recycling and secretion of exosomes or small extracellular vesicles (EVs). Although Rab11 is closely associated with the progression and metastasis of various cancer types, little is known about Rab11' role in head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the roles of Rab11a and Rab11b in HNSCC. METHODS: The clinical significance of Rab11 expression in HNSCC was investigated using a public database and tissue microarray analysis. Stable cell lines with loss and gain of Rab11a or Rab11b were originally established to investigate their roles in the proliferative, migratory, and invasive capabilities of HNSCC cells. RESULTS: Database analysis revealed a significant association between Rab11b mRNA expression and a favorable patient survival rate in HNSCC. Tissue microarray analysis revealed that Rab11b expression was the highest in normal tissues and gradually decreased across the stages of HNSCC progression. Overexpression of Rab11a or Rab11b resulted in a decrease in epidermal growth factor receptor (EGFR), Epithelial cell adhesion molecule (EpCAM) exosome secretion, and the migratory and invasive potential of HNSCC cells. The knockdown of Rab11a or Rab11b increased EpCAM/CD9 exosome secretion in addition to the migratory and invasive potential of HNSCC cells. CONCLUSIONS: Rab11 suppresses HNSCC by regulating EGFR recycling and EpCAM exosome secretion in HNSCC cells. Our results indicate that Rab11b is a superior prognostic indicator of HNSCC and holds promise for developing novel therapeutic strategies.

Multimodal Prediction of Cervical Lymph Node Metastasis and Recurrence in Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is the most common malignancy in the head/neck region, and cervical lymph node (CLN) metastasis is a strong poor-prognosis factor. In addition, many patients with OSCC experience recurrence despite multidisciplinary treatment. We sought to identify factors associated with CLN metastasis and recurrence in patients with OSCC. PATIENTS AND METHODS: We evaluated a total of 45 patients and 233 target CLNs. The longest diameter of the target CLN, the shortest diameter of the target CLN (LS), the area of the target CLN, and the relative computed tomography (CT) values of the target CLNs calculated based on the CT values of the internal jugular vein (LCT) were obtained from preoperative CT images, and the maximum standardized uptake values of the primary tumor (pSUV) and target CLN (nSUV) were obtained from preoperative 18F-fluorodeoxyglucose-positron emission tomography/CT images. We performed immunohistochemical staining for cytokeratin 13 (CK13) and 17 (CK17) on neck dissection tissues. RESULTS: A discrimination equation was used that can predict CLN metastasis with a 92.2% discrimination rate using LS, LCT, pSUV, and nSUV. The CLNs were divided into discrimination and non-discrimination groups based on discriminant equations and CK13 and CK17 were used as the objective variables. A significantly higher recurrence rate was observed in the non-discrimination group (CK13: 5-year recurrence rate 28.6% vs. 64.3%, p<0.01; CK17: 5-year recurrence rate 28.0% vs. 76.0%, p<0.01). CONCLUSION: CLN metastases in OSCC can be assessed by combining preoperative imaging. The combined use of CK13 and CK17 expression with imaging findings offers an integrated approach to predict OSCC recurrence.

Multiple Targeting of HSP Isoforms to Challenge Isoform Specificity and Compensatory Expression.

Heat shock proteins (HSPs) are molecular chaperones that assist in protein folding, trafficking, and metabolism. Intracellular chaperone functions of HSPs had been well-investigated, but extracellular and exosomal HSPs have been recently found. Exosomal HSPs are intercellularly transferred, while extracellular HSPs play cytokine-like roles called chaperokines. We have shown that exosomal HSPs play key roles in intercellular communication between tongue carcinoma and tumor-associated macrophages in the tumor microenvironment. Notably, HSP90 isoforms consist of HSP90alpha, HSP90beta, mitochondrial TRAP1, and GRP94 in the endoplasmic reticulum. Moreover, many pseudogenes of HSP90 can be transcribed into RNA. Besides, the function of HSP90 is defined by their cochaperones, such as CDC37 or AHA1. Therefore, isoform-specific small interfering RNA (siRNA) is necessary for precisely targeting each HSP90 isoform and cochaperone. Nevertheless, we often encountered compensatory expression of HSP90 isoforms in the knockdown studies. Here, we provide dual and triple knockdown methods to target multiple RNA for challenging isoform-specific roles and compensatory expression of intracellular, extracellular, and exosomal HSPs.

Proteomic Profiling of the Extracellular Vesicle Chaperone in Cancer.

Molecular chaperones are widely distributed intracellular proteins that play essential roles in maintaining proteome function by assisting in the folding of client proteins. Molecular chaperones, such as heat shock proteins (HSPs), are found intracellularly and extracellularly. Extracellular vesicles (EVs), such as exosomes, contain HSPs and horizontally transfer the functional chaperones into various recipient cells. Besides, mass spectrometry has enabled a comprehensive analysis of exosomal and EV proteins, which is useful in basic biomedical research to clinical biomarker search. We have performed deep proteome analysis of EVs, including exosomes, from metastatic tongue and prostate cancers and detected >700 protein types, including cytoplasmic, ER, mitochondrial, small, and large HSPs. Here, we provide protocols for isolating exosomes/EVs and deep proteome analysis to detect the EV chaperone.

Multiplex Immunostaining Method to Distinguish HSP Isoforms in Cancer Tissue Specimens.

Heat shock proteins (HSPs) are often expressed in all nucleated cells, but their expression profiles differ. In particular, HSP90α and HSP90ß have high sequence identity and have not been fully examined for their individual and compensatory functions as molecular chaperones, differences in client proteins, and extracellular distributions with exosomes. Immunohistochemical staining is a technique to visualize the presence and localization of target antigens using specific antibodies, of which the multiplex immunostaining method can reveal differences in protein expression in the same tumor tissue and the localization of proteins of interest within tumor tissue or single cells. The common multiplex immunostaining method uses multiple secondary antibodies of different reacting animal species to identify and detect different antigens, thus requiring different animals to be immunized with each primary antibody. Furthermore, the fluorescent-antibody method is the predominant multiplex staining method but has the critical disadvantage that permanent specimens cannot be prepared. Here, we outline a multiplex staining method for HSP90α and HSP90ß based on the enzyme-antibody method that allows permanent specimens to be prepared without the restriction of immunized animal species.

Large-Scale Databases and Portals on Cancer Genome to Analyze Chaperone Genes Correlated to Patient Prognosis.

Molecular chaperones, such as heat shock proteins (HSPs), have attracted attention as molecules involved in malignant events in cancers and are potential therapeutic targets and biomarkers for tumor therapy. Furthermore, mutations in chaperones can significantly impact cancer risk and prognosis. Bioinformatics is a particularly useful method for developing biomarkers as a practical consideration for the immediate clinical application of data. Many large-scale databases and portals on cancer genome are nowadays publicly available, including the International Cancer Genome Consortium (ICGC); The Cancer Genome Atlas (TCGA), renamed as Genomic Data Commons (GDC); Catalogue of Somatic Mutations in Cancer (COSMIC); and Cancer Cell Line Encyclopedia (CCLE). Referring to these databases, advanced web portals are publicized, including cBioPortal, Human Protein Atlas (HPA), Kaplan-Meier (KM) plotter, Gene Expression Profiling Interactive Analysis 2 (GEPIA2), Genomics of Drug Sensitivity in Cancer (GDSC), and Dependency Map (DepMap). Here, we assemble these databases and portals to clarify what is available and useful for current cancer research and provide protocols to utilize the HPA, KM plotter, and GEPIA2 for studies on chaperone genes in cancer patients. Utilizing these portals will reveal the correlation between tumor subtype-specific high expression of chaperone genes and patient prognosis. Our protocols are useful to increase systematic awareness of chaperones and find new biomarkers for diagnosis and prognosis and new targets for anticancer drugs.

Preservation of the Nerve to the Mylohyoid Muscle During Submental Island Flaps: An Anatomic Feasibility Study for Facial Nerve Reanimation Procedures.

The submental island flap is an axial pattern pedicle flap widely used in head and neck surgery because of its ease and success. Indications of the submental island flap range from reconstruction for the malignant tumor resection to loss of temporal bone and facial skin due to trauma. Whereas, intraoperative facial nerve injury is not uncommon. We verified whether it was possible to localize the nerve to the mylohyoid muscle and reanimate the facial nerve during submental island flap procedures by preserving the mylohyoid muscle using human fresh cadaveric specimens. Six cadaveric heads were dissected and the position of the nerve to the mylohyoid muscle identified to the mylohyoid triangle documented. We identified the nerve to the mylohyoid muscle on all sides within the mylohyoid triangle and were able to separate the nerve from the submental island flap completely. Our results suggest that facial nerve reanimation using the nerve to the mylohyoid muscle can be used while reconstructing with a submental island flap in cases of intraoperative facial nerve injury.

EpEX, the soluble extracellular domain of EpCAM, resists cetuximab treatment of EGFR-high head and neck squamous cell carcinoma.

OBJECTIVES: Cetuximab (Cmab) is a molecularly targeted monoclonal antibody drug for head and neck squamous cell carcinoma (HNSC), although cetuximab resistance is a serious challenge. Epithelial cell adhesion molecule (EpCAM) is an established marker for many epithelial tumors, while the soluble EpCAM extracellular domain (EpEX) functions as a ligand for epidermal growth factor receptor (EGFR). We investigated the expression of EpCAM in HNSC, its involvement in Cmab action, and the mechanism by which soluble EpEX activated EGFR and played key roles in Cmab resistance. MATERIALS AND METHODS: We first examined EPCAM expression in HNSCs and its clinical significance by searching gene expression array databases. We then examined the effects of soluble EpEX and Cmab on intracellular signaling and Cmab efficacy in HNSC cell lines (HSC-3 and SAS). RESULTS: EPCAM expression was found to be enhanced in HNSC tumor tissues compared to normal tissues, and the enhancement was correlated with stage progression and prognosis. Soluble EpEX activated the EGFR-ERK signaling pathway and nuclear translocation of EpCAM intracellular domains (EpICDs) in HNSC cells. EpEX resisted the antitumor effect of Cmab in an EGFR expression-dependent manner. CONCLUSION: Soluble EpEX activates EGFR to increase Cmab resistance in HNSC cells. The EpEX-activated Cmab resistance in HNSC is potentially mediated by the EGFR-ERK signaling pathway and the EpCAM cleavage-induced nuclear translocation of EpICD. High expression and cleavage of EpCAM are potential biomarkers for predicting the clinical efficacy and resistance to Cmab.

Lactate secreted via MCT4 from bone­colonizing breast cancer excites sensory neurons via GPR81.

Breast cancer (BC) bone metastasis causes bone pain (BP), which detrimentally damages the quality of life and outcome of patients with BC. However, the mechanism of BC­BP is poorly understood, and effective treatments are limited. The present study demonstrated a novel mechanism of BC­BP using a mouse model of bone pain, in which mouse (EO771) and human (MDA­MB­231) BC cells were injected in the bone marrow cavity of tibiae. Western blot analysis using sensory nerves, in vivo assessment of cancer pain and in vitro calcium flux analysis were performed. These mice developed progressive BC­BP in tibiae in conjunction with an upregulation of phosphorylated pERK1/2 and cAMP­response element­binding protein (pCREB), which are molecular indicators of neuron excitation, in the dorsal root ganglia (DRG) of sensory nerves. Importantly, mice injected with BC cells, in which the expression of the lactic acid transporter monocarboxylate transporter 4 (MCT4) was silenced, exhibited decreased BC­BP with downregulated expression of pERK1/2 and pCREB in the DRG and reduced circulating levels of lactate compared with mice injected with parental BC cells. Further, silencing of the cell­surface orphan receptor for lactate, G protein­coupled receptor 81 (GPR81), in the F11 sensory neuron cells decreased lactate­promoted upregulation of pERK1/2 and Ca2+ influx, suggesting that the sensory neuron excitation was inhibited. These results suggested that lactate released from BC cells via MCT4 induced BC­BP through the activation of GPR81 of sensory neurons. In conclusion, the activation of GPR81 of sensory neurons by lactate released via MCT4 from BC was demonstrated to contribute to the induction of BC­BP, and disruption of the interactions among lactate, MCT4 and GPR81 may be a novel approach to control BC­BP.

Lingual nerve impairment/injury after retrieval of the displaced mandibular third molar into the floor of the mouth.

Retrieval of the displaced mandibular third molar in the floor of the mouth is challenging as the lingual nerve is always at risk of injury. However, there are no available data to show the incidence of the injury caused by the retrieval. The goal of this review article is to provide the incidence of the iatrogenic lingual nerve impairment/injury caused by the retrieval based on the review of the existing literature. The retrieval cases were collected with the search words below using PubMed, Google Scholar, and CENTRAL Cochrane Library database on October 6, 2021. A total of 38 cases of lingual nerve impairment/injury in 25 studies were eligible and reviewed. Temporary lingual nerve impairment/injury due to retrieval was found in six cases (15.8%) and all recovered between three to six months after retrieval. General anaesthesia and local anaesthesia were used for retrieval in three cases each. The tooth was retrieved using a lingual mucoperiosteal flap in all six cases. The permanent iatrogenic lingual nerve impairment/injury due to retrieval of the displaced mandibular third molar is considered extremely rare as long as the appropriate surgical approach is chosen based on surgeons' clinical experience and anatomical knowledge.

Western Blot Protocols for Analysis of CCN Proteins and Fragments in Exosomes, Vesicle-Free Fractions, and Cells.

Cellular Communication Network (CCN) proteins are growth factors that play key roles in many pathophysiological events, including bone formation, wound healing, and cancer. CCN factors and fragments generated by metalloproteinases-dependent cleavage are often associated with extracellular matrix (ECM) or small extracellular vesicles (sEVs) such as exosomes or matrix-coated vesicles. We provide reliable methods and protocols for Western blotting to analyze CCN factors and fragments in cells, sEVs, and vesicle-free fractions.

Comprehensive Method for Exosome Isolation and Proteome Analysis for Detection of CCN Factors in/on Exosomes.

Cellular Communication Network (CCN) proteins are secretory growth factors often associated with extracellular matrix (ECM) and extracellular vesicles (EVs) such as exosomes or matrix-coated vesicles. CCN factors and fragments loaded on/in EVs may play key roles in cell communication networks in cancer biology, bone and cartilage metabolism, wound healing, and tissue regeneration. CCN proteins and EVs/exosomes are found in body fluids, such as blood, urine, milk, and supernatants of the two-dimensionally (2D) cultured cells and three-dimensionally (3D) cultured tissues, such as spheroids or organoids. More than ten methods to isolate exosomes or EVs have been developed with different properties. Here, we introduce comprehensive protocols for polymer-based precipitation, affinity purification, ultracentrifugation methods combined with the ultrafiltration method for isolating CCN-loaded exosomes/EVs from 2D and 3D cultured tissues, and proteome analysis using mass spectrometry for comprehensive analysis of CCN proteins.