BRAFV600E Mutation-Responsive miRNA-222-3p Promotes Metastasis of Papillary Thyroid Cancer Cells via Snail-Induced EMT.

BRAF mutation accounts for 50% of the PTC (papillary thyroid carcinoma) and is closely associated with high-risk clinicopathological characteristics. Increasing evidence implied that dysregulation of miRNA participated in carcinogenesis and progression of cancer. Clinical data showed the significant up-regulation of miR-222-3p in PTC; however, the role of miR-222-3p and possible relationship with BRAF mutation remained unclear. Here, we identified significant up-regulation of miR-222-3p in PTC tissues harboring BRAFV600E mutation compared with BRAF wild type (BRAFWT ) from collected PTC clinical samples. External validation performed with The Cancer Genome Atlas (TCGA) databases was consistent with the above result. Exogenous expression of BRAFV600E oncoprotein increased the expression of miR-222-3p in B-CPAP and TPC-1 cells. The treatment of BRAFV600E and MEK inhibitor, PLX4720 and PD0325901, decreased the expression of miR-222-3p in B-CPAP but not in TPC-1. Inhibition of miR-222-3p significantly suppressed the migration of B-CPAP and induced a mesenchymal-epithelial transition (MET) phenotype via the Snail transcription factor. Immunohistochemistry (IHC) analysis demonstrated the up-regulation of Snail correlated with lymph node metastasis and BRAFV600E mutation in PTC. Besides, in situ hybridization (ISH) and IHC analysis of PTC clinical samples confirmed the correlation between the expression of miR-222-3p and Snail. These results showed miR-222-3p conduced more aggressive clinical manifestation of PTC by promoting Snail-induced EMT.

Spontaneous Thyroid Hemorrhage Caused by Langerhans Cell Histiocytosis: A Case Report and Literature Review.

Purpose: Langerhans cell histiocytosis (LCH) is a rare clonal disorder of Langerhans antigen-presenting cells. However, thyroid LCH involvement is relatively rare. We present the first case of spontaneous thyroid hemorrhage due to LCH progression and discuss the clinical features, diagnosis, and treatments of thyroid LCH in a literature review. Methods: Clinical data were collected. Previously published articles on thyroid LCH involvement were reviewed to assess the clinical features, diagnosis, and treatments for thyroid LCH. Results: A 54-year-old female presented with a multi-system LCH, affecting the uterus, liver, pituitary gland, and thyroid gland. Clinical stability was achieved after systemic chemotherapy. After 7 years of regular follow up, the patient complained of a sudden painful neck swelling and progressive dyspnea. Computed Tomography revealed bilateral goiter with hematoma, and the patient was diagnosed with spontaneous thyroid bleeding based on her clinical symptoms and radiological findings. The patient was incubated to relieve airway compromise and partial thyroidectomy was performed for definitive treatment. Pathological evaluation further confirmed the diagnosis of thyroid LCH. The patient recovered well after surgery. Conclusion: Spontaneous thyroid bleeding due to thyroid LCH progression is extremely rare. Treatments for LCH vary depending on the severity of the disease. We suggest that, for patients with multi-system LCH with thyroid lesion, long-term active surveillance of thyroid hormone concentrations, and thyroid gland volume is required. Physicians should be alert of the potentially life-threatening spontaneous thyroid hemorrhage when aggravated diffuse goiter and hypothyroidism appear. Further investigation is required to establish the guidelines for thyroid LCH treatment.

Unilateral Multifocality and Bilaterality Could Be Two Different Multifocal Entities in Patients with Papillary Thyroid Microcarcinoma.

OBJECTIVE: Multifocality within an affected lobe (unilateral multifocality) or two lobes (bilaterality) is commonly denoted as multifocality without differentiation. Recently, there has been molecular evidence indicating that unilateral multifocality and bilaterality could be two different entities. However, few studies concerning the comparison between these two different multifocality entities have been reported. DESIGN: A retrospective cohort study. METHODS: From 2010 to 2013, in total, 949 consecutive patients with papillary thyroid microcarcinoma (PTMC) were enrolled and further divided into four groups based on multifocality status. Unilateral multifocality and bilaterality were analyzed by binary logistic regression along with other clinicopathological factors. RESULTS: Unilateral multifocality, instead of bilaterality, was correlated with central neck metastasis (CNM) in both univariate and multivariate analyses. Group IV (unilateral multifocality and bilaterality coexist) had the highest CNM rate. Group III (unilateral multifocality) had a higher CNM rate than group II (bilaterality, single lesion in each lobe), with a significant difference (p = 0.032). Similar lateral neck metastasis tendency was observed among the four groups. In the multivariate analysis, only unilateral multifocality and bilaterality which coexisted were correlated with CNM. Moreover, 9 cases had a recurrence, with the recurrence rate ranking top in group IV (3.6%), second in group III (2.8%), and third in group II (1.2%). The difference was significant (p = 0.021). CONCLUSION: Unilateral multifocality and bilaterality could be two different multifocal entities in patients with PTMC. Unilateral multifocality serving as a prognostic factor indicated a worse prognosis than bilaterality on neck metastasis. When the two factors coexisted in PTMC, patients had the highest risk of CNM and possibly local recurrence compared with those with either risk factor alone.

Finger-pressing: a simple and efficient way to stop chyle leak post neck dissection.

PURPOSE: Postoperative chyle leak (CL) is a rare but severe complication after neck dissection. Conservative first-line treatments, such as total parenteral nutrition (TPN), octreotide and pressure dressing, can significantly prolong the hospital stay. Efficient and well-tolerated conservative treatment options are still lacking. METHODS: We have developed a new maneuver to treat CL called "finger-pressing." A thumb is pressed against the point of leakage between the two muscle heads of the sternocleidomastoid. Finger-pressing continues for about 24 h until CL stops. We used this maneuver to treat six CL patients. Drainage output and duration of hospital stay were compared with another six CL patients treated using the traditional pressure dressing. RESULTS: The finger-pressing maneuver cured all six CL patients. Most CLs (5/6) could be controlled after about 24 h pressing. Only one high-output CL required 72 h of finger-pressing to stop the leak. All patients had their drainage tubes removed within 3 days after pressing started and all patients tolerated the treatment well. In the control group, the traditional pressure dressing time varied from 2 to 5 days, until the patient was no longer able to cooperate. Three patients underwent a second operation that cured the CL in two cases. The average hospital stay was significantly longer than that in the finger-pressing group (20 vs 9 days, p = 0.037). CONCLUSIONS: Finger-pressing is an efficient and well-tolerated method for treating CL post neck dissection. This maneuver could be a useful adjuvant therapy to traditional methods in treating CL.

miR-222 expression is correlated with the ATA risk stratifications in papillary thyroid carcinomas.

BACKGROUND: miR-222 is one of the most consistently overexpressed miRNAs in papillary thyroid carcinoma (PTC). Previous studies demonstrated that miR-222 overexpression conferred high-risk features in PTC patients, suggesting its value in risk-stratification. However, studies in term of miR-222's utility on stratifying PTCs are lacking. METHODS: One hundred patients including 10 with multinodular goiter and 90 with PTC were enrolled. Formalin-fixed paraffin-embedded samples were exploited for miR-222 quantitative reverse transcriptase- polymerase chain reaction (RT-PCR) analysis. Correlations between miR-222 expression and different clinicopathological features, Tumor-node-metastasis (TNM) staging and ATA risk level were analyzed. RESULTS: miR-222 expression of the PTC group was significantly higher than that of the goiter group (P < .001). Furthermore, miR-222 expression was significantly higher in PTCs with advanced features like larger tumor, capsular invasion, vascular invasion and lymph nodes metastasis. The majority of patients (61%) were in stage I group (similar to ATA low-risk) by TNM staging system. As to the ATA system, the majority (73%) were in intermediate-risk group (similar to TNM stage II and III roughly). Contrary to previous report, here we found that miR-222 expression was correlated with the ATA risk level (P < .001), but not with the TNM staging (P = .122). CONCLUSION: In the present study, we demonstrated that miR-222 overexpression was correlated with advanced features like capsular invasion, vascular invasion, larger tumor size and lymph node metastasis in PTCs. Most importantly, miR-222 expression was correlated with ATA risk levels, suggesting its potential value in PTC risk-stratification.

Appendiceal mucinous cystadenoma presenting as abdominal internal hernia: a case report.

Appendiceal mucinous neoplasm is a rare form of appendix disorder of all appendectomies. Patients may present with many non-special symptoms which the right low quadrant pain is the mostly reported. We reported a case of 70-year-old man who was admitted to emergency for abdominal pain and progressive aggravation of peritoneal irritation sign. Computed tomography (CT) revealed incomplete intestinal obstruction with volvulus of partial small intestine in right lower quadrant, and internal hernia was suspected and simple appendectomy was performed followed. The postoperative pathologic diagnosis was mucinous cystadenoma with negative cutting margin. During three-year follow-up, the patient was in good condition and there was no sign of recurrence.

Endoscopic thyroidectomy along with bilateral central neck dissection (ETBC) increases the risk of transient hypoparathyroidism for patients with thyroid carcinoma.

Increasing number of patients with thyroid carcinoma, especially young female patients, prefer to choose endoscopic thyroidectomy with bilateral central neck dissection (ETBC) for perfect cosmetic effects. However, the incidence of hypoparathyroidism after ETBC has not been well studied. Ninety six patients with papillary thyroid carcinoma were enrolled. All patients, including 49 ETBC and 47 open surgery patients, underwent total thyroidectomy with bilateral central neck dissection (CND). Some patients also underwent lateral neck dissection simultaneously. The incidence of hypoparathyroidism and parathyroid hormone (PTH) level were examined. Patients in the open surgery group had more advanced lesions, with larger tumor (p = 0.000), older age (p = 0.000), and more serious local involvement. The dissection extent of the open group was significantly larger than that of the ETBC group (p = 0.006). In contrast, the ETBC group with less dissection extent showed a significantly higher incidence of transient hypoparathyroidism than the open group (59.2 vs. 29.6 %, p = 0.004). The average PTH decline of the ETBC group was significantly higher than that of the open group on postoperative day 1 (POD1) (32.1 vs. 21.6 pg/ml, p = 0.010). Furthermore, the ETBC group had a significantly higher portion of patients with a PTH <10 pg/ml on POD1 (p = 0.001). One patient in the ETBC group developed permanent hypoparathyroidism. Autotransplantation and inadvertent removal rates of parathyroid did not differ between the two groups. Although generally considered a safe method for patients with thyroid carcinoma, ETBC may increase the risk of transient hypoparathyroidism compared with conventional open surgery.

[Low temperature freezing followed by dispersive solid phase extraction for the determination of 104 pesticide residues in vegetable oils using ultra-performance liquid chromatography-tandem mass spectrometry].

A method using liquid-liquid extraction (LLE) followed by clean-up steps of centrifugation, freezing and dispersive solid phase extraction (D-SPE) and ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) has been devel- oped for the determination of trace levels of 104 pesticides in vegetable oils. LLE has been optimized to extract these pesticide residues from oils to obtain the highest recoveries of pesticides and the lowest co-extract fat residue in the final extract. In addition, the centrifugation and freezing steps as well as D-SPE with primary secondary amine (PSA), graphite carbon black (GCB) and C18 were used as the clean-up steps to minimize the co-extract fat. The recoveries obtained ranged from 55% to 121% and the relative standard deviations (RSDs) ranged from 0. 47% to 19. 2% at the spiked levels of 0.01, 0.02 and 0.05 mg/kg. This method, combining with accurate and sensitive detection, allowed quantification and confirmation at levels as low as 1 µg/kg for 80% of the analytes. The limits of quantification (LOQs) of the most compounds were below the maximum residue limits (MRLs) established by the Chinese legislations for oils. The proposed method was applied successfully for the residue determination of the selected pesticides in oils.

Visual detection of formaldehyde by highly selective fluorophore labeling via gold(III) complex-mediated three-component coupling reaction.

A novel method for visual detection of formaldehyde with excellent selectivity via a gold(iii) complex-mediated three-component coupling reaction of resin-linked sterically bulky amines and fluorescent alkynes has been developed.

[Determination of docosahexaenoic acid in milk powder by gas chromatography using acid hydrolysis].

A method to determine docosahexenoic acid (DHA) in milk powder by gas chromatography was established. The milk powder samples were hydrolyzed with hydrochloric acid, extracted to get total fatty acids by Soxhlet extractor, then esterified with potassium hydroxide methanol solution to form methyl esters, and treated with sodium hydrogen sulfate. The optimal experiment conditions were obtained from orthogonal experiment L9(3(3)) which performed with three factors and three levels, and it requires the reaction performed with 1 mol/L potassium hydroxide solution at 25 degrees C for 5 min. The derivative treated with sodium hydrogen sulfate was separated on a column of SP-2560 (100 m x 0.25 mm x 0.20 µm), and determined in 55 min by temperature programming-gas chromatography. Good linearity was obtained in the range 5.0-300 mg/L with the correlation coefficient of 0.999 9. The relative standard deviations (RSDs) were 3.4%, 1.2% and 1.1% for the seven repeated experiments of 10, 50 and 100 mg/L of DHA, respectively. The limit of detection was 2 mg/kg, and the recoveries of DHA were in the range of 90.4%-93.5%. The results are satisfactory through the tests of practical samples.

Papillary thyroid microcarcinomas located at the middle part of the middle third of the thyroid gland correlates with the presence of neck metastasis.

BACKGROUND: Papillary thyroid microcarcinomas (PTMCs), located at upper poles of the thyroid, are associated with lateral neck metastasis (LNM) according to previous reports. Controversy remains regarding the correlation between the location of PTMCs and central neck metastasis (CNM). METHODS: Medical records of 949 patients with PTMCs diagnosed between 2010 and 2013 were reviewed retrospectively. With a subdivision of the middle third of the thyroid gland, correlations between tumor location and CNM/LNM along with other clinicopathologic factors were analyzed by binary logistic regression. RESULTS: PTMCs located in the middle part of the middle third of the thyroid gland (MPMT) showed the greatest rate of CNM (57.5%) among all locations. PTMCs located at isthmus showed the second greatest rate of CNM (44.3%). In the multivariate analysis, MPMT, tumor size >0.5 cm, young and middle age, male sex, multifocality within the affected lobe, and capsular invasion were correlated with CNM. PTMCs located at upper poles and MPMT showed comparatively high rates of LNM (8.6% and 8.3%). Consistent with previous reports, an upper pole location, MPMT, and a tumor size >0.5 cm greatly correlated with LNM in the multivariate analysis. Eleven patients had skip metastases, which only occurred with upper/lower pole locations and MPMT. CONCLUSION: PTMCs located in the MPMT correlated with both CNM and LNM. Tumor location along with other clinicopathologic factors such as young and middle age, male sex, and tumor size >0.5 cm could facilitate preoperative stratification and guide operative management for patients with PTMC.

Contrast-enhanced ultrasound (CEUS) facilitated US in detecting lateral neck lymph node metastasis of thyroid cancer patients: diagnosis value and enhancement patterns of malignant lymph nodes.

OBJECTIVES: To examine the value of CEUS as a non-invasive tool in detecting lateral neck metastasis (LNM) and the enhancement patterns of malignant lymph nodes (LN) for thyroid cancer patients. METHODS: Eighty-two consecutive patients, who underwent both preoperative non-enhanced US and CEUS examinations, were retrospectively reviewed. All patients underwent lateral neck dissection (LND). Enhancement patterns of 102 collected LNs matching to CEUS findings were analyzed. RESULTS: CEUS detected LNM in 53 of 65 patients, showing a higher sensitivity and accuracy than that of conventional US (p = 0.109 and p = 0.154, respectively). Thirteen patients' surgical procedures were altered by CEUS findings, including nine true positive and four false positive cases. Five patients' surgical procedures were altered by conventional US findings, including two true positive and three false positive cases. Heterogeneous enhancement, perfusion defects, microcalcification, and centripetal/hybrid enhancement were all specific criteria for malignant LNs in univariate analysis. In multivariate analysis, only heterogeneous enhancement and centripetal/hybrid enhancement were significantly related to LN metastasis (p = 0.000 and p = 0.037, respectively). CONCLUSIONS: CEUS may be a potential tool to facilitate conventional US in detecting LNM. Heterogeneous enhancement and centripetal/hybrid enhancement are useful criteria to distinguish between malignant and benign LNs. KEY POINTS: • CEUS findings facilitated conventional US in detecting LNM. • Heterogeneous, centripetal/hybrid enhancement, microcalcification and perfusion defects were specific criteria of malignant LNs. • Heterogeneous and centripetal/hybrid enhancement were significantly related to LN metastasis in multivariate analysis.

Influence of up-regulation of Notch ligand DLL4 on biological behaviors of human gastric cancer cells.

AIM: To investigate the potential roles of Delta-like ligand 4 (DLL4) on the biological behavior of gastric cancer cells and its molecular mechanisms. METHODS: A recombinant eukaryotic expression vector containing human DLL4 gene was constructed and transfected into the human gastric cancer cell line SGC7901. Clones with up-regulated DLL4 were selected and amplified. The effect of DLL4 up-regulation on gastric cancer cell growth was assessed using cell growth assay. The migration and invasion were assessed using a transwell migration assay and matrigel invasion assay. Matrix metalloproteinases were detected using the zymogram technique. Cells were implanted subcutaneously into male BALB/c nu/nu mice. Tumor volumes were then calculated and compared. DLL4 staining in the implanted tumor was performed using immunohistochemistry technique. RESULTS: Growth curves over a six-day time course showed significantly promoted cell proliferation of SGC7901 cells with up-regulated DLL4. DLL4 up-regulation in SGC7901 cells promoted the migration (205.4 ± 15.2 vs 22.3 ± 12.1, P < 0.05) and invasion (68.8 ± 5.3 vs 18.2 ± 6.0, P < 0.05) in vitro and tumorigenicity in vivo (2640.5 ± 923.6 mm(3) vs 1115.1 ± 223.8 mm(3), P < 0.05). Furthermore, significantly increased mRNA level and increased secretion of matrix metalloproteinase-2 (MMP-2) proenzyme were observed in SGC7901 cells with up-regulated DLL4. However, increased MMP-9 mRNA level but decreased extracellular MMP-9 proenzyme level was observed. CONCLUSION: Our observations indicated a mechanism by which activation of DLL4-mediated Notch signaling promotes the expression and secretion of MMP-2 proenzyme and influences the progress of gastric cancer.

Overexpression of human telomerase reverse transcriptase promotes the motility and invasiveness of HepG2 cells in vitro.

Recent studies have indicated that telomerase activity promotes cancer invasion and metastasis, but the underlying mechanism remains unclear. Several studies have shown that expression of exogenous human telomerase reverse transcriptase (hTERT) can promote motility and invasiveness among telomerase-negative tumor cells, and inhibition of endogenous telomerase activity can reduce invasiveness in tumor cells. However, whether overexpression of hTERT can further enhance the motility and invasiveness of telomerase­positive tumor cells has yet to be determined. In the present study, we showed that stable overexpression of hTERT can increase telomerase activity and telomere length, which significantly promotes the invasive and metastatic potential of telomerase­positive HepG2 cells but does not affect cell proliferation. Further analysis suggested that enhanced invasiveness and metastasis may act through corresponding upregulation of mRNA and protein expression of matrix metalloproteinase 9 (MMP9) and Ras homolog gene family member C (RhoC). Our study indicated that exogenous expression of hTERT may promote invasiveness and metastasis through upregulation of MMP9 and RhoC.

[Detection of foodborne Salmonella typhimurium and Salmonella enteritidis by diplex real-time PCR using TaqMan probe].

OBJECTIVE: To establish a method to detect Salmonella typhimurium (ST) and Salmonella enteritidis (SE) simultaneously with a dual real-time PCR assay using double-color fluorescent TaqMan probes. METHODS: The primers and probes were designed based on the conservative domain of STM4599 sequence of ST (GenBank: AERV01000023.1) and the specific sequence of SE ( GenBank: AF370707.1) respectively. The probes were labeled with reporter dye FAM for ST or VIC for SE at the 5' end. The dual real-time fluorescence PCR assay was set up and conditions were modified. RESULTS: The dual real-time fluorescence PCR method for ST and SE was developed successfully. ST and SE specific primers and probes amplified 16 SE and 15 ST strains, while other 28 different Sa serotypes and 17 negative control Proteus strains showed negative results. The amplification efficiency of ST and SE with the dual fluorescent PCR were all 94. 2% and R2 were 0. 998 and 0. 995 respectively, while the minimum detectable concentration reached 300 CFU/ml for ST and 260 CFU/ml for SE. The entire test can be completed within 31 hours. CONCLUSION: The method is highly specific, sensitive, and fast. The present study thus provides a rapid and effective method to detect ST and SE simultaneously from food samples.

Genetic analysis of chikungunya viruses imported to mainland China in 2008.

BACKGROUND: Chikungunya virus (CHIKV) has caused large outbreaks worldwide in recent years, especially on the islands of the Indian Ocean and India. The virus is transmitted by mosquitoes (Aedes aegypti), which are widespread in China, with an especially high population density in southern China. Analyses of full-length viral sequences revealed the acquisition of a single adaptive mutation providing a selective advantage for the transmission of CHIKV by this species. No outbreaks due to the local transmission of CHIKV have been reported in China, and no cases of importation were detected on mainland China before 2008. We followed the spread of imported CHIKV in southern China and analyzed the genetic character of the detected viruses to evaluate their potential for evolution. RESULTS: The importation of CHIKV to mainland China was first detected in 2008. The genomic sequences of four of the imported viruses were identified, and phylogenetic analysis demonstrated that the sequences were clustered in the Indian Ocean group; however, seven amino acid changes were detected in the nonstructural protein-coding region, and five amino acid changes were noted in the structural protein-coding regions. In particular, a novel substitution in E2 was detected (K252Q), which may impact the neurovirulence of CHIKV. The adaptive mutation A226V in E1 was observed in two imported cases of chikungunya disease. CONCLUSIONS: Laboratory-confirmed CHIKV infections among travelers visiting China in 2008 were presented, new mutations in the viral nucleic acids and proteins may represent adaptive mutations for human or mosquito hosts.

[Develop a statistics analysis software in population genetics using VBA language].

OBJECTIVE: To develop a statistics analysis software that can be used in STR population genetics for the purpose of promoting and fastening the basic research of STR population genetics. METHODS: Selecting the Microsoft VBA for Excel, which is simple and easy to use, as the program language and using its macro function to develop a statistics analysis software used in STR population genetics. RESULTS: The software "Easy STR Genetics" based on VBA language, by which the population genetic analysis of STR data can be made, were developed. CONCLUSION: The developed software "Easy STR Genetics" based on VBA language, can be spread in the domain of STR population genetics research domestically and internationally, due to its feature of full function, good compatibility for different formats of input data, distinct and easy to understand outputs for statistics and calculation results.

STR data for the AmpFlSTR SGM Plus loci from Shantou (south of China).

Allele frequencies for the ten STRs included in the AmpFlSTR SGM Plus kit were obtained from a sample of 132 unrelated Han individuals born in the region of Shantou (south of China).