Simultaneous detection of two subtypes of extracellular vesicles using ultrabright fluorescent nanosphere-based test strips.

In recent years, the cargo profiles of extracellular vesicles (EVs), which were inherited from their parent cells, have emerged as a reliable biomarker for liquid biopsy (LB) in disease diagnosis, prognosis, and treatment monitoring. EVs secreted by different cells exhibit distinct characteristics, particularly in terms of disease diagnosis and prediction. However, currently available techniques for the quantitative analysis of EV cargoes, including enzyme-linked immunosorbent assay (ELISA), cannot specifically identify the cellular origin of EVs, thus seriously affecting the accuracy of EV-based liquid biopsy. In light of this, we here developed ultrabright fluorescent nanosphere (FNs)-based test strips which have the unique capability to specifically assess the levels of PD-L1-positive EVs (PD-L1+ EVs) derived from both tumor cells and immune cells in bodily fluids. The levels of PD-L1+ EV subpopulations in human saliva were quantified using the ultrabright fluorescent nanosphere-based test strips with more convenience and higher efficiency (detection time <30 min). Results demonstrated that the fluorescence intensity of the test line exhibited a good linear relationship respectively with the PD-L1 levels of tumor cell- (R2 = 0.993) and immune cell-derived EVs (R2 = 0.982) in human saliva. By assessing the levels of PD-L1+ EV subpopulations, our test strips hold immense potential for advancing the application of PD-L1+ EV subpopulation-based predictions in tumor diagnosis and prognosis evaluation. In summary, by integrating the benefits of FNs and lateral flow chromatography, we here provide a strategy to accurately measure the cargo levels of EVs originating from diverse cell sources in bodily fluids.

Association of DAZ and DAZLA Gene Loci with Spermatogenesis in Patients with Idiopathic Azoospermia and Severe Oligospermia.

Aim: To explore the relationship between DAZ (Deleted in Azoospermia, DAZ) and DAZLA (Deleted in Azoospermia-like autosomal) gene deletion and male idiopathic azoospermia and oligozoospermia. Methods: 80 patients with azoospermia (azoospermia group) and 80 patients with oligozoospermia (oligozoospermia group) who were treated at our hospital from April 2021 to April 2023, and male volunteers who underwent health examinations at our hospital during the same period were selected as the control group, The incidence of DAZ and DAZLA gene locus deletion in three groups of men was detected by polymerase chain reaction (PCR), and the differences of reproductive hormone levels and main semen parameters among the three groups were compared. The azoospermia were stratified according to whether DAZ and DAZLA gene locus deletion occurred. Results: DAZ gene locus deletion rate in azoospermia and oligospermia groups was considerably higher than in the control group (P < .05). The DAZLA gene locus deletion rate in the azoospermia group was apparently higher than that in the oligospermia and control groups (P < .05). The semen volume was compared between azoospermia and oligospermia patients and controls (P > .05). Sperm concentration, sperm survival rate, the proportion of normal morphological sperm, and the proportion of progressive motility sperm in the oligospermia group were lower than those in the control group (P < .05). The levels of serum T (Testosterone, T) and T/LH in the azoospermiaspermia group were lower than those in the control group (P < .05). Serum LH (Luteinizing Hormone) and FSH (Follicular Stimulating Hormone) in azoospermia group and oligospermia group were higher than those in the control group (P < .05). The Serum LH determination value of the azoospermia group is higher than the oligospermia group (P < .05). Serum T/LH in the azoospermia group was lower than in the oligospermia group (P < .05). The serum T and T/LH values in azoospermia male patients with DAZ and DAZLA gene deletion were lower than those without deletion (P < .05). Sperm concentration and survival rate of oligospermatism male patients with DAZ gene deletion were lower than those without deletion (P < .05). Sperm and serum T and T/LH values of oligospermatism male patients with DAZ gene deletion were lower than those without deletion (P < .05). Conclusion: The incidence of DAZ and DAZLA gene locus deletion in male patients with idiopathic azoospermia and oligozoospermia was higher than in normal males. The gene locus deletion was related to decreased androgen level, sperm count and motility.

Spatiotemporal characteristics of tissue derived small extracellular vesicles is associated with tumor relapse and anti-PD-1 response.

Small extracellular vesicles (sEVs) residing at tumor tissues are valuable specimens for biopsy. Tumor heterogeneity is common across all cancer types, but the heterogeneity of tumor tissue-derived sEVs (Ti-sEVs) is undefined. This study aims to discover the spatial distributions of Ti-sEVs in oral squamous cell carcinoma (OSCC) tissues and explore how these vesicle distributions affect the patients' prognosis. Multi-regional sampling enabled us to uncover that Ti-sEVs' accumulation at peritumoral sites correlates with a higher disease-free survival rate, and conversely, sparse peritumoral Ti-sEVs tend to forecast a higher risk of relapse. Of those relapsed patients, Ti-sEVs strongly bind to extracellular matrix and subsequently degrade it for allowing themselves enter the bloodstream rather than staying in situ. In advanced OSCC patients, the quantity and spatial distribution of Ti-sEVs prior to anti-PD-1 treatment, as well as the temporal variance of Ti-sEVs before and after immunotherapy, strongly map the clinical response and can help to distinguish the patients with shrinking tumors from those with growing tumors. Our work elucidates the correlation of spatiotemporal features of Ti-sEVs with patients' therapeutic outcomes and exhibit the potential for using Ti-sEVs as a predictor to forecast prognosis and screen the responders to anti-PD-1 therapy.

Tolerogenic Nano-/Microparticle Vaccines for Immunotherapy.

Autoimmune diseases, allergies, transplant rejections, generation of antidrug antibodies, and chronic inflammatory diseases have impacted a large group of people across the globe. Conventional treatments and therapies often use systemic or broad immunosuppression with serious efficacy and safety issues. Tolerogenic vaccines represent a concept that has been extended from their traditional immune-modulating function to induction of antigen-specific tolerance through the generation of regulatory T cells. Without impairing immune homeostasis, tolerogenic vaccines dampen inflammation and induce tolerogenic regulation. However, achieving the desired potency of tolerogenic vaccines as preventive and therapeutic modalities calls for precise manipulation of the immune microenvironment and control over the tolerogenic responses against the autoantigens, allergens, and/or alloantigens. Engineered nano-/microparticles possess desirable design features that can bolster targeted immune regulation and enhance the induction of antigen-specific tolerance. Thus, particle-based tolerogenic vaccines hold great promise in clinical translation for future treatment of aforementioned immune disorders. In this review, we highlight the main strategies to employ particles as exciting tolerogenic vaccines, with a focus on the particles' role in facilitating the induction of antigen-specific tolerance. We describe the particle design features that facilitate their usage and discuss the challenges and opportunities for designing next-generation particle-based tolerogenic vaccines with robust efficacy to promote antigen-specific tolerance for immunotherapy.

Aptamer-Assisted Traceless Isolation of PD-L1-Positive Small Extracellular Vesicles for Dissecting Their Subpopulation Signature and Function.

Small extracellular vesicles (sEVs) are heterogeneous membrane-bound vesicles that carry numerous bioactive molecules. Studies have reported that sEVs carrying PD-L1 on the surface could contribute to immunosuppression; however, the precise mechanisms are unclear. To fully dissect their mode of action, it requires qualified methods to specifically isolate natural PD-L1-positive sEVs from heterogeneous sEVs. This study reported an aptamer-assisted capture-and-release strategy for traceless isolation of PD-L1-positive sEVs. The PD-L1 aptamer-anchored magnetic microspheres enable the specific capture of PD-L1-positive sEVs. The traceless release of captured PD-L1-positive sEVs was triggered by competition of complementary oligonucleotides, endowing the obtained label-free PD-L1-positive sEVs with natural properties. Benefited from this traceless isolation strategy, the distinct molecule profiles in adhesion and immuno-regulation between PD-L1-positive and PD-L1-negative sEVs were revealed. Compared to PD-L1-negative sEVs, PD-L1-positive sEVs were much more concentrated in cadherin binding, accompanied by increased adhesion to lymphatic endothelial cells and T cells but decreased adhesion to the extracellular matrix. Moreover, PD-L1-positive sEVs could transfer their enriched immunosuppressive "synapse"-related proteins to antigen-presenting cells, thereby inducing a tolerogenic-like phenotype. In summary, the present work dissects the subpopulation signature and action mode of PD-L1-positive sEVs for the first time and provides a general approach to the traceless isolation of sEV subpopulations.

Interferon-γ induces immunosuppression in salivary adenoid cystic carcinoma by regulating programmed death ligand 1 secretion.

Interferon-γ (IFN-γ), a key effector molecule in anti-tumor immune response, has been well documented to correlate with the intratumoral infiltration of immune cells. Of interest, however, a high level of IFN-γ has been reported in salivary adenoid cystic carcinoma (SACC), which is actually a type of immunologically cold cancer with few infiltrated immune cells. Investigating the functional significance of IFN-γ in SACC would help to explain such a paradoxical phenomenon. In the present study, we revealed that, compared to oral squamous cell carcinoma cells (a type of immunologically hot cancer), SACC cells were less sensitive to the growth-inhibition effect of IFN-γ. Moreover, the migration and invasion abilities of SACC cells were obviously enhanced upon IFN-γ treatment. In addition, our results revealed that exposure to IFN-γ significantly up-regulated the level of programmed death ligand 1 (PD-L1) on SACC cell-derived small extracellular vesicles (sEVs), which subsequently induced the apoptosis of CD8+ T cells through antagonizing PD-1. Importantly, it was also found that SACC patients with higher levels of plasma IFN-γ also had higher levels of circulating sEVs that carried PD-L1 on their surface. Our study unveils a mechanism that IFN-γ induces immunosuppression in SACC via sEV PD-L1, which would account for the scarce immune cell infiltration and insensitivity to immunotherapy.

Untouched isolation enables targeted functional analysis of tumour-cell-derived extracellular vesicles from tumour tissues.

To accurately identify the functions of tumour-cell-derived extracellular vesicles (T-EVs), EVs directly isolated from tumour tissues are much preferred over those derived from in vitro cultured tumour cell lines. However, the functional analysis of T-EVs has still been severely limited by the difficulty in selective isolation of T-EVs from tissue-derived heterogeneous EVs, which also contain non-tumour cell-derived EVs. We here establish an untouched isolation strategy that specifically collects natural T-EVs from tumour tissues by removing non-tumour-cell-derived EVs. Different from traditional immunomagnetic separation, our isolation materials are directly bound to undesired non-tumour-cell-derived EVs, preserving the natural properties of T-EVs. Using this strategy, we reveal the distinct performances of tissue-derived T-EVs in organotropism to lymph nodes, immunosuppression and angiogenesis. The present work, which takes an extraordinary step forward in the isolation of EV subpopulation from tumour tissues, would dramatically accelerate the investigation of EV heterogeneity.

Facile synthesis of peanut-like Sn-doped silica nano-adsorbent for affinity separation of proteins.

A peanut-like hollow silica (denoted as p-l-hSiO2) adsorbent is prepared in a facile method, which is composed of several silica nanospheres and has an average diameter of 22 nm, with thickness of 5 nm. Its Brunauer-Emmett-Teller (BET) surface area, pore volume and pore size are 258.9 m2 g-1, 1.56 cm3 g-1 and 3.9 nm, respectively. Then the afforded p-l-hSiO2/GSH adsorbent is applied to purify glutathione S-transferases-tagged (denoted as GST-tagged) proteins. It is found that the p-l-hSiO2 adsorbent exhibits a specific adsorption, a high binding capacity (6.80 mg g-1), good recycling performance and high recovery (90.1%) to the target proteins, showing promising potential for the affinity separation of GST-tagged proteins.

[Expression and clinical significance of lymphocyte subsets and NK cell activating receptors in peripheral blood of patients with non-small cell lung cancer].

Objective To explore the expression and clinical significance of lymphocyte subsets distribution, NK cell activating receptors and regulatory T cells (Tregs) in the peripheral blood of patients with non-small cell lung cancer (NSCLC). Methods A total of 42 NSCLC patients were selected and divided into stage I+II group and stage III+IV group according to tumor stage, or well-differentiated group, moderately-differentiated group and poorly-differentiated group according to the degree of differentiation. Noncancerous lesion group and healthy control group were also set up. Flow cytometry was used to detect the expression levels of lymphocyte subsets, NK cell cytotoxic receptor 3 (NCR3/NKp30), NCR1/NKp46 and Tregs in the peripheral blood. Results Compared with the healthy control group, the proportion of CD3+ T cells significantly increased in the stage III+IV group, the proportion of CD3+CD4+ T cells also significantly increased in the stage III+IV group and the well-differentiated group, and the proportion of Treg in CD4+ T cells went up in all stage groups and poorly-differentiated group. The proportion of NK cells was significantly up-regulated in the stage I+II group and the moderately-differentiated group. The expression of NKp30 and NKp46 on the surface of NK cells was significantly reduced in all stage groups, moderately- and poorly-differentiated groups. Conclusion The proportions of T cells, Tregs and NK cells increase, but the levels of activating receptors on NK cells decrease in the peripheral blood of patients with NSCLC.

Feasibility and quality control of robotic sleeve lobectomy and bronchoplasty / 中国胸心血管外科临床杂志

@#Objective    To explore the feasibility of robotic sleeve lobectomy and bronchoplasty and to summarize the experience of quality control and technical process management. Methods    From January to December 2018, our hospital completed robotic sleeve lobectomy and bronchoplasty for 5 patients, including the upper right lung lobe in 2 patients, the middle right lung lobe in 1 patient and the lower left lung lobe in 2 patients. There were 3 males and 2 females with an age of 56.6 (39-75) years. The surgical approach was the same as the surgical incision of the robotic lobectomy. During the operation, the lobes were separated, all enlarged mediastinal lymph nodes were cleaned, pulmonary hilum was dissected, pulmonary arteriovenous vessels and bronchi were exposed, and pulmonary vessels were treated. After exposing the main bronchi, the bronchi were cut off at the distal end of the lesion, and the lobes where the lesion was located (including lesions) were excised by sleeve type and the bronchi were continuously sutured with 3-0 Prolene from the back wall for anastomosis. After the anastomosis, no air leakage was found in the expanded lung, and the anastomosis was no longer wrapped. Results    The operation time was 147.4 (100-192) min, including bronchial anastomosis time 17.6 (14-25) min. Intraoperative blood loss was 60.0 (20-100) mL, and 20 (9-37) lymph nodes were dissected. Three patients had squamous cell carcinoma, 1 adenocarcinoma, and 1 neuroendocrine tumor. All patients showed negative results in the freezing pathology of bronchial stump during operation. All patients recovered well after surgery, without perioperative complications, and the anastomosis was smooth. Postoperative hospital stay was 10.8 (7-14) days. The patients were followed up for 6 to 12 months without anastomotic stenosis or other complications. Conclusion    Since the robot system is a special instrument with 3D vision and 7 degrees of freedom for movable joints, the robotic bronchial suture is more flexible and accurate. The robotic sleeve lobectomy and bronchoplasty are safe and feasible.

Long-term outcome of robotic versus video-assisted thoracic surgery for stageⅠ lung adenocarcinoma: A propensity score matching study / 中国胸心血管外科临床杂志

@#Objective    To compare the the effectiveness of robot-assisted thoracic surgery (RATS) with video-assisted thoracic surgery (VATS), in stageⅠ lung adenocarcinoma. Methods    From January 2012 to December 2018, 291  patients were included. The patients were allocated into two groups including a RATS group with 125 patients and a VATS group with 166 patients. Two cohorts (RATS, VATS ) of clinical stageⅠ lung adenocarcinoma patients were matched by propensity score. Then there were 114 patients in each group (228 patients in total). There were 45 males and 69 females at age of 62±9 years in the RATS group; 44 males, 70 females at age of 62±8 years in the VATS group. Overall survival (OS) and disease-free survival (DFS) were assessed. Univariate and multivariate analyses were performed to identify factors associated with the outcomes. Results     Compared with the VATS group, the RATS group got less blood loss (P<0.05) and postoperative drainage (P<0.05) with a statistical difference. There was no statistical difference in drainage time (P>0.05) or postoperative hospital stay (P>0.05) between the two groups. The RATS group harvested more stations and number of the lymph nodes with a statistical difference (P<0.05). There was no statistical difference in 1-year, 3-year and 5-year OS and mean survival time (P>0.05). While there was a statistical difference in DFS between the two groups (1-year DFS: 94.1% vs. 95.6%; 3-year DFS: 92.6% vs. 75.2%; 5-year DFS: 92.6% vs. 68.4%, P<0.05; mean DFS time: 78 months vs. 63 months, P<0.05) between the two groups. The univariate analysis found that the number of the lymph nodes dissection was the prognostic factor for OS, and tumor diameter, surgical approach, stations and number of the lymph nodes dissection were the prognostic factors for DFS. However, multivariate analysis found that there was no independent risk factor for OS, but the tumor diameter and surgical approach were independently associated with DFS. Conclusion    There is no statistical difference in OS between the two groups, but the RATS group gets better DFS.

lncRNA LINC00460 promoted colorectal cancer cells metastasis via miR-939-5p sponging.

BACKGROUND: lncRNAs are widely involved in multiple malignancies including colorectal cancer (CRC). The expression and function of long intergenic non-protein coding RNA 460 (LINC00460) in CRC remains obscure. METHODS: In the present study, quantitative real-time PCR assays were applied to detect the expression changes of LINC00460 and microRNA-939-5p (miR-939-5p) in CRC tissue specimens and cell lines. Western blot assays were used to measure the changes of LIMK2. Bioinformatics analysis, luciferase assays, and RNA pull-down assays were applied to determine the targeting binding effect between LINC00460 and miR-939-5p as well as LIMK2 and miR-939-5p. Transwell assays were used to evaluate the metastatic ability changes of CRC line HT29 and LOVO cells. RESULTS: We found that LINC00460 was upregulated and closely correlated to clinicopathological features and poor prognosis of patients with CRC. Functionally, we elucidated that LINC00460 promoted metastasis in CRC cell lines HT29 and LOVO. Further, we showed that LIMK2 was a downstream effector in the LINC00460-induced promotion of metastasis in CRC cells HT29 and LOVO. Through online bioinformatics analysis, LINC00460 and LIMK2 were demonstrated to share similar microRNA response elements for miR-939-5p. Then, LINC00460 and LIMK2 were verified to be the targets of miR-939-5p via a luciferase assay and an RNA pull-down assay. Also, miR-939-5p was showed to suppress metastasis by targeting of LIMK2. Lastly, we revealed that LINC00460 promoted LIMK2-mediated metastasis via miR-939-5p sponging in CRC cells HT29 and LOVO. CONCLUSION: The findings of this study showed that LINC00460 works as an oncogene in CRC and promoted CRC cell metastasis via regulation of miR-939-3p/LIMK2 axial. The present study might provide a new target in treating CRC.

Retrospective analysis of mediastinal mass resection with totally no tube during da Vinci robotic surgery for 79 patients / 中国胸心血管外科临床杂志

@#Objective To analyze the feasibility of totally no tube (TNT) in da Vinci robotic mediastinal mass surgery and its significance for fast track surgery. Methods A total of 79 patients receiving robotic mediastinal TNT surgery in the General Hospital of Shenyang Military Command from January 2016 to December 2017 were enrolled as a TNT group; 35 patients receiving robotic mediastinal surgery in General Hospital of Shenyang Military Command from January 2014 to December 2017 and 54 patients receiving thoracoscopic mediastinal surgery during the same period were enrolled as a non-TNT group and a video-assisted thoracoscopic surgery (VATS) group. The muscle relaxation and tracheal intubation/laryngeal masking time, operation time, intraoperative blood loss, postoperative ICU stay, postoperative hospital stay, postoperative visual analogue scale (VAS), hospitalization costs and postoperative complications and other related indicators were retrospectively analyzed among the three groups. Results Surgeries were successfully completed in 168 patients with no transfer to thoracotomy, serious complications (postoperative complications in 9 patients) or death during the perioperative period. All patients were discharged. Compared with the non-TNT group, the TNT group had significantly less muscle relaxation-tracheal intubation/laryngeal masking time, operation time, intraoperative blood loss, VAS pain score, ICU stay, postoperative hospital stay (P<0.01); there was no significant difference in the total cost of hospitalization between the two groups (P>0.05). Between the non-TNT group and the VATS group, there was no significant difference in time of muscle relaxation and tracheal intubation, operation time and ICU stay (P>0.05). The non-TNT group was superior to the VATS group in terms of intraoperative blood loss, VAS pain scores on the following day after operation, chest drainage volume 1-3 days postoperatively, postoperative catheterization time and postoperative hospital stay (P<0.05); but the cost of hospitalization in the non-TNT group was significantly higher (P=0.000). Conclusion The da Vinci robot is safe and feasible for the treatment of mediastinal masses. At the same time, TNT is also safe and reliable on the basis of robotic surgery which has many advantages such as better comfort, less pain, ICU stay and hospital stay as well as faster recovery.

[Survival Analysis of Stage I Non-small Cell Lung Cancer Patients Treated with 
Da Vinci Robot-assisted Thoracic Surgery].

BACKGROUND: Da Vinci robotic surgery system is widely used in department of thoracic surgery. The aim of this study is to investigate the treatment outcome of stage I non-small cell lung cancer (NSCLC) via da Vinci Surgical System. METHODS: Clinical date of 347 stage I NSCLC patients, who underwent lobectomy and systematic node dissection from Jan. 2012 to Dec. 2017, were reviewed. 134 patients underwent robot-assisted thoracic surgery (RATS) and 213 patients underwent video-assisted thoracic surgery (VATS). To compare perioperative outcome (blood lose, postoperative drainage, drainage time, postoperative hospital stay, number of the LN dissection) and analyze overall survival (OS), disease free survival (DFS) of the two groups and prognostic factors. RESULTS: The RATS group got less blood lose [(49±39 mL for RATS vs (202±239) mL for VATS, P<0.05] and postoperative drainage [Day 1: (248±123) mL for RATS vs (350±213) mL for VATS; Day 2: (288±189) mL for RATS vs (338±189) mL for VATS, P<0.05]. There were no significant difference for drainage time (10±5 for RATS vs 11±8 for VATS, P<0.05) and postoperative hospital stay (13±6 for RATS vs 14±9 for VATS, P<0.05) between the two groups. The RATS group harvested a more number of mean stations (5±2 for RATS vs 4±2 for VATS) and amounts (18±9 for RATS vs 11±8 for VATS) of the lymph nodes, P<0.05. There was no statistically significant difference of OS between RATS and VATS group [1-year OS: 97.3% vs 96%; 3-year OS: 89.8% vs 83.1%; 5-year OS: 87.5 % vs 70.3%; overall survival time (mean): 61 months vs 59 months, P>0.05]; corresponding there had a statistically significant difference of DFS between the two groups [1-year DFS: 93.7% vs 91.3%; 3-year DFS: 87.7% vs 68.4%; 5-year DFS: 87.7% vs 52.5%; disease free survival time (mean): 61 months vs 50 months, P<0.05]. The univariate analysis found that the amounts of the lymph nodes dissection was the prognostic factor for OS and tumor diameter, surgical approach, stations and amounts of the lymph nodes dissection were respectively the prognostic factors for DFS. However, multivariate analysis found that there was not independently factors for OS, but the tumor diameter and surgical approach were independently associated with DFS. CONCLUSIONS: There was no significant difference about OS between the two groups, but the RATS got better DFS. RATS got more number of the LN dissection and less blood lose.

Surgical treatment of mediastinal tumors combined with myasthenia gravis: comparison of Da Vinci robot-assisted, video-assisted thoracoscopic surgery and median sternotomy / 中国胸心血管外科临床杂志

@#Objective To compare three surgical treatments for mediastinal mass with myasthenia gravis. Methods Retrospective analysis was performed on the clinical data of 53 patients who underwent extended thymectomy between January 2010 and December 2017 in our hospital. There were 29 males and 24 females, aged 17-73 years. Patients were divided into three groups according to the surgical methods: a group A (video-assisted thoracoscopic surgery with the da Vinci robotic system, n=22), a group B (video-assisted thoracoscopic surgery, n=12) and a group C (median sternotomy, n=19). The gender distribution, age, intraoperative blood loss, operation time, postoperative extubation time, postoperative hospital stay, Osserman classification of myasthenia gravis, postoperative myasthenic remission rate, etc were compared in three groups. Results No perioperative death was observed in 53 patients. One patient in the group C suffered from postoperative myasthenic crisis and improved after active treatment. One patient with video-assisted thoracoscopic surgery was converted to median sternotomy due to the intraoperative injury of the left brachiocephalic vein. Compared with the group B and group C, the group A had shorter operation time, less intraoperative blood loss and drainage on the first postoperative day and fewer days of extubation. Postoperative hospital stay was less in the group A than that in the group C (P<0.05). The postoperative myasthenic remission rate was higher in the group A than that in the other two groups, but there was no statistical difference. Conclusion Because of the robot’s unique minimally invasive advantage, in this study, the outcome of patients with myasthenia gravis treated with Da Vinci robots and thymectomy is better than that of the remaining two groups in terms of perioperative outcomes and myasthenic remission rate. But long-term results and a large of number matching experiments are needed to confirm. However, it is undeniable that robotic surgery must be the future of the minimally invasive surgery.