Serratia entomophila bet gene induction and the impact of glycine betaine accumulation on desiccation tolerance.

AIMS: The genes involved in choline transport and oxidation to glycine betaine in the biopesticidal bacterium Serratia entomophila were characterized, and the potential of osmoprotectants, coupled with increased NaCl concentrations, to improve the desiccation tolerance of this species was investigated. METHODS AND RESULTS: Serratia entomophila carries sequences similar to the Escherichia coli betTIBA genes encoding a choline transporter and dehydrogenase, a betaine aldehyde dehydrogenase and a regulatory protein. Disruption of betA abolished the ability of Ser. entomophila to utilize choline as a carbon source. Quantitative reverse-transcriptase PCR analysis revealed that betA transcription was reduced compared to that of the upstream genes in the operon, and that NaCl and choline induced bet gene expression. Glycine betaine and choline increased the NaCl tolerance of Ser. entomophila, and osmotically preconditioned cultures survived better than control cultures following desiccation and immediately after application to agricultural soil. CONCLUSIONS: Addition of glycine betaine and NaCl to growth medium can greatly enhance the desiccation survival of Ser. entomophila, and its initial survival in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Serratia entomophila is sensitive to desiccation and does not persist under low soil moisture conditions. Techniques described here for enhancing the desiccation survival of Ser. entomophila can be used to improve formulations of this bacterium, and allow its application under a wider range of environmental conditions.

The heavy metal content of skeletons from an ancient metalliferous polluted area in southern Jordan with particular reference to bioaccumulation and human health.

This paper considers pollution/toxicological science in an archaeological context. Copper mining was an important activity in southern Jordan, especially during the Bronze Age, Nabatean, Roman, and Byzantine periods, and the environmental legacy of such intensive mining and smelting activities exists today in the form of massive, ancient spoil and smelting tips. The environment was heavily polluted by copper, lead, and other cations during these early periods and the effects of such pollutants continue into modern times. Samples of goat, sheep, and Bronze Age and Byzantine skeletons have been analyzed and high metal loads, from uptake by diverse processes, are reported. Emphasis is placed on the importance of sampling procedure and sample location, bioaccumulation, and the partitioning of such elements. Implications of such pollutants in terms of environmental and human health in ancient and modern times are discussed. Teeth are found to provide excellent vehicles for the monitoring of pollution in both ancient and recent times. Bronze Age skeletons exhibited chemical fingerprints different from those of the Byzantine period.

Expression of multidrug resistance 1 and glutathione-S-transferase-Pi protein in nasopharyngeal carcinoma.

Radiotherapy is the modality of choice for the treatment of nasopharyngeal carcinoma (NPC). However, systemic chemotherapy has recently been found to play an increasing role in the treatment of advanced or metastatic disease. The status of drug resistance gene expression that has crucial impact on chemotherapy has not been fully addressed for patients with NPC. In this study, we examined the expression of multidrug resistance 1 (MDR-1) and glutathione-S-transferase-Pi (GST-Pi) in primary, recurrent, and metastatic NPC using results of immunohistochemical examinations. The results were correlated with the expression of Epstein-Barr virus (EBV) latent protein, latent membrane protein 1 (LMP1), and clinicopathologic features, including stage, histopathologic types, and survival rates. MDR-1 protein expression was detected in 18 (12.6%) of 143 patients with primary NPC, 14 (32.6%) of 43 with recurrent NPC, and O (0%) of 20 with metastatic NPC, whereas 83 (58%) of 143 patients with primary NPC, 30 (69.8%) of 43 with recurrent NPC, and 13 (65%) of 20 with metastatic NPC expressed GST-Pi. EBV-LMP1 was expressed in 59 (41.3%) of 143 patients with primary NPC, 23 (53.5%) of 43 with recurrent NPC, and 9 (45%) of 20 with metastatic NPC. Simultaneous expression of MDR1 and GST-Pi was observed in 13 (72.2%) of 18 patients with primary NPC and 12 (85.7%) of 14 with recurrent NPC. The expression of LMP1 was detected in only 6 of the 13 patients with primary NPC and 6 of the 12 with recurrent NPC. We concluded that the expression of GST-Pi was more frequent in NPC tumor tissues than the expression of MDR-1. The expression of MDR-1 correlated with clinicopathologic features of primary NPC, including the histopathologic types and survival rates, but not with disease stage. The expression of GST-Pi did not correlate with clinicopathologic features. The expression of MDR-1 and GST-Pi did not correlate with expression of EBV-LMP1 for patients with NPC.

Induction chemotherapy with mitomycin, epirubicin, cisplatin, fluorouracil, and leucovorin followed by radiotherapy in the treatment of locoregionally advanced nasopharyngeal carcinoma.

PURPOSE: Survival in advanced nasopharyngeal carcinoma (NPC) is compromised by distant metastasis. Because mitomycin is active against hypoxic and G0 cells, which may help to eradicate micrometastasis, we investigated the effect of mitomycin-containing cisplatin-based induction chemotherapy. PATIENTS AND METHODS: Recruited for this study were American Joint Committee on Cancer (AJCC) 1992 staging system stage IV NPC patients with the following adverse features: obvious intracranial invasion, supraclavicular or bilateral neck lymph node metastasis, large neck node (> 6 cm), or elevated serum lactate dehydrogenase (LDH) level. Patients were given three cycles of chemotherapy before radiotherapy. The chemotherapy comprised a 3-week cycle of mitomycin, epirubicin, and cisplatin on day 1 and fluorouracil and leucovorin on day 8 (MEPFL). RESULTS: From January 1994 to December 1997, 111 patients were recruited. The median follow-up period was 43 months. The actuarial 5-year overall survival rate was 70% (95% confidence interval [CI], 60% to 80%; n = 111). For patients having completed radiotherapy (n = 100), the 5-year locoregional control rate was 70% (95% CI, 55% to 84%) and the distant metastasis-free rate was 81% (95% CI, 73% to 89%). The 5-year distant metastasis-free rate of N3a and N3b disease of AJCC 1997 staging system were 79% (95% CI, 62% to 95%) and 74% (95% CI, 60% to 89%), respectively. By Cox multivariate analysis, high pretreatment serum LDH level (P = .04) and neck nodal enlargement before radiotherapy (P = .001) were adverse prognostic factors of survival. CONCLUSION: The good 5-year survival of N3 disease supports the effectiveness of induction MEPFL in the primary treatment of advanced NPC. Further investigation to incorporate concurrent chemoradiotherapy is warranted.

Specific IgA antibodies to recombinant early and nuclear antigens of Epstein-Barr virus in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma is a common cancer in Taiwan. The Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma. The sera of patients with nasopharyngeal carcinoma have IgA antibodies to a variety of EBV latent and replicated antigens. Recently, an enzyme-linked immunosorbent assay (ELISA) kit, combining both the EBV early antigen (EA) and nuclear antigen (EBNA-1) became commercially available. The purpose of this study was to assess its clinical application. Serum IgA antibodies to the EBV EA and EBNA-1 were measured by using the ELISA kit in various groups of subjects. Fluorescence antibody (FA) tests against EBV viral capsid antigen (VCA) and EA in the IgA and IgG classes were also studied for comparison. The DNA content analysis was also carried out to investigate the association with IgA antibody titres using ELISA. The sensitivity, specificity and accuracy of the ELISA test were 98.1%, 81.8% and 88.7% respectively. It was far better than any FA tests. The IgA antibody titres showed no association with DNA content analysis. Univariate analysis of various factors revealed that IgA antibody titres were statistically correlated to N stage (P = 0.0291) and M status (P = 0.001). However, there was no association with the age, sex, T stage and clinical stage. Multivariate analysis of various factors was found to be statistically significant in patients with T4 (P = 0.0133), N3 (P = 0.0244) or M1 (P = 0.001) respectively. Serial testing of antibody titres in 22 previously untreated patients found a trend of decreasing IgA antibody titres after initial treatment when the tumours disappeared (P = 0.0358). The ELISA kit to identify specific IgA antibodies with the combination of EBV EA and EBNA-1 recombinant antigens has high sensitivity and acceptable specificity and accuracy in the diagnosis of nasopharyngeal carcinoma. This assay should be useful for early diagnosis and mass screening of patients.

Soluble adhesion molecules and cytokines in tumor-associated tissue eosinophilia of nasopharyngeal carcinoma.

The phenomenon of tumor-associated tissue eosinophilia (TATE) is seen in some cases of nasopharyngeal carcinoma (NPC) and is characterized by the eosinophils breaking through the vascular wall and pervading the tumor stroma. The margination and trans-endothelial migration of eosinophils in a typical inflammatory reaction depend on the activating effects of certain cytokines and the expression of adhesion molecules on the eosinophils and endothelial cells. In order to investigate whether the adhesion molecules and activating cytokines play a role in eosinophil tumor infiltration, we measured the serum levels of 3 adhesion molecules, intercellular adhesion molecule-1, E-selectin and vascular cell adhesion molecule-1, and 2 cytokines, IL-3 and IL-5, in 60 NPC patients and 40 normal healthy subjects. We found that the NPC patients had higher serum levels of all three soluble adhesion molecules than the normal subjects but the levels of adhesion molecules failed to correlate with the TATE phenomenon. The levels of IL-3 and IL-5 appeared not to differ between the NPC and control groups. We postulate that the three soluble adhesion molecules do not play a major role in TATE and that their elevation in serum may be due to local and/or systemic immune responses.

Factors affecting the overall survival after salvage surgery in patients with recurrent nasopharyngeal carcinoma at the primary site: experience with 60 cases.

OBJECTIVE: To analyze the factors affecting overall survival after salvage surgery in patients with recurrent nasopharyngeal carcinoma at the primary site after a full course of radiotherapy. DESIGN: Retrospective analysis of 60 consecutive patients treated by surgical resection of the recurrent tumors, with a mean follow-up of 43.1 months (range, 19-96 months). SETTING: Academic tertiary referral center. RESULTS: The overall survival and locoregional relapse-free survival were 56% and 60% at 2 years, respectively, and 30% and 40% at 5 years. Twenty-nine (81%) of 36 patients died with uncontrolled local disease. The T stage of the recurrent tumors appeared to be an important prognostic factor. Age, sex, pathologic findings, and disease-free interval (time between previous radiotherapy and local recurrence) were not significant prognosis-affecting factors by the log-rank test. Multivariate analysis showed that patients with recurrent tumors of undifferentiated carcinoma, sarcoma, or small cell carcinoma had unfavorable prognoses. Uncontrolled local disease and the emergence of distant metastasis predicted grave results as well. Postoperative irradiation showed some benefit to patients, but the difference was not statistically significant. CONCLUSIONS: The T stage of the recurrence was the prominent prognosis-affecting factor in patients with recurrent nasopharyngeal carcinoma who received salvage surgery. Patients with local recurrence should be carefully selected for the salvage surgery. We recommend this surgery for patients with rT1, rT2, or limited rT3 lesions. The results of surgical resection in terms of local control and overall survival were slightly better than those of high-dose reirradiation, with fewer late complications.

Induction of c-Met proto-oncogene by Epstein-Barr virus latent membrane protein-1 and the correlation with cervical lymph node metastasis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is distinctive in head and neck carcinomas for its close association with Epstein-Barr virus and its highly metastatic nature. Up-regulation of cell motility is essential for enhancement of metastatic potential. The expression of c-Met proto-oncogene, a high-affinity receptor for hepatocyte growth factor/scatter factor, has been reported to correlate with metastatic ability of the tumor cell. We observed close association of c-Met expression with cervical lymph node metastasis (P = 0.0272) in 39 NPC specimens studied immunohistochemically. Epstein-Barr virus-encoding latent membrane protein-1 (LMP-1) is a primary oncogene and is suggested to enhance the metastatic property of NPC. Previously, we reported that LMP-1 enhanced the motility of Madin-Darby canine kidney (MDCK) epithelial cells that was mediated by activation of Ets-1 transcription factor. Therefore, we examined the interrelationships of LMP-1, Ets-1, and c-Met. In immunohistochemical studies, the expression of LMP-1, Ets-1, and c-Met correlated significantly with each other in NPC (LMP-1 versus Ets-1, P < 0.0001; Ets-1 versus c-Met, P = 0.0012; LMP-1 versus Met, P = 0.0005). Transfection of LMP-1-expressing plasmid in MDCK cells induced c-Met protein expression. The c-Met protein was also induced by Ets-1 expression, and induction of c-Met by LMP-1 was suppressed by introducing a dominant-negative form of Ets-1 in LMP-1-expressing MDCK cells. These results suggest that LMP-1 induces c-Met through the activation of Ets-1, which may contribute in part to the highly metastatic potential of NPC.

Induction of interleukin-8 by Epstein-Barr virus latent membrane protein-1 and its correlation to angiogenesis in nasopharyngeal carcinoma.

PURPOSE: The EBV latent membrane protein-1 (LMP-1) is a multifunctional protein. Recently, the contribution of LMP-1 to the metastasis of nasopharyngeal carcinoma (NPC) has been suggested. Angiogenesis is a key step for metastasis. Thus, the association of LMP-1 to neovascularization of NPC was examined in this study. EXPERIMENTAL DESIGN: The association of LMP-1 to angiogenesis in 39 patients with NPC was evaluated by immunohistochemical study, and then induction of angiogenic factors by LMP-1 was examined by ELISA and luciferase reporter assay. RESULTS: In an immunohistochemical study, the expression of LMP-1 was significantly correlated to microvessel counts (P = 0.0003), suggesting that LMP-1 may induce some angiogenic factors. Therefore, we studied the relationship between LMP-1 expression and interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) expression by immunohistochemical analysis. IL-8, VEGF, and bFGF expression were correlated to microvessel counts, but only IL-8 expression was significantly correlated to LMP-1 expression (P < 0.0001). Transfection with LMP-1 expression plasmid induced IL-8 protein expression in C33A cells. The expression of LMP-1 transactivated IL-8 promoter, as demonstrated by IL-8 promoter luciferase reporter assay. Mutation of the nuclear factor kappaB responsive element in the IL-8 promoter region completely abolished transactivation by LMP-1, whereas mutation of the activator protein responsive element did not affect promoter activity. CONCLUSION: These results suggested that LMP-1 induces expression of IL-8 through the nuclear factor kappaB binding site, which may contribute in part to angiogenesis in NPC.

Herniated blastomere following chemically assisted hatching may result in monozygotic twins.

OBJECTIVE: To explore a possible mechanism of the increasing incidence of monozygotic twins following assisted hatching of human embryos. DESIGN: Case report. SETTING: Clinical research center in a medical school teaching hospital. PATIENT: A 37-year-old infertile woman with repeated IVF failures. INTERVENTION(S): Assisted hatching of the day 3 embryos using acidic Tyrode's solution. MAIN OUTCOME MEASURE(S): The morphology of the zona-drilled embryos and the pregnancy outcome. RESULT(S): After assisted hatching, a herniated blastomere through an oversized opening in the zona pellucida was found in one embryo. The transfer of two zona-drilled embryos resulted in a triplet pregnancy. CONCLUSION(S): Large openings in the zona pellucida following chemically assisted hatching may cause premature hatching of the blastomeres and may be implicated in the occurrence of monozygotic twins.

Association of latent membrane protein 1 and matrix metalloproteinase 9 with metastasis in nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a highly metastatic carcinoma whose consistent association with Epstein-Barr virus (EBV) has been established. Latent membrane protein 1 (LMP1), an EBV membrane protein expressed in latent infection, is considered to be the EBV oncoprotein. Matrix metalloproteinase 9 (MMP9), one of the MMP families, degrades Type IV collagen, a major component of extracellular matrix and is believed to be crucial for cancer invasion and metastasis. Although MMP9 is reported to be expressed in a variety of cancers, no reports concerning NPC have been published to date to the authors' knowledge. Recently, the authors have shown that LMP1 induces MMP9 in vitro cell line, which suggests the possibility of a mechanism in which LMP1 of EBV contributes to the metastasis and tumorigenesis of NPC by the induction of MMP9. METHODS: The expressions of LMP1 and MMP9 were immunohistochemically examined in 38 NPC sections, and the relation of these proteins were statistically analyzed. The authors also analyzed the associations of these proteins with clinical features. RESULTS: Both LMP1 and MMP9 proteins were predominantly immunolocalized in cancer nests. The expression of MMP9 showed a significant positive correlation with the expression of LMP1 (r = 0.75; P < 0.0001). Also, the expression of MMP9 correlated with lymph node metastasis (P = 0. 0004). CONCLUSIONS: The results suggest that the induction of MMP9 by LMP1 contributes to the metastatic potential of NPC.

DNA content of nasopharyngeal carcinoma: an independent prognostic indicator.

The purpose of this study was to examine whether tumor DNA content correlated with prognosis in nasopharyngeal carcinoma (NPC). DNA flow-cytometric analysis in fresh specimens of nasopharyngeal biopsy from 123 patients with clinical suspicion of NPC was collected initially. Histopathologic study and successful flow-cytometric analysis had 28 lymphoid hyperplasias and 87 NPCs. Seventeen NPC patients were treated elsewhere and were excluded. A total of 98 patients, including 28 lymphoid hyperplasias and 70 NPCs, formed the materials of this study. There were 34 (49%) diploid and 36 (51%) aneuploid in NPC patients. No lymphoid hyperplasias were aneuploid. The mean of S-phase fraction was higher in NPC than in lymphoid hyperplasia (P < .001), indicating higher cellular activity in NPC. DNA content failed to associate with age, gender, pathology, distant metastasis, and stage, indicating that DNA content was an independent prognostic indicator and possibly a clinical parameter. The log-rank test of overall survival curves was significant for stage (P = .002) and DNA ploidy (P = .042); it was almost significant for S-phase fraction (P = .057). Because the follow-up duration was not long enough, univariate and multivariate analysis were not significant for stage, ploidy, and S-phase fraction, except for distant metastasis. It is also most likely colinearity of clinical stage and distant metastasis that explained why clinical stage could not show significance in prognosis. Interestingly, the DNA content appeared to be a potential prognostic parameter in overall survival, although it was not statistically significant (P = .052). Our data suggested that NPC patients with aneuploid DNA and high S-phase fraction tend to have poor prognosis and should be treated more aggressively, even in the early stage of the disease.

Upregulation of tyrosine kinase TKT by the Epstein-Barr virus transactivator Zta.

The Zta protein is a key transactivator involved in initiating the Epstein-Barr virus (EBV) lytic cascade. In addition to transactivating many viral genes, Zta has the capacity to influence host cellular signals by binding to promoter regions or by interacting with several important cellular factors. Based on the observation that tyrosine kinases play central roles in determining the fate of cells, a kinase display assay was used to investigate whether cells expressing Zta have an altered pattern of kinase expression. The assay revealed that TRK-related tyrosine kinase (TKT) is expressed at significant levels in Zta transfectants but not in control cells. Additional evidence was obtained from Northern and Western blotting. Importantly, the upregulation of phosphorylated TKT and TKT downstream effector matrix metalloproteinase 1 in Zta transfectants hinted that TKT might initiate a signaling cascade in Zta-expressing cells. In addition, deletion analysis of the Zta protein revealed that the transactivation and dimerization domains were both essential for the upregulation of TKT transcription. Moreover, correlation of expression levels of Zta and TKT transcripts in nasopharyngeal carcinoma biopsy specimens was clearly demonstrated by quantitative PCR (Q-PCR), which provides the first evidence for an effect of Zta on cellular gene expression in vivo. These findings offer insight into the virus-cell interactions and may help us elucidate the role of EBV in tumorigenesis.

Extraskeletal Ewing's sarcoma of the scalp.

Extraskeletal Ewing's sarcoma/primitive neuroectodermal tumour (EES/PNET) is a rare disease entity. Scalp EES/PNET has been reported rarely. We report a case of an 11-year-old boy who had painful and rapidly growing subcutaneous nodes over the scalp and neck. The final diagnosis was EES/PNET after biopsy and immunohistochemical assay. The patient underwent surgical excision, chemotherapy and radiotherapy with a dose of 2000 cGy. Now he has been free of disease for two years. Early awareness and treatment of this rare disease, and wide resection followed by chemotherapy and radiotherapy might improve patients' long-term survival.

Herpes zoster oticus treated with acyclovir and prednisolone: clinical manifestations and analysis of prognostic factors.

Herpes zoster oticus is a cranial polyneuropathy with facial nerve involvement as its main feature. The prognosis of the facial palsy is usually poor. Thirty patients with herpes zoster oticus suffering from facial palsy were admitted for parenteral acyclovir and oral prednisolone. Multiple regression analysis of improvement of facial palsy showed three significant covariates: age, multiple nerve palsies, and the initial grading of the palsy. The recovery of the facial palsy treated with acyclovir and prednisolone was good, and possibility of a good outcome was greater when the initial grade of the palsy was higher. Multiple nerve palsies and age had negative effects on the improvement.

Expression of the Epstein-Barr virus BHRF1 gene, a homologue of Bcl-2, in nasopharyngeal carcinoma tissue.

Epstein-Barr virus (EBV) infection is associated closely with the pathogenesis of nasopharyngeal carcinoma (NPC). The EBV gene product, BHRF1, has been demonstrated in vitro and is structurally and functionally similar to the oncogene bcl-2, that is able to protect cells from programmed cell death. To determine whether the BHRF1 gene is expressed in vivo, BHRF1 mRNA or protein were sought in tissues from NPC and non-NPC patients. BHRF1 transcripts were specifically detected in the NPC tumours (32 out of 44, 72.7%) rather than the non-NPC tissues (0 out of 25) by reverse transcription, polymerase chain reaction and Southern hybridization. Other EBV genes, such as the lytic gene BZLF1 and latent genes EBNA1 and LMP2A, were also investigated. BZLF1 transcripts also were found specifically in NPC tumours (33 out of 44, 75%). EBNA1 was expressed in 79.5% of NPC, and 28% of non- NPC, tissues and LMP2A was expressed in 70.5% of NPC, and 88% of non-NPC, tissues. BHRF1 protein was detected by immunohistochemistry in 4 metastatic NPC, of 36 NPC tissue sections available. The BHRF1 protein was distributed in both the nucleus and cytoplasm of the neoplastic epithelial cells. IgG antibody against the BHRF1 protein was detected in 6 of 17 (35. 3%) NPC plasma, but the protein and IgG were both absent from the non-NPC controls. BHRF1 DNA sequences were determined for 11 NPC and 3 non-NPC samples. No sequence was specific for the EBV isolates from NPC tissue. Amino acids 79 and 88 always appeared in the same form, however, for every tested isolate and both were valine or leucine. This particular characteristic was not present in the B95-8 strain or in the corresponding regions of homologues, Bcl-2 and Bcl-XL, and was regarded as unique to Oriental EBV strains.

A probability prediction rule for malignant cervical lymphadenopathy using sonography.

BACKGROUND: Our purpose was to weigh various sonographic parameters as predicting malignant cervical lymphadenopathy and build a reliable prediction rule. METHODS: One hundred and eighty-nine cervical lymph node lesions from 125 consecutive patients were used for building the prediction model. Sonographic variables, including 15 morphologic features of B-mode, 5 vascular parameters of color Doppler mode, along with age and sex, were analyzed with multivariate logistic regression to evaluate the joint effect of a set of independent variables. A prediction rule for malignant lymphadenopathy was established, and prospective validation was assessed on a new group consisting of 100 lymph nodes from another 60 consecutive patients. RESULTS: The association of heterogeneous content, long transverse diameter, pathologic vascular pattern, high vascular density, and older age provided the most robust prediction value. Scoring scale was designed as 1x (age) + 2x (vascularity index) + 3x (short axis) + 4x (vascular pattern) + 4x (internal echo) according to the parameter estimates of multivariate logistic regression analysis. Cut-off value of score >==10 as malignancy resulted in 89.2% sensitivity and 85.2% specificity. Prospective validation also showed satisfactory results (sensitivity, 82.9%; specificity, 86.2%). CONCLUSIONS: By measuring only 4 sonographic parameters and age, this prediction rule could provide the physician a nonconfusing and reliable probability reference for managing cervical lymphadenopathy.

Usefulness of the C1 transverse process as a reference guide in the dissection of the upper lateral neck.

In this investigation we dissected 3 cadavers with the lateral cervical approach to assess the usefulness of the transverse process of the atlas (TPA) as a reference guide in the upper lateral neck. Our results indicate that all the important structures in this space can be identified systematically. Lateral to the TPA sits the posterior belly of the digastric muscle, the stylohyoid muscle, and the occipital artery. Anterior to the TPA, the styloid process can be exposed. The internal jugular vein and cranial nerves X, XI, and XII sit between the styloid process and the TPA. Superior to the TPA, tracing the carotid sheath upward, the carotid canal and jugular foramen can be reached. Anteroinferior to the jugular foramen, the hypoglossal nerve emerges from the cranial cavity through the hypoglossal canal. Posterior to the TPA, the suboccipital triangle can be recognized. Within the triangle, the vertebral artery and its accompanying venous complex can be identified.