SARS-CoV-2 spike-specific TFH cells exhibit unique responses in infected and vaccinated individuals.

Long-term humoral immunity to SARS-CoV-2 is essential for preventing reinfection. The production of neutralizing antibody (nAb) and B cell differentiation are tightly regulated by T follicular help (TFH) cells. However, the longevity and functional role of TFH cell subsets in COVID-19 convalescents and vaccine recipients remain poorly defined. Here, we show that SARS-CoV-2 infection and inactivated vaccine elicited both spike-specific CXCR3+ TFH cell and CXCR3- TFH cell responses, which showed distinct response patterns. Spike-specific CXCR3+ TFH cells exhibit a dominant and more durable response than CXCR3- TFH cells that positively correlated with antibody responses. A third booster dose preferentially expands the spike-specific CXCR3+ TFH cell subset induced by two doses of inactivated vaccine, contributing to antibody maturation and potency. Functionally, spike-specific CXCR3+ TFH cells have a greater ability to induce spike-specific antibody secreting cells (ASCs) differentiation compared to spike-specific CXCR3- TFH cells. In conclusion, the persistent and functional role of spike-specific CXCR3+ TFH cells following SARS-CoV-2 infection and vaccination may play an important role in antibody maintenance and recall response, thereby conferring long-term protection. The findings from this study will inform the development of SARS-CoV-2 vaccines aiming to induce long-term protective immune memory.

Identification of an Arylnaphthalene Lignan Derivative as an Inhibitor against Dengue Virus Serotypes 1 to 4 (DENV-1 to -4) Using a Newly Developed DENV-3 Infectious Clone and Replicon.

Dengue virus (DENV) is the most widespread arbovirus, causing symptoms ranging from dengue fever to severe dengue, including hemorrhagic fever and shock syndrome. Four serotypes of DENV (DENV-1 to -4) can infect humans; however, no anti-DENV drug is available. To facilitate the study of antivirals and viral pathogenesis, here we developed an infectious clone and a subgenomic replicon of DENV-3 strains for anti-DENV drug discovery by screening a synthetic compound library. The viral cDNA was amplified from a serum sample from a DENV-3-infected individual during the 2019 epidemic; however, fragments containing the prM-E-partial NS1 region could not be cloned until a DENV-3 consensus sequence with 19 synonymous substitutions was introduced to reduce putative Escherichia coli promoter activity. Transfection of the resulting cDNA clone, plasmid DV3syn, released an infectious virus titer of 2.2 × 102 focus-forming units (FFU)/mL. Through serial passages, four adaptive mutations (4M) were identified, and addition of 4M generated recombinant DV3syn_4M, which produced viral titers ranging from 1.5 × 104 to 6.7 × 104 FFU/mL and remained genetically stable in transformant bacteria. Additionally, we constructed a DENV-3 subgenomic replicon and screened an arylnaphthalene lignan library, from which C169-P1 was identified as exhibiting inhibitory effects on viral replicon. A time-of-drug addition assay revealed that C169-P1 also impeded the internalization process of cell entry. Furthermore, we demonstrated that C169-P1 inhibited the infectivity of DV3syn_4M, as well as DENV-1, DENV-2, and DENV-4, in a dose-dependent manner. This study provides an infectious clone and a replicon for the study of DENV-3 and a candidate compound for future development against DENV-1 to -4 infections. IMPORTANCE Dengue virus (DENV) is the most prevalent mosquito-transmitted virus, and there is no an anti-dengue drug. Reverse genetic systems representative of different serotype viruses are invaluable tools for the study of viral pathogenesis and antiviral drugs. Here, we developed an efficient infectious clone of a clinical DENV-3 genotype III isolate. We successfully overcame the instability of flavivirus genome-length cDNA in transformant bacteria, an unsolved issue for construction of cDNA clones of flaviviruses, and adapted this clone to efficiently produce infectious viruses following plasmid transfection of cell culture. Moreover, we constructed a DENV-3 subgenomic replicon and screened a compound library. An arylnaphthalene lignan, C169-P1, was identified as an inhibitor of virus replication and cell entry. Finally, we demonstrated that C169-P1 exhibited a broad-spectrum antiviral effect against the infections with DENV-1 to -4. The reverse genetic systems and the compound candidate described here facilitate the study of DENV and related RNA viruses.

Bi2Se3 Growth on (001) GaAs Substrates for Terahertz Integrated Systems.

Terahertz (THz) technologies have been of interest for many years due to the variety of applications including gas sensing, nonionizing imaging of biological systems, security and defense, and so forth. To date, scientists have used different classes of materials to perform different THz functions. However, to assemble an on-chip THz integrated system, we must understand how to integrate these different materials. Here, we explore the growth of Bi2Se3, a topological insulator material that could serve as a plasmonic waveguide in THz integrated devices, on technologically important GaAs(001) substrates. We explore surface treatments and find that an atomically smooth GaAs surface is critical to achieving high-quality Bi2Se3 films despite the relatively weak film/substrate interaction. Calculations indicate that the Bi2Se3/GaAs interface is likely selenium-terminated and shows no evidence of chemical bonding between the Bi2Se3 and the substrate. These results are a guide for integrating van der Waals materials with conventional semiconductor substrates and serve as the first steps toward achieving an on-chip THz integrated system.

Ultrasonic Characteristics Improve Prediction of Central Lymph Node Metastasis in cN0 Unifocal Papillary Thyroid Cancer.

Background: Prediction of central lymph node metastasis (CLNM) is vital for clinical decision-making processes in clinically N0 (cN0) unifocal papillary thyroid carcinoma (PTC), but the sensitivity of preoperative detection of CLNM is limited. The aim of the present study was to determine whether there are ultrasonic (US) characteristics associated with CLNM. Methods: In total, 1657 PTC patients (514 men and 1143 women) were enrolled in the present study between January 2018 and May 2021. The patients met the following inclusion criteria based on preoperative detection: suspected nodule confirmed as PTC by biopsy; the nodule was unifocal and less than 4 cm in diameter; no prior neck radiation exposure; no extrathyroidal extension; and no CLNM or distant metastases on imaging. All the enrolled patients underwent total thyroidectomy with prophylactic central lymph node dissection (CLND). A postoperative pathological diagnosis was made. Results: CLNM was found in 58.4% of male patients and 36.9% of female patients. In univariate analysis, size, adjacent anterior capsule, distance to the lower pole and color Doppler flow imaging (CDFI) were considered risk factors for the male and female groups (p < 0.05). In multivariate analyses, size, adjacent anterior capsule, distance to the lower pole and CDFI were independent risk factors for male patients. For females, the independent risk factors included size, adjacent anterior capsule, distance to the lower pole and CDFI. Conclusion: In the present cohort, US imaging characteristics, including size, adjacent anterior capsule, distance to the lower pole and CDFI, were identified to be potentially beneficial in preoperative clinical decision-making processes for cN0 unifocal PTC patients.

Surgical Outcome and Malignant Risk Factors in Patients With Thyroid Nodule Classified as Bethesda Category III.

Background: Thyroid nodules are a very common finding in the general population. Fine-needle aspiration (FNA) has been recommended as the initial test for the evaluation of thyroid nodules. The trend on reporting as atypia of undetermined significance (AUS) has been significantly increased, but the malignant risk is still controversial among different studies. The aim of this study is to investigate the malignancy risk of thyroid nodules reported as Bethesda category III (AUS/FLUS) on initial FNA. Method: We reviewed 272 papillary thyroid cancer (PTC) patients with suspicious thyroid nodules who underwent fine-needle aspiration and received surgical treatment during 2019 to 2020. Results: One hundred ten (40.4%) patients were diagnosed with PTC. Multivariate analysis showed that microcalcification (p = 0.037, OR = 2.260, 95% CI: 1.051-4.860), shape (p = 0.003, OR = 4.367, 95% CI: 1.629-11.705), diameters (p = 0.002, OR = 0.278, 95% CI: 0.123-0.631), anti-thyroglobulin antibodies (TGAb) (p = 0.002, OR = 0.150, 95% CI: 0.046-0.494), anti-thyroid peroxidase antibody (A-TPO) (p = 0.009, OR = 4.784, 95% CI: 1.486-15.401), and nodule goiter (p < 0.001, OR = 0.100, 95% CI: 0.046-0.217) were independent malignant risk factors in patients with thyroid nodule classified as Bethesda category III. Conclusion: In this study, malignant risk factors in patients with thyroid nodule classified as Bethesda category III were significantly associated with preoperative serum TGAb, A-TPO, microcalcification, irregular shape, and nodule diameters. Nodules with malignant factors should be carefully elevated; surgery may be the better option for those patients.

Predictive Factor of Large-Volume Central Lymph Node Metastasis in Clinical N0 Papillary Thyroid Carcinoma Patients Underwent Total Thyroidectomy.

Large-volume central lymph node metastasis (large-volume CLNM) is associated with high recurrence rate in papillary thyroid carcinoma (PTC) patients. However, sensitivity in investigating large-volume CLNM on preoperative ultrasonography (US) is not high. The aim of this study is to investigate the clinical factors associated with large-volume CLNM in clinical N0 PTC patients. We reviewed 976 PTC patients undergoing total thyroidectomy with central lymph node dissection during 2017 to 2019. The rate of large-volume LNM was 4.1% (40 of 967 patients). Multivariate analysis showed that male gender and young age (age<45 years old) were independent risk factors for large-volume CLNM with odds ratios [(OR), 95% confidence interval (CI)] of 2.034 (1.015-4.073) and 2.997 (1.306-6.876), respectively. In papillary thyroid microcarcinoma (PTMC), capsule invasion was associated with large-volume CLNM with OR (95% CI) of 2.845 (1.110-7.288). In conventional papillary thyroid cancer (CPTC), tumor diameter (>2cm) was associated with large-volume CLNM, with OR (95% CI) 3.757 (1.061-13.310), by multivariate analysis. In ROC curve analysis on the diameter of the CPTC tumor, the Area Under Curve (AUC) =0.682(p=0.013), the best cut-off point was selected as 2.0cm. In conclusion, male gender and young age were predictors for large-volume CLNM of cN0 PTC. cN0 PTMC patient with capsule invasion and cN0 CPTC patient with tumor diameter >2cm were correlated with large-volume CLNM. Total thyroidectomy with central lymph node dissection may be a favorable primary treatment option for those patients.

Predictive factors of contralateral occult carcinoma in patients with papillary thyroid carcinoma: a retrospective study.

BACKGROUND: The surgical approach toward unilateral papillary thyroid carcinoma (PTC) has been in controversy. One of the concerns is the existence of contralateral occult carcinoma, which could cause relapse and even lead to re-operation if not dealt with. This study aims to find out risk factors related to contralateral occult PTC, in order to facilitate in surgical approach decision for PTC. METHODS: A total of 921 PTC patients who underwent total/near-total thyroidectomy and central lymph node dissection (CND) with/without lateral lymph node dissection (LND) from January 2014 to Sept 2017 in Guangdong General Hospital were assessed retrospectively. The relations between contralateral occult PTC and clinicopathologic characteristics of PTC were analyzed by univariate and multivariate logistic regression. RESULTS: The incidence of contralateral occult carcinoma in patients with PTC was 16.7% (154 of 921 cases). Univariate analysis showed that multifocality of the primary carcinoma (P=0.000), lymph node metastasis (P=0.001), pathologic tumor size (P=0.014) and contralateral benign nodule (P=0.000) were significantly associated with the increased incidence of contralateral occult PTC. No significant correlations were found between contralateral carcinoma and other variables such as gender (P=0.338), age (P=0.283), BRAF mutation (P=0.187) or extrathyroidal extension (P=0.423). Multivariate logistic regression analysis revealed that contralateral benign nodule (P=0.000), multifocality (P=0.000) and lymph node metastasis (P=0.009) were independent predictors of bilateral PTC of patients whose pre-operation ultrasound (US) show a unilateral carcinoma. CONCLUSIONS: Lymph node metastasis, contralateral benign nodule and multifocality are independent predictors of contralateral occult PTC. For unilateral PTC patients with one or more of these factors, total/near-total thyroidectomy should be considered when making surgical approach decisions.

Predictive factors of lateral lymph node metastasis in conventional papillary thyroid carcinoma.

BACKGROUND: Lymph node metastasis (LNM) has been a significant predictor for local recurrence and distant metastasis in papillary thyroid carcinoma (PTC). However, the clinical predictors for conventional papillary thyroid carcinoma (CPTC) with lateral lymph node metastasis (LLNM) have yet to be established. Our work aimed to effectively identify the risk factors of LLNM in CPTC, helping surgeons to devise better individualized therapy strategies. METHODS: We retrospectively analyzed 652 patients who were diagnosed with CPTC in the Department of General Surgery at Guangdong Provincial People's Hospital between October 2015 and June 2019. Univariate and multivariate logistic regression was used to analyze the correlation between clinicopathological characteristics and the LLNM of CPTC. RESULTS: The incidence of LLNM in CPTC was 29.75% (194 of 652 patients). Univariate analysis found that LLNM was significantly associated with gender, age, number of central lymph nodes (CLNs), primary tumor location, multifocality, maximum tumor diameter, and BRAF V600E mutation. Independent risk factors for LLNM were multifocality (P=0.017, OR =4.325, 95% CI: 1.298-14.408), maximum tumor diameter (P<0.001, OR =4.076, 95% CI: 1.945-8.540), primary tumor location (P=0.037, OR =2.127, 95% CI: 1.046-4.328), number of CLNs (P<0.001, OR =8.604, 95% CI: 3.630-20.390), and BRAF V600E mutation (P=0.001, OR =3.913, 95% CI: 1.803-8.496) by multivariate analysis. CONCLUSIONS: LLNM in CPTC was related to the large volume of central lymph node metastasis (CLNM), tumor located in the upper lobe, multifocality, tumor diameter >2 cm, and BRAF V600E mutation-negative, which should be considered when examining patients with suspected metastasis in the lateral compartment.

Preoperative serum thyroglobulin is a risk factor of skip metastasis in papillary thyroid carcinoma.

BACKGROUND: Lymph node metastasis of papillary thyroid carcinoma (PTC) was a significant risk factor of local recurrence and distant metastasis, and our study aimed to identify predictive factors of skip metastasis in PTC, helping surgeons to build a strategy when facing patients in N1b stage with clinical central lymph node-negative. METHODS: A total of 304 patients who underwent total thyroidectomy with central and lateral lymph node dissection and were diagnosed PTC with lateral lymph node metastasis (LLNM) in the Department of General Surgery at Guangdong Provincial People's Hospital were enrolled. We collected clinicopathological characteristics and analyzed their correlation with skip metastasis by univariate and multivariate analysis. RESULTS: The incidence of skip metastasis in PTC was 14.8%. Univariate analysis showed that age, tumor diameter, primary tumor location, and preoperative serum thyroglobulin (Tg) were risk factors. Age (P=0.049, OR =3.418), primary tumor location (P<0.001, OR =7.279), and Tg (P=0.038, OR =9.412) were independent predictors in PTC by multivariate analysis. CONCLUSIONS: Skip metastasis of PTC was significantly associated with preoperative serum Tg ≤77 ng/mL, tumor diameter ≤10 mm, age ≥55, and tumor located in the upper lobe. It is the first time to demonstrate that Tg is associated with skip metastasis of PTC.

Genomic ERBB2/ERBB3 mutations promote PD-L1-mediated immune escape in gallbladder cancer: a whole-exome sequencing analysis.

OBJECTIVES: Patients with gallbladder carcinoma (GBC) lack effective treatment methods largely due to the inadequacy of both molecular characterisation and potential therapeutic targets. We previously uncovered a spectrum of genomic alterations and identified recurrent mutations in the ErbB pathway in GBC. Here, we aimed to study recurrent mutations of genes and pathways in a larger cohort of patients with GBC and investigate the potential mechanisms and clinical significance of these mutations. DESIGN: We performed whole-exome sequencing (WES) in 157 patients with GBC. Functional experiments were applied in GBC cell lines to explore the oncogenic roles of ERBB2/ERBB3 hotspot mutations, their correlation with PD-L1 expression and the underlying mechanisms. ERBB inhibitors and a PD-L1 blocker were used to evaluate the anticancer activities in co-culture systems in vitro and in vivo. RESULTS: WES identified ERBB2 and ERBB3 mutations at a frequency of 7%-8% in the expanded cohort, and patients with ERBB2/ERBB3 mutations exhibited poorer prognoses. A set of in vitro and in vivo experiments revealed increased proliferation/migration on ERBB2/ERBB3 mutation. Ectopic expression of ERBB2/ERBB3 mutants upregulated PD-L1 expression in GBC cells, effectively suppressed normal T-cell-mediated cytotoxicity in vitro through activation of the PI3K/Akt signalling pathway and contributed to the growth and progression of GBC in vivo. Treatment with an ERBB2/ERBB3 inhibitor or a PD-L1 monoclonal antibody reversed these immunosuppressive effects, and combined therapy revealed promising therapeutic activities. CONCLUSIONS: ERBB2/ERBB3 mutations may serve as useful biomarkers in identifying patients who are sensitive to ERBB2/ERBB3 inhibitors and PD-L1 monoclonal antibody treatment. TRIAL REGISTRATION NUMBER: NCT02442414;Pre-results.

MicroRNA-30a-5p inhibits gallbladder cancer cell proliferation, migration and metastasis by targeting E2F7.

Gallbladder carcinoma (GBC), the most common malignant tumour of the bile duct, is highly aggressive and has a poor prognosis. MicroRNA-30a-5p (miR-30a-5p) is an important tumour suppressor that participates in many aspects of carcinogenesis and cancer development. However, the role of miR-30a-5p in GBC development remains to be determined, as do the mechanisms underlying its effects in GBC. Using samples collected from 42 subjects with gallbladder carcinoma (GBC), we showed decreased miR-30a-5p expression in the primary lesions vs. non-tumour adjacent tissues (NATs). Decreased miR-30a-5p was associated with shorter disease-free survival (DFS) and overall survival (OS). Inhibiting miR-30a-5p expression in 2 representative GBC cell lines (GBC-SD and NOZ) increased cell proliferation, migration, invasiveness, as well as ß-catenin nuclear translocation, vice versa. In nude mice, NOZ cells transfected with miR-30a-5p mimics grew slower (vs. miR-NC) upon subcutaneous inoculation, and had lower rate of hepatic metastasis upon spleen inoculation. Dual luciferase assay confirmed that E2F transcription factor 7 (E2F7) was a direct target of miR-30a-5p and antagonized the effects induced by miR-30a-5p downregulation in GBC cells. MiR-30a-5p attenuates the EMT and metastasis in GBC cells by targeting E2F7, suggesting miR-30a-5p is a tumour suppressor that may serve as a novel potential prognostic biomarker or molecular therapeutic target for GBC.

miR-143-3p targeting of ITGA6 suppresses tumour growth and angiogenesis by downregulating PLGF expression via the PI3K/AKT pathway in gallbladder carcinoma.

Gallbladder cancer (GBC) is the most common malignant tumour of the biliary track system. Angiogenesis plays a pivotal role in the development and progression of malignant tumours. miR-143-3p acts as a tumour suppressor in various cancers. Their role in GBC is however less well defined. Here we show that the expression levels of miR-143-3p were decreased in human GBC tissues compared with the non-tumour adjacent tissue (NAT) counterparts and were closely associated with overall survival. We discovered that miR-143-3p was a novel inhibitor of tumour growth and angiogenesis in vivo and in vitro. Our antibody array, ELISA and PLGF rescue analyses indicated that PLGF played an essential role in the antiangiogenic effect of miR-143-3p. Furthermore, we used miRNA target-prediction software and dual-luciferase assays to confirm that integrin α6 (ITGA6) acted as a direct target of miR-143-3p. Our ELISA and western blot analyses confirmed that the expression of PLGF was decreased via the ITGA6/PI3K/AKT pathway. In conclusion, miR-143-3p suppresses tumour angiogenesis and growth of GBC through the ITGA6/PI3K/AKT/PLGF pathways and may be a novel molecular therapeutic target for GBC.

STYK1 promotes cancer cell proliferation and malignant transformation by activating PI3K-AKT pathway in gallbladder carcinoma.

Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract with extremely poor prognosis. The malignant transformation of GBC is associated with cell proliferation, invasion, and epithelial-mesenchymal transition (EMT). However, the molecular mechanisms underlying GBC progression are poorly understood. We found that serine threonine tyrosine kinase 1 (STYK1) was elevated in GBC and was negatively correlated with clinical outcomes and prognosis. Overexpression of STYK1 in GBC cell lines gave rise to increased cell proliferation, colony formation, migration and invasion, thus committing cells to undergoing EMT. In contrast, silence of STYK1 led to opposite effects on cell transformation. Consistent with STYK1 gene knockdown, AKT specific inhibitor MK2206 abrogated tumor promoting action induced by STYK1, suggesting that PI3K/AKT pathway is essential for the oncogenic role of STYK1 in GBC. STYK1 shRNA in GBC cells inhibited development of xenografted tumors compared with control cells. Collectively, our findings suggest that STYK1 is a critical regulator of tumor growth and metastasis, and may serve as a potential target for GBC therapy.

Chloride intracellular channel 1 regulates the antineoplastic effects of metformin in gallbladder cancer cells.

Metformin is the most commonly used drug for type 2 diabetes and has potential benefit in treating and preventing cancer. Previous studies indicated that membrane proteins can affect the antineoplastic effects of metformin and may be crucial in the field of cancer research. However, the antineoplastic effects of metformin and its mechanism in gallbladder cancer (GBC) remain largely unknown. In this study, the effects of metformin on GBC cell proliferation and viability were evaluated using the Cell Counting Kit-8 (CCK-8) assay and an apoptosis assay. Western blotting was performed to investigate related signaling pathways. Of note, inhibition, knockdown and upregulation of the membrane protein Chloride intracellular channel 1 (CLIC1) can affect GBC resistance in the presence of metformin. Our data demonstrated that metformin apparently inhibits the proliferation and viability of GBC cells. Metformin promoted cell apoptosis and increased the number of early apoptotic cells. We found that metformin can exert growth-suppressive effects on these cell lines via inhibition of p-Akt activity and the Bcl-2 family. Notably, either dysfunction or downregulation of CLIC1 can partially decrease the antineoplastic effects of metformin while upregulation of CLIC1 can increase drug sensitivity. Our findings provide experimental evidence for using metformin as an antitumor treatment for gallbladder carcinoma.