Association between surgical extent and recurrence in unilateral intermediate- to high-risk papillary thyroid cancer.

BACKGROUND: Guidelines recommend total thyroidectomy (TT) to facilitate radioactive ablation and serological follow-up for intermediate- to high-risk papillary thyroid carcinoma (PTC). However, the association between surgical extent and tumor recurrence in these patients has not been well validated. We aimed to examine the association between the extent of surgery and recurrence in patients with completely resected unilateral intermediate- to high-risk PTC. METHODS: Patients with completely resected unilateral PTC from 2000 to 2017 in a single institute were reviewed. Those who had extrathyroidal extension (ETE) or lymph node metastasis (LNM, cN1 or pN1 > 5 lymph nodes involved) were included for analysis. Cox proportional hazards models were applied to measure the association between surgical extent and recurrence-free survival (RFS) while adjusting for patient demographic, clinicopathological and treatment variables. RESULTS: A total of 4550 patients (mean[SD] age, 43.0[11.7] years; 3379 women[74.3%]) were included. Of these patients, 2262(49.7%), 656(14.4%), 1032(22.7%), and 600 (13.2%) underwent lobectomy, TT, lobectomy + neck dissection (ND) and TT + ND, respectively. With a median follow-up period of 68 months, after multivariate adjustment, lobectomy was associated with a compromised RFS compared with other surgical extents (HR[95%CI], TT 0.537[0.333-0.866], P = 0.011, lobectomy + ND 0.531[0.392-0.720] P < 0.0001, TT + ND 0.446[0.286-0.697] P < 0.0001). RFS was similar between the two extents with ND (lobectomy + ND, HR [95%CI], 1.196 [0.759-1.885], P = 0.440). CONCLUSION: Lobectomy alone is associated with an elevated recurrence risk in patients with unilateral intermediate- to high-risk PTC compared with larger surgical extents. However, lobectomy and ND may provide similar tumor control compared with the conventional approach of TT and ND.

Correlation of DNA methylation and lymph node metastasis in papillary thyroid carcinoma.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer with a primarily good prognosis, and its 10-year survival rate is over 90%. However, PTC is prone to early lymph node metastasis. METHODS: Thyroid cancer tissues from PTC patients with lymphatic metastasis and normal tissues were collected for DNA methylation analysis. Different methylation sites, different methylation regions, gene-enriched pathways, and protein-protein interactions (PPIs) were analyzed. RESULTS: There were 1004 differentially methylated sites in the PTC group versus the control group; these involved 479 hypermethylated sites in 415 related genes, 525 hypomethylated sites in 482 related genes, 64 differentially methylated regions located in the CpG island region, 34 differentially methylated genes closely related to thyroid cancer, and 17 genes with differentially methylated genes in the DNA promoter region. CONCLUSION: NDRG4 hypermethylation and FOXO3, ZEB2, and CDK6 hypomethylation were associated with PTC lymph node metastasis.

Redox-Regulated Conformational Change of Disulfide-Rich Assembling Peptides.

Disulfide bond formation is a common mechanism for regulating conformational changes in proteins during oxidative folding. Despite extensive studies of the use of multiple disulfide bonds to constrain peptide conformation, few studies have explored their usage in developing self-assembling peptides. Herein, we report that a thiol-rich peptide could fold into an amphiphilic ß-hairpin conformation through the formation of two hetero-disulfide bonds upon oxidation, subsequently self-assembling into a mechanically rigid hydrogel. Breaking disulfide bonds under reductive condition, the hydrogel exhibited a transition from hydrogel to solution. Molecular simulation revealed that intermolecular interaction between two tryptophan residues was indispensable for hydrogelation. This work is the first case of the use of multiple disulfide bonds to control conformational change and self-assembly, and provides a cell-compatible hydrogel material for potential biomedical application.

Design and Ribosomal Incorporation of Noncanonical Disulfide-Directing Motifs for the Development of Multicyclic Peptide Libraries.

The engineering of naturally occurring disulfide-rich peptides (DRPs) has been significantly hampered by the difficulty of manipulating disulfide pairing. New DRPs that take advantage of fold-directing motifs and noncanonical thiol-bearing amino acids are easy-to-fold with expected disulfide connectivities, representing a new class of scaffolds for the development of peptide ligands and therapeutics. However, the limited diversity of the scaffolds and particularly the use of noncanonical amino acids [e.g., penicillamine (Pen)] that are difficult to be translated by ribosomes greatly hamper the further development and application of these DRPs. Here, we designed and synthesized noncanonical bisthiol motifs bearing sterically obstructed thiol groups analogous to the Pen thiol to direct the folding of peptides into specific bicyclic and tricyclic structures. These bisthiol motifs can be ribosomally incorporated into peptides through a commercially available PURE system integrated with genetic code reprograming, which enables, for the first time, the in vitro expression of bicyclic peptides with two noncanonical and orthogonal disulfide bonds. We further constructed a bicyclic peptide library encoded by mRNA, with which new bicyclic peptide ligands with nanomolar affinity to proteins were successfully selected. Therefore, this study provides a new, general, and robust method for discovering de novo DRPs with new structures and functions not derived from natural peptides, which would greatly benefit the field of peptide drug discovery.

Design and Synthesis of Disulfide-Rich Peptides with Orthogonal Disulfide Pairing Motifs.

Disulfide-rich peptides (DRPs) are a class of peptides that are constrained through two or more disulfide bonds. Though natural DRPs have been extensively exploited for developing protein binders or potential therapeutics, their synthesis and re-engineering to bind new targets are not straightforward due to difficulties in handling the disulfide pairing problem. Rationally designed DRPs with an intrinsically orthogonal disulfide pairing propensity provide an alternative to the natural scaffolds for developing functional DRPs. Herein we report the use of tandem CXPen/PenXC motifs ((C) cysteine; (Pen) penicillamine; (X) any residue) for directing the oxidative folding of peptides. Diverse tricyclic peptides were designed and synthesized by varying the pattern of C/Pen residues and incorporating a tandem CXPen/PenXC motif into peptides. The folding of these peptides was determined primarily by C/Pen patterns and tolerated to sequence manipulations. The applicability of the designed C/Pen-DRPs was demonstrated by designing protein binders using an epitope grafting strategy. This study thus demonstrates the potential of using orthogonal disulfide pairing to design DRP scaffolds with new structures and functions, which would greatly benefit the development of multicyclic peptide ligands and therapeutics.

Design and Synthesis of Cross-Link-Dense Peptides by Manipulating Regioselective Bisthioether Cross-Linking and Orthogonal Disulfide Pairing.

Existing disulfide-rich peptides, both naturally occurring and de novo designed, only represent a tiny amount of the possible sequence space because natural evolution and de novo design only keep sequences that are structurally approachable by correct disulfide pairings. To bypass this limitation for designing new peptide scaffolds beyond the natural sequence space, we dedicate to developing novel disulfide-rich peptides with predefined disulfide pairing patterns irrelevant to primary sequences. However, most of these designed peptides still suffer from disulfide rearrangements to at least one to three possible isomers. Here, we report a general and reliable strategy for the design and synthesis of a range of structurally diverse cross-link-dense peptide (CDP) scaffolds with two orthogonal disulfide bonds and a bisthioether bridge that are not subject to disulfide isomerizations. Altering the pattern of cysteine and penicillamine generates hundreds of different CDP scaffolds tolerant to extensive sequence manipulations. This work thus provides many useful scaffolds for the design of functional molecules such as protein binders with improved proteolytic stability (e.g., designed by epitope grafting).

Bleomycin inhibits proliferation and induces apoptosis in TPC-1 cells through reversing M2-macrophages polarization.

Papillary thyroid carcinoma (PTC) is one of the most common types of thyroid malignancy. Previous studies have demonstrated that the density of tumor-associated macrophages (TAMs) within the tumor microenvironment affects the progression of PTC due to the imbalance in M1/M2 macrophage subtypes. M2 macrophages induce anti-inflammatory effects and promote tumor progression, whereas M1 macrophages destroy tumor cells. Therefore, reversing TAM polarization to M1 may be a novel strategy for the treatment of cancer. Although bleomycin (BLM) is a commonly used anti-cancer drug, which regulates the secretion of relevant cytokines, high dose and long-term treatment with BLM may lead to pulmonary fibrosis. In the present study, flow cytometry data revealed that low dose treatment with BLM (5 or 10 mU/ml) facilitated the expression of the M1 phenotype markers cluster of differentiation (CD)80 and C-C chemokine receptor 7, and concurrently suppressed the M2 marker CD206 on M2-macrophages. Reverse transcription-quantitative polymerase chain reaction data revealed that the expression levels of tumor necrosis factor-α and interleukin-1ß markedly increased, whereas the expression of IL-10 decreased in M2 macrophages treated with BLM. A fluorescein isothiocyanate-dextran uptake experiment revealed that BLM increased the phagocytic capacity of M2, however not M1 or M0 macrophages. In addition, to verify the effect of BLM-treated M2 macrophages on thyroid carcinoma cells, a co-culture system of macrophages and the human PTC cell line TPC-1, was established. BLM-treated M2 macrophages increased the number of cells in early and late apoptosis and inhibited the migration, proliferation and invasion of TPC-1 cells. These results suggest that a low dose and indirect effect of BLM may induce suppressive effects on PTC by selectively reversing M2 macrophage polarization to M1, which may provide a novel strategy for cancer treatment.

Highly Sensitive Electrochemical Detection of Tumor Exosomes Based on Aptamer Recognition-Induced Multi-DNA Release and Cyclic Enzymatic Amplification.

Sensitive and specific detection of tumor exosomes is of great significance for early cancer diagnosis. In this paper, we report an aptamer strategy for exosome detection based on aptamer recognition-induced multi-DNA release and cyclic enzymatic amplification. First, we use aptamer-magnetic bead bioconjugates to capture tumor exosomes derived from LNCaP cells, leading to the release of three kinds of messenger DNAs (mDNAs). After magnetic separation, the released mDNAs hybridized with the probe DNAs immobilized on a gold electrode. Electroactive Ru(NH3)63+ was used as the signal reporter because of its electrostatic attraction to DNA. Subsequent Exo III cyclic digestion caused the electrochemical signal to "turn off". Because the electrochemical signal reflects the concentration of Ru(NH3)63+ and the concentration of Ru(NH3)63+ is correlated with the mDNA concentration, which is correlated with the exosome concentration, the tumor exosomes can be detected by examining the decrease in the peak current of Ru(NH3)63+. In this paper, the signal was amplified by the numerous mDNAs released from the magnetic bead and the Exo III-assisted mDNA recycling. Under the optimal conditions, a detection limit down to 70 particles/µL was achieved, which is lower than the LODs of most currently available methods. Furthermore, this assay can be used to detect tumor exosomes in complex biological samples, demonstrating potential application in real sample diagnosis.

An electrochemiluminescence assay for sensitive detection of methyltransferase activity in different cancer cells by hybridization chain reaction coupled with a G-quadruplex/hemin DNAzyme biosensing strategy.

In this work, a highly sensitive electrochemiluminescence (ECL) assay was fabricated for the detection of human DNA (cytosine-5)-methyltransferase1 (DNMT1) activity in cancer cells. The ECL assay coupled hybridization chain reaction with a G-quadruplex/hemin DNAzyme biosensing strategy. The ECL intensity changes (ΔI) allowed detection of DNMT1 activity down to 0.09 U mL-1, and ΔI was proportional to the logarithm of the activity of DNMT1 within the range of 1.0 to 30.0 U mL-1 in buffer solution. It also showed high sensitivity to DNMT1 activity in A549 cells, with a detection limit of about 2 cells. This ECL assay provides a promising platform for profiling of the mutational cells of tumors and shows a great potential for application to DNA methylation-related clinical diagnostics.

Sensitive Electrochemical Detection of Human Methyltransferase Based on a Dual Signal Amplification Strategy Coupling Gold Nanoparticle-DNA Complexes with Ru(III) Redox Recycling.

Effective detection of DNA methyltransferase (DNMT) activity is significant for cancer research. Herein, we developed a sensitive electroanalytical method to detect human DNA (cytosine-5)-methyltransferase 1 (DNMT1) from crude lysates of cancer cells. In this assay, capture DNA having a preferred DNMT1 methylation site was immobilized on a gold electrode and then hybridized with gold nanoparticle (Au NP)-DNA complexes. The modified electrodes were equilibrated with the lysate and then incubated with methylation-sensitive restriction enzyme. If the lysate was negative for DNMT1 activity, the Au NP-DNA complexes would be cut by the restriction enzyme and released from the electrode. Conversely, restriction enzyme cleavage would be blocked by the fully methylated duplexes, and the Au NP-DNA complexes would remain on the electrode. Electroactive Ru(NH3)63+ was used as the signal reporter, because of its electrostatic attraction to DNA, resulting in an electrochemical signal. Since the electrochemical signal reflects the amount of Ru(III) redox and the amount of Ru(III) redox is correlated with the activity of DNMT1, the activity of DNMT1 is proportional to the electrochemical signal. The signal could be amplified by the numerous DNAs on the Au NPs and further amplified by Ru(III) redox recycling. With this method, a detection limit down to 0.3 U/mL for pure DNMT1 and 8 MCF-7 cells was achieved. DNMT1 activities of different cell lines were also successfully evaluated.

A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity.

DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs.

G-quadruplex DNAzyme-based electrochemiluminescence biosensing strategy for VEGF165 detection: Combination of aptamer-target recognition and T7 exonuclease-assisted cycling signal amplification.

The expression profile of vascular endothelial growth factor (VEGF) is highly correlated with the occurrence and development of cancer. This work reports an electrochemiluminescence (ECL) approach for highly sensitive detection of VEGF165. This approach comprises aptamer-target recognition, T7 exonuclease (T7 Exo)-assisted cycling signal amplification and efficient quenching of ECL of CdS:Eu nanocrystals (NCs) by using DNAzyme. In this assay, CdS:Eu NCs were used as the ECL substrate, A guanine (G)-rich single-stranded DNA (ssDNA) sequence and VEGF165 aptamer were co-immobilized on the surface of the CdS:Eu NCs modified glassy carbon electrode. After recognition and binding to VEGF165, the aptamer moved away from the electrode surface and induced the proposed cyclic cleavage of the target DNA with T7 Exo. A large amount of G-rich ssDNA was released on the CdS:Eu film and folded into G-quadruplex/hemin DNAzyme in the presence of hemin and K(+), consequently decreasing the ECL intensity of CdS:Eu. A good linearity was obtained for VEGF165 detection within the range of 1 pM to 20 nM with a detection limit of 0.2 pM. This assay could be a universal and promising protocol for detection of various biomarkers for early clinical diagnosis.

[Surgical treatment and reconstruction of thyroid carcinoma invading cervical esophagus and trachea full-thickness].

OBJECTIVE: To summary the clinical therapy experience of thyroid carcinoma invading cervical esophagus and trachea full thickness and reconstruction of them to improve the survival rate and quality of life of these patients. METHOD: In 33 patients with thyroid carcinoma inviding cervical esophagus and trachea, 7 patients were operated with total thyroid resection, 22 patients were operated with one lobectomy and the other side subtotal thyroid resection,and 4 patients were operated with partial lobectomy. Trachea local recection and intubation were performed on 19 patients, partial laryngectomy and pyriform sinus resection with trachea local recection and intubation were performed on 4 patients,and 10 cases were operated with tracheal sleeve resection and end to end anastomos. RESULT: All patients were primary healing without tracheal anastomosis fistula, tracheal wall necrosis, esophageal fistula and pharyngeal fistula. One case had hypoparathyroidism after the operation and took a favorable turn a month later. Seven cases were dead (21. 21%). 1-year, 3-year, 5-year survival rates of the thyroid papillary carcinoma inviding cervical esophagus and trachea were 100.0%, 93.8% and 70.3%; 1-year, 3-year, 5-year survival rates of the thyroid carcinoma inviding cervical esophagus and trachea were 96.6%, 79.0% and 61.4%. CONCLUSION: Thyroid carcinoma invading cervical esophagus and trachea full-thickness can be treated with surgical methods, and tracheal sleeve resection and end to end anastomos are the suitable methods.

[Application of free anterolateral thigh flap in head and neck surgery].

OBJECTIVE: To evaluate the results of reconstruction by free anterolateral thigh flaps (ALT) after operation of head and neck tumors. METHODS: Forty-three cases underwent the reconstruction of postoperative defects with free anterolateral thigh flaps after head and neck cancer surgeries between November 2007 and June 2010 were reviewed. Ages of the patients ranged from 40 to 81 years, with a median of 56 years; 32 males and 11 females; 23 cases of oral carcinoma, 7 cases of tonsil carcinoma, 11 cases of hypopharyngeal carcinoma, and 2 cases of head skin cancer. TNM classified as follows: no case of distant metastasis; T1 9 cases; T2 17 cases; T3 11 cases; T4 6 cases. All patients were applied with ALT to restore swallowing and respiratory functions. The mean length of blood vessel pedicles of the ALT free flaps was 12.5 (8 - 18) cm. The flaps were 4 - 15 cm in width, 5 - 25 cm in length. RESULTS: In the 43 cases applied with ALT free flaps, 40 cases were successful and 3 cases unsuccessful. Two of the failed cases were reconstructed with pectoralis major flap. In 11 cases of hypopharyngeal carcinoma, except 3 cases with total laryngectomy, 8 cases (72.7%) had their laryngeal function been preserved. CONCLUSIONS: The successful rate of ALT free flaps is perfect. There were no serious complication in offered areas. The flap could be shaped into various forms. ALT free flap is an ideal flap to reconstruct the defect after surgery in some head and neck tumors.

[Preservation of parathyroid glands and their functions during total thyroidectomy].

OBJECTIVE: To explore the protection methods of parathyroid glands (PTGs) and their functions during total thyroidectomy. METHODS: The locations and the blood supplies of parathyroid glands in 292 cases underwent total thyroidectomy between February 1990 and December 2009 were studied. The protective measures for PTGs and their blood supplies during total thyroidectomy were analyzed. RESULTS: Total of 542 superior PTGs and 467 inferior PTGs were found in 296 cases of total thyroidectomy. Of the superior PTGs, 444 (81.9%) consistently located in the back sides of the thyroid glands and at the level of inferior edge of thyroid cartilage. The locations of the inferior PTGs were variable, 231 (49.5%) of them located in the inferior 1/3 part of the back sides of the thyroids and 116 (24.8%) at the inferior thyroid, in where inferior thyroid artery (ITA) branches enter thyroid. The fine dissections showed that the blood supplies to superior PTGs were mainly from the upper branch of ITA, accounting for 71 (68.3%) of 104 superior PTGs and the blood supplies to inferior PTGs were from the inferior branches of ITA system, accounting for 114 (80.3%) of 142 inferior PTGs. There was 13 cases with short-term hypocalcemia postoperatively, but no case with permanent hypoparathyroidism. CONCLUSIONS: The blood supplies of PTGs are associated with their locations. During total or subtotal thyroidectomy, parathyroid glands and their artery blood-supply should be exposed and preserved to prevent hypoparathyroidism after surgery.

[Level VI and II-V cervical lymph node metastasis in differentiated thyroid carcinoma].

OBJECTIVE: To explore the characters of the cervical lymph node metastasis in differentiated thyroid carcinoma, and to provide evidence for proper surgery of differentiated thyroid carcinoma. METHODS: From 1984 to 2000, 99 cases with differentiated thyroid carcinoma were performed thyroidectomy and neck dissection. The patients were followed up. A retrospective analysis was performed. Results In 99 cases with differentiated thyroid carcinoma, there were 61 papillary carcinoma, 13 papillary and follicular mixed carcinoma, 25 follicular carcinoma. According to 2002 UICC TNM classification, 60 cases were staged I, 1 case staged II, 5 cases staged III, 33 cases staged IV. Lobectomy and isthmectomy was performed in 80 cases, lobectomy was resected and opposite subtotal lobectomy in 15 cases, total thyroidectomy in 4 cases. One hundred and four neck dissection were performed in 99 cases (5 cases were bilateral neck dissection ). Among them, 66 (68 sides) were radical neck dissection, 33 (36 sides) were modified neck dissection. Pathological results showed that lymph nodes were positive in 86 sides of 83 cases. The rate of cervical lymph node metastasis was 83.8% (83/99). The positive rates of lymph node were 37.5% (39/104) in level VI and 76.9% (80/104) in II-V, which was statistically different (chi2 = 33.01, P < 0.01). The cervical lymph node metastasis in lateral area (level II-V) and that in VI had not relationship (chi2 = 2.08, P > 0.05). Ten and 15 year survival rates of all 99 cases were 88.3% and 84.5% respectively. CONCLUSIONS: The occurrence of lymph node metastasis in level VI and level II was different and no relationship .One can not judge whether lateral neck metastasis by the lymph node statue in level VI only . Although they all had good prognosis, patients with positive nodes in level VI were not worse than that in lateral neck (II-V).

[Operation for well-differentiated invading thyroid carcinoma].

OBJECTIVE: To analyze the surgical data of well-differentiated invading thyroid carcinoma (WITC) , for acquiring the clinical experience. METHODS: A retrospective analysis was made in 201 cases with well-differentiated invading thyroid carcinoma in Liaoning Tumour Hospital from 1984 to 2000. The data were statistically treated for survival curves according to the Kaplan-Meier method. The Log-Rank tests were employed to assess the statistical significance of various groups. RESULTS: There were 3 cases with total laryngectomy and partial cervical trachea resection, 6 cases with partial cervical trachea resection, 67 cases with shaving off tumor from its surface, 9 cases with recurrent laryngeal nerve resection, 26 cases with shaving off tumor from the surface of recurrent laryngeal nerve. Other local structures invaded in 90 cases was resected with the thyroid tumour en bloc. One hundred and eighty nine cases with one thyroid lobe and isthmus ectomy, 5 cases one lobe and opsite subtotal ectomy. One hundred and twenty nine cases simultaneous neck dissection (5 cases bilateral neck dissection), in them, 75 neck radical neck dissection, 59 neck modified dissection. The 5-, 10- and 15-year living rates of well-differentiated thyroid carcinoma patients were 85.6% , 77.3% and 69.4% respectively. Multivariate analysis showed that patients' age, tumour invading structure were independent prognostic factors. CONCLUSIONS: With proper operation, a better cure will be made in WITC.

[Re-operation for thyroid carcinoma].

OBJECTIVE: To evaluate the results of re-operation of thyroidectomy for patients of thyroid carcinoma. METHODS: 268 patients of thyroid carcinoma received completion thyroidectomy were retrospectively reviewed from 1984 to 2000. There were 59 males and 209 female cases. 256 cases had received nodule enucleating or partial thyroidectomy in other hospitals and 12 cases had unilateral subtotal thyroidectomy in this hospital. The types of reoperation were: Total thyroidectomy for bilateral thyroid carcinoma (6 cases); expand isthmectomy for isthmus carcinoma (1 case). Lobectomy plus isthmectomy for unilateral disease (261) Simultaneous neck dissections were performed in 196 cases. Among them, 94 cases had classical neck dissection, 102 cases had modified neck dissection. RESULTS: Pathological results confirmed that there were 78 cases with residual thyroid carcinoma. The rate of residual carcinoma was 29. 1% (78/268). There were 95 cases with lymphnode metastasis. The rate of lymphnode metastasis was 48.5% (95/196). The rate of recurrent laryngeal nerve injury was 1.1% (3/268). The 5-year and 10-year survival rates of patients were 94. 0% (251/267) and 85. 2% (127/149) respectively. CONCLUSIONS: The residual carcinoma of completing thyroidectomy was high. Therefore re-operations of thyroid in selected cases were necessary.