Single-cell and bulk RNA sequencing reveal heterogeneity and diagnostic markers in papillary thyroid carcinoma lymph-node metastasis.

PURPOSE: Papillary thyroid carcinoma (PTC) is characterized by lymph-node metastasis (LNM), which affects recurrence and prognosis. This study analyzed PTC LNM by single-cell RNA sequencing (scRNA-seq) data and bulk RNA sequencing (RNA-seq) to find diagnostic markers and therapeutic targets. METHODS: ScRNA-seq data were clustered and malignant cells were identified. Differentially expressed genes (DEGs) were identified in malignant cells of scRNA-seq and bulk RNA-seq, respectively. PTC LNM diagnostic model was constructed based on intersecting DEGs using glmnet package. Next, PTC samples from 66 patients were used to validate the two most significant genes in the diagnostic model, S100A2 and type 2 deiodinase (DIO2) by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemical (IHC). Further, the inhibitory effect of DIO2 on PTC cells was verified by cell biology behavior, western blot, cell cycle analysis, 5-ethynyl-2'-deoxyuridine (EdU) assay, and xenograft tumors. RESULTS: Heterogeneity of PTC LNM was demonstrated by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis. A total of 19 differential genes were used to construct the diagnostic model. S100A2 and DIO2 differ significantly at the RNA (p < 0.01) and protein level in LNM patient tissues (p < 0.001). And differed in PTC tissues with different pathologic typing (p < 0.001). Further, EdU (p < 0.001) and cell biology behavior revealed that PTC cells overexpressed DIO2 had reduced proliferative capacity. Cell cycle proteins were reduced and cells are more likely to be stuck in G2/M phase (p < 0.001). CONCLUSIONS: This study explored the heterogeneity of PTC LNM using scRNA-seq. By combining with bulk RNA-seq data, diagnostic markers were explored and the model was established. Clinical diagnostic efficacy of S100A2 and DIO2 was validated and the treatment potential of DIO2 was discovered.

[A comparative study for the efficacies of transaxillary non-inflatable endoscopic surgery versus traditional surgery for papillary thyroid carcinoma].

Objective: To compare the efficacies between open surgery and axillary non-inflatable endoscopic surgery in papillary thyroid carcinoma (PTC). Methods: A retrospective analysis was performed on 343 patients with unilateral PTC treated by traditional open surgery (201 cases) and transaxillary non-inflating endoscopic surgery (142 cases) from May 2019 to December 2021 in the Head and Neck Surgery of Sichuan Cancer Hospital. Among them, 97 were males and 246 were females, aged 20-69 years. 1∶1 propensity score matching (PSM) was performed on the enrolled patients, and the basic characteristics, perioperative clinical outcomes, postoperative complications, postoperative quality of life (Thyroid Cancer-Specific Quality of Life), aesthetic satisfaction and other aspects of the two groups were compared after successful matching. SPSS 26.0 software was used for statistical analysis. Results: A total of 190 patients were enrolled after PSM, with 95 cases in open group and 95 cases in endoscopic group. Intraoperative blood losses for endoscopic and open groups were [20 (20) ml vs. 20 (10) ml, M (IQR), Z=-2.22], postoperative drainage volumes [170 (70)ml vs. 101 (55)ml, Z=-7.91], operative time [135 (35)min vs. 95 (35)min, Z=-7.34], hospitalization cost [(28 188.7±2 765.1)yuan vs. (25 643.5±2 610.7)yuan, x¯±s, t=0.73], postoperative hospitalization time [(3.1±0.9)days vs. (2.6±0.9)days, t=-3.24], and drainage tube placement time [(2.5±0.8) days vs. (2.0±1.0)days, t=-4.16], with statistically significant differrences (all P<0.05). There was no significant difference in surgical complications (P>0.05). There were significant diffferences between two groups in the postoperative quality of life scores in neuromuscular, psychological, scar and cold sensation (all P<0.05), while there were no statistically significant differences in other quality of life scores (all P>0.05). In terms of aesthetic satisfaction 6 months after surgery, the endoscopic group was better than the open group, with statistically significant difference (χ2=41.47, P<0.05). Conclusion: Endoscopic thyroidectomy by a gasless unilateral axillary approach is a safe and reliable surgical method, which has remarkable cosmetic effect and can improve the postoperative quality of life of patients compared with the traditional thyroidectomy.

[Clinical application of the gasless unilateral axillary approach in endoscopic thyroid surgery].

Objective: To investigate the efficacy, safety and advantages of gasless unilateral axillary approach (GUAA) in endoscopic thyroid surgery. Methods: A total of 334 patients who underwent the GUAA endoscopic thyroid surgery (GUAA group) or conventional open thyroid surgery (OS group) in the Department of Head and Neck Surgery of Zhejiang Cancer Hospital from January 2017 to June 2018 were retrospectively analyzed. There were 45 males and 289 females, aged from 12 to 72 years old, of whom 139 patients were assigned to GUAA group and 195 patients to OS group. Pathological results included papillary thyroid carcinoma (282 cases), nodular goiter (41 cases) and thyroid adenoma (11 cases). Surgical exploration development curve of GUAA group was drawn and was divided into two parts: the technical exploration stage and the technical stable stage. Surgical efficiency, incidences of complications, and incision satisfaction were compared between GUAA group in technical stable stage and OS group. SPSS 25.0 software was adopted for statistical analysis. Results: The mean age in GUAA group was younger than that in OS group, with a significant difference [(35.3±9.5) years vs. (48.1±10.6) years, t=11.31, P<0.01]. The cases in the endoscope group were divided into technical exploration stage for 51 cases and technical stable stage for 88 cases according to the exploration and development curve. In unilateral radical thyroidectomy and unilateral thyroid lobectomy, the mean operation time [(90.6±18.6) min and (93.5±22.0) min] and postoperative drainage volumes [(121.5±87.6) ml and (155.5±69.1) ml] of GUAA group in the stable stage were more than those of OS group [(61.6±15.6) min and (46.5±8.4) min] and [(93.2±42.3) ml and (78.9±48.7) ml]. The difference was statistically significant (t=12.28, 7.23, 3.35 and 3.05 respectively, all P<0.05), but there were no significant differences in surgical bleeding volumes between two groups [(12.7±6.8) ml vs. (13.5±7.7) ml, t=0.74, P>0.05 and (16.3±14.1) ml vs. (11.9±5.1) ml, t=1.05, P>0.05]. Compared with OS group, GUAA group had the lower incidence of anterior cervical discomfort during swallowing (2.3% vs. 29.2%, P<0.01) and the higher incision satisfaction score (1.1±0.5 vs. 2.8±0.7, t=21.12, P<0.01), however, GUAA group had the higher incidence of supraclavicular (or infraclavicular) numbness after surgery (5.7% vs. 0, P<0.01). And there was no significant difference in the incidences of temporary recurrent laryngeal nerve injury, bleeding, hematoma, infection, lymphatic leakage or chylous leakage after surgery between two groups (P>0.05). Conclusion: GUAA endoscopic thyroid surgery is a safe method with high cosmetic satisfaction.

[The bisphenol A-enhanced activity of thyroid carcinoma cell line B-CPAP is inhibited by Icarrin].

Objective: To investigate the effect of icariin (ICA) on the bisphenol A (BPA)-enhanced proliferation function of thyroid carcinoma cell B-CPAP and underlying mechanism. Methods: The proliferation of Gastric B-CPAP cell line was evaluated by cell counting kit-8 (CCK-8). Apoptosis and ROS expression in B-CPAP cells were detected by flow cytometry. The expression of superoxide dismutase (SOD) and malondialdehyde (MDA) in B-CPAP cells were measured by individual assay kits. The expressions of Bcl-2 and γ-HA2X were detected by Western blot. SPSS 18.0 software was used to analyze the data. Results: B-CPAP cell activity was promoted by treatment with 3×10(-7)mol/L BPA for 48 h, with significant difference in absorbance between BPA and control groups (1.089±0.053 vs 0.935±0.010, P<0.05). The cell activities of BPA+ ICA(25), BPA+ ICA(50), BPA+ ICA(100) and BPA+ ICA(200) groups was 0.780±0.036, 1.007±0.050, 0.958±0.033 and 0.625±0.064, respectively (all P<0.01). The proliferation of B-CPAP cells treated with BPA for 72 hours showed a similar trend to 48 hours. There was no significant difference between all treatment groups in 24 hours. The apoptosis rate was (19.272±0.186)% in BPA-treated cells, and was (22.412±0.238)% in control cells (P<0.05). The apoptosis rates of BPA+ ICA(50) and BPA+ ICA(200) groups were (23.688±0.412)% and (30.270±0.696)%, respectively (P<0.01). The intracellular accumulation of ROS in BPA, BPA+ ICA(50), and BPA+ ICA(200) groups were 806±21, 1 772±37, 2 041±16, respectively (P<0.01). The expressions of anti-apoptotic protein Bcl-2 in control, BPA, BPA+ ICA(50), BPA+ ICA(200) groups were 7 120±151, 9 801±286, 5 902±171 and 4 203±216, respectively (P<0.01). Conclusion: BPA can promote the proliferation of thyroid carcinoma B-CPAP cells and decrease the apoptosis of cells, and this effect can be inhibited by ICA. The possible mechanism is to induce high expression of intracellular ROS and inhibit the expression of antioxidase system, leading to cell oxidative damage, thereby inducing apoptosis.

Frequency thresholds of rat cochlear nerve fibers.

Activities of single cochlear nerve fibers of Wistar rats were recorded extracellularly. Best frequencies (BF) distributed from 0.50 to 62.6 kHz. The audiogram was made as the minimum boundary of the BF threshold distribution. The range of audible frequency was 0.54-63 kHz at 60 dB SPL and 0.15-67 kHz at 100 dB SPL. The lowest trough of the audiogram was 5 dB SPL at 41.2 kHz. There was the second trough of 10 dB SPL at 7.01 kHz leaving a notch between the two troughs. The shapes of the frequency-threshold curves (FTCs) of fibers were evaluated quantitatively and typical FTCs were shown as a function of BF.

[Activities of single cochlear nerve fibers in rats].

Activities of 325 single cochlear nerve fibers in response to pure tones at various frequencies including ultrasonic sounds were observed in rats. The stimulus sounds were measured with a probe tube inserted into an ear canal and a microphone and expressed in dB SPL. The best frequency (BF, characteristic frequency) and the threshold at BF were determined in every fiber. The measured BF ranged from 0.58 kHz to 62.6 kHz. BF and the threshold at BF of a fiber with the lowest BF-threshold were 27.49kHz and 6dB SPL respectively. The highest sensitivity was found in fibers with BF from 20 to 50 kHz. The tuning curve rose from BF and its slope was steeper for the high side than the low one as observed in other animals. The sharpness of the tuning curve was expressed by QN values, where QN is the ratio of BF to the band width at N dB above the BF threshold. Mean and the variance of Q10dB, Q20dB, Q30dB, Q40dB, Q50dB, all increased as the BF did. Almost all fibers had spontaneous discharge. The discharge rate increased with the increase in sound intensity and saturated at about 30 dB above the threshold. The auditory threshold curve obtained from distribution of the lowest BF-threshold of 325 fibers was consistent with the audiograms determined behaviorally.