LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of L-plastin.

Mice with disruption of Lrrk1 and patients with nonfunctional mutant Lrrk1 exhibit severe osteopetrosis phenotypes because of osteoclast cytoskeletal dysfunction. To understand how Lrrk1 regulates osteoclast function by modulating cytoskeleton rearrangement, we examined the proteins that are differentially phosphorylated in wild-type mice and Lrrk1-deficient osteoclasts by metal affinity purification coupled liquid chromatography/mass spectrometry (LC/MS) analyses. One of the candidates that we identified by LC/MS is L-plastin, an actin bundling protein. We found that phosphorylation of L-plastin at serine (Ser) residues 5 was present in wild-type osteoclasts but not in Lrrk1-deficient cells. Western blot analyses with antibodies specific for Ser5 phosphorylated L-plastin confirmed the reduced L-plastin Ser5 phosphorylation in Lrrk1 knockout (KO) osteoclasts. micro computed tomography (Micro-CT) analyses revealed that the trabecular bone volume of the distal femur was increased by 27% in the 16 to 21-week-old L-plastin KO females as compared with the wild-type control mice. The ratio of bone volume to tissue volume and connectivity density were increased by 44% and 47% (both P < 0.05), respectively, in L-plastin KO mice. Our data suggest that targeted disruption of L-plastin increases trabecular bone volume, and phosphorylation of Ser5 in L-plastin in the Lrrk1 signaling pathway may in part contribute to actin assembly in mature osteoclasts.

Role of MRI in the early diagnosis of tubal ectopic pregnancy.

OBJECTIVE: To determine the role of MRI in the early diagnosis of tubal ectopic pregnancy (EP). METHODS: Clinical and MRI features of 27 cases of tubal pregnancy were reviewed. RESULTS: A thick-walled gestational sac (GS)-like structure was demonstrated lateral to the uterus in all cases. On T2-weighted images, the thick wall typically exhibited 3 discrete rings in 22 cases (81 %), among which 17 cases (63 %) displayed small vessels and 6 cases (33 %) exhibited small areas of fresh haemorrhage inside the thick wall. The contents demonstrated non-specific liquid in 26 %, papillary solid components in 56 %, and fresh blood or fluid-fluid level in 19 % of the cases. Dilatation of the affected fallopian tube associated with hematosalpinx was demonstrated in 18 cases (67 %) and marked enhancement of the tubal wall was observed in 22 cases (81 %). No correlation was found between the size of the GS and the estimated gestational age (r = 0.056). CONCLUSION: MRI plays an important role in the early diagnosis and management of tubal pregnancy. The characteristic MRI features include a GS-like structure with a "three rings" appearance on T2-weighted images, presence of solid components in the sac, dilatation of the affected fallopian tube with hematosalpinx, and tubal wall enhancement. KEY POINTS: • MR imaging has served as a problem-solving procedure in ectopic pregnancy. • MR imaging features can be criteria for early diagnosis of tubal pregnancy. • Detailed assessment of ectopic implantation is necessary for management decision-making.

In-depth analysis of local recurrence of giant cell tumour of bone with soft tissue extension after intralesional curettage.

PURPOSE: The aim of this study was to assess the local recurrence rate of giant cell tumour of bone (GCTB) with soft tissue extension, to identify characteristics of the soft tissue extension that can best indicate recurrence of GCTB after intralesional curettage. MATERIALS AND METHODS: A total of 48 cases of GCTB with soft tissue extension after intralesional curettage were recruited. Patients were divided into two groups based on various objective features of soft tissue extension including size, number, margins, involvement of adjacent tissues, signal intensity, static enhancement and Jaffe grade. The local recurrence rate was compared using the Chi-square test and Chi-square value correction for continuity. Risk factors were assessed by multivariate logistic regression analysis. RESULTS: The local recurrence rate was significantly different according to soft tissue extension size, number and margins (p < 0.05). There was no significant difference in the groups of adjacent tissue involvement and Jaffe grade (p > 0.05). Size, number and margins of the soft tissue extension were independent risk factors of local recurrence of GCTB after intralesional curettage (p < 0.05). CONCLUSIONS: The local recurrence rate of GCTB with soft tissue extension after intralesional curettage is higher if the soft tissue extension is large, multiple and lacking bone envelope integrity. For cases with the above-mentioned features, we suggest that the higher recurrence rate can be taken into full consideration when choosing appropriate surgical procedures.

Giant cell tumours of the mobile spine: characteristic imaging features and differential diagnosis.

PURPOSE: The aim of this study was to investigate the characteristic imaging features of giant cell tumours (GCTs) of the mobile spine. MATERIALS AND METHODS: Thirty pathologically proven GCTs of the mobile spine were reviewed. X-ray (n = 18), computed tomography (CT) (n = 24) and magnetic resonance (MR) (n = 21) images were retrospectively evaluated. RESULTS: Five tumours were located in the cervical spine, 15 tumours were located in the thoracic spine and 10 tumours in the lumbar spine. The characteristic X-ray findings included an osteolytic and expansile lesion with a "soap bubble" or purely lytic appearance. Cortical destruction was commonly seen. Margin sclerosis was seen in two lesions. No mineralised tumour matrix or periosteal reaction appeared. The CT findings were similar but outlined the cortical alterations in a more accurate way. The characteristic MR findings included a well-defined and expansile mass with heterogeneous low-to-iso signal intensity on T2-weighted images. Cystic areas were commonly seen in 17 cases. Five cases presented fluid-fluid levels, suggesting the development of aneurysmal bone cyst. The solid portions of the tumours were enhanced with a very heterogeneous signal pattern reflecting high blood supply after contrast-enhanced scan. Tumour involvement in the epidural space occurred in 12 cases, causing spinal cord and/or nerve root compression. Involvement of intervertebral discs and/or adjacent vertebrae appeared in two cases. CONCLUSIONS: Although rare, GCT can occur in the mobile spine as a kind of benign but locally aggressive tumour. Radiologists should be familiar with its characteristic imaging features in order to make a correct diagnosis and to help preoperative evaluations.

Treatment of congenital middle ear cholesteatoma in children using endoscopic and microscopic ear surgeries: a case series.

Introduction: Surgical removal is widely employed in children with congenital middle ear cholesteatoma (CMEC). Here, we report the surgical outcomes of CMEC removal via endoscopic ear surgery (EES) and microscopic ear surgery (MES) in children. Methods: Children with CMEC who underwent preoperative medical history inquiry, hearing test, endoscopic evaluation, and radiology imaging before receiving EES or MES were included. Postoperative audiological outcomes and recurrence rates were collected. Results: Seventeen children (20 ears) with stage II-IV CMEC were included. Of those, 11 ears (55.0%) underwent EES, and 9 ears (45.0%) underwent MES. The follow-up time was 35 ± 13.5 months. One child in the EES group with stage III CMEC had a recurrence during the follow-up period. In the EES group, the average minimum diameter of the external auditory canal on the affected side was 5.8 mm (4.3-8.0 mm). No linear association was found between age and the minimum diameter of the external auditory canal. Discussion: EES is a promising treatment option for children with early-stage CMEC because of its low recurrence rate and minimally invasive nature. The minimum diameter of the external auditory canal on the affected side should be meticulously examined when performing EES in children.

MicroRNA-126 inhibits osteosarcoma cells proliferation by targeting Sirt1.

Numerous studies have recently suggested that miRNAs contribute to the development of various types of human cancer as well as to their proliferation and metastasis. The aim of this study was to investigate the functional significance of miR-126 and to identify its possible target genes in osteosarcoma (OS) cells. Here, we found that expression level of miR-126 was reduced in osteosarcoma cells in comparison with the adjacent normal tissues. The enforced expression of miR-126 was able to inhibit cell proliferation in U2OS and MG63 cells, while miR-126 antisense oligonucleotides (antisense miR-126) promoted cell proliferation. At the molecular level, our results further revealed that expression of Sirt1, a member of histone deacetylase, was negatively regulated by miR-126. Therefore, the data reported here demonstrate that miR-126 is an important regulator in osteosarcoma, which will contribute to better understanding of the important misregulated miRNAs in osteosarcoma cells.

Leucine Repeat Rich Kinase 1 Controls Osteoclast Activity by Managing Lysosomal Trafficking and Secretion.

We previously demonstrated that mice with targeted deletion of the leucine repeat rich kinase 1 (Lrrk1) gene were osteopetrotic due to the failure of osteoclasts to resorb bone. To determine how LRRK1 regulates osteoclast activity, we examined the intracellular and extracellular acidification with an acidotropic probe, acridine orange, in live osteoclasts on bone slices. We examined lysosome distribution in osteoclasts by localization of LAMP-2, cathepsin K, and v-ATPase by immunofluorescent staining with specific antibodies. We found that both vertical and horizontal cross-sectional images of the wild-type (WT) osteoclasts showed orange-staining of the intracellular acidic vacuoles/lysosomes dispersed to the ruffled border. By contrast, the LRRK1 deficient osteoclasts exhibited fluorescent orange staining in the cytoplasm away from the extracellular lacunae because of an altered distribution of the acidic vacuoles/lysosomes. In addition, WT osteoclasts displayed a peripheral distribution of LAMP-2 positive lysosomes with a typical actin ring. The clustered F-actin constitutes a peripheral sealing zone and a ruffled border which was stretched out into a resorption pit. The LAMP-2 positive lysosomes were also distributed to the sealing zone, and the cell was associated with a resorption pit. By contrast, LRRK1-deficient osteoclasts showed diffused F-actin throughout the cytoplasm. The sealing zone was weak and not associated with a resorption pit. LAMP-2 positive lysosomes were also diffuse in the cytoplasm and were not distributed to the ruffled border. Although the LRRK1-deficient osteoclast expressed normal levels of cathepsin K and v-ATPase, the lysosomal-associated cathepsin K and v-ATPase were not accumulated at the ruffled border in Lrrk1 KO osteoclasts. Our data indicate that LRRK1 controls osteoclast activity by regulating lysosomal distribution, acid secretion, and protease exocytosis.

A Prognostic Model Generated from an Apparent Diffusion Coefficient Ratio Reliably Predicts the Outcomes of Oral Tongue Squamous Cell Carcinoma.

This study aimed to develop an apparent diffusion coefficient (ADC) ratio-based prognostic model to predict the recurrence and disease-free survival (DFS) of oral tongue squamous cell carcinoma (OTSCC). A total of 188 patients with cT1-2 oral tongue squamous cell carcinoma were enrolled retrospectively. Clinical and laboratory data were extracted from medical records. The ADC values were measured at the regions of interest of the tumor and non-tumor tissues of the MRI images, and the ADC ratio was used for comparison between the patient with recurrence (n = 83 case, 44%) and patients without recurrence (n = 105 cases, 56%). Cox proportional hazards models were generated to analyze the risk factors of cancer recurrence. A nomogram was developed based on significant risk factors to predict 1-, 5- and 10-year DFS. The receiver operator characteristic (ROC) curves of predictors in the multivariable Cox proportional hazards prognostic model were generated to predict the recurrence and DFS. The integrated areas under the ROC curve were calculated to evaluate discrimination of the models. The ADC ratio, tumor thickness and lymph node ratio were reliable predictors in the final prognostic model. The final model had a 71.1% sensitivity and an 81.0% specificity. ADC ratio was the strongest predictor of cancer recurrence in prognostic performance. Discrimination and calibration statistics were satisfactory with C-index above 0.7 for both model development and internal validation. The calibration curve showed that the 5- and 10-year DFS predicted by the nomogram agreed with actual observations.

Application of Multi-Slice Computed Tomography for the Preoperative Diagnosis and Classification of Pulmonary Cystic Echinococcosis.

Pulmonary cystic echinococcosis remains a serious threat to public health. A standardized, imaging-based classification method for pulmonary echinococcosis has not yet been developed despite the existence of a standardized ultrasound classification method and treatment plan for hepatic cystic echinococcosis. Chest computed tomography (CT) images from 34 cases of pulmonary cystic echinococcosis with 46 lesions were used for classification based on the World Health Organization (WHO) standardized ultrasound classification of hepatic cystic echinococcosis. CT findings were compared with intraoperative observations and postoperative pathological results to assess accuracy. Pulmonary cystic echinococcosis was common in women (14/34, 41.2%) and children (14/34, 41.2%) with a single cyst (28/46, 60.9%). Most lesions were classified as cystic echinococcosis 1(CE1, 19/46) or cystic echinococcosis 3(CE3, 21/46). Blood leukocytosis was mostly observed in CE3 lesions (100%, 9/9) (p < 0.05). The preoperative CT diagnosis of pulmonary cystic echinococcosis had an accuracy rate of 100%. The preoperative CT typing, and postoperative pathological typing had a coincidence rate of 97.8% (45/46). Our study provided a classification method based on CT imaging for pulmonary cystic echinococcosis that can be used during pre-surgical planning to reduce patient's postoperative complications and mortality.

A small molecular inhibitor of LRRK1 identified by homology modeling and virtual screening suppresses osteoclast function, but not osteoclast differentiation, in vitro.

We used TGFß activation kinase 1 as a template to build a 3D structure of the human LRRK1 kinase domain (hLRRK1 KD) and performed small molecule docking. One of the chemicals (IN04) that docked into the pocket was chosen for evaluation of biological effects on osteoclasts (OCs) in vitro. INO4 at 16 nM completely blocked ATP binding to hLRRK1 KD in an in vitro pulldown assay. In differentiation and pit assays, while the number of OCs on bone slices were comparable for OCs treated with IN04 and DMSO, IN04 treatment of OCs significantly impaired their ability to resorb bone. The area of pits on bone slices was reduced by 43% at 5 µM and 83% at 10 µM as compared to DMSO. Individual pits appeared smaller and shallower. F-actin staining revealed that DMSO-treated OCs displayed clear actin rings, and F-actin forms a peripheral sealing zone. By contrast, IN04-treated OCs showed disarranged F-actin in the cytoplasm, and F-actin failed to form a sealing zone on bone slices. IN04 treatment had no effects on OC-derived coupling factor production nor on osteoblast nodule formation. Our data indicate IN04 is a potent inhibitor of LRRK1, suppressing OC function with no effect on OC formation.

Thin-section computed tomography-histopathologic comparisons of pulmonary focal interstitial fibrosis, atypical adenomatous hyperplasia, adenocarcinoma in situ, and minimally invasive adenocarcinoma with pure ground-glass opacity.

OBJECTIVE: To retrospectively compare focal interstitial fibrosis (FIF), atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), and minimally invasive adenocarcinoma (MIA) with pure ground-glass opacity (GGO) using thin-section computed tomography (CT). MATERIALS AND METHODS: Sixty pathologically confirmed cases were reviewed including 7 cases of FIF, 17 of AAH, 23of AIS, and 13 of MIA. All nodules kept pure ground glass appearances before surgical resection and their last time of thin-section CT imaging data before operation were collected. Differences of patient demographics and CT features were compared among these four types of lesions. RESULTS: FIF occurred more frequently in males and smokers while the others occurred more frequently in female nonsmokers. Nodule size was significant larger in MIA (P<0.001, cut-off value=7.5mm). Nodule shape (P=0.045), margin characteristics (P<0.001), the presence of pleural indentation (P=0.032), and vascular ingress (P<0.001) were significant factors that differentiated the 4 groups. A concave margin was only demonstrated in a high proportion of FIF at 85.7% (P=0.002). There were no significant differences (all P>0.05) in age, malignant history, attenuation value, location, and presence of bubble-like lucency. CONCLUSION: A nodule size >7.5mm increases the possibility of MIA. A concave margin could be useful for differentiation of FIF from the other malignant or pre-malignant GGO nodules. The presence of spiculation or pleural indentation may preclude the diagnosis of AAH.

Triple-phase dynamic MRI: a new clue to predict malignant transformation of giant cell tumor of bone.

OBJECTIVE: Our purpose was, through the comparison of the characteristics of time-intensity curve on triple-phase dynamic contrast-enhanced MRI among groups of giant cell tumor of bone (GCTB), recurrent benign giant cell tumor of bone (RBGCTB), and secondary malignant giant cell tumor of bone (SMGCTB), to find clues to predict the malignant transformation of GCTB. SUBJECTS AND METHODS: 21 patients diagnosed as GCTB were included in this study. All cases took recurrence after intralesional curettage. 9 cases were confirmed as SMGCTB and 12 cases were confirmed as RBGCTB. Cases were divided into four groups: group A, GCTB (n=9); group B, SMGCTB (n=9); group C, GCTB (n=12); group D, RBGCTB (n=12). Enhancement index(EI) of lesions on DCEMRI was calculated using formula: EI(t)=[S(t)-S(0)]/S(0), where S(0) was signal intensity of lesion on non-contrast-enhanced T1-weighted images and S(t) was signal intensity of lesion on DCEMRI (t=30, 60, 180s). Enhancement index of each group in each phase was compared using One-Way ANOVA analysis. Slope values of time-intensity curve were compared by the same way. RESULTS: Time-intensity curve of SMGCTB was characterized by a steep upward slope followed by an early and rapid washout phase. Time-intensity curve of GCTB and RBGCTB was characterized by a steep slope followed by a relatively slow washout phase. No significant difference in enhancement index was found in the first phase (p>0.05). There was significant difference in the second and the third phase (p<0.05). Enhancement index of group B (SMGCTB) was smaller. There was no difference in rising slope value (p>0.05). CONCLUSIONS: Dynamic contrast-enhanced MRI appears a helpful method to find new clues to predict malignant transformation of GCTB.

Differentiation of primary chordoma, giant cell tumor and schwannoma of the sacrum by CT and MRI.

OBJECTIVE: To evaluate criteria to differentiate sacral chordoma (SC), sacral giant cell tumor (SGCT) and giant sacral schwannoma (GSS) with CT and MRI. MATERIALS AND METHODS: CT and MR images of 22 SCs, 19 SGCTs and 8 GSSs were reviewed. The clinical and imaging features of each tumor were analyzed. RESULTS: The mean ages of SC, SGCT and GSS were 55.1 ± 10.7, 34.3 ± 10.7 and 42.4 ± 15.7 years old. SCs (77.3%) were predominantly located in the midline of lower sacrum, while most SGCTs (73.7%) and GSSs (87.5%) were eccentrically located in upper sacrum. There were significant differences in age, location, eccentricity, morphology of bone residues, intratumoral bleeding and septations. Multiple small cysts were mainly observed in SGCTs (73.7%) with large central cysts in GSSs (87.5%). SGCTs expanded mainly inside sacrum while SCs and GSSs often extended into pelvic cavity (P = 0.0022). Involvement of sacroiliac joints and muscles were also different. Ascending extension within sacral canal was only displayed in SCs. The preservation of intervertebral discs showed difference between large and small tumors (P = 0.0002), regardless of tumor type (P = 0.095). No significant difference was displayed in gender (P = 0.234) or tumor size (P = 0.0832) among three groups. CONCLUSION: Age, epicenter of the lesion (midline vs. eccentric and upper vs. lower sacral vertebra), bone residues, cysts, bleeding, septation, expanding pattern, muscles and sacroiliac joint involvement can be criteria for diagnosis. Fluid-fluid level is specific for SGCTs and ascending extension within the sacral canal for SCs. The preservation of intervertebral discs is related to tumor size rather than tumor type.

Noninvasive assessment of response to neoadjuvant chemotherapy in osteosarcoma of long bones with diffusion-weighted imaging: an initial in vivo study.

OBJECTIVES: The purpose of our study is to investigate whether diffusion-weighted imaging (DWI) is useful for monitoring the therapeutic response after neoadjuvant chemotherapy in osteosarcoma of long bones. MATERIALS AND METHODS: Conventional magnetic resonance imaging (MRI) and DWI were obtained from 35 patients with histologically proven osteosarcomas. MR examinations were performed in all patients before and after 4 courses of preoperative neoadjuvant chemotherapy. Apparent diffusion coefficients (ADC) were measured. The degree of tumor necrosis was assessed macroscopically and histologically by two experienced pathologists after operation. Student's t test was performed for testing changes in ADC value. Pearson's correlation coefficient was used to estimate the correlation between necrosis rate and post- neoadjuvant chemotherapy ADC values. P<0.05 was considered to denote a significant difference. RESULTS: The difference of the whole osteosarcoma between pre- neoadjuvant chemotherapy ADC value (1.24±0.17×10(-3) mm(2)/s) and post- (1.93±0.39×10(-3) mm(2)/s) was significant difference (P<0.01). Regarding in patients with good response, the post- neoadjuvant chemotherapy values were significantly higher than the pre- neoadjuvant chemotherapy values (P<0.01). The post- neoadjuvant chemotherapy ADC value in patients with good response was higher than that of poor response (t = 8.995, P<0.01). The differences in post- neoadjuvant chemotherapy ADC between viable (1.03±0.17×10(-3) mm(2)/s) and necrotic (2.38±0.25×10(-3) mm(2)/s) tumor was highly significant (t = 23.905, P<0.01). A positive correlation between necrosis rates and the whole tumor ADC values (r = 0.769, P<0.01) was noted, but necrosis rates were not correlated with the ADC values of necrotic (r = -0.191, P = 0.272) and viable tumor areas (r = 0.292, P = 0.089). CONCLUSIONS: DWI can identify residual viable tumor tissues and tumor necrosis induced by neoadjuvant chemotherapy in osteosarcoma. The ADC value can directly reflect the degree of tumor necrosis, and it is useful to evaluate the preoperative neoadjuvant chemotherapy response in patients with osteosarcoma.